Fragile X syndrome (FXS) is the most common inherited form of human mental retardation, and it is caused by expansion of CGG repeat in the FMR1 gene. The resulting epigenetic silencing causes the loss of the fragile X mental retardation protein (FMRP) with defects in the regulation of dendritic spine morphology and synaptogenesis.
FXS is widely studied into 2D cell culture differentiating human iPSCs into neuronal population to characterize the disease phenotype taking advantages of molecular and functional analysis. However, conventional 2D cell culture fails to recapitulate the complex neural environment revealing itself as a not reliable in vitro model system to fully characterize the pathology. In this direction novel 2D and 3D model systems have been proposed for dissecting the molecular and cellular processes underling FXS.
Several 3D protocols are available to better mimicking the cell complexity and architecture of the brain tissue, however the lack of non-neural cell types such as microglia still hinders their exploitation for the study of the neuro-immune axis in neurodevelopmental diseases.
The aim of our study is to create an in vitro 3D model based on patient-specific induced pluripotent stem cells (iPSCs) with the purpose of deciphering the neurobiological phenotypes associated with FXS. Specifically, we propose to co-culture iPSC-derived dorso-cortical organoids and isogenic iPSC-derived microglia to generate a disease-relevant and tailored platform for the investigation of neuro-immune interaction during brain development. Indeed, microglia plays a prominent role in shaping synaptic circuitries during neurodevelopment and its presence might unveil possible neural-immune interplay at the basis of FXS and the establishment of a mature synaptic transmission.
In the last few years, iPSCs have led to important improvements for the study of neurological diseases. In fact, given the inaccessibility of neural tissue, researchers have been forced to develop new and robust model system to study these pathologies.
In particular, the development of effective drugs for FXS is challenging because of the limited understanding of its pathophysiology, the differences between patients and animal models, and the difficulties in modelling FXS in vitro and in vivo. Several animal models, including drosophila, zebrafish, knock-out mice and knock-out rats, have been used for the study of FXS and its mechanisms. Although the attempts to turn the findings obtained using these animal models into therapies for patients did not give the desired results, these models provided significantly understandings of FXS features. Moreover, the differences in epigenetic mechanisms between mice and humans raise several critical reasons why the use of human cellular models is so necessary to study the FXS features. For this purpose, the use of human pluripotent stem cells carrying the disease-causing mutations represent a useful and alternative approach for FXS disease modelling. The advances in cellular reprogramming make possible the in vitro generation of neurons and other brain cell types from an individual¿s unique genetic background.
Nevertheless, the limitations of stem cell-derived monolayer cultures have pressured the researchers to develop more sophisticated in vitro models that could better recapitulate the structural and functional complexity of the human brain such as brain organoids. However, this model system still has its limitations as it only represents the neuroectodermal lineage without taking into account other cellular types, such as microglia cells. Indeed, while originating from the mesodermal lineage, microglia play a key role in several crucial processes during brain development and circuitry formation.
In this scenario, the purpose of this project is to develop a strong and reliable platform to characterize the FSX disease starting from FXS-patient derived iPSCs. These cells will be used for the production of cortical brain organoids and microglia, which will be cultured together with the aim to generate a unique in vitro model system for studying microglia physiology and function.
One of the strengths of this model system is the use of FXS patient derived iPSCs, still poorly used in FXS modeling. These cells displaying the genome and the molecular phenotype of the affected individuals, allows to develop a more robust disease model to investigate the pathology at network and cellular level.
Moreover, the presence of isogenic microglia inside cortical organoids will allow us to recapitulate in vitro the first stages of neurodevelopment. In literature just few experiments were done coculturing microglia with brain organoids and the data shows that microglia exhibit phagocytic activity and synaptic pruning function.
Thanks to the combination between microglia and FXS cortical organoids, we will have the opportunity to see the effect of FMRP deficiency both on synaptic shaping and excitation/inhibition ratio during the first stages of neurodevelopment.