Biochemical and Biophysical Research Communications
Subcellular localization and putative role of VPS13A/chorein in dopaminergic neuronal cells
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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Current state of knowledge in Chorea-Acanthocytosis as core Neuroacanthocytosis syndrome
2018, European Journal of Medical GeneticsCitation Excerpt :Expression analysis showed Vps13a expression in many brain regions; subcellular analysis of brain lysates showed high level of Vps13a in the microsomal and synaptosomal fraction (Kurano et al., 2007). This would fit to a report in PC12 cells showing that Vps13a and synaptotagmin I were located in dense-core vesicles (DCVs), which interestingly also contained dopamine (Hayashi et al., 2012). GABAA receptor γ2-subunit and gephrin were found to be increased in the striatum of Vps13a deficient mice (Kurano et al., 2006).
Defective mitochondrial and lysosomal trafficking in chorea-acanthocytosis is independent of Src-kinase signaling
2018, Molecular and Cellular NeuroscienceCitation Excerpt :Even though the physiological role of chorein and its orthologues in yeast have begun to be understood, the pathophysiological implication of its deficit in humans and especially in human neurons remains largely elusive (Park et al., 2016; Park and Neiman, 2012). The first connection of chorein protein and neuronal tissue was achieved by Hayashi and co-workers, who were able to detect the chorein protein in the fraction of dense core vesicles of dopaminergic neurons upon gradient centrifugation (Hayashi et al., 2012). However, a first functional implication of the lack of chorein in human MSNs was just recently described by Stanslowsky and colleagues revealing a hyper-excitability phenotype of ChAc MSNs that was reversible upon PP2-induced Src kinase inhibition (Stanslowsky et al., 2016).
Chorein, the protein responsible for chorea-acanthocytosis, interacts with β-adducin and β-actin
2013, Biochemical and Biophysical Research CommunicationsCitation Excerpt :Human chorein contains a chorein_N motif, a proposed leucine zipper, a DUF1162 motif (conserved within vacuolar protein sorting-related proteins), and an ATG_C motif that may function by targeting it to vacuoles. Chorein also contains 10 Tetratrico Peptide Repeat motifs (Swiss-Prot entry Q96RL7) that potentially interact with other binding partners, and is predicted to have a coiled-coil structure by the online program PSORT II [13,35]. However, chorein does not contain an actin-binding domain.
TBC1D1 interacting proteins, VPS13A and VPS13C, regulate GLUT4 homeostasis in C2C12 myotubes
2020, Scientific ReportsCombined Dystonia With Self-Mutilation in 6-Pyruvoyl-Tetrahydropterin Synthase (PTPS) Deficiency: A Case Report
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They contributed equally to this study.