Antiglycative Activity and RAGE Expression in Rett Syndrome

Abstract

Rett syndrome (RTT) is a human neurodevelopmental disorder, whose pathogenesis has been linked to both oxidative stress and subclinical inflammatory status (OxInflammation). Methylglyoxal (MG), a glycolytic by-product with cytotoxic and pro-oxidant power, is the major precursor in vivo of advanced glycation end products (AGEs), which are known to exert their detrimental effect via receptor- (e.g., RAGE) or non-receptor-mediated mechanisms in several neurological diseases. On this basis, we aimed to compare fibroblasts from healthy subjects (CTR) with fibroblasts from RTT patients (N = 6 per group), by evaluating gene/protein expression patterns, and enzymatic activities of glyoxalases (GLOs), along with the levels of MG-dependent damage in both basal and MG-challenged conditions. Our results revealed that RTT is linked to an alteration of the GLOs system (specifically, increased GLO2 activity), that ensures unchanged MG-dependent damage levels. However, RTT cells underwent more pronounced cell death upon exogenous MG-treatment, as compared to CTR, and displayed lower RAGE levels than CTR, with no alterations following MG-treatment, thus suggesting that an adaptive response to dicarbonyl stress may occur. In conclusion, besides OxInflammation, RTT is associated with reshaping of the major defense systems against dicarbonyl stress, along with an altered cellular stress response towards pro-glycating insults.

Keywords: MeCP2; advanced glycation end products; dicarbonyl stress; fibroblasts; glyoxalases; methylglyoxal.

Figure 1

Evaluation of glyoxalase 1 pattern. (A) GLO1 specific activity; (B) GLO1 protein levels, with representative (inverted) Western blots; (C) glo1 gene expression levels. Data of real time RT-PCR were given as 2^−ΔΔCt, using rpl13a as the reference, and one of the controls as the internal calibrator. All the data were expressed as means ± SD. CTR, control; RTT, Rett syndrome. Data were analyzed by a t-test for independent groups.

Figure 2

Assessment of glyoxalase 2 pattern. (A) GLO2 specific activity; (B) GLO2 protein levels, with representative (inverted) Western blots; (C) glo2 gene expression levels. Data of real time RT-PCR were given as 2^−ΔΔCt, using rpl13a as the reference, and one of the controls as the internal calibrator. All the data were expressed as means ± SD. CTR, control; RTT, Rett syndrome. ** p < 0.01. Data were analyzed by a t-test for independent groups.

Figure 3

Cell survival from a 24-h exogenous MG challenge. (A) Cell viability of CTR and RTT fibroblasts, upon MG treatment; (B) cell death of CTR and RTT fibroblasts following MG challenge. Values were expressed as means ± SD. The chosen MG concentration (650 μM) represented the 30% reduction of live cells (IC₃₀, indicated by the arrow), calculated through a 4P-logistic regression curve derived from a dose-response curve obtained by incubating cells with MG concentrations ranging from 0 to 2 mM (inset diagram). CTR, control; RTT, Rett syndrome; MG, methylglyoxal. * p < 0.05; ** p < 0.01; *** p < 0.001. Results were analyzed by Two-way ANOVA, with post hoc Tukey’s multiple comparisons test.

Figure 4

Assessment of methylglyoxal-derived hydroimidazolone 1 (MG-H1) levels, following a 24 h exogenous MG challenge. Data were given as means ± SD. CTR, control; RTT, Rett syndrome; MG, methylglyoxal. * p < 0.05. Results were analyzed by two-way ANOVA, with post hoc Tukey’s multiple comparisons test.

Figure 5

Evaluation of cellular RAGE protein levels in MG-challenged fibroblasts. Data were expressed as means ± SD. Representative (inverted) Western blots were reported. CTR, control; RTT, Rett syndrome; MG, methylglyoxal. * p < 0.05; ** p < 0.01; *** p < 0.001. Results were analyzed by two-way ANOVA, with post hoc Tukey’s multiple comparisons test.

Figure 6

Assessment of dicarbonyl damage by the analysis of MG-H1 levels in plasma from RTT patients. Data were given as means ± SD. CTR, control; RTT, Rett syndrome. * p < 0.05. Data were analyzed by a t-test for independent groups.