Judd M. Aiken

Mitochondrial DNA deletion mutations colocalize with segmental electron transport system abnormalities, muscle fiber atrophy, fiber splitting, and oxidative damage in sarcopenia

The in vivo cellular impact of age‐associated mitochondrial DNA mutations is unknown. We hypothesized that mitochondrial DNA deletion mutations contribute to the fiber atrophy and loss that cause sarcopenia, the age‐related decline of muscle mass and function. We examined 82,713 rectus femoris muscle fibers from Fischer 344 ×Brown Norway F1 hybrid rats of ages 5, 18, and 38 months through 1000 microns by serial cryosectioning and histochemical staining for cytochrome c oxidase and succinate dehydrogenase. Between 5 and 38 months of age, the rectus femoris muscle in the hybrid rat demonstrated a 33% decrease in mass concomitant with a 30% decrease in total fibers at the muscle mid‐belly. We observed significant increases in the number of mitochondrial abnormalities with age from 289 ± 8 ETS abnormal fibers in the entire 5‐month‐old rectus femoris to 1094 ± 126 in the 38‐month‐old as calculated from the volume density of these abnormalities. Segmental mitochondrial abnormalities contained mitochondrial DNA deletion mutations as revealed by laser capture microdissection and whole mitochondrial genome amplification. Muscle fibers harboring mitochondrial deletions often displayed atrophy, splitting and increased steady‐state levels of oxidative nucleic damage. These data suggest a causal role for age‐associated mitochondrial DNA deletion mutations in sarcopenia.—Wanagat, J., Cao, Z., Pathare, P., Aiken, J. M. Mitochondrial DNA deletion mutations colocalize with segmental electron transport system abnormalities, muscle fiber atrophy, fiber splitting, and oxidative damage in sarcopenia. FASEB J. 15, 322‐332 (2001)

Mitochondrial DNA–Deletion Mutations Accumulate Intracellularly to Detrimental Levels in Aged Human Skeletal Muscle Fibers

Skeletal muscle-mass loss with age has severe health consequences, yet the molecular basis of the loss remains obscure. Although mitochondrial DNA (mtDNA)-deletion mutations have been shown to accumulate with age, for these aberrant genomes to be physiologically relevant, they must accumulate to high levels intracellularly and be present in a significant number of cells. We examined mtDNA-deletion mutations in vastus lateralis (VL) muscle of human subjects aged 49-93 years, using both histologic and polymerase-chain-reaction (PCR) analyses, to determine the physiological and genomic integrity of mitochondria in aging human muscle. The number of VL muscle fibers exhibiting mitochondrial electron-transport-system (ETS) abnormalities increased from an estimated 6% at age 49 years to 31% at age 92 years. We analyzed the mitochondrial genotype of 48 single ETS-abnormal, cytochrome c oxidase-negative/succinate dehydrogenase-hyperreactive (COX-/SDH++) fibers from normal aging human subjects and identified mtDNA-deletion mutations in all abnormal fibers. Deletion mutations were clonal within a fiber and concomitant to the COX-/SDH++ region. Quantitative PCR analysis of wild-type and deletion-containing mtDNA genomes within ETS-abnormal regions of single fibers demonstrated that these deletion mutations accumulate to detrimental levels (>90% of the total mtDNA).

Caloric restriction reduces fiber loss and mitochondrial abnormalities in aged rat muscle.

The influence of caloric restriction (CR) initiated at 17 months of age was investigated on selected age‐associated measures in skeletal muscle. Tissue from young (3–4 months) ad libitum‐fed, old (30–32 months) restricted (35% and 50% CR, designated CR35 and CR50, respectively), and old ad libitum‐fed rats (29 months) was studied. CR preserved fiber number and fiber type composition in the vastus lateralis muscle of the CR50 rats. In the old rats from all groups, individual fibers were found with either no detectable cytochrome c oxidase activity (COX−), hyperreactivity for succinate dehydrogenase activity (SDH++; also known as ragged red fibers [RRF]), or both COX− and SDH++. Muscle from the CR50 rats contained significantly fewer COX− and SDH++ fibers than did the muscle from CR35 rats. CR50 rats also had significantly lower numbers of mtDNA deletion products in two (adductor longus and soleus) of the four muscles examined compared to CR35 rats. These data indicate that CR begun in late middle age can retard age‐associated fiber loss and fiber type changes, as well as increases in the number of skeletal muscle fibers showing mitochondrial enzyme abnormalities. CR also decreased the accumulation of mtDNA deletions.—Aspnes, L. E., Lee, C. M., Weindruch, R., Chung, S. S., Roecker, E. B., Aiken, J. M. Caloric restriction reduces fiber loss and mitochondrial abnormalities in aged rat muscle. FASEB J. 11, 573–581 (1997)

Oral Transmissibility of Prion Disease Is Enhanced by Binding to Soil Particles

Soil may serve as an environmental reservoir for prion infectivity and contribute to the horizontal transmission of prion diseases (transmissible spongiform encephalopathies [TSEs]) of sheep, deer, and elk. TSE infectivity can persist in soil for years, and we previously demonstrated that the disease-associated form of the prion protein binds to soil particles and prions adsorbed to the common soil mineral montmorillonite (Mte) retain infectivity following intracerebral inoculation. Here, we assess the oral infectivity of Mte- and soil-bound prions. We establish that prions bound to Mte are orally bioavailable, and that, unexpectedly, binding to Mte significantly enhances disease penetrance and reduces the incubation period relative to unbound agent. Cox proportional hazards modeling revealed that across the doses of TSE agent tested, Mte increased the effective infectious titer by a factor of 680 relative to unbound agent. Oral exposure to Mte-associated prions led to TSE development in experimental animals even at doses too low to produce clinical symptoms in the absence of the mineral. We tested the oral infectivity of prions bound to three whole soils differing in texture, mineralogy, and organic carbon content and found soil-bound prions to be orally infectious. Two of the three soils increased oral transmission of disease, and the infectivity of agent bound to the third organic carbon-rich soil was equivalent to that of unbound agent. Enhanced transmissibility of soil-bound prions may explain the environmental spread of some TSEs despite the presumably low levels shed into the environment. Association of prions with inorganic microparticles represents a novel means by which their oral transmission is enhanced relative to unbound agent.

Reversibility of Scrapie Inactivation Is Enhanced by Copper

The only known difference between the cellular (PrPC) and scrapie-specific (PrPSc) isoforms of the prion protein is conformational. Because disruption of PrPSc structure decreases scrapie infectivity, restoration of the disease-specific conformation should restore infectivity. In this study, disruption of PrPSc (as monitored by the loss of proteinase K resistance) by guanidine hydrochloride (GdnHCl) resulted in decreased infectivity. Upon dilution of the GdnHCl, protease resistance of PrP was restored and infectivity was regained. The addition of copper facilitated restoration of both infectivity and protease resistance of PrP in a subset of samples that did not renature by the simple dilution of the GdnHCl. These data demonstrate that loss of scrapie infectivity can be a reversible process and that copper can enhance this restoration of proteinase K resistance and infectivity.

Persistence and expression of Microplitis demolitor polydnavirus in Pseudoplusia includens.

Persistence and expression of Microplitis demolitor polydnavirus (MdPDV) was examined in parasitized and virus-injected Pseudoplusia includens larvae. Viral DNA persisted in P. includens larvae for 6 days, but no increase in the amount of viral DNA present was detected. Viral transcripts were observed in parasitized and virus-injected larvae 4 h post-parasitism and expression continued for 6 days. When specific host tissues were examined, more viral DNA and RNA was detected in haemocytes than in the gut, nervous system and fat body. 32P-labelled MdPDV DNA hybridized to approximately six different size classes of mRNAs on Northern blots of RNA from haemocytes of parasitized larvae. MdPDV transcription was first detected in haemocytes at 4 h post-parasitism and continued for 6 days. Similar transcripts were observed in haemocytes from larvae that had been injected with calyx fluid or MdPDV plus venom. First-strand cDNA probes of haemocyte-specific MdPDV transcripts hybridized to only certain MdPDV viral DNAs, suggesting that only part of the MdPDV genome is expressed in this host cell type.

High levels of mitochondrial DNA deletions in skeletal muscle of old rhesus monkeys

Mitochondrial DNA (mtDNA) deletions increase in abundance with age in many tissues, however, their calculated low levels (usually < 0.1%) in samples from tissue homogenates containing thousands of cells argue against physiologic significance. Through the analysis of defined numbers of cells (skeletal muscle fibers) from rhesus monkeys, we report that the calculated abundance of specific mtDNA deletions is dependent upon the number of fibers analyzed: as the number of fibers decreases, the calculated deletion abundance increases. Also, most mtDNA deletions appear to occur in a mosaic pattern, varying from cell to cell in size, number and abundance. These data support the hypothesis that mtDNA deletions can focally accumulate to high levels contributing to declines in mass and function of aging skeletal muscle.

Adaptation and Selection of Prion Protein Strain Conformations following Interspecies Transmission of Transmissible Mink Encephalopathy

ABSTRACT Interspecies transmission of the transmissible spongiform encephalopathies (TSEs), or prion diseases, can result in the adaptation and selection of TSE strains with an expanded host range and increased virulence such as in the case of bovine spongiform encephalopathy and variant Creutzfeldt-Jakob disease. To investigate TSE strain adaptation, we serially passaged a biological clone of transmissible mink encephalopathy (TME) into Syrian golden hamsters and examined the selection of distinct strain phenotypes and conformations of the disease-specific isoform of the prion protein (PrPSc). The long-incubation-period drowsy (DY) TME strain was the predominate strain, based on the presence of its strain-specific PrPSc following interspecies passage. Additional serial passages in hamsters resulted in the selection of the hyper (HY) TME PrPSc strain-dependent conformation and its short incubation period phenotype unless the passages were performed with a low-dose inoculum (e.g., 10−5 dilution), in which case the DY TME clinical phenotype continued to predominate. For both TME strains, the PrPSc strain pattern preceded stabilization of the TME strain phenotype. These findings demonstrate that interspecies transmission of a single cloned TSE strain resulted in adaptation of at least two strain-associated PrPScconformations that underwent selection until one type of PrPSc conformation and strain phenotype became predominant. To examine TME strain selection in the absence of host adaptation, hamsters were coinfected with hamster-adapted HY and DY TME. DY TME was able to interfere with the selection of the short-incubation HY TME phenotype. Coinfection could result in the DY TME phenotype and PrPSc conformation on first passage, but on subsequent passages, the disease pattern converted to HY TME. These findings indicate that during TSE strain adaptation, there is selection of a strain-specific PrPSc conformation that can determine the TSE strain phenotype.

ASSOCIATION OF AGE-RELATED MITOCHONDRIAL ABNORMALITIES WITH SKELETAL MUSCLE FIBER ATROPHY

The hypothesis that mitochondrial dysfunction contributes to the senescent loss of skeletal muscle was investigated in quadriceps from 2- to 39-year old rhesus monkeys. Histological approaches, both cross-sectional (a single cross-section of the muscle) and longitudinal (multiple cross-sections of individual fibers spanning a 350-1600 microm region), were used to identify muscle fibers with abnormal mitochondrial electron transport system (ETS) enzyme activities and mitochondrial DNA deletions. Fibers were examined for two ETS activities, succinate dehydrogenase (SDH, ETS complex II) and cytochrome c oxidase (COX, ETS complex IV). The number of individual fibers containing ETS abnormalities (predominately negative for cytochrome c oxidase activity and/or hyperreactive for succinate dehydrogenase) increased with age. Deletions of the mitochondrial genome were observed in 89% of these ETS abnormal fibers. Longitudinal analysis allowed characterization of the ETS abnormal phenotype along their length. A decrease in cross-sectional area in 14% of the ETS abnormal fibers supports the hypothesis that deleted mitochondrial genomes may contribute to age-related fiber atrophy.

Oxidative stress and aging reduce COX I RNA and cytochrome oxidase activity in Drosophila

Drosophila melanogaster displays an age-associated increase in oxidative damage and a decrease in mitochondrial transcripts. To determine if these changes result in energy production deficiencies, we measured the electron transport system (ETS) enzyme activity, and ATP levels with age. No statistically significant influences of age on activities of complexes I and II or citrate synthase were observed. In contrast, from 2 to 45 days post-eclosion, declines were found in complex IV cytochrome c oxidase activity (COX, 40% decline) and ATP abundance (15%), while lipid peroxidation increased 71%. We next examined flies that were either genetically or chemically oxidatively stressed to determine the effect on levels of mitochondrial-encoded cytochrome oxidase I RNA (coxI) and COX activity. A catalase null mutant line had 48% of coxI RNA compared to the wild type. In Cu/Zn superoxide dismutase (cSOD) null flies, the rate of coxI RNA decline was greater than in controls. CoxI RNA also declined with increasing hydrogen peroxide (H2O2) treatment, which was reflected in reduced cytochrome c oxidase (COX) activity. These results show that oxidative stress is closely associated with reductions in mitochondrial transcript levels and support the hypothesis that oxidative stress may contribute to mitochondrial dysfunction and aging in D. melanogaster.