Continued 26S proteasome dysfunction in mouse brain cortical neurons impairs autophagy and the Keap1-Nrf2 oxidative defence pathway

Abstract

The ubiquitin–proteasome system (UPS) and macroautophagy (autophagy) are central to normal proteostasis and interdependent in that autophagy is known to compensate for the UPS to alleviate ensuing proteotoxic stress that impairs cell function. UPS and autophagy dysfunctions are believed to have a major role in the pathomechanisms of neurodegenerative disease. Here we show that continued 26S proteasome dysfunction in mouse brain cortical neurons causes paranuclear accumulation of fragmented dysfunctional mitochondria, associated with earlier recruitment of Parkin and lysine 48-linked ubiquitination of mitochondrial outer membrane (MOM) proteins, including Mitofusin-2. Early events also include phosphorylation of p62/SQSTM1 (p62) and increased optineurin, as well as autophagosomal LC3B and removal of some mitochondria, supporting the induction of selective autophagy. Inhibition of the degradation of ubiquitinated MOM proteins with continued 26S proteasome dysfunction at later stages may impede efficient mitophagy. However, continued 26S proteasome dysfunction also decreases the levels of essential autophagy proteins ATG9 and LC3B, which is characterised by decreases in their gene expression, ultimately leading to impaired autophagy. Intriguingly, serine 351 phosphorylation of p62 did not enhance its binding to Keap1 or stabilise the nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factor in this neuronal context. Nrf2 protein levels were markedly decreased despite transcriptional activation of the Nrf2 gene. Our study reveals novel insights into the interplay between the UPS and autophagy in neurons and is imperative to understanding neurodegenerative disease where long-term proteasome inhibition has been implicated.

P62/SQSTM1 is a novel leucine-rich repeat kinase 2 (LRRK2) substrate that enhances neuronal toxicity

“…When we investigated if p62 modulated this phenotype, we indeed found a synergistic toxic effect of mutant LRRK2 and p62 expression, dependent on Thr138 (Figure 9). It is possible that, in this context, overexpressed hyperphosphorylated p62, co-expressed with G2019S-LRRK2, fails to activate the Nrf2 cytoprotective pathway similar to that recently reported in cultured neurons in which elevated levels of phosphorylated p62 (at Ser351) are observed following proteasome inhibition [126]. In fact, it was recently shown that up-regulating Nrf activity decreased LRRK2 neuronal toxicity [79].…”

SOD1A4V aggregation alters ubiquitin homeostasis in a cell model of ALS

“…Recent studies have revealed that mitochondrial impairment occurs soon after proteasome inhibition (Maharjan et al, 2014). In fact, continued proteasome dysfunction in mouse brain cortical neurons inhibited the degradation of ubiquitylated mitochondrial proteins and led to the accumulation of dysfunctional mitochondria (Ugun-Klusek et al, 2017). Misfolded proteins such as mutant SOD1 have also been shown to interact with mitochondrial proteins and translocate into the mitochondrial intermembrane space and mitochondrial matrix, where they accumulate and induce mitochondrial dysfunction (Vijayvergiya et al, 2005;Jaarsma et al, 2001;Ruan et al, 2017;Igoudjil et al, 2011;Fischer et al, 2011).…”

Promiscuous Roles of Autophagy and Proteasome in Neurodegenerative Proteinopathies

“…To date, plenty of evidence exists linking alterations of either autophagy or UPS with the aggregation, spreading and toxicity of so called prionoids, including α-syn, Aβ, tau, huntingtin, SOD1, TDP-43 and FUS [19,29,[52][53][54][55][56][57][58][59][60]. Albeit being both autophagy and UPS implicated in the clearance of the above-mentioned proteins, recent biochemical and morphological studies have added to this scenario, focusing on the cross-talk between these two systems [61][62][63][64][65][66][67][68][69][70][71][72][73]. In fact, a coordinated, either synergistic or compensatory interplay occurs between autophagy and the UPS, which has attracted the attention of many investigators while challenging the traditional view of two segregated and independent degradation systems.…”

“…Besides sharing prion-like protein substrates, autophagy and UPS converge at both biochemical and morphological levels [61][62][63][64][65][66], though the functional significance of such an interplay remains to be fully elucidated. A growing number of biochemical pathways are being shown to operate at the crossroad between autophagy and UPS, orchestrating the specificity and magnitude of the two degradation systems for effective protein homeostasis [61][62][63][64][65][66][67][68][69][70][71][72][73][74].…”