Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA, USA.
Neurobiol Dis. 2011 Jun;42(3):459-67. doi: 10.1016/j.nbd.2011.02.008. Epub 2011 Feb 18.
R6/2 transgenic mice with expanded CAG repeats (>300) have a surprisingly prolonged disease progression and longer lifespan than prototypical parent R6/2 mice (carrying 150 CAGs); however, the mechanism of this phenotype amelioration is unknown. We compared gene expression profiles in the striatum of R6/2 transgenic mice carrying ~300 CAG repeats (R6/2(Q300) transgenic mice) to those carrying ~150 CAG repeats (R6/2(Q150) transgenic mice) and littermate wildtype controls in order to identify genes that may play determinant roles in the time course of phenotypic expression in these mice. Of the top genes showing concordant expression changes in the striatum of both R6/2 lines, 85% were decreased in expression, while discordant expression changes were observed mostly for genes upregulated in R6/2(Q300) transgenic mice. Upregulated genes in the R6/2(Q300) mice were associated with the ubiquitin ligase complex, cell adhesion, protein folding, and establishment of protein localization. We qPCR-validated increases in expression of genes related to the latter category, including Lrsam1, Erp29, Nasp, Tap1, Rab9b, and Pfdn5 in R6/2(Q300) mice, changes that were not observed in R6/2 mice with shorter CAG repeats, even in late stages (i.e., 12 weeks of age). We further tested Lrsam1 and Erp29, the two genes showing the greatest upregulation in R6/2(Q300) transgenic mice, for potential neuroprotective effects in primary striatal cultures overexpressing a mutated human huntingtin (htt) fragment. Overexpression of Lrsam1 prevented the loss of NeuN-positive cell bodies in htt171-82Q cultures, concomitant with a reduction of nuclear htt aggregates. Erp29 showed no significant effects in this model. This is consistent with the distinct pattern of htt inclusion localization observed in R6/2(Q300) transgenic mice, in which smaller cytoplasmic inclusions represent the major form of insoluble htt in the cell, as opposed to large nuclear inclusions observed in R6/2(Q150) transgenic mice. We suggest that the prolonged onset and disease course observed in R6/2 mice with greatly expanded CAG repeats might result from differential upregulation of genes related to protein localization and clearance. Such genes may represent novel therapeutic avenues to decrease htt aggregate toxicity and cell death in HD patients, with Lrsam1 being a promising, novel candidate disease modifier.
R6/2 转基因小鼠具有扩展的 CAG 重复 (>300),其疾病进展和寿命比典型亲本 R6/2 小鼠 (携带 150 个 CAG) 长得多;然而,这种表型改善的机制尚不清楚。我们比较了携带 ~300 个 CAG 重复的 R6/2 转基因小鼠 (R6/2(Q300)转基因小鼠)和携带 ~150 个 CAG 重复的 R6/2 转基因小鼠 (R6/2(Q150)转基因小鼠)以及同窝野生型对照的纹状体中的基因表达谱,以确定可能在这些小鼠表型表达的时间过程中起决定作用的基因。在这两种 R6/2 系的纹状体中,具有一致表达变化的前基因中有 85%的基因表达下调,而在 R6/2(Q300)转基因小鼠中上调的基因则观察到不一致的表达变化。R6/2(Q300) 小鼠中上调的基因与泛素连接酶复合物、细胞黏附、蛋白质折叠和蛋白质定位建立有关。我们通过 qPCR 验证了与后一类基因相关的基因表达增加,包括 R6/2(Q300)小鼠中的 Lrsam1、Erp29、Nasp、Tap1、Rab9b 和 Pfdn5,而在携带较短 CAG 重复的 R6/2 小鼠中,甚至在晚期 (即 12 周龄) 也未观察到这些基因的表达增加。我们进一步测试了 Lrsam1 和 Erp29,这两个基因在 R6/2(Q300)转基因小鼠中表现出最大的上调,以检测它们在过表达突变型人类亨廷顿蛋白 (htt) 片段的原代纹状体培养物中的潜在神经保护作用。Lrsam1 的过表达可防止 htt171-82Q 培养物中 NeuN 阳性细胞体的丢失,同时减少核 htt 聚集。Erp29 在该模型中没有显示出显著的作用。这与 R6/2(Q300)转基因小鼠中观察到的 htt 包涵体定位的不同模式一致,其中较小的细胞质包涵体代表细胞中可溶性 htt 的主要形式,而不是在 R6/2(Q150)转基因小鼠中观察到的大核包涵体。我们认为,在具有大大扩展的 CAG 重复的 R6/2 小鼠中观察到的疾病发病和病程延长可能是由于与蛋白质定位和清除相关的基因的差异上调所致。这些基因可能代表减少 HD 患者 htt 聚集毒性和细胞死亡的新的治疗途径,其中 Lrsam1 是一种有前途的新型候选疾病修饰剂。