c-Abl phosphorylates α-synuclein and regulates its degradation: implication for α-synuclein clearance and contribution to the pathogenesis of Parkinson's disease

Increasing evidence suggests that the c-Abl protein tyrosine kinase could play a role in the pathogenesis of Parkinson's disease (PD) and other neurodegenerative disorders. c-Abl has been shown to regulate the degradation of two proteins implicated in the pathogenesis of PD, parkin and α-synuclein (α-syn). The inhibition of parkin's neuroprotective functions is regulated by c-Abl-mediated phosphorylation of parkin. However, the molecular mechanisms by which c-Abl activity regulates α-syn toxicity and clearance remain unknown. Herein, using NMR spectroscopy, mass spectrometry, in vitro enzymatic assays and cell-based studies, we established that α-syn is a bona fide substrate for c-Abl. In vitro studies demonstrate that c-Abl directly interacts with α-syn and catalyzes its phosphorylation mainly at tyrosine 39 (pY39) and to a lesser extent at tyrosine 125 (pY125). Analysis of human brain tissues showed that pY39 α-syn is detected in the brains of healthy individuals and those with PD. However, only c-Abl protein levels were found to be upregulated in PD brains. Interestingly, nilotinib, a specific inhibitor of c-Abl kinase activity, induces α-syn protein degradation via the autophagy and proteasome pathways, whereas the overexpression of α-syn in the rat midbrains enhances c-Abl expression. Together, these data suggest that changes in c-Abl expression, activation and/or c-Abl-mediated phosphorylation of Y39 play a role in regulating α-syn clearance and contribute to the pathogenesis of P

c-Abl phosphorylates α-synuclein and regulates its degradation: implication for α-synuclein clearance and contribution to the pathogenesis of Parkinson's disease

Increasing evidence suggests that the c-Abl protein tyrosine kinase could play a role in the pathogenesis of Parkinson's disease (PD) and other neurodegenerative disorders. c-Abl has been shown to regulate the degradation of two proteins implicated in the pathogenesis of PD, parkin and α-synuclein (α-syn). The inhibition of parkin's neuroprotective functions is regulated by c-Abl-mediated phosphorylation of parkin. However, the molecular mechanisms by which c-Abl activity regulates α-syn toxicity and clearance remain unknown. Herein, using NMR spectroscopy, mass spectrometry, in vitro enzymatic assays and cell-based studies, we established that α-syn is a bona fide substrate for c-Abl. In vitro studies demonstrate that c-Abl directly interacts with α-syn and catalyzes its phosphorylation mainly at tyrosine 39 (pY39) and to a lesser extent at tyrosine 125 (pY125). Analysis of human brain tissues showed that pY39 α-syn is detected in the brains of healthy individuals and those with PD. However, only c-Abl protein levels were found to be upregulated in PD brains. Interestingly, nilotinib, a specific inhibitor of c-Abl kinase activity, induces α-syn protein degradation via the autophagy and proteasome pathways, whereas the overexpression of α-syn in the rat midbrains enhances c-Abl expression. Together, these data suggest that changes in c-Abl expression, activation and/or c-Abl-mediated phosphorylation of Y39 play a role in regulating α-syn clearance and contribute to the pathogenesis of PD

Alix, un lien entre le système endolysosomal et la mort cellulaire.

Alix is an adaptor protein involved in the regulation of the endolysosomal system through binding to endophilins and CIN85, proteins involved in the receptor endocytosis and to ESCRT proteins involved in the endosomal function. Several observations suggest a role for Alix in controlling neuronal cell death through its interaction with ALG-2 protein. I wanted to test whether Alix may influence cell death of motoneurons (MTN) in vivo. The C-terminal part of Alix (Alix-CT) prevents early programmed cell death (PCD) in cervical MTN at day 4.5 of chick embryo development. This effect depends on the Alix-CT interaction with ALG-2 and ESCRT-I protein. Our results suggest that the interaction of the ALG-2/Alix complex with ESCRT proteins is necessary for the naturally occurring death of MTN. Therefore, Alix/ALG-2 complex could make a link between endosomes and a signalling or an execution step of neuronal death. I have shown that the deletion of CIN85 binding site, a protein involved in the TNFR1 endocytosis, in Alix-CT totally abolished the capacity of the latter to block the early death of MTN in vivo. I have found evidence that Alix functions downstream of the death-inducing receptor TNFR1, both in early MTN death in ovo and in vitro in cultured cells induced to die by TNFR1 overexpression. I have shown that Alix/ALG-2 complex interacts with TNFR1 localized on endosomes. Alix, together with ALG-2 seems to help recruiting and activating elements of the death machinery such as caspase 8 onto "death-inducing endosomes" containing activated TNFR1.Alix est une protéine adaptatrice impliquée dans la régulation du système endolysosomal grâce à son interaction avec des protéines comme CIN85 et les endophilines, protéines participant à l'endocytose de récepteurs ou les protéines des complexes ESCRT nécessaires à la maturation des compartiments endosomaux, Alix pourrait également contrôler la mort neuronale en se fixant à la protéine ALG-2. Le but de mon projet de thèse a été de tester si Alix intervient dans la mort des motoneurones au cours du développement in vivo et de définir les mécanismes moléculaires sous-jacents. J'ai montré que l'expression d'une protéine tronquée, correspondant à la moitié C-terminale d'Alix (Alix-CT), protège les motoneurones cervicaux de la mort cellulaire programmée précoce (PCD). Cette protection dépend de la liaison d'Alix-CT à ALG-2 et à ESCRT-I mettant en évidence l'implication du complexe Alix/ALG-2 avec les protéines ESCRT dans la mort naturelle des motoneurones : Alix ferait le lien entre la voie endolysosomale et la machinerie de mort cellulaire. J'ai montré que la délétion du site de liaison à CIN85, connue pour participer à l'endocytose du récepteur au TNF, le TNFR1, dans Alix-CT, inhibe sa capacité protectrice sur la PCD. La suite de mon travail démontre qu'Alix fonctionne en aval de la signalisation du TNFR1 à la fois in vivo et in vitro. Alix/ALG-2 interagit avec le TNFR1 au niveau des endosomes et pourrait permettre la formation d'une plateforme d'activation de la caspase-8 qui serait recrutée au niveau des « endosome de mort » contenant le TNFR1

Isolation of ORCTL3 in a novel genetic screen for tumor-specific apoptosis inducers

We have established a systematic high-throughput screen for genes that cause cell death specifically in transformed tumor cells. In a first round of screening, cDNAs that induce apoptosis in a transformed human cell line are detected. Positive genes are subsequently tested in a synthetic lethal screen in normal cells versus their isogenic counterparts that have been transformed by a particular oncogene. In this way, the organic cation transporter-like 3 (ORCTL3) gene was found to be inactive in normal rat kidney (NRK) cells, but to induce apoptosis in NRK cells transformed by oncogenic H-ras. ORCTL3 also causes cell death in v-src-transformed cells and in various human tumor cell lines but not in normal cells or untransformed cell lines. Although ORCTL3 is a member of the organic cation transporter gene family, our data indicate that this gene induces apoptosis independently of its putative transporter activity. Rather, various lines of evidence suggest that ORCTL3 brings about apoptosis by an endoplasmic reticulum stress-mediated mechanism. Finally, we detected ORCTL3 to be downregulated in human kidney tumors

Alix, making a link between apoptosis-linked gene-2, the endosomal sorting complexes required for transport, and neuronal death in vivo.

International audienceAlix/apoptosis-linked gene-2 (ALG-2)-interacting protein X is an adaptor protein involved in the regulation of the endolysosomal system through binding to endophilins and to endosomal sorting complexes required for transport (ESCRT) proteins, TSG101 and CHMP4b. It was first characterized as an interactor of ALG-2, a calcium-binding protein necessary for cell death, and several observations suggest a role for Alix in controlling cell death. We used electroporation in the chick embryo to test whether overexpressed wild-type or mutated Alix proteins influence cell death in vivo. We show that Alix overexpression is sufficient to induce cell death of neuroepithelial cells. This effect is strictly dependent on its capacity to bind to ALG-2. On the other hand, expression of Alix mutants lacking the ALG-2 or the CHMP4b binding sites prevents early programmed cell death in cervical motoneurons at day 4.5 of chick embryo development. This protection afforded by Alix mutants was abolished after deletion of the TSG101, but not of the endophilin, binding sites. Our results suggest that the interaction of the ALG-2/Alix complex with ESCRT proteins is necessary for naturally occurring death of motoneurons. Therefore, Alix represents a molecular link between the endolysosomal system and the cell death machinery

Health hazards of methylammonium lead iodide based perovskites: cytotoxicity studies

New technologies launch novel materials; besides their performances in products, their health hazards must be tested. This applies to the lead halide perovskite CH3NH3PbI3 as well, which offers fulgurate applications in photovoltaic devices. We report the effects of CH3NH3PbI3 photovoltaic perovskites in human lung adenocarcinoma epithelial cells (A549), human dopaminergic neuroblastoma cells (SH-SY5Y) and murine primary hippocampal neurons by using multiple assays and electron microscopy studies. In cell culture media the major part of the dissolved CH3NH3PbI3 has a strong cell-type dependent effect. Hippocampal primary neurons and neuroblastoma cells suffer a massive apoptotic cell death, whereas exposure to lung epithelial cells dramatically alters the kinetics of proliferation, metabolic activity and cellular morphology without inducing noticeable cell death. Our findings underscore the critical importance of conducting further studies to investigate the effect of short and long-term exposure to CH3NH3PbI3 on health and environment

Discovery and characterization of novel stable tau oligomeric complexes: Implications for the role of Tau/phospholipid interactions in regulating its functions in health and disease

The microtubule-associated protein Tau plays a central role in the pathogenesis of Alzheimer's disease. Although Tau interaction with membranes is thought to affect some of its physiological functions and its aggregation properties, the sequence determinants and the structural and functional consequences of such interactions remain poorly understood. Here, we report that the interaction of Tau with vesicles results in the formation of highly stable protein/phospholipid complexes. These complexes are toxic to primary hippocampal cultures and are detected by MC-1, an antibody recognizing pathological Tau conformations. The core of these complexes is comprised of the PHF6* and PHF6 hexapeptide motifs, the latter in a β-strand conformation. Studies using Tau-derived peptides enabled the design of mutants that disrupt Tau interactions with phospholipids without interfering with its ability to form fibrils, thus providing powerful tools for uncoupling these processes and investigating the role of membrane interactions in regulating Tau function, aggregation and toxicity

The Nt17 Domain and its Helical Conformation Regulate the Aggregation, Cellular Properties and Neurotoxicity of Mutant Huntingtin Exon 1

Converging evidence points to the N-terminal domain comprising the first 17 amino acids of the Huntingtin protein (Nt17) as a key regulator of its aggregation, cellular properties and toxicity. In this study, we further investigated the interplay between Nt17 and the polyQ domain repeat length in regulating the aggregation and inclusion formation of exon 1 of the Huntingtin protein (Httex1). In addition, we investigated the effect of removing Nt17 or modulating its local structure on the membrane interactions, neuronal uptake, and toxicity of monomeric or fibrillar Httex1. Our results show that the polyQ and Nt17 domains synergistically modulate the aggregation propensity of Httex1 and that the Nt17 domain plays important roles in shaping the surface properties of mutant Httex1 fibrils and regulating their poly-Q-dependent growth, lateral association and neuronal uptake. Removal of Nt17 or disruption of its transient helical conformations slowed the aggregation of monomeric Httex1 in vitro, reduced inclusion formation in cells, enhanced the neuronal uptake and nuclear accumulation of monomeric Httex1 proteins, and was sufficient to prevent cell death induced by Httex1 72Q overexpression. Finally, we demonstrate that the uptake of Httex1 fibrils into primary neurons and the resulting toxicity are strongly influenced by mutations and phosphorylation events that influence the local helical propensity of Nt17. Altogether, our results demonstrate that the Nt17 domain serves as one of the key master regulators of Htt aggregation, internalization, and toxicity and represents an attractive target for inhibiting Htt aggregate formation, inclusion formation, and neuronal toxicity

Alix and ALG-2 make a link between endosomes and neuronal death.

International audienceAlix [ALG-2 (apoptosis-linked gene 2)-interacting protein X] is a ubiquitinous adaptor protein first described for its capacity to bind to the calcium-binding protein, ALG-2. Alix regulates neuronal death in ways involving interactions with ALG-2 and with proteins of the ESCRT (endosomal sorting complex required for transport). Even though all Alix interactors characterized to date are involved in endosomal trafficking, the genuine function of the protein in this process remains unclear. We have demonstrated recently that Alix and ALG-2 form in the presence of calcium, a complex with apical caspases and with the endocytosed death receptor TNFR1 (tumour necrosis factor alpha receptor 1), thus suggesting a molecular coupling between endosomes and the cell death machinery