Walter J. Brecht

Apolipoprotein E fragments present in Alzheimer's disease brains induce neurofibrillary tangle-like intracellular inclusions in neurons.

Human apolipoprotein (apo) E4, a major risk factor for Alzheimers disease (AD), occurs in amyloid plaques and neurofibrillary tangles (NFTs) in AD brains; however, its role in the pathogenesis of these lesions is unclear. Here we demonstrate that carboxyl-terminal-truncated forms of apoE, which occur in AD brains and cultured neurons, induce intracellular NFT-like inclusions in neurons. These cytosolic inclusions were composed of phosphorylated tau, phosphorylated neurofilaments of high molecular weight, and truncated apoE. Truncated apoE4, especially apoE4(Δ272–299), induced inclusions in up to 75% of transfected neuronal cells, but not in transfected nonneuronal cells. ApoE4 was more susceptible to truncation than apoE3 and resulted in much greater intracellular inclusion formation. These results suggest that apoE4 preferentially undergoes intracellular processing, creating a bioactive fragment that interacts with cytoskeletal components and induces NFT-like inclusions containing phosphorylated tau and phosphorylated neurofilaments of high molecular weight in neurons.

Neuron-Specific Apolipoprotein E4 Proteolysis Is Associated with Increased Tau Phosphorylation in Brains of Transgenic Mice

Apolipoprotein E (apoE) is found in amyloid plaques and neurofibrillary tangles (NFTs) in Alzheimers disease (AD) brains, but its role in their pathogenesis is unclear. Previously, we found C-terminal-truncated fragments of apoE in AD brains and showed that such fragments can cause neurodegeneration and can induce NFT-like inclusions in cultured neuronal cells and in transgenic mice. Here, we analyzed apoE fragmentation in brain tissue homogenates from transgenic mice expressing apoE3 or apoE4 in neurons [neuron-specific enolase (NSE)-apoE] or astrocytes [glial fibrillary acidic protein (GFAP)-apoE] by Western blotting. The C-terminal-truncated fragments of apoE accumulated, in an age-dependent manner, in the brains of NSE-apoE4 and, to a significantly lesser extent, NSE-apoE3 mice; however, no fragments were detected in GFAP-apoE3 or GFAP-apoE4 mice. In NSE-apoE mice, the pattern of apoE fragmentation resembled that seen in AD brains, and the fragmentation was specific for certain brain regions, occurring in the neocortex and hippocampus, which are vulnerable to AD-related neurodegeneration, but not in the less vulnerable cerebellum. Excitotoxic challenge with kainic acid significantly increased apoE fragmentation in NSE-apoE4 but not NSE-apoE3 mice. Phosphorylated tau (p-tau) also accumulated in an age-dependent manner in NSE-apoE4 mice and, to a much lesser extent, in NSE-apoE3 mice but not in GFAP-apoE3 or GFAP-apoE4 mice. Intraneuronal p-tau inclusions in the hippocampus were prominent in 21-month-old NSE-apoE4 mice but barely detectable in NSE-apoE3 mice. Thus, the accumulation of potentially pathogenic C-terminal-truncated fragments of apoE depends on both the isoform and the cellular source of apoE. Neuron-specific proteolytic cleavage of apoE4 is associated with increased phosphorylation of tau and may play a key role in the development of AD-related neuronal deficits.

Carboxyl-terminal-truncated apolipoprotein E4 causes Alzheimer's disease-like neurodegeneration and behavioral deficits in transgenic mice

Apolipoprotein (apo) E4 increases the risk and accelerates the onset of Alzheimers disease (AD). However, the underlying mechanisms remain to be determined. We previously found that apoE undergoes proteolytic cleavage in AD brains and in cultured neuronal cells, resulting in the accumulation of carboxyl-terminal-truncated fragments of apoE that are neurotoxic. Here we show that this fragmentation is caused by proteolysis of apoE by a chymotrypsin-like serine protease that cleaves apoE4 more efficiently than apoE3. Transgenic mice expressing the carboxyl-terminal-cleaved product, apoE4(Δ272–299), at high levels in the brain died at 2–4 months of age. The cortex and hippocampus of these mice displayed AD-like neurodegenerative alterations, including abnormally phosphorylated tau (p-tau) and Gallyas silver-positive neurons that contained cytosolic straight filaments with diameters of 15–20 nm, resembling preneurofibrillary tangles. Transgenic mice expressing lower levels of the truncated apoE4 survived longer but showed impaired learning and memory at 6–7 months of age. Thus, carboxyl-terminal-truncated fragments of apoE4, which occur in AD brains, are sufficient to elicit AD-like neurodegeneration and behavioral deficits in vivo. Inhibiting their formation might inhibit apoE4-associated neuronal deficits.

Overexpression of Hepatic Lipase in Transgenic Mice Decreases Apolipoprotein B-containing and High Density Lipoproteins EVIDENCE THAT HEPATIC LIPASE ACTS AS A LIGAND FOR LIPOPROTEIN UPTAKE

To determine the mechanisms by which human hepatic lipase (HL) contributes to the metabolism of apolipoprotein (apo) B-containing lipoproteins and high density lipoproteins (HDL)in vivo, we developed and characterized HL transgenic mice. HL was localized by immunohistochemistry to the liver and to the adrenal cortex. In hemizygous (hHLTg +/0) and homozygous (hHLTg +/+) mice, postheparin plasma HL activity increased by 25- and 50-fold and plasma cholesterol levels decreased by 80% and 85%, respectively. In mice fed a high fat, high cholesterol diet to increase endogenous apoB-containing lipoproteins, plasma cholesterol decreased 33% (hHLTg +/0) and 75% (hHLTg +/+). Both apoB-containing remnant lipoproteins and HDL were reduced. To extend this observation, the HL transgene was expressed in human apoB transgenic (huBTg) and apoE-deficient (apoE −/−) mice, both of which have high plasma levels of apoB-containing lipoproteins. (Note that thehuBTg mice that were used in these studies were all hemizygous for the human apoB gene.) In both thehuBTg,hHLTg +/0 mice and theapoE −/−,hHLTg +/0mice, plasma cholesterol decreased by 50%. This decrease was reflected in both the apoB-containing and the HDL fractions. To determine if HL catalytic activity is required for these decreases, we expressed catalytically inactive HL (HL-CAT) in apoE −/−mice. The postheparin plasma HL activities were similar in theapoE −/− and theapoE −/−,HL-CAT +/0mice, reflecting the activity of the endogenous mouse HL and confirming that the HL-CAT was catalytically inactive. However, the postheparin plasma HL activity was 20-fold higher in theapoE −/−,hHLTg +/0mice, indicating expression of the active human HL. Immunoblotting demonstrated high levels of human HL in postheparin plasma of bothapoE −/−,hHLTg +/0and apoE −/−,HL-CAT +/0mice. Plasma cholesterol and apoB-containing lipoprotein levels were ∼60% lower inapoE −/−,HL-CAT +/0mice than in apoE −/− mice. However, the HDL were only minimally reduced. Thus, the catalytic activity of HL is critical for its effects on HDL but not for its effects on apoB-containing lipoproteins. These results provide evidence that HL can act as a ligand to remove apoB-containing lipoproteins from plasma.

Overexpression of Apolipoprotein E3 in Transgenic Rabbits Causes Combined Hyperlipidemia by Stimulating Hepatic VLDL Production and Impairing VLDL Lipolysis

The differential effects of overexpression of human apolipoprotein (apo) E3 on plasma cholesterol and triglyceride metabolism were investigated in transgenic rabbits expressing low (<10 mg/dL), medium (10 to 20 mg/dL), or high (>20 mg/dL) levels of apoE3. Cholesterol levels increased progressively with increasing levels of apoE3, whereas triglyceride levels were not significantly affected at apoE3 levels up to 20 mg/dL but were markedly increased at levels of apoE3 >20 mg/dL. The medium expressers had marked hypercholesterolemia (up to 3- to 4-fold over nontransgenics), characterized by an increase in low density lipoprotein (LDL) cholesterol, while the low expressers had only slightly increased plasma cholesterol levels. The medium expressers displayed an 18-fold increase in LDL but also had a 2-fold increase in hepatic very low density lipoprotein (VLDL) triglyceride production, an 8-fold increase in VLDL apoB, and a moderate decrease in the ability of the VLDL to be lipolyzed. However, plasma clearance of VLDL was increased, likely because of the increased apoE3 content. The increase in LDL appears to be due to an enhanced competition of VLDL for LDL receptor binding and uptake, resulting in the accumulation of LDL. The combined hyperlipidemia of the apoE3 high expressers (>20 mg/dL) was characterized by a 19-fold increase in LDL cholesterol but also a 4-fold increase in hepatic VLDL triglyceride production associated with a marked elevation of plasma VLDL triglycerides, cholesterol, and apoB100 (4-, 9-, and 25-fold over nontransgenics, respectively). The VLDL from the high expressers was much more enriched in apoE3 and markedly depleted in apoC-II, which contributed to a >60% inhibition of VLDL lipolysis. The combined effects of stimulated VLDL production and impaired VLDL lipolysis accounted for the increases in plasma triglyceride and VLDL concentrations in the apoE3 high expressers. The hyperlipidemic apoE3 rabbits have phenotypes similar to those of familial combined hyperlipidemia, in which VLDL overproduction is a major biochemical feature. Overall, elevated expression of apoE3 appears to determine plasma lipid levels by stimulating hepatic VLDL production, enhancing VLDL clearance, and inhibiting VLDL lipolysis. Thus, the differential expression of apoE may, within a rather narrow range of concentrations, play a critical role in modulating plasma cholesterol and triglyceride levels and may represent an important determinant of specific types of hyperlipoproteinemia.

Bioactive TGF‐β can associate with lipoproteins and is enriched in those containing apolipoprotein E3

Transforming growth factor‐β1 (TGF‐β1) has central functions in development, tissue maintenance, and repair and has been implicated in major diseases. We discovered that TGF‐β1 contains several amphipathic helices and hydrophobic domains similar to apolipoprotein E (apoE), a protein involved in lipoprotein metabolism. Indeed, TGF‐β1 associates with lipoproteins isolated from human plasma, cultured liver cells, or astrocytes, and its bioactivity was highest in high‐density lipoprotein preparations. Importantly, lipoproteins containing the apoE3 isoform had higher TGF‐β levels and bioactivity than those containing apoE4, a major genetic risk factor for atherosclerosis and Alzheimer’s disease. Because TGF‐β1 can be protective in these diseases an association with apoE3 may be beneficial. Association of TGF‐β with different types of lipoproteins may facilitate its diffusion, regulate signaling, and offer additional specificity for this important growth factor.