Pathological roles of the VEGF/SphK pathway in Niemann–Pick type C neurons

Abstract

Sphingosine is a major storage compound in Niemann–Pick type C disease (NP–C), although the pathological role(s) of this accumulation have not been fully characterized. Here we found that sphingosine kinase (SphK) activity is reduced in NP–C patient fibroblasts and NP–C mouse Purkinje neurons (PNs) due to defective vascular endothelial growth factor (VEGF) levels. Sphingosine accumulation due to inactivation of VEGF/SphK pathway led to PNs loss via inhibition of autophagosome–lysosome fusion in NP–C mice. VEGF activates SphK by binding to VEGFR2, resulting in decreased sphingosine storage as well as improved PNs survival and clinical outcomes in NP–C cells and mice. We also show that induced pluripotent stem cell (iPSC)-derived human NP–C neurons are generated and the abnormalities caused by VEGF/SphK inactivity in these cells are corrected by replenishment of VEGF. Overall, these results reveal a pathogenic mechanism in NP–C neurons where defective SphK activity is due to impaired VEGF levels.

Mutations in Vps15 perturb neuronal migration in mice and are associated with neurodevelopmental disease in humans

“…Antibodies targeting phosphoproteins were incubated with 1 mM sodium orthovanadate in 0.5% BSA/TBS. The following primary antibodies were used: 1:2,000 Vps15 for mouse studies (Novus Biologicals NBP1-30463) 39 , 1:1,000 VPS15 for human studies (Abcam ab124817, Abcam ab128903) 41 , 1:500 Vps34 (Cell Signaling, #3811) 42,43 , 1:1,000 Beclin1 (Cell Signaling #3738) 44 , 1:500 p62 (Novus Biologicals, H00008878-M01) 45 , 1:2,000 GAPDH (Millipore, MAB3 74) 26,46 , 1:1,000 ubiquitin (Santa Cruz, sc-8017) 47 , 1:10,000 α-tubulin (Sigma Aldrich, T6199) 48 , 1:100 LC3 (Nanotools, 0260-100/LC3-2G6) 49 , 1:600 EGFR (Cell Signaling #4267 S) 50 , 1:200 Nischarin (Santa Cruz, sc-365364) 21 , 1:500 PAK1 T423 (Cell Signaling, #2601 S) 51 and 1:500 N -terminal Vps15 antibody (Proteintech, 17894-1-AP). Western blots were quantified using ImageJ.…”

Sphingosine Kinase 1 Cooperates with Autophagy to Maintain Endocytic Membrane Trafficking

“…Similar to our phenotype, NP-C fibroblasts accumulate LEs enriched in sphingolipids and cholesterol (Sarkar et al, 2014). As SphK activity is suppressed in NP-C (Lee et al, 2014), we propose that a failure to recruit SphK to endosomal membranes may drive Sph accumulation during the pathogenesis of NP-C. Despite functional lysosomes, NP-C cells have impaired autophagic flux due to the failure to recruit autophagic SNARE machinery to LEs (Sarkar et al, 2013).…”

Neuropathophysiology of Lysosomal Storage Diseases: Synaptic Dysfunction as a Starting Point for Disease Progression

“…Niemann-pick type C-neurological impairments of early onset NPC1 include cerebellar ataxia, strongly implicating the loss of Purkinje cells in the cerebellum [144]. Although, there have been no published reports yet exclusively on iPSC derived Purkinje neurons, a study by Lee et al [145], reported that iPSCs-derived neurons from NPC1 patients accumulated cholesterol and sphingosine, and had reduced levels of sphingosine kinase (SphK) activity and VEGF (vesicular endothelial growth factor) levels. The authors proposed that sphingosine accumulation was caused by inactivation of the VEGF/SphK activity, leading to inhibition of autophagosome-lysosome fusion.…”