RESUMEN

Images of brain metabolism and measurements of activities of components of the electron transport chain support earlier studies that suggest that brain glucose oxidation is inherently abnormal in a significant proportion of persons with schizophrenia. Therefore, we measured the activities of enzymes of the tricarboxylic (TCA) cycle in dorsolateral-prefrontal-cortex from schizophrenia patients (N=13) and non-psychiatric disease controls (N=13): the pyruvate dehydrogenase complex (PDHC), citrate synthase (CS), aconitase, isocitrate dehydrogenase (ICDH), the alpha-ketoglutarate dehydrogenase complex (KGDHC), succinate thiokinase (STH), succinate dehydrogenase (SDH), fumarase and malate dehydrogenase (MDH). Activities of aconitase (18.4%, p<0.05), KGDHC (26%) and STH (28.2%, p<0.05), enzymes in the first half of the TCA cycle, were lower, but SDH (18.3%, p<0.05) and MDH (34%, p<0.005), enzymes in the second half, were higher than controls. PDHC, CS, ICDH and fumarase activities were unchanged. There were no significant correlations between enzymes of TCA cycle and cognitive function, age or choline acetyl transferase activity, except for aconitase activity which decreased slightly with age (r=0.55, p=003). The increased activities of dehydrogenases in the second half of the TCA cycle may reflect a compensatory response to reduced activities of enzymes in the first half. Such alterations in the components of TCA cycle are adequate to alter the rate of brain metabolism. These results are consistent with the imaging studies of hypometabolism in schizophrenia. They suggest that deficiencies in mitochondrial enzymes can be associated with mental disease that takes the form of schizophrenia.

Asunto(s)

Aconitato Hidratasa/metabolismo , Encéfalo/metabolismo , Ciclo del Ácido Cítrico , Malato Deshidrogenasa/metabolismo , Esquizofrenia/metabolismo , Succinato Deshidrogenasa/metabolismo , Anciano , Anciano de 80 o más Años , Animales , Encéfalo/enzimología , Femenino , Glucosa/metabolismo , Humanos , Masculino , Ratones , Persona de Mediana Edad , Oxidación-Reducción , Corteza Prefrontal/metabolismo , Caracteres Sexuales , Factores de Tiempo

RESUMEN

Mitochondrial defects including reduction of a key mitochondrial tricarboxylic acid cycle enzyme alpha-ketoglutarate-dehydrogenase complex (KGDHC) are characteristic of many neurodegenerative diseases. KGDHC consists of alpha-ketoglutarate dehydrogenase, dihydrolipoyl succinyltransferase (E2k), and dihydrolipoamide dehydrogenase (Dld) subunits. We investigated whether Dld or E2k deficiency influences adult brain neurogenesis using immunohistochemistry for the immature neuron markers, doublecortin (Dcx) and polysialic acid-neural cell adhesion molecule, as well as a marker for proliferation, proliferating cell nuclear antigen (PCNA). Both Dld- and E2k-deficient mice showed reduced Dcx-positive neuroblasts in the subgranular zone (SGZ) of the hippocampal dentate gyrus compared with wild-type mice. In the E2k knockout mice, increased immunoreactivity for the lipid peroxidation marker, malondialdehyde occurred in the SGZ. These alterations did not occur in the subventricular zone (SVZ). PCNA staining revealed decreased proliferation in the SGZ of E2k-deficient mice. In a transgenic mouse model of Alzheimer's disease, Dcx-positive cells in the SGZ were also reduced compared with wild type, but Dld deficiency did not exacerbate the reduction. In the malonate lesion model of Huntington's disease, Dld deficiency did not alter the lesion-induced increase and migration of Dcx-positive cells from the SVZ into the ipsilateral striatum. Thus, the KGDHC subunit deficiencies associated with elevated lipid peroxidation selectively reduced the number of neuroblasts and proliferating cells in the hippocampal neurogenic zone. However, these mitochondrial defects neither exacerbated certain pathological conditions, such as amyloid precursor protein (APP) mutation-induced reduction of SGZ neuroblasts, nor inhibited malonate-induced migration of SVZ neuroblasts. Our findings support the view that mitochondrial dysfunction can influence the number of neural progenitor cells in the hippocampus of adult mice.

Asunto(s)

Encéfalo/patología , Proliferación Celular , Mitocondrias/enzimología , Enfermedades Neurodegenerativas/enzimología , Enfermedades Neurodegenerativas/patología , Neuronas/ultraestructura , Aciltransferasas/deficiencia , Análisis de Varianza , Animales , Dihidrolipoamida Deshidrogenasa/deficiencia , Modelos Animales de Enfermedad , Proteínas de Dominio Doblecortina , Proteína Doblecortina , Regulación Enzimológica de la Expresión Génica/genética , Peroxidación de Lípido/genética , Malondialdehído/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/metabolismo , Enfermedades Neurodegenerativas/genética , Neuronas/metabolismo , Neuronas/patología , Neuropéptidos/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Tinción con Nitrato de Plata/métodos

RESUMEN

Thiamine-dependent enzymes are diminished in multiple neurodegenerative diseases. Thiamine deficiency (TD) reduces the activity of thiamine dependent-enzymes [e.g., the alpha-ketoglutarate dehydrogenase complex (KGDHC)], induces regional selective neurodegeneration and serves as a model of a mild impairment of oxidative metabolism. The current experiments tested whether changes in KGDHC protein subunits (E1k, E2k and E3) or activity or message levels underlie the selective loss of neurons in particular brain regions. Thus, TD-induced changes in these variables in the brain region most vulnerable to TD [the sub-medial thalamic nucleus (SmTN)] were compared to those in a region that is relatively resistant to TD (cortex) at stages of TD when the neuron loss in SmTN is not present, minimal or severe. Impaired motor performance on rotarod was apparent by 8 days of TD (-32%) and was severe by 10 days of TD (-97%). At TD10, the overall KGDHC activity measured by an in situ histochemical staining method declined 52% in SmTN but only 20% in cortex. Reductions in the E2k and E3 mRNA in SmTN occurred as early as TD6 (-28 and -18%, respectively) and were more severe by TD10 (-61 and -66%, respectively). On the other hand, the level of E1k mRNA did not decline in SmTN until TD10 (-48%). In contrast, TD did not alter mRNA levels of the subunits in cortex at late stages. Western blots and immunocytochemistry revealed different aspects of the changes in protein levels. In SmTN, the immunoreactivity of E1k and E3 by Western blotting increased 34 and 40%, respectively, only at TD8. In cortex, the immunoreactivity of the three subunits was not altered. Immunocytochemical staining of brain sections from TD10 mice indicated a reduction in the immunoreactivity of all subunits in SmTN, but not in cortex. These findings demonstrate that the response of the KGDHC activity, mRNA and immunoreactivity of E1k, E2k and E3 to TD is region and time dependent. Loss of KGDHC activity in cortex is likely related to post-translational modification rather than a loss of protein, whereas in SmTN transcriptional and post-translational modifications may account for diminished KGDHC activity. Moreover, the earlier detection in TD induced-changes of the transcripts of KGDHC indicates that transcriptional modification of the two subunits (E2k and E3) of KGDHC may be one of the early events in the cascade leading to selective neuronal death.

Asunto(s)

Encéfalo/enzimología , Complejo Cetoglutarato Deshidrogenasa/metabolismo , Mitocondrias/enzimología , Desempeño Psicomotor/fisiología , Deficiencia de Tiamina/enzimología , Animales , Modelos Animales de Enfermedad , Complejo Cetoglutarato Deshidrogenasa/genética , Masculino , Ratones , Ratones Endogámicos C57BL , ARN/genética , ARN/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética

RESUMEN

Parkinson's disease (PD) is associated with mitochondrial dysfunction, specifically a deficiency of complex I of the electron transport chain. Most, although not all, studies indicate that this deficiency is limited to brain regions with neurodegeneration. The current studies tested for deficiencies in other mitochondrial components in PD brain in a neuropathologically unaffected region where the abnormality cannot be attributed to secondary effects of neurodegeneration. The activity of a key (and arguably rate-limiting) tricarboxylic acid cycle enzyme, the alpha-ketoglutarate dehydrogenase complex (KGDHC), was measured in the cerebellum of patients with PD. Activity in 19 PD brains was 50.5% of that in 18 controls matched for age, sex, post-mortem interval, and method of preservation (P<0.0019). The protein subunits of KGDHC were present in normal amounts in PD brains, indicating a relatively discrete abnormality in the enzyme. The activities of another mitochondrial enzyme, glutamate dehydrogenase (GDH), were normal in PD brains. These results demonstrate that specific reductions in KGDHC occur even in pathologically unaffected areas in PD, where the decline is unlikely to be a non-specific result of neurodegeneration. Reductions in the activity of this enzyme, if widespread in the brain, may predispose vulnerable regions to further damage.

Asunto(s)

Encéfalo/metabolismo , Ciclo del Ácido Cítrico , Trastornos Parkinsonianos/metabolismo , Adolescente , Adulto , Cadáver , Causas de Muerte , Niño , Femenino , Glutamato Deshidrogenasa/metabolismo , Humanos , Complejo Cetoglutarato Deshidrogenasa/metabolismo , Masculino , Persona de Mediana Edad , Complejos Multienzimáticos/metabolismo , NADH NADPH Oxidorreductasas/metabolismo , Valores de Referencia

RESUMEN

Abnormalities in energy metabolism and oxidative stress accompany many neurodegenerative diseases, including progressive supranuclear palsy (PSP). Previously, we showed decreased activities of a mitochondrial enzyme complex, alpha-ketoglutarate dehydrogenase complex (KGDHC), and marked increases in tissue malondialdehyde levels in post-mortem superior frontal cortex from the patients with PSP. The current study demonstrates that KGDHC is also significantly diminished (-58%) in the cerebellum from patients with PSP (n = 14), compared to age-matched control brains (n = 13). In contrast to cortex, markers of oxidative stress, such as malondialdehyde, tyrosine nitration or general protein carbonyl modification, did not increase in cerebellum. Furthermore, the protein levels of the individual components of KGDHC did not decline. The activities of two other mitochondrial enzymes were measured to determine whether the changes in KGDHC were selective. The activity of aconitase, a mitochondrial enzyme with an iron/sulfur cluster, is also significantly diminished (-50%), whereas glutamate dehydrogenase activity is unchanged. The present results suggest that the interaction of metabolic impairment and oxidative stress is region-specific in PSP brain. In cerebellum, reductions in KGDHC occur in the absence of increases in common measures of oxidative stress, and may underlie the metabolic deficits and contribute to pathological and clinical manifestation related to the cerebellum in patients with PSP.

Asunto(s)

Cerebelo/enzimología , Regulación hacia Abajo/fisiología , Metabolismo Energético/fisiología , Complejo Cetoglutarato Deshidrogenasa/metabolismo , Mitocondrias/enzimología , Estrés Oxidativo/fisiología , Parálisis Supranuclear Progresiva/enzimología , Tirosina/análogos & derivados , Aconitato Hidratasa/metabolismo , Animales , Encefalopatías Metabólicas/enzimología , Encefalopatías Metabólicas/fisiopatología , Cerebelo/fisiopatología , Glutamato Deshidrogenasa/metabolismo , Humanos , Immunoblotting , Ratones , Mitocondrias/patología , Enfermedades Mitocondriales/enzimología , Enfermedades Mitocondriales/fisiopatología , Óxido Nítrico/metabolismo , Fracciones Subcelulares/metabolismo , Parálisis Supranuclear Progresiva/fisiopatología , Tirosina/metabolismo

RESUMEN

Thiamine deficiency (TD) is a model of chronic impairment of oxidative metabolism that leads to neurodegeneration. TD induces oxidative stress and death in neurons, but does not kill astrocytes, microglia or brain endothelial cells. TD primary hippocampal neurons were either cultured alone, or co-cultured with primary astrocytes or microglia. After 7 days of TD, 50% of the neurons died, and the processes of many of the surviving neurons were severely truncated. When TD neurons were co-cultured with astrocytes or microglia, neurons did not die nor show decreased neurite outgrowth. Thus, neuronal-glial interactions are critical for maintaining neuronal homeostasis during chronic metabolic impairment.

Asunto(s)

Astrocitos/metabolismo , Microglía/metabolismo , Deficiencia de Tiamina/fisiopatología , Tiamina/fisiología , Animales , Astrocitos/citología , Astrocitos/efectos de los fármacos , Astrocitos/fisiología , Recuento de Células , Supervivencia Celular/efectos de los fármacos , Técnicas de Cocultivo , Hipocampo/citología , Ratones , Ratones Endogámicos ICR , Microglía/citología , Microglía/efectos de los fármacos , Microglía/fisiología , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/fisiología , Tiamina/farmacología

RESUMEN

Dichloroacetate (DCA) stimulates pyruvate dehydrogenase complex (PDHC) activity and lowers cerebral lactate concentrations. In the R6/2 and N171-82Q transgenic mouse models of Huntington's disease (HD), DCA significantly increased survival, improved motor function, delayed loss of body weight, attenuated the development of striatal neuron atrophy, and prevented diabetes. The percentage of PDHC in the active form was significantly reduced in R6/2 mice at 12 weeks of age, and DCA ameliorated the deficit. These results provide further evidence for a role of energy dysfunction in HD pathogenesis and suggest that DCA may exert therapeutic benefits in HD.

Asunto(s)

Ácido Dicloroacético/uso terapéutico , Enfermedad de Huntington/tratamiento farmacológico , Animales , Conducta Animal/efectos de los fármacos , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Transgénicos , Factores de Tiempo

RESUMEN

BACKGROUND: Basal cell carcinoma (BCC) occurring on non-sun-exposed sites, especially the perianal and genital regions, is very rare. OBJECTIVE: We analyzed the incidence, clinical and pathologic features, and etiologic and prognostic factors of all non-nevoid perianal and genital BCCs diagnosed at our institution within a defined period (January 1985-September 1996). METHODS: A retrospective review was performed with the use of patient clinical records and dermatopathologic slides. Cutaneous biopsy samples were tested for the presence of human papillomavirus (HPV) by in situ hybridization using biotinylated pan-HPV and serotype-specific (6, 11, 16, 18, 31, 33, 51) probes. RESULTS: Of all non-nevoid BCC syndrome cases, 51 BCCs (0.27%) were located within the perianal and genital regions. The average age of the patients was 73 years. Nine perianal BCCs occurred in men, 6 in women. Ten BCCs occurred in the pubic area, 18 on the vulva, 6 on the scrotum, and 2 on the penis. Three patients had 2 tumor sites. The average size of BCC was 1.95 cm; 29.4% were ulcerated. Seventeen patients (36%) had a history of skin cancer on sun-exposed sites and 10 (21%) had a possibly relevant associated condition. HPV was not detected in the specimens tested. Treatments included wide excision (n = 32), electrodesiccation and curettage (n = 10), Mohs micrographic surgery (n = 8), and carbon dioxide laser (n = 1). Of 30 patients with 5 years' follow-up or longer, 1 recurrence was noted 7 years after wide excision. There were no metastases. CONCLUSION: BCC of the perianal and genital skin is rare and exhibits clinical and histologic heterogeneity. Advancing age and local trauma may contribute to the pathogenesis of BCC at these sites.

Asunto(s)

Carcinoma Basocelular/patología , Neoplasias de los Genitales Femeninos/patología , Neoplasias de los Genitales Masculinos/patología , Neoplasias Cutáneas/patología , Anciano , Anciano de 80 o más Años , Envejecimiento , Canal Anal/patología , Carcinoma Basocelular/epidemiología , Carcinoma Basocelular/cirugía , Femenino , Neoplasias de los Genitales Femeninos/epidemiología , Neoplasias de los Genitales Femeninos/cirugía , Neoplasias de los Genitales Masculinos/epidemiología , Neoplasias de los Genitales Masculinos/cirugía , Humanos , Incidencia , Masculino , Persona de Mediana Edad , Cirugía de Mohs , Pronóstico , Recurrencia , Estudios Retrospectivos , Neoplasias Cutáneas/epidemiología , Neoplasias Cutáneas/cirugía

RESUMEN

Neurons may be particularly susceptible to oxidative damage, which has been proposed to induce somatic mutations, particularly in mitochondrial DNA (mtDNA). Therefore, acquired mtDNA mutations might preferentially accumulate in the brain and could play a role in aging and neurodegenerative disorders. Recently, a somatic T to G mtDNA mutation at noncoding nucleotide position 414 was reported in fibroblasts specifically from elderly subjects, with mutational burdens of up to 50%. We screened for this mutation in brain-derived mtDNA from 8 Alzheimer's disease patients, 27 Parkinson's disease patients, 4 multiple system atrophy patients, and 44 controls using up to three RFLP analyses. A total of 73 of these subjects were over the age of 65. The 414 mutation was absent in all cases. Next, individual mtDNA fragments from 6 elderly subjects were cloned, and a total of 70 clones were sequenced. The 414 mutation was absent in all clones, though occasional sequence variations were identified at other sites in single clones. The 414 mutation also was absent in blood (n = 6) and fibroblasts (n = 11) from elderly subjects. Our data suggest that it is rare for any one particular acquired mtDNA mutation to reach levels in the brain that are functionally significant. This does not exclude the possibility that the cumulative burden of multiple, individually rare, acquired mutations impairs mitochondrial function.

Asunto(s)

Encefalopatías/genética , ADN Mitocondrial/genética , Mutación Puntual , Anciano , Envejecimiento/genética , Enfermedad de Alzheimer/genética , Sangre , Análisis Mutacional de ADN , Fibroblastos , Frecuencia de los Genes/genética , Humanos , Estrés Oxidativo , Polimorfismo de Longitud del Fragmento de Restricción

RESUMEN

Recent etiological study in twins (Tanner et al. 1999) strongly suggests that environmental factors play an important role in typical, non-familial Parkinson's disease (PD), beginning after age 50. Epidemiological risk factor analyses of typical PD cases have identified several neurotoxicants, including MPP(+) (the active metabolite of MPTP), paraquat, dieldrin, manganese and salsolinol. Here, we tested the hypothesis that these neurotoxic agents might induce cell death in our nigral dopaminergic cell line, SN4741 (Son et al. 1999) through a common molecular mechanism. Our initial experiments revealed that treatment with both MPP(+) and the other PD-related neurotoxicants induced apoptotic cell death in SN4741 cells, following initial increases of H(2)O(2)-related ROS activity and subsequent activation of JNK1/2 MAP kinases. Moreover, we have demonstrated that during dopaminergic cell death cascades, MPP(+), the neurotoxicants and an oxidant, H(2)O(2) equally induce the ROS-dependent events. Remarkably, the oxidant treatment alone induced similar sequential molecular events: ROS increase, activation of JNK MAP kinases, activation of the PITSLRE kinase, p110, by both Caspase-1 and Caspase-3-like activities and apoptotic cell death. Pharmacological intervention using the combination of the antioxidant Trolox and a pan-caspase inhibitor Boc-(Asp)-fmk (BAF) exerted significant neuroprotection against ROS-induced dopaminergic cell death. Finally, the high throughput cDNA microarray screening using the current model identified downstream response genes, such as heme oxygenase-1, a constituent of Lewy bodies, that can be the useful biomarkers to monitor the pathological conditions of dopaminergic neurons under neurotoxic insult.

Asunto(s)

1-Metil-4-fenilpiridinio/toxicidad , Dopamina/metabolismo , Neuronas/metabolismo , Neurotoxinas/toxicidad , Oxidantes/toxicidad , Animales , Apoptosis/efectos de los fármacos , Caspasa 1/metabolismo , Caspasa 3 , Caspasas/metabolismo , Línea Celular , Relación Dosis-Respuesta a Droga , Peróxido de Hidrógeno/farmacología , Dosificación Letal Mediana , Ratones , Proteína Quinasa 8 Activada por Mitógenos , Proteína Quinasa 9 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neuronas/citología , Neuronas/efectos de los fármacos , Enfermedad de Parkinson Secundaria/etiología , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Especies Reactivas de Oxígeno/metabolismo , Tirosina 3-Monooxigenasa/metabolismo

RESUMEN

Mitochondrial membrane potentials (MMP) reflect the functional status of mitochondria within cells. Fluorescent probes to estimate these potentials within cells have been available for some time, but measurements of populations of mitochondria are not possible by existing methods. Therefore, comparisons between different cell types (e.g., fibroblasts and neuroblastoma), fibroblast cell lines from different patients, or even the same cell following various experimental paradigms are not feasible. The current approach estimates populations of MMP within living cells at 37 degrees C using the combination of conventional fluorescence microscopy and three-dimensional deconvolution by exhaustive photon reassignment. With this method, raw images are acquired rapidly with low-intensity (nonlaser) light with minimal concentrations of fluorescent dye. The method uses the fluorescent dye tetramethylrhodamine methyl ester, which equilibrates in cells according to the Nernst equation and provides a numerical, replicable estimate of MMP for populations of cellular mitochondria. This method can detect either increases or decreases in MMP as small as 5%. Furthermore, MMP in different cell types appear distinct. Values in fibroblasts (-105 +/- 0.9 mV) and N2a cells (-81 +/- 0.7 mV) were very different by this method. This approach bridges investigations of individual mitochondria to those that assess MMP by examining global fluorescence from cells.

Asunto(s)

Membranas Intracelulares/fisiología , Mitocondrias/fisiología , Línea Celular , Espectroscopía de Resonancia por Spin del Electrón , Transporte de Electrón , Fibroblastos/fisiología , Humanos , Procesamiento de Imagen Asistido por Computador , Ionóforos/metabolismo , Potenciales de la Membrana/fisiología , Microscopía Fluorescente , Neuroblastoma/metabolismo , Nigericina/metabolismo , Rodaminas , Piel/metabolismo

RESUMEN

Experimental thiamine deficiency (TD) is a model of impaired oxidative metabolism associated with region-selective neuronal loss in the brain. Oxidative stress is a prominent feature of TD neuropathology, as evidenced by the accumulation of heme oxygenase-1 (HO-1), ferritin, reactive iron and superoxide dismutase in microglia, nitrotyrosine and 4-hydroxynonenal in neurons, as well as induction of endothelial nitric oxide synthase within the vulnerable areas. Dietary restriction (DR) reduces oxidative stress in several organ systems including the brain. DR increases lifespan and reduces neurodegeneration in a variety of models of neuronal injury. The possibility that DR can protect vulnerable neurons against TD-induced oxidative insults has not been tested. The current studies tested whether approximately 3 months of DR (60% of ad libitum intake) altered the response to TD. Six month-old ad libitum-fed or dietary restricted C57BL/6 mice received a thiamine-deficient diet either ad libitum, or under a DR regimen respectively for eleven days. The TD mice also received daily injections of the thiamine antagonist pyrithiamine. Control ad libitum-fed or DR mice received an unlimited amount, or 60% of ad libitum intake, respectively, of thiamine-supplemented diet. As in past studies, TD produced region-selective neuronal loss (-60%), HO-1 induction, and IgG extravasation in the thalamus of ad libitum-fed mice. DR attenuated the TD-induced neuronal loss (-30%), HO-1 induction and IgG extravasation in the thalamus. These studies suggest that oxidative damage is critical to the pathogenesis of TD, and that DR modulates the extent of free radical damage in the brain. Thus, TD is an important model for studying the relationship between aging, oxidative stress and nutrition.

Asunto(s)

Barrera Hematoencefálica/fisiología , Ingestión de Energía/fisiología , Hemo Oxigenasa (Desciclizante)/metabolismo , Degeneración Nerviosa/metabolismo , Neuronas/enzimología , Deficiencia de Tiamina/metabolismo , Envejecimiento/metabolismo , Envejecimiento/patología , Animales , Conducta Animal/fisiología , Muerte Celular/fisiología , Hemo Oxigenasa (Desciclizante)/análisis , Hemo Oxigenasa (Desciclizante)/inmunología , Hemo-Oxigenasa 1 , Inmunoglobulina G/metabolismo , Inmunoglobulina G/farmacología , Proteínas de la Membrana , Ratones , Ratones Endogámicos C57BL , Degeneración Nerviosa/patología , Neuronas/patología , Estrés Oxidativo/fisiología , Tálamo/irrigación sanguínea , Tálamo/citología , Tálamo/metabolismo , Deficiencia de Tiamina/patología

RESUMEN

Oxidative stress occurs in brains of Alzheimer's disease (AD) patients. A major question in AD research is whether the oxidative stress is just secondary to neurodegeneration. To test whether oxidative stress is an inherent property of AD tissues, the ability of cultured fibroblasts bearing the AD Presenilin-1 246 Ala-->Glu mutation to handle reactive oxygen species (ROS) was compared to controls. Although ROS in cells from AD subjects were only slightly less than cells from controls under basal conditions (-10%) or after exposure to H(2)O(2) (-16%), treatment with antioxidants revealed clear differences. Pretreatment with DMSO, a hydroxyl radical scavenger, reduced basal and H(2)O(2)-induced ROS levels significantly more in cells from controls (-22%, -22%) than in those from AD subjects (-4%, +14%). On the other hand, pretreatment with Trolox diminished H(2)O(2)-induced ROS significantly more in cells from AD (-60%) than control subjects (-39%). In summary, cells from AD patients have greater Trolox sensitive ROS and less DMSO sensitive ROS than controls. The results demonstrate that fibroblasts bearing this PS-1 mutation have altered means of handling oxidative stress and appear useful for determining the mechanism underlying the altered redox metabolism.

Asunto(s)

Enfermedad de Alzheimer/metabolismo , Antioxidantes/metabolismo , Proteínas de la Membrana/farmacología , Adulto , Anciano , Enfermedad de Alzheimer/genética , Antioxidantes/farmacología , Células Cultivadas , Cromanos/farmacología , Dimetilsulfóxido/farmacología , Femenino , Fibroblastos/efectos de los fármacos , Fluoresceínas , Colorantes Fluorescentes , Depuradores de Radicales Libres/farmacología , Humanos , Peróxido de Hidrógeno , Concentración de Iones de Hidrógeno , Masculino , Proteínas de la Membrana/genética , Persona de Mediana Edad , Mutación , Estrés Oxidativo , Presenilina-1 , Especies Reactivas de Oxígeno/metabolismo

RESUMEN

Brain metabolism and the activity of the alpha-ketoglutarate dehydrogenase complex (KGDHC), a mitochondrial enzyme, are diminished in brains from patients with Alzheimer's disease (AD). In 109 subjects, the Clinical Dementia Rating (CDR) score was highly correlated with brain KGDHC activity. In AD patients who carried the epsilon 4 allele of the apolipoprotein E gene (ApoE4), the CDR score correlated better with KGDHC activity than with the densities of neuritic plaques or neuritic tangles. In contrast, in patients without ApoE4, the CDR score correlated significantly better with tangles and plaques than with KGDHC activity. The results suggest that mitochondrial/oxidative damage may be more important for the cognitive dysfunction in AD patients who carry ApoE4 than in those who do not.

Asunto(s)

Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Apolipoproteínas E/genética , Mitocondrias/genética , Mitocondrias/patología , Anciano , Análisis de Varianza , Encéfalo/patología , Femenino , Genotipo , Humanos , Masculino

RESUMEN

Alzheimer disease (AD) is a form of the dementia syndrome. AD appears to have a variety of fundamental etiologies that lead to the neuropathological manifestations which define the disease. Patients who are at high risk to develop AD typically show impairments of cerebral metabolic rate in vivo even before they show any evidence of the clinical disease on neuropsychological, electrophysiological, and neuroimaging examinations. Therefore, impairment in energy metabolism in AD can not be attributed to loss of brain substance or to electrophysiological abnormalities. Among the characteristic abnormalities in the AD brain are deficiencies in several enzyme complexes which participate in the mitochondrial oxidation of substrates to yield energy. There include the pyruvate dehydrogenase complex (PDHC), the alpha-ketoglutarate dehydrogenase complex (KGDHC), and Complex IV of the electron transport chain (COX). The deficiency of KGDHC may be due to a mixture of causes including damage by free radicals and perhaps to genetic variation in the DLST gene encoding the core protein of this complex. Inherent impairment of glucose oxidation by the AD brain may reasonably be expected to interact synergistically with an impaired supply of oxygen and glucose to the AD brain, in causing brain damage. These considerations lead to the hypothesis that cerebrovascular compromise and inherent abnormalities in the brain's ability to oxidize substrates can interact to favor the development of AD, in individuals who are genetically predisposed to develop neuritic plaques.

Asunto(s)

Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/fisiopatología , Encéfalo/metabolismo , Circulación Cerebrovascular/fisiología , Metabolismo Energético , Encéfalo/irrigación sanguínea , Encéfalo/fisiopatología , Humanos , Complejo Cetoglutarato Deshidrogenasa/metabolismo , Modelos Neurológicos , Degeneración Nerviosa , Consumo de Oxígeno

RESUMEN

Free radical production in vascular endothelial cells and inflammatory responses in perivascular microglia accompany the selective neuronal death induced by TD. Lipid peroxidation and tyrosine nitration occur in neurons within susceptible areas. Thus, region- and cell-specific oxidative stress contributes to selective neurodegeneration during TD. These data are consistent with the hypothesis that in TD, vascular factors constitute a critical part of a cascade of events leading to increases in blood-brain barrier permeability to nonneuronal proteins and iron, leading to inflammation and oxidative stress. Inflammatory cells may release deleterious compounds or cytokines that exacerbate the oxidative damage to metabolically compromised neurons. Similar mechanisms may operate in the pathophysiology of neurodegenerative diseases in which vascular factors, inflammation and oxidative stress are implicated including AD.

Asunto(s)

Endotelio Vascular/metabolismo , Inflamación/fisiopatología , Neuronas/patología , Deficiencia de Tiamina/fisiopatología , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Animales , Circulación Cerebrovascular , Modelos Animales de Enfermedad , Radicales Libres/metabolismo , Humanos , Óxido Nítrico Sintasa/metabolismo , Deficiencia de Tiamina/patología

RESUMEN

Lipid alterations have been reported in brain regions affected by Alzheimer disease (AD). The mechanisms causing these changes are poorly understood because it is difficult to study dynamic, biochemical processes in post-mortem brain. Fibroblasts derived from AD patients offer an alternative model to study disease-related alterations in lipid metabolism. Therefore, we measured the phospholipid levels and composition of fibroblasts from individuals bearing two different presenilin-1 mutations and compared these values to appropriate control fibroblasts. There were no differences between groups in phospholipid composition or in individual phospholipid levels, including the plasmalogens. Cholesterol levels and the cholesterol/phospholipid ratio were not different between presenilin-1 mutation bearing and control fibroblasts. Although these presenilin-1 mutation bearing fibroblasts have a number of biochemical changes related to AD, the absence of a change in phospholipid levels suggests that under these conditions, these cells are not useful in studying the mechanisms underlying the alterations in brain phospholipid levels associated with AD. However, these results do not preclude the possible use of other fibroblasts bearing AD-related mutations, e.g., APP mutations, to examine AD-related changes in brain lipid metabolism, or of these fibroblasts under different conditions.

Asunto(s)

Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Proteínas de la Membrana/genética , Fosfolípidos/metabolismo , Mutación Puntual , Células Cultivadas , Colesterol/análisis , Colesterol/metabolismo , Salud de la Familia , Fibroblastos/química , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Fosfolípidos/análisis , Plasmalógenos/análisis , Plasmalógenos/metabolismo , Presenilina-1

RESUMEN

Thiamine deficiency (TD) models the cellular and molecular mechanisms by which chronic oxidative deficits lead to death of select neurons in brain. Region- and cell-specific oxidative stress and vascular changes accompany the TD-induced neurodegeneration. The current studies analyzed the role of oxidative stress in initiating these events by testing the role of intercellular adhesion molecule-1 (ICAM-1) and endothelial nitric oxide synthase (eNOS) in the selective neuronal loss that begins in the submedial thalamic nucleus of mice. Oxidative stress to microvessels is known to induce eNOS and ICAM-1. TD increased ICAM-1 immunoreactivity in microvessels within the submedial nucleus and adjacent regions 1 day prior to the onset of neuronal loss. On subsequent days, the pattern of ICAM-1 induction overlapped that of neuronal loss, and of induction of the oxidative stress marker heme oxygenase-1 (HO-1). The intensity and extent of ICAM-1 and HO-1 induction progressively spread in parallel with the neuronal death in the thalamus. Targeted disruption of ICAM-1 or eNOS gene, but not the neuronal NOS gene, attenuated the TD-induced neurodegeneration and HO-1 induction. TD induced ICAM-1 in eNOS knockout mice, but did not induce eNOS in mice lacking ICAM-1. These results demonstrate that in TD, an ICAM-1-dependent pathway of eNOS induction leads to oxidative stress-mediated death of metabolically compromised neurons. Thus, TD provides a useful model to help elucidate the role of ICAM-1 and eNOS in the selective neuronal death in diseases in which oxidative stress is implicated.

Asunto(s)

Molécula 1 de Adhesión Intercelular/genética , Neuronas/enzimología , Óxido Nítrico Sintasa/genética , Estrés Oxidativo/fisiología , Deficiencia de Tiamina/metabolismo , Animales , Anticuerpos Monoclonales/farmacología , Conducta Animal , Biomarcadores , Barrera Hematoencefálica/fisiología , Peso Corporal , Eliminación de Gen , Genotipo , Hemo Oxigenasa (Desciclizante)/metabolismo , Hemo-Oxigenasa 1 , Inmunoglobulina G/farmacología , Molécula 1 de Adhesión Intercelular/análisis , Molécula 1 de Adhesión Intercelular/inmunología , Proteínas de la Membrana , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutagénesis/fisiología , Degeneración Nerviosa/metabolismo , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/inmunología , Óxido Nítrico Sintasa/análisis , Óxido Nítrico Sintasa/inmunología , Óxido Nítrico Sintasa de Tipo I , Óxido Nítrico Sintasa de Tipo II , Óxido Nítrico Sintasa de Tipo III , Peroxidasa/análisis , Peroxidasa/inmunología , Tálamo/irrigación sanguínea , Tálamo/enzimología

RESUMEN

Altered energy metabolism is characteristic of many neurodegenerative disorders. Reductions in the key mitochondrial enzyme complex, the alpha-ketoglutarate dehydrogenase complex (KGDHC), occur in a number of neurodegenerative disorders including Alzheimer's Disease (AD). The reductions in KGDHC activity may be responsible for the decreases in brain metabolism, which occur in these disorders. KGDHC can be inactivated by several mechanisms, including the actions of free radicals (Reactive Oxygen Species, ROS). Other studies have associated specific forms of one of the genes encoding KGDHC (namely the DLST gene) with AD, Parkinson's disease, as well as other neurodegenerative diseases. Reductions in KGDHC activity can be plausibly linked to several aspects of brain dysfunction and neuropathology in a number of neurodegenerative diseases. Further studies are needed to assess mechanisms underlying the sensitivity of KGDHC to oxidative stress and the relation of KGDHC deficiency to selective vulnerability in neurodegenerative diseases.

Asunto(s)

Complejo Cetoglutarato Deshidrogenasa , Enfermedades Neurodegenerativas/enzimología , Encéfalo/enzimología , Encéfalo/patología , Encéfalo/fisiopatología , Regulación de la Expresión Génica , Humanos , Complejo Cetoglutarato Deshidrogenasa/análisis , Complejo Cetoglutarato Deshidrogenasa/genética , Complejo Cetoglutarato Deshidrogenasa/metabolismo , Enfermedades Neurodegenerativas/patología , Enfermedades Neurodegenerativas/fisiopatología

RESUMEN

Abnormalities in oxidative metabolism and inflammation accompany many neurodegenerative diseases. Thiamine deficiency (TD) is an animal model in which chronic oxidative stress and inflammation lead to selective neuronal death, whereas other cell types show an inflammatory response. Therefore, the current studies determined the response of different brain cell types to TD and/or inflammation in vitro and tested whether their responses reflect inherent properties of the cells. The cells that have been implicated in TD-induced neurotoxicity, including neurons, microglia, astrocytes, and brain endothelial cells, as well as neuroblastoma and BV-2 microglial cell lines, were cultured in either thiamine-depleted media or in normal culture media with amprolium, a thiamine transport inhibitor. The activity levels of a key mitochondrial enzyme, alpha-ketoglutarate dehydrogenase complex (KGDHC), were uniquely distributed among different cell types: The highest activity was in the endothelial cells, and the lowest was in primary microglia and neurons. The unique distribution of the activity did not account for the selective response to TD. TD slightly inhibited general cellular dehydrogenases in all cell types, whereas it significantly reduced the activity of KGDHC exclusively in primary neurons and neuroblastoma cells. Among the cell types tested, only in neurons did TD induce apoptosis and cause the accumulation of 4-hydroxy-2-nonenal, a lipid peroxidation product. On the other hand, chronic lipopolysaccharide-induced inflammation significantly inhibited cellular dehydrogenase and KGDHC activities in microglia and astrocytes but not in neurons or endothelial cells. The results demonstrate that the selective cell changes during TD in vivo reflect inherent properties of the different brain cell types.

Asunto(s)

Encéfalo/metabolismo , Neuronas/fisiología , Estrés Oxidativo/fisiología , Aldehídos/metabolismo , Amprolio , Animales , Apoptosis , Encéfalo/citología , Muerte Celular/fisiología , Células Cultivadas , Colorantes , Complejo Cetoglutarato Deshidrogenasa/antagonistas & inhibidores , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos C57BL , Neuronas/clasificación , Neuronas/enzimología , Neuronas/metabolismo , Oxidorreductasas/metabolismo , Sales de Tetrazolio/farmacocinética , Deficiencia de Tiamina/inducido químicamente , Deficiencia de Tiamina/enzimología , Tiazoles/farmacocinética