RESUMO
Although a disease-modifying therapy for classic late infantile neuronal ceroid lipofuscinosis (CLN2 disease) exists, poor understanding of cellular pathophysiology has hampered the development of more effective and persistent therapies. Here, we investigated the nature and progression of neurological and underlying neuropathological changes in Cln2R207X mice, which carry one of the most common pathogenic mutations in human patients but are yet to be fully characterized. Long-term electroencephalography recordings revealed progressive epileptiform abnormalities, including spontaneous seizures, providing a robust, quantifiable, and clinically relevant phenotype. These seizures were accompanied by the loss of multiple cortical neuron populations, including those stained for interneuron markers. Further histological analysis revealed early localized microglial activation months before neuron loss started in the thalamocortical system and spinal cord, which was accompanied by astrogliosis. This pathology was more pronounced and occurred in the cortex before the thalamus or spinal cord and differed markedly from the staging seen in mouse models of other forms of neuronal ceroid lipofuscinosis. Neonatal administration of adeno-associated virus serotype 9-mediated gene therapy ameliorated the seizure and gait phenotypes and prolonged the life span of Cln2R207X mice, attenuating most pathological changes. Our findings highlight the importance of clinically relevant outcome measures for judging preclinical efficacy of therapeutic interventions for CLN2 disease.
Assuntos
Neurônios , Convulsões , Animais , Humanos , Camundongos , Neurônios/patologia , Convulsões/genética , Convulsões/terapia , Convulsões/patologia , Gliose/patologia , Interneurônios/patologia , Tálamo/patologia , Modelos Animais de DoençasRESUMO
CLN1 disease, also called infantile neuronal ceroid lipofuscinosis (NCL) or infantile Batten disease, is a fatal neurodegenerative lysosomal storage disorder resulting from mutations in the CLN1 gene encoding the soluble lysosomal enzyme palmitoyl-protein thioesterase 1 (PPT1). Therapies for CLN1 disease have proven challenging because of the aggressive disease course and the need to treat widespread areas of the brain and spinal cord. Indeed, gene therapy has proven less effective for CLN1 disease than for other similar lysosomal enzyme deficiencies. We therefore tested the efficacy of enzyme replacement therapy (ERT) by administering monthly infusions of recombinant human PPT1 (rhPPT1) to PPT1-deficient mice (Cln1-/-) and CLN1R151X sheep to assess how to potentially scale up for translation. In Cln1-/- mice, intracerebrovascular (i.c.v.) rhPPT1 delivery was the most effective route of administration, resulting in therapeutically relevant CNS levels of PPT1 activity. rhPPT1-treated mice had improved motor function, reduced disease-associated pathology, and diminished neuronal loss. In CLN1R151X sheep, i.c.v. infusions resulted in widespread rhPPT1 distribution and positive treatment effects measured by quantitative structural MRI and neuropathology. This study demonstrates the feasibility and therapeutic efficacy of i.c.v. rhPPT1 ERT. These findings represent a key step toward clinical testing of ERT in children with CLN1 disease and highlight the importance of a cross-species approach to developing a successful treatment strategy.
Assuntos
Lipofuscinoses Ceroides Neuronais , Animais , Criança , Modelos Animais de Doenças , Terapia de Reposição de Enzimas , Humanos , Camundongos , Mutação , Lipofuscinoses Ceroides Neuronais/tratamento farmacológico , Lipofuscinoses Ceroides Neuronais/genética , OvinosRESUMO
Cannabidiol (CBD) has gained attention as a therapeutic agent and is purported to have immunomodulatory, neuroprotective, and anti-seizure effects. Here, we determined the effects of chronic CBD administration in a mouse model of CLN1 disease (Cln1-/-) that simultaneously exhibits neuroinflammation, neurodegeneration, and spontaneous seizures. Proteomic analysis showed that putative CBD receptors are expressed at similar levels in the brains of Cln1-/- mice compared to normal animals. Cln1-/- mice received an oral dose (100 mg/kg/day) of CBD for six months and were evaluated for changes in pathological markers of disease and seizures. Chronic cannabidiol administration was well-tolerated, high levels of CBD were detected in the brain, and markers of astrocytosis and microgliosis were reduced. However, CBD had no apparent effect on seizure frequency or neuron survival. These data are consistent with CBD having immunomodulatory effects. It is possible that a higher dose of CBD could also reduce neurodegeneration and seizure frequency.
Assuntos
Canabidiol , Doença Enxerto-Hospedeiro , Animais , Canabidiol/farmacologia , Canabidiol/uso terapêutico , Modelos Animais de Doenças , Doença Enxerto-Hospedeiro/tratamento farmacológico , Camundongos , Doenças Neuroinflamatórias , Lipofuscinoses Ceroides Neuronais , ProteômicaRESUMO
Human genetics have defined a new neurodevelopmental syndrome caused by loss-of-function mutations in MYT1L, a transcription factor known for enabling fibroblast-to-neuron conversions. However, how MYT1L mutation causes intellectual disability, autism, ADHD, obesity, and brain anomalies is unknown. Here, we developed a Myt1l haploinsufficient mouse model that develops obesity, white-matter thinning, and microcephaly, mimicking common clinical phenotypes. During brain development we discovered disrupted gene expression, mediated in part by loss of Myt1l gene-target activation, and identified precocious neuronal differentiation as the mechanism for microcephaly. In contrast, in adults we discovered that mutation results in failure of transcriptional and chromatin maturation, echoed in disruptions in baseline physiological properties of neurons. Myt1l haploinsufficiency also results in behavioral anomalies, including hyperactivity, muscle weakness, and social alterations, with more severe phenotypes in males. Overall, our findings provide insight into the mechanistic underpinnings of this disorder and enable future preclinical studies.
Assuntos
Deficiência Intelectual , Proteínas do Tecido Nervoso/genética , Fatores de Transcrição/genética , Animais , Encéfalo/metabolismo , Humanos , Deficiência Intelectual/genética , Masculino , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Neurogênese , Fenótipo , Fatores de Transcrição/metabolismoRESUMO
Lysosomal storage diseases (LSDs) are typically caused by a deficiency in a soluble acid hydrolase and are characterized by the accumulation of undegraded substrates in the lysosome. Determining the role of specific cell types in the pathogenesis of LSDs is a major challenge due to the secretion and subsequent uptake of lysosomal hydrolases by adjacent cells, often referred to as "cross-correction." Here we create and validate a conditional mouse model for cell-autonomous expression of galactocerebrosidase (GALC), the lysosomal enzyme deficient in Krabbe disease. We show that lysosomal membrane-tethered GALC (GALCLAMP1) retains enzyme activity, is able to cleave galactosylsphingosine, and is unable to cross-correct. Ubiquitous expression of GALCLAMP1 fully rescues the phenotype of the GALC-deficient mouse (Twitcher), and widespread deletion of GALCLAMP1 recapitulates the Twitcher phenotype. We demonstrate the utility of this model by deleting GALCLAMP1 specifically in myelinating Schwann cells in order to characterize the peripheral neuropathy seen in Krabbe disease.
Assuntos
Galactosilceramidase/metabolismo , Leucodistrofia de Células Globoides/patologia , Lisossomos/enzimologia , Proteínas Recombinantes de Fusão/metabolismo , Células de Schwann/patologia , Animais , Células Cultivadas , Modelos Animais de Doenças , Feminino , Fibroblastos , Galactosilceramidase/genética , Técnicas de Silenciamento de Genes , Humanos , Membranas Intracelulares/metabolismo , Leucodistrofia de Células Globoides/diagnóstico , Leucodistrofia de Células Globoides/genética , Proteínas de Membrana Lisossomal/genética , Proteínas de Membrana Lisossomal/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Cultura Primária de Células , Proteínas Recombinantes de Fusão/genéticaRESUMO
Infantile globoid cell leukodystrophy (GLD, Krabbe disease) is a fatal demyelinating disorder caused by a deficiency in the lysosomal enzyme galactosylceramidase (GALC). GALC deficiency leads to the accumulation of the cytotoxic glycolipid, galactosylsphingosine (psychosine). Complementary evidence suggested that psychosine is synthesized via an anabolic pathway. Here, we show instead that psychosine is generated catabolically through the deacylation of galactosylceramide by acid ceramidase (ACDase). This reaction uncouples GALC deficiency from psychosine accumulation, allowing us to test the long-standing "psychosine hypothesis." We demonstrate that genetic loss of ACDase activity (Farber disease) in the GALC-deficient mouse model of human GLD (twitcher) eliminates psychosine accumulation and cures GLD. These data suggest that ACDase could be a target for substrate reduction therapy (SRT) in Krabbe patients. We show that pharmacological inhibition of ACDase activity with carmofur significantly decreases psychosine accumulation in cells from a Krabbe patient and prolongs the life span of the twitcher (Twi) mouse. Previous SRT experiments in the Twi mouse utilized l-cycloserine, which inhibits an enzyme several steps upstream of psychosine synthesis, thus altering the balance of other important lipids. Drugs that directly inhibit ACDase may have a more acceptable safety profile due to their mechanistic proximity to psychosine biogenesis. In total, these data clarify our understanding of psychosine synthesis, confirm the long-held psychosine hypothesis, and provide the impetus to discover safe and effective inhibitors of ACDase to treat Krabbe disease.
Assuntos
Ceramidase Ácida/genética , Deleção de Genes , Leucodistrofia de Células Globoides/genética , Leucodistrofia de Células Globoides/metabolismo , Psicosina/metabolismo , Animais , Linhagem Celular Tumoral , Citocinas/metabolismo , Metilação de DNA , Modelos Animais de Doenças , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Leucodistrofia de Células Globoides/tratamento farmacológicoRESUMO
Here we have characterized the functional impairments resulting from conditional knockout of the ubiquitin-conjugating E2 enzyme (UBC13) in rodent cerebellar granule neurons, which greatly increases the parallel fiber presynaptic boutons and functional parallel fiber/Purkinje cell synapses. We report that conditional UBC13 knockout mice exhibit reliable deficits on several gait-related variables when their velocity of ambulation is tightly controlled by a moving treadmill and by restricting space for movement. Selected gait parameters and movement patterns related to spontaneous exploration in an open field may also be affected in conditional UBC13 knockout mice. Analysis of open-field data as a function of test session half using force-plate actometer instrumentation suggest that conditional UBC13 knockout mice have alterations in emotionality, possibly affecting gait and movement variables. These findings suggest that conditional UBC13 knockout mice represent a valuable platform for assessing the effects of disturbances in cerebellar granule cell circuitry on gait and other aspects of locomotion. Also, the possibility that psychological factors such as altered emotionality may impact gait and movement patterns in these mice suggest that these mice may provide a useful model for evaluating analogous behavioral impairments in autism spectrum disorders and other neurodevelopmental syndromes associated with deregulation of ubiquitin signaling.
Assuntos
Comportamento Exploratório/fisiologia , Marcha/fisiologia , Locomoção/fisiologia , Enzimas de Conjugação de Ubiquitina/metabolismo , Análise de Variância , Animais , Feminino , Marcha/genética , Locomoção/genética , Masculino , Camundongos , Camundongos Knockout , Enzimas de Conjugação de Ubiquitina/deficiência , Enzimas de Conjugação de Ubiquitina/genéticaRESUMO
Medulloblastoma is the most common malignant brain cancer of childhood. Further understanding of tumorigenic mechanisms may define new therapeutic targets. Geminin maintains genome fidelity by controlling re-initiation of DNA replication within a cell cycle. In some contexts, Geminin inhibition induces cancer-selective cell cycle arrest and apoptosis and/or sensitizes cancer cells to Topoisomerase IIα inhibitors such as etoposide, which is used in combination chemotherapies for medulloblastoma. However, Geminin's potential role in medulloblastoma tumorigenesis remained undefined. Here, we found that Geminin is highly expressed in human and mouse medulloblastomas and in murine granule neuron precursor (GNP) cells during cerebellar development. Conditional Geminin loss significantly enhanced survival in the SmoA1 mouse medulloblastoma model. Geminin loss in this model also reduced numbers of preneoplastic GNPs persisting at one postnatal month, while at two postnatal weeks these cells exhibited an elevated DNA damage response and apoptosis. Geminin knockdown likewise impaired human medulloblastoma cell growth, activating G2 checkpoint and DNA damage response pathways, triggering spontaneous apoptosis, and enhancing G2 accumulation of cells in response to etoposide treatment. Together, these data suggest preneoplastic and cancer cell-selective roles for Geminin in medulloblastoma, and suggest that targeting Geminin may impair tumor growth and enhance responsiveness to Topoisomerase IIα-directed chemotherapies.
RESUMO
Infantile neuronal ceroid lipofuscinosis (INCL, or CLN1 disease) is an inherited neurodegenerative storage disorder caused by a deficiency of the lysosomal enzyme palmitoyl protein thioesterase 1 (PPT1). It was widely believed that the pathology associated with INCL was limited to the brain, but we have now found unexpectedly profound pathology in the human INCL spinal cord. Similar pathological changes also occur at every level of the spinal cord of PPT1-deficient (Ppt1-/- ) mice before the onset of neuropathology in the brain. Various forebrain-directed gene therapy approaches have only had limited success in Ppt1-/- mice. Targeting the spinal cord via intrathecal administration of an adeno-associated virus (AAV) gene transfer vector significantly prevented pathology and produced significant improvements in life span and motor function in Ppt1-/- mice. Surprisingly, forebrain-directed gene therapy resulted in essentially no PPT1 activity in the spinal cord, and vice versa. This leads to a reciprocal pattern of histological correction in the respective tissues when comparing intracranial with intrathecal injections. However, the characteristic pathological features of INCL were almost completely absent in both the brain and spinal cord when intracranial and intrathecal injections of the same AAV vector were combined. Targeting both the brain and spinal cord also produced dramatic and synergistic improvements in motor function with an unprecedented increase in life span. These data show that spinal cord pathology significantly contributes to the clinical progression of INCL and can be effectively targeted therapeutically. This has important implications for the delivery of therapies in INCL, and potentially in other similar disorders.
Assuntos
Encéfalo/patologia , Terapia Genética/métodos , Proteínas de Membrana/farmacologia , Lipofuscinoses Ceroides Neuronais/terapia , Medula Espinal/patologia , Tioléster Hidrolases/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Criança , Modelos Animais de Doenças , Vetores Genéticos/administração & dosagem , Vetores Genéticos/farmacologia , Humanos , Injeções Intraventriculares/métodos , Injeções Espinhais , Proteínas de Membrana/administração & dosagem , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Neuroglia/patologia , Lipofuscinoses Ceroides Neuronais/patologia , Neurônios/patologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Medula Espinal/efeitos dos fármacos , Tioléster Hidrolases/administração & dosagem , Tioléster Hidrolases/genética , Tioléster Hidrolases/metabolismoRESUMO
"Cross-correction," the transfer of soluble lysosomal enzymes between neighboring cells, forms the foundation for therapeutics of lysosomal storage disorders (LSDs). However, "cross-correction" poses a significant barrier to studying the role of specific cell types in LSD pathogenesis. By expressing the native enzyme in only one cell type, neighboring cell types are invariably corrected. In this study, we present a strategy to limit "cross-correction" of palmitoyl-protein thioesterase-1(PPT1), a lysosomal hydrolase deficient in Infantile Neuronal Ceroid Lipofuscinosis (INCL, Infantile Batten disease) to the lysosomal membrane via the C-terminus of lysosomal associated membrane protein-1 (LAMP1). Tethering PPT1 to the lysosomal membrane prevented "cross-correction" while allowing PPT1 to retain its enzymatic function and localization in vitro. A transgenic line harboring the lysosomal membrane-tethered PPT1 was then generated. We show that expression of lysosome-restricted PPT1 in vivo largely rescues the INCL biochemical, histological, and functional phenotype. These data suggest that lysosomal tethering of PPT1 via the C-terminus of LAMP1 is a viable strategy and that this general approach can be used to study the role of specific cell types in INCL pathogenesis, as well as other LSDs. Ultimately, understanding the role of specific cell types in the disease progression of LSDs will help guide the development of more targeted therapeutics. One Sentence Synopsis: Tethering PPT1 to the lysosomal membrane is a viable strategy to prevent "cross-correction" and will allow for the study of specific cellular contributions in INCL pathogenesis.
RESUMO
Infantile neuronal ceroid lipofuscinosis (INCL, Infantile Batten disease) is an invariably fatal neurodegenerative pediatric disorder caused by an inherited mutation in the PPT1 gene. Patients with INCL lack the lysosomal enzyme palmitoyl protein thioesterase-1 (PPT1, EC 3.1.2.22), resulting in intracellular accumulation of autofluorescent storage material and subsequent neuropathology. The Ppt1(-/-) mouse is deficient in PPT1 activity and represents a useful animal model of INCL that recapitulates most of the clinical and pathological aspects of the disease. Preclinical therapeutic experiments performed in the INCL mouse include CNS-directed gene therapy and recombinant enzyme replacement therapy; both seek to re-establish therapeutic levels of the deficient enzyme. We present a novel method for the histochemical localization of PPT1 activity in the Ppt1(-/-) mouse. By utilizing the substrate CUS-9235, tissues known to be positive for PPT1 activity turn varying intensities of blue. Presented here are histochemistry data showing the staining pattern in Ppt1(-/-), wild type, and Ppt1(-/-) mice treated with enzyme replacement therapy or AAV2/9-PPT1-mediated gene therapy. Results are paired with quantitative biochemistry data that confirm the ability of CUS-9235 to detect and localize PPT1 activity. This new method complements the current tools for the study of INCL and evaluation of effective therapies.
Assuntos
Tioléster Hidrolases/metabolismo , Animais , Feminino , Terapia Genética , Humanos , Rim/enzimologia , Fígado/enzimologia , Masculino , Camundongos Knockout , Miocárdio/enzimologia , Lipofuscinoses Ceroides Neuronais/terapia , Especificidade de Órgãos , Tioléster Hidrolases/genéticaRESUMO
Infantile neuronal ceroid lipofuscinosis (INCL, Infantile Batten disease) is a neurodegenerative lysosomal storage disease caused by a deficiency in palmitoyl protein thioesterase-1 (PPT1). The PPT1-deficient mouse (Cln1(-/-)) is a useful phenocopy of human INCL. Cln1(-/-) mice display retinal dysfunction, seizures, motor deficits, and die at ~8 months of age. However, little is known about the cognitive and behavioral functions of Cln1(-/-) mice during disease progression. In the present study, younger (~1-2 months of age) Cln1(-/-) mice showed minor deficits in motor/sensorimotor functions while older (~5-6 months of age) Cln1(-/-) mice exhibited more severe impairments, including decreased locomotor activity, inferior cued water maze performance, decreased running wheel ability, and altered auditory cue conditioning. Unexpectedly, certain cognitive functions such as some learning and memory capabilities seemed intact in older Cln1(-/-) mice. Younger and older Cln1(-/-) mice presented with walking initiation defects, gait abnormalities, and slowed movements, which are analogous to some symptoms reported in INCL and parkinsonism. However, there was no evidence of alterations in dopaminergic markers in Cln1(-/-) mice. Results from this study demonstrate quantifiable changes in behavioral functions during progression of murine INCL and suggest that Parkinson-like motor/sensorimotor deficits in Cln1(-/-) mice are not mediated by dopamine deficiency.
Assuntos
Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Lipofuscinoses Ceroides Neuronais/metabolismo , Doença de Parkinson Secundária/metabolismo , Convulsões/metabolismo , Tioléster Hidrolases/genética , Animais , Modelos Animais de Doenças , Progressão da Doença , Neurônios Dopaminérgicos/patologia , Feminino , Expressão Gênica , Humanos , Masculino , Aprendizagem em Labirinto , Camundongos , Camundongos Knockout , Atividade Motora , Lipofuscinoses Ceroides Neuronais/genética , Lipofuscinoses Ceroides Neuronais/patologia , Doença de Parkinson Secundária/genética , Doença de Parkinson Secundária/patologia , Reconhecimento Fisiológico de Modelo , Convulsões/genética , Convulsões/patologia , Transdução de Sinais , Tioléster Hidrolases/deficiênciaRESUMO
Infantile neuronal ceroid lipofuscinosis (INCL) is an inherited neurodegenerative lysosomal storage disease (LSD) caused by a deficiency in palmitoyl protein thioesterase-1 (PPT1). Studies in Ppt1(-/-) mice demonstrate that glial activation is central to the pathogenesis of INCL. Astrocyte activation precedes neuronal loss, while cytokine upregulation associated with microglial reactivity occurs before and concurrent with neurodegeneration. Therefore, we hypothesized that cytokine cascades associated with neuroinflammation are important therapeutic targets for the treatment of INCL. MW01-2-151SRM (MW151) is a blood-brain barrier penetrant, small-molecule anti-neuroinflammatory that attenuates glial cytokine upregulation in models of neuroinflammation such as traumatic brain injury, Alzheimer's disease, and kainic acid toxicity. Thus, we used MW151, alone and in combination with CNS-directed, AAV-mediated gene therapy, as a possible treatment for INCL. MW151 alone decreased seizure susceptibility. When combined with AAV-mediated gene therapy, treated INCL mice had increased life spans, improved motor performance, and eradication of seizures. Combination-treated INCL mice also had decreased brain atrophy, astrocytosis, and microglial activation, as well as intermediary effects on cytokine upregulation. These data suggest that MW151 can attenuate seizure susceptibility but is most effective when used in conjunction with a therapy that targets the primary genetic defect.
Assuntos
Barreira Hematoencefálica/metabolismo , Terapia Genética , Microglia/metabolismo , Lipofuscinoses Ceroides Neuronais/terapia , Tioléster Hidrolases/genética , Animais , Anti-Inflamatórios/farmacocinética , Anti-Inflamatórios/uso terapêutico , Barreira Hematoencefálica/efeitos dos fármacos , Citocinas/genética , Citocinas/metabolismo , Dependovirus/genética , Locomoção , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Piridazinas/farmacocinética , Piridazinas/uso terapêutico , Pirimidinas/farmacocinética , Pirimidinas/uso terapêutico , Convulsões/terapia , Tioléster Hidrolases/metabolismoRESUMO
Apolipoprotein E (apoE) is the strongest known genetic risk factor for late onset Alzheimer's disease (AD). It influences amyloid-ß (Aß) clearance and aggregation, which likely contributes in large part to its role in AD pathogenesis. We recently found that HJ6.3, a monoclonal antibody against apoE, significantly reduced Aß plaque load when given to APPswe/PS1ΔE9 (APP/PS1) mice starting before the onset of plaque deposition. To determine whether the anti-apoE antibody HJ6.3 affects Aß plaques, neuronal network function, and behavior in APP/PS1 mice after plaque onset, we administered HJ6.3 (10 mg/kg/week) or PBS intraperitoneally to 7-month-old APP/PS1 mice for 21 weeks. HJ6.3 mildly improved spatial learning performance in the water maze, restored resting-state functional connectivity, and modestly reduced brain Aß plaque load. There was no effect of HJ6.3 on total plasma cholesterol or cerebral amyloid angiopathy. To investigate the underlying mechanisms of anti-apoE immunotherapy, HJ6.3 was applied to the brain cortical surface and amyloid deposition was followed over 2 weeks using in vivo imaging. Acute exposure to HJ6.3 affected the course of amyloid deposition in that it prevented the formation of new amyloid deposits, limited their growth, and was associated with occasional clearance of plaques, a process likely associated with direct binding to amyloid aggregates. Topical application of HJ6.3 for only 14 d also decreased the density of amyloid plaques assessed postmortem. Collectively, these studies suggest that anti-apoE antibodies have therapeutic potential when given before or after the onset of Aß pathology.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Anticorpos/uso terapêutico , Apolipoproteínas E/imunologia , Encéfalo/metabolismo , Doença de Alzheimer/sangue , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/efeitos dos fármacos , Precursor de Proteína beta-Amiloide/genética , Amiloidose/tratamento farmacológico , Amiloidose/metabolismo , Amiloidose/patologia , Animais , Encéfalo/efeitos dos fármacos , Colesterol/sangue , Modelos Animais de Doenças , Feminino , Hemorragia/tratamento farmacológico , Hemorragia/etiologia , Coxeadura Animal/tratamento farmacológico , Coxeadura Animal/etiologia , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Transgênicos , Mutação/genética , Presenilina-1/genéticaRESUMO
Nicotinamide adenine dinucleotide (NAD(+)) is an enzyme cofactor or cosubstrate in many essential biological pathways. To date, the primary source of neuronal NAD(+) has been unclear. NAD(+) can be synthesized from several different precursors, among which nicotinamide is the substrate predominantly used in mammals. The rate-limiting step in the NAD(+) biosynthetic pathway from nicotinamide is performed by nicotinamide phosphoribosyltransferase (Nampt). Here, we tested the hypothesis that neurons use intracellular Nampt-mediated NAD(+) biosynthesis by generating and evaluating mice lacking Nampt in forebrain excitatory neurons (CaMKIIαNampt(-/-) mice). CaMKIIαNampt(-/-) mice showed hippocampal and cortical atrophy, astrogliosis, microgliosis, and abnormal CA1 dendritic morphology by 2-3 months of age. Importantly, these histological changes occurred with altered intrahippocampal connectivity and abnormal behavior; including hyperactivity, some defects in motor skills, memory impairment, and reduced anxiety, but in the absence of impaired sensory processes or long-term potentiation of the Schaffer collateral pathway. These results clearly demonstrate that forebrain excitatory neurons mainly use intracellular Nampt-mediated NAD(+) biosynthesis to mediate their survival and function. Studying this particular NAD(+) biosynthetic pathway in these neurons provides critical insight into their vulnerability to pathophysiological stimuli and the development of therapeutic and preventive interventions for their preservation.
Assuntos
Córtex Cerebral/metabolismo , Cognição/fisiologia , Hipocampo/metabolismo , Neurônios/metabolismo , Nicotinamida Fosforribosiltransferase/metabolismo , Animais , Atrofia/genética , Atrofia/metabolismo , Atrofia/patologia , Comportamento Animal/fisiologia , Córtex Cerebral/patologia , Gliose/metabolismo , Gliose/patologia , Hipocampo/patologia , Memória/fisiologia , Camundongos , Camundongos Knockout , Atividade Motora/fisiologia , Rede Nervosa/metabolismo , Rede Nervosa/patologia , Neurônios/patologia , Nicotinamida Fosforribosiltransferase/genéticaRESUMO
Brain aging is associated with diminished circadian clock output and decreased expression of the core clock proteins, which regulate many aspects of cellular biochemistry and metabolism. The genes encoding clock proteins are expressed throughout the brain, though it is unknown whether these proteins modulate brain homeostasis. We observed that deletion of circadian clock transcriptional activators aryl hydrocarbon receptor nuclear translocator-like (Bmal1) alone, or circadian locomotor output cycles kaput (Clock) in combination with neuronal PAS domain protein 2 (Npas2), induced severe age-dependent astrogliosis in the cortex and hippocampus. Mice lacking the clock gene repressors period circadian clock 1 (Per1) and period circadian clock 2 (Per2) had no observed astrogliosis. Bmal1 deletion caused the degeneration of synaptic terminals and impaired cortical functional connectivity, as well as neuronal oxidative damage and impaired expression of several redox defense genes. Targeted deletion of Bmal1 in neurons and glia caused similar neuropathology, despite the retention of intact circadian behavioral and sleep-wake rhythms. Reduction of Bmal1 expression promoted neuronal death in primary cultures and in mice treated with a chemical inducer of oxidative injury and striatal neurodegeneration. Our findings indicate that BMAL1 in a complex with CLOCK or NPAS2 regulates cerebral redox homeostasis and connects impaired clock gene function to neurodegeneration.
Assuntos
Fatores de Transcrição ARNTL/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Encéfalo/patologia , Proteínas CLOCK/fisiologia , Ritmo Circadiano/fisiologia , Gliose/genética , Degeneração Neural/fisiopatologia , Proteínas do Tecido Nervoso/fisiologia , Neurônios/metabolismo , Fatores de Transcrição ARNTL/deficiência , Envelhecimento/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/deficiência , Encéfalo/fisiopatologia , Proteínas CLOCK/deficiência , Córtex Cerebral/patologia , Ritmo Circadiano/genética , Corpo Estriado/patologia , Regulação da Expressão Gênica/fisiologia , Gliose/patologia , Hipocampo/patologia , Homeostase/genética , Homeostase/fisiologia , Locomoção/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes Neurológicos , Degeneração Neural/genética , Proteínas do Tecido Nervoso/deficiência , Neuroglia/metabolismo , Neuroglia/patologia , Neurônios/patologia , Oxirredução , Estresse Oxidativo , Proteínas Circadianas Period/deficiência , Proteínas Circadianas Period/fisiologia , Interferência de RNA , Transtornos do Sono do Ritmo Circadiano/fisiopatologiaRESUMO
Children with neurofibromatosis type 1 (NF1) frequently have cognitive and behavioral deficits. Some of these deficits have been successfully modeled in Nf1 genetically-engineered mice that develop optic gliomas (Nf1 OPG mice). In the current study, we show that abnormal motivational influences affect the behavior of Nf1 OPG mice, particularly with regard to their response to novel environmental stimuli. For example, Nf1 OPG mice made fewer spontaneous alternations in a Y-maze and fewer arm entries relative to WT controls. However, analysis of normalized alternation data demonstrated that these differences were not due to a spatial working memory deficit. Other reported behavioral results (e.g., open-field test, below) suggest that differential responses to novelty and/or other motivational influences may be more important determinants of these kinds of behavior than simple differences in locomotor activity/spontaneous movements. Importantly, normal long-term depression was observed in hippocampal slices from Nf1 OPG mice. Results from elevated plus maze testing showed that differences in exploratory activity between Nf1 OPG and WT control mice may be dependent on the environmental context (e.g., threatening or non-threatening) under which exploration is being measured. Nf1 OPG mice also exhibited decreased exploratory hole poking in a novel holeboard and showed abnormal olfactory preferences, although L-dopa (50 mg/kg) administration resolved the abnormal olfactory preference behaviors. Nf1 OPG mice displayed an attenuated response to a novel open field in terms of decreased ambulatory activity and rearing but only during the first 10 min of the session. Importantly, Nf1 OPG mice demonstrated investigative rearing deficits with regard to a novel hanging object suspended on one side of the field which were not rescued by L-dopa administration. Collectively, our results provide new data important for evaluating therapeutic treatments aimed at ameliorating NF1-associated cognitive/behavioral deficits.
Assuntos
Comportamento Animal/efeitos dos fármacos , Modelos Animais de Doenças , Dopaminérgicos/administração & dosagem , Levodopa/administração & dosagem , Motivação/efeitos dos fármacos , Neurofibromatose 1/tratamento farmacológico , Neurofibromina 1/fisiologia , Animais , Depressão/diagnóstico , Depressão/tratamento farmacológico , Depressão/etiologia , Eletrofisiologia , Feminino , Humanos , Integrases/metabolismo , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Transtornos da Memória/diagnóstico , Transtornos da Memória/tratamento farmacológico , Transtornos da Memória/etiologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Neurofibromatose 1/complicações , Neurofibromatose 1/metabolismoRESUMO
Previous studies suggest that loss of γ-secretase activity in postnatal mouse brains causes age-dependent memory impairment and neurodegeneration. Due to the diverse array of γ-secretase substrates, it remains to be demonstrated whether loss of cleavage of any specific substrate(s) is responsible for these defects. The bulk of the phenotypes observed in mammals deficient for γ-secretase or exposed to γ-secretase inhibitors are caused by the loss of Notch receptor proteolysis. Accordingly, inhibition of Notch signaling is the main cause for untoward effects for γ-secretase inhibitors as therapeutics for Alzheimer's disease. Therefore, we wished to determine if loss of canonical Notch signaling is responsible for the age-dependent neurodegeneration observed upon γ-secrectase deficiency in the mouse brain. We generated postnatal forebrain-specific RBPj conditional knockout (cKO) mice using the CamKII-Cre driver and examined behavior and brain pathology in 12-18 month old animals. Since all four mammalian Notch receptor homologues signal via this DNA binding protein, these mice lack canonical Notch signaling. We found that loss of RBPj in mature excitatory neurons was well tolerated, with no evidence for neurodegeneration or of learning and memory impairment in mice aged up to 18 months. The only phenotypic deficit we observed in the RBPj-deficient mice was a subtle abnormality in olfactory preferences, particularly in females. We conclude that the loss of canonical Notch signaling through the four receptors is not responsible for age-dependent neurodegeneration or learning and memory deficits seen in γ-secretase deficient mice.
Assuntos
Envelhecimento/fisiologia , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Transtornos da Memória/metabolismo , Doenças Neurodegenerativas/metabolismo , Neurônios/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Feminino , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Masculino , Transtornos da Memória/genética , Camundongos , Camundongos Knockout , Doenças Neurodegenerativas/genética , Prosencéfalo/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismoRESUMO
The androgenic adrenal steroids dehydroepiandrosterone (DHEA) and 4α-androstenedione (4-A) have significant biological activity, but it is unclear if the behavioral effects are unique or only reflections of the effects of testosterone (TS). Gonadally intact male Long-Evans rats were assigned to groups to receive supplements of DHEA, 4-A, TS, corticosteroid (CORT), all at 400 µg steroid/kg of body weight, or vehicle only for 5 weeks. All males were tested in a paradigm for sexual motivation that measures time and urinary marks near an inaccessible receptive female. It was found that DHEA and 4-A supplements failed to influence time near the estrous female in the same way TS supplements did, and, indeed, 5 weeks of 4-A administration reduced the time similar to the suppressive effects of CORT after 3 weeks. Further, animals treated with DHEA or 4-A left fewer urinary marks near an estrous female than TS and control groups. These results suggest that DHEA and 4-A are not merely precursors of sex hormones, and provide support for these steroids influencing the brain and behavior in a unique fashion that is dissimilar from the effects of TS on male sexual behavior.