Excessive alcohol drinking in rodents induces increased mTORC1-dependent Prosapip1 translation in the nucleus accumbens

We previously found that excessive alcohol consumption activates a PI3K/AKT signaling pathway that ultimately lead to the activation of the mechanistic target of rapamycin complex 1 (mTORC1) activity in the nucleus accumbens (Nac) of rodents (1, 2). mTORC1 is localized in dendrites, promotes the translation of synaptic proteins and plays an important role in synaptic plasticity (3). Together, these findings suggest that mTORC1-dependent mRNA to protein translation could play a major role in neuroadaptations induced by alcohol drinking that underlie the development and/or maintenance of excessive consumption. To identify novel gene products whose translation is induced in the Nac in response to the alcohol-dependent mTORC1 activation, we utilized the high throughput RNA sequencing (RNA seq) approach. Specifically, mice underwent an intermittent access to 20% alcohol or water only using 2-bottle choice paradigm for 8 weeks, and were then treated with rapamycin or vehicle. The NAc was dissected, polysomes (e.g. RNA undergoing translation) were isolated, and RNA seq was performed. Among the 12 identified candidates whose translation was dependent of mTORC1 was ProSAP-interacting protein 1 (Prosapip1), a synaptic protein that interacts with the scaffolding protein ProSAP2/Shank3 and the Rap GAP SPAR (4). Using quantitative Real-Time PCR (qRT-PCR), we first confirmed the RNAseq data and showed that the translation of Prosapip1 was induced in response to excessive alcohol drinking in an mTORC1-dependent manner. We further obtained data suggesting that the increase in mRNA of Prosapip1 was due solely to a translation and not a transcription event, as Prosapip1mRNA expression was only increased in the polysomal mRNA fraction and not in the total mRNA fraction. By western blot analysis, we found that Prosapip1 protein expression is significantly increased in the Nac of mice after a 4 hours binge drinking session and was maintained even after 24 hours of withdrawal. Prosapip1 levels were not altered in other brain regions where mTORC1 is not activated by alcohol. Finally, similar findings were obtained in rats undergoing an intermittent 20% alcohol 2-bottle choice paradigm. Together our data suggest that Propapip1 is a novel mTORC1-dependent gene product whose levels are induced by voluntary alcohol intake in the Nac. Functional consequences of the molecular findings will also be described.

Supported by NIH P50 AA017072 (DR)

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