Darya Kiryushko

Regulators of Neurite Outgrowth: Role of Cell Adhesion Molecules

Abstract: Neuronal differentiation is a fundamental event in the development of the nervous system as well as in the regeneration of damaged nervous tissue. The initiation and guidance of a neurite are accomplished by positive (permissive or attractive), negative (inhibitory or repulsive), or guiding (affecting the advance of the growth cone) signals from the extracellular space. The signals may arise from either the extracellular matrix (ECM) or the surface of other cells, or be diffusible secreted factors. Based on this classification, we briefly describe selected positive, negative, and guiding signaling cues focusing on the role of cell adhesion molecules (CAMs). CAMs not only regulate cell‐cell and cell‐ECM adhesion “mechanically,” they also trigger intracellular signaling cascades launching neurite outgrowth. Here, we describe the structure, function, and signaling of three key CAMs found in the nervous system: N‐cadherin and two Ig‐CAMs, L1 and the neural cell adhesion molecule NCAM.

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.

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.

Epidermal growth factor receptor ligands as new extracellular targets for the metastasis-promoting S100A4 protein.

The function of S100A4, a member of the calcium‐binding S100 protein family, has been associated with tumor invasion and metastasis. Although an essential pro‐metastatic role of extracellular S100A4 in tumor progression has been demonstrated, the identification of the precise underlying mechanisms and protein partners (receptors) has remained elusive. To identify putative targets for extracellular S100A4, we screened a phage display peptide library using S100A4 as bait. We identified three independent peptide motifs with varying affinities for the S100A4 protein. Sequence analyses indicated that the most abundant peptide mimicked the F/YCC motif present in the epidermal growth factor domain of ErbB receptor ligands. S100A4 selectively interacted with a number of epidermal growth factor receptor (EGFR) ligands, demonstrating highest affinity for amphiregulin. Importantly, we found that S100A4 stimulated EGFR/ErbB2 receptor signaling and enhanced the amphiregulin‐mediated proliferation of mouse embryonic fibroblasts. S100A4‐neutralizing antibodies, as well as EGFR‐ and ErbB2 receptor‐specific tyrosine kinase inhibitors, blocked these effects. The present results suggest that extracellular S100A4 regulates tumor progression by interacting with EGFR ligands, thereby enhancing EGFR/ErbB2 receptor signaling and cell proliferation.

Neuroprotective properties of a novel, non-haematopoietic agonist of the erythropoietin receptor

Erythropoietin, a member of the type 1 cytokine superfamily, controls proliferation and differentiation of erythroid progenitor cells through binding to and dimerization of the erythropoietin receptor. Both erythropoietin and its receptor are also expressed in the central nervous system, where they are involved in tissue protection. However, the use of erythropoietin as a neuroprotective agent may be hampered by its erythropoietic activity. Therefore, developing non-haematopoietic erythropoietin mimetics is important. Based on the crystal structure of the complex of erythropoietin and its receptor, we designed a peptide, termed Epotris, corresponding to the C α-helix region (amino-acid residues 92-111) of human erythropoietin. The peptide specifically bound to the erythropoietin receptor and promoted neurite outgrowth and survival of primary neurons with the same efficiency as erythropoietin, but with 10(3)-fold lower potency. Knockdown of the erythropoietin receptor or interference with its downstream signalling inhibited the Epotris-induced neuritogenic and pro-survival effect. Similarly to erythropoietin, Epotris penetrated the blood-brain barrier. Moreover, treatment with the peptide attenuated seizures, decreased mortality and reduced neurodegeneration in an in vivo model of kainic acid-induced neurotoxicity. In contrast to erythropoietin, Epotris did not stimulate erythropoiesis upon chronic administration. Thus, Epotris is a novel neuroprotective non-haematopoietic erythropoietin mimetic that may offer new opportunities for the treatment of neurological disorders.

Neuroplastin-55 binds to and signals through the fibroblast growth factor receptor

Neuroplastin (Np) is a glycoprotein belonging to the immunoglobulin superfamily of cell adhesion molecules (CAMs) and existing in two isoforms, Np55 and Np65, named according to their molecular weights. The extracellular part of Np65 contains three immunoglobulin (Ig)‐like modules (Igl, Ig2, and Ig3), whereas Np55 lacks the Igl module. Of these two isoforms, only Np65 is involved in homophilic interactions resulting in cell adhesion, whereas the role of Np55 is poorlyunderstood. The present studyreports for the first time the crystal structure of the ectodomain of Np55 at 1.95‐Å resolution and demonstrates that Np55 binds to and activates the fibroblast growth factor receptor 1 (FGFR1). Furthermore, we identify a sequence motif in the Ig2 module of Np55 interacting with FGFR1 and show that a synthetic peptide encompassing this motif, termed narpin, binds to and activates FGFR1. We show that both Np55 and the narpin peptide induce neurite outgrowth through FGFR1 activation and that Np55 increases synaptic calcium concentration in an FGFR1‐dependent manner. Moreover, we demonstrate that narpin has an antidepressive‐like effect in rats subjected to the forced swim test, suggesting that Np55‐induced signaling may be involved in synaptic plasticity in vivo. Owczarek, S., Kiryushko, D., Larsen, M. H., Kastrup, J. S., Gajhede, M., Sandi, C., Berezin, V., Bock, E., Soroka, V. Neuroplastin‐55 binds to and signals through the fibroblast growth factor receptor. FASEB J. 24, 1139–1150 (2010). www.fasebj.org

The metastasis-promoting S100A4 protein confers neuroprotection in brain injury

Identification of novel pro-survival factors in the brain is paramount for developing neuroprotective therapies. The multifunctional S100 family proteins have important roles in many human diseases and are also upregulated by brain injury. However, S100 functions in the nervous system remain unclear. Here we show that the S100A4 protein, mostly studied in cancer, is overexpressed in the damaged human and rodent brain and released from stressed astrocytes. Genetic deletion of S100A4 exacerbates neuronal loss after brain trauma or excitotoxicity, increasing oxidative cell damage and downregulating the neuroprotective protein metallothionein I+II. We identify two neurotrophic motifs in S100A4 and show that these motifs are neuroprotective in animal models of brain trauma. Finally, we find that S100A4 rescues neurons via the Janus kinase/STAT pathway and, partially, the interleukin-10 receptor. Our data introduce S100A4 as a therapeutic target in neurodegeneration, and raise the entire S100 family as a potentially important factor in central nervous system injury.

Neuroplastin‐65 and a mimetic peptide derived from its homophilic binding site modulate neuritogenesis and neuronal plasticity

J. Neurochem. (2011) 117, 984–994.

A new agonist of the erythropoietin receptor, Epobis, induces neurite outgrowth and promotes neuronal survival

J. Neurochem. (2012) 121, 915–923.

A metallothionein mimetic peptide protects neurons against kainic acid-induced excitotoxicity

Metallothioneins I and II (MTI/II) are metal‐binding proteins overexpressed in response to brain injury. Recently, we have designed a peptide, termed EmtinB, which is modeled after the β‐domain of MT‐II and mimics the biological effects of MTI/II in vitro. Here, we demonstrate the neuroprotective effect of EmtinB in the in vitro and in vivo models of kainic acid (KA)‐induced neurotoxicity. We show that EmtinB passes the blood–brain barrier and is detectable in plasma for up to 24 hr. Treatment with EmtinB significantly attenuates seizures in C57BL/6J mice exposed to moderate (20 mg/kg) and high (30 mg/kg) KA doses and tends to decrease mortality induced by the high KA dose. Histopathological evaluation of hippocampal (CA3 and CA1) and cortical areas of mice treated with 20 mg/kg KA shows that EmtinB treatment reduces KA‐induced neurodegeneration in the CA1 region. These findings establish EmtinB as a promising target for therapeutic development.