Α-syn Pathology Research Articles

Neuroprotection of low dose carbon monoxide in Parkinson’s disease models commensurate with the reduced risk of Parkinson’s among smokers

Paradoxically, cigarette smoking is associated with a reduced risk of Parkinson’s Disease (PD). This led us to hypothesize that carbon monoxide (CO) levels, which are constitutively but modestly elevated in smokers, might contribute to neuroprotection. Using rodent models of PD based on α-synuclein (αSyn) accumulation and oxidative stress, we show that low-dose CO mitigates neurodegeneration and reduces αSyn pathology. Oral CO administration activated signaling cascades mediated by heme oxygenase-1 (HO-1), which have been implicated in limiting oxidative stress, and in promoting αSyn degradation, thereby conferring neuroprotection. Consistent with the neuroprotective effect of smoking, HO-1 levels in cerebrospinal fluid were higher in human smokers compared to nonsmokers. Moreover, in PD brain samples, HO-1 levels were higher in neurons without αSyn pathology. Thus, CO in rodent PD models reduces pathology and increases oxidative stress responses, phenocopying possible protective effects of smoking evident in PD patients. These data highlight the potential for low-dose CO-modulated pathways to slow symptom onset and limit pathology in PD patients.

Motor Cortical Neuronal Hyperexcitability Associated with α-Synuclein Aggregation.

Dysfunction of the cerebral cortex is thought to underlie motor and cognitive impairments in Parkinson disease (PD). While cortical function is known to be suppressed by abnormal basal ganglia output following dopaminergic degeneration, it remains to be determined how the deposition of Lewy pathology disrupts cortical circuit integrity and function. Moreover, it is also unknown whether cortical Lewy pathology and midbrain dopaminergic degeneration interact to disrupt cortical function in late-stage. To begin to address these questions, we injected α-synuclein (αSyn) preformed fibrils (PFFs) into the dorsolateral striatum of mice to seed αSyn pathology in the cortical cortex and induce degeneration of midbrain dopaminergic neurons. Using this model system, we reported that αSyn aggregates accumulate in the motor cortex in a layer- and cell-subtype-specific pattern. Particularly, intratelencephalic neurons (ITNs) showed earlier accumulation and greater extent of αSyn aggregates relative to corticospinal neurons (CSNs). Moreover, we demonstrated that the intrinsic excitability and inputs resistance of αSyn aggregates-bearing ITNs in the secondary motor cortex (M2) are increased, along with a noticeable shrinkage of cell bodies and loss of dendritic spines. Last, neither the intrinsic excitability of CSNs nor their thalamocortical input was altered by a partial striatal dopamine depletion associated with αSyn pathology. Our results documented motor cortical neuronal hyperexcitability associated with αSyn aggregation and provided a novel mechanistic understanding of cortical circuit dysfunction in PD.

Alpha-synuclein fine-tunes neuronal response to pro-inflammatory cytokines

Pro-inflammatory cytokines are emerging as neuroinflammatory mediators in Parkinson’s disease (PD) due to their ability to act through neuronal cytokine receptors. Critical questions persist regarding the role of cytokines in neuronal dysfunction and their contribution to PD pathology. Specifically, the potential synergy of the hallmark PD protein alpha-synuclein (α-syn) with cytokines is of interest. We therefore investigated the direct impact of pro-inflammatory cytokines on neurons and hypothesized that α-syn pathology exacerbates cytokine-induced neuronal deficits in PD.iPSC-derived cortical neurons (CNs) from healthy controls and patients with α-syn gene locus duplication (SNCA dupl) were stimulated with IL-17A, TNF-α, IFN-γ, or a combination thereof. For rescue experiments, CNs were pre-treated with α-syn anti-oligomerisation compound NPT100-18A prior to IL-17A stimulation. Cytokine receptor expression, microtubule cytoskeleton, axonal transport and neuronal activity were assessed.SNCA dupl CNs displayed an increased IL-17A receptor expression and impaired IL-17A-mediated cytokine receptor regulation. Cytokines exacerbated the altered distribution of tubulin post-translational modifications in SNCA dupl neurites, with SNCA dupl-specific IL-17A effects. Tau pathology in SNCA dupl CNs was also aggravated by IL-17A and cytokine mix. Cytokines slowed down mitochondrial axonal transport, with IL-17A-mediated retrograde slowing in SNCA dupl only. The pre-treatment of SNCA dupl CNs with NPT100-18A prevented the IL-17A-induced functional impairments in axonal transport and neural activity.Our work elucidates the detrimental effects of pro-inflammatory cytokines, particularly IL-17A, on human neuronal structure and function in the context of α-syn pathology, suggesting that cytokine-mediated inflammation represents a second hit to neurons in PD which is amenable to disease modifying therapies that are currently in clinical trials.

Association of CSF α-Synuclein Seeding Amplification Assay Results With Clinical Features of Possible and Probable Dementia With Lewy Bodies.

The clinical diagnosis of dementia with Lewy bodies (DLB) depends on identifying significant cognitive decline accompanied by core features of parkinsonism, visual hallucinations, cognitive fluctuations, and REM sleep behavior disorder (RBD). Hyposmia is one of the several supportive features. α-Synuclein seeding amplification assays (αSyn-SAAs) may enhance diagnostic accuracy by detecting pathologic αSyn seeds in CSF. In this study, we examine how different clinical features associate with CSF αSyn-SAA positivity in a large group of clinically diagnosed participants with DLB. Cross-sectional and longitudinal CSF samples from the multicentered observational cohort study of the DLB Consortium and similar studies within the Parkinson's Disease Biomarker Program, contributed by academic medical centers in the United States, underwent αSyn-SAA testing. Participants included those clinically diagnosed with DLB and 2 control cohorts. Associations between core DLB features and olfaction with αSyn-SAA positivity were evaluated using logistic regression. CSF samples from 191 participants diagnosed with DLB (mean age 69.9 ± 6.8, 15% female), 50 age-matched and sex-matched clinical control participants, and 49 younger analytical control participants were analyzed. Seventy-two percent (137/191) of participants with DLB had positive αSyn-SAAs vs 4% of the control groups. Among participants with DLB, those who were αSyn-SAA-positive had lower Montreal Cognitive Assessment scores (18.8 ± 5.7 vs 21.2 ± 5.2, p = 0.01), had worse parkinsonism on the Movement Disorders Society Unified Parkinson's Disease Rating Scale part III (33.8 ± 15.1 vs 25.6 ± 16.4, p = 0.001), were more likely to report RBD (114/133 [86%] vs 33/53 [62%], p < 0.0001), and had worse hyposmia on the University of Pennsylvania Smell Identification Test (UPSIT) (94/105 [90%] below 15th percentile vs 14/44 [32%], p < 0.0001). UPSIT percentile had the highest area under the curve (0.87, 95% CI 0.81-0.94) in predicting αSyn-SAA positivity and participants scoring at or below the 15th percentile of age and sex normative values had 18.3 times higher odds (95% CI 7.52-44.6) of having a positive αSyn-SAA test. Among 82 participants with longitudinal CSF samples, 81 (99%) had the same αSyn-SAA result for initial and follow-up specimens. A substantial proportion of clinically diagnosed participants with DLB had negative αSyn-SAA results. Hyposmia was the strongest clinical predictor of αSyn-SAA positivity. Hyposmia and αSyn-SAA may have utility in improving the diagnostic assessment of individuals with potential DLB. This study provided Class III evidence that CSF αSyn-SAA distinguishes patients with clinically diagnosed DLB from normal controls.

Discovery and Evaluation of Imidazo[2,1-b][1,3,4]thiadiazole Derivatives as New Candidates for α-Synuclein PET Imaging.

α-synuclein (α-syn) pathologies are central to the development of synucleinopathies including Parkinson's disease (PD). Positron emission tomography (PET) imaging of α-syn pathologies is one strategy to facilitate the diagnosis, understanding, and treatment of synucleinopathies, but has been restricted by the lack of specific α-syn PET probes. In this work, we identified 2,6-disubstituted imidazo[2,1-b][1,3,4]thiadiazole (ITA) as a new α-syn-binding scaffold. Through autoradiography studies, we discovered an iodinated lead compound [125I]ITA-3, with moderate binding affinity (IC50 = 55 nM) to α-syn pathologies in human PD brain sections. Modified from [125I]ITA-3, we developed a potential PET tracer, [18F]FITA-2 (radiochemical yield >25%, molar activity >110 GBq/μmol), which demonstrated clear signals in α-syn-rich regions in human PD brain tissues (IC50 = 245 nM), good brain uptake (SUVpeak = 2.80 ± 0.45), and fast clearance rate in rats. Overall, [18F]FITA-2 appears to be a promising candidate for α-syn PET imaging and merits further development.

MRI Signature of α-Synuclein Pathology in Asymptomatic Stages and a Memory Clinic Population

The lack of an in vivo measure for α-synuclein (α-syn) pathology until recently has limited thorough characterization of its brain atrophy pattern, especially during early disease stages. To assess the association of state-of-the-art cerebrospinal fluid (CSF) seed amplification assays (SAA) α-syn positivity (SAA α-syn+) with magnetic resonance imaging (MRI) structural measures, across the continuum from clinically unimpaired (CU) to cognitively impaired (CI) individuals, in 3 independent cohorts, and separately in CU and CI individuals, the latter reflecting a memory clinic population. Cross-sectional data were used from the Swedish BioFINDER-2 study (inclusion, 2017-2023) as the discovery cohort and the Swedish BioFINDER-1 study (inclusion, 2007-2015) and Alzheimer's Disease Neuroimaging Initiative (ADNI; inclusion 2005-2022) as replication cohorts. All cohorts are from multicenter studies, but the BioFINDER cohorts used 1 MRI scanner. CU and CI individuals fulfilling inclusion criteria and without missing data points in relevant metrics were included in the study. All analyses were performed from 2023 to 2024. Presence of α-syn pathology, estimated by baseline CSF SAA α-syn. The primary outcomes were cross-sectional structural MRI measures either through voxel-based morphometry (VBM) or regions of interest (ROI) including an automated pipeline for cholinergic basal forebrain nuclei CH4/4p (nucleus basalis of Meynert [NBM]) and CH1/2/3. Secondary outcomes were domain-specific cross-sectional cognitive measures. Analyses were adjusted for CSF biomarkers of Alzheimer pathology. A total of 2961 participants were included in this study: 1388 (mean [SD] age, 71 [10] years; 702 female [51%]) from the BioFINDER-2 study, 752 (mean [SD] age, 72 [6] years; 406 female [54%]) from the BioFINDER-1 study, and 821 (mean [SD] age, 75 [8] years; 449 male [55%]) from ADNI. In the BioFINDER-2 study, VBM analyses in the whole cohort revealed a specific association between SAA α-syn+ and the cholinergic NBM, even when adjusting for Alzheimer copathology. ROI-based analyses in the BioFINDER-2 study focused on regions involved in the cholinergic system and confirmed that SAA α-syn+ was indeed independently associated with smaller NBM (β = -0.271; 95% CI, -0.399 to -0.142; P <.001) and CH1/2/3 volumes (β = -0.227; 95% CI, -0.377 to -0.076; P =.02). SAA α-syn+ was also independently associated with smaller NBM volumes in the separate CU (β = -0.360; 95% CI, -0.603 to -0.117; P =.03) and CI (β = -0.251; 95% CI, -0.408 to -0.095; P =.02) groups. Overall, the association between SAA α-syn+ and NBM volume was replicated in the BioFINDER-1 study and ADNI cohort. In CI individuals, NBM volumes partially mediated the association of SAA α-syn+ with attention/executive impairments in all cohorts (BioFINDER-2, β = -0.017; proportion-mediated effect, 7%; P =.04; BioFINDER-1, β = -0.096; proportion-mediated effect, 19%; P =.04; ADNI, β = -0.061; proportion-mediated effect, 20%; P =.007). In this cohort study, SAA α-syn+ was consistently associated with NBM atrophy already during asymptomatic stages. Further, in memory clinic CI populations, SAA α-syn+ was associated with NBM atrophy, which partially mediated α-syn-induced attention/executive impairment.

Gba1 E326K renders motor and non-motor symptoms with pathologic α-syn, tau and glial activation.

Mutations in the GBA1 gene are common genetic risk factors for Parkinson's disease (PD), disrupting enzymatic activity and causing lysosomal dysfunction, leading to elevated α-synuclein (α-syn) levels. While GBA1's role in synucleinopathy is well-established, recent research underscores neuroinflammation as a significant pathogenic mechanism in GBA1 deficiency. This study investigates neuroinflammation in Gba1 E326K knock-in mice, a model associated with increased PD and dementia risk. At 9 and 24 months, we assessed GBA1 protein and activity, α-synuclein pathology, neurodegeneration, motor deficits, and gliosis in the ventral midbrain and hippocampus using immunohistochemistry (IHC), Western blot (WB), and GCase assays. Additionally, primary microglia from WT and GBA1E326K/E326K mice were treated with α-syn preformed fibrils (PFF) to study microglia activation, pro-inflammatory cytokines, reactive astrocyte formation, and neuronal death through qPCR, WB, and immunocytochemistry analyses. We also evaluated the effects of gut inoculation of α-syn PFF in Gba1 E326K mice at 7 months and striatal inoculation at 10 months, assessing motor/non-motor symptoms, α-syn pathology, neuroinflammation, gliosis, and neurodegeneration via behavioural tests, IHC, and WB assays. At 24 months, Gba1 E326K knock-in mice showed reduced GCase enzymatic activity and glucosylceramide build-up in the ventral midbrain and hippocampus. Increased pro-inflammatory cytokines and reactive astrocytes were observed in microglia and astrocytes from Gba1 E326K mice treated with pathologic α-syn PFF. Gut inoculation of α-syn PFF increased Lewy body accumulation in the hippocampal dentate gyrus, with heightened microglia and astrocyte activation and worsened non-motor symptoms. Intrastriatal α-syn preformed fibril injection induced motor deficits, reactive glial protein accumulation, and tauopathy in the prefrontal cortex and hippocampus of Gba1 E326K mice. GBA1 deficiency due to the Gba1 E326K mutation exacerbates neuroinflammation and promotes pathogenic α-synuclein transmission, intensifying disease pathology in PD models. This study enhances our understanding of how the Gba1 E326K mutation contributes to neuroinflammation and the spread of pathogenic α-syn in the brain, suggesting new therapeutic strategies for PD and related synucleinopathies.

The Pathobiology of Behavioral Changes in Multiple System Atrophy: An Update.

While cognitive impairment, which was previously considered a red flag against the clinical diagnosis of multiple system atrophy (MSA), is a common symptom of this rare neurodegenerative disorder, behavioral disorders are reported in 30 to 70% of MSA patients. They include anxiety, apathy, impaired attention, compulsive and REM sleep behavior disorders (RBD), and these conditions, like depression, are early and pervasive features in MSA, which may contribute to disease progression. Despite changing concepts of behavioral changes in this synucleinopathy, the underlying pathophysiological and biochemical mechanisms are poorly understood. While specific neuropathological data are unavailable, neuroimaging studies related anxiety disorders to changes in the cortico-limbic system, apathy (and depression) to dysfunction of prefrontal-subcortical circuits, and compulsive behaviors to impairment of basal ganglia networks and involvement of orbito-frontal circuits. Anxiety has also been related to α-synuclein (αSyn) pathology in the amygdala, RBD to striatal monoaminergic deficit, and compulsive behavior in response to dopamine agonist therapy in MSA, while the basic mechanisms of the other behavioral disorders and their relations to other non-motor dysfunctions in MSA are unknown. In view of the scarcity of functional and biochemical findings in MSA with behavioral symptoms, further neuroimaging and biochemical studies are warranted in order to obtain better insight into their pathogenesis as a basis for the development of diagnostic biomarkers and future adequate treatment modalities of these debilitating comorbidities.

Excitatory synaptic structural abnormalities produced by templated aggregation of α-syn in the basolateral amygdala

Parkinson's disease (PD) and Dementia with Lewy bodies (DLB) are characterized by neuronal α-synuclein (α-syn) inclusions termed Lewy Pathology, which are abundant in the amygdala. The basolateral amygdala (BLA), in particular, receives projections from the thalamus and cortex. These projections play a role in cognition and emotional processing, behaviors which are impaired in α-synucleinopathies. To understand if and how pathologic α-syn impacts the BLA requires animal models of α-syn aggregation. Injection of α-syn pre-formed fibrils (PFFs) into the striatum induces robust α-syn aggregation in excitatory neurons in the BLA that corresponds with reduced contextual fear conditioning. At early time points after aggregate formation, cortico-amygdala excitatory transmission is abolished. The goal of this project was to determine if α-syn inclusions in the BLA induce synaptic degeneration and/or morphological changes. In this study, we used C57BL/6 J mice injected bilaterally with PFFs in the dorsal striatum to induce α-syn aggregate formation in the BLA. A method was developed using immunofluorescence and three-dimensional reconstruction to analyze excitatory cortico-amygdala and thalamo-amygdala presynaptic terminals closely juxtaposed to postsynaptic densities. The abundance and morphology of synapses were analyzed at 6- or 12-weeks post-injection of PFFs. α-Syn aggregate formation in the BLA did not cause a significant loss of synapses, but cortico-amygdala and thalamo-amygdala presynaptic terminals and postsynaptic densities with aggregates of α-syn show increased volumes, similar to previous findings in human DLB cortex, and in non-human primate models of PD. Transmission electron microscopy showed that asymmetric synapses in mice with PFF-induced α-syn aggregates have reduced synaptic vesicle intervesicular distances, similar to a recent study showing phospho-serine-129 α-syn increases synaptic vesicle clustering. Thus, pathologic α-syn causes major alterations to synaptic architecture in the BLA, potentially contributing to behavioral impairment and amygdala dysfunction observed in synucleinopathies.

Evaluation of the Effect of β-Wrapin AS69 in a Mouse Model Based on Alpha-Synuclein Overexpression.

Aggregation of the protein α-Synuclein (αSyn) is a hallmark of Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple systems atrophy, and alleviating the extent of αSyn pathology is an attractive strategy against neurodegeneration. The engineered binding protein β-wrapin AS69 binds monomeric αSyn. AS69 reduces primary and secondary nucleation as well as fibril elongation in vitro. It also mitigates aSyn pathology in a mouse model based on intrastriatal injection of aSyn pre-formed fibrils (PFFs). Since the PFF-based model does not represent all aspects of PD, we tested here whether AS69 can reduce neurodegeneration resulting from αSyn overexpression. Human A53T-αSyn was overexpressed in the mouse Substantia nigra (SN) by using recombinant adeno-associated viral vector (rAAV). AS69 was also expressed by rAAV transduction. Behavioral tests and immunofluorescence staining were used as outcomes. Transduction with rAAV-αSyn resulted in αSyn pathology as reported by phospho-αSyn staining and caused degeneration of dopaminergic neurons in the SN. The co-expression of rAAV-AS69 did not reduce αSyn pathology or the degeneration of dopaminergic neurons. We conclude that αSyn monomer binding by rAAV-AS69 was insufficient to protect from aSyn pathology resulting from αSyn overexpression.

Target engagement and immunogenicity of an active immunotherapeutic targeting pathological α-synuclein: a phase 1 placebo-controlled trial

Investigational therapeutics that target toxic species of α-synuclein (αSyn) aim to slow down or halt disease progression in patients with Parkinson’s disease (PD). Here this 44-week, randomized, placebo-controlled, double-blind, single-center phase 1 study investigated safety, tolerability and immunogenicity of UB-312, an active immunotherapeutic targeting pathological αSyn, in patients with PD. The primary outcome measures were adverse event frequency and change in anti-αSyn antibody titers in blood and cerebrospinal fluid (CSF). Exploratory outcomes were changes in clinical scales and biomarker-based target engagement as measured by seed amplification assays. Twenty patients were randomized 7:3 (UB-312:placebo) into 300/100/100 μg or 300/300/300 μg (weeks 1, 5 and 13) intramuscular prime-boost dose groups. Safety was similar across groups; adverse events were mostly mild and transient. Two patients experienced three serious adverse events in total, one possibly treatment related; all resolved without sequalae. Anti-αSyn antibodies in serum from 12/13 and CSF from 5/13 patients who received three UB-312 doses confirmed immunogenicity. Mean serum titers (in log-dilution factor) increased from baseline by 1.398 and 1.354, and peaked at week 29 at 2.520 and 2.133, for 300/100/100 μg and 300/300/300 μg, respectively. CSF titers were 0 at baseline and were 0.182 and 0.032 at week 21, respectively. Exploratory analyses showed no statistical differences in clinical scales but a significant reduction of αSyn seeds in CSF of a subset of UB-312-treated patients. These data support further UB-312 development. ClinicalTrials.gov:NCT04075318.

Selection of DNA aptamers that prevent the fibrillization of α-synuclein protein in cellular and mouse models

A neuropathological hallmark of Parkinson's Disease (PD) is the aggregation and spreading of misfolded α-synuclein (αSyn) protein. In this study, a selection method was developed to identify aptamers that showed affinity for monomeric αSyn and inhibition of αSyn aggregation. Aptamer a-syn-1 exhibited strong inhibition of αSyn aggregation in vitro by transmission electron microscopy and thioflavin T fluorescence. A-syn-1-treated SH-SY5Y cells incubated with pre-formed fibrils (PFFs) showed less intracellular aggregation of αSyn in comparison to a scrambled oligonucleotide control, as observed with fluorescent microscopy. Systemic delivery of a-syn-1 to the brain was achieved using a liposome vehicle and confirmed with fluorescence microscopy and qPCR. Transgenic mice overexpressing the human A53T variant of αSyn protein were injected with a-syn-1 loaded liposomes at 5 months of age both acutely (single i.p injection) and repeatedly (5 i.p. injections over 5 days). Western blot protein quantification revealed that both acute and repeated injections of a-syn-1 decreased levels of the aggregated form of αSyn in the transgenic mice in the prefrontal cortex, caudate, and substania nigra (SNc). These results provide in vitro and in vivo evidence that a-syn-1 can inhibit pathological αSyn aggregation and may have implications in treatment strategies to target dysregulation in PD.

A multiverse of α-synuclein: investigation of prion strain properties with carboxyl-terminal truncation specific antibodies in animal models

Synucleinopathies are a group of neurodegenerative disorders characterized by the presence of misfolded α-Synuclein (αSyn) in the brain. These conditions manifest with diverse clinical and pathophysiological characteristics. This disease diversity is hypothesized to be driven by αSyn strains with differing biophysical properties, potentially influencing prion-type propagation and consequentially the progression of illness. Previously, we investigated this hypothesis by injecting brain lysate (seeds) from deceased individuals with various synucleinopathies or human recombinant αSyn preformed fibrils (PFFs) into transgenic mice overexpressing either wild type or A53T human αSyn. In the studies herein, we expanded on these experiments, utilizing a panel of antibodies specific for the major carboxyl-terminally truncated forms of αSyn (αSynΔC). These modified forms of αSyn are found enriched in human disease brains to inform on potential strain-specific proteolytic patterns. With monoclonal antibodies specific for human αSyn cleaved at residues 103, 114, 122, 125, and 129, we demonstrate that multiple system atrophy (MSA) seeds and PFFs induce differing neuroanatomical spread of αSyn pathology associated with host specific profiles. Overall, αSyn cleaved at residue 103 was most widely present in the induced pathological inclusions. Furthermore, αSynΔC-positive inclusions were present in astrocytes, but more frequently in activated microglia, with patterns dependent on host and inoculum. These findings support the hypothesis that synucleinopathy heterogeneity might stem from αSyn strains with unique biochemical properties that include proteolytic processing, which could result in dominant strain properties.

Computer-Based Evaluation of α-Synuclein Pathology in Multiple System Atrophy as a Novel Tool to Recognize Disease Subtypes

Multiple system atrophy (MSA) is a neurodegenerative disorder with variable disease course and distinct constellations of clinical (cerebellar [MSA-C] or parkinsonism [MSA-P]) and pathological phenotypes, suggestive of distinct α-synuclein (αSyn) strains. Neuropathologically, MSA is characterized by the accumulation of αSyn in oligodendrocytic glial cytoplasmic inclusions (GCI). Using a novel computer-based method, this study quantified the size of GCIs, density of all αSyn pathology, density of only the GCIs, and number of GCIs in MSA cases (n = 20). The putamen and cerebellar white matter were immunostained with the disease-associated 5G4 anti-αSyn antibody. Following digital scanning and image processing, total 5G4-immunoreactive pathology (ie, neuronal, neuritic, and glial) and GCIs were optically dissected for inclusion size and density measurement and then evaluated applying a novel computer-based method using ImageJ. GCI size varied between cases and brain regions (P < .0001), and heterogeneity in the density of all αSyn pathology including the density and number of GCIs were observed between regions and across cases, where MSA-C cases had a significantly higher density of all αSyn pathology in the cerebellar white matter (P = .049). Some region-specific morphologic variables inversely correlated with the age of onset and death, suggestive of an underlying aging-related cellular mechanism. Unsupervised K-means cluster analysis classified MSA cases into 3 distinct groups based on region-specific morphologic variables. In conclusion, we developed a novel computer-based method that is easily accessible, providing a first step to developing artificial intelligence–based evaluation strategies for large scale comparative studies. Our observations on the variability of morphologic variables between brain regions and cases highlight (1) the importance of computer-based approaches to detect features not considered in the routine diagnostic practice, and (2) novel aspects for the identification of previously unrecognized MSA subtypes that do not necessarily reflect the current clinical classification of MSA-C or MSA-P.

Ciita Regulates Local and Systemic Immune Responses in a Combined rAAV-α-synuclein and Preformed Fibril-Induced Rat Model for Parkinson's Disease.

Parkinson's disease (PD) is characterized by alpha-synuclein (α-Syn) pathology, neurodegeneration and neuroinflammation. Human leukocyte antigen (HLA) variants associated with PD and α-Syn specific CD4+ T lymphocytes in PD patients highlight the importance of antigen presentation in PD etiology. The class II transactivator (CIITA) regulates major histocompatibility complex class II (MHCII) expression. Reduced Ciita levels significantly increase α-Syn pathology, nigrostriatal neurodegeneration and behavioral deficits in α-Syn-induced rat PD models. Characterize immune profiles associated with enhanced PD-like pathology observed in rats expressing lower Ciita levels (DA.VRA4) compared to the background strain (DA). To model PD, we combined rAAV-mediated α-Syn overexpression in the substantia nigra with striatal injection of α-Syn preformed fibrils. Immune profiles in brain and blood were analyzed by flow cytometry and multiplexed ELISA in naïve rats, 4- and 8 weeks post rAAV injection. Flow cytometry showed Ciita-dependent regulation of MHCII on microglia, brain macrophages and circulating myeloid cells. The MHCII-dependent microglial response was highest at 4 weeks post rAAV injection, whereas the MHCII levels in circulating myeloid cells was highest at 8 weeks. There was no major infiltration of macrophages or T lymphocytes into the CNS in response to α-Syn and only subtle Ciita- and/or α-Syn-dependent changes in the T lymphocyte compartment. Lower Ciita levels were consistently associated with higher TNF levels in serum. Ciita regulates susceptibility to PD-like pathology through minor but detectable changes in resident and peripheral immune cells and TNF levels, indicating that mild immunomodulatory therapies could have therapeutic effects in PD.

The immune system in Parkinson's disease: what we know so far.

Parkinson's disease is characterized neuropathologically by the degeneration of dopaminergic neurons in the ventral midbrain, the accumulation of α-synuclein (α-syn) aggregates in neurons and chronic neuroinflammation. In the past two decades, in vitro, ex vivo and in vivo studies have consistently shown the involvement of inflammatory responses mediated by microglia and astrocytes, which may be elicited by pathological α-syn or signals from affected neurons and other cell types, and are directly linked to neurodegeneration and disease development. Apart from the prominent immune alterations seen in the CNS, including the infiltration of T cells into the brain, more recent studies have demonstrated important changes in the peripheral immune profile within both the innate and adaptive compartments, particularly involving monocytes, CD4+ and CD8+ T cells. This review aims to integrate the consolidated understanding of immune-related processes underlying the pathogenesis of Parkinson's disease, focusing on both central and peripheral immune cells, neuron-glia crosstalk as well as the central-peripheral immune interaction during the development of Parkinson's disease. Our analysis seeks to provide a comprehensive view of the emerging knowledge of the mechanisms of immunity in Parkinson's disease and the implications of this for better understanding the overall pathogenesis of this disease.

SNCA and TPPP transcripts increase in oligodendroglial cytoplasmic inclusions in multiple system atrophy

Multiple system atrophy (MSA) is characterized by glial cytoplasmic inclusions (GCIs) containing aggregated α-synuclein (α-syn) in oligodendrocytes. The origin of α-syn accumulation in GCIs is unclear, in particular whether abnormal α-syn aggregates result from the abnormal elevation of endogenous α-syn expression in MSA or ingested from the neuronal source. Tubulin polymerization promoting protein (TPPP) has been reported to play a crucial role in developing GCI pathology. Here, the total cell body, nucleus, and cytoplasmic area density of SNCA and TPPP transcripts in neurons and oligodendrocytes with and without various α-syn pathologies in the pontine base in autopsy cases of MSA (n = 4) and controls (n = 2) were evaluated using RNAscope with immunofluorescence. Single-nucleus RNA-sequencing data for TPPP was evaluated using control frontal cortex (n = 3). SNCA and TPPP transcripts were present in the nucleus and cytoplasm of oligodendrocytes in both controls and diseased, with higher area density in GCIs and glial nuclear inclusions in MSA. Area densities of SNCA and TPPP transcripts were lower in neurons showing cytoplasmic inclusions in MSA. Indeed, TPPP transcripts were unexpectedly found in neurons, while the anti-TPPP antibody failed to detect immunoreactivity. Single-nucleus RNA-sequencing revealed significant TPPP transcript expression predominantly in oligodendrocytes, but also in excitatory and inhibitory neurons. This study addressed the unclear origin of accumulated α-syn in GCIs, proposing that the elevation of SNCA transcripts may supply templates for misfolded α-syn. In addition, the parallel behavior of TPPP and SNCA transcripts in GCI development highlights their potential synergistic contribution to inclusion formation. In conclusion, this study advances our understanding of MSA pathogenesis, offers insights into the dynamics of SNCA and TPPP transcripts in inclusion formation, and proposes regulating their transcripts for future molecular therapy to MSA.

Alpha-synuclein aggregates are phosphatase resistant

Alpha-synuclein (αsyn) is an intrinsically disordered protein that aggregates in the brain in several neurodegenerative diseases collectively called synucleinopathies. Phosphorylation of αsyn at serine 129 (PSER129) was considered rare in the healthy human brain but is enriched in pathological αsyn aggregates and is used as a specific marker for disease inclusions. However, recent observations challenge this assumption by demonstrating that PSER129 results from neuronal activity and can be readily detected in the non-diseased mammalian brain. Here, we investigated experimental conditions under which two distinct PSER129 pools, namely endogenous-PSER129 and aggregated-PSER129, could be detected and differentiated in the mammalian brain. Results showed that in the wild-type (WT) mouse brain, perfusion fixation conditions greatly influenced the detection of endogenous-PSER129, with endogenous-PSER129 being nearly undetectable after delayed perfusion fixation (30-min and 1-h postmortem interval). Exposure to anesthetics (e.g., Ketamine or xylazine) before perfusion did not significantly influence endogenous-PSER129 detection or levels. In situ, non-specific phosphatase calf alkaline phosphatase (CIAP) selectively dephosphorylated endogenous-PSER129 while αsyn preformed fibril (PFF)-seeded aggregates and genuine disease aggregates (Lewy pathology and Papp–Lantos bodies in Parkinson’s disease and multiple systems atrophy brain, respectively) were resistant to CIAP-mediated dephosphorylation. The phosphatase resistance of aggregates was abolished by sample denaturation, and CIAP-resistant PSER129 was closely associated with proteinase K (PK)-resistant αsyn (i.e., a marker of aggregation). CIAP pretreatment allowed for highly specific detection of seeded αsyn aggregates in a mouse model that accumulates non-aggregated-PSER129. We conclude that αsyn aggregates are impervious to phosphatases, and CIAP pretreatment increases detection specificity for aggregated-PSER129, particularly in well-preserved biological samples (e.g., perfusion fixed or flash-frozen mammalian tissues) where there is a high probability of interference from endogenous-PSER129. Our findings have important implications for the mechanism of PSER129-accumulation in the synucleinopathy brain and provide a simple experimental method to differentiate endogenous-from aggregated PSER129.

Aplp1 interacts with Lag3 to facilitate transmission of pathologic α-synuclein

Pathologic α-synuclein (α-syn) spreads from cell-to-cell, in part, through binding to the lymphocyte-activation gene 3 (Lag3). Here we report that amyloid β precursor-like protein 1 (Aplp1) interacts with Lag3 that facilitates the binding, internalization, transmission, and toxicity of pathologic α-syn. Deletion of both Aplp1 and Lag3 eliminates the loss of dopaminergic neurons and the accompanying behavioral deficits induced by α-syn preformed fibrils (PFF). Anti-Lag3 prevents the internalization of α-syn PFF by disrupting the interaction of Aplp1 and Lag3, and blocks the neurodegeneration induced by α-syn PFF in vivo. The identification of Aplp1 and the interplay with Lag3 for α-syn PFF induced pathology deepens our insight about molecular mechanisms of cell-to-cell transmission of pathologic α-syn and provides additional targets for therapeutic strategies aimed at preventing neurodegeneration in Parkinson’s disease and related α-synucleinopathies.

Advancing Parkinson's Disease Diagnostics: The Potential of Arylpyrazolethiazole Derivatives for Imaging α-Synuclein Aggregates.

The development of positron emission tomography (PET) tracers capable of detecting α-synuclein (α-syn) aggregates in vivo would represent a breakthrough for advancing the understanding and enabling the early diagnosis of Parkinson's disease and related disorders. It also holds the potential to assess the efficacy of therapeutic interventions. However, this remains challenging due to different structures of α-syn aggregates, the need for selectivity over other structurally similar amyloid proteins, like amyloid-β (Aβ), which frequently coexist with α-syn pathology, and the low abundance of the target in the brain that requires the development of a high-affinity ligand. To develop a successful PET tracer for the central nervous system (CNS), stringent criteria in terms of polarity and molecular size must also be considered, as the tracer must penetrate the blood-brain barrier and have low nonspecific binding to brain tissue. Here, we report a series of arylpyrazolethiazole (APT) derivatives, rationally designed from a structure-activity relationship study centered on existing ligands for α-syn fibrils, with a particular focus on the selectivity toward α-syn fibrils and control of physicochemical properties suitable for a CNS PET tracer. In vitro competition binding assays performed against [3H]MODAG-001 using recombinant α-syn and Aβ1-42 fibrils revealed APT-13 with an inhibition constant of 27.8 ± 9.7 nM and a selectivity of more than 3.3 fold over Aβ. Radiolabeled [11C]APT-13 demonstrated excellent brain penetration in healthy mice with a peak standardized uptake value of 1.94 ± 0.29 and fast washout from the brain (t 1/2 = 9 ± 1 min). This study highlights the potential of APT-13 as a lead compound for developing PET tracers to detect α-syn aggregates in vivo.