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

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.The three isoforms of human apoE (apoE2, apoE3, and apoE4) differ at amino acid positions 112 or 158 or both (1, 2). ApoE4 is the major genetic risk factor for Alzheimer disease (AD) 2 (3-5), and apoE4 carriers account for 65-80% of all AD cases, highlighting the importance of apoE4 in AD pathogenesis (6). Two biophysical properties that distinguish apoE4 from the other isoforms likely hold the key to a mechanistic understanding of its association with AD. First, apoE4 is more unstable and tends to form a molten globule state (7,8). Second, the amino-terminal domain (amino acids 1-191) of apoE4 interacts with its carboxyl-terminal domain (amino acids 223-299) (9, 10). This domain interaction occurs predominantly in apoE4, in which positively charged Arg-112 repels the side chain of Arg-61 in the amino-terminal domain, allowing the formation of a salt bridge between Arg-61 and the negatively charged Glu-255 in the carboxyl-terminal domain (9, 10). Domain interaction occurs to a significantly lesser extent in apoE2 and apoE3 because both have Cys-112, resulting in a different conformation of Arg-61 (11). Importantly, only human apoE has Arg-61; the 17 other species in which the apoE gene has been sequenced have 11). Mutation of Arg-61 to Thr in apoE4 prevents domain interaction, converting apoE4 to an apoE3-like molecule (9 -12). ApoE4 domain interaction occurs on lipoprotein particles in vitro in human plasma, in cultured Neuro-2a cells, and in Arg-61 knock-in mice, in wh...

Small Molecule Structure Correctors Abolish Detrimental Effects of Apolipoprotein E4 in Cultured Neurons

“…Previously, we demonstrated that the intracellular trafficking of apoE4 is impaired in the endoplasmic reticulum and Golgi apparatus of Neuro-2a cells expressing apoE4 compared with apoE3 trafficking, as determined by fluorescence recovery after photobleaching (24). ApoE4-R61T, which lacks domain interaction, gave results comparable with those for apoE3.…”

“…domain interaction between Arg-61 and Glu-255) markedly alters its structure and accounts for many of the functional features that distinguish apoE4 from apoE3 (6,8,9,12,13). The neuropathological features of apoE4 include (a) impaired neurite outgrowth (17)(18)(19); (b) disruption of the cytoskeleton in neurons (38), including increased Tau hyperphosphorylation (39,40); (c) mitochondrial dysfunction in neurons, including altered mitochondrial membrane potential (36) and decreased respiratory enzyme levels and activity (16); (d) impaired neuronal mitochondrial motility (current study); (e) impaired synaptogenesis (20,24); (f) enhanced susceptibility to neuron-specific proteolysis and neurotoxic fragment formation (39 -41), (g) increased amyloid ␤ production (21); (h) increased neuronal lysosomal leakage and apoptosis (42); (i) CNS neuropathology and impaired behavioral activity (26,27,41,43), and (j) enhanced amyloid ␤ deposition (44 -47). All of these abnormal features (a through i above) that distinguish apoE4 from apoE3 are corrected or significantly improved by blocking apoE4 domain interaction by mutation of Arg-61 to threonine, small molecule structure correctors, or engineering of mouse apoE to alter domain interaction (8 -11, 24).…”

“…Treatment of neuronal cells with GIND-25 reduced the apoE4-enhanced amyloid ␤ production (21) and restored mtCOX1 levels in apoE4-expressing cells (16). In addition, GIND-25 and a small molecule, PH-002, restored endoplasmic reticulum and Golgi apparatus transit of apoE4 in cultured neurons to levels equivalent to apoE3 and apoE4-R61T and rescued apoE4-caused impairment of neurite outgrowth and synaptogenesis (24). In the present study, we identified additional potent small molecules that inhibit domain interaction and thus reverse the detrimental effects of apoE4, using a high throughput, cell-based FRET primary screening assay that quantifies the change in apoE4 domain interaction and a panel of secondary cell-and function-based assays in neuronal cells.…”

“…The sp-GFP-apoE3-eDHFR construct was created from an sp-GFP-apoE4-eDHFR plasmid by mutagenesis using a QuikChange kit (Stratagene) and oligomers of 5Ј-catggaggacgtgtgcggccgcc-3Ј and 5Ј-ggcggccgcacacgtcctccatg-3Ј, following the manufacturer's protocol. Neuro-2a cells were transfected with sp-GFP-apoE4-eDHFR, sp-GFP-apoE3-eDHFR, or a mixture of sp-GFP-apoE4 and eDHFR pLL1 (for expression of GFP-apoE4 and eDHFR from separate constructs) to establish stable transfectants as described (24,25).…”

“…6). We have previously demonstrated that PH-002 (100 nM) increased dendritic spine development in primary neurons from NSE-apoE4 trans- genic mice to levels comparable with those in NSE-apoE3 primary neurons (apoE3-expressing primary neurons treated with PH-002 gave results identical to untreated primary neurons (24)). EPR Demonstrating Direct Target Engagement-To demonstrate that PH-002 affected apoE4 structure through a direct binding mechanism, the apoE4 amino terminus spin-labeled at position 76 was incubated with PH-002 at two molar ratios, 2:1 and 4:1 (PH-002/apoE), and the effect on the motility of the Cys-76 spin label was determined.…”