RESUMEN
A series of 1,3,4-oxadiazole tethered capsaicin derivatives was prepared by using one point modification at the vanillyl-hydroxyl group of capsaicin. All the prepared capsaicinoids were evaluated for their antiproliferative activity against NCI-60 human cancer cell lines at 10 µM. Among the compounds tested, compound 20a exhibited good cytotoxic activity against HCT-116, NCI-H460, and SKOV3 cell lines with IC50 8.55 µΜ, 5.41 µΜ, and 6.4 µΜ, respectively, compared to the parent natural product capsaicin. Further on, it significantly inhibited the colony formation in NCI-H460 in a dose dependent manner and enhanced the ROS effect. It also caused cell arrest at the S phase and induced apoptosis via suppressing the Pro parp marker. Compound 20a exhibited an antimigratory property and suppressed the expression of the VEGF marker in a dose dependent manner. Furthermore, compound 20a also suppressed the effects of the p-Erk, p-p38, and P-CNA makers. In silico studies supported the interaction of this class of compounds with the VEGFR2 protein.
RESUMEN
Amyloid beta (Aß) peptide aggregation and cholinergic neurodegeneration are involved in the development of cognitive impairment. Therefore, in this article, we examined rosuvastatin (RSV), an oral hypolipidemic drug, to determine its potential as a dual inhibitor of acetylcholinesterase (AChE) and Aß peptide aggregation for the treatment of cognitive impairment. Molecular docking study was done to examine the affinity of RSV with Aß1-42 and AChE in silico. We also employed neurobehavioral activity tests, biochemical estimation, and histopathology to study the anti-Aß1-42 aggregation capability of RSV in vivo. Molecular docking study provided evidence that RSV has the best binding conformer at its receptor site or active site of an enzyme. The cognitive impairment in female Wistar rats was induced by high-salt and cholesterol diet (HSCD) ad libitum for 8 weeks. RSV ameliorated serum cholesterol level, AChE activity, and Aß1-42 peptide aggregations in HSCD induced cognitive impairment. In addition, RSV-treated rats showed greater scores in the open field (locomotor activity) test. Moreover, the histopathological studies in the hippocampus and cortex of rat brain also supported that RSV markedly reduced the cognitive impairment and preserved the normal histoarchitectural pattern of the hippocampus and cortex. Taken together, these data indicate that RSV may act as a dual inhibitor of AChE and Aß1-42 peptide aggregation, therefore suggesting a therapeutic strategy for cognitive impairment treatment.
Asunto(s)
Péptidos beta-Amiloides/antagonistas & inhibidores , Conducta Animal/efectos de los fármacos , Encéfalo/efectos de los fármacos , Colesterol en la Dieta , Inhibidores de la Colinesterasa/farmacología , Cognición/efectos de los fármacos , Disfunción Cognitiva/prevención & control , Fragmentos de Péptidos/antagonistas & inhibidores , Agregación Patológica de Proteínas , Rosuvastatina Cálcica/farmacología , Cloruro de Sodio Dietético , Acetilcolinesterasa/química , Acetilcolinesterasa/metabolismo , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/metabolismo , Animales , Encéfalo/metabolismo , Encéfalo/patología , Encéfalo/fisiopatología , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Corteza Cerebral/fisiopatología , Inhibidores de la Colinesterasa/química , Disfunción Cognitiva/enzimología , Disfunción Cognitiva/patología , Disfunción Cognitiva/psicología , Modelos Animales de Enfermedad , Femenino , Proteínas Ligadas a GPI/antagonistas & inhibidores , Proteínas Ligadas a GPI/química , Proteínas Ligadas a GPI/metabolismo , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/patología , Hipocampo/fisiopatología , Locomoción/efectos de los fármacos , Simulación del Acoplamiento Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Ratas Wistar , Rosuvastatina Cálcica/química , Relación Estructura-ActividadRESUMEN
Amyotrophic lateral sclerosis (ALS) is a generally fatal neurodegenerative disease of adults that produces weakness and atrophy due to dysfunction and death of upper and lower motor neurons. We used RNA-sequencing (RNA-seq) to analyze expression of all mitochondrial DNA (mtDNA)-encoded respiratory genes in ALS and CTL human cervical spinal cords (hCSC) and isolated motor neurons. We analyzed with RNA-seq mtDNA gene expression in human neural stem cells (hNSC) exposed to recombinant human mitochondrial transcription factor A (rhTFAM), visualized in 3-dimensions clustered gene networks activated by rhTFAM, quantitated their interactions with other genes and determined their gene ontology (GO) families. RNA-seq and quantitative PCR (qPCR) analyses showed reduced mitochondrial gene expression in ALS hCSC and ALS motor neurons isolated by laser capture microdissection (LCM), and revealed that hNSC and CTL human cervical spinal cords were similar. Rats treated with i.v. rhTFAM showed a dose-response increase in brain respiration and an increase in spinal cord mitochondrial gene expression. Treatment of hNSC with rhTFAM increased expression of mtDNA-encoded respiratory genes and produced one major and several minor clusters of gene interactions. Gene ontology (GO) analysis of rhTFAM-stimulated gene clusters revealed enrichment in GO families involved in RNA and mRNA metabolism, suggesting mitochondrial-nuclear signaling. In postmortem ALS hCSC and LCM-isolated motor neurons we found reduced expression of mtDNA respiratory genes. In hNSC's rhTFAM increased mtDNA gene expression and stimulated mRNA metabolism by unclear mechanisms. rhTFAM may be useful in improving bioenergetic function in ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Médula Cervical/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Mitocondriales/metabolismo , Neuronas Motoras/metabolismo , Factores de Transcripción/metabolismo , Animales , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Células Cultivadas , ADN Mitocondrial , Proteínas de Unión al ADN/administración & dosificación , Expresión Génica , Glucosa/metabolismo , Humanos , Captura por Microdisección con Láser , Masculino , Proteínas Mitocondriales/administración & dosificación , Células-Madre Neurales/metabolismo , Ratas Sprague-Dawley , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de ARN , Factores de Transcripción/administración & dosificaciónRESUMEN
Mitochondria are responsible for the majority of energy production in energy-intensive tissues like brain, modulate Ca+2 signaling and control initiation of cell death. Because of their extensive use of oxygen and lack of protective histone proteins, mitochondria are vulnerable to oxidative stress (ROS)-induced damage to their genome (mtDNA), respiratory chain proteins and ROS repair enzymes. Animal and cell models of PD use toxins that impair mitochondrial complex I activity. Maintenance of mitochondrial mass, mitochondrial biogenesis (mitobiogenesis), particularly in high-energy brain, occurs through complex signaling pathways involving the upstream "master regulator" PGC-1alpha that is transcriptionally and post-translationally regulated. Alzheimer disease (AD) and Parkinson disease (PD) brains have reduced respiratory capacity and impaired mitobiogenesis, which could result in beta-amyloid plaques and neurofibrillary tangles. Aggregated proteins in genetic and familial AD and PD brains impair mitochondrial function, and mitochondrial dysfunction is involved in activated neuroinflammation. Mitochondrial ROS can activate signaling pathways that mediate cell death in neurodegenerative diseases. The available data support restoration of mitochondrial function to reduce disease progression and restore lost neuronal function in AD and PD.
Asunto(s)
Enfermedad de Alzheimer/fisiopatología , Mitocondrias/fisiología , Enfermedad de Parkinson/fisiopatología , Animales , Encéfalo/fisiopatología , Calcio/metabolismo , ADN Mitocondrial/genética , Humanos , Inflamación/fisiopatología , Mitofagia , Mutación , Biogénesis de Organelos , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Transducción de SeñalRESUMEN
The aim of the present study was to assess the Tacrolimus (TL) loaded nanostructured lipid carrier for enhancement in solubilisation potential, pharmacokinetic parameters and lymphatic distribution profile. The solubilisation potential of TL-NLC was determined via application of dynamic lipolysis models, which simulate the GIT environment and the intestinal conditions. The in vitro lipolysis studies revealed significantly high solubilisation (***p<0.001) of TL-NLCs in aqueous phase (69.3%) in contrast to TL suspension (1.6%) and the results were very well corroborated with in vivo AUC values of the corresponding formulations (R2>0.99). The in vivo, lymphatic and organ distribution studies were performed in albino wistar rats. There was marked increase of 7.2 folds in relative bioavailability of TL-NLC in comparison to TL suspension. Furthermore, study findings demonstrated that the lymphatic distribution of TL-NLC was enhanced by 19.25 folds in comparison to TL suspension. The results of dynamic lipolysis, bioavailability, lymphatic and other organ distribution studies confirmed that incorporation of TL in hybrid of lipids with different fatty acid chain length could substantially improve its in vivo prospect.
Asunto(s)
Portadores de Fármacos/química , Composición de Medicamentos/métodos , Lípidos/química , Nanoestructuras/química , Tacrolimus/administración & dosificación , Administración Oral , Animales , Área Bajo la Curva , Disponibilidad Biológica , Cromatografía Líquida de Alta Presión , Diseño de Fármacos , Estabilidad de Medicamentos , Ácidos Grasos/química , Femenino , Inmunosupresores/administración & dosificación , Inmunosupresores/química , Inmunosupresores/farmacocinética , Cinética , Lipólisis , Ganglios Linfáticos/efectos de los fármacos , Masculino , Tamaño de la Partícula , Ratas , Ratas Wistar , Programas Informáticos , Solubilidad , Tacrolimus/farmacocinéticaRESUMEN
Tacrolimus (TL), which is currently the mainstay of immunosuppressive therapy faces significant hurdles subsequent to its oral administration attributable to its poor aqueous solubility and extensive intestinal and hepatic first pass metabolism. Therefore, the present study aimed to design the stable nanostructured lipid carrier (NLC) of TL that would be able to overcome such hurdles. Capmul MCMC8 and Compritol 888ATO in 3:2 were selected as binary lipid phase on the basis of solubility study. An exhaustive screening of surfactants is done by aqueous titration to select the surfactant with best emulsifying potential and to optimize the concentration of lipids and surfactants in NLC. Different methods of preparation were explored and compared to optimize NLC which could have the best characteristic properties. TL-NLC was characterized for particle size, drug entrapment efficiency, crystal state, surface morphology and drug release. The obtained particle size, PDI and % drug entrapment efficiency of optimized formulations i.e., NLC-C2 and NLC-N2 were 70±5.42nm, 98±7.52nm; 0.43±0.081, 0.2±0.029 and 87±2.34%, 94±3.18%, respectively. The results of in vitro release studies showed significantly increased (***p<0.001) and sustained release of TL from NLC dispersions as compared to drug suspension (95.73% from NLC-C2, 99.86% from NLC-N2 and 9.27% drug suspension in pH 1.2 in 24h; 93.11% from NLC-C2, 96.65% from NLC-N2 and 10.2% drug suspension in pH 6.8 in 24h). The study demonstrated that proper selection of excipients (by aqueous titration) and modification of method of preparation (by inclusion of cold step) would lead to production of NLC with best characteristic properties.
Asunto(s)
Sistemas de Liberación de Medicamentos , Lípidos/química , Nanopartículas/química , Tacrolimus/administración & dosificación , Administración Oral , Rastreo Diferencial de Calorimetría , Cromatografía Líquida de Alta Presión , Portadores de Fármacos/química , Liberación de Fármacos , Ácidos Grasos/química , Glicéridos/química , Concentración de Iones de Hidrógeno , Microscopía Electrónica de Transmisión , Tamaño de la Partícula , Polietilenglicoles/química , Solubilidad , Solventes/química , Espectroscopía Infrarroja por Transformada de Fourier , Tensoactivos/química , Temperatura , ViscosidadRESUMEN
Ten years ago we first proposed the Alzheimer's disease (AD) mitochondrial cascade hypothesis. This hypothesis maintains that gene inheritance defines an individual's baseline mitochondrial function; inherited and environmental factors determine rates at which mitochondrial function changes over time; and baseline mitochondrial function and mitochondrial change rates influence AD chronology. Our hypothesis unequivocally states in sporadic, late-onset AD, mitochondrial function affects amyloid precursor protein (APP) expression, APP processing, or beta amyloid (Aß) accumulation and argues if an amyloid cascade truly exists, mitochondrial function triggers it. We now review the state of the mitochondrial cascade hypothesis, and discuss it in the context of recent AD biomarker studies, diagnostic criteria, and clinical trials. Our hypothesis predicts that biomarker changes reflect brain aging, new AD definitions clinically stage brain aging, and removing brain Aß at any point will marginally impact cognitive trajectories. Our hypothesis, therefore, offers unique perspective into what sporadic, late-onset AD is and how to best treat it.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Mitocondrias/metabolismo , Modelos Biológicos , Transducción de Señal , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/patología , Animales , Biomarcadores/metabolismo , HumanosRESUMEN
Two series of Benzimidazole clubbed with triazolo-thiadiazoles (5a-q, 5r, 5s and 5x-a(1)) and triazolo-thiadiazines (5t-w) were synthesized with an aim to produce promising anticancer agents. In vitro anticancer activities of synthesized compounds were investigated at the National Cancer Institute (NCI) against NCI 60 cell line panel; results showed good to remarkable broad-spectrum anticancer activity. Among them, the compound 5h (NCS: 760452, 1-(1H-benzo [d] imidazol-2-yl)-3-(6-(2,4-dichlorophenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-3-yl) propan-1-one) exhibited significant growth inhibition with GI50 values ranging from 0.20 to 2.58 µM and found superior selectivity for the leukemia cell lines and further screened at 10-fold dilutions of five different concentrations (0.01, 0.1, 1, 10 and 100 µM). The 5h may possibly be used as lead compound for developing new anticancer agents.
Asunto(s)
Antineoplásicos/farmacología , Bencimidazoles/química , Tiadiazinas/química , Tiadiazoles/química , Triazoles/química , Antineoplásicos/síntesis química , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Mitochondrial function declines with age in postmitotic tissues such as brain, heart and skeletal muscle. Despite weekly exercise, aged mice showed substantial losses of mtDNA gene copy numbers and reductions in mtDNA gene transcription and mitobiogenesis signaling in brain and heart. We treated these mice with weekly intravenous injections of recombinant human mitochondrial transcription factor A (rhTFAM). RhTFAM treatment for one month increased mitochondrial respiration in brain, heart and muscle, POLMRT expression and mtDNA gene transcription in brain, and PGC-1 alpha mitobiogenesis signaling in heart. RhTFAM treatment reduced oxidative stress damage to brain proteins, improved memory in Morris water maze performance and increased brain protein levels of BDNF and synapsin. Microarray analysis showed co-expression of multiple Gene Ontology families in rhTFAM-treated aged brains compared to young brains. RhTFAM treatment reverses age-related memory impairments associated with loss of mitochondrial energy production in brain, increases levels of memory-related brain proteins and improves mitochondrial respiration in brain and peripheral tissues.
Asunto(s)
Proteínas de Unión al ADN/farmacología , Expresión Génica/efectos de los fármacos , Memoria/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Proteínas Mitocondriales/farmacología , Factores de Transcripción/farmacología , Envejecimiento , Animales , Western Blotting , Respiración de la Célula/efectos de los fármacos , ADN Mitocondrial/efectos de los fármacos , Humanos , Aprendizaje por Laberinto/efectos de los fármacos , Ratones , Reacción en Cadena de la Polimerasa Multiplex , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosforilación Oxidativa/efectos de los fármacos , Proteínas Recombinantes/farmacologíaRESUMEN
A series of 1,6-dihydropyrimidine-2-amine derivatives and 1,6-dihydropyrimidine-2-thiol derivatives were synthesized by the reaction of substituted 1,3-diphenylprop-2-en-1-one (chalcones) with guanidine hydrochloride and thiourea, respectively. All the synthesized compounds were in good agreement with elemental and spectral data. The synthesized compounds were screened in vivo for diuretic activity. The four compounds 2d, 2e, 3d and 3e, which showed moderate to good diuretic activity, were evaluated for their toxicity studies and none of the compounds showed any toxicity of the liver as compared with control. However, compounds 3e and 3d showed diuretic properties more than that of standard (acetazolamide) and were long acting. Overall, compound 3e, 6-(2,6-dichlorophenyl)-4-(pyridin-2-yl)-1,6-dihydropyrimidine-2-thiol, was found to be the most promising candidate of the series.
Asunto(s)
Diuréticos/farmacología , Diuréticos/orina , Pirimidinas/farmacología , Pirimidinas/orina , Animales , Cloruros/orina , Diuréticos/química , Pirimidinas/química , Ratas , Sodio/orinaRESUMEN
Recombinant human mitochondrial transcription factor A protein (rhTFAM) was evaluated for its acute effects on cultured cells and chronic effects in mice. Fibroblasts incubated with rhTFAM acutely increased respiration in a chloramphenicol-sensitive manner. SH-SY5Y cells showed rhTFAM concentration-dependent reduction of methylpyridinium (MPP(+))-induced oxidative stress and increases in lowered ATP levels and viability. Mice treated with weekly i.v. rhTFAM showed increased mitochondrial gene copy number, complex I protein levels and ATP production rates; oxidative damage to proteins was decreased ~50%. rhTFAM treatment improved motor recovery rate after treatment with MPTP and dose-dependently improved survival in the lipopolysaccharide model of endotoxin sepsis.
Asunto(s)
Adenosina Trifosfato/biosíntesis , Proteínas de Unión al ADN/farmacología , Endotoxemia/mortalidad , Mitocondrias/metabolismo , Proteínas Mitocondriales/farmacología , Actividad Motora/fisiología , Estrés Oxidativo/efectos de los fármacos , Proteínas Recombinantes/farmacología , Factores de Transcripción/farmacología , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/administración & dosificación , Animales , Línea Celular Tumoral , Respiración de la Célula/efectos de los fármacos , Supervivencia Celular , ADN Mitocondrial/efectos de los fármacos , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Proteínas de Unión al ADN/administración & dosificación , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endotoxemia/tratamiento farmacológico , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/fisiología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Proteínas Mitocondriales/administración & dosificación , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Actividad Motora/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/fisiología , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factores de Transcripción/administración & dosificación , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
We first proposed the mitochondrial cascade hypothesis of sporadic Alzheimer's disease (AD) in 2004. Our core assumptions were a person's genes determine baseline mitochondrial function and durability, this durability determines how mitochondria change with advancing age, and critical changes in mitochondrial function initiate other pathologies characteristic of AD. Since then several lines of investigation report data consistent with or supportive of our hypothesis. In particular, AD endophenotype studies suggest a strong maternal genetic contribution, and links between mitochondrial function, tau phosphorylation, and amyloid-beta (Abeta) amyloidosis are increasingly recognized. As predicted, AD therapies designed to reduce Abeta thus far have had at best very limited clinical benefits; our hypothesis identifies alternative therapeutic targets. While placing mitochondria at the apex of an AD cascade certainly remains controversial, it is increasingly accepted by the AD research community that mitochondria play an important role in the late-onset forms of the disease. Even if the mitochondrial cascade hypothesis proves incorrect, considering its assumptions could potentially advance our understanding of sporadic, late-onset AD.
Asunto(s)
Enfermedad de Alzheimer/etiología , Enfermedad de Alzheimer/patología , Mitocondrias/patología , Envejecimiento/metabolismo , Envejecimiento/patología , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/fisiopatología , Péptidos beta-Amiloides/metabolismo , Animales , ADN Mitocondrial/genética , Complejo IV de Transporte de Electrones/metabolismo , Humanos , Mitocondrias/genética , Mitocondrias/metabolismo , Modelos BiológicosRESUMEN
In 2004 we proposed the mitochondrial cascade hypothesis of sporadic Alzheimer's disease (AD). Our hypothesis assumed sporadic and autosomal dominant AD are not etiologically homogeneous, considered evidence that AD pathology is not brain-limited, and incorporated aging theory. The mitochondrial cascade hypothesis asserted: (1) inheritance determines mitochondrial baseline function and durability; (2) mitochondrial durability influences how mitochondria change with age; and (3) when mitochondrial change reaches a threshold, AD histopathology and symptoms ensue. We now review the reasoning used to formulate the hypothesis, discuss pertinent interim data, and update its tenants. Readers are invited to consider the conceptual strengths and weaknesses of this hypothesis.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Amiloidosis/patología , Mitocondrias/metabolismo , Proteínas tau/metabolismo , Envejecimiento , Enfermedad de Alzheimer/etiología , Amiloidosis/metabolismo , Animales , Encéfalo/metabolismo , Encéfalo/patología , Ciclo Celular , Humanos , Mitocondrias/patología , Factores de RiesgoRESUMEN
Neurodegeneration in Parkinson's disease (PD) affects mainly dopaminergic neurons in the substantia nigra, where age-related, increasing percentages of cells lose detectable respiratory activity associated with depletion of intact mitochondrial DNA (mtDNA). Replenishment of mtDNA might improve neuronal bioenergetic function and prevent further cell death. We developed a technology ("ProtoFection") that uses recombinant human mitochondrial transcription factor A (TFAM) engineered with an N-terminal protein transduction domain (PTD) followed by the SOD2 mitochondrial localization signal (MLS) to deliver mtDNA cargo to the mitochondria of living cells. MTD-TFAM (MTD = PTD + MLS = "mitochondrial transduction domain") binds mtDNA and rapidly transports it across plasma membranes to mitochondria. For therapeutic proof-of-principle we tested ProtoFection technology in Parkinson's disease cybrid cells, using mtDNA generated from commercially available human genomic DNA (gDNA; Roche). Nine to 11 weeks after single exposures to MTD-TFAM + mtDNA complex, PD cybrid cells with impaired respiration and reduced mtDNA genes increased their mtDNA gene copy numbers up to 24-fold, mtDNA-derived RNAs up to 35-fold, TFAM and ETC proteins, cell respiration, and mitochondrial movement velocities. Cybrid cells with no or minimal basal mitochondrial impairments showed reduced or no responses to treatment, suggesting the possibility of therapeutic selectivity. Exposure of PD but not control cybrid cells to MTD-TFAM protein alone or MTD-TFAM + mtDNA complex increased expression of PGC-1alpha, suggesting activation of mitochondrial biogenesis. ProtoFection technology for mitochondrial gene therapy holds promise for improving bioenergetic function in impaired PD neurons and needs additional development to define its pharmacodynamics and delineate its molecular mechanisms. It also is unclear whether single-donor gDNA for generating mtDNA would be a preferred therapeutic compared with the pooled gDNA used in this study.
Asunto(s)
Genes Mitocondriales , Terapia Genética , Mitocondrias/genética , Mitocondrias/patología , Enfermedad de Parkinson/fisiopatología , Enfermedad de Parkinson/terapia , Línea Celular Tumoral , Respiración de la Célula , ADN Mitocondrial/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Humanos , Proteínas Mitocondriales/química , Proteínas Mitocondriales/metabolismo , Enfermedad de Parkinson/genética , Señales de Clasificación de Proteína , Estructura Terciaria de Proteína , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
Quantitative real time PCR (qPCR) is commonly used to determine cell mitochondrial DNA (mtDNA) copy number. This technique involves obtaining the ratio of an unknown variable (number of copies of an mtDNA gene) to a known parameter (number of copies of a nuclear DNA gene) within a genomic DNA sample. We considered the possibility that mtDNA:nuclear DNA (nDNA) ratio determinations could vary depending on the method of genomic DNA extraction used, and that these differences could substantively impact mtDNA copy number determination via qPCR. To test this we measured mtDNA:nDNA ratios in genomic DNA samples prepared using organic solvent (phenol-chloroform-isoamyl alcohol) extraction and two different silica-based column methods, and found mtDNA:nDNA ratio estimates were not uniform. We further evaluated whether different genomic DNA preparation methods could influence outcomes of experiments that use mtDNA:nDNA ratios as endpoints, and found the method of genomic DNA extraction can indeed alter experimental outcomes. We conclude genomic DNA sample preparation can meaningfully influence mtDNA copy number determination by qPCR.
Asunto(s)
ADN Mitocondrial/análisis , Hepatocitos/química , Manejo de Especímenes/métodos , Animales , ADN/aislamiento & purificación , Hígado/química , Ratones , Reacción en Cadena de la Polimerasa/métodos , Reproducibilidad de los ResultadosRESUMEN
The past two decades have witnessed an evolving understanding of the mitochondrial genome's (mtDNA) role in basic biology and disease. From the recognition that mutations in mtDNA can be responsible for human disease to recent efforts showing that mtDNA mutations accumulate over time and may be responsible for some phenotypes of aging, the field of mitochondrial genetics has greatly benefited from the creation of cell and animal models of mtDNA mutation. In this review, we critically discuss the past two decades of efforts and insights gained from cell and animal models of mtDNA mutation. We attempt to reconcile the varied and at times contradictory findings by highlighting the various methodologies employed and using human mtDNA disease as a guide to better understanding of cell and animal mtDNA models. We end with a discussion of scientific and therapeutic challenges and prospects for the future of mtDNA transfection and gene therapy.
Asunto(s)
ADN Mitocondrial/fisiología , Mitocondrias/fisiología , Modelos Animales , Animales , Células Cultivadas , ADN Mitocondrial/genética , Terapia Genética , Humanos , Ratones , Mitocondrias/genética , Enfermedades Mitocondriales/genética , Mutación , TransfecciónRESUMEN
Although oxidative stress and mitochondrial dysfunction have been linked to neurodegenerative diseases such as Alzheimer's disease (AD), it remains unclear how mitochondrial oxidative stress may induce neuronal death. In a variety of tissues, cumulative oxidative stress, disrupted mitochondrial respiration, and mitochondrial damage are associated with, and may indeed promote cell death and degeneration. In this review, we examine current evidence supporting the involvement of mitochondria and mitochondrially generated stress signaling in AD and discuss potential implications for the mechanism of pathogenesis of this disease. Mitochondria are pivotal in controlling cell life and death not only by producing ATP, and sequestering calcium, but by also generating free radicals and serving as repositories for proteins which regulate the intrinsic apoptotic pathway. Perturbations in the physiological function of mitochondria inevitably disturb cell function, sensitize cells to neurotoxic insults and may initiate cell death, all significant phenomena in the pathogenesis of a number of neurodegenerative disorders including AD.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Encéfalo/metabolismo , Mitocondrias/metabolismo , Neuronas/metabolismo , Estrés Oxidativo/fisiología , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/fisiopatología , Animales , Apoptosis/fisiología , Encéfalo/patología , Encéfalo/fisiopatología , Humanos , Proteínas de la Membrana/fisiología , Mitocondrias/genética , Mitocondrias/patología , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Neuronas/patología , Especies Reactivas de Oxígeno/metabolismo , Serina Endopeptidasas/fisiologíaRESUMEN
We implicate a recently described form of mitochondrial mutation, mitochondrial microheteroplasmy, as a candidate for the principal component of aging. Microheteroplasmy is the presence of hundreds of independent mutations in one organism, with each mutation usually found in 1-2% of all mitochondrial genomes. Despite the low abundance of single mutations, the vast majority of mitochondrial genomes in all adults are mutated. This mutational burden includes inherited mutations, de novo germline mutations, as well as somatic mutations acquired either during early embryonic development or later in adult life. We postulate that microheteroplasmy is sufficient to explain the pathomechanism of several age-associated diseases, especially in conditions with known mitochondrial involvement, such as diabetes (DM), cardiovascular disease, Parkinson's disease (PD), and Alzheimer's disease (AD) and cancer. The genetic properties of microheteroplasmy reconcile the results of disease models (cybrids, hypermutable PolG variants and mitochondrial toxins), with the relatively low levels of maternal inheritance in the aforementioned diseases, and provide an explanation of their delayed, progressive course.
Asunto(s)
Envejecimiento/fisiología , ADN Mitocondrial/genética , Modelos Biológicos , Mutación/fisiología , Envejecimiento/genética , Animales , Enfermedad , Genoma , HumanosRESUMEN
Many "classic" mitochondrial diseases have been described that arise from single homoplasmic mutations in mitochondrial DNA (mtDNA). These diseases typically affect nonmitotic tissues (brain, retina, muscle), present with variable phenotypes, can appear sporadically, and are untreatable. Evolving evidence implicates mtDNA abnormalities in diseases such as Alzheimer's, Parkinson's, and type II diabetes, but specific causal mutations for these conditions remain to be defined. Understanding the mtDNA genotype-phenotype relationships and developing specific treatment for mtDNA-based diseases is hampered by inability to manipulate the mitochondrial genome. We present a novel protein transduction technology ("protofection") that allows insertion and expression of the human mitochondrial genome into mitochondria of living cells. With protofection, the mitochondrial genotype can be altered, or exogenous genes can be introduced to be expressed and either retained in mitochondria or be directed to other organelles. Protofection also delivers mtDNA in vivo, opening the way to rational development of mitochondrial gene replacement therapy of mtDNA-based diseases.
Asunto(s)
ADN Mitocondrial/administración & dosificación , ADN Mitocondrial/genética , Marcación de Gen/métodos , Terapia Genética/métodos , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/terapia , Transfección/tendencias , Animales , Humanos , Resultado del TratamientoRESUMEN
Alzheimer's disease (AD) includes etiologically heterogeneous disorders characterized by senile or presenile dementia, extracellular amyloid protein aggregations containing an insoluble amyloid precursor protein derivative, and intracytoplasmic tau protein aggregations. Recent studies also show excess neuronal aneuploidy, programmed cell death (PCD), and mitochondrial dysfunction. The leading AD molecular paradigm, the "amyloid cascade hypothesis", is based on studies of rare autosomal dominant variants and does not specify what initiates the common late-onset, sporadic form. We propose for late-onset, sporadic AD a "mitochondrial cascade hypothesis" that comprehensively reconciles seemingly disparate histopathologic and pathophysiologic features. In our model, the inherited, gene-determined make-up of an individual's electron transport chain sets basal rates of reactive oxygen species (ROS) production, which determines the pace at which acquired mitochondrial damage accumulates. Oxidative mitochondrial DNA, RNA, lipid, and protein damage amplifies ROS production and triggers three events: (1) a reset response in which cells respond to elevated ROS by generating the beta-sheet protein, beta amyloid, which further perturbs mitochondrial function, (2) a removal response in which compromised cells are purged via PCD mechanisms, and (3) a replace response in which neuronal progenitors unsuccessfully attempt to re-enter the cell cycle, with resultant aneuploidy, tau phosphorylation, and neurofibrillary tangle formation. In addition to defining a role for aging in AD pathogenesis, the mitochondrial cascade hypothesis also allows and accounts for histopathologic overlap between the sporadic, late-onset and autosomal dominant, early onset forms of the disease.