Functional Effect of GRIN2B Alterations in Humans Neuro Progenitor Cells, a Model for Intellectual Disabilities
Gene mutations and chromosomal rearrangements are found in a proportion of patients diagnosed with autism spectrum disorders (ASDs) or intellectual disabilities (IDs). Modifications at the DNA level are likely responsible for altering neurodevelopmental programs; however, the molecular mechanisms that links mutations with altered sub-cellular and cellular processes leading to neuronal dysfunctions are poorly understood. A new approach which may help to better understand such mechanisms involves using human neuroprogenitor cells (NPCs) derived from induced pluripotent stem cells (iPSC). In fact, proliferation and differentiation processes of such cells determined to become neurons, glia or oligodendrocytes can be easily observed in vitro, in physiological and pathological contexts.
Several studies have reported mutations, deletions, or disruptions of GRIN2B gene in cases diagnosed with ASDs or neurodevelopmental disorders. GRIN2Bgene is located on chromosome 12p12. That gene codes for an NMDA receptor subunit which plays important roles at pre and post natal stages in synaptic development and glutamatergic neurotransmission.
The main objective of that study is to understand how alterations in GRIN2B gene may lead to an altered neurodevelopmental program using neural progenitor cell models.
At a molecular level, we aim to identify the genes and gene networks that are deregulated in a model of GRIN2B silencing or in a NPC cell line from a patient with GRIN2B mutation.
We would determine if those changes at the DNA or at the RNA levels could affect cell morphology and electrophysiological properties.
DNA from patients diagnosed with ASD or ID were analysed using deep sequencing. NPCs producing sh-RNA that targets GRIN2B gene or derived from patients with a GRIN2B mutation were expanded and differentiated for 30 days. Whole transcritptome profiling was performed on the GRIN2Bsilenced cell lines using RNA sequencing. Differentially expressed genes were validated by quantitative real-time PCR. 3D reconstructions and electrophysiological recordings were done to assess morphology and glutamate receptor function, respectively.
Gene expression analysis performed on the model of GRIN2Bsilencing shows that differentially expressed genes are related to synapse functioning and ion transport. We have validated the differential expression of many mRNA glutamatergic subunits such as GRIK1, GRIK2, GRIA1, GRIA2 and GRIA4. Preliminary data of 3D reconstruction suggest that GRIN2B silenced cells present dendritic abnormalities.
These data suggest that reduced dosage of GRIN2B affects the expression of other mRNA coding glutamatergic subunits which potentially alter crosstalks between receptors and consequently dendritic spine development. These mechanisms may be crucial in the pathophysiology of ASDs and IDs.