A multi-hit hypothesis for an APOE4-dependent pathophysiological state

Abstract

The APOE gene encoding the Apolipoprotein E protein is the single most significant genetic risk factor for late-onset Alzheimer's disease. The APOE4 genotype confers a significantly increased risk relative to the other two common genotypes APOE3 and APOE2. Intriguingly, APOE4 has been associated with neuropathological and cognitive deficits in the absence of Alzheimer's disease-related amyloid or tau pathology. Here, we review the extensive literature surrounding the impact of APOE genotype on central nervous system dysfunction, focussing on preclinical model systems and comparison of APOE3 and APOE4, given the low global prevalence of APOE2. A multi-hit hypothesis is proposed to explain how APOE4 shifts cerebral physiology towards pathophysiology through interconnected hits. These hits include the following: neurodegeneration, neurovascular dysfunction, neuroinflammation, oxidative stress, endosomal trafficking impairments, lipid and cellular metabolism disruption, impaired calcium homeostasis and altered transcriptional regulation. The hits, individually and in combination, leave the APOE4 brain in a vulnerable state where further cumulative insults will exacerbate degeneration and lead to cognitive deficits in the absence of Alzheimer's disease pathology and also a state in which such pathology may more easily take hold. We conclude that current evidence supports an APOE4 multi-hit hypothesis, which contributes to an APOE4 pathophysiological state. We highlight key areas where further study is required to elucidate the complex interplay between these individual mechanisms and downstream consequences, helping to frame the current landscape of existing APOE-centric literature.

Keywords: APOE; ageing; multihit hypothesis; pathophysiology; review.

FIGURE 1

APOE4 drives a neurodegenerative phenotype via fragmentation, microtubule depolymerisation and somal ApoE4 sequestration. Path A: Under conditions of cellular stress and injury, neuronal ApoE expression is upregulated, and there is an increased propensity of ApoE4 to undergo proteolytic fragmentation which results in enhanced intracellular ApoE4 fragment accumulation. ApoE4 fragments disrupt mitochondrial function and induce ER stress, to which, GABA‐Ins are particularly sensitive and exhibit exacerbated cell death. Path B: Somal sequestration of ApoE4 and its associated lipoproteins alongside away from the dendritic cell surface poorer lipid availability inhibits effective dendritic repair, remodelling and maintenance. Path C: In addition, through action at surface LRP/HSPGs ApoE4 enhances microtubule depolymerisation in a mechanism thought to be driven by enhanced ERK signalling. The culmination of these pathways is a heightened state of neuronal vulnerability and dendritic degeneration, and thus a neurodegenerative phenotype. Abbreviations: ApoE4, apolipoprotein E4; ER, endoplasmic reticulum; ERK, extracellular signal‐related kinase; GABA‐INs, gamma aminobutyric acid (GABA) interneurons; LRP‐HSPG, low‐density lipoprotein receptor‐related protein;. Created with BioRender.com

FIGURE 2

ApoE4 drives neurovascular impairment. Path A: Increased NFAT drives APOE expression in pericytes, ultimately resulting in the accumulation of ApoE within pericytes, promoting pericyte degeneration. Path B: A reduced ability of ApoE4 to associate with LRP results in impaired suppression of CypA activity. Disinhibited CypA drives MMP9 activity through an NF‐kB dependant pathway. Elevated MMP9 activity has been directly linked to decreased microvascular length and degeneration of the blood brain barrier. Combined, these pathways represent significantly reduced maintenance of both the wider neurovascular system and result in a ‘leaky BBB’ phenotype. This leaky BBB in turn causes an increase in blood born proteins in the CSF, increasing neuroinflammation and oxidative stress. In addition, net cerebral blood flow and volume reductions have been observed likely linked to impaired oxidative responsivity and microvascular integrity Abbreviations: ApoE4, apolipoprotein E4; BBB, blood brain barrier; CNS, central nervous system; CSF, cerebrospinal fluid; CypA, cyclophilin A; LRP, low‐density lipoprotein receptor; MMP9, matrix metalloproteinase 9; NFAT, nuclear factor of activated T cells; NF‐kB, nuclear factor kappa B. Created with BioRender.com

FIGURE 3

An elevated pro‐inflammatory state is caused by ApoE4. Path A: ApoE4 fails to suppress pro‐inflammatory activation via a loss of activation of LDLR/LRP family signalling, resulting in chronic activation of JNK and P38 culminating in activation of the NF‐kB pathway, driving the release of pro‐inflammatory factors such as TNFa, while reducing anti‐inflammatory cytokine release. ApoE also interacts with TREM2 to induce a phagocytic state, although the isoform interaction is unclear. Path B: ApoE4 drives activation of lipid‐sensitive inflammatory pathways. L/RXR can supress NF‐kB activation, while promoting lipid efflux via increasing APOE and ABCA1 expression upon lipid loading. ApoE4 impairs lipid efflux and excessive lipid accumulation may subsequently increase activation of inflammatory factors including NF‐kB. TLR signalling is similarly sensitive to lipid loading, with the net effect being elevated NF‐kB. Impaired lipid trafficking likely mediated in part by endosomal accumulation as discussed in hit 5 of the review. These imbalances result in an overall pro‐inflammatory state that is also associated with aberrant glial behaviour including reduced clearance of cellular debris and microglia entering an aberrant amoeboid state. Abbreviations: ABCA1, adenosine triphosphate (ATP)‐binding membrane cassette transporter A1; ApoE4, apolipoprotein E4; JNK, c‐Jun N‐terminal kinase; L/RXR, liver/retinoid X receptors; LRP, low‐density lipoprotein receptor‐related protein; NF‐kB, nuclear factor kappa B; TLR, toll‐like receptor; TREM2, triggering receptor expressed on myeloid cells 2. Created with BioRender.com

FIGURE 4

ApoE4 associated oxidative stress. Path A: The presence of APOE4 impairs the basal expression of antioxidant proteins such as SOD, HSP, TRX1 and COXIV and may impair ability to buffer oxidative challenge. Path B: ApoE4 exhibits a reduced affinity for reactive oxygen species and lipid peroxidation byproducts, likely due to a decreased number of ROS binding cysteine residues relative to that of ApoE2 and ApoE3. Combined, these pathways result in elevated ROS accumulation causing lipid peroxidation enhanced oxidative toxicity, ultimately increasing susceptibility to cell death. Abbreviations: ApoE4, apolipoprotein E4; COXIV, cytochrome oxidase subunit 4; HSP, heat shock protein; SOD, superoxide dismutase; TRX, thioredoxin; ROS, reactive oxygen species. Created with BioRender.com

FIGURE 5

ApoE4 impairs endosomal trafficking. In both neurons and astrocytes, normal endosomal trafficking is stalled by accumulation of ApoE4 in endosomes, preventing not only their recycling but also the surface expression of multiple receptors including AMPAR, NMDAR, IR, APOER and ABCA1. However, cell‐specific differences are evident as described below; Path A: In neurons, endosomal pH is thought to be close to the altered isoelectric point of ApoE4 resulting in denaturation to a molten globule state promoting subsequent accumulation. Path B: In astrocytes, excessive HDAC4 activity prevents expression of the Na+/H + exchanger NHE6, lowering the endosomal pH and resulting in similar endosomal accumulation as seen in neurons. Inset box: Endosomal pH is regulated by the opposing actions of Na+/H + exchangers and V‐ATPases. Abbreviations: ABCA1, adenosine triphosphate binding cassette transporter A1; AMPAR, alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid receptor; APOER, ApoE receptor; ApoE4, apolipoprotein E4; HDAC4, histone deacetylase 4; IR, insulin receptor; NHE6, Na+/H + exchanger 6; NMDAR, N‐methyl‐D‐aspartate receptor. Created with BioRender.com

FIGURE 6

Metabolic dysfunction caused by ApoE4. Path A: ApoE4 impairs the activity of IDE, resulting in elevated extracellular levels of insulin. This causes receptor internalisation, that is, exacerbated by the previously described endosomal trafficking defects and leads to reduced surface expression of GLUT4 and impaired glucose uptake. This is likely exacerbated by reduced PPARy and GLUT4 expression, associated with ApoE4. Path B: ApoE4 shifts the mitochondrial metabolome away from effective oxidative phosphorylation while promoting intracellular lipid accumulation and lipid droplet formation through impaired lipid efflux. Path C: ApoE4 escaping the secretory pathway within neurons undergo proteolytic fragmentation. ApoE4 fragments accumulate in the mitochondria and ER, impairing mitochondrial complex function and inducing cellular toxicity. Abbreviations: ApoE4, apolipoprotein E4; GLUT4, glucose transporter 4; IDE, insulin degrading enzyme; PPARy, peroxisome proliferator‐activated receptor y.Created with BioRender.com

FIGURE 7

ApoE4 mediated impaired calcium homeostasis. Path A: Elevated calcium influx through L‐VGCCs and NMDARs results in an elevated level of intracellular calcium. A Dab1/SFK mediated pathway downstream of ApoE binding to APOERs promotes NMDAR mediated calcium influx. ApoE4 binding to LDLR/LRP family receptors upregulates MAPK signalling activation and is linked to activation of cFos/CREB synaptogenic signalling which may be chronically dysregulated. Path B: In astrocytes ApoE4 has been shown to induce the release of calcium from intracellular stores such as the mitochondria and endo/lysosomal compartments. The net effect of these pathways is a prolonged increase in intracellular calcium concentration, calcium signalling and subsequent cell death. Abbreviations: APOER, apolipoprotein E receptor; ApoE4, apolipoprotein E4; cFos, FBJ osteosarcoma oncogene; CREB, cyclic adenosine monophosphate (cAMP) response element binding protein; Dab1, disabled 1; L‐VGCCs, L type voltage gated calcium channels; LRP1, Lowdensity lipoprotein receptor‐related protein 1; MAPK, mitogen activated protein kinase; NMDAR, N‐methyl‐daspartate receptor; SFK, serine family kinases. Created with BioRender.com

FIGURE 8

The multihit hypothesis. APOE4 genotype promotes a pathophysiological state of vulnerability to further damage through multiple interacting hits. Cellular stress, ageing and an altered transcriptional landscape act as key moderators of many of the hits described and are thus highlighted in their own box (top right). There are multiple points of crosstalk between APOE4‐mediated hits (as described in Section 10). Including the following: lipid and metabolic homeostasis and neuronal maintenance; calcium homeostasis and transcriptional regulation; neuroinflammation, oxidative stress and neurovascular dysfunction; and endosomal trafficking, lipid homeostasis and neurodegeneration. Abbreviations: ApoE4, apolipoprotein E4; CNS, central nervous system. Created with BioRender.com