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1.
Genes Cells ; 28(2): 156-169, 2023 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-36530170

RESUMEN

Extended pluripotent stem cells (EPSCs) derived from mice and humans showed an enhanced potential for chimeric formation. By exploiting transcriptomic approaches, we assessed the differences in gene expression profile between extended EPSCs derived from mice and humans, and those newly derived from the common marmoset (marmoset; Callithrix jacchus). Although the marmoset EPSC-like cells displayed a unique colony morphology distinct from murine and human EPSCs, they displayed a pluripotent state akin to embryonic stem cells (ESCs), as confirmed by gene expression and immunocytochemical analyses of pluripotency markers and three-germ-layer differentiation assay. Importantly, the marmoset EPSC-like cells showed interspecies chimeric contribution to mouse embryos, such as E6.5 blastocysts in vitro and E6.5 epiblasts in vivo in mouse development. Also, we discovered that the perturbation of gene expression of the marmoset EPSC-like cells from the original ESCs resembled that of human EPSCs. Taken together, our multiple analyses evaluated the efficacy of the method for the derivation of marmoset EPSCs.


Asunto(s)
Callithrix , Células Madre Embrionarias , Animales , Humanos , Ratones , Células Madre Embrionarias/metabolismo , Diferenciación Celular , Perfilación de la Expresión Génica , Transcriptoma
2.
FASEB J ; 36(2): e22160, 2022 02.
Artículo en Inglés | MEDLINE | ID: mdl-35064699

RESUMEN

Dopamine in the prefrontal cortex is essential for the regulation of social behavior. However, stress-causing social withdrawal also promotes dopamine release in the prefrontal cortex. Thus, this evidence suggests opposite functions of dopamine in the prefrontal cortex. However, the influence of dopamine on prefrontal functions is yet to be fully understood. Here, we show that dopamine differentially modulated the neuronal activity triggered by social stimuli in the prefrontal cortex, depending on the duration of the dopamine activation (transient or sustained activation). Using chemogenetic techniques, we have found that social behavior was negatively regulated by a sustained increase in dopamine neuronal activity in the ventral tegmental area, while it was positively regulated by an acute increase. The duration of social interactions was positively correlated with the transient dopamine release triggered by social stimuli in the prefrontal cortex and negatively correlated with the sustained increase in prefrontal dopamine levels. Furthermore, the elevation of neural calcium signal, triggered by social stimuli, in the prefrontal cortex was attenuated by the persistent elevation of prefrontal dopamine levels, whereas an acute increase in dopamine levels enhanced it. Additionally, the chronic excess of dopamine suppressed c-Fos induction triggered by social stimuli in prefrontal neurons expressing dopamine D1 receptors, but not D2 receptors. These results suggest that sustained activation of prefrontal dopamine, at the opposite of its transient activation, can reduce prefrontal activity associated with social behavior, even for identical dopamine concentrations. Thus, dopamine plays opposite roles in modulating prefrontal activity depending on the duration of its action.


Asunto(s)
Dopamina/metabolismo , Corteza Prefrontal/metabolismo , Animales , Neuronas Dopaminérgicas/metabolismo , Masculino , Proteínas Proto-Oncogénicas c-fos/metabolismo , Ratas , Ratas Transgénicas/metabolismo , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/metabolismo , Conducta Social , Área Tegmental Ventral/metabolismo
3.
Int J Mol Sci ; 24(7)2023 Apr 02.
Artículo en Inglés | MEDLINE | ID: mdl-37047614

RESUMEN

We previously demonstrated that fatty acid-binding protein 3 null (FABP3-/-) mice exhibit resistance to nicotine-induced conditioned place preference (CPP). Here, we confirm that the FABP3 inhibitor, MF1 ((4-(2-(1-(2-chlorophenyl)-5-phenyl-1H-pyrazol-3-yl)phenoxy) butanoic acid), successfully reduces nicotine-induced CPP scores in mice. MF1 (0.3 or 1.0 mg/kg) was orally administered 30 min before nicotine, and CPP scores were assessed in the conditioning, withdrawal, and relapse phases. MF1 treatment decreased CPP scores in a dose-dependent manner. Failure of CPP induction by MF1 (1.0 mg/kg, p.o.) was associated with the inhibition of both CaMKII and ERK activation in the nucleus accumbens (NAc) and hippocampal CA1 regions. MF1 treatment reduced nicotine-induced increases in phosphorylated CaMKII and cAMP-response element-binding protein (CREB)-positive cells. Importantly, the increase in dopamine D2 receptor (D2R) levels following chronic nicotine exposure was inhibited by MF1 treatment. Moreover, the quinpirole (QNP)-induced increase in the level of CaMKII and ERK phosphorylation was significantly inhibited by MF1 treatment of cultured NAc slices from wild type (WT) mice; however, QNP treatment had no effect on CaMKII and ERK phosphorylation levels in the NAc of D2R null mice. Taken together, these results show that MF1 treatment suppressed D2R/FABP3 signaling, thereby preventing nicotine-induced CPP induction. Hence, MF1 can be used as a novel drug to block addiction to nicotine and other drugs by inhibiting the dopaminergic system.


Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Nicotina , Ratones , Animales , Nicotina/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Núcleo Accumbens/metabolismo , Transducción de Señal , Ratones Noqueados , Proteína 3 de Unión a Ácidos Grasos/metabolismo
4.
J Biol Chem ; 296: 100544, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33737022

RESUMEN

Dopamine (DA) exerts well-known functions in the brain as a neurotransmitter. In addition, it plays important physiological roles in peripheral organs, but it is largely unknown how and where peripheral DA is synthesized and regulated. Catecholamines in peripheral tissues are either produced within the tissue itself and/or derived from sympathetic neurons, which release neurotransmitters for uptake by peripheral tissues. To evaluate DA-producing ability of each peripheral tissue, we generated conditional KO mice (cKO mice) in which the tyrosine hydroxylase (TH) gene is ablated in the sympathoadrenal system, thus eliminating sympathetic neurons as a DA source. We then examined the alterations in the noradrenaline (NA), DA, and 3,4-dihydroxyphenylalanine (DOPA) contents in peripheral organs and performed immunohistochemical analyses of TH-expressing cells. In the heart and pancreas of cKO mice, both the TH protein and NA levels were significantly decreased, and the DA contents were decreased in parallel with NA contents, indicating that the DA supply originated from sympathetic neurons. We found TH-immunoreactive cells in the stomach and lung, where the TH protein showed a decreasing trend, but the DA levels were not decreased in cKO mice. Moreover, we found a significant correlation between the DA content in the kidney and the plasma DOPA concentration, suggesting that the kidney takes up DOPA from blood to make DA. The aforementioned data unravel differences in the DA biosynthetic pathway among tissues and support the role of sympathetic neurons as a DA supplier.


Asunto(s)
Glándulas Suprarrenales/metabolismo , Vías Biosintéticas , Catecolaminas/metabolismo , Dopamina/biosíntesis , Neuronas/metabolismo , Sistema Nervioso Simpático/metabolismo , Tirosina 3-Monooxigenasa/fisiología , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Especificidad de Órganos
5.
J Neurosci ; 39(38): 7551-7563, 2019 09 18.
Artículo en Inglés | MEDLINE | ID: mdl-31371425

RESUMEN

Mental disorders are caused by genetic and environmental factors. We here show that deficiency of an isoform of dopamine D2 receptor (D2R), D2LR, causes stress vulnerability in mouse. This occurs through dysfunction of serotonin [5-hydroxytryptamine (5-HT)] 1A receptor (5-HT1AR) on serotonergic neurons in the mouse brain. Exposure to forced swim stress significantly increased anxiety- and depressive-like behaviors in D2LR knock-out (KO) male mice compared with wild-type mice. Treatment with 8-OH-DPAT, a 5-HT1AR agonist, failed to alleviate the stress-induced behaviors in D2LR-KO mice. In forced swim-stressed D2LR-KO mice, 5-HT efflux in the medial prefrontal cortex was elevated and the expression of genes related to 5-HT levels was upregulated by the transcription factor PET1 in the dorsal raphe nucleus. Notably, D2LR formed a heteromer with 5-HT1AR in serotonergic neurons, thereby suppressing 5-HT1AR-activated G-protein-activated inwardly rectifying potassium conductance in D2LR-KO serotonergic neurons. Finally, D2LR overexpression in serotonergic neurons in the dorsal raphe nucleus alleviated stress vulnerability observed in D2LR-KO mice. Together, we conclude that disruption of the negative feedback regulation by the D2LR/5-HT1A heteromer causes stress vulnerability.SIGNIFICANCE STATEMENT Etiologies of mental disorders are multifactorial, e.g., interactions between genetic and environmental factors. In this study, using a mouse model, we showed that genetic depletion of an isoform of dopamine D2 receptor, D2LR, causes stress vulnerability associated with dysfunction of serotonin 1A receptor, 5-HT1AR in serotonergic neurons. The D2LR/5-HT1AR inhibitory G-protein-coupled heteromer may function as a negative feedback regulator to suppress psychosocial stress.


Asunto(s)
Encéfalo/metabolismo , Receptor de Serotonina 5-HT1A/metabolismo , Receptores de Dopamina D2/deficiencia , Neuronas Serotoninérgicas/metabolismo , Estrés Psicológico/metabolismo , Animales , Masculino , Ratones , Ratones Noqueados
6.
Neurobiol Dis ; 130: 104516, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31229688

RESUMEN

Spinocerebellar ataxia 42 (SCA42) is a neurodegenerative disorder recently shown to be caused by c.5144G > A (p.Arg1715His) mutation in CACNA1G, which encodes the T-type voltage-gated calcium channel CaV3.1. Here, we describe a large Japanese family with SCA42. Postmortem pathological examination revealed severe cerebellar degeneration with prominent Purkinje cell loss without ubiquitin accumulation in an SCA42 patient. To determine whether this mutation causes ataxic symptoms and neurodegeneration, we generated knock-in mice harboring c.5168G > A (p.Arg1723His) mutation in Cacna1g, corresponding to the mutation identified in the SCA42 family. Both heterozygous and homozygous mutants developed an ataxic phenotype from the age of 11-20 weeks and showed Purkinje cell loss at 50 weeks old. Degenerative change of Purkinje cells and atrophic thinning of the molecular layer were conspicuous in homozygous knock-in mice. Electrophysiological analysis of Purkinje cells using acute cerebellar slices from young mice showed that the point mutation altered the voltage dependence of CaV3.1 channel activation and reduced the rebound action potentials after hyperpolarization, although it did not significantly affect the basic properties of synaptic transmission onto Purkinje cells. Finally, we revealed that the resonance of membrane potential of neurons in the inferior olivary nucleus was decreased in knock-in mice, which indicates that p.Arg1723His CaV3.1 mutation affects climbing fiber signaling to Purkinje cells. Altogether, our study shows not only that a point mutation in CACNA1G causes an ataxic phenotype and Purkinje cell degeneration in a mouse model, but also that the electrophysiological abnormalities at an early stage of SCA42 precede Purkinje cell loss.


Asunto(s)
Canales de Calcio Tipo T/metabolismo , Cerebelo/metabolismo , Fenotipo , Células de Purkinje/metabolismo , Ataxias Espinocerebelosas/metabolismo , Anciano , Anciano de 80 o más Años , Animales , Canales de Calcio Tipo T/genética , Cerebelo/patología , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Ratones , Células de Purkinje/patología , Ataxias Espinocerebelosas/genética , Ataxias Espinocerebelosas/patología
7.
Biochem Biophys Res Commun ; 492(2): 199-205, 2017 10 14.
Artículo en Inglés | MEDLINE | ID: mdl-28821434

RESUMEN

BACKGROUND: The dystroglycan complex consists of two subunits: extracellular α-dystroglycan and membrane-spanning ß-dystroglycan, which provide a tight link between the extracellular matrix and the intracellular cytoskeleton. Previous studies showed that 43 kDa ß-dystroglycan is proteolytically cleaved into the 30 kDa fragment by matrix metalloproteinases (MMPs) in various non-muscle tissues, whereas it is protected from cleavage in muscles by the sarcoglycan complex which resides close to the dystroglycan complex. It is noteworthy that cleaved ß-dystroglycan is detected in muscles from patients with sarcoglycanopathy, sarcoglycan-deficient muscular dystrophy. In vitro assays using protease inhibitors suggest that both MMP-2 and MMP-9 contribute to the cleavage of ß-dystroglycan. However, this has remained uninvestigated in vivo. METHODS: We generated triple-knockout (TKO) mice targeting MMP-2, MMP-9 and γ-sarcoglycan to examine the status of ß-dystroglycan cleavage in the absence of the candidate matrix metalloproteinases in sarcoglycan-deficient muscles. RESULTS: Unexpectedly, ß-dystroglycan was cleaved in muscles from TKO mice. Muscle pathology was not ameliorated but worsened in TKO mice compared with γ-sarcoglycan single-knockout mice. The gene expression of MMP-14 was up-regulated in TKO mice as well as in γ-sarcoglycan knockout mice. In vitro assay showed MMP-14 is capable to cleave ß-dystroglycan. CONCLUSIONS: Double-targeting of MMP-2 and MMP-9 cannot prevent cleavage of ß-dystroglycan in sarcoglycanopathy. Thus, matrix metalloproteinases contributing to ß-dystroglycan cleavage are redundant, and MMP-14 could participate in the pathogenesis of sarcoglycanopathy.


Asunto(s)
Distroglicanos/metabolismo , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/genética , Músculo Esquelético/metabolismo , Sarcoglicanopatías/genética , Sarcoglicanos/genética , Animales , Eliminación de Gen , Humanos , Metaloproteinasa 14 de la Matriz/genética , Metaloproteinasa 14 de la Matriz/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones Noqueados , Músculo Esquelético/patología , Proteolisis , Sarcoglicanopatías/metabolismo , Sarcoglicanopatías/patología , Sarcoglicanos/metabolismo , Regulación hacia Arriba
8.
Learn Mem ; 23(7): 359-64, 2016 07.
Artículo en Inglés | MEDLINE | ID: mdl-27317196

RESUMEN

Considerable evidence has demonstrated a critical role for the nucleus accumbens (NAc) in the acquisition and flexibility of behavioral strategies. These processes are guided by the activity of two discrete neuron types, dopamine D1- or D2-receptor expressing medium spiny neurons (D1-/D2-MSNs). Here we used the IntelliCage, an automated group-housing experimental cage apparatus, in combination with a reversible neurotransmission blocking technique to examine the role of NAc D1- and D2-MSNs in the acquisition and reversal learning of a place discrimination task. We demonstrated that NAc D1- and D2-MSNs do not mediate the acquisition of the task, but that suppression of activity in D2-MSNs impairs reversal learning and increased perseverative errors. Additionally, global knockout of the dopamine D2L receptor isoform produced a similar behavioral phenotype to D2-MSN-blocked mice. These results suggest that D2L receptors and NAc D2-MSNs act to suppress the influence of previously correct behavioral strategies allowing transfer of behavioral control to new strategies.


Asunto(s)
Discriminación en Psicología/fisiología , Neuronas/fisiología , Núcleo Accumbens/fisiología , Receptores de Dopamina D2/fisiología , Aprendizaje Inverso/fisiología , Animales , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Isoformas de Proteínas/genética , Receptores de Dopamina D1/fisiología , Receptores de Dopamina D2/genética
9.
BMC Neurosci ; 17(1): 77, 2016 11 30.
Artículo en Inglés | MEDLINE | ID: mdl-27903237

RESUMEN

BACKGROUND: α-Synuclein (αSYN) has been genetically implicated in familial and sporadic Parkinson's disease (PD), and is associated with disease susceptibility, progression and pathology. Excess amounts of αSYN are toxic to neurons. In the brain, microglial αSYN clearance is closely related to neuronal survival. Leucine-rich repeat kinase 2 (LRRK2) is the one of the other genes implicated in familial and sporadic PD. While LRRK2 is known to be expressed in microglia, its true function remains to be elucidated. In this study, we investigated αSYN clearance by microglia isolated from LRRK2-knockout (KO) mice. RESULTS: In LRRK2-KO microglia, αSYN was taken up in larger amounts and cleared from the supernatant more effectively than for microglia isolated from wild-type (WT) mice. This higher clearance ability of LRRK2-KO microglia was thought to be due to an increase of Rab5-positive endosomes, but not Rab7- or Rab11-positive endosomes. Increased engagement between Rab5 and dynamin 1 was also observed in LRRK2-KO microglia. CONCLUSION: LRRK2 negatively regulates the clearance of αSYN accompanied by down-regulation of the endocytosis pathway. Our findings reveal a new functional role of LRRK2 in microglia and offer a new insight into the mechanism of PD pathogenesis.


Asunto(s)
Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Microglía/metabolismo , alfa-Sinucleína/metabolismo , Animales , Western Blotting , Encéfalo/metabolismo , Encéfalo/patología , Células Cultivadas , Dinamina I/metabolismo , Endocitosis/fisiología , Endosomas/metabolismo , Técnica del Anticuerpo Fluorescente , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Microglía/patología , Proteínas Recombinantes/metabolismo , Proteínas de Unión al GTP rab5/metabolismo
10.
Cereb Cortex ; 25(12): 4885-97, 2015 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-26443442

RESUMEN

In the basal ganglia (BG), dopamine plays a pivotal role in motor control, and dopamine deficiency results in severe motor dysfunctions as seen in Parkinson's disease. According to the well-accepted model of the BG, dopamine activates striatal direct pathway neurons that directly project to the output nuclei of the BG through D1 receptors (D1Rs), whereas dopamine inhibits striatal indirect pathway neurons that project to the external pallidum (GPe) through D2 receptors. To clarify the exact role of dopaminergic transmission via D1Rs in vivo, we developed novel D1R knockdown mice in which D1Rs can be conditionally and reversibly regulated. Suppression of D1R expression by doxycycline treatment decreased spontaneous motor activity and impaired motor ability in the mice. Neuronal activity in the entopeduncular nucleus (EPN), one of the output nuclei of the rodent BG, was recorded in awake conditions to examine the mechanism of motor deficits. Cortically evoked inhibition in the EPN mediated by the cortico-striato-EPN direct pathway was mostly lost during suppression of D1R expression, whereas spontaneous firing rates and patterns remained unchanged. On the other hand, GPe activity changed little. These results suggest that D1R-mediated dopaminergic transmission maintains the information flow through the direct pathway to appropriately release motor actions.


Asunto(s)
Núcleo Entopeduncular/fisiología , Actividad Motora , Corteza Motora/fisiología , Neuronas/fisiología , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D1/fisiología , Animales , Doxiciclina/farmacología , Estimulación Eléctrica , Núcleo Entopeduncular/efectos de los fármacos , Femenino , Técnicas de Silenciamiento del Gen , Masculino , Ratones , Ratones Endogámicos C57BL , Actividad Motora/efectos de los fármacos , Inhibición Neural/efectos de los fármacos , Vías Nerviosas/metabolismo , Vías Nerviosas/fisiología , Neuronas/efectos de los fármacos , Receptores de Dopamina D1/genética , Prueba de Desempeño de Rotación con Aceleración Constante
11.
Proc Natl Acad Sci U S A ; 110(1): 342-7, 2013 Jan 02.
Artículo en Inglés | MEDLINE | ID: mdl-23248274

RESUMEN

The basal ganglia-thalamocortical circuitry plays a central role in selecting actions that achieve reward-seeking outcomes and avoid aversive ones. Inputs of the nucleus accumbens (NAc) in this circuitry are transmitted through two parallel pathways: the striatonigral direct pathway and the striatopallidal indirect pathway. In the NAc, dopaminergic (DA) modulation of the direct and the indirect pathways is critical in reward-based and aversive learning and cocaine addiction. To explore how DA modulation regulates the associative learning behavior, we developed an asymmetric reversible neurotransmission-blocking technique in which transmission of each pathway was unilaterally blocked by transmission-blocking tetanus toxin and the transmission on the intact side was pharmacologically manipulated by local infusion of a receptor-specific agonist or antagonist. This approach revealed that the activation of D1 receptors and the inactivation of D2 receptors postsynaptically control reward learning/cocaine addiction and aversive learning in a direct pathway-specific and indirect pathway-specific manner, respectively. Furthermore, this study demonstrated that aversive learning is elicited by elaborate actions of NMDA receptors, adenosine A2a receptors, and endocannabinoid CB1 receptors, which serve as key neurotransmitter receptors in inducing long-term potentiation in the indirect pathway. Thus, reward and aversive learning is regulated by pathway-specific neural plasticity via selective transmitter receptors in the NAc circuit.


Asunto(s)
Aprendizaje por Asociación/fisiología , Reacción de Prevención/fisiología , Trastornos Relacionados con Cocaína/fisiopatología , Plasticidad Neuronal/fisiología , Núcleo Accumbens/fisiología , Recompensa , Análisis de Varianza , Animales , Humanos , Ratones , Vías Nerviosas/metabolismo , Receptor de Adenosina A2A/metabolismo , Receptor Cannabinoide CB1/metabolismo , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Toxina Tetánica
12.
Cereb Cortex ; 23(8): 1824-35, 2013 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-22705452

RESUMEN

Polypyrimidine tract-binding protein (PTB) is a well-characterized RNA-binding protein and known to be preferentially expressed in neural stem cells (NSCs) in the central nervous system; however, its role in NSCs in the developing brain remains unclear. To explore the role of PTB in embryonic NSCs in vivo, Nestin-Cre-mediated conditional Ptb knockout mice were generated for this study. In the mutant forebrain, despite the depletion of PTB protein, neither abnormal neurogenesis nor flagrant morphological abnormalities were observed at embryonic day 14.5 (E14.5). Nevertheless, by 10 weeks, nearly all mutant mice succumbed to hydrocephalus (HC), which was caused by a lack of the ependymal cell layer in the dorsal cortex. Upon further analysis, a gradual loss of adherens junctions (AJs) was observed in the ventricular zone (VZ) of the dorsal telencephalon in the mutant brains, beginning at E14.5. In the AJs-deficient VZ, impaired interkinetic nuclear migration and precocious differentiation of NSCs were observed after E14.5. These findings demonstrated that PTB depletion in the dorsal telencephalon is causally involved in the development of HC and that PTB is important for the maintenance of AJs in the NSCs of the dorsal telencephalon.


Asunto(s)
Uniones Adherentes/ultraestructura , Hidrocefalia/etiología , Proteína de Unión al Tracto de Polipirimidina/fisiología , Telencéfalo/embriología , Animales , Hidrocefalia/genética , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Células-Madre Neurales/ultraestructura , Proteína de Unión al Tracto de Polipirimidina/genética , Telencéfalo/anomalías
13.
J Biol Chem ; 287(17): 13859-67, 2012 Apr 20.
Artículo en Inglés | MEDLINE | ID: mdl-22375006

RESUMEN

Methyl CpG-binding protein 2 gene (MeCP2) mutations are implicated in Rett syndrome (RTT), one of the common causes of female mental retardation. Two MeCP2 isoforms have been reported: MeCP2_e2 (splicing of all four exons) and MeCP2_e1 (alternative splicing of exons 1, 3, and 4). Their relative expression levels vary among tissues, with MeCP2_e1 being more dominant in adult brain, whereas MeCP2_e2 is expressed more abundantly in placenta, liver, and skeletal muscle. In this study, we performed specific disruption of the MeCP2_e2-defining exon 2 using the Cre-loxP system and examined the consequences of selective loss of MeCP2_e2 function in vivo. We performed behavior evaluation, gene expression analysis, using RT-PCR and real-time quantitative PCR, and histological analysis. We demonstrate that selective deletion of MeCP2_e2 does not result in RTT-associated neurological phenotypes but confers a survival disadvantage to embryos carrying a MeCP2_e2 null allele of maternal origin. In addition, we reveal a specific requirement for MeCP2_e2 function in extraembryonic tissue, where selective loss of MeCP2_e2 results in placenta defects and up-regulation of peg-1, as determined by the parental origin of the mutant allele. Taken together, our findings suggest a novel role for MeCP2 in normal placenta development and illustrate how paternal X chromosome inactivation in extraembryonic tissues confers a survival disadvantage for carriers of a mutant maternal MeCP2_e2 allele. Moreover, our findings provide an explanation for the absence of reports on MeCP2_e2-specific exon 2 mutations in RTT. MeCP2_e2 mutations in humans may result in a phenotype that evades a diagnosis of RTT.


Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Proteína 2 de Unión a Metil-CpG/química , Alelos , Empalme Alternativo , Animales , Apoptosis , Supervivencia Celular , Modelos Animales de Enfermedad , Epigénesis Genética , Femenino , Proteína 2 de Unión a Metil-CpG/metabolismo , Ratones , Fenotipo , Placenta/metabolismo , Placenta/fisiología , Embarazo , Unión Proteica , Isoformas de Proteínas , Síndrome de Rett/genética , Síndrome de Rett/metabolismo
14.
Neurochem Int ; 165: 105517, 2023 05.
Artículo en Inglés | MEDLINE | ID: mdl-36913980

RESUMEN

NMDA-type glutamate receptors (NMDARs) are tetrameric channel complex composed of two subunits of GluN1, which is encoded by a single gene and diversified by alternative splicing, and two subunits from four subtypes of GluN2, leading to various combinations of subunits and channel specificities. However, there is no comprehensive quantitative analysis of GluN subunit proteins for relative comparison, and their compositional ratios at various regions and developmental stages have not been clarified. Here we prepared six chimeric subunits, by fusing an N-terminal side of the GluA1 subunit with a C-terminal side of each of two splicing isoforms of GluN1 subunit and four GluN2 subunits, with which titers of respective NMDAR subunit antibodies could be standardized using common GluA1 antibody, thus enabling quantification of relative protein levels of each NMDAR subunit by western blotting. We determined relative protein amounts of NMDAR subunits in crude, membrane (P2) and microsomal fractions prepared from the cerebral cortex, hippocampus and cerebellum in adult mice. We also examined amount changes in the three brain regions during developmental stages. Their relative amounts in the cortical crude fraction were almost parallel to those of mRNA expression, except for some subunits. Interestingly, a considerable amount of GluN2D protein existed in adult brains, although its transcription level declines after early postnatal stages. GluN1 was larger in quantity than GluN2 in the crude fraction, whereas GluN2 increased in the membrane component-enriched P2 fraction, except in the cerebellum. These data will provide the basic spatio-temporal information on the amount and composition of NMDARs.


Asunto(s)
Receptores de N-Metil-D-Aspartato , Transducción de Señal , Animales , Ratones , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Cerebelo/metabolismo , Encéfalo/metabolismo , Ácido Glutámico/metabolismo , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo
15.
Elife ; 122023 01 25.
Artículo en Inglés | MEDLINE | ID: mdl-36695811

RESUMEN

Extensive serotonin (5-hydroxytryptamine, 5-HT) innervation throughout the brain corroborates 5-HT's modulatory role in numerous cognitive activities. Volume transmission is the major mode for 5-HT transmission but mechanisms underlying 5-HT signaling are still largely unknown. Abnormal brain 5-HT levels and function have been implicated in autism spectrum disorder (ASD). Neurexin (Nrxn) genes encode presynaptic cell adhesion molecules important for the regulation of synaptic neurotransmitter release, notably glutamatergic and GABAergic transmission. Mutations in Nrxn genes are associated with neurodevelopmental disorders including ASD. However, the role of Nrxn genes in the 5-HT system is poorly understood. Here, we generated a mouse model with all three Nrxn genes disrupted specifically in 5-HT neurons to study how Nrxns affect 5-HT transmission. Loss of Nrxns in 5-HT neurons reduced the number of serotonin neurons in the early postnatal stage, impaired 5-HT release, and decreased 5-HT release sites and serotonin transporter expression. Furthermore, 5-HT neuron-specific Nrxn knockout reduced sociability and increased depressive-like behavior. Our results highlight functional roles for Nrxns in 5-HT neurotransmission, 5-HT neuron survival, and the execution of complex behaviors.


Asunto(s)
Trastorno del Espectro Autista , Serotonina , Ratones , Animales , Serotonina/metabolismo , Neuronas Serotoninérgicas , Trastorno del Espectro Autista/metabolismo , Transmisión Sináptica/fisiología , Encéfalo/metabolismo
16.
Cell Rep ; 42(5): 112398, 2023 05 30.
Artículo en Inglés | MEDLINE | ID: mdl-37083330

RESUMEN

Spatiotemporal control of gene expression is important for neural development and function. Here, we show that heterogeneous nuclear ribonucleoprotein (hnRNP) A/B is highly expressed in developing olfactory sensory neurons (OSNs), and its knockout results in reduction in mature OSNs and aberrant targeting of OSN axons to the olfactory bulb. RNA immunoprecipitation analysis reveals that hnRNP A/B binds to a group of mRNAs that are highly related to axon projections and synapse assembly. Approximately 11% of the identified hnRNP A/B targets, including Pcdha and Ncam2, encode cell adhesion molecules. In Hnrnpab knockout mice, PCDHA and NCAM2 levels are significantly reduced at the axon terminals of OSNs. Furthermore, deletion of the hnRNP A/B-recognition motif in the 3' UTR of Pcdha leads to impaired PCDHA expression at the OSN axon terminals. Therefore, we propose that hnRNP A/B facilitates OSN maturation and axon projection by regulating the local expression of its target genes at axon terminals.


Asunto(s)
Neuronas Receptoras Olfatorias , Animales , Ratones , Axones/metabolismo , Ratones Noqueados , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Neurogénesis/genética , Bulbo Olfatorio , Neuronas Receptoras Olfatorias/metabolismo , Terminales Presinápticos/metabolismo
17.
Sci Rep ; 13(1): 18196, 2023 10 24.
Artículo en Inglés | MEDLINE | ID: mdl-37875516

RESUMEN

The common marmoset (Callithrix jacchus) has attracted attention as a valuable primate model for the analysis of human diseases. Despite the potential for primate genetic modification, however, its widespread lab usage has been limited due to the requirement for a large number of eggs. To make up for traditional oocyte retrieval methods such as hormone administration and surgical techniques, we carried out an alternative approach by utilizing ovarian tissue from deceased marmosets that had been disposed of. This ovarian tissue contains oocytes and can be used as a valuable source of follicles and oocytes. In this approach, the ovarian tissue sections were transplanted under the renal capsules of immunodeficient mice first. Subsequent steps consist of development of follicles by hormone administrations, induction of oocyte maturation and fertilization, and culture of the embryo. This method was first established with rat ovaries, then applied to marmoset ovaries, ultimately resulting in the successful acquisition of the late-stage marmoset embryos. This approach has the potential to contribute to advancements in genetic modification research and disease modeling through the use of primate models, promoting biotechnology with non-human primates and the 3Rs principle in animal experimentation.


Asunto(s)
Callithrix , Ovario , Femenino , Animales , Ratones , Fertilización In Vitro , Oocitos , Callitrichinae , Hormonas
18.
Proc Natl Acad Sci U S A ; 106(34): 14426-31, 2009 Aug 25.
Artículo en Inglés | MEDLINE | ID: mdl-19706528

RESUMEN

Planar cell polarity (PCP) genes are essential for establishing planar cell polarity in both invertebrate and vertebrate tissues and are known to regulate cellular morphogenesis and cell movements during development. We focused on Prickle, one of the core components of the PCP pathway, and deleted one of two mouse prickle homologous genes, mpk1. We found that the deletion of mpk1 gene resulted in early embryonic lethality, between embryonic day (E)5.5 and E6.5, associated with failure of distal visceral endoderm migration and primitive streak formation. The mpk1(-/-) epiblast tissue was disorganized, and analyses at the cellular level revealed abnormal cell shapes, mislocalized extracellular matrix (ECM) proteins, and disrupted orientation of mitotic spindles, from which loss of apico-basal (AB) polarity of epiblast cells are suspected. Furthermore, we show mpk1 genetically interacts with another core PCP gene Vangl2/stbm in the epiblast formation, suggesting that PCP components are commonly required for the establishment and/or the maintenance of epiblast AB polarity. This was further supported by our finding that overexpression of DeltaPET/LIM (DeltaP/L), a dominant-negative Pk construct, in Xenopus embryo disrupted uniform localization of an apical marker PKCzeta, and expanded the apical domain of ectoderm cells. Our results demonstrate a role for mpk1 in AB polarity formation rather than expected role as a PCP gene.


Asunto(s)
Blastocisto/metabolismo , Proteínas Portadoras/genética , Embrión de Mamíferos/metabolismo , Proteínas del Tejido Nervioso/genética , Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Blastocisto/citología , Proteínas Portadoras/metabolismo , Polaridad Celular , Citoesqueleto/metabolismo , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Femenino , Regulación del Desarrollo de la Expresión Génica , Inmunohistoquímica , Hibridación in Situ , Proteínas con Dominio LIM , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Mutación , Proteínas del Tejido Nervioso/metabolismo , Proteína Quinasa C/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo
19.
Front Behav Neurosci ; 16: 751053, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35309682

RESUMEN

Dopaminergic neurotransmission via dopamine D1 receptors (D1Rs) is considered to play an important role not only in reward-based learning but also in aversive learning. The contextual and auditory cued fear conditioning tests involve the processing of classical fear conditioning and evaluates aversive learning memory. It is possible to evaluate aversive learning memory in two different types of neural transmission circuits. In addition, when evaluating the role of dopaminergic neurotransmission via D1R, to avoid the effects in D1R-mediated neural circuitry alterations during development, it is important to examine using mice who D1R expression in the mature stage is suppressed. Herein, we investigated the role of dopaminergic neurotransmission via D1Rs in aversive memory formation in contextual and auditory cued fear conditioning tests using D1R knockdown (KD) mice, in which the expression of D1Rs could be conditionally and reversibly controlled with doxycycline (Dox) treatment. For aversive memory, we examined memory formation using recent memory 1 day after conditioning, and remote memory 4 weeks after conditioning. Furthermore, immunostaining of the brain tissues of D1RKD mice was performed after aversive footshock stimulation to investigate the distribution of activated c-Fos, an immediate-early gene, in the hippocampus (CA1, CA3, dentate gyrus), striatum, amygdala, and prefrontal cortex during aversive memory formation. After aversive footshock stimulation, immunoblotting was performed using hippocampal, striatal, and amygdalar samples from D1RKD mice to investigate the increase in the amount of c-Fos and phosphorylated SNAP-25 at Ser187 residue. When D1R expression was suppressed using Dox, behavioral experiments revealed impaired contextual fear learning in remote aversion memory following footshock stimulation. Furthermore, expression analysis showed a slight increase in the post-stimulation amount of c-Fos in the hippocampus and striatum, and a significant increase in the amount of phosphorylated SNAP-25 in the hippocampus, striatum, and prefrontal cortex before and after stimulation. These findings indicate that deficiency in D1R-mediated dopaminergic neurotransmission is an important factor in impairing contextual fear memory formation for remote memory.

20.
Sci Rep ; 12(1): 3566, 2022 03 04.
Artículo en Inglés | MEDLINE | ID: mdl-35246592

RESUMEN

When writing an object's name, humans mentally construct its spelling. This capacity critically depends on use of the dual-structured linguistic system, in which meaningful words are represented by combinations of meaningless letters. Here we search for the evolutionary origin of this capacity in primates by designing dual-structured bigram symbol systems where different combinations of meaningless elements represent different objects. Initially, we trained Japanese macaques (Macaca fuscata) in an object-bigram symbolization task and in a visually-guided bigram construction task. Subsequently, we conducted a probe test using a symbolic bigram construction task. From the initial trial of the probe test, the Japanese macaques could sequentially choose the two elements of a bigram that was not actually seen but signified by a visually presented object. Moreover, the animals' spontaneous choice order bias, developed through the visually-guided bigram construction learning, was immediately generalized to the symbolic bigram construction test. Learning of dual-structured symbols by the macaques possibly indicates pre-linguistic adaptations for the ability of mentally constructing symbols in the common ancestors of humans and Old World monkeys.


Asunto(s)
Macaca fuscata , Macaca , Animales , Aprendizaje
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