Author: 久米広大

Expectations for new therapies and drug discovery for this disease.

【Key points】

• We have identified the CACNA1G gene as the causative gene of spinocerebellar degeneration (SCD) (1) in a Japanese family. Using dermal fibroblasts obtained from patients with the CACNA1G mutation, we established iPS cells and succeeded in differentiating them into cerebellar Purkinje cells for the first time in the world. It is expected to lead to therapies and drug discovery based on a new approach to this disease.We have identified the CACNA1G gene (*2) as the causative gene of spinocerebellar degeneration (SCD) (*1) in a Japanese family. Using dermal fibroblasts obtained from patients with the CACNA1G mutation, we established iPS cells and succeeded in differentiating them into cerebellar Purkinje cells for the first time in the world. It is expected to lead to therapies and drug discovery based on a new approach to this disease.

Summary

　A research group led by Associate Professor Hiroyuki Morino, Research Scientist Yukiko Matsuda, and Professor Hidefumi Kawakami at the Hiroshima University Research Institute for Radiation Biology and Medicine, together with Professor Koichi Hashimoto at the Graduate School of Medical, Dental and Health Sciences and RIKEN, has identified the gene responsible for spinocerebellar degeneration (SCD). They identified the causative gene of spinocerebellar degeneration (SCD) in a Japanese family and found that mutations in the CACNA1G gene, which encodes a calcium channel, cause autosomal dominant (*3) spinocerebellar degeneration. The researchers also established iPS cells using dermal fibroblasts obtained from patients with CACNA1G mutation, and succeeded in differentiating them into cerebellar Purkinje cells for the first time in the world. These results are expected to lead to therapies and drug discovery based on a new approach to this disease. The results of this research were published in the British international academic journal Molecular Brain at 21:00 (Japan time) on December 29, 2015.

Publication

Authors
Hiroyuki Morino*, Yukiko Matsuda, Keiko Muguruma, Ryosuke Miyamoto, Ryosuke Ohsawa, Toshiyuki Ohtake, Reiko Otobe, Masahiko Watanabe, Hirofumi Maruyama, Kouichi Hashimoto, Hideshi Kawakami*
* Corresponding author
Title
A mutation in the low voltage-gated calcium channel CACNA1G alters the physiological properties of the channel, causing spinocerebellar ataxia.
Journal
Molecular Brain 2015 8:89
DOI:10.1186/s13041-015-0180-4

Background

　Spinocerebellar degeneration (SCD) is an intractable disease with no established cure and is known to be genetically diverse. Many forms of the disease have been described, and 36 autosomal dominant forms have been reported, of which the causative gene has been identified in 31 forms. In many neurodegenerative diseases, findings from inherited forms have contributed to the elucidation of pathogenesis and the development of therapies. However, based on a genetic study of more than 2,000 SCD cases [1], the causative gene is unknown in about 30% of cases with dominant inheritance, and there are still many unanswered questions, and further elucidation of the pathogenesis is required.

Details of research results

　In this study, we genetically analyzed a large family lineage of dominantly inherited SCD, including 10 patients with the disease, collected in collaboration with Dr. Toshiyuki Otake, Chief of the Department of Neurology, Ebara Hospital, Tokyo Metropolitan Healthcare Corporation (Fig. 1). First, linkage analysis was performed based on the results of high-density single nucleotide polymorphism (SNP), and it was inferred that the causative genes existed with high probability on chromosomes 17 and 21. The causative mutations were identified using exome sequencing, in which only the protein-coding region of the gene is selectively amplified and sequenced using a next-generation sequencer. The gene with the causative mutation was CACNA1G, which encodes one of the calcium channels. Furthermore, the same mutation was also found in one other family with dominantly inherited SCD. The same mutation was reported in a French SCD family in November 2015 [2]. In this study, we identified the causative gene in a Japanese ancestry as an independent study. The calcium channel encoded by CACNA1G is CaV3.1, one of the T-type voltage-dependent calcium channels (T-type VDCCs), which has six transmembrane sites repeated four times The fourth transmembrane site in each repeat has a role as a potential sensor. The mutation identified in this study alters a key amino acid in this voltage-sensing region, and is therefore thought to alter physiological properties (Fig. 2). In order to capture the mutation-induced changes in channel function, they expressed wild-type and mutant CaV3.1 in cultured cells and analyzed them by the patch-clamp method with Professor Koichi Hashimoto of Tohoku University. As a result, the current change induced by prepulse in the mutant CaV3.1 was shifted toward the positive potential (Fig. 3). In addition, we established iPS cells using dermal fibroblasts obtained from skin donated by patients with genetic abnormalities of CACNA1G, together with Keiko Muguruma, a researcher at the RIKEN Center for Multicellular Systems Research, who recently succeeded in differentiating mouse and human ES cells into cerebellar Purkinje cells [3]. They also succeeded in differentiating iPS cells into cerebellar Purkinje cells by applying the differentiation method described above (Fig. 4). This is the first differentiation of patient-derived iPS cells into cerebellar Purkinje cells in the world. Compared with cerebellar Purkinje cells derived from normal control iPS cells, no difference was observed immunohistologically or morphologically, and this disease model cell is expected to be used for drug discovery in the future.

Future perspective

　CACNA1A, KCNC3, and KCND3 have been reported as the causative genes of SCD, and KCNA1, CACNA1A, and CACNB4 are known as the causative genes of periodic ataxia, a related disease. Abnormalities in ion channels are thought to be very important as a cause of clinical cerebellar ataxia, and this discovery is expected to lead to therapies and drug discovery based on new approaches.

Acknoledgement

　Part of this work was supported by JSPS Grants-in-Aid for Scientific Research (A) (Kawakami, H.; 26242085), Grant-in-Aid for Scientific Research on Innovative Areas (Kawakami, H.; 23111008), Challenging Budding Researchers (Morino, T.; 15K15083), and for research using iPS cells, by the Japan Science and Technology Agency (JST) and the National Institute for Biomedical Innovation (AMED) Network Program for Realization of Regenerative Medicine, “Research on Intractable Diseases Using Disease-Specific iPS Cells” project, “In vitro Modeling and Development of Therapeutic Methods for Intractable Diseases of the Nervous System and Visual System Using High-Quality Differentiated Cells and Tissues” (Keiko Muguruma). This is the project.

【参考資料】

Figure 1. Family tree of hereditary SCD. Family 1 has 10 affected individuals, including the originator (arrow), over three generations. Family 2 has 5 affected individuals, including the originator (arrow), over 3 generations. The genotype of the causative mutation in the affected person is A/G, indicating heterozygous mutation.

Figure 2. Structure of CaV3.1 and site of mutation.
The identified mutation was located in a critical part of the calcium channel as a potential sensor.

Figure 3. Mutation-induced changes in electrophysiological properties.
The mutation shifts the prepulse-induced current change toward the positive potential.

Figure 4. Cerebellar Purkinje cells induced from patient-derived iPS cells.
Purkinje cells induced from normal control (left) and patient-derived (right) iPS cells. Distinctive dendrites are observed.

References

[1] Sugihara K, Maruyama H, Morino H, et al. The clinical characteristics of spinocerebellar ataxia 36: a study of 2121 Japanese ataxia patients. Mov Disord. 2012;27:1158–63.
[2] Coutelier M, Blesneac I, Monteil A, et al. A Recurrent Mutation in CACNA1G Alters Cav3.1 T-Type Calcium-Channel Conduction and Causes Autosomal-Dominant Cerebellar Ataxia. Am J Hum Genet 2015;97:1–12.
[3] Muguruma K, Nishiyama A, Kawakami H, et al. Self-Organization of Polarized Cerebellar Tissue in 3D Culture of Human Pluripotent Stem Cells. Cell Rep. 2015;10:537–50.

Annotation

(*1) Spinocerebellar degeneration (SCD) SCD is an intractable neurological disease with no fundamental cure. The main symptoms are lightheadedness, difficulty speaking, and hand tremors. About 1/3 of patients have a history of inheritance, and hereditary forms are known to be autosomal dominant and recessive, and X-linked.

(*2) CACNA1G gene
A gene encoding CaV3.1, one of the voltage-gated calcium channels that opens upon cell depolarization and is of the low potential active (T-type) type. It is mainly expressed in the brain and heart, and is related to repetitive activities such as pacemaking.

(*3) Autosomal dominant inheritance 　　　　　　　　　　　　　　　　　　　　　　　　　　　A form of inheritance that occurs when at least one of a pair of homologous genes has a mutation.

Summary

A research group led by Professor Hidefumi Kawakami and Associate Professor Hirofumi Maruyama at Hiroshima University Research Institute for Radiation Biology and Medicine has elucidated a new causative gene for amyotrophic lateral sclerosis (1). Mutations in the gene Optineurin (OPTN) cause amyotrophic lateral sclerosis. This achievement has been published in the academic journal “Nature” (May 13, 2010 Volume 465 Issue 7295).

Although several causative genes of amyotrophic lateral sclerosis (ALS) have been elucidated, the causative genes of most hereditary ALS remain unknown. In this study, we focused on a family with autosomal recessive heredity (2) and identified a candidate region by analyzing high-density Single Nucleotide Polymorphism (SNP) (3). gene mutation in OPTN. One of the functions of OPTN is to suppress NF-κB, an intracellular signal transduction system, and it was found that the suppressive function was lost due to the gene mutation. NF-κB is known to be involved in inflammation and carcinogenesis, and loss of function of OPTN may cause excessive activation of NF-κB and affect motor nerves. Until now, the relationship between amyotrophic lateral sclerosis and NF-κB has not attracted much attention, and although NF-κB inhibitors are candidates for therapeutic agents, we aim to develop a therapeutic method based on such a pathogenic mechanism in the future.

Main collaborators

Graduate School of Medicine, The University of Tokushima, Yuishin Izumi

Department of Neurology, Kansai Medical University (now Department of Neurology, Kyoto University) Hidefumi Ito

Department of Respiratory Medicine, Saitama Medical University Hospital, Koichi Hagiwara

Detailed explanation

The causative genes for amyotrophic lateral sclerosis that have been discovered so far account for only 10-20% of the hereditary forms of the disease. In order to develop a fundamental treatment for the disease, it is necessary to discover new causative genes and elucidate the common pathogenic mechanism. In this study, we focused on cases of autosomal recessive inheritance, which occur in families with consanguineous marriages. 6 cases were selected, and their high-density Single Nucleotide Polymorphism (SNP) was analyzed. (Note 4) in each of the six cases, and extracted candidate regions for the causative gene by finding contiguous regions of common homozygosity. For this extraction, we used a method devised by our collaborator Professor Koichi Hagiwara of Saitama Medical University. By determining the nucleotide sequence of the gene that exists in the region, they found the gene mutation of OPTN. This OPTN gene was originally reported to be the cause of autosomal dominant normal tension glaucoma. We found three types of abnormalities in regions other than glaucoma in three cases from two families with autosomal recessive inheritance, one case with no genetic history, and four cases from two families with autosomal dominant inheritance. In each case, the mutation impaired protein function. One of the functions of OPTN is to inhibit NF-κB, an intracellular signal transduction system, and these mutations were found to cause loss of this inhibitory function. Glaucoma mutations did not result in loss of the inhibitory function, suggesting that the pathogenesis of glaucoma is different between the two (Fig. 1).

Fig. 1. activity of NF-κB is measured by luciferase. activity is not suppressed by dominant and recessive mutations of ALS.

We examined the distribution of OPTN in cultured cells and found that in normal cells, the granular material was in close proximity to the Golgi apparatus (Fig. 2a), but with autosomal dominant mutations, the number of granules was reduced and their proximity to the Golgi apparatus was also reduced (Fig. 2b). Glaucoma mutations resulted in larger granules and closer proximity to the Golgi apparatus (Fig. 2c), suggesting that changes in the subcellular distribution of OPTN may impair the function of these protein complexes. It is predicted that changes in the subcellular distribution of OPTN may impair the function of these protein complexes.

Fig.2. The subcellular distribution of OPTN is reduced by ALS mutation.

　When the distribution of OPTN was examined in the autopsy spinal cord of a patient with amyotrophic lateral sclerosis who had an autosomal dominant mutation, it produced aggregates in the cytoplasm that were stained with anti-OPTN antibodies (Fig. 3a). In addition, in the spinal cord of a solitary patient without an OPTN mutation (Fig. 3b) and in the spinal cord of a familial patient with a SOD1 mutation (Fig. 3c), we found OPTN-positive aggregates in the cytoplasm. These findings suggest that OPTN is closely involved in the pathogenesis of amyotrophic lateral sclerosis in general.

Fig. 3. Autopsy spinal cord. Both show aggregates stained with anti-OPTN antibody.

　Mutations in OPTN are predicted to cause abnormalities in the function of intracellular protein complexes and NF-κB, resulting in the death of motor cells. This achievement not only clarified one of the causative genes of amyotrophic lateral sclerosis, but also demonstrated that OPTN is involved in the common pathogenic mechanism of this disease. In the future, we aim to elucidate this mechanism and to develop therapeutic methods based on the pathogenic mechanism, such as NF-κB inhibitors.

Annotation

(1) Amyotrophic lateral sclerosis is also called Lou Gehrig’s disease. It is a disease that causes muscle weakness and muscle atrophy due to degeneration and loss of the motor nerves that control muscles. There is no fundamental cure, and without support such as a ventilator, death can occur in three to five years. Autosomal recessive inheritance: A form of inheritance that occurs when an abnormal gene is on an autosome and two mutant alleles are present. (3) Single Nucleotide Polymorphism (SNP): Human genes consist of 3 billion base pairs of DNA, but when comparing each individual, about 0.1% of them have differences in base sequences. This is called a genetic polymorphism. This is called a genetic polymorphism, and when one base changes into another, it is called a single nucleotide polymorphism (SNP). (4) Homozygous A pair of homologous chromosomes has the same nucleotide sequence at a specific locus.

Reference

Mutations of optineurin in amyotrophic lateral sclerosis
H Maruyama, H Morino, H Ito, Y Izumi, H Kato, Y Watanabe, Y Kinoshita, M Kamada, H Nodera, H Suzuki, O Komure, S Matsuura, K Kobatake, N Morimoto, K Abe, N Suzuki, M Aoki, A Kawata, T Hirai, T Kato, K Ogasawara, A Hirano, T Takumi, H Kusaka, K Hagiwara, R Kaji, H Kawakami
Nature, doi: 10.1038/nature08971