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Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient
Published on: September 3, 2014
Neuronal localization and association of Niemann Pick C2 protein (HE1/NPC2) with the postsynaptic density
W Y Ong1, R K Sundaram, E Huang
1Department of Anatomy, National University of Singapore, Singapore 119260.
Abstract:
Niemann-Pick disease type C (NP-C) is an inherited disorder that is characterized biochemically by cellular cholesterol and glycolipid storage, and clinically by progressive neurodegeneration. Most cases of NP-C are caused by inactivating mutations of the npc1 gene, but about 5% are linked to npc2, which encodes a soluble cholesterol binding protein, previously identified as epididymal secretory glycoprotein 1 (HE1). The present study was carried out to investigate the immunocytochemical localization of HE1/NPC2 protein in the mouse brain. Using an antibody against recombinant HE1/NPC2, we found HE1/NPC2 to be localized predominantly in neurons in the brain. Immunoreactivity for HE1/NPC2 was observed in pyramidal or projection neurons in the cerebral cortex and amygdala, and Purkinje neurons in the cerebellum. Neurons in the thalamus, hypothalamus, and globus pallidus were lightly labeled, or unlabeled. This regional pattern of expression of HE1/NPC2 is similar to our previous findings with NPC1, with a low level of expression of both NPC1 and HE1/NPC2 proteins in regions derived from the diencephalon, such as the thalamus and hypothalamus. In contrast to NPC1, however, which is predominantly in astrocytes, HE1/NPC2 was observed mainly in neurons. Electron microscopic immunocytochemistry showed that HE1/NPC2 is present in the cytosol of dendrites and on post-synaptic densities (PSD). The occurrence of HE1/NPC2 in the PSD was confirmed by Western blots of PSD-enriched brain subcellular fractions that showed the presence of HE1/NPC2 together with the PSD-associated protein, PSD-95. These results suggest that NPC1 and HE1/NPC2 are differentially enriched in astrocytes and neurons, respectively, and that HE1/NPC2 may function in supporting the integrity of the PSD of neurons.
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