bims-midneu 2021-06-20 papers

bims-midneu

Biomed News

on Mitochondrial dysfunction in neurodegeneration

Issue of 2021–06–20
eightteen papers selected by
Radha Desai, Merck Sharp & Dohme Corp.

Parkinson's disease-associated VPS35 mutant reduces mitochondrial membrane potential and impairs PINK1/Parkin-mediated mitophagy.
Hypoxic postconditioning promotes mitophagy against transient global cerebral ischemia via PINK1/Parkin-induced mitochondrial ubiquitination in adult rats.
Neuronal autophagy and mitophagy in Parkinson's disease.
Mitochondrial quality control: Epigenetic signatures and therapeutic strategies.
Ischemia Injury Induces Mitochondrial Permeability Transition Pore Opening by Reducing Sirt3.
Novel reporters of mitochondria-associated membranes (MAM), MAMtrackers, demonstrate MAM disruption as a common pathological feature in amyotrophic lateral sclerosis.
An autopsy case of PARK2 due to a homozygous exon 2 deletion of parkin and associated with α-synucleinopathy.
The potential role of Keap1-Nrf2 pathway in the pathogenesis of Alzheimer's disease, type 2 diabetes, and type 2 diabetes-related Alzheimer's disease.
The inhibition of gadolinium ion (Gd3+) on the mitochondrial F1FO-ATPase is linked to the modulation of the mitochondrial permeability transition pore.
Glitazones Activate PGC-1α Signaling via PPAR-γ: A Promising Strategy for Antiparkinsonism Therapeutics.
Stress granules inhibit fatty acid oxidation by modulating mitochondrial permeability.
Mitochondrial CMPK2 mediates immunomodulatory and antiviral activities through IFN-dependent and IFN-independent pathways.
Investigation of the correlation between mildly deleterious mtDNA Variations and the clinical progression of multiple sclerosis.
ALS-linked mutations impair UBQLN2 stress-induced biomolecular condensate assembly in cells.
Early-adolescent antibiotic exposure results in mitochondrial and behavioral deficits in adult male mice.
Role of the TRPM4 channel in mitochondrial function, calcium release, and ROS generation in oxidative stress.
Induced pluripotent stem cell-derived motor neurons of CMT type 2 patients reveal progressive mitochondrial dysfunction.
Phosphoproteomic identification of ULK substrates reveals VPS15-dependent ULK/VPS34 interplay in the regulation of autophagy.

Transl Neurodegener. 2021 Jun 15. 10(1): 19

Parkinson's disease-associated VPS35 mutant reduces mitochondrial membrane potential and impairs PINK1/Parkin-mediated mitophagy.

Kai Yu Ma, Michiel R Fokkens, Fulvio Reggiori, Muriel Mari, Dineke S Verbeek.

   BACKGROUND: Mitochondrial dysfunction plays a prominent role in the pathogenesis of Parkinson's disease (PD), and several genes linked to familial PD, including PINK1 (encoding PTEN-induced putative kinase 1 [PINK1]) and PARK2 (encoding the E3 ubiquitin ligase Parkin), are directly involved in processes such as mitophagy that maintain mitochondrial health. The dominant p.D620N variant of vacuolar protein sorting 35 ortholog (VPS35) gene is also associated with familial PD but has not been functionally connected to PINK1 and PARK2.
METHODS: To better mimic and study the patient situation, we used CRISPR-Cas9 to generate heterozygous human SH-SY5Y cells carrying the PD-associated D620N variant of VPS35. These cells were treated with a protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) to induce the PINK1/Parkin-mediated mitophagy, which was assessed using biochemical and microscopy approaches.
RESULTS: Mitochondria in the VPS35-D620N cells exhibited reduced mitochondrial membrane potential and appeared to already be damaged at steady state. As a result, the mitochondria of these cells were desensitized to the CCCP-induced collapse in mitochondrial potential, as they displayed altered fragmentation and were unable to accumulate PINK1 at their surface upon this insult. Consequently, Parkin recruitment to the cell surface was inhibited and initiation of the PINK1/Parkin-dependent mitophagy was impaired.
CONCLUSION: Our findings extend the pool of evidence that the p.D620N mutation of VPS35 causes mitochondrial dysfunction and suggest a converging pathogenic mechanism among VPS35, PINK1 and Parkin in PD.

Keywords:  Mitochondrial membrane potential; Mitophagy; PINK1; Parkin; Parkinson’s disease; VPS35

DOI:  https://doi.org/10.1186/s40035-021-00243-4
Cell Death Dis. 2021 Jun 18. 12(7): 630

Hypoxic postconditioning promotes mitophagy against transient global cerebral ischemia via PINK1/Parkin-induced mitochondrial ubiquitination in adult rats.

Haixia Wen, Luxi Li, Lixuan Zhan, Yunyan Zuo, Kongping Li, Meiqian Qiu, Heying Li, Weiwen Sun, En Xu.

Mitophagy alleviates neuronal damage after cerebral ischemia by selectively removing dysfunctional mitochondria. Phosphatase and tensin homolog (PTEN) induced putative kinase 1 (PINK1)/Parkin-mediated mitophagy is the most well-known type of mitophagy. However, little is known about the role of PINK1/Parkin-mediated mitophagy in ischemic tolerance induced by hypoxic postconditioning (HPC) with 8% O2 against transient global cerebral ischemia (tGCI). Hence, we aimed to test the hypothesis that HPC-mediated PINK1/Parkin-induced mitochondrial ubiquitination and promotes mitophagy, thus exerting neuroprotection in the hippocampal CA1 subregion against tGCI. We found that mitochondrial clearance was disturbed at the late phase of reperfusion after tGCI, which was reversed by HPC, as evidenced by the reduction of the translocase of outer mitochondrial membrane 20 homologs (TOMM20), translocase of inner mitochondrial membrane 23 (TIMM23) and heat shock protein 60 (HSP60) in CA1 after HPC. In addition, HPC further increased the ratio of LC3II/I in mitochondrial fraction and promoted the formation of mitophagosomes in CA1 neurons after tGCI. The administration of lysosome inhibitor chloroquine (CQ) intraperitoneally or mitophagy inhibitor (Mdivi-1) intracerebroventricularly abrogated HPC-induced mitochondrial turnover and neuroprotection in CA1 after tGCI. We also found that HPC activated PINK1/Parkin pathway after tGCI, as shown by the augment of mitochondrial PINK1 and Parkin and the promotion of mitochondrial ubiquitination in CA1. In addition, PINK1 or Parkin knockdown with small-interfering RNA (siRNA) suppressed the activation of PINK1/Parkin pathway and hampered mitochondrial clearance and attenuated neuroprotection induced by HPC, whereas PINK1 overexpression promoted PINK1/Parkin-mediated mitophagy and ameliorated neuronal damage in CA1 after tGCI. Taken together, the new finding in this study is that HPC-induced neuroprotection against tGCI through promoting mitophagy mediated by PINK1/Parkin-dependent pathway.

DOI: https://doi.org/10.1038/s41419-021-03900-8
Mol Aspects Med. 2021 Jun 12. pii: S0098-2997(21)00032-7. [Epub ahead of print] 100972

Neuronal autophagy and mitophagy in Parkinson's disease.

Britney N Lizama, Charleen T Chu.

  Autophagy is the process by which cells can selectively or non-selectively remove damaged proteins and organelles. As the cell's main means of sequestering damaged mitochondria for removal, mitophagy is central to cellular function and survival. Research on autophagy and mitochondrial quality control has increased exponentially in relation to the pathogenesis of numerous disease conditions, from cancer and immune diseases to chronic neurodegenerative diseases like Parkinson's disease (PD). Understanding how components of the autophagic/mitophagic machinery are affected during disease, as well as the contextual relationship of autophagy with determining neuronal health and function, is essential to the goal of designing therapies for human disease. In this review, we will summarize key signaling molecules that consign damaged mitochondria for autophagic degradation, describe the relationship of genes linked to PD to autophagy/mitophagy dysfunction, and discuss additional roles of both mitochondrial and cytosolic pools of PTEN-induced kinase 1 (PINK1) in mitochondrial homeostasis, dendritic morphogenesis and inflammation.

Keywords:  Autophagy; Mitochondria; Mitophagy; Neurodegeneration; PINK1; Parkinson's disease

DOI:  https://doi.org/10.1016/j.mam.2021.100972
Neurochem Int. 2021 Jun 08. pii: S0197-0186(21)00141-8. [Epub ahead of print]148 105095

Mitochondrial quality control: Epigenetic signatures and therapeutic strategies.

Tanuja Ambekar, Jyoti Pawar, Ramdev Rathod, Monica Patel, Valencia Fernandes, Rahul Kumar, Shashi Bala Singh, Dharmendra Kumar Khatri.

  Mitochondria are semi-autonomous organelle staging a crucial role in cellular stress response, energy metabolism and cell survival. Maintaining mitochondrial quality control is very important for its homeostasis. Pathological conditions such as oxidative stress and neurodegeneration, disrupt this quality control, and involvement of genetic and epigenetic materials in this disruption have been reported. These regulatory factors trigger mitochondrial imbalance, as seen in many neurodegenerative diseases like Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis, and Huntington's disease. The dynamic regulatory pathways i.e. mitophagy, biogenesis, permeability pore transitioning, fusion-fission are affected as a consequence and have been reviewed in this article. Moreover, several epigenetic mechanisms such as DNA methylation and histone modulation participating in such neurological disorders have also been discussed. Apart from it, therapeutic approaches targeting mitochondrial quality control have been tremendously explored showing ameliorative effects for these diseases, and have been discussed here with a novel perspective.

Keywords:  Biogenesis; DNA methylation; Histone acetylation; Mitochondrial quality control; Mitophagy; Neurodegeneration

DOI:  https://doi.org/10.1016/j.neuint.2021.105095
Neuroscience. 2021 Jun 11. pii: S0306-4522(21)00288-8. [Epub ahead of print]

Ischemia Injury Induces Mitochondrial Permeability Transition Pore Opening by Reducing Sirt3.

Yaping Yang, Ye Tian, Xiaosu Guo, Shiping Li, Weiping Wang, Jiong Shi.

  Mitochondrial permeability transition pore (mPTP) opening is critical to mitochondrial apoptosis during ischemic injury. Sirtuin 3 (Sirt3) is a mitochondrial deacetylase known to play a major role in stress resistance and cell death. Our previous studies have shown that Sirt3 activates superoxide dismutase 2 and forkhead box O3a to reduce cellular reactive oxygen species. However, it is unclear the interaction between Sirt3 and mPTP and the roles they play in ischemic stroke. We used the middle cerebral artery occlusion (MCAO) model, a mouse model of stroke, to examine Sirt3 and mPTP-related protein levels. We then applied lentivirus packaged Sirt3 overexpression in HT22 cells, a mouse hippocampal neuronal cell line, to investigate the underlying mechanism. We found Sirt3 protein level was decreased in the penumbra area in MCAO mice, along with an increase in mPTP related proteins, namely voltage-dependent anion channel 1 (VDAC1) and adenine nucleotide translocator 1 (ANT1). Sirt3 overexpression suppressed the increase in VDAC1, ANT1 and cleaved caspase 3 that were induced by the serum and glucose deprivation (SGD) condition. Our studies suggest that ischemic injury induced mPTP opening and apoptosis by reducing Sirt3. It helps to identify new therapeutic targets for ischemic stroke.

Keywords:  ANT1; Sirt3; VDAC1; apoptosis; ischemic injury; mPTP

DOI:  https://doi.org/10.1016/j.neuroscience.2021.06.003
FASEB J. 2021 Jul;35(7): e21688

Novel reporters of mitochondria-associated membranes (MAM), MAMtrackers, demonstrate MAM disruption as a common pathological feature in amyotrophic lateral sclerosis.

Shohei Sakai, Seiji Watanabe, Okiru Komine, Akira Sobue, Koji Yamanaka.

  The mitochondria-associated membrane (MAM) is a functional subdomain of the endoplasmic reticulum membrane that tethers to the mitochondrial outer membrane and is essential for cellular homeostasis. A defect in MAM is involved in various neurological diseases, including amyotrophic lateral sclerosis (ALS). Recently, we and others reported that MAM was disrupted in the models expressing several ALS-linked genes, including SOD1, SIGMAR1, VAPB, TARDBP, and FUS, suggesting that MAM disruption is deeply involved in the pathomechanism of ALS. However, it is still uncertain whether MAM disruption is a common pathology in ALS, mainly due to the absence of a simple, quantitative tool for monitoring the status of MAM. In this study, to examine the effects of various ALS-causative genes on MAM, we created the following two novel MAM reporters: MAMtracker-Luc and MAMtracker-Green. The MAMtrackers could detect MAM disruption caused by suppression of SIGMAR1 or the overexpression of ALS-linked mutant SOD1 in living cells. Moreover, the MAMtrackers have an advantage in their ability to monitor reversible changes in the MAM status induced by nutritional conditions. We used the MAMtrackers with an expression plasmid library of ALS-causative genes and noted that 76% (16/21) of the genes altered MAM integrity. Our results suggest that MAM disruption is a common pathological feature in ALS. Furthermore, we anticipate our MAMtrackers, which are suitable for high-throughput assays, to be valuable tools to understand MAM dynamics.

Keywords:  ER-mitochondria contact; amyotrophic lateral sclerosis (ALS); mitochondria-associated membrane (MAM); organelle; sigma-1 receptor (Sig1R)

DOI:  https://doi.org/10.1096/fj.202100137R
Neuropathology. 2021 Jun 13.

An autopsy case of PARK2 due to a homozygous exon 2 deletion of parkin and associated with α-synucleinopathy.

Mayuko Sakuwa, Tadashi Adachi, Kentaro Yoshida, Yoshiki Adachi, Toshiya Nakano, Ritsuko Hanajima.

  Lewy bodies (LBs) are usually detected in patients with idiopathic Parkinson's disease (PD), but there have been few reports of LBs in a familial form of early-onset PD associated with several mutations in parkin, a gene that encodes a ubiquitin E3 ligase involved in mitochondrial homeostasis, being also known as PARK2. Here, we report a case of PD with a PARK2 mutation characterized by a homozygous deletion of exon 2 and incidental LB pathology. A 60-year-old man developed tremor in the upper limbs. Although levodopa was initially effective, his symptoms slowly progressed. His cardiac uptake of 123 I-metaiodobenzylguanidine, as assessed by myocardial scintigraphy, decreased from an early stage after the onset. At the age of 81 years, he developed Legionella pneumonia and died of respiratory failure. Histopathological examination revealed a moderate loss of pigmented neurons, as well as gliosis in the substantia nigra and the locus coeruleus. Little LB-related pathology was found in the locus coeruleus, dorsal nucleus of vagal nerve, and basal nucleus of Meynert. The cardiac sympathetic nerve in the epicardium showed a reduction in the numbers of fibers immunoreactive for tyrosine hydroxylase and phosphorylated neurofilament protein. Genetic analysis of frozen brain materials revealed a homozygous deletion of exon 2 of parkin. To our knowledge, this is the first autopsy case with a homozygous deletion of exon 2 of parkin. The number of LBs was small, the age of disease onset was later than that in typical PARK2-associated PD patients, and cardiac sympathetic denervation was also present. Thus, we considered the LBs in our case as incidental and preclinical α-synucleinopathy.

Keywords:  MIBG; Parkinson; parkin gene; pathology; synuclein

DOI:  https://doi.org/10.1111/neup.12735
Metab Brain Dis. 2021 Jun 15.

The potential role of Keap1-Nrf2 pathway in the pathogenesis of Alzheimer's disease, type 2 diabetes, and type 2 diabetes-related Alzheimer's disease.

Ling He, Yi Sun.

  Kelch-like ECH associated-protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway is thought to be the key regulatory process defensing oxidative stress in multiple organs. Type 2 diabetes mellitus (T2DM) and Alzheimer's disease (AD) are both serious global health problems with high prevalence. A growing number of literatures have suggested a possible link between Keap1-Nrf2 signaling pathway and the pathological changes of T2DM, AD as well as T2DM-related AD. The current review mainly discusses how the damaged Keap1-Nrf2 signaling pathway leads to dysregulated redox molecular signaling, which may contribute to the pathogenesis of AD and T2DM-related cognitive dysfunction, as well as some compounds targeting this pathway. The further exploration of the mechanisms of this pathway could provide novel therapeutic strategies to improve cognitive function, through restoration of expression or translocation of Nrf2 and scavenging excessive free radicals.

Keywords:  Alzheimer’s disease; Antioxidant agents; Diabetes mellitus; Keap1-Nrf2 signaling pathway; Oxidative stress

DOI:  https://doi.org/10.1007/s11011-021-00762-z
Int J Biol Macromol. 2021 Jun 11. pii: S0141-8130(21)01271-X. [Epub ahead of print]

The inhibition of gadolinium ion (Gd3+) on the mitochondrial F1FO-ATPase is linked to the modulation of the mitochondrial permeability transition pore.

Cristina Algieri, Fabiana Trombetti, Alessandra Pagliarani, Micaela Fabbri, Salvatore Nesci.

  The mitochondrial permeability transition pore (PTP), which drives regulated cell death when Ca2+ concentration suddenly increases in mitochondria, was related to changes in the Ca2+-activated F1FO-ATPase. The effects of the gadolinium cation (Gd3+), widely used for diagnosis and therapy, and reported as PTP blocker, were evaluated on the F1FO-ATPase activated by Mg2+ or Ca2+ and on the PTP. Gd3+ more effectively inhibits the Ca2+-activated F1FO-ATPase than the Mg2+-activated F1FO-ATPase by a mixed-type inhibition on the former and by uncompetitive mechanism on the latter. Most likely Gd3+ binding to F1, is favoured by Ca2+ insertion. The maximal inactivation rates (Kinact) of pseudo-first order inactivation are similar either when the F1FO-ATPase is activated by Ca2+ or by Mg2+. The half-maximal inactivator concentrations (KI) are 2.35 ± 0.35 mM and 0.72 ± 0.11 mM, respectively. The potency of a mechanism-based inhibitor (Kinact/KI) also highlights a higher inhibition efficiency of Gd3+ on the Ca2+-activated F1FO-ATPase (0.59 ± 0.09 mM-1∙s-1) than on the Mg2+-activated F1FO-ATPase (0.13 ± 0.02 mM-1∙s-1). Consistently, the PTP is desensitized in presence of Gd3+. The Gd3+ inhibition on both the mitochondrial Ca2+-activated F1FO-ATPase and the PTP strengthens the link between the PTP and the F1FO-ATPase when activated by Ca2+ and provides insights on the biological effects of Gd3+.

Keywords:  Enzyme kinetics; F(1)F(O)-ATPase; Gadolinium ion; Metal cofactors; Mitochondria; Permeability transition pore

DOI:  https://doi.org/10.1016/j.ijbiomac.2021.06.065
ACS Chem Neurosci. 2021 Jun 14.

Glitazones Activate PGC-1α Signaling via PPAR-γ: A Promising Strategy for Antiparkinsonism Therapeutics.

Prabitha P, Antony Justin, T Durai Ananda Kumar, Mithuna Chinaswamy, B R Prashantha Kumar.

  Understanding various aspects of Parkinson's disease (PD) by researchers could lead to a better understanding of the disease and provide treatment alternatives that could significantly improve the quality of life of patients suffering from neurodegenerative disorders. Significant progress has been made in recent years toward this goal, but there is yet no available treatment with confirmed neuroprotective effects. Recent studies have shown the potential of PPARγ agonists, which are the ligand activated transcriptional factor of the nuclear hormone superfamily, as therapeutic targets for various neurodegenerative disorders. The activation of central PGC-1α mediates the potential role against neurogenerative diseases like PD, Huntington's disease, Alzheimer's disease, and amyotrophic lateral sclerosis. Further understanding the mechanism of neurodegeneration and the role of glitazones in the activation of PGC-1α signaling could lead to a novel therapeutic interventions against PD. Keeping this aspect in focus, the present review highlights the pathogenic mechanism of PD and the role of glitazones in the activation of PGC-1α via PPARγ for the treatment of neurodegenerative disorders.

Keywords:  Glitazones; PGC-1α; PPAR γ; Parkinson’s disease; inflammation; mitochondria dysfunction

DOI:  https://doi.org/10.1021/acschemneuro.1c00085
Cell Rep. 2021 Jun 15. pii: S2211-1247(21)00596-9. [Epub ahead of print]35(11): 109237

Stress granules inhibit fatty acid oxidation by modulating mitochondrial permeability.

Triana Amen, Daniel Kaganovich.

  The formation of stress granules (SGs) is an essential aspect of the cellular response to many kinds of stress, but its adaptive role is far from clear. SG dysfunction is implicated in aging-onset neurodegenerative diseases, prompting interest in their physiological function. Here, we report that during starvation stress, SGs interact with mitochondria and regulate metabolic remodeling. We show that SG formation leads to a downregulation of fatty acid β-oxidation (FAO) through the modulation of mitochondrial voltage-dependent anion channels (VDACs), which import fatty acids (FAs) into mitochondria. The subsequent decrease in FAO during long-term starvation reduces oxidative damage and rations FAs for longer use. Failure to form SGs, whether caused by the genetic deletion of SG components or an amyotrophic lateral sclerosis (ALS)-associated mutation, translates into an inability to downregulate FAO. Because metabolic dysfunction is a common pathological element of neurodegenerative diseases, including ALS, our findings provide a direction for studying the clinical relevance of SGs.

Keywords:  ALS; VDAC; fatty acid oxidation; lipid droplet; lipid metabolism; metabolic adaptation; mitochondria; porin; starvation; stress granule

DOI:  https://doi.org/10.1016/j.celrep.2021.109237
iScience. 2021 Jun 25. 24(6): 102498

Mitochondrial CMPK2 mediates immunomodulatory and antiviral activities through IFN-dependent and IFN-independent pathways.

Jenn-Haung Lai, De-Wei Wu, Chien-Hsiang Wu, Li-Feng Hung, Chuan-Yueh Huang, Shuk-Man Ka, Ann Chen, Zee-Fen Chang, Ling-Jun Ho.

  Mitochondria regulate the immune response after dengue virus (DENV) infection. Microarray analysis of genes identified the upregulation of mitochondrial cytidine/uridine monophosphate kinase 2 (CMPK2) by DENV infection. We used small interfering RNA-mediated knockdown (KD) and CRISPR-Cas9 knockout (KO) approaches, to investigate the role of CMPK2 in mouse and human cells. The results showed that CMPK2 was critical in DENV-induced antiviral cytokine release and mitochondrial oxidative stress and mitochondrial DNA release to the cytosol. The DENV-induced activation of Toll-like receptor (TLR)-9, inflammasome pathway, and cell migration was suppressed by CMPK2 depletion; however, viral production increased under CMPK2 deficiency. Examining mouse bone marrow-derived dendritic cells from interferon-alpha (IFN-α) receptor-KO mice and signal transducer and activator of transcription 1 (STAT1)-KO mice, we confirmed that CMPK2-mediated antiviral activity occurred in IFN-dependent and IFN-independent manners. In sum, CMPK2 is a critical factor in DENV-induced immune responses to determine innate immunity.

Keywords:  Immunology; Molecular biology; Virology

DOI:  https://doi.org/10.1016/j.isci.2021.102498
Mult Scler Relat Disord. 2021 May 29. pii: S2211-0348(21)00322-9. [Epub ahead of print]53 103055

Investigation of the correlation between mildly deleterious mtDNA Variations and the clinical progression of multiple sclerosis.

Ilse S Pienaar, Rean Mohammed, Rebecca Courtley, Michael R Gledson, Richard Reynolds, Richard Nicholas, Joanna L Elson.

   BACKGROUND: Evidence suggests that mitochondrial DNA (mtDNA) variation at a population level may influence susceptibility to, or the clinical progression of Multiple Sclerosis (MS).
OBJECTIVE: To determine if mtDNA population variation is linked to the clinical progress of MS.
METHODS: Using the complete mtDNA sequences of 217 MS patients, we applied the new 'variant load' model, designed as a framework by which to examine the role of mtDNA variation in the context of complex clinical disease.
RESULTS: No significant association was detected between mtDNA 'variant load'and the clinical measures of progression.
CONCLUSION: Our results suggest that mtDNA population variation does not play a substantial role in the clinical progression of MS; however, modest effects and/or effects in a subgroup of patients cannot be entirely excluded. Results do not exclude the possibility of detecting an association between variation and more strictly quantified variables obtained from histopathologically-stained specimens. The results further illustrate the method's applicabilityto other disease phenotypes.

Keywords:  Haplogroup; Mitochondrial DNA (mtDNA); Multiple sclerosis, Disease progression; Variant load model (VLM)

DOI:  https://doi.org/10.1016/j.msard.2021.103055
J Neurochem. 2021 Jun 15.

ALS-linked mutations impair UBQLN2 stress-induced biomolecular condensate assembly in cells.

Julia F Riley, Peter J Fioramonti, Amber K Rusnock, Heidi Hehnly, Carlos A Castañeda.

  Mutations in Ubiquilin-2 (UBQLN2), a ubiquitin-binding shuttle protein involved in several protein quality control processes, can lead to amyotrophic lateral sclerosis (ALS). We previously found that wild-type UBQLN2 forms dynamic, membraneless biomolecular condensates upon cellular stress, and undergoes liquid-liquid phase separation in vitro. However, the impact of ALS-linked mutations on UBQLN2 condensate formation in cells is unknown. Here, we employ live-cell imaging and photokinetic analysis to investigate how five patient-derived ALS-linked mutations in UBQLN2 impact stress-induced UBQLN2 condensate assembly and condensate material properties. Both wild-type and mutant UBQLN2 condensates are generally cytoplasmic and liquid-like. However, cells transfected with mutant UBQLN2 contain fewer stress-induced UBQLN2 condensates than those with wild-type UBQLN2. Most strikingly, exogenously expressed P506T UBQLN2 forms the lowest number of stress-induced condensates of all UBQLN2 mutants, and these condensates are significantly smaller than those of wild-type UBQLN2. Fluorescence recovery after photobleaching (FRAP) analysis of UBQLN2 condensates revealed higher immobile fractions for UBQLN2 mutants, especially P506T. P497S and P497H mutations differentially impact condensate properties, demonstrating that the effects of ALS-linked mutations are both position- and amino acid-dependent. Collectively, our data show that disease mutations hinder assembly and alter viscoelastic properties of stress-induced UBQLN2 condensates, potentially leading to aggregates commonly observed in ALS.

Keywords:  UBQLN2; amyotrophic lateral sclerosis (ALS); arsenite stress; biomolecular condensates; neurodegenerative disorders

DOI:  https://doi.org/10.1111/jnc.15453
Sci Rep. 2021 Jun 18. 11(1): 12875

Early-adolescent antibiotic exposure results in mitochondrial and behavioral deficits in adult male mice.

Anouk C Tengeler, Tim L Emmerzaal, Bram Geenen, Vivienne Verweij, Miranda van Bodegom, Eva Morava, Amanda J Kiliaan, Tamas Kozicz.

Exposure to antibiotic treatment has been associated with increased vulnerability to various psychiatric disorders. However, a research gap exists in understanding how adolescent antibiotic therapy affects behavior and cognition. Many antibiotics that target bacterial translation may also affect mitochondrial translation resulting in impaired mitochondrial function. The brain is one of the most metabolically active organs, and hence is the most vulnerable to impaired mitochondrial function. We hypothesized that exposure to antibiotics during early adolescence would directly affect brain mitochondrial function, and result in altered behavior and cognition. We administered amoxicillin, chloramphenicol, or gentamicin in the drinking water to young adolescent male wild-type mice. Next, we assayed mitochondrial oxidative phosphorylation complex activities in the cerebral cortex, performed behavioral screening and targeted mass spectrometry-based acylcarnitine profiling in the cerebral cortex. We found that mice exposed to chloramphenicol showed increased repetitive and compulsive-like behavior in the marble burying test, an accurate and sensitive assay of anxiety, concomitant with decreased mitochondrial complex IV activity. Our results suggest that only adolescent chloramphenicol exposure leads to impaired brain mitochondrial complex IV function, and could therefore be a candidate driver event for increased anxiety-like and repetitive, compulsive-like behaviors.

DOI: https://doi.org/10.1038/s41598-021-92203-1
Biochem Biophys Res Commun. 2021 Jun 15. pii: S0006-291X(21)00468-X. [Epub ahead of print]566 190-196

Role of the TRPM4 channel in mitochondrial function, calcium release, and ROS generation in oxidative stress.

Chen Wang, Jian Chen, Mengxue Wang, Keiji Naruse, Ken Takahashi.

  Ischemic heart disease is one of the most common causes of death worldwide. Mitochondrial dysfunction, excessive reactive oxygen species (ROS) generation, and calcium (Ca2+) overload are three key factors leading to myocardial death during ischemia-reperfusion (I/R) injury. Inhibition of TRPM4, a Ca2+-activated nonselective cation channel, protects the rat heart from I/R injury, but the specific mechanism underlying this effect is unclear. In this study, we investigated the mechanism of cardioprotection against I/R injury via TRPM4 using hydrogen peroxide (H2O2), a major contributor to oxidative stress, as an I/R injury model. We knocked out the TRPM4 gene in the rat cardiomyocyte cell line H9c2 using CRISPR/Cas9. Upon H2O2 treatment, intracellular Ca2+ level and ROS production increased in wild type (WT) cells but not in TRPM4 knockout (TRPM4KO) cells. With this treatment, two indicators of mitochondrial function, mitochondrial membrane potential (ΔΨm) and intracellular ATP levels, decreased in WT but not in TRPM4KO cells. Taken together, these findings suggest that blockade of the TRPM4 channel might protect the myocardium from oxidative stress by maintaining the mitochondrial membrane potential and intracellular ATP levels, possibly through preventing aberrant increases in intracellular Ca2+ and ROS.

Keywords:  CRISPR/Cas9; Calcium; Mitochondrial function; Myocardial ischemia-reperfusion injury; Reactive oxygen species; TRPM4

DOI:  https://doi.org/10.1016/j.bbrc.2021.03.077
Brain. 2021 Jun 15. pii: awab226. [Epub ahead of print]

Induced pluripotent stem cell-derived motor neurons of CMT type 2 patients reveal progressive mitochondrial dysfunction.

Jonas Van Lent, Peter Verstraelen, Bob Asselbergh, Elias Adriaenssens, Ligia Mateiu, Christophe Verbist, Vicky De Winter, Kristel Eggermont, Ludo Van Den Bosch, Winnok H De Vos, Vincent Timmerman.

  Axonal Charcot-Marie-Tooth neuropathies (CMT type 2) are caused by inherited mutations in various genes functioning in different pathways. The type of genes and multiplicity of mutations reflect the clinical and genetic heterogeneity in CMT2 disease, which complicates the diagnosis and has halted therapy development. Here, we used CMT2 patient-derived pluripotent stem cells (iPSCs) to identify common hallmarks of axonal degeneration shared by different CMT2 subtypes. We compared the cellular phenotypes of neurons differentiated from CMT2 patient iPSCs with those from healthy controls and a CRISPR/Cas9-corrected isogenic line. Our results demonstrate neurite network alterations along with extracellular electrophysiological abnormalities in the differentiated motor neurons. Progressive deficits in mitochondrial and lysosomal trafficking, as well as in mitochondrial morphology, were observed in all CMT2 patient lines. Differentiation of the same CMT2 iPSC-lines into peripheral sensory neurons, only gave rise to cellular phenotypes in subtypes with sensory involvement, supporting the notion that some gene mutations predominantly affect motor neurons. We revealed a common mitochondrial dysfunction in CMT2-derived motor neurons, supported by alterations in the expression pattern and oxidative phosphorylation, which could be recapitulated in the sciatic nerve tissue of a symptomatic mouse model. Inhibition of a dual leucine zipper kinase (DLK) could partially ameliorate the mitochondrial disease phenotypes in CMT2 subtypes. Altogether, our data reveals shared cellular phenotypes across different CMT2 subtypes and suggests that targeting such common pathomechanisms could allow the development of a uniform treatment for CMT2.

Keywords:  Charcot-Marie-Tooth neuropathy; dual leucine kinase inhibitor; iPSC-derived motor and sensory neurons; mitochondrial dysfunction; phenotyping

DOI:  https://doi.org/10.1093/brain/awab226
EMBO J. 2021 06 14. e105985

Phosphoproteomic identification of ULK substrates reveals VPS15-dependent ULK/VPS34 interplay in the regulation of autophagy.

Thomas John Mercer, Yohei Ohashi, Stefan Boeing, Harold B J Jefferies, Stefano De Tito, Helen Flynn, Shirley Tremel, Wenxin Zhang, Martina Wirth, David Frith, Ambrosius P Snijders, Roger Lee Williams, Sharon A Tooze.

  Autophagy is a process through which intracellular cargoes are catabolised inside lysosomes. It involves the formation of autophagosomes initiated by the serine/threonine kinase ULK and class III PI3 kinase VPS34 complexes. Here, unbiased phosphoproteomics screens in mouse embryonic fibroblasts deleted for Ulk1/2 reveal that ULK loss significantly alters the phosphoproteome, with novel high confidence substrates identified including VPS34 complex member VPS15 and AMPK complex subunit PRKAG2. We identify six ULK-dependent phosphorylation sites on VPS15, mutation of which reduces autophagosome formation in cells and VPS34 activity in vitro. Mutation of serine 861, the major VPS15 phosphosite, decreases both autophagy initiation and autophagic flux. Analysis of VPS15 knockout cells reveals two novel ULK-dependent phenotypes downstream of VPS15 removal that can be partially recapitulated by chronic VPS34 inhibition, starvation-independent accumulation of ULK substrates and kinase activity-regulated recruitment of autophagy proteins to ubiquitin-positive structures.

Keywords:  PIK3R4; PRKAG2; ULK1; VPS15; p62

DOI:  https://doi.org/10.15252/embj.2020105985