Regulation of S-Nitrosylation in Aging and Senescence

S-Nitrosylation Is Regulated by S-Nitrosoglutathione Reductase

S-nitrosylation is the post-translational oxidative modification of cysteine residues by nitric oxide (NO) to form S-nitrosothiols (SNOs). ^1,2 Steady state levels of S-nitrosylation result from a balance between protein nitrosylating enzymes called nitrosylases and denitrosylation catalyzed by denitrosylases. S-nitrosoglutathione reductase (GSNOR) is an important denitrosylase. Originally characterized as a class III alcohol dehydrogenase or a glutathione-dependent formaldehyde dehydrogenase, recent studies indicate that the highest catalytic activity of GSNOR is for the reduction of S-nitrosoglutathione (GSNO). ^3,4 Because free, soluble GSNO is in equilibrium with protein-bound SNOs, that is, the NO moiety moves back and forth through transnitrosylation, the activity of GSNOR is a critical control point of global protein S-nitrosylation. ⁵

Expression of GSNOR Decreases During Cell Senescence, but Ultimately Increases During Aging

GSNOR knockout mice (Gsnor-/-) exhibit a few characteristics of accelerated aging, ^{4,6 –11} such as reduced muscle mass and neuromuscular dysfunction. In contrast, Gsnor-/- mice can live to normal old age ¹² and have enhanced cardiac regenerative potential. ¹³ Overall, GSNOR deficiency affects homeostasis and should not be considered a model of accelerated aging.

Rizza et al. demonstrated that the brains of Gsnor-/- mice had markedly increased levels of ubiquitin and alpha-synuclein-positive aggregates that accompanied a decline in motor skills seen in young animals (2 months). ¹⁴ In normal mice mRNA, protein and enzymatic activity of GSNOR was shown to decrease >40% in the first year of life in peripheral blood mononuclear cell (PBMC) and brains. ¹⁴ In contrast, Zhang et al. found that hippocampal GSNOR mRNA levels increased significantly from middle age (7 months) to old age (22 months), data that are consistent with their human hippocampus expression data. ¹² Dr. Chang Chen, the corresponding author of Zhang et al., makes a strong point in noting that Rizza et al. only studied mice until middle age and reported no data on old mice unlike his group. ¹⁵

Interestingly, Rizza et al. observed a statistically relevant decrease in GSNOR and epigenetic regulator ten-eleven translocation (TET)1 mRNA of PBMCs among a cohort of healthy aged humans (65–86 years old) versus a younger cohort (18–30 years old). In contrast, GSNOR levels from PBMCs from individuals between ages 95 and 101 were maintained or possibly increased over values of young individuals. ¹⁴ Contrast this with Zhang et al. who report increased mRNA expression levels of GSNOR for aging human hippocampuses using publicly available expression databases. ¹² The simplest explanation may be that brain aging is different than PBMC aging, but a more unifying answer suggested by Dr Chen may be that if Rizza et al. considered their data as an unbiased series, rather than age-grouped segments, they might observe that the high GSNOR levels observed for very old people actually suggest that GSNOR levels do increase with age, especially old age. Without further data we favor this interpretation.

Interestingly, GSNOR expression was found to be reduced in primary senescent, that is, higher passage mouse embryonic fibroblasts (MEFs). Some biomarkers of senescence such as increased nuclear size and beta-galactosidase activity observed in Gsnor-/- fibroblasts or senescent MEFs could be reversed by pan-nitric oxide synthase (NOS) inhibitor, L-NAME (L-N^G-nitroarginine methyl ester). ¹⁴ These data suggest that increasing GSNOR activity or reducing NOS may be one way to modulate the senescent phenotype. Global reduction in GSNOR levels with elevated S-nitrosylation accompanies senescence, but what is cause and what is effect?

That GSNOR levels seem to first decrease in middle age (which may be artifactual) and then increase in old age (for PBMCs), increase steadily in hippocampuses with increasing age, but decrease in senescent cells suggests divergent GSNOR dysregulation with different types of aging (cell senescence vs. chronological aging), which may have profound implications for targeting NO signaling in aging.

Methylation Regulates GSNOR Expression Through TET 1 Protein

Several CpG islands are located adjacent to the transcriptional start site of the GSNOR gene, suggesting that epigenetic silencing by cytosine methylation might be important in the regulation of GSNOR expression. The TET proteins participate in the demethylation of methylated cytosines. ^16,17 The initial step catalyzed by TET proteins is the oxidation of 5-methylcytosine (5meC) to 5-hydroxymethylcytosine (5hmeC). Therefore, levels of 5hmC are a measure of active cytosine demethylation. Rizza et al. compared the relative levels of 5mC and 5hmC in the promoter region of Gsnor in MEFs and the brains of wild-type mice at various ages. Levels of 5meC increased and 5hmeC decreased for the first 6 months of life. TET1 but not TET2 was found to modulate Gsnor expression in culture and its levels were reduced with age, at least until middle age. ¹⁴ Although epigenetic silencing of GSNOR through increased DNA methylation associated with reduced TET1 may explain the increased S-nitrosylation found in senescent cells and middle-aged mice, how does reduced GSNOR potentially contribute to the cell dysfunction associated with senescence?

GSNOR Regulates Mitochondrial Function and Mitophagy

Compromised function of mitochondria is a hallmark of aging. Several recent studies have suggested a role for S-nitrosylation of target proteins in respiratory complexes I, III, and IV in the oxidative dysfunction of mitochondria. ^{18 –20} Rizza et al. showed that reduced GSNOR activity is accompanied by mitochondrial nitrosative stress, characterized by elevated S-nitrosylation of Drp1 and Parkin with the downstream effect of impaired mitophagy. Mitochondria in MEFs from Gsnor-/- exhibit increased fragmentation and a markedly reduced transmembrane potential. Furthermore, Gsnor-/- cells exhibit reduced mitophagy. ¹⁴ Under normal circumstances, the e3 ubiquitin ligase activity of Parkin enhances mitophagy. ^{21 –23} However, in aging this activity is compromised. Oh et al. reported that PINK1, a substrate of Parkin, is S-nitrosylated at Cys-658. This contributes to the compromised mitophagy observed in Parkinson's disease. ²⁴ Keep in mind that 22-month-old GSNOR-/- animals were apparently healthy, ¹² which suggests that the mitochondrial dysfunction associated with GSNOR KO described by Rizza et al. may not achieve a pathological threshold during normal aging. However, low GSNOR levels are consistent with mitochondrial dysfunction observed in cell senescence. ¹⁴ But what of GSNOR in normal aging?

Increased GSNOR Observed in Old Mice Results in Reduced Synaptic Plasticity and Long-Term Potentiation

What are the consequences of the increased hippocampal GSNOR levels observed by Zhang et al.? Mice that have high levels of GSNOR either by having been engineered to express high ectopic levels or simply by being old (22 months) have cognitive defects in classical mouse learning tests such as the Morris Water Maze, fear conditioning, and long-term-potentiation (LTP) as well as lower dendrite spine density. Significantly, old GSNOR-/- KO mice (22 months) did not show the cognitive or LTP defects seen in normal old mice, suggesting that loss of GSNOR in the mutant mice was protective for loss of synaptic plasticity seen in normal aging and that NO signaling deficiency resulting from increased GSNOR may be a key mediator or age-related cognitive decline. Zhang et al. then correlate an observed loss of calmodulin kinase IIa (CaMKIIa) synaptosomal accumulation with loss of S-nitrosated sites on the enzyme, which presumably is due to increased GSNOR. ¹² The hypothesis is that the presence of S-nitrosated CaMKIIa, which becomes autonomously activated by NO, at synaptosomes stimulates α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (glutamate) receptors, ²⁵ which contribute to synaptic plasticity.

If decreased net nitrosylation by GSNOR really is important, then stimulating NO signaling should be beneficial. Zhang et al. increased NO signaling by injecting l-arginine interperitoneally into GSNOR overexpressing transgenic mice. l-arginine restored cognitive function to near wild-type levels, suggesting that increasing NO signaling may help improve cognitive function when GSNOR hippocampal levels are high, ¹² such as in aged mammals, although it appears that this treatment was not tried on the old animals.

Medical Implications

A number of conditions, for example, neurodegeneration, are associated with increased activity of various NOS enzymes with subsequent augmentation of S-nitrosylation and downstream mitochondrial and cellular pathology. ^{26 –29} Moreover, a general connection between decreased GSNOR and cell senescence is intriguing. Interventions to reduce global S-nitrosylation of key target proteins might be a logical outcome of the basic science studies described by Rizza et al., except that a Goldilocks principle might apply given that chronological aging resulted in increased hippocampal GSNOR.

Several phenotypes observed in Gsnor-/- fibroblasts or senescent MEFs could be reversed by pan-NOS inhibitor, L-NAME. It should be noted that L-NAME and other nonspecific NOS inhibitors were evaluated many years ago with equivocal results. Safety and toxicity of a global inhibitor are also an open question. However, drugs that increase GSNOR expression and activity are potentially another avenue to decrease S-nitrosylation and possibly modify the senescent state of affected cells without destroying them.

In contrast, the increased GSNOR seen in cognitive dysfunction requires a therapeutic that stimulates NO or reduces GSNOR. Injection of l-arginine appeared to reverse at least some cognitive defects associated with excessive GSNOR, and might work similarly in aging animals, but toxicity associated with a direct NO door or nNOS activation agent might be problematic because of the number of potential targets. More specific nitrosating agents for the key targets might be superior candidates for clinical development. ³⁰ But again, the Goldilocks principle erects a substantial challenge.

Thus, whether these dueling observations can be translated to human conditions associated with aging remains an open question.

Summary

GSNOR, a critical denitrosylase, is downregulated in senescence by hypermethylation of its promoter by TET1 and upregulated in aging hippocampuses, leading to loss of synaptic plasticity and cognitive dysfunction. GSNOR deficiency increases global S-nitrosylation, whereas GSNOR excess decreases global S-nitrosylation. Among critical downstream target proteins for decreased GSNOR encountered in senescence, Drp1 and Parkin are important control points in mitochondrial homeostasis with resulting impairment of mitophagy. Likewise, CaMKIIa is a critical target of increased GSNOR through loss of nitrosylated residues. Loss of active CaMKIIa at synaptosomes due to reduced nitrosylation leads to cognitive dysfunction in aged mice and perhaps humans. Development of appropriate drugs to overcome GSNOR dysregulation will require threading the needle between differential GSNOR dysregulation in senescent and dysfunctional chronologically aged cells.