RESUMO
Single domain antibodies (VHHs) are potentially disruptive therapeutics, with important biological value for treatment of several diseases, including neurological disorders. However, VHHs have not been widely used in the central nervous system (CNS), largely because of their restricted blood-brain barrier (BBB) penetration. Here, we propose a gene transfer strategy based on BBB-crossing adeno-associated virus (AAV)-based vectors to deliver VHH directly into the CNS. As a proof-of-concept, we explored the potential of AAV-delivered VHH to inhibit BACE1, a well-characterized target in Alzheimer's disease. First, we generated a panel of VHHs targeting BACE1, one of which, VHH-B9, shows high selectivity for BACE1 and efficacy in lowering BACE1 activity in vitro. We further demonstrate that a single systemic dose of AAV-VHH-B9 produces positive long-term (12 months plus) effects on amyloid load, neuroinflammation, synaptic function, and cognitive performance, in the AppNL-G-F Alzheimer's mouse model. These results constitute a novel therapeutic approach for neurodegenerative diseases, which is applicable to a range of CNS disease targets.
Assuntos
Doença de Alzheimer , Secretases da Proteína Precursora do Amiloide , Ácido Aspártico Endopeptidases , Anticorpos de Domínio Único , Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/imunologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Ácido Aspártico Endopeptidases/imunologia , Ácido Aspártico Endopeptidases/metabolismo , Barreira Hematoencefálica , Dependovirus/genética , Modelos Animais de Doenças , Vetores Genéticos/uso terapêutico , Camundongos , Camundongos TransgênicosRESUMO
Higher-order telencephalic circuitry has been suggested to be especially vulnerable to irradiation or other developmentally toxic impact. This report details the adult effects of prenatal irradiation at a sensitive time point on clinically relevant brain functions controlled by telencephalic regions, hippocampus (HPC), and prefrontal cortex (PFC). Pregnant C57Bl6/J mice were whole-body irradiated at embryonic day 11 (start of neurogenesis) with X-ray intensities of 0.0, 0.5, or 1.0 Gy. Female offspring completed a broad test battery of HPC-/PFC-controlled tasks that included cognitive performance, fear extinction, exploratory, and depression-like behaviors. We examined neural functions that are mechanistically related to these behavioral and cognitive changes, such as hippocampal field potentials and long-term potentiation, functional brain connectivity (by resting-state functional magnetic resonance imaging), and expression of HPC vesicular neurotransmitter transporters (by immunohistochemical quantification). Prenatally exposed mice displayed several higher-order dysfunctions, such as decreased nychthemeral activity, working memory defects, delayed extinction of threat-evoked response suppression as well as indications of perseverative behavior. Electrophysiological examination indicated impaired hippocampal synaptic plasticity. Prenatal irradiation also induced cerebral hypersynchrony and increased the number of glutamatergic HPC terminals. These changes in brain connectivity and plasticity could mechanistically underlie the irradiation-induced defects in higher telencephalic functions.
Assuntos
Efeitos Tardios da Exposição Pré-Natal , Exposição à Radiação , Animais , Comportamento Animal/fisiologia , Extinção Psicológica , Medo/psicologia , Feminino , Hipocampo/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Plasticidade Neuronal , Gravidez , Efeitos Tardios da Exposição Pré-Natal/patologiaRESUMO
BACKGROUND: RASopathies are a group of disorders that result from mutations in genes coding for proteins involved in regulating the Ras-MAPK signaling pathway, and have an increased incidence of autism spectrum disorder (ASD). Legius syndrome is a rare RASopathy caused by loss-of-function mutations in the SPRED1 gene. The patient phenotype is similar to, but milder than, Neurofibromatosis type 1-another RASopathy caused by loss-of-function mutations in the NF1 gene. RASopathies exhibit increased activation of Ras-MAPK signaling and commonly manifest with cognitive impairments and ASD. Here, we investigated if a Spred1-/- mouse model for Legius syndrome recapitulates ASD-like symptoms, and whether targeting the Ras-MAPK pathway has therapeutic potential in this RASopathy mouse model. METHODS: We investigated social and communicative behaviors in Spred1-/- mice and probed therapeutic mechanisms underlying the observed behavioral phenotypes by pharmacological targeting of the Ras-MAPK pathway with the MEK inhibitor PD325901. RESULTS: Spred1-/- mice have robust increases in social dominance in the automated tube test and reduced adult ultrasonic vocalizations during social communication. Neonatal ultrasonic vocalization was also altered, with significant differences in spectral properties. Spred1-/- mice also exhibit impaired nesting behavior. Acute MEK inhibitor treatment in adulthood with PD325901 reversed the enhanced social dominance in Spred1-/- mice to normal levels, and improved nesting behavior in adult Spred1-/- mice. LIMITATIONS: This study used an acute treatment protocol to administer the drug. It is not known what the effects of longer-term treatment would be on behavior. Further studies titrating the lowest dose of this drug that is required to alter Spred1-/- social behavior are still required. Finally, our findings are in a homozygous mouse model, whereas patients carry heterozygous mutations. These factors should be considered before any translational conclusions are drawn. CONCLUSIONS: These results demonstrate for the first time that social behavior phenotypes in a mouse model for RASopathies (Spred1-/-) can be acutely reversed. This highlights a key role for Ras-MAPK dysregulation in mediating social behavior phenotypes in mouse models for ASD, suggesting that proper regulation of Ras-MAPK signaling is important for social behavior.
Assuntos
Transtorno do Espectro Autista , Neurofibromina 1 , Proteínas Adaptadoras de Transdução de Sinal/genética , Adulto , Animais , Humanos , Camundongos , Camundongos Knockout , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Neurofibromina 1/genética , Fenótipo , Comportamento SocialRESUMO
Microglia, the mononuclear phagocytes of the central nervous system (CNS), are important for the maintenance of CNS homeostasis, but also critically contribute to CNS pathology. Here we demonstrate that the nuclear factor kappa B (NF-κB) regulatory protein A20 is crucial in regulating microglia activation during CNS homeostasis and pathology. In mice, deletion of A20 in microglia increases microglial cell number and affects microglial regulation of neuronal synaptic function. Administration of a sublethal dose of lipopolysaccharide induces massive microglia activation, neuroinflammation, and lethality in mice with microglia-confined A20 deficiency. Microglia A20 deficiency also exacerbates multiple sclerosis (MS)-like disease, due to hyperactivation of the Nlrp3 inflammasome leading to enhanced interleukin-1ß secretion and CNS inflammation. Finally, we confirm a Nlrp3 inflammasome signature and IL-1ß expression in brain and cerebrospinal fluid from MS patients. Collectively, these data reveal a critical role for A20 in the control of microglia activation and neuroinflammation.
Assuntos
Inflamassomos/imunologia , Microglia/imunologia , Esclerose Múltipla/imunologia , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Encéfalo/imunologia , Encéfalo/patologia , Modelos Animais de Doenças , Feminino , Humanos , Interleucina-1beta/metabolismo , Lipopolissacarídeos/imunologia , Masculino , Camundongos , Microglia/patologia , Pessoa de Meia-Idade , Esclerose Múltipla/líquido cefalorraquidiano , Esclerose Múltipla/patologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Transdução de Sinais/imunologia , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/genética , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/imunologiaRESUMO
Type 2 diabetes (T2DM) and obesity might increase the risk for AD by 2-fold. Different attempts to model the effect of diet-induced diabetes on AD pathology in transgenic animal models, resulted in opposite conclusions. Here, we used a novel knock-in mouse model for AD, which, differently from other models, does not overexpress any proteins. Long-term high fat diet treatment triggers a reduction in hippocampal N-acetyl-aspartate/myo-inositol metabolites ratio and impairs long term potentiation in hippocampal acute slices. Interestingly, these alterations do not correlate with changes in the core neuropathological features of AD, i.e. amyloidosis and Tau hyperphosphorylation. The data suggest that AD phenotypes associated with high fat diet treatment seen in other models for AD might be exacerbated because of the overexpressing systems used to study the effects of familial AD mutations. Our work supports the increasing insight that knock-in mice might be more relevant models to study the link between metabolic disorders and AD.
Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/fisiopatologia , Dieta Hiperlipídica/efeitos adversos , Hipocampo/metabolismo , Hipocampo/fisiopatologia , Potenciação de Longa Duração/fisiologia , Doença de Alzheimer/patologia , Animais , Glicemia/metabolismo , Dieta Hiperlipídica/tendências , Hipocampo/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Técnicas de Cultura de ÓrgãosRESUMO
OBJECTIVE: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an adult-onset neurovascular disorder caused by stereotyped mutations in the NOTCH3 receptor. Elucidation of its pathobiology is still incomplete and remains a challenge, in part because the available preclinical mouse models to date do not reproduce the full spectrum of CADASIL pathology and clinical disease. METHODS AND RESULTS: Here, we report a novel knock-in mouse with Arg170Cys substitution in murine Notch3, corresponding to the prevalent Arg169Cys substitution in CADASIL. The Notch3(Arg170Cys) mice displayed late-onset, dominant CADASIL arteriopathy with typical granular osmiophilic material deposition and developed brain histopathology including thrombosis, microbleeds, gliosis, and microinfarction. Furthermore, Notch3(Arg170Cys) mice experienced neurological symptoms with motor defects such as staggering gait and limb paresis. CONCLUSIONS: This model, for the first time, phenocopies the arteriopathy and the histopathologic as well as clinical features of CADASIL and may offer novel opportunities to investigate disease pathogenesis.
Assuntos
Arginina , CADASIL/genética , CADASIL/patologia , Cistina , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Receptores Notch/genética , Animais , Encéfalo/patologia , CADASIL/fisiopatologia , Capilares/patologia , Fertilidade/fisiologia , Camundongos , Atividade Motora/fisiologia , Músculo Liso Vascular/patologia , Mutação/genética , Receptor Notch3RESUMO
Germline mutations in SPRED1, a negative regulator of Ras, have been described in a neurofibromatosis type 1 (NF1)-like syndrome (NFLS) that included learning difficulties in some affected individuals. NFLS belongs to the group of phenotypically overlapping neuro-cardio-facial-cutaneous syndromes that are all caused by germ line mutations in genes of the Ras/mitogen-activated protein kinase extracellular signal-regulated kinase (ERK) pathway and that present with some degree of learning difficulties or mental retardation. We investigated hippocampus-dependent learning and memory as well as synaptic plasticity in Spred1(-/-) mice, an animal model of this newly discovered human syndrome. Spred1(-/-) mice show decreased learning and memory performance in the Morris water maze and visual-discrimination T-maze, but normal basic neuromotor and sensory abilities. Electrophysiological recordings on brain slices from these animals identified defects in short- and long-term synaptic hippocampal plasticity, including a disequilibrium between long-term potentiation (LTP) and long-term depression in CA1 region. Biochemical analysis, 4 h after LTP induction, demonstrated increased ERK-phosphorylation in Spred1(-/-) slices compared with those of wild-type littermates. This indicates that deficits in hippocampus-dependent learning and synaptic plasticity induced by SPRED1 deficiency are related to hyperactivation of the Ras/ERK pathway.
Assuntos
Hipocampo/fisiologia , Aprendizagem/fisiologia , Plasticidade Neuronal/fisiologia , Proteínas Repressoras/metabolismo , Potenciais Sinápticos/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Animais Recém-Nascidos , Comportamento Animal , Biofísica , Dendritos/ultraestrutura , Aprendizagem por Discriminação/fisiologia , Estimulação Elétrica , Tomografia com Microscopia Eletrônica/métodos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Força da Mão/fisiologia , Hipocampo/citologia , Técnicas In Vitro , Masculino , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/ultraestrutura , Limiar da Dor/fisiologia , Estimulação Luminosa/métodos , Proteínas Repressoras/genética , Coloração pela Prata/métodosRESUMO
A deficiency of arylsulfatase A (ASA) causes metachromatic leukodystrophy (MLD), a lysosomal storage disorder characterized by accumulation of sulfatide, a severe neurological phenotype and early death. The efficacy of enzyme replacement therapy (ERT) has previously been determined in ASA knockout (ASA-/-) mice representing the only available animal model for MLD. Repeated intravenous injection of human ASA (hASA) improved the nervous system pathology and function, but also elicited a progressive humoral immune response leading to treatment resistance, anaphylactic reactions, and high mortality. In contrast to ASA-/- mice, most MLD patients express mutant hASA which may entail immunological tolerance to substituted wildtype hASA and thus protect from immunological complications. To test this notion, a cysteine-to-serine substitution was introduced into the active site of the hASA and the resulting inactive hASA-C69S variant was constitutively expressed in ASA-/- mice. Mice with sub-to supranormal levels of mutant hASA expression were analyzed. All mice, including those showing transgene expression below the limit of detection, were immunologically unresponsive to injected hASA. More than 100-fold overexpression did not induce an overt new phenotype except occasional intralysosomal deposition of minor amounts of glycogen in hepatocytes. Furthermore, long-term, low-dose ERT reduced sulfatide storage in peripheral tissues and the central nervous system indicating that high levels of extracellular mutant hASA do not prevent cellular uptake and lysosomal targeting of substituted wildtype hASA. Due to the tolerance to hASA and maintenance of the MLD-like phenotype, the novel transgenic strain may be particularly advantageous to assess the benefit and risk of long-term ERT.
Assuntos
Cerebrosídeo Sulfatase/uso terapêutico , Modelos Animais de Doenças , Tolerância Imunológica/genética , Leucodistrofia Metacromática/tratamento farmacológico , Substituição de Aminoácidos , Animais , Sítios de Ligação , Células Cultivadas , Cerebrosídeo Sulfatase/administração & dosagem , Cerebrosídeo Sulfatase/genética , Cerebrosídeo Sulfatase/metabolismo , Cerebrosídeo Sulfatase/ultraestrutura , Cricetinae , Esquema de Medicação , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Humanos , Injeções Intravenosas , Rim/citologia , Leucodistrofia Metacromática/etiologia , Leucodistrofia Metacromática/metabolismo , Leucodistrofia Metacromática/patologia , Fígado/efeitos dos fármacos , Fígado/enzimologia , Fígado/metabolismo , Fígado/patologia , Fígado/ultraestrutura , Camundongos , Camundongos Transgênicos , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/uso terapêutico , Serina/metabolismo , TransgenesRESUMO
Neutral antagonists and inverse agonists can produce different cellular responses in some systems. The effects of chronic (14-day) infusion of three ligands, ICI-118,551, carvedilol, and alprenolol were examined in cardiac tissue from wild-type and transgenic mice with cardiac-specific overexpression of the human beta2-adrenoceptor. These ligands vary in their negative efficacy at the human beta2-adrenoceptor, with two (ICI-118,551 and carvedilol) behaving as inverse agonists and one (alprenolol) behaving as a neutral antagonist. Cardiac tissue from the transgenic mice exhibited elevated levels of protein kinase A activity and G protein receptor kinase-2. Fourteen-day infusions of the three ligands lowered the elevated levels of protein kinase A activity of the transgenic hearts to control levels. Alprenolol and carvedilol also decreased G protein receptor kinase-2 amounts to control levels. The left atria from transgenic mice exhibited an impaired inotropic response to histamine relative to responses of wild-type mice atria. Infusions of the inverse agonists and a neutral antagonist at the beta2-adrenoceptor significantly restored the impaired histamine response. Restoration of protein kinase A activity and the impaired histamine responses in the atria from transgenic mice can be observed following 14-day infusions of both a neutral antagonist and inverse agonists. The reversal of the effects of the transgene by both inverse agonists and a neutral antagonist suggests that agonist occupancy, and not spontaneous activity, of the beta2-adrenoceptor is producing the elevated protein kinase A activity and the impaired histamine response.