W. C. Nichols PhD*, N. Pankratz PhD, D. K. Marek BS, M. W. Pauciulo MBA, V. E. Elsaesser BS, C. A. Halter MS, A. Rudolph PhD, J. Wojcieszek MD, R. F. Pfeiffer MD, T. Foroud PhD, For the Parkinson Study Group–PROGENI Investigators
Objective: To characterize sequence variation within the glucocerebrosidase (GBA) gene in a select subset of our sample of patients with familial Parkinson disease (PD) and then to test in our full sample whether these sequence variants increased the risk for PD and were associated with an earlier onset of disease.
Methods: We performed a comprehensive study of all GBA exons in one patient with PD from each of 96 PD families, selected based on the family-specific lod scores at the GBA locus. Identified GBA variants were subsequently screened in all 1325 PD cases from 566 multiplex PD families and in 359 controls.
Results: Nine different GBA variants, five previously reported, were identified in 21 of the 96 PD cases sequenced. Screening for these variants in the full sample identified 161 variant carriers (12.2%) in 99 different PD families. An unbiased estimate of the frequency of the five previously reported GBA variants in the familial PD sample was 12.6% and in the control sample was 5.3% (odds ratio 2.6; 95% confidence interval 1.5–4.4). Presence of a GBA variant was associated with an earlier age at onset (p = 0.0001). On average, those patients carrying a GBA variant had onset with PD 6.04 years earlier than those without a GBA variant.
Conclusions: This study suggests that GBA is a susceptibility gene for familial Parkinson disease (PD) and patients with GBA variants have an earlier age at onset than patients with PD without GBA variants.
Wednesday, November 19, 2008
Deep Pockets Being Opened for Parkinson's Research
By: Lara Endreszl
Published: Monday, 22 September 2008
Parkinson’s disease (PD) has affected the global awareness by striking people within the public eye that we look up to and can empathize with. In 2003 I saw Pope John Paul II in an audience at the Vatican and under the weight of Parkinson’s, the leader of the Catholic Church had no power against the will of his own body; his hands shook instead of waved, his body was hunched over in pain, and his voice trembled with instability. Stung like a bee with the diagnosis in 1984, Mohammed Ali, an honored man and boxing legend, is now confined to a wheelchair and while he still attends functions as a living sports legacy, he nods and twitches his head and hands not to the words being spoken or the songs being sung but to the beat of the Parkinson’s resounding in his head; he hasn’t won this fight yet. Michael J. Fox, a beloved character for years from television to the future on the big screen and back again, is most often recognized now as the brave young actor who was diagnosed at age 30 and is still fighting against the disease’s crippling effects. With the exposure of Parkinson’s disease reaching high-calibers, it’s no wonder that The National Institute of Environmental Health Sciences (NIEHS)—a division of The National Institutes of Health (NIH)—is reaching deep into their pockets. It looks like the global awareness of the PD has sparked an interest in the reasons behind the diagnoses and what we can do to find a cure.
Over the next five years, NIEHS will grant $21.25 million to three research schools in the United States to fund studies relating to how environmental factors contribute to the cause, prevention, and treatment of Parkinson’s disease. A central nervous system disorder, Parkinson’s disease affects over one million Americans each year and the disease progresses with age. In most cases, scientists don’t know specifically what brings on the disease, but some cases are known to be caused by severe head trauma (such is the speculation in Mohammed Ali’s case) or patterns of genetic abnormalities. Parkinson’s is thought to be a derivative of genetic mutations and outside environmental causes. For example, prolonged exposure to pesticides is thought to double a person’s risk for the disease.
The three grantees are from respected research schools around the country and are using their grants to cover a specific area of researching the disease. Gary Miller, Ph.D., at Emory University, Atlanta, Georgia, receives a grant for prolonged research of how environmental and genetic factors change dopamine cells within the brain that lead to Parkinson’s disease. The second grant goes to Marie-Françoise Chesselet, M.D., Ph.D., at the University of California, Los Angeles, who plans to specifically research pesticides that may be the main cause of sporadic Parkinson’s diagnoses and possibly come up with a prevention plan by cautioning the use of certain pesticides. Lastly, Stuart Lipton, M.D., Ph.D., Burnham Institute for Medical Research in La Jolla, California, will investigate free radical stress caused by environmental toxins that cause genetic mutations responsible for helping to progress the disease and hopefully be able to isolate the body’s proteins damaged in the process.
Acting director of the Division of Extramural Research and Training at NIEHS, Dennis Lang, Ph.D., said of the grantees, “The UCLA and Emory CNS grants will extend the exciting lines of research previously supported by NIEHS,…while the Burnham Institute grant will bring an important new perspective to research on gene-environment interplay in Parkinson's disease.”
Perhaps the most influential person in my life who lived with Parkinson’s disease wasn’t a public figure and wasn’t world-renown for anything special; she was my grandmother. She raised seven children in a modest home, she was a do-gooder and revered by the community for her cooking talent and quilting techniques. As the years rolled by and she wasn’t able to stand up much less get out of the house, my dad’s mother spent her remaining years cooped up in a nursing home shaking and drooling, but always believed that she would walk again. That faith was finally lost in April 2005 when—the same day as Pope John Paul II—my grandmother passed on. With the generous research grants from NIEHS dedicated to finding a cure, I hope continuing investigations will be able to finally give those living with Parkinson’s the second chance at life they deserve.
Published: Monday, 22 September 2008
Parkinson’s disease (PD) has affected the global awareness by striking people within the public eye that we look up to and can empathize with. In 2003 I saw Pope John Paul II in an audience at the Vatican and under the weight of Parkinson’s, the leader of the Catholic Church had no power against the will of his own body; his hands shook instead of waved, his body was hunched over in pain, and his voice trembled with instability. Stung like a bee with the diagnosis in 1984, Mohammed Ali, an honored man and boxing legend, is now confined to a wheelchair and while he still attends functions as a living sports legacy, he nods and twitches his head and hands not to the words being spoken or the songs being sung but to the beat of the Parkinson’s resounding in his head; he hasn’t won this fight yet. Michael J. Fox, a beloved character for years from television to the future on the big screen and back again, is most often recognized now as the brave young actor who was diagnosed at age 30 and is still fighting against the disease’s crippling effects. With the exposure of Parkinson’s disease reaching high-calibers, it’s no wonder that The National Institute of Environmental Health Sciences (NIEHS)—a division of The National Institutes of Health (NIH)—is reaching deep into their pockets. It looks like the global awareness of the PD has sparked an interest in the reasons behind the diagnoses and what we can do to find a cure.
Over the next five years, NIEHS will grant $21.25 million to three research schools in the United States to fund studies relating to how environmental factors contribute to the cause, prevention, and treatment of Parkinson’s disease. A central nervous system disorder, Parkinson’s disease affects over one million Americans each year and the disease progresses with age. In most cases, scientists don’t know specifically what brings on the disease, but some cases are known to be caused by severe head trauma (such is the speculation in Mohammed Ali’s case) or patterns of genetic abnormalities. Parkinson’s is thought to be a derivative of genetic mutations and outside environmental causes. For example, prolonged exposure to pesticides is thought to double a person’s risk for the disease.
The three grantees are from respected research schools around the country and are using their grants to cover a specific area of researching the disease. Gary Miller, Ph.D., at Emory University, Atlanta, Georgia, receives a grant for prolonged research of how environmental and genetic factors change dopamine cells within the brain that lead to Parkinson’s disease. The second grant goes to Marie-Françoise Chesselet, M.D., Ph.D., at the University of California, Los Angeles, who plans to specifically research pesticides that may be the main cause of sporadic Parkinson’s diagnoses and possibly come up with a prevention plan by cautioning the use of certain pesticides. Lastly, Stuart Lipton, M.D., Ph.D., Burnham Institute for Medical Research in La Jolla, California, will investigate free radical stress caused by environmental toxins that cause genetic mutations responsible for helping to progress the disease and hopefully be able to isolate the body’s proteins damaged in the process.
Acting director of the Division of Extramural Research and Training at NIEHS, Dennis Lang, Ph.D., said of the grantees, “The UCLA and Emory CNS grants will extend the exciting lines of research previously supported by NIEHS,…while the Burnham Institute grant will bring an important new perspective to research on gene-environment interplay in Parkinson's disease.”
Perhaps the most influential person in my life who lived with Parkinson’s disease wasn’t a public figure and wasn’t world-renown for anything special; she was my grandmother. She raised seven children in a modest home, she was a do-gooder and revered by the community for her cooking talent and quilting techniques. As the years rolled by and she wasn’t able to stand up much less get out of the house, my dad’s mother spent her remaining years cooped up in a nursing home shaking and drooling, but always believed that she would walk again. That faith was finally lost in April 2005 when—the same day as Pope John Paul II—my grandmother passed on. With the generous research grants from NIEHS dedicated to finding a cure, I hope continuing investigations will be able to finally give those living with Parkinson’s the second chance at life they deserve.
Blood related Genetic Mechanisms Found Important In Parkinson's Disease
ScienceDaily (July 22, 2008) — What does the genetics of blood cells have to do with brain cells related to Parkinson's disease? From an unusual collaboration of neurologists and a pharmacologist comes the surprising answer: Genetic mechanisms at play in blood cells also control a gene and protein that cause Parkinson's disease.
The finding, by scientists from the University of Wisconsin School of Medicine and Public Health (SMPH), Harvard University-affiliated Brigham and Women's Hospital and the University of Ottawa, may lead to new treatments for the neurological disorder that affects as many as 1.5 million Americans.
The study is published in the Proceedings of the National Academy of Sciences Online Early Edition the week of July 21-25, 2008.
Patients with Parkinson's disease (PD) have elevated levels of the protein called alpha-synuclein in their brains. As the protein clumps, or aggregates, the resulting toxicity causes the death of neurons that produce the brain chemical dopamine. Consequently, nerves and muscles that control movement and coordination are destroyed.
The researchers discovered that the activity of three genes that control the synthesis of heme, the major component of hemoglobin that allows red blood cells to carry oxygen, precisely matched the activity of the alpha-synuclein gene, suggesting a common switch controlling both.
The scientists then found that a protein called GATA-1, which turns on the blood-related genes, was also a major switch for alpha-synuclein expression, and that it induced a significant increase in alpha-synuclein protein. Finally, they demonstrated that a related protein -- GATA-2 -- was expressed in PD-vulnerable brain cells and directly controlled alpha-synuclein production.
"Very little was known previously about what turns on alpha-synuclein in brain cells and causes variations in its expression," says Emery Bresnick, a UW-Madison professor of pharmacology who is an expert on GATA factors and their functions in blood. "Understanding how GATA factors work in the brain may provide fundamental insights into the biology of Parkinson's disease."
The new knowledge also may allow scientists to design therapies that keep alpha-synuclein levels within the normal range.
"Simply lowering alpha-synuclein levels by 40 percent may be enough to treat some forms of Parkinson's disease," says Dr. Clemens Scherzer of Harvard. "So far, researchers have focused on ways to get rid of too much 'bad' alpha-synuclein in Parkinson patients' brains. Now we will be able to tackle the problem from the production site, and search for new therapies that lower alpha-synuclein production up front."
Scherzer and Dr. Michael Schlossmacher, now at Ottawa, had independently analyzed the blood of PD patients and controls in a search for genes that were active in the disease. They both were surprised to notice large amounts of alpha-synuclein in the blood. To understand what it was doing there, Scherzer's group used gene chip data to see whether any of the thousands of genes active in blood were linked to alpha-synuclein. They found a gene expression pattern composed of alpha-synuclein and the heme genes, one of which Bresnick had previously shown to be a direct GATA-1 target gene.
The neurologists contacted Bresnick. The UW group rapidly determined that GATA-1 directly activated the alpha-synuclein gene, and that finding led the collaborators to discover that GATA-2 is expressed in regions of the brain that are relevant to PD.
"We all were excited because we realized that GATA-2 was active in the relevant brain regions, and so there could be a connection," says Bresnick. Together the researchers set out to examine whether common mechanisms activated alpha-synuclein transcription in both the blood and nerve cells.
The studies showed that GATA-1 and GATA-2 proteins find the alpha-synuclein gene, stick to it and then directly control it.
"This is not an indirect pathway; it is direct regulation of the gene," says Bresnick. "This directness provides the simplest scenario for creating a therapeutic strategy."
Bresnick, Schlossmacher and Scherzer are working with geneticists to see if possible abnormalities in the GATA-2 gene may exist in PD patients, stimulating more production of alpha-syinuclein.
"The discovery of the link between GATA proteins and the alpha-synuclein gene is like finding a long-sought-after molecular switch," says Schlossmacher. "We were very fortunate to find in Emery Bresnick's team the ideal partner in this endeavor."
The family of GATA factors consists of six members, and some of them, beyond GATA-2, may also be influencing alpha-synuclein expression in the brain, adds Schlossmacher.
"Identifying these would further add to the complexity of regulating the production of the 'bad player' in Parkinson's disease," he says.
Says Bresnick, "The $10 million question will be does deregulation of the GATA mechanism in humans lead to alpha-synuclein overproduction and Parkinson's disease."
Adapted from materials provided by University of Wisconsin-Madison.
The finding, by scientists from the University of Wisconsin School of Medicine and Public Health (SMPH), Harvard University-affiliated Brigham and Women's Hospital and the University of Ottawa, may lead to new treatments for the neurological disorder that affects as many as 1.5 million Americans.
The study is published in the Proceedings of the National Academy of Sciences Online Early Edition the week of July 21-25, 2008.
Patients with Parkinson's disease (PD) have elevated levels of the protein called alpha-synuclein in their brains. As the protein clumps, or aggregates, the resulting toxicity causes the death of neurons that produce the brain chemical dopamine. Consequently, nerves and muscles that control movement and coordination are destroyed.
The researchers discovered that the activity of three genes that control the synthesis of heme, the major component of hemoglobin that allows red blood cells to carry oxygen, precisely matched the activity of the alpha-synuclein gene, suggesting a common switch controlling both.
The scientists then found that a protein called GATA-1, which turns on the blood-related genes, was also a major switch for alpha-synuclein expression, and that it induced a significant increase in alpha-synuclein protein. Finally, they demonstrated that a related protein -- GATA-2 -- was expressed in PD-vulnerable brain cells and directly controlled alpha-synuclein production.
"Very little was known previously about what turns on alpha-synuclein in brain cells and causes variations in its expression," says Emery Bresnick, a UW-Madison professor of pharmacology who is an expert on GATA factors and their functions in blood. "Understanding how GATA factors work in the brain may provide fundamental insights into the biology of Parkinson's disease."
The new knowledge also may allow scientists to design therapies that keep alpha-synuclein levels within the normal range.
"Simply lowering alpha-synuclein levels by 40 percent may be enough to treat some forms of Parkinson's disease," says Dr. Clemens Scherzer of Harvard. "So far, researchers have focused on ways to get rid of too much 'bad' alpha-synuclein in Parkinson patients' brains. Now we will be able to tackle the problem from the production site, and search for new therapies that lower alpha-synuclein production up front."
Scherzer and Dr. Michael Schlossmacher, now at Ottawa, had independently analyzed the blood of PD patients and controls in a search for genes that were active in the disease. They both were surprised to notice large amounts of alpha-synuclein in the blood. To understand what it was doing there, Scherzer's group used gene chip data to see whether any of the thousands of genes active in blood were linked to alpha-synuclein. They found a gene expression pattern composed of alpha-synuclein and the heme genes, one of which Bresnick had previously shown to be a direct GATA-1 target gene.
The neurologists contacted Bresnick. The UW group rapidly determined that GATA-1 directly activated the alpha-synuclein gene, and that finding led the collaborators to discover that GATA-2 is expressed in regions of the brain that are relevant to PD.
"We all were excited because we realized that GATA-2 was active in the relevant brain regions, and so there could be a connection," says Bresnick. Together the researchers set out to examine whether common mechanisms activated alpha-synuclein transcription in both the blood and nerve cells.
The studies showed that GATA-1 and GATA-2 proteins find the alpha-synuclein gene, stick to it and then directly control it.
"This is not an indirect pathway; it is direct regulation of the gene," says Bresnick. "This directness provides the simplest scenario for creating a therapeutic strategy."
Bresnick, Schlossmacher and Scherzer are working with geneticists to see if possible abnormalities in the GATA-2 gene may exist in PD patients, stimulating more production of alpha-syinuclein.
"The discovery of the link between GATA proteins and the alpha-synuclein gene is like finding a long-sought-after molecular switch," says Schlossmacher. "We were very fortunate to find in Emery Bresnick's team the ideal partner in this endeavor."
The family of GATA factors consists of six members, and some of them, beyond GATA-2, may also be influencing alpha-synuclein expression in the brain, adds Schlossmacher.
"Identifying these would further add to the complexity of regulating the production of the 'bad player' in Parkinson's disease," he says.
Says Bresnick, "The $10 million question will be does deregulation of the GATA mechanism in humans lead to alpha-synuclein overproduction and Parkinson's disease."
Adapted from materials provided by University of Wisconsin-Madison.
Subject: H. Pylori Infection Delays Levodopa Onset and Reduces On Time
Date: 8/19/2008Infection with the gastric ulcer-causing bacterium Helicobacter pylori delays the onset of symptomatic benefit from a dose of levodopa, and shortens “on” time, according to this study, and both effects can be mitigated with antibiotic treatment. These results confirm and extend previous findings on the effect of H. pylori on levodopa adsorption ( http://www.mdvu.org/emove/article.asp?ID=892 )
Infection status of patients was determined with the [13C] urea breath test, a non-invasive diagnostic test. Thirty-five infected and 30 non-infected patients were evaluated for time to “on” and duration of “on” via 72-hour home diary prior to the screening test. There were no significant differences between groups in disease duration, UPDRS motor “on” score, daily levodopa dose, or prevalence of peak-dose dyskinesia. Delay to “on” and duration of “on” did differ:
Delay to “on” Duration of “on”
Infected 78 min 210 min
Non-Infected 56 min 257 min
Antibiotics successfully eradicated H. pylori in 34 of 35 patients. Eradication did not significantly chance motor “on” scores or dyskinesias. It did improve both delay to “on” and duration of “on”:
Delay to “on” Duration of “on”
Pre-treatment 78 min 210 min
Post-treatment 58 min 234 min
“These data suggest that HP infection may alter L-dopa absorption and lead to motor fluctuations in PD patients,” the authors conclude. They note that the [13C] urea breath test has high sensitivity and specificity, and has become the most convenient way of detecting an active HP infection. Overall seropositivity for H. pylori in the elderly has been estimated at over 50%, and is often asymptomatic.
Helicobacter pylori infection and motor fluctuations in patients with Parkinson’s disease
WY Lee, WT Yoon, HY Shin, SH Jeon, PL Rhee
Movement Disorders 2008; epub ahead of print; doi: 10.1002/mds.22190
E-MOVE Editor: Richard Robinson, NASW, WE MOVE
Infection status of patients was determined with the [13C] urea breath test, a non-invasive diagnostic test. Thirty-five infected and 30 non-infected patients were evaluated for time to “on” and duration of “on” via 72-hour home diary prior to the screening test. There were no significant differences between groups in disease duration, UPDRS motor “on” score, daily levodopa dose, or prevalence of peak-dose dyskinesia. Delay to “on” and duration of “on” did differ:
Delay to “on” Duration of “on”
Infected 78 min 210 min
Non-Infected 56 min 257 min
Antibiotics successfully eradicated H. pylori in 34 of 35 patients. Eradication did not significantly chance motor “on” scores or dyskinesias. It did improve both delay to “on” and duration of “on”:
Delay to “on” Duration of “on”
Pre-treatment 78 min 210 min
Post-treatment 58 min 234 min
“These data suggest that HP infection may alter L-dopa absorption and lead to motor fluctuations in PD patients,” the authors conclude. They note that the [13C] urea breath test has high sensitivity and specificity, and has become the most convenient way of detecting an active HP infection. Overall seropositivity for H. pylori in the elderly has been estimated at over 50%, and is often asymptomatic.
Helicobacter pylori infection and motor fluctuations in patients with Parkinson’s disease
WY Lee, WT Yoon, HY Shin, SH Jeon, PL Rhee
Movement Disorders 2008; epub ahead of print; doi: 10.1002/mds.22190
E-MOVE Editor: Richard Robinson, NASW, WE MOVE
Subthalamic deep brain stimulation after anesthetic inhalation in Parkinson disease: a preliminary study
Sheng-Huang Lin, M.D., M.Sc.1, Tsung-Ying Chen, M.D.2, Shinn-Zong Lin, M.D., Ph.D.3, Ming-Hwang Shyr, M.D., Ph.D.2, Yu-Cheng Chou, M.D.3, Wanhua Annie Hsieh, Ph.D.4, Sheng-Tzung Tsai, M.D.3, and Shin-Yuan Chen, M.D., M.Sc.3
1Departments of Neurology, 2Anesthesiology, and 3Neurosurgery, Tzu Chi General Hospital, Tzu Chi University; and 4Institute of Aboriginal Health, Tzu Chi University, Hualien, Taiwan
Abbreviations used in this paper: DBS = deep brain stimulation; MAC = minimal alveolar concentration; MER = microelectrode recording; PD = Parkinson disease; SD = standard deviation; SNr = substantia pars nigra reticulata; STN = subthalamic nucleus; UPDRS = Unified Parkinson's Disease Rating Scale.
Object
The authors of this preliminary study investigated the outcome and feasibility of intraoperative microelectrode recording (MER) in patients with Parkinson disease (PD) undergoing deep brain stimulation of the subthalamic nucleus (STN) after anesthetic inhalation.
Methods
The authors conducted a retrospective analysis of 10 patients with PD who received a desflurane anesthetic during bilateral STN electrode implantation. The MERs were obtained as an intraoperative guide for final electrode implantation and the data were analyzed offline. The functional target coordinates of the electrodes were compared preoperatively with estimated target coordinates.
Results
Outcomes were evaluated using the Unified Parkinson's Disease Rating Scale 6 months after surgery. The mean improvement in total and motor Unified Parkinson's Disease Rating Scale scores was 54.27 ± 17.96% and 48.85 ± 16.97%, respectively. The mean STN neuronal firing rate was 29.7 ± 14.6 Hz. Typical neuronal firing patterns of the STN and substantia pars nigra reticulata were observed in each patient during surgery. Comparing the functional target coordinates, the z axis coordinates were noted to be significantly different between the pre- and postoperative coordinates.
Conclusions
The authors found that MER can be adequately performed while the patient receives a desflurane anesthetic, and the results can serve as a guide for STN electrode implantation. This may be a good alternative surgical method in patients with PD who are unable to tolerate deep brain stimulation surgery with local anesthesia.
Archives of Neurology
Vol. 65 N. 8 Aug. 2008
1Departments of Neurology, 2Anesthesiology, and 3Neurosurgery, Tzu Chi General Hospital, Tzu Chi University; and 4Institute of Aboriginal Health, Tzu Chi University, Hualien, Taiwan
Abbreviations used in this paper: DBS = deep brain stimulation; MAC = minimal alveolar concentration; MER = microelectrode recording; PD = Parkinson disease; SD = standard deviation; SNr = substantia pars nigra reticulata; STN = subthalamic nucleus; UPDRS = Unified Parkinson's Disease Rating Scale.
Object
The authors of this preliminary study investigated the outcome and feasibility of intraoperative microelectrode recording (MER) in patients with Parkinson disease (PD) undergoing deep brain stimulation of the subthalamic nucleus (STN) after anesthetic inhalation.
Methods
The authors conducted a retrospective analysis of 10 patients with PD who received a desflurane anesthetic during bilateral STN electrode implantation. The MERs were obtained as an intraoperative guide for final electrode implantation and the data were analyzed offline. The functional target coordinates of the electrodes were compared preoperatively with estimated target coordinates.
Results
Outcomes were evaluated using the Unified Parkinson's Disease Rating Scale 6 months after surgery. The mean improvement in total and motor Unified Parkinson's Disease Rating Scale scores was 54.27 ± 17.96% and 48.85 ± 16.97%, respectively. The mean STN neuronal firing rate was 29.7 ± 14.6 Hz. Typical neuronal firing patterns of the STN and substantia pars nigra reticulata were observed in each patient during surgery. Comparing the functional target coordinates, the z axis coordinates were noted to be significantly different between the pre- and postoperative coordinates.
Conclusions
The authors found that MER can be adequately performed while the patient receives a desflurane anesthetic, and the results can serve as a guide for STN electrode implantation. This may be a good alternative surgical method in patients with PD who are unable to tolerate deep brain stimulation surgery with local anesthesia.
Archives of Neurology
Vol. 65 N. 8 Aug. 2008
Subject: Mild Cognitive Impairment in PD (MDS 2008)
Date: 7/15/2008
E-MOVE reports from the 12th International Congress of Parkinson’s Disease and Movement Disorders, sponsored by the Movement Disorders Society and held in Chicago June 22-26, 2008. Abstract numbers and pages refer to abstracts published in Movement Disorders 2008;23(suppl 1).
Mild cognitive impairment is common even in newly diagnosed PD patients, and non-impaired patients are at higher risk than controls to develop MCI, according to two new studies.
Aarsland et al. examined cognitive function in 181 newly diagnosed PD patients without dementia or depressive disorder. Mean duration of symptoms was 2.3 years. As a group, PD patients were impaired on all neuropsychological tests compared to controls. Mild cognitive impairment (defined as a z score below -1.5 in at least one cognitive domain out of verbal memory, visuospatial function, and executive function) was found in 17.7% of PD patients, versus 8.2% of age-matched controls (p<0.001).
In those PD patients with MCI, impairments were as follows:
single-domain non-amnestic MCI 62.5%
multiple-domain non-amnestic 9.4%
single-domain amnestic 21.9%
multiple-domain, amnestic MCI 6.3%
“All cognitive domains assessed were impaired, but the majority of MCI had a non-memory type,” the authors conclude. “Cognitive impairment should be considered in PD even at the time of diagnosis.”
Adler et al. prospectively evaluated 44 PD patients, 72 tremor patients, and 164 controls, all with similar ages (mean 77-80 years) and education levels and without MCI. Median follow-up time was 3.1 years. After controlling for sex differences among the groups, the adjusted 5-year incidence of conversion to MCI was 50% for PD, 19% for tremor, and 17% for controls. The hazard ratio was 3.7 for PD vs. tremor and 3.0 for PD vs. controls. The risk for men was twice that as for women.
Prevalence and profile of mild cognitive impairment in early, untreated Parkinson’s disease – a community-based study
D. Aarsland, K. Bronnick, J.P. Larsen, O.B. Tysnes, G. Alves
257;S88
Parkinson’s disease increases the risk for developing mild cognitive impairment
C.H. Adler, D. Connor, M. Sabbagh, H. Shill, J. Hentz, V. Evidente, P. Mahant, J. Samanta, S. Burns, A. Ahmed, E. Driver-Dunckley, L. Vedders, B. Noble, J. Caviness
292;S100
E-MOVE Editor: Richard Robinson, NASW, WE MOVE
E-MOVE reports from the 12th International Congress of Parkinson’s Disease and Movement Disorders, sponsored by the Movement Disorders Society and held in Chicago June 22-26, 2008. Abstract numbers and pages refer to abstracts published in Movement Disorders 2008;23(suppl 1).
Mild cognitive impairment is common even in newly diagnosed PD patients, and non-impaired patients are at higher risk than controls to develop MCI, according to two new studies.
Aarsland et al. examined cognitive function in 181 newly diagnosed PD patients without dementia or depressive disorder. Mean duration of symptoms was 2.3 years. As a group, PD patients were impaired on all neuropsychological tests compared to controls. Mild cognitive impairment (defined as a z score below -1.5 in at least one cognitive domain out of verbal memory, visuospatial function, and executive function) was found in 17.7% of PD patients, versus 8.2% of age-matched controls (p<0.001).
In those PD patients with MCI, impairments were as follows:
single-domain non-amnestic MCI 62.5%
multiple-domain non-amnestic 9.4%
single-domain amnestic 21.9%
multiple-domain, amnestic MCI 6.3%
“All cognitive domains assessed were impaired, but the majority of MCI had a non-memory type,” the authors conclude. “Cognitive impairment should be considered in PD even at the time of diagnosis.”
Adler et al. prospectively evaluated 44 PD patients, 72 tremor patients, and 164 controls, all with similar ages (mean 77-80 years) and education levels and without MCI. Median follow-up time was 3.1 years. After controlling for sex differences among the groups, the adjusted 5-year incidence of conversion to MCI was 50% for PD, 19% for tremor, and 17% for controls. The hazard ratio was 3.7 for PD vs. tremor and 3.0 for PD vs. controls. The risk for men was twice that as for women.
Prevalence and profile of mild cognitive impairment in early, untreated Parkinson’s disease – a community-based study
D. Aarsland, K. Bronnick, J.P. Larsen, O.B. Tysnes, G. Alves
257;S88
Parkinson’s disease increases the risk for developing mild cognitive impairment
C.H. Adler, D. Connor, M. Sabbagh, H. Shill, J. Hentz, V. Evidente, P. Mahant, J. Samanta, S. Burns, A. Ahmed, E. Driver-Dunckley, L. Vedders, B. Noble, J. Caviness
292;S100
E-MOVE Editor: Richard Robinson, NASW, WE MOVE
ADAGIO Trial Results Show Teva's AZILECT(R) 1 mg Tablets Slow Progression of Parkinson's Disease
Results Presented at 12th Congress of European Federation of Neurological Societies
JERUSALEM, Aug 26, 2008 (BUSINESS WIRE) -- Teva Pharmaceutical Industries Ltd. (TEVA:
teva pharmaceutical inds ltd adr
TEVA 46.87, +0.29, +0.6%) announces that results of the phase III ADAGIO trial were presented today during the 12th Congress of European Federation of Neurological Societies (EFNS) in Madrid, Spain as part of a "Late Breaking News" session. The ADAGIO study showed that Parkinson's disease (PD) patients who took AZILECT(R) (rasagiline) 1mg tablets once-daily upon entry into the trial, demonstrated a significant improvement compared to those who initiated the drug 9months later. The 1mg dose met all three primary endpoints, as well as the secondary endpoint, with statistical significance.
The primary analysis included three hierarchical endpoints based on Total-UPDRS (Unified Parkinson's Disease Rating Scale) scores: A) superiority of slopes in weeks 12-36 (-0.05; p=0.013, 95%CI -0.08,-0.01), B) change from baseline to week 72 (-1.7 units; p=0.025, 95%CI -3.15,-0.21), and C) non-inferiority of slopes (0.15 margin) in weeks 48-72 (0.0; 90%CI -0.04,0.04). The safety profile of AZILECT(R) seen in the ADAGIO study was similar to previous experience with AZILECT(R).
Main results were presented at the congress by Professor Olivier Rascol, M.D., Ph.D., Department of Clinical Pharmacology, University Hospital, Toulouse, France, one of two principal investigators of the trial.
"The rigorous trial design and the fact that all three primary endpoints were met with statistical significance reinforce the quality of the data, supporting the potential for AZILECT(R) to have an effect on disease progression," said Prof. Rascol. "The successful outcome of the study provides further rationale for the early use of AZILECT(R) among Parkinson's disease patients," he added.
"Delaying disease progression is the most important unmet need in the management of Parkinson's disease," stated Prof. C. Warren Olanow, professor and chairman of the Department of Neurology at the Mount Sinai School of Medicine, New York, NY, and ADAGIO co-principal investigator. "The ADAGIO study, the first of its kind, was prospectively designed to demonstrate if AZILECT(R) can slow down the progression of Parkinson's disease. Results of the study show that early treatment with once-daily rasagiline 1mg tablets provided significant clinical benefits that were not obtained by those patients where initiation of AZILECT(R) therapy was delayed by nine months."
The ADAGIO study, one of the largest conducted in PD, included 1,176 patients with very early Parkinson's disease in 14 countries and 129 medical centers who were randomized to receive rasagiline 1 or 2 mg/day for 72 weeks (early start) or placebo for 36 weeks followed by rasagiline 1 or 2 mg/day for 36 weeks (delayed start).
Description of trial results can be found online ( http://www.abstracts2view.com/ana) in the abstract submitted by Prof. Olanow and Prof. Rascol to the 133rd Annual Meeting of the American Neurological Association, Salt Lake City, UT, September 21-24, 2008.
Prof. Olanow will be presenting these results during the Works in Progress poster session on Tuesday, September 23, 2008. The abstract was also chosen to be presented orally by Prof. Olanow on Tuesday from 11:45am-noon.
Teva intends to submit these results to the regulatory authorities in the U.S. and Europe. Based on these results, Teva will work with the regulatory authorities to incorporate the results into the label for AZILECT(R).
For more information on AZILECT(R), please visit www.azilect.com.
About the Study
ADAGIO is a randomized, multi-center, double-blind, placebo-controlled, parallel-group study prospectively examining rasagiline's potential disease-modifying effects in 1,176 patients with early, untreated Parkinson's disease. Patients from 129 centers in 14 countries were randomized to early-start treatment (72 weeks rasagiline 1 or 2 mg once daily) or delayed-start treatment (36 weeks placebo followed by 36 weeks rasagiline 1 or 2 mg once daily [active treatment phase]). The primary analyses of the trial were based on change in total UPDRS (Unified Parkinson's Disease Rating Scale) and included slope superiority of rasagiline over placebo in the placebo-controlled phase, change from baseline to week 72, and non-inferiority of early-start vs. delayed-start slopes during weeks 48-72 of the active phase. UPDRS is the most commonly used rating scale to assess disease status.
About AZILECT (R)
AZILECT(R) 1mg tablets (rasagiline tablets) are indicated for the treatment of the signs and symptoms of Parkinson's disease both as initial therapy alone and to be added to levodopa later in the disease. AZILECT(R) 1mg tablets are currently available in 30 countries, including the US, Canada, Israel, Mexico, and most of the EU countries.
About Parkinson's Disease
Parkinson's disease is an age-related degenerative disorder of the brain. Symptoms can include: tremor, stiffness, slowness of movement, and impaired balance. An estimated four million people worldwide suffer from the disease, which usually affects people over the age of 60.
JERUSALEM, Aug 26, 2008 (BUSINESS WIRE) -- Teva Pharmaceutical Industries Ltd. (TEVA:
teva pharmaceutical inds ltd adr
TEVA 46.87, +0.29, +0.6%) announces that results of the phase III ADAGIO trial were presented today during the 12th Congress of European Federation of Neurological Societies (EFNS) in Madrid, Spain as part of a "Late Breaking News" session. The ADAGIO study showed that Parkinson's disease (PD) patients who took AZILECT(R) (rasagiline) 1mg tablets once-daily upon entry into the trial, demonstrated a significant improvement compared to those who initiated the drug 9months later. The 1mg dose met all three primary endpoints, as well as the secondary endpoint, with statistical significance.
The primary analysis included three hierarchical endpoints based on Total-UPDRS (Unified Parkinson's Disease Rating Scale) scores: A) superiority of slopes in weeks 12-36 (-0.05; p=0.013, 95%CI -0.08,-0.01), B) change from baseline to week 72 (-1.7 units; p=0.025, 95%CI -3.15,-0.21), and C) non-inferiority of slopes (0.15 margin) in weeks 48-72 (0.0; 90%CI -0.04,0.04). The safety profile of AZILECT(R) seen in the ADAGIO study was similar to previous experience with AZILECT(R).
Main results were presented at the congress by Professor Olivier Rascol, M.D., Ph.D., Department of Clinical Pharmacology, University Hospital, Toulouse, France, one of two principal investigators of the trial.
"The rigorous trial design and the fact that all three primary endpoints were met with statistical significance reinforce the quality of the data, supporting the potential for AZILECT(R) to have an effect on disease progression," said Prof. Rascol. "The successful outcome of the study provides further rationale for the early use of AZILECT(R) among Parkinson's disease patients," he added.
"Delaying disease progression is the most important unmet need in the management of Parkinson's disease," stated Prof. C. Warren Olanow, professor and chairman of the Department of Neurology at the Mount Sinai School of Medicine, New York, NY, and ADAGIO co-principal investigator. "The ADAGIO study, the first of its kind, was prospectively designed to demonstrate if AZILECT(R) can slow down the progression of Parkinson's disease. Results of the study show that early treatment with once-daily rasagiline 1mg tablets provided significant clinical benefits that were not obtained by those patients where initiation of AZILECT(R) therapy was delayed by nine months."
The ADAGIO study, one of the largest conducted in PD, included 1,176 patients with very early Parkinson's disease in 14 countries and 129 medical centers who were randomized to receive rasagiline 1 or 2 mg/day for 72 weeks (early start) or placebo for 36 weeks followed by rasagiline 1 or 2 mg/day for 36 weeks (delayed start).
Description of trial results can be found online ( http://www.abstracts2view.com/ana) in the abstract submitted by Prof. Olanow and Prof. Rascol to the 133rd Annual Meeting of the American Neurological Association, Salt Lake City, UT, September 21-24, 2008.
Prof. Olanow will be presenting these results during the Works in Progress poster session on Tuesday, September 23, 2008. The abstract was also chosen to be presented orally by Prof. Olanow on Tuesday from 11:45am-noon.
Teva intends to submit these results to the regulatory authorities in the U.S. and Europe. Based on these results, Teva will work with the regulatory authorities to incorporate the results into the label for AZILECT(R).
For more information on AZILECT(R), please visit www.azilect.com.
About the Study
ADAGIO is a randomized, multi-center, double-blind, placebo-controlled, parallel-group study prospectively examining rasagiline's potential disease-modifying effects in 1,176 patients with early, untreated Parkinson's disease. Patients from 129 centers in 14 countries were randomized to early-start treatment (72 weeks rasagiline 1 or 2 mg once daily) or delayed-start treatment (36 weeks placebo followed by 36 weeks rasagiline 1 or 2 mg once daily [active treatment phase]). The primary analyses of the trial were based on change in total UPDRS (Unified Parkinson's Disease Rating Scale) and included slope superiority of rasagiline over placebo in the placebo-controlled phase, change from baseline to week 72, and non-inferiority of early-start vs. delayed-start slopes during weeks 48-72 of the active phase. UPDRS is the most commonly used rating scale to assess disease status.
About AZILECT (R)
AZILECT(R) 1mg tablets (rasagiline tablets) are indicated for the treatment of the signs and symptoms of Parkinson's disease both as initial therapy alone and to be added to levodopa later in the disease. AZILECT(R) 1mg tablets are currently available in 30 countries, including the US, Canada, Israel, Mexico, and most of the EU countries.
About Parkinson's Disease
Parkinson's disease is an age-related degenerative disorder of the brain. Symptoms can include: tremor, stiffness, slowness of movement, and impaired balance. An estimated four million people worldwide suffer from the disease, which usually affects people over the age of 60.
Hsp104 antagonizes α-synuclein aggregation and reduces dopaminergic degeneration in a rat model of Parkinson disease
Christophe Lo Bianco1,2, James Shorter3, Etienne Régulier2, Hilal Lashuel2, Takeshi Iwatsubo4, Susan Lindquist5 and Patrick Aebischer2
Published August 14, 2008
Received for publication March 28, 2008, and accepted in revised form July 7, 2008.
Parkinson disease (PD) is characterized by dopaminergic neurodegeneration and intracellular inclusions of α-synuclein amyloid fibers, which are stable and difficult to dissolve. Whether inclusions are neuroprotective or pathological remains controversial, because prefibrillar oligomers may be more toxic than amyloid inclusions. Thus, whether therapies should target inclusions, preamyloid oligomers, or both is a critically important issue. In yeast, the protein-remodeling factor Hsp104 cooperates with Hsp70 and Hsp40 to dissolve and reactivate aggregated proteins. Metazoans, however, have no Hsp104 ortholog. Here we introduced Hsp104 into a rat PD model. Remarkably, Hsp104 reduced formation of phosphorylated α-synuclein inclusions and prevented nigrostriatal dopaminergic neurodegeneration induced by PD-linked α-synuclein (A30P). An in vitro assay employing pure proteins revealed that Hsp104 prevented fibrillization of α-synuclein and PD-linked variants (A30P, A53T, E46K). Hsp104 coupled ATP hydrolysis to the disassembly of preamyloid oligomers and amyloid fibers composed of α-synuclein. Furthermore, the mammalian Hsp70 and Hsp40 chaperones, Hsc70 and Hdj2, enhanced α-synuclein fiber disassembly by Hsp104. Hsp104 likely protects dopaminergic neurons by antagonizing toxic α-synuclein assemblies and might have therapeutic potential for PD and other neurodegenerative amyloidoses.
Introduction
Abnormal protein aggregation in the brain characterizes several lethal neurodegenerative diseases (1), including Parkinson disease (PD). There are no cures for PD, the most common neurodegenerative movement disorder, which debilitates several million people worldwide (2). PD involves a progressive and selective elimination of dopaminergic neurons from the substantia nigra pars compacta, although neuropathology can extend into other brain regions (3). The signature lesions of PD are intracellular inclusions termed Lewy Bodies and Lewy neurites, which contain the small presynaptic protein, α-synuclein (α-syn) (4). Although PD is primarily a sporadic disorder, mutations in α-syn (e.g., A53T, A30P, E46K) and overexpression of the wild-type gene are linked with early-onset PD in rare familial forms of the disease (5).
The function of α-syn is uncertain, but various studies connect α-syn to synaptic vesicle pool regulation and dopamine release (5). Yet, how these potential functions might relate to PD is unclear. α-syn is natively unstructured in isolation, but gains α-helical structure upon association with phospholipid bilayers (6). Purified α-syn forms amyloid fibers in vitro, which bind to the diagnostic dyes Thioflavin-T (ThT) and Congo red and possess the generic amyloid “cross-β” conformation, in which the strands of the β-sheets run orthogonal to the fiber axis (7–9). Fibers assembled in vitro are very similar to α-syn filaments isolated from synucleinopathy patients (10, 11). Once initiated, α-syn amyloidogenesis can cascade out of control, because α-syn fibers self-template by recruiting non-amyloid α-syn conformers to fiber ends and converting them to the amyloid form (8). α-syn fibers are exceptionally stable (e.g., protease and detergent resistant) and extraordinarily difficult to clear (12, 13).
Prior to fibrillization, α-syn populates heterogeneous oligomeric states, which have not yet accessed the final cross-β form of mature fibers. This ensemble of preamyloid oligomeric states comprises transient species that are likely “on-pathway” to fiber formation as well as “off-pathway” forms (8, 14, 15). One of the transient oligomeric conformations populated by α-syn is common to many amyloidogenic proteins regardless of their primary sequence and is recognized by a conformation-specific Ab (14). Preamyloid α-syn oligomers may be more cytotoxic than fibers (14, 16), and sequestration of α-syn into fiber inclusions at the expense of preamyloid oligomers might even be neuroprotective (17, 18). Despite intense investigation, how the process of α-syn amyloidogenesis elicits the selective cell death that distinguishes PD and other synucleinopathies remains unclear.
PD-linked mutations in α-syn increase its propensity to access misfolded forms. A53T and E46K fibrillize more rapidly than wild-type α-syn, whereas A30P fibrillizes more slowly (7, 8, 19). By contrast, A30P and A53T access preamyloid oligomers more rapidly than wild-type α-syn, whereas E46K is less able to form these species (8, 20). Specific posttranslational modifications, such as phosphorylation at serine 129 and nitration, also promote α-syn fibrillization (21, 22). Phosphorylated and nitrated α-syn selectively and abundantly accumulates in α-synucleinopathy lesions in animal models and humans (5, 13, 18, 21). Together, this suggests that α-syn misfolding contributes to familial and sporadic PD. Thus, inhibiting α-syn misfolding and/or aggregation or promoting the clearance of α-syn aggregates may constitute promising therapeutic strategies for PD and other synucleinopathies.
Protein misfolding is a problem as ancient as life itself, and so too are the solutions that synergize to antagonize it. Thus, sophisticated molecular chaperones recognize misfolded proteins and prevent their aggregation, protein-remodeling factors resolve protein aggregates, osmolytes function as chemical chaperones, and degradation systems eradicate misfolded proteins. Upregulation of protein quality control safeguards may provide important therapeutic avenues (23). Indeed, Hsp70 and Hsp40 chaperones associate with Lewy Bodies and Lewy neurites in PD and other synucleinopathies (24) as well as polyglutamine aggregates in several diseases (25, 26). Overexpression of Hsp70 and Hsp40 can suppress polyglutamine aggregation (25) or ameliorate toxicity associated with α-syn aggregation (24). Yet, Hsp70 and Hsp40 have only very limited ability to resolve protein aggregates once they have formed (27). The ability to restore aggregated proteins to native structure and function would obviate the huge energetic cost of degrading and resynthesizing them. Further, this would simultaneously eliminate 3 malicious problems associated with protein aggregation that likely synergize in the etiology of various protein misfolding disorders: (a) the toxic gain of function of aggregated conformers; (b) the loss of function of the aggregated protein; and (c) the sequestration of other essential proteins that coprecipitate with the aggregated protein. However, whether mammals possess any activity that reverses protein aggregation and restores the functionality of aggregated proteins remains unclear.
By contrast, fungi, plants, and bacteria all express orthologs of Hsp104, a powerful protein-remodeling factor. All are hexameric AAA+ (ATPases associated with diverse activities) proteins with 2 AAA+ ATPase domains per monomer (28, 29). Hsp104 synergizes with Hsp70 and Hsp40 to resolve protein aggregates and return proteins to normal enzymatic activity (27, 30). This increases cell survival after multifarious stresses by up to 10,000-fold (31, 32). Hsp104 also possesses an unusually powerful amyloid-remodeling activity and rapidly disassembles amyloid fibers composed of the yeast prion proteins Sup35 and Ure2 (29, 33–37). Moreover, Hsp104 eliminates Sup35 preamyloid oligomers that adopt a conformation shared by many amyloidogenic proteins, including α-syn (14, 33, 34). Critically, even transient overexpression of Hsp104 can purge yeast of Sup35 prions (38). Given these remarkable activities, which would appear beneficial to all cells, it is puzzling why Hsp104 has been lost from metazoan lineages. This issue remains moot and unaddressed. Nevertheless, Hsp104 can synergize with the mammalian Hsp70 chaperone system to promote protein disaggregation and stress tolerance (39, 40).
Disassembly of yeast prions by Hsp104 raises awareness that amyloids can be resolved by a protein-remodeling factor (28). Amyloids share a common cross-β scaffold, where the β-sheet strands are aligned orthogonal to the fiber axis, irrespective of the primary sequence of the protein (41). Even though local steric details of different amyloids may vary enormously (41), this suggests that agents that antagonize amyloid fibers of one protein may also be active against amyloid fibers composed of another. Some preamyloid oligomers also share a common structure that is independent of primary sequence and distinct to that of fibers (14). Thus, can introduction of Hsp104 into metazoan systems prevent or reverse various amyloidoses? Answering this question will help clarify whether protein aggregation is protective or toxic in various disease settings.
The development of potential therapies for PD has been hampered by a paucity of animal models that recapitulate the selective loss of dopaminergic neurons. Here we employ a rat PD model based on the lentiviral-mediated expression of human α-syn A30P in the substantia nigra, which successfully recreates the progressive and selective degeneration of dopaminergic neurons and formation of phosphorylated α-syn inclusions that characterize PD (18, 42). We first evaluated whether Hsp104 affects α-syn toxicity and aggregation in this model. Because Hsp104 does not have general anti-apoptotic effects (e.g., in response to staurosporine or hydrogen peroxide) when expressed in mammalian cells (43), any reductions in toxicity will likely reflect direct effects on α-syn misfolding. We then employed pure proteins to delineate how Hsp104 modulates α-syn aggregation. Our studies highlight the therapeutic utility of Hsp104 for neurodegenerative disease and provide new insights into the controversial issues of how protein aggregation and inclusion formation contribute to PD pathogenesis and other diseases.
Results
Hsp104 reduces α-syn–induced toxicity. Overexpression of mutated human α-syn with viral vectors induces a progressive loss of nigral dopamine neurons in rodents and nonhuman primates (42, 44–46). To test whether Hsp104 modulates α-syn aggregation and α-syn–induced degeneration of dopamine neurons, we simultaneously overexpressed Hsp104 and A30P α-syn in the brain of rats. Viral suspensions containing lentiviral vectors coding either for A30P human α-syn and Hsp104 (lenti-A30P/lenti-Hsp104; see Methods) or for A30P human α-syn and yellow fluorescent protein (lenti-A30P/lenti-YFP; see Methods) were directly injected in the substantia nigra of rats. Brain slices from the substantia nigra were then stained for the dopaminergic marker tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis.
Animals injected with lentiviral vectors coding for A30P α-syn showed a 33% loss of TH-immunoreactive (TH-IR) neurons in the substantia nigra at 6 weeks after injection (42). Importantly, viral expression of 2 reporter proteins (YFP/GFP) does not induce any damage to the nigrostriatal pathway (18). In contrast, injection of lenti-A30P/lenti-YFP in the right side of the rat substantia nigra induced a marked loss of TH-IR cells compared with the noninjected side (Figure 1A), similar to the expression of A30P α-syn alone. Coexpression of Hsp104 rescued TH-IR neurons from A30P α-syn–induced toxicity (Figure 1A). Quantitation of neuronal loss was performed throughout the whole substantia nigra (Figure 1B). The contralateral noninjected side was used as an internal control. Animals expressing A30P/YFP revealed a 31.4% loss of TH-IR cells relative to the contralateral side. Expression of Hsp104 resulted in a significant reduction in cell loss (12.6%; P < 0.05; n = 7). Confocal analysis of animals expressing Hsp104/A30P revealed that surviving dopamine neurons still expressed abundant levels of human α-syn (Figure 1C).
Hsp104 prevents A30P α-syn–induced neurodegeneration. Nigral dopaminergic neurons project their nerve terminals directly to the striatum. Lentiviral-mediated expression of A30P α-syn in the substantia nigra of rats induces a significant loss of striatal nerve terminals (42). To determine whether the protective effect of Hsp104 on dopaminergic cells is accompanied by reductions in the loss of nerve terminals in the striatum, TH staining was performed on striatal brain slices from animals expressing either A30P/YFP or A30P/Hsp104 (Figure 2A).
Animals expressing A30P/YFP showed a decrease in the TH marker in the ipsilateral side (Figure 2A). Nigral expression of Hsp104 reduced the dopaminergic nerve terminal loss induced by the accumulation of A30P α-syn (Figure 2A). Quantitation of TH-IR fiber density was performed throughout the striatum. Consistent with the neuroprotective effect of Hsp104 at the cellular level, expression of Hsp104 significantly decreased the nerve terminal loss from 21.6% (A30P/YFP) to 7% (A30P/Hsp104) (P < 0.05; n = 7) (Figure 2B).
Next, we used silver staining as a marker for degenerating neurons in animals expressing A30P/YFP or A30P/Hsp104 (42, 47, 48). No silver staining was observed in the noninjected substantia nigra (Figure 3, A and D). In contrast, scattered neurons containing silver-positive dark structures were observed in animals injected with lenti-A30P/lenti-YFP, consistent with α-syn–induced degeneration (18, 42) (Figure 3B). Higher magnifications revealed that animals expressing A30P/YFP abundantly accumulated granular deposits in both cell bodies and axons at the substantia nigra level (Figure 3, E and G) (42). Clear neuritic pathology was also observed (Figure 3G). In stark contrast, coexpression of Hsp104 greatly reduced the appearance of silver-positive degenerating neurons and granular silver deposits (Figure 3, C and F), indicating that Hsp104 antagonizes A30P α-syn–induced neurodegeneration. Only a few sparse silver-positive neurons were still observed in the A30P/Hsp104 group. Although these observations were consistently observed in animals expressing Hsp104, the high variability in the signal/noise ratio observed with silver staining among sections, even within the same animal, prevents quantification.
Hsp104 reduces the formation of phosphorylated inclusions. To understand the mechanism by which Hsp104 prevents α-syn–induced neurodegeneration, we investigated how Hsp104 affects the α-syn aggregation in vivo. Two different Abs were used to detect α-syn in animals expressing either A30P/YFP or A30P/Hsp104 (Figure 4). The Ab RG syn recognizes both rat and human α-syn by western blot (42). However, this Ab only detects overexpression of α-syn and not endogenous levels of this protein in the rat brain (42). Stainings with LB509, an Ab that is specific for human α-syn, were also performed (Figure 4, D–I). A strong accumulation of human α-syn in both the perikarya and neurites is observed in the substantia nigra of animals expressing either A30P/YFP (Figure 4, B and E) or A30P/Hsp104 (Figure 4, C and F) as compared with noninjected animals (Figure 4, A, D, and G). Examination at higher magnification illustrates the difficulty in discriminating between α-syn aggregates and subcellular accumulation of this protein (Figure 4, H and I). High levels of soluble α-syn may mask the appearance of aggregates and render quantification very difficult. However, in some neurons, dense puncta were frequently observed in neurites, and these were less evident in animals expressing Hsp104. Furthermore, a more dense coloration inside the perikarya of α-syn–positive neurons indicates the presence of α-syn aggregates (Figure 4, H and I). Hsp104 significantly reduces the percentage of α-syn–positive neurons harboring dense structures in cell bodies or axons (54.8% ± 5.5% and 30.8% ± 4.4% for A30P/YFP and A30P/Hsp104, respectively; mean ± SEM; P < 0.05; n = 7). Interestingly, in animals expressing Hsp104, we detected a more diffuse and homogenous α-syn localization along the neuropil, indicating that Hsp104 antagonizes the dense structures or aggregates and helps maintain soluble forms of α-syn. These data suggest that Hsp104 may antagonize α-syn aggregation in vivo.
To evaluate more clearly, whether Hsp104 affected α-syn aggregation in vivo, we assessed phosphorylated α-syn inclusions. α-syn phosphorylated predominantly at serine 129 selectively and abundantly accumulates in both Lewy bodies of PD patients and inclusions of α-syn animal models (13, 18, 21). Therefore, we quantified the number of phosphorylated inclusions with the Pser129 Ab specific for phosphorylated α-syn at serine 129 (18, 21) (Figure 4, J–M). As described previously (18), expression of A30P α-syn leads to the formation of phosphorylated inclusions (Figure 4K) as compared with noninjected animals (Figure 4J). Strikingly, coexpression of Hsp104 with A30P α-syn resulted in a 57% reduction in the number of neurons containing phosphorylated inclusions (Figure 4, L and M), indicating that Hsp104 may protect dopaminergic cells by decreasing the numbers of phosphorylated α-syn aggregates.
Hsp104 inhibits α-syn amyloidogenesis. From our in vivo experiments, it is difficult to distinguish whether Hsp104 prevents aggregation of α-syn or resolubilizes α-syn after it has aggregated. Therefore, we determined how Hsp104 affects α-syn amyloidogenesis in vitro using pure proteins. In vitro, purified α-syn assembles into fibers, which are very similar to α-syn filaments isolated from synucleinopathy patients (9, 10). α-syn assembled into amyloid fibers after a lag of approximately 5 hours of incubation, and assembly was virtually complete after approximately 24 hours as assessed by ThT fluorescence (a diagnostic amyloid dye) (Figure 5A) and sedimentation analysis (Figure 5B). When Hsp104 was added at substoichiometric levels (α-syn monomers/Hsp104 hexamers, 80:0.2 μM) fibrillization was retarded (Figure 5, A and B). Remarkably, higher Hsp104 concentrations (α-syn monomers/Hsp104 hexamers, 80:0.8 μM or 80:1.6 μM) allowed very little fibrillization after 48 hours (Figure 5, A and B). EM confirmed that Hsp104 inhibited α-syn fibrillization (Figure 5C). Amorphous material accumulated in the presence of Hsp104 (Figure 5C). Inhibition of α-syn fibrillization by Hsp104 was stable until approximately 72 hours (data not shown). After 96 hours, Hsp104 began to lose activity and some α-syn fibrillization was able to occur (Figure 5D). However, if reactions were supplemented with additional Hsp104 at 72 hours, the inhibition of α-syn fibrillization was maintained (Figure 5D). Thus, the inhibition α-syn fibrillization is stable, provided there is a renewable source of active Hsp104.
Hsp104 afforded little inhibition of α-syn fibrillization in the absence of ATP and inhibition was reduced by the presence of the nonhydrolyzable ATP analogue, AMP-PNP (Figure 5A). We also employed Hsp104 carrying mutations in the highly conserved Walker A motifs of both AAA+ ATPase domains, Hsp104K218T:K620T, which is defective in ATP binding and hydrolysis at both sites (49). The mutant failed to inhibit α-syn fibrillization (Figure 5A). Thus, maximal inhibition of α-syn fibrillization requires ATP binding and hydrolysis by Hsp104.
Next, we tested whether Hsp104 could inhibit the fibrillization of PD-linked α-syn variants, including A30P, A53T, and E46K. A53T and E46K assemble into amyloid fibers more rapidly than wild-type α-syn, whereas A30P accesses preamyloid oligomers more rapidly than wild type but takes longer to form fibers (7, 19). Hsp104 inhibited A53T fibrillization at early times, but this inhibition was overcome by 48 hours of incubation (Figure 5E). Stable inhibition of A53T over the 48-hour time frame required higher concentrations of Hsp104 (Figure 5E), perhaps because A53T assembles more rapidly than any of the other α-syn variants. By contrast, Hsp104 inhibited fibrillization by A30P and E46K just as well as wild-type α-syn (Figure 5E). Thus, Hsp104 potently inhibits the assembly of the spectrum of PD-linked α-syn mutants.
In synucleinopathy patients, α-syn is phosphorylated on serine 129, which may stimulate α-syn fibrillization (21). We found that Hsp104 inhibits fibrillization of both α-syn S129A, which cannot be phosphorylated at position 129, and S129E, which may mimic α-syn phosphorylated at S129 (Figure 5E). Therefore, the reduction in phosphorylated A30P α-syn inclusions observed in vivo is likely due, at least in part, to the inhibition of their assembly by Hsp104.
Hsp104 remodels α-syn A30P preamyloid oligomers. Preamyloid oligomers may be the more cytotoxic species in many neurodegenerative amyloidoses, including synucleinopathies (14, 50). Further, they are likely important intermediates in the fibrillization process (8, 15, 33, 34). Thus, we determined if Hsp104 could remodel preformed A30P preamyloid oligomers. A30P preamyloid oligomers were formed and purified away from monomers by gel filtration (15). Purified A30P preamyloid oligomers are stable for long periods (~10 days) and do not dissociate into monomers or dimers (15). However, to ensure our starting material was 100% oligomeric, purified A30P preamyloid oligomers were treated immediately with Hsp104 or Hsp104K218T:K620T. Hsp104K218T:K620T was unable to remodel preamyloid A30P oligomers as determined by anti-oligomer immunoreactivity, EM, and retention by a 100-kDa filter (Figure 6). By contrast, Hsp104 reduced anti-oligomer immunoreactivity (Figure 6A), and EM revealed that Hsp104 disassembled A30P preamyloid oligomers (Figure 6B). A30P was now able traverse a 100-kDa filter (Figure 6, C and D). Thus, Hsp104 eradicates α-syn A30P preamyloid oligomers, which are potentially the most toxic species that arise during A30P amyloidogenesis (14).
Hsp104 remodels α-syn fibers. Finally, we tested whether Hsp104 remodeled fibers formed by wild-type α-syn as well as PD-linked mutants A30P, A53T, and E46K and the serine 129 mutants S129A and S129E. Remarkably, Hsp104 disassembled fibers composed of wild-type α-syn, A53T, A30P, and S129A as revealed by ThT fluorescence (Figure 7A) and turbidity (Figure 7B). Of these, A30P was the most susceptible to disassembly by Hsp104 (Figure 7, A–C). This required ATP hydrolysis by Hsp104 and was not observed with Hsp104K218T:K620T or in the presence of AMP-PNP (Figure 7, A and B). Intriguingly, S129E fibers were more resistant to disassembly by Hsp104, and E46K fibers were refractory to Hsp104. In sum, these data suggest that Hsp104 disassembles fibers composed of wild-type α-syn and some PD-linked α-syn variants.
Hsp104 combines with mammalian Hsp70 and Hsp40 chaperones to promote reactivation of denatured luciferase aggregates (27, 40). Thus, we tested whether mammalian Hsp70 and Hsp40 chaperones could assist Hsp104 in disassembling α-syn fibers. In contrast to Hsp104 alone, Hsp70 and Hsc70 alone or in combination with either Hdj1 or Hdj2 were unable to disassemble α-syn fibers over the time frame of the assay (Figure 7, D and E). Hsp104 combined with Hsc70 and Hdj2 to promote more α-syn fiber disassembly than Hsp104 alone as determined by ThT fluorescence (Figure 7D) and sedimentation analysis (Figure 7E). To a lesser extent, Hsp104, Hsp70, and Hdj2 promoted more α-syn fiber disassembly than Hsp104 alone (Figure 7, D and E), while other combinations were equally (Hsp104, Hsp70, and Hdj1) or slightly less (Hsp104, Hsc70, and Hdj1) effective than Hsp104 alone (Figure 7, D and E). Overall, these data demonstrate that Hsp104 antagonizes various α-syn conformations populated during amyloidogenesis, and that the mammalian Hsp70 chaperone system can assist Hsp104 in disassembling α-syn fibers.
Discussion
Here we show for what we believe to be the first time that augmentation of the mammalian protein quality control system with a protein-remodeling factor not ordinarily found in metazoa, Hsp104, dramatically reduces dopaminergic neurodegeneration and phosphorylated α-syn inclusion formation in a rat lentiviral model of PD. To help understand these events, we employed pure proteins to analyze how Hsp104 affects α-syn amyloidogenesis. Importantly, Hsp104 directly inhibits the fibrillization of α-syn as well as the PD-linked α-syn mutants A53T, A30P, and E46K and the serine 129 phosphorylation mutants S129A and S129E. Hsp104 inhibited α-syn fibrillization even when α-syn was 400-fold more abundant than Hsp104. This suggests that Hsp104 specifically antagonizes a rare or transient α-syn conformer, perhaps a specific oligomeric species, which nucleates α-syn fibrillization. Indeed, Hsp104 disassembled purified A30P preamyloid oligomers that adopted a toxic conformation common to many amyloidogenic proteins (14). Such α-syn preamyloid oligomers are toxic to human neuroblastoma SH-SY5Y cells (14), suggesting that Hsp104 eliminates toxic α-syn conformers. Crucially, Hsp104 disassembled preformed α-syn fibers. Specific mammalian Hsp70 and Hsp40 chaperones, most notably Hsc70 and Hdj2, increased α-syn fiber disassembly by Hsp104. This is to our knowledge the first demonstration of effective disassembly of both α-syn preamyloid oligomers and amyloid fibers (which are particularly stable structures) by any protein. These various α-syn–remodeling activities required ATP binding and hydrolysis by Hsp104 and explain how Hsp104 might reduce dopaminergic neurodegeneration and phosphorylated α-syn inclusion formation in the arena of the rat brain.
Two previous studies claim to provide evidence that Hsp104 can dissociate α-syn fibers, but no experiments with α-syn preamyloid oligomers were performed (51, 52). However, under the conditions employed, Hsp104 reduced ThT fluorescence of preformed α-syn fibers by only 5%–20% after 24 hours of incubation (51, 52). This low level of potential fiber disassembly was not corroborated by other methods (51, 52). Remarkably, evidence was presented that Hsp104 degraded α-syn (51). This is extraordinarily unlikely given that Hsp104 possesses no protease motifs or protease activity (27, 29, 30, 31, 53, 54). It is more probable that the findings of Kong et al. are due to a contaminating protease (51, 52). By contrast, we find absolutely no evidence of degradation of α-syn by Hsp104 (Figure 6C). Rather, Hsp104 rapidly disassembles α-syn fibers and preamyloid oligomers to yield soluble α-syn.
It is not clear why Hsp104 has been lost from metazoa (28). Indeed, it is unclear whether mammalian cells express an analogous protein disaggregase able to solubilize large protein aggregates and restore protein functionality. Initial attempts to isolate such activity have been unsuccessful (40). The metazoan quality control machinery may be more optimized to prevent protein aggregation than reverse it and relies more heavily on the Hsp70 chaperone system. This system powerfully suppresses protein aggregation but only has limited ability to resolve even small soluble protein aggregates (27). Despite this, mammalian cells do have mechanisms to clear protein aggregates as revealed by silencing the gene encoding the aggregated protein (55). However, these pathways reflect autophagy and other protein degradation pathways rather than protein reactivation (23, 56). This general inability to rescue aggregated proteins may contribute to the lethality of excessive protein aggregation in animal cells.
Remodeling of α-syn preamyloid oligomers and amyloid fibers by Hsp104 is surprising since α-syn shares no sequence similarity with Hsp104’s natural amyloidogenic substrates: Sup35, Ure2, and Rnq1 (57). Hence, Hsp104 may specifically engage and remodel generic aspects of the cross-β amyloid form (41) and the distinct generic structure of preamyloid oligomers (14). However, S129E fibers were more resistant to disassembly. Thus, Hsp104 may need to initially engage α-syn at amino acid 129, which is solvent accessible in assembled α-syn fibers (58). Mutation of serine 129 to glutamate (but not alanine) may disrupt this interaction. Furthermore, E46K fibers were refractory to disassembly by Hsp104. This may reflect the different morphology of E46K fibers, which form compact bundles and meshwork arrays not observed with wild-type α-syn (19).
In rescuing α-syn neurotoxicity, Hsp104 reduced the number of α-syn inclusions. By contrast, Hsp70 prevents dopaminergic neurodegeneration in α-syn transgenic flies without affecting inclusion formation (24). Similar observations were reported for polyglutamine disorders, underscoring the limited ability of Hsp70 chaperones to remove aggregates (26). The lack of correlation between inclusion formation and neurodegeneration suggests that formation of inclusions may represent a protective cellular mechanism for sequestrating toxic assemblies (e.g., preamyloid oligomers) into safe inclusions. Indeed, expression of parkin, an E3 ubiquitin ligase connected with juvenile parkinsonism (5), prevents dopaminergic degeneration induced by A30P in our rat lentiviral PD model, but this is accompanied by an increase in phosphorylated α-syn inclusions (18). This suggests that Hsp104 reduces the number of inclusions in a manner that does not generate dangerous levels of toxic conformers, presumably because Hsp104 can disassemble these toxic species. Indeed, Hsp104’s ability to safely disassemble protein aggregates is likely an adaptation that ensures cell survival and the dissolution of the entire aggregated proteome after environmental stress in yeast.
However, further study is needed to address whether Hsp104 reduces preamyloid oligomer levels in α-syn expressing midbrain dopamine neurons. The low amount of preamyloid oligomers (relative to amyloid fibers), the low percentage of virally transduced cells, and the difficulty in isolating dopamine neurons from tissue extracts make the extraction of detectable levels of preamyloid oligomers very difficult. Further, we simultaneously expressed Hsp104 and α-syn, and not Hsp104 after α-syn aggregation, mainly due to technical issues. We have shown that coinjection of 2 viral vectors leads to more than 70% of cotransduced cells (18). In contrast, sequential injection of 2 viral vectors at 2-week intervals increases the variability of targeting of nigral cells and greatly decreases the number of cotransduced cells (Lo Bianco et al., unpublished observations). This is due to the difficulty in performing 2 identical injections into the same brain at different times. Thus, further studies are required to assess whether Hsp104 reverses preformed α-syn aggregates in vivo.
Overexpression of human α-syn in yeast induces toxicity and cytoplasmic foci containing α-syn (59). Surprisingly, Hsp104 overexpression has little effect on α-syn toxicity in yeast (60). However, the α-syn foci in yeast result from colocalization of α-syn with a membrane compartment, which perturbs endoplasmic reticulum to Golgi transport and Rab-GTPase homeostasis (61, 62). That α-syn does not form genuine aggregates in yeast, in contrast to the rat lentiviral model, explains why Hsp104 has little effect on its toxicity. It is likely that α-syn toxicity in yeast mimics an early, underappreciated stage in PD, prior to the accumulation of large quantities of preamyloid oligomers and fibers (61–63).
Even though not ordinarily expressed in mammalian cells, Hsp104 is extremely well tolerated in both tissue culture cells (including neurons) and in the brains of transgenic rodents and can perform beneficial protein-remodeling functions (39, 40, 64–66). Our studies demonstrate that Hsp104 combats α-syn misfolding and associated dopaminergic degeneration in a rat model of PD. Hsp104’s ability to prevent and reverse pathogenic protein aggregation should be considered as a potential strategy for treating or reversing PD and other protein aggregation diseases. However, further study is required to evaluate the safety of long-term Hsp104 expression in neurons.
Methods
Lentiviral vectors. cDNA coding for nuclear-localized YFP (BD Biosciences — Clontech), A30P human α-syn, and Hsp104 (kindly provided by D. Picard, Département de Biologie Cellulaire, Université de Genève, Genève, Switzerland) were cloned in the SIN-W-PGK lentiviral transfer vector, and viral particles (lenti-YFP, lenti-A30P, and lenti-Hsp104) were produced as described (42, 67). The viral suspensions lenti-A30P/lenti-YFP and lenti-A30P/lenti-Hsp104 were prepared by mixing viruses at 1:1 ratios (18). Particle content was matched to 180,000 ng of p24/ml for each lentiviral vector (18).
Stereotaxic injection. Lentiviral vectors were stereotaxically injected in the right substantia nigra of adult female Wistar rats (Iffa-Credo) weighing 200 g. Viral suspensions were injected at 2 sites with a 10-μl Hamilton syringe at a speed of 0.2 μl/min with an automatic injector (Stoelting Co.), and the needle was left in place for an additional 10 minutes before withdrawal. Stereotaxic injections were performed in 2 sites (2.5 μl per site) with anterior, lateral, and ventral coordinates (4.8, 2, 7.7, and 5.5, 1.7, 7.7) as described (18, 42). Animals were sacrificed at 6 weeks after injection. Experiments were carried out in accordance with the European Community Council directive (86/609/EEC) for the care and use of laboratory animals. The experiments described in this article were approved by the Veterinarian Office as well as by the Commission for Animal Experimentation of the Canton of Vaud (Switzerland) and were carried out under the animal license 1653.
Immunohistochemistry. Animals were deeply anesthetized with sodium pentobarbital and perfused transcardially with 4% paraformaldehyde. Brains were removed and postfixed in 4% paraformaldehyde for approximately 24 hours, cryoprotected in 25% sucrose in 0.1 M phosphate buffer for 48 hours, and processed as described (18, 42).
The following primary Abs were used: a TH sheep Ab (1:500; Pel-Freez Biologicals), the RG syn α-syn polyclonal rabbit Ab (1:400; ref. 42), the LB509 human α-syn specific monoclonal Ab (1:500; Zymed), the Pser129 Ab specifically recognizing phosphorylated Ser 129 of α-syn (1:100; ref. 21), and a rabbit Ab to the C-terminus of Hsp104 (1:800; Stressgen). For light microscopy, sections were stained by the classical avidin-biotin complex method as described (42). For multiple fluorescent labeling, the secondary Abs conjugated to Cy3 (donkey anti-sheep) and Cy5 (donkey anti-mouse) were from Jackson ImmunoResearch Laboratories. Hsp104 expression was revealed with a TSA fluorescein system (PerkinElmer Life Sciences). Sections were then analyzed by confocal microscopy (Leica TCS SP2 AOBS).
Silver staining was performed to detect degenerating neurons on paraformaldehyde-fixed sections (18, 47). The FD NeuroSilver kit was used according to the manufacturer’s protocol (FD Neuro-Technologies).
Cell counting and TH fiber density. To determine the percentage of TH-IR cell loss in the substantia nigra, 9–10 coronal sections of 40-μm thickness per animal were stained by immunofluorescence for the TH marker. All TH-IR neurons were counted in the injected and noninjected side of the substantia nigra, and the results were expressed as the percentage of TH-IR cell loss relative to the noninjected side (18, 42, 67). The borders of the substantia nigra were defined in the rostrocaudal axis using the anatomical landmarks in a rat brain atlas (68). Since the delineation of the borders between the ventral tegmental area (VTA) (A10), retrorubral nucleus (A8), and substantia nigra pars compacta (A9) are not clear, the medial border between VTA and substantia nigra was defined by a vertical line passing through the medial tip of the cerebral peduncle (and by the medial terminal nucleus of the accessory nucleus of the optic tract, when present in the sections), thereby excluding the TH-IR cells in the VTA. The ventral border followed the dorsal border of the cerebral peduncle, thereby including the TH-IR cells in pars reticulata, and the area extended laterally to include the pars lateralis in addition to the pars compacta. The counting was carried out caudally until the pars reticulata disappeared, thus excluding the retrorubral nucleus. The sections used for quantification covered the entire substantia nigra from the rostral tip of the pars compacta back to the caudal end of the pars reticulata. Importantly, no significant difference was observed in the estimated volume of the substantia nigra using Cavalieri’s principle or the size of the cell bodies of the counted neurons within experimental groups (noninjected versus injected side) or between experimental groups.
To determine the density of TH-IR terminals, striatal fibers were stained for TH with the ABC kit (Vector Laboratories), and the corresponding optical densities were evaluated with NIH IMAGE 1.4 software ( http://rsbweb.nih.gov/nih-image/) (18, 42). For the numbers of neurons containing phosphorylated α-syn inclusions, 5 sections throughout the substantia nigra were stained with the Pser129 Ab with the avidin-biotin complex method.
Statistics. Statistical analysis was performed by 1-way ANOVA, followed by a Scheffé’s protected least significant difference (PLSD) post-hoc test (Statistica 5.1; Statsoft Inc.). The significance level was set at P < 0.05.
Proteins. α-syn bacterial expression plasmids were kindly provided by P. Lansbury (Brigham and Women’s Hospital and Harvard Medical School, Cambridge, Massachusetts, USA). α-syn proteins (wild type, A53T, A30P, E46K, S129A, and S129E) were purified as described (20). Hsp104 and Hsp104K218T:K620T were purified as described (33). Hsp104 concentrations refer to hexameric Hsp104. Hsc70, Hsp70, Hdj1, and Hdj2 were from Alexis Biochemicals.
α-syn preamyloid oligomer purification and disassembly. α-syn A30P preamyloid oligomers were purified by gel filtration (15). For disassembly experiments, A30P preamyloid oligomers (0.5 μM monomer) were incubated with either Hsp104 or Hsp104K218T:K620T (10 μM) in KHM buffer (40 mM HEPES-KOH, pH 7.4, 150 mM KCl, 20 mM MgCl2, 1 mM DTT) plus ATP (10 mM) and an ATP regeneration system (20 mM creatine phosphate and 0.001 mg/ml creatine kinase) for 1 hour at 37°C. Reactions were processed for dot blot (33) and probed with either anti-oligomer Ab (kindly provided by C. Glabe, University of California, Irvine, Irvine, California, USA) (14) or anti–α-syn Ab (BD Biosciences). Alternatively, reactions were buffer exchanged using Bio-Gel P-6 spin columns into 40 mM HEPES-KOH, pH 7.4, 150 mM KCl, and 20 mM NaEDTA, diluted 5-fold and incubated for 10 minutes at 25°C to disassemble Hsp104 hexamers, and processed for negative-stain EM (33). Other reactions were depleted of Hsp104 as described (33) and then fractionated through a Microcon YM-100 (100-kDa molecular weight cut off) filter (Milllipore). Retentate and filtrate fractions were TCA precipitated and processed for SDS-PAGE followed by Coomassie Brilliant Blue staining.
α-syn fiber assembly and disassembly. For fibrillization reactions, α-syn proteins (80 μM) were incubated in KHM plus ATP (10 mM) and regeneration system plus or minus Hsp104 or Hsp104K218T:K620T (0–1.6 μM) for 0–48 hours at 37°C, with rotation (80 rpm) on a Mini-rotator (Glas-Col). Every 8 hours, reactions were supplemented with fresh regeneration system to maintain ATP levels.
For disassembly reactions, α-syn fibers (0.5 μM monomer) were incubated in KHM plus ATP (10 mM) and regeneration system plus or minus Hsp104 or Hsp104K218T:K620T (10 μM) in the presence or absence of the indicated combinations of Hsp70, Hsc70, Hdj1, and Hdj2 (10 μM) for 1 hour at 37°C. In reactions containing AMP-PNP (1 mM), the regeneration system was omitted.
Fiber assembly or disassembly was determined by ThT fluorescence, sedimentation analysis, turbidity, or EM (8, 33). For ThT fluorescence, ThT in 50 mM glycine (pH 8.5) was added to give final concentrations of α-syn (0.25 μM) and ThT (10 μM). Fluorescence at 480 nm was measured after excitation at 450 nm. For sedimentation analysis, reactions were centrifuged at 436,000 g for 10 minutes at 25°C. Supernatant and pellet fractions were then resolved by SDS-PAGE and stained with Coomassie Brilliant Blue. The percentage of α-syn in the pellet was determined by densitometry and comparison to know quantities of α-syn. Turbidity was monitored by absorbance at 395 nm.
Acknowledgments
We thank D. Picard, P. Lansbury, and C. Glabe for generous provision of reagents; P. Colin, C. Sadeghi, and M. Rey for excellent technical assistance; A. Gitler for comments on the manuscript. C. Lo Bianco was supported by the Michael J. Fox Foundation, European Molecular Biology Organization, Swedish Parkinson Foundation, and the Swiss National Science Foundation. J. Shorter was supported by an American Heart Association Scientist Development Grant, University of Pennsylvania Institute on Aging pilot grant, and NIH Director’s New Innovator Award (DP2OD002177). P. Aebischer was supported by the Michael J. Fox Foundation and the Swiss National Science Foundation.
Footnotes
Nonstandard abbreviations used: PD, Parkinson disease; α-syn, α-synuclein; TH, tyrosine hydroxylase; TH-IR, TH-immunoreactive; ThT, Thioflavin-T; YFP, yellow fluorescent protein.
References
1. Skovronsky, D.M., Lee, V.M., Trojanowski, J.Q. 2006. Neurodegenerative diseases: new concepts of pathogenesis and their therapeutic implications. Annu. Rev. Pathol. 1:151-170.
2. Dorsey, E.R., et al. 2007. Projected number of people with Parkinson’s disease in the most populous nations, 2005 through 2030. Neurology. 68:384-386.
3. Braak, H., et al. 2003. Staging of brain pathology related to sporadic Parkinson’s disease. Neurobiol. Aging. 24:197-211.
4. Spillantini, M.G., et al. 1997. Alpha-synuclein in Lewy bodies. Nature. 388:839-840.
5. Moore, D.J., West, A.B., Dawson, V.L., Dawson, T.M. 2005. Molecular pathophysiology of Parkinson’s disease. Annu. Rev. Neurosci. 28:57-87.
6. Davidson, W.S., Jonas, A., Clayton, D.F., George, J.M. 1998. Stabilization of alpha-synuclein secondary structure upon binding to synthetic membranes. J. Biol. Chem. 273:9443-9449.
7. Conway, K.A., Harper, J.D., Lansbury, P.T. 1998. Accelerated in vitro fibril formation by a mutant alpha-synuclein linked to early-onset Parkinson disease. Nat. Med. 4:1318-1320.
8. Conway, K.A., et al. 2000. Acceleration of oligomerization, not fibrillization, is a shared property of both alpha-synuclein mutations linked to early-onset Parkinson’s disease: implications for pathogenesis and therapy. Proc. Natl. Acad. Sci. U. S. A. 97:571-576.
9. Serpell, L.C., Berriman, J., Jakes, R., Goedert, M., Crowther, R.A. 2000. Fiber diffraction of synthetic alpha-synuclein filaments shows amyloid-like cross-beta conformation. Proc. Natl. Acad. Sci. U. S. A. 97:4897-4902.
10. Crowther, R.A., Daniel, S.E., Goedert, M. 2000. Characterisation of isolated alpha-synuclein filaments from substantia nigra of Parkinson’s disease brain. Neurosci. Lett. 292:128-130.
11. Spillantini, M.G., et al. 1998. Filamentous alpha-synuclein inclusions link multiple system atrophy with Parkinson’s disease and dementia with Lewy bodies. Neurosci. Lett. 251:205-208.
12. Miake, H., Mizusawa, H., Iwatsubo, T., Hasegawa, M. 2002. Biochemical characterization of the core structure of alpha-synuclein filaments. J. Biol. Chem. 277:19213-19219.
13. Neumann, M., et al. 2002. Misfolded proteinase K-resistant hyperphosphorylated alpha-synuclein in aged transgenic mice with locomotor deterioration and in human alpha-synucleinopathies. J. Clin. Invest. 110:1429-1439.
14. Kayed, R., et al. 2003. Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis. Science. 300:486-489.
15. Lashuel, H.A., et al. 2002. Alpha-synuclein, especially the Parkinson’s disease-associated mutants, forms pore-like annular and tubular protofibrils. J. Mol. Biol. 322:1089-1102.
16. Sharon, R., et al. 2003. The formation of highly soluble oligomers of alpha-synuclein is regulated by fatty acids and enhanced in Parkinson’s disease. Neuron. 37:583-595.
17. Chen, L., Feany, M.B. 2005. Alpha-synuclein phosphorylation controls neurotoxicity and inclusion formation in a Drosophila model of Parkinson disease. Nat. Neurosci. 8:657-663.
18. Lo Bianco, C., et al. 2004. Lentiviral vector delivery of parkin prevents dopaminergic degeneration in an alpha-synuclein rat model of Parkinson’s disease. Proc. Natl. Acad. Sci. U. S. A. 101:17510-17515.
19. Choi, W., et al. 2004. Mutation E46K increases phospholipid binding and assembly into filaments of human alpha-synuclein. FEBS Lett. 576:363-368.
20. Fredenburg, R.A., et al. 2007. The impact of the E46K mutation on the properties of alpha-synuclein in its monomeric and oligomeric states. Biochemistry. 46:7107-7118.
21. Fujiwara, H., et al. 2002. alpha-Synuclein is phosphorylated in synucleinopathy lesions. Nat. Cell Biol. 4:160-164.
22. Norris, E.H., Giasson, B.I., Ischiropoulos, H., Lee, V.M. 2003. Effects of oxidative and nitrative challenges on alpha-synuclein fibrillogenesis involve distinct mechanisms of protein modifications. J. Biol. Chem. 278:27230-27240.
23. Balch, W.E., Morimoto, R.I., Dillin, A., Kelly, J.W. 2008. Adapting proteostasis for disease intervention. Science. 319:916-919.
24. Auluck, P.K., Chan, H.Y., Trojanowski, J.Q., Lee, V.M., Bonini, N.M. 2002. Chaperone suppression of alpha-synuclein toxicity in a Drosophila model for Parkinson’s disease. Science. 295:865-868.
25. Cummings, C.J., et al. 1998. Chaperone suppression of aggregation and altered subcellular proteasome localization imply protein misfolding in SCA1. Nat. Genet. 19:148-154.
26. Warrick, J.M., et al. 1999. Suppression of polyglutamine-mediated neurodegeneration in Drosophila by the molecular chaperone HSP70. Nat. Genet. 23:425-428.
27. Glover, J.R., Lindquist, S. 1998. Hsp104, Hsp70, and Hsp40: a novel chaperone system that rescues previously aggregated proteins. Cell. 94:73-82.
28. Shorter, J. 2008. Hsp104: a weapon to combat diverse neurodegenerative disorders. Neurosignals. 16:63-74.
29. Wendler, P., et al. 2007. Atypical AAA+ subunit packing creates an expanded cavity for disaggregation by the protein-remodeling factor Hsp104. Cell. 131:1366-1377.
30. Parsell, D.A., Kowal, A.S., Singer, M.A., Lindquist, S. 1994. Protein disaggregation mediated by heat-shock protein Hsp104. Nature. 372:475-478.
31. Sanchez, Y., Lindquist, S.L. 1990. HSP104 required for induced thermotolerance. Science. 248:1112-1115.
32. Sanchez, Y., Taulien, J., Borkovich, K.A., Lindquist, S. 1992. Hsp104 is required for tolerance to many forms of stress. EMBO J. 11:2357-2364.
33. Shorter, J., Lindquist, S. 2004. Hsp104 catalyzes formation and elimination of self-replicating Sup35 prion conformers. Science. 304:1793-1797.
34. Shorter, J., Lindquist, S. 2006. Destruction or potentiation of different prions catalyzed by similar Hsp104 remodeling activities. Mol. Cell. 23:425-438.
35. Doyle, S.M., et al. 2007. Asymmetric deceleration of ClpB or Hsp104 ATPase activity unleashes protein-remodeling activity. Nat. Struct. Mol. Biol. 14:114-122.
36. Narayanan, S., Walter, S., Reif, B. 2006. Yeast prion-protein, sup35, fibril formation proceeds by addition and substraction of oligomers. Chembiochem. 7:757-765.
37. Savistchenko, J., Krzewska, J., Fay, N., Melki, R. 2008. Molecular chaperones and the assembly of the prion Ure2p in vitro. J. Biol. Chem. 283:15732-15739.
38. Chernoff, Y.O., Lindquist, S.L., Ono, B., Inge-Vechtomov, S.G., Liebman, S.W. 1995. Role of the chaperone protein Hsp104 in propagation of the yeast prion-like factor [PSI+]. Science. 268:880-884.
39. Dandoy-Dron, F., et al. 2006. Infection by ME7 prion is not modified in transgenic mice expressing the yeast chaperone Hsp104 in neurons. Neurosci. Lett. 405:181-185.
40. Mosser, D.D., Ho, S., Glover, J.R. 2004. Saccharomyces cerevisiae Hsp104 enhances the chaperone capacity of human cells and inhibits heat stress-induced proapoptotic signaling. Biochemistry. 43:8107-8115.
41. Nelson, R., Eisenberg, D. 2006. Structural models of amyloid-like fibrils. Adv. Protein Chem. 73:235-282.
42. Lo Bianco, C., Ridet, J.L., Schneider, B.L., Deglon, N., Aebischer, P. 2002. alpha -Synucleinopathy and selective dopaminergic neuron loss in a rat lentiviral-based model of Parkinson’s disease. Proc. Natl. Acad. Sci. U. S. A. 99:10813-10818.
43. Bao, Y.P., Cook, L.J., O’Donovan, D., Uyama, E., Rubinsztein, D.C. 2002. Mammalian, yeast, bacterial, and chemical chaperones reduce aggregate formation and death in a cell model of oculopharyngeal muscular dystrophy. J. Biol. Chem. 277:12263-12269.
44. Kirik, D., et al. 2003. Nigrostriatal alpha-synucleinopathy induced by viral vector-mediated overexpression of human alpha-synuclein: a new primate model of Parkinson’s disease. Proc. Natl. Acad. Sci. U. S. A. 100:2884-2889.
45. Kirik, D., et al. 2002. Parkinson-like neurodegeneration induced by targeted overexpression of alpha-synuclein in the nigrostriatal system. J. Neurosci. 22:2780-2791.
46. Klein, R.L., King, M.A., Hamby, M.E., Meyer, E.M. 2002. Dopaminergic cell loss induced by human A30P alpha-synuclein gene transfer to the rat substantia nigra. Hum. Gene Ther. 13:605-612.
47. Beltramino C.A., de Olmos J.S., Gallyas F., Heimer L., Záborszky L.. 1993;Silver staining as a tool for neurotoxic assessment.. NIDA Res. Monogr. 136:101–126; discussion 126–132.126; discussion 126–132 . 82899098289909
48. Sandmann-Keil, D., Braak, H., Okochi, M., Haass, C., Braak, E. 1999. Alpha-synuclein immunoreactive Lewy bodies and Lewy neurites in Parkinson’s disease are detectable by an advanced silver-staining technique. Acta Neuropathol. 98:461-464.
49. Schirmer, E.C., Queitsch, C., Kowal, A.S., Parsell, D.A., Lindquist, S. 1998. The ATPase activity of Hsp104, effects of environmental conditions and mutations. J. Biol. Chem. 273:15546-15552.
50. Lashuel, H.A., Hartley, D., Petre, B.M., Walz, T., Lansbury (Jr.), P.T. 2002. Neurodegenerative disease: amyloid pores from pathogenic mutations. Nature. 418:291.
51. Kong, B., Chae, Y., Lee, K. 2005. Degradation of wild-type alpha-synuclein by a molecular chaperone leads to reduced aggregate formation. Cell Biochem. Funct. 23:125-132.
52. Kong, B., Chae, Y.K., Lee, K. 2003. Regulation of in vitro fibril formation of synuclein mutant proteins by Hsp104p. Protein Pept. Lett. 10:491-495.
53. Parsell, D.A., Sanchez, Y., Stitzel, J.D., Lindquist, S. 1991. Hsp104 is a highly conserved protein with two essential nucleotide-binding sites. Nature. 353:270-273.
54. Parsell, D.A., Taulien, J., Lindquist, S. 1993. The role of heat-shock proteins in thermotolerance. Philos. Trans. R. Soc. Lond. B Biol. Sci. 339:279-286.
55. Yamamoto, A., Lucas, J.J., Hen, R. 2000. Reversal of neuropathology and motor dysfunction in a conditional model of Huntington’s disease. Cell. 101:57-66.
56. Martin-Aparicio, E., et al. 2001. Proteasomal-dependent aggregate reversal and absence of cell death in a conditional mouse model of Huntington’s disease. J. Neurosci. 21:8772-8781.
57. Shorter, J., Lindquist, S. 2005. Prions as adaptive conduits of memory and inheritance. Nat. Rev. Genet. 6:435-450.
58. Del Mar, C., Greenbaum, E.A., Mayne, L., Englander, S.W., Woods (Jr.), V.L. 2005. Structure and properties of alpha-synuclein and other amyloids determined at the amino acid level. Proc. Natl. Acad. Sci. U. S. A. 102:15477-15482.
59. Outeiro, T.F., Lindquist, S. 2003. Yeast cells provide insight into alpha-synuclein biology and pathobiology. Science. 302:1772-1775.
60. Cashikar, A.G., Duennwald, M., Lindquist, S.L. 2005. A chaperone pathway in protein disaggregation. Hsp26 alters the nature of protein aggregates to facilitate reactivation by Hsp104. J. Biol. Chem. 280:23869-23875.
61. Cooper, A.A., et al. 2006. Alpha-synuclein blocks ER-Golgi traffic and Rab1 rescues neuron loss in Parkinson’s models. Science. 313:324-328.
62. Gitler, A.D., et al. 2008. The Parkinson’s disease protein alpha-synuclein disrupts cellular Rab homeostasis. Proc. Natl. Acad. Sci. U. S. A. 105:145-150.
63. Gitler, A.D., Shorter, J. 2007. Prime time for alpha-synuclein. J. Neurosci. 27:2433-2434.
64. Carmichael, J., et al. 2000. Bacterial and yeast chaperones reduce both aggregate formation and cell death in mammalian cell models of Huntington’s disease. Proc. Natl. Acad. Sci. U. S. A. 97:9701-9705.
65. Perrin, V., et al. 2007. Neuroprotection by Hsp104 and Hsp27 in lentiviral-based rat models of Huntington’s disease. Mol. Ther. 15:903-911.
66. Vacher, C., Garcia-Oroz, L., Rubinsztein, D.C. 2005. Overexpression of yeast hsp104 reduces polyglutamine aggregation and prolongs survival of a transgenic mouse model of Huntington’s disease. Hum. Mol. Genet. 14:3425-3433.
67. Deglon, N., et al. 2000. Self-inactivating lentiviral vectors with enhanced transgene expression as potential gene transfer system in Parkinson’s disease. Hum. Gene Ther. 11:179-190.
68. Paxinos, G., and Watson, C. 1998.The rat brain in stereotaxic coordinates. 4th edition. Academic Press. San Diego, California, USA. 256 pp.
Published August 14, 2008
Received for publication March 28, 2008, and accepted in revised form July 7, 2008.
Parkinson disease (PD) is characterized by dopaminergic neurodegeneration and intracellular inclusions of α-synuclein amyloid fibers, which are stable and difficult to dissolve. Whether inclusions are neuroprotective or pathological remains controversial, because prefibrillar oligomers may be more toxic than amyloid inclusions. Thus, whether therapies should target inclusions, preamyloid oligomers, or both is a critically important issue. In yeast, the protein-remodeling factor Hsp104 cooperates with Hsp70 and Hsp40 to dissolve and reactivate aggregated proteins. Metazoans, however, have no Hsp104 ortholog. Here we introduced Hsp104 into a rat PD model. Remarkably, Hsp104 reduced formation of phosphorylated α-synuclein inclusions and prevented nigrostriatal dopaminergic neurodegeneration induced by PD-linked α-synuclein (A30P). An in vitro assay employing pure proteins revealed that Hsp104 prevented fibrillization of α-synuclein and PD-linked variants (A30P, A53T, E46K). Hsp104 coupled ATP hydrolysis to the disassembly of preamyloid oligomers and amyloid fibers composed of α-synuclein. Furthermore, the mammalian Hsp70 and Hsp40 chaperones, Hsc70 and Hdj2, enhanced α-synuclein fiber disassembly by Hsp104. Hsp104 likely protects dopaminergic neurons by antagonizing toxic α-synuclein assemblies and might have therapeutic potential for PD and other neurodegenerative amyloidoses.
Introduction
Abnormal protein aggregation in the brain characterizes several lethal neurodegenerative diseases (1), including Parkinson disease (PD). There are no cures for PD, the most common neurodegenerative movement disorder, which debilitates several million people worldwide (2). PD involves a progressive and selective elimination of dopaminergic neurons from the substantia nigra pars compacta, although neuropathology can extend into other brain regions (3). The signature lesions of PD are intracellular inclusions termed Lewy Bodies and Lewy neurites, which contain the small presynaptic protein, α-synuclein (α-syn) (4). Although PD is primarily a sporadic disorder, mutations in α-syn (e.g., A53T, A30P, E46K) and overexpression of the wild-type gene are linked with early-onset PD in rare familial forms of the disease (5).
The function of α-syn is uncertain, but various studies connect α-syn to synaptic vesicle pool regulation and dopamine release (5). Yet, how these potential functions might relate to PD is unclear. α-syn is natively unstructured in isolation, but gains α-helical structure upon association with phospholipid bilayers (6). Purified α-syn forms amyloid fibers in vitro, which bind to the diagnostic dyes Thioflavin-T (ThT) and Congo red and possess the generic amyloid “cross-β” conformation, in which the strands of the β-sheets run orthogonal to the fiber axis (7–9). Fibers assembled in vitro are very similar to α-syn filaments isolated from synucleinopathy patients (10, 11). Once initiated, α-syn amyloidogenesis can cascade out of control, because α-syn fibers self-template by recruiting non-amyloid α-syn conformers to fiber ends and converting them to the amyloid form (8). α-syn fibers are exceptionally stable (e.g., protease and detergent resistant) and extraordinarily difficult to clear (12, 13).
Prior to fibrillization, α-syn populates heterogeneous oligomeric states, which have not yet accessed the final cross-β form of mature fibers. This ensemble of preamyloid oligomeric states comprises transient species that are likely “on-pathway” to fiber formation as well as “off-pathway” forms (8, 14, 15). One of the transient oligomeric conformations populated by α-syn is common to many amyloidogenic proteins regardless of their primary sequence and is recognized by a conformation-specific Ab (14). Preamyloid α-syn oligomers may be more cytotoxic than fibers (14, 16), and sequestration of α-syn into fiber inclusions at the expense of preamyloid oligomers might even be neuroprotective (17, 18). Despite intense investigation, how the process of α-syn amyloidogenesis elicits the selective cell death that distinguishes PD and other synucleinopathies remains unclear.
PD-linked mutations in α-syn increase its propensity to access misfolded forms. A53T and E46K fibrillize more rapidly than wild-type α-syn, whereas A30P fibrillizes more slowly (7, 8, 19). By contrast, A30P and A53T access preamyloid oligomers more rapidly than wild-type α-syn, whereas E46K is less able to form these species (8, 20). Specific posttranslational modifications, such as phosphorylation at serine 129 and nitration, also promote α-syn fibrillization (21, 22). Phosphorylated and nitrated α-syn selectively and abundantly accumulates in α-synucleinopathy lesions in animal models and humans (5, 13, 18, 21). Together, this suggests that α-syn misfolding contributes to familial and sporadic PD. Thus, inhibiting α-syn misfolding and/or aggregation or promoting the clearance of α-syn aggregates may constitute promising therapeutic strategies for PD and other synucleinopathies.
Protein misfolding is a problem as ancient as life itself, and so too are the solutions that synergize to antagonize it. Thus, sophisticated molecular chaperones recognize misfolded proteins and prevent their aggregation, protein-remodeling factors resolve protein aggregates, osmolytes function as chemical chaperones, and degradation systems eradicate misfolded proteins. Upregulation of protein quality control safeguards may provide important therapeutic avenues (23). Indeed, Hsp70 and Hsp40 chaperones associate with Lewy Bodies and Lewy neurites in PD and other synucleinopathies (24) as well as polyglutamine aggregates in several diseases (25, 26). Overexpression of Hsp70 and Hsp40 can suppress polyglutamine aggregation (25) or ameliorate toxicity associated with α-syn aggregation (24). Yet, Hsp70 and Hsp40 have only very limited ability to resolve protein aggregates once they have formed (27). The ability to restore aggregated proteins to native structure and function would obviate the huge energetic cost of degrading and resynthesizing them. Further, this would simultaneously eliminate 3 malicious problems associated with protein aggregation that likely synergize in the etiology of various protein misfolding disorders: (a) the toxic gain of function of aggregated conformers; (b) the loss of function of the aggregated protein; and (c) the sequestration of other essential proteins that coprecipitate with the aggregated protein. However, whether mammals possess any activity that reverses protein aggregation and restores the functionality of aggregated proteins remains unclear.
By contrast, fungi, plants, and bacteria all express orthologs of Hsp104, a powerful protein-remodeling factor. All are hexameric AAA+ (ATPases associated with diverse activities) proteins with 2 AAA+ ATPase domains per monomer (28, 29). Hsp104 synergizes with Hsp70 and Hsp40 to resolve protein aggregates and return proteins to normal enzymatic activity (27, 30). This increases cell survival after multifarious stresses by up to 10,000-fold (31, 32). Hsp104 also possesses an unusually powerful amyloid-remodeling activity and rapidly disassembles amyloid fibers composed of the yeast prion proteins Sup35 and Ure2 (29, 33–37). Moreover, Hsp104 eliminates Sup35 preamyloid oligomers that adopt a conformation shared by many amyloidogenic proteins, including α-syn (14, 33, 34). Critically, even transient overexpression of Hsp104 can purge yeast of Sup35 prions (38). Given these remarkable activities, which would appear beneficial to all cells, it is puzzling why Hsp104 has been lost from metazoan lineages. This issue remains moot and unaddressed. Nevertheless, Hsp104 can synergize with the mammalian Hsp70 chaperone system to promote protein disaggregation and stress tolerance (39, 40).
Disassembly of yeast prions by Hsp104 raises awareness that amyloids can be resolved by a protein-remodeling factor (28). Amyloids share a common cross-β scaffold, where the β-sheet strands are aligned orthogonal to the fiber axis, irrespective of the primary sequence of the protein (41). Even though local steric details of different amyloids may vary enormously (41), this suggests that agents that antagonize amyloid fibers of one protein may also be active against amyloid fibers composed of another. Some preamyloid oligomers also share a common structure that is independent of primary sequence and distinct to that of fibers (14). Thus, can introduction of Hsp104 into metazoan systems prevent or reverse various amyloidoses? Answering this question will help clarify whether protein aggregation is protective or toxic in various disease settings.
The development of potential therapies for PD has been hampered by a paucity of animal models that recapitulate the selective loss of dopaminergic neurons. Here we employ a rat PD model based on the lentiviral-mediated expression of human α-syn A30P in the substantia nigra, which successfully recreates the progressive and selective degeneration of dopaminergic neurons and formation of phosphorylated α-syn inclusions that characterize PD (18, 42). We first evaluated whether Hsp104 affects α-syn toxicity and aggregation in this model. Because Hsp104 does not have general anti-apoptotic effects (e.g., in response to staurosporine or hydrogen peroxide) when expressed in mammalian cells (43), any reductions in toxicity will likely reflect direct effects on α-syn misfolding. We then employed pure proteins to delineate how Hsp104 modulates α-syn aggregation. Our studies highlight the therapeutic utility of Hsp104 for neurodegenerative disease and provide new insights into the controversial issues of how protein aggregation and inclusion formation contribute to PD pathogenesis and other diseases.
Results
Hsp104 reduces α-syn–induced toxicity. Overexpression of mutated human α-syn with viral vectors induces a progressive loss of nigral dopamine neurons in rodents and nonhuman primates (42, 44–46). To test whether Hsp104 modulates α-syn aggregation and α-syn–induced degeneration of dopamine neurons, we simultaneously overexpressed Hsp104 and A30P α-syn in the brain of rats. Viral suspensions containing lentiviral vectors coding either for A30P human α-syn and Hsp104 (lenti-A30P/lenti-Hsp104; see Methods) or for A30P human α-syn and yellow fluorescent protein (lenti-A30P/lenti-YFP; see Methods) were directly injected in the substantia nigra of rats. Brain slices from the substantia nigra were then stained for the dopaminergic marker tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis.
Animals injected with lentiviral vectors coding for A30P α-syn showed a 33% loss of TH-immunoreactive (TH-IR) neurons in the substantia nigra at 6 weeks after injection (42). Importantly, viral expression of 2 reporter proteins (YFP/GFP) does not induce any damage to the nigrostriatal pathway (18). In contrast, injection of lenti-A30P/lenti-YFP in the right side of the rat substantia nigra induced a marked loss of TH-IR cells compared with the noninjected side (Figure 1A), similar to the expression of A30P α-syn alone. Coexpression of Hsp104 rescued TH-IR neurons from A30P α-syn–induced toxicity (Figure 1A). Quantitation of neuronal loss was performed throughout the whole substantia nigra (Figure 1B). The contralateral noninjected side was used as an internal control. Animals expressing A30P/YFP revealed a 31.4% loss of TH-IR cells relative to the contralateral side. Expression of Hsp104 resulted in a significant reduction in cell loss (12.6%; P < 0.05; n = 7). Confocal analysis of animals expressing Hsp104/A30P revealed that surviving dopamine neurons still expressed abundant levels of human α-syn (Figure 1C).
Hsp104 prevents A30P α-syn–induced neurodegeneration. Nigral dopaminergic neurons project their nerve terminals directly to the striatum. Lentiviral-mediated expression of A30P α-syn in the substantia nigra of rats induces a significant loss of striatal nerve terminals (42). To determine whether the protective effect of Hsp104 on dopaminergic cells is accompanied by reductions in the loss of nerve terminals in the striatum, TH staining was performed on striatal brain slices from animals expressing either A30P/YFP or A30P/Hsp104 (Figure 2A).
Animals expressing A30P/YFP showed a decrease in the TH marker in the ipsilateral side (Figure 2A). Nigral expression of Hsp104 reduced the dopaminergic nerve terminal loss induced by the accumulation of A30P α-syn (Figure 2A). Quantitation of TH-IR fiber density was performed throughout the striatum. Consistent with the neuroprotective effect of Hsp104 at the cellular level, expression of Hsp104 significantly decreased the nerve terminal loss from 21.6% (A30P/YFP) to 7% (A30P/Hsp104) (P < 0.05; n = 7) (Figure 2B).
Next, we used silver staining as a marker for degenerating neurons in animals expressing A30P/YFP or A30P/Hsp104 (42, 47, 48). No silver staining was observed in the noninjected substantia nigra (Figure 3, A and D). In contrast, scattered neurons containing silver-positive dark structures were observed in animals injected with lenti-A30P/lenti-YFP, consistent with α-syn–induced degeneration (18, 42) (Figure 3B). Higher magnifications revealed that animals expressing A30P/YFP abundantly accumulated granular deposits in both cell bodies and axons at the substantia nigra level (Figure 3, E and G) (42). Clear neuritic pathology was also observed (Figure 3G). In stark contrast, coexpression of Hsp104 greatly reduced the appearance of silver-positive degenerating neurons and granular silver deposits (Figure 3, C and F), indicating that Hsp104 antagonizes A30P α-syn–induced neurodegeneration. Only a few sparse silver-positive neurons were still observed in the A30P/Hsp104 group. Although these observations were consistently observed in animals expressing Hsp104, the high variability in the signal/noise ratio observed with silver staining among sections, even within the same animal, prevents quantification.
Hsp104 reduces the formation of phosphorylated inclusions. To understand the mechanism by which Hsp104 prevents α-syn–induced neurodegeneration, we investigated how Hsp104 affects the α-syn aggregation in vivo. Two different Abs were used to detect α-syn in animals expressing either A30P/YFP or A30P/Hsp104 (Figure 4). The Ab RG syn recognizes both rat and human α-syn by western blot (42). However, this Ab only detects overexpression of α-syn and not endogenous levels of this protein in the rat brain (42). Stainings with LB509, an Ab that is specific for human α-syn, were also performed (Figure 4, D–I). A strong accumulation of human α-syn in both the perikarya and neurites is observed in the substantia nigra of animals expressing either A30P/YFP (Figure 4, B and E) or A30P/Hsp104 (Figure 4, C and F) as compared with noninjected animals (Figure 4, A, D, and G). Examination at higher magnification illustrates the difficulty in discriminating between α-syn aggregates and subcellular accumulation of this protein (Figure 4, H and I). High levels of soluble α-syn may mask the appearance of aggregates and render quantification very difficult. However, in some neurons, dense puncta were frequently observed in neurites, and these were less evident in animals expressing Hsp104. Furthermore, a more dense coloration inside the perikarya of α-syn–positive neurons indicates the presence of α-syn aggregates (Figure 4, H and I). Hsp104 significantly reduces the percentage of α-syn–positive neurons harboring dense structures in cell bodies or axons (54.8% ± 5.5% and 30.8% ± 4.4% for A30P/YFP and A30P/Hsp104, respectively; mean ± SEM; P < 0.05; n = 7). Interestingly, in animals expressing Hsp104, we detected a more diffuse and homogenous α-syn localization along the neuropil, indicating that Hsp104 antagonizes the dense structures or aggregates and helps maintain soluble forms of α-syn. These data suggest that Hsp104 may antagonize α-syn aggregation in vivo.
To evaluate more clearly, whether Hsp104 affected α-syn aggregation in vivo, we assessed phosphorylated α-syn inclusions. α-syn phosphorylated predominantly at serine 129 selectively and abundantly accumulates in both Lewy bodies of PD patients and inclusions of α-syn animal models (13, 18, 21). Therefore, we quantified the number of phosphorylated inclusions with the Pser129 Ab specific for phosphorylated α-syn at serine 129 (18, 21) (Figure 4, J–M). As described previously (18), expression of A30P α-syn leads to the formation of phosphorylated inclusions (Figure 4K) as compared with noninjected animals (Figure 4J). Strikingly, coexpression of Hsp104 with A30P α-syn resulted in a 57% reduction in the number of neurons containing phosphorylated inclusions (Figure 4, L and M), indicating that Hsp104 may protect dopaminergic cells by decreasing the numbers of phosphorylated α-syn aggregates.
Hsp104 inhibits α-syn amyloidogenesis. From our in vivo experiments, it is difficult to distinguish whether Hsp104 prevents aggregation of α-syn or resolubilizes α-syn after it has aggregated. Therefore, we determined how Hsp104 affects α-syn amyloidogenesis in vitro using pure proteins. In vitro, purified α-syn assembles into fibers, which are very similar to α-syn filaments isolated from synucleinopathy patients (9, 10). α-syn assembled into amyloid fibers after a lag of approximately 5 hours of incubation, and assembly was virtually complete after approximately 24 hours as assessed by ThT fluorescence (a diagnostic amyloid dye) (Figure 5A) and sedimentation analysis (Figure 5B). When Hsp104 was added at substoichiometric levels (α-syn monomers/Hsp104 hexamers, 80:0.2 μM) fibrillization was retarded (Figure 5, A and B). Remarkably, higher Hsp104 concentrations (α-syn monomers/Hsp104 hexamers, 80:0.8 μM or 80:1.6 μM) allowed very little fibrillization after 48 hours (Figure 5, A and B). EM confirmed that Hsp104 inhibited α-syn fibrillization (Figure 5C). Amorphous material accumulated in the presence of Hsp104 (Figure 5C). Inhibition of α-syn fibrillization by Hsp104 was stable until approximately 72 hours (data not shown). After 96 hours, Hsp104 began to lose activity and some α-syn fibrillization was able to occur (Figure 5D). However, if reactions were supplemented with additional Hsp104 at 72 hours, the inhibition of α-syn fibrillization was maintained (Figure 5D). Thus, the inhibition α-syn fibrillization is stable, provided there is a renewable source of active Hsp104.
Hsp104 afforded little inhibition of α-syn fibrillization in the absence of ATP and inhibition was reduced by the presence of the nonhydrolyzable ATP analogue, AMP-PNP (Figure 5A). We also employed Hsp104 carrying mutations in the highly conserved Walker A motifs of both AAA+ ATPase domains, Hsp104K218T:K620T, which is defective in ATP binding and hydrolysis at both sites (49). The mutant failed to inhibit α-syn fibrillization (Figure 5A). Thus, maximal inhibition of α-syn fibrillization requires ATP binding and hydrolysis by Hsp104.
Next, we tested whether Hsp104 could inhibit the fibrillization of PD-linked α-syn variants, including A30P, A53T, and E46K. A53T and E46K assemble into amyloid fibers more rapidly than wild-type α-syn, whereas A30P accesses preamyloid oligomers more rapidly than wild type but takes longer to form fibers (7, 19). Hsp104 inhibited A53T fibrillization at early times, but this inhibition was overcome by 48 hours of incubation (Figure 5E). Stable inhibition of A53T over the 48-hour time frame required higher concentrations of Hsp104 (Figure 5E), perhaps because A53T assembles more rapidly than any of the other α-syn variants. By contrast, Hsp104 inhibited fibrillization by A30P and E46K just as well as wild-type α-syn (Figure 5E). Thus, Hsp104 potently inhibits the assembly of the spectrum of PD-linked α-syn mutants.
In synucleinopathy patients, α-syn is phosphorylated on serine 129, which may stimulate α-syn fibrillization (21). We found that Hsp104 inhibits fibrillization of both α-syn S129A, which cannot be phosphorylated at position 129, and S129E, which may mimic α-syn phosphorylated at S129 (Figure 5E). Therefore, the reduction in phosphorylated A30P α-syn inclusions observed in vivo is likely due, at least in part, to the inhibition of their assembly by Hsp104.
Hsp104 remodels α-syn A30P preamyloid oligomers. Preamyloid oligomers may be the more cytotoxic species in many neurodegenerative amyloidoses, including synucleinopathies (14, 50). Further, they are likely important intermediates in the fibrillization process (8, 15, 33, 34). Thus, we determined if Hsp104 could remodel preformed A30P preamyloid oligomers. A30P preamyloid oligomers were formed and purified away from monomers by gel filtration (15). Purified A30P preamyloid oligomers are stable for long periods (~10 days) and do not dissociate into monomers or dimers (15). However, to ensure our starting material was 100% oligomeric, purified A30P preamyloid oligomers were treated immediately with Hsp104 or Hsp104K218T:K620T. Hsp104K218T:K620T was unable to remodel preamyloid A30P oligomers as determined by anti-oligomer immunoreactivity, EM, and retention by a 100-kDa filter (Figure 6). By contrast, Hsp104 reduced anti-oligomer immunoreactivity (Figure 6A), and EM revealed that Hsp104 disassembled A30P preamyloid oligomers (Figure 6B). A30P was now able traverse a 100-kDa filter (Figure 6, C and D). Thus, Hsp104 eradicates α-syn A30P preamyloid oligomers, which are potentially the most toxic species that arise during A30P amyloidogenesis (14).
Hsp104 remodels α-syn fibers. Finally, we tested whether Hsp104 remodeled fibers formed by wild-type α-syn as well as PD-linked mutants A30P, A53T, and E46K and the serine 129 mutants S129A and S129E. Remarkably, Hsp104 disassembled fibers composed of wild-type α-syn, A53T, A30P, and S129A as revealed by ThT fluorescence (Figure 7A) and turbidity (Figure 7B). Of these, A30P was the most susceptible to disassembly by Hsp104 (Figure 7, A–C). This required ATP hydrolysis by Hsp104 and was not observed with Hsp104K218T:K620T or in the presence of AMP-PNP (Figure 7, A and B). Intriguingly, S129E fibers were more resistant to disassembly by Hsp104, and E46K fibers were refractory to Hsp104. In sum, these data suggest that Hsp104 disassembles fibers composed of wild-type α-syn and some PD-linked α-syn variants.
Hsp104 combines with mammalian Hsp70 and Hsp40 chaperones to promote reactivation of denatured luciferase aggregates (27, 40). Thus, we tested whether mammalian Hsp70 and Hsp40 chaperones could assist Hsp104 in disassembling α-syn fibers. In contrast to Hsp104 alone, Hsp70 and Hsc70 alone or in combination with either Hdj1 or Hdj2 were unable to disassemble α-syn fibers over the time frame of the assay (Figure 7, D and E). Hsp104 combined with Hsc70 and Hdj2 to promote more α-syn fiber disassembly than Hsp104 alone as determined by ThT fluorescence (Figure 7D) and sedimentation analysis (Figure 7E). To a lesser extent, Hsp104, Hsp70, and Hdj2 promoted more α-syn fiber disassembly than Hsp104 alone (Figure 7, D and E), while other combinations were equally (Hsp104, Hsp70, and Hdj1) or slightly less (Hsp104, Hsc70, and Hdj1) effective than Hsp104 alone (Figure 7, D and E). Overall, these data demonstrate that Hsp104 antagonizes various α-syn conformations populated during amyloidogenesis, and that the mammalian Hsp70 chaperone system can assist Hsp104 in disassembling α-syn fibers.
Discussion
Here we show for what we believe to be the first time that augmentation of the mammalian protein quality control system with a protein-remodeling factor not ordinarily found in metazoa, Hsp104, dramatically reduces dopaminergic neurodegeneration and phosphorylated α-syn inclusion formation in a rat lentiviral model of PD. To help understand these events, we employed pure proteins to analyze how Hsp104 affects α-syn amyloidogenesis. Importantly, Hsp104 directly inhibits the fibrillization of α-syn as well as the PD-linked α-syn mutants A53T, A30P, and E46K and the serine 129 phosphorylation mutants S129A and S129E. Hsp104 inhibited α-syn fibrillization even when α-syn was 400-fold more abundant than Hsp104. This suggests that Hsp104 specifically antagonizes a rare or transient α-syn conformer, perhaps a specific oligomeric species, which nucleates α-syn fibrillization. Indeed, Hsp104 disassembled purified A30P preamyloid oligomers that adopted a toxic conformation common to many amyloidogenic proteins (14). Such α-syn preamyloid oligomers are toxic to human neuroblastoma SH-SY5Y cells (14), suggesting that Hsp104 eliminates toxic α-syn conformers. Crucially, Hsp104 disassembled preformed α-syn fibers. Specific mammalian Hsp70 and Hsp40 chaperones, most notably Hsc70 and Hdj2, increased α-syn fiber disassembly by Hsp104. This is to our knowledge the first demonstration of effective disassembly of both α-syn preamyloid oligomers and amyloid fibers (which are particularly stable structures) by any protein. These various α-syn–remodeling activities required ATP binding and hydrolysis by Hsp104 and explain how Hsp104 might reduce dopaminergic neurodegeneration and phosphorylated α-syn inclusion formation in the arena of the rat brain.
Two previous studies claim to provide evidence that Hsp104 can dissociate α-syn fibers, but no experiments with α-syn preamyloid oligomers were performed (51, 52). However, under the conditions employed, Hsp104 reduced ThT fluorescence of preformed α-syn fibers by only 5%–20% after 24 hours of incubation (51, 52). This low level of potential fiber disassembly was not corroborated by other methods (51, 52). Remarkably, evidence was presented that Hsp104 degraded α-syn (51). This is extraordinarily unlikely given that Hsp104 possesses no protease motifs or protease activity (27, 29, 30, 31, 53, 54). It is more probable that the findings of Kong et al. are due to a contaminating protease (51, 52). By contrast, we find absolutely no evidence of degradation of α-syn by Hsp104 (Figure 6C). Rather, Hsp104 rapidly disassembles α-syn fibers and preamyloid oligomers to yield soluble α-syn.
It is not clear why Hsp104 has been lost from metazoa (28). Indeed, it is unclear whether mammalian cells express an analogous protein disaggregase able to solubilize large protein aggregates and restore protein functionality. Initial attempts to isolate such activity have been unsuccessful (40). The metazoan quality control machinery may be more optimized to prevent protein aggregation than reverse it and relies more heavily on the Hsp70 chaperone system. This system powerfully suppresses protein aggregation but only has limited ability to resolve even small soluble protein aggregates (27). Despite this, mammalian cells do have mechanisms to clear protein aggregates as revealed by silencing the gene encoding the aggregated protein (55). However, these pathways reflect autophagy and other protein degradation pathways rather than protein reactivation (23, 56). This general inability to rescue aggregated proteins may contribute to the lethality of excessive protein aggregation in animal cells.
Remodeling of α-syn preamyloid oligomers and amyloid fibers by Hsp104 is surprising since α-syn shares no sequence similarity with Hsp104’s natural amyloidogenic substrates: Sup35, Ure2, and Rnq1 (57). Hence, Hsp104 may specifically engage and remodel generic aspects of the cross-β amyloid form (41) and the distinct generic structure of preamyloid oligomers (14). However, S129E fibers were more resistant to disassembly. Thus, Hsp104 may need to initially engage α-syn at amino acid 129, which is solvent accessible in assembled α-syn fibers (58). Mutation of serine 129 to glutamate (but not alanine) may disrupt this interaction. Furthermore, E46K fibers were refractory to disassembly by Hsp104. This may reflect the different morphology of E46K fibers, which form compact bundles and meshwork arrays not observed with wild-type α-syn (19).
In rescuing α-syn neurotoxicity, Hsp104 reduced the number of α-syn inclusions. By contrast, Hsp70 prevents dopaminergic neurodegeneration in α-syn transgenic flies without affecting inclusion formation (24). Similar observations were reported for polyglutamine disorders, underscoring the limited ability of Hsp70 chaperones to remove aggregates (26). The lack of correlation between inclusion formation and neurodegeneration suggests that formation of inclusions may represent a protective cellular mechanism for sequestrating toxic assemblies (e.g., preamyloid oligomers) into safe inclusions. Indeed, expression of parkin, an E3 ubiquitin ligase connected with juvenile parkinsonism (5), prevents dopaminergic degeneration induced by A30P in our rat lentiviral PD model, but this is accompanied by an increase in phosphorylated α-syn inclusions (18). This suggests that Hsp104 reduces the number of inclusions in a manner that does not generate dangerous levels of toxic conformers, presumably because Hsp104 can disassemble these toxic species. Indeed, Hsp104’s ability to safely disassemble protein aggregates is likely an adaptation that ensures cell survival and the dissolution of the entire aggregated proteome after environmental stress in yeast.
However, further study is needed to address whether Hsp104 reduces preamyloid oligomer levels in α-syn expressing midbrain dopamine neurons. The low amount of preamyloid oligomers (relative to amyloid fibers), the low percentage of virally transduced cells, and the difficulty in isolating dopamine neurons from tissue extracts make the extraction of detectable levels of preamyloid oligomers very difficult. Further, we simultaneously expressed Hsp104 and α-syn, and not Hsp104 after α-syn aggregation, mainly due to technical issues. We have shown that coinjection of 2 viral vectors leads to more than 70% of cotransduced cells (18). In contrast, sequential injection of 2 viral vectors at 2-week intervals increases the variability of targeting of nigral cells and greatly decreases the number of cotransduced cells (Lo Bianco et al., unpublished observations). This is due to the difficulty in performing 2 identical injections into the same brain at different times. Thus, further studies are required to assess whether Hsp104 reverses preformed α-syn aggregates in vivo.
Overexpression of human α-syn in yeast induces toxicity and cytoplasmic foci containing α-syn (59). Surprisingly, Hsp104 overexpression has little effect on α-syn toxicity in yeast (60). However, the α-syn foci in yeast result from colocalization of α-syn with a membrane compartment, which perturbs endoplasmic reticulum to Golgi transport and Rab-GTPase homeostasis (61, 62). That α-syn does not form genuine aggregates in yeast, in contrast to the rat lentiviral model, explains why Hsp104 has little effect on its toxicity. It is likely that α-syn toxicity in yeast mimics an early, underappreciated stage in PD, prior to the accumulation of large quantities of preamyloid oligomers and fibers (61–63).
Even though not ordinarily expressed in mammalian cells, Hsp104 is extremely well tolerated in both tissue culture cells (including neurons) and in the brains of transgenic rodents and can perform beneficial protein-remodeling functions (39, 40, 64–66). Our studies demonstrate that Hsp104 combats α-syn misfolding and associated dopaminergic degeneration in a rat model of PD. Hsp104’s ability to prevent and reverse pathogenic protein aggregation should be considered as a potential strategy for treating or reversing PD and other protein aggregation diseases. However, further study is required to evaluate the safety of long-term Hsp104 expression in neurons.
Methods
Lentiviral vectors. cDNA coding for nuclear-localized YFP (BD Biosciences — Clontech), A30P human α-syn, and Hsp104 (kindly provided by D. Picard, Département de Biologie Cellulaire, Université de Genève, Genève, Switzerland) were cloned in the SIN-W-PGK lentiviral transfer vector, and viral particles (lenti-YFP, lenti-A30P, and lenti-Hsp104) were produced as described (42, 67). The viral suspensions lenti-A30P/lenti-YFP and lenti-A30P/lenti-Hsp104 were prepared by mixing viruses at 1:1 ratios (18). Particle content was matched to 180,000 ng of p24/ml for each lentiviral vector (18).
Stereotaxic injection. Lentiviral vectors were stereotaxically injected in the right substantia nigra of adult female Wistar rats (Iffa-Credo) weighing 200 g. Viral suspensions were injected at 2 sites with a 10-μl Hamilton syringe at a speed of 0.2 μl/min with an automatic injector (Stoelting Co.), and the needle was left in place for an additional 10 minutes before withdrawal. Stereotaxic injections were performed in 2 sites (2.5 μl per site) with anterior, lateral, and ventral coordinates (4.8, 2, 7.7, and 5.5, 1.7, 7.7) as described (18, 42). Animals were sacrificed at 6 weeks after injection. Experiments were carried out in accordance with the European Community Council directive (86/609/EEC) for the care and use of laboratory animals. The experiments described in this article were approved by the Veterinarian Office as well as by the Commission for Animal Experimentation of the Canton of Vaud (Switzerland) and were carried out under the animal license 1653.
Immunohistochemistry. Animals were deeply anesthetized with sodium pentobarbital and perfused transcardially with 4% paraformaldehyde. Brains were removed and postfixed in 4% paraformaldehyde for approximately 24 hours, cryoprotected in 25% sucrose in 0.1 M phosphate buffer for 48 hours, and processed as described (18, 42).
The following primary Abs were used: a TH sheep Ab (1:500; Pel-Freez Biologicals), the RG syn α-syn polyclonal rabbit Ab (1:400; ref. 42), the LB509 human α-syn specific monoclonal Ab (1:500; Zymed), the Pser129 Ab specifically recognizing phosphorylated Ser 129 of α-syn (1:100; ref. 21), and a rabbit Ab to the C-terminus of Hsp104 (1:800; Stressgen). For light microscopy, sections were stained by the classical avidin-biotin complex method as described (42). For multiple fluorescent labeling, the secondary Abs conjugated to Cy3 (donkey anti-sheep) and Cy5 (donkey anti-mouse) were from Jackson ImmunoResearch Laboratories. Hsp104 expression was revealed with a TSA fluorescein system (PerkinElmer Life Sciences). Sections were then analyzed by confocal microscopy (Leica TCS SP2 AOBS).
Silver staining was performed to detect degenerating neurons on paraformaldehyde-fixed sections (18, 47). The FD NeuroSilver kit was used according to the manufacturer’s protocol (FD Neuro-Technologies).
Cell counting and TH fiber density. To determine the percentage of TH-IR cell loss in the substantia nigra, 9–10 coronal sections of 40-μm thickness per animal were stained by immunofluorescence for the TH marker. All TH-IR neurons were counted in the injected and noninjected side of the substantia nigra, and the results were expressed as the percentage of TH-IR cell loss relative to the noninjected side (18, 42, 67). The borders of the substantia nigra were defined in the rostrocaudal axis using the anatomical landmarks in a rat brain atlas (68). Since the delineation of the borders between the ventral tegmental area (VTA) (A10), retrorubral nucleus (A8), and substantia nigra pars compacta (A9) are not clear, the medial border between VTA and substantia nigra was defined by a vertical line passing through the medial tip of the cerebral peduncle (and by the medial terminal nucleus of the accessory nucleus of the optic tract, when present in the sections), thereby excluding the TH-IR cells in the VTA. The ventral border followed the dorsal border of the cerebral peduncle, thereby including the TH-IR cells in pars reticulata, and the area extended laterally to include the pars lateralis in addition to the pars compacta. The counting was carried out caudally until the pars reticulata disappeared, thus excluding the retrorubral nucleus. The sections used for quantification covered the entire substantia nigra from the rostral tip of the pars compacta back to the caudal end of the pars reticulata. Importantly, no significant difference was observed in the estimated volume of the substantia nigra using Cavalieri’s principle or the size of the cell bodies of the counted neurons within experimental groups (noninjected versus injected side) or between experimental groups.
To determine the density of TH-IR terminals, striatal fibers were stained for TH with the ABC kit (Vector Laboratories), and the corresponding optical densities were evaluated with NIH IMAGE 1.4 software ( http://rsbweb.nih.gov/nih-image/) (18, 42). For the numbers of neurons containing phosphorylated α-syn inclusions, 5 sections throughout the substantia nigra were stained with the Pser129 Ab with the avidin-biotin complex method.
Statistics. Statistical analysis was performed by 1-way ANOVA, followed by a Scheffé’s protected least significant difference (PLSD) post-hoc test (Statistica 5.1; Statsoft Inc.). The significance level was set at P < 0.05.
Proteins. α-syn bacterial expression plasmids were kindly provided by P. Lansbury (Brigham and Women’s Hospital and Harvard Medical School, Cambridge, Massachusetts, USA). α-syn proteins (wild type, A53T, A30P, E46K, S129A, and S129E) were purified as described (20). Hsp104 and Hsp104K218T:K620T were purified as described (33). Hsp104 concentrations refer to hexameric Hsp104. Hsc70, Hsp70, Hdj1, and Hdj2 were from Alexis Biochemicals.
α-syn preamyloid oligomer purification and disassembly. α-syn A30P preamyloid oligomers were purified by gel filtration (15). For disassembly experiments, A30P preamyloid oligomers (0.5 μM monomer) were incubated with either Hsp104 or Hsp104K218T:K620T (10 μM) in KHM buffer (40 mM HEPES-KOH, pH 7.4, 150 mM KCl, 20 mM MgCl2, 1 mM DTT) plus ATP (10 mM) and an ATP regeneration system (20 mM creatine phosphate and 0.001 mg/ml creatine kinase) for 1 hour at 37°C. Reactions were processed for dot blot (33) and probed with either anti-oligomer Ab (kindly provided by C. Glabe, University of California, Irvine, Irvine, California, USA) (14) or anti–α-syn Ab (BD Biosciences). Alternatively, reactions were buffer exchanged using Bio-Gel P-6 spin columns into 40 mM HEPES-KOH, pH 7.4, 150 mM KCl, and 20 mM NaEDTA, diluted 5-fold and incubated for 10 minutes at 25°C to disassemble Hsp104 hexamers, and processed for negative-stain EM (33). Other reactions were depleted of Hsp104 as described (33) and then fractionated through a Microcon YM-100 (100-kDa molecular weight cut off) filter (Milllipore). Retentate and filtrate fractions were TCA precipitated and processed for SDS-PAGE followed by Coomassie Brilliant Blue staining.
α-syn fiber assembly and disassembly. For fibrillization reactions, α-syn proteins (80 μM) were incubated in KHM plus ATP (10 mM) and regeneration system plus or minus Hsp104 or Hsp104K218T:K620T (0–1.6 μM) for 0–48 hours at 37°C, with rotation (80 rpm) on a Mini-rotator (Glas-Col). Every 8 hours, reactions were supplemented with fresh regeneration system to maintain ATP levels.
For disassembly reactions, α-syn fibers (0.5 μM monomer) were incubated in KHM plus ATP (10 mM) and regeneration system plus or minus Hsp104 or Hsp104K218T:K620T (10 μM) in the presence or absence of the indicated combinations of Hsp70, Hsc70, Hdj1, and Hdj2 (10 μM) for 1 hour at 37°C. In reactions containing AMP-PNP (1 mM), the regeneration system was omitted.
Fiber assembly or disassembly was determined by ThT fluorescence, sedimentation analysis, turbidity, or EM (8, 33). For ThT fluorescence, ThT in 50 mM glycine (pH 8.5) was added to give final concentrations of α-syn (0.25 μM) and ThT (10 μM). Fluorescence at 480 nm was measured after excitation at 450 nm. For sedimentation analysis, reactions were centrifuged at 436,000 g for 10 minutes at 25°C. Supernatant and pellet fractions were then resolved by SDS-PAGE and stained with Coomassie Brilliant Blue. The percentage of α-syn in the pellet was determined by densitometry and comparison to know quantities of α-syn. Turbidity was monitored by absorbance at 395 nm.
Acknowledgments
We thank D. Picard, P. Lansbury, and C. Glabe for generous provision of reagents; P. Colin, C. Sadeghi, and M. Rey for excellent technical assistance; A. Gitler for comments on the manuscript. C. Lo Bianco was supported by the Michael J. Fox Foundation, European Molecular Biology Organization, Swedish Parkinson Foundation, and the Swiss National Science Foundation. J. Shorter was supported by an American Heart Association Scientist Development Grant, University of Pennsylvania Institute on Aging pilot grant, and NIH Director’s New Innovator Award (DP2OD002177). P. Aebischer was supported by the Michael J. Fox Foundation and the Swiss National Science Foundation.
Footnotes
Nonstandard abbreviations used: PD, Parkinson disease; α-syn, α-synuclein; TH, tyrosine hydroxylase; TH-IR, TH-immunoreactive; ThT, Thioflavin-T; YFP, yellow fluorescent protein.
References
1. Skovronsky, D.M., Lee, V.M., Trojanowski, J.Q. 2006. Neurodegenerative diseases: new concepts of pathogenesis and their therapeutic implications. Annu. Rev. Pathol. 1:151-170.
2. Dorsey, E.R., et al. 2007. Projected number of people with Parkinson’s disease in the most populous nations, 2005 through 2030. Neurology. 68:384-386.
3. Braak, H., et al. 2003. Staging of brain pathology related to sporadic Parkinson’s disease. Neurobiol. Aging. 24:197-211.
4. Spillantini, M.G., et al. 1997. Alpha-synuclein in Lewy bodies. Nature. 388:839-840.
5. Moore, D.J., West, A.B., Dawson, V.L., Dawson, T.M. 2005. Molecular pathophysiology of Parkinson’s disease. Annu. Rev. Neurosci. 28:57-87.
6. Davidson, W.S., Jonas, A., Clayton, D.F., George, J.M. 1998. Stabilization of alpha-synuclein secondary structure upon binding to synthetic membranes. J. Biol. Chem. 273:9443-9449.
7. Conway, K.A., Harper, J.D., Lansbury, P.T. 1998. Accelerated in vitro fibril formation by a mutant alpha-synuclein linked to early-onset Parkinson disease. Nat. Med. 4:1318-1320.
8. Conway, K.A., et al. 2000. Acceleration of oligomerization, not fibrillization, is a shared property of both alpha-synuclein mutations linked to early-onset Parkinson’s disease: implications for pathogenesis and therapy. Proc. Natl. Acad. Sci. U. S. A. 97:571-576.
9. Serpell, L.C., Berriman, J., Jakes, R., Goedert, M., Crowther, R.A. 2000. Fiber diffraction of synthetic alpha-synuclein filaments shows amyloid-like cross-beta conformation. Proc. Natl. Acad. Sci. U. S. A. 97:4897-4902.
10. Crowther, R.A., Daniel, S.E., Goedert, M. 2000. Characterisation of isolated alpha-synuclein filaments from substantia nigra of Parkinson’s disease brain. Neurosci. Lett. 292:128-130.
11. Spillantini, M.G., et al. 1998. Filamentous alpha-synuclein inclusions link multiple system atrophy with Parkinson’s disease and dementia with Lewy bodies. Neurosci. Lett. 251:205-208.
12. Miake, H., Mizusawa, H., Iwatsubo, T., Hasegawa, M. 2002. Biochemical characterization of the core structure of alpha-synuclein filaments. J. Biol. Chem. 277:19213-19219.
13. Neumann, M., et al. 2002. Misfolded proteinase K-resistant hyperphosphorylated alpha-synuclein in aged transgenic mice with locomotor deterioration and in human alpha-synucleinopathies. J. Clin. Invest. 110:1429-1439.
14. Kayed, R., et al. 2003. Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis. Science. 300:486-489.
15. Lashuel, H.A., et al. 2002. Alpha-synuclein, especially the Parkinson’s disease-associated mutants, forms pore-like annular and tubular protofibrils. J. Mol. Biol. 322:1089-1102.
16. Sharon, R., et al. 2003. The formation of highly soluble oligomers of alpha-synuclein is regulated by fatty acids and enhanced in Parkinson’s disease. Neuron. 37:583-595.
17. Chen, L., Feany, M.B. 2005. Alpha-synuclein phosphorylation controls neurotoxicity and inclusion formation in a Drosophila model of Parkinson disease. Nat. Neurosci. 8:657-663.
18. Lo Bianco, C., et al. 2004. Lentiviral vector delivery of parkin prevents dopaminergic degeneration in an alpha-synuclein rat model of Parkinson’s disease. Proc. Natl. Acad. Sci. U. S. A. 101:17510-17515.
19. Choi, W., et al. 2004. Mutation E46K increases phospholipid binding and assembly into filaments of human alpha-synuclein. FEBS Lett. 576:363-368.
20. Fredenburg, R.A., et al. 2007. The impact of the E46K mutation on the properties of alpha-synuclein in its monomeric and oligomeric states. Biochemistry. 46:7107-7118.
21. Fujiwara, H., et al. 2002. alpha-Synuclein is phosphorylated in synucleinopathy lesions. Nat. Cell Biol. 4:160-164.
22. Norris, E.H., Giasson, B.I., Ischiropoulos, H., Lee, V.M. 2003. Effects of oxidative and nitrative challenges on alpha-synuclein fibrillogenesis involve distinct mechanisms of protein modifications. J. Biol. Chem. 278:27230-27240.
23. Balch, W.E., Morimoto, R.I., Dillin, A., Kelly, J.W. 2008. Adapting proteostasis for disease intervention. Science. 319:916-919.
24. Auluck, P.K., Chan, H.Y., Trojanowski, J.Q., Lee, V.M., Bonini, N.M. 2002. Chaperone suppression of alpha-synuclein toxicity in a Drosophila model for Parkinson’s disease. Science. 295:865-868.
25. Cummings, C.J., et al. 1998. Chaperone suppression of aggregation and altered subcellular proteasome localization imply protein misfolding in SCA1. Nat. Genet. 19:148-154.
26. Warrick, J.M., et al. 1999. Suppression of polyglutamine-mediated neurodegeneration in Drosophila by the molecular chaperone HSP70. Nat. Genet. 23:425-428.
27. Glover, J.R., Lindquist, S. 1998. Hsp104, Hsp70, and Hsp40: a novel chaperone system that rescues previously aggregated proteins. Cell. 94:73-82.
28. Shorter, J. 2008. Hsp104: a weapon to combat diverse neurodegenerative disorders. Neurosignals. 16:63-74.
29. Wendler, P., et al. 2007. Atypical AAA+ subunit packing creates an expanded cavity for disaggregation by the protein-remodeling factor Hsp104. Cell. 131:1366-1377.
30. Parsell, D.A., Kowal, A.S., Singer, M.A., Lindquist, S. 1994. Protein disaggregation mediated by heat-shock protein Hsp104. Nature. 372:475-478.
31. Sanchez, Y., Lindquist, S.L. 1990. HSP104 required for induced thermotolerance. Science. 248:1112-1115.
32. Sanchez, Y., Taulien, J., Borkovich, K.A., Lindquist, S. 1992. Hsp104 is required for tolerance to many forms of stress. EMBO J. 11:2357-2364.
33. Shorter, J., Lindquist, S. 2004. Hsp104 catalyzes formation and elimination of self-replicating Sup35 prion conformers. Science. 304:1793-1797.
34. Shorter, J., Lindquist, S. 2006. Destruction or potentiation of different prions catalyzed by similar Hsp104 remodeling activities. Mol. Cell. 23:425-438.
35. Doyle, S.M., et al. 2007. Asymmetric deceleration of ClpB or Hsp104 ATPase activity unleashes protein-remodeling activity. Nat. Struct. Mol. Biol. 14:114-122.
36. Narayanan, S., Walter, S., Reif, B. 2006. Yeast prion-protein, sup35, fibril formation proceeds by addition and substraction of oligomers. Chembiochem. 7:757-765.
37. Savistchenko, J., Krzewska, J., Fay, N., Melki, R. 2008. Molecular chaperones and the assembly of the prion Ure2p in vitro. J. Biol. Chem. 283:15732-15739.
38. Chernoff, Y.O., Lindquist, S.L., Ono, B., Inge-Vechtomov, S.G., Liebman, S.W. 1995. Role of the chaperone protein Hsp104 in propagation of the yeast prion-like factor [PSI+]. Science. 268:880-884.
39. Dandoy-Dron, F., et al. 2006. Infection by ME7 prion is not modified in transgenic mice expressing the yeast chaperone Hsp104 in neurons. Neurosci. Lett. 405:181-185.
40. Mosser, D.D., Ho, S., Glover, J.R. 2004. Saccharomyces cerevisiae Hsp104 enhances the chaperone capacity of human cells and inhibits heat stress-induced proapoptotic signaling. Biochemistry. 43:8107-8115.
41. Nelson, R., Eisenberg, D. 2006. Structural models of amyloid-like fibrils. Adv. Protein Chem. 73:235-282.
42. Lo Bianco, C., Ridet, J.L., Schneider, B.L., Deglon, N., Aebischer, P. 2002. alpha -Synucleinopathy and selective dopaminergic neuron loss in a rat lentiviral-based model of Parkinson’s disease. Proc. Natl. Acad. Sci. U. S. A. 99:10813-10818.
43. Bao, Y.P., Cook, L.J., O’Donovan, D., Uyama, E., Rubinsztein, D.C. 2002. Mammalian, yeast, bacterial, and chemical chaperones reduce aggregate formation and death in a cell model of oculopharyngeal muscular dystrophy. J. Biol. Chem. 277:12263-12269.
44. Kirik, D., et al. 2003. Nigrostriatal alpha-synucleinopathy induced by viral vector-mediated overexpression of human alpha-synuclein: a new primate model of Parkinson’s disease. Proc. Natl. Acad. Sci. U. S. A. 100:2884-2889.
45. Kirik, D., et al. 2002. Parkinson-like neurodegeneration induced by targeted overexpression of alpha-synuclein in the nigrostriatal system. J. Neurosci. 22:2780-2791.
46. Klein, R.L., King, M.A., Hamby, M.E., Meyer, E.M. 2002. Dopaminergic cell loss induced by human A30P alpha-synuclein gene transfer to the rat substantia nigra. Hum. Gene Ther. 13:605-612.
47. Beltramino C.A., de Olmos J.S., Gallyas F., Heimer L., Záborszky L.. 1993;Silver staining as a tool for neurotoxic assessment.. NIDA Res. Monogr. 136:101–126; discussion 126–132.126; discussion 126–132 . 82899098289909
48. Sandmann-Keil, D., Braak, H., Okochi, M., Haass, C., Braak, E. 1999. Alpha-synuclein immunoreactive Lewy bodies and Lewy neurites in Parkinson’s disease are detectable by an advanced silver-staining technique. Acta Neuropathol. 98:461-464.
49. Schirmer, E.C., Queitsch, C., Kowal, A.S., Parsell, D.A., Lindquist, S. 1998. The ATPase activity of Hsp104, effects of environmental conditions and mutations. J. Biol. Chem. 273:15546-15552.
50. Lashuel, H.A., Hartley, D., Petre, B.M., Walz, T., Lansbury (Jr.), P.T. 2002. Neurodegenerative disease: amyloid pores from pathogenic mutations. Nature. 418:291.
51. Kong, B., Chae, Y., Lee, K. 2005. Degradation of wild-type alpha-synuclein by a molecular chaperone leads to reduced aggregate formation. Cell Biochem. Funct. 23:125-132.
52. Kong, B., Chae, Y.K., Lee, K. 2003. Regulation of in vitro fibril formation of synuclein mutant proteins by Hsp104p. Protein Pept. Lett. 10:491-495.
53. Parsell, D.A., Sanchez, Y., Stitzel, J.D., Lindquist, S. 1991. Hsp104 is a highly conserved protein with two essential nucleotide-binding sites. Nature. 353:270-273.
54. Parsell, D.A., Taulien, J., Lindquist, S. 1993. The role of heat-shock proteins in thermotolerance. Philos. Trans. R. Soc. Lond. B Biol. Sci. 339:279-286.
55. Yamamoto, A., Lucas, J.J., Hen, R. 2000. Reversal of neuropathology and motor dysfunction in a conditional model of Huntington’s disease. Cell. 101:57-66.
56. Martin-Aparicio, E., et al. 2001. Proteasomal-dependent aggregate reversal and absence of cell death in a conditional mouse model of Huntington’s disease. J. Neurosci. 21:8772-8781.
57. Shorter, J., Lindquist, S. 2005. Prions as adaptive conduits of memory and inheritance. Nat. Rev. Genet. 6:435-450.
58. Del Mar, C., Greenbaum, E.A., Mayne, L., Englander, S.W., Woods (Jr.), V.L. 2005. Structure and properties of alpha-synuclein and other amyloids determined at the amino acid level. Proc. Natl. Acad. Sci. U. S. A. 102:15477-15482.
59. Outeiro, T.F., Lindquist, S. 2003. Yeast cells provide insight into alpha-synuclein biology and pathobiology. Science. 302:1772-1775.
60. Cashikar, A.G., Duennwald, M., Lindquist, S.L. 2005. A chaperone pathway in protein disaggregation. Hsp26 alters the nature of protein aggregates to facilitate reactivation by Hsp104. J. Biol. Chem. 280:23869-23875.
61. Cooper, A.A., et al. 2006. Alpha-synuclein blocks ER-Golgi traffic and Rab1 rescues neuron loss in Parkinson’s models. Science. 313:324-328.
62. Gitler, A.D., et al. 2008. The Parkinson’s disease protein alpha-synuclein disrupts cellular Rab homeostasis. Proc. Natl. Acad. Sci. U. S. A. 105:145-150.
63. Gitler, A.D., Shorter, J. 2007. Prime time for alpha-synuclein. J. Neurosci. 27:2433-2434.
64. Carmichael, J., et al. 2000. Bacterial and yeast chaperones reduce both aggregate formation and cell death in mammalian cell models of Huntington’s disease. Proc. Natl. Acad. Sci. U. S. A. 97:9701-9705.
65. Perrin, V., et al. 2007. Neuroprotection by Hsp104 and Hsp27 in lentiviral-based rat models of Huntington’s disease. Mol. Ther. 15:903-911.
66. Vacher, C., Garcia-Oroz, L., Rubinsztein, D.C. 2005. Overexpression of yeast hsp104 reduces polyglutamine aggregation and prolongs survival of a transgenic mouse model of Huntington’s disease. Hum. Mol. Genet. 14:3425-3433.
67. Deglon, N., et al. 2000. Self-inactivating lentiviral vectors with enhanced transgene expression as potential gene transfer system in Parkinson’s disease. Hum. Gene Ther. 11:179-190.
68. Paxinos, G., and Watson, C. 1998.The rat brain in stereotaxic coordinates. 4th edition. Academic Press. San Diego, California, USA. 256 pp.
Assessment of sexual dysfunction in patients with Parkinson's disease: a case-control study.
Department of Psychiatry, Ankara University School of Medicine, Ankara, Turkey.
Background: Sexual dysfunction (SD) in patients with Parkinson's disease (PD) has not been very well studied, as most of the research has methodological restrictions like having no control group, using invalid assessment tools, unidimensional investigation of sexual functions and inclusion of males/females only. This study aimed to examine different sexual functions in patients with PD and compare with matched non-parkinsonian controls by using a valid instrument. Predicting factors of SD in PD were also investigated. Methods: The sample consisted of 45 patients with PD and 45 age- and sex-matched healthy controls. Sexual functions were evaluated by Arizona Sexual Experiences Scale (ASEX). Results: Female patients had reduced sexual drive and they were less satisfied with orgasm, while male patients had easier orgasms than did the controls. Regression analysis identified increased age and female sex predictive of reduced sexual drive and sexual arousal. Ability to reach orgasm and satisfaction with orgasm were associated with female sex, while erection/lubrication was associated with marital status. The severity and duration of PD, as well as the severity of anxiety and depression were not associated with SD. Conclusion: Using ASEX in the detection of SD in PD might be important in directing patients to further evaluation and treatment.
Background: Sexual dysfunction (SD) in patients with Parkinson's disease (PD) has not been very well studied, as most of the research has methodological restrictions like having no control group, using invalid assessment tools, unidimensional investigation of sexual functions and inclusion of males/females only. This study aimed to examine different sexual functions in patients with PD and compare with matched non-parkinsonian controls by using a valid instrument. Predicting factors of SD in PD were also investigated. Methods: The sample consisted of 45 patients with PD and 45 age- and sex-matched healthy controls. Sexual functions were evaluated by Arizona Sexual Experiences Scale (ASEX). Results: Female patients had reduced sexual drive and they were less satisfied with orgasm, while male patients had easier orgasms than did the controls. Regression analysis identified increased age and female sex predictive of reduced sexual drive and sexual arousal. Ability to reach orgasm and satisfaction with orgasm were associated with female sex, while erection/lubrication was associated with marital status. The severity and duration of PD, as well as the severity of anxiety and depression were not associated with SD. Conclusion: Using ASEX in the detection of SD in PD might be important in directing patients to further evaluation and treatment.
Thomson strikes note of caution at World Stem Cell Summit
Joe Vanden Plas
September 22, 2008
Madison, Wis. - James Thomson acknowledged that scientists are notoriously bad at predicting timelines, so when he was asked about the timing of potential therapeutic stem cell research breakthroughs at the World Stem Cell Summit, he was naturally cautious.
Thomson, speaking on the potential future benefits of induced pluripotent stem cells, has been more cautious than some stem cell research advocates when addressing its possibilities. It's not that he isn't enthusiastic about the potential of the research he ignited 10 years ago when he became the first scientist to isolate and develop methods to culture human embryonic stem cells, but he has no illusions about the degree of difficulty that lies ahead.
In developing the induced pluripotent stem cell technique, Thomson and other researchers reprogrammed human adult skin cells to act as human embryonic stem cells. The iPS cells are remarkably similar to human embryonic stem cells in that researchers can make as many of them as they want, and they can become any type of cell in the human body.
Thomson would not be surprised if successful stem cell therapies develop in five to 10 years, but he said they will be few and far between, and there will be many setbacks that the public should be prepared for.
“We need to roll up our sleeves and do a great deal of work here,” he said, “but it's not going to happen
Cell transplantation
Perhaps nowhere is this truer than with transplants. Thomson said both human embryonic stem cells and iPS cells could provide an unlimited source of cells for transplantation therapies. This is the area of stem cell research that has created the most interest, and the one Thomson is most cautious about.
There are several potential barriers to cell-based transplantation therapy using both iPS and human embryonic stem cells. Those barriers include:
* The ability to make the cell type of interest.
* Safety concerns such as cancer, immune rejection, and preventing a recurrence of the process that originally killed the cells.
* Integration into the body in a physiologically useful form.
While both the cardiovascular and central nervous systems are complex, Thomson believes that cell transplantation will be easier with the heart than with the central nervous system. He noted that scientists already can make heart cells from embryonic stem cells and iPS cells, and they already are screening these cells against potential drugs in ways that won't make the New York Times, but will help people with heart disease.
In contrast, the central nervous system is so complex that cell transplantation could take a long time, he said. In the short term, however, the cells could help scientists understand why Parkinson's disease, for example, occurs in first place. The cells also could lead to therapies to prevent the disease or arrest its progression so people can live productive lives, Thomson said.
Actual transplantation will be very challenging. “It's one thing to make tissue in a culture,” Thomson said. “It's another to get it into the body and re-establish function.”
Tough neighborhood
One scientist who is trying to tackle a nervous system challenge is Lawrence Goldstein, a professor of cellular and molecular medicine at the University of California-San Diego. Goldstein told the summit gathering that cells, including nerve cells, have an “interstate highway system” within them to move biological materials to the right place.
How materials are moved inside cells has led science to some new ideas about conditions like Alzheimer's and Huntington's Disease, and Goldstein said researchers almost have worn out what they can do with animal versions of the diseases using fruit flies and mice. The end of that road has led them to use human pluripotent stem cells, embryonic and induced, to understand how diseases work and how they might be better treated.
One project in Goldstein's lab involves amyotrophic lateral sclerosis, also known as Lou Gehrig's disease, which weakens the muscles by starving them of their nourishment. With this disease, cells called motoneurons, which control the ability of muscle to contract so that people can walk and swallow, die for reasons that are not completely understood. If cell replacement therapy is ever going to treat it, the obvious step is to replace motoneurons that are dying, he said.
“In practice, it is devilishly difficult to do that because some of these motoneuron cells have sizes that are a yard or more along the spinal cord,” Goldstein explained, “and run connections to fingers and toes and to our chest so we can breathe. How to rewire that is a difficult problem to contemplate.”
What his lab has learned in one mouse version of Lou Gehrig's disease is that even though motoneurons are dying, cells immediately surrounding them in the spinal cord can either poison the motoneurons or, if the are normal, rescue them from dying.
“Cells live in neighborhoods, and the quality of the neighborhood has a big impact on the health and viability and education of motoneurons that live in that neighborhood," Goldstein said. “We're trying to use human embryonic stem cells to make cells of the neighborhood and begin implanting them in rat models of Lou Gehrig's disease to see if we can rescue the dying motoneurons.”
September 22, 2008
Madison, Wis. - James Thomson acknowledged that scientists are notoriously bad at predicting timelines, so when he was asked about the timing of potential therapeutic stem cell research breakthroughs at the World Stem Cell Summit, he was naturally cautious.
Thomson, speaking on the potential future benefits of induced pluripotent stem cells, has been more cautious than some stem cell research advocates when addressing its possibilities. It's not that he isn't enthusiastic about the potential of the research he ignited 10 years ago when he became the first scientist to isolate and develop methods to culture human embryonic stem cells, but he has no illusions about the degree of difficulty that lies ahead.
In developing the induced pluripotent stem cell technique, Thomson and other researchers reprogrammed human adult skin cells to act as human embryonic stem cells. The iPS cells are remarkably similar to human embryonic stem cells in that researchers can make as many of them as they want, and they can become any type of cell in the human body.
Thomson would not be surprised if successful stem cell therapies develop in five to 10 years, but he said they will be few and far between, and there will be many setbacks that the public should be prepared for.
“We need to roll up our sleeves and do a great deal of work here,” he said, “but it's not going to happen
Cell transplantation
Perhaps nowhere is this truer than with transplants. Thomson said both human embryonic stem cells and iPS cells could provide an unlimited source of cells for transplantation therapies. This is the area of stem cell research that has created the most interest, and the one Thomson is most cautious about.
There are several potential barriers to cell-based transplantation therapy using both iPS and human embryonic stem cells. Those barriers include:
* The ability to make the cell type of interest.
* Safety concerns such as cancer, immune rejection, and preventing a recurrence of the process that originally killed the cells.
* Integration into the body in a physiologically useful form.
While both the cardiovascular and central nervous systems are complex, Thomson believes that cell transplantation will be easier with the heart than with the central nervous system. He noted that scientists already can make heart cells from embryonic stem cells and iPS cells, and they already are screening these cells against potential drugs in ways that won't make the New York Times, but will help people with heart disease.
In contrast, the central nervous system is so complex that cell transplantation could take a long time, he said. In the short term, however, the cells could help scientists understand why Parkinson's disease, for example, occurs in first place. The cells also could lead to therapies to prevent the disease or arrest its progression so people can live productive lives, Thomson said.
Actual transplantation will be very challenging. “It's one thing to make tissue in a culture,” Thomson said. “It's another to get it into the body and re-establish function.”
Tough neighborhood
One scientist who is trying to tackle a nervous system challenge is Lawrence Goldstein, a professor of cellular and molecular medicine at the University of California-San Diego. Goldstein told the summit gathering that cells, including nerve cells, have an “interstate highway system” within them to move biological materials to the right place.
How materials are moved inside cells has led science to some new ideas about conditions like Alzheimer's and Huntington's Disease, and Goldstein said researchers almost have worn out what they can do with animal versions of the diseases using fruit flies and mice. The end of that road has led them to use human pluripotent stem cells, embryonic and induced, to understand how diseases work and how they might be better treated.
One project in Goldstein's lab involves amyotrophic lateral sclerosis, also known as Lou Gehrig's disease, which weakens the muscles by starving them of their nourishment. With this disease, cells called motoneurons, which control the ability of muscle to contract so that people can walk and swallow, die for reasons that are not completely understood. If cell replacement therapy is ever going to treat it, the obvious step is to replace motoneurons that are dying, he said.
“In practice, it is devilishly difficult to do that because some of these motoneuron cells have sizes that are a yard or more along the spinal cord,” Goldstein explained, “and run connections to fingers and toes and to our chest so we can breathe. How to rewire that is a difficult problem to contemplate.”
What his lab has learned in one mouse version of Lou Gehrig's disease is that even though motoneurons are dying, cells immediately surrounding them in the spinal cord can either poison the motoneurons or, if the are normal, rescue them from dying.
“Cells live in neighborhoods, and the quality of the neighborhood has a big impact on the health and viability and education of motoneurons that live in that neighborhood," Goldstein said. “We're trying to use human embryonic stem cells to make cells of the neighborhood and begin implanting them in rat models of Lou Gehrig's disease to see if we can rescue the dying motoneurons.”
Oral creatine supplementation attenuates L-DOPA-induced dyskinesia in 6-hydroxydopamine-lesioned rats.
Valastro B, Dekundy A, Danysz W, Quack G.
Preclinical Research and Development, In vitro screening, Merz Pharmaceuticals GmbH, Altenhöferallee 3, 60438 Frankfurt am Main, Germany.
L-DOPA-induced dyskinesia (LID) is among the motor complications that arise in Parkinson patients after a prolonged treatment with levodopa (L-DOPA). Since previous transcriptome and proteomic studies performed in the rat model of LID suggested important changes in striatal energy-related components, we hypothesize that oral creatine supplementation could prevent or attenuate the occurrence of LID. In this study, 6-hydroxydopamine-lesioned rats received a 2% creatine-supplemented diet for 1 month prior to L-DOPA therapy. During the 21 days of L-DOPA treatment, significant reductions in abnormal involuntary movements (AIMs) have been observed in the creatine-supplemented group, without any worsening of parkinsonism. In situ hybridization histochemistry and immunohistochemistry analysis of the striatum also showed a reduction in the levels of prodynorphin mRNA and FosB/DeltaFosB-immunopositive cells in creatine-supplemented diet group, an effect that was dependant on the development of AIMs. Further investigation of the bioenergetics' status of the denervated striatum revealed significant changes in the levels of creatine both after L-DOPA alone and with the supplemented diet. In conclusion, we demonstrated that combining L-DOPA therapy with a diet enriched in creatine could attenuate LID, which may represent a new way to control the motor complication associated with L-DOPA therapy.
Preclinical Research and Development, In vitro screening, Merz Pharmaceuticals GmbH, Altenhöferallee 3, 60438 Frankfurt am Main, Germany.
L-DOPA-induced dyskinesia (LID) is among the motor complications that arise in Parkinson patients after a prolonged treatment with levodopa (L-DOPA). Since previous transcriptome and proteomic studies performed in the rat model of LID suggested important changes in striatal energy-related components, we hypothesize that oral creatine supplementation could prevent or attenuate the occurrence of LID. In this study, 6-hydroxydopamine-lesioned rats received a 2% creatine-supplemented diet for 1 month prior to L-DOPA therapy. During the 21 days of L-DOPA treatment, significant reductions in abnormal involuntary movements (AIMs) have been observed in the creatine-supplemented group, without any worsening of parkinsonism. In situ hybridization histochemistry and immunohistochemistry analysis of the striatum also showed a reduction in the levels of prodynorphin mRNA and FosB/DeltaFosB-immunopositive cells in creatine-supplemented diet group, an effect that was dependant on the development of AIMs. Further investigation of the bioenergetics' status of the denervated striatum revealed significant changes in the levels of creatine both after L-DOPA alone and with the supplemented diet. In conclusion, we demonstrated that combining L-DOPA therapy with a diet enriched in creatine could attenuate LID, which may represent a new way to control the motor complication associated with L-DOPA therapy.
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