Mutant SOD1 rats (lobsi-affy-rat-194438). Rattus norvegicus

Missense mutations in the gene for the ubiquitously expressed superoxide dismutase-1 (SOD1) are one of the causes of familial amyotrophic lateral sclerosis (ALS), the most common adult onset motor neuron disease in humans killing selectively large motor neurons. Mice and rats overexpressing mutant SOD1 develop an adult onset neurodegenerative disease with hindlimb-paralysis and subsequent death similar to the human condition. In order to analyze the effects of mutant SOD1 expression onto the most affected cell-type in ALS, a small subpopulation of spinal cord cells, we propose to use laser microdissection to isolate mouse lumbar motor neurons and to assess the changes onto the mRNA expression profile using Affymetrix GeneChips compared to control animals. While two studies applying a genomic approach on the ALS mouse models used the entire spinal cord, contributions of changes to motor neurons were masked by the inflammatory effects of mutant SOD1 and the much larger population of non-motor neuronal cells. What is therefore needed is a cell-type specific expression profile that could reveal dysregulations in the transcriptome of the affected motor neurons. In order to find and analyze disease relevant gene expression profile changes in the mutant vs. the wildtype overexpressing SOD1 mice, we propose to use two different mutant SOD1 lines. Both lines develop the same ALS-like motor neuron disease, however, line 1 (with a G37R mutation) retains the full activity of the SOD1 enzyme while line 2 (with a G85R mutation) has almost no SOD1 enzymatic activity. We believe that by using these two most extreme variants of ALS-inducing mutant SOD1 forms, candidate genes that will be similarly dysregulated in both mutant lines will have great potential to be a disease relevant hit. We will collect from both mutant SOD1 mouse lines lumbar motor neurons from three presymptomatic timepoints, equally distributed during their adult life and before obvious hindlimb-weakness or paralysis signs and compare them to two control lines, negative littermates and wildtype SOD1 overexpressing mice. Furthermore, to determine if their are already mutant SOD1-induced changes since the formation of the affected cells, we will also isolate embryonic spinal cord motor neurons and compare them between mutant SOD1 and wildtype SOD1 overexpressing animals. Both, mice deleted in wildtype SOD1 and mice overexpressing mutant human SOD1 clearly established that the toxic property of mutant SOD1 is based upon a gain-of-toxic function phenomenon rather than a loss-of-function effect, since the SOD1 knock-out mice are overall normal. Furthermore, mice overexpressing wildtype SOD1 are healthy and serve as ideal control animals. Therefore, one of the most important question in ALS is to determine what slowly acting mechanism, or build-up of toxic products is responsible for ultimately killing more than 50-70% of the large lumbar spinal cord motor neurons. We hypothezise that the toxic property of mutant SOD1 must already induce changes onto the transcriptome of the most at-risk cell-type, the large lumbar spinal cord motor neuron, very early on in the presymptomatic phase of the disease course, when the animal is still without any paralysis signs and moving normally around the cage. We will choose 3 timepoints before phenotypic disease-onset that is at 22 weeks for the SOD1G37R line (L42) and at 11 months for the SOD1G85R line (L148). The timepoints are 8, 15 and 18 weeks for SOD1G37R line and 4, 7.5 and 9 months for the SOD1G85R line all compared to age-matched control animals (wildtype SOD1 overexpressing mice (L76) for the SOD1G37R line (high mutant SOD1 levels) and negative littermates for the SOD1G85R line (low mutant SOD1 levels)). Using the Leica lasermicrodissection system on fresh frozen spinal cord sections, we process a single spinal cord sample in 3 days resulting in approximately 4000 ventral horn motor neurons collected from 150 consecutive 20 um sections of the most affected lumbar spinal cord region (L4-L6). To visualize the motor neurons, we have established a fast Nissel-staining protocol that is optimized for speed to preserve the integrity of the RNA. Before lasermicrodissection slides containing 5 spinal cord sections are dessicated for 1 hour and samples are collected directly into RNA-preserving lysis-buffer. Using Stratagene's NanoPrep RNA-extraction columns a yield of around 150 ng of total RNA was obtained (Ribogreen-quantification), that is enough for both the 2-round linear RNA amplification and for future candidate confirmation studies using real-time PCR analysis. We are using the Affymetrix Two-Cycle 3'-Amplification Kit, starting with 100 ng of total RNA and will hybridize on Affymetrix Mouse 430 2.0 GeneChips. Embryonic motor neurons are isolated from e14 mutant SOD1G93A overexpressing rat embryos and compared to wildtype SOD1 overexpressing embryos using a metrazimide gradient followed by a p75-antibody purification in combination with magnetic beads. Cells from one litter of embryos were directly collected into lysis-buffer, yielding at least 200-500 ng of total RNA, enough for one Rat 230 2.0 GeneChip (2-round amplification using 100 ng total RNA). Keywords: time-course

泛在表达的超氧化物歧化酶1（superoxide dismutase-1, SOD1）基因的错义突变是家族性肌萎缩侧索硬化症（familial amyotrophic lateral sclerosis, ALS）的诱因之一，而ALS是人类最常见的成人起病型运动神经元疾病，会选择性杀伤大型运动神经元。过表达突变型SOD1的小鼠和大鼠会出现成人起病的神经退行性疾病，表现为后肢瘫痪并最终死亡，与人类患者的病症相似。为了分析突变型SOD1表达对ALS中受累最显著的细胞类型——脊髓细胞的一小部分亚群——的影响，本研究拟采用激光显微切割（laser microdissection）技术分离小鼠腰段运动神经元，并利用Affymetrix基因芯片（Affymetrix GeneChips）对比对照组动物，评估其mRNA表达谱的变化。此前有两项针对ALS小鼠模型的基因组学研究采用了全脊髓组织作为样本，但由于突变型SOD1引发的炎症效应以及占比更高的非运动神经元细胞群体的干扰，运动神经元的表达变化被掩盖。因此，亟需获取细胞类型特异性的表达谱，以揭示受累运动神经元转录组的失调情况。为了在突变型与野生型过表达SOD1小鼠中筛选并分析疾病相关的基因表达谱变化，本研究拟使用两种不同的突变型SOD1品系。两种品系均会引发类似ALS的运动神经元疾病，但品系1（携带G37R突变）仍保留SOD1酶的完整活性，而品系2（携带G85R突变）几乎不具备SOD1酶活性。我们认为，通过使用这两种诱导ALS的极端突变型SOD1变体，在两种突变品系中均出现失调的候选基因，极有可能是与疾病相关的有效靶点。我们将从两种突变型SOD1小鼠品系中，于三个症状前时间点收集腰段运动神经元，这些时间点均匀分布于成年期且未出现明显的后肢无力或瘫痪症状，并将其与两种对照组（阴性同窝仔鼠及野生型SOD1过表达小鼠）进行对比。此外，为了明确突变型SOD1是否在受累细胞形成之初即已引发表达变化，我们还将分离胚胎期脊髓运动神经元，对比突变型SOD1与野生型SOD1过表达动物的样本。已有研究证实，敲除野生型SOD1的小鼠以及过表达突变型人SOD1的小鼠均表明，突变型SOD1的毒性效应基于毒性功能获得（gain-of-toxic function）机制，而非功能丧失（loss-of-function），因为SOD1敲除小鼠整体表型正常。此外，过表达野生型SOD1的小鼠健康状态良好，是理想的对照动物。因此，ALS研究中最重要的问题之一是：究竟是何种缓慢起效的致病机制，或是毒性产物的累积，最终导致超过50%-70%的腰段大型脊髓运动神经元死亡。我们提出假说：在疾病病程的症状前阶段，当动物尚未出现任何瘫痪症状且可正常活动时，突变型SOD1的毒性效应即已在受累风险最高的细胞类型——大型腰段脊髓运动神经元的转录组中引发变化。我们将选择三个表型发病前的时间点：对于SOD1G37R品系（L42）为22周龄前的8周、15周和18周；对于SOD1G85R品系（L148）为11月龄前的4月龄、7.5月龄和9月龄，所有时间点均与同年龄段对照组（SOD1G37R品系的对照组为野生型SOD1过表达小鼠（L76），该组突变型SOD1表达水平较高；SOD1G85R品系的对照组为阴性同窝仔鼠，该组突变型SOD1表达水平较低）匹配。我们将使用徕卡激光显微切割系统（Leica lasermicrodissection system）处理新鲜冰冻的脊髓切片，每例脊髓样本可在3天内完成处理，从受累最显著的腰段脊髓区域（L4-L6）的150张连续20μm切片中收集约4000个腹角运动神经元。为了可视化运动神经元，我们已建立一种快速尼氏染色（Nissel-staining）方案，该方案经过优化以保证染色速度并维持RNA的完整性。激光显微切割前，包含5张脊髓切片的玻片会被干燥1小时，样本将直接收集至RNA保存型裂解缓冲液中。使用Stratagene公司的NanoPrep RNA提取柱，可获得约150ng总RNA（经Ribogreen定量），其总量足够进行两轮线性RNA扩增，也可用于后续通过实时荧光定量PCR（real-time PCR）进行的候选基因验证研究。我们将采用Affymetrix双循环3'端扩增试剂盒（Affymetrix Two-Cycle 3'-Amplification Kit），以100ng总RNA为起始样本，在Affymetrix Mouse 430 2.0基因芯片上进行杂交。胚胎期运动神经元将从e14天的SOD1G93A突变型过表达大鼠胚胎中分离，通过美蓝梯度（metrazimide gradient）结合磁珠偶联p75抗体纯化的方案，与野生型SOD1过表达胚胎进行对比。同一窝胚胎的细胞将直接收集至裂解缓冲液中，可获得至少200-500ng总RNA，足够进行一次大鼠230 2.0基因芯片实验（以100ng总RNA为起始样本进行两轮扩增）。关键词：时间进程（time-course）