Accumulation of depolarized mitochondria within b-cells has been associated with oxidative damage and development of diabetes. To determine the source and fate of depolarized mitochondria, individual mitochondria were photolabeled and tracked through fusion and fission. Mitochondria were found to go through frequent cycles of fusion and fission in a 'kiss and run' pattern. Fission events often generated uneven daughter units: one daughter exhibited increased membrane potential (Dw m ) and a high probability of subsequent fusion, while the other had decreased membrane potential and a reduced probability for a fusion event. Together, this pattern generated a subpopulation of nonfusing mitochondria that were found to have reduced Dw m and decreased levels of the fusion protein OPA1. Inhibition of the fission machinery through DRP1 K38A or FIS1 RNAi decreased mitochondrial autophagy and resulted in the accumulation of oxidized mitochondrial proteins, reduced respiration and impaired insulin secretion. Pulse chase and arrest of autophagy at the pre-proteolysis stage reveal that before autophagy mitochondria lose Dw m and OPA1, and that overexpression of OPA1 decreases mitochondrial autophagy. Together, these findings suggest that fission followed by selective fusion segregates dysfunctional mitochondria and permits their removal by autophagy.

NLRP3 is an important pattern recognition receptor involved in mediating inflammasome activation in response to viral and bacterial infections as well as various proinflammatory stimuli associated with tissue damage or malfunction. Upon activation, NLRP3 assembles a multimeric inflammasome complex comprising the adaptor ASC and the effector pro-caspase-1 to mediate the activation of caspase-1. Although NLRP3 expression is induced by the NF-κB pathway, the posttranscriptional molecular mechanism controlling the activation of NLRP3 remains elusive. Using both pharmacological and molecular approaches, we show that the activation of NLRP3 inflammasome is regulated by a deubiquitination mechanism. We further identify the deubiquitinating enzyme, BRCC3, as a critical regulator of NLRP3 activity by promoting its deubiquitination and characterizing NLRP3 as a substrate for the cytosolic BRCC3-containing BRISC complex. Our results elucidate a regulatory mechanism involving BRCC3-dependent NLRP3 regulation and highlight NLRP3 ubiquitination as a potential therapeutic target for inflammatory diseases.

Multiple sclerosis (MS), a common neurodegenerative disease of the CNS, is characterized by the loss of oligodendrocytes and demyelination. TNFα, a proinflammatory cytokine implicated in MS, can activate necroptosis, a necrotic cell death pathway regulated by RIPK1 and RIPK3 under caspase-8 deficient conditions. Here, we demonstrate defective caspase-8 activation, as well as, activation of RIPK1, RIPK3 and MLKL, the hallmark mediators of necroptosis, in the cortical lesions of human MS pathological samples. Furthermore, we show that MS pathological samples are characterized by an increased insoluble proteome in common with other neurodegenerative diseases such as AD, PD and HD. Finally, we show that necroptosis mediates oligodendrocyte degeneration induced by TNFα, and inhibition of RIPK1 protects against oligodendrocyte cell death in two animal models of MS and in culture. Our findings demonstrate that necroptosis is involved in MS and suggest that targeting RIPK1 may represent a novel therapeutic strategy for MS.

Dysregulation of autophagy, a cellular catabolic mechanism essential for degradation of misfolded proteins, has been implicated in multiple neurodegenerative diseases. However, the mechanisms that lead to the autophagy dysfunction are still not clear. Based on the results of a genome-wide screen, we show that reactive oxygen species (ROS) serve as common mediators upstream of the activation of the type III PI3 kinase, which is critical for the initiation of autophagy. Furthermore, ROS play an essential function in the induction of the type III PI3 kinase and autophagy in response to amyloid β peptide, the main pathogenic mediator of Alzheimer's disease (AD). However, lysosomal blockage also caused by Aβ is independent of ROS. In addition, we demonstrate that autophagy is transcriptionally down-regulated during normal aging in the human brain. Strikingly, in contrast to normal aging, we observe transcriptional upregulation of autophagy in the brains of AD patients, suggesting that there might be a compensatory regulation of autophagy. Interestingly, we show that an AD drug and an AD drug candidate have inhibitory effects on autophagy, raising the possibility that decreasing input into the lysosomal system may help to reduce cellular stress in AD. Finally, we provide a list of candidate drug targets that can be used to safely modulate levels of autophagy without causing cell death.reactive oxygen species | type III PI3 kinase | neurodegeneration | signaling | transcriptional regulation A utophagy, a lysosome-dependent catabolic process mediating turnover of cellular components, plays an important role in regulating cellular homeostasis in the nervous system (1). Even in the absence of any other risk factors, autophagy deficiency in the CNS has been shown to lead to the accumulation of protein aggregates and progressive neurodegeneration (2). Thus, autophagy has been established as an important mechanism mediating degradation of misfolded proteins in the CNS. Because accumulation of misfolded proteins is a common feature in multiple human neurodegenerative diseases, activation of autophagy has been proposed as a strategy for combating neurodegeneration (3). However, little is currently known about how defects in autophagy might be involved in specific neurodegenerative diseases. Furthermore, as induction of autophagy is frequently associated with cell death, it remains a challenge to identify molecular targets whose inhibition can specifically activate autophagy without compromising cell viability.Pathological evidence supports the involvement of autophagy dysfunction in neurodegenerative diseases in humans. In Alzheimer's disease (AD), one of the earliest pathological changes include accumulation of autophagic vesicles (AVs) specifically within damaged neuritic processes and synaptic terminals (4). This phenotype is also observed in AD animal models and in cellbased models upon exposure to amyloid β peptide (Aβ). However, the mechanisms leading to the accumulation of AVs and the causal relationship to neurodegener...

Inflammation is triggered by a repertoire of receptors detecting infections and damages. Some of these receptors directly bind microbial ligands, while others recognize endogenous molecules exposed under stress conditions, including infections. Most of these receptors can be engaged by a relatively limited number of stimuli. Differently, NLRP3 acts as a broad sensor of cell homeostasis rupture and can be activated downstream of a plethora of stimuli. NLRP3 then assembles a multiprotein platform resulting in caspase-1 activation, which controls, by direct cleavage, the maturation of cytosolic pro-cytokines including pro-interleukin-1β. In addition, caspase-1 processes cytosolic gasdermin-D and unleashes its pore-forming N-terminal domain, leading to the release of mature cytosolic cytokines and alarmins, as well as pyroptotic cell lysis. Accumulating evidences of the aggravating role of NLRP3-mediated inflammation in various highly prevalent human conditions, including diabetes, neurodegenerative and cardiovascular diseases, raises a huge clinical interest. Nevertheless, the molecular mechanism governing NLRP3 activation remains insufficiently understood. In line with the detrimental consequences of NLRP3 activation illustrated by the aforementioned pathologies, this process is tightly regulated. In this review, we address the current understanding of the control of NLRP3 activity which can be divided into two coordinated processes referred to as priming and activation. In particular, we detail the emerging role of NLRP3 post-translational modifications critical in inflammasome assembly regulation.