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Altered Expression of Cellular Markers in Molecular Brain Aging
by
Anulika Nwakaeze
A thesis submitted in conformity with the requirements for the degree of Master of Science
Graduate Department of Pharmacology and Toxicology
Altered Expression of Cellular Markers in Molecular Brain Aging
Anulika Nwakaeze
Master of Science
Graduate Department of Pharmacology and Toxicology
University of Toronto
2016 ABSTRACT The molecular aging of the human brain encompasses pervasive transcriptome changes
associated with “normal” brain aging that occur in local cortical circuits comprised of GABA
neurons, pyramidal cells, and astrocytes. Cortical samples obtained from a novel postmortem
human cohort were analyzed using RT-qPCR technology to assess changes in the expression of
nine markers representative of neuropeptide signalling, synaptic function, calcium regulation,
and glial activation within cortical cellular networks that occur in congruence with a pervasive
molecular program mediating brain aging. Presynaptic interneuron markers displayed decreases
in expression in aged subjects compared to young controls, whilst postsynaptic interneuron
marker expression was increased, as was the expression of glial markers. Although changes in
extrasynaptic glutamate marker expression were not significantly different, there was a nominal
decrease in their expression in aged subjects. Altogether, these results replicate findings of
pervasive molecular changes in the aged cortex, and suggest consequences for cortical network
function.
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ACKNOWLEDGEMENTS
I would like to take this opportunity to express my sincere gratitude to my supervisor, Dr.
Etienne Sibille, for accepting me into his prestigious lab and for providing me with counselling
and guidance throughout my degree. Additionally, I am grateful to my advisor, Dr. Peter
McPherson, for his understanding and patience with me during difficult moments of my graduate
studies, and to my committee members for their valuable insights into my project.
I would also like to extend my appreciation to my colleagues in the Sibille lab, particularly to
Drs. Brad Rocco, Hyunjung Oh, and Yuliya Nikolova, who mentored me in this exciting project.
Their knowledge and expertise in molecular biological and data analytical techniques were
instrumental to my success in the program.
Finally, I am endlessly grateful to my parents for their unwavering support, love, and
encouragement.
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TABLE OF CONTENTS Abstract ........................................................................................................................................... II
Acknowledgements ....................................................................................................................... III
List of Tables ................................................................................................................................ VI
List of Figures .............................................................................................................................. VII
List of Abbreviations .................................................................................................................. VIII
List of Appendices ........................................................................................................................ IX
Chapter 1: Introduction and Background ........................................................................................ 1
Research objectives .................................................................................................................... 2
Literature review ........................................................................................................................ 2
An introduction to aging ..................................................................................................... 2
Normal brain aging ............................................................................................................. 3
Definition of normal brain aging ............................................................................ 3
Functional changes in normal brain aging .............................................................. 4
Molecular changes in normal brain aging ............................................................... 6
Altered neuropeptide gene expression in GABAergic neurons ................ 10
Altered intracellular calcium signalling in local cortical circuits ............. 12
Altered GABAergic and glutamatergic transmission across synapses ..... 14
Altered activity of glial cells ..................................................................... 16
Molecular aging of the prefrontal cortex .......................................................................... 18
GFAP (glial fibrillary acidic protein), ALDH1L1 (aldehyde dehydrogenase 1 family member
L1), and GLUL (glutamine synthase). Thirty samples of human grey matter from prefrontal
cortex tissue of Brodmann areas 11 and 47 (BA11 and BA47; orbitoventral prefrontal cortex)
that were obtained from the McGill University brain bank at the Douglas Institute were
processed into 100 µm thick sections using the Leica CM1950 cryostat system. Two grey
matter sections of 100 µm thickness were immersed in 350 µL of Buffer RLT (Qiagen
miRNeasy) for homogenization. Total RNA was extracted using the Qiagen RNeasy® Micro
protocol. Concentration and purity of RNA were determined by absorbance ratios at 260, 280,
and 230 nm using the Implen P360 NanoPhotometer. RNA integrity values were generated
using the Agilent 2100 BioAnalyzer microfluidics system. Subsequently, cDNA was
synthesized from 1 µg of total RNA using the Superscript VILOTM kit and gene expression was
quantified by real time polymerase chain reaction using the Bio-Rad Universal SYBR green kit
and Bio-Rad C1000 touch thermal cycler. Finally, gene enrichment was analyzed as a function
of the quantification cycle (Chq) values obtained from the real-time reaction and expression of
cellular markers were presented as a geometric mean of the ratio of the target mRNA copies to
mRNA copies of the reference genes beta-actin, GAPDH, and cyclophilin G. Data was
processed to determine differential expression of the target genes in aged vs. control subjects,
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and to demonstrate a linear effect of age on gene expression, as well as to suggest a concerted
movement of gene expression changes with age.
RT-QPCR EXPERIMENTAL PROTOCOL
Summary of RT-qPCR technology
Fluorescence detection modules contained within the RT-qPCR instrument monitor the
fluorescence signal as amplification proceeds. The measured fluorescent signal is proportional
to the total amount of amplicon, i.e., the amplified sequence of interest. The change in
fluorescence over time is used to determine the amount of amplicon produced in each PCR
cycle. RT-qPCR allows the user to assess the initial copy number of the target sequence with
accuracy and sensitivity and without the need for gel electrophoresis.
Data generated from RT-qPCR is displayed as an amplification plot consisting of the number
of PCR cycles on the x-axis, usually ranging from 0-40 cycles, and relative fluorescence units
on the y-axis. Amplification plots are typically divided into two phases – an exponential phase,
during which the amount of PCR product approximately doubles in each cycle, and a non-
exponential phase, in which the reaction slows and reaches a plateau due to reagent exhaustion.
Initially, fluorescence remains at background levels during the exponential phase until enough
amplified product accumulates to yield a detectable fluorescence signal. The cycle number at
which fluorescence crosses past the threshold is known as the quantification cycle, or Cq. The
Cq of a reaction is largely determined by the initial amount of nucleic acid template present at
the start of the amplification. The Cq of a sample is inversely proportional to the initial amount
of template available in the reaction, such that a low, or early, Cq is indicative of larger
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amounts of starting material present in the reaction because relatively fewer amplification
cycles are required to accumulate enough product to breach threshold and produce
fluorescence above background levels. This relationship forms the quantitative aspect of RT-
qPCR.
The RT-qPCR reagents utilized for this experiment were obtained from Bio-Rad and consisted
of the SsoAdvancedTM Universal SYBR® Green supermix, including buffer, DNA polymerase,
dNTPs, and a dsDNA-binding dye. Specific primer pairs were designed to optimize RNA
detection using the web-based Primer3Plus software, and cDNA was generated from extracted
RNA to serve as the template for the reaction. Specificity of the RT-qPCR assay was assessed
by confirming a single product peak in the melt curve analysis provided by the real-time
instrument.
Sample Collection for RT-qPCR
Fresh-frozen cortical blocks of Brodmann Areas BA11 and BA47, corresponding to the
orbitoventral prefrontal cortex (PFC), were obtained from the Douglas Institute Brain Bank
(Canada) from male subjects without a diagnosed neurodegenerative or neuropsychiatric
disorder. Thirty samples were used in this study and were previously delineated into two
groups on the basis of age, such that control subjects (n = 13) were grouped under age 45 and
aged subjects (n = 17) were grouped above age 60. Subject groups differed in mean age (p =
1.55E-15) but did not significantly differ in other demographic parametres of postmortem
interval (PMI; p = 0.24), brain pH (p = 0.06), or RNA integrity number (RIN; p = 0.84) as
determined by UNIANOVA (Figure 2).
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0.0#
20.0#
40.0#
60.0#
80.0#
Age#
Age$(years)$
Demographic$Differences$in$Age$
Comparison#
Aged#
p#=#1.55E915#
0.0#
5.0#
10.0#
15.0#
20.0#
25.0#
30.0#
PMI#
PMI$(ho
urs)$
Demographic$Differences$in$PMI$
Comparison#
Aged#
p#=#0.24##
1.0$
3.0$
5.0$
7.0$
9.0$
11.0$
13.0$
pH$
pH#
Demographic#Differences#in#pH#
Comparison$
Aged$
p$=$0.06$$
2.0$
4.0$
6.0$
8.0$
10.0$
RIN$
RIN$
Demographic$Differences$in$RIN$
Comparison$
Aged$
p$=$0.84$
Figure 3. Demographic characteristics of age-cohort.
Comparison and aged subjects were significantly different in mean age (p = 1.55E-15) but did
not significantly differ in PMI (p = 0.24), pH (p = 0.06), or RIN values (p = 0.84). Error bars
represent standard error.
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Grey matter was identified in the cortical blocks on the basis of gross anatomy and was
selectively harvested into 100 µm sections using a Leica CM1950 cryostat set to -20°C in an
RNAse-free environment. Cortical blocks were approximately 1.5 cm3 in volume, thus the
dimensions for the cryosections were comparable across subjects. Two grey matter
cryosections of 100 µm thickness and approximately 0.75 cm2area were collected for each
subject and immediately immersed in 350 µL of chilled Buffer RLT (Qiagen miRNeasy) for
manual homogenization using RNase-free disposable pellet pestles (Fisherbrand™). Control
and aged samples were collected in a semi-randomized (alternating) manner to minimize order
effects in sample collection93. Following homogenization, samples were transferred on dry ice
and stored at -80°C until further processing.
Demographic and postmortem characteristics of human subjects Comparison subjects Aged subjects
Case Sex Age (yrs)
PMI (hrs) pH RIN
Cause of death Case Sex
Age (yrs)
PMI (hrs) pH RIN
Cause of death
DH1025 M 41 14.5 5.89 5 Natural DH411 M 76 10.75 5.95 4.4 COPD DH787 M 43 23.75 6.25 2.8 Thrombosis DH476 M 73 27.5 6.17 4.4 COPD S11 M 19 32 6.35 2.3 Natural
DH505 M 69 23.5 6.04 2.9 CA
S15 M 30 30 6.22 4.4 Accident
DH530 M 67 24.75 6.23 6.5 AP S16 M 28 27 6.59 3.3 Natural
DH580 M 69 34.12 5.85 4.2 N/A
S17 M 41 24 5.95 2.4 Accident
DH598 M 79 21.92 6.4 4.3 PE S173 M 20 12 6.22 2.4 Natural
DH650 M 73 10 5.46 2.4 N/A
S20 M 31 29.5 6.49 5.6 Accident
DH724 M 70 32.75 6.02 2.8 HC S215 M 43 27 6.33 6.4 Accident
DH745 M 63 16.75 6.02 5 MI
S250 M 26 12 6.75 3.2 Natural
DH776 M 70 26 5.76 2.4 N/A S31 M 21 24 6.27 4.3 Accident
DH796 M 69 27.58 5.6 4.5 MI
S36 M 27 20.5 6.18 5 Natural
DH880 M 62 6.17 6.15 4.2 VH S94 M 15 27 5.95 4.7 Natural
DH974 M 72 24.5 6.36 6.1 MI
DH988 M 66 20.5 6.09 4.3 TD
S101 M 63 13 6.84 3.9 N/A
S187 M 71 17 6.2 3.8 N/A
Table 1. Demographic and postmortem information of study sample. Total of 30 subjects divided into 13 young comparison subjects and 17 aged subjects. M, male; PMI, postmortem interval; RIN, RNA integrity number; COPD, chronic obstructive pulmonary disease; CA, cardiac arrest; AP, acute peritonitis; PE, pulmonary edema; HC, hemorrhagic colitis; MI, myocardial infarction; VH, ventricular hypertrophy; TD, thromboembolic disease; N/A, not available
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RNA Isolation and Conversion to cDNA
Total RNA was isolated according to the Qiagen RNeasy® micro protocol. Briefly, tissue
samples were homogenized and subjected to centrifugation to separate the supernatant
containing total RNA. Genomic DNA (gDNA) was separated from the preparation using a
gDNA spin column provided in the kit. Subsequent steps purified the RNA, which was eluted
using RNase-free water. Approximately 12 µL of RNA was collected per sample.
Concentration and purity of RNA were determined using a nanospectrophotometer to assess
absorbance ratios. Samples with A260/A280 ratios greater than 1.9 were accepted for
downstream processing, as commonly acceptable A260/A280 ratios for nucleic acids is ~2.
A260/A230 ratios were lower than the commonly acceptable range of 2.0-2.2 (Appendix 1).
This may be attributed to residual RNA extraction buffers in the eluents. This did not,
however, impact the extraction of high concentration RNA to sufficiently create enough
reagents for cDNA synthesis. RNA integrity was assessed to determine the extent of RNA
degradation within the samples using an Agilent 2100 Bioanalyzer System. RNA integrity
numbers (RIN) were generated for each sample. The generally high level of degradation found
within the samples was noted for downstream measures to counteract possible processing
difficulties from degraded RNA.
1 µg of RNA per sample was used to synthesize cDNA using the Superscript VILOTM kit. The
required volumes of reagents were calculated according to the following paradigm:
Components Volume (µL) Nuclease-free water x
Reaction mix 4 Enzyme mix 2
RNA (1 µg) TOTAL 20
29
The reaction mix and the enzyme mix are set to 4 µL and 2 µL per 20 µL reaction, respectively.
The required volume of RNA that amounted to 1 µg of product was added to the reaction tube
and the volume of nuclease-free water was adjusted such that the final volume was 20 µL. A
no-reverse transcriptase control was included to control for genomic DNA (gDNA)
contamination. Reaction tubes were placed in a Bio-Rad T100 thermal cycler set to an adapted
Superscript VILOTM protocol. The program consisted of an initial incubation at 25˚C for 10
minutes, followed by incubation at 42˚C for 120 minutes to allow cDNA synthesis. Reaction
termination was set at 85˚C for 5 minutes, and the samples were held at 4˚C. The program is
summarized below.
Step Temp (˚C)
Time
Incubation 25 10 min Incubation 42 2 h
Termination 85 5 min Hold 4 ∞
Newly synthesized cDNA was diluted 1:4 with nuclease-free water by adding 80 µL into the
reaction tubes to bring the final volume to 100 µL. Samples were stored at -20˚C until further
processing.
Samples were then tested for viability with a validated in-house actin primer. Average Cq
values were calculated and standard errors less than 0.2 standard deviations from the mean of
the biological sample were used for analyses 94,95. Samples were standardized to an actin Cq
value of 20 by adding nuclease-free water according to the following formula(Appendix 2):
30
[ 2 !"!!"!"# − 1 𝑥 𝑉𝑜𝑙𝑢𝑚𝑒
Newly standardized samples were then stored at -20˚C until further processing.
RT-qPCR Primer Design, Preparation, and Testing
Primers were designed using the Primer3Plus software(Appendix 3). Primers were restricted to
80 base pairs to target short RNA fragments as a preemptive measure to account for RNA
degradation96,97. Sequences were selected that encoded the longest isoform of the transcript
variant. Optimal Tm was set to 60˚C. The first three available forward and reverse primer
sequence pairs representing non-overlapping base pairs were selected for testing and
validation.
100 µM stock solutions were prepared for all forward and reverse primers by adding the
amount of nuclease-free water equal to 10 times the nanomolar amount of the primer.
Lastly, 10 µL of the forward and 10 µL of the reverse primers were added to 980 µL of
nuclease-free water in a 1.5 mL Eppendorf tube to create a 1 mL working solution of each
primer. The working solutions were stored at -20˚C and at4˚C after thawing.
Primer sequences were tested for greatest affinity with an in-house validated actin primer and
stock human cDNA to determine which of the sequence pairs functioned most optimally. Each
primer pair was tested in triplicate to calculate an average actin Cq and standard errors less
than 0.2 standard deviations from the mean of the biological sample were used for analyses
94,95. No template controls were included to control for the presence of contaminants and
primer-dimer formation. Primer pairs that produced earlier amplifications of actin in the human
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sample, and thus lower Cq values, and also tested negative for contaminants or primer-dimers,
were selected for downstream use.
RT-qPCR Program and Assay
The RT-qPCR program used for the Bio-Rad C1000 touch thermal cycler was set according to
the Bio-Rad Universal SYBR green protocol. The program consisted of an initial denaturation
step carried out at 95˚C for 30 seconds. This was followed by 40 qPCR cycles consisting of
denaturation at 95˚C for 15 seconds and annealing/extension at 60˚C for 30 seconds, followed
by a plate read to generate a melting curve. Lastly, a final round of denaturation at 95˚C for 30
seconds, followed by a final extension at 65˚C for 30 seconds and a hold at 20˚C for 5 minutes.
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