Differential up-regulation of Vesl-1/Homer 1 protein isoforms associated with decline in visual performance in a preclinical glaucoma model

Abstract

Glaucoma is a multifactorial progressive ocular pathology, clinically presenting with damage to the retina and optic nerve, ultimately leading to blindness. Retinal ganglion cell loss in glaucoma ultimately results in vision loss. Vesl/Homer proteins are scaffolding proteins that are critical for maintaining synaptic integrity by clustering, organizing and functionally regulating synaptic proteins. Current anti-glaucoma therapies target IOP as the sole modifiable clinical parameters. Long-term pharmacotherapy and surgical treatment do not prevent gradual visual field loss as the disease progresses, highlighting the need for new complementary, alternative and comprehensive treatment approaches. Vesl/Homer expression was measured in the retinae of DBA/2J mice, a preclinical genetic glaucoma model with spontaneous mutations resulting in a phenotype reminiscent of chronic human pigmentary glaucoma. Vesl/Homer proteins were differentially expressed in the aged, glaucomatous DBA/2J retina, both at the transcriptional and translational level. Immunoreactivity for the long Vesl-1L/Homer 1c isoform, but not of the immediate early gene product Vesl-1S/Homer 1a was increased in the synaptic layers of the retina. This increased protein level of Vesl-1L/Homer 1c was correlated with phenotypes of increased disease severity and a decrease in visual performance. The increased expression of Vesl-1L/Homer 1c in the glaucomatous retina likely results in increased intracellular Ca(2+) release through enhancement of synaptic coupling. The ensuing Ca(2+) toxicity may thus activate neurodegenerative pathways and lead to the progressive loss of synaptic function in glaucoma. Our data suggest that higher levels of Vesl-1L/Homer 1c generate a more severe disease phenotype and may represent a viable target for therapy development.

Keywords: Calcium channel; DBA/2J; Glaucoma; HRP; IOP; Neurodegeneration; RGCs; Synaptic clustering; VASP/Ena-related gene up-regulated during seizure and LTP protein; Vesl; Vesl/Homer; horseradish peroxidase; intraocular pressure; retinal ganglion cells.

Figure 1. Increased IOP and impaired visual function in the glaucomatous DBA/2J retina

A) IOP is elevated in the aged cohort of 9 months-old DBA/2J mice, compared with young 6 week-old controls (n=5, P<0.001). B) Visual acuity as tested in the OptoMotry^™ system was significantly reduced in aged DBA/2J mice (n=5, P<0.001). C) Similarly, the contrast sensitivity threshold showed evidence of impaired vision in the aged cohort and was significantly increased (n=5, P<0.05). * P<0.05; *** P<0.001.

Figure 2. Vesl-1L/Homer 1c is increased at the transcriptional and translational level in the neural retina

A) Vesl-1S/Homer 1a mRNA levels were increased by 60% in the neural retina, however, this increase did not reach statistical significance (R_Q=1.000 ± 0.008 and R_Q=1.590 ± 0.437 for 6w and 9m, respectively; n=3, P=0.25). B) In contrast, normalized gene expression (R_Q) for Vesl-1L/Homer 1c was 4-fold higher in the aged, glaucomatous retina compared with young, healthy controls (n=3, P<0.01). C) Representative immunoblots from two animals per cohort showing expression of Vesl-1S/Homer 1a, Vesl-1L/Homer 1c and GAPDH. Expression of Vesl-1L/Homer 1c is increased in the neural retina in aged, glaucomatous DBA/2J mice. D) Protein levels of Vesl-1S/Homer 1a showed a trend towards an increase in the aged DBA/2J retina (n=5, P=0.07). E) Vesl-1L/Homer 1c expression was increased by 60% in the aged cohort, compared with young controls (n=5, P<0.01). ** P<0.01

Figure 3. Vesl-1L/Homer 1c expression in the neural retina correlates with markers of disease severity in DBA/2J mice

A–C) Vesl-1S/Homer 1a protein expression showed a positive association with IOP (n=5, P<0.05). In contrast, expression of the short Vesl/Homer isoform did neither correlate with visual acuity nor the contrast sensitivity threshold. D–F) In contrast, Vesl-1L/Homer 1c expression correlated strongly with markers of glaucoma disease severity. Specifically, Vesl-1L/Homer 1c levels were positively associated with IOP (n=5, P<0.01). Furthermore, the long constitutively expressed long Vesl/Homer isoform showed a statistically significant negative association with visual acuity (n=5, P<0.05) and a positive association with the contrast sensitivity threshold (n=5, P<0.05), indicative of progressively higher Vesl-1L/Homer 1c expression as disease severity increased.

Figure 4. Vesl-1/Homer 1 expression is markedly increased in the synaptic layers of the retina

A) Vesl-1S/Homer 1a immunoreactivity showed a typical expression pattern that did not change in the aged, glaucomatous retina. A representative single confocal section is shown. Scale bar: 20 μm. B) Magnified view of the region in the RGC layer indicated by a square in A). Scale bar: 2.5 μm. C) Quantification using microfluorimetry is presented as the mean intensity of 25 line scans across a length of 180 μm. Intensity of Vesl-1S/Homer 1a immunoreactivity was increased in the synaptic layers, by approx. 60% in the ganglion cell layer (GCL) and the inner plexiform layer (IPL), and approx. 2-fold in the outer plexiform layer (OPL). D) Immunolabeling with an antibody specific for the long Vesl-1L/Homer 1c isoform revealed a significant increase in immunoreactivity in the synaptic layers of the retina of aged DBA/2J mice compared with young controls as evident from a representative single confocal section. Scale bar: 20 μm. E) Magnified view of the region in the RGC layer indicated by a square in D). Scale bar: 2.5 μm. D) The intensity graph represents the mean intensity of 25 line scans across a 180 μm stretch of retina, showing a 3-fold increase in Homer 1c immunoreactivity in the GCL, IPL and OPL.

Figure 5. Proposed mechanism of Vesl-1/Homer 1-mediated changes in the neural retina

In the young, non-glaucomatous retina, Vesl-1S/Homer 1a prevents synaptic coupling of intracellular calcium channels (ICC) in the membranes of the endoplasmic reticulum with G-protein coupled receptors (GPCR) at the plasma membrane, resulting in tightly controlled intracellular Ca²⁺ release mediated by 2^nd messenger pathways (green arrow). Up-regulation of Vesl-1L/Homer 1c in the aged, glaucomatous retina increases synaptic coupling, leading to increased intracellular Ca²⁺ release due to increased strength of second messenger pathways (red arrow) and the direct modulation of ICCs by Vesl-1L/Homer 1c.