bims-axbals 2023-12-31 papers

bims-axbals

Biomed News

on Axonal biology and ALS

Issue of 2023–12–31
thirteen papers selected by
TJ Krzystek, ALS Therapy Development Institute

The importance of patient-centred drug development for amyotrophic lateral sclerosis.
Sex-Specific Early Retinal Dysfunction in Mutant TDP-43 Transgenic Mice.
Generation of a human induced pluripotent stem cell line (SMUSHi002-A) from an ALS patient carrying a heterozygous mutation c.1562G > A in the FUS gene.
Distinctive in vitro phenotypes in iPSC-derived neurons from patients with gain- and loss-of-function SCN2A developmental and epileptic encephalopathy.
A Microfluidic High-Capacity Screening Platform for Neurological Disorders.
Rhotekin regulates axon regeneration through the talin-Vinculin-Vinexin axis in Caenorhabditis elegans.
C9orf72 Repeat Expansion Discordance in 6 Multigenerational Kindreds.
The IRE1/Xbp1 axis restores ER and tissue homeostasis perturbed by excess Notch in Drosophila.
The role of lysine acetylation in the function of mitochondrial ribosomal protein L12.
Reproducible Differentiation of Human Pluripotent Stem Cells into Two-Dimensional Cortical Neuron Cultures with Checkpoints for Success.
Enhanced axon outgrowth of spinal motor neurons in co-culturing with dorsal root ganglions antagonizes the growth inhibitory environment.
Validation of automated pipeline for the assessment of a motor speech disorder in amyotrophic lateral sclerosis (ALS).
Mitochondrial electron transport chain, ceramide, and coenzyme Q are linked in a pathway that drives insulin resistance in skeletal muscle.

Neuropathol Appl Neurobiol. 2023 Dec;49(6): e12944

The importance of patient-centred drug development for amyotrophic lateral sclerosis.

Matthew C Kiernan, Glenda M Halliday, Dominic B Rowe, Rachel H Tan.

DOI: https://doi.org/10.1111/nan.12944
J Alzheimers Dis. 2023 Dec 18.

Sex-Specific Early Retinal Dysfunction in Mutant TDP-43 Transgenic Mice.

Ju Gao, Henri Leinonen, Evan J Wang, Mao Ding, George Perry, Krzysztof Palczewski, Xinglong Wang.

   BACKGROUND: Increasing evidence has highlighted retinal impairments in neurodegenerative diseases. Dominant mutations in TAR DNA-binding protein 43 (TDP-43) cause amyotrophic lateral sclerosis (ALS), and the accumulation of TDP-43 in the cytoplasm is a pathological hallmark of ALS, frontotemporal dementia (FTD), and many other neurodegenerative diseases.
OBJECTIVE: While homozygous transgenic mice expressing the disease-causing human TDP-43 M337V mutant (TDP-43M337V mice) experience premature death, hemizygous TDP-43M337V mice do not suffer sudden death, but they exhibit age-dependent motor-coordinative and cognitive deficits. This study aims to leverage the hemizygous TDP-43M337V mice as a valuable ALS/FTD disease model for the assessment also of retinal changes during the disease progression.
METHODS: We evaluated the retinal function of young TDP-43M337V mice by full field electroretinogram (ERG) recordings.
RESULTS: At 3-4 months of age, well before the onset of brain dysfunction at 8 months, the ERG responses were notably impaired in the retinas of young female TDP-43M337V mice in contrast to their male counterparts and age-matched non-transgenic mice. Mitochondria have been implicated as critical targets of TDP-43. Further investigation revealed that significant changes in the key regulators of mitochondrial dynamics and bioenergetics were only observed in the retinas of young female TDP-43M337V mice, while these alterations were not present in the brains of either gender.
CONCLUSIONS: Together our findings suggest a sex-specific vulnerability within the retina in the early disease stage, and highlight the importance of retinal changes and mitochondrial markers as potential early diagnostic indicators for ALS, FTD, and other TDP-43 related neurodegenerative conditions.

Keywords:  Alzheimer’s disease; OXPHOS; TDP-43; amyotrophic lateral sclerosis; frontotemporal dementia; mitochondrial dynamics; retina; sex differences

DOI:  https://doi.org/10.3233/JAD-231102
Stem Cell Res. 2023 Dec 16. pii: S1873-5061(23)00272-6. [Epub ahead of print]74 103286

Generation of a human induced pluripotent stem cell line (SMUSHi002-A) from an ALS patient carrying a heterozygous mutation c.1562G > A in the FUS gene.

Mei Tang, Min Xiong, Wenyan Zhou, Jiafan Lei, Min Huang, Chunyan Huang, Fei Wang, Jun Liu, Jun Li, Xueqing Xu.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease. Affected patients experience gradual loss of their spinal cord and cortical motor neurons with consequent muscle weakness and emaciation, and eventual respiratory failure. The pathogenesis of ALS remains largely unknown although the FUS (sarcoma fusion gene) gene is known to be one of the major pathogenic genes. We have generated an induced pluripotent stem cell line SMUSHi002-A from an ALS patient who carries a heterozygous mutation c.1562G > A in FUS. This cell line will serve as a useful model to investigate disease pathogenesis and develop potential therapeutic approaches for ALS.

DOI: https://doi.org/10.1016/j.scr.2023.103286
J Neurosci. 2023 Dec 26. pii: JN-RM-0692-23. [Epub ahead of print]

Distinctive in vitro phenotypes in iPSC-derived neurons from patients with gain- and loss-of-function SCN2A developmental and epileptic encephalopathy.

Miaomiao Mao, Cristiana Mattei, Ben Rollo, Sean Byars, Claire Cuddy, Geza Berecki, Jacqueline Heighway, Svenja Pachernegg, Trevelyan Menheniott, Danielle Apted, Linghan Jia, Kelley Dalby, Alex Nemiroff, Saul Mullen, Christopher A Reid, Snezana Maljevic, Steven Petrou.

SCN2A encodes NaV1.2, an excitatory neuron voltage-gated sodium channel and a major monogenic cause of neurodevelopmental disorders, including developmental and epileptic encephalopathies (DEE) and autism. Clinical presentation and pharmocosensitivity vary with the nature of SCN2A variant dysfunction and can be divided into gain-of-function (GoF) cases with pre- or peri-natal seizures and loss-of-function (LoF) patients typically having infantile spasms after 6 months of age.We established and assessed patient induced pluripotent stem cell (iPSC) - derived neuronal models for two recurrent SCN2A DEE variants with GoF R1882Q and LoF R853Q associated with early- and late-onset DEE, respectively. Two male patient-derived iPSC isogenic pairs were differentiated using Neurogenin-2 overexpression yielding populations of cortical-like glutamatergic neurons. Functional properties were assessed using patch clamp and multielectrode array recordings and transcriptomic profiles obtained with total mRNA sequencing after 2-4 weeks in culture.At 3 weeks of differentiation, increased neuronal activity at cellular and network levels was observed for R1882Q iPSC-derived neurons. In contrast, R853Q neurons showed only subtle changes in excitability after 4 weeks and an overall reduced network activity after 7 weeks in vitro Consistent with the reported efficacy in some GoF SCN2A patients, phenytoin (sodium channel blocker) reduced the excitability of neurons to the control levels in R1882Q neuronal cultures. Transcriptomic alterations in neurons were detected for each variant and convergent pathways suggested potential shared mechanisms underlying SCN2A DEE.In summary, patient iPSC-derived neuronal models of SCN2A GoF and LoF pathogenic variants causing DEE show specific functional and transcriptomic in vitro phenotypes.Significance statement SCN2A encodes one of the major brain voltage-gated sodium channels, NaV1.2, and is a major monogenic cause of neurodevelopmental disorders, including severe infantile epilepsy and autism. SCN2A pathogenic variants cause either gain or loss of channel function, which correlates well with the clinical phenotype. Gain of function variants are associated with early-onset seizures with or without developmental delay, whereas loss of function results in late-onset severe epilepsy and/or autism. We used patient-derived induced pluripotent stem cells to generate neuronal cultures for two recurring SCN2A variants causing early and late seizure onset epilepsy. Identified electrophysiological and transcriptome changes compared to the isogenic control lines can be correlated with the distinguishable clinical phenotype.

DOI: https://doi.org/10.1523/JNEUROSCI.0692-23.2023
ACS Chem Neurosci. 2023 Dec 27.

A Microfluidic High-Capacity Screening Platform for Neurological Disorders.

Lydia Moll, Johan Pihl, Mattias Karlsson, Paul Karila, Camilla I Svensson.

  Compartmentalized cell cultures (CCCs) provide the possibility to study mechanisms of neurodegenerative diseases, such as spreading of misfolded proteins in Alzheimer's or Parkinson's disease or functional changes in, e.g., chronic pain, in vitro. However, many CCC devices do not provide the necessary capacity for identifying novel mechanisms, targets, or drugs in a drug discovery context. Here, we present a high-capacity cell culture microtiter microfluidic plate compliant with American National Standard Institute of the Society for Laboratory Automation and Screening (ANSI/SLAS) standards that allows to parallelize up to 96 CCCs/experimental units, where each experimental unit comprises three microchannel-connected compartments. The plate design allows the specific treatment of cells in individual compartments through the application of a fluidic barrier. Moreover, the compatibility of the plate with neuronal cultures was confirmed with rodent primary as well as human-induced pluripotent stem cell-derived neurons of the central or peripheral nervous system for up to 14 days in culture. Using immunocytochemistry, we demonstrated that the plate design restricts neuronal soma to individual compartments, while axons, but not dendrites, can grow through the connecting microchannels to neighboring compartments. In addition, we show that neurons are spontaneously active and, as deemed by the appearance of synchronous depolarizations in neighboring compartments, are synaptically coupled. In summary, the design of the microfluidic plate allows for both morphological and functional studies of neurological in vitro cultures with increased capacity to support identification of novel mechanisms, targets, or drugs.

Keywords:  drug discovery; electrophysiology; in vitro systems; microfluidics; neurological disorders; plate-based screening

DOI:  https://doi.org/10.1021/acschemneuro.3c00409
PLoS Genet. 2023 Dec;19(12): e1011089

Rhotekin regulates axon regeneration through the talin-Vinculin-Vinexin axis in Caenorhabditis elegans.

Yoshiki Sakai, Tatsuhiro Shimizu, Mayuka Tsunekawa, Naoki Hisamoto, Kunihiro Matsumoto.

Axon regeneration requires actomyosin interaction, which generates contractile force and pulls the regenerating axon forward. In Caenorhabditis elegans, TLN-1/talin promotes axon regeneration through multiple down-stream events. One is the activation of the PAT-3/integrin-RHO-1/RhoA GTPase-LET-502/ROCK (Rho-associated coiled-coil kinase)-regulatory non-muscle myosin light-chain (MLC) phosphorylation signaling pathway, which is dependent on the MLC scaffolding protein ALP-1/ALP-Enigma. The other is mediated by the F-actin-binding protein DEB-1/vinculin and is independent of the MLC phosphorylation pathway. In this study, we identified the svh-7/rtkn-1 gene, encoding a homolog of the RhoA-binding protein Rhotekin, as a regulator of axon regeneration in motor neurons. However, we found that RTKN-1 does not function in the RhoA-ROCK-MLC phosphorylation pathway in the regulation of axon regeneration. We show that RTKN-1 interacts with ALP-1 and the vinculin-binding protein SORB-1/vinexin, and that SORB-1 acts with DEB-1 to promote axon regeneration. Thus, RTKN-1 links the DEB-1-SORB-1 complex to ALP-1 and physically connects phosphorylated MLC on ALP-1 to the actin cytoskeleton. These results suggest that TLN-1 signaling pathways coordinate MLC phosphorylation and recruitment of phosphorylated MLC to the actin cytoskeleton during axon regeneration.

DOI: https://doi.org/10.1371/journal.pgen.1011089
Neurol Genet. 2024 Feb;10(1): e200112

C9orf72 Repeat Expansion Discordance in 6 Multigenerational Kindreds.

Marie Ryan, Mark A Doherty, Ahmad Al Khleifat, Emmet Costello, Jennifer C Hengeveld, Mark Heverin, Ammar Al-Chalabi, Russell L Mclaughlin, Orla Hardiman.

Background and Objectives: A hexanucleotide repeat expansion in the noncoding region of the C9orf72 gene is the most common genetically identifiable cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia in populations of European ancestry. Pedigrees associated with this expansion exhibit phenotypic heterogeneity and incomplete disease penetrance, the basis of which is poorly understood. Relatives of those carrying the C9orf72 repeat expansion exhibit a characteristic cognitive endophenotype independent of carrier status. To examine whether additional shared genetic or environmental risks within kindreds could compel this observation, we have conducted a detailed cross-sectional study of the inheritance within multigenerational Irish kindreds carrying the C9orf72 repeat expansion.
Methods: One hundred thirty-one familial ALS pedigrees, 59 of which carried the C9orf72 repeat expansion (45.0% [95% CI 36.7-53.5]), were identified through the Irish population-based ALS register. C9orf72 genotyping was performed using repeat-primed PCR with amplicon fragment length analysis. Pedigrees were further investigated using SNP, targeted sequencing data, whole-exome sequencing, and whole-genome sequencing.
Results: We identified 21 kindreds where at least 1 family member with ALS carried the C9orf72 repeat expansion and from whom DNA was available from multiple affected family members. Of these, 6 kindreds (28.6% [95% CI 11.8-48.3]) exhibited discordant segregation. The C9orf72 haplotype was studied in 2 families and was found to segregate with the C9orf72-positive affected relative but not the C9orf72-negative affected relative. No other ALS pathogenic variants were identified within these discordant kindreds.
Discussion: Family members of kindreds associated with the C9orf72 repeat expansion may carry an increased risk of developing ALS independent of their observed carrier status. This has implications for assessment and counseling of asymptomatic individuals regarding their genetic risk.

DOI: https://doi.org/10.1212/NXG.0000000000200112
Dev Biol. 2023 Dec 23. pii: S0012-1606(23)00209-9. [Epub ahead of print]507 11-19

The IRE1/Xbp1 axis restores ER and tissue homeostasis perturbed by excess Notch in Drosophila.

Yu Li, Dongyue Liu, Haochuan Wang, Xuejing Zhang, Bingwei Lu, Shuangxi Li.

  Notch signaling controls numerous key cellular processes including cell fate determination and cell proliferation. Its malfunction has been linked to many developmental abnormalities and human disorders. Overactivation of Notch signaling is shown to be oncogenic. Retention of excess Notch protein in the endoplasmic reticulum (ER) can lead to altered Notch signaling and cell fate, but the mechanism is not well understood. In this study, we show that V5-tagged or untagged exogenous Notch is retained in the ER when overexpressed in fly tissues. Furthermore, we show that Notch retention in the ER leads to robust ER enlargement and elicits a rough eye phenotype. Gain-of-function of unfolded protein response (UPR) factors IRE1 or spliced Xbp1 (Xbp1-s) alleviates Notch accumulation in the ER, restores ER morphology and ameliorates the rough eye phenotype. Our results uncover a pivotal role of the IRE1/Xbp1 axis in regulating the detrimental effect of ER-localized excess Notch protein during development and tissue homeostasis.

Keywords:  Drosophila melanogaster; Endoplasmic reticulum (ER); IRE1; Notch; Unfolded protein response (UPR)

DOI:  https://doi.org/10.1016/j.ydbio.2023.12.007
Proteins. 2023 Dec 25.

The role of lysine acetylation in the function of mitochondrial ribosomal protein L12.

Katelynn V Paluch, Karlie R Platz, Emma J Rudisel, Ryan R Erdmann, Taylor R Laurin, Kristin E Dittenhafer-Reed.

  Mitochondria play a central role in energy production and cellular metabolism. Mitochondria contain their own small genome (mitochondrial DNA, mtDNA) that carries the genetic instructions for proteins required for ATP synthesis. The mitochondrial proteome, including the mitochondrial transcriptional machinery, is subject to post-translational modifications (PTMs), including acetylation and phosphorylation. We set out to determine whether PTMs of proteins associated with mtDNA may provide a potential mechanism for the regulation of mitochondrial gene expression. Here, we focus on mitochondrial ribosomal protein L12 (MRPL12), which is thought to stabilize mitochondrial RNA polymerase (POLRMT) and promote transcription. Numerous acetylation sites of MRPL12 were identified by mass spectrometry. We employed amino acid mimics of the acetylated (lysine to glutamine mutants) and deacetylated (lysine to arginine mutants) versions of MRPL12 to interrogate the role of lysine acetylation in transcription initiation in vitro and mitochondrial gene expression in HeLa cells. MRPL12 acetyl and deacetyl protein mimics were purified and assessed for their ability to impact mtDNA promoter binding of POLRMT. We analyzed mtDNA content and mitochondrial transcript levels in HeLa cells upon overexpression of acetyl and deacetyl mimics of MRPL12. Our results suggest that MRPL12 single-site acetyl mimics do not change the mtDNA promoter binding ability of POLRMT or mtDNA content in HeLa cells. Individual acetyl mimics may have modest effects on mitochondrial transcript levels. We found that the mitochondrial deacetylase, Sirtuin 3, is capable of deacetylating MRPL12 in vitro, suggesting a potential role for dynamic acetylation controlling MRPL12 function in a role outside of the regulation of gene expression.

Keywords:  acetylation; mitochondrial DNA; mitochondrial genome; mitochondrial proteins; post-translational protein modification; transcription

DOI:  https://doi.org/10.1002/prot.26654
Curr Protoc. 2023 Dec;3(12): e948

Reproducible Differentiation of Human Pluripotent Stem Cells into Two-Dimensional Cortical Neuron Cultures with Checkpoints for Success.

Elisa A Waxman, Lea V Dungan, Leah M DeFlitch, Julie P Merchant, Alyssa L Gagne, Ethan M Goldberg, Deborah L French.

  The patterning of excitatory cortical neurons from human pluripotent stem cells (hPSCs) is a desired technique for the study of neurodevelopmental disorders, as neurons can be created and compared from control hPSC lines, hPSC lines generated from patients, and CRISPR-modified hPSC lines. Therefore, this technique allows for the examination of disease phenotypes and assists in the development of potential new therapeutics for neurodevelopmental disorders. Many protocols, however, are optimized for use with specific hPSC lines or within a single laboratory, and they often provide insufficient guidance on how to identify positive stages in the differentiation or how to troubleshoot. Here, we present an efficient and reproducible directed differentiation protocol to generate two-dimensional cultures of hPSC-derived excitatory cortical neurons without intermediary embryoid body formation. This novel protocol is supported by our data generated with five independent hPSC lines and in two independent laboratories. Importantly, as neuronal differentiations follow a long time course to reach maturity, we provide extensive guidance regarding morphological and flow cytometry checkpoints allowing for early indications of successful differentiation. We also include extensive troubleshooting tips and support protocols to assist the operator. The goal of this protocol is to assist others in the successful differentiation of excitatory cortical neurons from hPSCs. © 2023 Wiley Periodicals LLC. Basic Protocol: Directed differentiation of hPSCs into excitatory cortical neurons Support Protocol 1: Harvesting and fixing cells for flow cytometry analyses Support Protocol 2: Performing flow cytometry analyses Support Protocol 3: Thawing NPCs from a cryopreserved stock Alternate Protocol 1: Continuing Expansion of NPCs Alternate Protocol 2: Treatment of neurons with Ara-C to ablate radial glia Support Protocol 4: Experimental methods for validation of excitatory cortical neurons.

Keywords:  cortical; differentiation; neural; neurons; stem cells

DOI:  https://doi.org/10.1002/cpz1.948
Regen Ther. 2024 Mar;25 68-76

Enhanced axon outgrowth of spinal motor neurons in co-culturing with dorsal root ganglions antagonizes the growth inhibitory environment.

Zi-Xing Xu, Dan Xu, Fang Fang, Ying-Juan Fan, Bing Wu, Yu-Fan Chen, Hao-En Huang, Xin-Hao Huang, Yue-Hong Zhuang, Wei-Hong Xu.

   Introduction: Forming a bridge made of functional axons to span the lesion is essential to reconstruct the motor circuitry following spinal cord injury (SCI). Dorsal root ganglion (DRG) axons are robust in axon growth and have been proved to facilitate the growth of cortical neurons in a process of axon-facilitated axon regeneration. However, whether DRG transplantation affects the axon outgrowth of spinal motor neurons (SMNs) that play crucial roles in motor circuitry remains unclear.
Methods: We investigated the axonal growth patterns of co-cultured DRGs and SMN aggregates (SMNAs) taking advantage of a well-designed 3D-printed in vitro system. Chondroitin sulphate proteoglycans (CSPG) induced inhibitory matrix was introduced to imitate the inhibitory environment following SCI. Axonal lengths of DRG, SMNA or DRG & SMNA cultured on the permissive or CSPG induced inhibitory matrix were measured and compared.
Results: Our results indicated that under the guidance of full axonal connection generated from two opposing populations of DRGs, SMNA axons were growth-enhanced and elongated along the DRG axon bridge to distances that they could not otherwise reach. Quantitatively, the co-culture increased the SMNA axonal length by 32.1 %. Moreover, the CSPG matrix reduced the axonal length of DRGs and SMNAs by 46.2 % and 17.7 %, respectively. This inhibitory effect was antagonized by the co-culture of DRGs and SMNAs. Especially for SMNAs, they extended the axons across the CSPG-coating matrix, reached the lengths close to those of SMNAs cultured on the permissive matrix alone.
Conclusions: This study deepens our understanding of axon-facilitated reconstruction of the motor circuitry. Moreover, the results support SCI treatment utilizing the enhanced outgrowth of axons to restore functional connectivity in SCI patients.

Keywords:  3D printing; Axonal bridging; Central nervous system regeneration; Dorsal root ganglia transplantation; Experimental modeling; Spinal cord injury; Spinal motor neuron aggregates

DOI:  https://doi.org/10.1016/j.reth.2023.11.013
Digit Health. 2023 Jan-Dec;9:9 20552076231219102

Validation of automated pipeline for the assessment of a motor speech disorder in amyotrophic lateral sclerosis (ALS).

Leif Er Simmatis, Jessica Robin, Timothy Pommée, Scotia McKinlay, Rupinder Sran, Niyousha Taati, Justin Truong, Bharatkumar Koyani, Yana Yunusova.

   Background and objective: Amyotrophic lateral sclerosis (ALS) frequently causes speech impairments, which can be valuable early indicators of decline. Automated acoustic assessment of speech in ALS is attractive, and there is a pressing need to validate such tools in line with best practices, including analytical and clinical validation. We hypothesized that data analysis using a novel speech assessment pipeline would correspond strongly to analyses performed using lab-standard practices and that acoustic features from the novel pipeline would correspond to clinical outcomes of interest in ALS.
Methods: We analyzed data from three standard speech assessment tasks (i.e., vowel phonation, passage reading, and diadochokinesis) in 122 ALS patients. Data were analyzed automatically using a pipeline developed by Winterlight Labs, which yielded 53 acoustic features. First, for analytical validation, data were analyzed using a lab-standard analysis pipeline for comparison. This was followed by univariate analysis (Spearman correlations between individual features in Winterlight and in-lab datasets) and multivariate analysis (sparse canonical correlation analysis (SCCA)). Subsequently, clinical validation was performed. This included univariate analysis (Spearman correlation between automated acoustic features and clinical measures) and multivariate analysis (interpretable autoencoder-based dimensionality reduction).
Results: Analytical validity was demonstrated by substantial univariate correlations (Spearman's ρ > 0.70) between corresponding pairs of features from automated and lab-based datasets, as well as interpretable SCCA feature groups. Clinical validity was supported by strong univariate correlations between automated features and clinical measures (Spearman's ρ > 0.70), as well as associations between multivariate outputs and clinical measures.
Conclusion: This novel, automated speech assessment feature set demonstrates substantial promise as a valid tool for analyzing impaired speech in ALS patients and for the further development of these technologies.

Keywords:  Motor speech; amyotrophic lateral sclerosis; digital biomarkers; speech; validation

DOI:  https://doi.org/10.1177/20552076231219102
Elife. 2023 Dec 27. pii: RP87340. [Epub ahead of print]12

Mitochondrial electron transport chain, ceramide, and coenzyme Q are linked in a pathway that drives insulin resistance in skeletal muscle.

Alexis Diaz-Vegas, Søren Madsen, Kristen C Cooke, Luke Carroll, Jasmine X Y Khor, Nigel Turner, Xin Y Lim, Miro A Astore, Jonathan C Morris, Anthony S Don, Amanda Garfield, Simona Zarini, Karin A Zemski Berry, Andrew P Ryan, Bryan C Bergman, Joseph T Brozinick, David E James, James G Burchfield.

  Insulin resistance (IR) is a complex metabolic disorder that underlies several human diseases, including type 2 diabetes and cardiovascular disease. Despite extensive research, the precise mechanisms underlying IR development remain poorly understood. Previously we showed that deficiency of coenzyme Q (CoQ) is necessary and sufficient for IR in adipocytes and skeletal muscle (Fazakerley et al., 2018). Here, we provide new insights into the mechanistic connections between cellular alterations associated with IR, including increased ceramides, CoQ deficiency, mitochondrial dysfunction, and oxidative stress. We demonstrate that elevated levels of ceramide in the mitochondria of skeletal muscle cells result in CoQ depletion and loss of mitochondrial respiratory chain components, leading to mitochondrial dysfunction and IR. Further, decreasing mitochondrial ceramide levels in vitro and in animal models (mice, C57BL/6J) (under chow and high-fat diet) increased CoQ levels and was protective against IR. CoQ supplementation also rescued ceramide-associated IR. Examination of the mitochondrial proteome from human muscle biopsies revealed a strong correlation between the respirasome system and mitochondrial ceramide as key determinants of insulin sensitivity. Our findings highlight the mitochondrial ceramide-CoQ-respiratory chain nexus as a potential foundation of an IR pathway that may also play a critical role in other conditions associated with ceramide accumulation and mitochondrial dysfunction, such as heart failure, cancer, and aging. These insights may have important clinical implications for the development of novel therapeutic strategies for the treatment of IR and related metabolic disorders.

Keywords:  biochemistry; cell biology; ceramides; chemical biology; coenzyme Q; human; insulin resistance; mitochondria; mouse; muscle; rat

DOI:  https://doi.org/10.7554/eLife.87340