## 4 Neuoprotective Peptides and Their Therapeutic Application in HIE
### Studies Using CARPs in Animal Models of HIE
#### 4.4.3 c-Jun N-Terminal Kinase (JNK) Inhibitors  
JNKI-1 is a 20-amino acid peptide (also referred to as JBD: RPKRPTTLNLFPQVPRSQDT\({}_{157-176}\); net charge +3), derived from the signaling adaptor protein c-Jun N-terminal kinase protein-1 (JNKI-1) [112]. JNKs are a member of the mitogen-activated protein kinases (MAPK) family, encoded by the _Jnk1_, _Jnk2_ and _Jnk3_ genes, with high expression levels in neurons, especially after pathological injury (e.g., excitotoxicity, stroke, epilepsy and HI) [113]. The JNKI-1 peptide can inhibit JNK interaction with JNK-interacting protein-1 (JIP-1), blocking JNK phosphorylation and activation, thereby inhibiting downstream pro-death cellular signaling pathways [112]. JNK has emerged as a central mediator of excitotoxic damage in the developing [114; 115] and developed CNS [116; 117]. The JNKI-1 peptide derivatives bound to TAT, such as TAT-JNKI-1L (YGRKKRRQRRR-PP-RPKRPTTLNLFPQVPRSQDT-NH\({}_{2}\), net charge +12) and its retro inverso D-enantiomer JNKI-1-TATD (tdqsrpvqpfnlttprkpr-pp-rrrqrrkkrgy-NH\({}_{2}\); net charge +12, lower case indicates D-isoform amino acids) have demonstrated in vitro and/or in vivo neuroprotective properties. Studies using the JNKI-1 peptide alone (D-JNKI-1) or when fused to TAT (TAT-JNKI-1L and JNKI-1-TATD) have been assessed in neonatal HIE models. The initial study examining the efficacy of D-JNKI-1 (tdqsrpvqpfnlttprkpr-NH\({}_{2}\); net charge +4) in a P7 rat model of HIE when administered (76 nmol/kg: IP) 30 min before and 3, 5, 8, 12 and 20 h after hypoxia did not reveal any reduction in cerebral infarction at 24 h [115], although there was evidence for reduced calpain, caspase-3 and autophagic activation. A subsequent study demonstrated that the TAT-fused peptide JNKI-1-TATL administered (2446 nmol/kg; IP) immediately and 3 h or 3 h after hypoxia, significantly reduced cerebral infarction at 48 h, while administration 6 h after hypoxia was ineffective [118]. When administered immediately and 3 h after hypoxia, functional benefits were observed 14 weeks post-HI. Despite improvements in cerebral infarct and functional outcomes, JNKI-1-TATL failed to prevent caspase-8-mediated cleavage of Bid, which was unexpected, as activated JNK is known to induce caspase-8 cleavage of Bid and promote mitochondrial pro-apoptotic cell death pathways; this suggests JNKI-1-TATL-mediated neuroprotection was occurring via mechanism independent of JNK suppression. In a second study, the \({}_{\rm D}\)-isoform peptide JNKI-1-TATD (2616 nmol/kg: IP) significantly reduced cerebral infarct volume when administered immediately, 3 or 6 h after hypoxia, but not when administered immediately and 3 h after hypoxia [119]. JNKI-1-TATD treatment also provided long-term functional improvements. It was also demonstrated that treatment with JNKI-1-TATD reduced mitochondrial levels of phosphorylated JNK, preserved mitochondrial integrity, and up-regulated anti-apoptotic proteins 24 h post-HI. The study also assessed a mitochondrial JNK scaffold inhibiting peptide, Sab\({}_{\text{KIM1}}\) (gfeslsvpspldlsgprvva-pp-rrrqrkkrg; net charge +7) and a scrambled control (lpsvfgdvgapssrlpevsls-pp-rrrqrrkkrg; net charge +7); Sab (SH3 domain-binding protein 5) is a mitochondrial scaffold protein required for JNK kinase activity. Administration of Sab\({}_{\text{KIM1}}\) (2700 and 5555 nmol/kg: IP) immediately after hypoxia was neuroprotective, whereas the scrambled peptide (2700 nmol/kg: IP) was ineffective. In our laboratory, administration of JNK-1-TATD (1000 nmol/kg; IP) immediately after hypoxia resulted in a positive trend in reduced total infarct volume (15% reduction) although it did not improve behavioral outcomes 48 h post-HI [22]. It was also demonstrated that in cultured cortical neurons JNK-1-TATD dose-dependently reduced glutamic acid mediated excitotoxic intracellular calcium influx.