Serine 1283 in extracellular matrix glycoprotein Reelin is crucial for Reelin′s function in brain development

Deficiency in the extracellular matrix glycoprotein Reelin severely affects migration of neurons during development. The function of serine at position 1283 in Reelin has remained uncertain. To explore its relevance we generated rlnA/A mice that carry alanine instead of serine at position 1283, thereby disrupting the putative casein kinase 2 (CK2) phosphorylation site S1283DGD. Mutated mice displayed reeler-like locomotor behavior, abnormal brain anatomy and decrease of Reelin RNA and protein levels during development and in adulthood. Since serine 1283 was previously proposed to mediate proteolysis of adhesion molecules, we investigated proteolysis of cell adhesion molecule L1 and found it normal in rlnA/A mice. Neuronal migration in the embryonic rlnA/A cerebral cortex was impaired, but rescued by in utero electroporation of the Reelin fragment N-R6 containing the putative CK2 phosphorylation site. In rlnA/A mice migration of cerebellar granule cells in vitro was promoted by application of wild-type but not by mutated Reelin. In cerebellar neuron cultures, Reelin expression was decreased upon inhibition of ecto-phosphorylation by CK2. Biochemically purified wild-type, but not mutated Reelin was found phosphorylated. Altogether, the results indicate that ecto-phosphorylation at serine 1283 rather than proteolytic processing of adhesion molecules by Reelin plays an important role in Reelin functions.

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Tissue specificity and regulation of the N-terminal diversity of reticulon 3

Biochemical Journal ◽

10.1042/bj20040458 ◽

2004 ◽

Vol 385 (1) ◽

pp. 125-134 ◽

Cited By ~ 24

Author(s):

Franck DI SCALA ◽

Luc DUPUIS ◽

Christian GAIDDON ◽

Marc DE TAPIA ◽

Natasa JOKIC ◽

...

Keyword(s):

Granule Cells ◽

Cerebellar Granule Cells ◽

Granule Neurons ◽

Cerebellar Granule ◽

Preferential Expression ◽

Protein Levels ◽

The Central Nervous System ◽

Cerebellum Development ◽

Promoter Usage

Over the last few years, the widely distributed family of reticulons (RTNs) is receiving renewed interest because of the implication of RTN4/Nogo in neurite regeneration. Four genes were identified in mammals and are referred to as RTN1, 2, 3 and the neurite outgrowth inhibitor RTN4/Nogo. In the present paper, we describe the existence of five new isoforms of RTN3 that differ in their N-termini, and analysed their tissue distribution and expression in neurons. We redefined the structure of human and murine rtn3 genes, and identified two supplementary exons that may generate up to seven putative isoforms arising by alternative splicing or differential promoter usage. We confirmed the presence of five of these isoforms at the mRNA and protein levels, and showed their preferential expression in the central nervous system. We analysed rtn3 expression in the cerebellum further, and observed increased levels of several of the RTN3 isoforms during cerebellum development and during in vitro maturation of cerebellar granule cells. This pattern of expression paralleled that shown by RTN4/Nogo isoforms. Specifically, RTN3A1 expression was down-regulated upon cell death of cerebellar granule neurons triggered by potassium deprivation. Altogether, our results demonstrate that the rtn3 gene generates multiple isoforms varying in their N-termini, and that their expression is tightly regulated in neurons. These findings suggest that RTN3 isoforms may contribute, by as yet unknown mechanisms, to neuronal survival and plasticity.

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The Expression of the Cancer-Associated lncRNA Snhg15 Is Modulated by EphrinA5-Induced Signaling

International Journal of Molecular Sciences ◽

10.3390/ijms22031332 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1332

Author(s):

Daniel Pensold ◽

Julia Gehrmann ◽

Georg Pitschelatow ◽

Asa Walberg ◽

Kai Braunsteffer ◽

...

Keyword(s):

Tyrosine Kinases ◽

Intracellular Signaling ◽

Granule Cells ◽

Cerebellar Granule Cells ◽

Membrane Receptors ◽

In Vitro Assays ◽

Eph Receptor ◽

Medulloblastoma Cell Line ◽

And Migration

The Eph receptor tyrosine kinases and their respective ephrin-ligands are an important family of membrane receptors, being involved in developmental processes such as proliferation, migration, and in the formation of brain cancer such as glioma. Intracellular signaling pathways, which are activated by Eph receptor signaling, are well characterized. In contrast, it is unknown so far whether ephrins modulate the expression of lncRNAs, which would enable the transduction of environmental stimuli into our genome through a great gene regulatory spectrum. Applying a combination of functional in vitro assays, RNA sequencing, and qPCR analysis, we found that the proliferation and migration promoting stimulation of mouse cerebellar granule cells (CB) with ephrinA5 diminishes the expression of the cancer-related lncRNA Snhg15. In a human medulloblastoma cell line (DAOY) ephrinA5 stimulation similarly reduced SNHG15 expression. Computational analysis identified triple-helix-mediated DNA-binding sites of Snhg15 in promoters of genes found up-regulated upon ephrinA5 stimulation and known to be involved in tumorigenic processes. Our findings propose a crucial role of Snhg15 downstream of ephrinA5-induced signaling in regulating gene transcription in the nucleus. These findings could be potentially relevant for the regulation of tumorigenic processes in the context of glioma.

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Deferoxamine antioxidant activity on cerebellar granule cells γ-irradiated in vitro

Neurotoxicology and Teratology ◽

10.1016/j.ntt.2004.02.001 ◽

2004 ◽

Vol 26 (3) ◽

pp. 477-483 ◽

Cited By ~ 21

Author(s):

L Guelman

Keyword(s):

Antioxidant Activity ◽

Granule Cells ◽

Cerebellar Granule Cells ◽

Cerebellar Granule

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Selective Modulation of Tonic and Phasic Inhibitions in Dentate Gyrus Granule Cells

Journal of Neurophysiology ◽

10.1152/jn.2002.87.5.2624 ◽

2002 ◽

Vol 87 (5) ◽

pp. 2624-2628 ◽

Cited By ~ 325

Author(s):

Zoltan Nusser ◽

Istvan Mody

Keyword(s):

Dentate Gyrus ◽

Granule Cells ◽

Cerebellar Granule Cells ◽

Hippocampal Slices ◽

Tonic Inhibition ◽

Adult Rats ◽

Adult Rat ◽

Phasic Inhibition ◽

The Mean

In some nerve cells, activation of GABAA receptors by GABA results in phasic and tonic conductances. Transient activation of synaptic receptors generates phasic inhibition, whereas tonic inhibition originates from GABA acting on extrasynaptic receptors, like in cerebellar granule cells, where it is thought to result from the activation of extrasynaptic GABAA receptors with a specific subunit composition (α6βxδ). Here we show that in adult rat hippocampal slices, extracellular GABA levels are sufficiently high to generate a powerful tonic inhibition in δ subunit–expressing dentate gyrus granule cells. In these cells, the mean tonic current is approximately four times larger than that produced by spontaneous synaptic currents occurring at a frequency of ∼10 Hz. Antagonizing the GABA transporter GAT-1 with NO-711 (2.5 μM) selectively enhanced tonic inhibition by 330% without affecting the phasic component. In contrast, by prolonging the decay of inhibitory postsynaptic currents (IPSCs), the benzodiazepine agonist zolpidem (0.5 μM) augmented phasic inhibition by 66%, while leaving the mean tonic conductance unchanged. These results demonstrate that a tonic GABAA receptor–mediated conductance can be recorded from dentate gyrus granule cells of adult rats in in vitro slice preparations. Furthermore, we have identified distinct pharmacological tools to selectively modify tonic and phasic inhibitions, allowing future studies to investigate their specific roles in neuronal function.

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Proteolytic processing of Semliki Forest virus-specific non-structural polyprotein by nsP2 protease

Journal of General Virology ◽

10.1099/0022-1317-82-4-765 ◽

2001 ◽

Vol 82 (4) ◽

pp. 765-773 ◽

Cited By ~ 54

Author(s):

Andres Merits ◽

Lidia Vasiljeva ◽

Tero Ahola ◽

Leevi Kääriäinen ◽

Petri Auvinen

Keyword(s):

Mammalian Cells ◽

Active Sites ◽

Insect Cells ◽

Semliki Forest Virus ◽

Point Mutations ◽

Proteolytic Processing ◽

Wild Type ◽

In Trans ◽

Polyprotein Precursor

The RNA replicase proteins of Semliki Forest virus (SFV) are translated as a P1234 polyprotein precursor that contains two putative autoproteases. Point mutations introduced into the predicted active sites of both proteases nsP2 (P2) and nsP4 (P4), separately or in combination, completely abolished virus replication in mammalian cells. The effects of these mutations on polyprotein processing were studied by in vitro translation and by expression of wild-type polyproteins P1234, P123, P23, P34 and their mutated counterparts in insect cells using recombinant baculoviruses. A mutation in the catalytic site of the P2 protease, C478A, (P2CA) completely abolished the processing of P12CA34, P12CA3 and P2CA3. Co-expression of P23 and P12CA34 in insect cells resulted in in trans cleavages at the P2/3 and P3/4 sites. Co-expression of P23 and P34 resulted in cleavage at the P3/4 site. In contrast, a construct with a mutation in the active site of the putative P4 protease, D6A, (P1234DA) was processed like the wild-type protein. P34 or its truncated forms were not processed when expressed alone. In insect cells, P4 was rapidly destroyed unless an inhibitor of proteosomal degradation was used. It is concluded that P2 is the only protease needed for the processing of SFV polyprotein P1234. Analysis of the cleavage products revealed that P23 or P2 could not cleave the P1/2 site in trans.

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Testase 1 (ADAM 24) a plasma membrane-anchored sperm protease implicated in sperm function during epididymal maturation or fertilization

Journal of Cell Science ◽

10.1242/jcs.114.9.1787 ◽

2001 ◽

Vol 114 (9) ◽

pp. 1787-1794 ◽

Cited By ~ 1

Author(s):

G.Z. Zhu ◽

D.G. Myles ◽

P. Primakoff

Keyword(s):

Plasma Membrane ◽

Protein Kinase C ◽

Protein Kinase ◽

Acrosome Reaction ◽

Phosphorylation Site ◽

Equatorial Region ◽

Proteolytic Processing ◽

Mature Sperm ◽

Kinase C

Plasma membrane-anchored proteases have key roles in cell signaling, migration and refashioning the cell surface and its surroundings. We report the first example of a plasma membrane-anchored protease on mature sperm, testase 1 (ADAM 24). Unlike other studied sperm ADAMs (fertilin (α) and (β), cyritestin) whose metalloprotease domains are removed during sperm development, we found testase 1 retains an active metalloprotease domain, suggesting it acts as a protease on mature sperm. Testase 1 is a glycoprotein (molecular mass 88 kDa), localized to the equatorial region of the plasma membrane of cauda epididymal sperm. Typically, proteolytic removal of the pro-domain is an initial activation step for ADAM proteases. The pro-domain of the testase 1 precursor (108 kDa) is proteolytically removed as sperm transit the caput epididymis to produce processed (mature) testase 1 (88 kDa). Testase 1 is unique among all studied ADAMs in that its proteolytic processing occurs on the sperm plasma membrane instead of at an intracellular site (the Golgi). Using GST-fusion proteins and a synthetic testase 1 C-terminal peptide, we found that the cytoplasmic tail of testase 1 could be phosphorylated in vitro by protein kinase C (PKC). Thus testase 1 apparently has a cytoplasmic PKC phosphorylation site(s). Protein kinase C is known to stimulate other ADAMs' protease activity. Because events of the acrosome reaction include PKC activation, we speculate that testase 1 protease function could be important in sperm penetration of the zona pellucida after sperm PKC is activated during the acrosome reaction.

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Tetrabromobisphenol A-induced depolarization of rat cerebellar granule cells: ex vivo and in vitro studies

Chemosphere ◽

10.1016/j.chemosphere.2019.02.032 ◽

2019 ◽

Vol 223 ◽

pp. 64-73 ◽

Cited By ~ 4

Author(s):

Dominik Diamandakis ◽

Elzbieta Zieminska ◽

Marcin Siwiec ◽

Krzysztof Tokarski ◽

Elzbieta Salinska ◽

...

Keyword(s):

Granule Cells ◽

Cerebellar Granule Cells ◽

Ex Vivo ◽

In Vitro Studies ◽

Tetrabromobisphenol A ◽

Cerebellar Granule

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Methylation determines the extracellular calcium sensitivity of the leak channel NALCN in hippocampal dentate granule cells

Experimental & Molecular Medicine ◽

10.1038/s12276-019-0325-0 ◽

2019 ◽

Vol 51 (10) ◽

pp. 1-14 ◽

Cited By ~ 1

Author(s):

Seul-Yi Lee ◽

Tuan Anh Vuong ◽

Xianlan Wen ◽

Hyeon-Ju Jeong ◽

Hyun-Kyung So ◽

...

Keyword(s):

Neuronal Excitability ◽

Granule Cells ◽

Phosphorylation Site ◽

Calcium Sensitivity ◽

Arginine Methylation ◽

Resting Membrane Potential ◽

Leak Channel ◽

Dentate Granule Cells ◽

Severe Intellectual Disability

Abstract The sodium leak channel NALCN is a key player in establishing the resting membrane potential (RMP) in neurons and transduces changes in extracellular Ca2+ concentration ([Ca2+]e) into increased neuronal excitability as the downstream effector of calcium-sensing receptor (CaSR). Gain-of-function mutations in the human NALCN gene cause encephalopathy and severe intellectual disability. Thus, understanding the regulatory mechanisms of NALCN is important for both basic and translational research. This study reveals a novel mechanism for NALCN regulation by arginine methylation. Hippocampal dentate granule cells in protein arginine methyltransferase 7 (PRMT7)-deficient mice display a depolarization of the RMP, decreased threshold currents, and increased excitability compared to wild-type neurons. Electrophysiological studies combined with molecular analysis indicate that enhanced NALCN activities contribute to hyperexcitability in PRMT7−/− neurons. PRMT7 depletion in HEK293T cells increases NALCN activity by shifting the dose-response curve of NALCN inhibition by [Ca2+]e without affecting NALCN protein levels. In vitro methylation studies show that PRMT7 methylates a highly conserved Arg1653 of the NALCN gene located in the carboxy-terminal region that is implicated in CaSR-mediated regulation. A kinase-specific phosphorylation site prediction program shows that the adjacent Ser1652 is a potential phosphorylation site. Consistently, our data from site-specific mutants and PKC inhibitors suggest that Arg1653 methylation might modulate Ser1652 phosphorylation mediated by CaSR/PKC-delta, leading to [Ca2+]e-mediated NALCN suppression. Collectively, these data suggest that PRMT7 deficiency decreases NALCN methylation at Arg1653, which, in turn, decreases CaSR/PKC-mediated Ser1652 phosphorylation, lifting NALCN inhibition, thereby enhancing neuronal excitability. Thus, PRMT7-mediated NALCN inhibition provides a potential target for the development of therapeutic tools for neurological diseases.

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