The role of the neuropsychiatric disorder-associated protein ARHGAP46/GMIP in the development and function of the neocortex

Background: Neurodevelopmental disorders, such as autism spectrum disorder (ASD) and schizophrenia, are often characterized by disruptions in neocortical structure and mitochondrial function. Genetic studies have implicated ARHGAP46 in multiple neuropsychiatric disorders. However, the function of ARHGAP46 in the mammalian brain is not understood. In this study, we investigate the role of ARHGAP46 in the development of the mammalian neocortex. Methods: I analysed ARHGAP46 expression in the developing murine neocortex using in situ hybridization, Western blotting, and reporter mice. Conditional deletion of ARHGAP46 in the murine neocortex (cKO) was then used to assess the impact of loss of ARHGAP46 on animal behaviour. I then evaluated the cerebral structure of cKO and control mice. Furthermore, I analysed how loss of ARHGAP46 impacts dendritic and synaptic development both in vivo and in cultured neurons. To understand the cellular roles of ARHGAP46, I used immunofluorescence to examine the subcellular localization of ARHGAP46. Finally, I studied how loss of ARHGAP46 affects mitochondrial structure and function as well as the interaction of mitochondria with the endoplasmic reticulum using several reporters. Results: I show that ARHGAP46 is strongly expressed in the developing neocortex from late embryonic until early postnatal stages. cKO mice display heightened anxiety-like behaviours and impaired learning. Furthermore, loss of ARHGAP46 in the neocortex reduced cortical thickness, impaired dendritic arborization and decreased synaptic density. At a cellular level, loss of ARHGAP46 results in mitochondrial dysfunction characterized by fragmented mitochondria, diminished ATP production, impaired mitochondrial membrane potential, increased oxidative stress, and enhanced mitophagy. I show that ARHGAP46 is localized to the ER, specifically to mitochondria-associated membranes (MAMs), the sites of interaction between the ER and mitochondria. Furthermore, I identify new binding partners of ARGHAP46 and show that it interacts with proteins involved in the ER: mitochondrial interaction, such as Miro1. cKO neurons show fewer but larger mitochondrial: ER contact sites (MERC). Notably, the reintroduction of ARHGAP46 or the expression of Miro1 in conditional knockout (cKO) neurons restores mitochondrial morphology, MERCs, and mitochondrial function, indicating that ARHGAP46 regulates mitochondrial and MERC stability, at least partially, through Miro1. Conclusion: ARHGAP46 is essential for dendritic development. At a cellular level, ARHGAP46 promotes mitochondrial function, most likely by regulating ER–mitochondria contact sites. These changes in dendritic development have important consequences for brain function and result in altered animal behaviour. This work identifies ARHGAP46 as a critical regulator of neurodevelopment and highlights its role in ER: mitochondrial contacts for correct neocortical development and function.