Parkinson’s disease (PD) is the second most common neurodegenerative disorder. Its cause(s) are predominantly unknown; however, a subset of cases has a genetic origin. Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common genetic cause of PD. Cases are clinically indistinguishable from idiopathic PD and display incomplete penetrance. Thereby, it is predicted that genetic vulnerability combined with environmental factors cause pathogenesis. However, the identity of these factors is unknown. Unfortunately, LRRK2`s native and pathogenic biological function(s) remain to be defined; owing to obstacles including a complex protein structure and the lack of pathological phenotypes in LRRK2 research models. To address the knowledge gap in LRRK2 biology, we set out to investigate the role of LRRK2 in the central nervous system (CNS). We generated and characterized a disease-mimicking D. melanogaster model of LRRK2-linked PD. This system was utilized to perform an in vivo, unbiased, high-throughput genetic screen to identify candidate interactors of LRRK2. Successful identification of a discrete number of genetic interactors was accomplished and, coupled with published evidence, highlighted the pursuit of subsequent mitochondrial-related investigations of LRRK2. These studies were performed using the M. musculus model system. Since LRRK2 murine models lack disease-relevant phenotypes, and LRRK2’s incomplete penetrance is predicted to be the result of gene-environment interaction, we employed the mitochondrial-targeting exogenous neurotoxin – MPTP/MPP+, to investigate neuronal mitochondrial phenotypes and subsequent survival in the context of LRRK2. Using the pathogenic R1441 GTPase-linked mutation, we did not observe altered neuronal mitochondrial length phenotypes or enhanced CNS sensitization to MPTP/MPP+-induced death; highlighting that MPTP-mediated, mitochondrial-centered mechanisms of action should be approached cautiously in the context of R1441-LRRK2. Collectively, the work presented herein has unveiled novel targets for the exploration of LRRK2 biological function and encourages the investigation of alternative pathogenic trigger mechanisms in the context of LRRK2-linked PD.

Amyotrophic Lateral Sclerosis (ALS) is a rare and fatal neurodegenerative disorder resulting in the loss of motor neurons from the spinal cord and frontal cortex. The patterns of neurodegeneration, affected regions, age of onset, and time course of disease progression are all highly variable between and within variants of the disease. Familial ALS (fALS), inherited versions of ALS due to genetic changes, accounts for between 5-20% of all ALS cases, while the rest are sporadic, with either no causative mutation identified or no familial history of ALS. Recently, the discovery of C9orf72 hexanucleotide repeat expansions have been identified as one of the most common causes of familial ALS, with some patients presenting with dual phenotypes of ALS and frontotemporal dementia, leading to new hypotheses about the nature of neurodegenerative diseases. Despite the continued discovery of new ALS causative genes, little is known about the pathogenesis of the disease. While almost all variants include the presence of intracellular protein inclusions, the site of the protein plaques and involved proteins varies between genetic and phenotypic variants of this disease. Due to the lack of clear pathogenic mechanisms, several hypotheses have been developed to explain the process of neurodegeneration. Autophagy, the process of self-eating, leading to destruction of damaged or excess proteins and organelles, has been implicated as being altered in ALS. Multiple variants have demonstrated altered mitochondrial morphology and cellular energetic dynamics, which could explain previous observations that implicate the process of apoptosis in cellular death. Many of the involved proteins in ALS have functional roles for intracellular, nucleocytoplasmic, and axonal transport of various proteins or RNA. These three competing hypotheses are currently the most prominent hypotheses in the pathogenesis of ALS, and have largely been considered as separate and competing in past research. Is there a chance that the true pathogenesis leading to neuronal destruction via apoptosis involve all three hypotheses? Altered protein and RNA transport dynamics could lead to changes in cellular stress responses or overload autophagy pathways, leading to exacerbated cellular stress responses, leading to alterations in mitochondrial morphology and eventually cell death via apoptosis.

Heat shock protein A12A (HSPA12A) is a newly discovered member of the Hsp70 family. The biological characteristics and functional roles of HSPA12A are poorly understood. This study investigated the effects of HSPA12A on ischaemic stroke in mice. Ischaemic stroke was induced by left middle cerebral artery occlusion for 1 h followed by blood reperfusion. We observed that HSPA12A was highly expressed in brain neurons, and neuronal HSPA12A expression was downregulated by ischaemic stroke and stroke-associated risk factors (aging, obesity and hyperglycaemia). To investigate the functional requirement of HSPA12A in protecting ischaemic brain injury, HSPA12A knockout mice (Hspa12a−/−) were generated. Hspa12a−/− mice exhibited an enlarged infarct volume and aggravated neurological deficits compared to their wild-type (WT) littermates after stroke. These aggravations in Hspa12a−/− mice were accompanied by more apoptosis and severer hippocampal morphological abnormalities in ischaemic hemispheres. Long-term examination revealed impaired motor function recovery and neurogenesis in stroke-affected Hspa12a−/− mice compared to stroke-affected WT controls. Significant reduced activation of GSK-3β/mTOR/p70S6K signalling was also observed in ischaemic hemispheres of Hspa12a−/− mice compared to WT controls. Administration with lithium (non-selective GSK-3β inhibitor) activated GSK-3β/mTOR/p70S6K signalling in stroke-affected Hspa12a−/− mice. Notably, lithium administration attenuated the HSPA12A deficiency-induced aggravation in infarct size, neurological deficits and neuronal death in Hspa12a−/− mice after stroke. Altogether, the findings suggest that HSPA12A expression encodes a critical novel prosurvival pathway during ischaemic stroke. We identified HSPA12A as a novel neuroprotective target for stroke patients.

No description available.

Este trabalho teve como objetivo analisar os efeitos comportamentais e morfológicos da exposição ao campo magnético contínuo em um modelo experimental de doença de Huntington. Foram utilizados 68 ratos Wistar, machos, divididos em 6 grupos: GC (controle, n=12), GS (sham, n=12), GSM (sham magnético, n=8), GL (lesão, n=12), GEPS (polo sul, n=12) e GEPN (polo norte, n=12). O animais passaram por habituação ao Rota Rod durante 3 dias pré-cirúrgicos e por habituação ao monitor de atividade 24 horas antes da cirurgia. Após procedimentos cirúrgicos adequados, os GL, GEPS e GEPN receberam administração de ácido quinolínico (120nmol/2L) no núcleo estriado esquerdo. Os GS e GSM receberam administração de 2L de salina na mesma região. Ainda, nos GEPS e GEPN foi implantado no crânio de cada animal um magneto circular de neodímio (8x3mm) com potência de 3200 Gauss e no GSM foi realizado implante do mesmo material, sem estar magnetizado. No 7º dia pós-cirúrgico, os animais foram avaliados em relação à atividade motora espontânea no monitor de atividades, após 5 minutos da injeção subcutânea de apomorfina (2,5 mg/Kg), sendo que os animais do GC não receberam esta injeção, e atividade motora forçada no Rota Rod. Posteriormente aos experimentos os animais foram perfundidos e os encéfalos retirados para histologia. Os resultados da avaliação comportamental espontânea evidenciaram, para o comportamento de distância percorrida, um aumento significativo do GEPS em relação aos GC, GL e GEPN, e também do GSM e GS em relação ao GC, GL e GEPN. Observamos também uma diminuição significativa do GEPN em relação aos GS, GSM, GL e GEPS [F(5,62) = 3,19; p0,05]. Para o tempo de atividade, um aumento significativo do GEPS em relação aos GC e GEPN, e também do GSM e GS em relação ao GC, GL e GEPN. Observamos também uma diminuição significativa do GEPN em relação aos GS, GSM, GL e GEPS [F(5,62) = 5,46; p0,05]. Para o comportamento de cruzamentos, um aumento significativo do GEPS em relação aos GC e GEPN e também do GSM e GS em relação ao GC, GL e GEPN. Observamos também uma diminuição significativa do GEPN em relação aos GS, GSM, GL, e GEPS [F(5,62) = 3,31; p0,05]. E para o comportamento de giros anti-horários (ipsilaterais a lesão) um aumento significativo dos GL, GEPS e GEPN em relação aos GC, GS e GSM. Observamos também uma diminuição significativa dos GEPN e GEPS em relação ao GL e ainda uma diminuição do GEPN em relação ao GEPS [F(5,62) = 16,01; p0,05]. Os resultados referentes ao Rota Rod (atividade motora forçada) revelaram diminuição significativa do tempo de permanência no aparato do GL em relação aos demais GC, GS, GSM, GEPS e GEPN [(F(5,62) = 5,46; p0,05)]. A análise histológica revelou uma perda significativa de neurônios no núcleo estriado esquerdo do GL em relação aos demais GC, GS, GSM, GEPS e GEPN [(F(5,66) = 5,13; p0,05)]. Dessa forma, os resultados obtidos sugerem que a estimulação magnética exerce efeito neuroprotetor, com reversão das alterações comportamentais e morfológicas promovidas pelo ácido quinolínico. / The aim of this study was to analyze the behavioral and morphologic effects of the static magnetic field exposition in an animal model of Huntingtons disease. Sixty- eight male Wistar rats were used, placed in 6 groups: GC (control group, n=12), GS (sham group, n=12), GSM (sham magnetic group, n=8), GL (lesion group, n=12), GEPS (south pole stimulated group, n=12) and GEPN (north pole stimulated group, n=12). The animals passed through habituation to Rota Rod, during the 3 days pre-surgical, and to habituation to the activity monitor, 24 hours before surgery. After appropriate surgical procedures GL, GEPS and GEPN received administration of quinolinic acid (120nmol/ 2L) in the left striatum. The GS and GSM received administration of 2L of saline in the same region. Also, in GEPS and GEPN was implanted, on the skull of each animal, a circular neodymium magnet (8x3mm) with a power of 3200 gauss, the GSM was performed the implant of the same material, without being magnetized. On the seventh after surgery day, the animals were evaluated referring to spontaneous motor activity in the activity monitor, 5 minutes after subcutaneous injection of apomorphine (2.5 mg / kg), whereas the animals of the CG did not receive this injection, and forced motor activity in Rota Rod. Subsequently the experiments the animals were perfused and their brains removed for histology. The results showed to spontaneous behavioral assessment, related to the behavior of distance travelled, significant increase in GEPS compared to GC, GL e GEPN, and also in GSM and GS compared to GC, GL, and GEPN, and a decrease in GEPN compared to GC, GS, GSM, GL e GEPS [F (5,62) = 3.19, p 0.05]; for time of activity, a significant increase in GEPS compared to GC and GEPN, and also in GSM and GS compared to GC, GL and GEPN, and a decrease in GEPN compared to GS, GSM, GL e GEPS [F (5,62) = 5.46, p 0.05]; for the behavior of crossings, a significant increase in GEPS compared to GC and GEPN, and also in GSM and GS compared to GC, GL and GEPN, and a decrease in GEPN compared to GS, GSM, GL, e GEPS [F (5,62) = 3.31, p 0.05]; and to the behavior of anti hourly rotations, significant increase in GL, GEPS and GEPN compared to GC, GS, GSM, significant decrease in GEPN and GEPS compared to GL and also a decrease in GEPN compared to GEPS [F (5 , 62) = 16.01, p 0.05]. The results for the Rota Rod indicated a significant decrease in the permanency time on apparatus to GL compared to GC, GS, GSM, GEPS and GEPN [(F (5, 62) = 5.46, p 0.05)]. The histological analysis revealed a significant reduction in the number of neurons in the animals of GL compared to the others groups [F (5, 66) = 5, 13, p 0.05]. Therefore, the results suggest that magnetic stimulation exerts neuroprotective effect, with reversal of behavioral and morphological changes caused by quinolinic acid.

Este trabalho teve como objetivo analisar os efeitos comportamentais e morfológicos da exposição ao campo magnético contínuo em um modelo experimental de doença de Huntington. Foram utilizados 68 ratos Wistar, machos, divididos em 6 grupos: GC (controle, n=12), GS (sham, n=12), GSM (sham magnético, n=8), GL (lesão, n=12), GEPS (polo sul, n=12) e GEPN (polo norte, n=12). O animais passaram por habituação ao Rota Rod durante 3 dias pré-cirúrgicos e por habituação ao monitor de atividade 24 horas antes da cirurgia. Após procedimentos cirúrgicos adequados, os GL, GEPS e GEPN receberam administração de ácido quinolínico (120nmol/2L) no núcleo estriado esquerdo. Os GS e GSM receberam administração de 2L de salina na mesma região. Ainda, nos GEPS e GEPN foi implantado no crânio de cada animal um magneto circular de neodímio (8x3mm) com potência de 3200 Gauss e no GSM foi realizado implante do mesmo material, sem estar magnetizado. No 7º dia pós-cirúrgico, os animais foram avaliados em relação à atividade motora espontânea no monitor de atividades, após 5 minutos da injeção subcutânea de apomorfina (2,5 mg/Kg), sendo que os animais do GC não receberam esta injeção, e atividade motora forçada no Rota Rod. Posteriormente aos experimentos os animais foram perfundidos e os encéfalos retirados para histologia. Os resultados da avaliação comportamental espontânea evidenciaram, para o comportamento de distância percorrida, um aumento significativo do GEPS em relação aos GC, GL e GEPN, e também do GSM e GS em relação ao GC, GL e GEPN. Observamos também uma diminuição significativa do GEPN em relação aos GS, GSM, GL e GEPS [F(5,62) = 3,19; p0,05]. Para o tempo de atividade, um aumento significativo do GEPS em relação aos GC e GEPN, e também do GSM e GS em relação ao GC, GL e GEPN. Observamos também uma diminuição significativa do GEPN em relação aos GS, GSM, GL e GEPS [F(5,62) = 5,46; p0,05]. Para o comportamento de cruzamentos, um aumento significativo do GEPS em relação aos GC e GEPN e também do GSM e GS em relação ao GC, GL e GEPN. Observamos também uma diminuição significativa do GEPN em relação aos GS, GSM, GL, e GEPS [F(5,62) = 3,31; p0,05]. E para o comportamento de giros anti-horários (ipsilaterais a lesão) um aumento significativo dos GL, GEPS e GEPN em relação aos GC, GS e GSM. Observamos também uma diminuição significativa dos GEPN e GEPS em relação ao GL e ainda uma diminuição do GEPN em relação ao GEPS [F(5,62) = 16,01; p0,05]. Os resultados referentes ao Rota Rod (atividade motora forçada) revelaram diminuição significativa do tempo de permanência no aparato do GL em relação aos demais GC, GS, GSM, GEPS e GEPN [(F(5,62) = 5,46; p0,05)]. A análise histológica revelou uma perda significativa de neurônios no núcleo estriado esquerdo do GL em relação aos demais GC, GS, GSM, GEPS e GEPN [(F(5,66) = 5,13; p0,05)]. Dessa forma, os resultados obtidos sugerem que a estimulação magnética exerce efeito neuroprotetor, com reversão das alterações comportamentais e morfológicas promovidas pelo ácido quinolínico. / The aim of this study was to analyze the behavioral and morphologic effects of the static magnetic field exposition in an animal model of Huntingtons disease. Sixty- eight male Wistar rats were used, placed in 6 groups: GC (control group, n=12), GS (sham group, n=12), GSM (sham magnetic group, n=8), GL (lesion group, n=12), GEPS (south pole stimulated group, n=12) and GEPN (north pole stimulated group, n=12). The animals passed through habituation to Rota Rod, during the 3 days pre-surgical, and to habituation to the activity monitor, 24 hours before surgery. After appropriate surgical procedures GL, GEPS and GEPN received administration of quinolinic acid (120nmol/ 2L) in the left striatum. The GS and GSM received administration of 2L of saline in the same region. Also, in GEPS and GEPN was implanted, on the skull of each animal, a circular neodymium magnet (8x3mm) with a power of 3200 gauss, the GSM was performed the implant of the same material, without being magnetized. On the seventh after surgery day, the animals were evaluated referring to spontaneous motor activity in the activity monitor, 5 minutes after subcutaneous injection of apomorphine (2.5 mg / kg), whereas the animals of the CG did not receive this injection, and forced motor activity in Rota Rod. Subsequently the experiments the animals were perfused and their brains removed for histology. The results showed to spontaneous behavioral assessment, related to the behavior of distance travelled, significant increase in GEPS compared to GC, GL e GEPN, and also in GSM and GS compared to GC, GL, and GEPN, and a decrease in GEPN compared to GC, GS, GSM, GL e GEPS [F (5,62) = 3.19, p 0.05]; for time of activity, a significant increase in GEPS compared to GC and GEPN, and also in GSM and GS compared to GC, GL and GEPN, and a decrease in GEPN compared to GS, GSM, GL e GEPS [F (5,62) = 5.46, p 0.05]; for the behavior of crossings, a significant increase in GEPS compared to GC and GEPN, and also in GSM and GS compared to GC, GL and GEPN, and a decrease in GEPN compared to GS, GSM, GL, e GEPS [F (5,62) = 3.31, p 0.05]; and to the behavior of anti hourly rotations, significant increase in GL, GEPS and GEPN compared to GC, GS, GSM, significant decrease in GEPN and GEPS compared to GL and also a decrease in GEPN compared to GEPS [F (5 , 62) = 16.01, p 0.05]. The results for the Rota Rod indicated a significant decrease in the permanency time on apparatus to GL compared to GC, GS, GSM, GEPS and GEPN [(F (5, 62) = 5.46, p 0.05)]. The histological analysis revealed a significant reduction in the number of neurons in the animals of GL compared to the others groups [F (5, 66) = 5, 13, p 0.05]. Therefore, the results suggest that magnetic stimulation exerts neuroprotective effect, with reversal of behavioral and morphological changes caused by quinolinic acid.

Calpains, a highly conserved family of calcium-dependent cysteine proteases, are divided in two groups; classical and non-conventional calpains. Calpain-1 and calpain-2, the classical ones, are ubiquitously expressed and abundant in the CNS. Findings through different experimental approaches, predominantly pharmacological calpain inhibitors, proposed the necessity of the proteases for the modulation of various biological events particularly in the CNS, or a functional link between calpain and neurodegeneration. Significant functions associated with calpain activity are neuronal proliferation/differentiation, signal transduction, apoptosis, and synaptic plasticity; or neuronal death in Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, and ischemic stroke. However, due to limited insights of the approaches taken, such as non-specificity of the inhibitors, the exact roles of calpains in the CNS and the key mechanisms underlying them remain controversial. Calpain-1/calpain-2 germline knock-out are embryonic lethal at a very early stage hindering the use of these lines as mouse models for CNS studies. Accordingly, this thesis research introduced a unique brain-specific calpain-1/calpain-2 knock-out and explored the role of the proteases in brain development/function and in neuronal death. The first set of analyses examined how the elimination of calpain-1/calpain-2 activities in mouse brain impacts CNS development in general and synaptic plasticity in CA1 neurons of hippocampus. CNS-specific elimination of CAPNS1, the common small subunit, abolished calpain-1/calpain-2 activities in mouse brain. In contrast to Calpain-1/calpain-2 germ line knock-outs, the brain-specific knock-outs are viable and the general development of mouse brain is normal. However, morphology of dendrites in pyramidal neurons of the hippocampal CA1 region showed significantly decreased dendritic branching complexity and spine density. Consistent with dendrite morphological abnormalities, electrophysiological analyses revealed a significant decrease in field excitatory postsynaptic potentials, long term potentiation, and learning and memory in the hippocampal CA1 neurons of the mutants. In the second part of this research we investigated the direct role of the calpains in neuronal death and their potential downstream targets in in vitro models of PD and ischemic stroke. Our findings indicated that ablation of calpains activity improves survival of different types of neurons against mitochondrial toxin 1-methyl-4-phenylpyridinium (MPP+), glutamate, and hypoxia. Importantly, we demonstrated an increase in p35-cleavage to p25, a cyclin dependent kinase 5 (Cdk5) activator, and that restoration of p25 significantly suppresses the neuronal survival associated with calpain deficiency. Taken together, this work unequivocally establishes two central roles of calpain-1/calpain-2 in CNS function in plasticity and neuronal death.

La maladie de Parkinson (MP) est caractérisée par une dégénérescence des neurones dopaminergiques de la substance noire (SN) en lien avec l’agrégation de l’α-synucléine (α-Syn) sous la forme d’inclusions intraneuronales. De nombreux travaux supportent l’idée que des interactions neuro-immunes, favorisées par des altérations du système vasculaire cérébral, sont impliquées dans les mécanismes pathologiques de la MP. Au cours de ce travail, nous nous sommes intéressés à une population myéloïde particulière qui borde les vaisseaux: les macrophages périvasculaires (MPVs). Nous mettons en évidence chez des patients parkinsoniens et des souris développant une synucléinopathie dégénérative, une augmentation proliférative des MPVs. Leur ablation entraîne une aggravation du phénotype dégénératif chez les souris synucléinopathiques, suggérant un rôle protecteur qui ne serait toutefois pas lié à une régulation de la microgliose. Nous observons néanmoins que l’absence de MPVs s’associe à une augmentation vasculaire de VCAM-1 et de l’infiltrat T CD4+ et CD8+. Par ailleurs, leur absence entraîne une accélération de la diffusion de la synucléinopathie suite à l’injection de fibrilles d’α-Syn. Nous faisons l’hypothèse que les MPVs sont essentiels dans la clairance des assemblages extracellulaires d’α-Syn en raison de leur activité de phagocytose mais aussi de leur importance dans la régulation des flux paravasculaires et lymphatiques, comme le montrent nos expériences d’infusion de traceurs fluorescents. Au total, nos travaux soulignent l’importance des MPVs dans la régulation de la toxicité et la diffusion de la synucléinopathie et en font une cible thérapeutique potentielle pour la MP. / Parkinson’s disease (PD) is characterized by the progressive loss of dopaminergic neurons in the substantia nigra (SN) associated to the aggregation of α-synuclein (α-Syn) that forms intraneuronal inclusions. In addition, mounting evidence suggest that neuro-immune interactions, favoured by cerebrovascular alterations, are involved in the pathomechanisms of PD. In this work, we focus on a myeloid population that surrounds blood vessels: perivascular macrophages (PVMs). We show that both in PD patients and in a mouse model of degenerative synucleinopathy, there is an increased proliferative recruitment of PVMs within the SN. We also found that specific ablation of PVMs aggravates the degenerative phenotype in synucleinopathic mice. PVMs-mediated neuroprotection is not likely linked to regulatory mechanisms of microglial cell response. Interestingly, the absence of PVMs is associated with higher vascular expression of VCAM-1 and enhanced infiltration of CD4+ and CD8+ T-cells, whose pathogenic role remains to be determined. We also observe that PVMs deficiency leads to an increased spreading of α-Syn pathology following striatal injections of α-Syn fibrils. We hypothesize that PVMs might be essential for the clearance of toxic α-Syn species due to their phagocytic activity, but also to their importance in regulating interstitial and cerebrospinal fluid (CSF) circulation, as shown by analysing CSF movement via the infusion of fluorescent tracers in PVMs-deficient conditions. Overall, our data highlight the importance of PVMs in regulating the toxicity and propagation of synucleinopathy and provide a rationale for therapeutic strategies aimed at boosting PVMs functions in PD.

Synucleins represent a conserved family of small proteins that include α-, β-, and γ- isoforms, which are highly expressed in neurons of the vertebrate nervous system. The normal function of these proteins is not well understood. However, in humans α- synuclein dysfunction is causatively linked to Parkinson’s Disease (PD), where it abnormally accumulates in neuronal cell bodies as protein aggregates that are associated with neuronal death. Although the associations between synuclein accumulation and cellular death are established in PD, the extent to which this occurs in other contexts, such as neuronal injury, is unknown. Furthermore, the effects of synuclein aggregation on the function of synapses, where synuclein is normally localized, are not well understood. To address these questions I took advantage of the experimentally accessible nervous system of the sea lamprey (Petromyzon marinus). I used molecular cloning and phylogenetic analyses to characterize three lamprey synuclein orthologues, one of which is highly expressed within a class of neurons called the giant reticulospinal (RS) neurons. Spinal cord injury induces the accumulation of synuclein protein only within a population of poor surviving RS neurons, and this accumulation is correlated with cellular death. Thus, similar to PD, the abundance of synuclein protein is associated with neuronal toxicity. In a related project, I demonstrated that elevating synuclein levels at synapses, such as occurs in PD, is deleterious to synaptic function through an inhibition of synaptic vesicle (SV) recycling. By injecting excess synuclein protein directly into the axons of giant RS neurons, and analyzing the ultrastructural morphology of synapses, I have shown that clathrin-mediated synaptic vesicle endocytosis was greatly inhibited. The conserved N-terminal domain was sufficient to inhibit vesicle recycling, and injecting synuclein mutants with disrupted N-terminal α-helices caused reduced defects in SV recycling. Therefore the α-helical structure of the N-terminus is necessary to inhibit SV recycling at early stages of clathrin-mediated endocytosis. Binding interactions with clathrin-mediated endocytosis components, such as the phosphoinositide lipid PI(4)P support this hypothesis. These studies provide a better understanding of the mechanisms by which synuclein dysfunction leads to neuronal death after injury and synaptic dysfunction in PD and other synuclein-associated diseases. / text

La conversion conformationnelle de la protéine prion cellulaire PrPC neuroprotectrice en protéine prion PrPSc infectieuse et pathogène caractérise les maladies à prions. Dans le cerveau infecté par les prions, la perte de PrPC, le gain de PrPSc neurotoxique et l’inflammation concourent à la mort neuronale par des mécanismes encore mal connus.Ces travaux valident les cultures organotypiques de cervelet de souris comme système expérimental ex vivo favorable à l’étude de ces mécanismes et montrent que l’absence de PrPC aussi bien que PrPSc activent des mécanismes apoptotiques et autophagiques qui conduisent à la mort des cellules de Purkinje du cervelet. Une deuxième étude in situ chez la souris montre que la compartimentation anatomo-fonctionnelle du cervelet est un paramètre endogène de la pathogenèse des prions de tremblante 22L. Une troisième série d’expériences in situ montre que les prions provoquent l’augmentation du récepteur TNFR1 de la cytokine pro-inflammatoire TNF-α à la membrane des astrocytes enveloppant les synapses excitatrices des cellules de Purkinje dans le cortex cérébelleux de souris infectées. Ceci implique une composante astrocytaire dans la réaction des complexes synaptiques aux prions. / The conversion of the protective cellular prion protein PrPC into an infectious, neurotoxic conformer PrPSc is a feature of prion diseases. In the prion-diseased brain, the loss of PrPC, the production of pathogenic PrPSc and inflammation contribute to neuronal death by still unknown mechanisms.The present results validate cerebellar organotypic cultures as a valuable experimental system to study ex vivo these mechanisms and provide insight into the apoptotic and autophagic processes activated by the absence of PrPC in Prnp-deficient mice and by PrPSc prions and lead to the death of the cerebellar Purkinje cells. A second line of research in situ showed that the anatomo-functional compartmentation of the mouse cerebellum is an endogenous parameter of the pathogenesis of the 22L scrapie prions. Finally, another in situ approach revealed that prions increase the levels of TNFR1, a receptor for the pro-inflammatory cytokine TNF-α at the membrane of the astrocytes enveloping Purkinje cell excitatory synapses in the cerebellar cortex of infected mice. This implies that the response of synaptic complexes to prions involves a glial component.