Abstract

Autophagy is essential for neuronal development and its deregulation contributes to neurodegenerative diseases. NDR1 and NDR2 are highly conserved kinases implicated in neuronal development, mitochondrial health and autophagy, but how they affect mammalian brain development in vivo is not known. Using single and double Ndr1/2 knockout mouse models we show that, dual, but not individual loss of Ndr1/2 in neurons causes neurodegeneration during brain development, but also in adult mice. Proteomic and phosphoproteomic comparisons between Ndr1/2 knockout and control brains revealed novel kinase substrates and indicated that endocytosis is significantly affected in the absence of NDR1/2. We validated the endocytic protein, Raph1/Lpd1 as a novel NDR1/2 substrate and showed that both NDR1/2 and Raph1 are critical for endocytosis and membrane recycling. In NDR1/2 knockout brains, we observed prominent accumulation of transferrin receptor, p62 and ubiquitinated proteins, indicative of a major impairment of protein homeostasis. Furthermore, the levels of LC3-positive autophagosomes were reduced in knockout neurons, implying that reduced autophagy efficiency mediates p62 accumulation and neurotoxicity. Mechanistically, pronounced mislocalisation of the transmembrane autophagy protein ATG9A at the neuronal periphery, impaired axonal ATG9A trafficking and increased ATG9A surface levels further confirm defects in membrane trafficking and could underlie the impairment in autophagy. We provide novel insight into the roles of NDR1/2 kinases in maintaining neuronal health. HighlightsO_LIDual neuronal Ndr1 and Ndr2 knockout during development or in adult mice causes neurodegeneration. C_LIO_LIPhosphoproteomics comparison of Ndr1/2 knockouts with control littermates shows endocytosis and membrane trafficking to be affected and reveals novel substrates. C_LIO_LIRaph1/Lamellipodin is a novel NDR1/2 substrate that is required for TfR endocytosis. C_LIO_LINdr1/2 knockout brains exhibit a severe defect in ubiquitinated protein clearance and reduced autophagy. C_LIO_LINDR1/2 and Raph1 are required for the trafficking of the only transmembrane autophagy protein, ATG9A. C_LI