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1.
PLoS Pathog ; 17(3): e1009324, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33735218

RESUMO

The development of safe subunit vaccines requires adjuvants that augment immunogenicity of non-replicating protein-based antigens. Current vaccines against infectious diseases preferentially induce protective antibodies driven by adjuvants such as alum. However, the contribution of antibody to host defense is limited for certain classes of infectious diseases such as fungi, whereas animal studies and clinical observations implicate cellular immunity as an essential component of the resolution of fungal pathogens. Here, we decipher the structural bases of a newly identified glycoprotein ligand of Dectin-2 with potent adjuvancy, Blastomyces endoglucanase-2 (Bl-Eng2). We also pinpoint the developmental steps of antigen-specific CD4+ and CD8+ T responses augmented by Bl-Eng2 including expansion, differentiation and tissue residency. Dectin-2 ligation led to successful systemic and mucosal vaccination against invasive fungal infection and Influenza A infection, respectively. O-linked glycans on Bl-Eng2 applied at the skin and respiratory mucosa greatly augment vaccine subunit- induced protective immunity against lethal influenza and fungal pulmonary challenge.


Assuntos
Anticorpos Antivirais/imunologia , Blastomyces/imunologia , Vacinas Fúngicas/imunologia , Infecções por Orthomyxoviridae/imunologia , Adjuvantes Imunológicos , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Celulase/imunologia , Vacinas contra Influenza/imunologia
2.
Curr Protoc Microbiol ; 59(1): e132, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33315302

RESUMO

Dimorphic fungi in the genera Blastomyces, Histoplasma, Coccidioides, and Paracoccidioides are important human pathogens that affect human health in many countries throughout the world. Understanding the biology of these fungi is important for the development of effective treatments and vaccines. Gene editing is a critically important tool for research into these organisms. In recent years, gene targeting approaches employing RNA-guided DNA nucleases, such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9), have exploded in popularity. Here, we provide a detailed description of the steps involved in applying CRISPR/Cas9 technology to dimorphic fungi, with Blastomyces dermatitidis in particular as our model fungal pathogen. We discuss the design and construction of single guide RNA and Cas9-expressing targeting vectors (including multiplexed vectors) as well as introduction of these plasmids into Blastomyces using Agrobacterium-mediated transformation. Finally, we cover the outcomes that may be expected in terms of gene-editing efficiency and types of gene alterations produced. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Construction of CRISPR/Cas9 targeting vectors Support Protocol 1: Choosing protospacers in the target gene Basic Protocol 2: Agrobacterium-mediated transformation of Blastomyces Support Protocol 2: Preparation of electrocompetent Agrobacterium Support Protocol 3: Preparation and recovery of Blastomyces frozen stocks.


Assuntos
Sistemas CRISPR-Cas , Fungos/genética , Edição de Genes/métodos , Agrobacterium , Sequência de Bases , Blastomyces/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Técnicas de Cocultura , Marcação de Genes/métodos , Técnicas Microbiológicas/métodos , Reação em Cadeia da Polimerase , RNA Guia de Cinetoplastídeos/genética
3.
Front Genet ; 11: 519206, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33193604

RESUMO

The retrotransposon long interspersed nuclear element-1 (LINE-1) can autonomously increase its copy number within a host genome through the retrotransposition process. LINE-1 is active in the germline and in neural progenitor cells, and its somatic retrotransposition activity has a broad impact on neural development and susceptibility to neuropsychiatric disorders. The method to quantify the genomic copy number of LINE-1 would be important in unraveling the role of retrotransposition, especially in the brain. However, because of the species-specific evolution of LINE-1 sequences, methods for quantifying the copy number should be independently developed. Here, we developed a quantitative PCR (qPCR) assay to measure the copy number of active LINE-1 subfamilies in mice. Using the assay, we investigated aging-associated alterations of LINE-1 copy number in several brain regions in wild-type mice and Polg+/D257A mice as a model for accelerated aging. We found that aged Polg+/D257A mice showed higher levels of the type GfII LINE-1 in the basal ganglia than the wild-type mice did, highlighting the importance of assays that focus on an individual active LINE-1 subfamily.

4.
Cell Host Microbe ; 27(4): 614-628.e6, 2020 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-32130954

RESUMO

Airway epithelium is the first body surface to contact inhaled irritants and report danger. Here, we report how epithelial cells recognize and respond to aeroallergen alkaline protease 1 (Alp1) of Aspergillus sp., because proteases are critical components of many allergens that provoke asthma. In a murine model, Alp1 elicits helper T (Th) cell-dependent lung eosinophilia that is initiated by the rapid response of bronchiolar club cells to Alp1. Alp1 damages bronchiolar cell junctions, which triggers a calcium flux signaled through calcineurin within club cells of the bronchioles, inciting inflammation. In two human cohorts, we link fungal sensitization and/or asthma with SNP/protein expression of the mechanosensitive calcium channel, TRPV4. TRPV4 is also necessary and sufficient for club cells to sensitize mice to Alp1. Thus, club cells detect junction damage as mechanical stress, which signals danger via TRPV4, calcium, and calcineurin to initiate allergic sensitization.


Assuntos
Aspergillus fumigatus/metabolismo , Asma/etiologia , Serina Endopeptidases/metabolismo , Canais de Cátion TRPV/metabolismo , Alérgenos/efeitos adversos , Alérgenos/metabolismo , Animais , Aspergillus fumigatus/imunologia , Bronquíolos/citologia , Calcineurina/metabolismo , Canais de Cálcio/metabolismo , Sinalização do Cálcio , Estudos de Coortes , Eosinofilia , Células Epiteliais/metabolismo , Humanos , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Camundongos Endogâmicos C57BL , Serina Endopeptidases/efeitos adversos , Linfócitos T/imunologia
5.
Mucosal Immunol ; 13(3): 518-529, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31900406

RESUMO

Priming at the site of natural infection typically elicits a protective T cell response against subsequent pathogen encounter. Here, we report the identification of a novel fungal antigen that we harnessed for mucosal vaccination and tetramer generation to test whether we can elicit protective, antigen-specific tissue-resident memory (Trm) CD4+ T cells in the lung parenchyma. In contrast to expectations, CD69+, CXCR3+, CD103- Trm cells failed to protect against a lethal pulmonary fungal infection. Surprisingly, systemic vaccination induced a population of tetramer+ CD4+ T cells enriched within the pulmonary vasculature, and expressing CXCR3 and CX3CR1, that migrated to the lung tissue upon challenge and efficiently protected mice against infection. Mucosal vaccine priming of Trm may not reliably protect against mucosal pathogens.


Assuntos
Antígenos/imunologia , Movimento Celular/imunologia , Resistência à Doença/imunologia , Fungos/imunologia , Interações Hospedeiro-Patógeno/imunologia , Memória Imunológica , Micoses/imunologia , Animais , Biomarcadores , Epitopos de Linfócito T/imunologia , Imunização , Imunofenotipagem , Interferon gama , Camundongos , Micoses/microbiologia , Micoses/prevenção & controle , Receptores CXCR3/metabolismo , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Vacinas/imunologia
6.
Sci Rep ; 10(1): 1296, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31992827

RESUMO

Mitochondrial mutations and dysfunction have been demonstrated in several age-related disorders including osteoarthritis, yet its relative contribution to pathogenesis remains unknown. Here we evaluated whether premature aging caused by accumulation of mitochondrial DNA mutations in PolgD275A mice predisposes to the development of knee osteoarthritis. Compared with wild type animals, homozygous PolgD275A mice displayed a specific bone phenotype characterized by osteopenia of epiphyseal trabecular bone and subchondral cortical plate. Trabecular thickness was significantly associated with osteocyte apoptosis rates and osteoclasts numbers were increased in subchondral bone tissues. While chondrocyte apoptosis rates in articular and growth plate cartilage were similar between groups, homozygous mitochondrial DNA mutator mice displayed elevated numbers of hypertrophic chondrocytes in articular calcified cartilage. Low grade cartilage degeneration, predominantly loss of proteoglycans, was present in all genotypes and the development of osteoarthritis features was not found accelerated in premature aging. Somatically acquired mitochondrial DNA mutations predispose to elevated subchondral bone turnover and hypertrophy in calcified cartilage, yet additional mechanical or metabolic stimuli would seem required for induction and accelerated progression of aging-associated osteoarthritis.


Assuntos
Senilidade Prematura , Doenças Ósseas Metabólicas , Condrócitos , DNA Polimerase gama , Mutação de Sentido Incorreto , Osteoartrite , Senilidade Prematura/enzimologia , Senilidade Prematura/genética , Senilidade Prematura/patologia , Substituição de Aminoácidos , Animais , Doenças Ósseas Metabólicas/enzimologia , Doenças Ósseas Metabólicas/genética , Doenças Ósseas Metabólicas/patologia , Condrócitos/enzimologia , Condrócitos/patologia , DNA Polimerase gama/genética , DNA Polimerase gama/metabolismo , Hipertrofia , Camundongos , Camundongos Mutantes , Osteoartrite/enzimologia , Osteoartrite/genética , Osteoartrite/patologia
7.
mBio ; 9(2)2018 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-29615501

RESUMO

Blastomyces dermatitidis is a human fungal pathogen of the lung that can lead to disseminated disease in healthy and immunocompromised individuals. Genetic analysis of this fungus is hampered by the relative inefficiency of traditional recombination-based gene-targeting approaches. Here, we demonstrate the feasibility of applying CRISPR/Cas9-mediated gene editing to Blastomyces, including to simultaneously target multiple genes. We created targeting plasmid vectors expressing Cas9 and either one or two single guide RNAs and introduced these plasmids into Blastomyces via Agrobacterium gene transfer. We succeeded in disrupting several fungal genes, including PRA1 and ZRT1, which are involved in scavenging and uptake of zinc from the extracellular environment. Single-gene-targeting efficiencies varied by locus (median, 60% across four loci) but were approximately 100-fold greater than traditional methods of Blastomyces gene disruption. Simultaneous dual-gene targeting proceeded with efficiencies similar to those of single-gene-targeting frequencies for the respective targets. CRISPR/Cas9 disruption of PRA1 or ZRT1 had a variable impact on growth under zinc-limiting conditions, showing reduced growth at early time points in low-passage-number cultures and growth similar to wild-type levels by later passage. Individual impairment of PRA1 or ZRT1 resulted in a reduction of the fungal burden in a mouse model of Blastomyces infection by a factor of ~1 log (range, up to 3 logs), and combined disruption of both genes had no additional impact on the fungal burden. These results underscore the utility of CRISPR/Cas9 for efficient gene disruption in dimorphic fungi and reveal a role for zinc metabolism in Blastomyces fitness in vivoIMPORTANCEBlastomyces is a human fungal pathogen that can cause serious, even fatal, lung infections. Genetic analysis of this fungus is possible but inefficient. We applied a recently developed gene editing technology, CRISPR/Cas9, to dramatically improve the efficiency with which gene disruptions are introduced into Blastomyces We used this system to disrupt genes involved in zinc uptake and found that this reduced the fitness of the fungus upon infection.


Assuntos
Blastomyces/crescimento & desenvolvimento , Blastomyces/metabolismo , Edição de Genes/métodos , Aptidão Genética , Zinco/metabolismo , Animais , Blastomyces/genética , Blastomicose/microbiologia , Proteína 9 Associada à CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Contagem de Colônia Microbiana , Modelos Animais de Doenças , Redes e Vias Metabólicas/genética , Camundongos , RNA Guia de Cinetoplastídeos/metabolismo
8.
PLoS One ; 12(2): e0171159, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28158260

RESUMO

Mitochondrial DNA (mtDNA) mutations are thought to have a causative role in age-related pathologies. We have shown previously that mitochondrial mutator mice (PolgD257A/D257A), harboring a proofreading-deficient version of the mtDNA polymerase gamma (POLG), accumulate mtDNA mutations in multiple tissues and display several features of accelerated aging. Calorie restriction (CR) is known to delay the onset of age-related diseases and to extend the lifespan of a variety of species, including rodents. In the current study we investigated the effects of CR on the lifespan and healthspan of mitochondrial mutator mice. Long-term CR did not increase the median or maximum lifespan of PolgD257A/D257A mice. Furthermore, CR did not reduce mtDNA deletions in the heart and muscle, accelerated sarcopenia, testicular atrophy, nor improve the alterations in cardiac parameters that are present in aged mitochondrial mutator mice. Therefore, our findings suggest that accumulation of mtDNA mutations may interfere with the beneficial action of CR in aging retardation.


Assuntos
Restrição Calórica , DNA Mitocondrial/genética , DNA Polimerase Dirigida por DNA/metabolismo , Animais , Peso Corporal/genética , Peso Corporal/fisiologia , DNA Polimerase Dirigida por DNA/genética , Ecocardiografia , Estimativa de Kaplan-Meier , Masculino , Camundongos , Músculo Esquelético/metabolismo , Mutação/genética , Tamanho do Órgão/genética , Tamanho do Órgão/fisiologia , Testículo/metabolismo
9.
Turk Neurosurg ; 26(4): 500-12, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27400095

RESUMO

AIM: We have applied diffusion tensor imaging (DTI) to interrogate microstructural changes in white matter integrity in a widely used middle cerebral artery occlusion (MCAO) model of cerebral ischemia. MATERIAL AND METHODS: We performed ex vivo DTI 35 days after 60 minutes transient focal ischemia in male spontaneously hypertensive rats and generated fractional anisotropy (FA), mean, axial and radial diffusivity maps. Regions of interest corresponding to external capsule (EC), corpus callosum (CC) and internal capsule (IC) were compared among sham and stroked rats. We compared tractographic projections of white matter fiber patterns and examined white matter integrity by Luxol fast blue histological analysis. We also determined infarct lesion volumes at 24 hours post-ischemia by T2-weighted magnetic resonance imaging (MRI) or at 35 days by histological staining with cresyl violet. RESULTS: We found alterations in EC and IC, but not CC, as represented by decreased FA and increased mean, axial and radial diffusivities. The size of the ischemic lesion detected subacutely by T2-weighted MRI or at 35 days by histological staining correlated with the decline in FA in the affected structures. Tractography revealed disruption of fiber trajectories through the EC and reorientation of fibers within the caudate/putamen of rats subjected to MCAO. Similarly, loss of white matter integrity in the EC and increased white matter density in the caudate/putamen along the infarct border zone was evidenced by Luxol fast blue staining. CONCLUSION: Diffusion tensor imaging therefore allows for monitoring of white matter injury and reorganization in hypertensive rats.


Assuntos
Encéfalo/diagnóstico por imagem , Corpo Caloso/diagnóstico por imagem , Imagem de Tensor de Difusão/métodos , Infarto da Artéria Cerebral Média/diagnóstico por imagem , Acidente Vascular Cerebral/diagnóstico por imagem , Animais , Anisotropia , Encéfalo/patologia , Corpo Caloso/patologia , Modelos Animais de Doenças , Infarto da Artéria Cerebral Média/patologia , Masculino , Ratos , Ratos Endogâmicos SHR , Acidente Vascular Cerebral/patologia
10.
Ann Clin Transl Neurol ; 1(11): 909-20, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25540805

RESUMO

OBJECTIVE: Mutations in nuclear-encoded mitochondrial DNA (mtDNA) polymerase (POLG) are known to cause autosomal dominant chronic progressive external ophthalmoplegia (adCPEO) with accumulation of multiple mtDNA deletions in muscles. However, no animal model with a heterozygous Polg mutation representing mtDNA impairment and symptoms of CPEO has been established. To understand the pathogenic mechanism of CPEO, it is important to determine the age dependency and tissue specificity of mtDNA impairment resulting from a heterozygous mutation in the Polg gene in an animal model. METHODS: We assessed behavioral phenotypes, tissue-specific accumulation of mtDNA deletions, and its age dependency in heterozygous Polg (D257A) knock-in mice carrying a proofreading-deficient mutation in the Polg. RESULTS: Heterozygous Polg (D257A) knock-in mice exhibited motor dysfunction in a rotarod test. Polg (+/D257A) mice had significant accumulation of multiple mtDNA deletions, but did not show significant accumulation of point mutations or mtDNA depletion in the brain. While mtDNA deletions increased in an age-dependent manner regardless of the tissue even in Polg (+/+) mice, the age-dependent accumulation of mtDNA deletions was enhanced in muscles and in the brain of Polg (+/D257A) mice. INTERPRETATION: Heterozygous Polg (D257A) knock-in mice showed tissue-specific, age-dependent accumulation of multiple mtDNA deletions in muscles and the brain which was likely to result in neuromuscular symptoms. Polg (+/D257A) mice may be used as an animal model of adCPEO associated with impaired mtDNA maintenance.

11.
Neurotoxicol Teratol ; 46: 62-7, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25450660

RESUMO

Mitochondrial DNA (mtDNA) mutations are hypothesized to play a pathogenic role in aging and age-related neurodegenerative diseases such as Parkinson's disease (PD). In support of this, high levels of somatic mtDNA mutations in "POLG mutator" mice carrying a proofreading-deficient form of mtDNA polymerase ã (Polg(D257A)) lead to a premature aging phenotype. However, the relevance of this finding to the normal aging process has been questioned as the number of mutations is greater even in young POLG mutator mice, which shows no overt phenotype, than levels achieved during normal aging in mice. Vulnerability of dopaminergic neurons to 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) increases with age, and we hypothesized that this may result in part from the accumulation with age of somatic mtDNA mutations. If correct, then levels of mutations in young (2­3 month old) POLG mutator mice should be sufficient to increase vulnerability to MPTP. In contrast, we find that susceptibility to MPTP in both heterozygous and homozygous POLG mutator mice at this young age is not different from that of wild type littermate controls as measured by levels of tyrosine hydroxylase positive (TH+) striatal terminals, striatal dopamine and its metabolites, a marker of oxidative damage, or stereological counts of TH+ and total substantia nigra neurons. These unexpected results do not support the hypothesis that somatic mtDNA mutations contribute to the age-related vulnerability of dopaminergic neurons to MPTP. It remains possible that somatic mtDNA mutations influence vulnerability to other stressors, or require additional time for the deleterious consequences to manifest. Furthermore, the impact of the higher levels of mutations present at older ages in these mice was not assessed in our study, although a prior study also failed to detect an increase in vulnerability to MPTP in older mice. With these caveats, the current data do not provide evidence for a role of somatic mtDNA mutations in determining the vulnerability to MPTP.


Assuntos
Envelhecimento/efeitos dos fármacos , DNA Mitocondrial/efeitos dos fármacos , DNA Polimerase Dirigida por DNA/genética , Intoxicação por MPTP , Mutação , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Animais , Contagem de Células , Corpo Estriado/química , Corpo Estriado/efeitos dos fármacos , DNA Polimerase gama , DNA Mitocondrial/genética , Dopamina/análise , Dopamina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Estresse Oxidativo/efeitos dos fármacos , Substância Negra/efeitos dos fármacos , Tirosina 3-Mono-Oxigenase/análise
12.
Mol Neurodegener ; 9: 16, 2014 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-24885175

RESUMO

BACKGROUND: The role of mitochondrial dysfunction has long been implicated in age-related brain pathology, including Alzheimer's disease (AD). However, the mechanism by which mitochondrial dysfunction may cause neurodegeneration in AD is unclear. To model mitochondrial dysfunction in vivo, we utilized mice that harbor a knockin mutation that inactivates the proofreading function of mitochondrial DNA polymerase γ (PolgA D257A), so that these mice accumulate mitochondrial DNA mutations with age. PolgA D257A mice develop a myriad of mitochondrial bioenergetic defects and physical phenotypes that mimic premature ageing, with subsequent death around one year of age. RESULTS: We crossed the D257A mice with a well-established transgenic AD mouse model (APP/Ld) that develops amyloid plaques. We hypothesized that mitochondrial dysfunction would affect Aß synthesis and/or clearance, thus contributing to amyloidogenesis and triggering neurodegeneration. Initially, we discovered that Aß42 levels along with Aß42 plaque density were increased in D257A; APP/Ld bigenic mice compared to APP/Ld monogenic mice. Elevated Aß production was not responsible for increased amyloid pathology, as levels of BACE1, PS1, C99, and C83 were unchanged in D257A; APP/Ld compared to APP/Ld mice. However, the levels of a major Aß clearance enzyme, insulin degrading enzyme (IDE), were reduced in mice with the D257A mutation, suggesting this as mechanism for increased amyloid load. In the presence of the APP transgene, D257A mice also exhibited significant brain atrophy with apparent cortical thinning but no frank neuron loss. D257A; APP/Ld mice had increased levels of 17 kDa cleaved caspase-3 and p25, both indicative of neurodegeneration. Moreover, D257A; APP/Ld neurons appeared morphologically disrupted, with swollen and vacuolated nuclei. CONCLUSIONS: Overall, our results implicate synergism between the effects of the PolgA D257A mutation and Aß in causing neurodegeneration. These findings provide insight into mechanisms of mitochondrial dysfunction that may contribute to the pathogenesis of AD via decreased clearance of Aß.


Assuntos
Envelhecimento/genética , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Encéfalo/patologia , DNA Mitocondrial/genética , Mutação , Envelhecimento/patologia , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Atrofia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Técnicas de Introdução de Genes , Humanos , Immunoblotting , Camundongos , Camundongos Transgênicos , Placa Amiloide/patologia
13.
Neurosurg Focus ; 36(1 Suppl): 1, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24380520

RESUMO

The retrosigmoid approach is a work-horse approach to the cerebellopontine angle (CPA), providing access from the foremen magnum to the tentorium. Indications for this approach are variable such as resection of meningiomas, acoustic neuromas and epidermoid tumors, treatment of vascular lesions of vertebrobasilar system, vascular decompression of cranial nerves (V, VII, IX, X), cranial nerve neurectomies, and intrinsic lesions of the cerebellum and brainstem. In this video, we demonstrate the use of retrosigmoid craniotomy for resection of a large CPA meningioma, delineating all steps including positioning, mapping. The video can be found here: http://youtu.be/kISkYS16Brk .


Assuntos
Neoplasias Cerebelares/cirurgia , Ângulo Cerebelopontino/cirurgia , Craniotomia/métodos , Neoplasias Meníngeas/cirurgia , Meningioma/cirurgia , Idoso , Neoplasias Cerebelares/diagnóstico , Feminino , Humanos , Neoplasias Meníngeas/diagnóstico , Meningioma/diagnóstico , Resultado do Tratamento
14.
PLoS One ; 8(7): e69327, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23935986

RESUMO

Mitochondrial DNA (mtDNA) mutations lead to decrements in mitochondrial function and accelerated rates of these mutations has been linked to skeletal muscle loss (sarcopenia). The purpose of this study was to investigate the effect of mtDNA mutations on mitochondrial quality control processes in skeletal muscle from animals (young; 3-6 months and older; 8-15 months) expressing a proofreading-deficient version of mtDNA polymerase gamma (PolG). This progeroid aging model exhibits elevated mtDNA mutation rates, mitochondrial dysfunction, and a premature aging phenotype that includes sarcopenia. We found increased expression of the mitochondrial biogenesis regulator peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) and its target proteins, nuclear respiratory factor 1 (NRF-1) and mitochondrial transcription factor A (Tfam) in PolG animals compared to wild-type (WT) (P<0.05). Muscle from older PolG animals displayed higher mitochondrial fission protein 1 (Fis1) concurrent with greater induction of autophagy, as indicated by changes in Atg5 and p62 protein content (P<0.05). Additionally, levels of the Tom22 import protein were higher in PolG animals when compared to WT (P<0.05). In contrast, muscle from normally-aged animals exhibited a distinctly different expression profile compared to PolG animals. Older WT animals appeared to have higher fusion (greater Mfn1/Mfn2, and lower Fis1) and lower autophagy (Beclin-1 and p62) compared to young WT suggesting that autophagy is impaired in aging muscle. In conclusion, muscle from mtDNA mutator mice display higher mitochondrial fission and autophagy levels that likely contribute to the sarcopenic phenotype observed in premature aging and this differs from the response observed in normally-aged muscle.


Assuntos
Senilidade Prematura/patologia , Mitocôndrias/metabolismo , Sarcopenia/patologia , Senilidade Prematura/metabolismo , Animais , Autofagia , DNA Polimerase gama , DNA Mitocondrial/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Modelos Animais de Doenças , Camundongos , Dinâmica Mitocondrial , Proteínas Mitocondriais/metabolismo , Músculos/enzimologia , Músculos/patologia , Transporte Proteico , Sarcopenia/metabolismo , Regulação para Cima
15.
Mitochondrion ; 13(4): 282-91, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23542163

RESUMO

The mitochondrial DNA (mtDNA) polymerase γ (POLG) mutator mice provide the first experimental evidence that high levels of somatic mtDNA mutations can be functionally significant. Here we report that older homozygous, but not heterozygous, POLG mice show significant reductions in striatal dopaminergic terminals as well as deficits in motor function. However, resting oxygen consumption, heat production, mtDNA content and mitochondrial electron transport chain activities are significantly decreased at older ages in both homozygous and heterozygous mice. These results indicate that high levels of somatic mtDNA mutations can contribute to dopaminergic dysfunction and to behavioral and metabolic deficits.


Assuntos
DNA Mitocondrial/metabolismo , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Mutação , Animais , DNA Polimerase gama , Transtornos Neurológicos da Marcha , Heterozigoto , Homozigoto , Temperatura Alta , Camundongos , Consumo de Oxigênio , Córtex Visual/patologia
16.
Am J Physiol Gastrointest Liver Physiol ; 302(9): G914-24, 2012 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-22345551

RESUMO

Changes in intestinal absorption of nutrients are important aspects of the aging process. To address this issue, we investigated the impact of accelerated mitochondrial DNA mutations on the stem/progenitor cells in the crypts of Lieberkühn in mice homozygous for a mitochondrial DNA polymerase gamma mutation, Polg(D257A), that exhibit accelerated aging phenotype. As early as 3-7 mo of age, the small intestine was significantly enlarged in the PolgD257A mice. The crypts of the PolgD257A mice contained 20% more cells than those of their wild-type littermates and exhibited a 10-fold increase in cellular apoptosis primarily in the stem/progenitor cell zones. Actively dividing cells were proportionally increased, yet a significantly smaller proportion of cells was in the S phase of the cell cycle. Stem cell-derived organoids from PolgD257A mice failed to develop fully in culture and exhibited fewer crypt units, indicating an impact of the mutation on the intestinal epithelial stem/progenitor cell maintenance. In addition, epithelial cell migration along the crypt-villus axis was slowed and less organized, and the ATP content in the villi was significantly reduced. On a high-fat, high-carbohydrate diet, PolgD257A mice showed significantly restricted absorption of excess lipids accompanied by an increase in fecal steatocrits. We conclude that the PolgD257A mutation causes cell cycle dysregulation in the crypts leading to the age-associated changes in the morphology of the small intestine and contributes to the restricted absorption of dietary lipids.


Assuntos
Ciclo Celular/genética , DNA Polimerase Dirigida por DNA/genética , Gorduras na Dieta/metabolismo , Absorção Intestinal/genética , Mutação/genética , Células-Tronco/citologia , Células-Tronco/fisiologia , Animais , Contagem de Células , DNA Polimerase gama , Absorção Intestinal/fisiologia , Masculino , Camundongos , Camundongos Transgênicos , Edição de RNA/genética
17.
Proc Natl Acad Sci U S A ; 108(21): 8779-84, 2011 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-21555558

RESUMO

Diabetes and the development of its complications have been associated with mitochondrial DNA (mtDNA) dysfunction, but causal relationships remain undetermined. With the objective of testing whether increased mtDNA mutations exacerbate the diabetic phenotype, we have compared mice heterozygous for the Akita diabetogenic mutation (Akita) with mice homozygous for the D257A mutation in mitochondrial DNA polymerase gamma (Polg) or with mice having both mutations (Polg-Akita). The Polg-D257A protein is defective in proofreading and increases mtDNA mutations. At 3 mo of age, the Polg-Akita and Akita male mice were equally hyperglycemic. Unexpectedly, as the Polg-Akita males aged to 9 mo, their diabetic symptoms decreased. Thus, their hyperglycemia, hyperphagia and urine output declined significantly. The decrease in their food intake was accompanied by increased plasma leptin and decreased plasma ghrelin, while hypothalamic expression of the orexic gene, neuropeptide Y, was lower and expression of the anorexic gene, proopiomelanocortin, was higher. Testis function progressively worsened with age in the double mutants, and plasma testosterone levels in 9-mo-old Polg-Akita males were significantly reduced compared with Akita males. The hyperglycemia and hyperphagia returned in aged Polg-Akita males after testosterone administration. Hyperglycemia-associated distal tubular damage in the kidney also returned, and Polg-D257A-associated proximal tubular damage was enhanced. The mild diabetes of female Akita mice was not affected by the Polg-D257A mutation. We conclude that reduced diabetic symptoms of aging Polg-Akita males results from appetite suppression triggered by decreased testosterone associated with damage to the Leydig cells of the testis.


Assuntos
Apetite/genética , DNA Mitocondrial/genética , DNA Polimerase Dirigida por DNA/genética , Diabetes Mellitus/genética , Camundongos Endogâmicos/genética , Mutação , Envelhecimento , Animais , DNA Polimerase gama , Diabetes Mellitus/patologia , Diabetes Mellitus/fisiopatologia , Feminino , Hiperglicemia , Células Intersticiais do Testículo/patologia , Masculino , Camundongos , Fenótipo , Edição de RNA/genética , Testículo/patologia , Testosterona/sangue
18.
Proc Natl Acad Sci U S A ; 108(10): 4135-40, 2011 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-21368114

RESUMO

A causal role for mitochondrial DNA (mtDNA) mutagenesis in mammalian aging is supported by recent studies demonstrating that the mtDNA mutator mouse, harboring a defect in the proofreading-exonuclease activity of mitochondrial polymerase gamma, exhibits accelerated aging phenotypes characteristic of human aging, systemic mitochondrial dysfunction, multisystem pathology, and reduced lifespan. Epidemiologic studies in humans have demonstrated that endurance training reduces the risk of chronic diseases and extends life expectancy. Whether endurance exercise can attenuate the cumulative systemic decline observed in aging remains elusive. Here we show that 5 mo of endurance exercise induced systemic mitochondrial biogenesis, prevented mtDNA depletion and mutations, increased mitochondrial oxidative capacity and respiratory chain assembly, restored mitochondrial morphology, and blunted pathological levels of apoptosis in multiple tissues of mtDNA mutator mice. These adaptations conferred complete phenotypic protection, reduced multisystem pathology, and prevented premature mortality in these mice. The systemic mitochondrial rejuvenation through endurance exercise promises to be an effective therapeutic approach to mitigating mitochondrial dysfunction in aging and related comorbidities.


Assuntos
Envelhecimento/fisiologia , DNA Mitocondrial/genética , Mitocôndrias/fisiologia , Condicionamento Físico Animal , Resistência Física , Mutação Puntual , Envelhecimento/genética , Animais , Apoptose , Dosagem de Genes , Camundongos , Camundongos Mutantes , Estresse Oxidativo
19.
J Vis Exp ; (48)2011 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-21339721

RESUMO

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide. In human stroke, there exists a highly variable clinical state; in the development of animal models of focal ischemia, however, achieving reproducibility of experimentally induced infarct volume is essential. The rat is a widely used animal model for stroke due to its relatively low animal husbandry costs and to the similarity of its cranial circulation to that of humans. In humans, the middle cerebral artery (MCA) is most commonly affected in stroke syndromes and multiple methods of MCA occlusion (MCAO) have been described to mimic this clinical syndrome in animal models. Because recanalization commonly occurs following an acute stroke in the human, reperfusion after a period of occlusion has been included in many of these models. In this video, we demonstrate the transient endovascular suture MCAO model in the spontaneously hypertensive rat (SHR). A filament with a silicon tip coating is placed intraluminally at the MCA origin for 60 minutes, followed by reperfusion. Note that the optimal occlusion period may vary in other rat strains, such as Wistar or Sprague-Dawley. Several behavioral indicators of stroke in the rat are shown. Focal ischemia is confirmed using T2-weighted magnetic resonance images and by staining brain sections with 2,3,5-triphenyltetrazolium chloride (TTC) 24 hours after MCAO.


Assuntos
Isquemia Encefálica/etiologia , Modelos Animais de Doenças , Artéria Cerebral Média/cirurgia , Animais , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Suturas
20.
PLoS One ; 5(7): e11468, 2010 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-20628647

RESUMO

BACKGROUND: Aging results in a progressive loss of skeletal muscle, a condition known as sarcopenia. Mitochondrial DNA (mtDNA) mutations accumulate with aging in skeletal muscle and correlate with muscle loss, although no causal relationship has been established. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the relationship between mtDNA mutations and sarcopenia at the gene expression and biochemical levels using a mouse model that expresses a proofreading-deficient version (D257A) of the mitochondrial DNA Polymerase gamma, resulting in increased spontaneous mtDNA mutation rates. Gene expression profiling of D257A mice followed by Parametric Analysis of Gene Set Enrichment (PAGE) indicates that the D257A mutation is associated with a profound downregulation of gene sets associated with mitochondrial function. At the biochemical level, sarcopenia in D257A mice is associated with a marked reduction (35-50%) in the content of electron transport chain (ETC) complexes I, III and IV, all of which are partly encoded by mtDNA. D257A mice display impaired mitochondrial bioenergetics associated with compromised state-3 respiration, lower ATP content and a resulting decrease in mitochondrial membrane potential (Deltapsim). Surprisingly, mitochondrial dysfunction was not accompanied by an increase in mitochondrial reactive oxygen species (ROS) production or oxidative damage. CONCLUSIONS/SIGNIFICANCE: These findings demonstrate that mutations in mtDNA can be causal in sarcopenia by affecting the assembly of functional ETC complexes, the lack of which provokes a decrease in oxidative phosphorylation, without an increase in oxidative stress, and ultimately, skeletal muscle apoptosis and sarcopenia.


Assuntos
Apoptose/fisiologia , DNA Mitocondrial/genética , Mitocôndrias/genética , Mitocôndrias/patologia , Músculo Esquelético/metabolismo , Sarcopenia/genética , Sarcopenia/patologia , Animais , Apoptose/genética , Caspase 3/metabolismo , Caspase 9/metabolismo , Feminino , Masculino , Potencial da Membrana Mitocondrial/fisiologia , Camundongos , Camundongos Transgênicos , Músculo Esquelético/patologia , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
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