Mechanisms leading to the formation of α-synuclein inclusions in Parkinson's disease (PD) and related α-synucleinopathies characterized predominantly by abundant fibrillary intracellular α-synuclein inclusions in neurons and their processes as well as in glial cells (e.g., in multiple system atrophy) remain largely unknown. However, insights from studies of familial autosomal dominant forms of these disorders suggest that overproduction of α-synuclein due to α-synuclein gene triplication, or the predisposition of mutant α-synuclein to fibrillize are plausible mechanisms underlying heritable α-synucleinopathies, but less traction has been established in defining the basis for sporadic neurodegenerative α-synucleinopathies. Nonetheless, there is little evidence for overproduction of α-synuclein as a possible cause of sporadic disease. On the other hand, since sporadic α-synucleinopathies, like many other sporadic neurodegenerative brain amyloidoses (CJD, AD, PSP, CBD, etc.), involve the abnormal aggregation and fibrillization of...  Read more

Mechanisms leading to the formation of α-synuclein inclusions in Parkinson's disease (PD) and related α-synucleinopathies characterized predominantly by abundant fibrillary intracellular α-synuclein inclusions in neurons and their processes as well as in glial cells (e.g., in multiple system atrophy) remain largely unknown. However, insights from studies of familial autosomal dominant forms of these disorders suggest that overproduction of α-synuclein due to α-synuclein gene triplication, or the predisposition of mutant α-synuclein to fibrillize are plausible mechanisms underlying heritable α-synucleinopathies, but less traction has been established in defining the basis for sporadic neurodegenerative α-synucleinopathies. Nonetheless, there is little evidence for overproduction of α-synuclein as a possible cause of sporadic disease. On the other hand, since sporadic α-synucleinopathies, like many other sporadic neurodegenerative brain amyloidoses (CJD, AD, PSP, CBD, etc.), involve the abnormal aggregation and fibrillization of normally soluble brain proteins that become insoluble and form brain deposits composed of similar appearing amyloid fibrils (even though the building blocks of amyloid fibrils vary in different diseases), one plausible mechanism to account for these sporadic disorders is the failure to degrade the disease brain proteins effectively when they reach a certain threshold level or concentration in a cell whether or not they are in a native or misfolded state. Indeed, impairments in proteasomal and lysosomal/autophagic degradation of disease proteins are under intense investigation, but the literature is somewhat contradictory. Thus, the recent papers by Cuervo et al. in Science and Rideout et al. in the Journal of Biological Chemistry add new insights into the role of autophagy in the degradation of α-synuclein, which may play a more direct role in α-synucleinopathies than the proteasome. Significantly, Cuervo et al. show that α-synuclein is degraded in lysosomes through a process known as the chaperone mediated autophagy (CMA) pathway, while the proteasome pathway did not appear to be involved, and known familial PD α-synuclein gene mutations blocked CMA of α-synuclein and other substrates. Further, the studies of Rideout et al. indicate that proteasomal inhibition can induce formation of ubiquitinated inclusions and apoptosis, but they showed that this can occur in neurons from α-synuclein knockout mice. These studies highlight the timeliness of intensifying research on the role of CMA, lysosomes, and the ubiquitin proteasome pathways in neurodegenerative brain amyloidoses, and it would not be surprising if impairments in more than one of these degradative pathways were involved simultaneously and to variable extents in specific forms of these disorders. Further clarity about these issues may lead to strategies for assisting the aging brain to better clear effete brain proteins so that disease proteins do not accumulate as brain amyloids and lead to brain dysfunction and degeneration.

View all comments by John Trojanowski

Impaired Degradation of Mutant α-Synuclein by Chaperone-Mediated Autophagy
Expression levels of α-synuclein are correlated with Parkinson’s disease in several ways. The most extreme example is the finding of a triplication of the normal wild-type gene in a family with dominant PD/Lewy body disease (Singleton et al., 2003). This leads to an approximate doubling of the protein load (Miller et al., 2004), which is sufficient to produce a fulminant brain disease. If a twofold increase in α-synuclein causes such a prominent, dominantly inherited disease, then perhaps smaller changes in protein expression might be associated with the risk of sporadic PD. There are other ways in which steady-state α-synuclein levels can be affected. For example, Mike Lee’s group has recently shown that turnover of α-synuclein may be affected by neuronal differentiation, and slows with aging (Li et al., 2004). Taken together, these different observations suggest that some of the complexities of sporadic synucleinopathies may be driven, in part, by effects on the...  Read more

Impaired Degradation of Mutant α-Synuclein by Chaperone-Mediated Autophagy
Expression levels of α-synuclein are correlated with Parkinson’s disease in several ways. The most extreme example is the finding of a triplication of the normal wild-type gene in a family with dominant PD/Lewy body disease (Singleton et al., 2003). This leads to an approximate doubling of the protein load (Miller et al., 2004), which is sufficient to produce a fulminant brain disease. If a twofold increase in α-synuclein causes such a prominent, dominantly inherited disease, then perhaps smaller changes in protein expression might be associated with the risk of sporadic PD. There are other ways in which steady-state α-synuclein levels can be affected. For example, Mike Lee’s group has recently shown that turnover of α-synuclein may be affected by neuronal differentiation, and slows with aging (Li et al., 2004). Taken together, these different observations suggest that some of the complexities of sporadic synucleinopathies may be driven, in part, by effects on the relatively simple parameter of protein stability.

In this context, the paper by Cuervo and colleagues is important in beginning to understand some of the ways in which α-synuclein protein half-life can be affected. The observation that chaperone-mediated autophagy is important adds to the general feeling that lysosomes are a more important outlet for α-synuclein degradation than other means, such as proteasomal degradation. The paper also indicates a way to decrease protein levels of α-synuclein by increasing expression of Hsc70, or its co-chaperones (hip, hop, bag-1, Hsp40, and Hsp90). There is already some literature suggesting that chaperones can mitigate α-synuclein toxicity (e.g., Auluck et al., 2002); one wonders how much of these effects are mediated through correcting the conformation of α-synuclein and how much through inducing CMA and thus reducing the amount of α-synuclein in the cytosol.

References:
Auluck PK, Chan HY, Trojanowski JQ, Lee VM and Bonini NM. (2002) Chaperone suppression of α-synuclein toxicity in a Drosophila model for Parkinson's disease. Science 295, 865-868. Abstract

Li W, Lesuisse C, Xu Y, Troncoso JC, Price DL and Lee MK. (2004) Stabilization of α-synuclein protein with aging and familial parkinson's disease-linked A53T mutation. J Neurosci 24, 9400-9409. Abstract

Miller DW, Hague SM, Clarimon J, Baptista M, Gwinn-Hardy K, Cookson MR and Singleton AB. (2004) α-synuclein in blood and brain from familial Parkinson disease with SNCA locus triplication. Neurology 62, 1835-1838. Abstract

Singleton AB, Farrer M, Johnson J, Singleton A, Hague S, Kachergus J, Hulihan M, Peuralinna T, Dutra A, Nussbaum R, Lincoln S, Crawley A, Hanson M, Maraganore D, Adler C, Cookson MR, Muenter M, Baptista M, Miller D, Blancato J, Hardy J and Gwinn-Hardy K. (2003) α-Synuclein locus triplication causes Parkinson's disease. Science 302, 841. Abstract

View all comments by Mark Cookson

The extreme scarcity of autophagic vacuoles in normal brain and their appearance in states of disease have previously led many to assume that autophagy in neurons is mainly an inducible process. Autophagy is solely responsible for organelle turnover, however, and the large cytoplasmic mass of neurons would suggest, therefore, that autophagy might have a significant constitutive component. The two papers by Komatsu et al. and Hara et al. have now provided elegant and definitive evidence in neurons for constitutive autophagy and have demonstrated that it is required for neuron survival. In fact, the results imply that the brain may actually be one of the tissues most vulnerable to a possible impairment of autophagy. These findings, therefore, offer insight into why neurons are preferentially victimized in diseases that disrupt the lysosomal system, even when the disease is a systemic one.

This new evidence for actively ongoing autophagy in neurons, which normally proceeds in the absence of readily detectable morphological intermediates (i.e., autophagic vacuoles), indicates...  Read more

The extreme scarcity of autophagic vacuoles in normal brain and their appearance in states of disease have previously led many to assume that autophagy in neurons is mainly an inducible process. Autophagy is solely responsible for organelle turnover, however, and the large cytoplasmic mass of neurons would suggest, therefore, that autophagy might have a significant constitutive component. The two papers by Komatsu et al. and Hara et al. have now provided elegant and definitive evidence in neurons for constitutive autophagy and have demonstrated that it is required for neuron survival. In fact, the results imply that the brain may actually be one of the tissues most vulnerable to a possible impairment of autophagy. These findings, therefore, offer insight into why neurons are preferentially victimized in diseases that disrupt the lysosomal system, even when the disease is a systemic one.

This new evidence for actively ongoing autophagy in neurons, which normally proceeds in the absence of readily detectable morphological intermediates (i.e., autophagic vacuoles), indicates that this process in healthy neurons is exceptionally efficient. Another implication from these observations is that autophagic vacuole accumulation in neurodegenerative disease states may signify a failing autophagy system, rather than simply an activation of autophagy as is frequently proposed. The findings are highly relevant to Alzheimer disease (AD) where autophagic function is impaired as evidenced by a massive build-up of autophagy intermediates especially within dystrophic dendrites of affected neurons. This indicates that the usually efficient progression of autophagosomes to lysosomes is impeded (Nixon et al. 2005). Autophagosome-lysosome fusion is already known to be slowed by normal cell aging (Martinez-Vincente et al. 2005) and additional risk factors for AD are likely to be found to impair autophagy. Autophagic vacuoles are highly enriched in γ-secretase and actively generate Aβ during autophagy (Yu et al., 2005). Although normally most of the generated Aβ would be degraded within lysosomes, in AD and transgenic AD models, the marked build-up of autophagic intermediates within an impaired autophagy pathway is a significant source and intracellular reservoir of Aβ (Yu et al., 2005). The two new papers, in the context of these other recent observations, therefore, support potential links between autophagic failure and neurodegeneration, amyloidogenesis, and possibly the intracellular accumulation of other disease-related proteins in AD. Therapeutic strategies based on facilitating efficient autophagy show glimpses of promise in neurodegenerative disease models (e.g., Ravikumar et al., 2004).

References:
Nixon RA, Wegiel J, Kumar A, Yu WH, Peterhoff C, Cataldo A, Cuervo AM. Extensive involvement of autophagy in Alzheimer disease: an immuno-electron microscopy study. J Neuropathol Exp Neurol 2005; 64:113-122. Abstract

Martinez-Vicente M, Sovak G, Cuervo AM. Protein degradation and aging. Exp Gerontol. 2005 Aug-Sep;40(8-9):622-33. Abstract

Yu WH, Cuervo AM, Kumar A, Peterhoff CM, Schmidt SD, Lee JH, Mohan PS, Mercken M, Farmery MR, Tjernberg LO, Jiang Y, Duff K, Uchiyama Y, Naslund J, Mathews PM, Cataldo AM, Nixon RA. Macroautophagy, a novel amyloid-beta (Abeta) peptide-generating pathway activated in Alzheimer's disease. JCB 2005; 171:87-98. Abstract

Ravikumar B, Vacher C, Berger Z, Davies JE, Luo S, Oroz LG, Scaravilli F, Easton DF, Duden R, O'Kane CJ, Rubinsztein DC. Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease. Nat Genet. 2004 Jun;36(6):585-95. Epub 2004 May 16. Abstract

View all comments by Ralph Nixon

The recent papers by the Mizushima and Tanaka labs provide compelling support of a role for autophagy in the constitutive turnover of cellular material and the importance of this process in maintenance of neuronal health. However, the conclusion that autophagy has no role in the clearance of inclusion bodies is premature. There is now strong evidence from conditional models of polyglutamine disease (e.g., Yamamoto et al., 2000 and Zu et al., 2004) to indicate that neurons can eliminate inclusion bodies and can recover from the toxic effects of aggregated protein—once expression is turned off. While there is no direct evidence yet that autophagy is required for this process, the Mizushima and Tanaka groups are now in an excellent position to test this hypothesis.

View all comments by Ron Kopito

This pair of papers shows that disruption of the autophagy pathway through deletion of the genes that encode critical components of the pathway (i.e., either Atg5 or Atg7) within neurons leads to behavioral abnormalities, neurodegeneration, and inclusion formation. The papers are interesting for several reasons.

First, although features of autophagy are known to be involved in normal protein turnover and may be part of a coping response to nutrient deficiency, it also is believed to be a programmed cell death pathway. Thus, it was unclear whether disruption of this pathway would lead to greater cell death because of impaired protein turnover or greater cell survival as seen when another cell death pathway, apoptosis, is disrupted. The fact that abnormal protein accumulation and greater cell death is seen indicates that autophagy plays a critical role in normal protein turnover in mammalian systems.

Second, ubiquitin immunoreactive inclusions were found in both Atg5- and Atg7-deficient mice, despite the fact that these mice were not known to otherwise harbor...  Read more

This pair of papers shows that disruption of the autophagy pathway through deletion of the genes that encode critical components of the pathway (i.e., either Atg5 or Atg7) within neurons leads to behavioral abnormalities, neurodegeneration, and inclusion formation. The papers are interesting for several reasons.

First, although features of autophagy are known to be involved in normal protein turnover and may be part of a coping response to nutrient deficiency, it also is believed to be a programmed cell death pathway. Thus, it was unclear whether disruption of this pathway would lead to greater cell death because of impaired protein turnover or greater cell survival as seen when another cell death pathway, apoptosis, is disrupted. The fact that abnormal protein accumulation and greater cell death is seen indicates that autophagy plays a critical role in normal protein turnover in mammalian systems.

Second, ubiquitin immunoreactive inclusions were found in both Atg5- and Atg7-deficient mice, despite the fact that these mice were not known to otherwise harbor a genetic mutation producing aggregation-prone proteins. Although not shown in these papers, inclusion formation can be observed following inhibition of the proteasome, the other major protein degradation pathway. One of the two groups examined proteasome function in the Atg7-deficient mice and found no proteasome impairment (although it would have been interesting to examine proteasome function in vivo). Taken at face value, these results add further support to the notion that inclusion formation is a downstream cellular response to the accumulation of proteins that are otherwise destined for degradation. Notably, the paper by Tanaka and colleagues found that the accumulation of "diffuse" cytosolic ubiquitin immunoreactivity occurred first, before inclusion formation, and was a more consistent phenotype of autophagy disruption than inclusion formation.

Although it is generally assumed that the proteins that are ubiquitinated and accumulate in inclusion bodies are misfolded and possibly non-functional, the finding here raises the provocative possibility that inclusions may form, in part, from normal proteins that accumulate when degradation is impaired. In such a scenario, pathogenesis might arise from having too much of a good thing.

View all comments by Steven Finkbeiner

Don’t Jeopardize New Therapies With Sham Surgery Control—Placebo Responses May Be Part of Therapies
I was the patient representative on the FDA advisory panel that reviewed deep brain stimulation (DBS) in March of 2000, and later I participated in the Medicare National Coverage decision for DBS on behalf of the requester (not Medtronics but an individual person with Parkinson's). From these engagements, I recall this treatment was shown to be very effective (upwards of 85 percent have 50 percent improvement in motor symptoms). Such dramatic and lasting improvements would need to be expected to offer a treatment that makes it worthwhile to take the risk of brain surgery. After a delay of more than four years from the initial advisory group, DBS has been available to patients, as a near breakthrough option once first-line treatment fails. Indeed, it is the only major new therapy for PD in the 40 years since levodopa was discovered. Now the recent study published in JAMA continues to show efficacy and also shows that its adverse side effects for important functions...  Read more

Don’t Jeopardize New Therapies With Sham Surgery Control—Placebo Responses May Be Part of Therapies
I was the patient representative on the FDA advisory panel that reviewed deep brain stimulation (DBS) in March of 2000, and later I participated in the Medicare National Coverage decision for DBS on behalf of the requester (not Medtronics but an individual person with Parkinson's). From these engagements, I recall this treatment was shown to be very effective (upwards of 85 percent have 50 percent improvement in motor symptoms). Such dramatic and lasting improvements would need to be expected to offer a treatment that makes it worthwhile to take the risk of brain surgery. After a delay of more than four years from the initial advisory group, DBS has been available to patients, as a near breakthrough option once first-line treatment fails. Indeed, it is the only major new therapy for PD in the 40 years since levodopa was discovered. Now the recent study published in JAMA continues to show efficacy and also shows that its adverse side effects for important functions like cognition are greater for DBS than with standard drug therapy. The DBS experience can be instructive for other surgical treatments for PD.

The question I want to raise regards the Ceregene 120 study, a gene therapy application of the nerve growth factor neurturin, NTN. The Phase 2 study "failed to meet primary endpoints" in comparison to a sham surgery placebo control. Similar to experiences with other surgically installed treatments, such as GDNF infusion pump and implantation of spheramine, a cell-based therapy using retinal dopaminergic cells, this latest placebo-controlled trial did not replicate the gains from the Phase 1 open-label study. The results of all of these studies are clouded from methodological issues such as differences in dosing between Phase 1 and Phase 2, dislodgement of pump connections, and differences in the use of other PD medications during the studies that may have affected the results. Even so, the fact remains that some study participants have experienced dramatic improvements lasting as long as six years and counting, and some have reduced their PD medications to near zero, being almost symptom-free after decades of increasing disability. A brain autopsy of one GDNF patient showed nerve growth in the side of the brain in which the treatment was administered during the trial. For all of these treatments, data point to improvements well beyond reasonable estimates of placebo effects.

Clearly placebo effects are very strong. Research on placebo response for a range of medical conditions including PD attributes these real physiological effects to expectations of benefit and conditioning established in the social context of administering a treatment. The greater the risk and notoriety of the intervention, and the more certain and authoritative the source, a greater placebo effect is produced. Maximum placebo effects, as would be expected, are found from brain surgery as well as from the safest form of sham brain surgery, where the brain is not penetrated but the patient goes through the same process including lengthy anesthesia. DBS patients report vast improvements in symptoms even before the stimulators are turned on. Clinical brain researchers (including more than 90 percent of the Parkinson's Study Group) agree that sham brain surgery is necessary to prove that improvements are attributable to the treatment beyond the placebo. On the other hand, an online survey of activist PD patients found that only 37 percent would participate in a sham surgery study. Closing this gap raises practical as well as ethical issues.

DBS was approved without sham surgery as a placebo control. So why aren't DBS's gains in motor scores attributed in part to a learned placebo response? Shouldn't the placebo effects that last multiple years be counted as part of the treatment, as they effectively are with DBS, and not written off as bias? The recent JAMA publication improved the evidence of efficacy for DBS by randomization to best medical treatment versus surgery. Why isn't random assignment to best medical practice a sufficient comparison for other surgical interventions?

Sham surgery is not a sugar pill; it is a powerful intervention, although you would probably be charged with fraud if you tried to sell it. Placebo studies on the experience of pain in fact demonstrate that the "bias" from patient hopes and expectations, a central element of all healing, is opposite of what has been assumed by science in experimental settings. That is, treatment effects are reduced and placebo effects are increased. That is so because random assignment dilutes positive effect of patients’ expectation that they will improve from the ongoing uncertainty about whether they are on the real thing, and conversely it elevates the placebo group’s expectation that they may be on the real thing. This biases the results toward type II errors (false negative), which are more important to patients with serious illness than are type I errors (false positive) that are the target of statistical models. The pain studies suggest that the placebo mechanism may be necessary to trigger the therapeutic effects of treatment. Elaborate deception to control this effect could be undermining its evaluation.

Sure, we need to control bias. But variability and bias can come from many sources, including, importantly the selection of participants and the variability of raters on subjective scales such as UPDRS. For example, are all study participants diagnosed correctly? And do they represent homogeneous types of patients? Depression medication trials, which also fail at high rates, have taught us that clearer distinction between treatment and placebo results from higher-quality central rating of subjective measurement scales. Multiple ratings of key measures reduce noise in data when averaged. Patients who have participated in PD clinical trials know that UPDRS "off" may describe different behavior on different days, and are not totally determined by the time since the last dose. Better understanding of these factors from the patient perspective is necessary to control this source of variability in the data.

Alternatively, where the sources of variability are unbiased, the problem can be fixed by increasing sample size to account for random fluctuations in the calculations of confidence of the result. This not only costs more, but it also may bump up against practical limitations including recruitment and FDA statisticians.

New Directions for the Twenty-first Century
As science progresses, we need to re-examine our assumptions about the standards for evidence in the assessment of safety and effectiveness. The gold standard of the randomized, prospective, double-blind placebo-controlled study cannot be applied as a one-size-fits-all to conditions on the cutting edge of medical science.

Medical miracles of the twentieth century mostly pertain to acute conditions, where linear assumptions of statistical models for hypothesis testing more closely approximate the relatively short-term interventions. As we deal with longer-term degenerative processes involving dynamic interaction and feedback to our conscious brain processes, assumptions from the experimental model become questionable. This is true even where all orthodoxies of statistics are followed and statistical significance is achieved.

Recent FDA law offers greater flexibility to align methods to the parameters of the specific case. Such alternative methods need to be qualified and used. Examples include Bayesian statistics for application to dose-finding tasks, or mathematical models of disease progression as historical controls. Crossover designs can detect differences in symptomatic benefits, and delayed start designs have shown promise to detect neuroprotection.

FDA law requires well-controlled randomized studies, not placebos. New policies put more emphasis on life cycle monitoring of treatments in real practice settings, and provide reimbursement coverage for access to new treatments while evidence of long-term safety and efficacy are established with greater certainty. Following patients more closely for a number of years to see the lasting effects can establish whether the treatment effects are purely placebo, at the same time that long-term safety is tracked.

Continuing failure of studies based on faulty assumptions about human behavior is not a viable option. A better understanding of placebo responses in the design of clinical trials points to new approaches in collaboration with patient advocates and communications to FDA.

Perry D. Cohen directs the Parkinson Pipeline Project.

View all comments by Perry Cohen