Parkin, an E3 ubiquitin ligase and a Parkinson's disease (PD) related gene, translocates to impaired mitochondria and drives their elimination via autophagy, a process known as mitophagy. Mitochondrial pro-fusion protein Mitofusins (Mfn1 and Mfn2) were found to be a target for Parkin mediated ubiquitination. Mfns are transmembrane GTPase embedded in the outer membrane of mitochondria, which are required on adjacent mitochondria to mediate fusion. In mammals, Mfn2 also forms complexes that are capable of tethering mitochondria to endoplasmic reticulum (ER), a structural feature essential for mitochondrial energy metabolism, calcium (Ca) transfer between the organelles and Ca dependent cell death. Despite its fundamental physiological role, the molecular mechanisms that control ER-mitochondria cross talk are obscure. Ubiquitination has recently emerged as a powerful tool to modulate protein function, via regulation of protein subcellular localization and protein ability to interact with other proteins. Ubiquitination is also a reversible mechanism, which can be actively controlled by opposing ubiquitination-deubiquitination events. In this work we found that in Parkin deficient cells and parkin mutant human fibroblasts, the tether between ER and mitochondria is decreased. We identified the site of Parkin dependent ubiquitination and showed that the non-ubiquitinatable Mfn2 mutant fails to restore ER-mitochondria physical and functional interaction. Finally, we took advantage of an established in vivo model of PD to demonstrate that manipulation of ER-mitochondria tethering by expressing an ER-mitochondria synthetic linker is sufficient to rescue the locomotor deficit associated to an in vivo Drosophila model of PD.
Mitochondrial autophagy or mitophagy is a key process that allows selective sequestration and degradation of dysfunctional mitochondria to prevent excessive reactive oxygen species, and activation of cell death. Recent studies revealed that ubiquitin–proteasome complex activity and mitochondrial membrane rupture are key steps preceding mitophagy, in combination with the ubiquitination of specific outer mitochondrial membrane (OMM) proteins. The deubiquitinating enzyme ubiquitin‐specific peptidase 14 (USP14) has been shown to modulate both proteasome activity and autophagy. Here, we report that genetic and pharmacological inhibition of USP14 promotes mitophagy, which occurs in the absence of the well‐characterised mediators of mitophagy, PINK1 and Parkin. Critical to USP14‐induced mitophagy is the exposure of the LC3 receptor Prohibitin 2 by mitochondrial fragmentation and mitochondrial membrane rupture. Genetic or pharmacological inhibition of USP14 in vivo corrected mitochondrial dysfunction and locomotion behaviour of PINK1/Parkin mutant Drosophila model of Parkinson's disease, an age‐related progressive neurodegenerative disorder that is correlated with diminished mitochondrial quality control. Our study identifies a novel therapeutic target that ameliorates mitochondrial dysfunction and in vivo PD‐related symptoms.
Aberrant mitochondrial dynamics disrupts mitochondrial function and contributes to disease conditions. A targeted RNA interference screen for deubiquitinating enzymes (DUBs) affecting protein levels of multifunctional mitochondrial fusion protein Mitofusin (MFN) identified USP8 prominently influencing MFN levels. Genetic and pharmacological inhibition of USP8 normalized the elevated MFN protein levels observed in PINK1 and Parkin-deficient models. This correlated with improved mitochondrial function, locomotor performance and life span, and prevented dopaminergic neurons loss in Drosophila PINK1 KO flies. We identified a novel target antagonizing pathologically elevated MFN levels, mitochondrial dysfunction, and dopaminergic neuron loss of a Drosophila model of mitochondrial dysfunction.
Dysregulations of mitochondria with alterations in trafficking and morphology of these organelles have been related to Parkinson's disease (PD), a neurodegenerative disorder characterized by brain accumulation of Lewy bodies (LB), intraneuronal inclusions mainly composed of α-synuclein (α-syn) fibrils. Experimental evidence supports that α-syn pathological aggregation can negatively impinge on mitochondrial functions suggesting that this protein may be crucially involved in the control of mitochondrial homeostasis. The aim of this study was to assay this hypothesis by analyzing mitochondrial function and morphology in primary cortical neurons from C57BL/6JOlaHsd α-syn null and C57BL/6J wild-type (wt) mice. Primary cortical neurons from mice lacking α-syn showed decreased respiration capacity measured with a Seahorse XFe24 Extracellular Flux Analyzer. In addition, morphological Airyscan superresolution microscopy showed the presence of fragmented mitochondria while real-time PCR and western blot confirmed altered expression of proteins involved in mitochondrial shape modifications in the primary cortical neurons of α-syn null mice. Transmission electron microscopy (TEM) studies showed that α-syn null neurons exhibited impaired mitochondria-endoplasmic reticulum (ER) physical interaction. Specifically, we identified a decreased number of mitochondria-ER contacts (MERCs) paralleled by a significant increase in ER-mitochondria distance (i.e., MERC length). These findings support that α-syn physiologically preserves mitochondrial functions and homeostasis. Studying α-syn/mitochondria interplay in health and disease is thus pivotal for understanding their involvement in PD and other LB disorders.
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