RESUMEN
Disruption of retromer-dependent endosomal trafficking is considered pathogenic in late-onset Alzheimer's disease (AD). Here, to investigate this disruption in the intact brain, we turn to a genetic mouse model where the retromer core protein VPS35 is depleted in hippocampal neurons, and then we replete VPS35 using an optimized viral vector protocol. The VPS35 depletion-repletion studies strengthen the causal link between the neuronal retromer and AD-associated neuronal phenotypes, including the acceleration of amyloid precursor protein cleavage and the loss of synaptic glutamate receptors. Moreover, the studies show that the neuronal retromer can regulate a distinct, dystrophic, microglia morphology, phenotypic of hippocampal microglia in AD. Finally, the neuronal and, in part, the microglia responses to VPS35 depletion were found to occur independent of tau. Showing that the neuronal retromer can regulate AD-associated pathologies in two of AD's principal cell types strengthens the link, and clarifies the mechanism, between endosomal trafficking and late-onset sporadic AD.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Microglía/patología , Neuronas/patología , Proteínas de Transporte Vesicular/metabolismo , Animales , Endosomas/metabolismo , Ratones , Microglía/metabolismo , Neuronas/metabolismo , Fenotipo , Transporte de Proteínas/fisiologíaRESUMEN
Brain lesions composed of pathological tau help to drive neurodegeneration in Alzheimer's disease (AD) and related tauopathies. Here, we identified the mammalian suppressor of tauopathy 2 (MSUT2) gene as a modifier of susceptibility to tau toxicity in two mouse models of tauopathy. Transgenic PS19 mice overexpressing tau, a model of AD, and lacking the Msut2 gene exhibited decreased learning and memory deficits, reduced neurodegeneration, and reduced accumulation of pathological tau compared to PS19 tau transgenic mice expressing Msut2 Conversely, Msut2 overexpression in 4RTauTg2652 tau transgenic mice increased pathological tau deposition and promoted the neuroinflammatory response to pathological tau. MSUT2 is a poly(A) RNA binding protein that antagonizes the canonical nuclear poly(A) binding protein PABPN1. In individuals with AD, MSUT2 abundance in postmortem brain tissue predicted an earlier age of disease onset. Postmortem AD brain tissue samples with normal amounts of MSUT2 showed elevated neuroinflammation associated with tau pathology. We observed co-depletion of MSUT2 and PABPN1 in postmortem brain samples from a subset of AD cases with higher tau burden and increased neuronal loss. This suggested that MSUT2 and PABPN1 may act together in a macromolecular complex bound to poly(A) RNA. Although MSUT2 and PABPN1 had opposing effects on both tau aggregation and poly(A) RNA tail length, we found that increased poly(A) tail length did not ameliorate tauopathy, implicating other functions of the MSUT2/PABPN1 complex in tau proteostasis. Our findings implicate poly(A) RNA binding proteins both as modulators of pathological tau toxicity in AD and as potential molecular targets for interventions to slow neurodegeneration in tauopathies.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Encéfalo/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Unión a Poli(A)/metabolismo , Proteínas tau/metabolismo , Enfermedad de Alzheimer/genética , Animales , Proteínas Portadoras/genética , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Transgénicos , Proteína I de Unión a Poli(A)/genética , Proteína I de Unión a Poli(A)/metabolismo , Proteínas de Unión a Poli(A)/genética , Proteínas tau/genéticaRESUMEN
Recent genome-wide association studies (GWAS) have identified copy number variations (CNVs) at chromosomal locus 7q36.3 that significantly contribute to the risk of schizophrenia, with all of the microduplications occurring within a single gene: vasoactive intestinal peptide receptor 2 (VIPR2). To confirm disease causality and translate such a genetic vulnerability into mechanistic and pathophysiological insights, we have developed a series of conditional VIPR2 bacterial artificial chromosome (BAC) transgenic mouse models of VIPR2 CNV. VIPR2 CNV mouse model recapitulates gene expression and signaling deficits seen in human CNV carriers. VIPR2 microduplication in mice elicits prominent dorsal striatal dopamine dysfunction, cognitive, sensorimotor gating, and social behavioral deficits preceded by an increase of striatal cAMP/PKA signaling and the disrupted early postnatal striatal development. Genetic removal of VIPR2 transgene expression via crossing with Drd1a-Cre BAC transgenic mice rescued the dopamine D2 receptor abnormality and multiple behavioral deficits, implicating a pathogenic role of VIPR2 overexpression in dopaminoceptive neurons. Thus, our results provide further evidence to support the GWAS studies that the dosage sensitivity intolerance of VIPR2 is disease causative to manifest schizophrenia-like dopamine, cognitive, and social behavioral deficits in mice. The conditional BAC transgenesis offers a novel strategy to model CNVs with a gain-of -copies and facilitate the genetic dissection of when/where/how the genetic vulnerabilities affect development, structure, and function of neural circuits. Our findings have important implications for therapeutic development, and the etiology-relevant mouse model provides a useful preclinical platform for drug discovery.
Asunto(s)
Receptores de Tipo II del Péptido Intestinal Vasoactivo/genética , Esquizofrenia/genética , Esquizofrenia/metabolismo , Animales , Cromosomas Artificiales Bacterianos/genética , Variaciones en el Número de Copia de ADN/genética , Modelos Animales de Enfermedad , Duplicación de Gen/genética , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Genotipo , Humanos , Ratones , Ratones Transgénicos , Fenotipo , Receptores de Tipo II del Péptido Intestinal Vasoactivo/metabolismoRESUMEN
In order to contribute to a better knowledge on the relationship between amyloid and tau pathology, and electroencephalography (EEG) disturbances, the aim of this study was to evaluate the effects of injection of beta amyloid Abeta(1-42) peptide, tau (a recombinant AAV (Adeno-Associated Virus) containing the human transgene tau with the P301 L mutation on rats and the combination of both, on the power of brain's rhythm (delta, theta, alpha, beta and gamma waves) during the different sleep/wake states of animals by EEG recording. Currently, no preclinical studies explore the effect of the tau pathology on EEG. The experimentations were performed 3 weeks and 3 months post injections. Beta amyloid deposits and hyperphosphorylated Tau are observed by immunohistofluorescence, only in the hippocampus. Furthermore, using a radial arm water maze, the main effect was observed on working memory which was significantly impaired in Abeta-Tau group only 3 months post injections. However, on EEG, as early as the 3rd week, an overall decrease of the EEG bands power was observed in the treated groups, particularly the theta waves during the rapid eye movement (REM) sleep. Beta amyloid was mainly involved in these perturbations. Obviously, EEG seems to be an interesting tool in the early diagnostic of amyloid and tau pathologies, with a good sensitivity and the possibility to perform a follow up during a large period.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Encéfalo/fisiopatología , Electroencefalografía , Fragmentos de Péptidos/metabolismo , Proteínas tau/metabolismo , Enfermedad de Alzheimer/diagnóstico , Enfermedad de Alzheimer/fisiopatología , Péptidos beta-Amiloides/administración & dosificación , Animales , Dermatoglifia del ADN , Dependovirus/genética , Modelos Animales de Enfermedad , Humanos , Himecromona , Masculino , Aprendizaje por Laberinto/fisiología , Memoria a Corto Plazo/fisiología , Fragmentos de Péptidos/administración & dosificación , Fosforilación , Ratas Sprague-Dawley , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sueño REM/fisiología , Proteínas tau/administración & dosificación , Proteínas tau/genéticaRESUMEN
BACKGROUND AND AIMS: The recombinant adeno-associated viral vector serotype 8 expressing the gain-of-function mutation of mouse proprotein convertase subtilisin/kexin type 9 (AAV8- PCSK9) is a new model for the induction of hypercholesterolemia. AAV8 preferentially infects hepatocytes and the incorporated liver-specific promoter should ensure expression of PCSK9 in the liver. Since tissue distribution of AAVs can differ between male and female mice, we investigated the differences in PCSK9 expression and hypercholesterolemia development between male and female mice using the AAV8-PCSK9 model. METHODS: Male and female C57BL/6 mice were injected with either a low-dose or high-dose of AAV8-PCSK9 and fed a high-fat diet. Plasma lipid levels were evaluated as a measure of the induction of hypercholesterolemia. RESULTS: Injection of mice with low dose AAV8-PCSK9 dramatically elevated both serum PCSK9 and cholesterol levels in male but not female mice. Increasing the dose of AAV8-PCSK9 threefold in female mice rescued the hypercholesterolemia phenotype but did not result in full restoration of AAV8-PCSK9 transduction of livers in female mice compared to the low-dose male mice. Our data demonstrate female mice respond differently to AAV8-PCSK9 injection compared to male mice. CONCLUSIONS: These differences do not hinder the use of female mice when AAV8-PCSK9 doses are taken into consideration. However, localization to and production of AAV8-PCSK9 in organs besides the liver in mice may introduce confounding factors into studies and should be considered during experimental design.
Asunto(s)
Hipercolesterolemia/metabolismo , Hígado/metabolismo , Proproteína Convertasa 9/biosíntesis , Factores Sexuales , Animales , Dependovirus , Modelos Animales de Enfermedad , Femenino , Técnicas de Transferencia de Gen , Masculino , Ratones , Ratones Endogámicos C57BL , MutaciónRESUMEN
A risk factor for cardiovascular disease (CVD), mutant PCSK9, was expressed in APP/PS1 mice to study the CVD-Alzheimer's disease inter-relationship. Cholesterol levels were elevated by 5-6-fold from 3 to 13 weeks after PCSK9 gene transfer. We tested whether hypercholesterolemia would increase amyloid-ß plaques at a relatively early stage of plaque deposition. Plaque burden was increased in the hippocampus of PCSK9 treated mice though the increase was modest compared to the large elevation in cholesterol. Elevating cholesterol via gene transfer could be valuable in a variety of disease models compared to making crosses with germ-line transgenic mouse models of CVD.
Asunto(s)
Colesterol/sangre , Hipercolesterolemia/genética , Placa Amiloide/genética , Proproteína Convertasa 9/genética , Proproteína Convertasa 9/metabolismo , Transducción Genética/métodos , Precursor de Proteína beta-Amiloide/genética , Amiloidosis/genética , Animales , Encéfalo/metabolismo , Encéfalo/patología , Proteínas de Unión al Calcio/metabolismo , Modelos Animales de Enfermedad , Proteína Ácida Fibrilar de la Glía/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hipercolesterolemia/etiología , Masculino , Ratones , Ratones Transgénicos , Proteínas de Microfilamentos/metabolismo , Mutación/genética , Presenilina-1/genética , Factores de TiempoRESUMEN
Engineered recombinant adeno-associated virus (AAV) vectors have advanced the transduction of neurons in the CNS on an expansive, wide-scale basis since the papers first using AAV9 for this purpose. Wide-scale CNS expression is relevant to gene therapy as well as indispensable for basic studies such as disease modeling. For example, the wide-scale gene transfer approach could expedite hypothesis testing in vivo relative to the generation of germ-line transgenic mice for all of the genes of interest. Wide-scale gene transfer is more efficient in neonates than in adults, so improving gene transfer efficiency in adults is an important goal. Here we characterized the relatively novel AAV PHP.EB vector for expansive gene transfer in the CNS of adult rats at three doses. The dose-response data were consistent; expression levels can be controlled in a reproducible manner in the rat from moderate to robust levels. Within the CNS, the AAV PHP.EB-derived expression was neuron-selective to neuron-specific, while outside the CNS, organs such as the liver and heart were transduced by the parenteral gene delivery. Though we demonstrated graded expression levels, only the high dose, 1.2 × 1014 vector genomes/kg, yielded efficient expression in spinal cord motor neurons of the adult rat, so this vector dose would be required for models of spinal cord motor neuron disease. The neuronal expression in the rat CNS was greater with AAV PHP.EB than the previous engineered vector AAV PHP.B. AAV PHP.EB is thus one of the most efficient AAV vectors in the field for CNS gene transfer.
Asunto(s)
Terapia Genética/métodos , Transducción Genética/métodos , Animales , Encéfalo/metabolismo , Línea Celular , Sistema Nervioso Central/inmunología , Sistema Nervioso Central/fisiología , Dependovirus/genética , Relación Dosis-Respuesta a Droga , Femenino , Técnicas de Transferencia de Gen , Vectores Genéticos , Ratones , Ratones Transgénicos , Neuronas Motoras/metabolismo , Ratas , Ratas Sprague-Dawley , Médula EspinalRESUMEN
Trace eyeblink conditioning is a hippocampus-dependent associative learning paradigm which is impaired in patients with Alzheimer's disease (AD) and animal AD models. Learning in this paradigm accompanies changes in oscillatory activity in forebrain regions, some of which are loci of pathogenic changes in prodromal AD stages. These observations motivated us to examine how cortical event-related potentials (ERPs) during this paradigm are affected by two features of the asymptomatic, AD-related brain abnormality, entorhinal tau accumulation and mild cholinergic deficit. Adult rats received viral overexpression of P301L mutant human tau in the entorhinal cortex, low-dose scopolamine treatment, or both. Electroencephalograms were recorded with epidural electrodes on the surface of the frontal, parietal, and temporal cortices during differential and reversal trace eyeblink conditioning. All rats developed conditioned responses to one of two stimuli (auditory or visual) paired with mild eyelid shock (CS+), but not to the other stimulus presented alone (CS-). They were also able to adjust the response when the stimulus contingency was reversed. With learning, the amplitude of several ERP components in the frontal and temporal cortices came to differentiate the CS+ from CS-. Scopolamine affected the learning-related change in temporal P2 and other learning-unrelated components in three regions. Entorhinal tau overexpression primary affected the amplitude of temporal visual ERPs and learning-unrelated frontal and temporal auditory ERP components. The double manipulation only affected two components of temporal auditory ERPs. Thus, cortical ERPs during differential associative learning are sensitive to asymptomatic brain abnormality associated with AD.
Asunto(s)
Enfermedad de Alzheimer/fisiopatología , Aprendizaje por Asociación/fisiología , Encéfalo/fisiopatología , Condicionamiento Palpebral/fisiología , Potenciales Evocados , Enfermedad de Alzheimer/diagnóstico , Enfermedad de Alzheimer/patología , Animales , Percepción Auditiva/fisiología , Encéfalo/patología , Diagnóstico por Computador , Electroencefalografía , Electrochoque , Potenciales Evocados/fisiología , Masculino , Mutación , Ratas Long-Evans , Escopolamina , Máquina de Vectores de Soporte , Percepción Visual/fisiología , Proteínas tau/administración & dosificación , Proteínas tau/genética , Proteínas tau/metabolismoRESUMEN
Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are progressive neurodegenerative disorders marked in most cases by the nuclear exclusion and cytoplasmic deposition of the RNA binding protein TDP43. We previously demonstrated that ALS-associated mutant TDP43 accumulates within the cytoplasm, and that TDP43 mislocalization predicts neurodegeneration. Here, we sought to prevent neurodegeneration in ALS/FTD models using selective inhibitor of nuclear export (SINE) compounds that target exportin-1 (XPO1). SINE compounds modestly extend cellular survival in neuronal ALS/FTD models and mitigate motor symptoms in an in vivo rat ALS model. At high doses, SINE compounds block nuclear egress of an XPO1 cargo reporter, but not at lower concentrations that were associated with neuroprotection. Neither SINE compounds nor leptomycin B, a separate XPO1 inhibitor, enhanced nuclear TDP43 levels, while depletion of XPO1 or other exportins had little effect on TDP43 localization, suggesting that no single exporter is necessary for TDP43 export. Supporting this hypothesis, we find overexpression of XPO1, XPO7 and NXF1 are each sufficient to promote nuclear TDP43 egress. Taken together, our results indicate that redundant pathways regulate TDP43 nuclear export, and that therapeutic prevention of cytoplasmic TDP43 accumulation in ALS/FTD may be enhanced by targeting several overlapping mechanisms.
Asunto(s)
Transporte Activo de Núcleo Celular/efectos de los fármacos , Esclerosis Amiotrófica Lateral/patología , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Demencia Frontotemporal/patología , Neuronas/patología , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Citoplasma/metabolismo , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Dependovirus/genética , Femenino , Demencia Frontotemporal/tratamiento farmacológico , Demencia Frontotemporal/metabolismo , Humanos , Masculino , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Recombinant adeno-associated virus (AAV) vectors are a popular genetic approach in neuroscience because they confer such efficient transgene expression in the brain and spinal cord. A number of studies have used AAV to express pathological disease-related proteins in the dopaminergic neurons of the substantia nigra in situ ( e.g., α-synuclein to model aspects of Parkinson's disease). The neuropathology and neurodegeneration of Parkinson's disease occur in a circumscribed pattern in the brain, and one of the most important goals of any gene transfer study is accurate, pinpoint targeting. By combining Cre recombinase-dependent AAVs in Cre-driver rats in which Cre is expressed only in the tyrosine hydroxylase neurons, we have achieved more highly targeted expression of several disease-relevant neuropathological proteins in the substantia nigra pars compacta than using constitutive expression AAV vectors. Alpha-synuclein, tau, transactive response DNA-binding protein of 43 kDa, or the control fluorescent protein yellow fluorescent protein was individually expressed to induce highly targeted, dopaminergic neuron-specific neurodegeneration models. The refined targeting foreshadows a next-generation disease modeling system for expressing neurodegenerative disease-related proteins in a disease-relevant manner. We foresee specific utilities of this in vivo AAV vector targeting of pathological proteins to a well-defined and well-demarcated cell population.-Grames, M. S., Dayton, R. D., Jackson, K. L., Richard, A. D., Lu, X., Klein, R. L. Cre-dependent AAV vectors for highly targeted expression of disease-related proteins and neurodegeneration in the substantia nigra.
Asunto(s)
Dependovirus/metabolismo , Vectores Genéticos/metabolismo , Integrasas/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Sustancia Negra/metabolismo , Animales , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/metabolismo , Femenino , Ratas , alfa-Sinucleína/metabolismoRESUMEN
BACKGROUND: Mitochondrial reactive oxygen species (ROS) contribute to inflammation and vascular remodeling during atherosclerotic plaque formation. C57BL/6N (6N) and C57BL/6J (6J) mice display distinct mitochondrial redox balance due to the absence of nicotinamide nucleotide transhydrogenase (NNT) in 6J mice. We hypothesize that differential NNT expression between these animals alters plaque development. METHODS: 6N and 6J mice were treated with AAV8-PCSK9 (adeno-associated virus serotype 8/proprotein convertase subtilisin/kexin type 9) virus leading to hypercholesterolemia, increased low-density lipoprotein, and atherosclerosis in mice fed a high-fat diet (HFD). Mice were co-treated with the mitochondria-targeted superoxide dismutase mimetic MitoTEMPO to assess the contribution of mitochondrial ROS to atherosclerosis. RESULTS: Baseline and HFD-induced vascular superoxide is increased in 6J compared to 6N mice. MitoTEMPO diminished superoxide in both groups demonstrating differential production of mitochondrial ROS among these strains. PCSK9 treatment and HFD led to similar increases in plasma lipids in both 6N and 6J mice. However, 6J animals displayed significantly higher levels of plaque formation. MitoTEMPO reduced plasma lipids but did not affect plaque formation in 6N mice. In contrast, MitoTEMPO surprisingly increased plaque formation in 6J mice. CONCLUSION: These data indicate that loss of NNT increases vascular ROS production and exacerbates atherosclerotic plaque development.
Asunto(s)
Aorta/enzimología , Enfermedades de la Aorta/enzimología , Aterosclerosis/enzimología , NADP Transhidrogenasa AB-Específica/deficiencia , Animales , Antioxidantes/farmacología , Aorta/efectos de los fármacos , Aorta/patología , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/patología , Aterosclerosis/genética , Aterosclerosis/patología , Colesterol/sangre , Modelos Animales de Enfermedad , Predisposición Genética a la Enfermedad , Hipercolesterolemia/enzimología , Hipercolesterolemia/genética , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Proteínas Mitocondriales/deficiencia , Proteínas Mitocondriales/genética , NADP Transhidrogenasa AB-Específica/genética , Compuestos Organofosforados/farmacología , Fenotipo , Piperidinas/farmacología , Placa Aterosclerótica , Proproteína Convertasa 9/genética , Proproteína Convertasa 9/metabolismo , Superóxidos/metabolismo , Factores de TiempoRESUMEN
OBJECTIVE: Macrophage proinflammatory responses induced by modified low-density lipoproteins (modLDL) contribute to atherosclerotic progression. How modLDL causes macrophages to become proinflammatory is still enigmatic. Macrophage foam cell formation induced by modLDL requires glycerolipid synthesis. Lipin-1, a key enzyme in the glycerolipid synthesis pathway, contributes to modLDL-elicited macrophage proinflammatory responses in vitro. The objective of this study was to determine whether macrophage-associated lipin-1 contributes to atherogenesis and to assess its role in modLDL-mediated signaling in macrophages. APPROACH AND RESULTS: We developed mice lacking lipin-1 in myeloid-derived cells and used adeno-associated viral vector 8 expressing the gain-of-function mutation of mouse proprotein convertase subtilisin/kexin type 9 (adeno-associated viral vector 8-proprotein convertase subtilisin/kexin type 9) to induce hypercholesterolemia and plaque formation. Mice lacking myeloid-associated lipin-1 had reduced atherosclerotic burden compared with control mice despite similar plasma lipid levels. Stimulation of bone marrow-derived macrophages with modLDL activated a persistent protein kinase Cα/ßII-extracellular receptor kinase1/2-jun proto-oncogene signaling cascade that contributed to macrophage proinflammatory responses that was dependent on lipin-1 enzymatic activity. CONCLUSIONS: Our data demonstrate that macrophage-associated lipin-1 is atherogenic, likely through persistent activation of a protein kinase Cα/ßII-extracellular receptor kinase1/2-jun proto-oncogene signaling cascade that contributes to foam cell proinflammatory responses. Taken together, these results suggest that modLDL-induced foam cell formation and modLDL-induced macrophage proinflammatory responses are not independent consequences of modLDL stimulation but rather are both directly influenced by enhanced lipid synthesis.
Asunto(s)
Aorta/enzimología , Enfermedades de la Aorta/enzimología , Aterosclerosis/enzimología , Mediadores de Inflamación/metabolismo , Inflamación/enzimología , Lipoproteínas LDL/sangre , Macrófagos/enzimología , Proteínas Nucleares/metabolismo , Fosfatidato Fosfatasa/metabolismo , Animales , Aorta/patología , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/patología , Aterosclerosis/genética , Aterosclerosis/patología , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Células Espumosas/enzimología , Células Espumosas/patología , Inflamación/genética , Inflamación/patología , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Nucleares/deficiencia , Proteínas Nucleares/genética , Fosfatidato Fosfatasa/deficiencia , Fosfatidato Fosfatasa/genética , Placa Aterosclerótica , Proteína Quinasa C beta/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Células RAW 264.7 , Transducción de SeñalRESUMEN
Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), optogenetics, cre-lox targeting, etc. The purpose of this manuscript is to aid the investigator attempting expansive central nervous system (CNS) gene transfer in the rat via tail vein injection of AAV. Wide-scale expression is relevant for conditions with widespread pathology, and a rat model is significant due to its greater size and physiologic similarities to humans compared to mice. In this example application, a wide-scale neuronal transduction is used to mimic a neurodegenerative disease that affects the entire spinal cord, amyotrophic lateral sclerosis (ALS). The efficient wide-scale CNS transduction can also be used to deliver therapeutic protein factors in pre-clinical studies. After a post-injection expression interval of several weeks, the effects of the transduction are evaluated. For a green fluorescent protein (GFP) control vector, the amount of GFP in the cerebellum is estimated quickly and reliably by a basic imaging program. For motor disease phenotypes that are induced by the ALS related protein transactive response DNA-binding protein of 43 kDa (TDP-43), the deficits are scored by escape reflex and rotarod. Beyond disease modeling and gene therapy, there are diverse potential applications for the wide-scale gene targeting described here. The expanded use of this method will aid in expediting hypothesis testing in the neurosciences and neurogenetics.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Sistema Nervioso Central/metabolismo , Técnicas de Transferencia de Gen , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Administración Intravenosa , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Femenino , Humanos , Ratas , Ratas Sprague-Dawley , Transducción GenéticaRESUMEN
A neural signature of asymptomatic preclinical Alzheimer's disease (AD) is disrupted connectivity between brain regions; however, its underlying mechanisms remain unknown. Here, we tested whether a preclinical pathologic feature, tau aggregation in the entorhinal cortex (EC) is sufficient to disrupt the coordination of local field potentials (LFPs) between its efferent regions. P301L-mutant human tau or green fluorescent protein (GFP) was virally overexpressed in the EC of adult rats. LFPs were recorded from the dorsal hippocampus and prelimbic medial prefrontal cortex while the rats underwent trace eyeblink conditioning where they learned to associate 2 stimuli separated by a short time interval. In GFP-expressing rats, the 2 regions strengthened phase-phase and amplitude-amplitude couplings of theta and gamma oscillations during the interval separating the paired stimuli. Despite normal memory acquisition, this learning-related, inter-region oscillatory coupling was attenuated in the tau-expressing rats while prefrontal phase-amplitude theta-gamma cross-frequency coupling was elevated. Thus, EC tau aggregation caused aberrant long-range circuit activity during associative learning, identifying a culprit for the neural signature of preclinical AD stages.
Asunto(s)
Corteza Entorrinal , Hipocampo/fisiopatología , Aprendizaje/fisiología , Corteza Prefrontal/fisiopatología , Tauopatías/fisiopatología , Potenciales de Acción/fisiología , Enfermedad de Alzheimer , Animales , Parpadeo/fisiología , Condicionamiento Palpebral/fisiología , Corteza Entorrinal/metabolismo , Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Masculino , Agregación Patológica de Proteínas , Ratas Long-Evans , Proteínas tau/genética , Proteínas tau/metabolismoRESUMEN
One of the proteins most frequently found in neuropathological lesions is the ubiquitin binding protein p62 (sequestosome 1). Post-mortem analysis of p62 is a defining diagnostic marker in several neurodegenerative diseases including amyotrophic lateral sclerosis and inclusion body myositis. Since p62 functions in protein degradation pathways including autophagy, the build-up of p62-positive inclusions suggests defects in protein clearance. p62 was expressed unilaterally in the rat substantia nigra with an adeno-associated virus vector (AAV9) in order to study p62 neuropathology. Inclusions formed within neurons from several days to several weeks after gene transfer. By electron microscopy, the inclusions were found to contain packed 10 nm thick filaments, and mitochondria cristae structure was disrupted, resulting in the formation of empty spaces. In corollary cell culture transfections, p62 clearly impaired mitochondrial function. To probe for potential effects on macroautophagy, we co-expressed p62 with a double fluorescent tagged reporter for the autophagosome protein LC3 in the rat. p62 induced a dramatic and specific dissociation of the two tags. By 12 weeks, a rotational behavior phenotype manifested, consistent with a significant loss of dopaminergic neurons analyzed post-mortem. p62 overexpression resulted in a progressive and robust pathology model with neuronal inclusions and neurodegeneration. p62 gene transfer could be a novel methodological probe to disrupt mitochondrial function or autophagy in the brain and other tissues in vivo.
Asunto(s)
Cuerpos de Inclusión/genética , Cuerpos de Inclusión/patología , Enfermedades Neurodegenerativas/genética , Proteína Sequestosoma-1/genética , Sustancia Negra/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Animales , Modelos Animales de Enfermedad , Femenino , Células HEK293 , Humanos , Miositis por Cuerpos de Inclusión/genética , Miositis por Cuerpos de Inclusión/patología , Enfermedades Neurodegenerativas/patología , Ratas , Ratas Sprague-Dawley , Ratas Transgénicas , Proteína Sequestosoma-1/fisiologíaRESUMEN
[This corrects the article on p. 116 in vol. 9, PMID: 27867348.].
RESUMEN
Widespread genetic modification of cells in the central nervous system (CNS) with a viral vector has become possible and increasingly more efficient. We previously applied an AAV9 vector with the cytomegalovirus/chicken beta-actin (CBA) hybrid promoter and achieved wide-scale CNS transduction in neonatal and adult rats. However, this method transduces a variety of tissues in addition to the CNS. Thus we studied intravenous AAV9 gene transfer with a synapsin promoter to better target the neurons. We noted in systematic comparisons that the synapsin promoter drives lower level expression than does the CBA promoter. The engineered adeno-associated virus (AAV)-PHP.B serotype was compared with AAV9, and AAV-PHP.B did enhance the efficiency of expression. Combining the synapsin promoter with AAV-PHP.B could therefore be advantageous in terms of combining two refinements of targeting and efficiency. Wide-scale expression was used to model a disease with widespread pathology. Vectors encoding the amyotrophic lateral sclerosis (ALS)-related protein transactive response DNA-binding protein, 43 kDa (TDP-43) with the synapsin promoter and AAV-PHP.B were used for efficient CNS-targeted TDP-43 expression. Intracerebroventricular injections were also explored to limit TDP-43 expression to the CNS. The neuron-selective promoter and the AAV-PHP.B enhanced gene transfer and ALS disease modeling in adult rats.
RESUMEN
BACKGROUND: Fused in sarcoma (FUS) is an RNA-binding protein associated with the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration. ALS manifests in patients as a progressive paralysis which leads to respiratory dysfunction and failure, the primary cause of death in ALS. We expressed human FUS in rats to determine if FUS would induce ALS relevant respiratory changes to serve as an early stage disease indicator. The FUS expression was initiated in adult rats by way of an intravenously administered adeno-associated virus vector serotype 9 (AAV9) providing an adult onset model. RESULTS: The rats developed progressive motor impairments observed as early as 2-3 weeks post gene transfer. Respiratory abnormalities manifested 4-7 weeks post gene transfer including increased respiratory frequency and decreased tidal volume. Rats with breathing abnormalities also had arterial blood acidosis. Similar detailed plethysmographic changes were found in adult rats injected with AAV9 TDP-43. FUS gene transfer to adult rats yielded a consistent pre-clinical model with relevant motor paralysis in the early to middle stages and respiratory dysfunction at the end stage. Both FUS and TDP-43 yielded a similar consistent disease state. CONCLUSIONS: This modeling method yields disease relevant motor and respiratory changes in adult rats. The reproducibility of the data supports the use of this method to study other disease related genes and their combinations as well as a platform for disease modifying interventional strategies.
Asunto(s)
Esclerosis Amiotrófica Lateral/fisiopatología , Modelos Animales de Enfermedad , Proteína FUS de Unión a ARN/metabolismo , Trastornos Respiratorios/fisiopatología , Acidosis/fisiopatología , Esclerosis Amiotrófica Lateral/complicaciones , Animales , Dependovirus/genética , Progresión de la Enfermedad , Reacción de Fuga/fisiología , Femenino , Vectores Genéticos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Hipoxia/fisiopatología , Actividad Motora/fisiología , Fuerza Muscular/fisiología , Parálisis/fisiopatología , Proteína FUS de Unión a ARN/genética , Ratas Sprague-Dawley , Respiración , Trastornos Respiratorios/etiología , TransfecciónRESUMEN
Green fluorescent protein (GFP) is a broadly used live cell reporter for gene transduction although side effects associated with GFP in gene transfer are reported. The present study was designed to systematically examine host responses, including inflammatory and immune responses, induced by persistent overexpression of the GFP gene mediated by adeno-associated viral vector serotype 9 (AAV9), and their effects on GFP gene transduction in rat striatum. Our results show that host responses against AAV9-GFP transduction, and GFP transgene expression in the striatum exhibited a temporal and dose-dependent pattern. Both muscular and striatal delivery of AAV9-GFP increased levels of inflammation and immune reactions against sequential AAV9-GFP transduction in the striatum, leading to reduced levels of GFP expression. We also observed that rat sera from sequential administrations of AAV9-GFP group had significantly higher levels of neutralizing antibody against AAV9 vectors when compared with the age-matched rats. As excessive GFP can trigger vigorous inflammation and intense immune response after GFP gene transduction, the use of GFP as a live cell marker protein should be deliberated, especially in repeated administration studies.