Current insights and assumptions on α-synuclein in Lewy body disease.

Lewy body disorders are heterogeneous neurological conditions defined by intracellular inclusions composed of misshapen α-synuclein protein aggregates. Although α-synuclein aggregates are only one component of inclusions and not strictly coupled to neurodegeneration, evidence suggests they seed the propagation of Lewy pathology within and across cells. Genetic mutations, genomic multiplications, and sequence polymorphisms of the gene encoding α-synuclein are also causally linked to Lewy body disease. In nonfamilial cases of Lewy body disease, the disease trigger remains unidentified but may range from industrial/agricultural toxicants and natural sources of poisons to microbial pathogens. Perhaps due to these peripheral exposures, Lewy inclusions appear at early disease stages in brain regions connected with cranial nerves I and X, which interface with inhaled and ingested environmental elements in the nasal or gastrointestinal cavities. Irrespective of its identity, a stealthy disease trigger most likely shifts soluble α-synuclein (directly or indirectly) into insoluble, cross-ß-sheet aggregates. Indeed, ß-sheet-rich self-replicating α-synuclein multimers reside in patient plasma, cerebrospinal fluid, and other tissues, and can be subjected to α-synuclein seed amplification assays. Thus, clinicians should be able to capitalize on α-synuclein seed amplification assays to stratify patients into potential responders versus non-responders in future clinical trials of α-synuclein targeted therapies. Here, we briefly review the current understanding of α-synuclein in Lewy body disease and speculate on pathophysiological processes underlying the potential transmission of α-synucleinopathy across the neuraxis.

Rapid iPSC inclusionopathy models shed light on formation, consequence, and molecular subtype of α-synuclein inclusions.

The heterogeneity of protein-rich inclusions and its significance in neurodegeneration is poorly understood. Standard patient-derived iPSC models develop inclusions neither reproducibly nor in a reasonable time frame. Here, we developed screenable iPSC "inclusionopathy" models utilizing piggyBac or targeted transgenes to rapidly induce CNS cells that express aggregation-prone proteins at brain-like levels. Inclusions and their effects on cell survival were trackable at single-inclusion resolution. Exemplar cortical neuron α-synuclein inclusionopathy models were engineered through transgenic expression of α-synuclein mutant forms or exogenous seeding with fibrils. We identified multiple inclusion classes, including neuroprotective p62-positive inclusions versus dynamic and neurotoxic lipid-rich inclusions, both identified in patient brains. Fusion events between these inclusion subtypes altered neuronal survival. Proteome-scale α-synuclein genetic- and physical-interaction screens pinpointed candidate RNA-processing and actin-cytoskeleton-modulator proteins like RhoA whose sequestration into inclusions could enhance toxicity. These tractable CNS models should prove useful in functional genomic analysis and drug development for proteinopathies.

Are Preformed Fibrils a Model of Parkinson's Disease?

 Pre-formed fibrils (PFFs) made from recombinant α-synuclein are broadly used throughout the field in cellular and animal models of Parkinson's disease. However, their ability to successfully recapitulate disease biology is a controversial topic. In this article, two researchers debate this issue with Amanda Woerman taking the view that PFFs are a model of synucleinopathy but not Parkinson's disease, while Kelvin Luk defends their use as an important tool in the field.

Parkinson's disease patients develop accumulations of misfolded proteins in the brain called Lewy bodies. An important protein found in Lewy bodies is α-synuclein, which aggregates into large fibrils that are thought to have toxic effects on neurons. To study the effect of α-synuclein fibrils, many scientists use pre-formed fibrils (PFFs), which are α-synuclein fibrils made in the lab under a variety of conditions. In this article, two researchers will debate the use of PFFs as a model of Parkinson's disease. Amanda Woerman discusses the data suggesting why PFFs are an inadequate tool for understanding Parkinson's disease. Kelvin Luk discusses the data supporting the use of PFFs as a Parkinson's disease model.

Positron Emission Tomography with [18F]ROStrace Reveals Progressive Elevations in Oxidative Stress in a Mouse Model of Alpha-Synucleinopathy.

The synucleinopathies are a diverse group of neurodegenerative disorders characterized by the accumulation of aggregated alpha-synuclein (aSyn) in vulnerable populations of brain cells. Oxidative stress is both a cause and a consequence of aSyn aggregation in the synucleinopathies; however, noninvasive methods for detecting oxidative stress in living animals have proven elusive. In this study, we used the reactive oxygen species (ROS)-sensitive positron emission tomography (PET) radiotracer [18F]ROStrace to detect increases in oxidative stress in the widely-used A53T mouse model of synucleinopathy. A53T-specific elevations in [18F]ROStrace signal emerged at a relatively early age (6-8 months) and became more widespread within the brain over time, a pattern which paralleled the progressive development of aSyn pathology and oxidative damage in A53T brain tissue. Systemic administration of lipopolysaccharide (LPS) also caused rapid and long-lasting elevations in [18F]ROStrace signal in A53T mice, suggesting that chronic, aSyn-associated oxidative stress may render these animals more vulnerable to further inflammatory insult. Collectively, these results provide novel evidence that oxidative stress is an early and chronic process during the development of synucleinopathy and suggest that PET imaging with [18F]ROStrace holds promise as a means of detecting aSyn-associated oxidative stress noninvasively.

ENGINEERED NANOBODIES WITH PROGRAMMABLE TARGET ANTIGEN PROTEOLYSIS (PTAP) FUSIONS REGULATE INTRACELLULAR ALPHA-SYNUCLEIN IN VITRO AND IN VIVO.

Alpha-synuclein (αSyn) aggregation and the formation of Lewy pathology (LP) is a foundational pathophysiological phenomenon in synucleinopathies. Delivering therapeutic single-chain and single-domain antibodies that bind pathogenic targets can disrupt intracellular aggregation. The fusion of antibody fragments to a negatively-charged proteasomal targeting motif (PEST) creates bifunctional constructs that enhance both solubility and turnover. With sequence-specific point mutations of PEST sequences that modulate proteasomal degradation efficiency, we report the creation of Programmable Target Antigen Proteolysis (PTAP) technology that can provide graded control over the levels of target antigens. We have previously demonstrated our lead anti-αSyn intrabody, VH14-PEST, is capable of reducing the pathological burden of synucleinopathy in vitro and in vivo. Here, we report a family of fully humanized VH14-PTAP constructs for controllable, therapeutic targeting of intracellular α-Syn. In cells, we demonstrate successful target engagement and efficacy of VH14-hPEST intrabodies, and validate proof-of-principle in human cells using 3D human organoids derived from PD-patient induced pluripotent stem cells (iPSC). In two synuclein-based rat models, PTAP intrabodies attenuated nigral αSyn pathology, preserved nigrostriatal dopaminergic tone, and slowed the propagation of αSyn pathology. These data demonstrate the potency of intracellular αSyn targeting as a method to alleviate pathology and highlight the potential clinical utility of PTAP intrabodies.

Alpha-synuclein inclusion responsive microglia are resistant to CSF1R inhibition.

BACKGROUND: Parkinson's disease (PD) is a neurodegenerative disorder that is characterized by the presence of proteinaceous alpha-synuclein (α-syn) inclusions (Lewy bodies), markers of neuroinflammation and the progressive loss of nigrostriatal dopamine (DA) neurons. These pathological features can be recapitulated in vivo using the α-syn preformed fibril (PFF) model of synucleinopathy. We have previously determined that microglia proximal to PFF-induced nigral α-syn inclusions increase in soma size, upregulate major-histocompatibility complex-II (MHC-II) expression, and increase expression of a suite of inflammation-associated transcripts. This microglial response is observed months prior to degeneration, suggesting that microglia reacting to α-syn inclusion may contribute to neurodegeneration and could represent a potential target for novel therapeutics. The goal of this study was to determine whether colony stimulating factor-1 receptor (CSF1R)-mediated microglial depletion impacts the magnitude of α-syn aggregation, nigrostriatal degeneration, or the response of microglial in the context of the α-syn PFF model. METHODS: Male Fischer 344 rats were injected intrastriatally with either α-syn PFFs or saline. Rats were continuously administered Pexidartinib (PLX3397B, 600 mg/kg), a CSF1R inhibitor, to deplete microglia for a period of either 2 or 6 months. RESULTS: CSF1R inhibition resulted in significant depletion (~ 43%) of ionized calcium-binding adapter molecule 1 immunoreactive (Iba-1ir) microglia within the SNpc. However, CSF1R inhibition did not impact the increase in microglial number, soma size, number of MHC-II immunoreactive microglia or microglial expression of Cd74, Cxcl10, Rt-1a2, Grn, Csf1r, Tyrobp, and Fcer1g associated with phosphorylated α-syn (pSyn) nigral inclusions. Further, accumulation of pSyn and degeneration of nigral neurons was not impacted by CSF1R inhibition. Paradoxically, long term CSF1R inhibition resulted in increased soma size of remaining Iba-1ir microglia in both control and PFF rats, as well as expression of MHC-II in extranigral regions. CONCLUSIONS: Collectively, our results suggest that CSF1R inhibition does not impact the microglial response to nigral pSyn inclusions and that CSF1R inhibition is not a viable disease-modifying strategy for PD.

MSUT2 regulates tau spreading via adenosinergic signaling mediated ASAP1 pathway in neurons.

Inclusions comprised of microtubule-associated protein tau (tau) are implicated in a group of neurodegenerative diseases, collectively known as tauopathies, that include Alzheimer's disease (AD). The spreading of misfolded tau "seeds" along neuronal networks is thought to play a crucial role in the progression of tau pathology. Consequently, restricting the release or uptake of tau seeds may inhibit the spread of tau pathology and potentially halt the advancement of the disease. Previous studies have demonstrated that the Mammalian Suppressor of Tauopathy 2 (MSUT2), an RNA binding protein, modulates tau pathogenesis in a transgenic mouse model. In this study, we investigated the impact of MSUT2 on tau pathogenesis using tau seeding models. Our findings indicate that the loss of MSUT2 mitigates human tau seed-induced pathology in neuron cultures and mouse models. In addition, MSUT2 regulates many gene transcripts, including the Adenosine Receptor 1 (A1AR), and we show that down regulation or inhibition of A1AR modulates the activity of the "ArfGAP with SH3 Domain, Ankyrin Repeat, and PH Domain 1 protein" (ASAP1), thereby influencing the internalization of pathogenic tau seeds into neurons resulting in reduction of tau pathology.

O-GlcNAc forces an α-synuclein amyloid strain with notably diminished seeding and pathology.

Amyloid-forming proteins such α-synuclein and tau, which are implicated in Alzheimer's and Parkinson's disease, can form different fibril structures or strains with distinct toxic properties, seeding activities and pathology. Understanding the determinants contributing to the formation of different amyloid features could open new avenues for developing disease-specific diagnostics and therapies. Here we report that O-GlcNAc modification of α-synuclein monomers results in the formation of amyloid fibril with distinct core structure, as revealed by cryogenic electron microscopy, and diminished seeding activity in seeding-based neuronal and rodent models of Parkinson's disease. Although the mechanisms underpinning the seeding neutralization activity of the O-GlcNAc-modified fibrils remain unclear, our in vitro mechanistic studies indicate that heat shock proteins interactions with O-GlcNAc fibril inhibit their seeding activity, suggesting that the O-GlcNAc modification may alter the interactome of the α-synuclein fibrils in ways that lead to reduce seeding activity in vivo. Our results show that posttranslational modifications, such as O-GlcNAc modification, of α-synuclein are key determinants of α-synuclein amyloid strains and pathogenicity.

Deficits in basal and evoked striatal dopamine release following alpha-synuclein preformed fibril injection: An in vivo microdialysis study.

Parkinson's disease (PD) is characterized by the accumulation of misfolded alpha-synuclein (α-syn) protein, forming intraneuronal Lewy body (LB) inclusions. The α-syn preformed fibril (PFF) model of PD recapitulates α-syn aggregation, progressive nigrostriatal degeneration and motor dysfunction; however, little is known about the time course of PFF-induced alterations in basal and evoked dopamine (DA). In vivo microdialysis is well suited for identifying small changes in neurotransmitter levels over extended periods. In the present study, adult male Fischer 344 rats received unilateral, intrastriatal injections of either α-syn PFFs or phosphate-buffered saline (PBS). At 4 or 8 months post-injection (p.i.), animals underwent in vivo microdialysis to evaluate basal extracellular striatal DA and metabolite levels, local KCl-evoked striatal DA release and the effects of systemic levodopa (l-DOPA). Post-mortem analysis demonstrated equivalent PFF-induced reductions in tyrosine hydroxylase (TH) immunoreactive nigral neurons (~50%) and striatal TH (~20%) at both time points. Compared with reduction in striatal TH, reduction in striatal dopamine transporter (DAT) was more pronounced and progressed between the 4- and 8-month p.i. intervals (36% âž” 46%). Significant PFF-induced deficits in basal and evoked striatal DA, as well as deficits in motor performance, were not observed until 8 months p.i. Responses to l-DOPA did not differ regardless of PBS or PFF treatment. These results suggest that basal and evoked striatal DA are maintained for several months following PFF injection, with loss of both associated with motor dysfunction. Our studies provide insight into the time course and magnitude of PFF-induced extracellular dopaminergic deficits in the striatum.

Tau maturation in the clinicopathological spectrum of Lewy body and Alzheimer's disease.

OBJECTIVE: Alzheimer's disease neuropathologic change and alpha-synucleinopathy commonly co-exist and contribute to the clinical heterogeneity of dementia. Here, we examined tau epitopes marking various stages of tangle maturation to test the hypotheses that tau maturation is more strongly associated with beta-amyloid compared to alpha-synuclein, and within the context of mixed pathology, mature tau is linked to Alzheimer's disease clinical phenotype and negatively associated with Lewy body dementia. METHODS: We used digital histology to measure percent area-occupied by pathology in cortical regions among individuals with pure Alzheimer's disease neuropathologic change, pure alpha-synucleinopathy, and a co-pathology group with both Alzheimer's and alpha-synuclein pathologic diagnoses. Multiple tau monoclonal antibodies were used to detect early (AT8, MC1) and mature (TauC3) epitopes of tangle progression. We used linear/logistic regression to compare groups and test the association between pathologies and clinical features. RESULTS: There were lower levels of tau pathology (ß = 1.86-2.96, p < 0.001) across all tau antibodies in the co-pathology group compared to the pure Alzheimer's pathology group. Among individuals with alpha-synucleinopathy, higher alpha-synuclein was associated with greater early tau (AT8 ß = 1.37, p < 0.001; MC1 ß = 1.2, p < 0.001) but not mature tau (TauC3 p = 0.18), whereas mature tau was associated with beta-amyloid (ß = 0.21, p = 0.01). Finally, lower tau, particularly TauC3 pathology, was associated with lower frequency of both core clinical features and categorical clinical diagnosis of dementia with Lewy bodies. INTERPRETATION: Mature tau may be more closely related to beta-amyloidosis than alpha-synucleinopathy, and pathophysiological processes of tangle maturation may influence the clinical features of dementia in mixed Lewy-Alzheimer's pathology.

Transcriptomic profiling of early synucleinopathy in rats induced with preformed fibrils.

Examination of early phases of synucleinopathy when inclusions are present, but long before neurodegeneration occurs, is critical to both understanding disease progression and the development of disease modifying therapies. The rat alpha-synuclein (α-syn) preformed fibril (PFF) model induces synchronized synucleinopathy that recapitulates the pathological features of Parkinson's disease (PD) and can be used to study synucleinopathy progression. In this model, phosphorylated α-syn (pSyn) inclusion-containing neurons and reactive microglia (major histocompatibility complex-II immunoreactive) peak in the substantia nigra pars compacta (SNpc) months before appreciable neurodegeneration. However, it remains unclear which specific genes are driving these phenotypic changes. To identify transcriptional changes associated with early synucleinopathy, we used laser capture microdissection of the SNpc paired with RNA sequencing (RNASeq). Precision collection of the SNpc allowed for the assessment of differential transcript expression in the nigral dopamine neurons and proximal glia. Transcripts upregulated in early synucleinopathy were mainly associated with an immune response, whereas transcripts downregulated were associated with neurotransmission and the dopamine pathway. A subset of 29 transcripts associated with neurotransmission/vesicular release and the dopamine pathway were verified in a separate cohort of males and females to confirm reproducibility. Within this subset, fluorescent in situ hybridization (FISH) was used to localize decreases in the Syt1 and Slc6a3 transcripts to pSyn inclusion-containing neurons. Identification of transcriptional changes in early synucleinopathy provides insight into the molecular mechanisms driving neurodegeneration.

Neuroinflammatory gene expression profiles of reactive glia in the substantia nigra suggest a multidimensional immune response to alpha synuclein inclusions.

Parkinson's disease (PD) pathology is characterized by alpha-synuclein (α-syn) aggregates, degeneration of dopamine neurons in the substantia nigra pars compacta (SNpc), and neuroinflammation. The presence of reactive glia correlates with deposition of pathological α-syn in early-stage PD. Thus, understanding the neuroinflammatory response of microglia and astrocytes to synucleinopathy may identify therapeutic targets. Here we characterized the neuroinflammatory gene expression profile of reactive microglia and astrocytes in the SNpc during early synucleinopathy in the rat α-syn pre-formed fibril (PFF) model. Rats received intrastriatal injection of α-syn PFFs and expression of immune genes was quantified with droplet digital PCR (ddPCR), after which fluorescent in situ hybridization (FISH) was used to localize gene expression to microglia or astrocytes in the SNpc. Genes previously associated with reactive microglia (Cd74, C1qa, Stat1, Axl, Casp1, Il18, Lyz2) and reactive astrocytes (C3, Gbp2, Serping1) were significantly upregulated in the SN of PFF injected rats. Localization of gene expression to SNpc microglia near α-syn aggregates identified a unique α-syn aggregate microglial gene expression profile characterized by upregulation of Cd74, Cxcl10, Rt-1a2, Grn, Csf1r, Tyrobp, C3, C1qa, Serping1 and Fcer1g. Importantly, significant microglial upregulation of Cd74 and C3 were only observed following injection of α-syn PFFs, not α-syn monomer, confirming specificity to α-syn aggregation. Serping1 expression also localized to astrocytes in the SNpc. Interestingly, C3 expression in the SNpc localized to microglia at 2- and 4-months post-PFF, but to astrocytes at 6-months post-PFF. We also observed expression of Rt1-a2 and Cxcl10 in SNpc dopamine neurons. Cumulatively our results identify a dynamic, yet reproducible gene expression profile of reactive microglia and astrocytes associated with early synucleinopathy in the rat SNpc.

Combining fibril-induced alpha-synuclein aggregation and 6-hydroxydopamine in a mouse model of Parkinson's disease and the effect of cerebral dopamine neurotrophic factor on the induced neurodegeneration.

The existent pre-clinical models of Parkinson's disease do not simultaneously recapitulate severe degeneration of dopamine neurons and the occurrence of alpha-synuclein (aSyn) aggregation in one study system. In this study, we injected aSyn pre-formed fibrils (PFF) and 6-hydroxydopamine (6-OHDA) unilaterally into the striatum of C57BL/6 wild-type male mice at an interval of 2 weeks to induce aggregation of aSyn protein and trigger the loss of dopamine neurons simultaneously in one model and studied the behavioural effects of the combination in these mice. 6-OHDA was tested at three different doses, and 2 µg of 6-OHDA combined with PFF-induced aSyn aggregation was found to produce the most optimal disease phenotype. At 14 weeks timepoint, mice injected with a combination of PFF and 6-OHDA sustained significant damage to the nigrostriatal pathway and exhibited aSyn-positive aggregation. Our data suggest that the neurons that formed large aSyn aggregates were particularly vulnerable to 6-OHDA-induced degeneration. We also demonstrate the manifestation of a relatively aggressive pathology in 2- to 4-month-old mice, as compared to younger 7- to 9-week-old ones. Furthermore, cerebral dopamine neurotrophic factor (CDNF) administered intrastriatally rescued dopamine neurons and motor behaviour of the animals to some extent from 6-OHDA toxicity. However, no such effect could be seen in the novel 6-OHDA + PFFs combination model. For the first time, we demonstrate the combined effect of PFF and 6-OHDA simultaneously in one model. We further discuss the scope for further optimizing this combination model to develop it as a promising pre-clinical platform for drug screening and development.

α-Synuclein aggregates amplified from patient-derived Lewy bodies recapitulate Lewy body diseases in mice.

Extraction of α-Synuclein (αSyn) aggregates from Lewy body disease (LBD) brains has been widely described yet templated fibrillization of LB-αSyn often fails to propagate its structural and functional properties. We recently demonstrated that aggregates amplified from LB-αSyn (ampLB) show distinct biological activities in vitro compared to human αSyn preformed fibrils (hPFF) formed de novo. Here we compare the in vivo biological activities of hPFF and ampLB regarding seeding activity, latency in inducing pathology, distribution of pathology, inclusion morphology, and cell-type preference. Injection of ampLB into mice expressing only human αSyn (male Thy1:SNCA/Snca-/- mice) induced pathologies similar to those of LBD subjects that were distinct from those induced by hPFF-injection or developing spontaneously with aging. Importantly, αSyn aggregates in ampLB-injected Thy1:SNCA/Snca-/- mice maintained the unique biological and conformational features of original LB-αSyn. These results indicate that ampLB-injection, rather than conventional PFF-injection or αSyn overexpression, faithfully models key aspects of LBD.

Developmental exposure to the Parkinson's disease-associated organochlorine pesticide dieldrin alters dopamine neurotransmission in α-synuclein pre-formed fibril (PFF)-injected mice.

Parkinson's disease (PD) is the fastest-growing neurological disease worldwide, with increases outpacing aging and occurring most rapidly in recently industrialized areas, suggesting a role of environmental factors. Epidemiological, post-mortem, and mechanistic studies suggest that persistent organic pollutants, including the organochlorine pesticide dieldrin, increase PD risk. In mice, developmental dieldrin exposure causes male-specific exacerbation of neuronal susceptibility to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and synucleinopathy. Specifically, in the α-synuclein (α-syn) pre-formed fibril (PFF) model, exposure leads to increased deficits in striatal dopamine (DA) turnover and motor deficits on the challenging beam. Here, we hypothesized that alterations in DA handling contribute to the observed changes and assessed vesicular monoamine transporter 2 (VMAT2) function and DA release in this dieldrin/PFF 2-hit model. Female C57BL/6 mice were exposed to 0.3 mg/kg dieldrin or vehicle every 3 days by feeding, starting at 8 weeks of age and continuing throughout breeding, gestation, and lactation. Male offspring from independent litters underwent unilateral, intrastriatal injections of α-syn PFFs at 12 weeks of age, and vesicular 3H-DA uptake assays and fast-scan cyclic voltammetry were performed 4 months post-PFF injection. Dieldrin-induced an increase in DA release in striatal slices in PFF-injected animals, but no change in VMAT2 activity. These results suggest that developmental dieldrin exposure increases a compensatory response to synucleinopathy-triggered striatal DA loss. These findings are consistent with silent neurotoxicity, where developmental exposure to dieldrin primes the nigrostriatal striatal system to have an exacerbated response to synucleinopathy in the absence of observable changes in typical markers of nigrostriatal dysfunction and degeneration.

A Novel Brain PET Radiotracer for Imaging Alpha Synuclein Fibrils in Multiple System Atrophy.

Abnormal α-synuclein (α-syn) aggregation characterizes α-synucleinopathies, including Parkinson's disease (PD) and multiple system atrophy (MSA). However, no suitable positron emission tomography (PET) radiotracer for imaging α-syn in PD and MSA exists currently. Our structure-activity relationship studies identified 4-methoxy-N-(4-(3-(pyridin-2-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)phenyl)benzamide (4i) as a PET radiotracer candidate for imaging α-syn. In vitro assays revealed high binding of 4i to recombinant α-syn fibrils (inhibition constant (Ki) = 6.1 nM) and low affinity for amyloid beta (Aß) fibrils in Alzheimer's disease (AD) homogenates. However, [3H]4i also exhibited high specific binding to AD, progressive supranuclear palsy, and corticobasal degeneration tissues as well as PD and MSA tissues, suggesting notable affinity to tau. Nevertheless, the specific binding to pathologic α-syn aggregates in MSA post-mortem brain tissues was significantly higher than in PD tissues. This finding demonstrated the potential use of [11C]4i as a PET tracer for imaging α-syn in MSA patients. Nonhuman primate PET studies confirmed good brain uptake and rapid washout for [11C]4i.

A 14-day pulse of PLX5622 modifies α-synucleinopathy in preformed fibril-infused aged mice of both sexes.

Reactive microglia are observed with aging and in Lewy body disorders, including within the olfactory bulb of men with Parkinson's disease. However, the functional impact of microglia in these disorders is still debated. Resetting these reactive cells by a brief dietary pulse of the colony-stimulating factor 1 receptor (CSF1R) inhibitor PLX5622 may hold therapeutic potential against Lewy-related pathologies. To our knowledge, withdrawal of PLX5622 after short-term exposure has not been tested in the preformed α-synuclein fibril (PFF) model, including in aged mice of both sexes. Compared to aged female mice, we report that aged males on the control diet showed higher numbers of phosphorylated α-synuclein+ inclusions in the limbic rhinencephalon after PFFs were injected in the posterior olfactory bulb. However, aged females displayed larger inclusion sizes compared to males. Short-term (14-day) dietary exposure to PLX5622 followed by control chow reduced inclusion numbers and levels of insoluble α-synuclein in aged males-but not females-and unexpectedly raised inclusion sizes in both sexes. Transient delivery of PLX5622 also improved spatial reference memory in PFF-infused aged mice, as evidenced by an increase in novel arm entries in a Y-maze. Superior memory was positively correlated with inclusion sizes but negatively correlated with inclusion numbers. Although we caution that PLX5622 delivery must be tested further in models of α-synucleinopathy, our data suggest that larger-sized-but fewer-α-synucleinopathic structures are associated with better neurological outcomes in PFF-infused aged mice.

O-GlcNAc modification forces the formation of an α-Synuclein amyloid-strain with notably diminished seeding activity and pathology.

The process of amyloid fibril formation remains one of the primary targets for developing diagnostics and treatments for several neurodegenerative diseases (NDDs). Amyloid-forming proteins such α-Synuclein and Tau, which are implicated in the pathogenesis of Alzheimer's and Parkinson's disease, can form different types of fibril structure, or strains, that exhibit distinct structures, toxic properties, seeding activities, and pathology spreading patterns in the brain. Therefore, understanding the molecular and structural determinants contributing to the formation of different amyloid strains or their distinct features could open new avenues for developing disease-specific diagnostics and therapies. In this work, we report that O-GlcNAc modification of α-Synuclein monomers results in the formation of amyloid fibril with distinct core structure, as revealed by Cryo-EM, and diminished seeding activity in seeding-based neuronal and rodent models of Parkinson's disease. Although the mechanisms underpinning the seeding neutralization activity of the O-GlcNAc modified fibrils remain unclear, our in vitro mechanistic studies indicate that heat shock proteins interactions with O-GlcNAc fibril inhibit their seeding activity, suggesting that the O-GlcNAc modification may alter the interactome of the α-Synuclein fibrils in ways that lead to reduce seeding activity in vivo. Our results show that post-translational modifications, such as O-GlcNAc modification, of α-Synuclein are key determinants of α-Synuclein amyloid strains and pathogenicity. These findings have significant implications for how we investigate and target amyloids in the brain and could possibly explain the lack of correlation between amyloid burden and neurodegeneration or cognitive decline in some subtypes of NDDs.