Altered zinc transport disrupts mitochondrial protein processing/import in fragile X-associated tremor/ataxia syndrome.

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 ß-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.

Basal bioenergetic abnormalities in skeletal muscle from ryanodine receptor malignant hyperthermia-susceptible R163C knock-in mice.

Malignant hyperthermia (MH) and central core disease in humans have been associated with mutations in the skeletal ryanodine receptor (RyR1). Heterozygous mice expressing the human MH/central core disease RyR1 R163C mutation exhibit MH when exposed to halothane or heat stress. Considering that many MH symptoms resemble those that could ensue from a mitochondrial dysfunction (e.g. metabolic acidosis and hyperthermia) and that MH-susceptible mice or humans have a higher than normal cytoplasmic Ca(2+) concentration at rest, we evaluated the role of mitochondria in skeletal muscle from R163C compared with wild type mice under basal (untriggered) conditions. R163C skeletal muscle exhibited a significant increase in matrix Ca(2+), increased reactive oxygen species production, lower expression of mitochondrial proteins, and higher mtDNA copy number. These changes, in conjunction with lower myoglobin and glycogen contents, Myh4 and GAPDH transcript levels, GAPDH activity, and lower glucose utilization suggested a switch to a compromised bioenergetic state characterized by both low oxidative phosphorylation and glycolysis. The shift in bioenergetic state was accompanied by a dysregulation of Ca(2+)-responsive signaling pathways regulated by calcineurin and ERK1/2. Chronically elevated resting Ca(2+) in R163C skeletal muscle elicited the maintenance of a fast-twitch fiber program and the development of insulin resistance-like phenotype as part of a metabolic adaptation to the R163C RyR1 mutation.

Evidence of mitochondrial dysfunction in fragile X-associated tremor/ataxia syndrome.

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.

Mitochondrial dysfunction in autism.

CONTEXT: Impaired mitochondrial function may influence processes highly dependent on energy, such as neurodevelopment, and contribute to autism. No studies have evaluated mitochondrial dysfunction and mitochondrial DNA (mtDNA) abnormalities in a well-defined population of children with autism. OBJECTIVE: To evaluate mitochondrial defects in children with autism. DESIGN, SETTING, AND PATIENTS: Observational study using data collected from patients aged 2 to 5 years who were a subset of children participating in the Childhood Autism Risk From Genes and Environment study in California, which is a population-based, case-control investigation with confirmed autism cases and age-matched, genetically unrelated, typically developing controls, that was launched in 2003 and is still ongoing. Mitochondrial dysfunction and mtDNA abnormalities were evaluated in lymphocytes from 10 children with autism and 10 controls. MAIN OUTCOME MEASURES: Oxidative phosphorylation capacity, mtDNA copy number and deletions, mitochondrial rate of hydrogen peroxide production, and plasma lactate and pyruvate. RESULTS: The reduced nicotinamide adenine dinucleotide (NADH) oxidase activity (normalized to citrate synthase activity) in lymphocytic mitochondria from children with autism was significantly lower compared with controls (mean, 4.4 [95% confidence interval {CI}, 2.8-6.0] vs 12 [95% CI, 8-16], respectively; P = .001). The majority of children with autism (6 of 10) had complex I activity below control range values. Higher plasma pyruvate levels were found in children with autism compared with controls (0.23 mM [95% CI, 0.15-0.31 mM] vs 0.08 mM [95% CI, 0.04-0.12 mM], respectively; P = .02). Eight of 10 cases had higher pyruvate levels but only 2 cases had higher lactate levels compared with controls. These results were consistent with the lower pyruvate dehydrogenase activity observed in children with autism compared with controls (1.0 [95% CI, 0.6-1.4] nmol × [min × mg protein](-1) vs 2.3 [95% CI, 1.7-2.9] nmol × [min × mg protein](-1), respectively; P = .01). Children with autism had higher mitochondrial rates of hydrogen peroxide production compared with controls (0.34 [95% CI, 0.26-0.42] nmol × [min × mg of protein](-1) vs 0.16 [95% CI, 0.12-0.20] nmol × [min × mg protein](-1) by complex III; P = .02). Mitochondrial DNA overreplication was found in 5 cases (mean ratio of mtDNA to nuclear DNA: 239 [95% CI, 217-239] vs 179 [95% CI, 165-193] in controls; P = 10(-4)). Deletions at the segment of cytochrome b were observed in 2 cases (ratio of cytochrome b to ND1: 0.80 [95% CI, 0.68-0.92] vs 0.99 [95% CI, 0.93-1.05] for controls; P = .01). CONCLUSION: In this exploratory study, children with autism were more likely to have mitochondrial dysfunction, mtDNA overreplication, and mtDNA deletions than typically developing children.

Pathology and diagnosis of proliferative and ulcerative dermatitis associated with Tunga penetrans infestation in cattle.

Tunga penetrans is the smallest biting flea known. In cattle, infestation by T. penetrans (tungiasis) typically affects the skin of the distal legs, udder, prepuce, and perianal area. A detailed clinical and pathologic description of bovine tungiasis, together with electron microscopy and molecular diagnostics to establish the identity of the parasite are described. Ninety percent of the cows and heifers and 80% of the bulls in a herd in northwest Argentina had proliferative and ulcerative skin lesions affecting the coronary band, interdigital space, heels, and rudimentary toes of the fore and/or rear limbs, teats, and/or prepuce. These proliferative lesions had multiple large cavities filled with hemorrhagic fluid, necrotic debris, and Tunga spp. parasites. Histologically, the skin showed diffuse papillary epithelial hyperplasia with severe orthokeratotic hyperkeratosis, and it was multifocally ulcerated and inflamed. Multifocally, sections of arthropod parasites were observed embedded in the epidermis and dermis with the posterior end toward the surface. Images of wet mounts and scanning electron microscopy of the parasite showed morphologic characteristics compatible with Tunga spp. Polymerase chain reaction followed by sequencing of the cytochrome c oxidase subunit II and the internal transcribed spacer region indicated 99% homology to published T. penetrans sequences. Tungiasis should be considered as a differential diagnosis for proliferative lesions in skin of cattle.

Development and validation of a multiplex quantitative PCR assay for the rapid detection of Grapevine virus A, B and D.

A single real-time multiplex quantitative PCR (qPCR) assay for the simultaneous detection of Grapevine virus A, B and D (GVA, GVB and GVD) was developed, using three different fluorescently labeled minor groove binding probes. This multiplex RT-qPCR was compared to singleplex RT-qPCR designed specifically for each virus and a conventional multiplex RT-PCR. The capacity of the multiplex RT-qPCR assay in detecting the three vitiviruses in mixed infections from a range of virus concentrations in the host was assessed. A series of cDNA derived from 48 different grapevine cultivars obtained from diverse geographical regions infected with various isolates and strains of GVA, GVB and GVD were subjected to singleplex, multiplex RT-qPCR, and conventional multiplex RT-PCR testing. The results showed that the developed multiplex RT-qPCR assay was a cost-effective diagnostic tool that could streamline the testing of grapevine vitiviruses, and replace the singleplex RT-qPCR assays, thus reducing time and labor while retaining the same sensitivity and specificity. In particular, no significant differences in detection limits were found between singleplex and multiplex RT-qPCR and specificity was not affected by the inclusion of the three primer/probe combinations within a multiplex RT-qPCR. Comparing the viral load for each virus using singleplex and multiplex RT-qPCR assays revealed no significant differences between the two assays in detecting GVB and GVD. However, while in detecting GVA using singleplex RT-qPCR assay, viral load was higher. Finally, the multiplex RT-qPCR assay was also more sensitive and time efficient than the conventional multiplex RT-PCR that was designed using degenerate primers to detect GVA, GVB and GVD. This multiplex RT-qPCR method could detect viruses in 95.83% of mixed infected samples as compared to 77.08% for multiplex RT-PCR.

Mitochondrial dysfunction in Pten haplo-insufficient mice with social deficits and repetitive behavior: interplay between Pten and p53.

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.