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Loss of NDR1/2 kinases impairs endomembrane trafficking and ATG9A localisation, reduces autophagy and leads to neurodegeneration

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.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Brain

SUBMITTER: Simeon Mihaylov

LAB HEAD: Sila K. Ultanir

PROVIDER: PXD035566 | Pride | 2022-11-21

REPOSITORIES: Pride

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			Action	DRS
	EF2439_mihayls_FeNTA_10pc.raw	Raw
	EF2439_mihayls_SMOAC_bRP_HCDMS2_F1.raw	Raw
	EF2439_mihayls_SMOAC_bRP_HCDMS2_F2.raw	Raw
	EF2439_mihayls_SMOAC_bRP_HCDMS2_F3.raw	Raw
	EF2439_mihayls_SMOAC_bRP_HCDMS2_F4.raw	Raw

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Project description:Huntington's disease (HD) is a dominantly inherited genetic disease caused by mutant huntingtin (htt) protein with expanded polyglutamine tracts. A neuropathological hallmark of HD is the presence of neuronal inclusions of mutant htt. p62 is an important regulatory protein in selective autophagy, a process by which aggregated proteins are degraded, and it is associated with several neurodegenerative disorders including HD. Here we investigated the effect of p62 depletion in three HD model mice: R6/2, HD190QG and HD120QG mice. We found that loss of p62 in these models led to longer lifespans and reduced nuclear inclusions, although cytoplasmic inclusions increased with polyglutamine length. In mouse embryonic fibroblasts (MEFs) with or without p62, mutant htt with a nuclear localization signal (NLS) showed no difference in nuclear inclusion between the two MEF types. In the case of mutant htt without NLS, however, p62 depletion increased cytoplasmic inclusions. Furthermore, to examine the effect of impaired autophagy in HD model mice, we crossed R6/2 mice with Atg5 conditional knockout mice. These mice also showed decreased nuclear inclusions and increased cytoplasmic inclusions, similar to HD mice lacking p62. These data suggest that the genetic ablation of p62 in HD model mice enhances cytoplasmic inclusion formation by interrupting autophagic clearance of polyQ inclusions. This reduces polyQ nuclear influx and paradoxically ameliorates disease phenotypes by decreasing toxic nuclear inclusions. Gene expression profiles were analyzed to examine the effects of p62 depletion in mouse with or without mutant huntingtin exon 1 To examine the effect of p62 depletion on the transcriptome of Huntington's disease model mice, we crossed p62 knockout mice with HD model mice. We extracted total RNA from the striatum of these mice at 8 weeks and used for a microaaray analysis. The samples are HD transgenic mice with p62 knockout (HD_p62KO), HD mice with normal p62 (HD_p62WT), non-HD-transgenic mice with p62 knockout (NT_p62KO), and non-HD-transgenic mice with normal p62 (NT_p62WT).

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Loss of NDR1/2 kinases impairs endomembrane trafficking and ATG9A localisation, reduces autophagy and leads to neurodegeneration

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