Elsevier

Neurobiology of Aging

Volume 30, Issue 10, October 2009, Pages 1523-1534
Neurobiology of Aging

Rapid communication
Astrocytes produce the antiinflammatory and neuroprotective agent hydrogen sulfide

https://doi.org/10.1016/j.neurobiolaging.2009.06.001Get rights and content

Abstract

Hydrogen sulfide (H2S) is an essential physiological product in brain. We investigated the expression of cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CGL), the two H2S synthesizing enzymes, in human cell lines and in human brain. Only astrocytes were strongly immunostained for CBS. Cultured astrocytes synthesized H2S at the rate of 15.06 μmol/g protein/h, which was 7.57 fold higher than microglial cells, 10.27 fold higher than SH-SY5Y cells and 11.32 fold higher than NT-2 cells. The H2S synthesis in all these cell types was inhibited by the CBS inhibitor hydroxylamine, but not by the CGL inhibitor propargylglycine (PAG). Synthesis of H2S by HUVEC cells was inhibited by PAG but not by hydroxylamine indicating that these vascular cells utilize CGL but not CBS. Inflammatory activation of microglia and astrocytes caused induction of NFκB, release of the inflammatory mediators TNFα, IL-6 and nitrite ions, down-regulation of CBS, and down-regulation of H2S synthesis. There was no effect of such treatment on HUVEC cells. The effects were partially reversed by pretreatment of cells with the H2S releasing agent NaSH. These data indicate that H2S is an endogenous antiinflammatory and neuroprotective agent under the synthetic control of CBS. H2S releasing drugs may have therapeutic potential in neurodegenerative disorders of aging such as Alzheimer disease and Parkinson disease.

Introduction

Hydrogen sulfide (H2S) is best known as a toxic gas with an extremely unpleasant odor. Yet it is a physiological product generated by all tissues of the body. Concentrations have been estimated to be as high as 50–160 μM (Qu et al., 2008, Warenycia et al., 1989). In keeping with such high apparent concentrations, several important biological actions of H2S have been identified (for reviews see Kamoun, 2004, Lowicka and Beltowski, 2007, Qu et al., 2008, Szabo, 2007). At synapses, it is a neuromodulator, facilitating the induction of hippocampal long-term potentiation (Kimura, 2002). In smooth muscle, it enhances the relaxing effect of nitric oxide (Hosoki et al., 1997). Pharmacologically, it has a direct effect on K-ATP channel currents (Zhao et al., 2001). In brain cell cultures, it stimulates production of cAMP with downstream effects on cAMP stimulated intracellular pathways (Kimura, 2000). So far, a possible role for H2S as an endogenous antiinflammatory agent in brain has not been extensively evaluated.

Evaluation involves an understanding of how H2S is synthesized in brain tissue. There are two differing routes of H2S synthesis, although both involve l-cysteine as the substrate. One utilizes cystathionine-β-synthase (CBS, EC 4.2.1.22) and the other cystathionine-γ-lyase (cystathionase, CGL, EC 4.4.1.1). CBS catalyses conversion of cysteine to serine with release of H2S, while CGL catalyses conversion of cysteine to pyruvate with release of H2S and NH3. These differing metabolic routes suggest that the enzymes involved may have differing biological roles even though they have a common end product in H2S. The cells expressing these separate enzymes may have distinct H2S related functions. This is indicated by reports that H2S production in brain is associated with CBS while that in vessels is associated with CGL. Brain H2S synthesis is reduced by the CBS inhibitor hydroxylamine, but not by the CGL inhibitor d,l-propargylglycine (PAG) (Abe and Kimura, 1996). In contrast, blockade of CGL by PAG completely abolishes H2S production in arteries (Zhao et al., 2001) but not in brain. In mouse models, CBS knockouts cannot synthesize H2S in brain (Eto et al., 2002), while CGL deletion results in a marked reduction of H2S in serum, heart and aorta (Yang et al., 2008).

To investigate further the role of H2S in brain, we first examined the expression of CBS and CGL in post mortem human brain. We found that CBS is strongly expressed by astrocytes while CGL is expressed only in some vessels. We then examined various human cell types for their expression of CBS and CGL and their ability to synthesize H2S. We found that astrocytes are the most powerful producers of H2S, with much smaller quantities being produced by microglia and neuroblastoma cells. We further investigated the influence of sodium hydrosulfide (NaSH), a known releaser of H2S, on the production of inflammatory mediators, the generation of neurotoxins, and on the induction of NFκB. We found that it attenuated all of these effects.

We conclude that H2S is an endogenous antiinflammatory and neuroprotective agent. It is a powerful antioxidant. Oxidation products have been implicated in the aging process and longevity (Bonnefoy et al., 2002, Calabrese et al., 2008), as well as the pathogenesis of Alzheimer disease (AD) (Butterfield et al., 2006, Pratico, 2008), Parkinson disease (PD) (Danielson and Andersen, 2008) and other neurodegenerative diseases (Sayre et al., 2001). A deficiency of H2S production could contribute to all of these conditions. Accordingly, we propose that H2S releasing drugs may have therapeutic potential in CNS disorders such as AD and PD that are characterized by a neuroinflammatory process.

Section snippets

Immunostaining of post mortem human brain tissue

Five cases without neurological disease were selected from our brain bank at the University of British Columbia (4M, 1F, aged 76–89 years). In all five cases the motor cortex and hippocampus were examined. In two cases, additional examination of the substantia nigra, basal ganglia, thalamus, amygdala, cerebellum, entorhinal cortex, occipital cortex and angular cortex was carried out.

Brain tissues had been fixed in 4% paraformaldehyde, and, after 3–4 days, transferred to a 15% buffered sucrose

Results

Fig. 1 shows the result of immunostaining of human brain with antibodies to CBS and CGL. In all brain areas examined, the antibody against CBS strongly stained astrocytes throughout white and grey matter (Fig. 1A and B). CBS labeling was present in the cell bodies and processes, extending all the way into the astrocytic end feet attached to capillaries (Fig. 1C). Some astrocytes were found to be in close contact with neurons (Fig. 1D). Astrocytes were stained in the substantia nigra,

Discussion

The results described here confirm previous reports that H2S is synthesized in brain primarily by the enzyme CBS (Abe and Kimura, 1996). They further demonstrate that astrocytes are the most active producers of H2S, with much smaller quantities being generated by microglia and by the NT-2 and SH-SY5Y neuronal cell lines. The relatively low levels of CBS expression by these other cell types probably accounts for the fact that only astrocytes can be detected expressing CBS by immunohistochemistry

Acknowledgments

This research was supported by a grant from the Pacific Alzheimer Research Foundation. We thank Dr. John Maguire of the UBC Department of Pathology and Laboratory Medicine for assistance in obtaining brain tissue specimens.

Disclosure statement: The authors report no conflicts of interest.

References (33)

  • M.W. Warenycia et al.

    Acute hydrogen sulfide poisoning. Demonstration of selective uptake of sulfide by the brainstem by measurement of brain sulfide levels

    Biochem. Pharmacol.

    (1989)
  • K. Abe et al.

    The possible role of hydrogen sulfide as an endogenous neuromodulator

    J. Neurosci.

    (1996)
  • M. Bonnefoy et al.

    Antioxidants to delay the process of aging: facts and figures

    Presse Medicale

    (2002)
  • V. Calabrese et al.

    Cellular stress response: a novel target for chemoprevention and nutritional neuroprotection in aging, neurodegenerative disorders and longevity

    Neurochem. Res.

    (2008)
  • K. Danielson et al.

    Oxidative and nitrative protein modifications in Parkinson's disease

    Free Radic. Biol. Med.

    (2008)
  • Y. Enokido et al.

    Cystathionine B-synthase, a key enzyme for homocysteine metabolism, is preferentially expressed in the radial glia/astrocyte lineage of developing mouse CNS

    FASEB J.

    (2005)
  • Cited by (177)

    • Novel near-infrared spectroscopic probe for visualizing hydrogen sulfide in lysosomes

      2022, Spectrochimica Acta - Part A: Molecular and Biomolecular Spectroscopy
    View all citing articles on Scopus
    View full text