Motor neuron loss and neuroinflammation in a model of ?-synuclein-induced neurodegeneration.
ABSTRACT: 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:It has been hypothesized that ?-synuclein (?S) misfolding may begin in peripheral nerves and spread to the central nervous system (CNS), leading to Parkinson disease and related disorders. Although recent data suggest that ?S pathology can spread within the mouse brain, there is no direct evidence for spread of disease from a peripheral site. In the present study, we show that hind limb intramuscular (IM) injection of ?S can induce pathology in the CNS in the human Ala53Thr (M83) and wild-type (M20) ?S transgenic (Tg) mouse models. Within 2-3 mo after IM injection in ?S homozygous M83 Tg mice and 3-4 mo for hemizygous M83 Tg mice, these animals developed a rapid, synchronized, and predictable induction of widespread CNS ?S inclusion pathology, accompanied by astrogliosis, microgliosis, and debilitating motor impairments. In M20 Tg mice, starting at 4 mo after IM injection, we observed ?S inclusion pathology in the spinal cord, but motor function remained intact. Transection of the sciatic nerve in the M83 Tg mice significantly delayed the appearance of CNS pathology and motor symptoms, demonstrating the involvement of retrograde transport in inducing ?S CNS inclusion pathology. Outside of scrapie-mediated prion disease, to our knowledge, this findiing is the first evidence that an entire neurodegenerative proteinopathy associated with a robust, lethal motor phenotype can be initiated by peripheral inoculation with a pathogenic protein. Furthermore, this facile, synchronized rapid-onset model of ?-synucleinopathy will be highly valuable in testing disease-modifying therapies and dissecting the mechanism(s) that drive ?S-induced neurodegeneration.
Project description:Transgenic mice line M83 that express the A53T mutant α-synuclein protein at six times the level of endogenous mice α-synuclein are a model of α-synucleinopathy found in Parkinson's disease (PD). This Hualpha-Syn (A53T) PD model is useful in assessing non-motor deficits at earlier stages of onset of PD. We report findings on metabolic changes using [<sup>18</sup>F]FDG PET/CT in the Hualpha-Syn (A53T) PD mouse model in comparison to non-carrier mice. Whole-body PET/CT imaging of male and female mice were carried out 2 h after [<sup>18</sup>F]FDG ip administration under 3% isoflurane anesthesia. Brain images were analyzed with PET images coregistered to a mouse brain MRI template. Hualpha-Syn (A53T) mice had significantly lower [<sup>18</sup>F]FDG uptake in several brain regions compared to the no-carrier mice. Significant hind limb muscle and lower spinal cord [<sup>18</sup>F]FDG hypometabolism at 9 months of age in A53T PD mice was also indicative of neurodegenerative disease, with a progressive motoric dysfunction leading to death. Significant decrease (up to 30%) in [<sup>18</sup>F]FDG uptake were observed in 9-month old male and female Hualpha-Syn (A53) mice. This is consistent with the cortical hypometabolism in PD patients. Hualpha-Syn (A53) mice may thus be a suitable model for studies related to PD α-synucleinopathy for the discovery of new biomarkers.
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:Neurofilaments are a major component of the axonal cytoskeleton in neurons and have been implicated in a number of neurodegenerative diseases due to their presence within characteristic pathological inclusions. Their contributions to these diseases are not yet fully understood, but previous studies investigated the effects of ablating the obligate subunit of neurofilaments, low molecular mass neurofilament subunit (NFL), on disease phenotypes in transgenic mouse models of Alzheimer's disease and tauopathy. Here, we tested the effects of ablating NFL in ?-synuclein M83 transgenic mice expressing the human pathogenic A53T mutation, by breeding them onto an NFL null background. The induction and spread of ?-synuclein inclusion pathology was triggered by the injection of preformed ?-synuclein fibrils into the gastrocnemius muscle or hippocampus in M83 versus M83/NFL null mice. We observed no difference in the post-injection time to motor-impairment and paralysis endpoint or amount and distribution of ?-synuclein inclusion pathology in the muscle injected M83 and M83/NFL null mice. Hippocampal injected M83/NFL null mice displayed subtle region-specific differences in the amount of ?-synuclein inclusions however, pathology was observed in the same regions as the M83 mice. Overall, we observed only minor differences in the induction and transmission of ?-synuclein pathology in these induced models of synucleinopathy in the presence or absence of NFL. This suggests that NFL and neurofilaments do not play a major role in influencing the induction and transmission of ?-synuclein aggregation.
Project description:Peripheral administration (oral, intranasal, intraperitoneal, intravenous) of assembled A53T α-synuclein induced synucleinopathy in heterozygous mice transgenic for human mutant A53T α-synuclein (line M83). The same was the case when cerebellar extracts from a case of multiple system atrophy with type II α-synuclein filaments were administered intraperitoneally, intravenously or intramuscularly. We observed abundant immunoreactivity for pS129 α-synuclein in nerve cells and severe motor impairment, resulting in hindlimb paralysis and shortened lifespan. Filaments immunoreactive for pS129 α-synuclein were in evidence. A 70% loss of motor neurons was present five months after an intraperitoneal injection of assembled A53T α-synuclein or cerebellar extract with type II α-synuclein filaments from an individual with a neuropathologically confirmed diagnosis of multiple system atrophy. Microglial cells changed from a predominantly ramified to a dystrophic appearance. Taken together, these findings establish a close relationship between the formation of α-synuclein inclusions in nerve cells and neurodegeneration, accompanied by a shift in microglial cell morphology. Propagation of α-synuclein inclusions depended on the characteristics of both seeds and transgenically expressed protein.
Project description:Apolipoprotein E (APOE) ?4 genotype is associated with increased risk of dementia in Parkinson's disease (PD), but the mechanism is not clear, because patients often have a mixture of ?-synuclein (?Syn), amyloid-? (A?), and tau pathologies. APOE ?4 exacerbates brain A? pathology, as well as tau pathology, but it is not clear whether APOE genotype independently regulates ?Syn pathology. In this study, we generated A53T ?Syn transgenic mice (A53T) on Apoe knockout (A53T/EKO) or human APOE knockin backgrounds (A53T/E2, E3, and E4). At 12 months of age, A53T/E4 mice accumulated higher amounts of brainstem detergent-insoluble phosphorylated ?Syn compared to A53T/EKO and A53T/E3; detergent-insoluble ?Syn in A53T/E2 mice was undetectable. By immunohistochemistry, A53T/E4 mice displayed a higher burden of phosphorylated ?Syn and reactive gliosis compared to A53T/E2 mice. A53T/E2 mice exhibited increased survival and improved motor performance compared to other APOE genotypes. In a complementary model of ?Syn spreading, striatal injection of ?Syn preformed fibrils induced greater accumulation of ?Syn pathology in the substantia nigra of A53T/E4 mice compared to A53T/E2 and A53T/EKO mice. In two separate cohorts of human patients with PD, APOE ?4/?4 individuals showed the fastest rate of cognitive decline over time. Our results demonstrate that APOE genotype directly regulates ?Syn pathology independent of its established effects on A? and tau, corroborate the finding that APOE ?4 exacerbates pathology, and suggest that APOE ?2 may protect against ?Syn aggregation and neurodegeneration in synucleinopathies.
Project description:Parkinson's disease (PD) and related synucleinopathies are characterized by chronic neuroinflammation leading to the premise that anti-inflammatory therapies could ameliorate synucleinopathy and associated sequelae. To test this idea, we used recombinant adeno-associated viruses (AAV) to express the anti-inflammatory cytokine, Interleukin (Il)-10, in Line M83 transgenic mice that expresses the PD-associated A53T mutant human α-synuclein (αSyn). Contrary to our expectations, we observed that intraspinal Il-10 expression initiated at birth upregulated microgliosis and led to early death in homozygous M83+/+ mice. We further observed that Il-10 preconditioning led to reduced lifespan in the hemizygous M83+/- mice injected with preformed αSyn aggregates in hindlimb muscles. To determine the mechanistic basis for these adverse effects, we took advantage of the I87A variant Il-10 (vIl-10) that has predominantly immunosuppressive properties. Sustained intraspinal expression of vIl-10 in preformed αSyn-aggregate seeded M83+/- mice resulted in earlier death, accelerated αSyn pathology, pronounced microgliosis, and increased apoptosis compared to control mice. AAV-vIl-10 expression robustly induced p62 and neuronal LC3B accumulation in these mice, indicating that Il-10 signaling mediated preconditioning of the neuraxis can potentially exacerbate αSyn accumulation through autophagy dysfunction in the neurons. Together, our data demonstrate unexpected adverse effects of both Il-10 and its immunosuppressive variant, vIl-10, in a mouse model of PD, highlighting the pleiotropic functions of immune mediators and their complex role in non-cell autonomous signaling in neurodegenerative proteinopathies.
Project description:Analysis of human pathology led Braak to postulate that ?-synuclein (?-syn) pathology could spread from the gut to brain via the vagus nerve. Here, we test this postulate by assessing ?-synucleinopathy in the brain in a novel gut-to-brain ?-syn transmission mouse model, where pathological ?-syn preformed fibrils were injected into the duodenal and pyloric muscularis layer. Spread of pathologic ?-syn in brain, as assessed by phosphorylation of serine 129 of ?-syn, was observed first in the dorsal motor nucleus, then in caudal portions of the hindbrain, including the locus coeruleus, and much later in basolateral amygdala, dorsal raphe nucleus, and the substantia nigra pars compacta. Moreover, loss of dopaminergic neurons and motor and non-motor symptoms were observed in a similar temporal manner. Truncal vagotomy and ?-syn deficiency prevented the gut-to-brain spread of ?-synucleinopathy and associated neurodegeneration and behavioral deficits. This study supports the Braak hypothesis in the etiology of idiopathic Parkinson's disease (PD).
Project description: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: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.