Recognizing a deficiency of indispensable amino acids (IAAs) for protein synthesis is vital for dietary selection in metazoans, including humans. Cells in the brain's anterior piriform cortex (APC) ...are sensitive to IAA deficiency, signaling diet rejection and foraging for complementary IAA sources, but the mechanism is unknown. Here we report that the mechanism for recognizing IAA-deficient foods follows the conserved general control (GC) system, wherein uncharged transfer RNA induces phosphorylation of eukaryotic initiation factor 2 (eIF2) via the GC nonderepressing 2 (GCN2) kinase. Thus, a basic mechanism of nutritional stress management functions in mammalian brain to guide food selection for survival.
Alaskan Husky encephalopathy (AHE1) is a fatal brain disease associated with a mutation in SLC19A3.1 (c.624insTTGC, c.625C>A). This gene encodes for a thiamine transporter 2 with a predominately ...(CNS) central nervous system distribution. Considering that brain is particularly vulnerable to thiamine deficiency because of its reliance on thiamine pyrophosphate (TPP)‐dependent metabolic pathways involved in energy metabolism and neurotransmitter synthesis, we characterized the impact of this mutation on thiamine status, brain bioenergetics and the contribution of oxidative stress to this phenotype. In silico modeling of the mutated transporter indicated a significant loss of alpha‐helices resulting in a more open protein structure suggesting an impaired thiamine transport ability. The cerebral cortex and thalamus of affected dogs were severely deficient in TPP‐dependent enzymes accompanied by decreases in mitochondrial mass and oxidative phosphorylation (OXPHOS) capacity, and increases in oxidative stress. These results along with the behavioral and pathological findings indicate that the phenotype associated with AHE is consistent with a brain‐specific thiamine deficiency, leading to brain mitochondrial dysfunction and increased oxidative stress. While some of the biochemical deficits, neurobehavior and affected brain areas in AHE were shared by Wernicke's and Korsakoff's syndromes, several differences were noted likely arising from a tissue‐specific vs. that from a whole‐body thiamine deficiency.
A number of studies have been conducted that link mitochondrial dysfunction (MD) to Huntington's disease (HD); however, contradicting results had resulted in a lack of a clear mechanism that links ...expression of mutant Huntingtin protein and MD. Mouse homozygous (HM) and heterozygous (HT) mutant striatal cells with two or one allele encoding for a mutant huntingtin protein with 111 polyGln repeats showed a significant impairment of the mitochondrial disulfide relay system (MDRS). This system (consisting of two proteins, Gfer and Mia40) is involved in the mitochondrial import of Cys-rich proteins. The Gfer-to-Mia40 ratio was significantly altered in HM cells compared with controls, along with the expression of mitochondrial proteins considered substrates of the MDRS. In progenitors and differentiated neuron-like HM cells, impairment of MDRS were accompanied by deficient oxidative phosphorylation, Complex I, IV and V activities, decreased mtDNA copy number and transcripts, accumulation of mtDNA deletions and changes in mitochondrial morphology, consistent with other MDRS-deficient biological models, thus providing a framework for the energy deficits observed in this HD model. The majority (>90%) of the mitochondrial outcomes exhibited a gene-dose dependency with the expression of mutant Htt. Finally, decreases in the mtDNA copy number, along with the accumulation of mtDNA deletions, provide a mechanism for the progressive neurodegeneration observed in HD patients.
FXTAS (fragile X-associated tremor/ataxia syndrome) is a late-onset neurodegenerative disorder that affects individuals who are carriers of premutation expansions (55-200 CGG repeats) in the 5' ...untranslated region of the FMR1 (fragile X mental retardation 1) gene. The role of MD (mitochondrial dysfunction) in FXTAS was evaluated in fibroblasts and brain samples from premutation carriers with and without FXTAS symptoms, with a range of CGG repeats. This study resulted in several important conclusions: (i) decreased NAD- and FAD-linked oxygen uptake rates and uncoupling between electron transport and synthesis of ATP were observed in fibroblasts from premutation carriers; (ii) a lower expression of mitochondrial proteins preceded both in age and in CGG repeats the appearance of overt clinical involvement; (iii) the CGG repeat size required for altered mitochondrial protein expression was also smaller than that required to produce brain intranuclear inclusions from individuals with the premutation who died, suggesting that MD is an incipient pathological process occurring in individuals who do not display overt features of FXTAS; and (iv) on the basis of the CGG repeats, MD preceded the increase in oxidative/nitrative stress damage, indicating that the latter is a late event. MD in carriers of small CGG repeats, even when the allele size is not sufficient to produce FXTAS, may predispose them to other disorders (e.g. Parkinson's disease) that are likely to involve MD, and to environmental stressors, which may trigger the development of FXTAS symptoms. Detection of MD is of critical importance to the management of FXTAS, since it opens up additional treatment options for this disorder.
Etiology of aberrant social behavior consistently points to a strong polygenetic component involved in fundamental developmental pathways, with the potential of being enhanced by defects in ...bioenergetics. To this end, the occurrence of social deficits and mitochondrial outcomes were evaluated in conditional Pten (Phosphatase and tensin homolog) haplo-insufficient mice, in which only one allele was selectively knocked-out in neural tissues. Pten mutations have been linked to Alzheimer's disease and syndromic autism spectrum disorders, among others. By 4-6 weeks of age, Pten insufficiency resulted in the increase of several mitochondrial Complex activities (II-III, IV and V) not accompanied by increases in mitochondrial mass, consistent with an activation of the PI3K/Akt pathway, of which Pten is a negative modulator. At 8-13 weeks of age, Pten haplo-insufficient mice did not show significant behavioral abnormalities or changes in mitochondrial outcomes, but by 20-29 weeks, they displayed aberrant social behavior (social avoidance, failure to recognize familiar mouse, and repetitive self-grooming), macrocephaly, increased oxidative stress, decreased cytochrome c oxidase (CCO) activity (50%) and increased mtDNA deletions in cerebellum and hippocampus. Mitochondrial dysfunction was the result of a downregulation of p53-signaling pathway evaluated by lower protein expression of p21 (65% of controls) and the CCO chaperone SCO2 (47% of controls), two p53-downstream targets. This mechanism was confirmed in Pten-deficient striatal neurons and, HCT 116 cells with different p53 gene dosage. These results suggest a unique pathogenic mechanism of the Pten-p53 axis in mice with aberrant social behavior: loss of Pten (via p53) impairs mitochondrial function elicited by an early defective assembly of CCO and later enhanced by the accumulation of mtDNA deletions. Consistent with our results, (i) SCO2 deficiency and/or CCO activity defects have been reported in patients with learning disabilities including autism and (ii) mutated proteins in ASD have been found associated with p53-signaling pathways.
Citrullinemia type I (CTLN1, OMIM# 215700) is an inherited urea cycle disorder that is caused by an argininosuccinate synthetase (ASS) enzyme deficiency. In this report, we describe two spontaneous ...hypomorphic alleles of the mouse Ass1 gene that serve as an animal model of CTLN1. These two independent mouse mutant alleles, also described in patients affected with CTLN1, interact to produce a range of phenotypes. While some mutant mice died within the first week after birth, others survived but showed severe retardation during postnatal development as well as alopecia, lethargy, and ataxia. Notable pathological findings were similar to findings in human CTLN1 patients and included citrullinemia and hyperammonemia along with delayed cerebellar development, epidermal hyperkeratosis, and follicular dystrophy. Standard treatments for CTLN1 were effective in rescuing the phenotype of these mutant mice. Based on our studies, we propose that defective cerebellar granule cell migration secondary to disorganization of Bergmann glial cell fibers cause cerebellar developmental delay in the hyperammonemic and citrullinemic brain, pointing to a possible role for nitric oxide in these processes. These mouse mutations constitute a suitable model for both mechanistic and preclinical studies of CTLN1 and other hyperammonemic encephalopathies and, at the same time, underscore the importance of complementing knockout mutations with hypomorphic mutations for the generation of animal models of human genetic diseases.
Protein synthesis requires a continuous supply of all of the indispensable (essential) amino acids (IAAs). If any IAA is deficient, animals must obtain the limiting amino acid by diet selection. ...Sensing of IAA deficiency requires an intact anterior piriform cortex (APC), but does it act alone? Shortly after rats begin eating an IAA-deficient diet, the meal ends and EPSPs are activated in the APC; from there, neurons project to feeding circuits; the meal ends within 20 min. Within the APC in vivo, uncharged tRNA activates the general amino acid control non-derepressing 2 (GCN2) enzyme system increasing phosphorylation of eukaryotic initiation factor (P-eIF2α), which blocks general protein synthesis. If this paleocortex is sufficient for sensing IAA depletion, both neuronal activation and P-eIF2α should occur in an isolated APC slice. We used standard techniques for electrophysiology and immunohistochemistry. After rats ate IAA-devoid or -imbalanced diets, their depleted slices responded to different stimuli with increased EPSP amplitudes. Slices from rats fed a control diet were bathed in artificial CSF replete with all amino acids with or without the IAA, threonine, or a tRNA synthetase blocker, l-threoninol, or its inactive isomer, d-threoninol. Thr depletion in vitro increased both EPSP amplitudes and P-eIF2α. l (but not d)-threoninol also increased EPSP amplitudes relative to control. Thus, we show independent excitation of the APC with responses parallel to those known in vivo. These data suggest a novel idea: in addition to classical processing of peripheral sensory input, direct primary sensing may occur in mammalian cortex.
Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder that affects individuals who are carriers of small CGG premutation expansions in the fragile X mental ...retardation 1 (FMR1) gene. Mitochondrial dysfunction was observed as an incipient pathological process occurring in individuals who do not display overt features of FXTAS (
1). Fibroblasts from premutation carriers had lower oxidative phosphorylation capacity (35% of controls) and Complex IV activity (45%), and higher precursor-to-mature ratios (P:M) of nDNA-encoded mitochondrial proteins (3.1-fold). However, fibroblasts from carriers with FXTAS symptoms presented higher FMR1 mRNA expression (3-fold) and lower Complex V (38%) and aconitase activities (43%). Higher P:M of ATPase β-subunit (ATPB) and frataxin were also observed in cortex from patients that died with FXTAS symptoms. Biochemical findings observed in FXTAS cells (lower mature frataxin, lower Complex IV and aconitase activities) along with common phenotypic traits shared by Friedreich's ataxia and FXTAS carriers (e.g. gait ataxia, loss of coordination) are consistent with a defective iron homeostasis in both diseases. Higher P:M, and lower ZnT6 and mature frataxin protein expression suggested defective zinc and iron metabolism arising from altered ZnT protein expression, which in turn impairs the activity of mitochondrial Zn-dependent proteases, critical for the import and processing of cytosolic precursors, such as frataxin. In support of this hypothesis, Zn-treated fibroblasts showed a significant recovery of ATPB P:M, ATPase activity and doubling time, whereas Zn and desferrioxamine extended these recoveries and rescued Complex IV activity.
Diets deficient in an indispensable amino acid are known to suppress food intake in rats. Few studies were focused at understanding how amino acid-deficient diets may elicit biochemical changes at ...the mitochondrial level. The goal of this study was to evaluate mitochondrial function in rats fed diets with 0.00, 0.18, 0.36, and 0.88% threonine (Thr) (set at 0, 30, 60, and 140% of Thr requirement for growth). Here, it is described for the first time that Thr-deficient diets induce a specific uncoupling of mitochondria in liver, especially with NADH-linked substrates, not observed in heart (except for Thr-devoid diet). The advantage of this situation would be to provide ATP to support growth and maintenance when high-quality protein food (or wealth of high-quality food in general) is available, whereas Thr-deficient diets (or deficient-quality protein food) promote the opposite, increasing mitochondrial uncoupling in liver. The uncoupling with NADH substrates would favor the use of nutrients as energy sources with higher FADH-to-NADH ratios, such as fat, minimizing the first irreversible NADH-dependent catabolism of many amino acids, including Thr, thus enhancing the use of the limiting amino acid for protein synthesis when a low quality protein source is available.
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.
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