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1.
BMC Genomics ; 23(1): 69, 2022 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-35062879

RESUMO

BACKGROUND: The increasing prevalence and expanding geographical range of the chronic wasting disease (CWD) panzootic in cervids is threatening human, animal, environmental and economic health. The pathogenesis of CWD in cervids is, however, not well understood. We used RNA sequencing (RNA-seq) to compare the brain transcriptome from white-tailed deer (WTD; Odocoileus virginianus) clinically affected with CWD (n = 3) to WTD that tested negative (n = 8) for CWD. In addition, one preclinical CWD+ brain sample was analyzed by RNA-seq. RESULTS: We found 255 genes that were significantly deregulated by CWD, 197 of which were upregulated. There was a high degree of overlap in differentially expressed genes (DEGs) identified when using either/both the reference genome assembly of WTD for mapping sequenced reads to or the better characterized genome assembly of a closely related model species, Bos taurus. Quantitative PCR of a subset of the DEGs confirmed the RNA-seq data. Gene ontology term enrichment analysis found a majority of genes involved in immune activation, consistent with the neuroinflammatory pathogenesis of prion diseases. A metagenomic analysis of the RNA-seq data was conducted to look for the presence of spiroplasma and other bacteria in CWD infected deer brain tissue. CONCLUSIONS: The gene expression changes identified highlight the role of innate immunity in prion infection, potential disease associated biomarkers and potential targets for therapeutic agents. An association between CWD and spiroplasma infection was not found.


Assuntos
Cervos , Príons , Doença de Emaciação Crônica , Animais , Bovinos , Cervos/genética , Humanos , Transcriptoma , Doença de Emaciação Crônica/genética
2.
J Biol Chem ; 295(15): 4985-5001, 2020 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-32111742

RESUMO

Chronic wasting disease (CWD) is caused by an unknown spectrum of prions and has become enzootic in populations of cervid species that express cellular prion protein (PrPC) molecules varying in amino acid composition. These PrPC polymorphisms can affect prion transmission, disease progression, neuropathology, and emergence of new prion strains, but the mechanistic steps in prion evolution are not understood. Here, using conformation-dependent immunoassay, conformation stability assay, and protein-misfolding cyclic amplification, we monitored the conformational and phenotypic characteristics of CWD prions passaged through deer and transgenic mice expressing different cervid PrPC polymorphisms. We observed that transmission through hosts with distinct PrPC sequences diversifies the PrPCWD conformations and causes a shift toward oligomers with defined structural organization, replication rate, and host range. When passaged in host environments that restrict prion replication, distinct co-existing PrPCWD conformers underwent competitive selection, stabilizing a new prion strain. Nonadaptive conformers exhibited unstable replication and accumulated only to low levels. These results suggest a continuously evolving diversity of CWD conformers and imply a critical interplay between CWD prion plasticity and PrPC polymorphisms during prion strain evolution.


Assuntos
Encéfalo/patologia , Adaptação ao Hospedeiro , Polimorfismo Genético , Proteínas PrPC/genética , Doença de Emaciação Crônica/genética , Animais , Encéfalo/metabolismo , Cervos , Camundongos , Camundongos Transgênicos , Doença de Emaciação Crônica/patologia
3.
Aging Clin Exp Res ; 33(7): 1811-1820, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32965609

RESUMO

BACKGROUND: Mitochondrial DNA (mtDNA) deletion mutations lead to electron transport chain-deficient cells and age-induced cell loss in multiple tissues and mammalian species. Accurate quantitation of somatic mtDNA deletion mutations could serve as an index of age-induced cell loss. Quantitation of mtDNA deletion molecules is confounded by their low abundance in tissue homogenates, the diversity of deletion breakpoints, stochastic accumulation in single cells, and mosaic distribution between cells. AIMS: Translate a pre-clinical assay to quantitate mtDNA deletions for use in human DNA samples, with technical and biological validation, and test this assay on human subjects of different ages. METHODS: We developed and validated a high-throughput droplet digital PCR assay that quantitates human mtDNA deletion frequency. RESULTS: Analysis of human quadriceps muscle samples from 14 male subjects demonstrated that mtDNA deletion frequency increases exponentially with age-on average, a 98-fold increase from age 20-80. Sequence analysis of amplification products confirmed the specificity of the assay for human mtDNA deletion breakpoints. Titration of synthetic mutation mixtures found a lower limit of detection of at least 0.6 parts per million. Using muscle DNA from 6-month-old mtDNA mutator mice, we measured a 6.4-fold increase in mtDNA deletion frequency (i.e., compared to wild-type mice), biologically validating the approach. DISCUSSION/CONCLUSIONS: The exponential increase in mtDNA deletion frequency is concomitant with the known muscle fiber loss and accelerating mortality that occurs with age. The improved assay permits the accurate and sensitive quantification of deletion mutations from DNA samples and is sufficient to measure changes in mtDNA deletion mutation frequency in healthy individuals across the lifespan and, therefore, patients with suspected mitochondrial diseases.


Assuntos
DNA Mitocondrial , Músculo Esquelético , Adulto , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/genética , Animais , DNA Mitocondrial/genética , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Mitocôndrias , Fibras Musculares Esqueléticas , Músculo Esquelético/metabolismo , Deleção de Sequência , Adulto Jovem
4.
BMC Vet Res ; 15(1): 50, 2019 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-30717795

RESUMO

BACKGROUND: Chronic wasting disease (CWD) is a prion disease affecting members of the Cervidae family. PrPC primary structures play a key role in CWD susceptibility resulting in extended incubation periods and regulating the propagation of CWD strains. We analyzed the distribution of abnormal prion protein (PrPCWD) aggregates in brain and peripheral organs from orally inoculated white-tailed deer expressing four different PRNP genotypes: Q95G96/Q95G96 (wt/wt), S96/wt, H95/wt and H95/S96 to determine if there are substantial differences in the deposition pattern of PrPCWD between different PRNP genotypes. RESULTS: Although we detected differences in certain brain areas, globally, the different genotypes showed similar PrPCWD deposition patterns in the brain. However, we found that clinically affected deer expressing H95 PrPC, despite having the longest survival periods, presented less PrPCWD immunoreactivity in particular peripheral organs. In addition, no PrPCWD was detected in skeletal muscle of any of the deer. CONCLUSIONS: Our data suggest that expression of H95-PrPC limits peripheral accumulation of PrPCWD as detected by immunohistochemistry. Conversely, infected S96/wt and wt/wt deer presented with similar PrPCWD peripheral distribution at terminal stage of disease, suggesting that the S96-PrPC allele, although delaying CWD progression, does not completely limit the peripheral accumulation of the infectious agent.


Assuntos
Encéfalo/patologia , Cervos , Proteínas Priônicas/genética , Doença de Emaciação Crônica/patologia , Animais , Cerebelo/patologia , Suscetibilidade a Doenças , Lobo Frontal/patologia , Genótipo , Intestinos/patologia , Rim/patologia , Tecido Linfoide/patologia , Músculo Esquelético/patologia , Pâncreas/patologia , Polimorfismo Genético/genética , Doenças Priônicas/patologia , Doenças Priônicas/veterinária , Glândulas Salivares/patologia
5.
Emerg Infect Dis ; 23(9): 1598-1600, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28820384

RESUMO

Human and mouse prion proteins share a structural motif that regulates resistance to common chronic wasting disease (CWD) prion strains. Successful transmission of an emergent strain of CWD prion, H95+, into mice resulted in infection. Thus, emergent CWD prion strains may have higher zoonotic potential than common strains.


Assuntos
Especificidade de Hospedeiro , Príons/química , Doença de Emaciação Crônica/transmissão , Animais , Cricetinae , Cervos , Humanos , Camundongos , Príons/metabolismo , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Estabilidade Proteica , Especificidade da Espécie , Doença de Emaciação Crônica/patologia
6.
J Virol ; 89(24): 12362-73, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26423950

RESUMO

UNLABELLED: Transmission of chronic wasting disease (CWD) between cervids is influenced by the primary structure of the host cellular prion protein (PrP(C)). In white-tailed deer, PRNP alleles encode the polymorphisms Q95 G96 (wild type [wt]), Q95 S96 (referred to as the S96 allele), and H95 G96 (referred to as the H95 allele), which differentially impact CWD progression. We hypothesize that the transmission of CWD prions between deer expressing different allotypes of PrP(C) modifies the contagious agent affecting disease spread. To evaluate the transmission properties of CWD prions derived experimentally from deer of four PRNP genotypes (wt/wt, S96/wt, H95/wt, or H95/S96), transgenic (tg) mice expressing the wt allele (tg33) or S96 allele (tg60) were challenged with these prion agents. Passage of deer CWD prions into tg33 mice resulted in 100% attack rates, with the CWD H95/S96 prions having significantly longer incubation periods. The disease signs and neuropathological and protease-resistant prion protein (PrP-res) profiles in infected tg33 mice were similar between groups, indicating that a prion strain (Wisc-1) common to all CWD inocula was amplified. In contrast, tg60 mice developed prion disease only when inoculated with the H95/wt and H95/S96 CWD allotypes. Serial passage in tg60 mice resulted in adaptation of a novel CWD strain (H95(+)) with distinct biological properties. Transmission of first-passage tg60CWD-H95(+) isolates into tg33 mice, however, elicited two prion disease presentations consistent with a mixture of strains associated with different PrP-res glycotypes. Our data indicate that H95-PRNP heterozygous deer accumulated two CWD strains whose emergence was dictated by the PrP(C) primary structure of the recipient host. These findings suggest that CWD transmission between cervids expressing distinct PrP(C) molecules results in the generation of novel CWD strains. IMPORTANCE: CWD prions are contagious among wild and captive cervids in North America and in South Korea. We present data linking the amino acid variant Q95H in white-tailed deer cellular prion protein (PrP(C)) to the emergence of a novel CWD strain (H95(+)). We show that, upon infection, deer expressing H95-PrP(C) molecules accumulated a mixture of CWD strains that selectively propagated depending on the PRNP genotype of the host in which they were passaged. Our study also demonstrates that mice expressing the deer S96-PRNP allele, previously shown to be resistant to various cervid prions, are susceptible to H95(+) CWD prions. The potential for the generation of novel strains raises the possibility of an expanded host range for CWD.


Assuntos
Genótipo , Proteínas PrPC/genética , Proteínas PrPC/metabolismo , Doença de Emaciação Crônica/genética , Doença de Emaciação Crônica/metabolismo , Animais , Cervos , Camundongos , Camundongos Transgênicos
7.
BMC Genomics ; 16: 682, 2015 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-26341492

RESUMO

BACKGROUND: Prions diseases are fatal neurodegenerative diseases of mammals. While the molecular responses to prion infection have been extensively characterized in the laboratory mouse, little is known in other rodents. To explore these responses and make comparisons, we generated a prion disease in the laboratory rat by successive passage beginning with mouse RML prions. RESULTS: We describe the accumulation of rat prions, associated pathology and the transcriptional impact throughout the disease course. Comparative transcriptional profiling between laboratory mice and rats suggests that similar molecular and cellular processes are unfolding in response to prion infection. At the level of individual transcripts, however, variability exists between mice and rats and many genes deregulated by prion infection in mice are not affected in rats. CONCLUSION: Our findings detail the molecular responses to prion disease in the rat and highlight the usefulness of comparative approaches to understanding neurodegeneration and prion diseases in particular.


Assuntos
Regulação da Expressão Gênica , Doenças Priônicas/genética , Transcriptoma , Animais , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Camundongos , Proteínas PrPC/genética , Proteínas PrPC/metabolismo , Doenças Priônicas/metabolismo , Doenças Priônicas/patologia , Príons/genética , Príons/metabolismo , Ratos
8.
PLoS Pathog ; 9(11): e1003755, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24244171

RESUMO

Prion diseases are driven by the strain-specific, template-dependent transconformation of the normal cellular prion protein (PrP(C)) into a disease specific isoform PrP(Sc). Cell culture models of prion infection generally use replicating cells resulting in lower levels of prion accumulation compared to animals. Using non-replicating cells allows the accumulation of higher levels of PrP(Sc) and, thus, greater amounts of infectivity. Here, we infect non-proliferating muscle fiber myotube cultures prepared from differentiated myoblasts. We demonstrate that prion-infected myotubes generate substantial amounts of PrP(Sc) and that the level of infectivity produced in these post-mitotic cells, 10(5.5) L.D.50/mg of total protein, approaches that observed in vivo. Exposure of the myotubes to different mouse-adapted agents demonstrates strain-specific replication of infectious agents. Mouse-derived myotubes could not be infected with hamster prions suggesting that the species barrier effect is intact. We suggest that non-proliferating myotubes will be a valuable model system for generating infectious prions and for screening compounds for anti-prion activity.


Assuntos
Proliferação de Células , Proteínas PrPSc/metabolismo , Animais , Linhagem Celular , Cricetinae , Camundongos , Fibras Musculares Esqueléticas , Especificidade da Espécie
9.
Pathogens ; 13(2)2024 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-38392876

RESUMO

Chronic wasting disease (CWD) is a prion disease affecting cervid species, both free-ranging and captive populations. As the geographic range continues to expand and disease prevalence continues to increase, CWD will have an impact on cervid populations, local economies, and ecosystem health. Mitigation of this "wicked" disease will require input from many different stakeholders including hunters, landowners, research biologists, wildlife managers, and others, working together. The NC1209 (North American interdisciplinary chronic wasting disease research consortium) is composed of scientists from different disciplines involved with investigating and managing CWD. Leveraging this broad breadth of expertise, the Consortium has created a state-of-the-science review of five key aspects of CWD, including current diagnostic capabilities for detecting prions, requirements for validating these diagnostics, the role of environmental transmission in CWD dynamics, and potential zoonotic risks associated with CWD. The goal of this review is to increase stakeholders', managers', and decision-makers' understanding of this disease informed by current scientific knowledge.

10.
PLoS Pathog ; 7(11): e1002391, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22114562

RESUMO

During prion infections of the central nervous system (CNS) the cellular prion protein, PrP(C), is templated to a conformationally distinct form, PrP(Sc). Recent studies have demonstrated that the Sprn gene encodes a GPI-linked glycoprotein Shadoo (Sho), which localizes to a similar membrane environment as PrP(C) and is reduced in the brains of rodents with terminal prion disease. Here, analyses of prion-infected mice revealed that down-regulation of Sho protein was not related to Sprn mRNA abundance at any stage in prion infection. Down-regulation was robust upon propagation of a variety of prion strains in Prnp(a) and Prnp(b) mice, with the exception of the mouse-adapted BSE strain 301 V. In addition, Sho encoded by a TgSprn transgene was down-regulated to the same extent as endogenous Sho. Reduced Sho levels were not seen in a tauopathy, in chemically induced spongiform degeneration or in transgenic mice expressing the extracellular ADan amyloid peptide of familial Danish dementia. Insofar as prion-infected Prnp hemizygous mice exhibited accumulation of PrP(Sc) and down-regulation of Sho hundreds of days prior to onset of neurologic symptoms, Sho depletion can be excluded as an important trigger for clinical disease or as a simple consequence of neuronal damage. These studies instead define a disease-specific effect, and we hypothesize that membrane-associated Sho comprises a bystander substrate for processes degrading PrP(Sc). Thus, while protease-resistant PrP detected by in vitro digestion allows post mortem diagnosis, decreased levels of endogenous Sho may trace an early response to PrP(Sc) accumulation that operates in the CNS in vivo. This cellular response may offer new insights into the homeostatic mechanisms involved in detection and clearance of the misfolded proteins that drive prion disease pathogenesis.


Assuntos
Encéfalo/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas PrPSc/biossíntese , Doenças Priônicas/metabolismo , Animais , Regulação para Baixo , Proteínas Ligadas por GPI/biossíntese , Proteínas Ligadas por GPI/genética , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/biossíntese , Proteínas PrPC/metabolismo , RNA Mensageiro/metabolismo
11.
Geroscience ; 45(1): 555-567, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36178599

RESUMO

Beta-guanidinopropionic acid (GPA) is a creatine analog suggested as a treatment for hypertension, diabetes, and obesity, which manifest primarily in older adults. A notable side effect of GPA is the induction of mitochondrial DNA deletion mutations. We hypothesized that mtDNA deletions contribute to muscle aging and used the mutation promoting effect of GPA to examine the impact of mtDNA deletions on muscles with differential vulnerability to aging. Rats were treated with GPA for up to 4 months starting at 14 or 30 months of age. We examined quadriceps and adductor longus muscles as the quadriceps exhibits profound age-induced deterioration, while adductor longus is maintained. GPA decreased body and muscle mass and mtDNA copy number while increasing mtDNA deletion frequency. The interactions between age and GPA treatment observed in the quadriceps were not observed in the adductor longus. GPA had negative mitochondrial effects in as little as 4 weeks. GPA treatment exacerbated mtDNA deletions and muscle aging phenotypes in the quadriceps, an age-sensitive muscle, while the adductor longus was spared. GPA has been proposed for use in age-associated diseases, yet the pharmacodynamics of GPA differ with age and include the detrimental induction of mtDNA deletions, a mitochondrial genotoxic stress that is pronounced in muscles that are most vulnerable to aging. Further research is needed to determine if the proposed benefits of GPA on hypertension, diabetes, and obesity outweigh the detrimental mitochondrial and myopathic side effects.


Assuntos
Creatina , Roedores , Ratos , Animais , Músculo Esquelético , DNA Mitocondrial/genética , Obesidade/genética , Dano ao DNA
12.
Aging Cell ; 22(6): e13842, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37132288

RESUMO

Mitochondrial DNA (mtDNA) deletion mutations cause many human diseases and are linked to age-induced mitochondrial dysfunction. Mapping the mutation spectrum and quantifying mtDNA deletion mutation frequency is challenging with next-generation sequencing methods. We hypothesized that long-read sequencing of human mtDNA across the lifespan would detect a broader spectrum of mtDNA rearrangements and provide a more accurate measurement of their frequency. We employed nanopore Cas9-targeted sequencing (nCATS) to map and quantitate mtDNA deletion mutations and develop analyses that are fit-for-purpose. We analyzed total DNA from vastus lateralis muscle in 15 males ranging from 20 to 81 years of age and substantia nigra from three 20-year-old and three 79-year-old men. We found that mtDNA deletion mutations detected by nCATS increased exponentially with age and mapped to a wider region of the mitochondrial genome than previously reported. Using simulated data, we observed that large deletions are often reported as chimeric alignments. To address this, we developed two algorithms for deletion identification which yield consistent deletion mapping and identify both previously reported and novel mtDNA deletion breakpoints. The identified mtDNA deletion frequency measured by nCATS correlates strongly with chronological age and predicts the deletion frequency as measured by digital PCR approaches. In substantia nigra, we observed a similar frequency of age-related mtDNA deletions to those observed in muscle samples, but noted a distinct spectrum of deletion breakpoints. NCATS-mtDNA sequencing allows the identification of mtDNA deletions on a single-molecule level, characterizing the strong relationship between mtDNA deletion frequency and chronological aging.


Assuntos
Sequenciamento por Nanoporos , Masculino , Humanos , Deleção de Sequência/genética , Envelhecimento/genética , Longevidade , DNA Mitocondrial/genética
13.
PLoS One ; 17(10): e0271850, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36288327

RESUMO

Remdesivir is a leading therapy in patients with moderate to severe coronavirus 2 (SARS-CoV-2) infection; the majority of whom are older individuals. Remdesivir is a nucleoside analog that incorporates into nascent viral RNA, inhibiting RNA-directed RNA polymerases, including that of SARS-CoV-2. Less is known about remdesivir's effects on mitochondria, particularly in older adults where mitochondria are known to be dysfunctional. Furthermore, its effect on age-induced mitochondrial mutations and copy number has not been previously studied. We hypothesized that remdesivir adversely affects mtDNA copy number and deletion mutation frequency in aged rodents. To test this hypothesis, 30-month-old male F333BNF1 rats were treated with remdesivir for three months. To determine if remdesivir adversely affects mtDNA, we measured copy number and mtDNA deletion frequency in rat hearts, kidneys, and skeletal muscles using digital PCR. We found no effects from three months of remdesivir treatment on mtDNA copy number or deletion mutation frequency in 33-month-old rats. These data support the notion that remdesivir does not compromise mtDNA quality or quantity at old age in mammals. Future work should focus on examining additional tissues such as brain and liver, and extend testing to human clinical samples.


Assuntos
COVID-19 , DNA Mitocondrial , Animais , Pré-Escolar , Humanos , Masculino , Ratos , Monofosfato de Adenosina/farmacologia , Alanina , Variações do Número de Cópias de DNA , DNA Mitocondrial/genética , RNA Polimerases Dirigidas por DNA/genética , Mamíferos/genética , Mitocôndrias/genética , Nucleosídeos , RNA Viral , SARS-CoV-2 , Deleção de Sequência
14.
Biology (Basel) ; 11(5)2022 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-35625395

RESUMO

Chronic wasting disease (CWD) is a contagious, fatal, neurodegenerative prion disease of cervids. The expanding geographical range and rising prevalence of CWD are increasing the risk of pathogen transfer and spillover of CWD to non-cervid sympatric species. As beavers have close contact with environmental and food sources of CWD infectivity, we hypothesized that they may be susceptible to CWD prions. We evaluated the susceptibility of beavers to prion diseases by challenging transgenic mice expressing beaver prion protein (tgBeaver) with five strains of CWD, four isolates of rodent-adapted prions and one strain of Creutzfeldt-Jakob disease. All CWD strains transmitted to the tgBeaver mice, with attack rates highest from moose CWD and the 116AG and H95+ strains of deer CWD. Mouse-, rat-, and especially hamster-adapted prions were also transmitted with complete attack rates and short incubation periods. We conclude that the beaver prion protein is an excellent substrate for sustaining prion replication and that beavers are at risk for CWD pathogen transfer and spillover.

15.
J Proteome Res ; 10(6): 2687-702, 2011 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-21469646

RESUMO

Mass spectrometry (MS) -- based proteomic approaches have evolved as powerful tools for the discovery of biomarkers. However, the identification of potential protein biomarkers from biofluid samples is challenging because of the limited dynamic range of detection. Currently there is a lack of sensitive and reliable premortem diagnostic test for prion diseases. Here, we describe the use of a combined MS-based approach for biomarker discovery in prion diseases from mouse plasma samples. To overcome the limited dynamic range of detection and sample complexity of plasma samples, we used lectin affinity chromatography and multidimensional separations to enrich and isolate glycoproteins at low abundance. Relative quantitation of a panel of proteins was obtained by a combination of isotopic labeling and validated by spectral counting. Overall 708 proteins were identified, 53 of which showed more than 2-fold increase in concentration whereas 58 exhibited more than 2-fold decrease. A few of the potential candidate markers were previously associated with prion or other neurodegenerative diseases.


Assuntos
Glicoproteínas/sangue , Doenças Priônicas/sangue , Sequência de Aminoácidos , Animais , Biomarcadores/sangue , Proteínas de Transporte/sangue , Cromatografia de Afinidade/métodos , Lectinas/química , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Proteômica , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem
16.
J Toxicol Environ Health A ; 74(2-4): 146-53, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21218343

RESUMO

Global gene expression analysis allows for the identification of transcripts that are differentially regulated during a disease state. Many groups, including our own, have identified hundreds of genes differentially regulated in response to prion infection. Eleven transcripts, upregulated in the brains of prion-infected animals, which were classified in the literature as stimulated by the cytokine interferon-gamma (IFN-γ), were identified. This is intriguing, as IFN-γ has recently been detected in the brains of prion-infected animals. Quantitation of several genes, categorized as IFN-γ inducible, by quantitative real-time polymerase chain reaction (qRT-PCR) confirms that these transcripts are upregulated. Future approaches for delineating the role of IFN-γ-induced transcripts and their function in prion infection are described.


Assuntos
Interferon gama/genética , Doenças Priônicas/genética , Animais , Encéfalo/metabolismo , Interferon gama/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos , Doenças Priônicas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima/genética
17.
Rejuvenation Res ; 24(6): 434-440, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34779265

RESUMO

Metformin, a commonly used well-tolerated treatment for type 2 diabetes, is being deployed in clinical trials to ameliorate aging in older nondiabetic humans. Concerningly, some experiments in model organisms have suggested that metformin use at old ages shortens life span and is toxic to mitochondria. The demonstrated safety of metformin therapy in humans and the conflicting data from model organisms compelled us to test the hypothesis that metformin treatment would be toxic to older rats. To define an effective dose in 30-month-old hybrid rats, we evaluated two doses of metformin (0.1%, 0.75% of the diet) and treated the rats for 4 months. Body mass decreased at the 0.75% dose. Neither dose affected mortality between 30 and 34 months of age. We assessed mitochondrial integrity by measuring mitochondrial DNA (mtDNA) copy number and deletion mutation frequency, and mitochondrial respiration in skeletal muscle and the heart. In skeletal muscle, we observed no effect of metformin on quadriceps mass, mtDNA copy number, or deletion frequency. In the heart, metformin-treated rats had higher mtDNA copy number, lower cardiac mass, with no change in mtDNA deletion frequency. Metformin treatment resulted in lower mitochondrial complex I-dependent respiration in the heart. We found that, in old rats, metformin did not compromise mtDNA integrity, did not affect mortality, and may have cardiac benefits. These data provide some reassurance that a metformin intervention in aged mammals is not toxic at appropriate doses.


Assuntos
Diabetes Mellitus Tipo 2 , Metformina , Envelhecimento , Animais , DNA Mitocondrial/genética , Diabetes Mellitus Tipo 2/tratamento farmacológico , Metformina/farmacologia , Mitocôndrias , Ratos
18.
Geroscience ; 43(3): 1253-1264, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33740224

RESUMO

Mitochondrial DNA (mtDNA) quality and quantity relate to two hallmarks of aging-genomic instability and mitochondrial dysfunction. Physical performance relies on mitochondrial integrity and declines with age, yet the interactions between mtDNA quantity, quality, and physical performance are unclear. Using a validated digital PCR assay specific for mtDNA deletions, we tested the hypothesis that skeletal muscle mtDNA deletion mutation frequency (i.e., a measure of mtDNA quality) or mtDNA copy number predicts physical performance in older adults. Total DNA was isolated from vastus lateralis muscle biopsies and used to quantitate mtDNA copy number and mtDNA deletion frequency by digital PCR. The biopsies were obtained from a cross-sectional cohort of 53 adults aged 50 to 86 years. Before the biopsy procedure, physical performance measurements were collected, including VO2max, modified physical performance test score, 6-min walk distance, gait speed, grip strength, and total lean and leg mass. Linear regression models were used to evaluate the relationships between age, sex, and the outcomes. We found that mtDNA deletion mutation frequency increased exponentially with advancing age. On average from ages 50 to 86, deletion frequency increased from 0.008 to 0.15%, an 18-fold increase. Females may have lower deletion frequencies than males at older ages. We also measured declines in VO2max and mtDNA copy number with age in both sexes. The mtDNA deletion frequency measured from single skeletal muscle biopsies predicted 13.3% of the variation in VO2max. Copy number explained 22.6% of the variation in mtDNA deletion frequency and 10.4% of the lean mass variation. We found predictive relationships between age, mtDNA deletion mutation frequency, mtDNA copy number, and physical performance. These data are consistent with a role for mitochondrial function and genome integrity in maintaining physical performance with age. Analyses of mtDNA quality and quantity in larger cohorts and longitudinal studies could extend our understanding of the importance of mitochondrial DNA in human aging and longevity.


Assuntos
Variações do Número de Cópias de DNA , DNA Mitocondrial , Idoso , Idoso de 80 Anos ou mais , Estudos Transversais , Variações do Número de Cópias de DNA/genética , DNA Mitocondrial/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mitocôndrias , Músculo Esquelético/metabolismo , Desempenho Físico Funcional , Deleção de Sequência/genética
19.
Cells ; 9(8)2020 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-32751058

RESUMO

"The main conclusions are that the ageing atrophy begins as early as around 25 years of age and thereafter accelerates and, for this muscle, is caused mainly by a loss of fibers and to a lesser extent by a reduction in fiber size [...].


Assuntos
Mitocôndrias , Junção Neuromuscular , Senescência Celular , Músculos , Junção Neuromuscular/metabolismo
20.
J Cytol Histol ; 11(2)2020.
Artigo em Inglês | MEDLINE | ID: mdl-32566369

RESUMO

Human muscle biopsies are increasingly important for diagnosis, research, and to monitor therapeutic trials. We examined the use of a self-contained, vacuum-assisted biopsy system and a novel muscle freezing technique to improve, simplify, and standardize human muscle biopsy collection and cryopreservation in older adults. The VACORA vacuum-assisted biopsy system was deployed in muscle biopsies of 12 individuals ranging in age from 57 to 80 years. This office-based approach was well tolerated as it is minimally invasive, uses only local anesthetic, and has a quick recovery. To maximize biopsy sample quality and reproducibility, we developed a novel muscle sample freezing protocol. Fresh muscle biopsy samples were placed into readily available tissue cassettes followed by direct freezing in liquid nitrogen. After this modified snap freezing protocol, frozen muscle samples were enrobed in embedding medium for cryosectioning. We examined the effect of this freezing approach in histological sections of rodent and human muscle samples. The VACORA Biopsy System provided as many as four skeletal muscle core samples from a single biopsy site. Biopsy samples from 12 older adults weighed an average of 147.5 ± 11 mg each and had a consistent size and shape. There were no complications, and the residual scar is less than 10 mm. The freezing method using standard tissue cassettes with direct freezing in liquid nitrogen yielded high quality cryopreserved muscle tissue suitable for histological analysis without the need for isopentane and with little to no freeze-thaw damage. These enhancements have streamlined and improved the consistency of our muscle biopsy protocol and provide sufficient high-quality sample for multi-dimensional downstream studies of human muscle in aging and disease.

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