THE EFFECTS OF PRENATAL LEAD EXPOSURE
ON THE DEVELOPMENT OF BIOMARKERS OF ALZHEIMER’S DISEASE
by
Andrew Wood
A Senior Honors Project Presented to the
Honors College
East Carolina University
In Partial Fulfillment of the
Requirements for
Graduation with Honors
by
Andrew Wood
Greenville, NC
May 2015
Approved by:
Jamie C. DeWitt
Department of Pharmacology and Toxicology
Brody School of Medicine
ABSTRACT - While often well known for its adverse effects on memory, Alzheimer’s disease
(AD) is also noteworthy for being the sixth leading cause of death in the United States. An
estimated 5.2 million Americans had the disease as of 2013, with the number of cases expected
to increase in coming decades. Research into the condition has not revealed its exact cause;
however, AD is believed to result from a combination of genetic vulnerabilities and
environmental influences. An important aspect of AD and this study is the development of
plaques or clumps of amyloid beta (AΒ) protein in the brains of those with AD, with increased
concentrations of AΒ indicating a more severe condition. Microglia, resident immune cells of
the brain, are thought to influence the level of these proteins in the brain. Prenatal exposure to
environmental contaminants may influence the actions of microglia during an organism’s
lifetime in a way that encourages the development of AD and its observable plaques. This study
explored both the separate and combined effects of environmental agents and genetic
predispositions to the disease. This was done by exposing both 3x-Tg-AD mice, that have a
genetic predisposition to developing AD, and wild-type mice to lead prenatally. The level of AΒ42 in brains was measured by ELISA at varying ages, and the differences in AΒ-42 was compared
between wild-type and transgenic animals between doses for each age and each sex. The final
results showed a significant increase in AB-42 concentration in fully mature 3x-Tg-AD mice that
were exposed to lead. This evidence supports our hypothesis that developmental exposure to
an exogenous contaminant prenatally would exacerbate the tendency of genetically vulnerable
organisms to develop the pathologies of AD.
1
Table of Contents
Introduction
3
The Pathologies of Alzheimer’s Disease
3
The Role of Microglia in Alzheimer’s Disease
3
Critical Window of Development
5
Hypothesis
5
Relevance
5
Methods
7
Animals and Dosing
7
AB Levels
8
Statistical Analysis
9
Results
9
Discussion
14
Literature Cited
16
2
Introduction
The Pathologies of Alzheimer’s Disease
Alzheimer’s disease (AD) is the most common cause of dementia, a condition in which
the patient experiences behavioral abnormalities and a loss of cognitive functioning (U.S.
Department of Health and Human Services [USDHHS], 2011). While these mental effects are
well known symptoms of AD, those with the disease also show physical abnormalities within
the brain itself. The first of these biological markers of AD is the formation of neurofibrillary
tangles within the neurons of the brain (USDHHS, 2011). These tangles are due to an
accumulation of abnormal tau protein. While this protein typically supports the inner transport
system of neurons, the brains of AD patients show a large number of these proteins breaking
away from their normal position and clumping together to form tangles (U.S. Department of
Health and Human Services [USDHHS], 2013-2014). The second major neuropathological
biomarker of AD is the abnormal accumulation of amyloid beta (AΒ) protein plaques within the
brain. Small clumps of AB along with the larger accumulated plaques may interfere with the
ability of neurons to function and communicate properly. A buildup of tangles and plaques can
result in the death of large numbers of neurons, eventually resulting in the loss of cognitive
function that is characteristic of patients with AD (USDHHS, 2013-2014). These biomarkers can
also be used to confirm the presence and extent of AD in a patient after death (USDHHS, 20132014).
The Role of Microglia in Alzheimer’s Disease
Microglia cells are the resident immune cells of the brain. Their normal functions include
directing the actions of the immune system locally and, when necessary, phagocytizing
3
damaged or dead cells and cellular debris (Solito & Sastre, 2012). These actions are important
to maintain the normal brain environment. However, there is also evidence that the microglial
response to inflammation can actually harm neurons. As an individual ages, microglia become
less efficient in function, becoming over-activated when stimulated and producing too harsh a
reaction to stimulation (Solito & Sastre, 2012). Such prolonged responses of inflammation are
detrimental to neurons and could exacerbate or even lead to an environment that culminates
in AD. Microglia therefore are thought to have positive and negative effects on the
development of AD. Microglia are being mentioned here because of the relationship that they
are thought to have with amyloid beta proteins. The “macrophage” role of microglia would
typically include the removal of excess AB protein found within the brain of a patient with AD
(Solito & Sastre, 2012). Accumulating evidence therefore suggests that the existence of AB
plaques in AD patients indicates some sort of error in microglia function that either limits
phagocytosis of AB or increases deposition of AB. Increased AB levels have then been shown to
start a vicious “feed-forward cycle” with microglia cells. These unnaturally high levels of AB are
thought to activate microglia to increase local inflammation of immune system activity (Solito &
Sastre, 2012). This increased inflammation may affect the transcription of the enzyme
responsible for AB generation, β-secretase (Solito & Sastre, 2012). While the exact role of
microglia in the brain of a patient with AD is not fully known, it is generally agreed upon that
they play an important role in the disease. While this part of the project does not specifically
address microglia, they are being assessed as part of the larger project. However, it is important
to consider the role that they might play in AB deposition and/or clearance.
4
Critical Window of Development
While changes in the immune and nervous systems occur naturally as an organism ages,
changes may also occur due to events that occur early in life. Microglia have been shown to be
sensitive to pre-natal exposure to exogenous contaminants (Diz-Chaves, Pernía, Carrero, &
Garcia-Segura, 2012). Exposure to such contaminants during a “critical window” of microglia
development could alter microglia in a way that increases the potential of neurodegenerative
disorders, such as AD.
Hypothesis
Our overall hypothesis is that early-life exposure to an exogenous stimulus, such as an
environmental chemical, reprograms microglia and changes their responsiveness to other
signals, leading to early onset and/or increased severity of AD pathologies. For this portion of
the project, changes in levels of AB in wild-type and transgenic 3xTgAD mice after prenatal
exposure to lead will be evaluated. These transgenic mice have multiple mutations, one of
which increases their potential to develop amyloid beta plaques within the brain as they age.
Relevance
AD is not a single origin disease. It is thought to occur due to a combination of both
genetic and environmental factors (USDHHS, 2013-2014). The novelty of this study is the idea of
a “double hit” model that compares and contrasts the effects of both of these origins. This
“double hit” is accomplished by using both transgenic mice to observe genetic effects (hit 1)
and lead exposure to observe the effect of environmental influences (hit 2) on the development
of the biomarkers for AD. A second important facet of this study is the link between pre-natal
exposures to exogenous contaminants and diseases seen later in life. While many studies have
5
shown a positive correlation between these events, ours will investigate the “how” behind this
correlation by observing the contaminant’s effect on the form and function of microglia, and
the resultant effect on AB levels. Understanding the mechanisms behind AD development
would be very helpful when attempting to design prevention and treatment strategies that
focus on the developmental process.
6
Methods
Animals and Dosing
The study involved two separate strains of mice, the 3xTgAD transgenic mice and the
associated wild-type. The transgenic mice are genetically predisposed to developing behavioral
abnormalities, neurofibrillary tangles, and AB plaques. Two female mice of each strain were
paired with the appropriate males (transgenic females with transgenic males, and wild-type
females with wild-type males) overnight each week. The day after each pairing was designated
gestational day one (GD1). Females were then assigned to either a treated or a control group
and were then gavaged with 100 mg/kg (as 0.1 uL/10 g of body weight) of lead acetate or a
vehicle control from gestational days 7-11. This period of development was chosen as it is one
of several critical periods for microglial development/migration.
Litters were culled at postnatal day (PND) five, to three male and three female pups
when possible. Pups were weaned at PND21 and were sorted into same-sex sibling trios when
possible; single housing also was avoided when possible. Adult offspring were culled at PND 50,
90, and 180. The PND50 samples served as controls for AB levels and as a time point for very
early onset of neuropathologies. The PND180 samples represented an age when the transgenic
mice should show signs of neuropathologies. The PND90 samples were an “in-between” age for
early onset of neuropathologies.
7
AB Levels
Brains were immediately removed upon euthanasia and separated into specific sections.
The right and left hemispheres of the brain were separated and the hippocampus of the left
hemisphere was removed. The right hemisphere with hippocampus intact was snap frozen on
dry ice and kept frozen at -80°C until analysis. This portion of the brain was gently homogenized
using glass homogenizers and a homogenization buffer that included a protease inhibitor. This
solution was then mixed with a saline solution containing diethylamine and homogenized again.
The resulting liquid mixture was run on a centrifuge for 1 hour at 14,000 rpm. The homogenate
was then extracted and frozen at -80°C.
The right half of each brain was analyzed for AB-42 by enzyme linked immunosorbent
assay (ELISA). The assays were performed using Colorimetric BetaMark Beta-Amyloid x-42 ELISA
kits. The protocol for these kits involved adding diluted samples and a standard solution to a 96
well plate coated with an antigen specific to this assay. The standard solution was used to
create sequentially diluted solutions that varied from 0 to 250 pg/mL final concentrations. The
standards were allotted to two columns of the plate, leaving 80 wells in which samples were
run in singlets or duplicates. A detection antibody was then added to all wells before the plate
was placed in a 4°C fridge to incubate overnight. After incubation, the plate was washed to
remove excess antibodies from solution. A TMB solution was then added to all wells to start a
reaction that would “color” each well based on the amount of AB-42 each sample contained.
After a short incubation period, the plates were analyzed at 620nm using a microplate reader.
The absorbance of each sample was converted to the AB-42 concentration from the data given
by the standard curve.
8
Statistical Analysis
Data were evaluated with pairwise comparisons (t-tests) within age and within sex.
Comparisons within age and sex included mean differences in AB-42 concentration between
control and treated wild-type animals, control and treated transgenic animals, control wild-type
and transgenic animals, and treated wild-type and transgenic animals. Differences across age
and between sexes were not evaluated for this aspect of the study.
Results
At PND50, transgenic male animals treated with lead acetate had approximately 20% higher
AB-42 levels relative to control transgenic male animals (Figure 1). All other groups differed by
about 15% or less at this age. In brains of animals collected at PND90, levels of AB-42 in brains
of all control animals were equal to or greater than levels of AB-42 in brains of all treated
animals (Figure 2). However, this difference was only statistically significant in the female wildtype animals. When evaluated at PND180 (Figure 3), levels of AB-42 in brains of wild-type
animals were relatively equal between control and treated animals (differed by less than 15%,
on average). In the transgenic animals, however, levels of AB-42 in treated female animals was
nearly 60% greater relative to control animals and in treated male animals, was 87% greater
relative to control animals. The variability in the results for the male animals prevented this
difference from being statistically significant. At postnatal days (PND) 50 and 90, levels of AB-42
did not differ appreciably between control and treated animals or between wild-type and
transgenic animals (Figure 4).
9
Figure 1: Mean AB-42 concentration (pg/mL) among animals collected at postnatal day 50. AB42 from the transgenic (Psen) males treated with lead acetate was elevated relative to the
control Psen males (*).
10
Figure 2: Mean AB-42 concentration (pg/mL) among animals collected at postnatal day 90. AB42 from the wild-type females treated with lead acetate was reduced relative to the control
wild-type females (*).
11
Figure 3: Mean AB-42 concentration (pg/mL) among animals collected at postnatal day 180. AB42 from the transgenic (Psen) females treated with lead acetate was elevated relative to the
control Psen females (*).
12
A
B
Figure 4: Mean AB-42 concentration (pg/mL) among animals collected at postnatal day 50, 90,
and 180. A) Males. B) Females.
13
Discussion
Our hypothesis for this experiment was that early-life exposure to an exogenous stimulus, such
as an environmental chemical, reprograms microglia and changes their responsiveness to other
signals, leading to early onset and/or increased severity of AD neuropathologies. While the role
(or lack thereof) of microglia in this particular facet of the experiment was not evaluated, the
results do support our hypothesis of increased presence of AD neuropathologies, as indicated
by increased AB-42 levels, after a prenatal exogenous stimulus. In the PND 180 groups, the
treated transgenic mice had a large increase in AB-42 concentration over the control transgenic
mice. While prenatal exposure to lead did not increase the average concentration of AB-42 in
wild-type mice, it did exacerbate the tendencies of transgenic mice to develop this observed
neuropathology. One result that we did not observe in this experiment was an early-onset
increase in AB-42 concentration. Control transgenic animals had increased AB-42 at PND180
relative to the wild-type animals, but treated wild-type animals did not have increased AB-42 at
any time point. A similar experiment measured the expression of the APP gene (which produces
the precursors of AB) in rats exposed to lead early in life and in a separate group of rats
exposed to lead in old age. Early life lead exposure was found to increase APP expression late in
life; however, exposure to lead late in life did not result in a similar increase in expression
(Basha, Wei, Bakheet, Benitez,Siddiqi, Ge, Lahiri, & Zawia 2005). These observations are in
agreement with our findings of increased AB expression late in life. As treated transgenic
animals did not have increases in AB-42 at either PND50 or PND90 (Figure 4), this suggests that
some critical event occurs in the treated transgenic animals that is not occurring in the treated
wild-type animals. While additional work is ongoing in our laboratory to understand why this is
14
occurring, we hypothesize that the microglia in the treated transgenic animals are hypo- or
hypersensitive to the altered neuronal microenvironment induced by the mutations.
Some of our conclusions were confounded by the high variability of our results. While
the increased variability could have been caused by a number of factors, it was likely due to
pipetting error or malfunction causing an uneven amount of sample to be put in each sample
well. Extra care should be taken during this critical step of pipetting samples into their
respective wells, and any user should test pipette precision and accuracy before starting
procedures. This variability could also possibly be caused by denaturation of AB during the
homogenization procedure. Ethanol was used to clean homogenizers, and users should ensure
the glassware is completely dry before processing samples.
The real world application of these results will be to help formulate ideas and future
studies on the origins of AD. Our results support the idea that exposure to immunotoxicants
during early development can increase the probability of genetically predisposed individuals to
developing AD in the future. However, increased AB levels are only one aspect of AD
development. Further research would be needed to see if there is actual evidence of mental
impairment or neuronal damage as a result of a prenatal toxicant exposure.
15
Literature Cited
Basha, R. M., Wei, W., Bakheet, S. A., Benitez, N., Siddiqi, H. K., Ge, Y.-W, Lahiri, D.K., & Zawia,
N. H. (2005). The fetal basis of amyloidogenesis: Exposure to lead and latent
overexpression of amyloid precursor protein and β-amyloid in the aging brain. The
Journal of Neuroscience, 25(4), 823-829. doi:10.1523/JNEUROSCI.4335-04.2005
Diz-Chaves, Y., Pernía, O., Carrero, P., & Garcia-Segura, L. M. (2012). Prenatal stress causes
alterations in the morphology of microglia and the inflammatory response of the
hippocampus of adult female mice. Journal of Neuroinflammation, 9(71).
doi:10.1186/1742-2094-9-71
Harrington, C. R. (2012). The molecular pathology of Alzheimer's disease. Neuroimaging Clinics
of North America, 22(1), 11-22. doi:10.1016/j.nic.2011.11.003
Solito, E., & Sastre, M. (2012). Microglia function in Alzheimer’s disease. Frontiers in
Pharmacology, 3(14). doi:10.3389/fphar.2012.00014
Thies, W., & Bleiler, L. (2013). 2013 Alzheimer's disease facts and figures. Alzheimer's and
Dementia, 9(2), 208-245. doi:10.1016/j.jalz.2013.02.003.
U.S. Department of Health and Human Services. National Institutes of Health. (2012,
September). Alzheimer's disease fact sheet. (NIH Publication No. 11-6423). Retrieved
from http://www.nia.nih.gov/sites/default/files/alzheimers_disease_fact_sheet_1.pdf
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U.S. Department of Health and Human Services. National Institutes of Health. (2013-2014).
2013-2014 Alzheimer's disease progress report. Retrieved from
http://www.nia.nih.gov/alzheimers/publication/2013-2014-alzheimers-diseaseprogress-report/primer-alzheimers-disease-and#causes
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