Contact Jennie Sims and Connie Wang
jennie.sims.r@gmail.com, kwwang@caltech.edu
Assessing Differences in Function and Expression
Between Young and Aged Microglia
SENS Foundation Academic Initiative Grant Proposal
OVERVIEW
Microglia are bone marrow-derived glial cells that serve as the phagocytic immune cells of the
central nervous system (CNS). Microglial dysfunction has been hypothesized to play a role in
many diseases. In particular, age-related loss of function may have a significant role in
Alzheimer’s disease (AD), the most frequent cause of dementia in the elderly.
In this proposal, we request a $3,000 grant to characterize loss of microglial function with age,
using a variety of functional assays and gene expression assays. The results of these assays may
provide insights into the mechanisms behind Alzheimer’s disease. They may also form a
baseline for the behavior of “normal” young and old microglia to aid in the assessment of
potential future therapies.
1
Contact Jennie Sims and Connie Wang
jennie.sims.r@gmail.com, kwwang@caltech.edu
TABLE OF CONTENTS
OVERVIEW……………………………………………………………………………………………… 1
BACKGROUND……………………………………………………………………………………….. 3
Microglia: Function and Senescence……………………………………………….. 3
Relationship to Alzheimer’s disease………………………………………………… 3
PREVIOUS RESEARCH……………………………………………………………………………… 5
RESEARCH PLAN………………………………………………………………………………..…… 6
SPECIFIC AIMS………………………………………………………………………………………… 8
TEAM AND FACILITY….…………………………………………………………………………… 9
BUDGET….…………………………………………………………………………………………..… 11
SCIENTIFIC REFERENCES………………………………………………………………………… 12
2
Contact Jennie Sims and Connie Wang
jennie.sims.r@gmail.com, kwwang@caltech.edu
BACKGROUND
Microglia: Function and Senescence
Microglia make up about 5-12% of total brain cells1, 2 and act as the main immune cells of the
CNS3. In healthy brains, microglia perform various neuroprotective functions by phagocytosing
protein plaques, sick neurons, and other pathogens4, as well as by secreting cytokines and
neurotrophic factors5. They typically take on a ramified, inactive form with long processes14,
changing to an amoeboid morphology and becoming phagocytic upon detection of injury1. As
one of the few neural cell types that undergo significant cell division 5, microglia are
hypothesized to be affected by replicative senescence.
Senescent rat microglia have been found to have shorter telomeres5 and larger, more
deramified morphologies6 with fragmented cytoplasms7. Older microglia have also been
observed in ex vivo studies to secrete higher amounts of inflammatory cytokines than younger
controls8, 16. This increase in inflammatory cytokine production leads to an increase in
acetylcholinesterase activity, which has been shown to negatively affect cognitive function 17.
These observed differences between young and old microglia are especially significant because
many microglial functions have the potential to be both neuroprotective and neurotoxic 9,
which has confounded the results of previous microglia studies. For example, oxidative burst, a
spike in ROS levels and production of lysosomal proteases, is an important microglial function
that protects the brain from pathogens1. However, this same mechanism has also been shown
to induce neuronal death in studies of old and dysfunctional microglia7, 10, 11.
Relationship to Alzheimer’s disease
Both the neuroprotective and neurotoxic roles of microglia have implicated them as important
factors in Alzheimer’s disease (AD). AD is an age-related disease characterized by memory loss,
extracellular amyloid-β (AB) deposition12, formation of intracellular neurofibrillary tau tangles10,
loss of neuronal synapses, and significant neuronal death5. AD is the most common cause of
dementia in the elderly, afflicting one in ten individuals over the age of 65 and nearly half of
those over the age of 8513.
In particular, amyloid-β plaques appear to play a key pathogenic role in AD15. Microglia have
been shown not only to be recruited to AB plaques5, but also to phagocytose and break down
AB in vitro12. Additionally, microglia isolated from mouse models of AD express lower levels of
3
Contact Jennie Sims and Connie Wang
jennie.sims.r@gmail.com, kwwang@caltech.edu
AB-degrading enzymes, as well as lower levels of AB receptor proteins (scavenger receptors) 16,
implying that AB degradation by microglia may play a major role in Alzheimer’s disease’s
pathology.
Tau protein tangles, another hallmark of Alzheimer’s disease, have also been associated with
microglia in various ways. AB buildup has been seen to immediately precede tau tangle
formation in AD models10, and neuroinflammation from microglia can exacerbate
phosphorylation and aggregation of tau protein18. In addition, microglial accumulation has been
observed to match the progression rate of AD16, and activated microglial expansion appears to
precede massive neural cell death19.
All of these studies suggest that microglia play a key role in the control of AD, its pathogenesis,
or both. Furthermore, the age-related nature of Alzheimer’s disease and the types of microglia
found around AD-associated plaques seem to indicate that aging microglia play a key role in the
pathology of AD16.
4
Contact Jennie Sims and Connie Wang
jennie.sims.r@gmail.com, kwwang@caltech.edu
PREVIOUS RESEARCH
Both morphological and secretory distinctions between young and old microglia have been
investigated in previous studies, suggesting that there are differences in functional efficiency
and expression profiles that should be characterized further5, 6, 7,8,16.
In particular, lysosomal acidification has been shown to be important in the breakdown of
amyloid-β (AB), and the lysosomal pH of active microglia has been shown to be lower than that
of inactive microglia3, 4. It is also significant that microglia from adult mice are less effective in
phagocytosing fibrillar AB than microglia from postnatal mice7. These studies are the
motivation behind our investigation of lysosomal acidification genes Ostm1 and Clc-7. Studies
have not yet compared the lysosomal acidities of young and old mouse populations.
Importantly, previous studies have shown that marker expression and actual functional capacity
do not always correlate1, indicating the need for functional assays as well as an analysis of gene
expression profiles.
5
Contact Jennie Sims and Connie Wang
jennie.sims.r@gmail.com, kwwang@caltech.edu
RESEARCH PLAN
In order to characterize microglia isolated from mice, it is necessary to determine if they are
able to perform phagocytosis, maintain adequate lysosomal acidity, and express an oxidative
burst. All of these features are key components of microglial function. A comparison will be
done with isolated mouse lymphocytes to assess similarity to non-neuronal phagocytic immune
cells.
Phase 1: Isolation of mouse microglia and lymphocytes.
Phase 2: Phagocytosis assays will be performed to examine differences in the functionality of
young and old microglia, and to confirm that microglia are still viable in accordance with
current theories. Microglia will be assessed for the ability to produce an oxidative burst,
phagocytose E. coli, and maintain lysosomal acidity.
Phase 3: Isolation of mRNA and qRT-PCR analysis. Once the functionality of the microglia has
been ascertained, mRNA will be isolated. Real time PCR analysis may show differences in gene
expression of housekeeping genes, lysosomal acidification genes such as Clc-7 and OSTM-1,
scavenger receptor genes such as SRA and CD36, protein degradation genes such as IDE and
neprilysin, and inflammatory cytokines such as IL-1B and IL-6. Savenger receptor genes that
remain constant with age, RAGE and MARCO, will be used as negative controls. Insulysin (IDE),
Neprilysin and MMP9 will be used as positive controls for genes that decrease in expression
with age. TNFα and IL-1β will be used as positive controls for genes that increase in expression
with age
Research Methods
Collection and Purification of Microglia
Prior to primary cell isolation, animals will be euthanized and brain tissue collected by the
supplier. Brain tissue will be shipped in media at 4°C.
Neural tissue will be collected from dissected mice and dissociated using a neural dissociation
kit (130-092-628, Miltenyi Biotec, CA). The procedure will be altered to use the same enzyme
mixes with longer incubation times and less harsh mechanical dissociation (using a 3mL plastic
transfer pipette) to reduce shearing. After tissue dissociation into single cell suspension,
6
Contact Jennie Sims and Connie Wang
jennie.sims.r@gmail.com, kwwang@caltech.edu
magnetic separation will be performed using magnetic CD11b microbeads and magnetic
separation columns (130-042-201, Miltenyi Biotec, CA).
Phagocytosis Assay
Microglia will be incubated for two hours at 37°C, followed by two hours in the presence of E.
coli that have been transformed with a GFP-containing plasmid. The SENS Foundation already
holds stocks of E. coli that have been confirmed by fluorescence microscopy to express GFP.
The presence of the GFP will be detected by flow cytometry in three conditions: E. coli alone,
microglia alone, and combined microglia/E. coli, for both young and old microglia. Fluorescence
detected in the microglia population of the experimental microglia/E. coli sample will indicate
that the microglia has phagocytosed GFP-expressing E. coli. Mononuclear effector cells from
peripheral blood will be enriched by lymphocyte separation medium according to the
manufacturer’s protocol, and used as a positive control.
Acidification Assay
The lysosome acidification assay (Invitrogen, P35361, NY) will be performed according to the
manufacturer’s instructions, and the results will be analyzed using the Becton Dickinson
FACScan flow cytometer.
Reactive Oxygen Species (ROS) - Oxidative Spike Assay
Cells will be incubated with dihydrorhodamine 123 (D-23806, Invitrogen, NY), which will be
taken up by the cell and oxidized to cationic rhodamine 123, causing green florescence that will
be measured using the Becton Dickinson FACScan flow cytometer.
7
Contact Jennie Sims and Connie Wang
jennie.sims.r@gmail.com, kwwang@caltech.edu
SPECIFIC AIMS



Characterize isolated microglia on the basis of gene expression using RT-PCR.
Confirm that isolated microglia retain the functional characteristics described in previous
studies.
Correlate the functional assays with gene expression data.
8
Contact Jennie Sims and Connie Wang
jennie.sims.r@gmail.com, kwwang@caltech.edu
TEAM AND FACILITY
Team
Kelsey Moody
Kelsey currently serves as a research scientist at SENS Foundation, and heads the Research
Center’s OncoSENS program. He has extensive experience in the methods described, having
worked as Chief Technology Officer at ImmunePath, Inc, a biotechnology start-up specializing in
cell-based therapies. He will serve as Principal Investigator on the project, and will directly
supervise the work of all members of the team.
Dave Halvorsen
After completing his BSc, David spent several months as an intern at ImmunePath, Inc. culturing
stem cells. He then interned at the SENS Foundation Research Center, producing lentivirus and
working with the flow cytometer. He currently works as a researcher on the OncoSENS project.
Jennie Sims
Jennie is a senior at the University of Colorado, Boulder, where she is pursuing a triple major in
molecular, cellular, and developmental biology, integrative physiology, and neuroscience, with
a minor in leadership. She has two years of molecular and cellular biology laboratory
experience.
Connie Wang
Connie is a junior at the California Institute of Technology, where she is pursuing a double
major in bioengineering and business, economics, and management. Connie has laboratory
experience in molecular biology and bioimaging.
Thomas Hunt
Thomas Hunt is an aspiring scientist who is interning at the SENS Foundation Research Center.
He has a broad understanding of lab procedures, including aseptic technique and cell and tissue
culturing.
9
Contact Jennie Sims and Connie Wang
jennie.sims.r@gmail.com, kwwang@caltech.edu
Facility
All work on this project will be conducted at the SENS Foundation Research Center in Mountain
View, California. The Research Center has a fluorescence microscope and Becton Dickinson
FACScan flow cytometer for fluorescence-based assays, a real-time PCR machine (ABI Prism
7700) for qRT-PCR, and mammalian cell culture equipment.
10
Contact Jennie Sims and Connie Wang
jennie.sims.r@gmail.com, kwwang@caltech.edu
BUDGET
Kits/Materials
Cost ea.
Qty
Total cost
Magnetic mRNA Isolation Kit
268.00
1
268.00
SYBR Master Mix kit
109.00
1
109.00
ROS Assay kit (Dihydrohodamine 123)
181.92
1
181.92
RT-PCR Primers
12.53
15
187.95
MultiScribe Reverse Transcriptase
74.00
2
148.00
pHrodo Phagocytosis Kit
873.99
1
873.99
Lymphocyte separation medium
31.78
1
31.78
Water Bath Sonicator
250.00
1
250.00
SUBTOTAL
2050.64
Husbandry
Cost ea.
Qty
Total cost
30 mo. C57/BL6 mice
168.00
5
840.00
1 mo. C57/BL6 mice
28.00
5
140.00
Food, bedding, etc.
50.00
1
50.00
SUBTOTAL
1030.00
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Contact Jennie Sims and Connie Wang
jennie.sims.r@gmail.com, kwwang@caltech.edu
SCIENTIFIC REFERENCES
TOTAL
1.
3080.64
Hinze, A. & Stolzing, A. Differentiation of
mouse bone marrow derived stem cells toward microglia-like cells. BMC Cell Biology 12,
35 (2011).
2. Kettenmann, H., Hanisch, U.-K., Noda, M. & Verkhratsky, A. Physiology of microglia.
Physiol. Rev. 91, 461–553 (2011).
3. Majumdar, A. et al. Activation of microglia acidifies lysosomes and leads to degradation
of Alzheimer amyloid fibrils. Mol. Biol. Cell 18, 1490–1496 (2007).
4. Majumdar, A., Capetillo-Zarate, E., Cruz, D., Gouras, G. K. & Maxfield, F. R. Degradation
of Alzheimer’s amyloid fibrils by microglia requires delivery of ClC-7 to lysosomes. Mol.
Biol. Cell 22, 1664–1676 (2011).
5. Flanary, B. The role of microglial cellular senescence in the aging and Alzheimer diseased
brain. Rejuvenation Res 8, 82–85 (2005).
6. Streit, W. J., Sammons, N. W., Kuhns, A. J. & Sparks, D. L. Dystrophic microglia in the
aging human brain. Glia 45, 208–212 (2004).
7. Floden, A. M. & Combs, C. K. β-Amyloid Stimulates Murine Postnatal and Adult Microglia
Cultures in a Unique Manner. J. Neurosci. 26, 4644–4648 (2006).
8. Njie, E. G. et al. Ex vivo cultures of microglia from young and aged rodent brain reveal
age-related changes in microglial function. Neurobiol. Aging 33, 195.e1–12 (2012).
9. Luo, X.-G., Ding, J.-Q. & Chen, S.-D. Microglia in the aging brain: relevance to
neurodegeneration. Molecular Neurodegeneration 5, 12 (2010).
10. Blasko, I. et al. How chronic inflammation can affect the brain and support the
development of Alzheimer’s disease in old age: the role of microglia and astrocytes.
Aging Cell 3, 169–176 (2004).
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Contact Jennie Sims and Connie Wang
jennie.sims.r@gmail.com, kwwang@caltech.edu
11. Parvathenani, L. K. et al. P2X7 Mediates Superoxide Production in Primary Microglia and
Is Up-regulated in a Transgenic Mouse Model of Alzheimer’s Disease. J. Biol. Chem. 278,
13309–13317 (2003).
12. Simard, A. R., Soulet, D., Gowing, G., Julien, J.-P. & Rivest, S. Bone marrow-derived
microglia play a critical role in restricting senile plaque formation in Alzheimer’s disease.
Neuron 49, 489–502 (2006).
13. Kawas, C., Gray, S., Brookmeyer, R., Fozard, J. & Zonderman, A. Age-specific incidence
rates of Alzheimer’s disease: the Baltimore Longitudinal Study of Aging. Neurology 54,
2072–2077 (2000).
14. Nimmerjahn, A., Kirchhoff, F. & Helmchen, F. Resting microglial cells are highly dynamic
surveillants of brain parenchyma in vivo. Science 308, 1314–1318 (2005).
15. Lee, J. K. et al. Intracerebral transplantation of bone marrow-derived mesenchymal stem
cells reduces amyloid-beta deposition and rescues memory deficits in Alzheimer’s
disease mice by modulation of immune responses. Stem Cells 28, 329–343 (2010).
16. Hickman, S. E., Allison, E. K. & El Khoury, J. Microglial dysfunction and defective betaamyloid clearance pathways in aging Alzheimer’s disease mice. J. Neurosci. 28, 8354–
8360 (2008).
17. Li, Y. et al. Neuronal–Glial Interactions Mediated by Interleukin-1 Enhance Neuronal
Acetylcholinesterase Activity and mRNA Expression. J. Neurosci. 20, 149–155 (2000).
18. Bhaskar, K. et al. Regulation of tau pathology by the microglial fractalkine receptor.
Neuron 68, 19–31 (2010).
19. Wada, R., Tifft, C. J. & Proia, R. L. Microglial activation precedes acute
neurodegeneration in Sandhoff disease and is suppressed by bone marrow
transplantation. Proc. Natl. Acad. Sci. U.S.A. 97, 10954–10959 (2000).
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