## 5 Do All CARPs Including TAT-Fused Peptides Share a Common Neuroprotective Mechanism of Action?  
Based on previous studies in our and other laboratories, examining the neuroprotective properties of various CARPs, we view it highly likely that the neuroprotection demonstrated by the CARPs described above (i.e., TAT-NBD, TAT-mGluR1, JNKI-1-TATL, JNKI-1-TATD, Sab\({}_{\text{KIM1}}\), TAT-BH4, P5-TAT, D-TAT-GESV, TAT-NR2B9c, R18, R18D, COG133, Apelin-36 and IDR-1018) is largely mediated by peptide arginine content and positive charge [19; 27; 29; 65; 66]. There are several lines of evidence that support this hypothesis, which we provide below. In addition, we previously provided an explanation why we believe a CARP-mediated neuroprotective mechanism of action is operating for the TAT-NR2B9c and JNKI-1-TATD peptides, rather than the mechanism of action in which they were originally developed [27].  
It has been demonstrated that CARPs inhibit neuronal excitotoxic calcium [19; 22; 23; 25; 27; 28] and potassium [64; 75] influx and in doing so inhibit many of the pathophysiological processes downstream from the ischemic excitotoxic cascade (e.g., calpain activation, mitochondrial dysfunction and ROS production, calcium induced cell signaling and lipase and DNase activation). Therefore, given that the vast majority of the proposed mechanisms of action of developed TAT-fused and other neuroprotective CARPs are molecular targets downstream from excitotoxicity (e.g., NF-kB, JNK and Cdk5), it is not surprising that they are effective and appear to be inhibiting their intended targets.  
Interestingly, in addition to the inhibition of excitotoxic cationic ion influx, CARPs also have the capacity to modulate other cellular processes that are neuroprotective following HI such as proteolytic enzyme activation (e.g., proteasome and MMPs), ERK-1/2 and Akt phosphorylation and the modulation of immune responses. For example, in the case of TAT-NBD, which was developed to block NF-kB activation, the ability of CARPs to inhibit the proteasome [84; 85; 176; 177] would have the effect of preventing NF-kB activation by preventing proteasomal degradation of the NF-kB inhibitor protein i-kB.  
In the case of Apelin-36, other CARPs, such as protamine and R9 (ALX40-4C) can also bind to the apelin receptor [178; 179]. Therefore, it is possible that CARPs, in addition to Apelin-36, may also be able to stimulate pro-cell survival ERK-1/2 and Akt pathways [180]. Although it should be mentioned that not surprisingly, Apelin-36 can also reduce excitotoxic calcium influx [169; 170; 171]. In the case of CARP-mediated immune modulation, mR18L (Ac-GFRRFLGSWARIYRAFVG-NH\({}_{2}\); net charge +4) is known to reduce LPS-induced systemic infection [181], dRK6 (RRKRRR; net charge +6) inhibits TNF\(\alpha\) and IL-6 production by monocytes [182] and hBD3-3 (GKCSTRGRKCCRRKK; net charge +8) downregulates NF-kB induced inflammation [183]. Thus, CARPs have the potential to suppress many of the toxic inflammatory responses associated with HIE.  
Future studies examining the efficacy of putative neuroprotective peptides fused to TAT would benefit with respect to confirming neuroprotective mechanisms of action by fusing the peptide to a non-cationic CPP (e.g., MTS; AAVALLPAVLLALLP) and/or replacing any negatively charged aspartate (D) and glutamate (E) residues within the peptide sequence with arginine residues; we predict these two manipulations will decrease and increase neuroprotective efficacy, respectively. It is also advisable to confirm if the TAT-fused peptide has the capacity to reduce excitotoxic calcium influx.