Hydrogen sulfide and reactive sulfur species are regulators of physiological functions, have antioxidant effects against oxidative stresses, and are endogenously generated from l-cysteine. Recently, a novel pathway that generates hydrogen sulfide and reactive sulfur species from d-cysteine has been identified. d-Amino acid oxidase (DAO) is involved in this pathway and, among the various brain regions, is especially abundant in the cerebellum. d-Cysteine has been found to be a better substrate in the generation of hydrogen sulfide in the cerebellum than l-cysteine. Therefore, d-cysteine might be a novel neuroprotectant against cerebellar diseases such as spinocerebellar ataxia (SCA). However, it remains unknown if d-cysteine affects cerebellar Purkinje cells (PCs), which are important for cerebellar functions and are frequently degenerated in SCA patients. In the present study, we investigated whether the production of hydrogen sulfide from d-cysteine affects the dendritic development of cultured PCs. d-Cysteine was found to enhance the dendritic development of PCs significantly, while l-cysteine impaired it. The effect of d-cysteine was inhibited by simultaneous treatment with DAO inhibitors and was reproduced by treatment with 3-mercaptopyruvate, a metabolite of d-cysteine produced by the action of DAO, and disodium sulfide, a donor of hydrogen sulfide. In addition, hydrogen sulfide was immediately produced in cerebellar primary cultures after treatment with d-cysteine and 3-mercaptopyruvate. These findings suggest that d-cysteine enhances the dendritic development of primary cultured PCs via the generation of hydrogen sulfide.

Chaperone-mediated autophagy (CMA) is a pathway in the autophagy-lysosome protein degradation system. CMA impairment has been implicated to play a role in spinocerebellar ataxia (SCA) pathogenesis. D-cysteine is metabolized by D-amino acid oxidase (DAO), leading to hydrogen sulfide generation in the cerebellum. Although D-cysteine alleviates the disease phenotypes in SCA-model mice, it remains unknown how hydrogen sulfide derived from D-cysteine exerts this effect. In the present study, we investigated the effects of D-cysteine and hydrogen sulfide on CMA activity using a CMA activity marker that we have established. D-cysteine activated CMA in Purkinje cells (PCs) of primary cerebellar cultures where DAO was expressed, while it failed to activate CMA in DAO-deficient AD293 cells. In contrast, Na2S, a hydrogen sulfide donor, activated CMA in both PCs and AD293 cells. Nuclear factor erythroid 2-related factor 2 (Nrf2) is known to be activated by hydrogen sulfide and regulate CMA activity. An Nrf2 inhibitor, ML385, prevented CMA activation triggered by D-cysteine and Na2S. Additionally, long-term treatment with D-cysteine increased the amounts of Nrf2 and LAMP2A, a CMA-related protein, in the mouse cerebellum. These findings suggest that hydrogen sulfide derived from D-cysteine enhances CMA activity via Nrf2 activation.

N-Methyl-d-aspartate (NMDA) receptors, which play an important role in neuronal excitotoxicity, require not only agonists at the glutamate-binding site but also co-agonists at the glycine site for their activation. Here we examined the role of endogenous agonists at the glycine site of NMDA receptors in excitotoxic retinal damage in vivo. To quantify the number of surviving retinal ganglion cells (RGCs), we injected a retrograde tracer, fluoro-gold, into the superior colliculus bilaterally and subsequently counted RGCs on whole-mounted retinas. Co-injection of 5,7-dichlorokynurenic acid (300 nmol), a competitive antagonist at the glycine site of NMDA receptors, rescued RGCs from damage induced by 200 nmol NMDA. On the other hand, RGC death induced by 20 nmol NMDA was enhanced by addition of glycine (10 nmol), D-serine (10 nmol) or a competitive glycine transporter-1 inhibitor, sarcosine (0.3 or 3 nmol). Moreover, application of d-serine-degrading enzyme, D-amino acid oxidase (30 mU), partially suppressed RGC death induced by 20 nmol NMDA. These results suggest that the severity of excitotoxic retinal damage in vivo depends on the levels of both glycine and D-serine.