MhcII Regulates Transmission of α-Synuclein-Seeded Pathology in Mice.
ABSTRACT: MHCII molecules, expressed by professional antigen-presenting cells (APCs) such as T cells and B cells, are hypothesized to play a key role in the response of cellular immunity to α-synuclein (α-syn). However, the role of cellular immunity in the neuroanatomic transmission of α-syn pre-formed fibrillar (PFF) seeds is undetermined. To illuminate whether cellular immunity influences the transmission of α-syn seeds from the periphery into the CNS, we injected preformed α-syn PFFs in the hindlimb of the Line M83 transgenic mouse model of synucleinopathy lacking MhcII. We showed that a complete deficiency in MhcII accelerated the appearance of seeded α-syn pathology and shortened the lifespan of the PFF-seeded M83 mice. To characterize whether B-cell and T-cell inherent MhcII function underlies this accelerated response to PFF seeding, we next injected α-syn PFFs in Rag1-/- mice which completely lacked these mature lymphocytes. There was no alteration in the lifespan or burden of endstage α-syn pathology in the PFF-seeded, Rag1-deficient M83+/- mice. Together, these results suggested that MhcII function on immune cells other than these classical APCs is potentially involved in the propagation of α-syn in this model of experimental synucleinopathy. We focused on microglia next, finding that while microglial burden was significantly upregulated in PFF-seeded, MhcII-deficient mice relative to controls, the microglial activation marker Cd68 was reduced in these mice, suggesting that these microglia were not responsive. Additional analysis of the CNS showed the early appearance of the neurotoxic astrocyte A1 signature and the induction of the Ifnγ-inducible anti-viral response mediated by MhcI in the MhcII-deficient, PFF-seeded mice. Overall, our data suggest that the loss of MhcII function leads to a dysfunctional response in non-classical APCs and that this response could potentially play a role in determining PFF-induced pathology. Collectively, our results identify the critical role of MhcII function in synucleinopathies induced by α-syn prion seeds.
Project description:BACKGROUND:Synucleinopathy is a group of neurodegenerative disorders characterized by neurodegeneration and accumulation of alpha-synuclein (?-syn) aggregates in various brain regions. The detailed mechanism of ?-syn-caused neurotoxicity remains obscure, which is partly due to the lack of a suitable model that retains the in vivo three-dimensional cellular network and allows a convenient dissection of the neurotoxic pathways. Recent studies revealed that the pre-formed recombinant ?-syn amyloid fibrils (PFFs) induce a robust accumulation of pathogenic ?-syn species in cultured cells and animals. OBJECTIVE:Our goal is to determine whether PFFs are able to induce the pathogenic ?-syn accumulation and neurotoxicity in organotypic brain slice culture, an ex vivo system that retains the in vivo three-dimensional cell-cell connections. METHODS/RESULTS:Adding PFFs to cultured wild-type rat or mouse brain slices induced a time-dependent accumulation of pathogenic ?-syn species, which was indicated by ?-syn phosphorylated at serine 129 (p?-syn). The PFF-induced p?-syn was abolished in brain slices prepared from ?-syn null mice, suggesting that the p?-syn is from the phosphorylation of endogenous ?-syn. Human PFFs also induced p?-syn in brain slices prepared from mice expressing human ?-syn on a mouse ?-syn-null background. Furthermore, the synaptophysin immunoreactivity was inversely associated with p?-syn accumulation and an increase of neuronal loss was detected. CONCLUSION:PFF-treatment of brain slices is able to induce key pathological features of synucleinopathy: p?-syn accumulation and neurotoxicity. This model will be useful for investigating the neurotoxic mechanism and evaluating efficacy of therapeutic approaches.
Project description:Animal models that accurately recapitulate the accumulation of alpha-synuclein (?-syn) inclusions, progressive neurodegeneration of the nigrostriatal system and motor deficits can be useful tools for Parkinson's disease (PD) research. The preformed fibril (PFF) synucleinopathy model in rodents generally displays these PD-relevant features, however, the magnitude and predictability of these events is far from established. We therefore sought to optimize the magnitude of ?-syn accumulation and nigrostriatal degeneration, and to understand the time course of both. Rats were injected unilaterally with different quantities of ?-syn PFFs (8 or 16??g of total protein) into striatal sites selected to concentrate ?-syn inclusion formation in the substantia nigra pars compacta (SNpc). Rats displayed an ?-syn PFF quantity-dependent increase in the magnitude of ipsilateral SNpc inclusion formation at 2?months and bilateral loss of nigral dopamine neurons at 6?months. Unilateral 16??g PFF injection also resulted in modest sensorimotor deficits in forelimb adjusting steps associated with degeneration at 6?months. Bilateral injection of 16??g ?-syn PFFs resulted in symmetric bilateral degeneration equivalent to the ipsilateral nigral degeneration observed following unilateral 16??g PFF injection (~50% loss). Bilateral PFF injections additionally resulted in alterations in several gait analysis parameters. These ?-syn PFF parameters can be applied to generate a reproducible synucleinopathy model in rats with which to study pathogenic mechanisms and vet potential disease-modifying therapies.
Project description:Mechanisms underlying ?-synuclein (?Syn) mediated neurodegeneration are poorly understood. Intramuscular (IM) injection of ?Syn fibrils in human A53T transgenic M83+/- mice produce a rapid model of ?-synucleinopathy with highly predictable onset of motor impairment. Using varying doses of ?Syn seeds, we show that ?Syn-induced phenotype is largely dose-independent. We utilized the synchrony of this IM model to explore the temporal sequence of ?Syn pathology, neurodegeneration and neuroinflammation. Longitudinal tracking showed that while motor neuron death and ?Syn pathology occur within 2?months post IM, astrogliosis appears at a later timepoint, implying neuroinflammation is a consequence, rather than a trigger, in this prionoid model of synucleinopathy. Initiating at 3?months post IM, immune activation dominates the pathologic landscape in terminal IM-seeded M83+/- mice, as revealed by unbiased transcriptomic analyses. Our findings provide insights into the role of neuroinflammation in ?Syn mediated proteostasis and neurodegeneration, which will be key in designing potential therapies.
Project description:α-Synuclein (α-syn) protein aggregation is associated with several neurodegenerative disorders collectively referred to as synucleinopathies, including Parkinson's disease. We used protein misfolding cyclic amplification (PMCA) to study α-syn aggregation in brain homogenates of wild-type or transgenic mice expressing normal (D line) or A53T mutant (M83 line) human α-syn. We found that sonication-incubation cycles of M83 mouse brain gradually produce large quantities of SDS-resistant α-syn aggregates, involving both human and mouse proteins. These PMCA products, containing partially proteinase K-resistant α-syn species, are competent to accelerate the onset of neurologic symptoms after intracerebral inoculation to young M83 mice and to seed aggregate formation of α-syn following PMCA, including in D and wild-type mouse brain substrates. PMCA seeding activity in the M83 diseased brain correlates positively with regions mostly targeted by the α-syn pathology in this model. Our data indicate that similar to prions, PMCA can reproduce some characteristics of α-syn aggregation and seeded propagation <i>in vitro</i> in a complex milieu. This opens new opportunities for the molecular study of synucleinopathies.-Nicot, S., Verchère, J., Bélondrade, M., Mayran, C., Bétemps, D., Bougard, D., Baron, T. Seeded propagation of α-synuclein aggregation in mouse brain using protein misfolding cyclic amplification.
Project description:<h4>Background</h4>α-Synuclein (α-syn) is the predominant protein in Lewy-body inclusions, which are pathological hallmarks of α-synucleinopathies, such as Parkinson's disease (PD) and multiple system atrophy (MSA). Other hallmarks include activation of microglia, elevation of pro-inflammatory cytokines, as well as the activation of T and B cells. These immune changes point towards a dysregulation of both the innate and the adaptive immune system. T cells have been shown to recognize epitopes derived from α-syn and altered populations of T cells have been found in PD and MSA patients, providing evidence that these cells can be key to the pathogenesis of the disease.ObjectiveTo study the role of the adaptive immune system with respect to α-syn pathology.<h4>Methods</h4>We injected human α-syn preformed fibrils (PFFs) into the striatum of immunocompromised mice (NSG) and assessed accumulation of phosphorylated α-syn pathology, proteinase K-resistant α-syn pathology and microgliosis in the striatum, substantia nigra and frontal cortex. We also assessed the impact of adoptive transfer of naïve T and B cells into PFF-injected immunocompromised mice.<h4>Results</h4>Compared to wildtype mice, NSG mice had an 8-fold increase in phosphorylated α-syn pathology in the substantia nigra. Reconstituting the T cell population decreased the accumulation of phosphorylated α-syn pathology and resulted in persistent microgliosis in the striatum when compared to non-transplanted mice.<h4>Conclusion</h4>Our work provides evidence that T cells play a role in the pathogenesis of experimental α-synucleinopathy.
Project description:Direct cell-to-cell transmission of proteopathic ?-synuclein (?-syn) aggregates is thought to underlie the progression of neurodegenerative synucleinopathies. However, the specific intracellular processes governing this transmission remain unclear because currently available model systems are limited. For example, in cell culture models of ?-syn-seeded aggregation, it is difficult to discern intracellular from extracellular exogenously applied ?-syn seed species. Herein, we employed fluorescently labeled ?-syn preformed fibrils (pffs) in conjunction with the membrane-impermeable fluorescence quencher trypan blue to selectively image internalized ?-syn seeds in cultured primary neurons and to quantitatively characterize the concentration dependence, time course, and inhibition of pff uptake. To study the long-term fates of exogenous ?-syn pffs in neurons, we developed a pff species labeled at amino acid residue 114 with the environmentally insensitive fluorophore BODIPY or the pH-sensitive dye pHrodo red. We found that pffs are rapidly trafficked along the endolysosomal pathway, where most of the material remains for days. We also found that brief pharmacological perturbation of lysosomes shortly after the pff treatment causes aberrations in intracellular processing of pff seeds concomitant with an increased rate of inclusion formation via recruitment of endogenous ?-syn to a relatively small number of exogenous seeds. Our results validate a quantitative assay for pff uptake in primary neurons, implicate lysosomal processing as the major fate of internalized proteopathic seeds, and suggest lysosomal integrity as a significant rate-determining step in the transmission of ?-syn pathology. Further, lysosomal processing of transmitted seeds may represent a new therapeutic target to combat the spread of synucleinopathies.
Project description:Human and animal studies have shown that exposure to the organochlorine pesticide dieldrin is associated with increased risk of Parkinson's disease (PD). Previous work showed that developmental dieldrin exposure increased neuronal susceptibility to MPTP toxicity in male C57BL/6 mice, possibly via changes in dopamine (DA) packaging and turnover. However, the relevance of the MPTP model to PD pathophysiology has been questioned. We therefore studied dieldrin-induced neurotoxicity in the α-synuclein (α-syn)-preformed fibril (PFF) model, which better reflects the α-syn pathology and toxicity observed in PD pathogenesis. Specifically, we used a "two-hit" model to determine whether developmental dieldrin exposure increases susceptibility to α-syn PFF-induced synucleinopathy. Dams were fed either dieldrin (0.3 mg/kg, every 3-4 days) or vehicle corn oil starting 1 month prior to breeding and continuing through weaning of pups at postnatal day 22. At 12 weeks of age, male and female offspring received intrastriatal α-syn PFF or control saline injections. Consistent with the male-specific increased susceptibility to MPTP, our results demonstrate that developmental dieldrin exposure exacerbates PFF-induced toxicity in male mice only. Specifically, in male offspring, dieldrin exacerbated PFF-induced motor deficits on the challenging beam and increased DA turnover in the striatum 6 months after PFF injection. However, male offspring showed neither exacerbation of phosphorylated α-syn aggregation (pSyn) in the substantia nigra (SN) at 1 or 2 months post-PFF injection, nor exacerbation of PFF-induced TH and NeuN loss in the SN 6 months post-PFF injection. Collectively, these data indicate that developmental dieldrin exposure produces a male-specific exacerbation of synucleinopathy-induced behavioral and biochemical deficits. This sex-specific result is consistent with both previous work in the MPTP model, our previously reported sex-specific effects of this exposure paradigm on the male and female epigenome, and the higher prevalence and more severe course of PD in males. The novel two-hit environmental toxicant/PFF exposure paradigm established in this project can be used to explore the mechanisms by which other PD-related exposures alter neuronal vulnerability to synucleinopathy in sporadic PD.
Project description:Accumulation of misfolded alpha-synuclein (?-syn) into Lewy bodies (LBs) and Lewy neurites (LNs) is a major hallmark of Parkinson's disease (PD) and dementia with LBs (DLB). Recent studies showed that synthetic preformed fibrils (pffs) recruit endogenous ?-syn and induce LB/LN pathology in vitro and in vivo, thereby implicating propagation and cell-to-cell transmission of pathological ?-syn as mechanisms for the progressive spread of LBs/LNs. Here, we demonstrate that ?-syn monoclonal antibodies (mAbs) reduce ?-syn pff-induced LB/LN formation and rescue synapse/neuron loss in primary neuronal cultures by preventing both pff uptake and subsequent cell-to-cell transmission of pathology. Moreover, intraperitoneal (i.p.) administration of mAb specific for misfolded ?-syn into nontransgenic mice injected intrastriatally with ?-syn pffs reduces LB/LN pathology, ameliorates substantia nigra dopaminergic neuron loss, and improves motor impairments. We conclude that ?-syn antibodies could exert therapeutic effects in PD/DLB by blocking entry of pathological ?-syn and/or its propagation in neurons.
Project description:Parkinson's disease (PD) and multiple system atrophy (MSA) are neurodegenerative diseases characterized by inclusions mainly composed of ?-synuclein (?-syn) aggregates. The objective of this study was to investigate if ?-synuclein (?-syn) overexpression could have beneficial effects by inhibiting the aggregation of ?-syn. The M83 transgenic mouse is a model of synucleinopathy, which develops severe motor symptoms associated with aggregation of ?-syn. M83 neonate or adult mice were injected with adeno-associated virus vectors carrying the human ?-syn gene (AAV?-syn) or green fluorescent protein gene (AAVGFP) using different injection sites. The M83 disease was - or not - accelerated using extracts of M83 brains injected with brain extract from mouse (M83) or human (MSA) origins. AAV vectors expression was confirmed using Western blot and ELISA technics. AAV mediated ?-syn overexpression did not delay the disease onset or reduce the ?-syn phosphorylated at serine 129 levels detected by ELISA, regardless of the AAV injection route and the inoculation of brain extracts. Instead, a proteinase-K resistant ?-syn staining was detected by immunohistochemistry, specifically in sick M83 mice overexpressing ?-syn after inoculation of AAV?-syn. This study indicated for the first time that viral vector-mediated ?-syn overexpression could form aggregates in a model of synucleinopathy.
Project description:Pathology consisting of intracellular aggregates of alpha-Synuclein (α-Syn) spread through the nervous system in a variety of neurodegenerative disorders including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. The discovery of structurally distinct α-Syn polymorphs, so-called strains, supports a hypothesis where strain-specific structures are templated into aggregates formed by native α-Syn. These distinct strains are hypothesised to dictate the spreading of pathology in the tissue and the cellular impact of the aggregates, thereby contributing to the variety of clinical phenotypes. Here, we present evidence of a novel α-Syn strain induced by the multiple system atrophy-associated oligodendroglial protein p25α. Using an array of biophysical, biochemical, cellular, and in vivo analyses, we demonstrate that compared to α-Syn alone, a substoichiometric concentration of p25α redirects α-Syn aggregation into a unique α-Syn/p25α strain with a different structure and enhanced in vivo prodegenerative properties. The α-Syn/p25α strain induced larger inclusions in human dopaminergic neurons. In vivo, intramuscular injection of preformed fibrils (PFF) of the α-Syn/p25α strain compared to α-Syn PFF resulted in a shortened life span and a distinct anatomical distribution of inclusion pathology in the brain of a human A53T transgenic (line M83) mouse. Investigation of α-Syn aggregates in brain stem extracts of end-stage mice demonstrated that the more aggressive phenotype of the α-Syn/p25α strain was associated with an increased load of α-Syn aggregates based on a Förster resonance energy transfer immunoassay and a reduced α-Syn aggregate seeding activity based on a protein misfolding cyclic amplification assay. When injected unilaterally into the striata of wild-type mice, the α-Syn/p25α strain resulted in a more-pronounced motoric phenotype than α-Syn PFF and exhibited a "tropism" for nigro-striatal neurons compared to α-Syn PFF. Overall, our data support a hypothesis whereby oligodendroglial p25α is responsible for generating a highly prodegenerative α-Syn strain in multiple system atrophy.