Single-cell eQTL mapping reveals convergent glial\u2013neuronal risk architecture in Parkinson\u2019s disease

Alpha-synuclein is a major component of Lewy bodies and glial cytoplasmic inclusions, pathological hallmarks of idiopathic Parkinson's disease and multiple system atrophy, and it is assumed to be aetiologically involved in these conditions. However, the quantitative status of brain alpha-synuclein in different Parkinsonian disorders is still unresolved and it is uncertain whether alpha-synuclein accumulation is restricted to regions of pathology. We compared membrane-associated, sodium dodecyl sulfate-soluble alpha-synuclein, both the full-length 17 kDa and high molecular weight species, by western blotting in autopsied brain of patients with Parkinson's disease (brainstem-predominant Lewy body disease: n = 9), multiple system atrophy (n = 11), progressive supranuclear palsy (n = 16), and of normal controls (n = 13). Brain of a patient with familial Parkinsonism-dementia due to alpha-synuclein locus triplication (as positive control) showed increased membrane-associated, sodium dodecyl sulfate-soluble alpha-synuclein levels with abundant high molecular weight immunoreactivity. In multiple system atrophy, a massive increase in 17 kDa membrane-associated, sodium dodecyl sulfate-soluble alpha-synuclein was observed in highly pathologically affected regions, including putamen (+1760%, range +625-2900%), substantia nigra [+1000% (+356-1850%)], and white matter of internal capsule [+2210% (+430-6830%)] together with numerous high molecular weight species. Levels of 17 kDa membrane-associated, sodium dodecyl sulfate-soluble alpha-synuclein were only modestly increased in less affected areas (cerebellar cortex, +95%; caudate, +30%; with both also showing numerous high molecular weight species) and were generally normal in cerebral cortices. In both Parkinson's disease and progressive supranuclear palsy, membrane-associated, sodium dodecyl sulfate-soluble alpha-synuclein levels were normal in putamen and frontal cortex whereas a trend was observed for variably increased 17 kDa membrane-associated, sodium dodecyl sulfate-soluble alpha-synuclein concentrations [+184% (-60% to +618%)] with additional high molecular weight species in Parkinson's disease substantia nigra. No obvious correlation was observed between nigral membrane-associated, sodium dodecyl sulfate-soluble alpha-synuclein accumulation and Lewy body density in Parkinson's disease. Two progressive supranuclear palsy cases had membrane-associated, sodium dodecyl sulfate-soluble alpha-synuclein accumulation in substantia nigra similar to multiple system atrophy. Several Parkinson's disease patients had very modest high molecular weight membrane-associated, sodium dodecyl sulfate-soluble alpha-synuclein accumulation in putamen. Levels of 17-kDa membrane-associated, sodium dodecyl sulfate-soluble alpha-synuclein were generally positively correlated with those of high molecular weight membrane-associated, sodium dodecyl sulfate-soluble alpha-synuclein and there was a trend for a positive correlation between striatal dopamine loss and 17-kDa membrane-associated, sodium dodecyl sulfate-soluble alpha-synuclein concentrations in multiple system atrophy. Brain membrane-associated, sodium dodecyl sulfate-soluble alpha-synuclein accumulations in Parkinson's disease and multiple system atrophy are regionally specific, suggesting that these sporadic alpha-synucleinopathies, unlike familial Parkinsonism-dementia, are not associated with a simple global over-expression of the protein. Despite a similar extent of dopamine depletion, the magnitude of brain membrane-associated, sodium dodecyl sulfate-soluble alpha-synuclein changes is disease specific, with multiple system atrophy clearly having the most severe accumulation. Literature discrepancies on alpha-synuclein status in 'Parkinson's disease' might be explained by inclusion of cases not having classic brainstem-predominant Lewy body disease and by variable alpha-synuclein accumulation within this diagnostic classification.

The Discovery of α-Synuclein in Lewy Pathology of Parkinson's Disease: The Inspiration of a Revolution.

Observational studies have documented association between rapid eye movement sleep behavior disorder (RBD) and neurodegenerative diseases, but the causal relationship remains to be established. In this study, we utilized a bidirectional 2-sample Mendelian randomization (MR) approach to assess the potential causal connection between RBD and 6 neurodegenerative conditions, including Alzheimer disease (AD), amyotrophic lateral sclerosis (ALS), dementia with Lewy bodies (DLB), frontotemporal dementia (FTD), multiple system atrophy (MSA), and Parkinson disease (PD). Inverse variance weighting (IVW) was performed as the main MR analysis, and additional 4 MR methods were performed to reinforce the robustness of the final MR estimates. Sensitivity analyses were conducted to detect possible heterogeneity or pleiotropy. Ten single-nucleotide polymorphisms (SNPs) for RBD, 54 SNPs for AD, 11 SNPs for ALS, 4 SNPs for DLB, 3 SNPs for MSA, and 12 SNPs for PD were selected as instrumental variables in this study. No suitable instrumental variables for FTD could be identified from genome-wide association studies dataset using a threshold of P < 5 × 10-8. Genetically predicted RBD was causally associated with an increased risk of PD (IVW: odds ratios = 1.093, 95% confidence intervals = [1.031–1.159], P = .003). In the reverse MR analysis, genetically predicted PD was found to be causally increased the incidence of RBD (IVW: odds ratios = 1.758, 95% confidence intervals = [1.355–2.281], P = 2.176 × 10-5). No heterogeneity or pleiotropy was observed in Cochran Q test, MR-Egger intercept test, or MR-PRESSSO Global test in the determination of the above 2 MR analyses. However, this bidirectional MR study did not identify any causal relationship between RBD and AD, ALS, DLB, FTD, and MSA. This MR study supported a bidirectional causal relationship between RBD and PD, mutually increasing the incidence of each. However, current genetic evidence did not support causal associations between RBD and AD, ALS, DLB, FTD, and MSA in either direction. However, these null findings should be interpreted with caution due to the limited sample sizes of the genome-wide association studies summary data used, highlighting the need for larger genetic studies to investigate these relationships further.

Together with Parkinson's disease (PD) and dementia with Lewy bodies, multiple system atrophy (MSA) is a member of a diverse group of neurodegenerative disorders termed α-synucleinopathies. Previously, it has been shown that α-synuclein, parkin, and synphilin-1 display disease-specific transcription patterns in frontal cortex in PD, dementia with Lewy bodies, and MSA, and thus may mediate the development of α-synucleinopathies. In this study, the differential expression of α-synuclein isoforms on transcriptional and translational levels was ascertained in MSA patients in comparison with PD cases and normal controls using isoform-specific primers and exon-specific antibodies in substantia nigra, striatum, cerebellar cortex, and nucleus dentatus. These regions are severely affected by α-synuclein pathology and neurodegeneration. Furthermore, we have also investigated transcript levels for parkin and synphilin-1 isoforms. In MSA brains, α-synuclein140 and α-synuclein 112 isoform levels were significantly increased, whereas levels of the α-synuclein 126 isoform were decreased in the substantia nigra, striatum, cerebellar cortex, and nucleus dentatus versus controls. Moreover, in MSA cases, we showed increased levels of parkin isoforms lacking the N-terminal ubiquitin-like domain and an aggregation-prone synphilin-1A isoform that causes neuronal toxicity in MSA. In PD brains, parkin transcript variant 3, 7, and 11 were significantly and specifically over-expressed in the striatum and cerebellar cortex, together with synphilin-1A and 1C. The changes of isoform expression profiles in neurodegenerative diseases suggest alterations in the regulation of transcription and/or splicing events, leading to regional/cellular events that may be important for the highly increased aggregation of α-synuclein in the brain. We report differential expression of α-synuclein, parkin, and synphilin-1 isoforms in multiple system atrophy (MSA) versus Parkinson's disease and normal control brains. We have focused on brain regions that are severely affected by α-synuclein pathology and neurodegeneration in MSA. The reported changes of isoform expression profiles suggest alterations in the regulation of transcription that may be important for aggregation of α-synuclein in the brain.

Parkinson's disease involves the aggregation of alpha-synuclein to form fibrils, which are the major constituent of intracellular protein inclusions (Lewy bodies and Lewy neurites) in dopaminergic neurons of the substantia nigra. Occupational exposure to specific metals, especially manganese, copper, lead, iron, mercury, zinc, aluminum, appears to be a risk factor for Parkinson's disease based on epidemiological studies. Elevated levels of several of these metals have also been reported in the substantia nigra of Parkinson's disease subjects. We examined the effect of various metals on the kinetics of fibrillation of recombinant alpha-synuclein and in inducing conformational changes, as monitored by biophysical techniques. Several di- and trivalent metal ions caused significant accelerations in the rate of alpha-synuclein fibril formation. Aluminum was the most effective, along with copper(II), iron(III), cobalt(III), and manganese(II). The effectiveness correlated with increasing ion charge density. A correlation was noted between efficiency in stimulating fibrillation and inducing a conformational change, ascribed to formation of a partially folded intermediate. The potential for ligand bridging by polyvalent metal ions is proposed to be an important factor in the metal-induced conformational changes of alpha-synuclein. The results indicate that low concentrations of some metals can directly induce alpha-synuclein fibril formation.

Parkinson disease (PD), Lewy body dementia (LBD), and multiple system atrophy (MSA) are synucleinopathies whose primary pathogenic event is the deposition of inclusions composed of aberrantly fibrillized α-synuclein.1 In PD and LBD, Lewy bodies are the key aggregate, whereas in MSA, α-synuclein accumulates in the form of oligodendroglial and neuronal cytoplasmic inclusions (GCIs and NCIs).2,3 Parkinsonian manifestations have been noted in a subset of patients with Gaucher disease and there is evidence that parkinsonism is more frequent among carrier relatives of patients with Gaucher disease.4 In a remarkable study, the glucocerebrosidase (GBA) gene was sequenced in an American PD brain bank series where GBA mutations were detected at a much higher frequently than in controls (PD 21% vs control 4.5%).5 These findings have since been replicated, mainly in Ashkenazi patient groups who have a higher mutation frequency but also in patients with clinically and pathologically diagnosed PD and LBD in a number of studies in different populations.4 In a study of 75 neuropathologically confirmed synucleinopathies, GBA mutations were found in 23% of the cases with Lewy bodies.6 The frequency of GBA mutations around the world between 2.3 and 31% (depending on population) indicates that GBA mutations are one of the commonest genetic risk factors for PD. GBA mutation carriers have a wide spectrum of phenotypes, ranging from classic l-dopa-responsive PD to LBD. In neuropathologic studies of PD/LBD cases, GBA mutations, α-synuclein inclusions, and Lewy bodies have been seen. This spectrum of clinical and pathologic features would suggest that MSA should also be a candidate to have GBA mutation.3 Only 12 cases of MSA have been analyzed for GBA mutations and defects were seen in this handful of cases.6 We extracted DNA from the brain tissue of 108 neuropathologically confirmed British MSA cases that had been diagnosed according to brain bank criteria and 257 normal British controls. Mean age at onset was 58.2 ± 10.7 years (range 34–83), mean age at death 64.5 ± 10.2 years (39–87), mean disease duration 6.8 ± 2.9 years (2–16), and 48% were men. All exons and flanking intronic regions of the GBA gene were sequenced in MSA and control cases. To avoid amplifying and sequencing the GBA pseudogene we employed long range GBA PCR and then BigDye sequencing as previously described.7 In our MSA study group of 108 cases, we identified one heterozygous GBA mutation (c.904C>T; R262H), giving a mutation frequency of 0.92%. In the British controls, three heterozygous mutations (V497L, N409S, and R269Q) out of 257 cases were identified (1.17%). There was no significant difference between the two groups (p = 0.66). The single MSA case with the heterozygous R262H mutation was a woman with an age at onset of 44 years. She had parkinsonian, cerebellar, and autonomic features (MSA–mixed type) with no family history. She died at age 51 years and the neuropathology revealed widespread GCIs and NCIs with a predominance in striatonigral structures. There were no Lewy bodies. One limitation of our study is the small sample size. Our study has a power of 80% to detect variants with an OR >1.61 or <0.63 at a significance level of 0.05. The results of this study indicate that GBA mutations are not common etiologic players in Caucasian patients with MSA. We cannot exclude that GBA mutations confer modest or low risk to disease. Furthermore, we did not sequence risk variants in regulatory regions (such as the promotor region or untranslated regions). Mutations in these regions would therefore have been missed. The unexpected role of GBA mutations has been demonstrated in several populations and is undoubtedly a highly significant risk factor for PD and LBD. More importantly, GBA mutations reveal a direct link between the lysosomal protein pathway and the clearance or the development of α-synuclein aggregates into Lewy bodies. Our study indicates that GBA mutations are not associated with MSA in the population that we analyzed, and that this branch of the ceramide pathway is unlikely to be associated with all types of primary α-synuclein deposition.

Involvement of autophagic protein DEF8 in Lewy bodies

LRP10 in α-synucleinopathies

A comprehensive, unbiased inventory of synuclein forms present in Lewy bodies from patients with dementia with Lewy bodies was carried out using two-dimensional immunoblot analysis, novel sandwich enzyme-linked immunosorbent assays with modification-specific synuclein antibodies, and mass spectroscopy. The predominant modification of alpha-synuclein in Lewy bodies is a single phosphorylation at Ser-129. In addition, there is a set of characteristic modifications that are present to a lesser extent, including ubiquitination at Lys residues 12, 21, and 23 and specific truncations at Asp-115, Asp-119, Asn-122, Tyr-133, and Asp-135. No other modifications are detectable by tandem mass spectrometry mapping, except for a ubiquitous N-terminal acetylation. Small amounts of Ser-129 phosphorylated and Asp-119-truncated alpha-synuclein are present in the soluble fraction of both normal and disease brains, suggesting that these Lewy body-associated forms are produced during normal metabolism of alpha-synuclein. In contrast, ubiquitination is only detected in Lewy bodies and is primarily present on phosphorylated synuclein; it therefore likely occurs after phosphorylated synuclein has deposited into Lewy bodies. This invariant pattern of specific phosphorylation, truncation, and ubiquitination is also present in the detergent-insoluble fraction of brain from patients with familial Parkinson's disease (synuclein A53T mutation) as well as multiple system atrophy, suggesting a common pathogenic pathway for both genetic and sporadic Lewy body diseases. These observations are most consistent with a model in which preferential accumulation of normally produced Ser-129 phosphorylated alpha-synuclein is the key event responsible for the formation of Lewy bodies in various Lewy body diseases.

Convergence of Heat Shock Protein 90 with Ubiquitin in Filamentous α-Synuclein Inclusions of α-Synucleinopathies

In this study, we used the Mendelian randomization (MR) method to systematically examine whether there is a bidirectional causal relationship between amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD), Parkinson's disease (PD), frontotemporal dementia (FTD), multiple system atrophy (MSA), and dementia with Lewy bodies (DLB). We analyzed data from 6,44,924 participants using MR to evaluate causality. We employed inverse variance weighted and MR-Egger regression tests for MR analysis. Additionally, we performed sensitivity analyses using the MR-Egger test and Mendelian Randomization Pleiotropy RESidual Sum and Outlier. The inverse variance weighted analysis found no evidence of a risk effect between ALS and the neurodegenerative diseases AD, PD, FTD, MSA, and DLB. However, the MR-Egger analysis showed that both AD (odds ratio: 1.079, 95% confidence interval: 1.017-1.145, P = .029) and PD (odds ratio: 1.210, 95% confidence interval: 1.046-1.401, P = .020) have a risk effect on ALS, indicating that AD and PD increase the risk of ALS. Our MR analysis suggests that AD and PD may have a potential causal relationship with ALS. Conversely, ALS does not appear to have a causal relationship with the other neurodegenerative diseases examined (FTD, MSA, DLB).

Conventional single tensor diffusion analysis models have provided mixed findings in the substantia nigra of Parkinson's disease, but recent work using a bi-tensor analysis model has shown more promising results. Using a bi-tensor model, free-water values were found to be increased in the posterior substantia nigra of Parkinson's disease compared with controls at a single site and in a multi-site cohort. Further, free-water increased longitudinally over 1 year in the posterior substantia nigra of Parkinson's disease. Here, we test the hypothesis that other parkinsonian disorders such as multiple system atrophy and progressive supranuclear palsy have elevated free-water in the substantia nigra. Equally important, however, is whether the bi-tensor diffusion model is able to detect alterations in other brain regions beyond the substantia nigra in Parkinson's disease, multiple system atrophy, and progressive supranuclear palsy and to accurately distinguish between these diseases. Free-water and free-water-corrected fractional anisotropy maps were compared across 72 individuals in the basal ganglia, midbrain, thalamus, dentate nucleus, cerebellar peduncles, cerebellar vermis and lobules V and VI, and corpus callosum. Compared with controls, free-water was increased in the anterior and posterior substantia nigra of Parkinson's disease, multiple system atrophy, and progressive supranuclear palsy. Despite no other changes in Parkinson's disease, we observed elevated free-water in all regions except the dentate nucleus, subthalamic nucleus, and corpus callosum of multiple system atrophy, and in all regions examined for progressive supranuclear palsy. Compared with controls, free-water-corrected fractional anisotropy values were increased for multiple system atrophy in the putamen and caudate, and increased for progressive supranuclear palsy in the putamen, caudate, thalamus, and vermis, and decreased in the superior cerebellar peduncle and corpus callosum. For all disease group comparisons, the support vector machine 10-fold cross-validation area under the curve was between 0.93-1.00 and there was high sensitivity and specificity. The regions and diffusion measures selected by the model varied across comparisons and are consistent with pathological studies. In conclusion, the current study used a novel bi-tensor diffusion analysis model to indicate that all forms of parkinsonism had elevated free-water in the substantia nigra. Beyond the substantia nigra, both multiple system atrophy and progressive supranuclear palsy, but not Parkinson's disease, showed a broad network of elevated free-water and altered free-water corrected fractional anisotropy that included the basal ganglia, thalamus, and cerebellum. These findings may be helpful in the differential diagnosis of parkinsonian disorders, and thereby facilitate the development and assessment of targeted therapies.

Mutations in alpha-synuclein gene cause familial form of Parkinson disease, and deposition of wild-type alpha-synuclein as Lewy bodies occurs as a hallmark lesion of sporadic Parkinson disease and dementia with Lewy bodies, implicating alpha-synuclein in the pathogenesis of Parkinson disease and related neurodegenerative diseases. Dopamine neurons in substantia nigra are the major site of neurodegeneration associated with alpha-synuclein deposition in Parkinson disease. Here we establish transgenic Caenorhabditis elegans (TG worms) that overexpresses wild-type or familial Parkinson mutant human alpha-synuclein in dopamine neurons. The TG worms exhibit accumulation of alpha-synuclein in the cell bodies and neurites of dopamine neurons, and EGFP labeling of dendrites is often diminished in TG worms expressing familial Parkinson disease-linked A30P or A53T mutant alpha-synuclein, without overt loss of neuronal cell bodies. Notably, TG worms expressing A30P or A53T mutant alpha-synuclein show failure in modulation of locomotory rate in response to food, which has been attributed to the function of dopamine neurons. This behavioral abnormality was accompanied by a reduction in neuronal dopamine content and was treatable by administration of dopamine. These phenotypes were not seen upon expression of beta-synuclein. The present TG worms exhibit dopamine neuron-specific dysfunction caused by accumulation of alpha-synuclein, which would be relevant to the genetic and compound screenings aiming at the elucidation of pathological cascade and therapeutic strategies for Parkinson disease.

Biomarkers to detect central dopamine deficiency and distinguish Parkinson disease from multiple system atrophy

Olfactory dysfunction in pure autonomic failure: Implications for the pathogenesis of Lewy body diseases