Vera Novitskaya

Molecular mechanisms of Ca2+ signaling in neurons induced by the S100A4 protein

ABSTRACT The S100A4 protein belongs to the S100 family of vertebrate-specific proteins possessing both intra- and extracellular functions. In the nervous system, high levels of S100A4 expression are observed at sites of neurogenesis and lesions, suggesting a role of the protein in neuronal plasticity. Extracellular oligomeric S100A4 is a potent promoter of neurite outgrowth and survival from cultured primary neurons; however, the molecular mechanism of this effect has not been established. Here we demonstrate that oligomeric S100A4 increases the intracellular calcium concentration in primary neurons. We present evidence that both S100A4-induced Ca2+ signaling and neurite extension require activation of a cascade including a heterotrimeric G protein(s), phosphoinositide-specific phospholipase C, and diacylglycerol-lipase, resulting in Ca2+ entry via nonselective cation channels and via T- and L-type voltage-gated Ca2+ channels. We demonstrate that S100A4-induced neurite outgrowth is not mediated by the receptor for advanced glycation end products, a known target for other extracellular S100 proteins. However, S100A4-induced signaling depends on interactions with heparan sulfate proteoglycans at the cell surface. Thus, glycosaminoglycans may act as coreceptors of S100 proteins in neurons. This may provide a mechanism by which S100 proteins could locally regulate neuronal plasticity in connection with brain lesions and neurological disorders.

The transcription factors CREB and c-Fos play key roles in NCAM-mediated neuritogenesis in PC12-E2 cells.

The neural cell adhesion molecule (NCAM) stimulates axonal outgrowth by activation of the Ras‐mitogen activated protein kinase (MAPK) pathway and by generation of arachidonic acid. We investigated whether the transcription factors, cyclic‐AMP response‐element binding protein (CREB) and c‐Fos play roles in this process by estimating NCAM‐dependent neurite outgrowth from PC12‐E2 cells grown in co‐culture with NCAM‐negative or NCAM‐positive fibroblasts. PC12‐E2 cells were transiently transfected with expression plasmids encoding wild‐type or dominant negative forms of CREB and c‐Fos or an activated form of the MAPK kinase, MEK2. Alternatively, PC12‐E2 cells were treated with arachidonic acid, the cAMP analogue dBcAMP, or protein kinase A (PKA) inhibitors. The negative forms of CREB and c‐Fos inhibited neurite outgrowth mediated by NCAM, arachidonic acid, dBcAMP, or MEK2. Neither CREB nor c‐Fos could compensate for the inactivation of the other, indicating that both factors are important in NCAM‐mediated neuritogenesis. Treatment of primary hippocampal neurons with a synthetic NCAM peptide ligand known to stimulate neurite outgrowth induced phosphorylation of CREB and expression of c‐fos. We thus present evidence that NCAM‐mediated neurite outgrowth involves a series of signal transduction pathways, including the cAMP/PKA pathway, targeting c‐Fos and CREB.

GAP‐43 regulates NCAM‐180‐mediated neurite outgrowth

The neural cell adhesion molecule (NCAM), and the growth‐associated protein (GAP‐43), play pivotal roles in neuronal development and plasticity and possess interdependent functions. However, the mechanisms underlying the functional association of GAP‐43 and NCAM have not been elucidated. In this study we show that (over)expression of GAP‐43 in PC12E2 cells and hippocampal neurons strongly potentiates neurite extension, both in the absence and in the presence of homophilic NCAM binding. This potentiation is crucially dependent on the membrane association of GAP‐43. We demonstrate that phosphorylation of GAP‐43 by protein kinase C (PKC) as well as by casein kinase II (CKII) is important for the NCAM‐induced neurite outgrowth. Moreover, our results indicate that in the presence of GAP‐43, NCAM‐induced neurite outgrowth requires functional association of NCAM‐180/spectrin/GAP‐43, whereas in the absence of GAP‐43, the NCAM‐140/non‐receptor tyrosine kinase (Fyn)‐associated signaling pathway is pivotal. Thus, expression of GAP‐43 presumably acts as a functional switch for NCAM‐180‐induced signaling. This suggests that under physiological conditions, spatial and/or temporal changes of the localization of GAP‐43 and NCAM on the cell membrane may determine the predominant signaling mechanism triggered by homophilic NCAM binding: NCAM‐180/spectrin‐mediated modulation of the actin cytoskeleton, NCAM‐140‐mediated activation of Fyn, or both.

S100A12 protein is a strong inducer of neurite outgrowth from primary hippocampal neurons

Several members of the S100 family of Ca2+ binding proteins are at present known to be secreted and to have extracellular activities. We have investigated the neurite inducing potential of extracellularly added S100A12. Human recombinant S100A12 was found to dramatically induce neuritogenesis of hippocampal cells isolated from 17 to 19 days old rat embryos. The response to S100A12 was dependent on the dose in a bell‐shaped manner. A 10‐fold increase in neurite outgrowth was observed upon treatment with S100A12 in concentrations between 0.1 and 2.0 µm already after 24 h. Exposure to S100A12 for only 15 min was enough to induce neuritogenesis when measured after 24 h, but to obtain a maximal response, S100A12 had to be present in the culture for at least 4 h. The response to S100A12 was abolished by inhibitors of phospholipase C (PLC), protein kinase C (PKC), Ca2+ flux, Ca2+/calmodulin dependent kinase II (CaMKII) or mitogen‐activated protein kinase kinase (MEK). Therefore, we suggest that extracellular S100A12 triggers intracellular signal transduction in neurons, involving the classical mitogen‐activated protein (MAP) kinase pathway and a phospholipase C‐generated second messenger pathway leading to an increase in intracellular Ca2+ and activation of PKC, ultimately resulting in neuronal differentiation.

The Mts1/S100A4 protein is a neuroprotectant

Mts1 (S100A4) is a calcium‐binding protein of the EF‐hand type, belonging to the S100 family of proteins. The mts1/S100A4 gene was originally isolated from tumor cell lines, and the protein is believed to play an important role in tumor progression. More recently, oligomeric, but not dimeric, forms of Mts1 have been shown to have a neuritogenic effect when added extracellularly to hippocampal neurons. Here we show increased neurite outgrowth in two other cell types, dopaminergic and cerebellar neurons, in response to treatment with Mts1 oligomers. Moreover, we demonstrate that Mts1 acts as a neuroprotectant in primary cerebellar, dopaminergic, and hippocampal neurons induced to undergo cell death. Interestingly, the survival of the cerebellar and hippocampal neurons increased as a result of treatment with Mts1 not only in oligomeric form but also—although to a lesser extent—in dimeric form. The inhibition of death in cerebellar neurons by Mts1 was accompanied by an inhibition of DNA fragmentation, but Mts1 did not affect the activity of caspases‐3 and ‐6. In hippocampal neurons, cell death induced by the amyloid‐β peptide (Aβ25–35) was characterized by an increase in caspase‐3 and ‐6 activity, but no DNA fragmentation was observed. As in cerebellar neurons, the induced increase in caspase activity in hippocampal neurons was not affected by Mts1.

NEURONAL PROTEIN GAP-43 IS A MEMBER OF NOVEL GROUP OF BRAIN ACID-SOLUBLE PROTEINS (BASPS)

A group of brain acid-soluble proteins (BASPs) is preliminarily characterized. In some respects BASPs are similar to high mobility group (HMG) proteins, but in contrast to HMG, all BASPs are very acidic (pI 4.4-4.6) and show abnormal mobility during SDS-PAGE electrophoresis. BASP2-1 and BASP2-2 are identified as the two forms of neuronal protein GAP-43 (B-50, pp46, F1, neuromodulin). BASP1 and BASP3 are apparently novel brain proteins.

Natural N-terminal fragments of brain abundant myristoylated protein BASP1

BASP1 (also known as CAP-23 and NAP-22) is a novel myristoylated calmodulin-binding protein, abundant in nerve terminals. It is considered as a signal protein participating in neurite outgrowth and synaptic plasticity. BASP1 is also present in significant amounts in kidney, testis, and lymphoid tissues. In this study, we show that BASP1 is accompanied by at least six BASP1 immunologically related proteins (BIRPs), which are present in all animal species studied (rat, bovine, human, chicken). BIRPs have lower molecular masses than that of BASP1. Similarly to BASP1, they are myristoylated. Peptide mapping and partial sequencing have shown that BIRPs represent a set of BASP1 N-terminal fragments devoid of C-terminal parts of different length. In a definite species, the same set of BASP1 fragments is present in both brain and other tissues. The sum amount of the fragments is about 50% of the BASP1 amount in a tissue. Obligatory accompanying of BASP1 by a set of specific fragments indicates that these fragments are of physiological significance.

Neural cell adhesion molecule-mediated neurite outgrowth is repressed by overexpression of HES-1.

The neural cell adhesion molecule (NCAM) stimulates neurite outgrowth by activating intracellular signaling cascades. We investigated the role of the transcriptional repressor HES‐1 in NCAM‐dependent neurite outgrowth by estimating neurite extension from PC12‐E2 cells grown in coculture with NCAM‐negative or NCAM‐positive fibroblasts. PC12‐E2 cells were transiently transfected with an expression plasmid encoding HES‐1. We found that expression of HES‐1 inhibited NCAM‐dependent neurite outgrowth. Treatment with arachidonic acid (an important messenger in NCAM‐dependent signaling) restored NCAM‐induced neurite outgrowth inhibited by HES‐1. These results suggest that HES‐1 is a regulator of intracellular signal transduction stimulated by cell adhesion molecules involved in neurite outgrowth.