Regulation of autophagy and ubiquitination by ARHGAP33/NOMA-GAP during normal and pathological development of the mammalian neocortex.

• Applicant: Professorin Dr. Marta de Rocha Rosário
• Subject Area: Developmental Neurobiology ; Molecular Biology and Physiology of Neurons and Glial Cells
• Term: since 2013
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 246199359

The cerebral neocortex processes higher brain functions such as decision-making and voluntary movement. The excitatory neurons of the neocortex possess highly elaborate dendritic trees with spines that form synaptic connections. Defects in the maturation of dendritic trees and their spines impair connectivity and information processing and can lead to neurodevelopmental disorders such as intellectual disability and autism spectrum disorders.We have identified a signalling protein, NOMA-GAP, encoded by the ARHGAP33 gene, that regulates dendritic maturation in the neocortex. Loss of NOMA-GAP results in simplified dendritic trees with immature spines and is associated with autism-like behavior in mice. Furthermore, a collaboration with clinical geneticists, has revealed first mutations in the human ARHGAP33 gene that are associated with severe intellectual disability. In preliminary work we have uncovered structural alterations in the dendrites of young NOMA-GAP-deficient mice previously only observed in aged rodents or in degenerative conditions such as Alzheimer’s disease. These autophagy-linked disturbances represent a previously unrecognized mechanism for pathological development of the neocortex. The goal of the current project is to identify disease-relevant cellular and molecular mechanisms by which NOMA-GAP promotes dendritic maturation during neocortex development.Our first step is to identify the cellular mechanism underlying developmental autophagy arrest. We will investigate the different stages of this protein/membrane degradation process to elucidate the function of NOMA-GAP and its disease variants.In a second step, we will build on further preliminary work. Through IP mass spectrometry screens, we have identified a novel function for NOMA-GAP in the regulation of ubiquitination. Ubiquitination is a protein modification that alters protein function or degradation and can promote autophagy. We will investigate the interaction between NOMA-GAP and its disease-variants with a new candidate molecule involved in ubiquitination and address the consequences of de-regulation of this pathway on developmental autophagy and dendritic maturation.To this end, we will use reporter assays, live imaging, enzymatic assays and co-localization studies, together with knockout mouse lines (deficient in expression of the candidate and/or NOMA-GAP), in utero electroporation and transfection of primary neurons. In addition, we will perform IP mass spectrometric analysis of changes in protein complex formation and in the ubiquitome of in these mice to reveal the functional and molecular significance. This project will deepen our understanding of neocortex development and reveal a new cellular and molecular mechanism for neurodevelopmental disorders.

• DFG Programme: Research Grants