Neuroprotective Effect of Piracetam against Cocaine-Induced Neuro Epigenetic Modification of DNA Methylation in Astrocytes

Abstract

Cocaine abuse is known to alter mitochondrial biogenesis and induce epigenetic modification linked with neuronal dysfunction. Cocaine-induced epigenetic modification of DNA methylation and the mitochondrial genome may affect mitochondrial DNA (mtDNA) and nuclear DNA (nDNA), as epigenetic DNA methylation is key to maintaining genomic integrity in the central nervous system (CNS). However, the impact of cocaine-mediated epigenetic changes in astrocytes has not yet been elucidated. In this study, we explored the neuroprotective effect of piracetam against cocaine-induced epigenetic changes in DNA methylation in astrocytes. To study our hypothesis, we exposed human astrocytes to cocaine alone or in combination with the nootropic drug piracetam. We examined the expression of the DNA methyltransferases (DNMTs) DNMT-1, DNMT-3A, and DNMT-3B; global DNA methylation levels of 5-methycytosine (5-mC); and induction of ten-eleven translocation (TET) enzymes in astrocytes. In addition, we analyzed mtDNA methylation by targeted next-generation bisulfite sequencing. Our data provide evidence that cocaine impairs DNMT activity and thereby has impacts on mtDNA, which might contribute to the neurodegeneration observed in cocaine users. These effects might be at least partially prevented by piracetam, allowing neuronal function to be maintained.

Keywords: DNMTs; TET and astrocytes; cocaine; piracetam.

Figure 1

Piracetam reversed the impact of cocaine on global DNA methylation (5-mc) and DNA methyltransferases’ (DNMTs’) gene expression. Human primary astrocytes (2 × 10⁶ cells/mL) were exposed to cocaine (1 µM) and/or piracetam (10 µM) for 24 h. Total cellular DNA were used to determine the global DNA methylation (5-mC) by ELISA (A) and the total RNA were analysed (B) DNMT-1, (C) DNMT-3A and (D) DNMT-3B mRNA expression by qRT-PCR. The housekeeping gene β-actin was used as a loading control. The results are expressed as the mean ± SD of the transcript accumulation index (TAI) of three independent experiments. *** p < 0.001, ** p < 0.01, * p < 0.05, NS—nonsignificant.

Figure 2

Piracetam reversed the impact of cocaine on DNMT protein expression in primary astrocytes. Human primary astrocytes were exposed to cocaine (1 µM) and/or piracetam (10 µM) for 24 h, and the total and nuclear Fractions were isolated. Representative blots (A,G) showing the expression of DNMT-1, (B,H) DNMT-3A and (C,I) DNMT-3B in the total and nuclear fractions. (D–F,J–L) Densitometric analysis of the level of each protein relative to the corresponding level of GAPDH or lamin as a loading control (fold change relative to the control). The data are expressed as the mean ± SD of three independent experiments. *** p < 0.001, ** p < 0.01, * p < 0.05, NS—nonsignificant.

Figure 3

Piracetam reversed the impact of cocaine on DNMT protein expression in the mitochondrial fraction. Human primary astrocytes (5 × 10⁶ cells/mL) were exposed to cocaine (1 µM) and/or piracetam (10 µM) for 24 h. Mitochondrial fraction were resolved by SDS-PAGE and analyzed by western blots showing the expression of (A) DNMT-1, (B) DNMT-3A and (C) DNMT-3B. (D–F) Densitometric analysis of the level of each protein relative to the level of COX-IV as a loading control (fold change relative to the control). The data are expressed as the mean ± SD of three independent experiments. *** p < 0.001, ** p < 0.01, * p < 0.05, NS—nonsignificant. (G) DNMT-1 immunostaining (red) and cell nuclei, which were stained with DAPI (blue), were observed by confocal microscopy (magnification 100x, scale bar 100 μm). (H) Quantification of DNMT-1 fluorescence intensity (CTCF).

Figure 4

Analysis of mtDNA methylation by targeted next generation bisulfite sequencing (TNGBS). Human primary astrocytes were exposed to cocaine (1 µM) and/or piracetam (10 µM) for 24 h. The mtDNA methylation profiles were determined by TNGBS. Results represent the protective effect of piracetam on cocaine-induced hypomethylation in different mitochondrial CpG sites mt-RNR1, mt-RNR2, ND1, ND4, ND5, mt-CO1, mt-CO2, mt-ATP6 and mt-CYB (A–D). The results are expressed average of methylation percentage ± SD, * p < 0.05.

Figure 5

Piracetam reversed the impact of cocaine on TET gene expression. Human primary astrocytes (2 × 10⁶ cells/mL) were exposed to cocaine (1 µM) and/or piracetam (10 µM) for 24 h. The mRNA expression of (A) TET-1, (B) TET-2 and (C) TET-3 was determined by qRT-PCR analysis. The housekeeping gene β-actin was used as a loading control. The results are expressed as the mean ± SD of the TAI of three independent experiments. * p < 0.05, NS—nonsignificant.

Figure 6

Piracetam reversed the impact of cocaine on TET protein expression. Human primary astrocytes (2.5 × 10⁶ cells/mL) were exposed to cocaine (1 µM) and/or piracetam (10 µM) for 24 h. Total cell lysates were resolved by SDS-PAGE and analyzed by western blots showing the expression of (A) TET-1, (B) TET-2 and (C) TET-3. (D–F) Densitometric analysis of the level of each protein relative to the level of GAPDH as a loading control (fold change relative to the control). The data are expressed as the mean ± SD of three independent experiments. *** p < 0.001, ** p < 0.01, * p < 0.05, NS—nonsignificant. (G) TET-1 (green) immunostaining and cell nuclei, which were stained with DAPI (blue), were observed by confocal microscopy (magnification 100× scale bar 100 μm). (H) Quantification of TET-1 fluorescence intensity (CTCF).