Citations

Abstract Oxidative phosphorylation (OXPHOS) is a central function and a key indicator of mitochondrial fitness, yet studies in human tissue remain limited. Inclusion body myositis (IBM) is a progressive myopathy that lies at the intersection of aging, inflammation and mitochondrial dysfunction. We aimed to perform a comprehensive profiling of mitochondrial respiration in muscle tissue from patients with IBM. A wide battery of complementary approaches from RNA level to high-resolution respirometry on permeabilized muscle fibers was employed. The relationship between mitochondrial respiration, mitochondrial content, mitochondrial DNA (mtDNA) abnormalities and mitophagy was examined, along with the correlation with various clinical parameters to determine their clinical relevance. The study included a total of 67 patients with IBM and 45 controls. On high resolution respirometry of permeabilized muscle fibers, IBM samples exhibited reduced maximal mitochondrial respiration per tissue weight in State 3 (high substrates, high ADP) and uncoupled state with decreased coupling efficiency and higher leak control ratios. When adjusting for citrate synthase reflecting mitochondrial content, male patients had decreased State 3 intrinsic respiration, whereas female patients had greater intrinsic respiration under leak states. Complex I activity was decreased mainly in female patients, in whom complex II control ratio positively correlated with disease duration and severity. IBM was further associated with decreased RNA levels of all complexes, and lower protein expression of complex I, III, IV and V, likely related to the lower mtDNA content seen in IBM samples. Regarding the production of reactive oxygen species, IBM samples exhibited lower maximal H2O2 emission, accompanied by a higher total antioxidant capacity that positively correlated with disease duration in female patients. Lastly, correlation analyses suggested that impaired mitochondrial respiration, altered mitophagy, and reduced mtDNA content are interconnected in IBM and maybe of clinical significance. IBM is characterized by multifaceted, clinically relevant impairments in mitochondrial respiration. Future studies should further explore underlying pathomechanisms and the variation of mitochondrial respiration by disease stage.

Abstract Background Oxidative phosphorylation (OXPHOS) is a central function and a key indicator of mitochondrial fitness, yet studies in human tissue remain limited. Inclusion body myositis (IBM) is a progressive myopathy that lies at the intersection of aging, inflammation and mitochondrial dysfunction. We aimed to perform a comprehensive profiling of mitochondrial respiration in muscle tissue from patients with IBM. Methods A wide battery of complementary tests from RNA level to high-resolution respirometry on permeabilized muscle fibers was performed. The relationship between respiration, mitochondrial content, mitochondrial DNA (mtDNA) abnormalities and mitophagy was examined, along with the correlation with various clinical parameters to determine the clinical significance of the findings. Results The study included a total of 67 patients with IBM and 45 controls. IBM muscle tissue exhibited reduced maximal respiration per tissue weight in State 3 (high substrates, high ADP) and uncoupled state with decreased coupling efficiency and higher leak control ratios. When adjusting for citrate synthase reflecting mitochondrial content, males had decreased State 3 intrinsic respiration, whereas females had greater intrinsic respiration in leak states. Complex II control ratio strongly correlated with disease duration and severity only in females. IBM was associated with decreased RNA and protein expression of OXPHOS complexes. Complex I activity was decreased mainly in females. IBM samples exhibited lower maximal H 2 O 2 emission, accompanied by a higher total antioxidant capacity that correlated with disease duration in females. In IBM, there was decreased mtDNA content, and impaired mitophagy, both of which strongly correlated with respirometry measures and markers of disease severity, indicating these pathways are likely interconnected and of clinical significance. Conclusion IBM is characterized by multilevel impairments in mitochondrial coupling efficiency, revealing several potential therapeutic targets to improve mitochondrial fitness, while accounting for sex-specific differences.

Abstract Background Early mechanisms of neuronal damage in Alzheimer's Disease (AD) include mitochondrial dysfunction and abnormal energy homeostasis. AD‐associated mitochondrial signaling was shown to occur upstream of amyloid precursor protein processing, Ab production, apolipoprotein E (APOE) expression, and tau pathology. However, whether alterations in mitochondrial function are sufficient to instigate AD remains unclear. Mitochondrial complex I (mtCI) is a rate‐limiting enzyme in the oxidative phosphorylation (OXPHOS) machinery. Changes in mtCI activity influence ATP and ROS production, cellular energy and redox homeostasis. We examined how reduced activity of mtCI due to a knockout of one of its accessory subunits Ndufs4 in mice affects transcriptional networks in the brain. We also determined whether reduced activity of mtCI abrogates neuroprotective properties of a small molecule CP2. Method RNA‐sequencing of the brain tissue was performed using Ndufs4 ‐/‐ and control mice ( n = 3–5 per group). The co‐expression modules of differentially expressed genes were compared to the AMP‐AD patient cohort data. Key mechanisms, including mitophagy signaling, were confirmed using ELISA and western blot analyses; the effect on mtCI activity was confirmed using functional assays. Results In Ndufs4 ‐/‐ mice, a residual activity of mtCI was ∼50% of the control. This loss of function led to a global disruption of mitochondrial homeostasis, energy metabolism, synaptic function, significant increase in mitophagy and a reduction in biogenesis. Transcriptomic signatures in brain tissue of male and female Ndufs4 ‐/‐ mice overlapped with changer reported for AD patients and AD mouse models with Ab and p ‐tau pathologies. These changes were partially rescued by a neuroprotective mitochondria‐targeting small molecule CP2. Consistent with studies in AD mice, CP2 treatment in Ndufs4 ‐/‐ mice augmented the expression of genes associated with mitochondrial biogenesis and turnover, synaptic activity, autophagy, redox balance, and reduced expression of genes related to inflammation. Sex‐specific differences included better adaptation to a reduced activity of mtCI and to CP2 treatment in female mice. Conclusion Reduced mtCI activity was sufficient to induce the AD‐like transcriptomic changes in brain. Residual mtCI function was sufficient to mediate neuroprotective effect of CP2, emphasizing a key role of mitochondria in AD development and treatment.

Abstract INTRODUCTION Mitochondrial dysfunction is implicated in Alzheimer's disease (AD), but whether it drives AD‐associated changes is unclear. We assessed transcriptomic alterations in the brains of Ndufs4 −/− mice, a model of mitochondrial complex I (mtCI) deficiency, and evaluated the therapeutic effects of the neuroprotective mtCI inhibitor CP2. METHODS Cortico‐hippocampal tissue from Ndufs4 −/− and wild‐type mice was subjected to transcriptomic analysis, followed by cross‐species comparisons to human late‐onset AD and familial AD mouse datasets. RESULTS Knockout of Ndufs4 ‐mediated mtCI deficiency disrupted mitochondrial homeostasis, energy metabolism, and synaptic gene expression, recapitulating transcriptomic signatures of AD. CP2 treatment partially reversed these changes, with female Ndufs4 −/− mice showing greater compensatory adaptations and treatment responses. DISCUSSION Loss of mtCI activity alone is sufficient to induce AD‐like molecular changes in the brain, independent of amyloid beta or phosphorylated tau. CP2‐mediated rescue highlights the potential of targeting mitochondria as a therapeutic strategy for AD. Sex‐specific responses suggest important considerations for personalized therapeutics. Highlights Activity of mitochondrial complex I (mtCI) affects broad mitochondrial and neuronal transcriptional networks. A reduction of mtCI activity is sufficient to induce transcriptomic changes reminiscent of those observed in late‐onset Alsheimer's disease (AD) patients and familial mouse models of AD. Pharmacological targeting of mtCI mediates neuroprotective signaling. Male and female mice have differential responses to the loss of mtCI activity and to the mitochondria‐targeted therapeutics. Mitochondria play a key role in AD development and treatment.

AbstractINTRODUCTIONPhosphorylated ubiquitin (p‐S65‐Ub) is generated during PINK1‐PRKN mitophagy as a specific marker of mitochondrial damage. Despite the widespread deposition of p‐S65‐Ub in aged and diseased human brain, the genetic contribution to its accumulation remains unclear.METHODSTo identify novel mitophagy regulators, we performed a genome‐wide association study using p‐S65‐Ub level as a quantitative trait in 1012 autopsy‐confirmed Lewy body disease (LBD) samples.RESULTSWe identified a significant genome‐wide association with p‐S65‐Ub for rs429358 (apolipoprotein E ε4 [APOE4]) and a suggestive association for rs6480922 (ZMIZ1). APOE4 was associated with higher p‐S65‐Ub levels and greater neuropathological burden. Functional validation in mouse and human induced pluripotent stem cell (iPSC) models confirmed APOE4‐mediated mitophagy alterations. Intriguingly, ZMIZ1 rs6480922 was associated with lower p‐S65‐Ub levels, reduced neuropathological load, and increased brain weight, indicating a potential protective role.DISCUSSIONOur findings underscore the importance of mitochondrial quality control in LBD pathogenesis and nominate regulators that may contribute to disease risk or resilience.Highlights p‐S65‐Ub levels were used as a quantitative marker of mitochondrial damage. A GWAS identified two genetic variants that modify mitophagy in LBD autopsy brain. APOE4 was associated with increased p‐S65‐Ub accumulation and neuropathology. APOE4 altered mitophagy via pathology‐dependent and pathology‐independent mechanisms. ZMIZ1 was linked to reduced p‐S65‐Ub and neuropathology indicative of protection.

Mutations in Leucine-rich repeat kinase 2 (LRRK2) and PTEN-induced kinase 1 (PINK1) are associated with familial Parkinson’s disease (PD). LRRK2 phosphorylates Rab guanosine triphosphatase (GTPases) within the Switch II domain while PINK1 directly phosphorylates Parkin and ubiquitin (Ub) and indirectly induces phosphorylation of a subset of Rab GTPases. Herein we have crossed LRRK2 [R1441C] mutant knock-in mice with PINK1 knock-out (KO) mice and report that loss of PINK1 does not impact endogenous LRRK2-mediated Rab phosphorylation nor do we see significant effect of mutant LRRK2 on PINK1-mediated Rab and Ub phosphorylation. In addition, we observe that a pool of the Rab-specific, protein phosphatase family member 1H phosphatase, is transcriptionally up-regulated and recruited to damaged mitochondria, independent of PINK1 or LRRK2 activity. Parallel signaling of LRRK2 and PINK1 pathways is supported by assessment of motor behavioral studies that show no evidence of genetic interaction in crossed mouse lines. Previously we showed loss of cilia in LRRK2 R1441C mice and herein we show that PINK1 KO mice exhibit a ciliogenesis defect in striatal cholinergic interneurons and astrocytes that interferes with Hedgehog induction of glial derived-neurotrophic factor transcription. This is not exacerbated in double-mutant LRRK2 and PINK1 mice. Overall, our analysis indicates that LRRK2 activation and/or loss of PINK1 function along parallel pathways to impair ciliogenesis, suggesting a convergent mechanism toward PD. Our data suggest that reversal of defects downstream of ciliogenesis offers a common therapeutic strategy for LRRK2 or PINK1 PD patients, whereas LRRK2 inhibitors that are currently in clinical trials are unlikely to benefit PINK1 PD patients.

ABSTRACT Background Inclusion body myositis (IBM) is the most prevalent muscle disease in adults for which no current treatment exists. The pathogenesis of IBM remains poorly defined. In this study, we aimed to explore the interplay between inflammation and mitochondrial dysfunction in IBM. Methods The study population consisted of 38 IBM patients and 22 age‐ and sex‐matched controls without a myopathy. Mean age was 62.9 years (SD = 9) in IBM group and 59.7 (10) in controls. Bulk RNA sequencing, Meso Scale Discovery electrochemiluminescence (ECL), western blotting, histochemistry and immunohistochemistry were performed on frozen muscle samples from the study participants. Results We demonstrated activation of the NLRP3 inflammasome in IBM muscle samples, with the NLRP3 inflammasome being the most upregulated pathway on RNA sequencing, along with increased expression of NLRP3 and ASC proteins in IBM group. NLRP3 RNA levels most strongly correlated with TLR7 (correlation coefficient ρ = 0.91) and complement activation‐related genes, and inversely correlated with several mitochondria‐related genes among others. On muscle histopathology, there was increased NRLP3 immunoreactivity in both inflammatory cells and muscle fibres. Mitophagy is critical for removing damaged mitochondria and preventing the formation of a vicious cycle of mitochondrial dysfunction—NLRP3 inflammasome activation. Herein, we showed altered mitophagy, as witnessed by the elevated levels of p‐S65‐Ubiquitin, a mitophagy marker, in muscle lysates from IBM patients compared to controls (median of 114.3 vs. 81.25 ECL units, p = 0.005). The p‐S65‐Ubiquitin levels were most significantly elevated in IBM males compared to male controls (136 vs. 83.5 ECL units; p = 0.013), whereas IBM females had milder nonsignificant elevation compared to female controls (97.25 vs. 69 ECL units, p = 0.31). On muscle histopathology, p‐S65‐Ubiquitin aggregates accumulated in muscle fibres that were mostly Type 2 and devoid of cytochrome‐c‐oxidase reactivity. NLRP3 RNA levels correlated with p‐S65‐Ubiquitin levels in both sexes (males: ρ = 0.48, females: ρ = 0.54) but with loss of muscle strength, as reflected by the manual motor test score, only in males (males: ρ = 0.62, females: ρ = −0.14). Lastly, we identified sex‐specific molecular pathways in IBM. Females had upregulation of pathways related to response to stress, which could conceivably offset some of the pathomechanisms of IBM, while males had upregulation of pathways related to cell adhesion and migration. Conclusions There is activation of the NLRP3 inflammasome in IBM, along with altered mitophagy, particularly in males, which is of potential therapeutic significance. These findings suggest sex‐specific mechanisms in IBM that warrant further investigation.