Adenovirus expressing an NPC1‐GFP fusion gene corrects neuronal and nonneuronal defects associated with Niemann pick type C disease

Abstract

Niemann Pick type C (NPC) disease is an autosomal recessive disorder characterized by abnormal cholesterol metabolism and accumulation in lysosomal and endosomal compartments. Although peripheral organs are affected, the progressive neurodegeneration in the brain is typically most deleterious, leading to dystonia, ataxia, seizures, and premature death. Although the two genes underlying this disorder in humans and mouse models of the disease have been identified (NPC1 in 95% and NPC2/HE1 in 5% of human cases), their cellular roles have not Been fully defined, and there is currently no effective treatment for this disorder. To help address these issues, we constructed a recombinant adenovirus, Ad(NPC1-GFP), which contains a cDNA encoding a mouse NPC1 protein with a green fluorescent protein (GFP) fused to its C-terminus. Fluorescence microscopy and cholesterol trafficking assays demonstrate that the GFP-tagged NPC1 protein is functional and detectable in cells from different species (hamster, mouse, human) and of different types (ovary-derived cells, fibroblasts, astrocytes, neurons from peripheral and central nervous systems) in vitro. Combined with results from time-lapse microscopy and in vivo brain injections, our findings suggest that this adenovirus offers advantages for expressing NPC1 and analyzing its cellular localization, movement, functional properties, and beneficial effects in vitro and in vivo.

The subcellular localization of the Niemann-Pick Type C proteins depends on the adaptor complex AP-3

“…5 and Fig. 6A), mNPC1-GFP expressed from an adenoviral vector recapitulated the known localization of NPC1 to LAMPIpositive late endosomal compartments (Paul et al, 2005). Adenovirally delivered mNPC1-GFP was co-localized with AP-3 by infecting immortalized AP-3 ␦ -/-mocha mouse fibroblasts transduced with a retrovirus encoding the ␦ subunit of AP-3 (AP-3 +/+ cells) or an empty retrovirus (AP-3 -/-cells) as a control.…”

“…To test this hypothesis, we first examined whether mNPC1 and the AP-3 complex co-localize. Because we lack an antibody capable of detecting endogenous mNPC1 by indirect immunofluorescence, we assessed the localization of mNPC1-GFP expressed from an adenovirus construct capable of rescuing Npc1 -/-phenotypes (Paul et al, 2005). Furthermore, and as shown below (see Fig.…”

The Niemann-Pick C1 protein in recycling endosomes of presynaptic nerve terminals

“…Alexa Fluor 488-labeled goat anti-rabbit IgG and Texas Red-labeled goat anti-mouse IgG were from Molecular Probes (Eugene, OR). Adenoviruses containing a NPC1-GFP cDNA (26) were provided by Dr. R. A. Maue (Dartmouth Medical School, Hanover, NH) (26).…”

“…2E). This NPC1-GFP construct has previously been characterized and drives the expression of a functional NPC1 protein that eliminates the late endosomal accumulation of cholesterol in mouse Npc1 2/2 sympathetic neurons (26). When neurons expressing NPC1-GFP were stained with anti-synaptophysin antibodies (Fig.…”

“…In previous work, we have shown that NPC1 is present not only in neuronal cell bodies, where late endosomes and lysosomes mainly reside (24,25), but also is abundant in distal axons (23). Furthermore, vesicles containing a chimeric protein of NPC1 fused to green fluorescent protein (GFP) move bidirectionally along distal axons of mouse sympathetic neurons (26). The role of NPC1 in distal axons is not clear because axons are not generally thought to be a major site of lysosomal, degradative pathways (24,25).…”