Abstract
The microtubule-associated axonal specification collapsin response mediator protein 2 (CRMP2) is a novel target for neuroprotection. A CRMP2 peptide (TAT-CBD3) conjugated to the HIV transactivator of transcription (TAT) protein’s cationic cell penetrating peptide (CPP) motif protected neurons in the face of toxic levels of Ca2+ influx leaked in via N-methyl-D-aspartate receptor (NMDAR) hyperactivation. Here we tested whether replacing the hydrophilic TAT motif with alternative cationic (nona-arginine (R9)), hydrophobic (membrane transport sequence (MTS) of k-fibroblast growth factor) or amphipathic (model amphipathic peptide (MAP)) CPPs could be superior to the neuroprotection bestowed by TAT-CBD3. In giant plasma membrane vesicles (GPMVs) derived from cortical neurons, the peptides translocated across plasma membranes with similar efficiencies. Cortical neurons, acutely treated with peptides prior to a toxic glutamate challenge, demonstrated enhanced efflux of R9-CBD3 compared to others. R9-CBD3 inhibited N-methyl-D-aspartate (NMDA)-evoked Ca2+ influx to a similar extent as TAT-CBD3 while MTS-CBD3 was ineffective which correlated with the ability of R9- and TAT-CBD3, but not MTS-CBD3, to block NMDAR interaction with CRMP2. Unrestricted Ca2+ influx through NMDARs leading to delayed calcium dysregulation and neuronal cell death was blocked by all peptides but MAP-CBD3. When applied acutely for 10 min, R9-CBD3 was more effective than TAT-CBD3 at neuroprotection while MTS- and MAP-CBD3 were ineffective. In contrast, long-term (>24 h) treatment with MTS-CBD3 conferred neuroprotection where TAT-CBD3 failed. Neither peptide altered surface trafficking of NMDARs. Neuroprotection conferred by MTS-CBD3 peptide is likely due to its increased uptake coupled with decreased efflux when compared to TAT-CBD3. Overall, our results demonstrate that altering CPPs can bestow differential neuroprotective potential onto the CBD3 cargo.
Highlights
Excitotoxicity is characterized as a pathological process by which a disproportionate exposure to the neurotransmitter glutamate leads to an overstimulation of its cognate membrane receptors
RATIONALE FOR SELECTION OF CATIONIC AND AMPHIPATHIC CELL PENETRATING PEPTIDES (CPPs) We recently reported that collapsin response mediator protein 2 (CRMP2) knockdown is neuroprotective (Brittain et al, 2011a) and that the CRMP2 peptide, TAT-channel binding domain 3 (CBD3), is able to prevent neuronal cell death from toxic glutamate exposure via inhibition of N-methyl-D-aspartate receptors (NMDARs)-mediated Ca2+-influx (Brittain et al, 2011a, 2012)
Three additional cell penetrating peptides (CPPs) were selected: (i) the nona-arginine (R9) which has a half-life (t1/2) of ∼2 h (Sarko et al, 2010) and a greater than 20-fold penetration compared to TAT (Wender et al, 2000) using macropinocytosis; (ii) the α-helical model amphipathic peptide (MAP) with a t1/2 of >72 h (Sarko et al, 2010) which translocates cargo into cells in a non-endocytic fashion using multiple non-specific, energy-dependent and -independent processes (Oehlke et al, 1998); and (iii) the membrane translocating sequence (MTS) of Kaposi fibroblast growth factor (k-FGF) receptor with a t1/2 of ∼48 h (Sarko et al, 2010) which has a hydrophobic stretch of residues necessary for import into cells that likely occurs via a non-endocytotic route using an energyand temperature-independent translocation process reliant on its interactions with the membrane (Lin et al, 1995)
Summary
Excitotoxicity is characterized as a pathological process by which a disproportionate exposure to the neurotransmitter glutamate leads to an overstimulation of its cognate membrane receptors. NMDAR has a number of sites that have been exploited pharmacologically, including the ion channel pore, the glutamate-binding site, the glycine-binding site and the polyamine interaction site, but since NMDAR activity is crucial for normal neuronal function, the efforts to develop NMDAR antagonists have unequivocally failed in clinical trials due to their toxicity (Ikonomidou et al, 2000; Ikonomidou and Turski, 2002; Muir, 2006) In this context, targeting the proteins regulating the NMDAR may offer an advantage in preventing excitotoxicity with the possibility of minimal side effects.
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