Neurological impairment in Niemann-Pick C disease: a study on the role of excitatory neurotrasmitter receptors and identification of peripheral cellular biomarkers

Changes in the functioning of neuronal plasma membranes are good candidates in the research of Niemann-Pick Disease type C (NPDc) pathophysiogenetic mechanisms. The loss of a correct dynamic of cholesterol-sphingolipids-enriched microdomains in the neuronal and glial plasma membrane, caused by an imbalance in the lipid trafficking due to NPDc gene mutation, might have a key role in neuronal dysfunction and the consequent clinical pathologies. Our previous results suggest that changes in the plasma membrane cholesterol content are particularly important in affecting lipid rafts, regulators of glutamate receptor functioning. We therefore evaluated through several experimental approaches whether the physiological properties and the neurotransmission of NPC neurons show differences from what observed in the Wild Type (WT). To this aim acute brain slices, primary neuronal cell cultures and synaptosomal preparation from WT and NPC mice, a well-established mouse model for the Niemann- Pick type C disease, were used; in some experiments the Methyl-beta-Cyclodextrin (MbetaCD), a molecule that dissolves the hydrophobic core of lipid rafts, was perfused. The electrophysiological data suggest an impairment of the excitatory neurotransmitter receptors, since a different response to kainic acid perfusion was observed: in fact in hippocampal slices from NPC mice the excitotoxic effect properly described for the WT slices was lacking. These data are in agreement with the results of WT MbetaCD-treated slices which respond to kainic acid application that partially resembles the NPC slices trend, confirming that lipid rafts manipulation counteracts kainate effect. Moreover the induction and maintenance of NMDA-dependent LTP in the CA1 region of NPC hippocampal slices were significantly reduced. The electrophysiological results are supported by data outcoming from cell culture experiments on excitatory aminoacid-induced intracellular calcium increase. Indeed, application of both NMDA and kainic acid in WT cell culture treated with MbetaCD significantly reduced calcium influx. Moreover, results from Western Blot analysis on synaptosomal membrane fractions revealed that levels of GluR6/7 kainate receptor subunits were about 30% reduced in synaptosomes from NPC slices as compared to wild-type. In order to develop a method for a rapid and suitable diagnosis, we used some fibroblast cell lines obtained from patients. Cell lines have been maintained in culture and the lipid composition was determined with standard procedures. Finally, we used different strategies to correlate sphingolipid/cholesterol membrane content with the pathological status of the fibroblast donor, based on the ability of some toxins to bind cell lipids. The results obtained encourage more detailed studies.