The Neuronal EGF-Related Gene Nell2 Interacts with Macf1 and Supports Survival of Retinal Ganglion Cells after Optic Nerve Injury

Abstract

Nell2 is a neuron-specific protein containing six epidermal growth factor-like domains. We have identified Nell2 as a retinal ganglion cell (RGC)-expressed gene by comparing mRNA profiles of control and RGC-deficient rat retinas. The aim of this study was to analyze Nell2 expression in wild-type and optic nerve axotomized retinas and evaluate its potential role in RGCs. Nell2-positive in situ and immunohistochemical signals were localized to irregularly shaped cells in the ganglion cell layer (GCL) and colocalized with retrogradely-labeled RGCs. No Nell2-positive cells were detected in 2 weeks optic nerve transected (ONT) retinas characterized with approximately 90% RGC loss. RT-PCR analysis showed a dramatic decrease in the Nell2 mRNA level after ONT compared to the controls. Immunoblot analysis of the Nell2 expression in the retina revealed the presence of two proteins with approximate MW of 140 and 90 kDa representing glycosylated and non-glycosylated Nell2, respectively. Both products were almost undetectable in retinal protein extracts two weeks after ONT. Proteome analysis of Nell2-interacting proteins carried out with MALDI-TOF MS (MS) identified microtubule-actin crosslinking factor 1 (Macf1), known to be critical in CNS development. Strong Macf1 expression was observed in the inner plexiform layer and GCL where it was colocalizied with Thy-1 staining. Since Nell2 has been reported to increase neuronal survival of the hippocampus and cerebral cortex, we evaluated the effect of Nell2 overexpression on RGC survival. RGCs in the nasal retina were consistently more efficiently transfected than in other areas (49% vs. 13%; n = 5, p<0.05). In non-transfected or pEGFP-transfected ONT retinas, the loss of RGCs was approximately 90% compared to the untreated control. In the nasal region, Nell2 transfection led to the preservation of approximately 58% more cells damaged by axotomy compared to non-transfected (n = 5, p<0.01) or pEGFP-transfected controls (n = 5, p<0.01).

The Molecular Mechanism of NELL2 Movement and Secretion in Hippocampal Progenitor HiB5 Cells

“…NELL2 associates with microtubule-actin crosslinking factor 1 (Macf1) in retinal ganglion cells (Munemasa et al, 2012). Based on these previous reports and our fluorescent imaging of NELL2-EGFP trafficking (Fig.…”

“…NELL2 acts on neuronal cells to promote differentiation, proliferation (Kim et al, 2010;Nelson et al, 2004), and survival through mitogen-activated protein kinases (Aihara et al, 2003). NELL2 also supports the survival of retinal ganglion cells after optic nerve injury (Munemasa et al, 2012), and NELL2 is involved in the formation of the sexually dimorphic nucleus of the preoptic area (Jeong et al, 2008). NELL2-deficient mice may have hippocampal neurons with altered differ- rentiation and/or signal transduction properties, which ultimately lead to long-term potentiation enhancement and spatial memory impairment (Matsuyama et al, 2004;2005).…”

“…In addition, a recent study showed that NELL2 interacts with Macf1 in retinal ganglion cells (Munemasa et al, 2012). Because Macf1 may be involved in the translocation of proteins along axons, and its associated complex (composed of Apc, β-catenin, and Gsk3β) moves from the cytoplasm to the cell membrane (Munemasa et al, 2012), NELL2 transport in HiB5 cells may also be regulated by these proteins. Further investigations are needed to resolve the detailed mechanisms of NELL2 transport.…”

Effects of NELL2 on the regulation of GnRH expression and puberty in female rats

“…The highest expression levels are observed in the hippocampus and cerebral cortex, followed by the olfactory bulb and hypothalamus, and finally by the thalamus, cerebellum, and medulla oblongata. Many studies have shown that NELL2 plays an important role in nerve growth, neural differentiation, neural plasticity, synaptic transport, and vesicle release (Nelson et al, 2004;Choi et al, 2010;Nakamura et al, 2012;Munemasa et al, 2012). Ha et al (2008) found that NELL2 mRNA was highly expressed in the medial basal hypothalamus in female rats during the pubescent growth period.…”

NELL2 Function in the Protection of Cells against Endoplasmic Reticulum Stress

“…Characteristically, NELL2 has Ca 2+ -binding sites in its six EGF-like repeat domains, suggesting a contribution to Ca tion, and protection of neural cells (Choi et al, 2010;Jeong et al, 2008;Nelson et al, 2002). Among its possible functions, a cell survival-promoting effect has been relatively well studied (Aihara et al, 2003;Choi et al, 2010;Jeong et al, 2008;Munemasa et al, 2012) and is mediated by an intracellular mitogen-activated protein kinase (MAPK) pathway (Aihara et al, 2003;Choi et al, 2010).…”