Apolipoprotein E (apoE) is a major protein of the lipoprotein transport system that plays critical roles in atherosclerosis, dyslipidemia and Alzheimer’s disease (AD) (Zannis et al. 2004; Mahley et al. 2006). In the brain, apoE is synthesised primarily by astrocytes and to a lesser extent by microglia and neurons (Pitas et al. 1987; Metzger et al. 1996; Mori et al. 2004). ApoE contains 299 residues and has three common isoforms (apoE2, apoE3, apoE4) each differing in the amino acid positions 112 and 158 (Zannis et al. 2004). ApoE4 has been associated with a variety of neuropathological processes, including AD (Mahley et al. 2006). ApoE4 is a major genetic risk factor for AD since 40% of all patients have at least one ε4 allele (Corder et al. 1993). Being homozygous or heterozygous for the ε4 allele increases the risk of AD four-fold and lowers the age of onset of late-onset AD (Corder et al. 1993; Myers et al. 1996).
Several studies have suggested that the production, oligomerization and deposition of amyloid beta peptide (Aβ) play central roles in AD (Haass and Selkoe 2007). Aβ is a ∼4 kDa peptide fragment generated by sequential proteolytic cleavage of the transmembrane amyloid precursor protein (APP). The Aβ peptides can vary in length; the most common forms contain 38, 40 or 42 amino acids (Aβ38, Aβ40 or Aβ42) (Haass and Selkoe 2007). 42-amino-acid Αβ variant (Aβ42) has been found to be more prone to fibril formation and more closely associated with the pathogenesis of AD than shorter Aβ forms (Haass and Selkoe 2007). According to the amyloid cascade hypothesis, Aβ forms soluble oligomers that affect synaptic structure and plasticity. In addition, Aβ forms long insoluble amyloid fibrils that accumulate in neuritic plaques in the brains of AD patients and lead to widespread neuronal dysfunction and ultimate cell death (Haass and Selkoe 2007). Growing evidence from studies with humans and experimental animals suggests that Aβ accumulates inside neurons. Aβ accumulation occurs prior to extracellular amyloid formation and has been implicated in the onset of early cognitive alterations and may contribute to the pathological cascade of events that lead to neuronal dysfunction and eventually to AD (LaFerla et al. 2007; Bayer and Wirths 2008). Furthermore, Aβ42 constitutes the majority of intraneuronal Aβ (LaFerla et al. 2007).
A large number of studies have examined the association of apoE4 with sporadic AD. These studies suggested that apoE4 is involved in the modulation of plaque formation and clearance of Aβ, affects cholesterol homeostasis, alters phosphorylation of tau that leads to formation of neurofibrillary tangles, disrupts cytoskeleton structure and cause dysregulation of various signaling pathways (reviewed in Mahley et al. 2006). The molecular mechanisms of these pathological processes are still unclear. It is possible that several parallel pathways contribute to the pathogenic role of apoE4 in AD.
Apolipoprotein E is sensitive to proteolytic cleavage. Bioactive carboxy-terminal truncated fragments of apoE4, which is much more susceptible to proteolysis than apoE3, have been found in brains of AD patients (25–30 and 14–22kDa) and transgenic mice that express apoE4 in neurons (29–30 and 14–20kDa) (Huang et al. 2001; Harris et al. 2003; Brecht et al. 2004). Primary proteolytic cleavage sites of apoE have been proposed to be located at residues 268/272 (Harris et al. 2003), close to residue 187 (Wellnitz et al. 2005) or after residue 160 (Cho et al. 2001). The carboxy-terminal truncated apoE4 fragment apoE4[Δ(272-299)] has been associated with increased phosphorylation of tau, mitochondrial dysfunction and neurotoxicity in cultured neuronal cells and transgenic mice and could play a key role in the development of neuronal degeneration observed in AD (Huang et al. 2001; Brecht et al. 2004; Chang et al. 2005). In contrast, apoE4[Δ(241-299)] has been found to not be associated with increased mitochondrial dysfunction and neurotoxicity (Harris et al. 2003; Chang et al. 2005). This latter finding indicates that not all truncated apoE4 fragments have the same biological effects and that specific apoE4 fragments may be involved in separate processes associated with AD pathogenesis. Interestingly, the presence of apoE4 fragments in transgenic mice that express apoE4 in neurons is observed at 1 month of age, while the accumulation of phosphorylated tau starts at 5 months of age (Brecht et al. 2004).
Since apoE4 fragmentation has been suggested to be an early event in the pathogenesis of AD, we asked whether truncated apoE4 forms have any effect on Aβ intracellular accumulation, an event that has been linked to early pathological processes that lead to AD. We examined the effect of two truncated apoE4 forms, apoE4[Δ(186-299)] (designated thereafter as apoE4-185) and apoE4[Δ(166-299)] (designated thereafter as apoE4-165) with molecular weights of 21 and 19 kDa, respectively, on APP processing and Aβ levels in human neuroblastoma SK-N-SH and human embryonic kidney 293 cells (HEK293) transfected with human APP and on the uptake of exogenously added Aβ. We found that lipid-free apoE4-165, but not wild-type (WT) apoE4 or apoE4-185, significantly reduced the extracellular levels of 40-amino-acid Αβ variant (Aβ40) and Aβ42, without affecting β-secretase activity. Furthermore, apoE4-165 stimulated the uptake of exogenously added Aβ40 or Aβ42 by SK-N-SH cells. Following uptake, approximately half of the internalized Αβ42 remained in cells after 24 h and led to formation of reactive oxygen species (ROS), while Αβ40 is rapidly eliminated and did not lead to ROS formation. Our results indicate that a specific apoE4 fragment can promote the intracellular Αβ42 accumulation, an event that has been linked to the pathogenesis of AD.
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Although apoE4 is considered a major risk factor for AD its exact role in the pathogenesis of AD has not been elucidated. ApoE4 has been shown to be more susceptible to proteolysis than apoE2 and apoE3 and carboxy-terminal truncated fragments of apoE4 accumulate in brains of AD patients and neurons of apoE4-transgenic mice (Huang et al. 2001; Harris et al. 2003; Brecht et al. 2004). Furthermore, studies in transgenic mice expressing apoE4 in neurons suggested that apoE proteolysis occurs in the secretory and not in the internalization pathway in neurons and that this apoE4 fragmentation is an early event in the pathogenesis of AD (Brecht et al. 2004).
In the current study we determined the effect of two truncated apoE4 forms, apoE4-185 and apoE4-165 on Aβ40 and Aβ42 metabolism and intracellular levels of SK-N-SH neuroblastoma cells. These apoE4 truncated forms have a molecular weight of 21 and 19 kDa, respectively, both within the range of molecular weights of carboxy-terminal apoE4 fragments found in brains of AD patients and apoE4 transgenic mice (25–30 and 14–22 kDa) (Huang et al. 2001; Harris et al. 2003; Brecht et al. 2004). We show that one of the truncated apoE4 forms studied, apoE4-165, leads to intracellular Aβ42 accumulation, an event that has been previously suggested to be an early pathologic feature of AD (LaFerla et al. 2007; Bayer and Wirths 2008). Approximately 50% of the internalized Aβ42 persisted in the cells for at least 24 h, while the internalized Αβ40 was eliminated at a rapid rate. This is consistent with previous studies in nerve growth factor-differentiated PC12 cells and mouse cultured neurons which had also demonstrated that part of internalized Aβ42 accumulates intracellularly, while Aβ40 is eliminated (Burdick et al. 1997; Ditaranto et al. 2001). The majority of Aβ internalized by SK-N-SH cells and of Aβ42 accumulated in SK-N-SH cells can be found throughout the whole cell, outside of lysosomes, late endosomes or mitochondria.
It has been proposed that apoE can bind Aβ and undergo endocytosis via LRP (Gylys et al. 2003; Zerbinatti et al. 2006). Furthermore, it has been suggested that LRP also binds and endocytoses Aβ directly, facilitating Aβ cellular uptake (Zerbinatti et al. 2006). Therefore, we examined whether the Aβ uptake is mediated by direct interactions with apoE4-165 as well as LRP. We found that apoE4-165, as well as apoE4-185, failed to bind to Aβ, in accordance with previous studies showing that the carboxy-terminal region of apoE is necessary for Aβ binding (Aleshkov et al. 1999; Pillot et al. 1999). In addition, WT or truncated apoE4 forms did not have any effect on cellular LRP levels. These data suggest that uptake of Aβ from SK-N-SH cells treated with apoE4-165 is unlikely to proceed via direct binding of Aβ to apoE4-165 or LRP up-regulation.
Previous studies had suggested that changes in brain cellular lipid levels are associated with the internalization of Aβ and the pathogenesis of AD (Cutler et al. 2004; Saavedra et al. 2007; Bandaru et al. 2009). We demonstrated that apoE4-165 leads to 20% reduction of cellular sphingomyelin levels, while WT apoE4 and apoE4-185 have no such effect. Furthermore, we detected changes in the micro-fluidic properties of the cellular membrane of SK-N-SH neuroblastoma cells only when they were treated with apoE4-165. These two observations, taken together, suggest that exogenous apoE4-165 can affect the composition, biophysical state and possibly the functionally of the cellular membrane. In an earlier study it was shown that reduction of cellular sphingolipids by the ceramide synthesis inhibitor Fumonisin B1 led to increased Aβ42 internalization by rat primary neurons (Saavedra et al. 2007). In that study the authors suggested that when Aβ is not complexed with apoE its internalization is not mediated by members of the low-density lipoprotein receptor family, but rather by an endocytic process affected by cellular cholesterol and sphingolipids levels (Saavedra et al. 2007). Decreased levels of sphingomyelin have also been measured in a brain region with extensive Aβ plaques and neurofibrillary tangles (middle frontal gyrus) of AD patients compared to controls (Cutler et al. 2004), as well as in the middle frontal gyrus grey matter of apoE4 AD patients compared to apoE3 AD patients (Bandaru et al. 2009). In another study it was proposed that Aβ40 and Aβ42, located mostly in cytoplasm of rat primary hippocampal neurons and differentiated PC12 cells, could be internalized via passive diffusion (Kandimalla et al. 2009). The passive diffusion mechanism of Aβ internalization was supported by studies showing that Aβ40 can intercalate into the phospholipid bilayer of neuronal plasma membrane (Mason et al. 1999) and that interactions of Aβ with lipid bilayers are affected by the bilayer lipid composition (Waschuk et al. 2001). In the present study we showed by fluorescence confocal laser scanning microscopy that Aβ42 and Aβ40 internalized by SK-N-SH cells after incubation with apoE4-165 and Aβ for 24 h were partially localized to lysosomes, indicating an endocytic uptake. However, a large fraction of Aβ42 and a larger fraction of Aβ40 were found in the cytoplasm of SK-N-SH cells, separate from LysoTracker-labeled intact lysosomes or other acidic organelles. This latter observation suggests a non-endocytotic uptake. Taken together our results suggest that the uptake of Aβ by SK-N-SH cells in the presence of apoE4-165 proceeds either by a LRP-independent endocytotic mechanism or by passive diffusion. However, internalization of Aβ by SK-N-SH cells through another receptor not identified in this study or a combination of the above mechanisms cannot be ruled out.
Oxidative stress has been proposed to be one of the earliest events in AD that plays important roles in the onset and progression of the disease. This stress has been suggested to be chronic in neurons (Zhu et al. 2003). Furthermore, Aβ has been shown to contribute to the oxidative stress in neurons (Smith et al. 2007). It has been proposed that ROS, that are considered markers of oxidative stress, are generated in the cytoplasm of neurons by the interaction of mitochondria, redox transition metals and other factors and contribute to the pathogenesis of AD (Zhu et al. 2003). We show that incubation of SK-N-SH cells with apoE4-165 and Aβ42 for 24 h, but not Aβ40, leads to a 50% increase in intracellular ROS levels. These levels remained unchanged for at least 24 h after the removal of Aβ42 and apoE4-165 from the cell medium. It is thus possible that the apoE4-165-induced uptake and accumulation of Aβ42 from SK-N-SH neuroblastoma cells and chronic oxidative stress are linked.
Interestingly, the striking differences between the ability of apoE4-165 to promote Aβ uptake, compared to the apoE4-185 fragment can be correlated to structural differences between the two apoE4 fragments. ApoE in the lipid-free state is folded into two independent structural domains (Wetterau et al. 1988). Digestion with thrombin produces a 22kD amino-terminal fragment (residues 1 to 191) and a 10kD carboxy-terminal fragment (residues 216–299) (Wetterau et al. 1988). X-ray crystallographic analysis of the apoE amino-terminal domain (residues 1–191) has revealed a 4 helix bundle spanning residues 24–164 that segregates the hydrophobic core of the four helices from the solvent (Wilson et al. 1991). Unfolding of this amino-terminal domain is thought to constitute a necessary conformational change for lipid binding and apoE function (Wilson et al. 1991). In a previous study, we characterized the two apoE4 fragments by biophysical techniques and discovered that apoE4-165 is destabilized compared to apoE4-185 (Chroni et al. 2008). Furthermore, an additional solvent-exposed hydrophobic site was detected on apoE4-165 (Chroni et al. 2008). This site may be responsible for apoE4-165 destabilization and can mediate initial lipid interactions that subsequently lead to further conformational changes. It is conceivable that the lack of the 166–185 region may facilitate amino-terminal core domain unfolding leading to a truncated apoE4 molecule that can interact more readily with hydrophobic sites of membrane-bound Aβ and lipids on the cell surface. Such interactions between apoE4-165 and the cellular membrane may effect changes in the membrane’s structural and fluidic properties that mediate passive Aβ diffusion. Overall, the results described here are consistent with our previous study that suggested that apoE4 carboxy-terminal truncations can have complex effects on the stability and dynamics of the remaining fragment (Chroni et al. 2008). It is therefore possible that not all apoE4 fragments found in AD patient’s brains are equally bioactive and specific fragments alone can promote the pathogenesis of the disease.
Overall our findings provide an association between two molecular events, the proteolysis of apoE4 and the intraneuronal presence of Aβ, both of which are considered to be early events in the pathogenesis of AD. Intracellular accumulation of Aβ42 in SK-N-SH neuroblastoma cells incubated in the presence of apoE4 truncated form apoE4-165 is associated with ROS formation and therefore increased oxidative stress, which is also an early event in AD. We therefore propose that specific short apoE4 proteolytic fragments produced in the brain under pathologic conditions may promote intraneuronal accumulation of Aβ42 leading to neuronal dysfunction.
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Figure S1. Effect of WT apoE4 and truncated apoE4 forms, apoE4-185 and apoE4-165, on cellular APP, secreted sAPPa and secreted Ab levels in SK-N-SH or HEK293 cells expressing human APP.
Figure S2. Uptake of exogenous added Ab40 by HEK293 cells in the presence of WT apoE4 and truncated apoE4 forms apoE4-185 and apoE4-165.
Figure S3. Uptake of exogenous added Ab40 by SK-N-SH cells in the presence of WT apoE4 and truncated apoE4 form apoE4-165.
Figure S4. LRP1 and phospholipid levels in SK-N-SH cells incubated with WT and truncated apoE4 forms.
Figure S5. Determination of mitochondrial localization of Ab40 and Ab42 in SK-N-SH cells incubated with apoE4-165.
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