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1.
J Cell Mol Med ; 26(2): 364-374, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34845842

RESUMO

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels function as pacemaker channels in spontaneously active cells. We studied the existence of HCN channels and their functional roles in the interstitial cells of Cajal (ICC) from the mouse colon using electrophysiological, immunohistochemical and molecular techniques. HCN1 and HCN3 channels were detected in anoctamin-1 (Ca2+ -activated Cl- channel; ANO1)-positive cells within the muscular and myenteric layers in colonic tissues. The mRNA transcripts of HCN1 and HCN3 channels were expressed in ANO1-positive ICC. In the deletion of HCN1 and HCN3 channels in colonic ICC, the pacemaking potential frequency was reduced. Basal cellular adenylate cyclase activity was decreased by adenylate cyclase inhibitor in colonic ICC, whereas cAMP-specific phosphodiesterase inhibitors increased it. 8-Bromo-cyclic AMP and rolipram increased spontaneous intracellular Ca2+ oscillations. In addition, Ca2+ -dependent adenylate cyclase 1 (AC1) mRNA was detected in colonic ICC. Sulprostone, a PGE2 -EP3 agonist, increased the pacemaking potential frequency, maximum rate of rise of resting membrane in pacemaker potentials and basal cellular adenylate cyclase activity in colonic ICC. These results indicate that HCN channels exist in colonic ICC and participate in generating pacemaking potentials. Thus, HCN channels may be therapeutic targets in disturbed colonic motility disorders.


Assuntos
Células Intersticiais de Cajal , Animais , Colo , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Células Intersticiais de Cajal/fisiologia , Camundongos
2.
Mol Psychiatry ; 26(12): 7538-7549, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34253863

RESUMO

Heterogeneity in the etiopathology of autism spectrum disorders (ASD) limits the development of generic remedies, requires individualistic and patient-specific research. Recent progress in human-induced pluripotent stem cell (iPSC) technology provides a novel platform for modeling ASDs for studying complex neuronal phenotypes. In this study, we generated telencephalic induced neuronal (iN) cells from iPSCs derived from an ASD patient with a heterozygous point mutation in the DSCAM gene. The mRNA of DSCAM and the density of DSCAM in dendrites were significantly decreased in ASD compared to control iN cells. RNA sequencing analysis revealed that several synaptic function-related genes including NMDA receptor subunits were downregulated in ASD iN cells. Moreover, NMDA receptor (R)-mediated currents were significantly reduced in ASD compared to control iN cells. Normal NMDA-R-mediated current levels were rescued by expressing wild-type DSCAM in ASD iN cells, and reduced currents were observed by truncated DSCAM expression in control iN cells. shRNA-mediated DSCAM knockdown in control iN cells resulted in the downregulation of an NMDA-R subunit, which was rescued by the overexpression of shRNA-resistant DSCAM. Furthermore, DSCAM was co-localized with NMDA-R components in the dendritic spines of iN cells whereas their co-localizations were significantly reduced in ASD iN cells. Levels of phospho-ERK1/2 were significantly lower in ASD iN cells, suggesting a potential mechanism. A neural stem cell-specific Dscam heterozygous knockout mouse model, showing deficits in social interaction and social memory with reduced NMDA-R currents. These data suggest that DSCAM mutation causes pathological symptoms of ASD by dysregulating NMDA-R function.


Assuntos
Transtorno do Espectro Autista , Moléculas de Adesão Celular/genética , Receptores de N-Metil-D-Aspartato , Animais , Transtorno do Espectro Autista/metabolismo , Humanos , Camundongos , Camundongos Knockout , Mutação/genética , Neurônios/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo
3.
Mol Brain ; 14(1): 100, 2021 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-34183057

RESUMO

Autophagy is a lysosomal degradation pathway that regulates cellular homeostasis. It is constitutively active in neurons and controls the essential steps of neuronal development, leading to its dysfunction in neurodevelopmental disorders. Although mTOR-associated impaired autophagy has previously been reported in neurodevelopmental disorders, there is lack of information about the dysregulation of mTOR-independent autophagy in neurodevelopmental disorders. In this study, we investigated whether the loss of Epac2, involved in the mTOR-independent pathway, affects autophagy activity and whether the activity of autophagy is associated with social-behavioral phenotypes in mice with Epac2 deficiencies. We observed an accumulation of autophagosomes and a significant increase in autophagic flux in Epac2-deficient neurons, which had no effect on mTOR activity. Next, we examined whether an increase in autophagic activity contributed to the social behavior exhibited in Epac2-/- mice. The social recognition deficit observed in Epac2-/- mice recovered in double transgenic Epac2-/-: Atg5+/- mice. Our study suggests that excessive autophagy due to Epac2 deficiencies may contribute to social recognition defects through an mTOR-independent pathway.


Assuntos
Autofagia , Comportamento Animal , Fatores de Troca do Nucleotídeo Guanina/deficiência , Comportamento Social , Animais , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Camundongos , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo
4.
Mol Brain ; 14(1): 1, 2021 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-33402211

RESUMO

CCCTC-binding factor (CTCF) is a transcription factor that is involved in organizing chromatin structure. A reduction of CTCF expression is known to develop distinct clinical features. Furthermore, conditional knock out (cKO) study revealed reactive gliosis of astrocytes and microglia followed by age-dependent cell death in the excitatory neurons of CTCF cKO mice. To assess the cognitive ability in CTCF cKO mice of over 20 weeks of age, we examined pairwise discrimination (PD), PD reversal learning (PDr), and different paired-associate learning (dPAL) tasks using a touch screen apparatus. We found cognitive impairment in dPAL touch screen tests, suggesting that prolonged Ctcf gene deficiency results in cognitive deficits.


Assuntos
Fator de Ligação a CCCTC/deficiência , Transtornos Cognitivos/metabolismo , Neurônios/metabolismo , Animais , Comportamento Animal , Fator de Ligação a CCCTC/metabolismo , Camundongos Knockout , Fenótipo
5.
Genes Brain Behav ; 20(2): e12701, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-32909350

RESUMO

CCCTC-binding factor (CTCF) is a genome organizer that regulates gene expression through transcription and chromatin structure regulation. CTCF also plays an important role during the developmental and adult stages. Cell-specific CTCF deletion studies have shown that a reduction in CTCF expression leads to the development of distinct clinical features and cognitive disorders. Therefore, we knocked out Ctcf (CTCF cKO) in the excitatory neurons of the forebrain in a Camk2a-Cre mouse strain to examine the role of CTCF in cell death and gliosis in the cortex. CTCF cKO mice were viable, but they demonstrated an age-dependent increase in reactive gliosis of astrocytes and microglia in the anterior cingulate cortex (ACC) from 16 weeks of age prior to neuronal loss observed at over 20 weeks of age. Consistent with these data, qRT-PCR analysis of the CTCF cKO ACC revealed changes in the expression of inflammation-related genes (Hspa1a, Prokr2 and Itga8) linked to gliosis and neuronal death. Our results suggest that prolonged Ctcf gene deficiency in excitatory neurons results in neuronal cell death and gliosis, possibly through functional changes in inflammation-related genes.


Assuntos
Fator de Ligação a CCCTC/genética , Gliose/genética , Giro do Cíngulo/metabolismo , Animais , Fator de Ligação a CCCTC/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Morte Celular , Feminino , Deleção de Genes , Gliose/metabolismo , Gliose/patologia , Giro do Cíngulo/patologia , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Cadeias alfa de Integrinas/genética , Cadeias alfa de Integrinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Neurônios/patologia , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Peptídeos/genética , Receptores de Peptídeos/metabolismo
6.
Neurosci Res ; 161: 8-17, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33007326

RESUMO

Successfully navigating dynamic environments requires balancing the decision to stay at an optimal choice with that to switch to an alternative to acquire new knowledge. However, the genetic factors and cellular activity shaping this "stay or switch" action decision remains largely unidentified. Here we find that mice carrying a deletion of the exchange protein directly activated by cAMP 2 (Epac2) gene, a putative autism locus, exhibit perseverative "stay" behavior in a dynamic foraging task. Anatomical analysis found that the loss of Epac2 resulted in a significant decrease in the density of PV-expressing interneurons in the ventrolateral orbitofrontal cortex (OFC) and dorsal striatum (dSTR). Further, in vitro whole cell patch clamp recordings of PV+ GABAergic interneurons in the dSTR revealed altered neural activity in Epac2 KO mice in response to dopamine. Our findings highlight a potential role of Epac2 in structural changes and neural responses of PV-expressing GABAergic interneurons in the ventrolateral OFC and dSTR during value-based reinforcement learning and link Epac2 function to abnormal decision-making processes and perseverative behaviors seen in autism.


Assuntos
Interneurônios , Recompensa , Animais , Tomada de Decisões , Dopamina , Camundongos , Técnicas de Patch-Clamp , Córtex Pré-Frontal
7.
J Clin Med ; 9(6)2020 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-32560273

RESUMO

Cohen syndrome (CS), a rare autosomal recessive disorder, has been associated with genetic mutations in the VPS13B gene, which regulates vesicle-mediated protein sorting and transport. However, the cellular mechanism underlying CS pathogenesis in patient-derived human neurons remains unknown. We identified a novel compound heterozygous mutation, due to homozygous variation of biparental origin and heterozygous variation inherited from the father, in the VPS13B gene in a 20-month-old female patient. To understand the cellular pathogenic mechanisms, we generated induced pluripotent stem cells (iPSCs) from the fibroblasts of the CS patient. The iPSCs were differentiated into forebrain-like functional glutamatergic neurons or neurospheres. Functional annotation from transcriptomic analysis using CS iPSC-derived neurons revealed that synapse-related functions were enriched among the upregulated and downregulated genes in the CS neurons, whereas processes associated with neurodevelopment were enriched in the downregulated genes. The developing CS neurospheres were small in size compared to control neurospheres, likely due to the reduced proliferation of SOX2-positive neural stem cells. Moreover, the number of SV2B-positive puncta and spine-like structures was significantly reduced in the CS neurons, suggesting synaptic dysfunction. Taking these findings together, for the first time, we report a potential cellular pathogenic mechanism which reveals the alteration of neurodevelopment-related genes and the dysregulation of synaptic function in the human induced neurons differentiated from iPSCs and neurospheres of a CS patient.

8.
Mol Brain ; 12(1): 113, 2019 12 23.
Artigo em Inglês | MEDLINE | ID: mdl-31870404

RESUMO

AIM: cAMP signal transduction cascade activation is important in regulating neurogenesis in adult rodents by increasing the proliferation of newborn cells. Although the ventricular-subventricular zone (V-SVZ) and subgranular zone (SGZ) both contain large populations of neural stem/precursor cells; it remains unclear whether an alternative target of cAMP, the exchange protein directly activated by cAMP (Epac2), is involved in adult neurogenesis in the V-SVZ and SGZ. Here, we investigated the cell-specific expression of Epac2 protein in the V-SVZ and SGZ of the adult mouse brain. METHODS: Immunohistochemical analyses were performed using antibodies against Epac2, glial fibrillary acidic protein (GFAP), doublecortin (DCX), and beta-catenin, to examine the co-localization of Epac2 protein and neural stem/precursor cells in the V-SVZ and SGZ in three 8-week-old male mice. RESULTS: In the V-SVZ of the lateral ventricle, most GFAP-positive adult neural stem cells (NSC, defined as type B cells) and 75% of DCX-positive migrating neuroblasts (type A cells) expressed Epac2 proteins. Ninety-three percent of beta-catenin-positive ependymal cells (type E cells), which are in direct contact with NSCs and the ventricles, also expressed Epac2 protein. Similarly, in the SGZ of the hippocampus, Epac2-immunopositive signals were shown by 83% of GFAP-positive radial-glia-like NSCs (type 1 cells), 86% of DCX-positive transiently amplifying cells (type 2 and type 3 cells), and 71% of DCX-positive immature neurons. The present data suggest that a PKA-independent cAMP signaling pathway via Epac2 may be party to adult neurogenesis in the V-SVZ and the SGZ.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Ventrículos Laterais/citologia , Animais , Movimento Celular , Proteína Duplacortina , Masculino , Camundongos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurogênese
9.
BMB Rep ; 49(2): 128-33, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26645637

RESUMO

Astrocytes play a critical role in normal brain functions and maintaining the brain microenvironment, and defects in astrocytogenesis during neurodevelopment could give rise to severe mental illness and psychiatric disorders. During neuro-embryogenesis, astrocytogenesis involves astrocytic differentiation of neural precursor cells (NPCs) induced by signals from ciliary neurotrophic factor (CNTF) or pituitary adenylate cyclase-activating peptide (PACAP). However, in contrast to the CNTF signaling pathway, the exact mechanism underlying astrocytic differentiation induced by PACAP is unknown. In the present study, we aimed to verify a signaling pathway specific to PACAP-induced astrocytogenesis, using exchange protein directly activated by cAMP2 (Epac2)-knockout mice. We found that PACAP could trigger astrocytic differentiation of NPCs via Epac2 activation and an increase in the intracellular calcium concentration via a calcium ion influx. Taken together, we concluded that astrocytogenesis stimulated by PACAP occurs through a novel signaling pathway independent from CNTF-JAK/STAT signaling, that is the well-known pathway of astrocytogenesis. [BMB Reports 2016; 49(2): 128-133].


Assuntos
Astrócitos/citologia , Astrócitos/metabolismo , Cálcio/metabolismo , Diferenciação Celular/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células-Tronco Neurais/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Animais , Animais Recém-Nascidos , Astrócitos/efeitos dos fármacos , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Fatores de Troca do Nucleotídeo Guanina/deficiência , Espaço Intracelular/metabolismo , Íons , Camundongos Knockout , Células-Tronco Neurais/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
10.
Mol Brain ; 8: 38, 2015 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-26104314

RESUMO

BACKGROUND: Guanine nucleotide exchange factors (GEFs) activate small GTPases that are involved in several cellular functions. cAMP-guanine nucleotide exchange factor II (cAMP-GEF II) acts as a target for cAMP independently of protein kinase A (PKA) and functions as a GEF for Rap1 and Rap2. Although cAMP-GEF II is expressed abundantly in several brain areas including the cortex, striatum, and hippocampus, its specific function and possible role in hippocampal synaptic plasticity and cognitive processes remain elusive. Here, we investigated how cAMP-GEF II affects synaptic function and animal behavior using cAMP-GEF II knockout mice. RESULTS: We found that deletion of cAMP-GEF II induced moderate decrease in long-term potentiation, although this decrease was not statistically significant. On the other hand, it produced a significant and clear impairment in NMDA receptor-dependent long-term depression at the Schaffer collateral-CA1 synapses of hippocampus, while microscopic morphology, basal synaptic transmission, and depotentiation were normal. Behavioral testing using the Morris water maze and automated IntelliCage system showed that cAMP-GEF II deficient mice had moderately reduced behavioral flexibility in spatial learning and memory. CONCLUSIONS: We concluded that cAMP-GEF II plays a key role in hippocampal functions including behavioral flexibility in reversal learning and in mechanisms underlying induction of long-term depression.


Assuntos
Comportamento Animal , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Hipocampo/metabolismo , Potenciação de Longa Duração , Animais , Encéfalo/metabolismo , Eletrochoque , Fatores de Troca do Nucleotídeo Guanina/deficiência , Aprendizagem , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapses/metabolismo
11.
Mol Pain ; 11: 28, 2015 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-25981600

RESUMO

BACKGROUND: Pain is the most prominent non-motor symptom observed in patients with Parkinson's disease (PD). However, the mechanisms underlying the generation of pain in PD have not been well studied. We used a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of PD to analyze the relationship between pain sensory abnormalities and the degeneration of dopaminergic neurons. RESULTS: The latency to fall off the rotarod and the total distance traveled in round chamber were significantly reduced in MPTP-induced PD mice, consistent with motor dysfunction. MPTP-treated mice also showed remarkably shorter nociceptive response latencies compared to saline-treated mice and the subcutaneous injection of L-3,4-dihydroxyphenylalanine (L-DOPA) partially reversed pain hypersensitivity induced by MPTP treatment. We found that degeneration of cell bodies and fibers in the substantia nigra pars compacta and the striatum of MPTP-treated mice. In addition, astrocytic and microglial activation was seen in the subthalamic nucleus and neuronal activity was significantly increased in the striatum and globus pallidus. However, we did not observe any changes in neurons, astrocytes, and microglia of both the dorsal and ventral horns in the spinal cord after MPTP treatment. CONCLUSIONS: These results suggest that the dopaminergic nigrostriatal pathway may have a role in inhibiting noxious stimuli, and that abnormal inflammatory responses and neural activity in basal ganglia is correlated to pain processing in PD induced by MPTP treatment.


Assuntos
1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/farmacologia , Atividade Motora/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Percepção da Dor/efeitos dos fármacos , Doença de Parkinson , Animais , Astrócitos/efeitos dos fármacos , Corpo Estriado/efeitos dos fármacos , Modelos Animais de Doenças , Dopamina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Microglia/metabolismo , Neurônios/metabolismo
12.
Mol Brain ; 8: 13, 2015 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-25761792

RESUMO

BACKGROUND: Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels help control the rhythmic activation of pacemaker neurons during brain development. However, little is known about the timing and cell type specificity of the expression of HCN isoforms during development of the hippocampus. RESULTS: Here we examined the developmental expression of the brain-enriched HCN1, HCN2, and HCN4 isoforms of HCN channels in mouse hippocampus from embryonic to postnatal stages. All these isoforms were expressed abundantly in the hippocampus at embryonic day 14.5 and postnatal day 0. Each HCN channel isoform showed subfield-specific expression within the hippocampus from postnatal day 7, and only HCN4 was found in glial cells in the stratum lacunosum moleculare at this developmental stage. At postnatal days 21 and 56, all HCN isoforms were strongly expressed in the stratum lacunosum moleculare and the stratum pyramidale of the Cornu Ammonis (CA), as well as in the hilus of the dentate gyrus, but not in the subgranular zone. Furthermore, the immunolabeling for all these isoforms was colocalized with parvalbumin immunolabeling in interneurons of the CA field and in the dentate gyrus. CONCLUSIONS: Our mapping data showing the temporal and spatial changes in the expression of HCN channels suggest that HCN1, HCN2, and HCN4 subunits may have distinct physiological roles in the developing hippocampus.


Assuntos
Hipocampo/crescimento & desenvolvimento , Hipocampo/metabolismo , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Subunidades Proteicas/metabolismo , Envelhecimento/metabolismo , Animais , Animais Recém-Nascidos , Proteínas do Domínio Duplacortina , Embrião de Mamíferos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/metabolismo , Neuropeptídeos/metabolismo , Parvalbuminas/metabolismo , Isoformas de Proteínas/metabolismo
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