Inhibition of Brain GTP Cyclohydrolase I Attenuates 3-Nitropropionic Acid-Induced Striatal Toxicity: Involvement of Mas Receptor/PI3k/Akt/CREB/ BDNF Axis

Abstract

GTP cyclohydrolase I (GTPCH I) is the rate-limiting enzyme for tetrahydrobiopterin (BH4) biosynthesis; the latter is an essential factor for iNOS activation that contributes neuronal loss in Huntington's disease (HD). The aim of the study was to investigate the neuroprotective effect of 2,4-diamino-6-hydroxypyrimidine (DAHP), GTPCH I enzyme inhibitor, against neuronal loss in 3-nitropropinic acid (3-NP)-induced HD in rats and to reveal the possible involved mechanisms mediated through PI3K/Akt axis and its correlation to Mas receptor (MasR). Rats received 3-NP (10 mg/kg/day; i.p.) with or without administration of DAHP (0.5 g/kg/day; i.p.) or wortmannin (WM), a PI3K inhibitor, (15 μg/kg/day; i.v.) for 14 days. DAHP improved cognitive, memory, and motor abnormalities induced by 3-NP, as confirmed by striatal histopathological specimens and immunohistochemical examination of GFAP. Moreover, DAHP treatment inhibited GTPCH I activity, resulting in decreased BH4 levels and iNOS activation. Also, DAHP upregulated the protein expression of survival protein; p85/p55 (pY458/199)-PI3K and pS473-Akt that, in turn, boosted the activation of striatal neurotrophic factors and receptor, pS133-CREB, BDNF and pY515-TrKB, which positively affect MasR protein expression and improve mitochondrial dysfunction, as indicated by enhancing both SDH and PGC-1α levels. Indeed, DAHP attenuates oxidative stress by increasing SOD activity and Nrf2 expression in addition to reducing neuro-inflammatory status by inhibiting NF-κB p65 and TNF-α expression. Interestingly, all the previous effects were blocked by co-administration of WM with DAHP. In conclusion, DAHP exerts neuroprotective effect against neuronal loss induced by 3-NP administration via inhibition of GTPCH I and iNOS activity and activation of MasR/PI3K/Akt/CREB/BDNF/TrKB axis besides its antioxidant and anti-inflammatory effect.

Keywords: 3-nitropropionic acid; DAHP 3; PI3K/AKT signaling; mas receptor; mitochondrial dysfunction.

GRAPHICAL ABSTRACT

SCHEME 1

Time schedule for DAHP and wortmannin administration and behavioral assessment in 3-Nitropropionic acid rat model.

FIGURE 1

Effect of DAHP on 3-NP-induced changes in (A) rearing frequency and (B) ambulation frequency in open field test and (C) fall off latency in rotarod test, as well as (D) time spent in target quadrant in Morris water maze test, (E) the exploration time of familiar and novel objects, (F) discrimination index and (G) Total time exploring both object in novel object recognition test. Rearing and ambulation frequency are presented as boxplots with median, 25th, and 75th percentile values using Kruskel–Wallis test followed by Dunn’s as a post-hoc test. Parametric data are presented as mean ± SD of 14 rats per group, using one-way ANOVA followed by Tukey’s post-hoc test. Differences in familiar and novel object exploration time for each group were tested using two-way ANOVA; F (4.65) = 3,667, F (4.65) = 36.3, F (4.65) = 32.7, F (4.65) = 44.7; p < 0.05. * vs. control, # vs. 3-NP, @ vs. 3-NP + DAHP. 3-NP, 3-nitropropionic acid; DAHP, 2,4-diamino-6-hydroxypyrimidine; WM, wortmannin.

FIGURE 2

Effect of DAHP on 3-NP induced alteration in striatal (A) GTPCH I activity, (B) BH4 and (C) iNOS level. Data are presented as mean ± SD of four to six rats per group, using one-way ANOVA followed by Tukey’s post-hoc test; F (4.15) = 52.5, F (4.15) = 75, F (4.25) = 65.6; p < 0.05. * vs. control, # vs. 3-NP, @ vs. 3-NP + DAHP. 3-NP, 3-nitropropionic acid; DAHP, 2, 4-diamino-6-hydroxypyrimidine; WM, wortmannin; GTPCH I, GTP cyclohydrolase I; BH4, tetrahydrobiopterin; iNOS, inducible nitric oxide synthase.

FIGURE 3

Effect of DAHP on 3-NP induced alteration in striatal (A) SDH level, (B) PGC-1α content, (C) SOD activity and (D) Nrf2 protein expression. Data are presented as mean ± SD of five to six rats per group, using one-way ANOVA followed by Tukey’s post hoc test; F (4.25) = 69.6, F (4.25) = 175.9, F (4.25) = 20.1, F (4.20) = 138.8; p < 0.05. * vs. control, # vs. 3-NP, @ vs. 3-NP + DAHP. 3-NP, 3-nitropropionic acid; DAHP, 2,4-diamino-6-hydroxypyrimidine; WM, wortmannin; SDH, succinate dehydrogenase; PGC-1α, proliferator-activated receptor gamma coactivator 1-alpha; SOD, superoxide dismutase; Nrf2, nuclear factor erythroid-2-related factor-2.

FIGURE 4

Effect of DAHP on 3-NP induced alteration in striatal (A) p65 NF-κB and (B) TNF-α content. Data are presented as mean ± SD of six rats per group, using one-way ANOVA followed by Tukey’s post-hoc test; F (4.25) = 418, F (4.25) = 91.2; p < 0.05. * vs. control, # vs. 3-NP, @ vs. 3-NP + DAHP. 3-NP, 3-nitropropionic acid; DAHP, 2,4-diamino-6-hydroxypyrimidine; WM, wortmannin; p65 NF-kB, p65 nuclear factor-kB; TNF-α, tumor necrosis factor-alpha.

FIGURE 5

Effect of DAHP on 3-NP induced alteration in striatal (A) MasR, (B) p-PI3K, (C) p-Akt, (D) p-CREB, (E) BDNF and (F) p-TrkB protein expression. Data are presented as mean± SD of five rats per group, using one-way ANOVA followed by Tukey’s post hoc test; F (4.20) = 100.4, F (4.20) = 96.7, F (4.20) = 200.5, F (4.20) = 254.4, F (4.20) = 359.7, F (4.20) = 73.6; p < 0.05. * vs control, # vs 3-NP, @ vs 3-NP + DAHP. 3-NP, 3-nitropropionic acid; DAHP, 2, 4-diamino-6-hydroxypyrimidine; WM, wortmannin; MasR, Mas receptor; p-P13K, phosphorylated phosphoinositide-3-kinase; p-Akt, phosphorylated protein kinase B; p-CREBm phosphorylated cAMP responsive element-binding protein; BDNF, brain-derived neurotrophic factor; p-TrkB, phosphorylated tyrosine kinase B.

FIGURE 6

Effect of DAHP on 3-NP induced striatal histopathological alterations. (A–J) photomicrographs represent staining of striatum with H&E (Scale bar 200 µm). (A) Control group, (B) DAHP alone treatment, (C) 3-NP group, (D) DAHP treated group and (E) WM treated group. Well organized apparent intact neurons (black arrow), degenerated neurons (red arrow), perineuronal edema (blue arrow) and severe astrogliosis (arrow head). 3-NP, 3-nitropropionic acid; DAHP, 2,4-diamino-6-hydroxypyrimidine; WM, wortmannin; H&E, hematoxylin and eosin.

FIGURE 7

Effect of DAHP on 3-NP induced striatal GFAP immunoreactivity. (A–J) photomicrographs represent immunohistochemical staining of GFAP in striatum (Scale bar 50 and 200 µm). (A–B) Control group, (C–D) DAHP alone treatment, (E–F) 3-NP group, (G–H) DAHP-treated group and (I–J) WM-treated group. (K) % area of GFAP immunoexpression. Data are presented as mean ± SD. of three rats per group, using one-way ANOVA followed by Tukey’s post hoc test; F (4.25) = 643; p < 0.05. * vs control, # vs 3-NP, @ vs 3-NP + DAHP. 3-NP, 3-nitropropionic acid; DAHP, 2,4-diamino-6-hydroxypyrimidine; WM, wortmannin, GFAP, glial fibrillary acidic protein.