Apolipoprotein E: Effects on Cytoskeletal Structure and Function in Alzheimer's Disease and Related Disorders

Yadong Huang, M.D., Ph.D.

Overview
Research in our laboratory focuses on the biological and pathophysiological functions of apolipoprotein (apo) E. A long-term goal of our research is to understand the molecular and cellular mechanisms by which apoE isoforms differentially modulate the pathogenesis of AlzheimerÕs disease (AD).

Human apoE has three major isoforms, apoE2, apoE3, and apoE4 (1, 2). ApoE4 has been linked to the pathogenesis of AD. ApoE is found in both neuritic amyloid plaques and neurofibrillary tanglesÑthe neuropathological hallmarks of ADÑbut its role in the pathogenesis of these two lesions is unclear.

During the past year, we investigated the interactions of apoE3 and apoE4 with cytoskeletal components and their potential roles in the formation of neurofibrillary tangles. We hypothesized that internalized or endogenously synthesized apoE isoforms differentially interact with (or modulate) the cytoskeleton, contributing to neurofibrillary tangle formation. Our results demonstrate that the carboxyl-terminal-truncated forms of apoE enter the cytosol and interact with the cytoskeletal components, resulting in the formation of neurofibrillary tangleølike structures in neurons (3). The truncated apoE4, which can be generated in cultured neurons and in the brains of AD patients, has a greater ability to induce neurofibrillary tangleølike structures than truncated apoE3 (4).

Expression of ApoE4 Induces Intracellular Inclusions in Neuro-2a Cells
Neuro-2a cells transiently transfected with apoE3 or apoE4 cDNA constructs were examined by double immunostaining with a polyclonal antibody against apoE and a monoclonal antibody against phosphorylated tau (p-tau) (Figure 1). ApoE and p-tau colocalized within intracellular inclusions (Figure 1AøC) in a subset (2.7 ± 1.2%) of apoE4-expressing cells (Figure 1D). These intracellular inclusions were also recognized by a monoclonal antibody against a phosphorylated neurofilament of high molecular weight (p-NF-H). These results suggest the presence of an intracellular complex composed of apoE, p-tau, and p-NF-H in a small number of apoE4-transfected cells. However, immunostaining of Neuro-2a cells expressing apoE3 failed to detect the formation of intracellular inclusions.

Since apoE4 appears to act in concert with other factors to accelerate AD progression, we determined if treatment with b-amyloid peptide (Ab) increased the formation of intracellular inclusions in apoE-expressing Neuro-2a cells. Ab1-42 treatment clearly increased the number of apoE4-expressing cells containing apoE and p-tau immunoreactive intracellular inclusions (2.7 ± 1.2% to 6.7 ± 1.5%, p < 0.001) and induced inclusion formation in a subset of apoE3-expressing cells (0% to 2.8 ± 0.6%, p < 0.001). Treatment of apoE-expressing cells with brefeldin A, an endoplasmic reticulum stressor, produced similar results. Thus, Ab1-42 and brefeldin A potentiate the formation of intracellular inclusions.

Lysates of apoE4-expressing cells treated with Ab1-42 or brefeldin A were immunoprecipitated with anti-p-tau to identify the components of the intracellular inclusions. Western blotting of the immunoprecipitated complexes with anti-p-tau revealed two to three proteins with molecular masses of 50ø60 kDa (Figure 1D, left). In addition, anti-p-NF-H detected a protein of molecular mass ~200 kDa, characteristic of p-NF-H (Figure 1D, center). However, anti-apoE western blotting did not reveal full-length apoE but rather a protein with a lower molecular mass (~29ø30 kDa instead of 34 kDa) (Figure 1D, right). These results confirm that the intracellular inclusions within the Neuro-2a cells are composed of p-tau and p-NF-H and suggest that the apoE4 in the complex may be a truncated form with a molecular mass 10ø15% lower than that of full-length apoE.

Carboxyl-Terminal-Truncated ApoE Induces Intracellular Inclusions in the Cytosol of Neuro-2a Cells
To determine whether truncation of apoE induced the formation of intracellular inclusions, we transfected Neuro-2a cells with constructs possessing either amino- or carboxyl-terminal truncations of apoE. Deletion of up to 28 amino acids (~10% of the molecular mass) from the amino terminus of apoE4 did not alter the number of cells displaying the intracellular inclusions (~3% of the cells were positive, which is similar to the number of positive cells observed after transfection with full-length apoE4). However, deletion of 28 amino acids from the carboxyl terminus of apoE4 [apoE4(D272ø299)] increased the number of transfected cells displaying the intracellular filamentous inclusions to 78 ± 8% (Figure 2B and C). Expression of apoE3 with the same carboxyl-terminal truncation [apoE3(D272ø299)] caused a significantly smaller increase in the number of cells with intracellular inclusions (32 ± 5%, p < 0.001) (Figure 2A and C).

The intracellular inclusions in Neuro-2a cells transfected with apoE4(D272ø299) were immunoreactive with anti-apoE (Figure 2B), anti-p-tau, and anti-p-NF-H. There was complete merging of the immunoreactivity of the inclusions for anti-apoE and anti-p-tau, indicating colocalization of apoE4(D272ø299) and p-tau. The intracellular inclusions induced by apoE3(D272ø299) were similar (Figure 2A) but were smaller and occurred in many fewer transfected cells (Figure 2C). These results suggest that carboxyl-terminal-truncated apoE, especially apoE4(D272ø299), interacts with cytosolic p-tau and p-NF-H to induce the formation of intracellular inclusions in Neuro-2a cells. The cytosolic localization of the inclusions was confirmed by anti-apoE immunofluorescence staining after treatment with streptolysin O, which permeabilizes the plasma membrane without disrupting subcellular organelle membranes. In permeabilized apoE4(D272ø299)-expressing cells, anti-apoE immunofluorescence staining revealed extensive filamentous inclusions, suggesting cytosolic distribution of the inclusions.

Intracellular Inclusions Induced by ApoE4(D272ø299) Have Ultrastructural and Biochemical Characteristics of Neurofibrillary Tangles Seen in Human AD Brains
The ultrastructure and composition of the intracellular inclusions of Neuro-2a cells expressing a green fluorescent protein (GFP)øapoE4(D272ø299) fusion protein in the cytosol were determined. Ultrastructural analysis by electron microscopy revealed filamentous aggregates in cells expressing the fusion protein (Figure 3B, arrows), but not in those expressing GFP alone (Figure 3A). The filaments were randomly oriented and were not membrane bound. High-power magnification revealed that the inclusions are composed of many straight filaments, with a diameter of 10ø20 nm (Figure 3C), which are similar to the straight filament tangles seen in human AD brains. The cells expressing GFP-apoE4(D272ø299) also showed many electron-dense, membrane-bound organelles, suggestive of degenerating organelles (Figure 3B, arrowheads).

To characterize the intracellular inclusions in more detail, two sets of monoclonal antibodies were used: anti-p-tau (AT8, AT100, AT180, and AT270) and anti-p-NF-H (RT97 and SM31). It has been reported that the tangles in AD brains contain both p-tau and p-NF-H. Immunofluorescent staining of Neuro-2a cells expressing GFP-apoE4(D272ø299) demonstrated that the inclusions were positive for some (AT8, AT180, and AT270), but not all (AT100), of the monoclonal antibodies that recognize p-tau in paired helical filaments in human AD brains. These findings suggest that some, but not all, serines or threonines of tau protein were phosphorylated in the intracellular inclusions in Neuro-2a cells. Immunostaining with anti-p-NF-H antibody demonstrated that the intracellular inclusions also contained p-NF-H (RT97 and SM31).

Carboxyl-Terminal-Truncated ApoE Can Be Generated Inside Neurons
Lysates from Neuro-2a cells transfected with constructs containing the gene for full-length apoE3 or apoE4 or from cells incubated with exogenous full-length apoE3 or apoE4 were immunoprecipitated with polyclonal antibodies against apoE. Anti-apoE western blotting revealed a protein corresponding to full-length apoE and a protein of lower molecular mass corresponding to apoE(D272ø299) (~30 kDa). More fragments were generated with apoE4 than with apoE3, and Ab1-42 treatment of the cells increased the generation of truncated apoE4 to a greater extent than that of truncated apoE3. Importantly, the lower-molecular-mass protein represents a carboxyl-terminal-truncated form of apoE, as detected by monoclonal antibody 6C5, which recognizes the amino-terminal 15 amino acids. Therefore, the shorter fragment is a carboxyl-terminal-truncated form of apoE composed of approximately 270 amino acids.

Carboxyl-Terminal-Truncated Forms of ApoE Are Found in AD Brains
To determine whether carboxyl-terminal-truncated forms of apoE can be generated in vivo in human brains, we analyzed brain lysates from normal or AD patients by western blotting with polyclonal antibodies against full-length apoE or the carboxyl-terminal portion of apoE (amino acids 272ø299). Polyclonal anti-apoE revealed full-length apoE in both the supernatant (Figure 4A) and the pellet (Figure 4C) of brain lysates from normal and AD patients. An apoE fragment of ~29 kDa was also recognized by polyclonal anti-apoE in the supernatant from both normal and AD brains, but to a greater extent in AD brains (Figure 4A). The 29-kDa apoE fragment was also found in the pellet of AD brains, but not in the pellet of normal brains (Figure 4C). In addition, smaller apoE fragments of ~14ø20 kDa were found in both the supernatant and the pellet of AD brains but not in normal brains (Figure 4A and C). However, western blotting with an antibody against the carboxyl-terminal portion of apoE (amino acids 272ø299) only revealed full-length apoE, but not apoE fragments, in the supernatant (Figure 4B) and the pellets (Figure 4D), suggesting that the fragments are carboxyl-terminal-truncated forms of apoE. Large complexes (>220 kDa) were detected in the pellets of AD brains by anti-full-length apoE (Figure 4C) and anti-p-tau (Figure 4E) but not by anti-carboxyl-terminal apoE (Figure 4D), suggesting that these large complexes contain carboxyl-terminal-truncated forms of apoE and p-tau. Taken together, these results suggest that carboxyl-terminal-truncated forms of apoE are generated to a much greater extent in AD brains than in normal brains, and most of these fragments are present in insoluble forms, probably forming complexes with p-tau.

Our findings indicate that a carboxyl-terminal-truncated, bioactive form of apoE interacts with cytosolic p-tau and p-NF-H to induce neurofibrillary tangleølike structures in neuronal cells. Importantly, we showed evidence that the carboxyl-terminal-truncated forms of apoE exist in the brains of AD patients and in neurofibrillary tangles. ApoE4 was more susceptible to truncation than apoE3 and had a greater capacity to induce cytoskeletal alterations. In addition, Ab1-42 treatment of neurons enhanced the generation of truncated apoE4 and increased the number of cells with the neurofibrillary tangleølike inclusions (compared with apoE3 plus Ab1-42 treatment). It will be of interest to identify the putative protease that cleaves apoE at its carboxyl terminus, as it may serve as a therapeutic target for prevention and treatment of neurodegenerative diseases associated with apoE4.

References
1. Huang Y, Mahley RW (1999) Apolipoprotein E and human disease. In: Plasma Lipids and Their Role in Disease (Barter PJ, Rye K-A, eds) Harwood Academic Publishers, Amsterdam, pp 257ø284.

2. Mahley RW, Huang Y (1999) Apolipoprotein E: From atherosclerosis to AlzheimerÕs disease and beyond. Curr. Opin. Lipidol. 10:207ø217.

3. Huang Y, Liu XQ, Wyss-Coray T, Brecht WJ, Sanan DA, Mahley RW (2000) Bioactive fragments of apolipoprotein E induce neurofibrillary tangles in cultured neurons. Soc. Neurosci. 26 (Part 1):540 (abstract).

4. Huang Y, Liu XQ, Wyss-Coray T, Brecht WJ, Sanan DA, Mahley RW (2001) Apolipoprotein E fragments present in AlzheimerÕs disease brains induce neurofibrillary tangle-like intracellular inclusions in neurons. Proc. Natl. Acad. Sci. USA 98:8838ø8843.

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