Hormone deprivation alters mitochondrial function and lipid profile in the hippocampus

Mitochondrial dysfunction is a common hallmark in aging. In the female, reproductive senescence is characterized by loss of ovarian hormones, many of whose neuroprotective effects converge upon mitochondria. The functional integrity of mitochondria is dependent on membrane fatty acid and phospholipid composition, which are also affected during aging. The effect of long-term ovarian hormone deprivation upon mitochondrial function and its putative association with changes in mitochondrial membrane lipid profile in the hippocampus, an area primarily affected during aging and highly responsive to ovarian hormones, is unknown. To this aim, Wistar adult female rats were ovariectomized or sham-operated. Twelve weeks later, different parameters of mitochondrial function (O2 uptake, ATP production, membrane potential and respiratory complex activities) as well as membrane phospholipid content and composition were evaluated in hippocampal mitochondria. Chronic ovariectomy reduced mitochondrial O2 uptake and ATP production rates and induced membrane depolarization during active respiration without altering the activity of respiratory complexes. Mitochondrial membrane lipid profile showed no changes in cholesterol levels but higher levels of unsaturated fatty acids and a higher peroxidizability index in mitochondria from ovariectomized rats. Interestingly, ovariectomy also reduced cardiolipin content and altered cardiolipin fatty acid profile leading to a lower peroxidizability index. In conclusion, chronic ovarian hormone deprivation induces mitochondrial dysfunction and changes in the mitochondrial membrane lipid profile comparable to an aging phenotype. Our study provides insights into ovarian hormone loss-induced early lipidomic changes with bioenergetic deficits in the hippocampus that may contribute to the increased risk of Alzheimer’s disease and other age-associated disorders observed in postmenopause.Fil: Zarate, Sandra Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: Astiz, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; ArgentinaFil: Magnani, Natalia Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; ArgentinaFil: Imsen, Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: Merino, Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: Alvarez, Silvia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; ArgentinaFil: Reines, Analia Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Seilicovich, Adriana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; Argentin

A role for human N-alpha acetyltransferase 30 (Naa30) in maintaining mitochondrial integrity

N-terminal acetylation (Nt-acetylation) by N-terminal acetyltransferases (NATs) is one of the most common protein modifications in eukaryotes. The NatC complex represents one of three major NATs of which the substrate profile remains largely unexplored. Here, we defined the in vivo human NatC Nt-acetylome on a proteome-wide scale by combining knockdown of its catalytic subunit Naa30 with positional proteomics. We identified 46 human NatC substrates, expanding our current knowledge on the substrate repertoire of NatC which now includes proteins harboring Met-Leu, Met-Ile, Met-Phe, Met-Trp, Met-Val, Met-Met, Met-His and Met-Lys N termini. Upon Naa30 depletion the expression levels of several organellar proteins were found reduced, in particular mitochondrial proteins, some of which were found to be NatC substrates. Interestingly, knockdown of Naa30 induced the loss of mitochondrial membrane potential and fragmentation of mitochondria. In conclusion, NatC N-tacetylates a large variety of proteins and is essential for mitochondrial integrity and function

Mrpl35, A Mitospecific Component of Mitoribosomes, Plays A Key Role in Cytochrome \u3cem\u3eC\u3c/em\u3e Oxidase Assembly

Mitoribosomes perform the synthesis of the core components of the oxidative phosphorylation (OXPHOS) system encoded by the mitochondrial genome. We provide evidence that MrpL35 (mL38), a mitospecific component of the yeast mitoribosomal central protuberance, assembles into a subcomplex with MrpL7 (uL5), Mrp7 (bL27), and MrpL36 (bL31) and mitospecific proteins MrpL17 (mL46) and MrpL28 (mL40). We isolated respiratory defective mrpL35 mutant yeast strains, which do not display an overall inhibition in mitochondrial protein synthesis but rather have a problem in cytochrome coxidase complex (COX) assembly. Our findings indicate that MrpL35, with its partner Mrp7, play a key role in coordinating the synthesis of the Cox1 subunit with its assembly into the COX enzyme and in a manner that involves the Cox14 and Coa3 proteins. We propose that MrpL35 and Mrp7 are regulatory subunits of the mitoribosome acting to coordinate protein synthesis and OXPHOS assembly events and thus the bioenergetic capacity of the mitochondria

Mitochondrial ribosome assembly in Neurospora. Structural analysis of mature and partially assembled ribosomal subunits by equilibrium centrifugation in CsCl gradients

In Neurospora, one protein associated with the mitochondrial small ribosomal subunit (S-5, Mr 52,000) is synthesized intramitochondrially and is assumed to be encoded by mtDNA. When mitochondrial protein synthesis is inhibited, either by chloramphenicol or by mutation, cells accumulate incomplete mitochondrial small subunits (CAP-30S and INC-30S particles) that are deficient in S-5 and several other proteins. To gain additional insight into the role of S-5 in mitochondrial ribosome assembly, the structures of Neurospora mitochondrial ribosomal subunits, CAP-30S particles, and INC-30S particles were analyzed by equilibrium centrifugation in CsCl gradients containing different concentrations of Mg+2. The results show (a) that S-5 is tightly associated with small ribosomal subunits, as judged by the fact that it is among the last proteins to be dissociated in CsCl gradients as the Mg+2 concentration is decreased, and (b) that CAP-30S and INC-30S particles, which are deficient in S-5, contain at most 12 proteins that are bound as tightly as in mature small subunits. The CAP-30S particles isolated from sucrose gradients contain a number of proteins that appear to be loosely bound, as judged by dissociation of these proteins in CsCl gradients under conditions in which they remain associated with mature small subunits. The results suggest that S-5 is required for the stable binding of a subset of small subunit ribosomal proteins

Distinctive pattern and translational control of mitochondrial protein synthesis in rat brain synaptic endings

Mitochondrial gene expression has been investigated in synaptic endings from rat cerebral cortex isolated at various stages during the postnatal development and maturation of the animal. The pattern of the mitochondrial translation products labeled in vitro in rat brain synaptosomes revealed some distinctive features when compared with the pattern observed in a rat fibroblast cell line, the most remarkable being the apparent absence of labeling of the ND5 product. This absence contrasted with the presence in synaptosomes of an amount of ND5 mRNA comparable with that found in the rat fibroblast cell line. The rate of mitochondrial protein synthesis per unit amount of mtDNA inbrain synaptosomes showed a characteristic reproducible burst at 10-13 days after birth, thereafter declining sharply in the 3rd week to reach a level that remained constant over a 2-year period. The postnatal burst of mitochondrial protein synthesis coincided with a sharp increase in cytochrome c oxidase activity, pointing to a phase of rapid assembly of respiratory complexes. A comparison of the levels of mitochondrial mRNAs with the corresponding rates of protein synthesis during the animal development and maturation showed a lack of correlation. These observations, together with the apparent lack of translation of the ND5 mRNA, indicate that translational control plays a major role in the regulation of gene expression in rat brain synaptic mitochondria

Survey of Human Mitochondrial Diseases Using New Genomic/Proteomic Tools

BACKGROUND. We have constructed Bayesian prior-based, amino-acid sequence profiles for the complete yeast mitochondrial proteome and used them to develop methods for identifying and characterizing the context of protein mutations that give rise to human mitochondrial diseases. (Bayesian priors are conditional probabilities that allow the estimation of the likelihood of an event - such as an amino-acid substitution - on the basis of prior occurrences of similar events.) Because these profiles can assemble sets of taxonomically very diverse homologs, they enable identification of the structurally and/or functionally most critical sites in the proteins on the basis of the degree of sequence conservation. These profiles can also find distant homologs with determined three-dimensional structures that aid in the interpretation of effects of missense mutations. RESULTS. This survey reports such an analysis for 15 missense mutations one insertion and three deletions involved in Leber's hereditary optic neuropathy, Leigh syndrome, mitochondrial neurogastrointestinal encephalomyopathy, Mohr-Tranebjaerg syndrome, iron-storage disorders related to Friedreich's ataxia, and hereditary spastic paraplegia. We present structural correlations for seven of the mutations. CONCLUSIONS. Of the 19 mutations analyzed, 14 involved changes in very highly conserved parts of the affected proteins. Five out of seven structural correlations provided reasonable explanations for the malfunctions. As additional genetic and structural data become available, this methodology can be extended. It has the potential for assisting in identifying new disease-related genes. Furthermore, profiles with structural homologs can generate mechanistic hypotheses concerning the underlying biochemical processes - and why they break down as a result of the mutations.United States Department of Energy (DE-FG02-98ER62558); National Science Foundation (DBI-9807993

The Mitochondrial Myopathy, Encephalopathy, Lactic Acidosis, and Stroke-like Episode Syndrome-associated Human Mitochondrial tRNALeu(UUR) Mutation Causes Aminoacylation Deficiency and Concomitant Reduced Association of mRNA with Ribosomes

The pathogenetic mechanism of the mitochondrial tRNALeu(UUR) A3243G transition associated with the mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome has been investigated in transmitochondrial cell lines constructed by transfer of mutant mitochondrial DNA (mtDNA)-carrying mitochondria from three genetically unrelated MELAS patients or of isogenic wild-type mtDNA-carrying organelles into human mtDNA-less cells. An in vivo footprinting analysis of the mtDNA segment within the tRNALeu(UUR) gene that binds the transcription termination factor failed to reveal any difference in occupancy of sites or qualitative interaction with the protein between mutant and wild-type mtDNAs. Cell lines nearly homoplasmic for the mutation exhibited a strong (70-75%) reduction in the level of aminoacylated tRNALeu(UUR) and a decrease in mitochondrial protein synthesis rate. The latter, however, did not show any significant correlation between synthesis defect of the individual polypeptides and number or proportion of UUR codons in their mRNAs, suggesting that another step, other than elongation, may be affected. Sedimentation analysis in sucrose gradient showed a reduction in size of the mitochondrial polysomes, while the distribution of the two rRNA components and of the mRNAs revealed decreased association of mRNA with ribosomes and, in the most affected cell line, pronounced degradation of the mRNA associated with slowly sedimenting structures. Therefore, several lines of evidence indicate that the protein synthesis defect in A3243G MELAS mutation-carrying cells is mainly due to a reduced association of mRNA with ribosomes, possibly as a consequence of the tRNALeu(UUR) aminoacylation defect

Strategies for maximizing ATP supply in the microsporidian Encephalitozoon cuniculi: direct binding of mitochondria to the parasitophorous vacuole and clustering of the mitochondrial porin VDAC

Microsporidia are obligate intracellular parasites with extremely reduced genomes and a dependence on host-derived ATP. The microsporidium Encephalitozoon cuniculi proliferates within a membranous vacuole and we investigated how the ATP supply is optimized at the vacuole–host interface. Using spatial EM quantification (stereology), we found a single layer of mitochondria coating substantial proportions of the parasitophorous vacuole. Mitochondrial binding occurred preferentially over the vegetative ‘meront’ stages of the parasite, which bulged into the cytoplasm, thereby increasing the membrane surface available for mitochondrial interaction. In a broken cell system mitochondrial binding was maintained and was typified by electron dense structures (<10 nm long) bridging between outer mitochondrial and vacuole membranes. In broken cells mitochondrial binding was sensitive to a range of protease treatments. The function of directly bound mitochondria, as measured by the membrane potential sensitive dye JC-1, was indistinguishable from other mitochondria in the cell although there was a generalized depression of the membrane potential in infected cells. Finally, quantitative immuno-EM revealed that the ATP-delivering mitochondrial porin, VDAC, was concentrated atthe mitochondria-vacuole interaction site. Thus E. cuniculi appears to maximize ATP supply by direct binding of mitochondria to the parasitophorous vacuole bringing this organelle within 0.020 microns of the growing vegetative form of the parasite. ATP-delivery is further enhanced by clustering of ATP transporting porins in those regions of the outer mitochondrial membrane lying closest to the parasite