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1.
Commun Biol ; 4(1): 461, 2021 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-33846537

RESUMO

Optogenetic tools such as channelrhodopsin-2 (ChR2) enable the manipulation and mapping of neural circuits. However, ChR2 variants selectively transported down a neuron's long-range axonal projections for precise presynaptic activation remain lacking. As a result, ChR2 activation is often contaminated by the spurious activation of en passant fibers that compromise the accurate interpretation of functional effects. Here, we explored the engineering of a ChR2 variant specifically localized to presynaptic axon terminals. The metabotropic glutamate receptor 2 (mGluR2) C-terminal domain fused with a proteolytic motif and axon-targeting signal (mGluR2-PA tag) localized ChR2-YFP at axon terminals without disturbing normal transmission. mGluR2-PA-tagged ChR2 evoked transmitter release in distal projection areas enabling lower levels of photostimulation. Circuit connectivity mapping in vivo with the Spike Collision Test revealed that mGluR2-PA-tagged ChR2 is useful for identifying axonal projection with significant reduction in the polysynaptic excess noise. These results suggest that the mGluR2-PA tag helps actuate trafficking to the axon terminal, thereby providing abundant possibilities for optogenetic experiments.

2.
Cell Struct Funct ; 45(2): 143-154, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32641624

RESUMO

Prickle2 has been identified in genetic studies of subjects with autism spectrum disorder (ASD) and epilepsy, but the pathological mechanism of Prickle2 remains to be fully understood. Proteomic analysis of Prickle2 with mass spectrometry revealed twenty-eight Prickle2 interactors, including immunoglobulin superfamily member 9b (Igsf9b), in the brain. Here, because Igsf9 family proteins are associated with psychiatric diseases and seizures, we studied the physiological interaction between Prickle2 and Igsf9b. Prickle2 colocalized with Igsf9b in cultured hippocampal neurons. Knockdown of Prickle2 affected the subcellular localization of Igsf9b. Interestingly, Igsf9b localized along axonal processes in a pattern opposite to the ASD-related molecule ANK3/AnkG. AnkG is a major component of the axon initial segment (AIS), where a variety of ASD and epilepsy susceptibility proteins accumulate. Igsf9b-knockdown neurons displayed altered AnkG localization. Prickle2 depletion caused defects in AnkG and voltage-gated Na+ channel localization, resulting in altered network activity. These results support the idea that Prickle2 regulates AnkG distribution by controlling the proper localization of Igsf9b. The novel function of Prickle2 in AIS cytoarchitecture provides new insights into the shared pathology of ASD and epilepsy.Key words: Prickle2, Igsf9b, axon initial segment, neuronal excitability, ASD.

3.
J Hum Genet ; 65(12): 1143-1147, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32694621

RESUMO

Recently, the expansion of an intronic AAGGG repeat in the replication factor C subunit 1 (RFC1) gene was reported to cause cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS). In Europeans, the expansion accounted for 22% of sporadic patients with late-onset ataxia. We genotyped 37 Japanese patients comprising 25 familial (autosomal recessive or undecided transmission) and 12 sporadic ones with late-onset ataxia. We found intronic repeat expansions in RFC1 in three (12%) of the familial patients and one (8.5%) of the sporadic ones. Although our cohort study was small, the disease frequency in Japanese patients with CANVAS might be lower than that in European ones. In addition, we found biallelic ACAGG repeat expansion in one patient, indicating ACAGG repeat expansion might cause CANVAS. Clinically, we found one patient with sleep apnea syndrome, which has not been reported previously. Thus, this study might expand the clinical and genetic spectrum of CANVAS.

4.
Neurosci Res ; 2020 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-32598973

RESUMO

Increasing evidence has shown that voltage-gated L-type Ca2+ channels (LTCCs) are crucial for neurodevelopmental events, including neuronal differentiation/migration and neurite morphogenesis/extension. However, the time course of their functional maturation during the development of excitatory neurons remains unknown. Using a combination of fluorescence in situ hybridization and in utero electroporation-based labeling, we found that the transcripts of Cacna1c and Cacna1d, which encode the LTCC pore-forming subunits, were upregulated in the intermediate zone (IZ) during radial migration. Ca2+ imaging using GCaMP6s in acute brain slices showed spontaneous Ca2+ transients in migrating neurons throughout the IZ. Neurons in the IZ upper layer, especially in the multipolar-to-bipolar transition layer (TL), exhibited more frequent Ca2+ transients than adjacent layers and responded to FPL64176, a potent activator of LTCC. Consistently, nimodipine, an LTCC blocker, inhibited spontaneous Ca2+ transients in neurons in the TL. Collectively, we showed a hitherto unknown increased prevalence of LTCC-dependent Ca2+ transients in the TL of the IZ upper layer during the radial migration of excitatory neurons, which could be essential for the regulation of Ca2+-dependent neurodevelopmental processes.

5.
Biochem Biophys Res Commun ; 528(2): 322-329, 2020 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-32423795

RESUMO

Phosphatase and actin regulator 3/nuclear scaffold-associated protein phosphatase 1-inhibiting protein (Phactr3/Scapinin) is an actin- and protein phosphatase 1 (PP1)-binding protein known to negatively regulate axon elongation. In this study, we examined the expression pattern of Phactr3/Scapinin in several tissues and investigated the effect of Phactr3/Scapinin on dendritic morphology of cortical neurons. Results showed that Phactr3/Scapinin expression was up-regulated in the developing brain and enriched in neurons and in the postsynaptic density fraction, but not in astrocytes. Overexpression of wild type or mutant Phactr3/Scapinin, which lacked actin-binding activity, resulted in increased dendritic complexity and percentage of spines with a mushroom or stubby shape, as well as a decrease in spine density. However, overexpression of mutant Phactr3/Scapinin that lacked PP1-binding activity did not. Taken together, these findings suggest that Phactr3/Scapinin expression is neuronal and might contribute to synaptic formation via distinct actin- and PP1-binding domains involved in dendritic and axonal morphology, respectively.

6.
J Physiol ; 598(12): 2431-2452, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32304329

RESUMO

KEY POINTS: CAST/ELKS are positive regulators of presynaptic growth and are suppressors of active zone expansion at the developing mouse calyx of Held. CAST/ELKS regulate all three CaV 2 subtype channel levels in the presynaptic terminal and not just CaV 2.1. The half-life of ELKS is on the timescale of days and not weeks. Synaptic transmission was not impacted by the loss of CAST/ELKS. CAST/ELKS are involved in pathways regulating morphological properties of presynaptic terminals during an early stage of circuit maturation. ABSTRACT: Many presynaptic active zone (AZ) proteins have multiple regulatory roles that vary during distinct stages of neuronal circuit development. The CAST/ELKS protein family are evolutionarily conserved presynaptic AZ molecules that regulate presynaptic calcium channels, synaptic transmission and plasticity in the mammalian CNS. However, how these proteins regulate synapse development and presynaptic function in a developing neuronal circuit in its native environment is unclear. To unravel the roles of CAST/ELKS in glutamatergic synapse development and in presynaptic function, we used CAST knockout (KO) and ELKS conditional KO (CKO) mice to examine how their loss during the early stages of circuit maturation impacted the calyx of Held presynaptic terminal development and function. Morphological analysis from confocal z-stacks revealed that combined deletion of CAST/ELKS resulted in a reduction in the surface area and volume of the calyx. Analysis of AZ ultrastructure showed that AZ size was increased in the absence of CAST/ELKS. Patch clamp recordings demonstrated a reduction of all presynaptic CaV 2 channel subtype currents that correlated with a loss in presynaptic CaV 2 channel numbers. However, these changes did not impair synaptic transmission and plasticity and synaptic vesicle release kinetics. We conclude that CAST/ELKS proteins are positive regulators of presynaptic growth and are suppressors of AZ expansion and CaV 2 subtype currents and levels during calyx of Held development. We propose that CAST/ELKS are involved in pathways regulating presynaptic morphological properties and CaV 2 channel subtypes and suggest there is developmental compensation to preserve synaptic transmission during early stages of neuronal circuit maturation.

7.
Sci Rep ; 10(1): 5238, 2020 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-32251313

RESUMO

Although sociological studies affirm the importance of parental care in the survival of offspring, maltreatment-including child neglect-remains prevalent in many countries. While child neglect is well known to affect child development, the causes of maternal neglect are poorly understood. Here, we found that female mice with a deletion mutation of CAST (a presynaptic release-machinery protein) showed significantly reduced weaning rate when primiparous and a recovered rate when multiparous. Indeed, when nurturing, primiparous and nulliparous CAST knock out (KO) mice exhibited less crouching time than control mice and moved greater distances. Contrary to expectations, plasma oxytocin (OXT) was not significantly reduced in CAST KO mice even though terminals of magnocellular neurons in the posterior pituitary expressed CAST. We further found that compared with control mice, CAST KO mice drank significantly less water when nurturing and had a greater preference for sucrose during pregnancy. We suggest that deficiency in presynaptic release-machinery protein impairs the facilitation of some maternal behaviours, which can be compensated for by experience and learning.

8.
Mol Brain ; 13(1): 13, 2020 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-31996256

RESUMO

Presynaptic active zone cytomatrix proteins are essential elements of neurotransmitter release machinery that govern neural transmission. Among active zone proteins, cytomatrix at the active zone-associated structural protein (CAST) is known to regulate active zone size in retinal photoreceptors and neurotransmitter release by recruiting Ca2+ channels at various synapses. However, the role of ELKS-a protein from the same family as CAST-and the synergistic roles of CAST/ELKS have not been thoroughly investigated, particularly with regard to mouse behavior. Here, we generated ELKS conditional KO in mouse forebrain synapses by crossing ELKS flox mice with a CaMKII promoter-induced Cre line. Results showed that CAST is dominant at these synapses and that ELKS can support CAST function, but is less effective in the ELKS single KO. Pups of CAST/ELKS double KO in the forebrain were born in Mendelian rations but resulted in eventual death right after the birth. Anatomically, the forebrain neuronal compositions of CAST KO and CAST/ELKS double KO mice were indistinguishable, and the sensory neural network from whiskers on the face was identified as barrelette-like patches in the spinal trigeminal nucleus. Therefore, depletion of CAST and ELKS disrupts neurotransmission from sensory to motor networks, which can lead to deficits in exploration and failure to suckle.

9.
Mol Genet Genomic Med ; 8(3): e1108, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31876103

RESUMO

BACKGROUND: Alterations of vacuolar protein sorting-associated protein 13 (VPS13) family members including VPS13A, VPS13B, and VPS13C lead to chorea acanthocytosis, Cohen syndrome, and parkinsonism, respectively. Recently, VPS13D mutations were identified as a cause of VPS13D-related movement disorders, which show several phenotypes including chorea, dystonia, spastic ataxia, and spastic paraplegia. METHODS: We applied whole-exome analysis for a patient with a complicated form of hereditary spastic paraplegia (HSP) and her unaffected parents. Then, we screened the candidate genes in 664 Japanese families with HSP in Japan. RESULTS: We first found a compound heterozygote VPS13D mutation and a heterozygote ABHD4 variation in a sporadic patient with spastic paraplegia. Then, we found three patients with VPS13D mutations in two Japanese HSP families. The three patients with homozygous mutations (p.Thr1118Met/p.Thr1118Met and p.Thr2945Ala/p.Thr2945Ala) in the VPS13D showed an adult onset pure form of HSP. Meanwhile, the patient with a compound heterozygous mutation (p.Ser405Arg/p.Arg3141Ter) in the VPS13D showed a childhood onset complicated form of HSP associated with cerebellar ataxia, cervical dystonia, cataracts, and chorioretinal dystrophy. CONCLUSION: In the present study, we found four patients in three Japanese families with novel VPS13D mutations, which may broaden the clinical and genetic findings for VPS13D-related disorders.

10.
J Hum Genet ; 64(11): 1055-1065, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31515522

RESUMO

We aimed to find a new causative gene and elucidate the molecular mechanisms underlying a new type of hereditary spastic paraplegia (HSP). Patients with HSP were recruited from the Japan Spastic Paraplegia Research Consortium (JASPAC). Exome sequencing of genomic DNA from patients in four families was carried out, followed by Sanger sequencing of the UBAP1 gene. A mouse homolog of one UBAP1 frameshift mutation carried by one of the patients was created as a disease model. Functional properties of the UBAP1 wild type and UBAP1-mutant in mouse hippocampus neurons were examined. We identified three novel heterozygous loss of function mutations (c.425_426delAG, c.312delC, and c.535G>T) in the UBAP1 gene as the genetic cause of a new type of HSP (SPG80). All the patients presented identical clinical features of a pure type of juvenile-onset HSP. Functional studies on mouse hippocampal neurons revealed that the C-terminal deletion UBAP1-mutant of our disease model had lost its ability to bind ubiquitin in vitro. Overexpression of the UBAP1 wild type interacts directly with ubiquitin on enlarged endosomes, while the UBAP1-mutant cannot be recruited to endosome membranes. Our study demonstrated that mutations in the UBAP1 gene cause a new type of HSP and elucidated its pathogenesis. The full-length UBAP1 protein is involved in endosomal dynamics in neurons, while loss of UBAP1 function may perturb endosomal fusion and sorting of ubiquitinated cargos. These effects could be more prominent in neurons, thereby giving rise to the phenotype of a neurodegenerative disease such as HSP.


Assuntos
Proteínas de Transporte/genética , Predisposição Genética para Doença , Doenças Neurodegenerativas/genética , Paraplegia Espástica Hereditária/genética , Adolescente , Adulto , Idade de Início , Idoso , Idoso de 80 Anos ou mais , Animais , Grupo com Ancestrais do Continente Asiático , Criança , Modelos Animais de Doenças , Endossomos/genética , Feminino , Mutação da Fase de Leitura/genética , Humanos , Japão , Masculino , Camundongos , Pessoa de Meia-Idade , Doenças Neurodegenerativas/fisiopatologia , Neurônios/metabolismo , Neurônios/patologia , Linhagem , Fenótipo , Paraplegia Espástica Hereditária/fisiopatologia , Sequenciamento Completo do Exoma
11.
Mol Metab ; 27S: S81-S91, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31500835

RESUMO

BACKGROUND: Insulin is stored within large dense-core granules in pancreatic beta (ß)-cells and is released by Ca2+-triggered exocytosis with increasing blood glucose levels. Polarized and targeted secretion of insulin from ß-cells in pancreatic islets into the vasculature has been proposed; however, the mechanisms related to cellular and molecular localization remain largely unknown. Within nerve terminals, the Ca2+-dependent release of a polarized transmitter is limited to the active zone, a highly specialized area of the presynaptic membrane. Several active zone-specific proteins have been characterized; among them, the CAST/ELKS protein family members have the ability to form large protein complexes with other active zone proteins to control the structure and function of the active zone for tight regulation of neurotransmitter release. Notably, ELKS but not CAST is also expressed in ß-cells, implying that ELKS may be involved in polarized insulin secretion from ß-cells. SCOPE OF REVIEW: This review provides an overview of the current findings regarding the role(s) of ELKS and other active zone proteins in ß-cells and focuses on the molecular mechanism underlying ELKS regulation within polarized insulin secretion from islets. MAJOR CONCLUSIONS: ELKS localizes at the vascular-facing plasma membrane of ß-cells in mouse pancreatic islets. ELKS forms a potent insulin secretion complex with L-type voltage-dependent Ca2+ channels on the vascular-facing plasma membrane of ß-cells, enabling polarized Ca2+ influx and first-phase insulin secretion from islets. This model provides novel insights into the functional polarity observed during insulin secretion from ß-cells within islets at the molecular level. This active zone-like region formed by ELKS at the vascular side of the plasma membrane is essential for coordinating physiological insulin secretion and may be disrupted in diabetes.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Humanos
12.
Sci Rep ; 9(1): 2912, 2019 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-30814664

RESUMO

The PET and LIM domain-containing protein, Prickle, plays a key role in planar cell polarity (PCP) in Drosophila. It has been reported that mutations in the PRICKLE2 gene, which encodes one of the human orthologues of Prickle, are associated with human diseases such as epilepsy and autism spectrum disorder. To develop preventive and therapeutic strategies for these intractable diseases, we studied the regulation of Prickle2 protein levels in transfected HEK293T cells. Prickle2 levels were negatively regulated by a physical interaction with another PCP protein, Van Gogh-like 2 (Vangl2). The Vangl2-mediated reduction in Prickle2 levels was, at least in part, relieved by proteasome inhibitors or by functional inhibition of the Cullin-1 E3 ubiquitin ligase. Furthermore, the expression of Vangl2 enhanced the polyubiquitination of Prickle2. This ubiquitination was partially blocked by co-expression of a ubiquitin mutant, which cannot be polymerised through their Lys48 residue to induce target proteins toward proteasomal degradation. Together, these results suggest that Prickle2 is polyubiquitinated by the Vangl2 interaction in a Cullin-1-dependent manner to limit its expression levels. This regulation may play a role in the local and temporal fine-tuning of Prickle protein levels during PCP signal-dependent cellular behaviours.


Assuntos
Transtorno do Espectro Autista/genética , Epilepsia/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas com Domínio LIM/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Animais , Polaridade Celular/genética , Proteínas Culina/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Células HEK293 , Humanos , Proteínas com Domínio LIM/metabolismo , Ligação Proteica , Proteólise , Ubiquitinação
13.
Cell Rep ; 26(5): 1213-1226.e7, 2019 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-30699350

RESUMO

Pancreatic ß cells secrete insulin by Ca2+-triggered exocytosis. However, there is no apparent secretory site similar to the neuronal active zones, and the cellular and molecular localization mechanism underlying polarized exocytosis remains elusive. Here, we report that ELKS, a vertebrate active zone protein, is used in ß cells to regulate Ca2+ influx for insulin secretion. ß cell-specific ELKS-knockout (KO) mice showed impaired glucose-stimulated first-phase insulin secretion and reduced L-type voltage-dependent Ca2+ channel (VDCC) current density. In situ Ca2+ imaging of ß cells within islets expressing a membrane-bound G-CaMP8b Ca2+ sensor demonstrated initial local Ca2+ signals at the ELKS-localized vascular side of the ß cell plasma membrane, which were markedly decreased in ELKS-KO ß cells. Mechanistically, ELKS directly interacted with the VDCC-ß subunit via the GK domain. These findings suggest that ELKS and VDCCs form a potent insulin secretion complex at the vascular side of the ß cell plasma membrane for polarized Ca2+ influx and first-phase insulin secretion from pancreatic islets.


Assuntos
Cálcio/metabolismo , Células Secretoras de Insulina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Subunidades Proteicas/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Vasos Sanguíneos/efeitos dos fármacos , Vasos Sanguíneos/metabolismo , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Citosol/efeitos dos fármacos , Citosol/metabolismo , Glucose/farmacologia , Humanos , Insulina/metabolismo , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Ativação do Canal Iônico/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Proteínas do Tecido Nervoso/deficiência , Ligação Proteica/efeitos dos fármacos , Proteínas rab de Ligação ao GTP/deficiência
14.
Parkinsonism Relat Disord ; 61: 57-63, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30528172

RESUMO

INTRODUCTION: Glucocerebrosidase gene (GBA) variants are associated with Parkinson's disease (PD) and dementia with Lewy bodies (DLB). The molecular mechanisms underlying these diseases with GBA variants, however, are not well understood. In order to determine the effect of a deletion mutation in GBA, we performed a neuroimaging, genetic, and enzymatic study in a Japanese family with a gross deletion of exons 3 to 11 in GBA. METHODS: We performed [123I] FP-CIT SPECT and [123I] N-isopropyl-p-iodoamphetamine SPECT (IMP-SPECT), and determined GBA expression and glucocerebrosidase (GCase) activity in leukocytes in two GBA-associated PD patients and nine unaffected individuals (including four mutation carriers) in a Japanese family with a heterozygous gross deletion mutation in the GBA gene. RESULTS: The two PD patients and two of the four clinically unaffected carriers showed decreased [123I] FP-CIT uptake. IMP-SPECT showed a pattern like that in DLB in one patient. When we compared PD patients with GBA mutations with clinically unaffected carriers, there was a poor correlation between the development of PD and the expression level of GBA or GCase activity. CONCLUSION: We confirmed the gross deletion mutation in the GBA gene, which appeared to be associated with the PD or reduced [123I] FP-CIT in this family. However, since we cannot conclude whether a reduction of GCase activity is directly correlated with the pathogenesis of PD or not, longitudinal follow-up of this family is needed.


Assuntos
Encéfalo/diagnóstico por imagem , Glucosilceramidase/genética , Doença de Parkinson/genética , Idoso , Idoso de 80 Anos ou mais , Grupo com Ancestrais do Continente Asiático , Éxons , Família , Feminino , Deleção de Genes , Glucosilceramidase/metabolismo , Humanos , Iofetamina , Japão , Leucócitos , Masculino , Pessoa de Meia-Idade , Neuroimagem , Doença de Parkinson/diagnóstico por imagem , Doença de Parkinson/enzimologia , Linhagem , Compostos Radiofarmacêuticos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tomografia Computadorizada de Emissão de Fóton Único , Tropanos
15.
J Cell Biol ; 217(11): 3993-4006, 2018 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-30190286

RESUMO

At the presynaptic active zone (AZ), the related cytomatrix proteins CAST and ELKS organize the presynaptic release machinery. While CAST is known to regulate AZ size and neurotransmitter release, the role of ELKS and the integral system of CAST/ELKS together is poorly understood. Here, we show that CAST and ELKS have both redundant and unique roles in coordinating synaptic development, function, and maintenance of retinal photoreceptor ribbon synapses. A CAST/ELKS double knockout (dKO) mouse showed high levels of ectopic synapses and reduced responses to visual stimulation. Ectopic formation was not observed in ELKS conditional KO but progressively increased with age in CAST KO mice with higher rates in the dKO. Presynaptic calcium influx was strongly reduced in rod photoreceptors of CAST KO and dKO mice. Three-dimensional scanning EM reconstructions showed structural abnormalities in rod triads of CAST KO and dKO. Remarkably, AAV-mediated acute ELKS deletion after synapse maturation induced neurodegeneration and loss of ribbon synapses. These results suggest that CAST and ELKS work in concert to promote retinal synapse formation, transmission, and maintenance.


Assuntos
Sinalização do Cálcio , Proteínas de Transporte/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Sinapses/metabolismo , Transmissão Sináptica , Envelhecimento/genética , Envelhecimento/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas do Citoesqueleto/genética , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Células Fotorreceptoras de Vertebrados/citologia , Sinapses/genética , Proteínas rab de Ligação ao GTP
16.
Cell Rep ; 24(2): 284-293.e6, 2018 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-29996090

RESUMO

In the presynaptic terminal, the magnitude and location of Ca2+ entry through voltage-gated Ca2+ channels (VGCCs) regulate the efficacy of neurotransmitter release. However, how presynaptic active zone proteins control mammalian VGCC levels and organization is unclear. To address this, we deleted the CAST/ELKS protein family at the calyx of Held, a CaV2.1 channel-exclusive presynaptic terminal. We found that loss of CAST/ELKS reduces the CaV2.1 current density with concomitant reductions in CaV2.1 channel numbers and clusters. Surprisingly, deletion of CAST/ELKS increases release probability while decreasing the readily releasable pool, with no change in active zone ultrastructure. In addition, Ca2+ channel coupling is unchanged, but spontaneous release rates are elevated. Thus, our data identify distinct roles for CAST/ELKS as positive regulators of CaV2.1 channel density and suggest that they regulate release probability through a post-priming step that controls synaptic vesicle fusogenicity.


Assuntos
Canais de Cálcio Tipo N/metabolismo , Proteínas de Transporte/metabolismo , Proteínas do Citoesqueleto/metabolismo , Ativação do Canal Iônico , Proteínas do Tecido Nervoso/metabolismo , Sinapses/metabolismo , Potenciais de Ação/fisiologia , Animais , Proteínas do Citoesqueleto/deficiência , Cinética , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/deficiência , Neurotransmissores/metabolismo , Probabilidade , Sinapses/ultraestrutura , Transmissão Sináptica/fisiologia , Proteínas rab de Ligação ao GTP
18.
Sci Rep ; 8(1): 727, 2018 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-29335431

RESUMO

The megakaryoblastic leukaemia (MKL) family are serum response factor (SRF) coactivators, which are highly expressed in the brain. Accordingly, MKL plays important roles in dendritic morphology, neuronal migration, and brain development. Further, nucleotide substitutions in the MKL1 and MKL2 genes are found in patients with schizophrenia and autism spectrum disorder, respectively. Thus, studies on the precise synaptic localisation and function of MKL in neurons are warranted. In this study, we generated and tested new antibodies that specifically recognise endogenously expressed MKL1 and MKL2 proteins in neurons. Using these reagents, we biochemically and immunocytochemically show that MKL1 and MKL2 are localised at synapses. Furthermore, shRNA experiments revealed that postsynaptic deletion of MKL1 or MKL2 reduced the percentage of mushroom- or stubby-type spines in cultured neurons. Taken together, our findings suggest that MKL1 and MKL2 are present at synapses and involved in dendritic spine maturation. This study may, at least in part, contribute to better understanding of the molecular mechanisms underlying MKL-mediated synaptic plasticity and neurological disorders.


Assuntos
Espinhas Dendríticas/metabolismo , Neurônios/química , Neurônios/citologia , Sinapses/química , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Imuno-Histoquímica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transativadores/genética , Fatores de Transcrição/genética
19.
Sci Rep ; 8(1): 819, 2018 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-29339765

RESUMO

Clinical diagnosis of progressive supranuclear palsy (PSP) is sometimes difficult because various phenotypes have been identified. Here, we report a mutation in the bassoon (BSN) gene in a family with PSP-like syndrome. Their clinical features resembled not only those of PSP patients but also those of individuals with multiple system atrophy and Alzheimer's disease. The neuropathological findings showed a novel three + four repeat tauopathy with pallido-luysio-nigral degeneration and hippocampal sclerosis. Whole-exome analysis of this family identified a novel missense mutation in BSN. Within the pedigree, the detected BSN mutation was found only in affected individuals. Further genetic analyses were conducted in probands from four other pedigrees with PSP-like syndrome and in 41 sporadic cases. Three missense mutations in BSN that are very rarely listed in databases of healthy subjects were found in four sporadic cases. Western blot analysis of tau following the overexpression of wild-type or mutated BSN revealed the possibility that wild-type BSN reduced tau accumulation, while mutated BSN lost this function. An association between BSN and neurological diseases has not been previously reported. Our results revealed that the neurodegenerative disorder associated with the original proband's pedigree is a novel tauopathy, differing from known dementia and parkinsonism syndromes, including PSP.


Assuntos
Saúde da Família , Mutação de Sentido Incorreto , Proteínas do Tecido Nervoso/genética , Paralisia Supranuclear Progressiva/genética , Paralisia Supranuclear Progressiva/patologia , Hipocampo/patologia , Humanos
20.
Neurosci Res ; 127: 25-32, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29275163

RESUMO

Our brain functions rely on sophisticated communication between synapses in the nervous system. Most synapses utilize a specialized submembranous structure, the so-called 'active zone', for the efficient transmission of chemical signals. The presynaptic active zone plays pivotal roles in the precise regulation of neurotransmitter release from the nerve terminals in a temporally and spatially coordinated manner. During the last two decades, several active zone-specific proteins have been isolated and characterized, including Bassoon, Piccolo/Aczonin, RIM, Munc13-1, ELKS, and CAST. The CAST/ELKS family is capable of potent direct interactions with other active zone proteins, forming a large protein complex that seems to be a molecular basis for electron density in the presynaptic active zone. The molecular details of the integrity of the active zone been well studied, however, we are just beginning to understanding its physiological significance in higher brain functions such as learning and memory, emotion, and consciousness. Focusing on the CAST/ELKS protein family, this review describes their biochemical properties, physiological functions in brain areas such as the hippocampus, and the significance of CAST phosphorylation in presynaptic short-term plasticity.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Plasticidade Neuronal/fisiologia , Terminações Pré-Sinápticas/metabolismo , Sinapses/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Humanos , Estrutura Molecular , Fosforilação/fisiologia , Transdução de Sinais/fisiologia
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