Ataxia-telangiectasia (A-T) is an autosomal recessive disorder characterized by many clinical manifestations including neurodegeneration, most notably in the cerebellum resulting in gait ataxia and cancer predisposition. The mutated gene responsible for A-T is ATM (ataxia telangiectasia mutated), which encodes a kinase that activates multiple signal transduction pathways in response to DNA damage. Defects in survival signaling and the DNA damage response in neurons may cause the neurodegenerative pathology of A-T, but ATM substrates in the central nervous system are as yet unclear. Using protein sequence analysis, I have identified four potential ATM consensus phosphorylation motifs and one ATM -interaction motif in the neuronal pro-survival transcription factor, myocyte enhancer factor 2D (MEF2D). MEF2 represents a family of MADS (MCM1-agamous-deficiens- serum response factor) domain-containing transcription factors and plays a critical role in the nervous system regulating neurogenesis, synaptic plasticity and neuronal survival. There are four members of the MEF2 family, MEF2A to -D. MEF2A and -D predominate in the cerebellum, which is most affected in A-T. The activity of MEF2 proteins is governed in part by phosphorylation. Using in vitro immunocomplex kinase assays, I found that ATM phosphorylates MEF2A, -C and -D. DNA damaging agents that induce double-strand breaks increased phosphorylation of MEF2A and -D in cerebellar granule cell neurons. MEF2D phosphorylation was detectable in Atm wild-type cells but not in Atm-deficient cells. GAL4-dependent luciferase reporter gene assays revealed that ATM activates MEF2A and -D activity, but attenuates MEF2C activity. In addition, MEF2-dependent luciferase reporter gene assays showed that ATM increases endogenous MEF2 activity, and the potentiation of endogenous MEF2 activity is abolished either by RNA interference targeting ATM or by a small molecule inhibitor of ATM, KU-55933. Analysis by site- directed mutagenesis indicated that MEF2D is phosphorylated by ATMa t four ATM consensus phosphorylation sites : Thr²⁵⁹, Ser²⁷⁵, Ser²⁹⁴ and Ser³¹⁴. Knockdown of endogenous MEF2D expression by a short- hairpin RNA (shRNA) increased cellular sensitivity to etoposide-induced neuronal cell death. Interestingly, substitution of endogenous MEF2D expression with an shRNA- resistant phosphomimetic MEF2D mutant protected primary neurons from cell death after DNA damage, whereas an shRNA -resistant nonphosphorylatable MEF2D mutant did not. Coimmunoprecipitation studies indicated that ATM and MEF2D form a complex following DNA damage. Collectively, these results suggest that ATM associates with MEF2D and activates its activity via phosphorylation, thus promoting neuronal survival in response to DNA damage.

Redox-mediated posttranslational modifications represent a molecular switch that controls major mechanisms of cell function. Nitric oxide (NO) can mediate redox reactions via S-nitrosylation, representing transfer of an NO group to a critical protein thiol. NO is known to modulate neurogenesis and neuronal survival in various brain regions in disparate neurodegenerative conditions. However, a unifying molecular mechanism linking these phenomena remains unknown. Here, we report that S-nitrosylation of myocyte enhancer factor 2 (MEF2) transcription factors acts as a redox switch to inhibit both neurogenesis and neuronal survival. Structure-based analysis reveals that MEF2 dimerization creates a pocket, facilitating S-nitrosylation at an evolutionally conserved cysteine residue in the DNA binding domain. S-Nitrosylation disrupts MEF2-DNA binding and transcriptional activity, leading to impaired neurogenesis and survival in vitro and in vivo. Our data define a molecular switch whereby redox-mediated posttranslational modification controls both neurogenesis and neurodegeneration via a single transcriptional signaling cascade.

Transcription factor MEF2C regulates multiple genes linked to autism spectrum disorder (ASD), and human MEF2C haploinsufficiency results in ASD, intellectual disability, and epilepsy. However, molecular mechanisms underlying MEF2C haploinsufficiency syndrome remain poorly understood. Here we report that Mef2c ^+/-(Mef2c-het) mice exhibit behavioral deficits resembling those of human patients. Gene expression analyses on brains from these mice show changes in genes associated with neurogenesis, synapse formation, and neuronal cell death. Accordingly, Mef2c-het mice exhibit decreased neurogenesis, enhanced neuronal apoptosis, and an increased ratio of excitatory to inhibitory (E/I) neurotransmission. Importantly, neurobehavioral deficits, E/I imbalance, and histological damage are all ameliorated by treatment with NitroSynapsin, a new dual-action compound related to the FDA-approved drug memantine, representing an uncompetitive/fast off-rate antagonist of NMDA-type glutamate receptors. These results suggest that MEF2C haploinsufficiency leads to abnormal brain development, E/I imbalance, and neurobehavioral dysfunction, which may be mitigated by pharmacological intervention.