Dataset Information

The proteomic landscape of parkin-deficient and parkin-overexpressing rat nucleus accumbens: an insight into the role of parkin in methamphetamine use disorder

ABSTRACT: In recent years, methamphetamine (METH) misuse in the US has been rapidly increasing, and there is no FDA-approved pharmacotherapy for METH use disorder (MUD). We previously determined that ubiquitin-protein ligase parkin is involved in the regulation of METH addictive behaviors in rat models of MUD. Parkin is not yet a “druggable” drug target; therefore, this study aimed to determine which biological processes, pathways, and proteins downstream of parkin are likely drug targets against MUD. Employing young adult Long Evans male rats with parkin deficit or excess in the nucleus accumbens (NAc), label-free proteomics, and molecular biology, we determined that the pathways downstream of parkin that are candidates for regulating METH addictive behaviors in young adult male rats are mitochondrial respiration, oxidative stress, AMPA receptor trafficking, GABAergic neurotransmission, and actin cytoskeleton dynamics.

INSTRUMENT(S):

ORGANISM(S): Rattus Norvegicus (rat)

TISSUE(S): Brain, Nucleus Accumbens

SUBMITTER: TuKiet Lam

LAB HEAD: TuKiet Lam

PROVIDER: PXD065059 | Pride | 2025-08-04

REPOSITORIES: Pride

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			Action	DRS
	HFX21-2209_Moszczynska_WT_NA_1472.raw	Raw
	HFX21-2213_Moszczynska_PO_NA_1463.raw	Raw
	HFX21-2221_Moszczynska_WT_NA_1476_20210722181052.raw	Raw
	HFX21-2229_Moszczynska_PO_NA_1464.raw	Raw
	HFX21-2233_Moszczynska_PO_NA_1460.raw	Raw

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The Proteomic Landscape of Parkin-Deficient and Parkin-Overexpressing Rat Nucleus Accumbens: An Insight into the Role of Parkin in Methamphetamine Use Disorder.

Sharma Akhil A Atasi Tarek T Collin Florine F Wang Weiwei W Lam TuKiet T TT Garcia-Milian Rolando R Arroum Tasnim T Pham Lucynda L Hüttemann Maik M Moszczynska Anna A

Biomolecules 20250703 7

In recent years, methamphetamine (METH) misuse in the US has been rapidly increasing, and there is no FDA-approved pharmacotherapy for METH use disorder (MUD). We previously determined that ubiquitin-protein ligase parkin is involved in the regulation of METH addictive behaviors in rat models of MUD. Parkin is not yet a "druggable" drug target; therefore, this study aimed to determine which biological processes, pathways, and proteins downstream of parkin are likely drug targets against MUD. Emp ...[more]

PMID: 40723830

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Project description:Purpose: The E3 ubiquitin ligase Parkin is a well-characterized regulator of mitochondrial autophagy (mitophagy); however, it is becoming increasingly appreciated to perform additional roles in various compartments of the cell. Our laboratory confirmed the presence of Parkin in the nucleus of various tissues (biochemical fractionations) and cell types (immunofluorescent imaging). Hypoxia-induced nuclear translocation of Parkin occured independent of the mitophagy regulator PINK1, and Parkinson's disease-associated mutants were restricted from the nuclueus. Accordingly, we inserted a nuclear localization sequence (NLS) at the n-terminus of Parkin and overexpressed both NLS Parkin and the wild-type protein in HeLa cells cultured at normoxia and hypoxia. Next-generation RNA-sequencing (RNA-seq) was used to determine the effect of nuclear Parkin on cellular transcription. Methods: mCherry-tagged NLS and wild-type Parkin were overexpressed in HeLa cells. Differential expression analyses were performed on Parkin vs. mCherry control cells and NLS Parkin vs. mCherry control cells at normoxia and following 12hr of hypoxia (n=3/group/condition). Paired-end sequencing was performed using the HiSeq4000. FastQC v0.11.3 was used for quality control, Trimmomatic v0.36 was used to trim reads which were aligned to the human genome (GRCh37.p13) using the STAR aligner v2.5.3a. Read quantification was performed with RSEM v 1.3.0 and the Gencode release 19. The R BioConductor packages edgeR and limma were used to implement the limma-voom method for differential expression analysis. Results: During normoxia, Parkin had no effect on basal transcription; however, overexpression of NLS Parkin was associated with 168 differentially expressed genes (DEGs: fold-change </= 1.5, FDR < 0.05) relative to mCherry control cells. Following hypoxia, the transcriptome associated with the overexpression of wild-type Parkin more closely resembled that of NLS Parkin. Along these lines, Parkin overexpression during hypoxia coincided with a total of 158 DEGs, 37% of which were shared with NLS Parkin. Overlapping and shared DEGs among Parkin and NLS Parkin were implicated in cellular metabolism, HIF1 signaling and survival. Our follow up co-immunoprecipitation and real-time quantitative PCR studies demonstrated that Parkin interacts with the Estrogen Related Receptor Alpha (ERRa) to promote the induction of its downstream target genes. Conclusions: Nuclear translocation of Parkin is a novel means by which this cytoprotective protein contributes to cellular homeostasis and especially critical during hypoxia.

Project description:Methamphetamine (METH) addiction is associated with a plethora of neuropsychiatric symptoms. In rodents, administration of the drug is accompanied by complex changes in gene expression in the dorsal striatum. Very little is known about the effects of the drug on gene expression and epigenetic modifications in the nucleus accumbens (NAc). The present study was conducted in order to investigate the effects of a single injection of METH on gene expression in that structure. We also queried whether changes in transcript levels were accompanied by alterations in histone acetylation as well as in the expression of the histone acetyltransferase (HAT), ATF2, and of the histone deacetylases (HDACs), HDAC1 and HDAC2. Microarray analyses revealed that METH caused significant time-dependent increases in the expression of several genes that had previously been implicated in the acute and longterm effects of psychostimulants in the brain. These include several immediate early genes (c-fos, Egrs, c-jun, and Nurr1) and corticotropin-releasing factor (Crf). There were also increases in the levels of neuromedin U, Tnf-alpha, and Kcnk18, among others. In contrast, the METH injection caused decreases in the expression of many genes including Npas4 and cholecystokin (Cck). Pathway analyses showed that differentially affected genes participate in behavioral performance, cell-to-cell signaling and interactions, and regulation of gene expression. Other differentially affected genes are known to be involved in cellular compromise and death pathways. PCR analyses were used to confirm the changes in the expression of c-fos, fosB, c-jun, junB, Crf, NmU, Cck, and Npas4 transcripts. Western blot analyses also identified METH-mediated decreases in the acetylation of histone H3 at lysine 9 (H3K9) and lysine 18 (H3K18) as well as in histone 4 at lysine 16 (H4K16). In contrast, the METH injection caused time-dependent increases in the acetylation of H4K8 and H4K12. The changes in histone acetylation were also accompanied by decreased expression of HDAC1 but increased expression of ATF2 and of HDAC2. These results suggest that METH-induced alterations in global gene expression might be due, in part, to diverse effects of METH-induced histone acetylation secondary to changes in HAT and HDAC expression. At the indicated time after the METH or saline injections, rats (n = 5 per group) were euthanized and the NAc was dissected and immediately put on dry ice. The tissues were kept at -70 oC until RNA extraction. Total RNA was isolated from the nucleus accumbens according to the manufacturer’s manual using Qiagen RNeasy mini kit (Qiagen, Valencia, CA, USA). RNA integrity was detected using an Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA, USA).