RESUMEN
Huntington's disease (HD) results from expansion of a polyglutamine tract (polyQ) in mutant huntingtin (mHTT) protein, but mechanisms underlying polyQ expansion-mediated toxic gain-of-mHTT function remain elusive. Here, deletion and antibody-based experiments revealed that a proline-rich domain (PRD) adjacent to the polyQ tract is necessary for mutant huntingtin (mHTT) to inhibit fast axonal transport and promote axonal pathology in cultured mammalian neurons. Further, polypeptides corresponding to subregions of the PRD sufficed to elicit the toxic effect on fast axonal transport, which was mediated by JNK kinases and involved PRD binding to one or more SH3-domain containing proteins. Collectively, these data suggested a mechanism whereby polyQ tract expansion in mHTT promotes aberrant PRD exposure and interactions of this domain with SH3 domain-containing proteins including some involved in activation of JNK kinases. In support, biochemical and immunohistochemical experiments linked aberrant PRD exposure to increased JNK activation in striatal tissues of the zQ175 mouse model and from post-mortem HD patients. Collectively, these findings support a critical role of PRD on mHTT toxicity, suggesting a novel framework for the potential development of therapies aimed to halt or reduce axonal pathology in HD.
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Pathologic tau modifications are characteristic of Alzheimer's disease and related dementias, but mechanisms of tau toxicity continue to be debated. Inherited mutations in tau cause early onset frontotemporal lobar dementias (FTLD-tau) and are commonly used to model mechanisms of tau toxicity in tauopathies. Previous work in the isolated squid axoplasm model demonstrated that several pathogenic forms of tau inhibit axonal transport through a mechanism involving activation of protein phosphatase 1 (PP1). Here, we determined that P301L and R5L FTLD mutant tau proteins elicit a toxic effect on axonal transport as monomeric proteins. We evaluated interactions of wild-type or mutant tau with specific PP1 isoforms (α, ß, and γ) to examine how the interaction contributes to this toxic effect using primary rat hippocampal neurons from both sexes. Pull-down and bioluminescence resonance energy transfer experiments revealed selective interactions of wild-type tau with PP1α and PP1γ isoforms, but not PP1ß, which were significantly increased by the P301L tau mutation. The results from proximity ligation assays confirmed the interaction in primary hippocampal neurons. Moreover, expression of FTLD-linked mutant tau in these neurons enhanced levels of active PP1, also increasing the pausing frequency of fluorescently labeled vesicles in both anterograde and retrograde directions. Knockdown of PP1γ, but not PP1α, rescued the cargo-pausing effects of P301L and R5L tau, a result replicated by deleting a phosphatase-activating domain in the amino terminus of P301L tau. These findings support a model of tau toxicity involving aberrant activation of a specific PP1γ-dependent pathway that disrupts axonal transport in neurons.SIGNIFICANCE STATEMENT Tau pathology is closely associated with neurodegeneration in Alzheimer's disease and other tauopathies, but the toxic mechanisms remain a debated topic. We previously proposed that pathologic tau forms induce dysfunction and degeneration through aberrant activation of a PP1-dependent pathway that disrupts axonal transport. Here, we show that tau directly interacts with specific PP1 isoforms, increasing levels of active PP1. Pathogenic tau mutations enhance this interaction, further increasing active PP1 levels and impairing axonal transport in isolated squid axoplasm and primary hippocampal neurons. Mutant-tau-mediated impairment of axonal transport was mediated by PP1γ and a phosphatase-activating domain located at the amino terminus of tau. This work has important implications for understanding and potentially mitigating tau-mediated neurotoxicity in tauopathies.
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Transporte Axonal/efectos de los fármacos , Demencia Frontotemporal , Neuronas/metabolismo , Proteína Fosfatasa 1/metabolismo , Proteínas tau/farmacología , Animales , Células Cultivadas , Decapodiformes , Femenino , Hipocampo , Humanos , Masculino , Mutación , Neuronas/efectos de los fármacos , Ratas , Proteínas tau/genéticaRESUMEN
Mutations of various genes cause hereditary spastic paraplegia (HSP), a neurological disease involving dying-back degeneration of upper motor neurons. From these, mutations in the SPAST gene encoding the microtubule-severing protein spastin account for most HSP cases. Cumulative genetic and experimental evidence suggests that alterations in various intracellular trafficking events, including fast axonal transport (FAT), may contribute to HSP pathogenesis. However, the mechanisms linking SPAST mutations to such deficits remain largely unknown. Experiments presented here using isolated squid axoplasm reveal inhibition of FAT as a common toxic effect elicited by spastin proteins with different HSP mutations, independent of microtubule-binding or severing activity. Mutant spastin proteins produce this toxic effect only when presented as the tissue-specific M1 isoform, not when presented as the ubiquitously-expressed shorter M87 isoform. Biochemical and pharmacological experiments further indicate that the toxic effects of mutant M1 spastins on FAT involve casein kinase 2 (CK2) activation. In mammalian cells, expression of mutant M1 spastins, but not their mutant M87 counterparts, promotes abnormalities in the distribution of intracellular organelles that are correctable by pharmacological CK2 inhibition. Collectively, these results demonstrate isoform-specific toxic effects of mutant M1 spastin on FAT, and identify CK2 as a critical mediator of these effects.
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Adenosina Trifosfatasas/genética , Transporte Axonal/genética , Adenosina Trifosfatasas/metabolismo , Animales , Transporte Axonal/fisiología , Quinasa de la Caseína II/metabolismo , Células Cultivadas , Decapodiformes , Modelos Animales de Enfermedad , Fibroblastos , Humanos , Microtúbulos/metabolismo , Neuronas Motoras/metabolismo , Proteínas Mutantes/metabolismo , Mutación , Isoformas de Proteínas/genética , Transporte de Proteínas/fisiología , Ratas , Paraplejía Espástica Hereditaria/genética , Paraplejía Espástica Hereditaria/metabolismo , EspastinaRESUMEN
OBJECTIVE: The goal of this work is to study the changes in white matter integrity in R6/2, a well-established animal model of Huntington's disease (HD) that are captured by ex vivo diffusion imaging (DTI) using a high field MRI (17.6 T). MATERIALS AND METHODS: DTI and continuous time random walk (CTRW) models were used to fit changes in the diffusion-weighted signal intensity in the corpus callosum of controls and in R6/2 mice. RESULTS: A significant 13% decrease in fractional anisotropy, a 7% increase in axial diffusion, and a 33% increase in radial diffusion were observed between R6/2 and control mice. No change was observed in the CTRW beta parameter, but a significant decrease in the alpha parameter (- 21%) was measured. Histological analysis of the corpus callosum showed a decrease in axonal organization, myelin alterations, and astrogliosis. Electron microscopy studies demonstrated ultrastructural changes in degenerating axons, such as an increase in tortuosity in the R6/2 mice. CONCLUSIONS: DTI and CTRW diffusion models display quantitative changes associated with the microstructural alterations observed in the corpus callosum of the R6/2 mice. The observed increase in the diffusivity and decrease in the alpha CTRW parameter providing support for the use of these diffusion models for non-invasive detection of white matter alterations in HD.
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Axones , Imagen de Difusión Tensora , Enfermedad de Huntington/diagnóstico por imagen , Imagen por Resonancia Magnética , Animales , Anisotropía , Cuerpo Calloso/diagnóstico por imagen , Femenino , Masculino , Ratones , Microscopía Fluorescente , Vaina de Mielina , Sustancia Blanca/diagnóstico por imagenRESUMEN
Tau is a microtubule-associated protein that is involved in both normal and pathological processes in neurons. Since the discovery and characterization of tau over 40 years ago, our understanding of tau's normal functions and toxic roles in neurodegenerative tauopathies has continued to expand. Fast axonal transport is a critical process for maintaining axons and functioning synapses, critical subcellular compartments underlying neuronal connectivity. Signs of fast axonal transport disruption are pervasive in Alzheimer's disease and other tauopathies and various mechanisms have been proposed for regulation of fast axonal transport by tau. Post-translational modifications of tau including phosphorylation at specific sites, FTDP-17 point mutations, and oligomerization, confer upon tau a toxic effect on fast axonal transport. Consistent with the well-established dependence of axons on fast axonal transport, these disease-related modifications are closely associated temporally and spatially with axonal degeneration in the early disease stages. These factors position tau as a potentially critical factor mediating the disruption of fast axonal transport that precedes synaptic dysfunction and axonal degeneration at later disease stages. In this chapter, we review the evidence that tau affects fast axonal transport and examine several potential mechanisms proposed to underlie this toxicity.
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Transporte Axonal , Tauopatías/metabolismo , Tauopatías/patología , Proteínas tau/metabolismo , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Humanos , Fosforilación , Proteínas tau/químicaRESUMEN
Neurons affected in a wide variety of unrelated adult-onset neurodegenerative diseases (AONDs) typically exhibit a "dying back" pattern of degeneration, which is characterized by early deficits in synaptic function and neuritic pathology long before neuronal cell death. Consistent with this observation, multiple unrelated AONDs including Alzheimer's disease, Parkinson's disease, Huntington's disease, and several motor neuron diseases feature early alterations in kinase-based signaling pathways associated with deficits in axonal transport (AT), a complex cellular process involving multiple intracellular trafficking events powered by microtubule-based motor proteins. These pathogenic events have important therapeutic implications, suggesting that a focus on preservation of neuronal connections may be more effective to treat AONDs than addressing neuronal cell death. While the molecular mechanisms underlying AT abnormalities in AONDs are still being analyzed, evidence has accumulated linking those to a well-established pathological hallmark of multiple AONDs: altered patterns of neuronal protein phosphorylation. Here, we present a short overview on the biochemical heterogeneity of major motor proteins for AT, their regulation by protein kinases, and evidence revealing cell type-specific AT specializations. When considered together, these findings may help explain how independent pathogenic pathways can affect AT differentially in the context of each AOND.
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Transporte Axonal/fisiología , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Motoras Moleculares/metabolismo , Enfermedades Neurodegenerativas , Animales , Humanos , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Enfermedades Neurodegenerativas/fisiopatología , Transducción de Señal/fisiologíaRESUMEN
Cumulative evidence indicates that the onset and severity of Huntington's disease (HD) symptoms correlate with connectivity deficits involving specific neuronal populations within cortical and basal ganglia circuits. Brain imaging studies and pathological reports further associated these deficits with alterations in cerebral white matter structure and axonal pathology. However, whether axonopathy represents an early pathogenic event or an epiphenomenon in HD remains unknown, nor is clear the identity of specific neuronal populations affected. To directly evaluate early axonal abnormalities in the context of HD in vivo, we bred transgenic YFP(J16) with R6/2 mice, a widely used HD model. Diffusion tensor imaging and fluorescence microscopy studies revealed a marked degeneration of callosal axons long before the onset of motor symptoms. Accordingly, a significant fraction of YFP-positive cortical neurons in YFP(J16) mice cortex were identified as callosal projection neurons. Callosal axon pathology progressively worsened with age and was influenced by polyglutamine tract length in mutant huntingtin (mhtt). Degenerating axons were dissociated from microscopically visible mhtt aggregates and did not result from loss of cortical neurons. Interestingly, other axonal populations were mildly or not affected, suggesting differential vulnerability to mhtt toxicity. Validating these results, increased vulnerability of callosal axons was documented in the brains of HD patients. Observations here provide a structural basis for the alterations in cerebral white matter structure widely reported in HD patients. Collectively, our data demonstrate a dying-back pattern of degeneration for cortical projection neurons affected in HD, suggesting that axons represent an early and potentially critical target for mhtt toxicity.
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Axones/patología , Encéfalo/patología , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/patología , Anciano , Animales , Axones/metabolismo , Encéfalo/metabolismo , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Femenino , Expresión Génica , Genes Reporteros , Humanos , Enfermedad de Huntington/diagnóstico , Masculino , Ratones , Ratones Transgénicos , Microscopía Confocal , Persona de Mediana Edad , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/patología , Neuronas/metabolismo , Neuronas/patología , Agregación Patológica de Proteínas , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Índice de Severidad de la EnfermedadRESUMEN
Mutant human Cu/Zn superoxide dismutase 1 (SOD1) is associated with motor neuron toxicity and death in an inherited form of amyotrophic lateral sclerosis (ALS; Lou Gehrig disease). One aspect of toxicity in motor neurons involves diminished fast axonal transport, observed both in transgenic mice and, more recently, in axoplasm isolated from squid giant axons. The latter effect appears to be directly mediated by misfolded SOD1, whose addition activates phosphorylation of p38 MAPK and phosphorylation of kinesin. Here, we observe that several different oligomeric states of a fusion protein, comprising ALS-associated human G85R SOD1 joined with yellow fluorescent protein (G85R SOD1YFP), which produces ALS in transgenic mice, inhibited anterograde transport when added to squid axoplasm. Inhibition was blocked both by an apoptosis signal-regulating kinase 1 (ASK1; MAPKKK) inhibitor and by a p38 inhibitor, indicating the transport defect is mediated through the MAPK cascade. In further incubations, we observed that addition of the mammalian molecular chaperone Hsc70, abundantly associated with G85R SOD1YFP in spinal cord of transgenic mice, exerted partial correction of the transport defect, associated with diminished phosphorylation of p38. Most striking, the addition of the molecular chaperone Hsp110, in a concentration substoichiometric to the mutant SOD1 protein, completely rescued both the transport defect and the phosphorylation of p38. Hsp110 has been demonstrated to act as a nucleotide exchange factor for Hsc70 and, more recently, to be able to cooperate with it to mediate protein disaggregation. We speculate that it can cooperate with endogenous squid Hsp(c)70 to mediate binding and/or disaggregation of mutant SOD1 protein, abrogating toxicity.
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Transporte Axonal/fisiología , Proteínas del Choque Térmico HSP110/farmacología , Proteínas Recombinantes de Fusión/metabolismo , Superóxido Dismutasa/metabolismo , Vesículas Transportadoras/metabolismo , Animales , Proteínas Bacterianas/metabolismo , Decapodiformes , Perfilación de la Expresión Génica , Proteínas del Choque Térmico HSP110/metabolismo , Humanos , Proteínas Luminiscentes/metabolismo , MAP Quinasa Quinasa Quinasa 5/antagonistas & inhibidores , Ratones , Ratones Transgénicos , Mutación Missense/genética , Fosforilación/efectos de los fármacos , Pliegue de Proteína , Proteómica , Médula Espinal/citología , Médula Espinal/metabolismo , Superóxido Dismutasa/química , Superóxido Dismutasa/genética , Superóxido Dismutasa-1 , Vesículas Transportadoras/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
Loss of function of galactosylceramidase lysosomal activity causes demyelination and vulnerability of various neuronal populations in Krabbe disease. Psychosine, a lipid-raft-associated sphingolipid that accumulates in this disease, is thought to trigger these abnormalities. Myelin-free in vitro analyses showed that psychosine inhibited fast axonal transport through the activation of axonal PP1 and GSK3ß in the axon. Abnormal levels of activated GSK3ß and abnormally phosphorylated kinesin light chains were found in nerve samples from a mouse model of Krabbe disease. Administration of GSK3ß inhibitors significantly ameliorated transport defects in vitro and in vivo in peripheral axons of the mutant mouse. This study identifies psychosine as a pathogenic sphingolipid able to block fast axonal transport and is the first to provide a molecular mechanism underlying dying-back degeneration in this genetic leukodystrophy.
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Transporte Axonal/efectos de los fármacos , Glucógeno Sintasa Quinasa 3/metabolismo , Leucodistrofia de Células Globoides/patología , Proteínas Motoras Moleculares/metabolismo , Neuronas/patología , Psicosina/farmacología , Análisis de Varianza , Animales , Animales Recién Nacidos , Células Cultivadas , Corteza Cerebral/patología , Modelos Animales de Enfermedad , Embrión de Mamíferos , Inhibidores Enzimáticos/farmacología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/genética , Glucógeno Sintasa Quinasa 3 beta , Leucodistrofia de Células Globoides/tratamiento farmacológico , Leucodistrofia de Células Globoides/genética , Microdominios de Membrana/efectos de los fármacos , Microdominios de Membrana/enzimología , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Microscopía Electrónica de Transmisión , Mitocondrias/efectos de los fármacos , Mitocondrias/fisiología , Proteínas del Tejido Nervioso/metabolismo , Neuronas/efectos de los fármacos , Neuronas/ultraestructura , Nervio Ciático/patología , Factores de TiempoRESUMEN
BACKGROUND: Cardiomyopathy and distal symmetrical polyneuropathy (DSPN), including sensory and autonomic dysfunction, often co-occur in diabetic mellitus (DM) patients. However, the temporal relationship and progression between these two complications has not been investigated. Using a streptozotocin DM animal model that develops insensate neuropathy, our aim was to examine in parallel the development of DSPN and DM-associated changes in cardiac structure and function as well as potential mechanisms, such as autonomic dysfunction, evaluated by changes in urinary and myocardial norepinephrine content and myocardial neuronal markers. METHODS: Sensory neuropathy was measured by behavioral tests using Von Frey filaments and Hargreaves methods. Echocardiography was used to evaluate myocardial structure and function. Autonomic function was evaluated by measuring urinary and myocardial norepinephrine (NE) levels by enzyme-linked immunosorbent assay and high-performance liquid chromatography/mass spectrometry. Quantitative immunohistochemistry was used to measure the myocardial neuronal markers, calcitonin gene-related peptide (CGRP) and general neuronal protein gene product 9.5 (PGP 9.5). RESULTS: The DM group developed tactile and thermal insensate neuropathy 4-5 weeks after DM onset. Cardiovascular changes were found between 4 and 12 weeks after DM onset and included bradycardia, diastolic and systolic dysfunction and cardiac dilation. There was a 2.5-fold reduction in myocardial NE levels and a 5-fold increase in urinary NE levels in the DM group. Finally, there was a 2.3-fold increase in myocardial CGRP levels in the DM group and no change in PGP9.5 levels. CONCLUSIONS: Cardiovascular structural and functional changes developed early in the course of DM and in combination with insensate neuropathy. In parallel, signs of cardiac autonomic dysfunction were also found and included decreased myocardial NE levels and altered CGRP levels. These results may indicate the need for early cardiovascular evaluation in DM patients with insensate neuropathy.
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Diabetes Mellitus Experimental/diagnóstico , Cardiomiopatías Diabéticas/diagnóstico , Neuropatías Diabéticas/diagnóstico , Modelos Animales de Enfermedad , Animales , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/orina , Cardiomiopatías Diabéticas/complicaciones , Cardiomiopatías Diabéticas/orina , Neuropatías Diabéticas/complicaciones , Neuropatías Diabéticas/orina , Masculino , Ratas , Ratas Endogámicas F344RESUMEN
The progressive loss of the nigrostriatal pathway is a distinguishing feature of Parkinson's disease. As terminal field loss seems to precede cell body loss, we tested whether alterations of axonal transport motor proteins would be early features in Parkinson's disease. There was a decline in axonal transport motor proteins in sporadic Parkinson's disease that preceded other well-known nigral cell-related pathology such as phenotypic downregulation of dopamine. Reductions in conventional kinesin levels precede the alterations in dopaminergic phenotypic markers (tyrosine hydroxylase) in the early stages of Parkinson's disease. This reduction was significantly greater in nigral neurons containing α-synuclein inclusions. Unlike conventional kinesin, reductions in the levels of the cytoplasmic dynein light chain Tctex type 3 subunit were only observed at late Parkinson's disease stages. Reductions in levels of conventional kinesin and cytoplasmic dynein subunits were recapitulated in a rat genetic Parkinson's disease model based on over-expression of human mutant α-synuclein (A30P). Together, our data suggest that α-synuclein aggregation is a key feature associated with reductions of axonal transport motor proteins in Parkinson's disease and support the hypothesis that dopaminergic neurodegeneration following a 'dying-back' pattern involving axonal transport disruption.
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Transporte Axonal/fisiología , Dineínas Citoplasmáticas/metabolismo , Dineínas/metabolismo , Cinesinas/metabolismo , Trastornos Parkinsonianos/metabolismo , alfa-Sinucleína/metabolismo , Anciano , Anciano de 80 o más Años , Animales , Estudios de Casos y Controles , Neuronas Dopaminérgicas/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Ratas , Ratas Sprague-Dawley , Ratas Transgénicas , Sustancia Negra/metabolismo , Tirosina 3-Monooxigenasa/metabolismo , alfa-Sinucleína/genéticaRESUMEN
The amyloid precursor protein (APP) is a key molecular component of Alzheimer's disease (AD) pathogenesis. Proteolytic APP processing generates various cleavage products, including extracellular amyloid beta (Aß) and the cytoplasmic APP intracellular domain (AICD). Although the role of AICD in the activation of kinase signaling pathways is well established in the context of full-length APP, little is known about intracellular effects of the AICD fragment, particularly within discrete neuronal compartments. Deficits in fast axonal transport (FAT) and axonopathy documented in AD-affected neurons prompted us to evaluate potential axon-autonomous effects of the AICD fragment for the first time. Vesicle motility assays using the isolated squid axoplasm preparation revealed inhibition of FAT by AICD. Biochemical experiments linked this effect to aberrant activation of selected axonal kinases and heightened phosphorylation of the anterograde motor protein conventional kinesin, consistent with precedents showing phosphorylation-dependent regulation of motors proteins powering FAT. Pharmacological inhibitors of these kinases alleviated the AICD inhibitory effect on FAT. Deletion experiments indicated this effect requires a sequence encompassing the NPTY motif in AICD and interacting axonal proteins containing a phosphotyrosine-binding domain. Collectively, these results provide a proof of principle for axon-specific effects of AICD, further suggesting a potential mechanistic framework linking alterations in APP processing, FAT deficits, and axonal pathology in AD.
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Enfermedad de Alzheimer , Precursor de Proteína beta-Amiloide , Humanos , Precursor de Proteína beta-Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Transporte Axonal , Enfermedad de Alzheimer/metabolismo , Axones/metabolismoRESUMEN
Aggregated filamentous forms of hyperphosphorylated tau (a microtubule-associated protein) represent pathological hallmarks of Alzheimer's disease (AD) and other tauopathies. While axonal transport dysfunction is thought to represent a primary pathogenic factor in AD and other neurodegenerative diseases, the direct molecular link between pathogenic forms of tau and deficits in axonal transport remain unclear. Recently, we demonstrated that filamentous, but not soluble, forms of wild-type tau inhibit anterograde, kinesin-based fast axonal transport (FAT) by activating axonal protein phosphatase 1 (PP1) and glycogen synthase kinase 3 (GSK3), independent of microtubule binding. Here, we demonstrate that amino acids 2-18 of tau, comprising a phosphatase-activating domain (PAD), are necessary and sufficient for activation of this pathway in axoplasms isolated from squid giant axons. Various pathogenic forms of tau displaying increased exposure of PAD inhibited anterograde FAT in squid axoplasm. Importantly, immunohistochemical studies using a novel PAD-specific monoclonal antibody in human postmortem tissue indicated that increased PAD exposure represents an early pathogenic event in AD that closely associates in time with AT8 immunoreactivity, an early marker of pathological tau. We propose a model of pathogenesis in which disease-associated changes in tau conformation lead to increased exposure of PAD, activation of PP1-GSK3, and inhibition of FAT. Results from these studies reveal a novel role for tau in modulating axonal phosphotransferases and provide a molecular basis for a toxic gain-of-function associated with pathogenic forms of tau.
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Transporte Axonal/genética , Axones/patología , Encéfalo/patología , Cinesinas/metabolismo , Fosfotransferasas/metabolismo , Proteínas tau/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Análisis de Varianza , Animales , Transporte Axonal/efectos de los fármacos , Axones/efectos de los fármacos , Axones/metabolismo , Decapodiformes , Inhibidores Enzimáticos/farmacología , Ensayo de Inmunoadsorción Enzimática , Glucógeno Sintasa Quinasa 3/metabolismo , Humanos , Técnicas In Vitro , Cinesinas/genética , Modelos Biológicos , Mutagénesis/genética , Fragmentos de Péptidos/metabolismo , Isótopos de Fósforo/farmacocinética , Fosfotransferasas/genética , Proteínas Proto-Oncogénicas c-jun/farmacocinética , Receptores de Neuropéptido Y/metabolismo , Transducción de Señal/genética , Tauopatías/genética , Tauopatías/patología , Proteínas tau/genéticaRESUMEN
Krabbe Disease (KD) is a lysosomal storage disorder characterized by the genetic deficiency of the lysosomal enzyme ß-galactosyl-ceramidase (GALC). Deficit or a reduction in the activity of the GALC enzyme has been correlated with the progressive accumulation of the sphingolipid metabolite psychosine, which leads to local disruption in lipid raft architecture, diffuse demyelination, astrogliosis, and globoid cell formation. The twitcher mouse, the most used animal model, has a nonsense mutation, which limits the study of how different mutations impact the processing and activity of GALC enzyme. To partially address this, we generated two new transgenic mouse models carrying point mutations frequently found in infantile and adult forms of KD. Using CRISPR-Cas9 gene editing, point mutations T513M (infantile) and G41S (adult) were introduced in the murine GALC gene and stable founders were generated. We show that GALC T513M/T513M mice are short lived, have the greatest decrease in GALC activity, have sharp increases of psychosine, and rapidly progress into a severe and lethal neurological phenotype. In contrast, GALC G41S/G41S mice have normal lifespan, modest decreases of GALC, and minimal psychosine accumulation, but develop adult mild inflammatory demyelination and slight declines in coordination, motor skills, and memory. These two novel transgenic lines offer the possibility to study the mechanisms by which two distinct GALC mutations affect the trafficking of mutated GALC and modify phenotypic manifestations in early- vs adult-onset KD.
RESUMEN
Aggregation and accumulation of the microtubule-associated protein tau are associated with cognitive decline and neuronal degeneration in Alzheimer's disease and other tauopathies. Thus, preventing the transition of tau from a soluble state to insoluble aggregates and/or reversing the toxicity of existing aggregates would represent a reasonable therapeutic strategy for treating these neurodegenerative diseases. Here we demonstrate that molecular chaperones of the heat shock protein 70 (Hsp70) family are potent inhibitors of tau aggregation in vitro, preventing the formation of both mature fibrils and oligomeric intermediates. Remarkably, addition of Hsp70 to a mixture of oligomeric and fibrillar tau aggregates prevents the toxic effect of these tau species on fast axonal transport, a critical process for neuronal function. When incubated with preformed tau aggregates, Hsp70 preferentially associated with oligomeric over fibrillar tau, suggesting that prefibrillar oligomeric tau aggregates play a prominent role in tau toxicity. Taken together, our data provide a novel molecular basis for the protective effect of Hsp70 in tauopathies.
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Transporte Axonal , Regulación hacia Abajo , Proteínas HSP70 de Choque Térmico/metabolismo , Tauopatías/metabolismo , Proteínas tau/química , Proteínas tau/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Humanos , Chaperonas Moleculares/metabolismo , Polimerizacion , Unión Proteica , Proteínas tau/antagonistas & inhibidores , Proteínas tau/genéticaRESUMEN
Experience in complex environments induces numerous forms of brain plasticity, improving structure and function. It has been long debated whether brain plasticity can be induced under neuropathological conditions, such as Alzheimer's disease (AD), to an extent that would reduce neuropathology, rescue brain structure, and restore its function. Here we show that experience in a complex environment rescues a significant impairment of hippocampal neurogenesis in transgenic mice harboring familial AD-linked mutant APPswe/PS1DeltaE9. Proliferation of hippocampal cells is enhanced significantly after enrichment, and these proliferating cells mature to become new neurons and glia. Enhanced neurogenesis was accompanied by a significant reduction in levels of hyperphosphorylated tau and oligomeric Abeta, the precursors of AD hallmarks, in the hippocampus and cortex of enriched mice. Interestingly, enhanced expression of the neuronal anterograde motor kinesin-1 was observed, suggesting enhanced axonal transport in hippocampal and cortical neurons after enrichment. Examination of synaptic physiology revealed that environmental experience significantly enhanced hippocampal long-term potentiation, without notable alterations in basal synaptic transmission. This study suggests that environmental modulation can rescue the impaired phenotype of the Alzheimer's brain and that induction of brain plasticity may represent therapeutic and preventive avenues in AD.
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Enfermedad de Alzheimer/prevención & control , Péptidos beta-Amiloides/fisiología , Neurogénesis , Plasticidad Neuronal , Neuronas/metabolismo , Presenilina-1/fisiología , Enfermedad de Alzheimer/patología , Animales , Western Blotting , Encéfalo/citología , Encéfalo/metabolismo , Proliferación Celular , Modelos Animales de Enfermedad , Electrofisiología , Hipocampo/citología , Hipocampo/metabolismo , Técnicas para Inmunoenzimas , Potenciación a Largo Plazo , Masculino , Ratones , Ratones Transgénicos , Neuronas/citología , Fosforilación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas tau/metabolismoRESUMEN
Over the last several decades, a number of mouse models have been generated for mechanistic and preclinical therapeutic research on Alzheimer's disease (AD)-like behavioral impairments and pathology. Acceptance or rejection of these models by the scientific community is playing a prominent role in how research findings are viewed and whether grants get funded and manuscripts published. The question of whether models are useful has become an exceptionally contentious issue. Much time and effort have gone into investigators debating comments such as "there are no mouse models of AD," " nice work but needs to be tested in another mouse model," or "only data from humans is valid." This leads to extensive written justifications for the choice of a model in grant applications, to the point of almost apologizing for the use of models. These debates also lead to initiatives to create new, better models of AD without consideration of what "better" may mean in this context. On the "other side," an argument supporting the use of mouse models is one cannot dissect a biological mechanism in postmortem human tissue. In this chapter, we examine issues that we believe must be addressed if in vivo AD research is to progress. We opine that it is not the models that are the issue, but rather a lack of understanding the aspects of AD-like pathology the models were designed to mimic. The goal here is to improve the utilization of models to address critical issues, not to offer a critique of existing models or make endorsements.
Asunto(s)
Enfermedad de Alzheimer , Animales , Modelos Animales de Enfermedad , Ratones , Ratones Transgénicos , Proteínas tauRESUMEN
Over four decades ago, in vitro experiments showed that tau protein interacts with and stabilizes microtubules in a phosphorylation-dependent manner. This observation fueled the widespread hypotheses that these properties extend to living neurons and that reduced stability of microtubules represents a major disease-driving event induced by pathological forms of tau in Alzheimer's disease and other tauopathies. Accordingly, most research efforts to date have addressed this protein as a substrate, focusing on evaluating how specific mutations, phosphorylation, and other post-translational modifications impact its microtubule-binding and stabilizing properties. In contrast, fewer efforts were made to illuminate potential mechanisms linking physiological and disease-related forms of tau to the normal and pathological regulation of kinases and phosphatases. Here, we discuss published work indicating that, through interactions with various kinases and phosphatases, tau may normally act as a scaffolding protein to regulate phosphorylation-based signaling pathways. Expanding on this concept, we also review experimental evidence linking disease-related tau species to the misregulation of these pathways. Collectively, the available evidence supports the participation of tau in multiple cellular processes sustaining neuronal and glial function through various mechanisms involving the scaffolding and regulation of selected kinases and phosphatases at discrete subcellular compartments. The notion that the repertoire of tau functions includes a role as a signaling hub should widen our interpretation of experimental results and increase our understanding of tau biology in normal and disease conditions.
RESUMEN
Adult-onset neurodegenerative diseases (AONDs) comprise a heterogeneous group of neurological disorders characterized by a progressive, age-dependent decline in neuronal function and loss of selected neuronal populations. Alterations in synaptic function and axonal connectivity represent early and critical pathogenic events in AONDs, but molecular mechanisms underlying these defects remain elusive. The large size and complex subcellular architecture of neurons render them uniquely vulnerable to alterations in axonal transport (AT). Accordingly, deficits in AT have been documented in most AONDs, suggesting a common defect acquired through different pathogenic pathways. These observations suggest that many AONDs can be categorized as dysferopathies, diseases in which alterations in AT represent a critical component in pathogenesis. Topics here address various molecular mechanisms underlying alterations in AT in several AONDs. Illumination of such mechanisms provides a framework for the development of novel therapeutic strategies aimed to prevent axonal and synaptic dysfunction in several major AONDs.
Asunto(s)
Transporte Axonal/fisiología , Enfermedades Neurodegenerativas/patología , Enfermedades Neurodegenerativas/fisiopatología , Neuronas/patología , Péptidos beta-Amiloides/metabolismo , Animales , Modelos Animales de Enfermedad , Humanos , Ratones , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Modelos Biológicos , Enfermedades Neurodegenerativas/genética , Transducción de Señal/fisiología , Sinapsis/fisiología , Proteínas tau/metabolismoRESUMEN
Abnormal expansion of a polyglutamine tract in huntingtin (Htt) protein results in Huntington's disease (HD), an autosomal dominant neurodegenerative disorder involving progressive loss of motor and cognitive function. Contrasting with the ubiquitous tissue expression of polyglutamine-expanded Htt, HD pathology is characterized by the increased vulnerability of specific neuronal populations within the striatum and the cerebral cortex. Morphological, biochemical, and functional characteristics of neurons affected in HD that might render these cells more vulnerable to the toxic effects of polyglutamine-Htt are covered in this review. The differential vulnerability of neurons observed in HD is discussed in the context of various major pathogenic mechanisms proposed to date, and in line with evidence showing a 'dying-back' pattern of degeneration in affected neuronal populations.