Mitophagy Eliminates the Accumulation of SARM1 on the Mitochondria, Alleviating Programmed Axon Death in Acrylamide Neuropathy

Sterile-α and toll/interleukin 1 receptor motif containing protein 1 (SARM1) is the central executioner of programmed axon death (Wallerian degeneration). Although it has been confirmed to have a mitochondrial targeting sequence and can bind to and stabilize PINK1 on mitochondria, the biological significance for mitochondrial localization of SARM1 is still unclear. The relationship between mitochondrial quality control mechanisms and programmed axon death also needs to be clarified. Chronic acrylamide (ACR) intoxication cause typical pathology of axon degeneration involving early axon loss. Here, we demonstrated that the SARM1 dependent Wallerian axon self-destruction pathway was activated following ACR intoxication. Moreover, increased SARM1 was observed on the mitochondria, which interfered with the mitochondrial quality control mechanisms. As a protective response to stress, mitochondrial components enriched in SARM1 were isolated from the mitochondrial network through an increased fission process and were degraded in an autophagy-dependent manner. Importantly, rapamycin (RAPA) administration eliminated mitochondrial accumulated SARM1 and inhibited axon loss. Thus, mitochondrial localization of SARM1 may be complement to the coordinated activity of NMNAT2 and SARM1, and may be part of the self-limiting molecular mechanisms of programmed axon death. In the early latent period, the mitochondrial localization of SARM1 will help it to be isolated by the mitochondrial network and to be degraded through mitophagy to maintain local axon homeostasis. When the mitochondrial quality control mechanisms are broken down, SARM1 will cause irreversible damage for axon death.

Exploring Mitochondrial Localization of SARS-CoV-2 RNA by Padlock Assay. A Pilot Study in Human Placenta

The ongoing COVID-19 pandemic dictated new priorities in biomedicine research. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, is a single-stranded positive-sense RNA virus. In this pilot study, we optimized our padlock assay to visualize genomic/subgenomic regions using formalin-fixed paraffin-embedded placental samples obtained from a confirmed case of COVID-19. SARS-CoV-2 RNA was localized in trophoblastic cells. We also checked the presence of the virion by immunolocalization of its glycoprotein spike. In addition, we imaged mitochondria of placental villi keeping in mind that the mitochondrion has been suggested as a potential residence of the SARS-CoV-2 genome. Indeed, we observed a substantial overlapping of SARS-CoV-2 RNA and mitochondria in trophoblastic cells. This intriguing linkage correlated with an aberrant mitochondrial network. Overall, to our knowledge, this is the first study that provides the evidence of a co-localization of the SARS-CoV-2 genome and mitochondria in SARS-CoV-2 infected tissue. These findings also support the notion that SARS-CoV-2 infection could reprogram mitochondrial activity in highly specialized maternal/fetal interface.

SARS-CoV-2 Causes Mitochondrial Dysfunction and Mitophagy Impairment

Mitochondria, which is essential for adequate innate immune response, energy metabolism and mitochondria reactive oxygen species (ROS) production, might be in the cross fire of Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and host cell defense. However, little is known about interactions between mitochondria and SARS-CoV-2. We performed fluorescent microscopy and found an enrichment of SARS-CoV-2 replication products double stranded RNA (dsRNA) within mitochondria. The entry process of dsRNA might be mediated by Tom20 as observed by reduced mitochondrial localization of SARS-CoV-2 dsRNA in Tom20 knockdown cells. Importantly, decreased mitochondrial localization of dsRNA, as well as mitochondrial membrane stabilizers mdivi-1 and cyclosporin A, inhibited viral load in cells. Next, we detected mitochondrial dysfunction caused by SARS-CoV-2 infection, including mitochondrial membrane depolarization, mitochondrial permeability transition pore opening and increased ROS release. In response to mitochondrial damage, we observed an increase in expression and mitochondrial accumulation of Pink1 and Parkin proteins, as well as Pink-1-mediated recruitment of P62 to mitochondria, suggesting initiated mitophagy for mitochondrial quality control and virus clearance. Nevertheless, we observed that mitophagy was inhibited and stayed in early stage with an unchanged Hsp60 expression post SARS-CoV-2 infection. This might be one of the anti-autophagy strategies of SARS-CoV-2 and we used co-immunoprecipitation to found that SARS-CoV-2 infection inhibited P62 and LC3 binding which plays a critical role in selective envelopment of substrates into autophagosomes. Our results suggest that mitochondria are closely involved in SARS-CoV-2 replication and mitochondrial homeostasis is disrupted by SARS-CoV-2 in the virus-cell confrontation.

Mitochondrial Localization of CB1 in Progesterone-producing Cells of Ovarian Interstitial Glands of Adult Mice

Localization of cannabinoid receptor type 1 (CB1) immunoreactivity on mitochondrial membranes, at least their outer membranes distinctly, was detected in progesterone-producing cells characterized by mitochondria having tubular cristae and aggregations of lipid droplets in ovarian interstitial glands in situ of adult mice. Both immunoreactive and immunonegative mitochondria were contained in one and the same cell. Considering that the synthesis of progesterone is processed in mitochondria, the mitochondrial localization of CB1 in the interstitial gland cells suggests the possibility that endocannabinoids modulate the synthetic process of progesterone in the cells through CB1:

Mitophagy Eliminates the Accumulation of SARM1 on the Mitochondria, Alleviating Axon Degeneration in Acrylamide Neuropathy

Background: Sterile-α and toll/interleukin 1 receptor motif containing protein 1 (SARM1) is the central executioner of axon degeneration. Although it has been confirmed to have a mitochondrial targeting sequence and can bind to and stabilize PINK1 on depolarized mitochondria, the biological significance for mitochondrial localization of SARM1 is still unclear. Chronic acrylamide (ACR) intoxication can cause typical pathology of axonal injury, owning the potential to explore the interaction between mitochondria and SARM1 during the latent period of axon destruction.Methods: The expression and the mitochondria distribution of SARM1 were evaluated in in vivo and in vitro ACR neuropathy models. Transmission electron microscopy, immunoblotting, and immunofluorescence were performed to evaluate mitochondrial dynamics and PINK1-dependent mitophagy. LC3 turnover experiment and live cell imaging were conducted to further assess the state of mitophagy flux. In order to verify the effect of mitophagy in SARM1-mediated axon degeneration, low-dose and low-frequency rapamycin was administered in ACR-exposed rats to increase basal autophagy.Results: In a time- and dose-dependent manner, ACR induced peripheral nerve injury in rats and truncated axons of differentiated N2a cell. Moreover, the severity of this axon damage was consistent with the up-regulation of SARM1. SARM1 prominently accumulated on mitochondria, and at the same time mitophagy was activated. Importantly, rapamycin (RAPA) administration eliminated mitochondrial accumulated SARM1 and alleviated SARM1 dependent axonal degeneration.Conclusions: Complementing to the coordinated activity of NMNAT2 and SARM1, mitochondrial localization of SARM1 may be part of the self-limiting molecular mechanisms of Wallerian axon destruction. In the early latent period of axon damage, the mitochondrial localization of SARM1 will help it to be isolated by the mitochondrial network and to be degraded through PINK1-dependent mitophagy to maintain local axon homeostasis. When the mitochondrial quality control mechanisms are broken down, SARM1 will cause irreversible damage for axon degeneration. Moderate autophagy activation can be invoked as potential strategies to alleviate axon degeneration in ACR neuropathy and even other axon degeneration diseases.

AMBRA1 promotes apoptosis induced by dsRNA and virus through interacting with and stabilizing MAVS

Apoptosis is an important cellular response to viral infection. In current study, we identified activating molecule in Beclin1-regulated autophagy protein 1 (AMBRA1) as a positive regulator of apoptosis triggered by dsRNA. Depletion of AMBRA1 by gene editing significantly reduced dsRNA-induced apoptosis, which was largely restored by trans-complementation of AMBRA1. Mechanistically, AMBRA1 interacts with mitochondrial antiviral-signaling protein (MAVS), a key mitochondrial adaptor in the apoptosis pathway induced by dsRNA and viral infection. Further Co-IP analysis demonstrated that the mitochondrial localization of MAVS was essential for their interaction. The impact of AMBRA1 on dsRNA-induced apoptosis relied on the presence of MAVS and caspase-8. AMBRA1 was involved in the stabilization of MAVS through preventing its proteasomal degradation induced by dsRNA. Consistently, AMBRA1 upregulated the apoptosis induced by Semliki Forest virus infection. Taken together, our work illustrated a role of AMBRA1 in the virus-induced apoptosis through interacting with and stabilizing MAVS.

Mitochondrial Localization Signal of Porcine Circovirus Type 2 Capsid Protein Plays a Critical Role in Cap-Induced Apoptosis

Porcine circovirus 2 (PCV2), considered one of the most globally important porcine pathogens, causes postweaning multisystemic wasting syndrome (PMWS). This virus is localized in the mitochondria in pigs with PMWS. Here, we identified, for the first time, a mitochondrial localization signal (MLS) in the PCV2 capsid protein (Cap) at the N-terminus. PK-15 cells showed colocalization of the MLS-EGFP fusion protein with mitochondria. Since the PCV2 Cap also contained a nuclear localization signal (NLS) that mediated entry into the nucleus, we inferred that the subcellular localization of the PCV2 Cap is inherently complex and dependent on the viral life cycle. Furthermore, we also determined that deletion of the MLS attenuated Cap-induced apoptosis. More importantly, the MLS was essential for PCV2 replication, as absence of the MLS resulted in failure of virus rescue from cells infected with infectious clone DNA. In conclusion, the MLS of the PCV2 Cap plays critical roles in Cap-induced apoptosis, and MLS deletion of Cap is lethal for virus rescue.

MIEF1/2 orchestrate mitochondrial dynamics through direct engagement with both the fission and fusion machineries

Background Mitochondrial dynamics is the result of a dynamic balance between fusion and fission events, which are driven via a set of mitochondria-shaping proteins. These proteins are generally considered to be binary components of either the fission or fusion machinery, but potential crosstalk between the fission and fusion machineries remains less explored. In the present work, we analyzed the roles of mitochondrial elongation factors 1 and 2 (MIEF1/2), core components of the fission machinery in mammals. Results We show that MIEFs (MIEF1/2), besides their action in the fission machinery, regulate mitochondrial fusion through direct interaction with the fusion proteins Mfn1 and Mfn2, suggesting that MIEFs participate in not only fission but also fusion. Elevated levels of MIEFs enhance mitochondrial fusion in an Mfn1/2- and OPA1-dependent but Drp1-independent manner. Moreover, mitochondrial localization and self-association of MIEFs are crucial for their fusion-promoting ability. In addition, we show that MIEF1/2 can competitively decrease the interaction of hFis1 with Mfn1 and Mfn2, alleviating hFis1-induced mitochondrial fragmentation and contributing to mitochondrial fusion. Conclusions Our study suggests that MIEFs serve as a central hub that interacts with and regulates both the fission and fusion machineries, which uncovers a novel mechanism for balancing these opposing forces of mitochondrial dynamics in mammals.