The Effects Of Histone Acetylation On Methamphetamine-induced Behavioral Sensitization

Addiction is a worldwide problem, and exacts an enormous emotional, medicaland financial toll on society in the form of overdose and family disintegration, healthcomplications, loss of employment, and crime. In the recent years, abuse ofamphetamine-type stimulants (ATS) is quickly spreading, and methamphetamine(METH) accounts for the majority percent of global ATS seizures. Therefore, it isurgent to investigate the neurobiology mechanism and effective therapies for METHaddiction.Addiction is a chronic psychiatric disorder characterized by compulsive drugseeking and taking. A person succumbed to addiction can hardly get rid of the drugs.They are always vulnerable to drug relapse—a lifelong struggle even after abstinentfor long durations of time. The recent studies suggest that addiction can be consideredas maladaptation of neural plasticity in vulnerable individuals in response to repeateddrugs administration. Emerging evidences have demonstrated that epigenetics is animportant regulator of neural plasticity. It is a series of biochemical processes whichchanged the gene expression throughout the lifecycle of an organism without anychange in DNA sequence. The epigenetic modifications are very stable, which makesthem ideal mediators both of addiction vulnerability and of drug-induced maladaptiveneural plasticity that underlie addiction behaviors. The recent studies have confirmedthe relationship between epigenetics and drug addiction. Acute or repeatedadministration of cocaine or other drugs increased global levels of acetylated histoneH3and H4in the nucleus accumbens (NAc), a key region of reward system. Anddrugs can also alter series of epigenetic related enzymes.In the recent years, more and more researchers have begun to investigate themechanism of METH addiction since it is abused seriously. Therefore, this study wasaim to investigate the relationship between METH addiction and histone acetylation,one of the epigenetic modifications, to further understand the addiction mechanism.Our findings are as follows:1. METH-induced behavioral sensitization modelRepeated drugs exposure can cause nervous system hypersensitivity to drugs and the related stumulis, which mediats durg-seeking and drug-takeng. This infers thatsensitisitzation is critical in drug relapse. And sensitization and relapse share the sameprojection from prefrontal cortex (PFC) to ventral tegmental area (VTA) and NAc.Therefore, behavioral sensitization is identified in this article. The behavioralsensitization can be divided into three periods, including development, transfer andexpression periods. To establish this model,0.25mg/kg-5.0mg/kg METH was testedin development and expression periods. We found that, the locomotor activity insensitizated group was significantly higher than acute1.0mg/kg group (about30%)by5.0mg/kg METH (s.c., qd) for7days, withdrawal for7days, and then1.0mg/kgMETH (s.c.) on day15, suggesting that this model was successfully established.2. The screen of mRNAexpression difference and histone modification differenceThis article firstly detected the changes in METH-induced behavioralsensitization by using chips of mRNAexpression and histone modification.The mRNA expression chips showed that about505genes expressed differentlyafter METH treatment, in which>50%gene expressions increased significantly; andmore genes exhibited enriched mRNA in expression period, inferring that METHpotentiated the transcription. GO analysis divided these genes into several classes,including metal ion binding related molecules, cell surface receptor linked signaltransduction related molecules, transcription related molecules, chromosomeorganization and DNA binding related molecules, plasma membrance relatedmolecules, nervous system development related molecules, cell apoptosis relatedmolecules and cancer related molecules, majority of which may be critical inaddiction.Many genes were modified differently in histone modification chips. Histoneacetylation is the major change locus. There were2491genes modified at H3acetylation, and5551genes at H4acetylation; however, H3methylation was almostnot changed. These results suggested that acetylation was the major modification toregulate changes in METH-induced behavioral sensitization.There were more genes active than repressive to transcribe in mRNA chip, andhistone modification chips also exhibited enriched acetylation on H3and H4,suggesting that histone acetylation can increase transcription. We selected Anp32a、Arrdc1、Atp5i、Avp、Bal2l1、BDNF、Cadm3、Camk2n1、Cox6a1、Cox8a、E2f3、Egr1、Eml2、Exog、Galnt9、Hira、Limk1、Lnx2、Metrn、Ndst4、Nmur1、Omal、Pou3f2、Shoc2、Stk32c、Stx2、Syt8、Tacr2、Trim17、Usp9x、Zfp36as the candidate molecules to be validated further on the base of comprehensive analysis of mRNAexpression and/or histone modification, gene functions, signaling pathway and theabsolute quantity of gene mRNAin PFC.3. The analysis of transcription upstreamHistone acetylation is dynamicly modified by histone acetyl transferase (HAT)and histone deacetylase (HDAC). The results showed that CBP decreased to80%after challenge. HDAC1、2(type I HDAC) decreased mainly after acute and7days’administration, while HDAC4、5(type II HDAC) decreased after withdrawal andchallenge. These indicated that different types of Hdac played different functions indifferent periods of METH-induced behavioral sensitization. HDAC activityattenuated after7days’ administration, while increased in transfer and expressionperiods. The histone modification chips inferred hyperacetylation after METHtreatment, and1.0mg/kg METH challenge increased histone acetylation significantly.The contrary between HDAC total acticity and histone acetylation may be caused by(a) other HATs and HDACs that were not studied in this article participated in thismodel;(b) one or several but not all HDAC subtypes regulated this model. And theseoffered cues and evidences for further study.4. Validation of candidate molecules and the dynamic transcription andpre-transcription changes of Pou3f2etc. in METH-induced behavioralsensitizationAnp32a、Egr1、Eml2and Pou3f2were selected to study further after be validatedon mRNA and histone modification. The four genes were investigated in variousperiods of METH-induced behavioral sensitization. The changes at histonemodifications of Anp32a and Pou3f2were accordant with their mRNAs changes.Anp32a and Pou3f2increased after acute and7days’ administration, recovered to thecontrol level after7days’ withdrawal, and increased significantly after1.0mg/kgchallenge. However, the changes at histone modifications of Egr1and Eml2were notcompletely consistent with their mRNAs. The histone acetylation at H4of Egr1increased after acute and7days’ administration, oppsiting to its mRNA; the histoneacetylation and mRNA did not change significantly after withdrawal and challenge.The histone acetylation at H4of Eml2increased after acute treatment, howevermRNA decreased significantly; H4acetylation and mRNA increased slightly after7days’ administration; H3/H4acetylation both increased, howerer its mRNA did notchange after7days’ withdrawal; and the histone acetylation and mRNA did not change significantly after challenge. These results suggested that histonemodifications partly regulated the transcription.From the above results, Pou3f2exhibited significant changes in response towithdrawal and challenge of METH-induced behavioral sensitization. As atranscription factor expressed specially in brain, Pou3f2is closely related with themolecules and pathways (such as cAMP pathways, Notch pathway and MAPKpathway, etc.) involved in addiction. However, no documents have reported the effectof Pou3f2in addiction. Therefore, Pou3f2was selected as the preferred molecule forstudying its relationship with addiction.5. The relationship between Pou3f2and METH-induced behavioral sensitizationThis article successfully established lentivirus to interfere Pou3f2. The lentiviruswas injected into the subregion of PFC-PL, and then rats were transferred toesthablish METH-induced behavioral sensitization. The preliminary study revealedthat the lentivirus reduced Pou3f2mRNA effectively, and the locomotor activityreduced about50%at the same time. These results suggested that Pou3f2was animportant molecule involved in the model, and was likely to play an important role indrug addiction.In conclusion, the present research confirmed that the genome-wide histoneacetylation in PFC increased and mediated the open expression of downstream genesin METH-induced behavioral sensitization; and in the pre-transcription, the mRNAand activity of CBP and HDAC were changed by METH and dynamicly regulatedhistone acetylation, inferring that the histone acetylation in PFC played a role in theprogress of METH-induced behavioral sensitization. Besides, the histone modificationand mRNA changes of Pou3f2in PFC induced by METH may be the important basisof the establishment of behavioral sensitization.