The Neuroprotective Efficacy of Cell-Penetrating Peptides TAT, Penetratin, Arg-9, and Pep-1 in Glutamic Acid, Kainic Acid, and In Vitro Ischemia Injury Models Using Primary Cortical Neuronal Cultures

Abstract

Cell-penetrating peptides (CPPs) are small peptides (typically 5-25 amino acids), which are used to facilitate the delivery of normally non-permeable cargos such as other peptides, proteins, nucleic acids, or drugs into cells. However, several recent studies have demonstrated that the TAT CPP has neuroprotective properties. Therefore, in this study, we assessed the TAT and three other CPPs (penetratin, Arg-9, Pep-1) for their neuroprotective properties in cortical neuronal cultures following exposure to glutamic acid, kainic acid, or in vitro ischemia (oxygen-glucose deprivation). Arg-9, penetratin, and TAT-D displayed consistent and high level neuroprotective activity in both the glutamic acid (IC50: 0.78, 3.4, 13.9 μM) and kainic acid (IC50: 0.81, 2.0, 6.2 μM) injury models, while Pep-1 was ineffective. The TAT-D isoform displayed similar efficacy to the TAT-L isoform in the glutamic acid model. Interestingly, Arg-9 was the only CPP that displayed efficacy when washed-out prior to glutamic acid exposure. Neuroprotection following in vitro ischemia was more variable with all peptides providing some level of neuroprotection (IC50; Arg-9: 6.0 μM, TAT-D: 7.1 μM, penetratin/Pep-1: >10 μM). The positive control peptides JNKI-1D-TAT (JNK inhibitory peptide) and/or PYC36L-TAT (AP-1 inhibitory peptide) were neuroprotective in all models. Finally, in a post-glutamic acid treatment experiment, Arg-9 was highly effective when added immediately after, and mildly effective when added 15 min post-insult, while the JNKI-1D-TAT control peptide was ineffective when added post-insult. These findings demonstrate that different CPPs have the ability to inhibit neurodamaging events/pathways associated with excitotoxic and ischemic injuries. More importantly, they highlight the need to interpret neuroprotection studies when using CPPs as delivery agents with caution. On a positive note, the cytoprotective properties of CPPs suggests they are ideal carrier molecules to deliver neuroprotective drugs to the CNS following injury and/or potential neuroprotectants in their own right.

Neuroprotective peptides fused to arginine-rich cell penetrating peptides: Neuroprotective mechanism likely mediated by peptide endocytic properties

“…However, several years ago, we and others demonstrated that TAT possesses intrinsic neuroprotective properties both in vitro in neurons exposed to excitotoxicity and oxygen-glucose deprivation (OGD) and in vivo following cerebral ischaemia in P12 rats after intraventricular injection (Craig et al, 2011;Meade et al, 2010a, b;Vaslin et al, 2009b;Xu et al, 2008). We subsequently showed that poly-arginine-9 (R9), penetratin and Pep-1 also display neuroprotective actions in in vitro excitotoxic and/or OGD models (Meloni et al, 2014). Furthermore, our data showed that R9 and penetratin were 17-and 4.6-fold respectively more neuroprotective than TAT (Meloni et al, 2014).…”

Inhibition of N-Methyl-d-aspartate-induced Retinal Neuronal Death by Polyarginine Peptides Is Linked to the Attenuation of Stress-induced Hyperpolarization of the Inner Mitochondrial Membrane Potential

“…In in vitro studies, cell-penetrating peptides were recently reported to protect dissociated cortical neurons against excitotoxic doses of NMDA (50,51). Furthermore, Meloni et al (49) reported that only long Arg-rich peptides, containing nine or more arginines, demonstrated neuroprotective properties.…”

Viral and Other Cell-Penetrating Peptides as Vectors of Therapeutic Agents in Medicine

“…Further studies on arginine-rich peptides showed a key role of guanidium side chains of arginine residues for cellular uptake (Futaki et al, 2013). Recently, a new class of cell-penetrating peptides represented by the short peptide Xentry (LCLRPVG) derived from an N-terminal region of the X protein of Hepatitis B virus has been described (Meloni et al, 2014) (Fig. 1).…”