HDL Mimetic Peptides Affect Apolipoprotein E Metabolism: Equal Supplement or Functional Enhancer?

Abstract

ε4 allele of ApoE is the strongest genetic risk factor for late onset Alzheimer’s disease (AD) and it is suggested that loss-of-protective function caused by apoE4 is likely involved in AD pathogenesis. Supplementation of ApoE proteins or mimetics has been pursued for drug developments against AD. An HDL mimetic peptide 4F was shown to alleviate AD-related deficits in APP transgenic mice, and this editorial highlights a study by Chernick et al. who use both mouse and human neuroglial cells to explore the mechanism underlying beneficial effects of this peptide. The authors demonstrate that 4F peptide significantly increased the secretion and lipidation of ApoE in the absence and presence of Aβ independent of de novo transcription/translation, but requiring ABCA1 and the integrity of the secretory pathway between ER and Golgi. This study reveales a novel mechanism of HDL mimetic peptide as a functional ApoE enhancer and support further development of ApoA-I 4F peptide as effective ApoE modulating agents against AD.

Graphical Abstract

ε4 allele of ApoE is the strongest genetic risk factor for late onset Alzheimer’s disease (AD). Supplementation of ApoE proteins or mimetics has been pursued for drug developments against AD. An very low-density lipoprotein (HDL) mimetic peptide 4F was shown to alleviate AD-related deficits in APP transgenic mice, and this editorial highlights a study by Chernick et al. who use both mouse and human neuroglial cells to explore the mechanism underlying beneficial effects of this peptide. The authors demonstrate that 4F peptide significantly increased the secretion and lipidation of ApoE in the absence and presence of Aβ independent of de novo transcription/translation, but requiring ABCA1 and the integrity of the secretory pathway between ER and Golgi. This study reveales a novel mechanism of HDL mimetic peptide as a functional ApoE enhancer and support further development of ApoA-I 4F peptide as effective ApoE modulating agents against AD.

Alzheimer’s disease (AD) is the most common neurodegenerative diseases in the elderly which is estimated to affect approximately 47.5 million people worldwide. In addition to selective neuronal loss in the cortex and hippocampus, senile plaques and neurofibrillary tangles are the two hallmark pathologies of the disease. Despite intense research efforts, there is no cure for this devastating disease which is largely due to the lack of clear understanding of the pathogenic mechanism(s) of the disease. Mutations in the genes of PS1, PS2, and APP cause the less common form of early-onset autosomal dominant familial AD (fAD), which develops symptoms before the age of 65 years. The majority of AD cases (>95%) are the sporadic form that occurs later in life and known as late-onset AD (LOAD). While environmental factors likely play a larger role in the occurrence of LOAD, genetic factors are also strongly involved. In humans, three polymorphic alleles of apolipoprotein E (ApoE) gene (ε2, ε3, and ε4) exist with ε3 being the most common and ε2 the least common (Liu et al. 2013). Much evidence revealed that ε4 allele of ApoE is the strongest and most validated genetic risk factor for AD as ε4 both significantly increases the risk for AD and shifts age of onset of AD years earlier in a dose-dependent manner compared to non-ε4 carriers (Liu et al. 2013, Corder et al. 1993, Sando et al. 2008). On the contrary, the presence of ε2 allele is protective (Liu et al. 2013). Therefore, studies on ApoE hold potential to deepen our understanding of the pathogenic mechanism and offer novel perspectives for the treatment of this disease and thus remains a hot area of research in the field.

The human ApoE protein is a secreted glycoprotein of 299 amino acids with a molecular weight of around 34 kDa in both the periphery and the central nervous system (CNS) (Kim et al. 2009). The polymorphism in ApoE causes differences at amino acid residues 112 and 158 in ApoE isoforms (ApoE2: Cys112/Cys158; ApoE3: Cys112/Arg158; ApoE4: Arg112/Arg158) which profoundly affect the structure and function of ApoE (Liu et al. 2013). In the periphery, ApoE proteins are mainly synthesized by hepatocytes in the liver which are isolated from the CNS because of limited permeability through blood-brain barrier (BBB). In the brain, they are mainly expressed in nonneuronal cells such as astrocytes and to some extent microglia. ApoE is one of the key lipoproteins in liproprotein complexes that mediate the transport of cholesterol and other lipids through cell surface ApoE receptors including the low-density lipoprotein (LDL) receptor family members (Kim et al. 2009). While ApoE isoform-specific lipoprotein preferences with ApoE4 being preferentially associated with LDL/very low density lipoprotein (VLDL) and ApoE2/3 with high-density lipoproteins (HDL) were noted in the plasma, in the brain, ApoE is associated predominantly with cholesterol and phospholipid-rich, HDL particles without any known isoform specificity (Yamazaki et al. 2016), suggesting a pathogenic mechanism that is not directly linked to isoform-specific effects on lipid metabolism in the brain.

Amyloid-β (Aβ) is considered to play a major role in the pathogenesis of AD and convincing evidence demonstrated isoform-specific and lipidation status-dependent binding of ApoE to Aβ (Kanekiyo et al. 2014). While the primary mechanism linking ApoE4 to neurodegeneration and AD remains elusive, multiple studies demonstrated that it is a strong modulator of Aβ metabolism and pathology and in fact both loss-of-neuroprotective function and gain-of-toxic function compared to ApoE3 are likely involved (Kanekiyo et al. 2014). ApoE4 caused reduced ApoE levels and impaired lipidation of ApoE proteins as well as reduced ApoE/Aβ interaction and thus is less effective in preventing Aβ aggregation/accumulation (i.e., seeding inhibition or clearance enhancement), or more likely to promote Aβ fibrillogenesis (i.e., through its self-aggregating propensity) or both (Kanekiyo et al. 2014). Therefore, it would be expected that supplementation of non-risk ApoE proteins (i.e., ApoE2/3), which would compensate for the reduced neuroprotective function of ApoE either due to decreased levels or lipidation, is a promising target for drug development and therapy against AD (Yamazaki et al. 2016). Two common approaches were proposed: one approach is to transduce engineered adenoviral vectors to express human ApoE2 or ApoE3 proteins; another one is to supplement functional equivalents mimetic peptides of holo-proteins. Considering the biosafety concerns of viral delivery and brain injection associated with the former approach, the latter approach appears more advantageous since functional mimetic peptides could be administrated peripherally which could reach the brain parenchyma across BBB. Among several ApoE mimetics, ApoE133–149 (COG133) (Vitek et al. 2012) and Ac-hE18A-NH2 (Handattu et al. 2013) were found beneficial in vivo in the treatment of transgenic mouse model of AD: COG133 improved animal behavior and reduced inflammation and amyloid pathology likely by increasing protein phosphate 2A activity, while Ac-hE18A-NH2 could directly enhance ApoE levels in the brains in vivo in addition to the anti-inflammation effects. Consistently, Ac-hE18A-NH2 also restored ApoE levels inhibited by ox-PAPC or Aβ₄₂ treatments in vitro (Handattu et al. 2013).

In addition to the mimetic peptides of ApoE, mimetics of ApoA-I were also studied for therapeutic potential in AD. ApoA-I mimetic peptides were developed based on the presence of lipid-associating amphipathic α-helical domains in ApoA-I, which is the major apolipoprotein of plasma HDL (Segrest et al. 1992). The most extensively studied ApoA-I peptide is 4F peptide that consists of 18 amino acids with the sequence of Ac-DWFKAFYDKVAEKFKEAF-NH2 (Segrest et al. 1992, Getz & Reardon 2011). Initial studies demonstrated that the administration of ApoA-I mimetic 4F decreased brain arteriole inflammation and improved cognition performance in LDL receptor-null mice on Western diet (Buga et al. 2006). Later, it was reported that oral application of 4F peptide improved cognition function and reduced amyloid burden in APP^Swe-PS1^ΔE9 mice (Handattu et al. 2009). Despite the demonstration of beneficial effects of ApoA-I mimetic 4F in vivo, it is still unclear how mimetic peptides work. Specifically, are they only acting as equal functional supplements of holo-ApoE proteins in the brain? Or can they also rescue the impaired endogenous ApoE proteins which were changed at the levels of expression and modification in AD brains (Figure 1)? In this issue, Chernick et al. further explore the mechanism underlying the beneficial effects of ApoA-I 4F peptide to address these critical issues in vitro.

Figure 1:

Supplementation of ApoE proteins or mimetics has been pursued for drug developments against AD. Direct HDL mimetic peptides can increase apoE levels or its functional equivalents. ApoA-I 4F peptide was found to act as a functional enhancer by increasing ApoE secretion and lipidation.

Chernick et al. focused on the primary culture of mouse and, more importantly, human neuroglia cells with the treatment of 4F peptide. Compared with the scramble peptides, 4F peptides specifically increased the ApoE secretion and lipidation in the culture medium in a dose- and time-dependent manner in the in vitro astrocyte cultures. Importantly, they found 4F peptide could alleviate the inhibition of ApoE secretion and lipidation induced by Aβ treatment, suggesting that this mimetic peptide has the potential to break the vicious feed-forward cycle between ApoE function and Aβ aggregation. They further demonstratef that the secretion of ApoE promoted by 4F is dependent on the availability of intracellular ApoE proteins and is less dependent on transcription and translation system of cultured glias. Furthermore, they delicately showed enhanced secretion of ApoE proteins by 4F peptides is in part through the protein transport pathway from the endoplasmic reticulum to the Golgi apparatus and requires lipid transporter ABCA1. Thus, this study demonstrated a novel molecular mechanism of apolipoprotein peptides in addition to their role as equal supplements of holo-proteins: the mimetic peptides can also enhance the metabolism of the endogenous holo-proteins (Figure 1).

However, one important question left unaddressed is how ApoA-I 4F peptide impacts the metabolism of ApoE4 compared with ApoE2 or E3. Can 4F peptide also increase the secretion of ApoE4 protein? This is important because increased ApoE4 can cause some toxic functions that may limit the therapeutic use of 4F peptide among ε4 carriers. In fact, this selectivity issue will be a hurdle for all these mimetic peptides before they can move forward. While Chernick et al. showed that 4F peptide mitigates Aβ₄₂-induced inhibition of ApoE secretion and lipidation, it remains to be distinguished whether this is due to enhanced ApoE secretion after 4F peptide treatment or competitive neutralization of Aβ₄₂ aggregates minimalizing their toxic effects on ApoE metabolism. Another question raised by the current study is that while ApoA-I 4F peptide as an ApoE enhancer increases ApoE secretion and mitigates Aβ-induced inhibition of ApoE secretion and lipidation, it relies on the availability of existing ApoE proteins. Its efficacy may be limited when the expression level of ApoE is significantly reduced such as in the case of ApoE knockout model (Nayyar et al. 2012) or in AD brain and it may be best used in combination with those treatments that increase the ApoE expression.

Abbreviations:

AD

Alzheimer’s disease

PS1

presenilin 1

APP

amyloid precursor protein

LOAD

late onset AD

ApoE

apolipoprotein E

CNS

central nervous system

BBB

blood-brain barrier

LDL

low-density lipoprotein

vLDL

very low-density lipoprotein

HDL

high-density lipoproteins

Aβ

Amyloid-β