Propagation and identification of <italic style="font-style: italic">Alg13</italic>  mutant epilepsy mice

Peng GAO; Junming HUO; Baorui GUO; Baoli YU; Ji WU; Juncheng WANG; Hui MA; Tao SUN; Jing ZHANG

doi:10.16016/j.2097-0927.202205051

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Propagation and identification of Alg13 mutant epilepsy mice

Neurosciences | 更新时间：2026-02-13

- Propagation and identification of Alg13 mutant epilepsy mice
- Journal of Army Medical University Vol. 44, Issue 22, Pages: 2256-2265(2022)
- 作者机构：
  
  1.750004 银川，宁夏医科大学总医院：神经外科
  2.750004 银川，宁夏医科大学：颅脑疾病重点实验室
  3.014010 内蒙古包头，内蒙古科技大学包头医学院第一附属医院心血管外科
  4.215400 江苏太仓，依科赛生物科技(太仓)有限公司
  5.200030 上海，上海交通大学Bio-X研究院遗传发育与精神神经疾病教育部重点实验室
  6.750004 银川，宁夏医科大学：生育力保持教育部重点实验室
  7.750002 银川，宁夏回族自治区人民医院神经外科
  8.750004 银川，宁夏医科大学总医院：外科学研究室
- 作者简介：
- 基金信息：
  
  the National Natural Science Foundation for Young Scholars of China(82001215);the Key Research and Development Program of Ningxia Science and Technology Department(2020BEB04033);the First-class Discipline Construction Project of Ningxia Higher Education Institutions (the Clinical Medicine Project of Domestic First-class Construction Discipline in Ningxia Medical University)(2020年)
- DOI：10.16016/j.2097-0927.202205051
  CLC： R-332;R394.1;R742.1
- Received：11 May 2022，
  
  Revised：2022-06-16，
  
  Published：30 November 2022
- 稿件说明：
移动端阅览
Peng GAO, Junming HUO, Baorui GUO, et al. Propagation and identification of Alg13 mutant epilepsy mice[J]. Journal of Army Medical University, 2022, 44(22): 2256-2265.
DOI：

Peng GAO, Junming HUO, Baorui GUO, et al. Propagation and identification of Alg13 mutant epilepsy mice[J]. Journal of Army Medical University, 2022, 44(22): 2256-2265. DOI： 10.16016/j.2097-0927.202205051.

摘要

目的

对

Alg13

基因敲除(knockout

KO)小鼠进行繁育和鉴定，为研究

Alg13

在癫痫中的作用提供一种理想的动物模型。

方法

本研究选用C57BL/6J小鼠，分为

Alg13

KO组及野生型(wildtype，WT)组，包括4周龄

Alg13

KO小鼠12只(雌雄各6只，体质量6~7 g)，WT小鼠12只(雌雄各6只，体质量7~8 g)，8周龄

Alg13

KO雄性小鼠18只(体质量21~23 g)，WT雄性小鼠18只(体质量22~24 g)。使用PCR、凝胶电泳、DNA测序、TA克隆和蓝白斑筛选综合鉴定

Alg13

KO小鼠基因型。免疫组织化学法和免疫印迹法对比验证

Alg13

KO小鼠ALG13蛋白的缺失情况。利用同步化视频脑电系统监测

Alg13

KO小鼠癫痫发作的行为学表现及脑电特征。免疫印迹分析电压门控钠离子通道α亚基1 (sodium channel alpha subunit 1，SCN1A)蛋白表达情况。

结果

凝胶电泳、DNA测序及蓝白斑筛选结果均显示

Alg13

KO小鼠中存在5个碱基的缺失。与WT组相比，免疫组织化学结果证实

Alg13

KO小鼠的皮质、小脑和海马区ALG13表达量显著降低(

0.001；

0.05；

0.001)，且蛋白免疫印迹结果也说明

Alg13

KO小鼠的皮质、小脑和海马区ALG13表达更少(

0.001)。视频脑电结果显示：部分

Alg13

KO小鼠出现频繁的自发性癫痫发作，具有典型癫痫发作的行为学表现和脑电改变；相较于WT组，所有的

Alg13

KO组小鼠脑电δ和θ波能量值均显著升高(

0.001)。

Alg13

KO小鼠海马和皮质中SCN1A的表达水平显著高于WT小鼠(

0.05；

0.01)。

结论

成功繁育

Alg13

基因敲除小鼠模型，该模型表现出显著的癫痫易感性，可用于遗传异常导致癫痫的机制研究。

Abstract

Objective

To breed an

d identify the

Alg13

knockout (KO) mice to provide an ideal animal model for exploring the role of

Alg13

in epilepsy.

Methods

Alg13

KO and wildtype (WT) mice with C57BL/6J background were used

including 4-week-old

Alg13

KO mice (

=12; 6 males and 6 females; 6~7 g)

4-week-old WT mice (

=12; 6 males and 6 females; 7~8 g)

8-week-old

Alg13

KO male mice (

=18; 21~23 g) and 8-week-old WT male mice (

=18; 22~24 g). The genotype of

Alg13

KO mice was identified using PCR

gel electrophoresis

DNA sequencing

TA cloning and blue-white selection method. Immunohistochemical analysis and Western blotting were applied to verify the deletion of ALG13 protein in

Alg13

KO mice. The behavioral manifestations and characteristics of seizures in

Alg13

KO mice were evaluated using a synchronized video EEG system. The expression level of voltage-gated sodium channel alpha subunit 1 (SCN1A) protein was measured by Western blot assay.

Results

The genotyping methods including the gel electrophoresis

DNA sequencing and blue-white selection confirmed a 5-base deletion in

Alg13

KO mice. Compared with WT mice

Alg13

KO mice showed a significantly lower ALG13 expression level in the cortical

cerebellar and hippocampal regions in the immunohistochemical (

0.001

0.05

0.001) and Western blot assay (

0.001). Besides

some

Alg13

KO mice developed frequent spontaneous seizures with typical behavioral manifestations and EEG changes of the epileptic seizure.

Alg13

KO mice had significantly higher δ and θ wave energy value of EEG compared with the WT group (

0.001). The expression levels of S

CN1A in the hippocampus and cortex were remarkably higher in

Alg13

KO mice than those in WT mice (

0.05

0.01).

Conclusion

The

Alg13

knockout mouse model can be obtained successfully

which shows significant epilepsy susceptibility and lays a foundation for studying the mechanism of the epilepsy caused by genetic abnormalities.

关键词

Keywords

references

GAO X D, TACHIKAWA H, SATO T, et al . Alg14 recruits Alg13 to the cytoplasmic face of the endoplasmic Reticulum to form a novel bipartite UDP-N-acetylglucosamine transferase required for the second step of N-linked glycosylation[J ] . J Biol Chem, 2005, 280(43): 36254-36262. DOI: 10.1074/jbc.M507569200..

HELENIUS A, AEBI M. Roles of N-linked glycans in the endoplasmic reticulum[J]. Annu Rev Biochem, 2004, 73: 1019-1049. DOI: 10.1146/annurev.biochem.73.011303.073752.

KWON S E, CHAPMAN E R. Glycosylation is dispensable for sorting of synaptotagmin 1 but is critical for targeting of SV 2 and synaptophysin to recycling synaptic vesicles[J ] . J Biol Chem, 2012, 287(42): 35658-35668. DOI: 10.1074/jbc.M112.398883.

BERRY G T, FREEZE H H, MORAVA E. Is X-linked, infantile onset ALG13-related developmental and epileptic encephalopathy a congenital disorder of glycosylation?[J]. Epilepsia, 2021, 62(2): 335-336. DOI: 10.1111/epi.16817.

CONSORTIUM E, PROJECT E P, ALLEN A S, et al . De novo mutations in epileptic encephalopathies[J ] . Nature, 2013, 501(7466): 217-221. DOI: 10.1038/nature12439.

SMITH-PACKARD B, MYERS S M, WILLIAMS M S. Girls with seizures due to the c. 320A>G variant in ALG13 do not show abnormal glycosylation pattern on standard testing[J]. JIMD Rep, 2015, 22: 95-98. DOI: 10.1007/8904_2015_416.

DATTA A N, BAHI-BUISSON N, BIENVENU T, et al . The phenotypic spectrum of X-linked, infantile onset ALG13-related developmental and epileptic encephalopathy[J ] . Epilepsia, 2021, 62(2): 325-334. DOI: 10.1111/epi.16761.

THIJS R D, SURGES R, O'BRIEN T J, et al . Epilepsy in adults[J ] . Lancet, 2019, 393(10172): 689-701. DOI: 10.1016/S0140-6736(18)32596-0.

CATTERALL W A, KALUME F, OAKLEY J C. NaV 1.1 channels and epilepsy[J ] . J Physiol, 2010, 588(Pt 11): 1849-1859. DOI: 10.1113/jphysiol.2010.187484.

MENG H, XU H Q, YU L, et al . The SCN1A mutation database: updating information and analysis of the relationships among genotype, functional alteration, and phenotype[J ] . Hum Mutat, 2015, 36(6): 573-580. DOI: 10.1002/humu.22782.

YU B L, ZHU G, LI S G, et al . CRISPR/Cas9-mediated mutation of asparagine-linked glycosylation 13 transcript variant 1 causes epilepsy in mice[J ] . J Bio X Res, 2020, 3(1): 6-12. DOI: 10.1097/jbr.0000000000000059.

HODGE R D, BAKKEN T E, MILLER J A, et al . Conserved cell types with divergent features in human versus mouse cortex[J ] . Nature, 2019, 573(7772): 61-68. DOI: 10.1038/s41586-019-1506-7.

LÖSCHER W. Animal models of seizures and epilepsy: past, present, and future role for the discovery of antiseizure drugs[J]. Neurochem Res, 2017, 42(7): 1873-1888. DOI: 10.1007/s11064-017-2222-z.

CAO F, LIU J J, ZHOU S S, et al . Neuroligin 2 regulates absence seizures and behavioral arrests through GABAergic transmission within the thalamocortical circuitry[J ] . Nat Commun, 2020, 11(1): 3744. DOI: 10.1038/s41467-020-17560-3.

陈睿, 薛国芳. 难治性癫痫动物模型的研究进展[J]. 癫痫杂志, 2021, 7(5): 431-435.

CHEN R, XUE G F. Research progress on animal models of drug-resistance epilepsy[J]. J Epilepsy, 2021, 7(5): 431-435.

KÖHLING R, WOLFART J. Potassium channels in epilepsy[J]. Cold Spring Harb Perspect Med, 2016, 6(5): a022871. DOI: 10.1101/cshperspect.a022871.

SCHMIDT J W, CATTERALL W A. Palmitylation, sulfation, and glycosylation of the alpha subunit of the sodium channel. Role of post-translational modifications in channel assembly[J]. J Biol Chem, 1987, 262(28): 13713-13723.

VERHEIJEN J, TAHATA S, KOZICZ T, et al . Therapeutic approaches in congenital disorders of glycosylation (CDG) involving N-linked glycosylation: an update[J ] . Genet Med, 2020, 22(2): 268-279. DOI: 10.1038/s41436-019-0647-2.

NG B G, EKLUND E A, SHIRYAEV S A, et al . Predominant and novel de novo variants in 29 individuals with ALG13 deficiency: clinical description, biomarker status, biochemical analysis, and treatment suggestions[J ] . J Inherit Metab Dis, 2020, 43(6): 1333-1348. DOI: 10.1002/jimd.12290.

GAO P, WANG F, HUO J M, et al . ALG13 deficiency ass-ociated with increased seizure susceptibility and severity[J ] . Neuroscience, 2019, 409: 204-221. DOI: 10.1016/j.neuroscience.2019.03.009.

WANG W Q, FRANKEL W N. Overlaps, gaps, and complexities of mouse models of developmental and epileptic encephalopathy[J]. Neurobiol Dis, 2021, 148: 105220. DOI: 10.1016/j.nbd.2020.105220.

ALBACH C, KLEIN R A, SCHMITZ B. Do rodent and human brains have different n-glycosylation patterns?[J]. Biol Chem, 2001, 382(2): 187-194. DOI: 10.1515/BC.2001.026.

REILY C, STEWART T J, RENFROW M B, et al . Glycosylation in health and disease[J ] . Nat Rev Nephrol, 2019, 15(6): 346-366. DOI: 10.1038/s41581-019-0129-4.

FANG P, XIE J J, SANG S M, et al . Multilayered n-glycoproteome profiling reveals highly heterogeneous and dys-regulated protein n-glycosylation related to Alzheimer's disease[J ] . Anal Chem, 2020, 92(1): 867-874. DOI: 10.1021/acs.analchem.9b03555.

LAZNIEWSKA J, WEISS N. Glycosylation of voltage-gated calcium channels in health and disease[J]. Biochim Biophys Acta Biomembr, 2017, 1859(5): 662-668. DOI: 10.1016/j.bbamem.2017.01.018.

BOLL I, JENSEN P, SCHWÄMMLE V, et al . Depolarization-dependent induction of site-specific changes in sialylation on n-linked glycoproteins in rat nerve terminals[J ] . Mol Cell Proteomics, 2020, 19(9): 1418-1435. DOI: 10.1074/mcp.RA119.001896.

HAMICI S, BASTAKI F, KHALIFA M. Exome sequence identified a c. 320A>G ALG13 variant in a female with infantile epileptic encephalopathy with normal glycosylation and random X inactivation: review of the literature[J]. Eur J Med Genet, 2017, 60(10): 541-547. DOI: 10.1016/j.ejmg.2017.07.014.

MADAAN P, NEGI S, SHARMA R, et al . X-linked Alg13 gene variant as a cause of epileptic encephalopathy in girls[J ] . Indian J Pediatr, 2019, 86(11): 1072-1073. DOI: 10.1007/s12098-019-03059-3..

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⁰

Propagation and identification of Alg13 mutant epilepsy mice

DOI：10.16016/j.2097-0927.202205051

摘要

Abstract

关键词

Keywords

references