Keywords

5.1 Introduction

Aging is a complex of biological long-lasting processes that result being unavoidable. Aging and diseases are closely related as aging is the largest risk factor for multiple chronic diseases. Increasing evidence suggests a certain degree of heritability of lifespan. Recently, genome-wide association studies (GWAS) candidate gene studies (CGAS) have identified variation in two genes (Fortney et al. 2015; Johnson et al. 2015), fork head box O3 (FOXO3) and apolipoprotein E (ApoE), to be consistently associated with human longevity, while some other genes have inconsistence (Blanche et al. 2001; Deelen et al. 2011; Schachter et al. 1994; Zhang et al. 1998). Furthermore, ApoE, which is involved in lipoprotein metabolism, is the only age-related gene confirmed in human (Bao et al. 2014; Fortney et al. 2015).

ApoE is a 34 kDa lipid-binding protein which was first discovered by Shore in 1973 in very-low-density lipoprotein (VLDL) (Shore and Shore 1974). It is mainly distributed in VLDL, chylomicron (CM), and their wreckage. ApoE plays an important role in lipoprotein metabolism. It not only can bind to LDL receptor but also bind to the hepatic cell membrane chylomicrons (CM), VLDL debris, and some HDL (which contains ApoE) receptors. The function of ApoE is transportation of triglycerides and cholesterol in multiple tissues (Bu 2009; Leduc et al. 2010; Puglielli et al. 2003; Wang et al. 2006).

Based on the pivotal role of ApoE protein in lipoprotein metabolism in the brain and in the periphery, its expression regulation and expression types have an important connection with Alzheimer’s disease (AD), Parkinson’s disease (PD), multiple sclerosis (MS), vascular dementia (VD), cardiovascular diseases (CVD), type 2 diabetes mellitus (T2DM), and other diseases (Fig. 5.1).

Fig. 5.1
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Diseases associated with ApoE

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5.1.1 The Structure and Physical Functions of ApoE

The molecular weight of ApoE is 34 kDa, consisting of 299 amino acid residues, rich in arginine with a single glycosylation site at threonine-194 (Lee et al. 2010; Rall et al. 1982; Weisgraber 1994). The secondary structure of ApoE was constituted of α-helix, β-turn β-sheet, and a “hinge region” which divides ApoE into two independent domains: the N-terminal domain (amino acids 1–191), two thirds of ApoE, contains the lipoprotein receptor-binding region (amino acids 136–150), and the C-terminal domain (amino acids 225–299) contains the lipid-binding region (amino acids 244–272) (Rasmussen 2016; Weisgraber 1994). X-ray crystallography solved the tertiary structure of the N-terminal domains of ApoE which consists of four helices arranged in antiparallel fashion (Weisgraber 1994), the lipoprotein receptor-binding region (amino acids 136–150) is in the fourth helix.

In contrast to other mammals, humans present three isoforms of ApoE, named ApoE2, ApoE3, and ApoE4, which are expressed by the polymorphic alleles: ɛ2, ɛ3, and ɛ4 (Tudorache et al. 2017). The substitution of Arg and Cys, two amino acid residues at positions 112 and 158 of the ApoE amino acid sequence, determines the type of isoforms: ApoE4 is Arg at both positions; ApoE2 is Cys; Cys at position 112 and Arg at position 158 are ApoE3 subtypes. The isoforms of ApoE display preferences for specific classes of lipoproteins, and ApoE4 prefers large, triglyceride-rich VLDL particles, whereas ApoE3 and ApoE2 associate preferentially with the small, phospholipid-rich HDL (Huang and Mahley 2014). The strange thing is the residues that differ the ApoE isoforms are in the N-terminal (E4, arginine 112; E3 and ApoE2, cysteine 112). However, the lipid-binding region is in the C-terminal (amino acids 244–272). This suggests that there may be a domain interaction between the N- and C-terminal domains in ApoE4; arginine 112 may orient the side chain of arginine 61 into the aqueous environment and then interact with glutamic acid 255, which determines the preference of ApoE4 for VLDL and of ApoE3 and ApoE2 for HDL (Huang and Mahley 2014) (Fig. 5.2).

Fig. 5.2
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Schematic illustration of structures of ApoE isoforms and its functional regions. The structure of ApoE constituted of two independent domains: the N-terminal domain (amino acids 1–191) contains the lipoprotein receptor-binding region (amino acids 136–150), and the C-terminal domain (amino acids 225–299) contains the lipid-binding region (amino acids 244–272). In ApoE4, two amino at position 112 and 158 differs the type of ApoE. The two arginines at position 112 and 158 in the N-terminal domain form a domain interaction with glutamic at position 255, which may determine the prior choice of ApoE4 for VLDL

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In the 1970s, scientists found that ApoE is a component of a key modulator of lipoprotein, plasma lipoprotein, and cholesterol concentrations. Up to 75% of ApoE in plasma is synthesized by hepatic parenchymal cells (Mahley 1988); however, there are other organs and tissues producing a large amount of ApoE, most notably the brain, as well as the spleen, kidney, macrophages, and adipocytes (Ang et al. 2008; Getz and Reardon 2009; Williams et al. 1985). Physically, ApoE acts as cholesterol transporter, the key regulator to redistribute cholesterol within cells and to mobilize cholesterol between cells. These functions of ApoE transport cholesterol are essential for keeping myelin and neuronal membranes maintain both in the central and peripheral nervous systems (Leduc et al. 2010).

5.1.2 The Polymorphism of apoE Gene

The human apoE gene, 3.6 kb long, is located on the long arm of chromosome 19 and consists of four exons (Weisgraber 1994). Utermann first observed the polymorphism of apoE in 1975. Subsequent confirmation of the cDNA sequence directly tested revealed that there are three isoforms of apoE gene: ε2, ε3, and ε4. Some people only contain one major subtype, which is homozygous; some people can contain two main subtypes, namely, heterozygotes. Thus, there are six different phenotypes in the population, and all were readily detectable in human subjects: three homozygous phenotypes (ε4/4, ε3/3, and ε2/2) and three heterozygous phenotypes (ε4/3, ε3/2, and ε4/2) (Utermann et al. 1978, 1979a, b; Utermann and Beisiegel 1979).

In natural populations, apoE ɛ3 allele is the most common (77.9%), ɛ2 allele the least common (8.4%), and ɛ4 in the medium (13.7%) (Farrer et al. 1997). The gene frequencies of ApoE in Chinese population are 0.88, 0.05, and 0.06. At the same time, ApoE is also involved in the normal growth of the nervous system and repair process after injury; the nervous system has a wide range of physiological and pathological effects. Because ɛ3 appears to have the highest frequency, it is considered “wild type,” apoE 2 and apoE 4 are due to its mutation, variant receptor binding than “wild type” decreased, ApoE2 receptor-binding activity was reduced to 1% of the activity of ApoE3, and the decrease in ApoE2 receptor binding is closely related to inherited lipid disorders. The mutation of the gene apoE is involved in the pathogenesis of some of the primary cases of Alzheimer’s disease. apoE 2 has protective effects on vascular integrity; apoE 3 is moderate, while apoE 4 causes a fivefold increase in vascular inflammatory factor CypA, making blood vessels brittle, and also increases the risk of getting Alzheimer’s disease. However, people who have this genetic variant do not necessarily have Alzheimer’s disease. On the contrary, those who do not have this genetic variant are equally likely to have Alzheimer’s disease. So apart from genes, scientists suspect there must be more environmental factors that contribute to the development of Alzheimer’s disease.

5.2 ApoE and Alzheimer’s Disease

5.2.1 Alzheimer’s Disease

Alzheimer’s disease (AD) is a common neurodegenerative disease among elder people which cannot be cured. The most adverse effects are cognitive decline and memory loss. Approximately 13% of elder people over the age of 65 and 45% over the age of 85 presently are affected by AD (Assoc, A 2012). There are at least 30 million AD patients around the world, and it will reach 131 million in 2050 (ADI 2016; Hung et al. 2016). Due to an increasing elder population, AD becomes one of the greatest health issues of this century (Hickman et al. 2016) and is definitely the sixth leading cause of death in the USA (Assoc, A 2015). In 2016, the total health-care costs including long-term care and hospice services, for people aged over 65 years with dementia, are estimated to be $236 billion, and this number will be doubled in 2030 (Association 2016) .

Compared with the healthy brain, AD patients’ brain has severe shrinkage, especially in the hippocampus. Histopathology shows that extracellular senile plaques and intracellular neurofibrillary tangles are two hallmarks of AD pathology (Kanekiyo et al. 2014). Senile plaque involves amyloid-β peptide’s (Aβ) abnormal accumulation and aggregation between the neurons and later forms depositions in the gray matter of the brain, mainly in the hippocampus (which involves in new memory formation) and neocortex (Luo et al. 2017), while neurofibrillary tangles are associated with tau hyper-phosphorylation. However, due to the complex genetic, epigenetic, and environmental factors that may influence the development of AD, the mechanisms of AD have not been fully studied. Strong evidence suggest that human apoE gene is the strongest genetic risk factor for LOAD known so far. Among the three isoforms, ε2, ε3, and ε4, the risk ranking of suffering AD is ε4 > ε3 > ε2. ApoE ε4 increases AD risk about ~3 (single allele)- and 15-fold (double allele), respectively, while ApoE ε2 can decrease the possibility of AD (Kim and Tsai 2009; Koffie et al. 2012; Saunders et al. 1993).

5.2.2 Role of ApoE in Alzheimer’ Disease

Amyloid cascade hypothesis (ACH) has been proposed in 1992. The hypothesis mainly stands for the point of view that the deposition of Aβ, which is the major component of the senile plaques formed in AD patients’ brains, is the upstream initiation factor of AD pathology. Aβ deposition finally induces neurofibrillary tangles, neuronal loss, cell death, and dementia (Hardy and Higgins 1992). Currently, a new modified ACH has been proposed by Karran E (Karran and De Strooper 2016). The modified ACH suggests that tau dysfunction may run in parallel with the deposition of Aβ, but the key event in AD pathology is still Aβ deposition (Ricciarelli and Fedele 2017). However, others proposed different views: Moir suggests Aβ plaque may not be responsible for AD occurrence; on the contrary, Aβ wraps harmful pathogens to prevent them from infecting the brain, it is like the body’s immune response, rather than the killer (Kumar et al. 2016).

Abundance of evidences has suggested apoE gene is the strongest genetic risk factor for LOAD, but the role ApoE plays in AD hasn’t been fully explained. ApoE is primarily produced by the liver and macrophages in peripheral tissues, while it is produced by astrocyte or glia cells in the brain (Liu et al. 2013), both in humans and animals, and serves as a cholesterol carrier and mediates the uptake of lipoprotein particles (Hirsch-Reinshagen et al. 2009). ApoE mediates cholesterol metabolism in an isoform-dependent manner (Kanekiyo et al. 2014). It was demonstrated that ApoE4 has preference to very-low-density lipoproteins (VLDL), while ApoE3 and ApoE2 have a preference for small high-density lipoproteins (HDLs) due to their different structure sequence (Huang and Mahley 2014; Mahoney-Sanchez et al. 2016).

In human, compared to ε2 and ε3, the presence of ε4 is associated with increased risk for suffering both EOAD and LOAD, especially LOAD. Studies reveal that there is a clear relevance between apoE ε4 and the neural disorder pathology in AD (Mahley et al. 2006). Genetic studies demonstrate that, among persons who inherit double ε4 alleles, the risk of suffering from AD by 85 years of age is 50–90%, and among persons with one ε4 allele is 45% (Xu et al. 2006).

Although there is a clear correlation between apoE ε4 gene and the elevated risk of AD, the mechanism for effect of ApoE in AD is complex and multi-angled. ApoE is associated with many aspects of AD (Arbor et al. 2016), both in Aβ-dependent and Aβ-independent ways, including Aβ metabolism, Aβ plaque formation, cytoskeletal structure and mitochondrial function impairment, synaptic plasticity loss, and blood-brain barrier (BBB) integrity impairment (Fig. 5.3).

Fig. 5.3
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Aβ metabolism in the brain and Aβ-dependent/Aβ-independent effects of ApoE on Alzheimer’s disease. (1) Non-amyloid metabolic pathway of amyloid precursor protein (APP); (2) amyloid metabolic pathway of APP produces amyloid-β peptide (Aβ); (3) Aβ is degraded by insulin-degrading enzyme (IDE) and neprilysin (NEP). (4) The major Aβ clearance pathways include receptor (LRP/LDLR)-mediated uptake into astrocyte/microglia cell or through the blood-brain barrier (BBB). (5) Extreme Aβ accumulation and aggregation can promote Aβ oligomers and Aβ plaque formation which leads to AD. (6) Apolipoprotein E (ApoE) is mainly produced by astrocyte in the brain. (7–9) Aβ-dependent effects of ApoE on AD: (7) ApoE directly interacts with Aβ and interferes Aβ clearance. (8) ApoE4 competes with Aβ for the same receptor LRP and LDLR, which interferences the cellular uptake pathways of Aβ. (9) ApoE4 inhibits Aβ-degrading enzymes to downregulate Aβ degradation. (10–12) Aβ-independent effects of ApoE on AD: (10) ApoE4 can directly damage neuron and leads to AD; (11) C-terminal of ApoE4 enters cytosol causing mitochondrion dysfunction; (12) ApoE4 impairs blood-brain barrier (BBB) integrity

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5.2.2.1 APOE4 Affects AD in Aβ-Dependent Way

Aβ production and clearance disturbance may play a central role in AD pathogenesis. Obviously, ApoE relates to Aβ metabolism in AD in isoform-dependent manner. There are evidences to indicate that levels of soluble Aβ are increased with ApoE4, providing a potential mechanism of ApoE4-induced AD risk (Tai et al. 2014). However, the pathways by which ApoE4 may increase Aβ levels are unclear. Based on the existing evidences, ApoE may affect Aβ by the pathways of forming complexes, interfering Aβ clearance, altering Aβ degradation enzyme, and facilitating Aβ plaque formation.

Some research suggest that ApoE can directly interact with Aβ. Histological analyses of AD patients’ brains show that ApoE is co-deposited with Aβ in amyloid plaques (Namba et al. 1991). Epitope mapping demonstrates that residues 144–148 in the ApoE N-terminal region can interact with residues 13–17 in Aβ, forming the ApoE/Aβ complexes (Cho et al. 2001) which interfere Aβ uptake ways. Purified ApoE4 can bind to Aβ with a higher affinity than ApoE3 and E2 (Ladu et al. 1994). Researches have shown that ApoE increases the level of Aβ oligomers in an isoform-dependent manner (E4 > E3 >E2) (Hashimoto et al. 2012; Youmans et al. 2012). Moreover, blocking the ApoE/Aβ interaction can relieve Aβ-related pathology including brain Aβ accumulation, co-accumulation of ApoE within Aβ plaques, and neurodegeneration in both APP/E2 and APP/E4 mice (Pankiewicz et al. 2014).

ApoE can also modulate Aβ clearance way as a competitor. All three isoforms of ApoE can bind to the receptors and transporters such as low-density lipoprotein (LDL) receptor-related protein (LRP) in astrocytes that supposed to bind Aβ, which form a competition of Aβ cellular uptake pathway (Verghese et al. 2013). Interestingly, compared to ApoE4, ApoE2 and ApoE3 cleared more Aβ in transgenic mice (Dodart et al. 2005; Hudry et al. 2013).

Our previous research has shown that ApoE can also regulate Aβ metabolism by affecting its degrading enzyme IDE extracellularly. ApoE4 significantly downregulates the expression of IDE, while ApoE3 could rescue these effects in ApoE knockout mice (Du et al. 2009a). Keeney’ research also demonstrated that ApoE4 mice exhibited downregulated peroxisome proliferator-activated receptor (PPARγ) levels and IDE expression (Keeney et al. 2015). In another research of our lab, we suggest that PPARγ could transcriptionally activate IDE gene expression (Du et al. 2009b). These results indicate that ApoE4 may decrease IDE expression by inhibiting PPARγ.

Furthermore, some studies suggest that ApoE isoforms on AD pathogenesis are through plaque formation. Holtzman’s researches provide evidences that APPsw mice carried two apoE (+/+) and one (+/−) presented more Aβ plaques than no copies (−/−) of normal mice apoE gene (Holtzman et al. 2000b). In addition, they further demonstrate that these effects of ApoE are isoform specific (E4>E3) (Holtzman et al. 2000a).

5.2.2.2 APOE4 Affects AD in Aβ-Independent Way

In addition, both in vivo and in vitro studies also suggest ApoE may affect AD in Aβ-independent ways in parallel with Aβ-independent ways, including synaptic plasticity, BBB integrity, cytoskeletal structure and mitochondrial function impairment, synaptic plasticity loss, and blood-brain barrier (BBB) integrity impairment.

ApoE4 causes neuronal and behavioral deficits in the absence of Aβ accumulation in transgenic mice. Transgenic mice expressing human ApoE3 or ApoE4 and lacking endogenous mouse ApoE have been established (Buttini et al. 1999; Raber et al. 1998). Among all these models, Aβ levels do not accumulate; however, ApoE4 mice show deficits in vertical exploratory behavior and impairment of spatial learning and memory, while ApoE3 mice and wild-type mice show no significant change, and these impairments of learning and memory are gender specific (female>male) (Buttini et al. 1999; Raber et al. 1998).

ApoE impairs synaptic plasticity in an isoform-dependent manner. As compared to ApoE3, ApoE4 decreases dendritic spine density in transgenic and gene-targeted mice (Jain et al. 2013). ApoE3 promotes neurite outgrowth and increases neuronal sprouting (Kim et al. 2014). However, the effect of ApoE4 on synaptic plasticity is inconsistent. A study reported that ApoE4 had prejudicial effects on neurite outgrowth (Teter et al. 2002), while another study suggested ApoE4 even had stimulating effects in the absence of Aβ (Puttfarcken et al. 1997).

Moreover, it has also been demonstrated that the C-terminal fragments of ApoE4 can enter the cytosol and cause neurotoxicity by disrupting the cytoskeleton (Huang et al. 2001). ApoE4 fragment also target the neuron mitochondrion, leading to mitochondrial dysfunction. Brodbeck’s research later demonstrate that ApoE decreases mitochondrial mobility in an isoform-specific manner (E4 fragment > E4 > E3) (Brodbeck et al. 2011; Chang et al. 2005).

On the other hand, ApoE also exhibits isoform-specific effects on BBB integrity in mouse models (Bell et al. 2012). In both human apoE gene knock-in and glial fibrillary acidic protein promoter transgenic mice, ApoE4 expression increases the susceptibility of BBB to injury in the absence of Aβ. It has been reported that pericytes express ApoE (Xu et al. 2006), which might lead to BBB damage in the context of ApoE4.

5.3 ApoE and Other Neurodegenerative Diseases

Although the linkage is not as strong as with AD, ApoE also associates with progression in other neurological or neurodegenerative diseases, including Parkinson’s disease (PD), vascular dementia (VD), multiple sclerosis (MS), traumatic brain injury (TBI), ischemic stroke (IS), etc.

5.3.1 ApoE and Parkinson’s Disease

Though PD has some clinical and neuropathological features that are similar with AD, there are still lots of inconsistent features. Compared to AD, PD progresses slowly in most people, affecting less of the population older than 65 years of age (PD 2% vs AD 13%) (Hughes et al. 1993). Until now, the association between ApoE and PD is still controversial. Hardy’s research notice a strong association between the apoE ɛ4 allele and AD but no association between the apoE ɛ4 allele and PD (Hardy et al. 1994). Also, apoE ɛ4 does not aggravate AD lesion in patient with PD (Egensperger et al. 1996). Li and Pulkes’s researches, however, demonstrate the association between ApoE and PD in CNS (Li et al. 2004; Pulkes et al. 2011). Another research demonstrate apoE ɛ2 is associated with higher risk of PD development (Huang et al. 2004). So far, the role of ApoE in PD remains a lot of inconclusive.

5.3.2 ApoE and Vascular Dementia

VD is a severe cognitive impairment caused by brain damage from impaired blood hypoperfusion in the brain and usually happens after suffering ischemic stroke, hemorrhagic stroke, and cerebrovascular diseases (Roman 2004). VD is one of the second common causes of dementia after Alzheimer’s disease, causing around 15% of cases (O’Brien and Thomas 2015). Clinically, VD presents pathological features such as the amyloid plaques, neurofibrillary tangles, and white matter lesions, same as AD (Kalaria 2003). There are many risk factors of VD, including hypertension, ischemic stroke, hemorrhagic stroke, atherosclerosis, and other metabolic disorders; in addition to the above, ApoE is also considered as an important risk factor for VD, but the conclusions are conflicting. Some studies demonstrate there is a positive association between apoE ɛ4 allele and increased risk of VD (Baum et al. 2006; Chuang et al. 2010; Yin et al. 2012); on the contrary, Kawamata’s research find no obvious association between apoE ɛ4 allele and VD in Japanese (Kawamata et al. 1994).

5.3.3 ApoE and Multiple Sclerosis

MS is the most common demyelinating disease of the central nervous system. MS usually occurs between the ages of 20 and 50 and more common in women than men. The lesions are characterized by multiple lesions, remissions, and recurrences in the optic nerve, spinal cord, and brain stem (Zephir 2018). So far, some researches demonstrate a negative association between apoE ɛ4 allele or ɛ2 allele and MS (Carmona et al. 2011; Ghaffar et al. 2010; Ramagopalan et al. 2007; Xuan et al. 2011; Zwemmer et al. 2004) or MS patients’ cognitive impairment (Portaccio et al. 2009), while some researches indicate apoE ɛ4 carriers with MS have worsening progression of cognitive deficits than noncarriers (Oliveri et al. 1999; Shi et al. 2011). In summary, the possible relationship between the apoE ɛ4 allele and cognitive dysfunction in MS patients is small and on balance suggests a link.

5.3.4 ApoE and Ischemic Stroke

Stroke is a medical condition in which poor blood flow to the brain results in cell death. There are two main types of stroke: ischemic stroke (IS), due to lack of blood flow, and hemorrhagic stroke (HS), due to bleeding. Due to the association between apoE ɛ4 allele and increased levels of LDL and cholesterol, ApoE may have an impact on IS occurrence; several meta-analyses report a significant association between IS and the apoE ɛ4 allele.(Das et al. 2016; McCarron et al. 1999; Wang et al. 2006; Xu et al. 2016). It has been demonstrated that apoE ɛ4 carrier patients have significantly greater risk of IS occurrence (Treger et al. 2003). Also, apoE ɛ4 allele is related to increasing carotid intima-media thickness, which is associated with IS (Paternoster et al. 2008). IS is a result of combination interactions between environmental and various genetic factors: mthfr, apoE, pon1, pde4d, etc. (Wei et al. 2017), and influence of each gene is not as strong as in AD but is expected to be modest. The influence of genetic factors may be obscured by the acquired risk factors in IS. However, apoE gene seems to be a strong candidate for studying the interplay between genetic and acquired risk factors (Van Giau et al. 2015).

5.4 Conclusion

ApoE is a kind of apolipoprotein closely related to the nervous system. Its genetic polymorphism is not only closely related to lipid metabolism but also closely related to various neurological or neurodegenerative diseases and cardiovascular diseases, such as AD, PD, VD, MS, and IS. This review highlighted the association between ApoE and neurodegenerative diseases. The association between ApoE (especially ApoE4) and AD is strong and has been known for decades; several theories have been proposed how ApoE plays its roles, both in Aβ-dependent and Aβ-independent pathways. On the contrary, the linkage between ApoE and other neurological or neurodegenerative diseases is not as strong as AD, the effect of ApoE expression and ApoE polymorphism is also controversial, and this may be explained by the complex of the influences of genetic factors and environment factors (acquired factors). In summary, the association between ApoE and the risk of pathogenesis is still not clear, but ApoE is a definite essential factor for diagnosis, risk assessment, prevention, and treatment of disease in humans.