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

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)

Visual Function and Survival of Injured Retinal Ganglion Cells in Aged Rbfox1 Knockout Animals.

Rbfox1 is a multifunctional RNA binding protein that regulates various aspects of RNA metabolism important for neuronal differentiation and normal physiology. Rbfox1 has been associated with neurodevelopmental and neurological conditions as well as age-related neurodegenerative diseases such as Alzheimer's and Parkinson's. We have shown that in mammalian retinas Rbfox1 is expressed in retinal ganglion cells (RGCs) and in amacrine cells (ACs). This study investigates the effect of advanced age (22-month-old mice) on visual function, retinal morphology and survival of injured retinal ganglion cells (RGC) in Rbfox1 knockout (KO) animals. A visual cliff test, which was used to evaluate visual function, showed that 22-month old Rbfox1 KO mice have profound depth perception deficiency. Retinal gross morphology in these animals appeared to be normal. Optic nerve crush (ONC) induced axonal injury resulted in approximately 50% of RGC loss in both Rbfox1 KO and age-matched control animals: the average RGC densities in uninjured control and Rbfox1 KO animals were 6274 ± 1673 cells/mm2 and 6004 ± 1531 cells/mm2, respectively, whereas 1 week after ONC, RGC numbers in the retinas of control and Rbfox1 KO mice were reduced to 2998 ± 858 cells/mm2 and 3036 ± 857 cells/mm2, respectively (Rbfox1 KO vs. Rbfox1 KO + ONC, p &lt; 0.0001 and control vs. control + ONC, p &lt; 0.0001). No significant difference between RGC numbers in Rbfox1 KO + ONC and age-matched control + ONC animals was observed, suggesting that Rbfox1 has no effect on the survival of injured RGCs. Interestingly, however, contrary to a commonly accepted view that the number of RGCs in old (18 month of age) compared to young animals is reduced by approximately 40%, the RGC densities in 22-month-old mice in this study were similar to those of 4-month-old counterparts

Quantitative Analysis of Retinal Ganglion Cell Survival with Rbpms Immunolabeling in Animal Models of Optic Neuropathies

The authors demonstrated that Rbpms can reliably be used as an RGC marker for quantitative evaluation in rat models of excitotoxicity, optic nerve crush, and experimental glaucoma

A reverse immunoprecipitation with a MACF1 antibody followed by immunoblot analysis with Nell2 antibodies and spatial expression of Macf1 in the retina.

<p>A. Two products with MW of approximately 90 and 140 kDa were detected by IB with Nell2 antibody following IP with anti Macf1. IB with MACF1 antibody of the lysate immunoprecipitated with Nell2 antibody showed a single high MW band corresponding to Macf1. B. Macf1 immunohistochemistry in retinal sections showed predominant Macf1 expression in the GCL and inner plexiform layerI (IPL). Expression of Macf1 in RGCs and their dendrites was shown by colocalization of Macf1 positive signals with Thy-1 staining, a commonly used marker for RGCs.</p

Expression of Nell2 splicing variants in the retina.

<p>A. RT-PCR analysis was performed to identify Nell2 isoforms. Only one ∼300 bp product corresponding to secreted Nell 2 isoform was detected (lane 1). Lane 2 is a 100 bp DNA ladder. B. Locations of the primers in the gene used for the PCR are indicated by arrows. The expected sizes of the products corresponding to secreted and non-secreted Nell2 isoforms are 308 bp and 179 bp, respectively.</p

Nell2 mRNA expression in control and ONT retinas.

<p>A and C. Nell2 mRNA expression was localized to the cells in the GCL. D. No Nell2 positive cells were detected in retinal sections two weeks after ONT that leads to RGC degeneration. A and B. All Nell2 <i>in situ</i> hybridization signals were colocalized with RGCs retrogradely labeled with FG.</p