Single-cell genetic expression of mutant GABAA receptors causing Human genetic epilepsy alters dendritic spine and GABAergic bouton formation in a mutation-specific manner.
Single-cell genetic expression of mutant GABAA receptors causing Human genetic epilepsy alters dendritic spine and GABAergic bouton formation in a mutation-specific manner.
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DOI：
10.3389/fncel.2014.00317
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发表时间：
2014
影响因子：
5.3
通讯作者：
Cossette P
中科院分区：
文献类型：
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作者：
Lachance-Touchette P;Choudhury M;Stoica A;Di Cristo G;Cossette P
编码GABAA受体亚单位的基因突变是遗传性全面性癫痫的一个公认的原因。GABA神经传递参与多种发育过程，包括轴突生长和突触形成。兴奋性/抑制性突触活动的改变在癫痫发病中起着关键作用，因此我们研究了GABA受体α1亚基突变是否会影响树突棘和GABA能突触的形成。特别是，我们检查了GABRA1基因的三个突变(D219N、A322D和K353-delins18X)的影响，这些突变是在一个患有遗传性全面性癫痫的法裔加拿大家庭中发现的。我们使用了一种新的单细胞遗传学方法，从GABRA1FLOX/FLOX小鼠制备皮质器官型培养物，同时灭活内源性GABRA1，并通过生物转染将突变的α1亚单位导入单个谷氨酸能锥体细胞和篮子GABA能中间神经元。我们发现，−/−GABA能细胞的神经支配野减少，α1-A322D和α1-WT共表达可以挽救这种神经支配野，而α1-D219N不能。我们进一步发现，最严重的GABRA1错义突变(α1-A322D)的表达导致锥体细胞内棘突密度显着增加，同时蘑菇样棘突的数量也增加。此外，α1-A322D在GABA能细胞中的表达略微增加了周围子的密度，而其他突变不改变周围子的形成。综上所述，这些结果表明，不同的GABAAR突变对GABA能转运子和树突棘形成的影响是该突变所特有的，不能总是用简单的功能丧失基因模型来解释。在器官培养中使用单细胞遗传操作可以更好地理解不同GABAA受体亚单位突变引起的特定和独特的神经回路变化，并有助于确定遗传性全身性癫痫综合征的病理生理学。
Mutations in genes encoding for GABAA receptor subunits is a well-established cause of genetic generalized epilepsy. GABA neurotransmission is implicated in several developmental processes including neurite outgrowth and synapse formation. Alteration in excitatory/inhibitory synaptic activities plays a critical role in epilepsy, thus here we investigated whether mutations in α1 subunit of GABAA receptor may affect dendritic spine and GABAergic bouton formation. In particular, we examined the effects of three mutations of the GABRA1 gene (D219N, A322D and K353delins18X) that were found in a cohort of French Canadian families with genetic generalized epilepsy. We used a novel single-cell genetic approach, by preparing cortical organotypic cultures from GABRA1flox/flox mice and simultaneously inactivating endogenous GABRA1 and transfecting mutant α1 subunits in single glutamatergic pyramidal cells and basket GABAergic interneurons by biolistic transfection. We found that GABRA1−/− GABAergic cells showed reduced innervation field, which was rescued by co-expressing α1-A322D and α1-WT but not α1-D219N. We further found that the expression of the most severe GABRA1 missense mutation (α1-A322D) induced a striking increase of spine density in pyramidal cells along with an increase in the number of mushroom-like spines. In addition, α1-A322D expression in GABAergic cells slightly increased perisomatic bouton density, whereas other mutations did not alter bouton formation. All together, these results suggest that the effects of different GABAAR mutations on GABAergic bouton and dendritic spine formation are specific to the mutation and cannot be always explained by a simple loss-of-function gene model. The use of single cell genetic manipulation in organotypic cultures may provide a better understanding of the specific and distinct neural circuit alterations caused by different GABAA receptor subunit mutations and will help define the pathophysiology of genetic generalized epilepsy syndromes.
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影响因子：
2.5
作者：
Bosman, LWJ;Heinen, K;Brussaard, AB
通讯作者：
Brussaard, AB
影响因子：
9.9
作者：
Carvill, Gemma L.;Weckhuysen, Sarah;Mefford, Heather C.
通讯作者：
Mefford, Heather C.
影响因子：
25
作者：
Carlson, G;Wang, Y;Alger, BE
通讯作者：
Alger, BE
影响因子：
5.3
作者：
Gaiarsa JL;Porcher C
通讯作者：
Porcher C
影响因子：
64.8
作者：
通讯作者：
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