PLPHP deficiency: clinical, genetic, biochemical, and mechanistic insights.

Biallelic pathogenic variants in PLPBP (formerly called PROSC) have recently been shown to cause a novel form of vitamin B6-dependent epilepsy, the pathophysiological basis of which is poorly understood. When left untreated, the disease can progress to status epilepticus and death in infancy. Here we present 12 previously undescribed patients and six novel pathogenic variants in PLPBP. Suspected clinical diagnoses prior to identification of PLPBP variants included mitochondrial encephalopathy (two patients), folinic acid-responsive epilepsy (one patient) and a movement disorder compatible with AADC deficiency (one patient). The encoded protein, PLPHP is believed to be crucial for B6 homeostasis. We modelled the pathogenicity of the variants and developed a clinical severity scoring system. The most severe phenotypes were associated with variants leading to loss of function of PLPBP or significantly affecting protein stability/PLP-binding. To explore the pathophysiology of this disease further, we developed the first zebrafish model of PLPHP deficiency using CRISPR/Cas9. Our model recapitulates the disease, with plpbp-/- larvae showing behavioural, biochemical, and electrophysiological signs of seizure activity by 10 days post-fertilization and early death by 16 days post-fertilization. Treatment with pyridoxine significantly improved the epileptic phenotype and extended lifespan in plpbp-/- animals. Larvae had disruptions in amino acid metabolism as well as GABA and catecholamine biosynthesis, indicating impairment of PLP-dependent enzymatic activities. Using mass spectrometry, we observed significant B6 vitamer level changes in plpbp-/- zebrafish, patient fibroblasts and PLPHP-deficient HEK293 cells. Additional studies in human cells and yeast provide the first empirical evidence that PLPHP is localized in mitochondria and may play a role in mitochondrial metabolism. These models provide new insights into disease mechanisms and can serve as a platform for drug discovery.

Unearthing the secrets of mitochondrial ROS and glutathione in bioenergetics.

During the cellular oxidation of fuels, electrons are used to power the proton pumps of the mitochondrial electron transport chain (ETC) and ultimately drive ATP synthesis and the reduction of molecular oxygen to water. During these oxidative processes, some electrons can 'spin off' during fuel oxidation and electron transport to univalently reduce O2, forming reactive oxygen species (ROS). In excess, ROS can be detrimental; however, at low concentrations oxyradicals are essential signaling molecules. Mitochondria thus use a battery of systems to finely control types and levels of ROS, including antioxidants. Several antioxidant systems depend on glutathione. Here, we review mitochondrial ROS homeostatic systems, including emerging knowledge about roles of glutathione in redox balance and the control of protein function by post-translational modification.

Glutaredoxin-2 is required to control oxidative phosphorylation in cardiac muscle by mediating deglutathionylation reactions.

Glutaredoxin-2 (Grx2) modulates the activity of several mitochondrial proteins in cardiac tissue by catalyzing deglutathionylation reactions. However, it remains uncertain whether Grx2 is required to control mitochondrial ATP output in heart. Here, we report that Grx2 plays a vital role modulating mitochondrial energetics and heart physiology by mediating the deglutathionylation of mitochondrial proteins. Deletion of Grx2 (Grx2(-/-)) decreased ATP production by complex I-linked substrates to half that in wild type (WT) mitochondria. Decreased respiration was associated with increased complex I glutathionylation diminishing its activity. Tissue glucose uptake was concomitantly increased. Mitochondrial ATP output and complex I activity could be recovered by restoring the redox environment to that favoring the deglutathionylated states of proteins. Grx2(-/-) hearts also developed left ventricular hypertrophy and fibrosis, and mice became hypertensive. Mitochondrial energetics from Grx2 heterozygotes (Grx2(+/-)) were also dysfunctional, and hearts were hypertrophic. Intriguingly, Grx2(+/-) mice were far less hypertensive than Grx2(-/-) mice. Thus, Grx2 plays a vital role in modulating mitochondrial metabolism in cardiac muscle, and Grx2 deficiency leads to pathology. As mitochondrial ATP production was restored by the addition of reductants, these findings may be relevant to novel redox-related therapies in cardiac disease.

Impaired activity of CCA-adding enzyme TRNT1 impacts OXPHOS complexes and cellular respiration in SIFD patient-derived fibroblasts.

BACKGROUND: SIFD (Sideroblastic anemia with B-cell immunodeficiency, periodic fevers, and developmental delay) is a novel form of congenital sideroblastic anemia associated with B-cell immunodeficiency, periodic fevers, and developmental delay caused by mutations in the CCA-adding enzyme TRNT1, but the precise molecular pathophysiology is not known. RESULTS: We show that the disease causing mutations in patient-derived fibroblasts do not affect subcellular localization of TRNT1 and show no gross morphological differences when compared to control cells. Analysis of cellular respiration and oxidative phosphorylation (OXPHOS) complexes demonstrates that both basal and maximal respiration rates are decreased in patient cells, which may be attributed to an observed decrease in the abundance of select proteins of the OXPHOS complexes. CONCLUSIONS: Our data provides further insight into cellular pathophysiology of SIFD.

Detailed Biochemical and Bioenergetic Characterization of FBXL4-Related Encephalomyopathic Mitochondrial DNA Depletion.

Mutations of FBXL4, which encodes an orphan mitochondrial F-box protein, are a recently identified cause of encephalomyopathic mtDNA depletion. Here, we describe the detailed clinical and biochemical phenotype of a neonate presenting with hyperlactatemia, leukoencephalopathy, arrhythmias, pulmonary hypertension, dysmorphic features, and lymphopenia. Next-generation sequencing in the proband identified a homozygous frameshift, c.1641_1642delTG, in FBXL4, with a surrounding block of SNP marker homozygosity identified by microarray. Muscle biopsy showed a paucity of mitochondria with ultrastructural abnormalities, mitochondrial DNA depletion, and profound deficiency of all respiratory chain complexes. Cell-based mitochondrial phenotyping in fibroblasts showed mitochondrial fragmentation, decreased basal and maximal respiration, absence of ATP-linked respiratory and leak capacity, impaired survival under obligate aerobic respiration, and reduced mitochondrial inner membrane potential, with relative sparing of mitochondrial mass. Cultured fibroblasts from the patient exhibited a more oxidized glutathione ratio, consistent with altered cellular redox poise. High-resolution respirometry of permeabilized muscle fibers showed marked deficiency of oxidative phosphorylation using a variety of mitochondrial energy substrates and inhibitors. This constitutes the fourth and most detailed report of FBXL4 deficiency to date. In light of our patient's clinical findings and genotype (homozygous frameshift), this phenotype likely represents the severe end of the FBXL4 clinical spectrum.

DNM1L-related mitochondrial fission defect presenting as refractory epilepsy.

Mitochondrial fission and fusion are dynamic processes vital to mitochondrial quality control and the maintenance of cellular respiration. In dividing mitochondria, membrane scission is accomplished by a dynamin-related GTPase, DNM1L, that oligomerizes at the site of fission and constricts in a GTP-dependent manner. There is only a single previous report of DNM1L-related clinical disease: a female neonate with encephalopathy due to defective mitochondrial and peroxisomal fission (EMPF; OMIM #614388), a lethal disorder characterized by cerebral dysgenesis, seizures, lactic acidosis, elevated very long chain fatty acids, and abnormally elongated mitochondria and peroxisomes. Here, we describe a second individual, diagnosed via whole-exome sequencing, who presented with developmental delay, refractory epilepsy, prolonged survival, and no evidence of mitochondrial or peroxisomal dysfunction on standard screening investigations in blood and urine. EEG was nonspecific, showing background slowing with frequent epileptiform activity at the frontal and central head regions. Electron microscopy of skeletal muscle showed subtle, nonspecific abnormalities of cristal organization, and confocal microscopy of patient fibroblasts showed striking hyperfusion of the mitochondrial network. A panel of further bioenergetic studies in patient fibroblasts showed no significant differences versus controls. The proband's de novo DNM1L variant, NM_012062.4:c.1085G>A; NP_036192.2:p.(Gly362Asp), falls within the middle (oligomerization) domain of DNM1L, implying a likely dominant-negative mechanism. This disorder, which presents nonspecifically and affords few diagnostic clues, can be diagnosed by means of DNM1L sequencing and/or confocal microscopy.

Severe Neonatal Presentation of Mitochondrial Citrate Carrier (SLC25A1) Deficiency.

Mutations of the mitochondrial citrate carrier (CIC) SLC25A1 cause combined D-2- and L-2-hydroxyglutaric aciduria (DL-2HGA; OMIM #615182), a neurometabolic disorder characterized by developmental delay, hypotonia, and seizures. Here, we describe the female child of consanguineous parents who presented neonatally with lactic acidosis, periventricular frontal lobe cysts, facial dysmorphism, recurrent apneic episodes, and deficient complex IV (cytochrome c oxidase) activity in skeletal muscle. Exome sequencing revealed a homozygous SLC25A1 missense mutation [NM_005984.4: c.593G>A; p.(Arg198His)] of a ubiquitously conserved arginine residue putatively situated within the substrate-binding site I of CIC. Retrospective review of the patient's organic acids confirmed the D- and L-2-hydroxyglutaric aciduria typical of DL-2HGA to be present, although this was not appreciated on initial presentation. Cultured patient skin fibroblasts showed reduced survival in culture, diminished mitochondrial spare respiratory capacity, increased glycolytic flux, and normal mitochondrial bulk, inner membrane potential, and network morphology. Neither cell survival nor cellular respiratory parameters were improved by citrate supplementation, although oral citrate supplementation did coincide with amelioration of lactic acidosis and apneic attacks in the patient. This is the fifth clinical report of CIC deficiency to date. The clinical features in our patient suggest that this disorder, which can potentially be recognized either by molecular means or based on its characteristic organic aciduria, should be considered in the differential diagnosis of pyruvate dehydrogenase deficiency and respiratory chain disorders. One-Sentence Summary A novel homozygous missense substitution in SLC25A1 was identified in a neonate presenting with lactic acidosis, intracerebral cysts, and an apparent mitochondrial complex IV defect in muscle.

Competition of nuclear factor-erythroid 2 factors related transcription factor isoforms, Nrf1 and Nrf2, in antioxidant enzyme induction.

Although the Nrf2 (nuclear factor-erythroid 2 p45 subunit-related factor 2) regulated expression of multiple antioxidant and cytoprotective genes through the electrophile responsive element (EpRE) is well established, interaction of Nrf2/EpRE with Nrf1, a closely-related transcription factor, is less well understood. Due to either proteolysis or alternative translation, Nrf1 has been found as proteins of varying size, p120, p95, and p65, which have been described as either activators of EpRE or competitive inhibitors of Nrf2. We investigated the effect of Nrf1 on EpRE-regulated gene expression using the catalytic and modifier subunits of glutamate cysteine ligase (GCLC and GCLM) as models and explored the potential role of Nrf1 in altering their expression in aging and upon chronic exposure to airborne nano-sized particulate matter (nPM). Nrf1 knockout resulted in the increased expression of GCLC and GCLM in human bronchial epithelial (HBE1) cells. Overexpression Nrf2 in combination with either p120 or p65 diminished or failed to further increase the GCLC- and GLCM-EpRE luciferase activity. All known forms of Nrf1 protein, remained unchanged in the lungs of mice with age or in response to nPM. Our study shows that Nrf1 could inhibit EpRE activity in vitro, whereas the precise role of Nrf1 in vivo requires further investigations. We conclude that Nrf1 may not be directly responsible for the loss of Nrf2-dependent inducibility of antioxidant and cytoprotective genes observed in aged animals.

The Nrf1 CNC-bZIP protein is regulated by the proteasome and activated by hypoxia.

BACKGROUND: Nrf1 (nuclear factor-erythroid 2 p45 subunit-related factor 1) is a transcription factor mediating cellular responses to xenobiotic and pro-oxidant stress. Nrf1 regulates the transcription of many stress-related genes through the electrophile response elements (EpREs) located in their promoter regions. Despite its potential importance in human health, the mechanisms controlling Nrf1 have not been addressed fully. PRINCIPAL FINDINGS: We found that proteasomal inhibitors MG-132 and clasto-lactacystin-ß-lactone stabilized the protein expression of full-length Nrf1 in both COS7 and WFF2002 cells. Concomitantly, proteasomal inhibition decreased the expression of a smaller, N-terminal Nrf1 fragment, with an approximate molecular weight of 23 kDa. The EpRE-luciferase reporter assays revealed that proteasomal inhibition markedly inhibited the Nrf1 transactivational activity. These results support earlier hypotheses that the 26 S proteasome processes Nrf1 into its active form by removing its inhibitory N-terminal domain anchoring Nrf1 to the endoplasmic reticulum. Immunoprecipitation demonstrated that Nrf1 is ubiquitinated and that proteasomal inhibition increased the degree of Nrf1 ubiquitination. Furthermore, Nrf1 protein had a half-life of approximately 5 hours in COS7 cells. In contrast, hypoxia (1% O(2)) significantly increased the luciferase reporter activity of exogenous Nrf1 protein, while decreasing the protein expression of p65, a shorter form of Nrf1, known to act as a repressor of EpRE-controlled gene expression. Finally, the protein phosphatase inhibitor okadaic acid activated Nrf1 reporter activity, while the latter was repressed by the PKC inhibitor staurosporine. CONCLUSIONS: Collectively, our data suggests that Nrf1 is controlled by several post-translational mechanisms, including ubiquitination, proteolytic processing and proteasomal-mediated degradation as well as by its phosphorylation status.