Nidia Quillinan

Androgen and PARP-1 regulation of TRPM2 channels after ischemic injury

The calcium-permeable transient receptor potential M2 (TRPM2) ion channel was recently demonstrated to have a sexually dimorphic contribution to ischemic brain injury, with inhibition or knockdown of the channel protecting male brain preferentially. We tested the hypothesis that androgen signaling is required for this male-specific cell-death pathway. Additionally, we tested the hypothesis that differential activation of the enzyme poly (ADP-ribose) polymerase-1 (PARP-1) is responsible for male-specific TRPM2 channel activation and neuronal injury. We observed that administration of the TRPM2 inhibitor clotrimazole (CTZ) 2 hours after onset of ischemia reduced infarct volume in male mice and that protection from ischemic damage by CTZ was abolished by removal of testicular androgens (castration; CAST) and rescued by androgen replacement. Male PARP-1 knockout mice had reduced ischemic damage compared with WT mice and inhibition of TRPM2 with CTZ failed to reduce infarct size. Lastly, we observed that ischemia increased PARP activity in the peri-infarct region of male mice to a greater extent than female mice and the difference was abolished in CAST male mice. Data presented in the current study indicate that TRPM2-mediated neuronal death in the male brain requires intact androgen signaling and PARP-1 activity.

Androgens and stroke: good, bad or indifferent?

Cerebral ischemia caused by loss of blood supply to the brain during cardiac arrest or stroke are major causes of death and disability. Biological sex is an important factor in predicting vulnerability of the brain to an ischemic insult, with males being at higher risk for cardio-cerebrovascular events than females of the same age. However, relative incidence of stroke between the genders appears to normalize at advanced ages. Therefore, many scientists have focused on the mechanisms of sex differences in outcome following brain ischemic injury, with a particular emphasis on the role of sex steroids. The majority of studies indicate that female sex steroids, such as estrogen and progesterone, play important roles in the relative neuroprotection following cerebral ischemia observed in females. However, less is known about male sex steroids and brain damage. This review describes the state of our knowledge of androgen-related contributions to neurological injury and recovery following cerebral ischemia that occurs following stroke. Experimental studies examining the effects of castration, androgenic agonists and antagonists and aging provide valuable insights into the role of androgens in clinical outcome following cerebrovascular events.

Increasing small conductance Ca2+‐activated potassium channel activity reverses ischemia‐induced impairment of long‐term potentiation

Global cerebral ischemia following cardiac arrest and cardiopulmonary resuscitation (CA/CPR) causes injury to hippocampal CA1 pyramidal neurons and impairs cognition. Small conductance Ca2+‐activated potassium channels type 2 (SK2), expressed in CA1 pyramidal neurons, have been implicated as potential protective targets. Here we showed that, in mice, hippocampal long‐term potentiation (LTP) was impaired as early as 3 h after recovery from CA/CPR and LTP remained impaired for at least 30 days. Treatment with the SK2 channel agonist 1‐Ethyl‐2‐benzimidazolinone (1‐EBIO) at 30 min after CA provided sustained protection from plasticity deficits, with LTP being maintained at control levels at 30 days after recovery from CA/CPR. Minimal changes in glutamate release probability were observed at delayed times after CA/CPR, implicating post‐synaptic mechanisms. Real‐time quantitative reverse transcriptase‐polymerase chain reaction indicated that CA/CPR did not cause a loss of N‐methyl‐D‐aspartate (NMDA) receptor mRNA at 7 or 30 days after CA/CPR. Similarly, no change in synaptic NMDA receptor protein levels was observed at 7 or 30 days after CA/CPR. Further, patch‐clamp experiments demonstrated no change in functional synaptic NMDA receptors at 7 or 30 days after CA/CPR. Electrophysiology recordings showed that synaptic SK channel activity was reduced for the duration of experiments performed (up to 30 days) and that, surprisingly, treatment with 1‐EBIO did not prevent the CA/CPR‐induced loss of synaptic SK channel function. We concluded that CA/CPR caused alterations in post‐synaptic signaling that were prevented by treatment with the SK2 agonist 1‐EBIO, indicating that activators of SK2 channels may be useful therapeutic agents to prevent ischemic injury and cognitive impairments.

Neuropathophysiology of Brain Injury

Every year in the United States, millions of individuals incur ischemic brain injury from stroke, cardiac arrest, or traumatic brain injury. These acquired brain injuries can lead to death or long-term neurologic and neuropsychological impairments. The mechanisms of ischemic and traumatic brain injury that lead to these deficiencies result from a complex interplay of interdependent molecular pathways, including excitotoxicity, acidotoxicity, ionic imbalance, oxidative stress, inflammation, and apoptosis. This article reviews several mechanisms of brain injury and discusses recent developments. Although much is known from animal models of injury, it has been difficult to translate these effects to humans.

Extended therapeutic window of a novel peptide inhibitor of TRPM2 channels following focal cerebral ischemia.

INTRODUCTION TRPM2 channels have been suggested to play a role in ischemic neuronal injury, specifically in males. A major hindrance to TRPM2 research has been the lack of specific TRPM2 inhibitors. The current study characterized the specificity and neuroprotective efficacy of a novel TRPM2 inhibitor. METHODS Fluorescent calcium imaging (Fluo5F) was used to determine inhibitor efficacy of the TRPM2 peptide inhibitor (tat-M2NX) in HEK293 cells stably expressing hTRPM2. Adult (2-3months) and aged (18-20months) mice were subjected to 60min middle cerebral artery occlusion (MCAO) and injected with tat-M2NX, control scrambled peptide (tat-SCR) or clotrimazole (CTZ) either 20min prior or 3h after reperfusion. Infarct size was assessed using TTC staining. RESULTS TRPM2 inhibition by tat-M2NX was observed by decreased Ca(2+) influx following H2O2 exposure human TRPM2 expressing cells. Male mice pre-treated with tat-M2NX had smaller infarct volume compared to tat-SCR. No effect of tat-M2NX on infarct size was observed in female mice. Importantly, male TRPM2(-/-) mice were not further protected by tat-M2NX, demonstrating selectivity of tat-M2NX. Administration of tat-M2NX 3h after reperfusion provided significant protection to males when analyzed at 24h or 4days after MCAO. Finally, we observed that tat-M2NX reduced ischemic injury in aged male mice. CONCLUSIONS These data demonstrate the development of a new peptide inhibitor of TRPM2 channels that provides protection from ischemic stroke in young adult and aged male animals with a clinically relevant therapeutic window.

A novel mouse model of pediatric cardiac arrest and cardiopulmonary resuscitation reveals age-dependent neuronal sensitivities to ischemic injury.

BACKGROUND Pediatric sudden cardiac arrest (CA) is an unfortunate and devastating condition, often leading to poor neurologic outcomes. However, little experimental data on the pathophysiology of pediatric CA is currently available due to the scarcity of animal models. NEW METHOD We developed a novel experimental model of pediatric cardiac arrest and cardiopulmonary resuscitation (CA/CPR) using postnatal day 20-25 mice. Adult (8-12 weeks) and pediatric (P20-25) mice were subjected to 6min CA/CPR. Hippocampal CA1 and striatal neuronal injury were quantified 3 days after resuscitation by hematoxylin and eosin (H&E) and Fluoro-Jade B staining, respectively. RESULTS Pediatric mice exhibited less neuronal injury in both CA1 hippocampal and striatal neurons compared to adult mice. Increasing ischemia time to 8 min CA/CPR resulted in an increase in hippocampal injury in pediatric mice, resulting in similar damage in adult and pediatric brains. In contrast, striatal injury in the pediatric brain following 6 or 8 min CA/CPR remained extremely low. As observed in adult mice, cardiac arrest causes delayed neuronal death in pediatric mice, with hippocampal CA1 neuronal damage maturing at 72 h after insult. Finally, mild therapeutic hypothermia reduced hippocampal CA1 neuronal injury after pediatric CA/CPR. COMPARISON WITH EXISTING METHOD This is the first report of a cardiac arrest and CPR model of global cerebral ischemia in mice. CONCLUSIONS Therefore, the mouse pediatric CA/CPR model we developed is unique and will provide an important new tool to the research community for the study of pediatric brain injury.

Sirtuin-2 mediates male specific neuronal injury following experimental cardiac arrest through activation of TRPM2 ion channels.

OBJECTIVE Sirtuins (Sirt) are a class of deacetylase enzymes that play an important role in cell proliferation. Sirt2 activation produces O-acetylated-ADPribose (OAADPr) which can act as a ligand for transient receptor potential cation channel, M2 (TRPM2). We tested the hypothesis that Sirt2 is activated following global cerebral ischemia and contributes to neuronal injury through activation of TRPM2. METHODS Adult male and female mice (8-12 weeks old) C57Bl/6 and TRPM2 knock-out mice were subjected to 8 min of cardiac arrest followed by cardiopulmonary resuscitation (CA/CPR). The Sirt2 inhibitor AGK-2 was administered intravenously 30 min after resuscitation. Hippocampal CA1 injury was analyzed at 3 days after CA/CPR. Acute Sirt2 activity was analyzed at 3 and 24 h after CA/CPR. Long-term hippocampal function was assessed using slice electrophysiology 7 days after CA/CPR. RESULTS AGK-2 significantly reduced CA1 injury in WT but not TRPM2 knock-out males and had no effect on CA1 injury in females. Elevated Sirt2 activity was observed in hippocampal tissue from males at 24 h after cardiac arrest and was reduced by AGK-2. In contrast, Sirt2 activity in females was increased at 3 but not 24 h. Finally, we observed long-term benefit of AGK-2 on hippocampal function, with a protection of long-term potentiation at CA1 synapses at 7 and 30 days after ischemia. CONCLUSIONS In summary, we observed a male specific activation of Sirt2 that contributes to neuronal injury and functional deficits after ischemia specifically in males. These results are consistent with a role of Sirt2 in activating TRPM2 following global ischemia in a sex specific manner. These results support the growing body of literature showing that oxidative stress mechanisms predominate in males and converge on TRPM2 activation as a mediator of cell death.

Experimental Pediatric Arterial Ischemic Stroke Model Reveals Sex-specific Estrogen Signaling

Background and Purpose— Pediatric stroke, birth to 18 years, is a significant cause of long-term disability in the United States; however, there is currently little experimental data on the pathophysiology of childhood stroke owing to lack of animal models. We developed a novel mouse model of experimental childhood-onset arterial ischemic stroke to characterize the sex-specific response of the adolescent brain to cerebral ischemia and assess the neuroprotective effect of estrogen at this developmental stage. Methods— Postnatal day 20 to 25 mice were subjected to 90 minutes experimental stroke via the intraluminal filament middle cerebral artery occlusion model and ischemic damage assessed 22 hours after reperfusion. Real-time quantitative real-time polymerase chain reaction was performed 22 hours after middle cerebral artery occlusion to determine the effects of ischemia and estrogen treatment on the proapoptotic gene Bax. Results— Ischemic injury did not differ between male and female juvenile (postnatal day 20–25) mice after middle cerebral artery occlusion. However, estrogen reduced ischemic injury in female mice, whereas having no effect in juvenile males. No differences in estrogen receptor expression were observed on postnatal day between 20 males and females. In contrast, estrogen minimized the ischemia-induced increase in the proapoptotic gene Bax in female mice, whereas having no effect on Bax induction in the male brain. Conclusions— Focal ischemia has fundamentally different effects in the juvenile brain compared with the adult, as evidenced by the lack of sex difference in ischemic injury in the murine postnatal day 20 to 25 middle cerebral artery occlusion model and the sexually dimorphic response to estrogen neuroprotection.

Alterations in Purkinje cell GABAA receptor pharmacology following oxygen and glucose deprivation and cerebral ischemia reveal novel contribution of β1‐subunit‐containing receptors

Cerebellar Purkinje cells (PCs) are particularly sensitive to cerebral ischemia, and decreased GABAA receptor function following injury is thought to contribute to PC sensitivity to ischemia‐induced excitotoxicity. Here we examined the functional properties of the GABAA receptors that are spared following ischemia in cultured Purkinje cells from rat and in vivo ischemia in mouse. Using subunit‐specific positive modulators of GABAA receptors, we observed that oxygen and glucose deprivation (OGD) and cardiac arrest‐induced cerebral ischemia cause a decrease in sensitivity to the β2/3‐subunit‐preferring compound, etomidate. However, sensitivity to propofol, a β‐subunit‐acting compound that modulates β1–3‐subunits, was not affected by OGD. The α/γ‐subunit‐acting compounds, diazepam and zolpidem, were also unaffected by OGD. We performed single‐cell reverse transcription–polymerase chain reaction on isolated PCs from acutely dissociated cerebellar tissue and observed that PCs expressed the β1‐subunit, contrary to previous reports examining GABAA receptor subunit expression in PCs. GABAA receptor β1‐subunit protein was also detected in cultured PCs by western blot and by immunohistochemistry in the adult mouse cerebellum and levels remained unaffected by ischemia. High concentrations of loreclezole (30 μm) inhibited PC GABA‐mediated currents, as previously demonstrated with β1‐subunit‐containing GABAA receptors expressed in heterologous systems. From our data we conclude that PCs express the β1‐subunit and that there is a greater contribution of β1‐subunit‐containing GABAA receptors following OGD.

Autonomous CaMKII Activity as a Drug Target for Histological and Functional Neuroprotection after Resuscitation from Cardiac Arrest

SUMMARY The Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a major mediator of physiological glutamate signaling, but its role in pathological glutamate signaling (excitotoxicity) remains less clear, with indications for both neurotoxic and neuro-protective functions. Here, the role of CaMKII in ischemic injury is assessed utilizing our mouse model of cardiac arrest and cardiopulmonary resuscitation (CA/CPR). CaMKII inhibition (with tatCN21 or tatCN19o) at clinically relevant time points (30 min after resuscitation) greatly reduces neuronal injury. Importantly, CaMKII inhibition also works in combination with mild hypothermia, the current standard of care. The relevant drug target is specifically Ca2+-independent “autonomous” CaMKII activity generated by T286 autophosphorylation, as indicated by substantial reduction in injury in autonomy-incompetent T286A mutant mice. In addition to reducing cell death, tatCN19o also protects the surviving neurons from functional plasticity impairments and prevents behavioral learning deficits, even at extremely low doses (0.01 mg/kg), further highlighting the clinical potential of our findings.