Gyu Song

Mitochondrial Citrate Transporter-dependent Metabolic Signature in the 22q11.2 Deletion Syndrome

Background: The most common microdeletion congenital disorder, 22qDS, is the second risk factor for schizophrenia. Results: Plasma metabolomics in 22qDS consisted of an oxidative phosphorylation-to-glycolysis shift with altered 2-hydroxyglutaric acid, cholesterol, and fatty acid concentrations. Conclusion: Despite the haploinsufficiency of ∼30 genes, the 22qDS metabolic signature chiefly reflected deficits in the mitochondrial citrate transporter. Significance: Biochemical profiling of 22qDS unveiled the impact of SLC25A1 haploinsufficiency. The congenital disorder 22q11.2 deletion syndrome (22qDS), characterized by a hemizygous deletion of 1.5–3 Mb on chromosome 22 at locus 11.2, is the most common microdeletion disorder (estimated prevalence of 1 in 4000) and the second risk factor for schizophrenia. Nine of ∼30 genes involved in 22qDS have the potential of disrupting mitochondrial metabolism (COMT, UFD1L, DGCR8, MRPL40, PRODH, SLC25A1, TXNRD2, T10, and ZDHHC8). Deficits in bioenergetics during early postnatal brain development could set the basis for a disrupted neuronal metabolism or synaptic signaling, partly explaining the higher incidence in developmental and behavioral deficits in these individuals. Here, we investigated whether mitochondrial outcomes and metabolites from 22qDS children segregated with the altered dosage of one or several of these mitochondrial genes contributing to 22qDS etiology and/or morbidity. Plasma metabolomics, lymphocytic mitochondrial outcomes, and epigenetics (histone H3 Lys-4 trimethylation and 5-methylcytosine) were evaluated in samples from 11 22qDS children and 13 age- and sex-matched neurotypically developing controls. Metabolite differences between 22qDS children and controls reflected a shift from oxidative phosphorylation to glycolysis (higher lactate/pyruvate ratios) accompanied by an increase in reductive carboxylation of α-ketoglutarate (increased concentrations of 2-hydroxyglutaric acid, cholesterol, and fatty acids). Altered metabolism in 22qDS reflected a critical role for the haploinsufficiency of the mitochondrial citrate transporter SLC25A1, further enhanced by HIF-1α, MYC, and metabolite controls. This comprehensive profiling served to clarify the biochemistry of this disease underlying its broad, complex phenotype.

Increased susceptibility to skin carcinogenesis associated with a spontaneous mouse mutation in the palmitoyl transferase Zdhhc13 gene

Here we describe a spontaneous mutation in the Zdhhc13 (zinc finger, DHHC domain containing 13) gene (also called Hip14l), one of 24 genes encoding palmitoyl acyltransferase (PAT) enzymes in the mouse. This mutation (Zdhhc13luc) was identified as a nonsense base substitution, which results in a premature stop codon that generates a truncated form of the ZDHHC13 protein, representing a potential loss-of-function allele. Homozygous Zdhhc13luc/Zdhhc13luc mice developed generalized hypotrichosis, associated with abnormal hair cycle, epidermal and sebaceous gland hyperplasia, hyperkeratosis and increased epidermal thickness. Increased keratinocyte proliferation and accelerated transit from basal to more differentiated layers were observed in mutant compared to wild-type epidermis, in untreated skin and after short-term 12-O-tetradecanoyl-phorbol-13-acetate (TPA) treatment and acute UVB exposure. Interestingly, this epidermal phenotype was associated with constitutive activation of NF-κB (RelA) and increased neutrophil recruitment and elastase activity. Furthermore, tumor multiplicity and malignant progression of papillomas after chemical skin carcinogenesis were significantly higher in mutant mice than wild-type littermates. To our knowledge, this is the first report of a protective role for a PAT in skin carcinogenesis.

Altered redox mitochondrial biology in the neurodegenerative disorder fragile X-tremor/ataxia syndrome: use of antioxidants in precision medicine.

A 55–200 expansion of the CGG nucleotide repeat in the 5′-UTR of the fragile X mental retardation 1 gene (FMR1) is the hallmark of the triplet nucleotide disease known as the “premutation” as opposed to those with >200 repeats, known as the full mutation or fragile X syndrome. Originally, premutation carriers were thought to be free of phenotypic traits; however, some are diagnosed with emotional and neurocognitive issues and, later in life, with the neurodegenerative disease fragile X-associated tremor/ataxia syndrome (FXTAS). Considering that mitochondrial dysfunction has been observed in fibroblasts and post-mortem brain samples from carriers of the premutation, we hypothesized that mitochondrial dysfunction-derived reactive oxygen species (ROS) may result in cumulative oxidative-nitrative damage. Fibroblasts from premutation carriers (n = 31, all FXTAS-free except 8), compared with age- and sex-matched controls (n = 25), showed increased mitochondrial ROS production, impaired Complex I activity, lower expression of MIA40 (rate-limiting step of the redox-regulated mitochondrial-disulfide-relay-system), increased mtDNA deletions and increased biomarkers of lipid and protein oxidative-nitrative damage. Most of the outcomes were more pronounced in FXTAS-affected individuals. Significant recovery of mitochondrial mass and/or function was obtained with superoxide or hydroxyl radicals’ scavengers, a glutathione peroxidase analog, or by overexpressing MIA40. The effects of ethanol (a hydroxyl radical scavenger) were deleterious, while others (by N-acetyl-cysteine, quercetin and epigallocatechin-3-gallate) were outcome- and/or carrier-specific. The use of antioxidants in the context of precision medicine is discussed with the goal of improving mitochondrial function in carriers with the potential of decreasing the morbidity and/or delaying FXTAS onset.

Premutation in the Fragile X Mental Retardation 1 (FMR1) Gene Affects Maternal Zn-milk and Perinatal Brain Bioenergetics and Scaffolding

Fragile X premutation alleles have 55–200 CGG repeats in the 5′ UTR of the FMR1 gene. Altered zinc (Zn) homeostasis has been reported in fibroblasts from >60 years old premutation carriers, in which Zn supplementation significantly restored Zn-dependent mitochondrial protein import/processing and function. Given that mitochondria play a critical role in synaptic transmission, brain function, and cognition, we tested FMRP protein expression, brain bioenergetics, and expression of the Zn-dependent synaptic scaffolding protein SH3 and multiple ankyrin repeat domains 3 (Shank3) in a knock-in (KI) premutation mouse model with 180 CGG repeats. Mitochondrial outcomes correlated with FMRP protein expression (but not FMR1 gene expression) in KI mice and human fibroblasts from carriers of the pre- and full-mutation. Significant deficits in brain bioenergetics, Zn levels, and Shank3 protein expression were observed in the Zn-rich regions KI hippocampus and cerebellum at PND21, with some of these effects lasting into adulthood (PND210). A strong genotype × age interaction was observed for most of the outcomes tested in hippocampus and cerebellum, whereas in cortex, age played a major role. Given that the most significant effects were observed at the end of the lactation period, we hypothesized that KI milk might have a role at compounding the deleterious effects on the FMR1 genetic background. A higher gene expression of ZnT4 and ZnT6, Zn transporters abundant in brain and lactating mammary glands, was observed in the latter tissue of KI dams. A cross-fostering experiment allowed improving cortex bioenergetics in KI pups nursing on WT milk. Conversely, WT pups nursing on KI milk showed deficits in hippocampus and cerebellum bioenergetics. A highly significant milk type × genotype interaction was observed for all three-brain regions, being cortex the most influenced. Finally, lower milk-Zn levels were recorded in milk from lactating women carrying the premutation as well as other Zn-related outcomes (Zn-dependent alkaline phosphatase activity and lactose biosynthesis—whose limiting step is the Zn-dependent β-1,4-galactosyltransferase). In premutation carriers, altered Zn homeostasis, brain bioenergetics and Shank3 levels could be compounded by Zn-deficient milk, increasing the risk of developing emotional and neurological/cognitive problems and/or FXTAS later in life.

Warburg effect linked to cognitive-executive deficits in FMR1 premutation

A55–200 CGG repeat expansion in the 5′‐UTR of the fragile X mental retardation 1 (FMR1) gene is known as a premutation. Some carriers are affected by the neurodegenerative disorder fragile X‐associated tremor/ataxia syndrome (FXTAS), primary ovarian insufficiency, and neurobehavioral impairments. Based on the mitochondrial dysfunction observed in fibroblasts and brain samples from carriers, as well as in neurons and brains from a mouse model of the premutation, we evaluated the presence of the Warburg effect in peripheral blood mononuclear cells (PBMCs) from 30 premutation carriers with either a rebalance of the metabolism [increasing glycolysis while decreasing oxidative phosphorylation (oxphos)] or a metabolic amplification (increasing glycolysis while maintaining/increasing oxphos). Deficits in oxphos—more pronounced in FXTAS‐affected subjects—were accompanied by a shift toward glycolysis, suggesting increased glycolysis despite aerobic conditions. Differential proteomics extended these findings, unveiling a decreased antioxidant response, translation, and disrupted extra‐cellular matrix and cytoskeleton organization with activation of prosenescence pathways. Lower bioenergetics segregated with increased incidence of low executive function, tremors, below‐average IQ, and FXTAS. The combination of functional and proteomic data unveiled new mechanisms related to energy production in the premutation, showing the potential of being applicable to other psychiatric disorders to identify endophenotypespecific responses relevant to neurobiology.—Napoli, E., Song, G., Schneider, A., Hagerman, R., Eldeeb, M.A.A.A., Azarang, A., Tassone, F., Giulivi, C. Warburg effect linked to cognitive‐executive deficits in FMR1 premutation. FASEB J. 30, 3334–3351 (2016). www.fasebj.org

Zdhhc13-dependent Drp1 S-palmitoylation impacts brain bioenergetics, anxiety, coordination and motor skills

Protein S-palmitoylation is a reversible post-translational modification mediated by palmitoyl acyltransferase enzymes, a group of Zn2+-finger DHHC-domain-containing proteins (ZDHHC). Here, for the first time, we show that Zdhhc13 plays a key role in anxiety-related behaviors and motor function, as well as brain bioenergetics, in a mouse model (luc) carrying a spontaneous Zdhhc13 recessive mutation. At 3 m of age, mutant mice displayed increased sensorimotor gating, anxiety, hypoactivity, and decreased motor coordination, compared to littermate controls. Loss of Zdhhc13 in cortex and cerebellum from 3- and 24 m old hetero- and homozygous male mutant mice resulted in lower levels of Drp1 S-palmitoylation accompanied by altered mitochondrial dynamics, increased glycolysis, glutaminolysis and lactic acidosis, and neurotransmitter imbalances. Employing in vivo and in vitro models, we identified that Zdhhc13-dependent Drp1 S-palmitoylation, which acting alone or in concert, enables the normal occurrence of the fission-fusion process. In vitro and in vivo direct Zdhhc13-Drp1 protein interaction was observed, confirming Drp1 as a substrate of Zdhhc13. Abnormal fission-fusion processes result in disrupted mitochondria morphology and distribution affecting not only mitochondrial ATP output but neurotransmission and integrity of synaptic structures in the brain, setting the basis for the behavioral abnormalities described in the Zdhhc13-deficient mice.

Two insulin-like peptides differentially regulate malaria parasite infection in the mosquito through effects on intermediary metabolism

Insulin-like peptides (ILPs) play important roles in growth and metabolic homeostasis, but have also emerged as key regulators of stress responses and immunity in a variety of vertebrates and invertebrates. Furthermore, a growing literature suggests that insulin signaling-dependent metabolic provisioning can influence host responses to infection and affect infection outcomes. In line with these studies, we previously showed that knockdown of either of two closely related, infection-induced ILPs, ILP3 and ILP4, in the mosquito Anopheles stephensi decreased infection with the human malaria parasite Plasmodium falciparum through kinetically distinct effects on parasite death. However, the precise mechanisms by which ILP3 and ILP4 control the response to infection remained unknown. To address this knowledge gap, we used a complementary approach of direct ILP supplementation into the blood meal to further define ILP-specific effects on mosquito biology and parasite infection. Notably, we observed that feeding resulted in differential effects of ILP3 and ILP4 on blood-feeding behavior and P. falciparum development. These effects depended on ILP-specific regulation of intermediary metabolism in the mosquito midgut, suggesting a major contribution of ILP-dependent metabolic shifts to the regulation of infection resistance and parasite transmission. Accordingly, our data implicate endogenous ILP signaling in balancing intermediary metabolism for the host response to infection, affirming this emerging tenet in host-pathogen interactions with novel insights from a system of significant public health importance.

Beyond autophagy: a novel role for autism-linked Wdfy3 in brain mitophagy

WD repeat and FYVE domain-containing 3 (WDFY3; also known as Autophagy-Linked FYVE or Alfy) is an identified intellectual disability, developmental delay and autism risk gene. This gene encodes for a scaffolding protein that is expressed in both the developing and adult central nervous system and required for autophagy and aggrephagy with yet unexplored roles in mitophagy. Given that mitochondrial trafficking, dynamics and remodeling have key roles in synaptic plasticity, we tested the role of Wdfy3 on brain bioenergetics by using Wdfy3+/lacZ mice, the only known Wdfy3 mutant animal model with overt neurodevelopmental anomalies that survive to adulthood. We found that Wdfy3 is required for sustaining brain bioenergetics and morphology via mitophagy. Decreased mitochondrial quality control by conventional mitophagy was partly compensated for by the increased formation of mitochondria-derived vesicles (MDV) targeted to lysosomal degradation (micromitophagy). These observations, extended through proteomic analysis of mitochondria-enriched cortical fractions, showed significant enrichment for pathways associated with mitophagy, mitochondrial transport and axon guidance via semaphorin, Robo, L1cam and Eph-ephrin signaling. Collectively, our findings support a critical role for Wdfy3 in mitochondrial homeostasis with implications for neuron differentiation, neurodevelopment and age-dependent neurodegeneration.

Impact of FMR1 Premutation on Neurobehavior and Bioenergetics in Young Monozygotic Twins

Mitochondrial dysfunction (MD) has been identified in lymphocytes, fibroblasts and brain samples from adults carrying a 55–200 CGG expansion in the fragile X mental retardation 1 (FMR1) gene (premutation; PM); however, limited data are available on the bioenergetics of pediatric carriers. Here we discuss a case report of three PM carriers: two monozygotic twins (aged 8 years) harboring an FMR1 allele with 150–180 CGG repeats, with no cognitive or intellectual issues but diagnosed with depression, mood instability and ADHD, and their mother (asymptomatic carrier with 78 CGG repeats). Fibroblasts and lymphocytes from the twins presented a generalized OXPHOS deficit, altered mitochondrial network, accumulation of depolarized mitochondria, and increased mitochondrial ROS production, outcomes distinct and more severe than the mother’s ones, suggesting the involvement of modulatory effects mediated by CGG expansion, X-activation ratio, sex hormones and epigenetic factors (chronic inflammation, consequence of Lyme disease). The degree of the severity of MD appeared to segregate with the morbidity of the phenotype. The mitochondrial ROS-mediated HIF-1α stabilization was identified as a key player at contributing to the MD, pointing it as a novel target for future therapeutical intervention.