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Objective: It is well established that early initiation to alcohol use is common among adolescents and strongly predicts alcohol abuse in adulthood. Although adolescence is known as a critical stage that is especially sensitive to alcohol, the molecular mechanisms underlying the long-lasting effects of alcohol remain unknown. The current study is based on the notion that dopaminergic imbalance is a key risk factor for the development of addiction and the hypothesis that α-Synuclein (α-Syn) can function as an activity-dependent negative regulator of DA neurotransmission. Therefore, we investigated the long-lasting effects of alcohol exposure during adolescence on the brain mRNA levels of α-Syn and tyrosine hydroxylase (TH) - the first rate-limiting enzyme in the DA biosynthetic pathway.
Methods: Midbrain and striatum were obtained from 58-d-old male Wister rats that had undergone 8 injections of 10% ethanol (3 g/kg, i.p., once daily starting from the 30th day of life) (experimental group, n=7) or vehicle (saline, control group, n=7). Levels of mRNA coding for α-Syn and TH were studied using quantitative RT-PCR. Total RNA was extracted using “RNeasy Lipid Tissue MiniKit” (Qiagen). The first cDNA strand was generated by reverse transcribing 1 µg of total RNA using “RevertAid First Strand cDNA Kit” (Fermentas). SYBR Green Quantitative real-time PCR analysis was performed using Multicolor Real-Time PCR Detection System iQTW5 (BioRad). Expression of each gene was normalized to the expression of the β-actin housekeeping gene and relative expression was obtained according to the 2-∆∆Ct method. The RT-PCR data were analyzed with two-sample t-tests.
Results: The mRNA level of α-Syn varied among structures and in controls was 2.5-fold higher in the striatum compared to the midbrain. mRNA coding for TH was highly expressed in the midbrain. At the same time, an extremely low but clearly detectable level of TH mRNA was found in the striatum (6% relative to midbrain). Thus α-Syn mRNA is detected at high level in the striatum – a brain region that does not normally contain DA neurons. Alcohol exposure during adolescence had differential effects on the regional expression of α-Syn mRNA. The estimated relative expression α-Syn was significantly higher in the striatum of alcohol-treated rats compared to controls (2-∆∆Ct =1.75+0.20 vs. 2-∆∆Ct =1.02+0.09, p<0.05), however, no significant changes were found in the midbrain (2-∆∆Ct =1.31+0.13 vs. 2-∆∆Ct =1.07+0.17). No significant changes in TH mRNA levels were found in either structure between the alcohol-treated group and controls. Based on these data, it can be suggested that the up-regulation of α-Syn mRNA in the striatum associated with the alcoholtreated group may result from α-Syn mRNA accumulation in the presynaptic terminals of dopaminergic midbrain neurons, rather than additional DAergic differentiation in the striatal neurons as a consequence of synaptic activation by ethanol during adolescence.
Conclusion: The changes revealed in the levels of mRNA coding for α-synuclein confirm the conclusion to assume an important role of these molecules and support the idea that exposure to alcohol during adolescence may edit gene expression profiles in the brain and serve as a precursor for alcohol abuse later in life.