Updated 27 September 2001. The Biology of α-Synuclein and Lewy body Disease Workshop Meeting
Cosponsored by NIA and NINDS, Bethesda, Maryland,
July 16-17, 2001.

Meeting organizers: Tony Phelps (NIA) and Dianne Murphy (NINDS)
Chair: John Trojanowski

Participants: D. Galasko; I. McKeith; B. Hyman; M. Higuchi; D. Dickson; G. Perry; D. Selkoe; T. Iwatsubo; P. Lansbury; T. Dawson; J. George; C. Pickart; M. Polymeropoulos; M. Farrer; V. Lee; D. Clayton; M. Feany; M. Lee; E. Masliah

D. Galasko (Cortical Lewy Body Pathology and Dementia)
Presented data that implicate α-synuclein pathology in the form of Lewy bodies (LBs) or neurites as a marker of dementia. About 15%-25% of patients with Alzheimer's disease (AD) have widespread, or diffuse LB pathology, affecting areas such as the hippocampus, entorhinal cortex, cingulate, and frontal and parietal cortex. Despite a lower tangle burden, patients with AD plus LBs show equal or faster progression of dementia than patients with 'pure' AD. Psychometric testing shows a pattern of deficits that distinguish AD with LB pathology from pure AD: impaired attention, psychomotor speed and visuospatial/ constructional tasks. About 60% of AD patients have α-synuclein pathology in the amygdala, of uncertain clinical significance. Patients with Parkinson's disease (PD) who later develop dementia often have more severe and widespread α-synuclein pathology compared to those who remain nondemented, and often have amyloid pathology. Aging, stress (triggered by amyloid?) and genetic susceptibility are factors that may lead to widespread LB pathology and dementia.

I. McKeith (DLB: Diagnosis and Treatment)
Discussed clinical criteria for dementia with LBs (DLB) that were developed at a workshop in 1995, and suggested that these could be adapted to include patients with PD who later developed dementia. The clinical criteria have performed variably in research studies, although they usually have high specificity for DLB. He reviewed several methods to assist in the diagnosis of DLB. On computerized tests that measure reaction time and continuous vigilance, there was greater impairment and higher variability (fluctuation) in DLB than in AD. These deficits improved significantly in a placebo-controlled clinical trial of rivastigmine, a cholinesterase inhibitor. Volumetric MR studies showed a lesser degree of hippocampal lobe atrophy in patients with DLB compared to AD.

B. Hyman (Clinicopathological Correlations and Tissue Culture of Synuclein)
Described studies in primary neuronal cells and cell lines transfected with α-synuclein GFP. Remarkably, in this system α-synuclein GFP gets truncated at the c-terminal site of GFP site, and has a higher tendency to aggregate, while other GFP fusion constructs (tau, MT, synaptophysin) are not cleaved. Inhibitors of the proteosome also enhance accumulation of α-synuclein GFP supporting the contention that abnormally folded α-synuclein might be processed via the proteosome. Studies describing the distribution of α-synuclein GFP in neuronal cells have been recently published (Abstract; McLean et al 2001 Neuroscience)

M. Higuchi (Neuroimaging and Neuropathological Markers in DLB)
Discussed studies directed at evaluating the usefulness of diverse laboratory tests to differentiate LBD from AD. There is a potential use for combining CSF biomarkers with PET and genotyping. In the DLB there is mild increase in tau in CSF; however, levels of a-synuclein are not altered (Higuchi et al. Ann Neurol, in press). Furthermore, levels of F-DOPA by PET are markedly reduced in striatum in DLB vs. AD. However, the most significant data showed that by PET in AD, there was reduced glucose metabolism in the temporo-parietal region, while in DLB reduced glucose metabolism was most prominent in the occipital cortex (Abstract: Prog Neuropsychopharmacol and Biol Psych 2001; Abstract; Exp Neurol 2000). Interestingly, these alterations were strongly correlated with gliosis of the white matter in the occipital that was unrelated to LB formation or α-synuclein accumulation.

D. Dickson (Neuropathological Spectrum of Synucleopathies)
Described the patterns of distribution and neurodegeneration in sporadic and familial PD. The amygdala is a site of high vulnerability to LB pathology not only in DLB but also in AD. In AD cases with high Braak stage, there are far more LB's in the amygdala than in AD with low Braak stage. LBs in amygdala colocalizes with tau and shows two types of filaments. Neuropathological analysis of the first case with familial parkinsonism (A53T mutant) showed CA2/3 neuronal loss and spongiosis, the sustantia nigra (SN) showed abundant LB's. There was also involvement of other cell types such as glial inclusions in cerebellum and basal ganglia region, very similar to multiple system atrophy (MSA). Neuropathological analysis of the brain of a patient from the Iowa kindred (early onset PD) 4q neuronal also showed loss of neurons in CA2/3, extensive neuritic pathology in dentate gyrus. Thus, while familal PD with a-synuclein mutations show LB formation, cases with Parkin mutations do not.

G. Perry (Oxidative Response and Damage in PD and AD)
Discussed the role of oxidative stress in the pathogenesis of neurodegenerative disorders. Of the markers tested 8OHGuanosine is an excellent marker of RNA oxidation. In AD and Down syndrome, markers of oxidative stress precedes amyloid deposition and when amyloid deposition occurs, oxidative stress is reduced. 8OHGuanosine is increased and present in LB's. Oxidative stress might play a role at different stages of the neurodegenerative process.

D. Selkoe (Functional Interactions between Parkin and Synuclein in PD)
Discussed the potential interactions between Parkin and α-synuclein. Studies in transgenic mice and human brains showed that α-synuclein is very rich in S1 and S370 (high speed supernatant), temperature and time increases high MW α-synuclein species. Furthermore, fatty acid extraction increases resulting α-synuclein aggregation. Analysis of α-synuclein sequence revealed that aa’s 2-19 and 123-120 are consensus motifs for cytosolic fatty acid binding. Double immunostaining of PD brains showed that α-synuclein is in the corona of the LB and Parkin in the center. Parkin antibodies immunoprecipitates α-synuclein22, a new o-glycosilated variant that is only observed in ARPD brain not in AD, PD or NL brains. Parkin which is an E3 ubiquitin ligase, conjugates ubiquitin into α-synuclein22. Cytosolic o-glycosilation of proteins is very rare and most occur extracytoplasmic. Interestingly, α-synuclein22 is not ubiquitinated in autosomal recessive PD (ARPD) and Parkin expression and probably activity are completely absent in ARPD. Dr. Selkoe concluded that the relation between Parkin and α-synuclein in ARPD might be similar to the one of PS and APP in familial AD.

T. Iwatsubo (Phosphorylation of Synculein in Synucleopathy)
Discussed the role of α-synuclein post-transcriptional modifications in PD. In particular he focused on the potential role of α-synuclein phosphorylation in DLB. α-synuclein is phosphorylated at the c-terminal region 128-140 maybe at ser129. The urea soluble fraction in DLB brains is phosphorylated at ser129 and it's not detected in normal brains. α-synuclein is phosphorylated at low levels in rat brains and dephosphorylated very rapidly postmortem. In DLB 90% of the α-synuclein remains phosphorylated while in normal brain only 4%. Antibodies against phospho-ser α-synuclein only labels LB's, LB neurites, and glial cells in MSA. More recently, Dr. Iwatsubo also found that phosph-ser 129 α-synuclein antibody recognizes inclusions in α-synuclein tg mice drosophila. α-synuclein is probably phosphorylated by CK2.

P. Lansbury (Synuclein Fibrillization and Neurodegneration)
Discussed the biophysical characteristics of native and folded α-synuclein using atomic tunneling microcopy and the search for compounds that will reduce fibril formation. He found that aggregated α-synuclein progress from spherical protofibrils 4nmà protofibrilsà fibrils 8nm. Interestingly, murine α-synuclein fibrillates more rapidly than human wt or mutant α-synuclein. More recently he has initiated screening studies toward discovering anti-aggregation factors using thio T assay assays and commercially available libraries. This study showed that cathecols inhibit α-synuclein fibril formation while dopamine promotes fibril formation.

T. Dawson (How Mutations in Syn and Parkin Cause PD)
Discussed the mechanisms by which Parkin mutations might lead to ARPD. Most Parkin mutations are in the IBR-ring finger domain and since the Parkin is an E3 ubiquitin ligase, mutations in this gene results in decreased α-synuclein ubiquitinization. In addition to α-synuclein, Parkin also ubiquitinates CDCrel-1 and accelerates degradation of this synaptic vesicle protein. Overall, Parkin mutations result in decreased activity in CDCrel-1. This protein plays an important role in DOPA release. In contrast to Dr. Selkoe's studies, Dawson was not able to co-IP α-synuclein with Parkin or detect 0-glycosilation However he was able to demonstrate that synphylin is in the center of LB's and that synphylin co-IP with Parkin. Furthermore, Parkin ubiquitinates synphilin and mutations in Parkin affect binding to synphilin.

J. George (Lipids Induce Irreversible Multimerization of Synuclein)
Discussed the interactions of α-synuclein with fatty acids (FA). This is probably one of the most clear physiological interactions of α-synuclein. Long chain PUFA promotes aggregation of α-synuclein. Deletion of any one exon does not block this function. α-, β- and γ -synuclein show multimerization, wt α-synuclein multimerize similar to mutant with FA and α-synuclein becomes more acidic (- charge) with PUFA.

C. Pickart (Ubiquitin, Proteosome and Neurodegeneration)
Discussed general principles as to how the proteosome and ubiquitinization systems operate and how they are related. E3 facilitates targeting, E2 conjugating, E1 activating The 26S proteosome requires unfolding of the protein for degradation and branched chain is a universal proteolytic signal. There is a UCHL-like mutation in rat that results in Gracile axonal dystrophy (GAD). Classical examples of diseases associated with ubiquitinated proteins that are targeted to the proteosome include -cystic fibrosis and trinucleotide repeat diseases. However, it is unclear if ubiquitinated- α-synuclein or -TAU are targeted to proteosome, this complicates understanding the role of parkin and UCHL1 in ARPD. It is important to remember that long ubiquitin chain usually are shuttled to proteososme while short ubiquitin chain are not. Monoubiquitinated membrane proteins are targeted for endocytosis. An interesting exception is histone 2A which is ubiquitinated but is not targeted to proteosome.

M. Polymeropoulos (Genetics of PD)
Described a new family with ARPD. A member of this family has advanced parkinsonism. The patient is 22 y/o started at age 16 who did not have mutations in synuclein, UCHL1 or parkin and has a mutation gly336ser in neurofilament M gene which results in Fsp I restriction site. This is a completely new gene associated with PD.

M. Farrer (Haplotype Analysis in PD)
Discussed previous and new results characterizing the neurogenetics of ARPD. PARK 1 Dom 4q21 α-syn
PARK2 Res 6q25.2 parkin
PARK3 Dom, reduced penetrance 2p13
PARK4 Res 4p15
PARK5 Res 4p14 UCHL1
PARK6 1p35
PARK7 1p
There is a new class of of genetic mechanism that might contribute to PD pathogenesis. This is association with dinucleotide repeats TC, TC, TA, CA.

In particular, α-synuclein haplotypes

distribution Rep1; -770; -116; IVS4+66A>G; IVS4+78ins
(2) (2) (1) (1 ) (2)

This particular α-synuclein haplotype is overepresented in LBD and PD patients (Hum Molec Genet, in press 2001). He also described a new and unique parkin mutation (R275W) that differs with the classical parkin mutations in that the patient has classical and abundant LB's and is probably dominant. In this mutation there is some residual Parkin activity, compared to other ARPD parkin mutants. Thus, it is possible that LB's can be a feature of parkin proven disease. UCHL1 S18Y polymorphism carriers have reduced risk of Parkinson's and overall UCHL1 poorly active at releasing ubiq and promoting degradation

V. Lee (Oxidative/nitrate Stress in Synculeopathies)
Discussed the role of oxidation and nitriation on α-synuclein fibrillation. Decreased complex I activity results in increased ROS, leading to peroxynitrite formation which inactivates TH and promotes α-synuclein aggregation. In this case, all 4 tyr residues tyr are target for nitration. The process of peroxynitrate addition results in stabilization of α-synuclein filaments. In PD, nitrated α-synuclein is found in halo and native in the core of LB's. Nitrated α-synuclein found in LB's and GCI's is recoverable in insoluble fraction. α-synuclein and ubiquitin are not colocalized when transfected cells are treated with oxidants and proteosome inhibitors. The half life of α-synuclein is about 48 hrs (very slow) no differences mut vs. WT in HEK293. α-synuclein is probably degraded in lysosomes. The extent and effects of oxidation and nitration of α-synuclein in DLB suggests a major role of this process in the pathogenesis of neurodegeneration.

D. Clayton (Effects of Ectopic Synuclein Expression in Yeast)
Discussed the potential physiological role of α-synuclein by combining expression in yeast of canary α-synuclein and microarray analysis. Remarkably, 25 genes were upregulated; most of these genes involved in lipid metabolism PDR16, regulates PLD; PI-specific lipid Peroxisomal b-oxidation; turn-over of long chain FA carnitine O-acetyltransferase peroxisomal catalase peroxisomal transferase These results a possible role for α-synuclein in massive membrane turnover, activation of peroxisomal b-oxidation and a probable role in osmotic stress. Furthermore, α-synuclein promotes decrease in long FA and an increase in short FA, leading to changes in phospholipid composition.

M. Feany (Modeling PD in Fly)
Described new developments in the a-synuclein Drosophila model. An important observation was that this model shows retinal degeneration, locomotor abnormalities, but no systemic toxicity. The retinal findings are being used in collaboration with Lansbury's lab as a screen for compounds with anti- α-synuclein activity. In addition, she found that dorsomedial cluster dopaminergic neurons are lost in a time dependent fashion. The effects of WT are similar to MUT α-synuclein. There was no evidence of generalized neuronal loss only TH+ cells. There was no evidence of glial inclusions in fly, α-synuclein does not promote cholinergic degeneration in fly, in contrast, htau promotes cholinergic loss in fly. Heat shock (29 c) decreases inclusion formation in α-synuclein fly BUT neurons are lost and in fact are lost faster, suggesting that inclusion formation might be a "protective" strategy for neurons.

M. Lee (Synucleopathy in tg Mice)
Described new findings in the Prp α-synuclein tg mice. While A30P and WT display no or very little alterations, mice expressing α-synuclein A53T at 10-20-fold endogenous levels show severe motor alterations at age of 10m, and they and die within 21 d. Other lower expresser PrP A53T α-synuclein tg display changes at 14-18m. Mice develop sudden onset, ataxia, rapid progression, there was accumulation of α-synuclein (A53T) in deep nuclei in the dentate in the cerebellum, accumulation of NF-H in cerebellum and neurons were ubiquitin positive.

E. Masliah (Development of New Treatments for PD in tg Mice)
Discussed potential uses of newly developed α-synuclein and APP tg mice for anti-DLB treatment studies. PDGFb- α-synuclein tg mice develop motor alterations and inclusion formation similar to PD, however Thy-1 α-synuclein show some accumulation of α-synuclein in neurons but not an overt phenotype. Crosses of APP with α-synuclein tg results in earlier onset of PD pathology. Amyloid β 1-42 promotes α-synuclein accumulation and this might help explain the pathogenesis of LB formation in familial AD, DS, and DLB. The association between AD and LB pathology are more than chance and suggests a pathogenic relationship. β-synculein, a close homologue of the synuclein family shows the remarkable ability of blocking α-synuclein aggregation in vitro and in vivo in tg mice. This suggests that β-synuclein might be a natural negative regulator of α-synuclein accumulation that might it might be possible to use it as a tool for treatment development.

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