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Wolfram syndrome is a rare genetic disease caused by mutations in the WFS1 or CISD2 gene. A primary defect in Wolfram syndrome involves poor ER Ca 2+ handling, but how this disturbance leads to the disease is not known. The current study, performed in primary neurons, the most affected and disease-relevant cells, involving both Wolfram syndrome genes, explains how the disturbed ER Ca 2+ handling compromises mitochondrial function and affects neuronal health. Loss of ER Ca 2+ content and impaired ER-mitochondrial contact sites in the WFS1- or CISD2-deficient neurons is associated with lower IP 3 R-mediated Ca 2+ transfer from ER to mitochondria and decreased mitochondrial Ca 2+ uptake. In turn, reduced mitochondrial Ca 2+ content inhibits mitochondrial ATP production leading to an increased NADH/NAD + ratio. The resulting bioenergetic deficit and reductive stress compromise the health of the neurons. Our work also identifies pharmacological targets and compounds that restore Ca 2+ homeostasis, enhance mitochondrial function and improve neuronal health., (© 2024. The Author(s).)

When ROS production exceeds the cellular antioxidant capacity, the cell needs to eliminate the defective mitochondria responsible for excessive ROS production. It has been proposed that the removal of these defective mitochondria involves mitophagy, but the mechanism of this regulation remains unclear. Here, we demonstrate that moderate mitochondrial superoxide and hydrogen peroxide production oxidates KEAP1, thus breaking the interaction between this protein and PGAM5, leading to the inhibition of its proteasomal degradation. Accumulated PGAM5 interferes with the processing of the PINK1 in the mitochondria leading to the accumulation of PINK1 on the outer mitochondrial membrane. In turn, PINK1 promotes Parkin recruitment to mitochondria and sensitizes mitochondria for autophagic removal. We also demonstrate that inhibitors of the KEAP1-PGAM5 protein-protein interaction (including CPUY192018) mimic the effect of mitochondrial ROS and sensitize mitophagy machinery, suggesting that these inhibitors could be used as pharmacological regulators of mitophagy. Together, our results show that KEAP1/PGAM5 complex senses mitochondrially generated superoxide/hydrogen peroxide to induce mitophagy., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

The Parkinson disease-associated proteins PINK1 and PRKN coordinate the ubiquitination of mitochondrial outer membrane proteins to tag them either for degradation or for autophagic clearance of the mitochondrion. The proteins include the mitochondrial trafficking proteins RHOT1 and RHOT2, the removal of which may be required for immobilization of mitochondria prior to mitophagy. Here, we demonstrate that RHOT1 and RHOT2 are not only substrates for PINK1-PRKN-dependent degradation but that they also play an active role in the process of mitophagy. RHOT1, and likely also RHOT2, may act as a docking site for inactive PRKN prior to mitochondrial damage, thus keeping PRKN in close proximity to its potential substrates and thereby facilitating mitophagy. We also show that RHOT1 functions as a calcium-sensing docking site for PRKN, and we suggest that calcium binding to RHOT is a key step in the calcium-dependent activation of mitophagy machinery.

The Parkinson's disease-associated protein kinase PINK1 and ubiquitin ligase Parkin coordinate the ubiquitination of mitochondrial proteins, which marks mitochondria for degradation. Miro1, an atypical GTPase involved in mitochondrial trafficking, is one of the substrates tagged by Parkin after mitochondrial damage. Here, we demonstrate that a small pool of Parkin interacts with Miro1 before mitochondrial damage occurs. This interaction does not require PINK1, does not involve ubiquitination of Miro1 and also does not disturb Miro1 function. However, following mitochondrial damage and PINK1 accumulation, this initial pool of Parkin becomes activated, leading to the ubiquitination and degradation of Miro1. Knockdown of Miro proteins reduces Parkin translocation to mitochondria and suppresses mitophagic removal of mitochondria. Moreover, we demonstrate that Miro1 EF-hand domains control Miro1's ubiquitination and Parkin recruitment to damaged mitochondria, and they protect neurons from glutamate-induced mitophagy. Together, our results suggest that Miro1 functions as a calcium-sensitive docking site for Parkin on mitochondria., (© 2018 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

The Ca 2+ -binding protein parvalbumin (PV) and mitochondria play important roles in Ca 2+ signaling, buffering and sequestration. Antagonistic regulation of PV and mitochondrial volume is observed in in vitro and in vivo model systems. Changes in mitochondrial morphology, mitochondrial volume and dynamics (fusion, fission, mitophagy) resulting from modulation of PV were investigated in MDCK epithelial cells with stable overexpression/downregulation of PV. Increased PV levels resulted in smaller, roundish cells and shorter mitochondria, the latter phenomenon related to reduced fusion rates and decreased expression of genes involved in mitochondrial fusion. PV-overexpressing cells displayed increased mitophagy, a likely cause for the decreased mitochondrial volumes and the smaller overall cell size. Cells showed lower mobility in vitro, paralleled by reduced protrusions. Constitutive PV down-regulation in PV-overexpressing cells reverted mitochondrial morphology and fractional volume to the state present in control MDCK cells, resulting from increased mitochondrial movement and augmented fusion rates. PV-modulated, bi-directional and reversible mitochondrial dynamics are key to regulation of mitochondrial volume.

Variants in the SPATA5 gene were recently described in a cohort of patients with global developmental delay, sensorineural hearing loss, seizures, cortical visual impairment and microcephaly. SPATA5 protein localizes predominantly in the mitochondria and is proposed to be involved in mitochondrial function and brain developmental processes. However no functional studies have been performed. This study describes five patients with psychomotor developmental delay, microcephaly, epilepsy and hearing impairment, who were thought clinically to have a mitochondrial disease with subsequent whole-exome sequencing analysis detecting compound heterozygous variants in the SPATA5 gene. A summary of clinical data of all the SPATA5 patients reported in the literature confirms the characteristic phenotype. To assess SPATA5's role in mitochondrial dynamics, functional studies were performed on rat cortical neurons. SPATA5-deficient neurons had a significant imbalance in the mitochondrial fusion-fission rate, impaired energy production and short axons. In conclusion, SPATA5 protein has an important role in mitochondrial dynamics and axonal growth. Biallelic variants in the SPATA5 gene can affect mitochondria in cortical neurons and should be considered in patients with a neurodegenerative disorder and/or with clinical presentation resembling a mitochondrial disorder.

Deficiency of the protein Wolfram syndrome 1 (WFS1) is associated with multiple neurological and psychiatric abnormalities similar to those observed in pathologies showing alterations in mitochondrial dynamics. The aim of this study was to examine the hypothesis that WFS1 deficiency affects neuronal function via mitochondrial abnormalities. We show that down-regulation of WFS1 in neurons leads to dramatic changes in mitochondrial dynamics (inhibited mitochondrial fusion, altered mitochondrial trafficking, and augmented mitophagy), delaying neuronal development. WFS1 deficiency induces endoplasmic reticulum (ER) stress, leading to inositol 1,4,5-trisphosphate receptor (IP3R) dysfunction and disturbed cytosolic Ca2+ homeostasis, which, in turn, alters mitochondrial dynamics. Importantly, ER stress, impaired Ca2+ homeostasis, altered mitochondrial dynamics, and delayed neuronal development are causatively related events because interventions at all these levels improved the downstream processes. Our data shed light on the mechanisms of neuronal abnormalities in Wolfram syndrome and point out potential therapeutic targets. This work may have broader implications for understanding the role of mitochondrial dynamics in neuropsychiatric diseases.

The autophagy protein BECN1/Beclin 1 is known to play a central role in autophagosome formation and maturation. The results presented here demonstrate that BECN1 interacts with the Parkinson disease-related protein PARK2. This interaction does not require PARK2 translocation to mitochondria and occurs mostly in cytosol. However, our results suggest that BECN1 is involved in PARK2 translocation to mitochondria because loss of BECN1 inhibits CCCP- or PINK1 overexpression-induced PARK2 translocation. Our results also demonstrate that the observed PARK2-BECN1 interaction is functionally important. Measurements of the level of MFN2 (mitofusin 2), a PARK2 substrate, demonstrate that depletion of BECN1 prevents PARK2 translocation-induced MFN2 ubiquitination and loss. BECN1 depletion also rescues the MFN2 loss-induced suppression of mitochondrial fusion. In sum, our results demonstrate that BECN1 interacts with PARK2 and regulates PARK2 translocation to mitochondria as well as PARK2-induced mitophagy prior to autophagosome formation.

Mitochondria are the power houses of the cell, but unlike the static structures portrayed in textbooks, they are dynamic organelles that move about the cell to deliver energy to locations in need. These organelles fuse with each other then split apart; some appear anchored and others more free to move around, and when damaged they are engulfed by autophagosomes. Together, these processes-mitochondrial trafficking, fusion and fission, and mitophagy-are best described by the term "mitochondrial dynamics". The molecular machineries behind these events are relatively well known yet the precise dynamics in neurons remains under debate. Neurons pose a peculiar logistical challenge to mitochondria; how do these energy suppliers manage to traffic down long axons to deliver the requisite energy supply to distant parts of the cell? To date, the majority of neuronal mitochondrial dynamics studies have used cultured neurons, Drosophila larvae, zebrafish embryos, with occasional experiments in resting mouse nerves. However, a new study in this issue of PLOS Biology from Marija Sajic and colleagues provides an in vivo look at mitochondrial dynamics along resting and electrically active neurons of live anaesthetized mice., Competing Interests: The authors have declared that no competing interests exist.

Mitochondrial fusion-fission dynamics play a crucial role in many important cell processes. These dynamics control mitochondrial morphology, which in turn influences several important mitochondrial properties including mitochondrial bioenergetics and quality control, and they appear to be affected in several neurodegenerative diseases. However, an integrated and quantitative understanding of how fusion-fission dynamics control mitochondrial morphology has not yet been described. Here, we took advantage of modern visualisation techniques to provide a clear explanation of how fusion and fission correlate with mitochondrial length and motility in neurons. Our main findings demonstrate that: (1) the probability of a single mitochondrion splitting is determined by its length; (2) the probability of a single mitochondrion fusing is determined primarily by its motility; (3) the fusion and fission cycle is driven by changes in mitochondrial length and deviations from this cycle serves as a corrective mechanism to avoid extreme mitochondrial length; (4) impaired mitochondrial motility in neurons overexpressing 120Q Htt or Tau suppresses mitochondrial fusion and leads to mitochondrial shortening whereas stimulation of mitochondrial motility by overexpressing Miro-1 restores mitochondrial fusion rates and sizes. Taken together, our results provide a novel insight into the complex crosstalk between different processes involved in mitochondrial dynamics. This knowledge will increase understanding of the dynamic mitochondrial functions in cells and in particular, the pathogenesis of mitochondrial-related neurodegenerative diseases.

Parkinson disease is characterized by the accumulation of aggregated α-synuclein as the major component of the Lewy bodies. α-Synuclein accumulation in turn leads to compensatory effects that may include the up-regulation of autophagy. Another common feature of Parkinson disease (PD) is mitochondrial dysfunction. Here, we provide evidence that the overactivation of autophagy may be a link that connects the intracellular accumulation of α-synuclein with mitochondrial dysfunction. We found that the activation of macroautophagy in primary cortical neurons that overexpress mutant A53T α-synuclein leads to massive mitochondrial destruction and loss, which is associated with a bioenergetic deficit and neuronal degeneration. No mitochondrial removal or net loss was observed when we suppressed the targeting of mitochondria to autophagosomes by silencing Parkin, overexpressing wild-type Mitofusin 2 and dominant negative Dynamin-related protein 1 or blocking autophagy by silencing autophagy-related genes. The inhibition of targeting mitochondria to autophagosomes or autophagy was also partially protective against mutant A53T α-synuclein-induced neuronal cell death. These data suggest that overactivated mitochondrial removal could be one of the contributing factors that leads to the mitochondrial loss observed in PD models.

Recent studies indicate that regulation of cellular oxidative capacity through enhancing mitochondrial biogenesis may be beneficial for neuronal recovery and survival in human neurodegenerative disorders. The peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) has been shown to be a master regulator of mitochondrial biogenesis and cellular energy metabolism in muscle and liver. The aim of our study was to establish whether PGC-1alpha and PGC-1beta control mitochondrial density also in neurons and if these coactivators could be up-regulated by deacetylation. The results demonstrate that PGC-1alpha and PGC-1beta control mitochondrial capacity in an additive and independent manner. This effect was observed in all studied subtypes of neurons, in cortical, midbrain, and cerebellar granule neurons. We also observed that endogenous neuronal PGC-1alpha but not PGC-1beta could be activated through its repressor domain by suppressing it. Results demonstrate also that overexpression of SIRT1 deacetylase or suppression of GCN5 acetyltransferase activates transcriptional activity of PGC-1alpha in neurons and increases mitochondrial density. These effects were mediated exclusively via PGC-1alpha, since overexpression of SIRT1 or suppression of GCN5 was ineffective where PGC-1alpha was suppressed by short hairpin RNA. Moreover, the results demonstrate that overexpression of PGC-1beta or PGC-1alpha or activation of the latter by SIRT1 protected neurons from mutant alpha-synuclein- or mutant huntingtin-induced mitochondrial loss. These evidences demonstrate that activation or overexpression of the PGC-1 family of coactivators could be used to compensate for neuronal mitochondrial loss and suggest that therapeutic agents activating PGC-1 would be valuable for treating neurodegenerative diseases in which mitochondrial dysfunction and oxidative damage play an important pathogenic role.

Calcium oscillations suppress mitochondrial movements along the microtubules to support on-demand distribution of mitochondria. To activate this mechanism, Ca(2+) targets a yet unidentified cytoplasmic factor that does not seem to be a microtubular motor or a kinase/phosphatase. Here, we have studied the dependence of mitochondrial dynamics on the Miro GTPases that reside in the mitochondria and contain two EF-hand Ca(2+)-binding domains, in H9c2 cells and primary neurons. At resting cytoplasmic [Ca(2+)] ([Ca(2+)](c)), movements of the mitochondria were enhanced by Miro overexpression irrespective of the presence of the EF-hands. The Ca(2+)-induced arrest of mitochondrial motility was also promoted by Miro overexpression and was suppressed when either the Miro were depleted or their EF-hand was mutated. Miro also enhanced the fusion state of the mitochondria at resting [Ca(2+)](c) but promoted mitochondrial fragmentation at high [Ca(2+)](c). These effects of Miro on mitochondrial morphology seem to involve Drp1 suppression and activation, respectively. In primary neurons, Miro also caused an increase in dendritic mitochondrial mass and enhanced mitochondrial calcium signaling. Thus, Miro proteins serve as a [Ca(2+)](c)-sensitive switch and bifunctional regulator for both the motility and fusion-fission dynamics of the mitochondria.

Organelle transport in neuronal processes is central to the organization, developmental fate, and functions of neurons. Organelles must be transported through the slender, highly branched neuronal processes, making the axonal transport vulnerable to any perturbation. However, some intracellular structures like mitochondria are able to considerably modify their volume. We therefore hypothesized that swollen mitochondria could impair the traffic of other organelles in neurite shafts. To test this hypothesis, we have investigated the effects of mitochondrial swellers on the organelle traffic. Our data demonstrate that treatment of neurons with potassium ionophore valinomycin led to the fast time-dependent inhibition of organelle movement in cerebellar granule neurons. Similar inhibition was observed in neurons treated with the inhibitors of the mitochondrial respiratory chain, sodium azide and antimycin, which also induced swelling. No decrease in the motility of organelles was observed in cultures treated with inhibitors of ATP production or transport, oligomycin or bongkrekic acid, suggesting that inhibition of the ATP-generating activity itself without swelling does not affect the motility of organelles. The effect of swellers on the traffic was more important in thin processes, thus indicating the role of steric hindrance of swollen mitochondria. We propose that the size and morphology of the transported cargo is also relevant for seamless axonal transport and speculate that mitochondrial swelling could be one of the reasons for impaired organelle transport in neuronal processes.

Mitochondrial volume homeostasis is a housekeeping cellular function essential for maintaining the structural integrity of the organelle. Changes in mitochondrial volume have been associated with a wide range of important biological functions and pathologies. Mitochondrial matrix volume is controlled by osmotic balance between cytosol and mitochondria. Any dysbalance in the fluxes of the main intracellular ion, potassium, will thus affect the osmotic balance between cytosol and the matrix and promote the water movement between these two compartments. It has been hypothesized that activity of potassium efflux pathways exceeds the potassium influx in functioning mitochondria and that potassium concentration in matrix could be actually lower than in cytoplasm. This hypothesis provides a clear-cut explanation for the mitochondrial swelling observed after mitochondrial depolarization, mitochondrial calcium overload, or opening of permeability transition pore. It should also be noted that the rate of water flux into or out of the mitochondrion is determined not only by the osmotic gradient that acts as the driving force for water transport but also by the water permeability of the inner membrane. Recent data suggest that the mitochondrial inner membrane has also specific water channels, aquaporins, which facilitate water movement between cytoplasm and matrix. This review discusses different phases of mitochondrial swelling and summarizes the potential effects of mitochondrial swelling on cell function.