Structure-dependent Impairment of Intracellular Apolipoprotein E4 Trafficking and Its Detrimental Effects Are Rescued by Small-molecule Structure Correctors

Dah-Eun Jeong,Mike Pleiss,Scott Peterson,Casey Mccomas,Yadong Huang,Zhaoping Liu,Maureen E Balestra,David Witter,Karl H Weisgraber,Nigel Liverton,Anke Meyer-Franke,Stephen Freedman,Jens Brodbeck,Matthew Childers,Jim Mcguire,Mark Goulet,Richard Hargreaves,Fred Hess,Robert W Mahley

doi:10.1074/jbc.m110.217380

Abstract

Apolipoprotein (apo) E4 is the major genetic risk factor for Alzheimer disease (AD) and likely contributes to neuropathology through various pathways. Here we report that the intracellular trafficking of apoE4 is impaired in Neuro-2a cells and primary neurons, as shown by measuring fluorescence recovery after photobleaching. In Neuro-2a cells, more apoE4 than apoE3 molecules remained immobilized in the endoplasmic reticulum (ER) and the Golgi apparatus, and the lateral motility of apoE4 was significantly lower in the Golgi apparatus (but not in the ER) than that of apoE3. Likewise, the immobile fraction was larger, and the lateral motility was lower for apoE4 than apoE3 in mouse primary hippocampal neurons. ApoE4 with the R61T mutation, which abolishes apoE4 domain interaction, was less immobilized, and its lateral motility was comparable with that of apoE3. The trafficking impairment of apoE4 was also rescued by disrupting domain interaction with the small-molecule structure correctors GIND25 and PH002. PH002 also rescued apoE4-induced impairments of neurite outgrowth in Neuro-2a cells and dendritic spine development in primary neurons. ApoE4 did not affect trafficking of amyloid precursor protein, another AD-related protein, through the secretory pathway. Thus, domain interaction renders more newly synthesized apoE4 molecules immobile and slows their trafficking along the secretory pathway. Correcting the pathological structure of apoE4 by disrupting domain interaction is a potential therapeutic approach to treat or prevent AD related to apoE4.

Highlights

Apolipoprotein E4 is the major genetic risk factor for Alzheimer disease (AD) and likely contributes to neuropathology through various pathways
In Neuro-2a cells, more apoE4 than apoE3 molecules remained immobilized in the endoplasmic reticulum (ER) and the Golgi apparatus, and the lateral motility of apoE4 was significantly lower in the Golgi apparatus than that of apoE3
enhanced green fluorescent protein (EGFP)-apoE Localizes to the ER and Golgi Apparatus in Neuro-2a Cells—Neuro-2a cells stably expressing EGFP-tagged apoE3 or apoE4 were established by transfection of cDNAs encoding the respective fusion protein starting with the signal peptide of apoE

Summary

EXPERIMENTAL PROCEDURES

Expression Constructs—cDNAs of apoE3 and apoE4 were subcloned into pcDNA3 expression vectors in-frame with the amino-terminal apoE signal sequence followed by EGFP and apoE with a stop codon to generate EGFP-apoE3 and EGFPapoE4. Neuro-2a cells stably expressing human apoE3 or apoE4 at similar levels were generated in our laboratory [24, 26]. At 13 days in vitro (DIV) culture, primary neurons were transduced with Sindbis virus expressing EGFPapoE3, EGFP-apoE4, or EGFP-apoE4-R61T. Fluorescence levels were normalized to their pre-bleach base lines, and the extent of recovery was determined [32]. At 5 DIV, primary neurons were transiently transfected with EGFP-␤-actin to highlight dendritic spines [34]. At 11 DIV, some of the primary neurons expressing apoE3 or apoE4 were treated with PH002 (100 nM for 3 days). At 14 DIV, all cells were fixed with ice-cold 4% paraformaldehyde in PBS (pH 7.4) and mounted on microscope slides with Vectashield (Vector Laboratories). Differences between means were assessed by t test, MannWhitney U test, or one-factor analysis of variance. p Ͻ 0.05 was considered statistically significant

RESULTS

GFP in cytoplasm GFP in ER lumen

DISCUSSION