RESUMEN
Alzheimer's disease is the most common neurodegenerative disease of the central nervous system characterized by a progressive loss in memory and deterioration of cognitive functions. In this study the transgenic mouse TgCRND8, which encodes a mutant form of the amyloid precursor protein 695 with both the Swedish and Indiana mutations and develops extracellular amyloid beta-peptide deposits as early as 2-3 months, was investigated. Extract from eight brain regions (cortex, frontal cortex, cerebellum, hippocampus, olfactory bulb, pons, midbrain and striatum) were studied using (1)H NMR spectroscopy. Analysis of the NMR spectra discriminated control from APP695 tissues in hippocampus, cortex, frontal cortex, midbrain and cerebellum, with hippocampal and cortical region being most affected. The analysis of the corresponding loading plots for these brain regions indicated a decrease in N-acetyl-L-aspartate, glutamate, glutamine, taurine (exception hippocampus), gamma-amino butyric acid, choline and phosphocholine (combined resonances), creatine, phosphocreatine and succinate in hippocampus, cortex, frontal cortex (exception gamma-amino butyric acid) and midbrain of affected animals. An increase in lactate, aspartate, glycine (except in midbrain) and other amino acids including alanine (exception frontal cortex), leucine, iso-leucine, valine and water soluble free fatty acids (0.8-0.9 and 1.2-1.3 ppm) were observed in the TgCRND8 mice. Our findings demonstrate that the perturbations in metabolism are more widespread and include the cerebellum and midbrain. Furthermore, metabolic perturbations are associated with a wide range of metabolites which could improve the diagnosis and monitoring of the progression of Alzheimer's disease.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Modelos Animales de Enfermedad , Metabolómica/métodos , Enfermedad de Alzheimer/diagnóstico , Animales , Biomarcadores/metabolismo , Encéfalo/metabolismo , Encéfalo/patología , Femenino , Humanos , Masculino , Metaboloma/genética , Ratones , Ratones TransgénicosRESUMEN
The role of cellular phosphatidylinositol 5-phosphate (PtdIns5P), as a signalling molecule or as a substrate for the production of small, compartmentalized pools of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)], may be dependent on cell type and subcellular localization. PtdIns5P levels are primarily regulated by the PtdIns5P 4-kinases (type II PIP kinases or PIP4Ks), and we have investigated the expression and localization in the brain of the least-studied PIP4K isoform, PIP4Kgamma. In situ hybridization and immunohistochemistry, using antisense oligonucleotide probes and a PIP4Kgamma-specific antibody, revealed that this isoform has a restricted CNS expression profile. The use of antibodies to different cell markers showed that this expression is limited to neurons, particularly the cerebellar Purkinje cells, pyramidal cells of the hippocampus, large neuronal cell types in the cerebral cortex including pyramidal cells, and mitral cells in the olfactory bulb and is not expressed in cerebellar, hippocampal formation, or olfactory bulb granule cells. In neurons expressing this enzyme, PIP4Kgamma has a vesicular distribution and shows partial colocalization with markers of cellular compartments of the endomembrane trafficking pathway. The PIP4Kgamma isoform expression is established after day 7 of postnatal development. Overall, our data suggest that PIP4Kgamma may have a role in neuron function, specifically in the regulation of vesicular transport, in specific regions of the developed brain.
Asunto(s)
Encéfalo/enzimología , Encéfalo/crecimiento & desarrollo , Neuronas/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Animales , Animales Recién Nacidos , Encéfalo/metabolismo , Células Cultivadas , Vesículas Citoplasmáticas/enzimología , Vesículas Citoplasmáticas/metabolismo , Ganglios Espinales/enzimología , Ganglios Espinales/metabolismo , Isoenzimas/metabolismo , Masculino , Ratones , Ratones Endogámicos , Neuronas/metabolismo , Médula Espinal/enzimología , Médula Espinal/metabolismo , Fracciones Subcelulares/enzimología , Fracciones Subcelulares/metabolismo , Factores de TiempoRESUMEN
Missense mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common causes of both familial and sporadic forms of Parkinson disease and are also associated with diverse pathological alterations. The mechanisms whereby LRRK2 mutations cause these pathological phenotypes are unknown. We used immunohistochemistry with 3 distinct anti-LRRK2 antibodies to characterize the expression of LRRK2 in the brains of 21 subjects with various neurodegenerative disorders and 7 controls. The immunoreactivity of LRRK2 was localized in a subset of brainstem-type Lewy bodies (LBs) but not in cortical-type LBs, tau-positive inclusions, or TAR-DNA-binding protein-43-positive inclusions. The immunoreactivity of LRRK2 frequently appeared as enlarged granules or vacuoles within neurons of affected brain regions, including the substantia nigra, amygdala, and entorhinal cortex in patients with Parkinson disease or dementia with LBs. The volumes of LRRK2-positive granular structures in neurons of the entorhinal cortex were significantly increased in dementia with LBs brains compared with age-matched control brains (p < 0.05). Double immunolabeling demonstrated that these LRRK2-positive granular structures frequently colocalized with the late-endosomal marker Rab7B and occasionally with the lysosomal marker, the lysosomal-associated membrane protein 2. These results suggest that LRRK2 normally localizes to the endosomal-lysosomal compartment within morphologically altered neurons in neurodegenerative diseases, particularly in the brains of patients with LB diseases.
Asunto(s)
Encéfalo/patología , Endosomas/patología , Enfermedad por Cuerpos de Lewy/patología , Lisosomas/patología , Neuronas/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Anciano , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Encéfalo/metabolismo , Demencia/metabolismo , Demencia/patología , Endosomas/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , Cuerpos de Inclusión/metabolismo , Cuerpos de Inclusión/patología , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Enfermedad por Cuerpos de Lewy/metabolismo , Lisosomas/metabolismo , Masculino , Persona de Mediana Edad , Neuronas/metabolismo , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Enfermedad de Pick/metabolismo , Enfermedad de Pick/patología , Parálisis Supranuclear Progresiva/metabolismo , Parálisis Supranuclear Progresiva/patologíaRESUMEN
We have shown that somatostatin released from activated capsaicin-sensitive nociceptive nerve endings during inflammatory processes elicits systemic anti-inflammatory and analgesic effects. With the help of somatostatin receptor subtype 4 gene-deleted mice (sst(4)(-/-)), we provide here several lines of evidence that this receptor has a protective role in a variety of inflammatory disease models; several symptoms are more severe in the sst(4) knockout animals than in their wild-type counterparts. Acute carrageenan-induced paw edema and mechanical hyperalgesia, inflammatory pain in the early phase of adjuvant-evoked chronic arthritis, and oxazolone-induced delayed-type hypersensitivity reaction in the skin are much greater in mice lacking the sst(4) receptor. Airway inflammation and consequent bronchial hyperreactivity elicited by intranasal lipopolysaccharide administration are also markedly enhanced in sst(4) knockouts, including increased perivascular/peribronchial edema, neutrophil/macrophage infiltration, mucus-producing goblet cell hyperplasia, myeloperoxidase activity, and IL-1beta, TNF-alpha, and IFN-gamma expression in the inflamed lung. It is concluded that during these inflammatory conditions the released somatostatin has pronounced counterregulatory effects through sst(4) receptor activation. Thus, this receptor is a promising novel target for developing anti-inflammatory, analgesic, and anti-asthmatic drugs.
Asunto(s)
Hiperreactividad Bronquial/etiología , Hiperalgesia/etiología , Inflamación/etiología , Receptores de Somatostatina/fisiología , Animales , Hiperreactividad Bronquial/prevención & control , Dermatitis Alérgica por Contacto/etiología , Femenino , Hiperalgesia/prevención & control , Inflamación/prevención & control , Lipopolisacáridos/toxicidad , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oxazolona/toxicidad , Receptores de Somatostatina/deficiencia , Receptores de Somatostatina/genéticaRESUMEN
A hypofunction of the N-methyl-D-aspartate (NMDA) receptor has been implicated in the pathophysiology of schizophrenia. Compelling evidence of altered NMDA receptor subunit expression in the schizophrenic brain has not, however, so far emerged. Rats reared in isolation exhibit several characteristics, including disturbed sensory gating, which resemble those seen in schizophrenia. To explore the possibility that NMDA receptor dysfunction may contribute to the behavioral and neurochemical consequences of rearing rats in isolation, we compared NMDA receptor subunit expression in brains of rats which were housed in isolation and which displayed a deficit in prepulse inhibition of the acoustic startle response with that of socially housed controls. An initial microarray analysis revealed a 1.26-fold increase in NR2A transcript in the prefrontal cortex, but not in the nucleus accumbens, of rats reared in isolation compared with those housed socially. In contrast, NR1, NR2B, NR2C, NR2D, NR3A, and NR3B subunit expression was unchanged in either brain area. In a second cohort of animals, in situ hybridization revealed increased NR2A mRNA expression in the medial prefrontal cortex, an observation that was substantiated by increased [(3)H]CGP39653 binding suggesting that NR2A receptor subunit protein expression was also elevated in the medial prefrontal cortex of the same animals. No changes in expression of NR1 or NR2B subunits were observed at both mRNA and protein level. Altered NR2A subunit expression in the medial prefrontal cortex of rats reared in isolation suggests that NMDA receptor dysfunction may contribute to the underlying pathophysiology of this preclinical model of aspects of schizophrenia.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Corteza Prefrontal/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Aislamiento Social , 2-Amino-5-fosfonovalerato/análogos & derivados , 2-Amino-5-fosfonovalerato/metabolismo , Estimulación Acústica/efectos adversos , Animales , Animales Recién Nacidos , Perfilación de la Expresión Génica/métodos , Indoles/metabolismo , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Unión Proteica/fisiología , Ensayo de Unión Radioligante/métodos , Ratas , Receptores de N-Metil-D-Aspartato/genética , Reflejo de Sobresalto/fisiología , Tritio/metabolismoRESUMEN
PIP4Ks (type II phosphatidylinositol 4-phosphate kinases) are phosphatidylinositol 5-phosphate (PtdIns5P) 4-kinases, believed primarily to regulate cellular PtdIns5P levels. In this study, we investigated the expression, localization, and associated biological activity of the least-studied PIP4K isoform, PIP4Kgamma. Quantitative RT-PCR and in situ hybridization revealed that compared with PIP4Kalpha and PIP4Kbeta, PIP4Kgamma is expressed at exceptionally high levels in the kidney, especially the cortex and outer medulla. A specific antibody was raised to PIP4Kgamma, and immunohistochemistry with this and with antibodies to specific kidney cell markers showed a restricted expression, primarily distributed in epithelial cells in the thick ascending limb and in the intercalated cells of the collecting duct. In these cells, PIP4Kgamma had a vesicular appearance, and transfection of kidney cell lines revealed a partial Golgi localization (primarily the matrix of the cis-Golgi) with an additional presence in an unidentified vesicular compartment. In contrast to PIP4Kalpha, bacterially expressed recombinant PIP4Kgamma was completely inactive but did have the ability to associate with active PIP4Kalpha in vitro. Overall our data suggest that PIP4Kgamma may have a function in the regulation of vesicular transport in specialized kidney epithelial cells.
Asunto(s)
Riñón/enzimología , Nefronas/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Vesículas Transportadoras/enzimología , Animales , Acuaporina 1/genética , Acuaporina 1/metabolismo , Autoantígenos/genética , Autoantígenos/metabolismo , Western Blotting , Células COS , Línea Celular , Chlorocebus aethiops , Expresión Génica , Aparato de Golgi/enzimología , Aparato de Golgi/metabolismo , Células HeLa , Humanos , Hibridación in Situ , Isoenzimas/genética , Isoenzimas/metabolismo , Riñón/citología , Riñón/metabolismo , Corteza Renal/citología , Corteza Renal/enzimología , Corteza Renal/metabolismo , Médula Renal/citología , Médula Renal/enzimología , Médula Renal/metabolismo , Asa de la Nefrona/citología , Asa de la Nefrona/enzimología , Asa de la Nefrona/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos , Antígenos de Histocompatibilidad Menor , Mucoproteínas/genética , Mucoproteínas/metabolismo , Nefronas/citología , Nefronas/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Vesículas Transportadoras/metabolismo , UromodulinaRESUMEN
The vesicular monoamine transporter 2 (VMAT2) controls the loading of dopamine (DA) into vesicles and therefore determines synaptic properties such as quantal size, receptor sensitivity, and vesicular and cytosolic DA concentration. Impairment of proper DA compartmentalization is postulated to underlie the sensitivity of DA neurons to oxidative damage and degeneration. It is known that DA can auto-oxidize in the cytosol to form quinones and other oxidative species and that this production of oxidative stress is thought to be a critical factor in DA terminal loss after methamphetamine (METH) exposure. Using a mutant strain of mice (VMAT2 LO), which have only 5-10% of the VMAT2 expressed by wild-type animals, we show that VMAT2 is a major determinant of METH toxicity in the striatum. Subsequent to METH exposure, the VMAT2 LO mice show an exacerbated loss of dopamine transporter and tyrosine hydroxylase (TH), as well as enhanced astrogliosis and protein carbonyl formation. More importantly, VMAT2 LO mice show massive argyrophilic deposits in the striatum after METH, indicating that VMAT2 is a regulator of METH-induced neurodegeneration. The increased METH neurotoxicity in VMAT2 LO occurs in the absence of any significant difference in basal temperature or METH-induced hyperthermia. Furthermore, primary midbrain cultures from VMAT2 LO mice show more oxidative stress generation and a greater loss of TH positive processes than wild-type cultures after METH exposure. Elevated markers of neurotoxicity in VMAT2 LO mice and cultures suggest that the capacity to store DA determines the amount of oxidative stress and neurodegeneration after METH administration.
Asunto(s)
Dopamina/metabolismo , Gliosis/inducido químicamente , Metanfetamina/toxicidad , Degeneración Nerviosa/inducido químicamente , Vesículas Sinápticas/efectos de los fármacos , Proteínas de Transporte Vesicular de Monoaminas/efectos de los fármacos , Trastornos Relacionados con Anfetaminas/metabolismo , Trastornos Relacionados con Anfetaminas/fisiopatología , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Biomarcadores/análisis , Biomarcadores/metabolismo , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/fisiopatología , Compartimento Celular/efectos de los fármacos , Compartimento Celular/fisiología , Células Cultivadas , Modelos Animales de Enfermedad , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/efectos de los fármacos , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Inhibidores de Captación de Dopamina/toxicidad , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Fiebre/inducido químicamente , Fiebre/metabolismo , Fiebre/fisiopatología , Gliosis/metabolismo , Gliosis/fisiopatología , Ratones , Ratones Noqueados , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/fisiopatología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Vesículas Sinápticas/metabolismo , Proteínas de Transporte Vesicular de Monoaminas/genéticaRESUMEN
Dopamine is a potentially toxic neurotransmitter that has long been speculated to contribute to the pathogenesis of Parkinson's disease (PD). Recent work has demonstrated the importance of proper storage of dopamine in vesicles to maintain dopamine homeostasis, thus protecting neurons from the detrimental effects of dopamine accumulation and breakdown in the cytosol. These studies suggest that factors which affect dopamine storage might increase the susceptibility of dopamine neurons to further environmental or genetic insults, exacerbating the neuronal degeneration that characterizes PD. This review seeks to revisit the pathogenicity of cytosolic dopamine and further address the critical role of neurotransmitter storage in dopamine-mediated neurotoxicity.
Asunto(s)
Dopamina/metabolismo , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Animales , Humanos , Neuronas/metabolismo , Enfermedad de Parkinson/genética , Sinucleínas/metabolismo , Proteínas de Transporte Vesicular de Monoaminas/genéticaRESUMEN
The vesicular monoamine transporter 2 (VMAT2) sequesters monoamines into synaptic vesicles in preparation for neurotransmission. Samples of cerebellum, cortex, hippocampus, substantia nigra and striatum from VMAT2-deficient mice were compared to age-matched control mice. Multivariate statistical analyses of (1)H NMR spectral profiles separated VMAT2-deficient mice from controls for all five brain regions. Although the data show that metabolic alterations are region- and age-specific, in general, analyses indicated decreases in the concentrations of taurine and creatine/phosphocreatine and increases in glutamate and N-acetyl aspartate in VMAT2-deficient mouse brain tissues. This study demonstrates the efficacy of metabolomics as a functional genomics phenotyping tool for mouse models of neurological disorders, and indicates that mild reductions in the expression of VMAT2 affect normal brain metabolism.
Asunto(s)
Envejecimiento/metabolismo , Encéfalo/metabolismo , Proteínas de Transporte Vesicular de Monoaminas/genética , Animales , Femenino , Masculino , Ratones , Resonancia Magnética Nuclear BiomolecularRESUMEN
Huntington's disease (HD) is caused by a CAG repeat expansion in the HD gene, but how this mutation causes neuronal dysfunction and degeneration is unclear. Inhibition of glutamate uptake, which could cause excessive stimulation of glutamate receptors, has been found in animals carrying very long CAG repeats in the HD gene. In seven HD patients with moderate CAG expansions (40-52), repeat expansion and HD grade at autopsy were strongly correlated (r=0.88, p=0.0002). Uptake of [(3)H]glutamate was reduced by 43% in prefrontal cortex, but the level of synaptic (synaptophysin, AMPA receptors) and astrocytic markers (GFAP, glutamate transporter EAAT1) were unchanged. Glutamate uptake correlated inversely with CAG repeat expansion (r= -0.82, p=0.015). The reducing agent dithiothreitol improved glutamate uptake in controls, but not in HD brains, suggesting irreversible oxidation of glutamate transporters in HD. We conclude that impairment of glutamate uptake may contribute to neuronal dysfunction and degeneration in HD.
Asunto(s)
Ácido Glutámico/metabolismo , Enfermedad de Huntington/metabolismo , Corteza Prefrontal/metabolismo , Astrocitos/patología , Transporte Biológico , Humanos , Corteza Prefrontal/patología , Repeticiones de TrinucleótidosRESUMEN
The vesicular monoamine transporter 2 (VMAT2; SLC18A2) is responsible for packaging dopamine into vesicles for subsequent release and has been suggested to serve a neuroprotective role in the dopamine system. Here, we show that mice that express approximately 5% of normal VMAT2 (VMAT2 LO) display age-associated nigrostriatal dopamine dysfunction that ultimately results in neurodegeneration. Elevated cysteinyl adducts to L-DOPA and DOPAC are seen early and are followed by increased striatal protein carbonyl and 3-nitrotyrosine formation. These changes were associated with decreased striatal dopamine and decreased expression of the dopamine transporter and tyrosine hydroxylase. Furthermore, we observed an increase in alpha-synuclein immunoreactivity and accumulation and neurodegeneration in the substantia nigra pars compacta in aged VMAT2 LO mice. Thus, VMAT2 LO animals display nigrostriatal degeneration that begins in the terminal fields and progresses to eventual loss of the cell bodies, alpha-synuclein accumulation, and an L-DOPA responsive behavioral deficit, replicating many of the key aspects of Parkinson's disease. These data suggest that mishandling of dopamine via reduced VMAT2 expression is, in and of itself, sufficient to cause dopamine-mediated toxicity and neurodegeneration in the nigrostriatal dopamine system. In addition, the altered dopamine homeostasis resulting from reduced VMAT2 function may be conducive to pathogenic mechanisms induced by genetic or environmental factors thought to be involved in Parkinson's disease.
Asunto(s)
Dopamina/metabolismo , Neostriado/metabolismo , Degeneración Nerviosa/metabolismo , Sustancia Negra/metabolismo , Vesículas Sinápticas/metabolismo , Proteínas de Transporte Vesicular de Monoaminas/metabolismo , Animales , Dopamina/genética , Dopamina/fisiología , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neostriado/patología , Degeneración Nerviosa/genética , Degeneración Nerviosa/patología , Sustancia Negra/patología , Vesículas Sinápticas/genética , Proteínas de Transporte Vesicular de Monoaminas/antagonistas & inhibidores , Proteínas de Transporte Vesicular de Monoaminas/genéticaRESUMEN
Abnormalities in microtubule-associated tau protein are a key neuropathological feature of both Alzheimer's disease and many frontotemporal dementias (FTDs), including hereditary FTD with Parkinsonism linked to chromosome 17 (FTDP-17). In these disorders, tau becomes aberrantly phosphorylated, leading to the development of filamentous neurofibrillary tangles in the brain. Here we report, in a longitudinal ageing study, the sensorimotor and cognitive assessment of transgenic mice expressing the human tau(V337M) ('Seattle Family A') FTDP-17 mutation, which we have previously shown to demonstrate abnormalities in brain tau phosphorylation. The data indicated highly specific effects of transgene expression on the ability to withhold responding in a murine version of the 5-choice serial reaction time task, behaviour consistent with deficits in impulse control. Ageing exacerbated these effects. In young tau(V337M) mice, increased impulsivity was present under task conditions making inhibition of premature responding more difficult (longer inter-trial intervals) but not under baseline conditions. However, when older, the tau(V337M) mice showed further increases in premature responding, including under baseline conditions. These impulse control deficits were fully dissociable from sensorimotor or motivation effects on performance. The findings recapitulate core abnormalities in impulsive responding observed in both frontal variant FTD and FTDP-17 linked to the tau(V337M) mutation in humans.
Asunto(s)
Demencia/genética , Trastornos Disruptivos, del Control de Impulso y de la Conducta/genética , Perfilación de la Expresión Génica , Mutación , Proteínas tau/genética , Factores de Edad , Envejecimiento , Animales , Encéfalo/metabolismo , Cromosomas Humanos Par 17 , Demencia/metabolismo , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Transgénicos , Médula Espinal/metabolismo , Factores de Tiempo , Proteínas tau/metabolismoRESUMEN
In the basal ganglia the effects of gamma-aminobutyrate (GABA) are mediated by both ionotropic (GABA(A)) and metabotropic (GABA(B)) receptors. Although the existence and widespread distribution in the CNS of the GABA(B) receptor had been established in the 1980s the field of GABA(B) research was revolutionized with the discovery that two related G-protein-coupled receptors (GPCRs) needed to dimerize to form the functional GABA(B) receptor at the cell surface. This finding lead to a number of studies of oligomerization in GPCRs and detailed pharmacological studies of the cloned receptors and their splice variants. Particular interest has focused on the proteins interacting with the receptor which may be important in mediating the longer term signalling effects of the receptor and modifying its cellular localization or physiology. The cloning of the GABA(B) receptors also lead to the identification of the first compounds interacting in an allosteric fashion with the receptor some of which may have therapeutic value. Most recently "knockouts" of both the GABA(B) subunits have been produced where in general as expected there is a loss of the majority of the inhibitory effects of the GABA(B) receptor.
Asunto(s)
Ganglios Basales/metabolismo , Receptores de GABA-B/fisiología , Regulación Alostérica/efectos de los fármacos , Regulación Alostérica/fisiología , Animales , Ganglios Basales/anatomía & histología , Moduladores del GABA/farmacología , Humanos , Estructura Molecular , Inhibición Neural/efectos de los fármacos , Inhibición Neural/fisiología , Subunidades de Proteína/efectos de los fármacos , Subunidades de Proteína/genética , Subunidades de Proteína/fisiología , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/efectos de los fármacos , Receptores Acoplados a Proteínas G/genética , Receptores de GABA-B/efectos de los fármacos , Receptores de GABA-B/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
Mutations in the LRRK2 gene cause autosomal dominant, late-onset parkinsonism, which presents with pleomorphic pathology including alpha-synucleopathy. To promote our understanding of the biological role of LRRK2 in the brain we examined the distribution of LRRK2 mRNA and protein in postmortem human brain tissue from normal and neuropathological subjects. In situ hybridization and immunohistochemical analysis demonstrate the expression and localization of LRRK2 to various neuronal populations in brain regions implicated in Parkinson's disease (PD) including the cerebral cortex, caudate-putamen and substantia nigra pars compacta. Immunofluorescent double labeling studies additionally reveal the prominent localization of LRRK2 to cholinergic-, calretinin- and GABA(B) receptor 1-positive, dopamine-innervated, neuronal subtypes in the caudate-putamen. The distribution of LRRK2 in brain tissue from sporadic PD and dementia with Lewy bodies (DLB) subjects was also examined. In PD brains, LRRK2 immunoreactivity localized to nigral neuronal processes is dramatically reduced which reflects the disease-associated loss of dopaminergic neurons in this region. However, surviving nigral neurons occasionally exhibit LRRK2 immunostaining of the halo structure of Lewy bodies. Moreover, LRRK2 immunoreactivity is not associated with Lewy neurites or with cortical Lewy bodies in sporadic PD and DLB brains. These observations indicate that LRRK2 is not a primary component of Lewy bodies and does not co-localize with mature fibrillar alpha-synuclein to a significant extent. The localization of LRRK2 to key neuronal populations throughout the nigrostriatal dopaminergic pathway is consistent with the involvement of LRRK2 in the molecular pathogenesis of familial and sporadic parkinsonism.
Asunto(s)
Encéfalo/enzimología , Trastornos Parkinsonianos/enzimología , Proteínas Serina-Treonina Quinasas/genética , Encéfalo/patología , Núcleo Caudado/enzimología , Núcleo Caudado/patología , Corteza Cerebral/enzimología , Corteza Cerebral/patología , Cuerpo Estriado/enzimología , Cuerpo Estriado/patología , Humanos , Hibridación in Situ , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Cuerpos de Lewy/enzimología , Cuerpos de Lewy/patología , Mutación , Neuronas/enzimología , Neuronas/patología , Trastornos Parkinsonianos/genética , Trastornos Parkinsonianos/patología , Putamen/enzimología , Putamen/patología , Valores de ReferenciaRESUMEN
Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) have been identified as the cause of familial Parkinson's disease (PD) at the PARK8 locus. To begin to understand the physiological role of LRRK2 and its involvement in PD, we have investigated the distribution of LRRK2 mRNA and protein in the adult mouse brain. In situ hybridization studies indicate sites of mRNA expression throughout the mouse brain, with highest levels of expression detected in forebrain regions, including the cerebral cortex and striatum, intermediate levels observed in the hippocampus and cerebellum, and low levels in the thalamus, hypothalamus and substantia nigra. Immunohistochemical studies demonstrate localization of LRRK2 protein to neurones in the cerebral cortex and striatum, and to a variety of interneuronal subtypes in these regions. Furthermore, expression of LRRK2 mRNA in the striatum of VMAT2-deficient mice is unaltered relative to wild-type littermate controls despite extensive dopamine depletion in this mouse model of parkinsonism. Collectively, our results demonstrate that LRRK2 is present in anatomical brain regions of direct relevance to the pathogenesis of PD, including the nigrostriatal dopaminergic pathway, in addition to other regions unrelated to PD pathology, and is likely to play an important role in the normal function of telencephalic forebrain neurones and other neuronal populations.
Asunto(s)
Encéfalo/metabolismo , Expresión Génica/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Monoaminas Biogénicas/metabolismo , Encéfalo/anatomía & histología , Recuento de Células/métodos , Proteínas Fluorescentes Verdes/biosíntesis , Inmunohistoquímica/métodos , Hibridación in Situ/métodos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Ratones , Ratones Mutantes , Proteínas del Tejido Nervioso/metabolismo , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Proteínas de Transporte Vesicular de Monoaminas/genéticaRESUMEN
The successfully functioning brain is a heavy user of metabolic energy. Alzheimer's disease, in which cognitive faculties decline, may be due, at least in part, to metabolic insufficiency. Using microarray analysis and quantitative RT-PCR, the expression of mRNA transcripts involved in glucose metabolism was investigated in Alzheimer's diseased post-mortem human hippocampal samples. Of the 51 members of the glycolytic, tricarboxylic acid cycle, oxidative phosphorylation, and associated pathways investigated by qPCR, 15 were confirmed to be statistically significantly (p<0.05) down-regulated in Alzheimer's disease. This finding suggests that reductions in the levels of transcripts encoded by genes that participate in energy metabolism may be involved in Alzheimer's disease.
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Enfermedad de Alzheimer/enzimología , Enfermedad de Alzheimer/genética , Encéfalo/enzimología , Enzimas/genética , Regulación Enzimológica de la Expresión Génica/genética , Redes y Vías Metabólicas/genética , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/fisiopatología , Encéfalo/fisiopatología , Ciclo del Ácido Cítrico/genética , Regulación hacia Abajo/genética , Metabolismo Energético/genética , Femenino , Perfilación de la Expresión Génica , Glucólisis/genética , Humanos , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosforilación Oxidativa , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Dopamine cytotoxicity is thought to contribute towards the selective loss of substantia nigra pars compacta dopamine neurons and disease progression in Parkinson's disease. However, the long-term toxicity of dopamine in vivo has not previously been established. The vesicular monoamine transporter 2 (VMAT2) sequesters monoamines into synaptic vesicles, a process that, in addition to being important in normal transmission, may also act to keep intracellular levels of monoamine neurotransmitters below potentially toxic thresholds. The homozygous VMAT2-hypomorphic mouse has an insertion in the VMAT2 gene (Slc18a2). Consequently, VMAT2-deficient mice (VD(-/-)) have an approximately 95% reduction in VMAT2 expression and an equivalent level of dopamine depletion in the striatum which results in moderate motor impairment. Here, we show that L-DOPA induces locomotor hyperactivity in VD(-/-) mice and reverses the deficit in motor coordination and balance as tested with the rotarod. We report that evidence for cytosolic accumulation of dopamine in substantia nigra neurons in these mice is two-fold: firstly, there is reduced phosphorylation of tyrosine hydroxylase at the residue associated with catechol feedback inhibition; and, secondly, there are increased rates of dopamine turnover at 6, 12 and 24 months of age. These animals exhibit a progressive decline in striatal monoamine levels and rotarod performance with increasing age. However, despite these data, there was no loss of nigral dopamine neurons as estimated by quantification of tyrosine hydroxylase-immunoreactive cells in the substantia nigra pars compacta of old VD(-/-) mice (24-month-old), implying that these age-dependent manifestations may be due to senescence alone.
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Envejecimiento , Cuerpo Estriado/metabolismo , Dopamina/metabolismo , Actividad Motora/fisiología , Enfermedad de Parkinson/fisiopatología , Animales , Antiparkinsonianos/farmacología , Western Blotting , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/patología , Modelos Animales de Enfermedad , Inmunohistoquímica , Levodopa/farmacología , Masculino , Ratones , Ratones Mutantes , Actividad Motora/efectos de los fármacos , Destreza Motora/efectos de los fármacos , Destreza Motora/fisiología , Degeneración Nerviosa/patología , Fosforilación , Sustancia Negra/patología , Tirosina 3-Monooxigenasa/efectos de los fármacos , Tirosina 3-Monooxigenasa/metabolismo , Proteínas de Transporte Vesicular de Monoaminas/deficienciaRESUMEN
OBJECTIVE: The PARK8 gene responsible for late-onset autosomal dominant Parkinson's disease encodes a large novel protein of unknown biological function termed leucine-rich repeat kinase 2 (LRRK2). The studies herein explore the localization of LRRK2 in the mammalian brain. METHODS: Polyclonal antibodies generated against the amino or carboxy termini of LRRK2 were used to examine the biochemical, subcellular, and immunohistochemical distribution of LRRK2. RESULTS: LRRK2 is detected in rat brain as an approximate 280kDa protein by Western blot analysis. Subcellular fractionation demonstrates the presence of LRRK2 in microsomal, synaptic vesicle-enriched and synaptosomal cytosolic fractions from rat brain, as well as the mitochondrial outer membrane. Immunohistochemical analysis of rat and human brain tissue and primary rat cortical neurons, with LRRK2-specific antibodies, shows widespread neuronal-specific labeling localized exclusively to punctate structures within perikarya, dendrites, and axons. Confocal colocalization analysis of primary cortical neurons shows partial yet significant overlap of LRRK2 immunoreactivity with markers specific for mitochondria and lysosomes. Furthermore, ultrastructural analysis in rodent basal ganglia detects LRRK2 immunoreactivity associated with membranous and vesicular intracellular structures, including lysosomes, endosomes, transport vesicles, and mitochondria. INTERPRETATION: The association of LRRK2 with a variety of membrane and vesicular structures, membrane-bound organelles, and microtubules suggests an affinity of LRRK2 for lipids or lipid-associated proteins and may suggest a potential role in the biogenesis and/or regulation of vesicular and membranous intracellular structures within the mammalian brain.
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Encéfalo/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas R-SNARE/genética , Proteínas R-SNARE/metabolismo , Animales , Afinidad de Anticuerpos , Transporte Biológico , Western Blotting , Encéfalo/citología , Humanos , Inmunohistoquímica , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Ratones , Neuronas/metabolismo , Ratas , Fracciones Subcelulares/metabolismoRESUMEN
Dysfunction of the 140 aa protein alpha-synuclein plays a central role in Lewy body disorders, including Parkinson's disease, as well as in multiple system atrophy. Here, we show that the expression of truncated human alpha-synuclein(1-120), driven by the rat tyrosine hydroxylase promoter on a mouse alpha-synuclein null background, leads to the formation of pathological inclusions in the substantia nigra and olfactory bulb and to a reduction in striatal dopamine levels. At the behavioral level, the transgenic mice showed a progressive reduction in spontaneous locomotion and an increased response to amphetamine. These findings suggest that the C-terminal of alpha-synuclein is an important regulator of aggregation in vivo and will help to understand the mechanisms underlying the pathogenesis of Lewy body disorders and multiple system atrophy.
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Dopamina/fisiología , Cuerpos de Lewy/patología , Enfermedad por Cuerpos de Lewy/patología , Neuronas/patología , Bulbo Olfatorio/patología , Sustancia Negra/patología , alfa-Sinucleína/genética , Animales , Humanos , Enfermedad por Cuerpos de Lewy/genética , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Transgénicos , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Regiones Promotoras Genéticas , Ratas , Tirosina 3-Monooxigenasa/genéticaRESUMEN
Although chronic stress is known to be linked with memory and other neurological disorders, little is known about the relationship between chronic stress and the onset or development of Alzheimer's disease (AD). In this study, we investigated the effects of long-term stress on the onset and severity of cognitive deficits and pathological changes in APPV717I-CT100 mice overexpressing human APP-CT100 containing the London mutation (V717I) after exposure to immobilization stress. We found that chronic immobilization stress accelerated cognitive impairments, as accessed by the Passive avoidance and the Social Transfer of Food Preference (STFP) tests. Moreover, the numbers and densities of vascular and extracellular deposits containing amyloid beta peptide (Abeta) and carboxyl-terminal fragments of amyloid precursor protein (APP-CTFs), which are pathologic markers of AD, were significantly elevated in stressed animals, especially in the hippocampus. Moreover, stressed animals, also showed highly elevated levels of neurodegeneration and tau phosphorylation and increased intraneuronal Abeta and APP-CTFs immunoreactivities in the hippocampus and in the entorhinal and piriform cortex. This study provides the first evidence that chronic stress accelerates the onset and severity of cognitive deficits and that these are highly correlated with pathological changes, which thus indicates that chronic stress may be an important contributor to the onset and development of AD.