Frontotemporal dementia (FTD) composes a heterogeneous group of devastating neurodegenerative disorders presenting distinct changes in behavior, language and motor function. Despite increasing knowledge on the genetic basis of FTD, including the recent discovery of new disease-associated genes, the wide spectrum of genetic and clinical manifestations suggests the presence of an extremely complex disease. So far, the hypothesized disease mechanisms include abnormal protein aggregation, deregulation of RNA translation, impairment of proteasome and lysosomal activity and a crucial role of chronic neuroinflammation, all converging to compromise synaptic transmission and neuronal survival. This multitude of pathological signatures are not completely recapitulated in the available animal models and constitute an important challenge in FTD, directly impacting the translation of preclinical findings to human clinical trials. Having all the above in consideration, we propose to employ patient-derived brain organoids seeded with microglia cells, to explore the molecular mechanisms through which mutations in the GRN gene, one of the most common genetic causes of FTD, contribute to the pathophysiology of the disease. Our goal is to investigate how GRN mutations in microglia contribute to TDP-43 proteinopathy, change microglia homeostatic activity and promote neuronal network dysfunction. Our first aim is to clarify how GRN haploinsufficiency shapes lysosomal function and complement production, which are crucial not only to the clearance of protein aggregates but also to the maintenance of correct network function and circuit refinement through synaptic pruning. A second aim is to investigate the contribution of microglia bearing GRN mutations to the altered network activity reported to occur in iPSC-derived neurons and FTD patients. Towards this purpose, we will evaluate cortical development and perform a thorough characterization of electrophysiological parameters in immunocompetent organoids in the presence of healthy or mutated microglia. Overall, this proposal will shed light on the underlying mechanisms behind the immune and synaptic dysfunction observed in GRN-associated FTD, while simultaneously generating and characterizing a new and useful tool to test disease-modifying therapeutic strategies.
In order to clarify the contribution of FTD-associated mutations in the GRN gene to the pathophysiology of the disease and to establish the foundations for future therapy-driven research, we have established a set of aims that will allow to generate and characterize new models of FTD that better mimic the complexity of the human disease. Aim 1. Establish immunocompetent human brain organoids and microglia-like cells from FTD patient-derived IPSCs. With support from the AFTD, we propose to generate novel iPSC lines from FTD patients bearing mutations in the progranulin (GRN) gene, that will be serve the double purpose of generating patient-derived human brain organoids and microglia-like cells, which will be available to perform functional studies. Aim 2. Determine the impact of GRN mutations on microglia function using patient-derived immunocompetent brain organoids. We will grow patient-derived brain organoids bearing GRN mutations in the presence of human microglia derived from iPSCs from the same patients or from healthy controls, in order to understand the contribution of GRN haploinsufficiency in microglia to TDP-43 accumulation, synaptic pruning and neuronal death. These experiments will help dissect how microglia lysosomal and secretory activity becomes compromised in the presence of GRN mutations and how this impacts the microglia:neuron interplay that helps shape neuronal development and cortical patterning. Aim 3. Evaluate changes in brain organoid single-cell and network activity in the presence of healthy or mutated microglia. We will evaluate synaptic activity, E/I ratios and network synchrony using calcium imaging and single-cell patch-clamp electrophysiology recordings in patient-derived organoids, in the presence of healthy or mutated microglia. This characterization will help dissect the contribution of GRN mutations to network dysfunction in a realistic human model of FTD, while shedding light on the specific contribution of microglia dysfunction to overall disease pathophysiology.