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THEME I: CLINICOPATHOLOGICAL CORRELATIONS
John Trojanowski, M.D., Ph.D.
Dr. Trojanowski provided an overview of a number of diverse hereditary and sporadic neurodegenerative diseases that lead to motor symptoms that are characteristic of Parkinsonism. These disorders form a heterogeneous group that may have commonalities at a cellular level, as many are characterized by accumulations of misfolded proteins that aggregate into fibrillar deposits containing a substance called amyloid (see Table below). Adding further complexity to the current classification and understanding of these neurodegenerative parkinsonian disorders is that some may involve deposits of one, two, or three different forms of amyloid. For example, the most common subtype of Alzheimer's disease (AD) - the Lewy body (LB) variant of AD - appears to involve accumulations of three types of amyloid: 1) LBs formed of alpha-synuclein fibrils, 2) senile plaques formed of aß fibrils, and 3) neurofibrillary tangles formed by tau protein. For these reasons, there is a growing need to ensure that the nomenclature used for the different parkinsonian disorders reflects our rapidly growing insights into the genetic and cell biological mechanisms underlying these conditions. This, in turn, will promote greater diagnostic accuracy, facilitate communications between researchers, clinicians and patients and enhance the pace of research to identify more effective therapies for these diseases.
TABLE: Neurodegenerative Parkinsonian Disorders
- Phenotypic Classification (Single, Double, Triple Brain Amyloidoses)
- Classical Parkinson's Disease
- Dementia With Lewy Bodies
- Multiple System Atrophy
- Neurodegeneration With Brain Iron Accumulation-1
- Alzheimer's Disease
- Corticobasal Degeneration
- Progressive Supranuclear Palsy
- Guam/Kii Peninsula Parkinsonism Dementia Complex
- Genetic Classification
- Park 1 through ~10
Dennis Dickson, M.D.
Dr. Dickson reviewed the new neuropathology of parkinsonian disorders and emphasized that clinical parkinsonism reflects the relative distribution of pathology in the brain, as opposed to the nature of the underlying type of pathology. For example, improved antibodies to alpha-synuclein have now improved the ability to label Lewy Bodies (LBs, fibrillar aggregates of alpha-synuclein and other proteins). Staining shows that Lewy Bodies may have a wider distribution than was appreciated previously, suggesting that the location of these inclusions in specific brain regions, rather than their specific composition, may be more indicative of how a disease will manifest itself clinically. Efforts in several laboratories to identify different stages of PD based on regional brain pathology have provided additional information as where neuronal pathology may occur as the disease progresses. For example, "typical PD" shows primary pathology in the nigrostriatal regions of the brain, while other PD clinical manifestations such as depression and inability to smell are probably due to pathology in the brainstem or olfactory bulb.
Lewy Bodies and abnormal (Lewy) neurites may now be classified into 3 general types: brainstem, cortical, and diffuse, and can occur throughout neocortex and subcortical regions as well as in glial support cells. Other types of amyloid (e.g., inclusions containing abnormal tau proteins) are also commonly seen.
Several other points discussed include:
- The concurrence of both tau and alpha-synuclein pathology occurs more frequently than was previously suspected, which provides evidence for a possible mechanistic relationship between the two proteins. The existence of synuclein pathology appears to increase the likelihood of tau pathology. This is also true of amyloid-beta and alpha-synuclein, although it was noted that the plaques in LBD are structurally different than plaques in AD.
- Alpha-synuclein pathology in the striatum (a brain region affected in parkinsonian syndromes) is a new and increasingly common feature of dementia with LBs, PD and PD with dementia.
- The convergence of fibrillar deposits of alpha-synuclein, tau and amyloid-beta are increasingly appreciated as common features of LB diseases.
In summary it was noted that there is an "imperfect match" between the clinical syndrome and the type of pathology, while there is a better match between the clinical syndrome and the anatomical location of pathology.
THEME II: GENETICS OF PD AND RELATED DISORDERS
Peter Heutinck, Ph.D.
Dr. Heutinck reviewed events leading to the discovery of the Park 7 familial Parkinson's disease (PD) gene locus, and the recent identification of mutations in the DJ-1 gene in both Dutch and Italian families with hereditary PD. The mutations of the DJ-1 gene are different in these two families: in the Dutch family, portions of the gene have been rearranged, while the gene appears to have undergone a single small ("point") mutation in the Italian family. The phenotype in these patients seems to be similar to the phenotype seen in parkin families, that is, an early onset, slow progression, and variability of severity with some dystonic features and psychiatric disturbances. A number of ongoing studies of DJ-1 in other PD families are include: analysis of the DJ-1 gene, better definition of the DJ-1 neurodegenerative disease phenotype, the role of DJ-1 in male fertility, the function of DJ-1 protein, the role of DJ-1 in stress responses, and the role of DJ-1 in mechanisms of neurodegenerative disease. Other mutations in this gene appear to have been identified by other investigators. Dr. Heutink also presented new data on the biology of DJ-1, including evidence of DJ-1 in mitochondria, the ability of DJ-1 to dimerize (form "doublets"), and the half-life of the normal and mutant DJ-1 proteins. Taken together, this preliminary evidence suggests that DJ-1 may be involved in anti-oxidant pathways, and/or the control of gene expression. Researchers also believe that DJ-1 may be involved in specific hormone pathways. Although present data suggest that DJ-1 does not interact with alpha-synuclein, pathological studies do show that DJ-1 antibodies label many neurofibrillary tangles and other cellular inclusions in tau-related neurodegenerative diseases, such as AD and Pick's disease.
John Hardy, Ph.D.
Dr. Hardy made several points in his introduction that he wished to emphasize: A) The size of proteins is relevant to neurodegeneration; B) Diseases are processes rather than specific entities like cats and dogs; C) Selective vulnerability is a significant but unexplained aspect of all of neurodegenerative diseases including those associated with parkinsonism. Following these points, a review and update on genetic causes of and genetic risk factors for dementing and parkinsonian neurodegenerative diseases was provided. John then presented evidence that increased levels of pathological proteins expressed in neurodegenerative disease may be causative for these diseases (as background for the subsequent presentation). For example, people who make more APP protein are more likely to get late onset AD, and there is genetic evidence (haplotypes) linking those who make more tau or synuclein to PSP and PD, respectively. Evidence from the Nussbaum intramural lab has shown the individuals with a particular promoter that increases the expression of the synuclein gene are also linked to PD.
Parkin mutations in PD were also discussed. It is presumed that parkin mutations are loss of function mutations in early onset PD, however it appears that a single mutation in parkin is not sufficient to cause disease - most individuals with early onset PD have multiple mutations. These and additional studies would suggest that a single mutation (one allele is mutant, and one allele is normal) may be associated with later onset PD.
Andy Singleton, Ph.D.
Dr. Singleton provided novel updates on the genetics of the Iowa "Spellman/Muenter" family (the members of which have been clinically studied for almost 100 years) that exhibits both Lewy Body Dementia and early-onset Parkinson's disease. Fibrillar deposits of alpha-synuclein and/or tau proteins are present in the brains of these individuals. Initially this kindred was linked to the "4p" region of chromosome 4, but more recent studies corrected previous misinterpretations and show linkage of the disease to a different region, "4q", known to be close to the alpha-synuclein gene. Reanalysis of the DNA samples from this family led to the discovery that the entire alpha-synuclein gene is repeated three times on one chromosome of affected family members. Genes on either side of the alpha-synuclein gene do not appear to be repeated. Further analysis of blood samples from this family indicate that the triplication of the alpha-synuclein gene is associated with an approximate 2 fold increase in alpha-synuclein protein in the platelets of affected individuals.
These data are extremely important, as they emphasize the importance of overall synuclein levels and its relationship parkinsonian syndromes, much like the finding over a decade ago that triplicate copies of the amyloid precursor protein gene in individuals with Down syndrome cause AD. Moreover, it suggests that synuclein levels in platelets may be utilized as an important biomarker in the future to identify individuals at risk for PD and related synucleinopathies. Follow up will include studies of the mutational mechanism as well as screens in additional families which are as yet unsolved. (It should be noted that this familial mutation was previously annotated as PARK 4 - as this has been discovered to be the synuclein gene, PARK 4 no longer exists in the genetic nomenclature.)
THEME III: CELL BIOLOGY AND MODELS
Virginia Lee, M.B.A.
Dr. Lee reported on evidence for interactions of tau and alpha-synuclein in neurodegenerative diseases. Briefly, it is known that the alpha-synuclein and tau proteins can bind together to form amyloid fibrils and filament-filled cellular inclusions characteristic of neurodegenerative diseases. Alpha-synuclein can also assemble into fibrils when alone in vitro, while tau fibrillization requires co-factors. Recent studies suggest that co-incubation of tau and alpha-synuclein synergistically promotes fibrillization of both proteins. Alpha-synuclein and tau filamentous amyloid inclusions can be seen to be co-localized in human patients with disease, in mice with an alpha-synuclein mutation, and in mice with both alpha-synuclein and tau mutations. Together, these data suggest that interactions between alpha-synuclein and tau can promote their fibrillization and drive the formation of pathological inclusions in human neurodegenerative diseases. The similarities between tau and alpha-synuclein were enumerated; both are small, acidic and abundant neuronal proteins, both are amphipathic, soluble and very heat stable with long half-lives and both contain hydrophobic residues and phosphorylation sites. Moreover, point mutations in either of these proteins cause autosomal dominant neurodegenerative diseases. It was noted that there is impressive tau pathology in the Contursi kindred, where synuclein mutations were first identified. While tau and synuclein are not completely co-localized, they co-occur as different types of filaments at the EM level.
Dr. Lee also discussed recent findings on the Parkin protein, associated with autosomal recessive juvenile Parkinsonism (ARJP), an early-onset form of PD. Their lab has generated a set of parkin antibodies and have found that parkin cannot be detected in Lewy Bodies. Importantly, they have found that the commercial antibodies currently available show non-specific bands on western blots, indicating that these antibodies may not be accurate for use in parkin studies (for example, these antibodies show positive staining in knock-out mice which do not express the parkin protein). It was suggested that previous studies should be reconsidered in light of these findings.
Ted Dawson, M.D., Ph.D.
Dr. Dawson focused his presentation on Parkin, which is mutated in autosomal recessive juvenile Parkinsonism (ARJP). Parkin is believed to play a role in the ubiquitination of proteins - or the "tagging" of these proteins for disposal by the cell. One of the key issues for those individuals studying Parkin is the identification of substrates for the protein, if it is indeed involved in the ubiquitination process. Possible Parkin substrates include components of the neuronal cytoskeleton, alpha-synuclein and related proteins, and proteins that induce cell stress. It is thought degradation of the Parkin substrate via a structure called the proteasome follows substrate ubiquitination and provides a mechanism by which Parkin could contribute to the aggregation of cellular proteins and ultimately to neurodegeneration. Dr. Dawson's laboratory is currently studying mice in which the Parkin gene has been "knocked-out", in order to evaluate behaviors that might be altered by the loss of Parkin, as well as the cellular downstream effects. Thus far, the accumulation of Parkin substrates has not occurred, but the mice do exhibit a reduced startle response, and they show an age-dependent loss of nerve cells in an area of the brain called the locus coeruleus. Parkin may play a role in the formation of Lewy bodies (LBs), but very little tissue is available from families with AJRP to enable an evaluation of LBs, and the results to date are not sufficient to make any conclusions.
John Yewdell, M.D., Ph.D.
Dr. Yewdell discussed the protein economy of cells, and specifically described Defective Ribosomal Products (DRiPs). DRiPs are believed to be "mistakes" made through the faulty synthesis or misfolding of newly-created proteins. Some DRiPs appear to be short-lived, and destined immediately for degradation. Others may simply be present in excess of that needed (based on the levels of their interacting protein partners, etc,). In either circumstance, DRiPs are potentially toxic because they are misfolded, functionless, fail to be targeted correctly, etc. For example, when cells have been treated with inhibitors of the proteasome (described by Dr. Yewdell as a "cellular cuisinart") there is a 30% increase in proteins within 30 minutes, suggesting that these are DRiPs which are normally rapidly degraded by the proteasome. Certain heat shock proteins appear to modulate the DRiP fraction however, they act only the soluble portion. Dr. Yewdell reviewed critical measures that a cell needs to maintain its "protein economy," and speculated on the significance of this economy in neurodegenerative diseases associated with accumulations of misfolded proteins.
Peter Lansbury, Ph.D.
Dr. Lansbury described "proto-filaments" as those intermediate structures prior to the development of insoluble fibrils that are the hallmarks of many neurodegenerative diseases. These structures may be capable of forming pores in cellular membranes, with subsequent damaging effects. The current efforts of the laboratory are directed at purifying amyloid pores from peptides to test the toxicity of these proteins on cultured cells, since they are not stable and may be difficult to isolate from diseased brain tissue. Subsequently, they have examined several protein subunits (including a-beta, alpha-synuclein, SOD. etc.) and there is evidence that the pores can cause mitochondrial swelling by "poking" holes in organelle membranes, and increasing permeability that could alter mitochondrial function. Dr. Lansbury's lab has also analyzed numerous mutant forms of the alpha-synuclein protein in order to identify those that preferentially fibrillize versus those that make pores. These data will help determine if some mutant pore-forming species can be identified that do not fibrillize, so that the toxicity of pure pore amyloid can be assayed. Finally, different types of pores were viewed with atomic force microscopy demonstrating that, despite which peptide they were formed from, all had a similar diameter corresponding to a predicted "cut-off" diameter for channel/pore formation that causes cells to lyse (i.e. their size corresponds to other naturally occurring more structures).
Presently, the lab is making a cDNA library of alpha-synuclein variants, in an attempt to find those particular synuclein mutants that may be highly toxic. Presumably those forms of synuclein that fibrillize rapidly, and therefore the existence of protofibrils/pores is extremely brief, would be less toxic (i.e. protofibrils do not hang around long enough for pore damage to occur.)
Craig Blackstone, M.D., Ph.D.
Dr. Blackstone discussed his studies of the normal DJ-1 protein; mutations in this protein have recently been implicated in hereditary parkinsonism. When DJ-1 is introduced into cells, it localizes to nuclei, mitochondria, and the cytoplasm. Distribution of mutant (with "L166P" mutations) and normal DJ-1 were similar, but the normal DJ-1 exhibited greater stability. Proteasomes appear to degrade both types of DJ-1, although the mutant form may be preferentially targeted. The transfer of this mutant form of DJ-1 to cells in culture may ultimately prove to be a useful model for understanding the effects of DJ-1 mutations in humans with parkinsonian syndromes. Dr. Blackstone also confirmed the observations made by Dr. Heutink that DJ-1 can form dimmers, and its self association is much stronger than its ability to bind other proteins. This may be the reason why yeast 2-hybrid studies are proving ineffective in revealing potential DJ-1 binding partners.
Leo Pallanck, Ph.D.
Dr. Pallanck discussed work to identify fruit flies with mutations in the Parkin gene, and to generate flies with the gene completely "knocked-out." Once produced, the Parkin knock out (KO) flies exhibited several interesting features, including male sterility, a decreased life span (2/3 of normal), flight and climbing defects, and structural alterations in dopaminergic neurons. They have observed similar defects in flies with Parkin mutations as well. Although at a superficial level, these defects do not appear related, further examination by this laboratory has revealed that several of these changes may be related to mitochondrial abnormalities. For example, the flight defect is associated with the degeneration of specific flight muscles, and these muscles exhibit abnormal mitochondria. Further, the sterility of affected males may be linked to defects in mitochondrial derivatives that are important energy-generating elements of developing sperm. Dopamine neurons do not die in the parkin knockout flies but they do exhibit abnormal morphology and processes. When Parkin knockout flies are crossed with alpha-synuclein over-expressing flies, there is no exacerbation of the phenotype, but they still exhibit Lewy bodies. Over-expression of Parkin does not rescue alpha-synuclein transgenic flies from neurodegeneration. Current work in this laboratory focuses on the evaluation of chemical sensitivities of Parkin fly mutants to environmental exposures, such as iron and copper sulfates and oxidative stress.
David Sulzer, Ph.D.
Dr. Sulzer described the three types of autophagy, the process whereby a cell digests and degrades proteins:
- Macro-autophagy: entire organelles are "swallowed whole" within double membrane structures then typically transferred to lysosomes for degradation. This is usually induced by stress.
- Micro-autophagy: small amounts of material, i.e. single proteins, are phagocytosed from the membrane and combined with lysosomal structures
- Chaperone-mediated autophagy (CMA): a cell's self-digestion of specific proteins that are "tagged" for breakdown by the binding of other proteins called chaperones.
In terms of the involvement of CMA in PD, it is known that the alpha-synuclein protein harbors a sequence of amino acids that may be recognized by chaperones - thus this protein could be a substrate for lysosomal breakdown via CMA. It is unclear, however, whether this would also be true of mutated forms of alpha-synuclein, such as those that are present in some forms of PD. Indeed, data show that alpha-synuclein mutants appear to undergo appropriate binding for CMA/lysosomal breakdown to occur, but are not actually transported into the lysosomes. By contrast, normal alpha-synuclein can undergo both binding and transport. These data suggest that there may be a transport defect for mutant alpha-synuclein that could lead to the accumulation of this protein in an undegraded form, and furthermore, these mutants may block the CMA process from occurring for other proteins by occupying all the available binding sites. Ultimately, neurodegeneration may then follow.
Ben Wolozin, M.D., Ph.D.
Dr. Wolozin discussed the capacity of aggregated alpha-synuclein to promote neuronal pathology. Briefly, mice engineered with the "A30P" alpha-synuclein mutation exhibit age-dependent aggregated alpha-synuclein in addition to increased levels of modified tau proteins and tau aggregates. These changes can occur in the same brain regions, but do not always occur in the same neurons that harbored the alpha-synuclein aggregates (although there is the potential for modest overlap). Further, ongoing studies suggest that the alpha-synuclein pathology precedes the abnormal changes in tau. Aggregated alpha-synuclein appears to be a potent inhibitor of the proteasome, while beta-synuclein doesn't appear to inhibit at all.
Future plans include an analysis of the time course of the emergence of both the tau and alpha-synuclein pathology in A30P alpha-synuclein transgenic mice, and the breeding of these mice with others engineered to overexpress the Parkin protein. The latter offspring will aid researchers in understanding whether increases in Parkin protein can protect the brain from abnormal changes in alpha-synuclein. Other studies will explore the relationship of the synuclein proteins to proteasomes, to determine if they play a regulatory role.
Leonard Petrucelli, Ph.D.
Dr. Petrucelli reviewed the current understanding of a protein called "CHIP" (for carboxyl-terminus-of-Hsc70-interacting protein), which originally was identified as a chaperone protein that might mediate protein degradation in neurons. He discussed studies that have examined the possibility that tau is a CHIP substrate, and presented data showing that CHIP may "tag" the protein tau for breakdown through a process called ubiquitination. In addition, he showed that CHIP can stabilize at least one mutant form of tau, and antibodies to CHIP can stain some types of tau lesions in Alzheimer's disease as well as in other tauopathies (e.g., progressive supranuclear palsy, corticobasal degeneration, Pick's disease). However, there is no evidence available to date suggesting that CHIP accumulates in neurons with tau-positive pre-tangle structures or Lewy bodies. Finally, other ongoing studies seek to confirm and extend this work in order to understand the role that CHIP activity might play in the normal and abnormal degradation of tau, and how impaired CHIP function might contribute to mechanisms underlying the formation of tau pathology in neurodegenerative diseases.
Last updated February 09, 2005
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