Projeto Portugal 2030

Sumário

(A graphical abstract was included in 'Supplementary material_ROC' document associated with this proposal.) The overall aim of this proposal is to comprehend the function of the hnRNPH2 protein, the implications of its mutation in neurons, and its significance in the pathogenesis of MRXSB, with the goal of translating this knowledge into future clinical applications. Specifically, the key objectives of the project are: • Investigate the effect of hnRNPH2 mutations associated with MRXSB on the morphology, development, and function of cultured rat hippocampal neurons. We will analyze the consequences of hnRNPH2 mutations on i) dendritic arborization complexity and spine morphology, evaluating the presence of a morphological phenotype resembling ASD; ii) synaptic transmission and neuronal network activity in neurons expressing mutant hnRNPH2. • Identify the unknown mRNAs that directly interact with the RNA-binding protein hnRNPH2 and address whether mutations in hnRNPH2 affect this list of transcripts. This will be investigated with RNA Cross-Linking and Immunoprecipitation (CLIP) followed by next-generation sequencing (NGS), gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis. We will explore a putative association of specific mRNAs to the ASD-like behavior observed in MRXSB. • Develop and characterize new in vivo models of MRXSB. Using utero electroporation, we will replace the endogenous hnRNPH2 protein with MRXSB-associated mutant forms of human hnRNPH2 within a specific region of the mice embryo’s brain (i.e. hippocampus or cortex). Then, we will study the effects of hnRNPH2 mutations on neuronal migration, brain development, synaptic function, and perform behavioral analysis. • Assess the potential for rescuing synaptic alterations and structural changes from hnRNPH2 mutations, in vitro and in vivo. In vitro, we will examine how mutant hnRNPH2 affects total mRNA levels (via qPCR) for the transcripts of interest, obtained within this proposal. i) If reduced levels of mRNAs are found, we will knockdown corresponding proteins in WT neurons and assess the functional outcome. ii) In neurons expressing mutant hnRNPH2, we will gauge changes in protein expression linked to candidate mRNAs. In strategy i), we will seek similarities with the MRXSB phenotype; in ii) we will explore rescuing synaptic alterations and structural changes due to hnRNPH2 mutations. In vivo, we will test strategies i) and ii) in MRXSB models, analyzing neuronal morphology, synaptic function, brain structure and behavior. We will aim to rescuing MRXSB associated phenotype in vitro and in vivo.