Interrogating neurogenic defects in complex assembloid models of fragile X syndrome

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PROJECT SUMMARY/ABSTRACT Human neural development is a dynamic process that requires a precise orchestration of a sequence of cellular, molecular and genetic events allowing for the establishment of appropriate neural circuitry and functional connectivity in the brain. Disruptions to this process have profound consequences, and several neurodevelopmental disorders converge on molecular pathways that regulate protein synthesis, proliferation, migration and differentiation in neural cells. One such disorder is Fragile X syndrome (FXS), which is the leading form of inherited intellectual disability and the most common monogenic cause of autism. Transcriptional silencing of FMR1 and subsequent loss of the RNA-binding protein FMRP leads to altered proliferation, dysregulated protein synthesis, and disrupted signal transduction in animal models of FXS; however, the consequence of loss of FMRP on early events in neurogenesis in humans remains relatively unknown. While several highly promising preclinical studies have demonstrated that targeting key signaling pathways ameliorates multiple defects in animal models, most of these have not translated into successful human therapeutic interventions. We believe that a major gap in the preclinical phase may have been the lack of a human neuronal model to test drug efficacy in a developmentally-relevant manner. Recently, a phase 2 clinical trial using a phosphodiesterase inhibitor to inhibit degradation of cyclic adenosine monophosphate (cAMP) in FXS patients showed highly promising results on several outcome measures. This proposal aims to provide a novel human cellular platform with robust cellular and molecular readouts to further test the efficacy of therapeutic interventions. Here, we propose to use 3D organoids to determine whether cAMP signaling is disrupted throughout early brain development in FXS, and whether aberrant cAMP signaling underlies defects in neurogenesis and cell fate commitment. We will also investigate the therapeutic potential of targeting the microtubule-associated protein doublecortin (DCX), which is an mRNA target of FMRP as well as a downstream target of the cAMP intracellular cascade (Aim 1). We will further employ a novel assembloid system to study cell fate commitment of excitatory and inhibitory neurons as well as interneuron migration in FXS, and to determine the contribution of altered DCX expression and cAMP signaling to interneuron development and differentiation (Aim 2). These findings will provide critical insight into the underlying pathomechanisms in FXS, as well as into the biology of FMRP during early human development. Ultimately, this may aid in the development of targeted patient-specific therapeutic strategies that have broader implications for other neurodevelopmental disorders.