Investigating the involvement and the regulation of autophagy in Parkinson's disease.

Autophagy is a conserved process that allows the degradation of intracellular debris within specific organelles, called lysosomes. The correct function of this cellular mechanism is crucial for the survival of neurons. Accordingly, the detrimental contribution of autophagic defects in the onset and progression of neurodegenerative diseases is widely accepted. For these reasons, the study of the molecular mechanisms of autophagy that are involved in neuronal damage and the characterization of the regulatory pathways that affect this process in neurodegeneration represent an important field in the research. In the last few years, it has emerged that autophagy and lysosomal functions are able to sense the stimuli that originate from other organelles and modulate their activity accordingly. This notion may be of great relevance in the context of neurodegenerative diseases, which are characterized by impairment at different cellular levels. In this scenario, the investigation of the crosstalk between organelles may offer new insights for a better understanding of these pathologies and eventually lead to novel therapeutic approaches. The communication between mitochondria and lysosomes has been gaining increasing attention, due to the crucial role of these organelles in neurodegeneration, and in particular, in Parkinson’s disease (PD). In this framework, it has been shown that in the familial forms of PD, mutations of lysosomal proteins may cause secondary perturbations in mitochondrial homeostasis. Conversely, PD-associated proteins involved in mitochondrial dynamics may participate in the regulation of the autophagic machinery. Among them, DJ-1 is a protein linked to PD, whose activity promotes in the maintenance of mitochondrial quality control and in the protection against oxidative stress. In addition, DJ-1 has also been associated with autophagic alterations, although its precise role is still elusive. Therefore, we evaluated how DJ-1 affects the autophagy-lysosomal pathway exploiting Drosophila melanogaster as an in vivo system, and human cell models for a deeper molecular characterization. Then, we investigated one of the possible signaling pathways that mediate the crosstalk between mitochondria and lysosomes in DJ-1 loss of function models. Overall, our data demonstrate that DJ-1 influences the autophagic process at different levels, through a signaling cascade that involves the modulation of the AMPK-mTORC1 pathway, which responds to the DJ-1-mediated increase of reactive oxygen species. Since autophagy activation is considered a good therapeutic strategy to counteract neurodegeneration, in parallel with the study on DJ-1, we also investigated a novel neuronal-specific pathway possibly involved in the regulation of autophagy. More specifically, our lab recently demonstrated the capability of the neuronal-enriched kinase PAK6 to phosphorylate the family of chaperone proteins 14-3-3s, affecting their interactome. Therefore, we considered the hypothesis that PAK6 may represent a modulator of TFEB, the major transcriptional activator of autophagy, whose function is highly dependent on the binding with 14-3-3s. In accordance with this hypothesis, the results we obtained demonstrated that PAK6 activity promotes the nuclear translocation of TFEB and the induction of autophagy in different in vivo and in vitro models. Importantly, our data suggest that PAK6 may participate to the regulation of TFEB through different mechanisms, by phosphorylating 14-3-3s and preventing their binding with the transcription factor and by directly interacting with TFEB. In conclusion, with these projects we investigated autophagy in the context of neurodegeneration, addressing this topic from different perspectives and exploiting multiple models. We characterized the participation of DJ-1 in the autophagic pathway and we contributed to provide novel insights on the mechanisms of TFEB and autophagy regulation in neuronal cells.Autophagy is a conserved process that allows the degradation of intracellular debris within specific organelles, called lysosomes. The correct function of this cellular mechanism is crucial for the survival of neurons. Accordingly, the detrimental contribution of autophagic defects in the onset and progression of neurodegenerative diseases is widely accepted. For these reasons, the study of the molecular mechanisms of autophagy that are involved in neuronal damage and the characterization of the regulatory pathways that affect this process in neurodegeneration represent an important field in the research. In the last few years, it has emerged that autophagy and lysosomal functions are able to sense the stimuli that originate from other organelles and modulate their activity accordingly. This notion may be of great relevance in the context of neurodegenerative diseases, which are characterized by impairment at different cellular levels. In this scenario, the investigation of the crosstalk between organelles may offer new insights for a better understanding of these pathologies and eventually lead to novel therapeutic approaches. The communication between mitochondria and lysosomes has been gaining increasing attention, due to the crucial role of these organelles in neurodegeneration, and in particular, in Parkinson’s disease (PD). In this framework, it has been shown that in the familial forms of PD, mutations of lysosomal proteins may cause secondary perturbations in mitochondrial homeostasis. Conversely, PD-associated proteins involved in mitochondrial dynamics may participate in the regulation of the autophagic machinery. Among them, DJ-1 is a protein linked to PD, whose activity promotes in the maintenance of mitochondrial quality control and in the protection against oxidative stress. In addition, DJ-1 has also been associated with autophagic alterations, although its precise role is still elusive. Therefore, we evaluated how DJ-1 affects the autophagy-lysosomal pathway exploiting Drosophila melanogaster as an in vivo system, and human cell models for a deeper molecular characterization. Then, we investigated one of the possible signaling pathways that mediate the crosstalk between mitochondria and lysosomes in DJ-1 loss of function models. Overall, our data demonstrate that DJ-1 influences the autophagic process at different levels, through a signaling cascade that involves the modulation of the AMPK-mTORC1 pathway, which responds to the DJ-1-mediated increase of reactive oxygen species. Since autophagy activation is considered a good therapeutic strategy to counteract neurodegeneration, in parallel with the study on DJ-1, we also investigated a novel neuronal-specific pathway possibly involved in the regulation of autophagy. More specifically, our lab recently demonstrated the capability of the neuronal-enriched kinase PAK6 to phosphorylate the family of chaperone proteins 14-3-3s, affecting their interactome. Therefore, we considered the hypothesis that PAK6 may represent a modulator of TFEB, the major transcriptional activator of autophagy, whose function is highly dependent on the binding with 14-3-3s. In accordance with this hypothesis, the results we obtained demonstrated that PAK6 activity promotes the nuclear translocation of TFEB and the induction of autophagy in different in vivo and in vitro models. Importantly, our data suggest that PAK6 may participate to the regulation of TFEB through different mechanisms, by phosphorylating 14-3-3s and preventing their binding with the transcription factor and by directly interacting with TFEB. In conclusion, with these projects we investigated autophagy in the context of neurodegeneration, addressing this topic from different perspectives and exploiting multiple models. We characterized the participation of DJ-1 in the autophagic pathway and we contributed to provide novel insights on the mechanisms of TFEB and autophagy regulation in neuronal cells