Abstract
Loss-of-function mutations in NEK1 gene, which encodes a serine/threonine kinase, are involved in human developmental disorders and ALS. Here we show that NEK1 regulates retromer-mediated endosomal trafficking by phosphorylating VPS26B. NEK1 deficiency disrupts endosomal trafficking of plasma membrane proteins and cerebral proteome homeostasis to promote mitochondrial and lysosomal dysfunction and aggregation of α-synuclein. The metabolic and proteomic defects of NEK1 deficiency disrupts the integrity of blood–brain barrier (BBB) by promoting lysosomal degradation of A20, a key modulator of RIPK1, thus sensitizing cerebrovascular endothelial cells to RIPK1-dependent apoptosis and necroptosis. Genetic inactivation of RIPK1 or metabolic rescue with ketogenic diet can prevent postnatal lethality and BBB damage in NEK1 deficient mice. Inhibition of RIPK1 reduces neuroinflammation and aggregation of α-synuclein in the brains of NEK1 deficient mice. Our study identifies a molecular mechanism by which retromer trafficking and metabolism regulates cerebrovascular integrity, cerebral proteome homeostasis and RIPK1-mediated neuroinflammation.
Highlights
Loss-of-function mutations in NIMA-Related Kinase 1 (NEK1) gene, which encodes a serine/threonine kinase, are involved in human developmental disorders and ALS
We found that knockdown of VPS26B sensitized wild type (WT), but not Nek1Kat2J/Kat2J MEFs, to RIPK1-dependent apoptosis (RDA) and necroptosis; while knockdown of VPS29 or VPS26A sensitized both WT and Nek1Kat2J/Kat2J MEFs to RDA and necroptosis, which was inhibited by treatment with RIPK1 inhibitor Nec-1s (Fig. 4a, b; Supplementary Fig. 4c-f)
We further confirmed the increased and decreased plasma membrane levels of sorting nexin 27 (SNX27) and Sorting nexin 2 (SNX2), respectively, in Nek1Kat2J/Kat2J MEFs compared to that of WT by western blotting analysis after cell surface selective aminooxy-biotinylation (Fig. 5a). These results demonstrate that Nek[1] deficiency blocks the phosphorylation of S302/S304 VPS26B which in turn leads to mislocalization of SNX27- and SNX2-retromers
Summary
Loss-of-function mutations in NEK1 gene, which encodes a serine/threonine kinase, are involved in human developmental disorders and ALS. The metabolic and proteomic defects of NEK1 deficiency disrupts the integrity of blood–brain barrier (BBB) by promoting lysosomal degradation of A20, a key modulator of RIPK1, sensitizing cerebrovascular endothelial cells to RIPK1-dependent apoptosis and necroptosis. NEK1 has been implicated in mediating diverse cellular functions, including cell cycle, cilia formation, DNA-damage response, microtubule stability, and neural development[4]. It remains unclear how NEK1 may mediate these diverse functions at cellular levels; nor is it known why NEK1 deficiency in animals and humans can lead to early developmental disorders. In TNF stimulated cells, RIPK1 is rapidly recruited into complex I in association with the intracellular DD of TNFR1 where RIPK1 is extensively modulated by ubiquitination and phosphorylation which collectively decides whether
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