Identification of Brain-Specific Treatment Effects in NPC1 Disease by Focusing on Cellular and Molecular Changes of Sphingosine-1-Phosphate Metabolism.

Niemann-Pick type C1 (NPC1) is a lysosomal storage disorder, inherited as an autosomal-recessive trait. Mutations in the Npc1 gene result in malfunction of the NPC1 protein, leading to an accumulation of unesterified cholesterol and glycosphingolipids. Beside visceral symptoms like hepatosplenomegaly, severe neurological symptoms such as ataxia occur. Here, we analyzed the sphingosine-1-phosphate (S1P)/S1P receptor (S1PR) axis in different brain regions of Npc1-/- mice and evaluated specific effects of treatment with 2-hydroxypropyl-ß-cyclodextrin (HPßCD) together with the iminosugar miglustat. Using high-performance thin-layer chromatography (HPTLC), mass spectrometry, quantitative real-time PCR (qRT-PCR) and western blot analyses, we studied lipid metabolism in an NPC1 mouse model and human skin fibroblasts. Lipid analyses showed disrupted S1P metabolism in Npc1-/- mice in all brain regions, together with distinct changes in S1pr3/S1PR3 and S1pr5/S1PR5 expression. Brains of Npc1-/- mice showed only weak treatment effects. However, side effects of the treatment were observed in Npc1+/+ mice. The S1P/S1PR axis seems to be involved in NPC1 pathology, showing only weak treatment effects in mouse brain. S1pr expression appears to be affected in human fibroblasts, induced pluripotent stem cells (iPSCs)-derived neural progenitor and neuronal differentiated cells. Nevertheless, treatment-induced side effects make examination of further treatment strategies indispensable.

Plasticity-related gene 5 promotes spine formation in murine hippocampal neurons.

The transmembrane protein plasticity-related genes 3 and 5 (PRG3 and PRG5) increase filopodial formation in various cell lines, independently of Cdc42. However, information on the effects of PRG5 during neuronal development is sparse. Here, we present several lines of evidence for the involvement of PRG5 in the genesis and stabilization of dendritic spines. First, PRG5 was strongly expressed during mouse brain development from embryonic day 14 (E14), peaked around the time of birth, and remained stable at least until early adult stages (i.e. P30). Second, on a subcellular level, PRG5 expression shifted from an equal distribution along all neurites toward accumulation only along dendrites during hippocampal development in vitro. Third, overexpression of PRG5 in immature hippocampal neurons induced formation of spine-like structures ahead of time. Proper amino acid sequences in the extracellular domains (D1 to D3) of PRG5 were a prerequisite for trafficking and induction of spine-like structures, as shown by mutation analysis. Fourth, at stages when spines are present, knockdown of PRG5 reduced the number but not the length of protrusions. This was accompanied by a decrease in the number of excitatory synapses and, consequently, by a reduction of miniature excitatory postsynaptic current frequencies, although miniature excitatory postsynaptic current amplitudes remained similar. In turn, overexpressing PRG5 in mature neurons not only increased Homer-positive spine numbers but also augmented spine head diameters. Mechanistically, PRG5 interacts with phosphorylated phosphatidylinositols, phospholipids involved in dendritic spine formation by different lipid-protein assays. Taken together, our data propose that PRG5 promotes spine formation.