A subcellular analysis of genetic modulation of PINK1 on mitochondrial alterations, autophagy and cell death.

Mutations in the PTEN-induced putative kinase1 (PINK1) represent the second most frequent cause of autosomal recessive Parkinson's disease. The PINK1 protein mainly localizes to mitochondria and interacts with a variety of proteins, including the pro-autophagy protein beclin1 and the ubiquitin-ligase parkin. Upon stress conditions, PINK1 is known to recruit parkin at the surface of dysfunctional mitochondria and to activate the mitophagy cascade. Aim of this study was to use a simple and highly reproducible catecholamine cell model and transmission electron microscopy to characterize whether PINK1 could affect mitochondrial homeostasis, the recruitment of specific proteins at mitochondria, mitophagy and apoptosis. Samples were analyzed both in baseline conditions and following treatment with methamphetamine (METH), a neurotoxic compound which strongly activates autophagy and produces mitochondrial damage. Our data provide robust sub-cellular evidence that the modulation of PINK1 levels dramatically affects the morphology and number of mitochondria and the amount of cell death. In particular, especially upon METH exposure, PINK1 is able to increase the total number of mitochondria, concurrently recruit beclin1, parkin and ubiquitin and enhance the clearance of damaged mitochondria. In the absence of functional PINK1 and upon autophagy stress, we observe a failure of the autophagy system at large, with marked accumulation of dysfunctional mitochondria and dramatic increase of apoptotic cell death. These findings highlight the strong neuroprotective role of PINK1 as a key protein in the surveillance and regulation of mitochondrial homeostasis.

Mapping and targeted viral activation of pancreatic nerves in mice reveal their roles in the regulation of glucose metabolism.

A lack of comprehensive mapping of ganglionic inputs into the pancreas and of technology for the modulation of the activity of specific pancreatic nerves has hindered the study of how they regulate metabolic processes. Here we show that the pancreas-innervating neurons in sympathetic, parasympathetic and sensory ganglia can be mapped in detail by using tissue clearing and retrograde tracing (the tracing of neural connections from the synapse to the cell body), and that genetic payloads can be delivered via intrapancreatic injection to target sites in efferent pancreatic nerves in live mice through optimized adeno-associated viruses and neural-tissue-specific promoters. We also show that, in male mice, the targeted activation of parasympathetic cholinergic intrapancreatic ganglia and neurons doubled plasma-insulin levels and improved glucose tolerance, and that tolerance was impaired by stimulating pancreas-projecting sympathetic neurons. The ability to map the peripheral ganglia innervating the pancreas and to deliver transgenes to specific pancreas-projecting neurons will facilitate the examination of ganglionic inputs and the study of the roles of pancreatic efferent innervation in glucose metabolism.

The Parkinson-associated protein PINK1 interacts with Beclin1 and promotes autophagy.

Mutations in the PINK1 gene cause autosomal recessive Parkinson's disease. The PINK1 gene encodes a protein kinase that is mitochondrially cleaved to generate two mature isoforms. In addition to its protective role against mitochondrial dysfunction and apoptosis, PINK1 is also known to regulate mitochondrial dynamics acting upstream of the PD-related protein Parkin. Recent data showed that mitochondrial Parkin promotes the autophagic degradation of dysfunctional mitochondria, and that stable PINK1 silencing may have an indirect role in mitophagy activation. Here we report a new interaction between PINK1 and Beclin1, a key pro-autophagic protein already implicated in the pathogenesis of Alzheimer's and Huntington's diseases. Both PINK1 N- and C-terminal are required for the interaction, suggesting that full-length PINK1, and not its cleaved isoforms, interacts with Beclin1. We also demonstrate that PINK1 significantly enhances basal and starvation-induced autophagy, which is reduced by knocking down Beclin1 expression or by inhibiting the Beclin1 partner Vps34. A mutant, PINK1(W437X), interaction of which with Beclin1 is largely impaired, lacks the ability to enhance autophagy, whereas this is not observed for PINK1(G309D), a mutant with defective kinase activity but unaltered ability to bind Beclin1. These findings identify a new function of PINK1 and further strengthen the link between autophagy and proteins implicated in the neurodegenerative process.