Transcriptome Analysis Reveals That Midnolin Regulates mRNA Expression Levels of Multiple Parkinson’s Disease Causative Genes

Abstract

We recently found that 10.5% of sporadic Parkinson's disease (PD) patients lacked one copy of the midnolin (MIDN) gene. In addition, gene knock-down/out of MIDN caused down-regulation of parkin E3 ubiquitin ligase, indicating MIDN to be a novel PD-risk factor or causative gene. In this study, we performed RNA-sequencing and transcriptome analysis of Midn wild-type and knockout cells. Midn positively or negatively regulated the expression of a wide variety of genes, including causative familial PD genes, such as α-synuclein, parkin, and EIF4G1. However, EIF4G1 protein levels were not altered by the reduction of its mRNA by Midn loss, as seen that parkin protein levels were correlated to the mRNA down-regulation. Taken together, these findings indicate that MIDN regulates the expression of a wide variety of genes, including multiple PD-causative genes and is associated with PD onset. Key words midnolin (MIDN); Parkinson's disease (PD); transcriptome analysis; parkinParkinson's disease (PD) is a progressive neurodegenerative disease accompanied by loss of dopaminergic neurons in the substantia nigra region of the midbrain, which projects to the striatum, resulting in an extrapyramidal disorder. Approximately 10% of PD is familial, and many causative genes have been identified.1-3) However, the other 90% of PD occurs sporadically and its causes are poorly understood.Midnolin (Midn) was discovered using a gene trap approach in embryonic stem cells. 4) MIDN is strongly expressed in the midbrain, and is localized to the nucleus and nucleolus. However, its expression is not limited to the midbrain and it is widely expressed in major organs.4) MIDN interacts with glucokinase via an N-terminal ubiquitin-like domain with high homology to parkin ubiquitin E3 ligase, leading to a significant reduction in glucokinase activity and glucose-induced insulin secretion from MIN6 cells. 5) Overall, although MIDN was discovered many years ago, its pathophysiological roles remain unclear.We recently showed that MIDN expression is induced by nerve growth factor (NGF) in PC12 cells, a model rat cell line for catecholaminergic neurons and that NGF-stimulated neurite outgrowth is attenuated in MIDN-deficient cells.6) However, tyrosine hydroxylase protein levels were not significantly altered in the study, indicating MIDN may not be involved in catecholamine biosynthesis in these cells. 6) Surprisingly, 10.5% of patients with sporadic PD lacked one copy of the MIDN gene whereas no copy number variation was observed in healthy people, suggesting MIDN to be a novel PD-risk factor or causative gene. Moreover, parkin (Park2), a familial PD-causative gene, was down-regulated by Midn knockout/ down in PC12 cells. 6) MIDN is assumed to be a transcription modulator; therefore, examination of expression of other PDrelated genes in Midn-manipulated cells is warranted. Thus, a comprehensive transcriptome analysis was performed in wildtype and Midn-knockout PC12 cells. MATERIALS AND METHODSMIDN-positive and -negative PC12 cells were grown in Dulbecco'...

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“…K + channel-interacting proteins (KChIPs) are β-subunit for K v 4.2, and the A-type current is influenced by the expression of KChiPs [ 24 ]. We previously performed RNA-sequencing to examine the gene expression levels comprehensively in PC12 cells [ 25 ]. The RPKM values for KChIPs 1, 2, 3, and 4 were 0.021284, 0, 1.21248, and 0.067198, respectively ( n = 3).…”

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“…PARK2, which is activated by PINK1 (Gladkova et al, 2018), wastemporally downregulated during memory formation, consistent with no reduction in T cell frequency in absence of PARK2 during viral infection, as reported elsewhere (Li et al, 2019). Although the exact mechanism of Park2 downregulation during T cell memory formation needs to be further elucidated, one possible mechanism could be related to midnolin ( Midn ) expression, which mediates gene expression of Park2 (Obara and Ishii, 2018) and has also been reported to be expressed in T cells (Hashimoto et al, 2013). However, whether Park2 downregulation during T cell memory formation is regulated by midnolin is a subject for future studies.…”

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“…The increase in the PARK2 gene expression level was also observed in clinical cases of PD or PD models occurred through certain epigenetic processes. For example, certain mutation or decrease in the expression in midnolin (MIDN) gene, observed in almost 10.5% of so-called sporadic PD, can lead to the decrease in PARK2 mRNA [27].…”

“…Cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method [27, 28]. As a colorimetric assay, MTT assay can measure the activity of NAD(P)H-dependent cellular oxidoreductase enzyme, so that the enzyme reduces the tetrazolium dye, MTT, into insoluble formazan crystals which has a purple color.…”