RESUMO
Ongoing, early-stage clinical trials illustrate the translational potential of human pluripotent stem cell (hPSC)-based cell therapies in Parkinson's disease (PD). However, an unresolved challenge is the extensive cell death following transplantation. Here, we performed a pooled CRISPR-Cas9 screen to enhance postmitotic dopamine neuron survival in vivo. We identified p53-mediated apoptotic cell death as a major contributor to dopamine neuron loss and uncovered a causal link of tumor necrosis factor alpha (TNF-α)-nuclear factor κB (NF-κB) signaling in limiting cell survival. As a translationally relevant strategy to purify postmitotic dopamine neurons, we identified cell surface markers that enable purification without the need for genetic reporters. Combining cell sorting and treatment with adalimumab, a clinically approved TNF-α inhibitor, enabled efficient engraftment of postmitotic dopamine neurons with extensive reinnervation and functional recovery in a preclinical PD mouse model. Thus, transient TNF-α inhibition presents a clinically relevant strategy to enhance survival and enable engraftment of postmitotic hPSC-derived dopamine neurons in PD.
Assuntos
Sobrevivência Celular , Neurônios Dopaminérgicos , NF-kappa B , Fator de Necrose Tumoral alfa , Proteína Supressora de Tumor p53 , Neurônios Dopaminérgicos/metabolismo , Animais , Humanos , NF-kappa B/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Camundongos , Sobrevivência Celular/efeitos dos fármacos , Transdução de Sinais , Doença de Parkinson/metabolismo , Células-Tronco Pluripotentes/metabolismo , Apoptose , Modelos Animais de Doenças , Sistemas CRISPR-CasRESUMO
Idiopathic Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta, which is associated with neuroinflammation and reactive gliosis. The underlying cause of PD and the concurrent neuroinflammation are not well understood. In this study, we utilize human and murine neuronal lines, stem cell-derived dopaminergic neurons, and mice to demonstrate that three previously identified genetic risk factors for PD, namely SATB1, MIR22HG, and GBA, are components of a single gene regulatory pathway. Our findings indicate that dysregulation of this pathway leads to the upregulation of glucocerebrosides (GluCer), which triggers a cellular senescence-like phenotype in dopaminergic neurons. Specifically, we discovered that downregulation of the transcriptional repressor SATB1 results in the derepression of the microRNA miR-22-3p, leading to decreased GBA expression and subsequent accumulation of GluCer. Furthermore, our results demonstrate that an increase in GluCer alone is sufficient to impair lysosomal and mitochondrial function, thereby inducing cellular senescence. Dysregulation of the SATB1-MIR22-GBA pathway, observed in both PD patients and normal aging, leads to lysosomal and mitochondrial dysfunction due to the GluCer accumulation, ultimately resulting in a cellular senescence-like phenotype in dopaminergic neurons. Therefore, our study highlights a novel pathway involving three genetic risk factors for PD and provides a potential mechanism for the senescence-induced neuroinflammation and reactive gliosis observed in both PD and normal aging.
Assuntos
Proteínas de Ligação à Região de Interação com a Matriz , MicroRNAs , Doença de Parkinson , Humanos , Camundongos , Animais , Neurônios Dopaminérgicos/metabolismo , Proteínas de Ligação à Região de Interação com a Matriz/genética , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Glucosilceramidas/metabolismo , Gliose , Doenças Neuroinflamatórias , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Senescência Celular/genética , Fatores de Transcrição/metabolismo , FenótipoRESUMO
Idiopathic Parkinson's Disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta, which is associated with neuroinflammation and reactive gliosis. The underlying cause of PD and the concurrent neuroinflammation are not well understood. In this study, we utilized human and murine neuronal lines, stem cell-derived dopaminergic neurons, and mice to demonstrate that three previously identified genetic risk factors for PD, namely SATB1, MIR22HG, and GBA, are components of a single gene regulatory pathway. Our findings indicate that dysregulation of this pathway leads to the upregulation of glucocerebrosides (GluCer), which triggers a cellular senescence-like phenotype in dopaminergic neurons. Specifically, we discovered that downregulation of the transcriptional repressor SATB1 results in the derepression of the microRNA miR-22-3p, leading to decreased GBA expression and subsequent accumulation of GluCer. Furthermore, our results demonstrate that an increase in GluCer alone is sufficient to impair lysosomal and mitochondrial function, thereby inducing cellular senescence dependent on S100A9 and stress factors. Dysregulation of the SATB1-MIR22-GBA pathway, observed in both PD patients and normal aging, leads to lysosomal and mitochondrial dysfunction due to the GluCer accumulation, ultimately resulting in a cellular senescence-like phenotype in dopaminergic neurons. Therefore, our study highlights a novel pathway involving three genetic risk factors for PD and provides a potential mechanism for the senescence-induced neuroinflammation and reactive gliosis observed in both PD and normal aging.
RESUMO
Ongoing, first-in-human clinical trials illustrate the feasibility and translational potential of human pluripotent stem cell (hPSC)-based cell therapies in Parkinson's disease (PD). However, a major unresolved challenge in the field is the extensive cell death following transplantation with <10% of grafted dopamine neurons surviving. Here, we performed a pooled CRISPR/Cas9 screen to enhance survival of postmitotic dopamine neurons in vivo . We identified p53-mediated apoptotic cell death as major contributor to dopamine neuron loss and uncovered a causal link of TNFa-NFκB signaling in limiting cell survival. As a translationally applicable strategy to purify postmitotic dopamine neurons, we performed a cell surface marker screen that enabled purification without the need for genetic reporters. Combining cell sorting with adalimumab pretreatment, a clinically approved and widely used TNFa inhibitor, enabled efficient engraftment of postmitotic dopamine neurons leading to extensive re-innervation and functional recovery in a preclinical PD mouse model. Thus, transient TNFa inhibition presents a clinically relevant strategy to enhance survival and enable engraftment of postmitotic human PSC-derived dopamine neurons in PD. Highlights: In vivo CRISPR-Cas9 screen identifies p53 limiting survival of grafted human dopamine neurons. TNFα-NFκB pathway mediates p53-dependent human dopamine neuron deathCell surface marker screen to enrich human dopamine neurons for translational use. FDA approved TNF-alpha inhibitor rescues in vivo dopamine neuron survival with in vivo function.
RESUMO
Open Science has changed research by making data accessible and shareable, contributing to replicability to accelerate and disseminate knowledge. However, for rodent cognitive studies the availability of tools to share and disseminate data is scarce. Automated touchscreen-based tests enable systematic cognitive assessment with easily standardised outputs that can facilitate data dissemination. Here we present an integration of touchscreen cognitive testing with an open-access database public repository (mousebytes.ca), as well as a Web platform for knowledge dissemination (https://touchscreencognition.org). We complement these resources with the largest dataset of age-dependent high-level cognitive assessment of mouse models of Alzheimer's disease, expanding knowledge of affected cognitive domains from male and female mice of three strains. We envision that these new platforms will enhance sharing of protocols, data availability and transparency, allowing meta-analysis and reuse of mouse cognitive data to increase the replicability/reproducibility of datasets.
Assuntos
Cognição/fisiologia , Ciência dos Animais de Laboratório/instrumentação , Ciência dos Animais de Laboratório/métodos , Roedores , Doença de Alzheimer , Animais , Comportamento Animal , Comportamento de Escolha , Bases de Dados Factuais , Modelos Animais de Doenças , Feminino , Aprendizagem/fisiologia , Masculino , Memória/fisiologia , Camundongos , Testes Neuropsicológicos , Reprodutibilidade dos Testes , Roedores/genética , SoftwareRESUMO
Cellular senescence is a mechanism used by mitotic cells to prevent uncontrolled cell division. As senescent cells persist in tissues, they cause local inflammation and are harmful to surrounding cells, contributing to aging. Generally, neurodegenerative diseases, such as Parkinson's, are disorders of aging. The contribution of cellular senescence to neurodegeneration is still unclear. SATB1 is a DNA binding protein associated with Parkinson's disease. We report that SATB1 prevents cellular senescence in post-mitotic dopaminergic neurons. Loss of SATB1 causes activation of a cellular senescence transcriptional program in dopamine neurons both in human stem cell-derived dopaminergic neurons and in mice. We observed phenotypes that are central to cellular senescence in SATB1 knockout dopamine neurons in vitro and in vivo. Moreover, we found that SATB1 directly represses expression of the pro-senescence factor p21 in dopaminergic neurons. Our data implicate senescence of dopamine neurons as a contributing factor in the pathology of Parkinson's disease.
Assuntos
Envelhecimento/fisiologia , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Neurônios Dopaminérgicos/fisiologia , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Doença de Parkinson/metabolismo , Animais , Células Cultivadas , Senescência Celular , Inibidor de Quinase Dependente de Ciclina p21/genética , Repressão Epigenética , Técnicas de Silenciamento de Genes , Humanos , Proteínas de Ligação à Região de Interação com a Matriz/genética , Camundongos , Camundongos Knockout , Mitose , Doença de Parkinson/genética , Ligação ProteicaRESUMO
Cholinergic dysfunction has been associated with cognitive abnormalities in a variety of neurodegenerative and neuropsychiatric diseases. Here we tested how information processing is regulated by cholinergic tone in genetically modified mice targeting the vesicular acetylcholine transporter (VAChT), a protein required for acetylcholine release. We measured long-term potentiation of Schaffer collateral-CA1 synapses in vivo and assessed information processing by using a mouse touchscreen version of paired associates learning task (PAL). Acquisition of information in the mouse PAL task correlated to levels of hippocampal VAChT, suggesting a critical role for cholinergic tone. Accordingly, synaptic plasticity in the hippocampus in vivo was disturbed, but not completely abolished, by decreased hippocampal cholinergic signaling. Disrupted forebrain cholinergic signaling also affected working memory, a result reproduced by selectively decreasing VAChT in the hippocampus. In contrast, spatial memory was relatively preserved, whereas reversal spatial memory was sensitive to decreased hippocampal cholinergic signaling. This work provides a refined roadmap of how synaptically secreted acetylcholine influences distinct behaviors and suggests that distinct forms of cognitive processing may be regulated in different ways by cholinergic activity.
Assuntos
Acetilcolina/metabolismo , Hipocampo/fisiologia , Memória de Curto Prazo/fisiologia , Plasticidade Neuronal/fisiologia , Proteínas Vesiculares de Transporte de Acetilcolina/metabolismo , Animais , Potenciação de Longa Duração/fisiologia , Camundongos Transgênicos , Prosencéfalo/metabolismo , Memória Espacial/fisiologia , Sinapses/metabolismoRESUMO
The relationship between long-term cholinergic dysfunction and risk of developing dementia is poorly understood. Here we used mice with deletion of the vesicular acetylcholine transporter (VAChT) in the forebrain to model cholinergic abnormalities observed in dementia. Whole-genome RNA sequencing of hippocampal samples revealed that cholinergic failure causes changes in RNA metabolism. Remarkably, key transcripts related to Alzheimer's disease are affected. BACE1, for instance, shows abnormal splicing caused by decreased expression of the splicing regulator hnRNPA2/B1. Resulting BACE1 overexpression leads to increased APP processing and accumulation of soluble Aß1-42. This is accompanied by age-related increases in GSK3 activation, tau hyperphosphorylation, caspase-3 activation, decreased synaptic markers, increased neuronal death, and deteriorating cognition. Pharmacological inhibition of GSK3 hyperactivation reversed deficits in synaptic markers and tau hyperphosphorylation induced by cholinergic dysfunction, indicating a key role for GSK3 in some of these pathological changes. Interestingly, in human brains there was a high correlation between decreased levels of VAChT and hnRNPA2/B1 levels with increased tau hyperphosphorylation. These results suggest that changes in RNA processing caused by cholinergic loss can facilitate Alzheimer's-like pathology in mice, providing a mechanism by which decreased cholinergic tone may increase risk of dementia.
Assuntos
Acetilcolina/metabolismo , Doença de Alzheimer/patologia , Regulação da Expressão Gênica/genética , Hipocampo/metabolismo , RNA/metabolismo , Proteínas Vesiculares de Transporte de Acetilcolina/deficiência , Doença de Alzheimer/genética , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Embrião de Mamíferos , Inibidores Enzimáticos/farmacologia , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Hipocampo/citologia , Humanos , Deficiências da Aprendizagem/etiologia , Deficiências da Aprendizagem/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , RNA/genética , Tiazóis/farmacologia , Fator Nuclear 1 de Tireoide/genética , Fator Nuclear 1 de Tireoide/metabolismo , Ureia/análogos & derivados , Ureia/farmacologia , Proteínas Vesiculares de Transporte de Acetilcolina/genéticaRESUMO
UNLABELLED: Cholinergic vulnerability, characterized by loss of acetylcholine (ACh), is one of the hallmarks of Alzheimer's disease (AD). Previous work has suggested that decreased ACh activity in AD may contribute to pathological changes through global alterations in alternative splicing. This occurs, at least partially, via the regulation of the expression of a critical protein family in RNA processing, heterogeneous nuclear ribonucleoprotein (hnRNP) A/B proteins. These proteins regulate several steps of RNA metabolism, including alternative splicing, RNA trafficking, miRNA export, and gene expression, providing multilevel surveillance in RNA functions. To investigate the mechanism by which cholinergic tone regulates hnRNPA2/B1 expression, we used a combination of genetic mouse models and in vivo and in vitro techniques. Decreasing cholinergic tone reduced levels of hnRNPA2/B1, whereas increasing cholinergic signaling in vivo increased expression of hnRNPA2/B1. This effect was not due to decreased hnRNPA2/B1 mRNA expression, increased aggregation, or degradation of the protein, but rather to decreased mRNA translation by nonsense-mediated decay regulation of translation. Cell culture and knock-out mice experiments demonstrated that M1 muscarinic signaling is critical for cholinergic control of hnRNPA2/B1 protein levels. Our experiments suggest an intricate regulation of hnRNPA2/B1 levels by cholinergic activity that interferes with alternative splicing in targeted neurons mimicking deficits found in AD. SIGNIFICANCE STATEMENT: In Alzheimer's disease, degeneration of basal forebrain cholinergic neurons is an early event. These neurons communicate with target cells and regulate their long-term activity by poorly understood mechanisms. Recently, the splicing factor hnRNPA2/B, which is decreased in Alzheimer's disease, was implicated as a potential mediator of long-term cholinergic regulation. Here, we demonstrate a mechanism by which cholinergic signaling controls the translation of hnRNPA2/B1 mRNA by activation of M1 muscarinic type receptors. Loss of cholinergic activity can have profound effects in target cells by modulating hnRNPA2/B1 levels.
Assuntos
Agonistas Colinérgicos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Neurônios/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Receptor Muscarínico M1/metabolismo , Animais , Carbacol/farmacologia , Células Cultivadas , Colina O-Acetiltransferase/genética , Colina O-Acetiltransferase/metabolismo , Colinérgicos/farmacologia , Embrião de Mamíferos , Regulação da Expressão Gênica/genética , Hipocampo/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fator Nuclear 1 de Tireoide , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitinação/efeitos dos fármacos , Ubiquitinação/genética , Proteínas Vesiculares de Transporte de Acetilcolina/genética , Proteínas Vesiculares de Transporte de Acetilcolina/metabolismoRESUMO
Stress-inducible phosphoprotein I (STIP1, STI1 or HOP) is a co-chaperone intermediating Hsp70/Hsp90 exchange of client proteins, but it can also be secreted to trigger prion protein-mediated neuronal signaling. Some mothers of children with autism spectrum disorders (ASD) present antibodies against certain brain proteins, including antibodies against STIP1. Maternal antibodies can cross the fetus blood-brain barrier during pregnancy, suggesting the possibility that they can interfere with STIP1 levels and, presumably, functions. However, it is currently unknown whether abnormal levels of STIP1 have any impact in ASD-related behavior. Here, we used mice with reduced (50%) or increased STIP1 levels (fivefold) to test for potential ASD-like phenotypes. We found that increased STIP1 regulates the abundance of Hsp70 and Hsp90, whereas reduced STIP1 does not affect Hsp70, Hsp90 or the prion protein. Interestingly, BAC transgenic mice presenting fivefold more STIP1 show no major phenotype when examined in a series of behavioral tasks, including locomotor activity, elevated plus maze, Morris water maze and five-choice serial reaction time task (5-CSRTT). In contrast, mice with reduced STIP1 levels are hyperactive and have attentional deficits on the 5-CSRTT, but exhibit normal performance for the other tasks. We conclude that reduced STIP1 levels can contribute to phenotypes related to ASD. However, future experiments are needed to define whether it is decreased chaperone capacity or impaired prion protein signaling that contributes to these phenotypes.
Assuntos
Transtorno do Deficit de Atenção com Hiperatividade/metabolismo , Comportamento Animal , Proteínas de Choque Térmico/deficiência , Animais , Transtorno do Deficit de Atenção com Hiperatividade/genética , Transtorno do Deficit de Atenção com Hiperatividade/psicologia , Modelos Animais de Doenças , Predisposição Genética para Doença , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Choque Térmico/genética , Masculino , Aprendizagem em Labirinto , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora , Fenótipo , Proteínas PrPC/metabolismo , Tempo de Reação , Natação , Fatores de TempoRESUMO
BACKGROUND AND PURPOSE: Disruptions of executive function, including attentional deficits, are a hallmark of a number of diseases. ACh in the prefrontal cortex regulates attentive behaviour; however, the role of α7 nicotinic ACh receptor (α7nAChR) in attention is contentious. EXPERIMENTAL APPROACH: In order to probe attention, we trained both wild-type and α7nAChR knockout mice on a touch screen-based five-choice serial reaction time task (5-CSRT). Following training procedures, we then tested sustained attention using a probe trial experiment. To further differentiate the role of specific nicotinic receptors in attention, we then tested the effects of both α7nAChR and ß2nAChR agonists on the performance of both wild-type and knockout mice on the 5-CSRT task. KEY RESULTS: At low doses, α7nAChR agonists improved attentional performance of wild-type mice, while high doses had deleterious effects on attention. α7nAChR knockout mice displayed deficits in sustained attention that were not ameliorated by α7nAChR agonists. However, these deficits were completely reversed by the administration of a ß2nAChR agonist. Furthermore, administration of a ß2nAChR agonist in α7nAChR knockout mice elicited similar biochemical response in the prefrontal cortex as the administration of α7nAChR agonists in wild-type mice. CONCLUSIONS AND IMPLICATIONS: Our experiments reveal an intricate relationship between distinct nicotinic receptors to regulate attentional performance and provide the basis for targeting ß2nAChRs pharmacologically to decrease attentional deficits due to a dysfunction in α7nAChRs.
Assuntos
Atenção/fisiologia , Receptores Nicotínicos/fisiologia , Receptor Nicotínico de Acetilcolina alfa7/fisiologia , Animais , Atenção/efeitos dos fármacos , Comportamento Animal/efeitos dos fármacos , Ingestão de Alimentos/efeitos dos fármacos , Isoxazóis/farmacologia , Masculino , Camundongos Knockout , Agonistas Nicotínicos/farmacologia , Pirrolidinas/farmacologia , Receptor Nicotínico de Acetilcolina alfa7/agonistas , Receptor Nicotínico de Acetilcolina alfa7/deficiênciaRESUMO
Sperm storage is a common phenomenon in most female reptiles. Evidence of sperm storage is based on the observation that female fertilization occurs even when females are separated from males, as well as the presence of agglomerates of spermatozoa in specific regions of the oviducts. Lizards are capable of storing sperm in the uterine tube, vagina, or in both regions. However, representatives of the Gekkonidae family commonly store spermatozoa in the uterine tube, which is considered an ancestral character state for Squamates. Using comparative techniques of light, transmission and scanning electron microscopy, we observed stored sperm organized in compact bundles with their heads facing the bottom of the crypts of the uterine tube, indicating chemotactic attraction. The alignment and packing of spermatozoa in Hemidactylus mabouia indicates that the process of evacuation of the crypts for fertilization may be related to the passage of the egg that exerts mechanical pressure against the walls of the uterine tube, causing its distension and the release of spermatozoa. We conclude that the sperm storage region and the morphological organization of the crypts in the uterine tube of H. mabouia is similar to other previously studied species of lizards, supporting the notion that sperm storage is a common reproductive strategy among female reptiles.
Assuntos
Reprodução/fisiologia , Répteis/anatomia & histologia , Espermatozoides/fisiologia , Animais , Tubas Uterinas/fisiologia , Feminino , Fertilização/fisiologia , Masculino , Microscopia Eletrônica de Transmissão , Espermatozoides/ultraestruturaRESUMO
One of the key brain regions in cognitive processing and executive function is the prefrontal cortex (PFC), which receives cholinergic input from basal forebrain cholinergic neurons. We evaluated the contribution of synaptically released acetylcholine (ACh) to executive function by genetically targeting the vesicular acetylcholine transporter (VAChT) in the mouse forebrain. Executive function was assessed using a pairwise visual discrimination paradigm and the 5-choice serial reaction time task (5-CSRT). In the pairwise test, VAChT-deficient mice were able to learn, but were impaired in reversal learning, suggesting that these mice present cognitive inflexibility. Interestingly, VAChT-targeted mice took longer to reach criteria in the 5-CSRT. Although their performance was indistinguishable from that of control mice during low attentional demand, increased attentional demand revealed striking deficits in VAChT-deleted mice. Galantamine, a cholinesterase inhibitor used in Alzheimer's disease, significantly improved the performance of control mice, but not of VAChT-deficient mice on the 5-CSRT. In vivo magnetic resonance spectroscopy showed altered levels of two neurochemical markers of neuronal function, taurine and lactate, suggesting altered PFC metabolism in VAChT-deficient mice. The PFC of these mice displayed a drastic reduction in the splicing factor heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2/B1), whose cholinergic-mediated reduction was previously demonstrated in Alzheimer's disease. Consequently, several key hnRNPA2/B1 target transcripts involved in neuronal function present changes in alternative splicing in VAChT-deficient mice, including pyruvate kinase M, a key enzyme involved in lactate metabolism. We propose that VAChT-targeted mice can be used to model and to dissect the neurochemical basis of executive abnormalities.
Assuntos
Transtornos Cognitivos/genética , Transtornos Cognitivos/patologia , Função Executiva/fisiologia , Córtex Pré-Frontal/metabolismo , Córtex Pré-Frontal/patologia , Splicing de RNA/genética , Proteínas Vesiculares de Transporte de Acetilcolina/deficiência , Acetilcolina/metabolismo , Animais , Ácido Aspártico/análogos & derivados , Ácido Aspártico/metabolismo , Comportamento de Escolha/efeitos dos fármacos , Comportamento de Escolha/fisiologia , Colina/metabolismo , Inibidores da Colinesterase/farmacologia , Transtornos Cognitivos/tratamento farmacológico , Galantamina/farmacologia , Inositol/metabolismo , Ácido Láctico/metabolismo , Locomoção/efeitos dos fármacos , Locomoção/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Estimulação Luminosa , Córtex Pré-Frontal/efeitos dos fármacos , Desempenho Psicomotor/efeitos dos fármacos , Taurina/metabolismo , Proteínas Vesiculares de Transporte de Acetilcolina/genéticaRESUMO
Acetylcholine (ACh) is an important neuromodulator in the nervous system implicated in many forms of cognitive and motor processing. Recent studies have used bacterial artificial chromosome (BAC) transgenic mice expressing channelrhodopsin-2 (ChR2) protein under the control of the choline acetyltransferase (ChAT) promoter (ChAT-ChR2-EYFP) to dissect cholinergic circuit connectivity and function using optogenetic approaches. We report that a mouse line used for this purpose also carries several copies of the vesicular acetylcholine transporter gene (VAChT), which leads to overexpression of functional VAChT and consequently increased cholinergic tone. We demonstrate that these mice have marked improvement in motor endurance. However, they also present severe cognitive deficits, including attention deficits and dysfunction in working memory and spatial memory. These results suggest that increased VAChT expression may disrupt critical steps in information processing. Our studies demonstrate that ChAT-ChR2-EYFP mice show altered cholinergic tone that fundamentally differentiates them from wild-type mice.
Assuntos
Atenção/fisiologia , Colina O-Acetiltransferase/genética , Colina O-Acetiltransferase/metabolismo , Cognição/fisiologia , Resistência Física/genética , Resistência Física/fisiologia , Animais , Ansiedade/psicologia , Western Blotting , Channelrhodopsins , Imunofluorescência , Teste de Tolerância a Glucose , Força da Mão/fisiologia , Elevação dos Membros Posteriores , Aprendizagem em Labirinto/fisiologia , Metabolismo/genética , Metabolismo/fisiologia , Camundongos , Camundongos Transgênicos , Sistema Nervoso Parassimpático/fisiologia , Reação em Cadeia da Polimerase , Equilíbrio Postural/fisiologia , Desempenho Psicomotor/fisiologia , Tempo de Reação/fisiologia , Natação/fisiologia , Proteínas Vesiculares de Transporte de Acetilcolina/genética , Proteínas Vesiculares de Transporte de Acetilcolina/fisiologiaRESUMO
Acetylcholine, the first chemical to be identified as a neurotransmitter, is packed in synaptic vesicles by the activity of VAChT (vesicular acetylcholine transporter). A decrease in VAChT expression has been reported in a number of diseases, and this has consequences for the amount of acetylcholine loaded in synaptic vesicles as well as for neurotransmitter release. Several genetically modified mice targeting the VAChT gene have been generated, providing novel models to understand how changes in VAChT affect transmitter release. A surprising finding is that most cholinergic neurons in the brain also can express a second type of vesicular neurotransmitter transporter that allows these neurons to secrete two distinct neurotransmitters. Thus a given neuron can use two neurotransmitters to regulate different physiological functions. In addition, recent data indicate that non-neuronal cells can also express the machinery used to synthesize and release acetylcholine. Some of these cells rely on VAChT to secrete acetylcholine with potential physiological consequences in the periphery. Hence novel functions for the oldest neurotransmitter known are emerging with the potential to provide new targets for the treatment of several pathological conditions.