Endogenous Protein-Protein Interaction Network of the NPC Cholesterol Transporter 1 in the Cerebral Cortex

Abstract

NPC intracellular cholesterol transporter 1 (NPC1) is a multipass, transmembrane glycoprotein mostly recognized for its key role in facilitating cholesterol efflux. Mutations in the NPC1 gene result in Niemann-Pick disease, type C (NPC), a fatal, lysosomal storage disease. Due to the progressively expanding implications of NPC1-related disorders, we investigated endogenous NPC1 protein-protein interactions in the mouse cortex and human-derived iPSCs neuronal models of the disease through coimmunoprecipitation-coupled with LC-MS based proteomics. The current study investigated protein-protein interactions specific to the wild-type and the most prevalent NPC1 mutation (NPC1^I1061T) while filtering out any protein interactor identified in the Npc1^-/- mouse model. Additionally, the results were matched across the two species to map the parallel interactome of wild-type and mutant NPC1^I1061T. Most of the identified wild-type NPC1 interactors were related to cytoskeleton organization, synaptic vesicle activity, and translation. We found many putative NPC1 interactors not previously reported, including two SCAR/WAVE complex proteins that regulate ARP 2/3 complex actin nucleation and multiple membrane proteins important for neuronal activity at synapse. Moreover, we identified proteins important in trafficking specific to wild-type and mutant NPC1^I1061T. Together, the findings are essential for a comprehensive understanding of NPC1 biological functions in addition to its classical role in sterol efflux.

Keywords: Interactome; Lysosomal Storage Disorder; Neurodegenerative Disease; Niemann-Pick type C; Transmembrane Protein.

Figure 1.

(A) Putative NPC1 interactors detected only in wild type mouse cortex (disconnected nodes not shown) with known interactions. (B) The characterization of detected interactors by protein class. The top ten biological pathways (C) and subcellular compartments (D) with the highest fold enrichment identified from gene ontology (GO) enrichment analyses.

Figure 2.

Expression level and localization of ARP2/3 complex using ARPC2. (A) Expression of ARPC2 in different NPC1 disease models as determined by Western blot. U18666A-treated HT-22 (a pharmacological model of the disease), the cerebellar tissue lysate and cortical tissue lysate from wild type and control Npc1^−/− mice were compared. (B) Confocal images from-wild type and Npc1^−/− DIV14 primary neuron cultures. Some overlapping of the signal in the merged (bottom row) images was observed. The Mander’s coefficient was used to measure colocalization of ARPC2 and NPC1 in the primary neurons (N=10, individual neurons). Green = NPC1, Magenta = ARPC2

Figure 3.

Synaptic vesicle recycling in wild-type and Npc1^−/− primary mouse cortical neurons using live-cell pHluorin imaging. Graphs show the endocytic decay with trace for normalized pHluorin signal on the left and endocytic tau on the right measured in excitatory neurons. Statistical analysis for endocytic tau was done using unpaired two-tailed t-test with Welch’s correction. For excitatory neurons, N=3 biological replicates were used, with n=244 synapses for wild-type neurons and n=259 for Npc1^−/−.

Figure 4.

Comparison of wild-type and NPC1^I1061T interactomes in mouse cortical tissue and human iPSC neurons. In our analysis, we first removed any proteins that were detected in NPC1^−/− co-IP eluents (negative control) from our datasets, and then matched wild-type and NPC1^I1061T interactome in mouse cortex and human-derived iPSC neurons. (A) 22 interactors were shared between wild type and NPC1^I1061T. 14 interactors were only found in the wild-type models, and 17 interactors were only found in NPC1^I1061T mutant models. Thickness of lines shows confidence in interaction based by STRING analysis. (B) Top enriched biological pathways in wild-type and NPC1^I1061T interactomes with lowest FDR (<0.05) based on KEGG database search, curated by ShinyGo.

Fig 5.

Suggested landscape of NPC1 interactome in the cortical neurons based on the shared interactome between mouse cortex and human-derived iPSC neurons. The figures show a map of wild-type and mutant NPC1 putative interactors in their respective subcellular compartment. NPC1 is marked with yellow, located on the late endosome/lysosome membrane. (A) shared interactome of wild type NPC1 (B) shared interactome of NPC1^I1061T, the most prevalent missense point mutation of NPC1 found in 20% of patients of western European descent. (Created in BioRender.com)