Robert M. Dietz

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.

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.

Therapeutic hypothermia protects against ischemia-induced impairment of synaptic plasticity following juvenile cardiac arrest in sex-dependent manner

Pediatric cardiac arrest (CA) often leads to poor neurologic outcomes, including deficits in learning and memory. The only approved treatment for CA is therapeutic hypothermia, although its utility in the pediatric population remains unclear. This study analyzed the effect of mild therapeutic hypothermia after CA in juvenile mice on hippocampal neuronal injury and the cellular model of learning and memory, termed long-term potentiation (LTP). Juvenile mice were subjected to cardiac arrest and cardiopulmonary resuscitation (CA/CPR) followed by normothermia (37°C) and hypothermia (30°C, 32°C). Histological injury of hippocampal CA1 neurons was performed 3days after resuscitation using hematoxylin and eosin (H&E) staining. Field excitatory post-synaptic potentials (fEPSPs) were recorded from acute hippocampal slices 7days after CA/CPR to determine LTP. Synaptic function was impaired 7days after CA/CPR. Mice exposed to hypothermia showed equivalent neuroprotection, but exhibited sexually dimorphic protection against ischemia-induced impairment of LTP. Hypothermia (32°C) protects synaptic plasticity more effectively in females, with males requiring a deeper level of hypothermia (30°C) for equivalent protection. In conclusion, male and female juvenile mice exhibit equivalent neuronal injury following CA/CPR and hypothermia protects both males and females. We made the surprising finding that juvenile mice have a sexually dimorphic response to mild therapeutic hypothermia protection of synaptic function, where males may need a deeper level of hypothermia for equivalent synaptic protection.

The role of T‐type calcium channels in the subiculum: to burst or not to burst?

Pharmacological, molecular and genetic data indicate a prominent role of low‐voltage‐activated T‐type calcium channels (T‐channels) in the firing activity of both pyramidal and inhibitory interneurons in the subiculum. Pharmacological inhibition of T‐channels switched burst firing with lower depolarizing stimuli to regular spiking, and fully abolished hyperpolarization‐induced burst firing. Our molecular studies showed that CaV3.1 is the most abundantly expressed isoform of T‐channels in the rat subiculum. Consistent with this finding, both regular‐spiking and burst firing patterns were profoundly depressed in the mouse with global deletion of CaV3.1 isoform of T‐channels. Selective inhibition of T‐channels and global deletion of CaV3.1 channels completely suppressed development of long‐term potentiation (LTP) in the CA1–subiculum, but not in the CA3–CA1 pathway.

Mild myelin disruption elicits early alteration in behavior and proliferation in the subventricular zone

Myelin, the insulating sheath around axons, supports axon function. An important question is the impact of mild myelin disruption. In the absence of the myelin protein proteolipid protein (PLP1), myelin is generated but with age, axonal function/maintenance is disrupted. Axon disruption occurs in Plp1-null mice as early as 2 months in cortical projection neurons. High-volume cellular quantification techniques revealed a region-specific increase in oligodendrocyte density in the olfactory bulb and rostral corpus callosum that increased during adulthood. A distinct proliferative response of progenitor cells was observed in the subventricular zone (SVZ), while the number and proliferation of parenchymal oligodendrocyte progenitor cells was unchanged. This SVZ proliferative response occurred prior to evidence of axonal disruption. Thus, a novel SVZ response contributes to the region-specific increase in oligodendrocytes in Plp1-null mice. Young adult Plp1-null mice exhibited subtle but substantial behavioral alterations, indicative of an early impact of mild myelin disruption.

Oxidative stress diseases unique to the perinatal period: A window into the developing innate immune response

The innate immune system has evolved to play an integral role in the normally developing lung and brain. However, in response to oxidative stress, innate immunity, mediated by specific cellular and molecular programs and signaling, contributes to pathology in these same organ systems. Despite opposing drivers of oxidative stress, namely hyperoxia in neonatal lung injury and hypoxia/ischemia in neonatal brain injury, similar pathways—including toll‐like receptors, NFκB and MAPK cascades—have been implicated in tissue damage. In this review, we consider recent insights into the innate immune response to oxidative stress in both neonatal and adult models to better understand hyperoxic lung injury and hypoxic‐ischemic brain injury across development and aging. These insights support the development of targeted immunotherapeutic strategies to address the challenge of harnessing the innate immune system in oxidative stress diseases of the neonate.