Neuropeptide Y (NPY), a sympathetic neurotransmitter, is involved in various physiological functions, and its dysregulation is implicated in several neurodegenerative diseases. Glutamate excitotoxicity, endoplasmic reticulum (ER) stress, and oxidative stress are the common mechanisms associated with numerous neurodegenerative illnesses. The present study aimed to elucidate the protective effects of NPY against glutamate toxicity and tunicamycin-induced ER stress in the human neuroblastoma SH-SY5Y cell line. We exposed the SH-SY5Y cells to glutamate and tunicamycin for two different time points and analyzed the protective effects of NPY at different concentrations. The protective effects of NPY treatments were assessed by cell viability assay, and the signalling pathway changes were evaluated by biochemical techniques such as Western blotting and immunofluorescence assays. Our results showed that treatment of SH-SY5Y cells with NPY significantly increased the viability of the cells in both glutamate toxicity and ER stress conditions. NPY treatments significantly attenuated the glutamate-induced pro-apoptotic activation of ERK1/2 and JNK/BAD pathways. The protective effects of NPY were further evident against tunicamycin-induced ER stress. NPY treatments significantly suppressed the ER stress activation by downregulating BiP, phospho-eIF2α, and CHOP expression. In addition, NPY alleviated the Akt/FoxO3a pathway in acute oxidative conditions caused by glutamate and tunicamycin in SH-SY5Y cells. Our results demonstrated that NPY is neuroprotective against glutamate-induced cell toxicity and tunicamycin-induced ER stress through anti-apoptotic actions.

Glaucoma is a complex neurodegenerative disease characterized by optic nerve damage and apoptotic retinal ganglion cell (RGC) death, and is the leading cause of irreversible blindness worldwide. Among the sphingosine 1-phosphate receptors (S1PRs) family, S1PR1 is a highly expressed subtype in the central nervous system and has gained rapid attention as an important mediator of pathophysiological processes in the brain and the retina. Our recent study showed that mice treated orally with siponimod drug exerted neuroprotection via modulation of neuronal S1PR1 in experimental glaucoma. This study identified the molecular signaling pathway modulated by S1PR1 activation with siponimod treatment in RGCs in glaucomatous injury. We investigated the critical neuroprotective signaling pathway in vivo using mice deleted for S1PR1 in RGCs. Our results showed marked upregulation of the apoptotic pathway was associated with decreased Akt and Erk1/2 activation levels in the retina in glaucoma conditions. Activation of S1PR1 with siponimod treatment significantly increased neuroprotective Akt and Erk1/2 activation and attenuated the apoptotic signaling via suppression of c-Jun/Bim cascade and by increasing Bad phosphorylation. Conversely, deletion of S1PR1 in RGCs significantly increased the apoptotic cells in the ganglion cell layer in glaucoma and diminished the neuroprotective effects of siponimod treatment on Akt/Erk1/2 activation, c-Jun/Bim cascade, and Bad phosphorylation. Our data demonstrated that activation of S1PR1 in RGCs induces crucial neuroprotective signaling that suppresses the proapoptotic c-Jun/Bim cascade and increases antiapoptotic Bad phosphorylation. Our findings suggest that S1PR1 is a potential therapeutic target for neuroprotection of RGCs in glaucoma.

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.

Photoreceptor cells are highly susceptible to oxidative-stress-induced damage due to their high metabolic rate. Oxidative stress plays a key role in driving pathological events in several different ocular diseases, which lead to retinal degeneration and ultimately blindness. A growing number of studies have been performed to understand downstream events caused by ROS induced oxidative stress in photoreceptor cells; however, the underlying mechanisms of ROS toxicity are not fully understood. To shed light on ROS induced downstream pathological events, we employed a tandem mass tag (TMT) labelling-based quantitative mass-spectrometric approach to determine proteome changes in 661W photoreceptor cells following oxidative stress induction via the application of different concentrations of H₂O₂ at different time points. Overall, 5920 proteins were identified and quantified, and 450 differentially expressed proteins (DEPs) were identified, which were altered in a dose and time dependent manner in all treatment groups compared to the control group. These proteins were involved in several biological pathways, including spliceosome and ribosome response, activated glutathione metabolism, decreased ECM-receptor interaction, oxidative phosphorylation, abnormally regulated lysosome, apoptosis, and ribosome biogenesis. Our results highlighted ECM receptor interaction, oxidative phosphorylation and spliceosome pathways as the major targets of oxidative stress that might mediate vascular dysfunction and cellular senescence.