Glycine, the smallest amino acid, confers neuroprotection againstd-galactose-induced neurodegeneration and memory impairment by regulating c-Jun N-terminal kinase in the mouse brain

Abstract

Background Glycine is the smallest nonessential amino acid and has previously unrecognized neurotherapeutic effects. In this study, we examined the mechanism underlying the neuroprotective effect of glycine (Gly) against neuroapoptosis, neuroinflammation, synaptic dysfunction, and memory impairment resulting fromd-galactose-induced elevation of reactive oxygen species (ROS) during the onset of neurodegeneration in the brains of C57BL/6N mice. Methods After in vivo administration ofd-galactose (d-gal; 100 mg/kg/day; intraperitoneally (i/p); for 60 days) alone or in combination with glycine (1 g/kg/day in saline solution; subcutaneously; for 60 days), all of the mice were sacrificed for further biochemical (ROS/lipid peroxidation (LPO) assay, Western blotting, and immunohistochemistry) after behavioral analyses. An in vitro study, in which mouse hippocampal neuronal HT22 cells were treated with or without a JNK-specific inhibitor (SP600125), and molecular docking analysis were used to confirm the underlying molecular mechanism and explore the related signaling pathway prior to molecular and histological analyses. Results Our findings indicated that glycine (an amino acid) inhibitedd-gal-induced oxidative stress and significantly upregulated the expression and immunoreactivity of antioxidant proteins (Nrf2 and HO-1) that had been suppressed in the mouse brain. Both the in vitro and in vivo results indicated thatd-gal induced oxidative stress-mediated neurodegeneration primarily by upregulating phospho-c-Jun N-terminal kinase (p-JNK) levels. However,d-gal + Gly cotreatment reversed the neurotoxic effects ofd-gal by downregulating p-JNK levels, which had been elevated byd-gal. We also found that Gly reversedd-gal-induced neuroapoptosis by significantly reducing the protein expression levels of proapoptotic markers (Bax, cytochrome c, cleaved caspase-3, and cleaved PARP-1) and increasing the protein expression level of the antiapoptotic protein Bcl-2. Both the molecular docking approach and the in vitro study (in which the neuronal HT22 cells were treated with or without a p-JNK-specific inhibitor (SP600125)) further verified our in vivo findings that Gly bound to the p-JNK protein and inhibited its function and the JNK-mediated apoptotic pathway in the mouse brain and HT22 cells. Moreover, the addition of Gly alleviatedd-gal-mediated neuroinflammation by inhibiting gliosis via attenuation of astrocytosis (GFAP) and microgliosis (Iba-1) in addition to reducing the protein expression levels of various inflammatory cytokines (IL-1 beta eta and TNF alpha). Finally, the addition of Gly reversedd-gal-induced synaptic dysfunction by upregulating the expression of memory-related presynaptic protein markers (synaptophysin (SYP), syntaxin (Syn), and a postsynaptic density protein (PSD95)) and markedly improved behavioral measures of cognitive deficits ind-gal-treated mice. Conclusion Our findings demonstrate that Gly-mediated deactivation of the JNK signaling pathway underlies the neuroprotective effect of Gly, which reversesd-gal-induced oxidative stress, apoptotic neurodegeneration, neuroinflammation, synaptic dysfunction, and memory impairment. Therefore, we suggest that Gly (an amino acid) is a safe and promising neurotherapeutic candidate that might be used for age-related neurodegenerative diseases.