Siponimod exerts neuroprotective effects on the retina and higher visual pathway through neuronal S1PR1 in experimental glaucoma

Abstract

Sphingosine-1-phosphate receptor (S1PR) signaling regulates diverse pathophysiological processes in the central nervous system. The role of S1PR signaling in neurodegenerative conditions is still largely unidentified. Siponimod is a specific modulator of S1P1 and S1P5 receptors, an immunosuppressant drug for managing secondary progressive multiple sclerosis. We investigated its neuroprotective properties in vivo on the retina and the brain in an optic nerve injury model induced by a chronic increase in intraocular pressure or acute N-methyl-D-aspartate excitotoxicity. Neuronal-specific deletion of sphingosine-1-phosphate receptor (S1PR1) was carried out by expressing AAV-PHP.eB-Cre recombinase under Syn1 promoter in S1PR1^flox/flox mice to define the role of S1PR1 in neurons. Inner retinal electrophysiological responses, along with histological and immunofluorescence analysis of the retina and optic nerve tissues, indicated significant neuroprotective effects of siponimod when administered orally via diet in chronic and acute optic nerve injury models. Further, siponimod treatment showed significant protection against trans-neuronal degenerative changes in the higher visual center of the brain induced by optic nerve injury. Siponimod treatment also reduced microglial activation and reactive gliosis along the visual pathway. Our results showed that siponimod markedly upregulated neuroprotective Akt and Erk1/2 activation in the retina and the brain. Neuronal-specific deletion of S1PR1 enhanced retinal and dorsolateral geniculate nucleus degenerative changes in a chronic optic nerve injury condition and attenuated protective effects of siponimod. In summary, our data demonstrated that S1PR1 signaling plays a vital role in the retinal ganglion cell and dorsolateral geniculate nucleus neuronal survival in experimental glaucoma, and siponimod exerts direct neuroprotective effects through S1PR1 in neurons in the central nervous system independent of its peripheral immuno-modulatory effects. Our findings suggest that neuronal S1PR1 is a neuroprotective therapeutic target and its modulation by siponimod has positive implications in glaucoma conditions.

Keywords: glaucoma; intraocular pressure; neurodegeneration; neuroprotection; optic nerve injury; retinal ganglion cells; siponimod; sphingosine-1-phosphate.

Figure 1

Neuroprotective effects of siponimod on function and structure of the retina and optic nerve (ON) in high intraocular pressure (IOP) condition. (A) Schematic depicting the experimental design and analysis of visual pathway in ON injury model. dLGN: Dorsolateral geniculate nucleus. (B) Chronic elevation of IOP in C57BL/6 mice eyes was induced by intracameral microbead injections for 2 months. (C) Positive scotopic threshold responses (pSTR) of control and glaucomatous mice eyes treated with/without siponimod after 8 weeks of chronic elevated IOP. (D) Quantification of the pSTR amplitudes (**P < 0.01, n = 10 per group). (E) Hematoxylin and eosin staining of sagittal cross-section of the eyes (representative images, arrows indicate the changes in cell densities, scale bar: 50 µm. GCL: Ganglion cell layer; INL: inner nuclear layer; ONL: outer nuclear layer). (F) Cell density in the GCL from the retinal hematoxylin and eosin-stained images (***P < 0.001, n = 5 per group). (G) Immunofluorescence staining of ON cross-sections (scale bar: 50 µm) with pNFH (SIM-31) and immunoreactivity measurements (green, pNFH⁺ areas) from high magnified areas (boxes, scale bar: 10 µm) of ONs, and (H) their quantifications (**P < 0.01, n = 5 per group). Statistical significance was determined using one-way analysis of variance with Tukey’s multiple comparisons test (mean ± SD).

Figure 2

Neuroprotective effects of siponimod on function and structure of the retina and optic nerve (ON) against acute retinal N-methyl-D-aspartate (NMDA) excitotoxicity. (A) Positive scotopic threshold responses (pSTR) of the eyes after 7 days of NMDA excitotoxicity and (B) quantification of the pSTR amplitudes in acute retinal NMDA excitotoxicity (*P < 0.05, n = 10 per group). (C) Histological analysis of sagittal cross-sectional of the eyes stained with hematoxylin and eosin (representative images, arrows indicate the changes in cell densities, scale bar: 50 µm; GCL: ganglion cell layer; INL: inner nuclear layer; ONL: outer nuclear layer) and (D) cell counts in the GCL (*P < 0.05, n = 5 per group). (E) Immunofluorescence images of ON cross-sections stained with pNFH (green, representative, scale bar: 50 µm) and immunoreactivity measurements from magnified areas (boxes, scale bars: 10 µm) and (F) their quantitative analysis (*P < 0.05, n = 5 per group). Statistical significance was determined using one-way analysis of variance with Tukey’s multiple comparisons test (mean ± SD).

Figure 3

Protective effects of siponimod on the dorsolateral geniculate nucleus (dLGN) of the brain in chronic glaucoma condition (8 weeks). (A) Nissl staining of coronal sections (contralateral) of mouse brains (dLGN is marked, representative images, scale bar: 500 µm). (B) Neuronal density measured from magnified images (scale bar: 20 µm) of the dLGN. (C) Immunofluorescence images of coronal sections of mouse brains (contralateral, representative) stained with anti-NeuN (green) a neuronal marker and DAPI (blue) (scale bar: 200 µm), the dLGN region is marked. (D) Percentage of NeuN⁺ cells out of DAPI-positive cells in the dLGN from magnified areas (scale bar: 20 µm). **P < 0.01, one-way analysis of variance with Tukey’s multiple comparisons test, n = 5 per group.

Figure 4

Siponimod upregulates Akt and Erk1/2 phosphorylation in the retina and the brain. (A) Western blot analysis of Akt (phsopho-S473 antibody) and Erk1/2 (phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) phosphorylation levels in retinas and (B) in the dorsolateral geniculate nucleus (dLGN) of the brain tissues lysates (8 weeks of chronic elevated IOP) and their densitometric quantitative analysis (*P < 0.05, **P < 0.01, ***P < 0.001, one-way analysis of variance with Tukey’s multiple comparisons test, n = 4 per group).

Figure 5

Siponimod treatment reduces glial activation along the visual pathway in chronic optic nerve injury condition (8 weeks). (A) Immunofluorescence staining of eye sections stained with NeuN, a marker for neurons (green) and Iba1 (red) for microglia (representative images, arrows indicate the changes in expression, scale bar: 50 µm; GCL: ganglion cell layer; INL: inner nuclear layer; ONL: outer nuclear layer). (B) Quantification of Iba1 expression by western blotting analysis of retina lysates (normalized to β-actin). (C) Representative immunofluorescence images of coronal sections of mouse brains (contralateral, the dorsolateral geniculate nucleus (dLGN) is marked with dotted circles, scale bar: 100 µm) stained with Iba1 antibody (red). (D) Western blot analysis of microdissected dLGN and their quantitative analysis for Iba1 expression. (E) Immunofluorescence analysis of reactive gliosis (Muller glia) in the eye sections stained with GFAP (red) (scale bar: 50 µm). (F) Western blot analysis for GFAP expressions in retina lysates and their quantitative analysis. (G) Immunofluorescence staining for brain coronal sections with GFAP (red) (scale bar: 100 µm). (H) Western blot analysis and quantification for GFAP expression in the dLGN. **P < 0.01, ***P < 0.001, one-way analysis of variance with Tukey’s multiple comparisons test; n = 4 per group. GFAP: Glial fibrillary acidic protein; Iba1: ionized calcium-binding adaptor molecule 1; IOP: intraocular pressure.

Figure 6

The functional importance of S1PR1 in neurons and its deletion impairs the protective effect of siponimod on inner retinal function and structure in chronic optic nerve injury condition. (A) Inner retinal positive scotopic threshold responses (pSTR) responses and (B) quantification of the pSTR amplitudes of control and high intraocular pressure (IOP) eyes (8 weeks) of S1PR1^+/+ control (AAV-GFP) and Neu-S1PR1^–/– (AAV-GFP-Cre) mice groups. (C) Comparison of pSTR amplitudes loss among different mice groups (n = 10 per group). (D) Hematoxylin and eosin staining of eye cross-sections (arrows indicate the changes in cell densities, scale bar: 50 µm. GCL: Ganglion cell layer; INL: inner nuclear layer; ONL: outer nuclear layer) and (E) cell counts in the GCL. (F) Comparison of cell loss in the GCL among different mouse groups (n = 5 per group). *P < 0.05, **P < 0.01, ***P < 0.001, one-way analysis of variance with Tukey’s multiple comparisons test. NS: Not significant; S1PR: sphingosine-1-phosphate receptor.

Figure 7

Neuron-specific deletion of S1PR1 reduced the protective effect of siponimod against trans-neuronal degeneration in dLGN of the brain in chronic optic nerve injury condition. (A) Representative immunofluorescence images of coronal sections of S1PR1^+/+ control (AAV-GFP) and Neu-S1PR1^–/– (AAV-GFP-Cre) mice brains (contralateral) stained with anti-NeuN (green) and DAPI (blue) (representative, the dLGN is marked with dotted circles, scale bar: 100 µm). (B) Quantification of the percentage of NeuN⁺ cells out of DAPI-positive cells in the dLGN from magnified different areas (scale bar: 20 µm) among mice groups (8 weeks of chronic high IOP). *P < 0.05, **P < 0.01, one-way analysis of variance with Tukey’s multiple comparisons test (mean ± SD, n = 5 per group). DAPI: 4′,6-Diamidino-2-phenylindole; AAV: adeno-associated virus; dLGN: dorsolateral geniculate nucleus; IOP: intraocular pressure; S1PR: sphingosine-1-phosphate receptor.

Figure 8

Attenuation of the protective effect of siponimod on retinal microglial activation and reactive Müller glia in high IOP condition in Neu-S1PR1^–/– (AAV-GFP-Cre) mice. (A) Representative immunofluorescence images of control S1PR1^+/+ and Neu-S1PR1^–/– (AAV-GFP-Cre) mice eyes (8 weeks of chronic high IOP) sections stained with NeuN (green) and Iba1 (red) for microglia and (B) GFAP (red) for Müller glia (arrows indicate the changes in expression, scale bar: 50 µm; GCL: ganglion cell layer; INL: inner nuclear layer; ONL: outer nuclear layer). (C) Western blot analysis and quantification of Iba1 and GFAP expressions in retina lysates (normalized to β actin) (*P < 0.05, ***P < 0.001, n = 4 per group). Statistical significance was determined using one-way analysis of variance with Tukey’s multiple comparisons test (mean ± SD). AAV: Adeno-associated virus; GFAP: glial fibrillary acidic protein; Iba1: ionized calcium-binding adaptor molecule 1; IOP: intraocular pressure; NS: not significant; S1PR: sphingosine-1-phosphate receptor.