Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Jun 23:15:919199.
doi: 10.3389/fnmol.2022.919199. eCollection 2022.

Rare KCND3 Loss-of-Function Mutation Associated With the SCA19/22

Mengjie Li  1 , Fen Liu  1   2 , Xiaoyan Hao  1   2 , Yu Fan  1   2 , Jiadi Li  1   2 , Zhengwei Hu  1   2 , Jingjing Shi  1   3 , Liyuan Fan  1   2 , Shuo Zhang  1   2 , Dongrui Ma  1 , Mengnan Guo  1 , Yuming Xu  1   3   4   5   6   7 , Changhe Shi  1   4   5   6   7
Affiliations

Rare KCND3 Loss-of-Function Mutation Associated With the SCA19/22

Mengjie Li et al. Front Mol Neurosci. .

Abstract

Spinocerebellar ataxia 19/22 (SCA19/22) is a rare neurodegenerative disorder caused by mutations of the KCND3 gene, which encodes the Kv4. 3 protein. Currently, only 22 KCND3 single-nucleotide mutation sites of SCA19/22 have been reported worldwide, and detailed pathogenesis remains unclear. In this study, Sanger sequencing was used to screen 115 probands of cerebellar ataxia families in 67 patients with sporadic cerebellar ataxia and 200 healthy people to identify KCND3 mutations. Mutant gene products showed pathogenicity damage, and the polarity was changed. Next, we established induced pluripotent stem cells (iPSCs) derived from SCA19/22 patients. Using a transcriptome sequencing technique, we found that protein processing in the endoplasmic reticulum was significantly enriched in SCA19/22-iPS-derived neurons and was closely related to endoplasmic reticulum stress (ERS) and apoptosis. In addition, Western blotting of the SCA19/22-iPS-derived neurons showed a reduction in Kv4.3; but, activation of transcription factor 4 (ATF4) and C/EBP homologous protein was increased. Therefore, the c.1130 C>T (p.T377M) mutation of the KCND3 gene may mediate misfold and aggregation of Kv4.3, which activates the ERS and further induces neuron apoptosis involved in SCA19/22.

Keywords: KCND3 mutation; PERK-ATF4-CHOP pathway; SCA19/22; endoplasmic reticulum stress; iPS; neuron; transcriptome (RNA-seq).

PubMed Disclaimer

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
A schematic diagram of the molecular structure of Kv4.3. A Kv4.3 subunit containing six transmembrane segments (S1–S6). The arrows indicate that 22 variants have been reported worldwide.
Figure 2
Figure 2
Clinical characterization of KCND3 mutations. (A) Pedigree chart: there are four patients in this family. The asterisks indicate that the members have been sequenced. The arrows denote probands. The filled symbols represent symptomatic members. The open symbols indicate unaffected individuals. The circles indicate female participants. The squares indicate male participants. The diagonal lines refer to the deceased. WT/WT refers to wild-type, while MT/WT specifies heterozygous mutation. (B) Sequencing chromatograms. Sanger sequencing verified the c.1130 C>T (p.T377M) mutation of the KCND3 and showed pedigree co-separation in this family. This mutation of KCND3 was not found in the normal family members. (C) No cerebellar atrophy and white matter abnormalities were found in III-5's brain MRI.
Figure 3
Figure 3
Conservation analysis and protein model prediction: (A) The protein sequence alignment is shown on the left. (B) The protein model showed that it was highly conservative. Highly conservative areas are shown in red color; the higher the score, the darker the color. (C) The protein structure model: A Kv4.3 subunit containing six transmembrane segments (S1–S6). The transmembrane segments are colored in green (S1), blue (S2), red (S3), yellow (S4), orange (S5), and purple (S6). (D) The AlphaFold Protein Structure Database's Kv4.3 structure models indicate that replacing polar threonine with nonpolar methionine causes an enlargement in the mean volume at residue 377 compared with the wild model.
Figure 4
Figure 4
Characterization of representative SCA19/22-iPSCs and control iPSCs. Immunostaining shows (A) pluripotency-associated markers in representative iPSC colonies; (B) NPC-expressed markers such as NESTIN1 and SOX2; (C) neuron-expressed markers such as GFAP and MAP2. Scale bar: 100 μm for panels. iPSCs: induced pluripotent stem cells; NPC: neural progenitor cell.
Figure 5
Figure 5
A visualization of the transcriptome data analysis. The volcano map (A) and heatmap (B) of the gene expression. The red gene is upregulated and the green gene is downregulated. |log2 (FC)| >1 is considered to be significant. Significant enrichment of GO (BP, CC, and MF) (C); the KEGG pathway showed protein processing in the endoplasmic reticulum, and focal adhesion was significantly enriched (D).
Figure 6
Figure 6
(A) The mutant Kv4.3 instability increased and remained in the endoplasmic reticulum, which may activate the endoplasmic reticulum-associated degradation (ERAD) pathway and cause ERS. Increased expression of ATF4 and CHOP can eventually lead to neuronal death. (B) Total proteins derived from neurons were subject to immunoblotting analyses with the indicated antibodies. GAPDH expression was the loading control. (C) The Kv4.3, ATF4, and CHOP levels were quantified and normalized to the GAPDH. Western blotting showed that the c.1130 C>T (p.T377M) mutation of KCND3 led to a decrease in Kv4.3 protein compared with the healthy controls (p < 0.05), while there was a significant increase in ATF4 and CHOP (p < 0.05). (D) The mRNA levels of Kv4.3, ATF4, and CHOP were quantified and normalized to the GAPDH. RT-qPCR showed that the mRNA level of mutated KCND3 was not significantly different from that of the healthy controls. However, the mRNA levels of ATF4 and CHOP were significantly increased (p < 0.05). Data are expressed as the mean ± SD; The significance was calculated using Student's t-test (*p < 0.05; **p < 0.01).
Figure 7
Figure 7
The percentage of symptoms in the early-onset and late-onset cohort. ND, Neurodevelopmental disorders; CI, Cognitive impairment.
See this image and copyright information in PMC

References

    1. Abisambra J. F., Jinwal U. K., Blair L. J., O'Leary J. C., 3rd, Li Q., Brady S., Wang L., et al. . (2013). Tau accumulation activates the unfolded protein response by impairing endoplasmic reticulum-associated degradation. J. Neurosci. 33, 9498–9507. 10.1523/JNEUROSCI.5397-12.2013 - DOI - PMC - PubMed
    1. Adachi T., Kitayama M., Nakano T., Adachi Y., Kato S., Nakashima K. (2015). Autopsy case of spinocerebellar ataxia type 31 with severe dementia at the terminal stage. Neuropathology 35, 273–279. 10.1111/neup.12184 - DOI - PubMed
    1. Adzhubei I. A., Schmidt S., Peshkin L., Ramensky V. E., Gerasimova A., Bork P., et al. . (2010). A method and server for predicting damaging missense mutations. Nat. Methods 7, 248–249. 10.1038/nmeth0410-248 - DOI - PMC - PubMed
    1. Aimé P., Karuppagounder S. S., Rao A., Chen Y., Burke R. E., Ratan R. R., et al. . (2020). The drug adaptaquin blocks ATF4/CHOP-dependent pro-death Trib3 induction and protects in cellular and mouse models of Parkinson's disease. Neurobiol. Dis. 136, 104725. 10.1016/j.nbd.2019.104725 - DOI - PMC - PubMed
    1. Alves S., Cormier-Dequaire F., Marinello M., Marais T., Muriel M. P., Beaumatin F., et al. . (2014). The autophagy/lysosome pathway is impaired in SCA7 patients and SCA7 knock-in mice. Acta Neuropathol. 128, 705–722. 10.1007/s00401-014-1289-8 - DOI - PubMed