Supplementary MaterialsFile S1: Figure S1, HeLa cells were transiently transfected with scrambled siRNA or Red1 siRNA. 1. No significant changes appeared in cells kept in 5%.(DOC) pone.0095288.s004.doc (31K) GUID:?3A7784E2-7E4E-44BA-BBB4-8A25F643E757 Abstract The mitochondrial kinase PINK1 and the ubiquitin ligase Parkin are participating in quality control after CCCP- or ROS-induced mitochondrial damage, and their dysfunction is associated with the development and progression of Parkinson’s disease. Furthermore, Red1 expression is also induced by starvation indicating an additional role for Red1 in stress response. Therefore, the effects of Red1 deficiency within the autophago-lysosomal pathway during stress were investigated. Under trophic deprivation SH-SY5Y cells with stable Red1 knockdown showed downregulation of important autophagic genes, including Beclin, LC3 and Light-2. In good agreement, protein levels of LC3-II and Light-2 but not of Light-1 were reduced in different cell model systems with Red1 knockdown or knockout after addition of different stressors. This downregulation of autophagic factors caused improved apoptosis, which could become rescued by overexpression of LC3 or Red1. Taken collectively, the Red1-mediated reduction of autophagic important factors during MCM2 stress resulted in improved cell death, thus defining an additional pathway that could contribute to the N6022 progression of Parkinson’s disease in individuals with Red1 mutations. Intro Parkinson’s disease (PD) is the second most common neurodegenerative disorder after Alzheimer’s disease and both are age-progressive disorders. PD individuals are characterized by a typical impairment of their motions and resting tremor caused mainly by degeneration of the dopaminergic neurons, which project from your substantia nigra in the midbrain to the striatum. Another hallmark N6022 of PD is the event of multi-protein aggregates in the affected neurons, the so-called Lewy body that contain the PD-associated protein alpha-synuclein and many additional proteins. Most PD instances happen sporadically, with aging becoming the main risk element for PD. However, an increasing quantity of gene mutations are becoming associated with PD. At the moment 18 gene loci are described as PD-associated, amongst others mutations in the genes PARKIN and PTEN induced putative kinase 1 (Red1) result in autosomal recessive PD variants PARK2 and PARK6 . Different causes are hypothesized to initiate or contribute to neuronal cell death in individuals N6022 with PARK6 mutations: oxidative stress , impaired bioenergetics , , dysregulation of neuronal Ca2+ , , reduced mitochondrial dynamics  and dysfunctional degradation of damaged mitochondria and/or protein aggregates , . All these hypotheses implicate a progressive mitochondrial dysfunction as common denominator, which could become enforced by stress and/or impaired quality control, finally resulting in cell death. Dopaminergic neurons seem to react especially sensitively to mitochondrial dysfunction, maybe because of the low glycolytic capacity , but also non-neuronal cells as e.g. pores and skin fibroblasts from PARK6 individuals demonstrate impaired mitochondrial function , . Damaged mitochondria can be either repaired by mitochondrial dynamics (fusion and fission) or degraded by mitophagy/macroautophagy. The selection of the appropriate pathway N6022 depends on the extent N6022 of mitochondrial damage. A strong reduction of mitochondrial membrane potential induces the Red1-controlled translocation of Parkin to these mitochondria, tagging them for degradation C. The actual autophagic process is definitely mediated and regulated from the proteins of the ATG family. It starts with the engulfment of a damaged mitochondrion or protein aggregate with an expanding membrane that is characterized by the presence of the autophagosomal marker protein LC3-II (ATG8). The adult autophagosome fuses consequently with endosomes and lysosomes to form an autolysosome. In the autolysosome the content is definitely degraded by lysosomal hydrolases, therefore providing the cell with amino acids. Lysosomes and autolysosomes are characterized by the presence of the proteins LAMP-1 and LAMP-2. The recently emerging important roles of PINK1 and Parkin in mitophagy imply that dysfunctional mitochondrial degradation is usually contributing to the progression of the autosomal recessive PD variants PARK2 and PARK6, which might be enhanced by additional stressors as e.g. aging. In accordance with this hypothesis the loss of functional PINK1 or Parkin results in impaired mitophagy after stress and an accumulation of damaged mitochondria C. In addition to targeted mitophagy, PINK1 and Parkin are also involved in.