PTD4 Peptide Increases Neural Viability in an In Vitro Model of Acute Ischemic Stroke

Abstract

Ischemic stroke is a disturbance in cerebral blood flow caused by brain tissue ischemia and hypoxia. We optimized a multifactorial in vitro model of acute ischemic stroke using rat primary neural cultures. This model was exploited to investigate the pro-viable activity of cell-penetrating peptides: arginine-rich Tat(49-57)-NH₂ (R⁴⁹KKRRQRRR⁵⁷-amide) and its less basic analogue, PTD4 (Y⁴⁷ARAAARQARA⁵⁷-amide). Our model included glucose deprivation, oxidative stress, lactic acidosis, and excitotoxicity. Neurotoxicity of these peptides was excluded below a concentration of 50 μm, and PTD4-induced pro-survival was more pronounced. Circular dichroism spectroscopy and molecular dynamics (MD) calculations proved potential contribution of the peptide conformational properties to neuroprotection: in MD, Tat(49-57)-NH₂ adopted a random coil and polyproline type II helical structure, whereas PTD4 adopted a helical structure. In an aqueous environment, the peptides mostly adopted a random coil conformation (PTD4) or a polyproline type II helical (Tat(49-57)-NH₂) structure. In 30% TFE, PTD4 showed a tendency to adopt a helical structure. Overall, the pro-viable activity of PTD4 was not correlated with the arginine content but rather with the peptide's ability to adopt a helical structure in the membrane-mimicking environment, which enhances its cell membrane permeability. PTD4 may act as a leader sequence in novel drugs for the treatment of acute ischemic stroke.

Keywords: PTD4; Tat(49–57)-NH2; arginine-rich peptides; cell-penetrating peptides; excitotoxicity; ischemic stroke; neural viability; neuroprotection; neurotoxicity; peptide conformation.

Figure 1

(A) Amino acid sequences of the “canonical” variant of HIV-1 Tat (P04608-1 identifier of the UniProt database), (B) Tat(49–57)-NH₂ with underlined Ala-substituted residues, and (C) PTD4. Asterisks (*) indicate positions of sequence homology (R, R, Q, R).

Figure 2

CD spectra of Tat(49–57)-NH₂ and PTD4 peptides in 10 mM PBS, pH 7.0 and 30% (v/v) TFE.

Figure 3

Spatial structures of the two most dominant clusters calculated for Tat(49–57)-NH₂ in water (A) and 30% TFE (C), and for PTD4 in water (B) and 30% TFE (D). On the left, cluster 1; on the right, cluster 2.

Figure 4

Experimental designs: peptide neurotoxicity (A), in vitro model of AIS (B), and peptide neuroprotection against AIS (C).

Figure 5

Peptide neurotoxicity in the cortical culture. Non-insulted cells were treated as 100% control. Data demonstrate relative measurements to control ± SD. Two-way ANOVA statistics: n = 3; * p < 0.05, ** p < 0.001.

Figure 6

Optimization of an in vitro model of AIS. Red bars indicate conditions causing a 30–40% decrease in neural viability. These conditions were then selected for assessments of the neuroprotective activity of tested peptides. Non-insulted cells were treated as 100% control. Two-way ANOVA statistics: n = 3; * p < 0.05, ** p < 0.001. Each statistical significance asterisk refers to a respective control (white bar) ± SD.

Figure 7

Pro-viable activity of Tat(49–57)-NH₂ and PTD4 peptides in a multifactorial in vitro model of AIS. Non-insulted cells were treated as 100% control. Two-way ANOVA statistics: n = 3; * p < 0.05. Each statistical significance asterisk refers to a respective no-peptide control (white bar) ± SD.

Figure 8

Conjectured mechanisms of PTD4-mediated neuroprotection based on the results of the study. PTD4–Y⁴⁷ARAAARQARA⁵⁷-amide; CaV2.2–N-type voltage-gated calcium channel; CaV3.3–T-type voltage-gated calcium channel; NCX3–sodium-calcium exchanger 3; TRPV1–transient receptor potential cation channel subfamily V member 1; Cyt C–cytochrome C; ATP–adenosine triphosphate; NOS–nitric oxide synthase; NO–nitric oxide; PSD-95–post-synaptic density protein 95; KAR–kainic acid receptor; NMDAR–N-methyl-D-aspartic acid receptor; TCA–tricarboxylic acid cycle.