Skeletal Muscle Mitochondrial and Lipid Droplet Volume Density: Validity of Electron Microscopy Point-counting Measurements

Mitochondrial (M) and lipid droplet (L) volume density (vd) are often used in exercise research. Vd is the volume of muscle occupied by M and L. The means of calculating these percents are accomplished by applying a grid to a 2D image taken with transmission electron microscopy; however, it is not known which grid best predicts these values. PURPOSE: To determine the grid with the least variability of Mvd and Lvd in human skeletal muscle. METHODS: Muscle biopsies were taken from vastus lateralis of 10 healthy adults, trained (N=6) and untrained (N=4). Samples of 5-10mg were fixed in 2.5% glutaraldehyde and embedded in EPON. Longitudinal sections of 60 nm were cut and 20 images were taken at random at 33,000x magnification. Vd was calculated as the number of times M or L touched two intersecting grid lines (called a point) divided by the total number of points using 3 different sizes of grids with squares of 1000x1000nm sides (corresponding to 1µm2), 500x500nm (0.25µm2) and 250x250nm (0.0625µm2). Statistics included coefficient of variation (CV), 1 way-BS ANOVA and spearman correlations. RESULTS: Mean age was 67 ± 4 yo, mean VO2peak 2.29 ± 0.70 L/min and mean BMI 25.1 ± 3.7 kg/m2. Mean Mvd was 6.39% ± 0.71 for the 1000nm squares, 6.01% ± 0.70 for the 500nm and 6.37% ± 0.80 for the 250nm. Lvd was 1.28% ± 0.03 for the 1000nm, 1.41% ± 0.02 for the 500nm and 1.38% ± 0.02 for the 250nm. The mean CV of the three grids was 6.65% ±1.15 for Mvd with no significant differences between grids (P>0.05). Mean CV for Lvd was 13.83% ± 3.51, with a significant difference between the 1000nm squares and the two other grids (P<0.05). The 500nm squares grid showed the least variability between subjects. Mvd showed a positive correlation with VO2peak (r = 0.89, p < 0.05) but not with weight, height, or age. No correlations were found with Lvd. CONCLUSION: Different size grids have different variability in assessing skeletal muscle Mvd and Lvd. The grid size of 500x500nm (240 points) was more reliable than 1000x1000nm (56 points). 250x250nm (1023 points) did not show better reliability compared with the 500x500nm, but was more time consuming. Thus, choosing a grid with square size of 500x500nm seems the best option. This is particularly relevant as most grids used in the literature are either 100 points or 400 points without clear information on their square size.

Species identification in mammals from mixed biological samples based on mitochondrial DNA control region length polymorphism.

The ability to identify the species origin of an unknown biological sample is relevant in the fields of human and wildlife forensics. However, the detection of several species mixed in the same sample still remains a challenge. We developed and tested a new approach for mammal DNA identification in mixtures of two or three species, based on the analysis of mitochondrial DNA control region interspecific length polymorphism followed by direct sequencing. Contrary to other published methods dealing with species mixtures, our protocol requires a single universal primer pair and is not based on a pre-defined panel of species. Amplicons can be separated either on agarose gels or using CE. The advantages and limitations of the assay are discussed under different conditions, such as variable template concentration, amplicon sizes and size difference among the amplicons present in the mixture. For the first time, this protocol provides a simple, reliable and flexible method for simultaneous identification of multiple mammalian species from mixtures, without any prior knowledge of the species involved.

Biogeographic origin and radiation of the Old World crocidurine shrews (Mammalia: Soricidae) inferred from mitochondrial and nuclear genes.

The crocidurine shrews include the most speciose genus of mammals, Crocidura. The origin and evolution of their radiation is, however, poorly understood because of very scant fossil records and a rather conservative external morphology between species. Here, we use an alignment of 3560 base pairs of mitochondrial and nuclear DNA to generate a phylogenetic hypothesis for the evolution of Old World shrews of the subfamily Crocidurinae. These molecular data confirm the monophyly of the speciose African and Eurasian Crocidura, which also includes the fossorial, monotypic genus Diplomesodon. The phylogenetic reconstructions give further credit to a paraphyletic position of Suncus shrews, which are placed into at least two independent clades (one in Africa and sister to Sylvisorex and one in Eurasia), at the base of the Crocidura radiation. Therefore, we recommend restricting the genus Suncus to the Palaearctic and Oriental taxa, and to consider all the African Suncus as Sylvisorex. Using molecular dating and biogeographic reconstruction analyses, we suggest a Palaearctic-Oriental origin for Crocidura dating back to the Upper Miocene (6.8 million years ago) and several subsequent colonisations of the Afrotropical region by independent lineages of Crocidura.

Mitochondrial targeting adaptation of the hominoid-specific glutamate dehydrogenase driven by positive Darwinian selection.

Many new gene copies emerged by gene duplication in hominoids, but little is known with respect to their functional evolution. Glutamate dehydrogenase (GLUD) is an enzyme central to the glutamate and energy metabolism of the cell. In addition to the single, GLUD-encoding gene present in all mammals (GLUD1), humans and apes acquired a second GLUD gene (GLUD2) through retroduplication of GLUD1, which codes for an enzyme with unique, potentially brain-adapted properties. Here we show that whereas the GLUD1 parental protein localizes to mitochondria and the cytoplasm, GLUD2 is specifically targeted to mitochondria. Using evolutionary analysis and resurrected ancestral protein variants, we demonstrate that the enhanced mitochondrial targeting specificity of GLUD2 is due to a single positively selected glutamic acid-to-lysine substitution, which was fixed in the N-terminal mitochondrial targeting sequence (MTS) of GLUD2 soon after the duplication event in the hominoid ancestor approximately 18-25 million years ago. This MTS substitution arose in parallel with two crucial adaptive amino acid changes in the enzyme and likely contributed to the functional adaptation of GLUD2 to the glutamate metabolism of the hominoid brain and other tissues. We suggest that rapid, selectively driven subcellular adaptation, as exemplified by GLUD2, represents a common route underlying the emergence of new gene functions.

Bihippocampal damage with emotional dysfunction: impaired auditory recognition of fear.

A right-handed man developed a sudden transient, amnestic syndrome associated with bilateral hemorrhage of the hippocampi, probably due to Urbach-Wiethe disease. In the 3rd month, despite significant hippocampal structural damage on imaging, only a milder degree of retrograde and anterograde amnesia persisted on detailed neuropsychological examination. On systematic testing of recognition of facial and vocal expression of emotion, we found an impairment of the vocal perception of fear, but not that of other emotions, such as joy, sadness and anger. Such selective impairment of fear perception was not present in the recognition of facial expression of emotion. Thus emotional perception varies according to the different aspects of emotions and the different modality of presentation (faces versus voices). This is consistent with the idea that there may be multiple emotion systems. The study of emotional perception in this unique case of bilateral involvement of hippocampus suggests that this structure may play a critical role in the recognition of fear in vocal expression, possibly dissociated from that of other emotions and from that of fear in facial expression. In regard of recent data suggesting that the amygdala is playing a role in the recognition of fear in the auditory as well as in the visual modality this could suggest that the hippocampus may be part of the auditory pathway of fear recognition.

Drug refractory epilepsy in brain damage: effect of dextromethorphan on EEG in four patients.

High doses of dextromethorphan (20-42 mg/kg/day) were given to four critically ill children with seizures and frequent epileptiform abnormalities in the EEG that were refractory to antiepileptic drugs. Their acute diseases (hypoxia, head trauma and hypoxia, neurodegenerative disease, hypoglycaemia) were thought to be due in part to N-methyl-D-aspartate (NMDA) receptor mediated processes. Treatment with dextromethorphan, an NMDA receptor antagonist, was started between 48 hours and 14 days after the critical incident. In three patients the EEG improved considerably within 48 hours and seizures ceased within 72 hours. In the patient with neurodegenerative disease the effect on the EEG was impressive, but the seizures were not controlled. Despite the improvement of the EEG the clinical outcome was poor in all children: three died in the critical period or due to the progressing disease; the patient with hypoglycaemia survived with severe neurological sequelae. Plasma concentrations of dextromethorphan varied between 74-1730 ng/ml and its metabolite dextrorphan varied between 349-3790 ng/ml. In one patient corresponding concentrations in CSF were lower than those in plasma. The suppression of epileptic discharges by the doses of dextromethorphan given suggests that such doses are sufficient to block NMDA receptors.

Genetic identity of the critically endangered Wimmer's shrew Crocidura wimmeri

Coastal primary rainforests have suffered damage in Côte d'Ivoire as a result of a lack of protection and urban pressures. Consequently, the highly endemic and critically endangered Wimmer's shrew, Crocidura wimmeri, known only from its type locality, Adiopodoumé, near Abidjan, was considered to have been extinct since 1976. Shrew species assignment is often problematic because of strong phenotypic similarities among many species. The phylogenetic position of C. wimmeri within the African Crocidura species should thus be clarified. In light of its recent rediscovery in the nearby small Banco National Park (34 km2), we investigated the genetic identity of seven specimens of C. wimmeri, based on 1020 bp of the mitochondrial DNA cytochrome b gene compared to other species sampled in the same region and published sequences from GenBank. Crocidura wimmeri formed a well-defined clade, the closest-related species being Crocidura sp., with a distance of 9.3%, a yet unknown species from Taï and Ziama forests. These results thus confirmed the validity of this species. This genetic characterization not only contributes to our knowledge of the evolution of West African shrews, but also may help in the discovery of additional populations of this critically endangered species.

Mitochondrial tRNA(Leu(UUR)) mutation m.3302A > G presenting as childhood-onset severe myopathy: threshold determination through segregation study.

Mitochondrial tRNA(Leu(UUR)) mutation m.3302A > G is associated with respiratory chain complex I deficiency and has been described as a rare cause of mostly adult-onset slowly progressive myopathy. Five families with 11 patients have been described so far; 5 of them died young due to cardiorespiratory failure. Here, we report on a segregation study in a family with an index patient who already presented at the age of 18 months with proximal muscular hypotonia, abnormal fatigability, and lactic acidosis. This early-onset myopathy was rapidly progressive. At 8 years, the patient is wheel-chair bound, requires nocturnal assisted ventilation, and suffers from recurrent respiratory infections. Severe complex I deficiency and nearly homoplasmy for m.3302A > G were found in muscle. We collected blood, hair, buccal swabs and muscle biopsies from asymptomatic adults in this pedigree and determined heteroplasmy levels in these tissues as well as OXPHOS activities in muscle. All participating asymptomatic adults had normal OXPHOS activities. In contrast to earlier reports, we found surprisingly little variation of heteroplasmy levels in different tissues of the same individual. Up to 45% mutation load in muscle and up to 38% mutation load in other tissues were found in non-affected adults. The phenotypic spectrum of tRNA(Leu(UUR)) m.3302A > G mutation seems to be wider than previously described. A threshold of more than 45% heteroplasmy in muscle seems to be necessary to alter complex I activity leading to clinical manifestation. The presented data may be helpful for prognostic considerations and counseling in affected families.

Inflammation-induced alteration of astrocyte mitochondrial dynamics requires autophagy for mitochondrial network maintenance.

Accumulating evidence suggests that changes in the metabolic signature of astrocytes underlie their response to neuroinflammation, but how proinflammatory stimuli induce these changes is poorly understood. By monitoring astrocytes following acute cortical injury, we identified a differential and region-specific remodeling of their mitochondrial network: while astrocytes within the penumbra of the lesion undergo mitochondrial elongation, those located in the core-the area invaded by proinflammatory cells-experience transient mitochondrial fragmentation. In brain slices, proinflammatory stimuli reproduced localized changes in mitochondrial dynamics, favoring fission over fusion. This effect was triggered by Drp1 phosphorylation and ultimately resulted in reduced respiratory capacity. Furthermore, maintenance of the mitochondrial architecture critically depended on the induction of autophagy. Deletion of Atg7, required for autophagosome formation, prevented the reestablishment of tubular mitochondria, leading to marked reactive oxygen species accumulation and cell death. Thus, our data reveal autophagy to be essential for regenerating astrocyte mitochondrial networks during inflammation.

Glutamate Transport Decreases Mitochondrial pH and Modulates Oxidative Metabolism in Astrocytes.

During synaptic activity, the clearance of neuronally released glutamate leads to an intracellular sodium concentration increase in astrocytes that is associated with significant metabolic cost. The proximity of mitochondria at glutamate uptake sites in astrocytes raises the question of the ability of mitochondria to respond to these energy demands. We used dynamic fluorescence imaging to investigate the impact of glutamatergic transmission on mitochondria in intact astrocytes. Neuronal release of glutamate induced an intracellular acidification in astrocytes, via glutamate transporters, that spread over the mitochondrial matrix. The glutamate-induced mitochondrial matrix acidification exceeded cytosolic acidification and abrogated cytosol-to-mitochondrial matrix pH gradient. By decoupling glutamate uptake from cellular acidification, we found that glutamate induced a pH-mediated decrease in mitochondrial metabolism that surpasses the Ca(2+)-mediated stimulatory effects. These findings suggest a model in which excitatory neurotransmission dynamically regulates astrocyte energy metabolism by limiting the contribution of mitochondria to the metabolic response, thereby increasing the local oxygen availability and preventing excessive mitochondrial reactive oxygen species production.

A Mitochondrial DNA Phylogeny Indicates Close Relationships between Populations of Lutzomyia whitmani (Diptera: Psychodidae, Phlebotominae) from the Rain-forest Regions of Amazônia and Northeast Brazil

Phylogenetic analysis of all 31 described mitochondrial (cytochrome b) haplotypes of Lutzomyia whitmani demonstrated that new material from the State of Rondônia, in southwest Amazônia, forms a clade within a lineage found only in the rain-forest regions of Brazil. This rain-forest lineage also contains two other clades of haplotypes, one from eastern Amazônia and one from the Atlantic forest zone of northeast Brazil (including the type locality of the species in Ilhéus, State of Bahia). These findings do not favour recognizing two allopatric cryptic species of L. whitmani, one associated with the silvatic transmission of Leishmania shawi in southeast Amazônia and the other with the peridomestic transmission of Le. braziliensis in northeast Brazil. Instead, they suggest that there is (or has been in the recent past) a continuum of inter-breeding populations of L. whitmani in the rain-forest regions of Brazil.

Mitochondrial DNA sequence variation within the remnant populations of the endangered numbat (Marsupialia: Myrmecobiidae: Myrmecobius fasciatus).

The numbat has been reduced to two populations in Western Australia. To better understand the effects of range reduction on gene flow and genetic variation, and to address questions crucial for the species' management, we analysed mitochondrial DNA (mtDNA) sequences of free-ranging individuals and museum specimens. The results suggest recent connectivity between the remnant populations, although one of those may have lost significant amounts of genetic diversity during the recent population size reduction. We propose that for management purposes the remnant populations should be treated as a single historical lineage and that, subject to certain caveats, consideration should be given to population augmentation by translocation.

Mitochondrial DNA variation of Triatoma infestans populations and its implication on the specific status of T. melanosoma

DNA sequence comparison of 412 base-pairs fragments of the mitochondrial cytochrome B gene was used to infer the genetic structure of nine geographical Triatoma infestans populations and their phylogenetic relationship with T. melanosoma and T. brasiliensis. T. infestans and T. melanosoma were compared by morphometry, allozyme and cytogenetic analyses, as well as subjected to reciprocal crosses, in order to clarify the taxonomic status of the latter. No differences were found to distinguish the two species and the crosses between them yielded progeny. T. infestans populations presented four haplotypes that could be separated in two clusters: one formed by the samples from Bolivia (Andes and Chaco) and the other formed by samples from Argentina and Brazil. Silvatic and domestic T. infestans populations from Bolivia (Andes) were genetically identical.

Hepatitis C virus proteins promote mitochondrial bioenergetic dysfunction and nitro-oxidative stress: insights into pathogenesis

HCV-infection induces a state of oxidative stress more pronounced than in many other inflammatory diseases. Here we propose a temporal sequence of events in the HCV-infected cell whereby the primary alteration consists in release of Ca2+ from the ER followed by uptake into mitochondria. This triggers successive mitochondrial dysfunctions leading to generation of ROS and to a progressive metabolic adaptive response. Pathogenetic implications of the model and new opportunities for therapeutic intervention are discussed.