Subcellular localization and putative role of VPS13A/chorein in dopaminergic neuronal cells

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Abstract

Chorea-acanthocytosis (ChAc) is a rare hereditary neurodegenerative disorder caused by loss of function mutations in the vacuolar protein sorting 13 homolog A (VPS13A) gene encoding chorein. Although a deficiency in chorein function leads to apoptosis of striatal neurons in ChAc model mouse, its detailed subcellular localization and physiological role remain unclear. In this study, we produced two anti-chorein polyclonal antibodies and examined the intracellular localization of endogenous chorein in neuronal cells. Immunocytochemically, chorein was observed in the termini of extended neurites and partially colocalized with synaptotagmin I in differentiated PC12 cells. Subcellular localization analysis by sucrose density gradient fractionation showed that chorein and synaptotagmin I were located in dense-core vesicles (DCVs), which contain dopamine. In addition, PC12 cells stably expressing carboxyterminal fragment of chorein increased K+-induced dopamine release. Taken together, these results suggest that chorein is involved in exocytosis of DCV.

Highlights

► Chorein was partially colocalized with Synaptotagmin I in PC12 cells. ► Chorein was localized to dense-core vesicle in mouse brain and PC12 cells. ► Expression of chorein C-terminus influenced dopamine release of PC12 cells.

Introduction

Chorea-acanthocytosis (ChAc; OMIM ID: 200150) is a rare autosomal recessive neurodegenerative disorder, which is characterized by adult-onset chorea and erythrocyte acanthocytosis. Clinically, ChAc patients show various symptoms such as psychiatric features, epilepsy, peripheral neuropathy, myopathy, and oral self-mutilation [1]. Their symptoms have been thought to resemble those of Huntington’s disease [2]. The main neuropathological feature of ChAc is degeneration of the striatum [3]. The inherited pattern of ChAc is considered to be autosomal recessive, and ChAc is caused by loss of function mutations in the VPS13A gene, encoding a protein named chorein [4], [5]. VPS13A is located on human chromosome 9q21, spanning approximately 250 kb. Concerning the structure of VPS13A, two main splicing variants have been reported: transcript A (exons 1–68, 70–73, GenBank Accession No. NM_033305) and transcript variant B (exons 1–69, GenBank Accession No. NM_015186) [4], [6]. Chorein is a 360 kDa protein that is absent or markedly reduced in ChAc patients with VPS13A mutations [7]. The null mutant of Saccharomyces cerevisiae homolog, VPS13, exhibits deficiency in vacuolar protein sorting [8]. A mutant TipC gene (an ortholog of Vps13p) in Dictyostelium discoideum has abnormality in cell-sorting behavior [9]. These findings suggest that chorein is involved in the cytoskeleton and intracellular transport system.

Chorein is highly expressed in testis, kidney, spleen, and brain. In wild-type mouse brain, chorein is predominantly localized to microsomal and synaptosomal fractions, the neuronal perinuclear region, cytoplasm, and fibers [10]. In ChAc model mouse, a deficiency in chorein function leads to apoptosis of striatal neurons [11]. Although there have been some findings about the biological function of chorein, its detailed subcellular localization and physiological role remain unclear.

In this study, we investigated the detailed subcellular localization and physiological role of chorein in neuronal cells to clarify the molecular pathogenesis of ChAc.

Section snippets

Plasmid constructions

We used partial cDNAs encoding human VPS13A (transcript variant B) (hVPS13A). pCold-TF/hVPS13A-(1-604) and pCold-TF/hVPS13A-(2610-3095) were constructed for bacterial His6-tagged protein expression using standard techniques. pcDNA-His-Flag/hVPS13A-(1-609) and pEF-BOS-HA/hVPS13A-(2610-3095) were constructed for mammalian expression using standard techniques.

Materials and chemicals

pcDNA-His-Flag vector and mouse N1E-115 neuroblastoma cells were provided by Dr. M. Fukata (National Institute for Physiological Sciences,

Detection of endogenous chorein

In this study, we produced two anti-chorein polyclonal antibodies. The anti-N-chorein and anti-C-chorein antibodies were produced and purified using bacterially expressed His6-tagged hVPS13A-(1-604) and hVPS13A-(2610-3095) as immunogens, respectively (Fig. 1A). Both antibodies recognized endogenous full-length chorein (approximately 360 kDa) in wild-type mouse brain, mouse N1E-115, rat PC12 cells, and human erythrocyte ghost samples, but not in the brain of the ChAc model mice (Fig. 1B).

These

Discussion

Chorein is thought to be involved in the cytoskeleton and intracellular transport system in non-mammalian species [8], [9]. However, its physiological role is poorly understood in mammals, except that it is absent or markedly reduced in ChAc patients. In this study, we have examined the detailed subcellular localization of chorein and neurotransmitter release in PC12 cells in order to clarify the physiological role of chorein, and demonstrated for the first time that chorein is involved in K+

Acknowledgments

We wish to thank Joint Research Laboratory, Kagoshima University Graduate School of Mental and Dental Sciences, for the use of their facilities. We thank all the staff members of the Institute of Laboratory Animal Sciences, Kagoshima University (Frontier Science Research Center) who kept the animals in good condition. This work was supported in part by Grants-in-Aid for Scientific Research (C) from the Ministry of Education, Culture, Sports, Sciences and Technology of Japan (S.K.), by grants

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    They contributed equally to this study.

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