RESUMO
Fragile X syndrome (FXS) is a neurodevelopmental disorder associated with intellectual disability, hyperactivity, and autism. FXS is due to the silencing of the X-linked FMR1 gene. Murine models of FXS, knock-out (KO) for the murine homolog Fmr1, have been generated, exhibiting CNS-related behavioral, and neuronal anomalies reminiscent of the human phenotypes. As a reflection of the almost ubiquitous expression of the FMR1 gene, FXS is also accompanied by physical abnormalities. This suggests that the FMR1-deficiency could impact skeletal ontogenesis. In the present study, we highlight that Fmr1-KO mice display changes in body composition with an increase in body weight, likely due to both increase of skeleton length and muscular mass along with reduced visceral adiposity. We also show that, while Fmr1-deficiency has no overt impact on cortical bone mineral density (BMD), cortical thickness was increased, and cortical eccentricity was decreased in the femurs from Fmr1-KO mice as compared to controls. Also, trabecular pore volume was reduced and trabecular thickness distribution was shifted toward higher ranges in Fmr1-KO femurs. Finally, we show that Fmr1-KO mice display increased physical activity. Although the precise molecular signaling mechanism that produces these skeletal and bone microstructure changes remains to be determined, our study warrants further investigation on the impact of FMR1-deficiency on whole-body composition, as well as skeletal and bone architecture.
RESUMO
There is a growing consensus that Alzheimer's disease (AD) involves failure of the homeostatic machinery, which underlies the firing stability of neural circuits. What are the culprits leading to neuron firing instability? The amyloid precursor protein (APP) is central to AD pathogenesis, and we recently showed that its intracellular domain (AICD) could modify synaptic signal integration. We now hypothesize that AICD modifies neuron firing activity, thus contributing to the disruption of memory processes. Using cellular, electrophysiological, and behavioral techniques, we show that pathological AICD levels weaken CA1 neuron firing activity through a gene-transcription-dependent mechanism. Furthermore, increased AICD production in hippocampal neurons modifies oscillatory activity, specifically in the γ-frequency range, and disrupts spatial memory task. Collectively, our data suggest that AICD pathological levels, observed in AD mouse models and in human patients, might contribute to progressive neuron homeostatic failure, driving the shift from normal aging to AD.
Assuntos
Potenciais de Ação/fisiologia , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/metabolismo , Região CA1 Hipocampal/fisiologia , Neurônios/fisiologia , Memória Espacial/fisiologia , Animais , Canais de Cálcio/metabolismo , Ritmo Gama/fisiologia , Humanos , Masculino , Camundongos Endogâmicos C57BL , Modelos Biológicos , Canais de Potássio/metabolismo , Domínios Proteicos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Transcrição GênicaRESUMO
Stressful life events are primary environmental factors that markedly contribute to depression by triggering brain cellular maladaptations. Dysregulation of ventral tegmental area (VTA) dopamine neurons has been causally linked to the appearance of social withdrawal and anhedonia, two classical manifestations of depression. However, the relevant inputs that shape these dopamine signals remain largely unknown. We demonstrate that chronic social defeat (CSD) stress, a preclinical paradigm of depression, causes marked hyperactivity of laterodorsal tegmentum (LDTg) excitatory neurons that project to the VTA. Selective chemogenetic-mediated inhibition of cholinergic LDTg neurons prevent CSD-induced VTA DA neurons dysregulation and depressive-like behaviors. Pro-depressant outcomes are replicated by pairing activation of LDTg cholinergic terminals in the VTA with a moderate stress. Prevention of CSD outcomes are recapitulated by blocking corticotropin-releasing factor receptor 1 within the LDTg. These data uncover a neuro-circuitry of depressive-like disorders and demonstrate that stress, via a neuroendocrine signal, profoundly dysregulates the LDTg.
Assuntos
Acetilcolina/metabolismo , Comportamento Animal , Depressão/psicologia , Neurônios Dopaminérgicos/patologia , Mesencéfalo/patologia , Ponte/patologia , Estresse Psicológico/complicações , Animais , Doença Crônica , Hormônio Liberador da Corticotropina/metabolismo , Depressão/patologia , Neurônios Dopaminérgicos/metabolismo , Inativação Gênica , Ácido Glutâmico/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Modelos Biológicos , Tegmento Pontino/patologia , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Transdução de Sinais , Área Tegmentar Ventral/patologiaRESUMO
Amyloid-ß is a peptide released by synapses in physiological conditions and its pathological accumulation in brain structures necessary for memory processing represents a key toxic hallmark underlying Alzheimer's disease. The oligomeric form of Amyloid-ß (Aßο) is now believed to represent the main Amyloid-ß species affecting synapse function. Yet, the exact molecular mechanism by which Aßο modifies synapse function remains to be fully elucidated. There is accumulating evidence that glucocorticoid receptors (GRs) might participate in Aßο generation and activity in the brain. Here, we provide evidence for an acute functional cross-talk between Aß and GRs at hippocampal excitatory synapses. Using live imaging and biochemical analysis of post-synaptic densities (PSD) in cultured hippocampal neurons, we show that synthetic Aßo (100â¯nM) increases GR levels in spines and PSD. Also, in these cultured neurons, blocking GRs with two different GR antagonists prevents Aßo-mediated PSD95 increase within the PSD. By analyzing long-term potentiation (LTP) and long-term depression (LTD) in ex vivo hippocampal slices after pharmacologically blocking GR, we also show that GR signaling is necessary for Aßo-mediated LTP impairment, but not Aßo-mediated LTD induction. The necessity of neuronal GRs for Aßo-mediated LTP was confirmed by genetically removing GRs in vivo from CA1 neurons using conditional GR mutant mice. These results indicate a tight functional interplay between GR and Aß activities at excitatory synapses.
Assuntos
Peptídeos beta-Amiloides/fisiologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Hipocampo/fisiologia , Fragmentos de Peptídeos/fisiologia , Receptor Cross-Talk/fisiologia , Receptores de Glucocorticoides/fisiologia , Sinapses/fisiologia , Animais , Células Cultivadas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
We previously reported that biphasic calcium phosphate (BCP) microparticles embedded in a blood clot induces ectopic bone formation in mice and repairs a critical femoral defect in rat. The present pilot study aimed to evaluate in dog and in two models of large defects the efficacy of this composite named "blood for reconstruction of bone" (BRB). We show here that BRB is a cohesive biomaterial easy to prepare from dog autologous blood and to mold to fill large bone defects. First in a model of cylindrical femoral condyle defect, the BRB was compared with BCP particles alone. After 8 weeks, this revealed that the amount of mature bone was slightly and significantly higher with BRB than with BCP particles. Second, in a model consisting in a 2 cm-long critical interruptive defect of the ulna, the BRB was compared with autologous bone. After 6 months, we observed that implantation of BRB can induce the complete reconstruction of the defect and that newly formed bone exhibits high regenerative potential. Comparison with the results obtained with autologous bone grafting strongly suggests that the BRB might be an efficient biomaterial to repair large bone defects, as an alternative or in addition to autologous bone. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1842-1850, 2018.
Assuntos
Sangue/metabolismo , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/patologia , Fosfatos de Cálcio/farmacologia , Microesferas , Animais , Regeneração Óssea/efeitos dos fármacos , Cães , Fêmur/diagnóstico por imagem , Fêmur/efeitos dos fármacos , Fêmur/patologia , Implantes Experimentais , Osteogênese/efeitos dos fármacos , Projetos Piloto , Ulna/diagnóstico por imagem , Ulna/efeitos dos fármacos , Ulna/patologia , Microtomografia por Raio-XRESUMO
The amyloid precursor protein (APP) harbors physiological roles at synapses and is central to Alzheimer's disease (AD) pathogenesis. Evidence suggests that APP intracellular domain (AICD) could regulate synapse function, but the underlying molecular mechanisms remain unknown. We addressed AICD actions at synapses, per se, combining in vivo AICD expression, ex vivo AICD delivery or APP knock-down by in utero electroporation of shRNAs with whole-cell electrophysiology. We report a critical physiological role of AICD in controlling GluN2B-containing NMDA receptors (NMDARs) at immature excitatory synapses, via a transcription-dependent mechanism. We further show that AICD increase in mature neurons, as reported in AD, alters synaptic NMDAR composition to an immature-like GluN2B-rich profile. This disrupts synaptic signal integration, via over-activation of SK channels, and synapse plasticity, phenotypes rescued by GluN2B antagonism. We provide a new physiological role for AICD, which becomes pathological upon AICD increase in mature neurons. Thus, AICD could contribute to AD synaptic failure.
Assuntos
Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/farmacologia , Precursor de Proteína beta-Amiloide/metabolismo , Hipocampo/patologia , Neurogênese/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapses/patologia , Precursor de Proteína beta-Amiloide/genética , Animais , Modelos Animais de Doenças , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Potenciação de Longa Duração/efeitos dos fármacos , Camundongos , Domínios Proteicos , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/genética , Sinapses/efeitos dos fármacos , Sinapses/metabolismoRESUMO
Cherubism is a rare autoinflammatory bone disorder that is associated with point mutations in the SH3-domain binding protein 2 (SH3BP2) gene, which encodes the adapter protein 3BP2. Individuals with cherubism present with symmetrical fibro-osseous lesions of the jaw, which are attributed to exacerbated osteoclast activation and defective osteoblast differentiation. Although it is a dominant trait in humans, cherubism appears to be recessively transmitted in mice, suggesting the existence of additional factors in the pathogenesis of cherubism. Here, we report that macrophages from 3BP2-deficient mice exhibited dramatically reduced inflammatory responses to microbial challenge and reduced phagocytosis. 3BP2 was necessary for LPS-induced activation of signaling pathways involved in macrophage function, including SRC, VAV1, p38MAPK, IKKα/ß, RAC, and actin polymerization pathways. Conversely, we demonstrated that the presence of a single Sh3bp2 cherubic allele and pathogen-associated molecular pattern (PAMP) stimulation had a strong cooperative effect on macrophage activation and inflammatory responses in mice. Together, the results from our study in murine genetic models support the notion that infection may represent a driver event in the etiology of cherubism in humans and suggest limiting inflammation in affected individuals may reduce manifestation of cherubic lesions.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Querubismo/genética , Inflamação/fisiopatologia , Ativação de Macrófagos/fisiologia , Mutação de Sentido Incorreto , Mutação Puntual , Actinas/química , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Transferência Adotiva , Substituição de Aminoácidos , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Heterozigoto , Humanos , Inflamação/microbiologia , Lipopolissacarídeos , Macrófagos Peritoneais/transplante , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutagênese Sítio-Dirigida , Osteoclastos/metabolismo , Osteoclastos/patologia , Fagocitose/fisiologia , Receptor 4 Toll-Like/efeitos dos fármacos , Receptor 4 Toll-Like/fisiologiaRESUMO
The early phase of Alzheimer's disease (AD) is characterized by hippocampus-dependent memory deficits and impaired synaptic plasticity. Increasing evidence suggests that stress and dysregulation of the hypothalamo-pituitary-adrenal (HPA) axis, marked by the elevated circulating glucocorticoids, are risk factors for AD onset. How these changes contribute to early hippocampal dysfunction remains unclear. Using an elaborated version of the object recognition task, we carefully monitored alterations in key components of episodic memory, the first type of memory altered in AD patients, in early symptomatic Tg2576 AD mice. We also combined biochemical and ex vivo electrophysiological analyses to reveal novel cellular and molecular dysregulations underpinning the onset of the pathology. We show that HPA axis, circadian rhythm, and feedback mechanisms, as well as episodic memory, are compromised in this early symptomatic phase, reminiscent of human AD pathology. The cognitive decline could be rescued by subchronic in vivo treatment with RU486, a glucocorticoid receptor antagonist. These observed phenotypes were paralleled by a specific enhancement of N-Methyl-D-aspartic acid receptor (NMDAR)-dependent LTD in CA1 pyramidal neurons, whereas LTP and metabotropic glutamate receptor-dependent LTD remain unchanged. NMDAR transmission was also enhanced. Finally, we show that, as for the behavioral deficit, RU486 treatment rescues this abnormal synaptic phenotype. These preclinical results define glucocorticoid signaling as a contributing factor to both episodic memory loss and early synaptic failure in this AD mouse model, and suggest that glucocorticoid receptor targeting strategies could be beneficial to delay AD onset.
Assuntos
Doença de Alzheimer/complicações , Doença de Alzheimer/patologia , Transtornos da Memória/etiologia , Memória Episódica , Plasticidade Neuronal/genética , Receptores de Glucocorticoides/metabolismo , Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Animais , Dexametasona/uso terapêutico , Modelos Animais de Doenças , Antagonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/genética , Glucocorticoides/sangue , Glucocorticoides/uso terapêutico , Hipocampo/patologia , Antagonistas de Hormônios/uso terapêutico , Humanos , Transtornos da Memória/tratamento farmacológico , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mifepristona/uso terapêutico , Mutação/genética , Plasticidade Neuronal/efeitos dos fármacos , Quinoxalinas/farmacologia , Reconhecimento Psicológico/efeitos dos fármacos , Valina/análogos & derivados , Valina/farmacologiaRESUMO
Achondroplasia is a rare genetic disease characterized by abnormal bone development, resulting in short stature. It is caused by a single point mutation in the gene coding for fibroblast growth factor receptor 3 (FGFR3), which leads to prolonged activation upon ligand binding. To prevent excessive intracellular signaling and rescue the symptoms of achondroplasia, we have developed a recombinant protein therapeutic approach using a soluble form of human FGFR3 (sFGFR3), which acts as a decoy receptor and prevents FGF from binding to mutant FGFR3. sFGFR3 was injected subcutaneously to newborn Fgfr3(ach/+) mice-the mouse model of achondroplasia-twice per week throughout the growth period during 3 weeks. Effective maturation of growth plate chondrocytes was restored in bones of treated mice, with a dose-dependent enhancement of skeletal growth in Fgfr3(ach/+) mice. This resulted in normal stature and a significant decrease in mortality and associated complications, without any evidence of toxicity. These results describe a new approach for restoring bone growth and suggest that sFGFR3 could be a potential therapy for children with achondroplasia and related disorders.
Assuntos
Acondroplasia/tratamento farmacológico , Desenvolvimento Ósseo/efeitos dos fármacos , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/uso terapêutico , Animais , Feminino , Humanos , Masculino , Camundongos , Transdução de Sinais/efeitos dos fármacosRESUMO
FOXO proteins are Akt-regulated transcription factors involved in the control of cell cycle, DNA repair, stress defense, apoptosis, and tumor suppression. We reported that plasmid-based overexpression of constitutively active FOXO3 in cells from chronic lymphocytic leukemia (CLL) reduced their survival, suggesting that increasing FOXO3 activity in hematologic malignancies may represent a promising therapeutic strategy. The transactivating transcription factor (TAT) protein transduction domain (PTD) derived from the HIV TAT protein was shown to efficiently deliver macromolecular cargo in various cell types. In this study, wild-type FOXO3 and FOXO3 mutated on Akt sites [FOXO3 T32A/S253A/S315A or TM (triple mutant)] were fused to the TAT-PTD. Using biochemical techniques, flow cytometry, and microscopy analysis, we found a rapid and dose-dependent cell penetration into leukemic cells of unlabeled and fluorescein isothiocyanate-labeled TAT-FOXO3 fusion proteins followed by their accumulation within nuclear and cytoplasmic compartments. Treatment with TAT-FOXO3 TM-but not wild-type TAT-FOXO3-proteins induced Jurkat and K562 leukemic cell death and affected cell viability of other hematologic malignancies including primary cells from CLL. Cell transduction with TAT-FOXO3 TM induced apoptotic cell death as shown by morphologic changes, Annexin V/7-AAD (7-amino-actinomycin D) staining, activation of effector caspases, and PARP cleavage, caspase blockade through the use of the inhibitor Z-VAD, and expression of Bim and p27(KIP1). By contrast, TAT-FOXO3 TM blocked cell proliferation of primary T cells, without affecting their viability. Together, our data show that cell penetrating TAT-FOXO3 TM fusion proteins constitute novel potential therapeutic agents in the treatment of lymphoproliferative disorders and hematologic malignancies.
Assuntos
Fatores de Transcrição Forkhead/farmacologia , Produtos do Gene tat/farmacologia , Leucemia/tratamento farmacológico , Proteínas de Fusão Oncogênica/farmacologia , Apoptose/efeitos dos fármacos , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p27/biossíntese , Inibidor de Quinase Dependente de Ciclina p27/imunologia , Relação Dose-Resposta a Droga , Fluoresceína-5-Isotiocianato/farmacocinética , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/farmacocinética , Produtos do Gene tat/genética , Produtos do Gene tat/farmacocinética , Humanos , Proteínas I-kappa B/biossíntese , Proteínas I-kappa B/imunologia , Células Jurkat , Células K562 , Leucemia/genética , Leucemia/metabolismo , Leucemia/patologia , Ativação Linfocitária/efeitos dos fármacos , Proteína Oncogênica v-akt/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/farmacocinética , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Transdução Genética , Células U937RESUMO
The adapter protein 3BP2 (also known as SH3BP2 and Abl SH3-binding protein 2) has been involved in leukocyte signaling and activation downstream immunoreceptors. Genetic studies have further associated 3BP2 mutations to the human disease cherubism and to inflammation and bone dysfunction in mouse. However, how wild type 3BP2 functions in macrophage differentiation remains poorly understood. In this study, using small interfering RNA-mediated silencing of 3BP2 in the RAW264.7 monocytic cell line, we show that 3BP2 was required for receptor activator of NFkappaB ligand (RANKL)-induced differentiation of RAW264.7 cells into multinucleated mature osteoclasts but not for granulocyte macrophage-colony stimulating factor/interleukin-4-induced differentiation into dendritic cells. 3BP2 silencing was associated with impaired activation of multiple signaling events downstream of RANK, including actin reorganization; Src, ERK, and JNK phosphorylation; and up-regulation of osteoclastogenic factors. In addition, 3BP2 knockdown cells induced to osteoclast by RANKL displayed a reduced increase of Src and nuclear factor of activated T cells (NFATc1) mRNA and protein expression. Importantly, 3BP2 interacted with Src, Syk, Vav, and Cbl in monocytic cells, and the introduction of constitutively active mutants of Src and NFATc1 in 3BP2-deficient cells restored osteoclast differentiation. Finally, the expression of a 3BP2 cherubism mutant was found to promote increased Src activity and NFAT-dependent osteoclast formation. Together, this study demonstrates that wild type 3BP2 is a key regulator of RANK-mediated macrophage differentiation into osteoclast through Src and NFATc1 activation.