Is not a surprise that Alzheimer’s

Apolipoprotein E disease is not a single entity which is caused by

gene, APOE a single factor, instead results from a variety of

genetic and non-genetic factors that converge

Francisco Monroig Torres to develop the disease. One of these genetic

Prof. Juan C. Martínez Cruzado factors that contribute the arousal of AD is the
September 20, 2012
presence of the epsilon-4 (ε4) allele of the
apolipoprotein (apoE) gene (APOE). This variant
Alzheimer’s disease (AD) is a
of the APOE gene increases the risk of
progressive neurodegenerative disorder and
developing the AD from 20% to 90%, and
the most common form of dementia in elderly,
decrease the mean age of onset from 84 to 68
affecting an estimated 2.4 million to 4.5 million
years. Even that it is evident that the presence
[6]
of Americans . Depending if you are the only
of this gene increase the risk of developing AD,
member with the disease it is considered as
cases are known where healthy individuals with
sporadic AD but if two or more members of one
the allele, even with a homozygous genotype,
family are diagnosed with the disease now it is
do not develop the disease, affirming that this
called familial AD. This disease has a tendency
disease is caused by a variety of genetic and
to appear in people older than 65 years. In
non-genetic factors.
cases where the patient develop the disease
before 65 years it is considered as early onset, The human APOE gene is positioned in
[11]
while the late-onset form appears after age 65. the long arm of chromosome 19(19q13.20 .

Scientists has found a variety of gene mutations Its physical location is from base pair

that can explain the early onset familial AD. The 45,409,038 to 45,412,649 base pair and consists
[15]
inheritance of one of these mutant alleles of four exons and three introns , figure 1.

always results in the development of the Some genes near APOE include TOMM40,

disease, even though this type of the disease APOC1, APOC2 and APOC1P1, more precise

accounts for less than 10% of all AD cases. The information is found in table 1.

most prevalent form of the disease, in

The APOE gene provides instructions for
approximately 90-95% of all AD cases, is the late
making a protein called apolipoprotein E. This
onset sporadic AD where the mean age of
protein is a member of a family of
developing the disease fluctuates between 70
Apolipoproteins, carrier proteins, which
to 80 years.
combine with lipids to form lipoprotein

1
particles, which have hydrophobic lipids at the
core and hydrophilic side chains made of amino
acids. There are several types of lipoproteins
ranging in density, from very low density
lipoproteins (VLDL), or very high density
[7]
lipoproteins (VHDL) . Apolipoprotein E is an
important constituent of atherogenic remnant
lipoproteins particles, including
chylomicrons and very low density lipoproteins
(VLDL), and functions as the ligand of the
receptor-mediated clearance of these particles
by the liver for excretion [7] .
In humans, the most common isoforms
of the ApoE protein are denoted as ApoE2,
ApoE3 and ApoE4 and are encoded by three
alleles at the same gene locus, named ϵ-2, ϵ-3
and ϵ-4, respectively. The differences of these
isoforms are caused by a single amino acid
substitution at residues 112 and 158. The most
common isoform of all, ApoE3, has a cysteine
residue at 112 and an arginine at 158. ApoE4
isoform is produced when cysteine at the 112
residue is substituted by arginine, meanwhile,
ApoE2 occurs when arginine in the 158 residue
[12]
is substituted by cysteine . Other variants of
the ApoE do exist but they are extremely rare,
these are named APOE ϵ-1, APOE ϵ-5 and APOE
ϵ-7. The polymorphic nature of the APOE gene
is not constrained to these aforementioned
SNP’s that cause the three major isoforms.
Several SNP’s have been identified in the
proximal promoter region, and sequencing of
the APOE gene has revealed as many as 21
SNP’s as well as one diallelic indel and one
[10]
multiallelic indel , which can cause a variety
of chronic diseases, table 2.
The first step in APOE gene expression
is the transcription to a 1163 bp mRNA. Then
translation of the apoE mRNA leads to the
lipolysed
production of a pre-apoE isoprotein of 317
amino acids, which contain an 18-amino amino
terminal signal peptide. It is the pre-apoE
isoprotein which co-translationally across the
membrane of the endoplasmic reticulum and
undergoes proteolytic processing and
glycosylation to generate the mature protein of
34.2kDa comprised of 299 amino acid, a major
component of various plasma lipoproteins [12].
The secondary structure of the
apolipoprotein E can be divided into three
main portions. The amino terminal end (up
to residue 165) is highly ordered, the next
35 residues make up a random structure,
and the carboxyl terminal portion becomes
highly ordered again. The majority of the
secondary structure, about 62%, is formed
from alpha helices, which are amphipathic
and important in lipid binding. The rest of
the secondary structure is made up of beta
sheets (9%), beta turns (11%), and random
structure (18%). The area of the protein
with the strongest lipid binding is found in
2
the carboxyl terminal portion,
residues 202-209. The five arginine and
three lysine residues between residues 140
and 160 are essential for binding to the LDL
(low-density lipoprotein) lipid receptor
Figure 2. The protein contains two distinct structural
domains: a 22-kDa amino-terminal domain and a 10-kDa
carboxyl-terminal domain.
The apoE gene has several single-
nucleotide polymorphisms,
[10],
distributed across the gene
of them with the capacity to induce
different types of diseases. However, in the
human population the most common SNP’s
are three alleles mentioned before, ϵ-2, ϵ-3
and ϵ-4. Although the three common isoforms
differ by only one or two amino acids at
from residues 112 or 158, these differences alter
apoE structure and function [9].
Studies have found that in all human
populations the ϵ-3 version of the APOE gene
[9].
was the most frequent [2]. The protein encoded
by this allele shows to carry out normally its
functions. The less common allele is the ϵ-2
version. In the figure 4 we can see how the
substitution of arginine to cysteine in the 158
residue disrupts the salt bridge that exist in the
apoE3 between Arg-158 and Aspartic in the 154
residue, causing Asp-154 to interact with Arg-
150. These structural differences reduce the
positive potential of the LDL receptor binding
site. Individuals with this allele increase
triglyceride and cholesterol levels caused by
delayed clearance of hepatic and intestinal
remnant lipoproteins, resulting in
Hyperlipoproteinemia, type III. By last, the ϵ-4
version of the gene caused by a substitution of
cysteine to arginine in the 112 residue, in
comparison with the “normal” APOE3 allele.
SNP’s, This amino acid substitution results in a salt
bridge between Arg-112 and Glu-109 which
and several
causes the Arg-61 chain to extend away from
[9]
the four-helix bundle . The orientation of
Arg-61 in apoE4 promotes domain
interaction by interacting with Glu-255, in
the C-terminal domain, within the lipid
binding region, causing apoE4 to have a
more compact conformation than apoE3.
3
Domain interaction is an important
structural property of apoE4 because it
alters the lipid binding region and, thus,
lipoprotein preference [14,5]. Because of
these lipoprotein preference this allele it
has been related with some diseases as
Hyperlipoproteinemia, type III, multiple
sclerosis and Alzheimer’s disease. ApoE3
and apoE2 are less likely to demonstrate
domain interaction.
ApoE is produced in several organs
like the spleen, heart, lungs, liver, brain,
adrenal and kidney. The liver is the major
site of ApoE’s mRNA expression and is
responsible for approximately three
quarters of the ApoE circulating in the
plasma. Within the liver, the primary cells
responsible for ApoE production are the
hepatic parenchymal cells. The second
highest level of ApoE’s mRNA is found in the
brain where studies have found that they
are predominantly expressed by astrocytes
and microglia cells. Even though neurons do
not synthesize this protein they have,
however, receptors to endocytose or
produce ApoE in certain situations, and the
presence of the protein in the vesicles of
these cells has been demonstrated []. In the
CNS, it is the most abundantly produced
apolipoprotein [9].
The protein encoded by APOE is a
multifunctional protein with diverse roles
through the body which uses different
metabolic pathways in the body. One of
these pathways is endocrine-like, and
involves the redistribution of lipids among
cells of different organs. It takes lipids from
the areas where are synthesized and
distributes them to other areas to be used
or stored. Another pathway is paracrine-
like, where the lipids are transported
among cells in the same organ or tissue.
Apo- E is also involved in various pathways
that are unrelated to lipid transport, such as
the stimulation of lymphocytes[9]. The steps
involved in the first two pathways
aforementioned begin with the interaction
of apoE with different types of lipoproteins
localized in the circulation, specifically HDL,
LDL and VLDP. When the protein is
synthesized by the liver as part of VLDL it
functions in the transport of triglycerides to
the liver tissue. It is also incorporated into
HDL (as HDL-E) and functions in cholesterol
distribution among cells. ApoE it can also be
incorporated into intestinally synthesized
cholymicrons and transports dietary
4
triglycerides and cholesterol. It is involved of gene alleles we can suggest that the

in lipid metabolism by mediating the hypermethylation of the APOE CpG island

located in the exon 4 can be an explanation of
receptor binding of apo-E lipoproteins to
the dominance of the major alleles of the
the LDL receptor. Receptor binding begins
gene.
the cellular uptake of lipoproteins to be
used in intracellular cholesterol metabolism
[9].

Analysis of the APOE gene using the

UCSC genome browser revealed that APOE
belongs to a group of genes that do not
possess a classical CpG island in their
promoters, but rather a low-CpG density
region [weber]. However, the gene contains a
high-density CGI at its 3’ end that covers exon
4, which contains the sequences for the major
Figure 5. APOE gene CpG islands, one
haplotypes (ε2, ε3 and ε4), which determine
hypomethylated in the 5’ end and one hypermethylated at
risk to develop AD [weber], figure 5. The the 3’ end.
impact of DNA methylation on APOE
I have chosen this gene because of
promoter activity itself is unknown since the
different reasons. The first is that every
regulation of APOE is highly complex and
individual posseses this gene and one of its
does not only rely on the 5′-promoter, it also
variations increases enormously the risk of
requires an interaction of proximal and distal
developing the Alzheimer’s disease, one of the
regulatory regions with transcription factors
most common diseases in the world,
to impart a net effect on APOE expression
including Puerto Rico, while other variation
[weber]. The ε4 sequence (that is usually
at the same gene locus seems to provide
associated with a higher risk of LOAD) may
certain protection to the disease. Even though
change the epigenetic function of the
this gene is not sufficient to produce the
methylated 3′-CGI since the ε4 allele
disease by itself, it has been found a
introduces a C → T transition that is
numerous ways by which contributes to the
associated with a loss of a methylatable CpG
principal pathologies of the disease. Another
unit [weber]. Because DNA methylation is
reason that interested me to study this gene
known to be involved in parental imprinting

5
is that its frequency and effect in AD patients
varies within ethnic groups. The frequency of
the APOE ε-4 allele is higher in Caucasian
populations but lower in both African-
American and Hispanics populations [2]. The
diversity of this gene within populations
makes Puerto Rico a perfect place to study
the prevalence of this gene in the general
population and how the differences in genetic
admixture among individuals and geographic
localization affect the distribution of this
gene.

6
 Table 1: Genes that surround APOE

Gene Start End Direction Function

PVRL2 45349393 45392485 Forward This gene
encodes a single-
pass type 1
membrane
glycoprotein. This
protein is one of
the plasma
membrane
components of
adherences
junctions.
TOMM40 45394477 45406946 Forward This protein is
the channel-
forming subunit
of the
mitochondrial
outer membrane
complex that is
essential for
protein import to
mitochondria.
APOC1 45417921 45422606 Forward The protein
encoded by this
gene is a member
of the
apolipoprotein C1
family. This gene
is expressed
primarily in the
liver.
APOC1P1 45430060 45434643 Forward APOC1
pseudogene
APOC2 45449239 45452822 Forward This gene
encodes a lipid-
binding protein
belonging to the
apolipoprotein
gene family.

7
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