Application of single cell sequencing to study astrocyte-mediated neuronal degeneration in a stem cell model of frontotemporal dementia

尽管痴呆症在世界范围内的发病率很高，但神经退行性变的机制却很差， 因此，仍然不存在治愈性治疗选择。越来越多的证据表明，非细胞- 神经元变性的自主机制在疾病发展过程中起重要作用， 神经元和星形胶质细胞都可能对患者大脑的病理变化有重要作用。在 在这项研究中，我们将应用单细胞测序来分析星形胶质细胞介导的对干细胞中神经元的影响。 额颞叶痴呆（FTD）细胞模型，并假设FTD患者的星形胶质细胞具有 在体外和体内对邻近神经元的有害影响。我们将从细胞中产生神经元和星形胶质细胞 在编码蛋白质的基因中携带N279 K突变的患者来源的诱导多能干细胞 tau（MAPT）作为FTD的模型。与此同时，我们将从CRISPR/CAS9- 1中分化神经元和星形胶质细胞。 基因校正的同基因对照（Ctrl）干细胞，其携带与亲本相同的遗传信息 MAPT N279 K细胞，除了致病突变。FTD和Ctrl神经元将与 FTD或对照星形胶质细胞以及对神经元存活和全转录组谱的影响将 测定星形胶质细胞介导的基因表达变化将在单细胞水平上进行评价， 将单细胞RNA测序应用于共培养的神经元和星形胶质细胞。我们将特别强调 在这种情况下，细胞质蛋白MAGEH-1的作用，这是已知的结合神经生长 受体（p75 NTR），我们发现其在FTD iPS细胞衍生的神经元中上调，同时伴随沿着 p75 NTR，试图防止对受损细胞的进一步损害。我们还将确定 通过将FTD星形胶质细胞或具有神经元的对照星形胶质细胞移植到 免疫受损的小鼠。移植后10周，将对移植物进行组织学检查， 体内单细胞RNA测序以评估细胞存活和细胞死亡以及单细胞RNA表达 体内分布。我们相信，这个项目有很强的潜力，以更好地了解星形胶质细胞的作用， FTD中的神经元变性。

Despite the high prevalence of dementias worldwide, mechanisms of neurodegeneration are only poorly understood and thus, curative treatment options still do not exist. There is growing evidence that non-cell- autonomous mechanisms of neuronal degeneration play an important role during disease development and that both neurons and astrocytes could significantly contribute to pathologic changes in patients' brains. In this study, we will apply single cell sequencing to analyze astrocyte-mediated effects on neurons in a stem cell model of frontotemporal dementia (FTD) and hypothesize that astrocytes from patients with FTD have detrimental effects on adjacent neurons in vitro and in vivo. We will generate neurons and astrocytes from patient-derived induced pluripotent stem cells carrying the N279K mutation in the gene encoding the protein tau (MAPT) as a model for FTD. In parallel, we will differentiate neurons and astrocytes from CRISPR/CAS9- gene corrected isogenic control (Ctrl) stem cells that carry an identical genetic information as the parental MAPT N279K cells except for the disease-causing mutation. FTD and Ctrl neurons will be co-cultured with either FTD or Ctrl astrocytes and effects on neuronal survival and whole transcriptome profiles will be determined. Astrocyte-mediated changes in gene expression will be evaluated at a single cell level by applying single cell RNA sequencing on co-cultured neurons and astrocytes. We will put a special emphasis on the role of the cytoplasmic protein MAGEH-1 in this context, which is known to bind to the nerve growth receptor (p75NTR) and which we found to be upregulated in FTD iPS cell-derived neurons along with p75NTR in an attempt to prevent further damage to compromised cells. We will also determine the effects of FTD astrocytes in vivo by transplanting FTD astrocytes or Ctrl astrocytes with neurons into the brain of immunocompromised mice. Grafts will be examined 10 weeks after transplantation histologically and via ex vivo single cell RNA sequencing to evaluate cell survival and cell death as well as single cell RNA expression profiles in vivo. We believe that this project has strong potential to better understand the role of astrocytes on neuronal degeneration in FTD.