Heterogeneity in α-synuclein fibril activity correlates to disease phenotypes in Lewy body dementia.

α-Synuclein aggregation underlies pathological changes in Lewy body dementia. Recent studies highlight structural variabilities associated with α-synuclein aggregates in patient populations. Here, we develop a quantitative real-time quaking-induced conversion (qRT-QuIC) assay to measure permissive α-synuclein fibril-templating activity in tissues and cerebrospinal fluid (CSF). The assay is anchored through reference panels of stabilized ultra-short fibril particles. In humanized α-synuclein transgenic mice, qRT-QuIC identifies differential levels of fibril activity across the brain months before the deposition of phosphorylated α-synuclein in susceptible neurons. α-Synuclein fibril activity in cortical brain extracts from dementia with Lewy bodies (DLB) correlates with activity in matched ventricular CSF. Elevated α-synuclein fibril activity in CSF corresponds to reduced survival in DLB. α-Synuclein fibril particles amplified from cases with high fibril activity show superior templating in the formation of new inclusions in neurons relative to the same number of fibril particles amplified from DLB cases with low fibril activity. Our results highlight a previously unknown broad heterogeneity of fibril-templating activities in DLB that may contribute to disease phenotypes. We predict that quantitative assessments of fibril activities in CSF that correlate to fibril activities in brain tissue will help stratify patient populations as well as measure therapeutic responses to facilitate the development of α-synuclein-targeted therapeutics.

ß-d-N4-Hydroxycytidine Is a Potent Anti-alphavirus Compound That Induces a High Level of Mutations in the Viral Genome.

Venezuelan equine encephalitis virus (VEEV) is a representative member of the New World alphaviruses. It is transmitted by mosquito vectors and causes highly debilitating disease in humans, equids, and other vertebrate hosts. Despite a continuous public health threat, very few compounds with anti-VEEV activity in cell culture and in mouse models have been identified to date, and rapid development of virus resistance to some of them has been recorded. In this study, we investigated the possibility of using a modified nucleoside analog, ß-d-N4-hydroxycytidine (NHC), as an anti-VEEV agent and defined the mechanism of its anti-VEEV activity. The results demonstrate that NHC is a very potent antiviral agent. It affects both the release of genome RNA-containing VEE virions and their infectivity. Both of these antiviral activities are determined by the NHC-induced accumulation of mutations in virus-specific RNAs. The antiviral effect is most prominent when NHC is applied early in the infectious process, during the amplification of negative- and positive-strand RNAs in infected cells. Most importantly, only a low-level resistance of VEEV to NHC can be developed, and it requires acquisition and cooperative function of more than one mutation in nsP4. These adaptive mutations are closely located in the same segment of nsP4. Our data suggest that NHC is more potent than ribavirin as an anti-VEEV agent and likely can be used to treat other alphavirus infections.IMPORTANCE Venezuelan equine encephalitis virus (VEEV) can cause widespread epidemics among humans and domestic animals. VEEV infections result in severe meningoencephalitis and long-term sequelae. No approved therapeutics exist for treatment of VEEV infections. Our study demonstrates that ß-d-N4-hydroxycytidine (NHC) is a very potent anti-VEEV compound, with the 50% effective concentration being below 1 µM. The mechanism of NHC antiviral activity is based on induction of high mutation rates in the viral genome. Accordingly, NHC treatment affects both the rates of particle release and the particle infectivity. Most importantly, in contrast to most of the anti-alphavirus drugs that are under development, resistance of VEEV to NHC develops very inefficiently. Even low levels of resistance require acquisition of multiple mutations in the gene of the VEEV-specific RNA-dependent RNA polymerase nsP4.

Gut mucosal cells transfer α-synuclein to the vagus nerve.

Epidemiological and histopathological findings have raised the possibility that misfolded α-synuclein protein might spread from the gut to the brain and increase the risk of Parkinson's disease (PD). While past experimental studies in mouse models have relied on gut injections of exogenous recombinant α-synuclein fibrils to study gut to brain α-synuclein transfer, the possible origins of misfolded α-synuclein within the gut have remained elusive. We recently demonstrated that sensory cells of the gut mucosa express α-synuclein. In this study, we employed mouse intestinal organoids expressing human α-synuclein to observe the transfer of α-synuclein protein from gut epithelial cells in organoids co-cultured with vagal nodose neurons that are otherwise devoid of α-synuclein expression. In intact mice that express pathological human α-synuclein, but no mouse α-synuclein, α-synuclein fibril templating activity emerges in α-synuclein seeded fibril aggregation assays in tissues from the gut, vagus nerve, and dorsal motor nucleus. In newly engineered transgenic mice that restrict pathological human α-synuclein expression to intestinal epithelial cells, α-synuclein fibril-templating activity transfers to the vagus nerve and to the dorsal motor nucleus. Subdiaphragmatic vagotomy prior to the induction of α-synuclein expression in the gut epithelial cells effectively protects the hindbrain from the emergence of α-synuclein fibril templating activity. Overall, these findings highlight a novel potential non-neuronal source of fibrillar α-synuclein protein that might arise in gut mucosal cells.

Gut mucosal cells transfer α-synuclein to the vagus nerve.

Epidemiological and histopathological findings have raised the possibility that misfolded α-synuclein protein might spread from the gut to the brain and increase the risk of Parkinson's disease. Although past experimental studies in mouse models have relied on gut injections of exogenous recombinant α-synuclein fibrils to study gut-to-brain α-synuclein transfer, the possible origins of misfolded α-synuclein within the gut have remained elusive. We recently demonstrated that sensory cells of intestinal mucosa express α-synuclein. Here, we employed mouse intestinal organoids expressing human α-synuclein to observe the transfer of α-synuclein protein from epithelial cells in organoids to cocultured nodose neurons devoid of α-synuclein. In mice expressing human α-synuclein, but no mouse α-synuclein, α-synuclein fibril-templating activity emerged in α-synuclein-seeded fibril aggregation assays in intestine, vagus nerve, and dorsal motor nucleus. In newly engineered transgenic mice that restrict pathological human α-synuclein expression to intestinal epithelial cells, α-synuclein fibril-templating activity transfered to the vagus nerve and dorsal motor nucleus. Subdiaphragmatic vagotomy prior to induction of α-synuclein expression in intestinal epithelial cells effectively protected the hindbrain from emergence of α-synuclein fibril-templating activity. Overall, these findings highlight a potential non-neuronal source of fibrillar α-synuclein protein that might arise in gut mucosal cells.

Anionic nanoplastic contaminants promote Parkinson's disease-associated α-synuclein aggregation.

Recent studies have identified increasing levels of nanoplastic pollution in the environment. Here, we find that anionic nanoplastic contaminants potently precipitate the formation and propagation of α-synuclein protein fibrils through a high-affinity interaction with the amphipathic and non-amyloid component (NAC) domains in α-synuclein. Nanoplastics can internalize in neurons through clathrin-dependent endocytosis, causing a mild lysosomal impairment that slows the degradation of aggregated α-synuclein. In mice, nanoplastics combine with α-synuclein fibrils to exacerbate the spread of α-synuclein pathology across interconnected vulnerable brain regions, including the strong induction of α-synuclein inclusions in dopaminergic neurons in the substantia nigra. These results highlight a potential link for further exploration between nanoplastic pollution and α-synuclein aggregation associated with Parkinson's disease and related dementias.

Anionic Nanoplastic Contaminants Promote Parkinson's Disease-Associated α-Synuclein Aggregation.

Recent studies have identified increasing levels of nanoplastic pollution in the environment. Here we find that anionic nanoplastic contaminants potently precipitate the formation and propagation of α-synuclein protein fibrils through a high-affinity interaction with the amphipathic and non-amyloid component (NAC) domains in α-synuclein. Nanoplastics can internalize in neurons through clathrin-dependent endocytosis, causing a mild lysosomal impairment that slows the degradation of aggregated α-synuclein. In mice, nanoplastics combine with α-synuclein fibrils to exacerbate the spread of α-synuclein pathology across interconnected vulnerable brain regions, including the strong induction of α-synuclein inclusions in dopaminergic neurons in the substantia nigra. These results highlight a potential link for further exploration between nanoplastic pollution and α-synuclein aggregation associated with Parkinson's disease and related dementias.

Evaluation of ABT-888 in the amelioration of α-synuclein fibril-induced neurodegeneration.

The accumulation of α-synuclein inclusions in vulnerable neuronal populations pathologically defines Lewy body diseases including Parkinson's disease. Recent pre-clinical studies suggest poly(ADP-ribose) polymerase-1 activation and the subsequent generation of poly(ADP-ribose) polymer represent key steps in the formation of toxic α-synuclein aggregates and neurodegeneration. Several studies suggest that the inhibition of poly(ADP-ribose) polymerase-1 activity via the poly(ADP-ribose) polymerase-1/2 small molecule inhibitor ABT-888 (Veliparib), a drug in clinical trials for different cancers, may prevent or ameliorate α-synuclein fibril-induced aggregation, inclusion formation and dopaminergic neurodegeneration. Herein, we evaluated the effects of poly(ADP-ribose) polymer on α-synuclein fibrillization in vitro, the effects of ABT-888 on the formation of fibril-seeded α-synuclein inclusions in primary mouse cortical neurons and the effects of an in-diet ABT-888 dosage regimen with the intracranial injection of α-synuclein fibrils into the mouse dorsal striatum. We found that poly(ADP-ribose) polymer minimally but significantly increased the rate of spontaneously formed α-synuclein fibrils in vitro. Machine-learning algorithms that quantitatively assessed α-synuclein inclusion counts in neurons, both in primary cultures and in the brains of fibril-injected mice, did not reveal differences between ABT-888- and vehicle-treated groups. The in-diet administered ABT-888 molecule demonstrated outstanding brain penetration in mice; however, dopaminergic cell loss in the substantia nigra caused by α-synuclein fibril injections in the striatum was similar between ABT-888- and vehicle-treated groups. α-Synuclein fibril-induced loss of dopaminergic fibres in the dorsal striatum was also similar between ABT-888- and vehicle-treated groups. We conclude that additional pre-clinical evaluation of ABT-888 may be warranted to justify further exploration of ABT-888 for disease modification in Lewy body diseases.

Repetitive mild TBI causes pTau aggregation in nigra without altering preexisting fibril induced Parkinson's-like pathology burden.

Population studies have shown that traumatic brain injury (TBI) is associated with an increased risk for Parkinson's disease (PD) and among U.S. Veterans with a history of TBI this risk is 56% higher. The most common type of TBI is mild (mTBI) and often occurs repeatedly among athletes, military personnel, and victims of domestic violence. PD is classically characterized by deficits in fine motor movement control resulting from progressive neurodegeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc) midbrain region. This neurodegeneration is preceded by the predictable spread of characteristic alpha synuclein (αSyn) protein inclusions. Whether repetitive mTBI (r-mTBI) can nucleate PD pathology or accelerate prodromal PD pathology remains unknown. To answer this question, an injury device was constructed to deliver a surgery-free r-mTBI to rats and human-like PD pathology was induced by intracranial injection of recombinant αSyn preformed fibrils. At the 3-month endpoint, the r-mTBI caused encephalomalacia throughout the brain reminiscent of neuroimaging findings in patients with a history of mTBI, accompanied by astrocyte expansion and microglial activation. The pathology associated most closely with PD, which includes dopaminergic neurodegeneration in the SNpc and Lewy body-like αSyn inclusion burden in the surviving neurons, was not produced de novo by r-mTBI nor was the fibril induced preexisting pathology accelerated. r-mTBI did however cause aggregation of phosphorylated Tau (pTau) protein in nigra of rats with and without preexisting PD-like pathology. pTau aggregation was also found to colocalize with PFF induced αSyn pathology without r-mTBI. These findings suggest that r-mTBI induced pTau aggregate deposition in dopaminergic neurons may create an environment conducive to αSyn pathology nucleation and may add to preexisting proteinaceous aggregate burden.

Pathological α-synuclein recruits LRRK2 expressing pro-inflammatory monocytes to the brain.

BACKGROUND: Leucine rich repeat kinase 2 (LRRK2) and SNCA are genetically linked to late-onset Parkinson's disease (PD). Aggregated α-synuclein pathologically defines PD. Recent studies identified elevated LRRK2 expression in pro-inflammatory CD16+ monocytes in idiopathic PD, as well as increased phosphorylation of the LRRK2 kinase substrate Rab10 in monocytes in some LRRK2 mutation carriers. Brain-engrafting pro-inflammatory monocytes have been implicated in dopaminergic neurodegeneration in PD models. Here we examine how α-synuclein and LRRK2 interact in monocytes and subsequent neuroinflammatory responses. METHODS: Human and mouse monocytes were differentiated to distinct transcriptional states resembling macrophages, dendritic cells, or microglia, and exposed to well-characterized human or mouse α-synuclein fibrils. LRRK2 expression and LRRK2-dependent Rab10 phosphorylation were measured with monoclonal antibodies, and myeloid cell responses to α-synuclein fibrils in R1441C-Lrrk2 knock-in mice or G2019S-Lrrk2 BAC mice were evaluated by flow cytometry. Chemotaxis assays were performed with monocyte-derived macrophages stimulated with α-synuclein fibrils and microglia in Boyden chambers. RESULTS: α-synuclein fibrils robustly stimulate LRRK2 and Rab10 phosphorylation in human and mouse macrophages and dendritic-like cells. In these cells, α-synuclein fibrils stimulate LRRK2 through JAK-STAT activation and intrinsic LRRK2 kinase activity in a feed-forward pathway that upregulates phosphorylated Rab10. In contrast, LRRK2 expression and Rab10 phosphorylation are both suppressed in microglia-like cells that are otherwise highly responsive to α-synuclein fibrils. Corroborating these results, LRRK2 expression in the brain parenchyma occurs in pro-inflammatory monocytes infiltrating from the periphery, distinct from brain-resident microglia. Mice expressing pathogenic LRRK2 mutations G2019S or R1441C have increased numbers of infiltrating pro-inflammatory monocytes in acute response to α-synuclein fibrils. In primary cultured macrophages, LRRK2 kinase inhibition dampens α-synuclein fibril and microglia-stimulated chemotaxis. CONCLUSIONS: Pathologic α-synuclein activates LRRK2 expression and kinase activity in monocytes and induces their recruitment to the brain. These results predict that LRRK2 kinase inhibition may attenuate damaging pro-inflammatory monocyte responses in the brain.