TRPM2 and CaMKII Signaling Drives Excessive GABAergic Synaptic Inhibition Following Ischemia.

Excitotoxicity and the concurrent loss of inhibition are well-defined mechanisms driving acute elevation in excitatory/inhibitory (E/I) balance and neuronal cell death following an ischemic insult to the brain. Despite the high prevalence of long-term disability in survivors of global cerebral ischemia (GCI) as a consequence of cardiac arrest, it remains unclear whether E/I imbalance persists beyond the acute phase and negatively affects functional recovery. We previously demonstrated sustained impairment of long-term potentiation (LTP) in hippocampal CA1 neurons correlating with deficits in learning and memory tasks in a murine model of cardiac arrest/cardiopulmonary resuscitation (CA/CPR). Here, we use CA/CPR and an in vitro ischemia model to elucidate mechanisms by which E/I imbalance contributes to ongoing hippocampal dysfunction in male mice. We reveal increased postsynaptic GABAA receptor (GABAAR) clustering and function in the CA1 region of the hippocampus that reduces the E/I ratio. Importantly, reduced GABAAR clustering observed in the first 24 h rebounds to an elevation of GABAergic clustering by 3 d postischemia. This increase in GABAergic inhibition required activation of the Ca2+-permeable ion channel transient receptor potential melastatin-2 (TRPM2), previously implicated in persistent LTP and memory deficits following CA/CPR. Furthermore, we find Ca2+-signaling, likely downstream of TRPM2 activation, upregulates Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity, thereby driving the elevation of postsynaptic inhibitory function. Thus, we propose a novel mechanism by which inhibitory synaptic strength is upregulated in the context of ischemia and identify TRPM2 and CaMKII as potential pharmacological targets to restore perturbed synaptic plasticity and ameliorate cognitive function.

PKCδ Regulates Chromatin Remodeling and DNA Repair through SIRT6.

Irradiation (IR) is a highly effective cancer therapy; however, IR damage to tumor-adjacent healthy tissues can result in significant comorbidities and potentially limit the course of therapy. We have previously shown that protein kinase C delta (PKCδ) is required for IR-induced apoptosis and that inhibition of PKCδ activity provides radioprotection in vivo. Here we show that PKCδ regulates histone modification, chromatin accessibility, and double-stranded break (DSB) repair through a mechanism that requires Sirtuin 6 (SIRT6). Overexpression of PKCδ promotes genomic instability and increases DNA damage and apoptosis. Conversely, depletion of PKCδ increases DNA repair via nonhomologous end joining (NHEJ) and homologous recombination (HR) as evidenced by increased formation of DNA damage foci, increased expression of DNA repair proteins, and increased repair of NHEJ and HR fluorescent reporter constructs. Nuclease sensitivity indicates that PKCδ depletion is associated with more open chromatin, while overexpression of PKCδ reduces chromatin accessibility. Epiproteome analysis reveals increased chromatin associated H3K36me2 in PKCδ-depleted cells which is accompanied by chromatin disassociation of KDM2A. We identify SIRT6 as a downstream mediator of PKCδ. PKCδ-depleted cells have increased SIRT6 expression, and depletion of SIRT6 reverses changes in chromatin accessibility, histone modification and DSB repair in PKCδ-depleted cells. Furthermore, depletion of SIRT6 reverses radioprotection in PKCδ-depleted cells. Our studies describe a novel pathway whereby PKCδ orchestrates SIRT6-dependent changes in chromatin accessibility to regulate DNA repair, and define a mechanism for regulation of radiation-induced apoptosis by PKCδ. IMPLICATIONS: PKCδ controls sensitivity to irradiation by regulating DNA repair.

PKCδ regulates chromatin remodeling and DNA repair through SIRT6.

Protein kinase C delta (PKCδ) is a ubiquitous kinase whose function is defined in part by localization to specific cellular compartments. Nuclear PKCδ is both necessary and sufficient for IR-induced apoptosis, while inhibition of PKCδ activity provides radioprotection in vivo. How nuclear PKCδ regulates DNA-damage induced cell death is poorly understood. Here we show that PKCδ regulates histone modification, chromatin accessibility, and double stranded break (DSB) repair through a mechanism that requires SIRT6. Overexpression of PKCδ promotes genomic instability and increases DNA damage and apoptosis. Conversely, depletion of PKCδ increases DNA repair via non-homologous end joining (NHEJ) and homologous recombination (HR) as evidenced by more rapid formation of NHEJ (DNA-PK) and HR (Rad51) DNA damage foci, increased expression of repair proteins, and increased repair of NHEJ and HR fluorescent reporter constructs. Nuclease sensitivity indicates that PKCδ depletion is associated with more open chromatin, while overexpression of PKCδ reduces chromatin accessibility. Epiproteome analysis revealed that PKCδ depletion increases chromatin associated H3K36me2, and reduces ribosylation of KDM2A and chromatin bound KDM2A. We identify SIRT6 as a downstream mediator of PKCδ. PKCδ-depleted cells have increased expression of SIRT6, and depletion of SIRT6 reverses the changes in chromatin accessibility, histone modification and NHEJ and HR DNA repair seen with PKCδ-depletion. Furthermore, depletion of SIRT6 reverses radioprotection in PKCδ-depleted cells. Our studies describe a novel pathway whereby PKCδ orchestrates SIRT6-dependent changes in chromatin accessibility to increase DNA repair, and define a mechanism for regulation of radiation-induced apoptosis by PKCδ.

Tyrosine kinase inhibitors protect the salivary gland from radiation damage by increasing DNA double-strand break repair.

We have previously shown that the tyrosine kinase inhibitors (TKIs) dasatinib and imatinib can protect salivary glands from irradiation (IR) damage without impacting tumor therapy. However, how they induce this protection is unknown. Here we show that TKIs mediate radioprotection by increasing the repair of DNA double-stranded breaks. DNA repair in IR-treated parotid cells, but not oral cancer cells, occurs more rapidly following pretreatment with imatinib or dasatinib and is accompanied by faster formation of DNA damage-induced foci. Similar results were observed in the parotid glands of mice pretreated with imatinib prior to IR, suggesting that TKIs "prime" cells for DNA repair. Mechanistically, we observed that TKIs increased IR-induced activation of DNA-PK, but not ATM. Pretreatment of parotid cells with the DNA-PK inhibitor NU7441 reversed the increase in DNA repair induced by TKIs. Reporter assays specific for homologous recombination (HR) or nonhomologous end joining (NHEJ) verified regulatation of both DNA repair pathways by imatinib. Moreover, TKIs also increased basal and IR-induced expression of genes associated with NHEJ (DNA ligase 4, Artemis, XLF) and HR (Rad50, Rad51 and BRCA1); depletion of DNA ligase 4 or BRCA1 reversed the increase in DNA repair mediated by TKIs. In addition, TKIs increased activation of the ERK survival pathway in parotid cells, and ERK was required for the increased survival of TKI-treated cells. Our studies demonstrate a dual mechanism by which TKIs provide radioprotection of the salivary gland tissues and support exploration of TKIs clinically in head and neck cancer patients undergoing IR therapy.