Aims

We examined how exosomal cargo from Parkinson’s disease (PD) mouse models impacts the transmission of pathology observed in PD brains.

Methods

We utilized the preformed fibril (PFF) mouse model and isolated brain-derived exosomes using sucrose gradient purification. The proteomic profile of the isolated exosomes was assessed via LC/MS and bioinformatics analysis. Their pathogenicity in recipient wild type (WT) mice was evaluated, following intrastriatal injections. Effects on host brain were examined by RNAseq and transcriptomic analysis, imaging and 3D reconstruction image analysis. Calcium imaging was employed to address the effects of the exosomes on neuronal calcium homeostasis.

Results

PFF inoculation does not alter the morphology, size distribution, and quantity of brain exosomes. Proteomics revealed that PFFs trigger changes in exosomal cargo elated to synaptic, mitochondrial and metabolic function. Only exosomes derived from PFF-treated mice were able to induce α-Syn pathology in the nigrostriatal axis, two months following injection in WT host mouse brain. A significant induction of astrogliosis and a decrease of the postsynaptic marker PSD-95 and its co-localization with synaptobrevin2, in the ipsilateral striatum were observed. No degeneration of the nigorstriatal axis or behavioral deficits were detected. Endogenous α-Syn was crucial as exosomal cargo for the transfer of pathology, since exosomes from PFF-inoculated α-Syn knockout (KO) mice failed to confer any pathology. Transcriptomic analysis of striatal tissue from mice injected with either WT or KO exosomes from PFF-treated mice, exhibited alterations in synaptic function. α-Syn expressing exosomes had a greater impact and negatively affected the aforementioned pathways.

Conclusions

We show for the first time that PFFs, can impact the exosomal cargo, independent of endogenous α-Syn. However, endogenous α-Syn, following PFF intoxication, appears to be the rate-limiting factor for exosomes to mediate α-Syn prion-like spreading.

Aims

Alzheimer’s Disease is neuropathologically diagnosed by the occurrence of beta-Amyloid and tau (MAPT) deposits, whereas in up to 50% of cases, alpha-Synuclein (SNCA) aggregates are detected as well. Because this co-pathology was shown to result in faster functional and cognitive decline, we aimed to characterize the genomic processes leading up to this phenomenon.

Methods

Polygenic risk scores were calculated using whole genome sequencing and GWAS summary data. Post-mortem human cortices from patients with AD and AD+SNCA co-pathology underwent single-nucleus multiome sequencing. Gene expression and protein assays as well as immunohistochemistry were performed on cerebral organoids and human iPSC cultures from an SNCA triplication line as well as its isogenic control.

Results

AD+SNCA co-pathology cases showed lower polygenic risk scores for AD, but a stronger propensity for SNCA expression. Mechanistically, AD+SNCA co-pathology resulted almost exclusively in neuronal changes in gene expression. Enrichment analyses demonstrated differential expression of transcripts related to translation and localized to the synapse, suggesting impairments in the translation of genes relevant for synaptic function. SNCA overexpression led to increased MAPT transcription in neuronal cell culture and iPSC assays, which ultimately displayed a synaptic phenotype.

Conclusions

While adverse interactions of SNCA and MAPT have been well demonstrated on the protein side, our study focuses on the upstream regulatory mechanisms guided by transcriptional networks and informed by personal genetic risk. Our results suggest that SNCA and MAPT are part of a genetic network where higher expression of SNCA drives the development of synaptic deficits, in part by increasing MAPT transcript levels.

Aims

To examine alpha-synuclein seeding doses in Lewy Body disease (LBD) by stage and to explore concurrent tau seeding previously observed in LBD cases.

Methods

We utilized real-time quaking induced conversion (RT-QuIC) assay as an ultra-sensitive methodology to estimate alpha-synuclein and tau seeding doses in midfrontal cortex of individuals with LBD (N=21) compared to those with AD (N=14). Alpha-synuclein seeding was compared to Lewy body stage. We employed linear regression to evaluate co-occurring tau and alpha-synuclein seeds and devised a metric of relative seeding to test the relationship when co-occurring seeds in individual cases were observed.

Results

Alpha-synuclein seeding was detected on the order of 10³-10⁷ seeding doses in 100% of LBD cases, and noted with prevalence (50%) in AD, albeit typically at multi-log lower levels than alpha-synuclein seeding quantified in LBD. 100% of AD with amygdala-predominant alpha-synuclein (N=4) and 30% of AD cases without Lewy bodies (N=10) had detectable alpha-synuclein seeding. Linear regression revealed that alpha-synuclein seeding dose accounted for about half of variability in tau seeding dose in LBD (p=0.0004), but none in AD (p=0.4391). Assessment of relative seeding in individual AD cases with alpha-synuclein-tau co-occurrence showed multi-log higher tau seeding. By comparison, LBD exhibited only a slight alpha-synuclein predominance, harboring significant tau seeding doses across all cases examined in this study.

Conclusions

We observed pervasive, but quantitatively distinct alpha-synuclein seeding activity in frontal cortex in LBD and AD, including detectable alpha-synuclein seeds in some AD cases without identifiable Lewy body histopathology. Regression analysis and relative seeding evaluation of co-occurring seeds revealed quantitatively similar levels of alpha-synuclein and tau seeding in LBD suggesting a possible novel role for alpha-synuclein-tau interaction in LBD, which we will explore in future studies.

Aims

The defining feature of Parkinson's disease (PD) and Lewy body dementia (LBD) is the accumulation of alpha-synuclein (Asyn) fibrils in Lewy bodies and Lewy neurites. We aimed to develop and validate a method to amplify Asyn fibrils extracted from LBD postmortem tissue samples.

Methods

We combined fibrils extracted from postmortem brain tissue with human recombinant Asyn and utilized multiple cycles of sonication-mediated fragmentation followed by quiescent incubation to produce amplified fibrils. We assessed seeding properties in a cell culture system to guide method development by comparing the properties of amplified fibrils with tissue-derived fibrils. We utilized solid-state NMR (SSNMR) to determine the atomic resolution structure from one LBD case.

Results

Amplified LBD fibrils comprise a mix of single and two protofilament fibrils with very low twist. SSNMR analysis of structure indicates that the fold of LBD amplified fibrils is highly similar to the fold determined by a recent cryo-electron microscopy study for a minority population of twisted single protofilament fibrils extracted from LBD tissue. Analysis of 1D SSNMR spectra from two additional cases and 2D SSNMR spectra from one additional case show that the spectra are highly similar to that of the case used for full structure determination, indicating a high degree of structural similarity in their fibril cores.

Conclusions

These results expand the structural characterization of LBD Asyn fibrils. By substantially expanding LBD fibril quantities beyond what can be obtained from frozen postmortem tissue samples, amplification enables new approaches for studies of disease mechanisms, imaging agents and therapeutics targeting Asyn.

Aims

Several studies suggest that in Parkinson’s disease (PD) alpha-synuclein first accumulates at presynaptic terminals, subsequently leading to neurodegeneration. Here, we aimed at exploring the accumulation of different alpha-synuclein proteoforms in putaminal dopaminergic and glutamatergic synaptic terminals across the neuropathological stages of PD.

Methods

PD (n=27; Braak-stage 4-6), incidental Lewy body disease (iLBD, n=32; Braak-stage 1-6) and control brain donors (n=10) were included. DAT⁺, VGLUT1⁺ and PSD95⁺ synapses immunopositive to Syn-1 (binding to aa 91-99 alpha-synuclein), phosphorylated Ser129 (pSer129), and C-terminal truncated alpha-synuclein at position 122 (CTT122) were quantified in the putamen using confocal microscopy. Lewy body (LB) density was quantified in substantia nigra, anterior cingulate and prefrontal cortex. Quantification of alpha-synuclein proteoforms in putaminal synaptosomes of the same donors will be performed to confirm our results.

Results

DAT⁺, but not VGLUT1⁺ and PSD95⁺ synapses, severely decreased across Braak alpha-synuclein stages. DAT⁺ Syn-1⁺ synapses were reduced in PD compared to controls (-86%) but not in iLBD. DAT⁺ CTT122 alpha-synuclein⁺ synapses did not differ between groups, while DAT⁺ pSer129 alpha-synuclein⁺ synapses were higher in PD compared to controls (+1253%). Specifically, pSer129 alpha-synuclein accumulation peaked in DAT⁺ synapses at Braak stage 3, when only few LBs were present in the substantia nigra. Moreover, we observed a trend towards increase in VGLUT1⁺ and PSD95⁺ pSer129 alpha-synuclein⁺ synapses at Braak stage 3, that became statistically-significant at Braak stage 5.

Conclusions

pSer129, and not CTT122 or Syn-1 alpha-synuclein, accumulates in putaminal dopaminergic presynaptic terminals in prodromal (iLBD) PD, and in glutamatergic and post-synaptic terminals at later disease stages. pSer129 alpha-synuclein pre-synaptic accumulation might be an earlier and more relevant measure than LB density in capturing early pathological alterations in PD.

Aims

Background: Synuclein amplification assays (SAA) enables the verification of alpha-synuclein (αS) aggregates; a widely considered pathological hallmark of Parkinson's disease (PD). Yet, surprisingly, αS aggregation is only detected in ~50% of LRRK2-PD patients.

Objectives:We investigated the association between cerebrospinal(CSF) αS aggregates, MRI, and dopamine transporter spectral tomography (DaTscan^TM) imaging metrics in patients with PD with and without genetic mutations associated with the disease.

Methods

Forty-two PD patients (15 iPD, 16 GBA1-PD, and 15 LRRK2-PD; mean age ±SD = 63.16 ± 9.5 yrs; F/M: 13/29) were included. All participants underwent comprehensive neurological and cognitive assessments, lumbar puncture for CSF collection, DAT-SPECT and MRI scans. Quantitative measures including deep grey matter volumes, diffusion-tensor (DT) indices along white matter (WM) fibers, functional connectivity (FC), and striatal binding ratio (SBR) values were calculated.

Results

Nine (all LRRK2-PD) out of 42 patients were negative for αS based on SAA (SAA-). No significant differences in demographic variables were detected between SAA+ and SAA- groups. Although no differences in SBR values were detected between both groups; SAA+ PD patients demonstrated significantly reduced FC in the right and left caudate. In DTI, SAA– showed significantly lower fractional anisotropy (FA) along the left external capsule, and higher FA along the left fronto-occipital fasciculus (SFOF) compared to the SAA+ group.

Conclusions

In PD, αS aggregation is linked to reduced FC in the caudate nucleus, as well as different patterns of WM integrity alterations; these changes were less apparent in the SAA- subgroup suggesting potentially a more benign degeneration process.

Aims

Our primary objective was to assess possible α-synuclein (αSyn) co-pathology in corticobasal syndrome (CBS) and progressive supranuclear palsy (PSP), as detected in CSF using an αSyn seed amplification assay (αSyn-SAA). Secondary objectives were to evaluate the association of αSyn-SAA positivity with other biomarkers, including of Alzheimer’s disease (AD), and clinical presentation. We hypothesized that αSyn-SAA positivity would be detectable in CBS and PSP, and that it would be associated with AD biomarker positivity and Aβ42 levels; age; neurodegeneration as assessed by NfL levels; and motor and non-motor symptoms associated with synucleinopathies.

Methods

This cross-sectional study included clinically diagnosed CBS and PSP patients who underwent a lumbar puncture between 2012-2021 at the Toronto Western Hospital (Ontario, Canada). We tested the CSF of 41 CBS (19 females, 65.6±8.5 y/o) and 28 PSP (13 females, 72.5±8.7 y/o) patients, for: αSyn-SAA positivity (real-time quaking-induced (RT-QuIC) protocol), AD biomarkers, and neurofilament light-chain (NfL) levels. ASyn-SAAs were run blind to clinical information and AD status. Clinical data were derived from medical records.

Results

AD positive biomarkers were observed in 31.7% CBS and 10.7% PSP patients; and αSyn-SAA positivity in 35.9% CBS and 28.6% PSP patients. In young-onset, but not late-onset subjects, αSyn-SAA positivity and AD positivity were associated (p<0.05). Age at onset was related to Aβ42 only in αSyn-SAA positive subjects (p<0.05). REM-sleep behavioural disorder was associated with αSyn-SAA positivity (p<0.01).

Conclusions

We detected a frequency of αSyn-SAA positivity in vivo in line with pathological studies, highlighting the usefulness of SAAs for the in vivo detection of otherwise undetectable neuropathological processes. Our results also suggest a preferential relationship between AD (specifically Aβ42) and αSyn-SAA positivity in young-onset subjects. This opens new perspectives for the stratification of patients in clinical trials.

Aims

The aims of this study were: 1) to characterize the expression level and the localization of an endogenous protease (tissue plasminogen activator, tPA) in the substantia nigra (SN), 2) to explore its role in neuroinflammation and neurodegeneration in a human α-synuclein (hα-syn) mouse model of Parkinson’s Disease (PD), and 3) to evaluate the therapeutic effect of the inhibition of the interaction between tPA and the N-methyl-D-aspartate receptor (NMDAR) by glunomab, a monoclonal antibody designed to counteract blood-brain barrier leakage and NMDAR overactivation.

Methods

WT, tPA^-/- and mice overexpressing proteolytically inactive tPA were injected with AAV-empty or AAV-hα-syn in the SN. Mice were evaluated after 4 weeks for lateralized neglect using the corridor task, tPA expression, dopaminergic neurodegeneration, and immune cell markers by immunohistochemistry and RNAseq.

Results

We identified tPA⁺ GABAergic striatal neurons innervating SN dopaminergic neurons and increased tPA protein in the SN upon AAV-hα-syn injection. Both tPA deficiency or glunomab administration protected dopaminergic neurons from α-syn-induced degeneration, reduced microglia activation and T-cell infiltration and led to recovery of lateralized sensory-motor integration in the corridor task. RNAseq analysis in WT and tPA^-/- mice after AAV-hα-syn injection showed that overexpression of hα-syn in WT mice significantly upregulated genes involved in the innate and adaptive immune responses whereas their expression was not increased in tPA^-/- mice.

Conclusions

Our data shows that tPA promotes neuroinflammation and dopaminergic neurodegeneration induced by overexpression of α-syn, and that glunomab prevents neurodegeneration by inhibiting tPA-induced neuroinflammation, translated into improved locomotion in a hα-syn mouse model of PD. This previously unidentified pathway suggests that glunomab, through its humanized form, represents a promising novel immunotherapy for the treatment of PD.