Original Article
Affiliation:
1Department of Neurodevelopmental Disorder Genetics, Institute of Brain Science, Nagoya City University Graduate School of Medical Sciences, Nagoya 467-8601, Japan
2Laboratory for Neurogenetics, RIKEN Center for Brain Science (CBS), Wako 351-0198, Japan
†These authors contributed equally to this work.
Email: toshi@med.nagoya-cu.ac.jp
ORCID: https://orcid.org/0000-0003-0585-5692
,
Affiliation:
2Laboratory for Neurogenetics, RIKEN Center for Brain Science (CBS), Wako 351-0198, Japan
3Current address: Department of Biotechnology and Bioinformatics, North Eastern Hill University (NEHU), Shillong 793022, India
†These authors contributed equally to this work.
Affiliation:
4Laboratory for Cell Function Dynamics, RIKEN Center for Brain Science (CBS), Wako 351-0198, Japan
5Current address: Laboratory of Biomolecular Network Dynamics, Biochemistry, Molecular and Structural Biology Section, Department of Chemistry, KU Leuven, 3001 Leuven, Belgium
ORCID: https://orcid.org/0000-0002-6983-5255
,
Affiliation:
2Laboratory for Neurogenetics, RIKEN Center for Brain Science (CBS), Wako 351-0198, Japan
Affiliation:
6Calcium Oscillation Project, International Cooperative Research Project-Solution Oriented Research for Science and Technology, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan
7Laboratory for Developmental Neurobiology, RIKEN Center for Brain Science (CBS), Wako 351-0198, Japan
8Current address: Laboratory for Cell Calcium Signaling, Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai 201210, China
Affiliation:
4Laboratory for Cell Function Dynamics, RIKEN Center for Brain Science (CBS), Wako 351-0198, Japan
ORCID: https://orcid.org/0000-0002-0671-4376
,
Affiliation:
1Department of Neurodevelopmental Disorder Genetics, Institute of Brain Science, Nagoya City University Graduate School of Medical Sciences, Nagoya 467-8601, Japan
2Laboratory for Neurogenetics, RIKEN Center for Brain Science (CBS), Wako 351-0198, Japan
Email: yamakawa@med.nagoya-cu.ac.jp
ORCID: https://orcid.org/0000-0002-1478-4390
Explor Neurosci. 2025;4:100699 DOl: https://doi.org/10.37349/en.2025.100699
Received: April 23, 2025 Accepted: June 16, 2025 Published: July 09, 2025
Academic Editor: Suyue Pan, Southern Medical University, China
The article belongs to the special issue Advances in Epilepsy Research
Aim: Mutations in the EFHC1 gene have been identified in patients with various epilepsies, including juvenile myoclonic epilepsy (JME). Mice with Efhc1 deficiency also exhibit epileptic phenotypes. The protein myoclonin1, encoded by EFHC1, is not expressed in neurons but in cells with motile cilia, including choroid plexus and ependymal cells lining of brain ventricles. However, the molecular mechanisms by which EFHC1 mutations cause epilepsy remain unclear. Because of the involvement of inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) in epileptic phenotypes and the involvement of myoclonin1 in calcium ions (Ca2+) signaling, we investigated possible functional interplay between myoclonin1 and IP3R1.
Methods: We performed immunohistochemical staining of brain tissues and co-immunoprecipitation assay of myoclonin1 and IP3R1, and Ca2+ imaging analyses using human HeLa.S3, mouse embryonic fibroblasts, or glial cells derived from Efhc1 homozygous knockout (Efhc1–/–) and wild-type (WT) littermates.
Results: Myoclonin1 was revealed to be well co-expressed with IP3R1 at choroid plexus and ependymal cells, and these two proteins bound to each other. Endoplasmic reticulum (ER) of Efhc1-deficient mouse (Efhc1–/–) cells showed larger amounts of Ca2+ than that of WT mice, and IP3-induced Ca2+ release (IICR) from ER was higher in Efhc1–/– cells than that of WT. Furthermore, myoclonin1 was revealed to interact with beta subunit of glucosidase II (PRKCSH), also known as a protein kinase C substrate 80K-H, which interacts with IP3R1. Myoclonin1 further binds to IP3R2 and IP3R3.
Conclusions: These results indicate that myoclonin1 modulates ER-Ca2+ homeostasis through interactions with IP3Rs and PRKCSH, and suggest that myoclonin1 dysfunctions cause impaired intracellular Ca2+ mobilization. Its relevance to the epileptic phenotypes of patients with EFHC1 mutations is now of interest.
Human EFHC1 gene encodes a 640 amino acid protein, myoclonin1, harboring three tandemly repeated DM10 domains and one EF-hand calcium-binding motif at C-terminus [1]. We originally and mistakenly reported that myoclonin1 is expressed in neurons of mouse brain [1], but our subsequent studies using Efhc1–/– mouse and a newly-developed mouse monoclonal antibody for myoclonin1 (6A3-mAb) revealed that myoclonin1 is actually not expressed in neurons but dominantly expressed in choroid plexus at embryonic stages, and at motile cilia of ependymal cells, tracheal motile cilia, and sperm flagella at postnatal stages [2–4]. EFHC1 heterozygous missense mutations in patients with epilepsies, including juvenile myoclonic epilepsy (JME), have been repeatedly reported by us and many other groups [1, 5–15]. A missense mutation in EFHC1 has also been identified homozygously in patients with intractable epilepsy of infancy [16]. We further reported that Efhc1-deficient mice exhibited spontaneous myoclonic seizures, increased seizure susceptibility to chemo-convulsant pentylenetetrazol (PTZ), decreased beating frequency of ependymal cells’ cilia as well as that of choroid plexus epithelial one, and enlarged brain ventricles [3, 17].
We have shown that over-expression of myoclonin1 in mouse hippocampal primary culture neurons activates R-type voltage-dependent calcium channel (Cav2.3), resulting in excessive intracellular calcium ions (Ca2+) influx and rapid cell-death, and these effects were compromised by EFHC1 mutations found in JME patients [1]. We further reported that myoclonin1 interacts with transient receptor potential M2 (TRPM2) channel, which is Ca2+-permeable cation channel, and potentiates the channel activity [18]. It is of interest whether myoclonin1 interacts with any other proteins to modulate Ca2+ signaling and homeostasis.
Endoplasmic reticulum (ER)-Ca2+ homeostasis is maintained by 1,4,5-trisphosphate receptors (IP3Rs) and IP3, which induce release of Ca2+ from ER [19]. There are three subtypes of IP3Rs, IP3R1–3, in mammals with distinct regulation and distribution throughout the body. IP3R1 is a predominant subtype in brain, and is involved in diverse functions such as development, axon-guidance, and cognition [20–23]. Because of the involvement of IP3R1 in epileptic phenotypes [24] and the involvement of myoclonin1 in Ca2+ signaling, as also suggested by its EF-hand Ca2+ binding motif [1], we investigated possible functional interplay between myoclonin1 and IP3R1. Here we report that in mouse brain, IP3R1 is well co-expressed with myoclonin1 at choroid plexus and ependymal cells, and IP3R1 binds to myoclonin1. Myoclonin1-deficiency significantly increased levels of ER-Ca2+ store ([Ca2+]ER) and IP3-induced Ca2+ release (IICR) activity in cells from Efhc1–/– mice. In addition, we find that myoclonin1 binds to beta subunit of glucosidase II (PRKCSH), which has been known to interact with IP3R1 [25]. Our findings indicate that myoclonin1 regulates ER-Ca2+ homeostasis through interaction with IP3R1.
Efhc1- and Ip3r1-deficient mice used for this study have been developed and reported previously [3, 24]. The heterozygous mice were maintained on C57BL/6J background. Heterozygous knockout Efhc1+/– or Ip3r1+/– mice were interbred to obtain wild-type (WT), heterozygous, and homozygous knockout mice. Food and water were available ad libitum, and cages (of less than 5 animals) were kept at 23°C on a 12-h/12-h light/dark cycle, with the lights off at 20:00. When euthanasia of mice was necessary, cervical dislocation was performed in accordance with institutional guidelines.
Mouse 6A3-mAb was reported previously [2, 4]. Following antibodies were also used: IP3R1 [KM1112 [26], 18A10 [24], 10A6 [27], H-80 or C-20 (Santa Cruz Biotechnology, USA)], IP3R2 (KM1083), IP3R3 (KM1082 [26] and BD Transduction lab, USA), FLAG (Sigma, USA), Myc (Cell Signaling Technology, USA), GAPDH (Santa Cruz Biotechnology), PRKCSH (Santa Cruz Biotechnology), and DsRed (Invitrogen).
Mice were deeply anesthetized with three types of mixed anesthetic agents (0.3 mg/kg medetomidine, 4.0 mg/kg midazolam, and 5.0 mg/kg butorphanol) and perfused intracardially with 0.9% NaCl, followed by 4% paraformaldehyde (TAAB, UK) in phosphate buffered saline (PBS). Preparations of mouse sagittal brain sections (paraffin) from embryonic day 14 (E14), unknown sex, and postnatal day 15 (P15) male mice and immunohistochemical staining were carried out as described previously [2, 3]. The 18A10 antibody for IP3R1 and 6A3-mAb for myoclonin1 were used for staining. Colorimetric and fluorescence images were acquired using the AX80 light-microscope (Olympus, Japan) and TCS SP2 confocal laser scanning microscope (Leica, Germany), respectively.
EFHC1 clone was described previously [1]. We amplified parts of IP3R1 (GenBank: NM_002222), IP3R2 (NM_002223), IP3R3 (NM_002224), and PRKCSH (NM_002743) from human adult brain cDNA by PCR and cloned them into pcDNA3-MycN, pcDNA3-FlagN, or pcDNA3-monomeric red fluorescence protein (mRFP) vectors. We introduced mutations by using QuickChange Site-Directed Mutagenesis kit (Agilent Technologies, USA) and confirmed nucleotide changes as well as integrity of full sequences by DNA sequencing.
Mouse embryonic fibroblasts (MEFs) were prepared as described previously from E16 unknown sex mouse tail [28]. Once the cells were confluent in poly-L-Lysine coated 60 mm dish (IWAKI, Japan), it was stored at –80°C until needed. To obtain glial cells, culture medium of hippocampal neuron culture prepared from E16 unknown sex mouse brain [1, 2] was replaced with Dulbecco’s Modified Eagle Medium (D-MEM) + 10% fetal bovine serum (FBS) + 30 U/mL penicillin and 30 mg/mL streptomycin (P/S) at 2 days in vitro (DIV) and cultured for 18 DIV. During the culture of MEFs or glial cells, visual inspection revealed no differences in cell viability or morphology between cells derived from WT and Efhc1–/– mice. The glial cells were harvested and stored in –80°C for subsequent assay. MEFs and HeLa.S3 cells were transfected with expression constructs using Lipofectamine LTX and PLUS reagent (Thermo Fisher Scientific, USA) according to the manufacturer’s protocol. Briefly, the cells were exposed to the lipid-DNA complex in serum-free medium (Opti-MEM I; Thermo Fisher Scientific). Subsequently, the cells were incubated in D-MEM supplemented with 10% FBS. Transfected cells were analyzed 24–48 hours post-transfection. The HeLa.S3 cell line was originally obtained from the American Type Culture Collection and cultured under standard conditions. Cells were authenticated by short tandem repeat profiling and were free of mycoplasma contamination.
We washed transfected HeLa.S3 cells twice in PBS and lysed in lysis buffer [10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5 or 1% NP40 and supplemented with protease inhibitor (Complete, Roche, Switzerland)]. Co-immunoprecipitation (co-IP) studies were carried out as described previously [1]. We probed blot with anti-FLAG, anti-Myc, or anti-DsRed antibodies and developed it with Western Lightning kit (Perkin Elmer, USA). Antibodies, IP3R1 (KM1112, 10A6, H-80 or C-20, Santa Cruz Biotechnology), IP3R2 (KM1083), IP3R3 (KM1082 and BD Transduction lab), PRKCSH (Santa Cruz Biotechnology), myoclonin1 (6A3-mAb), and GAPDH (Santa Cruz Biotechnology) were used for western blot analysis. Wherever necessary, we stripped blots and re-probed with respective antibodies.
Ca2+ imaging analyses were done as described previously [29]. We placed the cells on an inverted microscope IX70 (Olympus) and observed through an objective lens UApoN40XO340 (Olympus), etc. Some of the images were acquired using Olympus IX81-ZDC. The cells were illuminated by a xenon lamp through excitation filters, 340AF15 (Omega, USA) and 380HT15 (Omega), alternately. We used a computer-controlled filter exchanger Lambda 10-2 (Sutter, USA) to switch filters. We used a dichroic mirror and an emission filter 430DCLP (Omega) and 510WB40 (Omega), respectively. To eliminate Ca2+ influx, we performed all experiments in absence of extracellular Ca2+. We measured IICR with addition of 5 µM histamine [30] for HeLa.S3 cells or 300 nM bradykinin (BK) [31] for MEFs, whereas [Ca2+]ER was measured with addition of 5 µM ionomycin [32]. All measurements shown were representative results from two to four independent experiments (used 3 dishes per condition were used in each experiment).
Data were presented as mean ± standard error of the mean (s.e.m.) and statistical significance of differences between means was tested using unpaired t-test. Statistical significance levels were defined as P < 0.05.
Immunohistochemical analyses revealed that myoclonin1 and IP3R1 were abundantly expressed in choroid plexus at E14 (Figure 1A–C). As reported previously [2], the expression of myoclonin1 in choroid plexus was very transient, appearing at embryonic stages (E14–18) and gradually switching off until P15 (Figure 1D). In contrast, the IP3R1 expression in choroid plexus was observed at E14 (Figure 1B and C) and postnatal period (Figure 1D), suggesting its continuous expression through embryonic and postnatal stages. At P15, myoclonin1 was prominently expressed in motile cilia of ependymal cells but also moderately in cell body (Figure 1D); the somatic expression was more prominent in the 3rd ventricle as reported previously [2]. The IP3R1 expression was mainly observed in the somata and not in the cilia of ependymal cells (Figure 1D). Altogether, these results indicate that myoclonin1 and IP3R1 proteins are co-expressed in embryonic choroid plexus and postnatal ependymal cells.
Co-expressions of myoclonin1 and IP3R1 in embryonic choroid plexus and postnatal ependymal cells. (A–C) Colorimetric or fluorescent immunohistochemical analyses showed co-expression of myoclonin1 (brown in A, magenta in C) and IP3R1 (brown in B, green in C) in choroid plexus of mouse brain at E14 (sagittal brain sections, n = 2 WT, 2 Efhc1–/–, 2 Ip3r1–/–). (D) Co-expressions of IP3R1 (green) and myoclonin1 (magenta) were observed in ependymal cells at P15 (sagittal sections, n = 2 WT, 2 Efhc1–/–, 2 Ip3r1–/–). Boxed areas are enlarged. CP: choroid plexus; CPu: caudate putamen; CTX: cortex; E14: embryonic day 14; IP3R1: 1,4,5-trisphosphate receptor 1; LV: lateral ventricle; P15: postnatal day 15; WT: wild-type. Scale bars = 1 mm (low-magnification images in A, B), 25 µm (high-magnification images in A, B), 100 µm (low-magnification images in C, D), 15 µm (high-magnification images in C), and 8 µm (high-magnification images in D)
We next investigated whether myoclonin1 binds to IP3Rs. Because a number of proteins interact with either N- or C-terminal cytosolic regions of IP3R1 and modulate its activity [19], we selected these regions to test their ability to interact with myoclonin1 for co-IP assay. It actually revealed that myoclonin1 binds to the C-terminal region of IP3R1 but not to the N-terminal region or to Endophilin, a randomly selected protein with a molecular weight of 50 kDa used as a negative control (Figure 2A, B and Figure S1). Using a series of C-terminal deletion constructs, we further narrowed down the interacting region to amino acid residues (a.a.) 2565–2625 by co-IP assay (Figure 2A and C). Inversely, co-IP analyses using a series of deletion fragments of myoclonin1 revealed that each of the three DM10 domains of myoclonin1 independently bound to IP3R1 (Figure 2D and E). C-terminal regions of three IP3R subtypes (IP3R1, IP3R2, IP3R3) are highly conserved (Figure S2A), and a co-IP assay revealed that all C-termini of these IP3R subtypes similarly bound to myoclonin1 (Figure S2B).
Myoclonin1 interacts with C-terminus of IP3R1. (A) Schematic diagrams of IP3R1, its deletion constructs, and binding activity. Bold black lines indicate IP3R1 N- (a.a. 1–600) and C- (a.a. 2535–2695) terminus (top). A segment (a.a. 2565–2625; black line, middle) in C-terminal region of IP3R1 contains a binding site for myoclonin1. (B) Western blots of co-IP analysis showing that myoclonin1 interacted with IP3R1 C-terminus but not with N-terminus and Endophilin (negative control). (C) The interacting region of IP3R1 C-terminus to myoclonin1 was narrowed down to a.a. 2565–2625. (D) Schematic diagram of myoclonin1 deletion constructs and binding activity. A bold black line (top) contains a binding site for IP3R1. (E) Each of the three DM10 domains of myoclonin1independently bound to IP3R1. The degree of interaction is indicated by +, ±, or – (A, D). /C: C-terminal; /N: N-terminal; a.a.: amino acid residues; co-IP: co-immunoprecipitation; IB: immunoblot; IP: immunoprecipitation; IP3: 1,4,5-trisphosphate; IP3R1: 1,4,5-trisphosphate receptor 1. Input: 5% of cell lysate
PRKCSH is a gene that encodes the PRKCSH, also known as 80K-H, involved in protein folding in the ER. A previous report showed that PRKCSH was identified as an interacting protein of IP3Rs by yeast two-hybrid screening and regulates its activity [25]. Because binding sites of IP3R1 for PRKCSH and myoclonin1 look overlapping, we investigated interactions among these three proteins. As previously reported, co-IP revealed that a.a. 2555–2594 of IP3R1 bound to PRKCSH (Figure S3). A co-IP of myoclonin1 and a series of PRKCSH deletion constructs, including two clones, a.a. 32–528 and 184–528, which were identified as fragments binding to IP3R1 [25] and additional newly designed one, revealed that a.a. 400–448 of PRKCSH binds to myoclonin1 (Figure 3A–E and Figure S1). Similarly to IP3R1 (Figure 2D and E), the three DM10 domains of myoclonin1 independently bound to PRKCSH (Figure 3F and G).
Myoclonin1 interacts with PRKCSH at its interaction site for IP3R1. (A) Schematic diagram of PRKCSH deletion constructs and binding activity. A short segment (a.a. 400–448; black line) in PRKCSH contains binding site for myoclonin1. Other one (a.a. 365–418; gray line) has been reported as a binding site for IP3Rs [25]. (B–E) A region of PRKCSH a.a. 400–448 is critical to bind to myoclonin1. (F) Schematic diagram of myoclonin1 deletion constructs and binding activity. A bold black line (top) contains a binding site for PRKCSH. (G) PRKCSH is bound to myoclonin1 similarly to IP3R1. The degree of interaction is indicated by +, ±, or – (A, F). a.a.: amino acid residues; IB: immunoblot; IP: immunoprecipitation; IP3R1: 1,4,5-trisphosphate receptor 1; PRKCSH: beta subunit of glucosidase II. Input: 5% of cell lysate; ∆: internal deletion
We subsequently measured levels of [Ca2+]ER in MEFs and glial cells derived from Efhc1–/– and WT littermates by applying ionomycin, an ionophore that induces formation of Ca2+-permeable pores in cellular membranes, leading to complete emptying of [Ca2+]ER independently of IP3Rs activation [32]. The assay showed that [Ca2+]ER levels of the Efhc1–/– cells were significantly higher compared to WT (Figure 4A and B). We also measured IICR by addition of BK, which stimulates phospholipase C (PLC) metabolism of phosphatidylinositol-4,5-bisphosphate (PIPP) to IP3 [31], and observed a higher IICR level in Efhc1–/– MEFs than in WT (Figure 4C). Western blot analyses revealed that myoclonin1 expression was abrogated in the Efhc1–/– MEFs, while those of IP3Rs and PRKCSH remained unchanged (Figure S4). These results indicate that myoclonin1 deficiency enhances [Ca2+]ER and IICR.
Myoclonin1 deficiency enhances [Ca2+]ER and IICR. Ionomycin releasable [Ca2+]ER was significantly higher in Efhc1–/– cells than in WT. (A) n = 49 WT, 135 Efhc1–/– MEFs, unpaired t-test, WT vs. Efhc1–/–, P = 3.89 × 10–6, df = 182. There was a significant difference between the mean values. (B) n = 74 WT, 73 Efhc1–/– glial cells, unpaired t-test, WT vs. Efhc1–/–, P = 6.18 × 10–8, df = 145. There was a significant difference between the mean values. (C) IICR induced by BK was significantly higher in Efhc1–/– MEFs than in WT (n = 192 WT, 181 Efhc1–/–, unpaired t-test, WT vs. Efhc1–/–, P = 6.73 × 10–6, df = 371). There was a significant difference between the mean values. All measurements shown were representative results from two to four independent experiments (used 3 dishes, cells derived from 2 independent animals per genotype, were used in each experiment). Arrows indicate time point of addition of ionomycin or BK. df: degrees of freedom; n: total number of cells measured; P: P-value; IICR: 1,4,5-trisphosphate-induced calcium ions release; WT: wild-type; [Ca2+]ER: endoplasmic reticulum-calcium ions store; MEFs: mouse embryonic fibroblasts; BK: bradykinin
We further investigated whether myoclonin1 reduces [Ca2+]ER and IICR. Human HeLa.S3 cells were transfected with mRFP-fused myoclonin1, which did not affect expression of IP3Rs and PRKCSH (Figure S5A). Ca2+ imaging revealed that [Ca2+]ER by applying ionomycin and IICR by histamine, which generates IP3 through activation of PLC [30], were significantly decreased by myoclonin1 over-expression (Figure S5B and S5C). To confirm the effect, myoclonin1 was further re-introduced into Efhc1–/– MEFs. Ca2+ imaging also revealed that both [Ca2+]ER by ionomycin and IICR by BK were significantly attenuated in mRFP-myoclonin1 expressing MEFs compared to control one (Figure S5D and S5E). Together, these results indicate that myoclonin1 lowers [Ca2+]ER and is involved in maintenance of ER-Ca2+ homeostasis.
Here in this study, we showed that myoclonin1 forms a protein complex with IP3Rs and PRKCSH, and regulates IP3-mediated Ca2+ release. Together with our previous observations of the bindings of myoclonin1 with Cav2.3 [1] and with TRPM2 [18], the results suggest that myoclonin1 is involved in intracellular Ca2+ mobilization.
A notable observation is that myoclonin1 and IP3R1 are co-expressed in ciliated cells, such as choroid plexus and ependymal cells in the brain. We also found that myoclonin1 deficiency enhances [Ca2+]ER and IICR in cells from Efhc1-deficient mice. It has previously been reported that (1) Ca2+ initiates beating of cilia and flagella [33], and (2) elevation of cytosolic Ca2+ contributes to increased cilia beating frequency (CBF) of ependymal cells [34, 35]. Moreover, blockage of cilia-mediated cerebrospinal fluid (CSF) inflow has been known to be sufficient to predispose another mouse model to seizures [36]. These may let us assume that reduced CBF by decreased cytosolic Ca2+ causes seizures in the Efhc1-deficient mice. However, in our previous study of the mice, we showed that reduced CBF was observed only in homozygous Efhc1–/– mice but not in the heterozygous ones (Efhc1+/–), which still showed seizure phenotypes such as frequent spontaneous myoclonus and increased seizure susceptibility to PTZ [3]. These results indicate that there is considerable inconsistency between seizure phenotypes and CBF in heterozygous Efhc1+/– mice. Taken together, these observations may suggest that blockage of cilia-mediated CSF inflow and reduction of CBF themselves may not or minimally contribute directly to the seizure phenotypes observed in the Efhc1-deficient mice, and an alternative pathway (see below) would be required to explain the molecular basis of epilepsies in the mice.
We have reported that myoclonin1 is dominantly expressed in choroid plexus epithelial cells at embryonic stage [2], and in this study, we found that myoclonin1 and IP3R1 are well co-expressed in those cells as well, and these two proteins bind each other as mentioned above. In addition, we also have reported that CBF of neonatal choroid plexus epithelial cells from Efhc1-deficient mice was significantly lower than that of WT mice [17]. Based on these findings, we assume that myoclonin1 possibly plays critical role in choroid plexus epithelial cells. Further, the cells synthesize neurotrophic factors and other signaling molecules, including insulin, that are secreted in response to increased intracellular Ca2+ levels [37]. Previous study has shown that IP3R1-deficient mice suffer from epilepsy [24]. In our Ca2+ imaging analyses, we observed increased [Ca2+]ER and enhanced IICR through IP3R1 in cells from Efhc1-deficient mice. These findings suggested that impaired IP3R1-mediated Ca2+ signaling may disrupt the secretion of signaling molecules from choroid plexus cells. Such disruption could lead to alterations in synaptic plasticity (long-term potentiation/long-term depression) and abnormal neural circuit formation, which may also underlie the epileptic phenotypes in these mice.
In resting cells, [Ca2+]ER is tightly regulated by a balance between Ca2+ release via IP3Rs, reuptake by SERCA pumps, and the ER translocon [38–40]. In myoclonin1 deficient models, elevated [Ca2+]ER and enhanced IICR suggest that myoclonin1 may modulate ER-Ca2+ homeostasis by influencing either Ca2+ leak or SERCA activity. Myoclonin1 deficiency could reduce ER-Ca2+ leak, leading to excessive [Ca2+]ER accumulation, which may then trigger a compensatory downregulation of SERCA activity to restore Ca2+ balance. This mechanism is consistent with previous report that SERCA function is sensitive to luminal Ca2+ levels [38]. Thus, myoclonin1 likely fine-tunes ER-Ca2+ dynamics through its influence on leak and reuptake mechanisms.
Although we previously reported that myoclonin1 interacts with Cav2.3 [1] and TRPM2 [18], Cav2.3 is mainly expressed in neurons and may not be much expressed in choroid plexus and ependymal cells. Therefore, we currently assume that it may not be critically involved in the JME pathology. Meanwhile, TRPM2 is expressed in ependymal cells and therefore it is highly possible that TRPM2 is also involved in the pathology through myoclonin1-IP3R1 pathway.
In addition to EFHC1, we have identified and reported multiple pathogenic mutations of CILK1 (ciliogenesis associated kinase 1) gene, formerly known as ICK (intestinal-cell kinase), in patients affected with JME from a number of families [41]. High level of CILK1 expression was observed in ependymal and choroid plexus cells in postnatal mouse brain [41, 42]. Moreover, pathogenic mutations of several other ciliogenesis associated proteins, CDKL5/STK9 [43, 44], SDCCAG8 [45, 46], PRICKLE1 and PRICKLE2 [47, 48] have also been reported in patients with epilepsies, including JME. These observations support the notion that functional impairments of the cells with motile cilia in brain, such as cell-cell communication, synthesis of neurotrophic factors and signaling molecules, secretion of the molecules, and CSF or ion homeostasis, are likely the basis of epileptic seizure phenotypes in patients with EFHC1 mutations.
To further explore the link between seizures and altered intracellular Ca2+ signaling in non-neural cells such as choroid plexus and ependymal cells, future studies should examine neural activity and Ca2+ imaging in acute brain slices and in vivo. Furthermore, testing whether pharmacological modulation of IP3Rs can restore Ca2+ homeostasis and attenuate seizure phenotypes may provide mechanistic insights and identify potential therapeutic targets for JME. Given that JME is characterized by thalamocortical circuit hypersynchrony [49], future investigations at the circuit level would be of great interest to further elucidate how intracellular calcium dysregulation contributes to network-level pathophysiology.
Our results presented here indicate that myoclonin1 participates in controlling intracellular Ca2+ mobilization in a manner that involves IP3Rs. This could be molecular basis underlying pathology of epilepsies caused by EFHC1 mutations and potential candidate for prevention strategies and treatments of epilepsies.
[Ca2+]ER: endoplasmic reticulum-calcium ions store
6A3-mAb: monoclonal antibody for myoclonin1
a.a.: amino acid residues
BK: bradykinin
Ca2+: calcium ions
Cav2.3: R-type voltage-dependent calcium channel
CBF: cilia beating frequency
CILK1: ciliogenesis associated kinase 1
co-IP: co-immunoprecipitation
CSF: cerebrospinal fluid
DIV: days in vitro
D-MEM: Dulbecco’s Modified Eagle Medium
E14: embryonic day 14
ER: endoplasmic reticulum
FBS: fetal bovine serum
IICR: 1,4,5-trisphosphate-induced calcium ions release
IP3: 1,4,5-trisphosphate
IP3Rs: 1,4,5-trisphosphate receptors
JME: juvenile myoclonic epilepsy
MEFs: mouse embryonic fibroblasts
mRFP: monomeric red fluorescence protein
P15: postnatal day 15
PBS: phosphate buffered saline
PLC: phospholipase C
PRKCSH: beta subunit of glucosidase II
PTZ: pentylenetetrazol
TRPM2: transient receptor potential M2
WT: wild-type
The supplementary figures for this article are available at: https://www.explorationpub.com/uploads/Article/file/100699_sup_1.pdf.
We thank members of Laboratories for Cell Function Dynamics, Developmental Neurobiology, and Neurogenetics at RIKEN Center for Brain Science (CBS), Department of Neurodevelopmental Disorder Genetics, and animal facility at the Nagoya City University (NCU). We also thank Dr. Takeshi Nakamura, who passed away on July 23rd, 2006, for his contributions to experimental design, research execution, and the provision of animal materials. We are indebted to the Support Unit for Bio-material Analysis, Research Resources Division (RRD) at the RIKEN CBS and the Research Equipment Sharing Center at the NCU. We are also grateful to RIKEN CBS-Olympus Collaboration Center.
TS: Conceptualization, Investigation, Writing—original draft, Visualization, Funding acquisition. KA: Conceptualization, Investigation, Writing—original draft, Visualization. HM: Conceptualization, Methodology, Investigation, Resources, Writing—review & editing, Visualization. II: Investigation, Writing—review & editing. KM and AM: Resources, Writing—review & editing, Funding acquisition. KY: Conceptualization, Resources, Writing—review & editing, Supervision, Funding acquisition. All authors read and approved the submitted version.
The authors declare that they have no conflicts of interest.
The animal study was approved by the Animal Experiment Committee of RIKEN (W2019-1-006) and by the Institutional Animal Care and Use Committee of the Nagoya City University (NCU; 23-025). All animal breeding and experimental procedures were performed in accordance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines and regulations of the RIKEN and the NCU. This animal study adheres to the Guide for the Care and Use of Laboratory Animals.
Not applicable.
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The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.
This work was supported by grants from RIKEN CBS; Nagoya City University; JSPS (Japan Society for the Promotion of Science) KAKENHI Grant Numbers [20790866] and [22791154]; the Japan Epilepsy Research Foundation; and Grant-in-Aid for Outstanding Research Group Support Program in Nagoya City University [2401101]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
© The Author(s) 2025.
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