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

Abstract

SUMMARYThe 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.

Simultaneous Live Imaging of Multiple Endogenous Proteins Reveals a Mechanism for Alzheimer’s-Related Plasticity Impairment

“…Thus, we decided to test if CaMKII inhibition also abolishes the Aβ-induced block of CaMKII movement after a cLTP stimulus. In presence of either of two mechanistically distinct CaMKII inhibitors, KN93 or tatCN21 (Deng et al, 2017; Sumi et al, 1991; Vest et al, 2007), Aβ no longer blocked the cLTP-induced CaMKII movement (Figure 4D). Notably, either of the two inhibitors is also sufficient to prevent the Aβ-induced LTP impairment (Opazo et al, 2018).…”

“…Experiments were performed as previously described (Coultrap et al, 2014; Deng et al, 2017), however, dissociated rat hippocampal neurons (plated in 10 cm dishes, 2,500,000–3,000,000 neurons/dish) were used instead of whole hippocampi. Neurons were maintained as described and were treated in the same conditions as used for imaging then washed 3X in fresh ACSF before fractionation.…”

Analysis of the CaMKIIα and β splice-variant distribution among brain regions reveals isoform-specific differences in holoenzyme formation

“…For tissue isolation an preparation, mice were transcardially perfused under isoflurane anesthesia with phosphate-buffered saline for five minutes, followed by five minutes of fixation with 4% paraformaldehyde. Their brains were removed, allowed to post-fix in 4% PFA for 24 hours, and embedded in paraffin, as previously described 60 , 61 . Coronal sections were cut in 6 µm thickness, and every sixth section was mounted in series onto slides for further processing.…”

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

“…; Deng et al . ). The amount of potentiation was calculated as the percentage change from baseline (the averaged 10 min slope value from 50 to 60 min post‐TBS divided by the averaged slope value at 10 min prior to TBS).…”

“…fEPSPs were monitored as above. LTP for both the CA3-CA1 and CA1-subiculum pathway was induced by delivering a theta burst stimulation (TBS) train (four pulses delivered at 100 Hz in 30 ms bursts, repeated 10 times with 200 ms interburst intervals) as described previously (Orfila et al 2014;Dietz et al 2016;Deng et al 2017). The amount of potentiation was calculated as the percentage change from baseline (the averaged 10 min slope value from 50 to 60 min post-TBS divided by the averaged slope value at 10 min prior to TBS).…”