NDR1/2 kinases regulate membrane trafficking, enable efficient autophagy and prevent neurodegeneration

NDR1 and NDR2 are highly conserved kinases that regulate neuronal development, mitochondrial health and autophagy, but their roles in the mammalian brain in vivo are unexplored. Using constitutive Ndr1 and neuron-specific Ndr2 knockout mice we show that dual loss of Ndr1/2 in neurons causes neurodegeneration. Phosphoproteomic comparisons between Ndr1/2 knockout and control brains revealed novel kinase substrates and highlighted changes in endocytosis in the absence of NDR1/2. The endocytic protein Raph1/Lpd1 is a new NDR1/2 substrate and both NDR1/2 and Raph1 are critical for endocytosis and membrane recycling. NDR1/2 knockout brains exhibited a prominent accumulation of transferrin receptor, p62 and ubiquitinated proteins, indicative of a major impairment in protein homeostasis. Furthermore, autophagosome levels were reduced in knockout neurons, implying that reduced autophagy efficiency mediates p62 accumulation. 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.