RESUMO
Reelin, a large extracellular glycoprotein, plays a critical role in prenatal brain development and postnatally in synaptic plasticity, learning and memory. Dysregulation of Reelin signalling has been implicated in several neuropsychiatric disorders including schizophrenia, autism, depression and Alzheimer's disease. Previous studies have demonstrated that Reelin's central fragment, R3456, binds to ApoER2, inducing ApoER2 clustering and subsequent intracellular signalling. We previously reported the development of a novel luciferase complementation assay, which we used to demonstrate that R3456 can lead to ApoER2 receptor dimerization. Using this same assay, we explored various smaller fragments and combinations from R3456, and we identified a construct of repeats 3 and 6 (R36), which could still elicit equivalent receptor dimerization. The purpose of this study was to test R36 for biological effects in vitro and in vivo. We show that R36 was capable of initiating intracellular signalling in primary neuronal cultures. In addition, we demonstrate that a single intracerebroventricular injection of R36 protein into a model of Reelin deficiency, the heterozygous reeler mice, can significantly improve cognition. These data support a role for the new construct R36 to enhance the Reelin pathway, and the future possibility of exploring gene therapy approaches with R36 in diseases characterized by reduced levels of Reelin.
Assuntos
Moléculas de Adesão Celular Neuronais , Proteínas da Matriz Extracelular , Camundongos , Animais , Proteínas da Matriz Extracelular/genética , Camundongos Mutantes Neurológicos , Moléculas de Adesão Celular Neuronais/genética , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas de TransporteRESUMO
We recently generated a novel Angelman syndrome (AS) rat model with a complete Ube3a gene deletion, that recapitulates the loss of UBE3A protein and shows cognitive and EEG deficits. We also recently published the identification of extracellular UBE3A protein within the brain using microdialysis. Here we explored the effects of supplementation of exogenous UBE3A protein to hippocampal slices and intrahippocampal injection of AS rats. We report that the AS rat model demonstrates deficits in hippocampal long-term potentiation (LTP) which can be recovered with the application of exogenous UBE3A protein. Furthermore, injection of recombinant UBE3A protein into the hippocampus of the AS rat can rescue the associative learning and memory deficits seen in the fear conditioning task. These data suggest that extracellular UBE3A protein may play a role in synaptic function, LTP induction and hippocampal-dependent memory formation.
Assuntos
Síndrome de Angelman , Síndrome de Angelman/tratamento farmacológico , Síndrome de Angelman/genética , Síndrome de Angelman/metabolismo , Animais , Suplementos Nutricionais , Modelos Animais de Doenças , Hipocampo/metabolismo , Potenciação de Longa Duração , Ratos , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Treatment for Angelman syndrome (AS) is currently limited to symptomatic interventions. A mouse model of AS has reduced calcium/calmodulin-dependent kinase II activity due to excessive phosphorylation of specific threonine residues, leading to diminished long-term potentiation. In a rat model of Parkinson disease, levodopa reduced phosphorylation of various proteins, including calcium/calmodulin-dependent kinase II. Further studies demonstrated that AS mice treated with levodopa performed better on rotarod testing than untreated AS mice. We conducted a multi-center double-blind randomized placebo-controlled 1-year trial of levodopa / carbidopa with either 10 or 15 mg/kg/day of levodopa in children with AS. The outcome of this intervention was assessed using either the Bayley Scales of Infant Development or the Mullen Scales of Early Learning, as well as the Vineland Adaptive Behavior Scales, and the Aberrant Behavior Checklist. Of the 78 participants enrolled, 67 participants received study medication (33 on levodopa, 34 on placebo), and 55 participants (29 on levodopa, 26 on placebo) completed the 1-year study. There were no clinically or statistically significant changes in any of the outcome measures over a 1-year period comparing the levodopa and placebo groups. The number of adverse events reported, including the more serious adverse events, was similar in both groups, but none were related to treatment with levodopa. Our data demonstrate that levodopa is well-tolerated by children with AS. However, in the doses used in this study, it failed to improve their neurodevelopment or behavioral outcome.
Assuntos
Síndrome de Angelman/tratamento farmacológico , Levodopa/uso terapêutico , Síndrome de Angelman/diagnóstico , Síndrome de Angelman/fisiopatologia , Síndrome de Angelman/psicologia , Animais , Biomarcadores , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Modelos Animais de Doenças , Humanos , Levodopa/administração & dosagem , Potenciação de Longa Duração , Camundongos , Testes Neuropsicológicos , Resultado do TratamentoRESUMO
BACKGROUND: Angelman syndrome (AS) leads to clinical manifestations that include intellectual impairments, developmental delay and poor motor function. Initiatives to develop therapeutics implie an urgent need to identify methods that accurately measure the motor abilities. METHODS: Six children with AS (6 to 9 years old) walked on an instrumented walkway to get spatiotemporal parameters (STPs) and center of pressure (CoP). These outcomes were compared to typically developing children (TD): 44 TD 6 to 9 years old and 20 TD 4 to 5 years old. RESULTS: Analysis revealed differences in all STPs and gait variability index when compared to TD individuals. When AS participants were compared to younger TD individuals, except step length, STPs were different. Analysis of the CoP pathway revealed a less consistent and efficient pathway in AS. CONCLUSIONS: We could delineate the functional difference between children with AS and TD children. The variability of STP and the CoP were the most valuable components in gait to be considered in AS.
Assuntos
Síndrome de Angelman/fisiopatologia , Marcha/fisiologia , Equilíbrio Postural/fisiologia , Fenômenos Biomecânicos , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Projetos PilotoRESUMO
BACKGROUND: Angelman syndrome is a rare disorder in which most individuals do not develop speech. Testing of communication ability using traditional neuropsychological measures reveals a performance level at or near the floor of the instrument resulting in an inability to detect change when experimental therapeutics are applied. METHODS: Nine individuals, with molecularly confirmed AS, ranging in age from 34 to 126 months, and a single healthy control child (age 16 months) were audio and video-recorded while interacting with a licensed speech-language pathologist in an attempt to elicit vocalization and non-verbal communication. Thirty-minute audio recordings were transcribed and categorized per the Stark Assessment of Early Vocal Development-Revised and a phonetic inventory was created. Using video recordings, gestures were classified by function, either behavioral regulation or social interaction and further categorized as deictic or representational (i.e., behavioral regulation) and joint attention or shared engagement (i.e., social interaction). RESULTS: The range of vocalizations produced by the children with AS was characteristic of children between 0-6 months and none of the children with AS used advanced forms of vocalizations. The mean frequency of reflexive vocalizations, control of phonation and expansion far exceeded the number of uses of canonical syllables, consistant with the characteristics of children around 12 months of age. Most vocalizations were either laughter or isolated vowels, only three children with AS produced consonant-vowel combinations. Children with AS tended to use central and low vowels with few producing high vowels, suggesting the presence of childhood apraxia of speech. CONCLUSION: Our results show the utilization of video-recorded behavioral observations provides a feasible and reliable alternative for quantification of communication ability in this patient population and may be employed during future clinical studies of potential therapeutics.
Assuntos
Síndrome de Angelman/psicologia , Comunicação , Fala/fisiologia , Criança , Pré-Escolar , Feminino , Gestos , Humanos , Lactente , MasculinoRESUMO
Angelman syndrome (AS) is a rare genetic and neurological disorder presenting with seizures, developmental delay, ataxia, and lack of speech. Previous studies have indicated that oxidative stress-dependent metabolic dysfunction may underlie the phenotypic deficits reported in the AS mouse model. While the ketogenic diet (KD) has been used to protect against oxidative stress and has successfully treated refractory epilepsy in AS case studies, issues arise due to its strict adherence requirements, in addition to selective eating habits and weight issues reported in patients with AS. We hypothesized that ketone ester supplementation would mimic the KD as an anticonvulsant and improve the behavioral and synaptic plasticity deficits in vivo. AS mice were supplemented R,S-1,3-butanediol acetoacetate diester (KE) ad libitum for eight weeks. KE administration improved motor coordination, learning and memory, and synaptic plasticity in AS mice. The KE was also anticonvulsant and altered brain amino acid metabolism in AS treated animals. Our findings suggest that KE supplementation produces sustained ketosis and ameliorates many phenotypes in the AS mouse model, and should be investigated further for future clinical use.
Assuntos
Síndrome de Angelman/complicações , Ésteres/farmacologia , Ésteres/uso terapêutico , Comportamento Exploratório/efeitos dos fármacos , Hipocampo/patologia , Plasticidade Neuronal/efeitos dos fármacos , Convulsões , Estimulação Acústica/efeitos adversos , Potenciais de Ação/efeitos dos fármacos , Síndrome de Angelman/sangue , Animais , Condicionamento Psicológico/efeitos dos fármacos , Modelos Animais de Doenças , Ésteres/sangue , Agonistas de Aminoácidos Excitatórios/toxicidade , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Feminino , Ácido Caínico/toxicidade , Cetonas/sangue , Cetonas/farmacologia , Cetonas/uso terapêutico , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Desempenho Psicomotor/efeitos dos fármacos , Convulsões/tratamento farmacológico , Convulsões/etiologia , Convulsões/patologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
ApoE Receptor 2 (ApoER2) and the very low density lipoprotein receptor (VLDLR) are type I transmembrane proteins belonging to the LDLR family of receptors. They are neuronal proteins found in synaptic compartments that play an important role in neuronal migration during development. ApoER2 and VLDLR bind to extracellular glycoproteins, such as Reelin and F-spondin, which leads to phosphorylation of adaptor proteins and subsequent activation of downstream signaling pathways. It is thought that ApoER2 and VLDLR undergo clustering upon binding to their ligands, but no direct evidence of clustering has been shown. Here we show strong clustering of ApoER2 induced by the dimeric ligands Fc-RAP, F-spondin, and Reelin but relatively weak clustering with the ligand apoE in the absence of lipoproteins. This clustering involves numerous proteins besides ApoER2, including amyloid precursor protein and the synaptic adaptor protein PSD-95. Interestingly, we did not observe strong clustering of ApoER2 with VLDLR. Clustering was modulated by both extracellular and intracellular domains of ApoER2. Together, our data demonstrate that several multivalent ligands for ApoER2 induce clustering in transfected cells and primary neurons and that these complexes included other synaptic molecules, such as APP and PSD-95.
Assuntos
Proteínas Relacionadas a Receptor de LDL/metabolismo , Animais , Células COS , Movimento Celular , Chlorocebus aethiops , Proteínas Relacionadas a Receptor de LDL/fisiologia , Ligantes , Camundongos , Neurônios/fisiologia , Fosforilação , Proteína ReelinaRESUMO
The Reelin signaling pathway is implicated in processes controlling synaptic plasticity and hippocampus-dependent learning and memory. A single direct in vivo application of Reelin enhances long-term potentiation, increases dendritic spine density and improves associative and spatial learning and memory. Angelman syndrome (AS) is a neurological disorder that presents with an overall defect in synaptic function, including decreased long-term potentiation, reduced dendritic spine density, and deficits in learning and memory, making it an attractive model in which to examine the ability of Reelin to recover synaptic function and cognitive deficits. In this study, we investigated the effects of Reelin administration on synaptic plasticity and cognitive function in a mouse model of AS and demonstrated that bilateral, intraventricular injections of Reelin recover synaptic function and corresponding hippocampus-dependent associative and spatial learning and memory. Additionally, we describe alteration of the Reelin profile in tissue from both the AS mouse and post-mortem human brain.
Assuntos
Síndrome de Angelman/fisiopatologia , Síndrome de Angelman/psicologia , Moléculas de Adesão Celular Neuronais/administração & dosagem , Proteínas da Matriz Extracelular/administração & dosagem , Hipocampo/efeitos dos fármacos , Potenciação de Longa Duração/efeitos dos fármacos , Proteínas do Tecido Nervoso/administração & dosagem , Serina Endopeptidases/administração & dosagem , Síndrome de Angelman/tratamento farmacológico , Animais , Moléculas de Adesão Celular Neuronais/metabolismo , Córtex Cerebral/metabolismo , Espinhas Dendríticas/efeitos dos fármacos , Modelos Animais de Doenças , Proteínas da Matriz Extracelular/metabolismo , Feminino , Células HEK293 , Hipocampo/fisiopatologia , Hipocampo/ultraestrutura , Humanos , Injeções Intraventriculares , Masculino , Camundongos , Atividade Motora/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Proteína Reelina , Serina Endopeptidases/metabolismo , Aprendizagem Espacial/efeitos dos fármacos , Memória Espacial/efeitos dos fármacosRESUMO
Angelman Syndrome (AS) is a devastating neurological disorder caused by disruption of the maternal UBE3A gene. Ube3a protein is identified as an E3 ubiquitin ligase that shows neuron-specific imprinting. Despite extensive research evaluating the localization and basal expression profiles of Ube3a in mouse models, the molecular mechanisms whereby Ube3a deficiency results in AS are enigmatic. Using in vitro and in vivo systems we show dramatic changes in the expression of Ube3a following synaptic activation. In primary neuronal culture, neuronal depolarization was found to increase both nuclear and cytoplasmic Ube3a levels. Analogous up-regulation in maternal and paternal Ube3a expression was observed in Ube3a-YFP reporter mice following fear conditioning. Absence of Ube3a led to deficits in the activity-dependent increases in ERK1/2 phosphorylation, which may contribute to reported deficits in synaptic plasticity and cognitive function in AS mice. Taken together, our findings provide novel insight into the regulation of Ube3a by synaptic activity and its potential role in kinase regulation.
Assuntos
Síndrome de Angelman/fisiopatologia , Encéfalo/fisiopatologia , Neurônios/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Síndrome de Angelman/enzimologia , Animais , Núcleo Celular/metabolismo , Células Cultivadas , Condicionamento Psicológico , Citoplasma/metabolismo , Medo/fisiologia , Feminino , Técnicas In Vitro , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Potenciais da Membrana , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pais , Transmissão Sináptica , Ubiquitina-Proteína Ligases/genéticaRESUMO
Disabled-1 (Dab1) is an adaptor protein that is an obligate effector of the Reelin signaling pathway, and is critical for neuronal migration and dendrite outgrowth during development. Components of the Reelin pathway are highly expressed during development, but also continue to be expressed in the adult brain. Here we investigated in detail the expression pattern of Dab1 in the postnatal and adult forebrain, and determined that it is expressed in excitatory as well as inhibitory neurons. Dab1 was found to be localized in different cellular compartments, including the soma, dendrites, presynaptic and postsynaptic structures. Mice that are deficient in Dab1, Reelin, or the Reelin receptors ApoER2 and VLDLR exhibit severely perturbed brain cytoarchitecture, limiting the utility of these mice for investigating the role of this signaling pathway in the adult brain. In this study, we developed an adult forebrain-specific and excitatory neuron-specific conditional knock-out mouse line, and demonstrated that Dab1 is a critical regulator of synaptic function and hippocampal-dependent associative and spatial learning. These dramatic abnormalities were accompanied by a reduction in dendritic spine size, and defects in basal and plasticity-induced Akt and ERK1/2 signaling. Deletion of Dab1 led to no obvious changes in neuronal positioning, dendrite morphology, spine density, or synaptic composition. Collectively, these data conclusively demonstrate an important role for Reelin-Dab1 signaling in the adult forebrain, and underscore the importance of this pathway in learning and memory.
Assuntos
Aprendizagem , Proteínas do Tecido Nervoso/metabolismo , Plasticidade Neuronal , Animais , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Dendritos/metabolismo , Dendritos/fisiologia , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Hipocampo/metabolismo , Hipocampo/fisiologia , Proteínas Relacionadas a Receptor de LDL/genética , Proteínas Relacionadas a Receptor de LDL/metabolismo , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Prosencéfalo/citologia , Prosencéfalo/metabolismo , Prosencéfalo/fisiologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de LDL/genética , Receptores de LDL/metabolismo , Proteína Reelina , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Sinapses/metabolismo , Sinapses/fisiologiaRESUMO
Granulocyte colony-stimulating factor (G-CSF) is a hematopoietic cytokine that also possesses neurotrophic and antiapoptotic properties. G-CSF has been reported to decrease amyloid burden significantly, promote hippocampal neurogenesis, and improve spatial learning in a mouse model of Alzheimer's disease. To understand better the effects of G-CSF on hippocampal-dependent learning, the present study focused on electrophysiological correlates of neuroplasticity, long-term potentiation (LTP), and long-term depression (LTD). Two cohorts of transgenic APP/PS1 mice, with or without prior bone marrow transplantation from Tg GFP mice, were treated in vivo for 2 weeks with G-CSF or vehicle. After completion of the treatments, hippocampal slices were prepared for electrophysiological studies of LTP and LTD. LTP was induced and maintained in both G-CSF-treated and vehicle-treated groups of Tg APP/PS1. In contrast, LTD could not be induced in vehicle-treated Tg APP/PS1 mice, but G-CSF treatment restored LTD. The LTP and LTD results obtained from the cohort of bone marrow-grafted Tg APP/PS1 mice did not differ from those from nongrafted Tg APP/PS1 mice. The mechanism by which G-CSF restores LTD is not known, but it is possible that its capacity to reduce amyloid plaques results in increased soluble oligomers of amyloid-ß (A-ß), which in turn may facilitate LTD. This mechanism would be consistent with the recent report that soluble A-ß oligomers promote LTD in hippocampal slices.
Assuntos
Doença de Alzheimer/fisiopatologia , Fator Estimulador de Colônias de Granulócitos/farmacologia , Hipocampo/efeitos dos fármacos , Depressão Sináptica de Longo Prazo/efeitos dos fármacos , Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Animais , Modelos Animais de Doenças , Eletrofisiologia , Hipocampo/fisiopatologia , Potenciação de Longa Duração/efeitos dos fármacos , Potenciação de Longa Duração/fisiologia , Depressão Sináptica de Longo Prazo/fisiologia , Camundongos , Camundongos Transgênicos , Presenilina-1/genéticaRESUMO
BACKGROUND: Minocycline, a member of the tetracycline family, has a low risk of adverse effects and an ability to improve behavioral performance in humans with cognitive disruption. We performed a single-arm open-label trial in which 25 children diagnosed with Angelman syndrome (AS) were administered minocycline to assess the safety and tolerability of minocycline in this patient population and determine the drug's effect on the cognitive and behavioral manifestations of the disorder. METHODS: Participants, age 4-12 years old, were randomly selected from a pool of previously screened children for participation in this study. Each child received 3 milligrams of minocycline per kilogram of body weight per day for 8 weeks. Participants were assessed during 3 study visits: baseline, after 8-weeks of minocycline treatment and after an 8-week wash out period. The primary outcome measure was the Bayley Scales of Infant and Toddler Development 3rd Edition (BSID-III). Secondary outcome measures included the Clinical Global Impressions Scale (CGI), Vineland Adaptive Behavior Scales 2nd Edition (VABS-II), Preschool Language Scale 4th Edition (PLS-IV) and EEG scores. Observations were considered statistically significant if p < 0.05 using ANOVA and partial eta squared (η(2)) was calculated to show effect size. Multiple comparisons testing between time points were carried out using Dunnett's post hoc testing. RESULTS: Significant improvement in the mean raw scores of the BSID-III subdomains communication and fine motor ability as well as the subdomains auditory comprehension and total language ability of the PLS-IV when baseline scores were compared to scores after the washout period. Further, improvements were observed in the receptive communication subdomain of the VABS-II after treatment with minocycline. Finally, mean scores of the BSID-III self-direction subdomain and CGI scale score were significantly improved both after minocycline treatment and after the wash out period. CONCLUSION: The clinical and neuropsychological measures suggest minocycline was well tolerated and causes improvements in the adaptive behaviors of this sample of children with Angelman syndrome. While the optimal dosage and the effects of long-term use still need to be determined, these findings suggest further investigation into the effect minocycline has on patients with Angelman syndrome is warranted. TRIAL REGISTRATION: NCT01531582 - clinicaltrials.gov.
Assuntos
Síndrome de Angelman/tratamento farmacológico , Antibacterianos/farmacologia , Transtornos Cognitivos/tratamento farmacológico , Minociclina/farmacologia , Síndrome de Angelman/complicações , Antibacterianos/administração & dosagem , Antibacterianos/efeitos adversos , Criança , Pré-Escolar , Transtornos Cognitivos/etiologia , Feminino , Humanos , Masculino , Minociclina/administração & dosagem , Minociclina/efeitos adversos , Projetos Piloto , Resultado do TratamentoRESUMO
The apolipoprotein E4 (APOE-ε4) allele is the strongest genetic risk factor for developing late-onset Alzheimer's disease, and may predispose individuals to Alzheimer's-related cognitive decline by affecting normal brain function early in life. To investigate the impact of human APOE alleles on cognitive performance in mice, we trained 3-mo-old APOE targeted replacement mice (E2, E3, and E4) in the Barnes maze to locate and enter a target hole along the perimeter of the maze. Long-term spatial memory was probed 24 h and 72 h after training. We found that young E4 mice exhibited significantly impaired spatial learning and memory in the Barnes maze compared to E3 mice. Deficits in spatial cognition were also present in a second independent cohort of E4 mice tested at 18 mo of age. In contrast, cognitive performance in the hidden platform water maze was not as strongly affected by APOE genotype. We also examined the dendritic morphology of neurons in the medial entorhinal cortex of 3-mo-old TR mice, neurons important to spatial learning functions. We found significantly shorter dendrites and lower spine densities in basal shaft dendrites of E4 mice compared to E3 mice, consistent with spatial learning and memory deficits in E4 animals. These findings suggest that human APOE-ε4 may affect cognitive function and neuronal morphology early in life.
Assuntos
Apolipoproteína E4/genética , Espinhas Dendríticas/metabolismo , Córtex Entorrinal/metabolismo , Aprendizagem em Labirinto/fisiologia , Memória/fisiologia , Neurônios/metabolismo , Animais , Apolipoproteína E4/metabolismo , Comportamento Animal , Dendritos/genética , Dendritos/metabolismo , Espinhas Dendríticas/genética , Camundongos , Camundongos Transgênicos , Comportamento Espacial/fisiologiaRESUMO
APOE4 is the greatest risk factor for Alzheimer disease (AD) and synergistic effects with amyloid-ß peptide (Aß) suggest interactions among apoE isoforms and different forms of Aß accumulation. However, it remains unclear how the APOE genotype affects plaque morphology, intraneuronal Aß, soluble Aß42, and oligomeric Aß (oAß), particularly in vivo. As the introduction of human APOE significantly delays amyloid deposition in transgenic mice expressing familial AD (FAD) mutations (FAD-Tg), 5xFAD-Tg mice, which exhibit amyloid deposition by age 2 months, were crossed with apoE-targeted replacement mice to produce the new EFAD-Tg mice. Compared with 5xFAD mice, Aß deposition was delayed by â¼4 months in the EFAD mice, allowing detection of early changes in Aß accumulation from 2-6 months. Although plaque deposition is generally greater in E4FAD mice, E2/E3FAD mice have significantly more diffuse and E4FAD more compact plaques. As a first report in FAD-Tg mice, the APOE genotypes had no effect on intraneuronal Aß accumulation in EFAD mice. In E4FAD mice, total apoE levels were lower and total Aß levels higher than in E2FAD and E3FAD mice. Profiles from sequential three-step extractions (TBS, detergent, and formic acid) demonstrated that the lower level of total apoE4 is reflected only in the detergent-soluble fraction, indicating that less apoE4 is lipoprotein-associated, and perhaps less lipidated, compared with apoE2 and apoE3. Soluble Aß42 and oAß levels were highest in E4FAD mice, although soluble apoE2, apoE3, and apoE4 levels were comparable, suggesting that the differences in soluble Aß42 and oAß result from functional differences among the apoE isoforms. Thus, APOE differentially regulates multiple aspects of Aß accumulation.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Apolipoproteína E4/metabolismo , Genótipo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/genética , Animais , Apolipoproteína E4/genética , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Transgênicos , Mutação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMO
Hippocampal neurogenesis is the lifelong production of new neurons in the central nervous system (CNS), and affects many physiological and pathophysiological conditions, including neurobehavioral disorders. The early postnatal stage is the most prominent neurogenesis period; however, the functional role of neurogenesis in this developing stage has not been well characterized. To understand the role of hippocampal neurogenesis in the postnatal developing period, we analyzed secretin, a neuropeptide, which is expressed significantly higher in the development stage. Secretin is a pleiotropic neuropeptide hormone that belongs to the secretin/VIP/glucagon peptide family. Although secretin was originally isolated in the gastrointestinal system, it has been found that secretin itself acts as a neuropeptide in the CNS. Here, we report a new function of secretin as a survival factor for neural progenitor cells in the hippocampus. We found that secretin-deficient mice exhibit decreased numbers of BrdU-labeled new neurons and dramatically increased apoptosis of doublecortin-positive neural progenitor cells in the subgranular zone of the dentate gyrus (DG) during the early postnatal period. Furthermore, we found that reduced survival of neural progenitor cells leads to decreased volume of DG, reduced long-term potentiation and impaired spatial learning ability in adults. Our studies demonstrate that secretin has important implications for neurogenesis in postnatal development, and affects neurobehavioral function in the adult mouse.
Assuntos
Neurônios/citologia , Neurônios/metabolismo , Secretina/deficiência , Células-Tronco/citologia , Células-Tronco/metabolismo , Animais , Apoptose , Sobrevivência Celular , Giro Denteado/citologia , Giro Denteado/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Secretina/genéticaRESUMO
The goal of these studies was to use quantitative (q)EEG techniques on data from children with Angelman syndrome (AS) using spectral power analysis, and to evaluate this as a potential biomarker and quantitative method to evaluate therapeutics. Although characteristic patterns are evident in visual inspection, using qEEG techniques has the potential to provide quantitative evidence of treatment efficacy. We first assessed spectral power from baseline EEG recordings collected from children with AS compared to age-matched neurotypical controls, which corroborated the previously reported finding of increased total power driven by elevated delta power in children with AS. We then retrospectively analyzed data collected during a clinical trial evaluating the safety and tolerability of minocycline (3 mg/kg/d) to compare pretreatment recordings from children with AS (4-12 years of age) to EEG activity at the end of treatment and following washout for EEG spectral power and epileptiform events. At baseline and during minocycline treatment, the AS subjects demonstrated increased delta power; however, following washout from minocycline treatment the AS subjects had significantly reduced EEG spectral power and epileptiform activity. Our findings support the use of qEEG analysis in evaluating AS and suggest that this technique may be useful to evaluate therapeutic efficacy in AS. Normalizing EEG power in AS therefore may become an important metric in screening therapeutics to gauge overall efficacy. As therapeutics transition from preclinical to clinical studies, it is vital to establish outcome measures that can quantitatively evaluate putative treatments for AS and neurological disorders with distinctive EEG patterns.
Assuntos
Síndrome de Angelman , Criança , Humanos , Síndrome de Angelman/diagnóstico , Síndrome de Angelman/tratamento farmacológico , Eletroencefalografia , Minociclina/uso terapêutico , Estudos Retrospectivos , Resultado do TratamentoRESUMO
Amyloid precursor protein (APP) plays an important role in the pathogenesis of Alzheimer's disease (AD), but the normal function of APP at synapses is poorly understood. We and others have found that APP interacts with Reelin and that each protein is individually important for dendritic spine formation, which is associated with learning and memory, in vitro. However, whether Reelin acts through APP to modulate dendritic spine formation or synaptic function remains unknown. In the present study, we found that Reelin treatment significantly increased dendritic spine density and PSD-95 puncta number in primary hippocampal neurons. An examination of the molecular mechanisms by which Reelin regulates dendritic spinogenesis revealed that Reelin enhanced hippocampal dendritic spine formation in a Ras/ERK/CREB signaling-dependent manner. Interestingly, Reelin did not increase dendritic spine number in primary hippocampal neurons when APP expression was reduced or in vivo in APP knockout (KO) mice. Taken together, our data are the first to demonstrate that Reelin acts cooperatively with APP to modulate dendritic spine formation and suggest that normal APP function is critical for Reelin-mediated dendritic spinogenesis at synapses.
RESUMO
Reelin and its receptor, ApoER2, play important roles in prenatal brain development and postnatally in synaptic plasticity, learning, and memory. Previous reports suggest that reelin's central fragment binds to ApoER2 and receptor clustering is involved in subsequent intracellular signaling. However, limitations of currently available assays have not established cellular evidence of ApoER2 clustering upon binding of the central reelin fragment. In the present study, we developed a novel, cell-based assay of ApoER2 dimerization using a "split-luciferase" approach. Specifically, cells were co-transfected with one recombinant ApoER2 receptor fused to the N-terminus of luciferase and one ApoER2 receptor fused to the C-terminus of luciferase. Using this assay, we directly observed basal ApoER2 dimerization/clustering in transfected HEK293T cells and, significantly, an increase in ApoER2 clustering in response to that central fragment of reelin. Furthermore, the central fragment of reelin activated intracellular signal transduction of ApoER2, indicated by increased levels of phosphorylation of Dab1, ERK1/2, and Akt in primary cortical neurons. Functionally, we were able to demonstrate that injection of the central fragment of reelin rescued phenotypic deficits observed in the heterozygous reeler mouse. These data are the first to test the hypothesis that the central fragment of reelin contributes to facilitating the reelin intracellular signaling pathway through receptor clustering.
Assuntos
Proteínas da Matriz Extracelular , Serina Endopeptidases , Camundongos , Animais , Humanos , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Células HEK293 , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais/fisiologia , Modelos Animais de Doenças , Luciferases/metabolismo , Cognição , Receptores de LDL/metabolismoRESUMO
The cellular and molecular mechanisms responsible for the development of inner retinal circuitry are poorly understood. Reelin and apolipoprotein E (apoE), ligands of apoE receptor 2 (ApoER2), are involved in retinal development and degeneration, respectively. Here we describe the function of ApoER2 in the developing and adult retina. ApoER2 expression was highest during postnatal inner retinal synaptic development and was considerably lower in the mature retina. Both during development and in the adult, ApoER2 was expressed by A-II amacrine cells. ApoER2 knock-out (KO) mice had rod bipolar morphogenic defects, altered A-II amacrine dendritic development, and impaired rod-driven retinal responses. The presence of an intact ApoER2 NPxY motif, necessary for binding Disabled-1 and transducing the Reelin signal, was also necessary for development of the rod bipolar pathway, while the alternatively spliced exon 19 was not. Mice deficient in another Reelin receptor, very low-density lipoprotein receptor (VLDLR), had normal rod bipolar morphology but altered A-II amacrine dendritic development. VLDLR KO mice also had reductions in oscillatory potentials and delayed synaptic response intervals. Interestingly, age-related reductions in rod and cone function were observed in both ApoER2 and VLDLR KOs. These results support a pivotal role for ApoER2 in the establishment and maintenance of normal retinal synaptic connectivity.
Assuntos
Proteínas Relacionadas a Receptor de LDL/fisiologia , Retina/crescimento & desenvolvimento , Transmissão Sináptica/fisiologia , Animais , Animais Recém-Nascidos , Feminino , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes Neurológicos , Vias Neurais/metabolismo , Vias Neurais/fisiologia , Proteína Reelina , Retina/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/fisiologiaRESUMO
The protective/neurotoxic role of fractalkine (CX3CL1) and its receptor CX3C chemokine receptor 1 (CX3CR1) signaling in neurodegenerative disease is an intricate and highly debated research topic and it is becoming even more complicated as new studies reveal discordant results. It appears that the CX3CL1/CX3CR1 axis plays a direct role in neurodegeneration and/or neuroprotection depending on the CNS insult. However, all the above studies focused on the role of CX3CL1/CX3CR1 signaling in pathological conditions, ignoring the relevance of CX3CL1/CX3CR1 signaling under physiological conditions. No approach to date has been taken to decipher the significance of defects in CX3CL1/CX3CR1 signaling in physiological condition. In the present study we used CX3CR1â»/â», CX3CR1âº/â», and wild-type mice to investigate the physiological role of CX3CR1 receptor in cognition and synaptic plasticity. Our results demonstrate for the first time that mice lacking the CX3CR1 receptor show contextual fear conditioning and Morris water maze deficits. CX3CR1 deficiency also affects motor learning. Importantly, mice lacking the receptor have a significant impairment in long-term potentiation (LTP). Infusion with IL-1ß receptor antagonist significantly reversed the deficit in cognitive function and impairment in LTP. Our results reveal that under physiological conditions, disruption in CX3CL1 signaling will lead to impairment in cognitive function and synaptic plasticity via increased action of IL-1ß.