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.
This study sets out to investigate the role of astrocytes on neuronal degeneration in a stem cell model of frontotemporal dementia using patient-derived induced pluripotent stem cells and gene-corrected, isogenic control cells. Our preliminary data show that patient-derived astrocytes cause oxidative stress in co-cultured neurons. To look into the mechanisms of astrocyte-mediated changes, we will apply single cell RNA sequencing on astrocyte/neuron co-cultures and on mixed grafts 10 weeks after transplantation into the brains of adult immunocompromised mice.
