Investigating the neuroprotective effect of AAV-mediated ?-synuclein overexpression in a transgenic model of synucleinopathy.
ABSTRACT: 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:Parkinson's disease is a synucleinopathy that is characterized by motor dysfunction, death of midbrain dopaminergic neurons and accumulation of ?-synuclein (?-Syn) aggregates. Evidence suggests that ?-Syn aggregation can originate in peripheral tissues and progress to the brain via autonomic fibers. We tested this by inoculating the duodenal wall of mice with ?-Syn preformed fibrils. Following inoculation, we observed gastrointestinal deficits and physiological changes to the enteric nervous system. Using the AAV-PHP.S capsid to target the lysosomal enzyme glucocerebrosidase for peripheral gene transfer, we found that ?-Syn pathology is reduced due to the increased expression of this protein. Lastly, inoculation of ?-Syn fibrils in aged mice, but not younger mice, resulted in progression of ?-Syn histopathology to the midbrain and subsequent motor defects. Our results characterize peripheral synucleinopathy in prodromal Parkinson's disease and explore cellular mechanisms for the gut-to-brain progression of ?-Syn pathology.
Project description:Neurodegenerative disorders with alpha-synuclein (?-syn) accumulation (synucleinopathies) include Parkinson's disease (PD), PD dementia, dementia with Lewy bodies and multiple system atrophy (MSA). Due to the involvement of toxic ?-syn aggregates in the molecular origin of these disorders, developing effective therapies targeting ?-syn is a priority as a disease-modifying alternative to current symptomatic treatments. Importantly, the clinical and pathological attributes of MSA make this disorder an excellent candidate as a synucleinopathy model for accelerated drug development. Recent therapeutic strategies targeting ?-syn in in vivo and in vitro models of MSA, as well as in clinical trials, have been focused on the pathological mechanisms of ?-syn synthesis, aggregation, clearance, and/or cell-to-cell propagation of its neurotoxic conformers. Here we summarize the most relevant approaches in this direction, with emphasis on their potential as general synucleinopathy modifiers, and enumerate research areas for potential improvement in MSA drug discovery.
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:The synucleinopathy underlying multiple system atrophy (MSA) is characterized by the presence of abundant amyloid inclusions containing fibrillar ?-synuclein (?-syn) aggregates in the brains of the patients and is associated with an extensive neurodegeneration. In contrast to Parkinson's disease (PD) where the pathological ?-syn aggregates are almost exclusively neuronal, the ?-syn inclusions in MSA are principally observed in oligodendrocytes (OLs) where they form glial cytoplasmic inclusions (GCIs). This is intriguing because differentiated OLs express low levels of ?-syn, yet pathogenic amyloid ?-syn seeds require significant amounts of ?-syn monomers to feed their fibrillar growth and to eventually cause the buildup of cytopathological inclusions. One of the transgenic mouse models of this disease is based on the targeted overexpression of human ?-syn in OLs using the PLP promoter. In these mice, the histopathological images showing a rapid emergence of S129-phosphorylated ?-syn inside OLs are considered as equivalent to GCIs. Instead, we report here that they correspond to the accumulation of phosphorylated ?-syn monomers/oligomers and not to the appearance of the distinctive fibrillar ?-syn aggregates that are present in the brains of MSA or PD patients. In spite of a propensity to co-sediment with myelin sheath contaminants, the phosphorylated forms found in the brains of the transgenic animals are soluble (>80%). In clear contrast, the phosphorylated species present in the brains of MSA and PD patients are insoluble fibrils (>95%). Using primary cultures of OLs from PLP-?Syn mice we observed a variable association of S129-phosphorylated ?-syn with the cytoplasmic compartment, the nucleus and with membrane domains suggesting that OLs functionally accommodate the phospho-?-syn deriving from experimental overexpression. Yet and while not taking place spontaneously, fibrillization can be seeded in these primary cultures by challenging the OLs with ?-syn preformed fibrils (PFFs). This indicates that a targeted overexpression of ?-syn does not model GCIs in mice but that it can provide a basis for seeding aggregation using PFFs. This approach could help establishing a link between ?-syn aggregation and the development of a clinical phenotype in these transgenic animals.
Project description:Synucleinopathies, neurodegenerative disorders with alpha-synuclein (α-syn) accumulation, are the second leading cause of neurodegeneration in the elderly, however no effective disease-modifying alternatives exist for these diseases. Multiple system atrophy (MSA) is a fatal synucleinopathy characterized by the accumulation of toxic aggregates of α-syn within oligodendroglial cells, leading to demyelination and neurodegeneration, and the reduction of this accumulation might halt the fast progression of MSA. In this sense, the involvement of microRNAs (miRNAs) in synucleinopathies is yet poorly understood, and the potential of manipulating miRNA levels as a therapeutic tool is underexplored. In this study, we analyzed the levels of miRNAs that regulate the expression of autophagy genes in MSA cases, and investigated the mechanistic correlates of miRNA dysregulation in <i>in vitro</i> models of synucleinopathy. We found that microRNA-101 (miR-101) was significantly increased in the striatum of MSA patients, together with a reduction in the expression of its predicted target gene <i>RAB5A</i>. Overexpression of miR-101 in oligodendroglial cell cultures resulted in a significant increase in α-syn accumulation, along with autophagy deficits. Opposite results were observed upon expression of an antisense construct targeting miR-101. Stereotaxic delivery of a lentiviral construct expressing anti-miR-101 into the striatum of the MBP-α-syn transgenic (tg) mouse model of MSA resulted in reduced oligodendroglial α-syn accumulation and improved autophagy. These results suggest that miRNA dysregulation contributes to MSA pathology, with miR-101 alterations potentially mediating autophagy impairments. Therefore, therapies targeting miR-101 may represent promising approaches for MSA and related neuropathologies with autophagy dysfunction.
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:?-synuclein (?S) is the major component of several types of brain pathological inclusions that define neurodegenerative diseases termed synucleinopathies. Central nervous system (CNS) inoculation studies using either in vitro polymerized ?S fibrils or in vivo derived lysates containing ?S aggregates to induce the progressive spread of ?S inclusion pathology in animal disease models have supported the notion that ?S mediated progressive neurodegeneration can occur by a prion-like mechanism. We have previously shown that neonatal brain inoculation with preformed ?S fibrils in hemizygous M20<sup>+/-</sup> transgenic mice expressing wild type human ?S and to a lesser extent in non-transgenic mice can result in a concentration-dependent progressive induction of CNS ?S pathology. Recent studies using brain lysates from patients with multiple system atrophy (MSA), characterized by ?S inclusion pathology in oligodendrocytes, indicate that these may be uniquely potent at inducing ?S pathology with prion-like strain specificity. We demonstrate here that brain lysates from MSA patients, but not control individuals, can induce ?S pathology following neonatal brain inoculation in transgenic mice expressing A53T human ?S (M83 line), but not in transgenic expressing wild type human ?S (M20 line) or non-transgenic mice within the timeframe of the study design. Further, we show that neuroanatomical and immunohistochemical properties of the pathology induced by MSA brain lysates is very similar to what is produced by the neonatal brain injection of preformed human ?S fibrils in hemizygous M83<sup>+/-</sup> transgenic mice. Collectively, these findings reinforce the idea that the intrinsic traits of the M83 mouse model dominates over any putative prion-like strain properties of MSA ?S seeds that can induce pathology.
Project description:Synucleinopathies are neurodegenerative disorders including Parkinson disease (PD), dementia with Lewy body (DLB), and multiple system atrophy (MSA) that involve deposits of the protein alpha-synuclein (α-syn) in the brain. The inoculation of α-syn aggregates derived from synucleinopathy or preformed fibrils (PFF) formed in vitro induces misfolding and deposition of endogenous α-syn. This is referred to as prion-like transmission, and the mechanism is still unknown. In this study, we label α-syn PFF with quantum dots and visualize their movement directly in acute slices of brain tissue inoculated with α-syn PFF seeds. Using this system, we find that the trafficking of α-syn seeds is dependent on fast axonal transport and the seed spreading is dependent on endocytosis and neuronal activity. We also observe pharmacological effects on α-syn seed spreading; clinically available drugs including riluzole are effective in reducing the spread of α-syn seeds and this effect is also observed in vivo. Our quantum-dot-labeled α-syn seed assay system combined with in vivo transmission experiment reveals an early phase of transmission, in which uptake and spreading of seeds occur depending on neuronal activity, and a later phase, in which seeds induce the propagation of endogenous misfolded α-syn.
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:We aimed to characterize in vivo ?-synuclein (?-syn) aggregates in skin nerves to ascertain: 1) the optimal marker to identify them; 2) possible differences between synucleinopathies that may justify the clinical variability. We studied multiple skin nerve ?-syn deposits in 44 patients with synucleinopathy: 15 idiopathic Parkinson's disease (IPD), 12 dementia with Lewy Bodies (DLB), 5 pure autonomic failure (PAF) and 12 multiple system atrophy (MSA). Ten healthy subjects were used as controls. Antibodies against native ?-syn, C-terminal ?-syn epitopes such as phosphorylation at serine 129 (p-syn) and to conformation-specific for ?-syn mature amyloid fibrils (syn-F1) were used. We found that p-syn showed the highest sensitivity and specificity in disclosing skin ?-syn deposits. In MSA abnormal deposits were only found in somatic fibers mainly at distal sites differently from PAF, IPD and DLB displaying ?-syn deposits in autonomic fibers mainly at proximal sites. PAF and DLB showed the highest p-syn load with a widespread involvement of autonomic skin nerve fibers.<h4>In conclusion</h4>1) p-syn in skin nerves was the optimal marker for the in vivo diagnosis of synucleinopathies; 2) the localization and load differences of aggregates may help to identify specific diagnostic traits and support a different pathogenesis among synucleinopathies.