Identification of a muscle-specific isoform of VMA21 as a potent actor in X-linked myopathy with excessive autophagy pathogenesis.

Defective lysosomal acidification is responsible for a large range of multi-systemic disorders associated with impaired autophagy. Diseases caused by mutations in the VMA21 gene stand as exceptions, specifically affecting skeletal muscle (X-linked Myopathy with Excessive Autophagy, XMEA) or liver (Congenital Disorder of Glycosylation). VMA21 chaperones vacuolar (v-) ATPase assembly, which is ubiquitously required for proper lysosomal acidification. The reason VMA21 deficiencies affect specific, but divergent tissues remains unknown. Here, we show that VMA21 encodes a yet-unreported long protein isoform, in addition to the previously described short isoform, which we name VMA21-120 and VMA21-101, respectively. In contrast to the ubiquitous pattern of VMA21-101, VMA21-120 was predominantly expressed in skeletal muscle, and rapidly up-regulated upon differentiation of mouse and human muscle precursors. Accordingly, VMA21-120 accumulated during development, regeneration and denervation of mouse skeletal muscle. In contrast, neither induction nor blockade of autophagy, in vitro and in vivo, strongly affected VMA21 isoform expression. Interestingly, VMA21-101 and VMA21-120 both localized to the sarcoplasmic reticulum of muscle cells, and interacted with the v-ATPase. While VMA21 deficiency impairs autophagy, VMA21-101 or VMA21-120 overexpression had limited impact on autophagic flux in muscle cells. Importantly, XMEA-associated mutations lead to both VMA21-101 deficiency and loss of VMA21-120 expression. These results provide important insights into the clinical diversity of VMA21-related diseases and uncover a muscle-specific VMA21 isoform that potently contributes to XMEA pathogenesis.

Small mitochondrial protein NERCLIN regulates cardiolipin homeostasis and mitochondrial ultrastructure.

Cardiolipin (CL) is an essential phospholipid for mitochondrial structure and function. Here, we present a small mitochondrial protein, NERCLIN, as a negative regulator of CL homeostasis and mitochondrial ultrastructure. Primate-specific NERCLIN is expressed ubiquitously from the GRPEL2 locus on a tightly regulated low level. NERCLIN overexpression severely disrupts mitochondrial cristae structure and induces mitochondrial fragmentation. Proximity labeling and immunoprecipitation analysis suggested interactions of NERCLIN with CL synthesis and prohibitin complexes on the matrix side of the inner mitochondrial membrane. Lipid analysis indicated that NERCLIN regulates mitochondrial CL content. Furthermore, NERCLIN is responsive to heat stress ensuring OPA1 processing and cell survival. Thus, we propose that NERCLIN contributes to the stress-induced adaptation of mitochondrial dynamics. Our findings add NERCLIN to the group of recently identified small mitochondrial proteins with important regulatory functions.

Inter-organellar and systemic responses to impaired mitochondrial matrix protein import in skeletal muscle.

Effective protein import from cytosol is critical for mitochondrial functions and metabolic regulation. We describe here the mammalian muscle-specific and systemic consequences to disrupted mitochondrial matrix protein import by targeted deletion of the mitochondrial HSP70 co-chaperone GRPEL1. Muscle-specific loss of GRPEL1 caused rapid muscle atrophy, accompanied by shut down of oxidative phosphorylation and mitochondrial fatty acid oxidation, and excessive triggering of proteotoxic stress responses. Transcriptome analysis identified new responders to mitochondrial protein import toxicity, such as the neurological disease-linked intermembrane space protein CHCHD10. Besides communication with ER and nucleus, we identified crosstalk of distressed mitochondria with peroxisomes, in particular the induction of peroxisomal Acyl-CoA oxidase 2 (ACOX2), which we propose as an ATF4-regulated peroxisomal marker of integrated stress response. Metabolic profiling indicated fatty acid enrichment in muscle, a shift in TCA cycle intermediates in serum and muscle, and dysregulated bile acids. Our results demonstrate the fundamental importance of GRPEL1 and provide a robust model for detecting mammalian inter-organellar and systemic responses to impaired mitochondrial matrix protein import and folding.

A spontaneous mitonuclear epistasis converging on Rieske Fe-S protein exacerbates complex III deficiency in mice.

We previously observed an unexpected fivefold (35 vs. 200 days) difference in the survival of respiratory chain complex III (CIII) deficient Bcs1lp.S78G mice between two congenic backgrounds. Here, we identify a spontaneous homoplasmic mtDNA variant (m.G14904A, mt-Cybp.D254N), affecting the CIII subunit cytochrome b (MT-CYB), in the background with short survival. We utilize maternal inheritance of mtDNA to confirm this as the causative variant and show that it further decreases the low CIII activity in Bcs1lp.S78G tissues to below survival threshold by 35 days of age. Molecular dynamics simulations predict D254N to restrict the flexibility of MT-CYB ef loop, potentially affecting RISP dynamics. In Rhodobacter cytochrome bc1 complex the equivalent substitution causes a kinetics defect with longer occupancy of RISP head domain towards the quinol oxidation site. These findings represent a unique case of spontaneous mitonuclear epistasis and highlight the role of mtDNA variation as modifier of mitochondrial disease phenotypes.

Alternative oxidase-mediated respiration prevents lethal mitochondrial cardiomyopathy.

Alternative oxidase (AOX) is a non-mammalian enzyme that can bypass blockade of the complex III-IV segment of the respiratory chain (RC). We crossed a Ciona intestinalis AOX transgene into RC complex III (cIII)-deficient Bcs1lp.S78G knock-in mice, displaying multiple visceral manifestations and premature death. The homozygotes expressing AOX were viable, and their median survival was extended from 210 to 590 days due to permanent prevention of lethal cardiomyopathy. AOX also prevented renal tubular atrophy and cerebral astrogliosis, but not liver disease, growth restriction, or lipodystrophy, suggesting distinct tissue-specific pathogenetic mechanisms. Assessment of reactive oxygen species (ROS) production and damage suggested that ROS were not instrumental in the rescue. Cardiac mitochondrial ultrastructure, mitochondrial respiration, and pathological transcriptome and metabolome alterations were essentially normalized by AOX, showing that the restored electron flow upstream of cIII was sufficient to prevent cardiac energetic crisis and detrimental decompensation. These findings demonstrate the value of AOX, both as a mechanistic tool and a potential therapeutic strategy, for cIII deficiencies.

NAD+ repletion produces no therapeutic effect in mice with respiratory chain complex III deficiency and chronic energy deprivation.

Biosynthetic precursors of NAD+ can replenish a decreased cellular NAD+ pool and, supposedly via sirtuin (SIRT) deacetylases, improve mitochondrial function. We found decreased hepatic NAD+ concentration and downregulated biosynthesis in Bcs1lp.S78G knock-in mice with respiratory chain complex III deficiency and mitochondrial hepatopathy. Aiming at ameliorating disease progression via NAD+ repletion and improved mitochondrial function, we fed these mice nicotinamide riboside (NR), a NAD+ precursor. A targeted metabolomics verified successful administration and suggested enhanced NAD+ biosynthesis in the treated mice, although hepatic NAD+ concentration was unchanged at the end point. In contrast to our expectations, NR did not improve the hepatopathy, hepatic mitochondrial respiration, or survival of Bcs1lp.S78G mice. We linked this lack of therapeutic effect to NAD+-independent activation of SIRT-1 and -3 via AMPK and cAMP signaling related to the starvation-like metabolic state of Bcs1lp.S78G mice. In summary, we describe an unusual metabolic state with NAD+ depletion accompanied by energy deprivation signals, uncompromised SIRT function, and upregulated oxidative metabolism. Our study highlights that the knowledge of the underlying complex metabolic alterations is critical when designing therapies for mitochondrial dysfunction.-Purhonen, J., Rajendran, J., Tegelberg, S., Smolander, O.-P., Pirinen, E., Kallijärvi, J., Fellman, V. NAD+ repletion produces no therapeutic effect in mice with respiratory chain complex III deficiency and chronic energy deprivation.

Ketogenic diet attenuates hepatopathy in mouse model of respiratory chain complex III deficiency caused by a Bcs1l mutation.

Mitochondrial disorders are among the most prevalent inborn errors of metabolism but largely lack treatments and have poor outcomes. High-fat, low-carbohydrate ketogenic diets (KDs) have shown beneficial effects in mouse models of mitochondrial myopathies, with induction of mitochondrial biogenesis as the suggested main mechanism. We fed KD to mice with respiratory chain complex III (CIII) deficiency and progressive hepatopathy due to mutated BCS1L, a CIII assembly factor. The mutant mice became persistently ketotic and tolerated the KD for up to 11 weeks. Liver disease progression was attenuated by KD as shown by delayed fibrosis, reduced cell death, inhibition of hepatic progenitor cell response and stellate cell activation, and normalization of liver enzyme activities. Despite no clear signs of increased mitochondrial biogenesis in the liver, CIII assembly and activity were improved and mitochondrial morphology in hepatocytes normalized. Induction of hepatic glutathione transferase genes and elevated total glutathione level were normalized by KD. Histological findings and transcriptome changes indicated modulation of liver macrophage populations by the mutation and the diet. These results reveal a striking beneficial hepatic response to KD in mice with mitochondrial hepatopathy and warrant further investigations of dietary modification in the management of these conditions in patients.

Effect of High-Carbohydrate Diet on Plasma Metabolome in Mice with Mitochondrial Respiratory Chain Complex III Deficiency.

Mitochondrial disorders cause energy failure and metabolic derangements. Metabolome profiling in patients and animal models may identify affected metabolic pathways and reveal new biomarkers of disease progression. Using liver metabolomics we have shown a starvation-like condition in a knock-in (Bcs1lc.232A>G) mouse model of GRACILE syndrome, a neonatal lethal respiratory chain complex III dysfunction with hepatopathy. Here, we hypothesized that a high-carbohydrate diet (HCD, 60% dextrose) will alleviate the hypoglycemia and promote survival of the sick mice. However, when fed HCD the homozygotes had shorter survival (mean ± SD, 29 ± 2.5 days, n = 21) than those on standard diet (33 ± 3.8 days, n = 30), and no improvement in hypoglycemia or liver glycogen depletion. We investigated the plasma metabolome of the HCD- and control diet-fed mice and found that several amino acids and urea cycle intermediates were increased, and arginine, carnitines, succinate, and purine catabolites decreased in the homozygotes. Despite reduced survival the increase in aromatic amino acids, an indicator of liver mitochondrial dysfunction, was normalized on HCD. Quantitative enrichment analysis revealed that glycine, serine and threonine metabolism, phenylalanine and tyrosine metabolism, and urea cycle were also partly normalized on HCD. This dietary intervention revealed an unexpected adverse effect of high-glucose diet in complex III deficiency, and suggests that plasma metabolomics is a valuable tool in evaluation of therapies in mitochondrial disorders.

Obesity-linked homologues TfAP-2 and Twz establish meal frequency in Drosophila melanogaster.

In all animals managing the size of individual meals and frequency of feeding is crucial for metabolic homeostasis. In the current study we demonstrate that the noradrenalin analogue octopamine and the cholecystokinin (CCK) homologue Drosulfakinin (Dsk) function downstream of TfAP-2 and Tiwaz (Twz) to control the number of meals in adult flies. Loss of TfAP-2 or Twz in octopaminergic neurons increased the size of individual meals, while overexpression of TfAP-2 significantly decreased meal size and increased feeding frequency. Of note, our study reveals that TfAP-2 and Twz regulate octopamine signaling to initiate feeding; then octopamine, in a negative feedback loop, induces expression of Dsk to inhibit consummatory behavior. Intriguingly, we found that the mouse TfAP-2 and Twz homologues, AP-2ß and Kctd15, co-localize in areas of the brain known to regulate feeding behavior and reward, and a proximity ligation assay (PLA) demonstrated that AP-2ß and Kctd15 interact directly in a mouse hypothalamus-derived cell line. Finally, we show that in this mouse hypothalamic cell line AP-2ß and Kctd15 directly interact with Ube2i, a mouse sumoylation enzyme, and that AP-2ß may itself be sumoylated. Our study reveals how two obesity-linked homologues regulate metabolic homeostasis by modulating consummatory behavior.

Regulation of aggression by obesity-linked genes TfAP-2 and Twz through octopamine signaling in Drosophila.

In Drosophila, the monoamine octopamine, through mechanisms that are not completely understood, regulates both aggression and mating behavior. Interestingly, our study demonstrates that the Drosophila obesity-linked homologs Transcription factor AP-2 (TfAP-2; TFAP2B in humans) and Tiwaz (Twz; KCTD15 in humans) interact to modify male behavior by controlling the expression of Tyramine ß-hydroxylase and Vesicular monanime transporter, genes necessary for octopamine production and secretion. Furthermore, we reveal that octopamine in turn regulates aggression through the Drosophila cholecystokinin satiation hormone homolog Drosulfakinin (Dsk). Finally, we establish that TfAP-2 is expressed in octopaminergic neurons known to control aggressive behavior and that TfAP-2 requires functional Twz for its activity. We conclude that genetically manipulating the obesity-linked homologs TfAP-2 and Twz is sufficient to affect octopamine signaling, which in turn modulates Drosophila male behavior through the regulation of the satiation hormone Dsk.