Disruption of NMDAR-CRMP-2 signaling protects against focal cerebral ischemic damage in the rat middle cerebral artery occlusion model

Abstract

Collapsin response mediator protein 2 (CRMP-2), traditionally viewed as an axon/dendrite specification and axonal growth protein, has emerged as nidus in regulation of both pre- and post-synaptic Ca ( 2+) channels. Building on our discovery of the interaction and regulation of Ca ( 2+) channels by CRMP-2, we recently identified a short sequence in CRMP-2 which, when appended to the transduction domain of HIV TAT protein, suppressed acute, inflammatory and neuropathic pain in vivo by functionally uncoupling CRMP-2 from the Ca ( 2+) channel. Remarkably, we also found that this region attenuated Ca ( 2+) influx via N-methylD-Aspartate receptors (NMDARs) and reduced neuronal death in a moderate controlled cortical impact model of traumatic brain injury (TBI). Here, we sought to extend these findings by examining additional neuroprotective effects of this peptide (TAT-CBD3) and exploring the biochemical mechanisms by which TAT-CBD3 targets NMDARs. We observed that an intraperitoneal injection of TAT-CBD3 peptide significantly reduced infarct volume in an animal model of focal cerebral ischemia. Neuroprotection was observed when TAT-CBD3 peptide was given either prior to or after occlusion but just prior to reperfusion. Surprisingly, a direct biochemical complex was not resolvable between the NMDAR subunit NR2B and CRMP-2. Intracellular application of TAT-CBD3 failed to inhibit NMDAR current. NR2B interactions with the post synaptic density protein 95 (PSD-95) remained intact and were not disrupted by TAT-CBD3. Peptide tiling of intracellular regions of NR2B revealed two 15-mer sequences, in the carboxyl-terminus of NR2B, that may confer binding between NR2B and CRMP-2 which supports CRMP-2's role in excitotoxicity and neuroprotection.

Keywords: CRMP-2; MCAO model; N-methylD-aspartate receptor; focal cerebral ischemia; peptide.

Figure 1. TAT-CBD3 reduces infarct volume following focal cerebral ischemia. (A) Ten day old Sprague-Dawley rats were challenged with focal cerebral ischemia by MCAO. The occlusion lasted for 2 h and infarct volume was assessed 48 h after the onset of reperfusion. (B) Animals were pre-treated with either 20 mg/kg TAT-CBD3 of saline 1 h prior to occlusion. Representative images of TTC stained cortices are shown. (C) Bar graph representation of surviving brain volume (% ischemic vs. contralateral) compared between saline and TAT-CBD3 pre-treated animals. (D) Animals were treated with either 20 mg/kg TAT-CBD3 or saline 15 min prior to reperfusion. (E) Bar graph representation of surviving brain volume (% ischemic vs. contralateral) compared between animals treated with TAT-CBD3 or saline 15 min prior to reperfusion.

Figure 2. Protective effect of TAT-CBD3 against glutamate-induced Ca²⁺ dysregulation and mitochondrial depolarization. Tat-CBD3 failed to affect the resting cytosolic Ca²⁺ and mitochondrial membrane potential (Δψ). In (A–D), the representative individual (thin, gray traces obtained from individual neurons) and average Fura-2FF (F₃₄₀/F₃₈₀, thick gray traces) (A) and (C) and Rhodamine 123 (Rh123, F/F₀, thick black traces) fluorescence signals (B) and (D). Neurons were treated with 25 μM glutamate (plus 10μM glycine) as indicated. In (C) and (D), neurons were pre-incubated for 10 min with 10 μM TAT-CBD3.

Figure 5. Two NMDAR carboxyl-terminus peptides bind to CRMP-2. Peptides (15-mers), with a moving window of three amino acids, were generated for intracellular regions of the rat NMDAR. The SPOTS membranes were overlaid with rat brain lysates and then probed with polyclonal CRMP-2 and monoclonal phospho-CRMP-2 (3F4) antibodies. The intensity of each spot was measured using the Odyssey infrared imaging system utilizing the Li-Cor Biosciences one-dimensional software. Graphs represent normalized binding of poly (top) and mono (bottom) CRMP-2 antibodies to NMDAR peptides. Peptides were defined to bind if binding with both antibodies was observed above an arbitrary threshold of 0.6 of maximum. Using these criteria, two carboxyl-terminal peptides bound CRMP-2. The sequences of these peptides are highlighted in boldface font.

Figure 3. TAT-CBD3 does not intracellularly modulate NMDA current. The time course of NMDA current rundown in the condition that the pipette solution was supplemented with or without 10 μM TAT-CBD3. Peptide TAT-CBD3 was internally microdialyzed via the patch pipette. Current amplitude was normalized to the current evoked by the first application of NMDA (50 μM) at Time 0. No significant difference of current rundown between two conditions during the 15 min time course.

Figure 4. CRMP-2 is not found in a complex with NR2B. The biochemical interaction between CRMP-2 and NR2B was investigated using in vitro binding assays. (A) Lysates from E19 DIV 8 cortical neurons were pre-incubated with vehicle (0.5% DMSO), TAT-Control (100 μM), or TAT-CBD3 (100 μM) prior to performing co-immunoprecipitations (IP w/) using an anti-NR2B antibody and immunoblotted for NR2B, PSD-95, and CRMP-2 as indicated. The commercial suppliers of the antibodies are indicated in parentheses. (B) Postnatal day 2 rat brain fractions were used to perform co-immunoprecipitations using anti-CRMP-2 C4G followed by western blot (WB) analyses. (C) Cortical neuron lysates were used to coimmunoprecipitate NR2B using two additional NR2B antibodies antibody and immunoblotted as in (A). (D) Bacterially expressed purified GST (control) or CRMP-2 GST proteins were utilized in pull downs (PD) from cortical neuron lysates along with either a monoclonal IgG (control) or NR2B antibody, and then the immune-captured complexes were blotted with NR2B and CRMP-2 antibodies. Both GST tagged and endogenous CRMP-2 are indicated. Molecular weight markers are indicated in kilodaltons (kDa).