Lit Review

UNIVERSITY OF GUELPH
Apolipoprotein E4
The Greatest Genetic Risk Factor for Alzheimer’s
Disease
Gbolahan Olarewaju 0587370
11/29/2010
A review of pivotal studies in Alzheimer’s Disease research with emphasis on the genetic risk factor,
ApolipoproteinE4.

ApolipoproteinE4:The GreatestGeneticRiskFactorforAlzheimer’sDisease
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TABLE OF CONTENTS Page #
 Introduction 3
 Clinical Criteria for AD Diagnosis 4
o Neurofibrillary Tangles 5
o Neuritic Plaques 5
 Possible Causes for Alzheimer’s Disease 7
o Risk Factors for Alzheimer’s Disease 7
 Early-Onset/Familial Alzheimer’s Disease (FAD) 8
 Late-Onset/Sporadic/Senile Alzheimer’s Disease 10
 Apolipoprotein E 10
 Apolipoprotein E4 14
o The Evolutionary Perspective 16
o The Pathological Perspective 19
 Conclusion (Where does this leave us?) 26
 Works Cited 27

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1. Introduction
Among the many diseases or syndromes that often come with old age, Alzheimer’s
disease (AD) is definitely one of the most researched. This disease, marked with progressive
cognitive decline, was discovered by a German psychiatrist named Dr. Alois Alzheimer in 1906
[1]. Following the death of one of his patients (aged 51 years), a brain autopsy revealed the
presence of twisted bands of fibers and dense deposits around nerve cells, as well as some
neuronal cell loss [1]. At this time nothing was known of the disease, but these signs, now known
to be neurofibrillary tangles (NFT) and amyloid/neuritic plaques, are hallmarks of the disease
[2]. The disease, now called Alzheimer’s affects about 2% of the population in Canada above 65
years [3]. It is the leading form of dementia in the elderly. Currently in the United States, 4.5
million people are afflicted by AD. This is expected to double every 5 years [2]. Over the course
of a hundred years the disease has been investigated at length to provide an explanation for
observed signs that can be used to diagnose AD in living patients. Some of these signs include
cognitive decline, memory loss and gradual decline of physical co-ordination, unexpected mood
changes and behavior that is out of character [3].
All these changes in patients are signs of the effects of AD on the brain. As AD
progresses these functions decline further as different areas of the brain become affected. At
present, once any ability is lost, it is not known to return. Some research suggests that relearning
is possible. Neuronal cells are the main type of cells that are affected by the diseases. AD leads
to nerve cells loss and shrinkage of the brain which affects its functions over time, as can be
evidenced in the observed symptoms. In the Alzheimer’s brain, the cortex shrivels up, thus
affecting areas involved in cognition, planning and remembering. The ventricles, fluid-filled
spaces in the brain, grow larger as a result [3]. The worst effect by far is the shrinkage of the

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hippocampus, a ridge in the floor of the lateral ventricles that is involved strongly in the memory
process [4]. The result of all this shrinkage is that there are fewer neurons and significant
synaptic loss in the AD brain as compared to a healthy brain. The cause of cell death in the AD
brain is not fully known but there are some theories that will be discussed. This review will
examine what work has been done to elucidate the causative agents and risk factors of
Alzheimer’s disease. Specific attention will be paid to the Apolipoprotein E4 polymorphism as it
is a notable factor that seems to play a huge role in AD pathogenesis.
2. Clinical Criteria for AD Diagnosis
In 1984 the National Institute of Communicative Diseases and Stroke and the Alzheimer's
disease and Related Disorders Association (NINCDS-ADRDA) published the clinical criteria for
AD diagnosis based on patient’s history, physical and neurological exams and brain imaging.
These criteria needed to be exclusive to AD and not directly related to any other causes of
dementia or cognitive decline [5] such as lewy body or vascular dementia [2]. A patient that
fulfils these set criteria can only be diagnosed with probable AD as definite AD can only be
confirmed by light microscopic examinations of brain sections obtained by brain biopsy or
during autopsy. Of all the neuropathological changes that have been observed over the years
such as synaptic and neuronal loss, inflammation, neurotransmitter deficits [2], the criteria that
have been chosen as the core of AD are the presence of neurofibrillary tangles (NFT) and most
especially dense neuritic plaques. These are congruent with Dr. Alzheimer’s original findings.
These neuropathologies first affect the entorhinal cortex which is the main input to the
hippocampus and is important for the familiarity and pre-processing of input signals [4]. The

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effect spreads from the hippocampus to other limbic structures and finally the neocortex [2]. This
pattern contributes the different stages of cognitive decline in AD; from early to late. The
relationship connecting the NFT and plaques to AD is still under investigation but it is clear that
they are phenotypic manifestations of the disease.
a. Neurofibrillary Tangles
NFT are dense bundles of un-branched filaments that are often found as paired helical
filaments in neuronal cytoplasm. They are sometimes found in neurites (neuronal cell body
projections). These filaments are mostly composed of a microtubule-associated protein called
Tau [5]. The function of Tau in the cell is to maintain the microtubule conformation by
stabilization and regulation of polymerization of tubulin (a single unit protein of microtubules).
In neurons microtubules are responsible for axonal transport of materials and neurite extension
and maintenance [4]. In an AD situation, Tau becomes abnormally phosphorylated and self-
assembles into the paired helical filaments which become the characteristic tangles. NFT are
obstructive to the transport mechanism of axons and as such would be a logical choice to explain
the etiology of cell death in the AD brain. However, NFT may be found in other
neurodegenerative diseases [5]. This may explain a common pathological origin between these
diseases and Alzheimer’s. This origin is yet to be explained.
b. Neuritic Plaques
This neuropathological structure is required for the diagnosis of definite AD. Neuritic
plaques, otherwise known as senile/amyloid plaques are abnormal extracellular accumulations of
protein fragments. The plaques are made up of a host of different proteins, the most abundant of
which is the beta-amyloid peptide (Aβ) [5]. Beta-amyloid is a 39-43 amino acid peptide that is
produced by the regular proteolytic cleavage of Amyloid Precursor Protein (APP) [2]. APP is a

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transmembrane glycoprotein found in many tissues and neurons, especially concentrated in
synapses, which can produce the peptides that aggregate to form amyloid proteins [4] which in
turn, may contribute to the neuritic plaques. APP may be cleaved either by alpha-secretase and
gamma-secretase to release p3 peptides or by BACE1 (Beta-site APP Cleaving Enzyme 1) and
gamma-secretase to release Aβ [6]. The p3 peptides are relatively benign but the Aβ peptides
form the bulk of the plaques [2]. The BACE1/gamma-secretase pathway is referred to as the
amyloidogenic pathway [2]. A soluble form of Aβ was found in cerebrospinal fluid and plasma
of both healthy and AD patients [7]. Other than Aβ, neuritic plaques contain APP itself,
Apolipoprotein E (ApoE), IgG, amyloid P, glycosaminoglycans and other proteins [5]. Neither
the complete structure of the plaques nor how they are assembled is known. Their contribution to
the pathology of AD is also unclear but it is possible that they block cell to cell
neurotransmission at synapses and they may trigger an inflammatory immune system response
[8]. For neuropathological diagnosis of AD, a certain number of neuritic plaques per microscopic
field are required. Some patients may present as probably AD patients but may lack the plaque
count necessary for definite AD diagnosis [5].
Plaques and tangles spread through the cortex in the pattern alluded to earlier as the
disease progresses. It should be recognized that amyloid may be necessary for AD but may not
be sufficient to cause the disease. Other factors such as NFT, gliosis, and synaptic loss are
probably equally important in AD pathology. The rate of progression depends in part on age as
well as other risk factors that shall be discussed in the following sections.

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3. Possible Causes of Alzheimer’s disease
It is not fully understood what causes AD but its neuropathological manifestations are
being researched. However, researchers have realized that although AD is common with age, it is
not a part of normal aging, as statistics suggest. It is not a gender specific disease. It has also
been discovered that AD is neither caused by stress nor vascular pathologies [3]. The most
plausible causes are internal factors, external factors and family history [9]. The internal factors
most probably relate to a possible immune problem. Among the external factors studied,
aluminum was one that much research was carried out on. There is no evidence of a link with
AD [3]. Many AD cases suggest a relationship between family genetics and AD risk. A lot of
work is being done in this area but the connection is not fully understood. The disease appears to
be as a result of not a single, but a collection of risk factors [9] which will be discussed next.
a. Risk Factors for Alzheimer’s Disease
As previously noted, there is a fair bit of genetics involved in Alzheimer’s disease. It has
been noted that the risk of AD increases 2-4 fold if a person has first degree relatives with the
disease [2]. Some degree of aging is required for AD symptoms to begin manifesting. Age is the
greatest risk factor, although the related factors are unknown. Risk factors for stroke may
increase risk, albeit they are confounders for vascular dementia [2]. A low level of education and
occupation may increase risk as well as a history of head trauma that was sufficient enough to
cause unconsciousness [2]. Being female slightly increases risk. It has been suggested that this is
a result of the lack of post-menopausal estrogen [2]. Down’s syndrome, the presence of all or
part of an extra chromosome 21 (trisomy 21) has proven to be a risk factor [6]. This lies in the
fact that Amyloid Precursor Protein is encoded on chromosome 21q and as such an extra
chromosome would result in an excess in APP production. This excess implies an excess of Aβ

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which contributes greatly to one of the neuropathologies of AD. Thus this effect of Down’s
syndrome is referred to as a gene dosage effect [2]. APP mutations and polymorphisms may
increase risk as well. Although genetics play an important role, it should be noted that only 7%
of AD cases have conclusive evidence of gene-associated pathology [2]. These cases are the
early-onset or familial AD situations. The majority of AD cases are late-onset or sporadic and
there may be some gene associated pathologies involved, but other risk factors like age come
into play. Although age is the most prominent known risk factor for AD, most people do not
develop the disease as they age. Even in the more common late-onset AD, there have been an
amount of cases diagnosed in their 40’s and 50’s. This further accentuates the fact that
Alzheimer’s is not a normal part of aging and is the result of the interactions and contributions of
quite a few factors [9]. Among these factors, the one that will be the focus of this review is
genetics, as it relates to AD classification and the inherent pathologies associated with AD
clinical criteria.
4. Early-Onset/Familial Alzheimer’s Disease (FAD)
As previously noted, only 7% of AD cases are classified as early-onset or familial AD
(FAD). The pathology of this class is very gene-dependent. After pedigree and proband analyses,
much is known about the supposed cause of AD. At this time, about 158 mutations in 3 genes
from 326 families have been recognized as AD related [2]. These genetic patterns are extremely
similar to those observed in late-onset AD meaning that the mechanisms of the disease can be
projected towards late-onset AD. The earlier onset cases however, may cause symptoms that
aren’t typical to late-onset AD.

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The first mutations discovered in FAD were mutations that changed the codon for one
amino acid to that of another on the gene for APP; otherwise known as missense mutations.
These mutations cluster near the beta and gamma cleavage sites of APP, which are a part of the
amyloidogenic pathway [2]. This suggests that the mechanism favors the production of Aβ from
APP. An example of such a mutation is the double missense mutation at the gamma-cleavage site
that promotes the production of Aβ40 and Aβ42 which are forms of the Aβ peptide that differ by
the length of the peptide sequence. Of these two, Aβ42 is the form most abundantly found in AD
patients and the form with the greatest propensity for spontaneously forming Aβ fibrils [2].
Single missense mutations of the same kind promote Aβ42 secretion. The discovery of these
mutations was what led to the amyloid hypothesis which describes the presence of amyloid
aggregates as the defining factor for AD. Pathogenic mutations within the actual Aβ sequence
result in a much bigger Aβ load in AD patients. An example is the intrinsic Aβ missense
mutation near the alpha-cleavage site within the Aβ sequence [2]. The fact that they are intrinsic
to Aβ makes these mutations alter the propensity of the peptide to switch from soluble to
insoluble fibrils. Other discovered mutations are on chromosome 14 for presenilin-1 (PS-1) and
on chromosome 1 for PS-2, both of which are part of the gamma-secretase complex. PS-1 is
associated with presenile dementia (<60-65 years) and its mutation is responsible for about 85%
of FAD cases [2]. PS mutations specifically promote Aβ42 generation as well as increase
susceptibility of neurons to programmed death. The most common characteristic of FAD,
therefore, is the increased production of Aβ42.

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5. Late-Onset/Sporadic/Senile Alzheimer’s Disease
Sporadic AD is the more common form of the disease. Its pathology is directly related to
Apolipoprotein E polymorphisms, which will be the focus of this review. ApoE polymorphisms
on chromosome 19 are associated with an increased risk of developing this classification of AD.
One of such polymorphisms (ApoE4) plays a particularly huge role in the pathogenesis of the
disease. The mechanisms of the relationship are still under investigation but common knowledge
is that it acts as a pathological chaperone in AD by promoting the formation of insoluble fibrils
[10, 11]; much like PS-1 mutations in familial AD. Some research also suggests that ApoE4 may
be detrimental to people with other brain ailments [2]. Having one or 2 ApoE4 alleles has been
proven to increase the risk of sporadic AD as well as decrease the age of onset by a gene dosage
effect [12]. The genetic risks may be additive. For example, the age of onset is decreased in
patients who have an ApoE4 allele as well as Down’s syndrome. ApoE polymorphisms are the
only clear substance in a multitude of genetic associations with AD and will be further discussed
in detail.
6. Apolipoprotein E
In 1975, Gerd Utermann confirmed the existence of an arginine-rich, water-soluble
apolipoprotein that was separate from the known polypeptides of very low-density lipoproteins
(VLDL), apoB and apoC [13]. This polypeptide was isolated from human plasma VLDL by
column chromatography. It represented approximately 10% of the VLDL mass and had an
apparent relative molecular mass (Mr) of 39000 [13]. This apolipoprotein was called ApoE.
ApoE was further isolated in 1976 by Curry et al via electro-immunoassay [14]. Over the next
five years, Utermann was a very prominent name in ApoE description and characterization and

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in 1980, a paper in the American Journal of Human Genetics was published under his name [15].
This paper explained the genetics of ApoE in humans among other things. One of the first
notable explanations of ApoE polymorphisms can be found in this paper. Utermann explained
that ApoE polymorphism is governed by two co-dominant alleles, ApoEn and ApoEd, and two
other alleles, ApoE0 and ApoE4+. The latter should not be confused with the actual polymorph
and is the dominant allele. Both the ApoE-N/D and ApoE4 loci are closely linked and various
combinations of their alleles produce the three most prominent polymorphisms of the ApoE
lipoprotein; ApoE2, ApoE3, and ApoE4 [15]. The three forms of the lipoprotein are similar in
Mr.
The next pivotal step in ApoE research is sequencing. This was done by Rall et al in
1982. Rall concluded ApoE as a 299 amino-acid polypeptide with a molecular weight of
34,145Da [16]. Determination of its sequence provided a bridge between the structure of ApoE
and its function. The amino terminal of the protein is the receptor binding domain and the
carboxyl terminal binds the lipids [16]. This structure explains the function of ApoE in
lipoprotein transport to cells involved in cholesterol and triglyceride metabolism [17]. It interacts
with receptors on peripheral cells (apolipoprotein B, E LDL receptors [24]) as well as on the
liver. The differences between the isomers were also noted. It was stated that the difference
between E2 and E3 is the substitution of cysteine in E2 for arginine in E3 at position 158 [16].
ApoE4 other the other hand has arginine-158 and arginine-112 as compared to the cysteines in
both E2 and E3 [18]. These differences have a huge effect of ApoE domain interaction and
protein stability [19]. This will be discussed later. One of the first instances of ApoE mentioned
beyond its lipid metabolism roles was in 1983 [20]. Skene and Shooter discovered that after
injury to peripheral nerves, non-neuronal cells secrete an acidic protein of similar Mr,

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approximately 37kDa, to ApoE. It was noted that the protein is probably synthesized by
Schwann cells and was possibly involved in some aspect of nerve repair.
In 1984 Elshourbagy et al made a groundbreaking discovery in ApoE research. It was
discovered that the brains of rats, marmosets and humans exhibit ApoE mRNA at levels second
only to the liver where the protein is produces [21]. This began the speculations about the
importance of ApoE in the brain. More work was done on the subject and in 1986, congruent
with Skene and Shooter, Snipes et al confirmed the synthesis and secretion of a 37kDa protein in
the central and peripheral nervous system from astrocytes and Schwann cells respectively [22]. It
was explained that the regulation this phenomenon involved functional interaction between these
cells and the damaged axons. This study stipulated that ApoE may play a role in neuron growth
with regards to lipid metabolism. Ignatius et al further confirmed these findings [23]. In their
study, it was noted that in the central nervous system, the synthesis of a protein confirmed to be
ApoE is induced by nerve injury but its accumulation is less than in the peripheral nervous
system. The role of ApoE in clearing lipid/cholesterol axonal debris was also outlined. The
possible role of ApoE in axonal growth was further solidified by Ignatius et al the next year in
their study with pheochromocytoma (PC12) cells [24]. PC12 cells were used as models to
simulate axonal growth because they could be stimulated to produce neurites in vitro. It was
noted that the growth cones of the neurites internalized ApoE-containing lipid particles via
apolipoprotein B, E (LDL) receptor uptake, thus inferring that ApoE may deliver lipids to
growing axons for membrane biosynthesis. It is now known that ApoE is critical in cholesterol
and lipid trafficking during normal nervous tissue growth as well as repair [10], especially
regarding myelination of axons. Although it has been confirmed that Schwann cells, astrocytes
and microglia (in CNS) are sources of ApoE, a new source was discovered in 1999. In their

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study, Boschert et al discovered ApoE hybridization in cells that had the morphological
characteristics of pyramidal neurons [25]. Further testing of these cells revealed that the cells
were positive for Microtubule Associated Protein 2 (MAP2), a neuronal marker. It was also
observed that these cells tested negative for GFAP (Glial Fibrillary Acidic Protein) and OX-42,
astrocytic and microglia markers respectively. The distribution pattern of the ApoE
hybridizations were discovered to be the same as a transcription factor that is expressed in
neurons under stress. The conclusion was that when under stress, neurons express ApoE to
counteract neurodegeneration. This has been confirmed by many studies [26].
In ’91 Hallman and his associates carried out a genomic study of ApoE polymorphism
allele frequencies in nine populations across the world. The results of the study showed The
ApoE3 allele to be the most abundant followed by E4 and then E2 [18]. The results were
relatively similar across the populations studied thus proving that the ApoE alleles act in a
relatively uniform manner in different populations regardless of genetic background and
environmental factors. ApoE4 is now known to have the strongest effect on AD pathology [27].
ApoE3 does not have as strong an effect, but E2 has been classified as being a protective isoform
[2, 5]. In the same year another pivotal study was carried out. Yoshio Namba, a very prominent
scientist in the study of Alzheimer’s disease, made the first association between ApoE and the
neuropathological lesions that were considered the sine qua non of the disease [28]. Namba et al
detected ApoE immunoreactivity in neuritic plaques as well as intracellular and extracellular
tangles. This discovery was congruent with Ignatius et al in 1987 in that the neurons internalized
serum proteins, which would include ApoE, via specific receptors [24]. The conclusion of the
study stated that ApoE might be among the component of cerebral amyloid deposits, a notion
that was investigated in Sanan et al, 1994 [17]. In 1992, Wisniewski and Frangione found that

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ApoE immunoreactivity was not limited to cerebral amyloid but to systemic amyloid as well
[29]. Systemic amyloid was found to be relatively less fibrillogenic by virtue of the fact that
Aβ40 was the more abundant form present; recall that Aβ42 is more spontaneously fibrillogenic
than Aβ40 [2]. In Sanan et al’s study in ’94, it was observed that ApoE binds avidly to beta-
amyloid protein, a major component of neuritic plaques in AD, to yield monofibrils [17]. It is to
be noted, however that the formation of fibrils from Aβ occurs spontaneously but is merely
accelerated by ApoE interaction [30]. Sanan et al also explained the different abilities of ApoE
isoforms, especially E4, in relation to monofibril formation. This discovery went on to help
elucidate the effect of ApoE4 on AD risk; a notion that must be discussed in detail.
7. Apolipoprotein E4
After it was established that ApoE is highly associated with the risk of developing AD, an
intense search for its role in brain pathology ensued. It was in that process that ApoE4 was
discovered. As has been stated prior, the ApoE4 isoform is the one with the greatest connection
with AD pathology. In a typical control population, approximately 20% of the individuals carry
at least on ApoE4 allele. This percentage rises 65% in non-related sporadic AD patients and 80%
in FAD patients [27]. There have been nearly 2000 studies that have investigated and proved the
ApoE4-AD connection. ApoE4 is a protein that is different from the other isoforms of the ApoE
protein in that it has an arginine at position 112, where the other two common forms have a
cysteine, and arginine at position 158 of its amino acid sequence [16]. Population studies have
shown that the allele that encodes this isoform is second most common of the three major ApoE
isoforms [18]. Many studies have shown that ApoE4 increases the risk of developing AD.
Strittmatter et al studied ApoE4 and discovered that its association with AD, especially late-

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onset/familial AD, was significantly increased when compared to control populations [31]. This
finding is consistent with many other studies. The discovery of an association between ApoE4
and AD prompted extensive research into all possibilities. Various hypotheses were tested; a
prominent one of which is the effect of ApoE4 on Aβ fibrillogenesis. It has already been stated
that amyloid fibrils form plaques which are considered a determining factor in AD diagnosis.
Studies such as Wisniewski’s in 1994 and Aleshkov’s in 1997 concluded that ApoE4 was indeed
a promoting factor for the formation of these fibrils [10, 11]. Aleshkov made mention of the fact
that of all the isoforms, ApoE4 bound Aβ the best and inferred that their binding abilities were
inversely proportionate to the risk of developing familial AD [11]. This then gives rise to the
question of whether this means that the age of onset of AD is reduced in the case of ApoE4, or
that the risk of developing AD at all is reduced. The answer to this question can be found in
Strittmatter’s review in 1995. In the review, Strittmatter stated that the mean age of onset of AD
in patients who were homozygous for the ApoE4 allele was less than 70 yrs. On the contrary,
patients with genotype E2/E3 had a mean age of onset of >90 years [12]. This shows that carriers
of the ApoE4 allele are at great risk for developing the less common early-onset/sporadic AD or
developing AD at a younger age. Studies have been done to investigate the effect of the ApoE4
allele on other disease pathologies. In 1993, Katzel studied silent myocardial ischemia (SI) in
men and concluded that even in the presence of normal LDL cholesterol, men carrying the
ApoE4 allele are predisposed to developing SI [32]. There are other studies exploring the
cardiovascular effects of ApoE4 but they are not relevant to the purpose of this review.
There are a few ways to look at the effects of ApoE4 on Alzheimer’s disease such as its
association with neuropathological lesions discussed earlier and its contribution to nerve death or
repair. However, while reviewing studies on the subject, it is quite clear that researchers are

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viewing ApoE4 from two different perspectives, the evolutionary and the pathological. By
dividing studies into these two categories, a clear picture of the ApoE4 phenomenon can be
visualized.
a. The Evolutionary Perspective
In July 2001, there was a debate on ApoE4 held in Cincinnati. The ideas discussed were
whether the presence of ApoE4 is bad or its absence of good [33]. There were some interesting
perspectives considered and those gave rise to the notion that ApoE4 is merely a more primitive
version of the other two isoforms of ApoE. In support of the evolutionary perspective it was put
forward that the E2/E3 isoforms were more evolved forms of ApoE. It was stated that they were
more efficient in carrying out the functions required of the protein than ApoE4, and that ApoE4
is merely inadequate and therefore viewed as a negative factor.
Several studies have shown this to be a possibility. In 1994, Mortimer et al investigated
the effect of human ApoE4 on the clearance of chylomicron-like lipid emulsions and
atherogenesis in transgenic mice [34]. Mortimer fed ApoE4 transgenic mice a diet rich in
cholesterol and other atherogenic factors and observed the effects of a sustained increase in
plasma ApoE4 concentration. It was discovered that fatty lesion streaks in the transgenic mice
decreased in comparison with non-transgenic mice. This shows that in mice, human ApoE4 is
effective in carrying out its lipid trafficking duties. This may support the evolutionary
perspective by virtue of the fact that mice are less evolved than humans. There are studies such
as Katzel’s in 1993 that portray ApoE4 as ineffective in that role [32]. Katzel showed that
patients with an E4 allele, when compared with E2 or E3, had higher cholesterol and LDL levels.
It is now known that ApoE4 is less efficient in delivering to and removing cholesterol from
neurons by virtue of its interaction with receptors. These studies show E2 and E3 to be more

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effective in lipid transport, thus supporting the notion that they are more evolved than ApoE4.
Another area that was looked at was Tauopathy; that is the generation of neurofibrillary tangles
in AD. In 1994, Strittmatter et al looked at Tau pathology as it related to ApoE and discovered
that in vitro, ApoE3 forms a stable complex with Tau possibly slowing down the rate of
hyperphosphorylation of the protein [35]. It has already been explained that hyperphosphorylated
Tau does not bind to microtubules well and may for paired helical filaments which develop into
the tangles that are seen in AD patients [12]. The binding domain for ApoE3/2 on Tau is close to
the binding site for microtubules and as such, it was postulated that ApoE3/2 could modulate and
stabilize the microtubule association of Tau and prevent phosphorylation and self-association,
thereby preventing tangle formation. ApoE4, on the other hand, does not affect Tau this way and
this inability could be another explanation for the increased risk of developing AD in E4 allele
carriers. It could also be an explanation for the nerve death observed in AD as failure of
microtubule structure and function can lead to loss of normal transport mechanisms for required
materials and eventually neuronal death [12]. Furthermore, Wisniewski’s study on fibril
formation by ApoE [10] also speculated that the effect of ApoE4 on Tauopathy is probably due
to the lack of a protective factor that ApoE3 contains. It has now been confirmed that neuronal
ApoE4 and its fragments stimulate Tau phosphorylation, thus contributing to AD pathogenesis
[26]. Considering lipid trafficking and Tauopathy, the evolutionary perspective seems to be valid
regarding ApoE4 and AD pathology.
Another theory, quite similar to the poor functionality theory, was one put forward by
Wisniewski in his 1994 study [10]. He proposed that the ApoE4 isoform may be less efficient in
the role that ApoE plays in CNS cholesterol mobilization and distribution as result of the
observed high affinity of ApoE4 associated lipoproteins for the LDL receptors on nerves. As a

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result, lipid and cholesterol transport to the cells that require them may be hindered by
“blockage” of the passageway, thus leading to defective reinervation.
As will be discussed, countless studies have been done regarding Aβ pathologies and
ApoE4. The observed trend is that ApoE4 promotes Aβ fibrillogenesis in one way or the other.
These studies view ApoE4 from a pathological point of view. There are, however some
researchers that beg to differ. For example, Evans in 1995 claimed that neither ApoE3 nor E4
inhibit amyloid seeded growth and they do not affect amyloid solubility or structure either;
therefore not thermodynamic inhibitors [36]. Evans observed that both isoforms were inhibitors
of amyloid nucleation (the pivotal step in amyloid aggregation) and that ApoE3 performed this
role better than E4. She therefore proposed that the link between ApoE4 and AD could be
insufficient amyloid formation inhibition. This supports the claim to evolution. In 1994, Ma et al
did a study on amyloid fibril formation. It was observed that, congruent with the popular
pathological perspective, a 10-20 fold increase in Aβ polymerization was present with the ApoE4
isoform. However, it was also observed that in a heterozygous E2/E4 situation, amyloid filament
formation was inhibited [30]. This is consistent with the notion that ApoE2 serves a protective
role in vivo [2]. The absence of this role in ApoE4 further solidifies the idea of its primitiveness.
Still on the amyloid front, in a 2001 review by Selkoe, it was stated that analyses of mouse
models suggest that inheritance of ApoE4 leads to a rise in Aβ in the brain either by enhancing
fibrillogenic potential or decreasing its clearance from the brain’s extracellular space [37]. He
stated that the clearance hypothesis is more likely and that notion supports the evolutionary
perspective. Studies have shown that ApoE4 inhibits Aβ clearance [64, 65].
An innovative study in 2001 by Giuseppina Carrieri was focused on an entirely different
aspect of the effect of ApoE4 on AD. The study considered oxidative cell death and concluded

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that ApoE isoforms have an allele-specific effect on neuronal oxidative cell death [38]. Carrieri
et al compared mitochondrial DNA (mtDNA) haplogroup frequencies in AD patients in relation
to ApoE4. It was observed that the frequency distribution of mtDNA differed between E4 allele
carriers and non-carriers. In non-AD patients, however, no such difference was observed. The
inference from this is that ApoE/mtDNA interaction is restricted to AD; which might imply that
some haplogroups are able to modulate susceptibility to AD by influencing other factors like
ApoE4. Some mtDNA haplogroups are associated with reactive oxygen species production,
which can lead to oxidative cell death. Studies have shown that ApoE plays an antioxidative role,
but of all the isoforms, ApoE4 and E2 are the least and most protective respectively [39]. This
points to the idea of ApoE4 being the lesser evolved of the species and further solidifies E2 as a
protective isoform. All these studies provide strong arguments for several beneficial roles of
ApoE, which would be expected from the evolutionary perspective that a protein that is found
across many species should be mainly advantageous. It appears that ApoE3 and E2 may be good
proteins that help prevent cognitive dysfunction as human beings age and are evolved versions of
the now detrimental ApoE4 which can be found in a similar from throughout mammals.
b. The Pathological Perspective
In 2002, a participant of the Cincinnati debate, Bruce Teter, published a comprehensive
paper explaining that the observed association of ApoE4 with AD is not because of the absence
of the other “more evolved” isoforms of the protein but because of the detrimental effects of
ApoE4 [40]. There is a lot of evidence to support this argument. In fact, the bulk of research has
proven that the detrimental effects of ApoE4 overpower whatever positive effects the other
alleles may provide. Even if it were true that ApoE4 is primitive, its presence still affects the
patient greatly. There is not a lot of evidence on ApoE2 as it has been deemed a protective allele.

Page 20 of 31
Therefore the best way to look at this is to compare ApoE3 and E4 in all three major aspects of
AD pathology: amyloidogenesis, Tauopathy and neuronal death.
The bulk of ApoE4 research is on amyloidogenesis; the formation of amyloid plaques. As
has been mentioned, in 1994, Sanan et al discovered that ApoE/Aβ coincubates yield monofibrils
and ApoE4 does this at a greater capacity [17]; 10-20 fold greater according to Ma 1994 [30]. In
Wisniewski’s study on fibrillogenesis [10], it was noted that peptides like Aβ can spontaneously
form fibrils in solution, but ApoE4 merely enhances this process. Castano performed a
thioflavone-T assay for fibril formation and found that ApoE promotes fibrillogenesis as well as
amyloid formation [41]. The latter is an issue that was investigated in ’96 by Blennow et al. The
focus of their study was the synaptic path of AD [42]. The conclusion was that neither ApoE4
nor neurofibrillary tangles affected synaptic loss, but it was also observed that Aβ levels do not
seem to differ with regards to the presence or absence of the E4 allele. However, it is now known
that the unique structure of ApoE4 contributes to its ability to enhance Aβ production [64]. In the
same year, McGreer et al noticed that ApoE4 enhances complement activation for inflammation
by Aβ in vitro [8]. It was observed that this degenerative pathology by ApoE4 is exclusive to
Aβ-type amyloidosis. The common conclusion from all these studies was that ApoE4 plays a
huge Aβ-dependent role in AD pathology by virtue of its strong interaction with Aβ. Some
studies such as LaDu 1994 had different observations. In 1994, LaDu et al found that ApoE3
bound more avidly to Aβ than ApoE4 [43]. The same authors however, made an interesting
observation in 1995. Congruent with their prior study, they stated that unpurified or recombinant
forms of ApoE4 and E3 showed a greater ApoE3/Aβ complex [44]. They went on to explain that
when purified, the isoforms expressed similar avidity towards Aβ. These observed interactions
are probably due to the fact that ApoE3 promotes Aβ clearance in vivo [65]. Regarding purified

Page 21 of 31
ApoE isoforms there is some truth to LaDu’s findings. With recombinant ApoE, however, the
vast majority of studies show Aβ association in favor of ApoE4. Ming Li’s study in 1997 [45],
for example, used recombinant ApoE isoforms and observed a 3-fold higher binding activity in
favor of ApoE4.
After establishing that ApoE4 has a strong Aβ-associated role in AD, the next step was to
look at the underlying mechanism. One of the pivotal studies on topic is Golabek’s study on the
interaction between Aβ and ApoE [7]. After re-establishing that ApoE interacts with Aβ and
observing that Aβ sequence determinants are not decisive for Aβ/ApoE binding, Golabek et al
looked at the conformational aspect of things. They inferred that ApoE has a greater affinity for
Aβ peptides with high beta-sheet content from observations that Aβ in senile plaques, which are
predominantly beta-sheet in conformation, are complexed to ApoE and ApoE carboxyl
fragments. The idea put forward was that ApoE4 modulates Aβ molecules to adopt or stabilize a
pathological beta-sheet. Observations of ApoE structure show a 30% beta-sheet content and it
was proposed that this beta-sheet motif could act as a chaperone for random coil Aβ peptides.
This interaction with random coil Aβ is critical in the sense that, although it has lower affinity,
ApoE binding random coil Aβ will promote more fibril formation. This is solidified by the fact
that ApoE/beta-sheet Aβ interaction is not very fibrillogenic. All these observations have been
confirmed by numerous studies which have concluded that with regards to Aβ plaque formation,
Aβ secondary conformation is important. The study used purified isoforms of ApoE and no
isoform-specific differences were observed; this gives some truth to LaDu’s observation in 1995.
It is now understood that ApoE4 enhances Aβ deposition [19].
In 2005, Cheng et al studied the functional interaction between ApoE4 and low density
lipoproteins (LDL) in AD [46]. It was observed that the ligand binding domain of LDL receptors

Page 22 of 31
interacts with lipoproteins containing ApoE and ApoE4 binds the receptor with higher affinity
(congruent with Wisniewski 1994). This higher affinity binding, as has been stated before can
reduce the transfer of lipoproteins into the cell. The result of this is more cholesterol in plasma
which was seen to increase Aβ formation. This characteristic of ApoE4 was noted in the
evolutionary perspective as a sign of ApoE4’s primitiveness. This study by Cheng goes to show
that even in relation to functionality, ApoE4 indirectly affects amyloidosis and thus AD
pathology. Though most studies show that ApoE4 promotes Aβ fibrillogenesis and deposition,
some studies show that it promotes Aβ production from APP [64]. A study by Yadong Huang,
one of the most prolific personalities in AD research, showed a 60% increase in Aβ production in
ApoE4 cells as compared to ApoE3’s 30% [64, 66]. These studies confirm that ApoE4 promote
amyloidosis via increased Aβ production and Aβ fibrillogenesis as well as decreased Aβ
clearance.
There has been some focus, more recently, on neuronal death as it relates to AD
pathology. The bulk of these studies focused on the effects of neuronal fragments of ApoE4. In
1997, Tolar et al associated neurotoxicity with a 22kDa amino-terminal cleavage fragment of
ApoE. They observed that the ApoE4 isoform fragment was significantly more toxic than that of
ApoE3 [47]. They went on to assess the effectiveness of ApoE receptor blockers on this
phenomenon in chick and rat nerves in vitro. What was discovered was that when receptors were
blocked effectively, neurotoxicity was decreased, suggesting that the neurotoxicity of the
fragment is receptor-mediated. The same authors compared ApoE and its 22kDa fragment in
1999 [48]. They used protease inhibitors to prevent ApoE cleavage and noticed a reduction in
neurotoxicity of ApoE. This suggested that the fragments may account for its toxicity. They also
observed increased intracellular calcium levels in both situations which were reduced by

Page 23 of 31
blocking the receptors for ApoE. Calcium influx has been associated with neurotoxicity [49].
Michikawa et al, in 1998, investigated isoform specific effects of ApoE on neurons. They
suppressed de novo cholesterol synthesis by an inhibitor and observed the effect of ApoE [50]. It
was observed that ApoE4 bound to beta-migrating VLDL (an atherogenic lipoprotein) caused
premature neuronal cell death with apoptotic characteristics. This effect was not observed in
ApoE3. Michikawa went on to add cholesterol metabolites and observed that neurons were
rescued. It was concluded that a decrease in the level of endogenously synthesized cholesterol
seem like a prerequisite for ApoE4 induced death. Congruent with these studies was Hagiwara’s
in 2000. Hagiwara et al confirmed that ApoE4 causes significant neuronal cell death [51]. The
point of the study was to figure out whether this was related to its receptor-binding domain or to
its LDL receptor-related protein (LRP) binding domain. It was observed that if the LRP-binding
domain is cleaved, ApoE4 still exerts neurotoxicity. Recall that the amino-terminus is the
receptor-binding domain of ApoE. These findings by Hagiwara further prove the neurotoxicity
of N-terminal ApoE4 fragment that Tolar studied [14, 15]. Still on the neuronal death aspect, a
study was carried out by Ji et al in 2002 on the relationship between ApoE4 and lysosomal
leakage. Ji noted that in vitro studies show that Aβ, particularly Aβ42, induces neuronal death in
part via apoptotic pathways [52]. It was observed that in ApoE4 secreting cells, the effects of
Aβ42 were significantly greater than in ApoE3 cells. It was speculated that the underlying
mechanism has something to do with oxygen radicals which oxidize other molecules to induce
damage to intracellular membranes. Some studies have shown that to reduce toxicity, Aβ either
undergoes extracellular proteolysis or is taken up by receptor-mediated endocytosis for
lysosomal degradation [53, 54]. It was shown that the receptors required a ligand that forms a
complex with Aβ; one such ligand is ApoE. The problem with lysosomal degradation of Aβ is

Page 24 of 31
that Aβ42 is resistant to lysosomes and its accumulation in the vesicles results in leakage and
eventually apoptosis. It was observed that after the removal of extracellular sources of ApoE4,
the apoptotic effects were still present. This led to the inference that the potentiation of apoptosis
is dependent on intracellular ApoE, presumably in lysosomes. Their speculation that ApoE4
forms a reactive intermediate that, in concert with Aβ42, causes disruption of membranes and
leakage is supported by other studies. One of such studies showed that at a low pH, much like in
lysosomes, ApoE4 is more reactive; much more than ApoE4 [19]. As further confirmation that
neuronal/intracellular ApoE4 is the most pathogenic. Buttini et al did a study on ApoE4 and
neuronal exitotoxicity [55]. It was demonstrated that astrocyte-derived ApoE4 is exitotoxic in
vivo, whereas neuronal ApoE4 resulted in the loss of cortical neurons by promoting exitotoxic
death. It was speculated that an imbalance between the two sources may increase susceptibility
to diseases involving exitotoxic mechanisms. These are just a few studies and trials that prove
that ApoE4 is detrimental in AD.
There are not as many studies on Tauopathy as there are on Aβ interactions, but the few
that are available provide a clear idea of how the presence of ApoE4 is more detrimental than the
absence of the other isoforms. Some studies even show that ApoE3 is pathogenic, but just at a
lower level than ApoE4. As of 1999, it was confirmed that ApoE relates to Tau and Aβ but the
mechanisms were not very clear. DeMattos et al experimented by nucleus targeting of ApoE to
find out if ApoE had access to the cytosol [56]. It was discovered that cytosolic expression of
ApoE is cytotoxic. It was observed that the nucleus-targeted ApoE did not get to the nucleus;
thereby suggesting that in natural settings, ApoE remains in the endocytic pathway. The result of
their findings argued that the effects of ApoE on the cytoskeleton, as mentioned in Strittmatter
1995 were not mediated by cytosolic interaction but rather by actions originating at the surface

Page 25 of 31
(receptor-mediated signaling cascades). These findings are concurrent with Tolar’s research on
neurotoxicity of ApoE but their argument on cytoskeleton effects were proved invalid by a study
in 2001 by Huang et al [57]. In that study, further work was done on truncated ApoE. Huang et al
proved that removing the carboxyl terminal of ApoE (leaving the amino-terminal) induces NFT-
like inclusions in neuronal cells. It was observed that ApoE4 did this at a greater rate than ApoE3
because ApoE4 is more susceptible to truncation. They localized the determining factor to the
sequence containing amino acid 112 where the two isoforms differ. This sequence is responsible
for the domain interaction between ApoE4’s arginine-61 and glu-255 that many researchers
believe to be the crux of the detrimental effects of ApoE4 [7, 19, 26] as it makes the isoform
more susceptible to proteolytic cleavage. As such, they concluded that ApoE4 preferentially
undergoes processing to bioactive amino-terminal fragments and interacts with cytoskeleton
components, thereby causing neurotoxicity. This offers an explanation for the observed
cytotoxicity in DeMattos’ study. It is now known that ApoE3 binds preferentially to non-
phosporylated Tau while ApoE4 stimulates Tau phosphorylation which leads to NFT formation
[26]. The toxic effects of ApoE4 have been shown to be limited to neuronal cells. In 2010, Leoni
et al confirmed a direct correlation between ApoE, brain cholesterol and Tau. They stated that
cholesterol metabolism may be involved in the generation of both Tau and Aβ and that ApoE
may be released by astrocytes to counteract this ongoing process [58]. More work is to be done
in this regard. Andrews-Zwilling observed that ApoE4 causes and age and Tau-dependent
impairment of GABAergic (Gamma-Aminobutyric acid) interneurons leading to memory and
learning deficits in mice [59]. A study by Chang et al in 2005 showed that the amino terminal (or
receptor binding domain) is responsible for ApoE4 escaping the endocytic pathway while the
carboxyl terminal mediates mitochondrial interaction [60]. Other studies show that the positively

Page 26 of 31
charged amino acids in the receptor-binding domain allow ApoE4 fragments to translocate
across the membrane compartments of endocytic or secretory pathways [26]. The lipid-binding
domain has been show to be the cause of the mitochondrial dysfunction observed in AD via
interaction with components of the respiratory complex. Tesseur et al observed that transgenic
mice expressing human ApoE4 in neurons developed axonal degradation as a result of
impairment of axonal transport; a phenomenon not observed in ApoE3 mice [61]. This includes
axonal transport of mitochondria which would result in energy starvation in areas that need it and
local disruption of calcium homeostasis [62, 63]. Thus ApoE4 contributes to AD pathology by
mitochondrial dysfunction. These are just a few of the thousands of studies that prove that the
presence of ApoE4 is more detrimental than the absence of ApoE3.
It is clear from all these studies that there is more hard evidence supporting the claim to
pathology as compared with the evolutionary perspective. The evolutionary perspective may
have some truth, but most of it is based on speculation. From the research on ApoE4 it is now
clear that ApoE4 plays both Aβ-dependent and Aβ-independent roles in the pathogenesis of
Alzheimer’s disease. The Aβ-dependent roles are increased production and deposition, and poor
clearance of Aβ [19, 64, 65]. The Aβ-independent roles include its detrimental effects on
neuronal plasticity, Tauopathy, mitochondrial impairment and anomalous proteolysis to produce
neurotoxic fragments [26, 64]. These studies provide evidence that through a variety of
interactions and functional effects, ApoE genotype polymorphism may affect the rate of AD
progression and potentially, response to treatment. ApoE should be included as a covariate in AD
therapeutic trials. This would reduce the variability and as such increase the likelihood of

Page 27 of 31
developing an appropriate and beneficial treatment. The relevance of these studies to AD
treatment or prevention would involve aiming to inhibit or antagonize ApoE function.
8. Conclusion (Where does this leave us?)
There have been various ideas for treatment of Alzheimer’s disease. Among them are
drugs that inhibit cholinesterase (as a result of the 1976 discovery of cholinergic deficits
stemming from cholinergic neuron loss in AD), antioxidant treatments, drugs that treat
behavioral and psychiatric symptoms and acetylcholinesterase inhibitors [2]. All of these seem to
be somewhat beneficial but their focus is on the symptoms of AD. There is now speculation
about targeting the underlying mechanism such as APP cleavage and Aβ immunization. The
discovery of ApoE4 as the greatest genetic risk factor for AD gives new hope for the possibility
of preventative therapies that will target its effects.
Drugs that target the Aβ-dependent effects of ApoE4 are being researched, but in addition
to these, more attention should be paid to the Aβ-independent effects of the isoform. A potential
treatment for ApoE4-related AD was proposed by Andrews-Zwilling in 2010. It was proposed
that reducing Tau and enhancing GABA signaling may work [59]. More research is required for
this to be a possible treatment. A potential strategy is to inhibit the domain interaction that is
believed to be the root of ApoE4’s detrimental effects. A study is underway to disrupt ApoE4’s
domain interaction thereby making it structurally and functionally closer to ApoE3 [26]. Another
strategy is to inhibit the enzyme that mediated ApoE4 fragmentation. The identity and
characterization of this protease is required but protease inhibition is undoubtedly an important
possibility for drug development. Considering Tauopathy, a means to block ApoE4 fragment
interaction with cytoskeletal elements could be a promising area of research. Such a discovery

Page 28 of 31
would lead to elimination of the fragment-induced cytotoxicity that has been confirmed. Before
any of these can be achieved there are some obvious questions that need to be answered. For
example, what are the characteristics of the ApoE-cleaving enzyme? What kinase causes
hyperphosphorylation of Tau? How do ApoE4 fragments promote Tau phosphorylation? These
are just a few unanswered questions in the field of Alzheimer’s research that if answered, would
prove invaluable to the future development of successful therapies. Despite the missing pieces, it
is clear that potential combination therapies which utilize drugs targeting Aβ-dependent and
independent pathologies of ApoE4 will be extremely effective.
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