Overlaps and divergences between tauopathies and synucleinopathies: a duet of neurodegeneration.

Proteinopathy, defined as the abnormal accumulation of proteins that eventually leads to cell death, is one of the most significant pathological features of neurodegenerative diseases. Tauopathies, represented by Alzheimer's disease (AD), and synucleinopathies, represented by Parkinson's disease (PD), show similarities in multiple aspects. AD manifests extrapyramidal symptoms while dementia is also a major sign of advanced PD. We and other researchers have sequentially shown the cross-seeding phenomenon of α-synuclein (α-syn) and tau, reinforcing pathologies between synucleinopathies and tauopathies. The highly overlapping clinical and pathological features imply shared pathogenic mechanisms between the two groups of disease. The diagnostic and therapeutic strategies seemingly appropriate for one distinct neurodegenerative disease may also apply to a broader spectrum. Therefore, a clear understanding of the overlaps and divergences between tauopathy and synucleinopathy is critical for unraveling the nature of the complicated associations among neurodegenerative diseases. In this review, we discuss the shared and diverse characteristics of tauopathies and synucleinopathies from aspects of genetic causes, clinical manifestations, pathological progression and potential common therapeutic approaches targeting the pathology, in the aim to provide a timely update for setting the scheme of disease classification and provide novel insights into the therapeutic development for neurodegenerative diseases.

Characterization of Novel Human ß-glucocerebrosidase Antibodies for Parkinson's Disease Research.

BACKGROUND: Mutations in GBA1, which encodes the lysosome enzyme ß-glucocerebrosidase (also referred to as acid ß-glucosidase or GCase), are the most common genetic risk factor for Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Evidence also suggests that loss of GCase activity is implicated in PD without GBA1 mutations. Consequently, therapies targeting GCase are actively being pursued as potential strategies to modify the progression of PD and related synucleinopathies. Despite this significant interest in GCase as a therapeutic target, the lack of well-characterized GCase antibodies continues to impede progress in the development of GCase-targeted therapies. OBJECTIVE: This study aims to independently evaluate human GCase (hGCase) antibodies to provide recommendations for western blot, immunofluorescence, immunoprecipitation, and AlphaLISA (Amplified Luminescent Proximity Homogeneous Assay) assays. METHODS: Two mouse monoclonal antibodies, hGCase-1/17 and hGCase-1/23, were raised against hGCase using imiglucerase, the recombinant enzyme developed to treat patients, as the antigen. These novel antibodies, alongside commonly used antibodies in the field, underwent evaluation in a variety of assays. RESULTS: The characterization of hGCase-1/17 and hGCase-1/23 using genetic models including GBA1 loss-of-function human neuroglioma H4 line and neurons differentiated from human embryonic stem cells revealed their remarkable specificity and potency in immunofluorescence and immunoprecipitation assays. Furthermore, a hGCase AlphaLISA assay with excellent sensitivity, a broad dynamic range, and suitability for high throughput applications was developed using hGCase-1/17 and hGCase-1/23, which enabled a sandwich assay configuration. CONCLUSIONS: The hGCase immunofluorescence, immunoprecipitation, and AlphaLISA assays utilizing hGCase-1/17 and hGCase-1/23 will not only facilitate improved investigations of hGCase biology, but can also serve as tools to assess the distribution and effectiveness of GCase-targeted therapies for PD and related synucleinopathies.

Synucleinopathies: Intrinsic and Extrinsic Factors.

α-Synucleinopathies are a group of neurodegenerative disorders characterized by alterations in α-synuclein (α-syn), a protein associated with membrane phospholipids, whose precise function in normal cells is still unknown. These kinds of diseases are caused by multiple factors, but the regulation of the α-syn gene is believed to play a central role in the pathology of these disorders; therefore, the α-syn gene is one of the most studied genes. α-Synucleinopathies are complex disorders that derive from the interaction between genetic and environmental factors. Here, we offer an update on the landscape of the epigenetic regulation of α-syn gene expression that has been linked with α-synucleinopathies. We also delve into the reciprocal influence between epigenetic modifications and other factors related to these disorders, such as posttranslational modifications, microbiota participation, interactions with lipids, neuroinflammation and oxidative stress, to promote α-syn aggregation by acting on the transcription and/or translation of the α-syn gene.

HLA in isolated REM sleep behavior disorder and Lewy body dementia.

Synucleinopathies-related disorders such as Lewy body dementia (LBD) and isolated/idiopathic REM sleep behavior disorder (iRBD) have been associated with neuroinflammation. In this study, we examined whether the human leukocyte antigen (HLA) locus plays a role in iRBD and LBD. In iRBD, HLA-DRB1*11:01 was the only allele passing FDR correction (OR = 1.57, 95% CI = 1.27-1.93, p = 2.70e-05). We also discovered associations between iRBD and HLA-DRB1 70D (OR = 1.26, 95%CI = 1.12-1.41, p = 8.76e-05), 70Q (OR = 0.81, 95%CI = 0.72-0.91, p = 3.65e-04) and 71R (OR = 1.21, 95%CI = 1.08-1.35, p = 1.35e-03). Position 71 (pomnibus = 0.00102) and 70 (pomnibus = 0.00125) were associated with iRBD. Our results suggest that the HLA locus may have different roles across synucleinopathies.

Cerebral Microvascular Injury Induced by Lag3-Dependent α-Synuclein Fibril Endocytosis Exacerbates Cognitive Impairment in a Mouse Model of α-Synucleinopathies.

The pathological accumulation of α-synuclein (α-Syn) and the transmission of misfolded α-Syn underlie α-synucleinopathies. Increased plasma α-Syn levels are associated with cognitive impairment in Parkinson's disease, multiple system atrophy, and dementia with Lewy bodies, but it is still unknown whether the cognitive deficits in α-synucleinopathies have a common vascular pathological origin. Here, it is reported that combined injection of α-Syn preformed fibrils (PFFs) in the unilateral substantia nigra pars compacta, hippocampus, and cerebral cortex results in impaired spatial learning and memory abilities at 6 months post-injection and that this cognitive decline is related to cerebral microvascular injury. Moreover, insoluble α-Syn inclusions are found to form in primary mouse brain microvascular endothelial cells (BMVECs) through lymphocyte-activation gene 3 (Lag3)-dependent α-Syn PFFs endocytosis, causing poly(ADP-ribose)-driven cell death and reducing the expression of tight junction proteins in BMVECs. Knockout of Lag3 in vitro prevents α-Syn PFFs from entering BMVECs, thereby reducing the abovementioned response induced by α-Syn PFFs. Deletion of endothelial cell-specific Lag3 in vivo reverses the negative effects of α-Syn PFFs on cerebral microvessels and cognitive function. In short, this study reveals the effectiveness of targeting Lag3 to block the spread of α-Syn fibrils to endothelial cells in order to improve cognition.

The G51D SNCA mutation generates a slowly progressive α-synuclein strain in early-onset Parkinson's disease.

Unique strains of α-synuclein aggregates have been postulated to underlie the spectrum of clinical and pathological presentations seen across the synucleinopathies. Whereas multiple system atrophy (MSA) is associated with a predominance of oligodendroglial α-synuclein inclusions, α-synuclein aggregates in Parkinson's disease (PD) preferentially accumulate in neurons. The G51D mutation in the SNCA gene encoding α-synuclein causes an aggressive, early-onset form of PD that exhibits clinical and neuropathological traits reminiscent of both PD and MSA. To assess the strain characteristics of G51D PD α-synuclein aggregates, we performed propagation studies in M83 transgenic mice by intracerebrally inoculating patient brain extracts. The properties of the induced α-synuclein aggregates in the brains of injected mice were examined using immunohistochemistry, a conformational stability assay, and by performing α-synuclein seed amplification assays. Unlike MSA-injected mice, which developed a progressive motor phenotype, G51D PD-inoculated animals remained free of overt neurological illness for up to 18 months post-inoculation. However, a subclinical synucleinopathy was present in G51D PD-inoculated mice, characterized by the accumulation of α-synuclein aggregates in restricted regions of the brain. The induced α-synuclein aggregates in G51D PD-injected mice exhibited distinct properties in a seed amplification assay and were much more stable than those present in mice injected with MSA extract, which mirrored the differences observed between human MSA and G51D PD brain samples. These results suggest that the G51D SNCA mutation specifies the formation of a slowly propagating α-synuclein strain that more closely resembles α-synuclein aggregates associated with PD than MSA.

α-Synuclein conformers reveal link to clinical heterogeneity of α-synucleinopathies.

α-Synucleinopathies, such as Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy, are a class of neurodegenerative diseases exhibiting intracellular inclusions of misfolded α-synuclein (αSyn), referred to as Lewy bodies or oligodendroglial cytoplasmic inclusions (Papp-Lantos bodies). Even though the specific cellular distribution of aggregated αSyn differs in PD and DLB patients, both groups show a significant pathological overlap, raising the discussion of whether PD and DLB are the same or different diseases. Besides clinical investigation, we will focus in addition on methodologies, such as protein seeding assays (real-time quaking-induced conversion), to discriminate between different types of α-synucleinopathies. This approach relies on the seeding conversion properties of misfolded αSyn, supporting the hypothesis that different conformers of misfolded αSyn may occur in different types of α-synucleinopathies. Understanding the pathological processes influencing the disease progression and phenotype, provoked by different αSyn conformers, will be important for a personalized medical treatment in future.

New SNCA mutation and structures of α-synuclein filaments from juvenile-onset synucleinopathy.

A 21-nucleotide duplication in one allele of SNCA was identified in a previously described disease with abundant α-synuclein inclusions that we now call juvenile-onset synucleinopathy (JOS). This mutation translates into the insertion of MAAAEKT after residue 22 of α-synuclein, resulting in a protein of 147 amino acids. Both wild-type and mutant proteins were present in sarkosyl-insoluble material that was extracted from frontal cortex of the individual with JOS and examined by electron cryo-microscopy. The structures of JOS filaments, comprising either a single protofilament, or a pair of protofilaments, revealed a new α-synuclein fold that differs from the folds of Lewy body diseases and multiple system atrophy (MSA). The JOS fold consists of a compact core, the sequence of which (residues 36-100 of wild-type α-synuclein) is unaffected by the mutation, and two disconnected density islands (A and B) of mixed sequences. There is a non-proteinaceous cofactor bound between the core and island A. The JOS fold resembles the common substructure of MSA Type I and Type II dimeric filaments, with its core segment approximating the C-terminal body of MSA protofilaments B and its islands mimicking the N-terminal arm of MSA protofilaments A. The partial similarity of JOS and MSA folds extends to the locations of their cofactor-binding sites. In vitro assembly of recombinant wild-type α-synuclein, its insertion mutant and their mixture yielded structures that were distinct from those of JOS filaments. Our findings provide insight into a possible mechanism of JOS fibrillation in which mutant α-synuclein of 147 amino acids forms a nucleus with the JOS fold, around which wild-type and mutant proteins assemble during elongation.

miR-101a-3p Impairs Synaptic Plasticity and Contributes to Synucleinopathy.

BACKGROUND: Synucleinopathies are disorders characterized by the abnormal accumulation of α-synuclein (aSyn). Synaptic compromise is observed in synucleinopathies parallel to aSyn aggregation and is accompanied by transcript deregulation. OBJECTIVE: We sought to identify microRNAs associated with synaptic processes that may contribute to synaptic dysfunction and degeneration in synucleinopathies. METHODS: We performed small RNA-sequencing of midbrain from 6-month-old transgenic mice expressing A30P mutant aSyn, followed by comparative expression analysis. We then used real-time quantitative polymerase chain reaction (qPCR) for validation. Functional analysis was performed in primary neurons by biochemical assays and imaging. RESULTS: We found several deregulated biological processes linked to the synapse. miR-101a-3p was validated as a synaptic miRNA upregulated in aSyn Tg mice and in the cortex of dementia with Lewy bodies patients. Mice and primary cultured neurons overexpressing miR-101a-3p showed downregulation of postsynaptic proteins GABA Ab2 and SAPAP3 and altered dendritic morphology resembling synaptic plasticity impairments and/or synaptic damage. Interestingly, primary cultured neuron exposure to recombinant wild-type aSyn species efficiently increased miR-101a-3p levels. Finally, a dynamic role of miR-101a-3p in synapse plasticity was shown by identifying downregulation of miR-101a-3p in a condition of enhanced synaptic plasticity modelled in Wt animals housed in enriched environment. CONCLUSION: To conclude, we correlated pathologic aSyn with high levels of miR-101a-3p and a novel dynamic role of the miRNA in synaptic plasticity.

Mitochondrial function-associated genes underlie cortical atrophy in prodromal synucleinopathies.

Isolated rapid eye movement sleep behaviour disorder (iRBD) is a sleep disorder characterized by the loss of rapid eye movement sleep muscle atonia and the appearance of abnormal movements and vocalizations during rapid eye movement sleep. It is a strong marker of incipient synucleinopathy such as dementia with Lewy bodies and Parkinson's disease. Patients with iRBD already show brain changes that are reminiscent of manifest synucleinopathies including brain atrophy. However, the mechanisms underlying the development of this atrophy remain poorly understood. In this study, we performed cutting-edge imaging transcriptomics and comprehensive spatial mapping analyses in a multicentric cohort of 171 polysomnography-confirmed iRBD patients [67.7 ± 6.6 (49-87) years; 83% men] and 238 healthy controls [66.6 ± 7.9 (41-88) years; 77% men] with T1-weighted MRI to investigate the gene expression and connectivity patterns associated with changes in cortical thickness and surface area in iRBD. Partial least squares regression was performed to identify the gene expression patterns underlying cortical changes in iRBD. Gene set enrichment analysis and virtual histology were then done to assess the biological processes, cellular components, human disease gene terms, and cell types enriched in these gene expression patterns. We then used structural and functional neighbourhood analyses to assess whether the atrophy patterns in iRBD were constrained by the brain's structural and functional connectome. Moreover, we used comprehensive spatial mapping analyses to assess the specific neurotransmitter systems, functional networks, cytoarchitectonic classes, and cognitive brain systems associated with cortical changes in iRBD. All comparisons were tested against null models that preserved spatial autocorrelation between brain regions and compared to Alzheimer's disease to assess the specificity of findings to synucleinopathies. We found that genes involved in mitochondrial function and macroautophagy were the strongest contributors to the cortical thinning occurring in iRBD. Moreover, we demonstrated that cortical thinning was constrained by the brain's structural and functional connectome and that it mapped onto specific networks involved in motor and planning functions. In contrast with cortical thickness, changes in cortical surface area were related to distinct genes, namely genes involved in the inflammatory response, and to different spatial mapping patterns. The gene expression and connectivity patterns associated with iRBD were all distinct from those observed in Alzheimer's disease. In summary, this study demonstrates that the development of brain atrophy in synucleinopathies is constrained by specific genes and networks.

GBA1 Gene Mutations in α-Synucleinopathies-Molecular Mechanisms Underlying Pathology and Their Clinical Significance.

α-Synucleinopathies comprise a group of neurodegenerative diseases characterized by altered accumulation of a protein called α-synuclein inside neurons and glial cells. This aggregation leads to the formation of intraneuronal inclusions, Lewy bodies, that constitute the hallmark of α-synuclein pathology. The most prevalent α-synucleinopathies are Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). To date, only symptomatic treatment is available for these disorders, hence new approaches to their therapy are needed. It has been observed that GBA1 mutations are one of the most impactful risk factors for developing α-synucleinopathies such as PD and DLB. Mutations in the GBA1 gene, which encodes a lysosomal hydrolase ß-glucocerebrosidase (GCase), cause a reduction in GCase activity and impaired α-synuclein metabolism. The most abundant GBA1 gene mutations are N370S or N409S, L444P/L483P and E326K/E365K. The mechanisms by which GCase impacts α-synuclein aggregation are poorly understood and need to be further investigated. Here, we discuss some of the potential interactions between α-synuclein and GCase and show how GBA1 mutations may impact the course of the most prevalent α-synucleinopathies.

Striatal-Inoculation of α-Synuclein Preformed Fibrils Aggravated the Phenotypes of REM Sleep without Atonia in A53T BAC-SNCA Transgenic Mice.

Accumulation of α-synuclein (α-syn) is the pathological hallmark of α-synucleinopathy. Rapid eye movement (REM) sleep behavior disorder (RBD) is a pivotal manifestation of α-synucleinopathy including Parkinson's disease (PD). RBD is clinically confirmed by REM sleep without atonia (RWA) in polysomnography. To accurately characterize RWA preceding RBD and their underlying α-syn pathology, we inoculated α-syn preformed fibrils (PFFs) into the striatum of A53T human α-syn BAC transgenic (A53T BAC-SNCA Tg) mice which exhibit RBD-like phenotypes with RWA. RWA phenotypes were aggravated by PFFs-inoculation in A53T BAC-SNCA Tg mice at 1 month after inoculation, in which prominent α-syn pathology in the pedunculopontine nucleus (PPN) was observed. The intensity of RWA phenotype could be dependent on the severity of the underlying α-syn pathology.

Loss of tau expression attenuates neurodegeneration associated with α-synucleinopathy.

BACKGROUND: Neuronal dysfunction and degeneration linked to α-synuclein (αS) pathology is thought to be responsible for the progressive nature of Parkinson's disease and related dementia with Lewy bodies. Studies have indicated bidirectional pathological relationships between αS pathology and tau abnormalities. We recently showed that A53T mutant human αS (HuαS) can cause post-synaptic and cognitive deficits that require microtubule-associated protein tau expression. However, the role of tau in the development of αS pathology and subsequent neuronal dysfunction has been controversial. Herein, we set out to determine the role of tau in the onset and progression of αS pathology (α-synucleinopathy) using a transgenic mouse model of α-synucleinopathy lacking mouse tau expression. METHODS: Transgenic mice expressing A53T mutant HuαS (TgA53T) were crossed with mTau-/- mice to generate TgA53T/mTau-/-. To achieve more uniform induction of α-synucleinopathy in mice, we used intramuscular injections of αS preformed fibrils (PFF) in non-transgenic (nTg), TgA53T, TgA53T/mTau-/-, and mTau-/- mice. Motor behavior was analyzed at 70 days post inoculation (dpi) of PFF and tissues for biochemical and neuropathological analysis were collected at 40 dpi, 70 dpi, and end stage. RESULTS: Loss of tau expression significantly delayed the onset of motor deficits in the TgA53T model and the progression of α-synucleinopathy disease, as evidenced by a significant reduction in histopathological and behavioral markers of neurodegeneration and disease, and a significant improvement in survival. In vitro application of PFF to primary mouse hippocampal neurons demonstrated no changes in PFF uptake and processing or pS129 αS aggregation as a function of tau expression. However, PFF-induced neurotoxicity, including morphological deficits in nTg neurons, was prevented with tau removal. CONCLUSIONS: Collectively, our data suggest that tau is likely acting downstream of αS pathology to affect neuronal homeostasis and survival. This work further supports the investigation of tau in α-synucleinopathies to identify novel disease-modifying therapeutic strategies.

Modeling Parkinson's disease-related symptoms in alpha-synuclein overexpressing mice.

BACKGROUND: Intracellular deposition of alpha-synuclein (α-syn) as Lewy bodies and Lewy neurites is a central event in the pathogenesis of Parkinson's disease (PD) and other α-synucleinopathies. Transgenic mouse models overexpressing human α-syn, are useful research tools in preclinical studies of pathogenetic mechanisms. Such mice develop α-syn inclusions as well as neurodegeneration with a topographical distribution that varies depending on the choice of promoter and which form of α-syn that is overexpressed. Moreover, they display motor symptoms and cognitive disturbances that to some extent resemble the human conditions. PURPOSE: One of the main motives for assessing behavior in these mouse models is to evaluate the potential of new treatment strategies, including their impact on motor and cognitive symptoms. However, due to a high within-group variability with respect to such features, the behavioral studies need to be applied with caution. In this review, we discuss how to make appropriate choices in the experimental design and which tests that are most suitable for the evaluation of PD-related symptoms in such studies. METHODS: We have evaluated published results on two selected transgenic mouse models overexpressing wild type (L61) and mutated (A30P) α-syn in the context of their validity and utility for different types of behavioral studies. CONCLUSIONS: By applying appropriate behavioral tests, α-syn transgenic mouse models provide an appropriate experimental platform for studies of symptoms related to PD and other α-synucleinopathies.

Mutation Screening of TFG in α-Synucleinopathy and Amyotrophic Lateral Sclerosis.

BACKGROUND: Recently, p.R383H in TFG was identified as the disease cause in a family with α-synucleinopathy and amyotrophic lateral sclerosis (ALS). However, no further replication has been conducted in larger cohorts. OBJECTIVE: The aim was to explore the genetic role of TFG in α-synucleinopathy and ALS. METHODS: We analyzed the rare protein-coding variants in patients with Parkinson's disease (PD), ALS, multiple system atrophy (MSA), spastic paraplegia (N = 2709), and 7536 controls with whole-exome sequencing. RESULTS: Nine rare variants were identified in PD and two in MSA. One PD patient carried the same variant p.R383H. Similarly, this patient developed early-onset PD with bradykinesia and rigidity on the left side as the initial symptoms. However, at the gene level, rare variants of TFG were not enriched in patients. CONCLUSIONS: Rare variants of TFG were not enriched in α-synucleinopathy and ALS. However, we could not deny the potential pathogenicity of specific variants such as p.R383H. Further exploration is still necessary. © 2022 International Parkinson and Movement Disorder Society.

Brain atrophy in prodromal synucleinopathy is shaped by structural connectivity and gene expression.

Isolated REM sleep behaviour disorder (iRBD) is a synucleinopathy characterized by abnormal behaviours and vocalizations during REM sleep. Most iRBD patients develop dementia with Lewy bodies, Parkinson's disease or multiple system atrophy over time. Patients with iRBD exhibit brain atrophy patterns that are reminiscent of those observed in overt synucleinopathies. However, the mechanisms linking brain atrophy to the underlying alpha-synuclein pathophysiology are poorly understood. Our objective was to investigate how the prion-like and regional vulnerability hypotheses of alpha-synuclein might explain brain atrophy in iRBD. Using a multicentric cohort of 182 polysomnography-confirmed iRBD patients who underwent T1-weighted MRI, we performed vertex-based cortical surface and deformation-based morphometry analyses to quantify brain atrophy in patients (67.8 years, 84% male) and 261 healthy controls (66.2 years, 75%) and investigated the morphological correlates of motor and cognitive functioning in iRBD. Next, we applied the agent-based Susceptible-Infected-Removed model (i.e. a computational model that simulates in silico the spread of pathologic alpha-synuclein based on structural connectivity and gene expression) and tested if it recreated atrophy in iRBD by statistically comparing simulated regional brain atrophy to the atrophy observed in patients. The impact of SNCA and GBA gene expression and brain connectivity was then evaluated by comparing the model fit to the one obtained in null models where either gene expression or connectivity was randomized. The results showed that iRBD patients present with cortical thinning and tissue deformation, which correlated with motor and cognitive functioning. Next, we found that the computational model recreated cortical thinning (r = 0.51, P = 0.0007) and tissue deformation (r = 0.52, P = 0.0005) in patients, and that the connectome's architecture along with SNCA and GBA gene expression contributed to shaping atrophy in iRBD. We further demonstrated that the full agent-based model performed better than network measures or gene expression alone in recreating the atrophy pattern in iRBD. In summary, atrophy in iRBD is extensive, correlates with motor and cognitive function and can be recreated using the dynamics of agent-based modelling, structural connectivity and gene expression. These findings support the concepts that both prion-like spread and regional susceptibility account for the atrophy observed in prodromal synucleinopathies. Therefore, the agent-based Susceptible-Infected-Removed model may be a useful tool for testing hypotheses underlying neurodegenerative diseases and new therapies aimed at slowing or stopping the spread of alpha-synuclein pathology.

Unique seeding profiles and prion-like propagation of synucleinopathies are highly dependent on the host in human α-synuclein transgenic mice.

α-synuclein (αSyn) is an intrinsically disordered protein which can undergo structural transformations, resulting in the formation of stable, insoluble fibrils. αSyn amyloid-type nucleation can be induced by misfolded 'seeds' serving as a conformational template, tantamount to the prion-like mechanism. Accumulation of αSyn inclusions is a key feature of dementia with Lewy bodies (DLB) and multiple system atrophy (MSA), and are found as additional pathology in Alzheimer's disease (AD) such as AD with amygdala predominant Lewy bodies (AD/ALB). While these disorders accumulate the same pathological protein, they exhibit heterogeneity in clinical and histological features; however, the mechanism(s) underlying this variability remains elusive. Accruing data from human autopsy studies, animal inoculation modeling, and in vitro characterization experiments, have lent credence to the hypothesis that conformational polymorphism of the αSyn amyloid-type fibril structure results in distinct "strains" with categorical infectivity traits. Herein, we directly compare the seeding abilities and outcome of human brain lysates from these diseases, as well as recombinant preformed human αSyn fibrils by the intracerebral inoculation of transgenic mice overexpressing either human wild-type αSyn or human αSyn with the familial A53T mutation. Our study has revealed that the initiating inoculum heavily dictates the phenotypic and pathological course of disease. Interestingly, we have also established relevant host-dependent distinctions between propagation profiles, including burden and spread of inclusion pathology throughout the neuroaxis, as well as severity of neurological symptoms. These findings provide compelling evidence supporting the hypothesis that diverse prion-type conformers may explain the variability seen in synucleinopathies.

STING mediates neurodegeneration and neuroinflammation in nigrostriatal α-synucleinopathy.

In idiopathic Parkinson's disease (PD), pathologic αSyn aggregates drive oxidative and nitrative stress that may cause genomic and mitochondrial DNA damage. These events are associated with activation of the cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING) immune pathway, but it is not known whether STING is activated in or contributes to α-synucleinopathies. Herein, we used primary cell cultures and the intrastriatal αSyn preformed fibril (αSyn-PFF) mouse model of PD to demonstrate that αSyn pathology causes STING-dependent neuroinflammation and dopaminergic neurodegeneration. In microglia-astrocyte cultures, αSyn-PFFs induced DNA double-strand break (DSB) damage response signaling (γH2A.X), as well as TBK1 activation that was blocked by STING inhibition. In the αSyn-PFF mouse model, we similarly observed TBK1 activation and increased γH2A.X within striatal microglia prior to the onset of dopaminergic neurodegeneration. Using STING-deficient (Stinggt) mice, we demonstrated that striatal interferon activation in the α-Syn PFF model is STING-dependent. Furthermore, Stinggt mice were protected from α-Syn PFF-induced motor deficits, pathologic αSyn accumulation, and dopaminergic neuron loss. We also observed upregulation of STING protein in the substantia nigra pars compacta (SNpc) of human PD patients that correlated significantly with pathologic αSyn accumulation. STING was similarly upregulated in microglia cultures treated with αSyn-PFFs, which primed the pathway to mount stronger interferon responses when exposed to a STING agonist. Our results suggest that microglial STING activation contributes to both the neuroinflammation and neurodegeneration arising from α-synucleinopathies, including PD.

Spreading of Aggregated α-Synuclein in Sagittal Organotypic Mouse Brain Slices.

The accumulation of α-synuclein (α-syn) in the brain plays a role in synucleinopathies and it is hypothesized to spread in a prion-like fashion between connected brain regions. In the present study, we aim to investigate this spreading in well-characterized sagittal organotypic whole brain slices taken from postnatal wild type (WT) and transgenic mice overexpressing human α-syn under the promoter of proteolipid protein (PLP). Collagen hydrogels were loaded with monomers of human α-syn, as well as human and mouse pre-formed fibrils (PFFs), to allow local application and slow release. The spreading of α-syn was evaluated in different brain regions by immunohistochemistry for total α-syn and α-syn phosphorylated at the serine129 position (α-syn-P). The application of human and mouse PFFs of α-syn caused the aggregation and spreading of α-syn-P in the brain slices, which was pronounced the most at the region of hydrogel application and surrounding striatum, as well as along the median forebrain bundle. The organotypic slices from transgenic mice showed significantly more α-syn pathology than those from WT mice. The present study demonstrates that seeding with α-syn PFFs but not monomers induced intracellular α-syn pathology, which was significantly more prominent in brain slices with α-syn overexpression. This is consistent with the prion-like spreading theory of α-syn aggregates. The sagittal whole brain slices characterized in this study carry the potential to be used as a novel model to study α-syn pathology.