RESUMEN
Understanding how specific proteins are degraded by neurons in living animals is a fundamental question with relevance to many neurodegenerative diseases. Dysfunction in the ubiquitin-proteasome system (UPS) specifically has been implicated in several important neurodegenerative diseases including Alzheimer's Disease, Parkinson's Disease, and amyotrophic lateral sclerosis. Research in this area has been limited by the fact that many inhibitors of the UPS given systemically do not cross the blood-brain barrier (BBB) in appreciable levels. This limits the ability to easily test in vivo specific hypotheses generated in reduced systems, like brain slice or dissociated cell culture, about whether the UPS may degrade a particular protein of interest. Although several techniques including intracerebral application via direct syringe injection, catheter-pump systems and drug-eluting beads are available to introduce BBB-impermeant drugs into brain they each have certain limitations and new approaches could provide further insights into this problem. In order to test the role of the UPS in protein degradation in vivo we have developed a strategy to treat mouse cortex with the UPS inhibitor clasto-lactacystin beta-lactone (CLBL) via a "cranial window" and recover the treated tissue for immunoblot analysis. This approach can be used in several different cranial window configurations including single window and double hemi-window arrangements that are tailored for different applications. We have also developed two different strategies for recovering treated cortical tissue including a vibratome/laser capture microscopy (LCM)-based and a vibratome only-based approach, each with its own specific advantages. We have documented UPS inhibition >600µm deep into the cortex with this strategy. This set of techniques in the living mammalian brain is complementary to previously developed approaches and extends the repertoire of tools that can be used to the study protein degradation pathways relevant to neurodegenerative disease.
Asunto(s)
Enfermedades Neurodegenerativas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina/metabolismo , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacología , Animales , Western Blotting , Craneotomía , Inhibidores de Cisteína Proteinasa/farmacología , Modelos Animales de Enfermedad , Humanos , Ratones , Microscopía Confocal , MicrotomíaRESUMEN
Senile plaques are a major pathological hallmark of Alzheimer's disease (AD). Compelling evidence suggests that senile plaques lead to structural alterations of neuronal processes and that local toxicity may be mediated by increased oxidative stress. Anti-oxidant therapy can alleviate the neuronal abnormalities in APP mice, but the time-course of this beneficial effect is unknown. We used multiphoton microscopy to assess in vivo the characteristics of antioxidant treatment on senile plaques and neurites in AD model mice (APPswe/PS1dE9). We observed that α-phenyl-N-tert-butyl nitrone (PBN), Ginkgo biloba extract (EGb 761) and Trolox had no effect on the size of existing senile plaques. However, all anti-oxidants had a straightening effect on curved neurites. This effect was detected as soon as 4 days after commencing the treatment, and was maintained after 1 month of daily treatment, with no further increase in the effect. The straightening of neurites persisted 15 days after stopping the treatment. These data indicate that neuronal plasticity is fast and still active in adult animals, and suggest that amelioration of the neuritic distortions associated with senile plaques with antioxidants is both rapid and long lasting.
Asunto(s)
Precursor de Proteína beta-Amiloide/genética , Antioxidantes/farmacología , Estrés Oxidativo/efectos de los fármacos , Placa Amiloide/tratamiento farmacológico , Placa Amiloide/patología , Presenilina-1/genética , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Precursor de Proteína beta-Amiloide/antagonistas & inhibidores , Animales , Cromanos/farmacología , Óxidos N-Cíclicos/farmacología , Modelos Animales de Enfermedad , Depuradores de Radicales Libres/antagonistas & inhibidores , Depuradores de Radicales Libres/farmacología , Ginkgo biloba , Ratones , Ratones Transgénicos , Estrés Oxidativo/genética , Extractos Vegetales/farmacología , Placa Amiloide/genética , Presenilina-1/antagonistas & inhibidores , Factores de TiempoRESUMEN
Pigmented orthochromatic leukodystrophy and hereditary diffuse leukoencephalopathy with spheroids are two adult onset leukodystrophies with neuroaxonal spheroids presenting with prominent neurobehavioral, cognitive and motor symptoms. These are familial or sporadic disorders characterized by cerebral white matter degeneration including myelin and axonal loss, gliosis, macrophages and axonal spheroids. We report clinical, neuroimaging and pathological correlations of four women ages 34-50 years with adult onset leukodystrophy. Their disease course ranged from 1.5-8 years. Three patients had progressive cognitive and behavioral changes; however, one had acute onset. Neuroimaging revealed white matter abnormalities characterized by symmetric, bilateral, T2 hyperintense and T1 hypointense Magnetic Resonance Imaging signal involving frontal lobe white matter in all patients. Extensive laboratory investigations were negative apart from abnormalities in some mitochondrial enzymes and immunologic parameters. Autopsies demonstrated severe leukodystrophy with myelin and axonal loss, axonal spheroids and macrophages with early and severe frontal white matter involvement. The extent and degree of changes outside the frontal lobe appeared to correlate with disease duration. The prominent neurobehavioral deficits and frontal white matter disease provide clinical-pathologic support for association pathways linking distributed neural circuits sub-serving cognition. These observations lend further support to the notion that white matter disease alone can account for dementia.
Asunto(s)
Encefalopatías/patología , Encéfalo/patología , Leucoencefalopatía Multifocal Progresiva/patología , Adulto , Axones/patología , Axones/ultraestructura , Encéfalo/ultraestructura , Femenino , Lóbulo Frontal/patología , Lóbulo Frontal/ultraestructura , Humanos , Microscopía Electrónica , Persona de Mediana Edad , Distrofias Neuroaxonales/patologíaRESUMEN
Leukotrienes (LTs) are signaling molecules derived from arachidonic acid that initiate and amplify innate and adaptive immunity. In turn, how their synthesis is organized on the nuclear envelope of myeloid cells in response to extracellular signals is not understood. We define the supramolecular architecture of LT synthesis by identifying the activation-dependent assembly of novel multiprotein complexes on the outer and inner nuclear membranes of mast cells. These complexes are centered on the integral membrane protein 5-Lipoxygenase-Activating Protein, which we identify as a scaffold protein for 5-Lipoxygenase, the initial enzyme of LT synthesis. We also identify these complexes in mouse neutrophils isolated from inflamed joints. Our studies reveal the macromolecular organization of LT synthesis.
Asunto(s)
Araquidonato 5-Lipooxigenasa/metabolismo , Proteínas Portadoras/metabolismo , Leucotrienos/biosíntesis , Proteínas de la Membrana/metabolismo , Complejos Multiproteicos/análisis , Membrana Nuclear/metabolismo , Proteínas Activadoras de la 5-Lipooxigenasa , Animales , Artritis/enzimología , Artritis/metabolismo , Proteínas de la Membrana/análisis , Ratones , Células Mieloides/química , Células Mieloides/metabolismo , Neutrófilos/química , Neutrófilos/metabolismo , Membrana Nuclear/químicaRESUMEN
Alzheimer disease (AD) is characterized both by senile plaques and neurodegeneration, although the details of the relationship between the 2 are not well understood. We postulated that oxidative stress resulting from senile plaques may mediate plaques' effects on local neuronal processes. Using multiphoton microscopy, we directly demonstrate the generation of reactive oxygen species by senile plaques. After screening of several natural antioxidants ex vivo, we assessed in vivo the effect of 2 orally administered antioxidants in APPswe/PS1d9 transgenic mice. Both Ginkgo biloba extract and vitamin E reduced the oxidative stress resulting from senile plaques in vivo as monitored with intracranial imaging. Both treatments also lead to a progressive reversal of the structural changes in dystrophic neurites associated with senile plaques. These results suggest a causal relationship between plaque-associated oxidative stress and neuritic alterations and demonstrate for the first time that the focal neurotoxicity associated with the senile plaques of AD is partially reversible with antioxidant therapies. The quantitative ex vivo screen combined with in vivo monitoring of efficacy should lead to more effective clinical therapies for the prevention of oxidative stress and neurotoxicity in AD.