Wu Jun, Ryskamp Daniel, Birnbaumer Lutz, Bezprozvanny Ilya
Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX, USA.
Neurobiology Laboratory, NIEHS, Research Triangle Park, NC, USA.
J Huntingtons Dis. 2018;7(1):35-50. doi: 10.3233/JHD-170266.
Huntington disease (HD) is a dominantly inherited neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. We previously discovered that mutant Huntingtin sensitizes type 1 inositol 1,4,5-trisphosphate receptor (InsP3R1) to InsP3. This causes calcium leakage from the endoplasmic reticulum (ER) and a compensatory increase in neuronal store-operated calcium (nSOC) entry. We previously demonstrated that supranormal nSOC leads to synaptic loss in striatal medium spiny neurons (MSNs) in YAC128 HD mice.
We sought to identify calcium channels supporting supranormal nSOC in HD MSNs and to validate these channels as potential therapeutic targets for HD.
Cortico-striatal cultures were established from wild type and YAC128 HD mice and the density of MSN spines was quantified. The expression of candidate nSOC components was suppressed by RNAi knockdown and by CRISPR/Cas9 knockout. TRPC1 knockout mice were crossed with YAC128 HD mice for evaluation of motor performance in a beamwalk assay.
RNAi-mediated knockdown of TRPC1, TRPC6, Orai1, or Orai2, but not other TRPC isoforms or Orai3, rescued the density of YAC128 MSN spines. Knockdown of stromal interaction molecule 1 (STIM1), an ER calcium sensor and nSOC activator, also rescued YAC128 MSN spines. Knockdown of the same targets suppressed supranormal nSOC in YAC128 MSN spines. These channel subunits co-immunoprecipitated with STIM1 and STIM2 in synaptosomal lysates from mouse striata. Crossing YAC128 mice with TRPC1 knockout mice improved motor performance and rescued MSN spines in vitro and in vivo, indicating that inhibition of TRPC1 may serve as a neuroprotective strategy for HD treatment.
TRPC1 channels constitute a potential therapeutic target for treatment of HD.
亨廷顿舞蹈症(HD)是一种由亨廷顿基因中CAG重复序列扩增引起的常染色体显性遗传神经退行性疾病。我们之前发现突变型亨廷顿蛋白使1型肌醇1,4,5-三磷酸受体(InsP3R1)对肌醇三磷酸(InsP3)敏感。这导致内质网(ER)钙泄漏以及神经元储存操纵性钙(nSOC)内流代偿性增加。我们之前证明,超常的nSOC会导致YAC128 HD小鼠纹状体中等棘状神经元(MSN)突触丢失。
我们试图确定支持HD小鼠MSN超常nSOC的钙通道,并验证这些通道作为HD潜在治疗靶点的可能性。
从野生型和YAC128 HD小鼠建立皮质-纹状体培养物,并对MSN棘突密度进行定量分析。通过RNA干扰敲低和CRISPR/Cas9基因敲除抑制候选nSOC成分的表达。将瞬时受体电位阳离子通道亚家族C成员1(TRPC1)基因敲除小鼠与YAC128 HD小鼠杂交,在横梁行走试验中评估运动能力。
RNA干扰介导的TRPC1、TRPC6、Orai1或Orai2敲低,但不是其他TRPC亚型或Orai3,挽救了YAC128 MSN棘突密度。敲低基质相互作用分子1(STIM1),一种内质网钙传感器和nSOC激活剂,也挽救了YAC128 MSN棘突。相同靶点的敲低抑制了YACl28 MSN棘突中的超常nSOC。这些通道亚基在小鼠纹状体突触体裂解物中与STIM1和STIM2共同免疫沉淀。将YAC128小鼠与TRPC1基因敲除小鼠杂交可改善运动能力,并在体外和体内挽救MSN棘突,表明抑制TRPC1可能是HD治疗的一种神经保护策略。
TRPC1通道是HD治疗的潜在靶点。