Li-Ming Dong

Stable Expression and Secretion of Apolipoproteins E3 and E4 in Mouse Neuroblastoma Cells Produces Differential Effects on Neurite Outgrowth

Previously, we demonstrated in cultured dorsal root ganglion neurons that, in the presence of β-migrating very low density lipoproteins (β-VLDL), apolipoprotein (apo) E4, but not apoE3, suppresses neurite outgrowth. In the current studies, murine neuroblastoma cells (Neuro-2a) were stably transfected with human apoE3 or apoE4 cDNA, and the effect on neurite outgrowth was examined. The stably transfected cells secreted nanogram quantities of apoE (44-89 ng/mg of cell protein in 48 h). In the absence of lipoproteins, neurite outgrowth was similar in the apoE3- and apoE4-secreting cells. The apoE4-secreting cells, when incubated with β-VLDL, VLDL, cerebrospinal fluid lipoproteins (d < 1.21 g/ml), or with triglyceride/phospholipid (2.7:1 (w/w)) emulsions, showed a reduction in the number of neurites/cell, a decrease in neurite branching, and an inhibition of neurite extension, whereas in the apoE3-secreting cells in the presence of a lipid source, neurite extension was increased. Uptake of β-VLDL occurred to a similar extent in both the apoE3- and apoE4-secreting cells. With low density lipoproteins or with dimyristoylphosphatidylcholine emulsions, either alone or complexed with cholesterol, no differential effect on neurite outgrowth was observed. A slight differential effect was observed with apoE-containing high density lipoproteins. The differential effect of apoE3 and apoE4 in the presence of β-VLDL was blocked by incubation of the cells with heparinase and chlorate, with lactoferrin, or with receptor-associated protein, all of which prevent the uptake of lipoproteins by the low density lipoprotein receptor-related protein (LRP). The data suggest that the secreted and/or cell surface-bound apoE interact with the lipoproteins and facilitate their internalization via the heparan sulfate proteoglycan-LRP pathway. The mechanism by which apoE3 and apoE4 exert differential effects on neurite outgrowth remains speculative. However, the data suggest that apoE4, which has been shown to be associated with late onset familial and sporadic Alzheimers disease, may inhibit neuronal remodeling and contribute to the progression of the disease.

Human Apolipoprotein E4 Domain Interaction ARGININE 61 AND GLUTAMIC ACID 255 INTERACT TO DIRECT THE PREFERENCE FOR VERY LOW DENSITY LIPOPROTEINS

Human apolipoprotein (apo) E contains an amino- and a carboxyl-terminal domain, which are connected by a hinge region (approximately residues 165 to 215). The interaction of the two domains has been suggested to be responsible for the apoE4-binding preference for very low density lipoproteins (VLDL). In the absence of this interaction in apoE3, the preference is for high density lipoproteins (HDL). To exclude the possibility that the interaction of apoE with other apolipoproteins on the native particles may contribute to the isoform-specific preferences, VLDL-like emulsion particles were incubated with apoE, and the lipid-bound apoE was separated from free apoE on a Superose 6 column. The apoE4 bound more effectively to these particles than did apoE3, indicating that the apoE4 preference for VLDL is due not to interactions with other apolipoproteins but to an intrinsic property of apoE4, likely related to domain interaction. Previously, arginine 61 was shown to be critical for the isoform preferences, suggesting that it interacted with an acidic residue(s) in the carboxyl terminus. Substitution of arginine 61 with lysine did not alter the preference of apoE4 for VLDL, demonstrating that a positive charge rather than a specific requirement for arginine is critical for domain interaction. To identify the acidic residue(s) in the carboxyl terminus interacting with arginine 61, the six acidic residues (244, 245, 255, 266, 270, and 271) in a region known to be important for both lipoprotein association and isoform-specific preferences were substituted individually with alanine in apoE4. Only substitution of glutamic acid 255 altered the preference of apoE4 from VLDL to HDL, indicating that this was the sole residue in the carboxyl terminus that interacts with arginine 61. The participation of the hinge region in domain interaction was examined with internal deletion mutants. Deletion of the residues 186-202 or 186-223, representing major portions of the hinge region, had no effect on the apoE4 preference for VLDL. This suggests that the hinge region may act as a spacer that connects the two domains. Further deletion into the carboxyl-terminal domain (to residue 244) results in a loss of apoE4 VLDL binding. These studies establish that interaction of arginine 61 and glutamic acid 255 mediates apoE4 domain interaction.

Introduction of human apolipoprotein E4 "domain interaction" into mouse apolipoprotein E.

Human apolipoprotein E4 (apoE4) binds preferentially to lower density lipoproteins, including very low density lipoproteins, and is associated with increased risk of atherosclerosis and neurodegenerative disorders, including Alzheimers disease. This binding preference is the result of the presence of Arg-112, which causes Arg-61 in the amino-terminal domain to interact with Glu-255 in the carboxyl-terminal domain. ApoE2 and apoE3, which have Cys-112, bind preferentially to high density lipoproteins (HDL) and do not display apoE4 domain interaction. Mouse apoE, like apoE4, contains the equivalent of Arg-112 and Glu-255, but lacks the critical Arg-61 equivalent (it contains Thr-61). Thus, mouse apoE does not display apoE4 domain interaction and, as a result, behaves like human apoE3, including preferential binding to HDL. To assess the potential role of apoE4 domain interaction in atherosclerosis and neurodegeneration, we sought to introduce apoE4 domain interaction into mouse apoE. Replacing Thr-61 in mouse apoE with arginine converted the binding preference from HDL to very low density lipoproteins in vitro, suggesting that apoE4 domain interaction could be introduced into mouse apoE in vivo. Using gene targeting in embryonic stem cells, we created mice expressing Arg-61 apoE. Heterozygous Arg-61/wild-type apoE mice displayed two phenotypes found in human apoE4/E3 heterozygotes: preferential binding to lower density lipoproteins and reduced abundance of Arg-61 apoE in the plasma, reflecting its more rapid catabolism. These findings demonstrate the successful introduction of apoE4 domain interaction into mouse apoE in vivo. The Arg-61 apoE mouse model will allow the effects of apoE4 domain interaction in lipoprotein metabolism, atherosclerosis, and neurodegeneration to be determined.