Adult olfactory neuron turnover and the association
between fractalkine and microglia
Rebecca Fernandes Mello1, Dr. Kathleen Guthrie2
1Honors
2 Department
Thesis Undergraduate Program, Department of Biological Sciences, Charles E. Schmidt College of Science
of Biomedical Science, Charles E. Schmidt College of Medicine, Florida Atlantic University, Boca Raton, Florida, 33431
Abstract
In the adult mouse brain, there are neuroblasts born in the forebrain subventricular
zone (SVZ). These migrate to the olfactory bulb (OB), where they mature and replace
older neurons that are eliminated by programmed cell death (PCD). Continuous
neuronal turnover in the OB is important for mouse behaviors since they rely on odor
signals to survive. What initiates PCD in the OB is unknown. Microglia participate in
PCD by phagocytising apoptotic neurons. Microglia express the fractalkine receptor
(CX3CR1) and neurons express fractalkine (CX3CL1), a known ligand protein from the
chemokine family which functions in the immune system. However, its function in the
adult brain is unclear. Our hypothesis is that dying olfactory neurons upregulate
fractalkine during PCD, which then signals microglia to help eliminate that cell. If
fractalkine is informing the microglia to eliminate pre-existing neurons, then it may have
a role in olfactory neuron turnover in adult brain.
Summary of Findings
Results
Presence of fractalkine and microglia within the hippocampus
Figure 1: Immunofluorescent images of brain
tissue collected by confocal microscopy.
Hippocampal dentate gyrus shows microglia
with Iba1-IR (immunoreactivity, top right) and
fractalkine positive cell bodies in the granule
cell (GC) layer (bottom left). A labeled
microglial cell with processes is indicated by
the white arrow in the top right image. Dapi
staining of cell nuclei is shown in the upper left
Background
image. As reported by previous studies,
fractalkine is present within the hippocampal
Previous Studies:
Adult neurogenesis subgranular zone & subventricular zone [3,4]
Hippocampus & olfactory bulb gain new neurons
Synaptic pruning within the hippocampus done by microglia [6]
dentate gyrus.
Olfactory Bulb Neurons:
Constant cell turnover, when older neurons die and are replaced by new neurons [3]
Neurogenesis in the olfactory bulb: production of new neurons from stem cells in the
subventricular zone, these then integrate within the olfactory bulb granule cell layer [4]
Programed cell death takes place regularly in the adult olfactory bulb [3,4]
Fractalkine (CX3CL1):
Chemokine family of proteins [1]
High concentration in the brain,
including olfactory bulb [5]
Previous report that it is expressed by a few bulb granule cells
and in the hippocampus [8]
Neurons may signal to microglia
through fractalkine [5]
Presence of fractalkine and microglia within the olfactory bulb (OB)
CX3CR1
This model depicts the hypothetical role of fractalkine in membrane-bound form and secreted form and its
interaction between neurons and microglial cells. Adapted from Nishiyori et al., FEBS Letters 1998.429:
167-172.
Future Work
Figure 2: Deep granule cell layer of the OB with fractalkine-positive cell bodies shown in the left panel. Microglia
expressing Iba-1 are shown in the middle panel. The white arrows indicate colocalization of fractalkine with
microglia (merged image). However, there is also fractalkine expressed throughout the granule cell layer.
Diagram showing the rostral migratory stream
(RMS) of a rodent, with stem cells and
neuroblasts (1,2) migrating along the RMS (3)
to the adult olfactory bulb (4). From Ming and
Song, Ann Rev Neurosci 2005.28:223-250.
RESEARCH POSTER PRESENTATION DESIGN © 2012
The confocal microscopic image is shown as
an orthogonal projection of a stack of images
collected through the depth of the cell.
DAPI
Anti-Caspase 3
Immunofluorescence
Staining
Figure 4: A dying neuron in the bulb granule
Confocal Imaging
Confocal Zeiss LSM 700
Microglia
cell layer expressing activated caspase-3
(green) in the cell cytoplasm. This neuron will
go through programmed cell death and we
Immunofluorescent
Staining
Compare labeling
in Confocal Images
Confocal Zeiss LSM 700
H
Healthy
lth
mice
References
1. Bruserud Ø, Kittang AO. (2010) The Chemokine System in Experimental and Clinical Hematology. Curr. Top
Microbiol. Immunol. 341, 3–12.
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Research Reviews 20 (3): 269–287.
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Growth Differ. 51:37986
4. Ma DK, Bonaguidi MA, Ming G, and Song H. (2009) Adult neural stem cells in the mammalian central nervous
system. Cell Research 19: 67282.
5. Nishiyori A, Minami M, Ohtani Y, Takami S, Yamamoto J, Kawaguchi N, Kume T, Akaike A, Satoh M. (1998)
Localization of fractalkine and CX3CR1 mRNAs in rat brain: does fractalkine play a role in signaling from
neuron to microglia? FEBS Lett 429: 167–172.
6. Paolicelli, RC, Bolasco G, Pagani F, Maggi L, Scianni M, Panzanelli P, Giustetto M, Ferreira TA, Guiducci E,
Dumas L, Ragozzino D, and Gross CT. (2011) Synaptic pruning by microglia Is necessary for normal brain
development. Science 333: 1456458.
7. Ribak CE, Shapiro LA, Perez ZD, and Spigelman I. (2009) Microglia associated granule cell death in the normal
adult dentate gyrus. Brain Struct. Funct. 214: 2535.
8. Tarozzo G, Bortolazzi S, Crochemore C, Chen SC, Lira AS, Abrams JS, and Beltramo M. (2003) Fractalkine
protein localization and gene expression in mouse brain. J. Neurosci. Res.73: 8188.
assume it will later be phagocytosed by
microglia. Fractalkine expression (red) is not
seen at this stage in this cell.
Fractalkine
Anti-fractalkine
www.PosterPresentations.com
Figure 3: A microglial cell in the OB granule
Neuron dying by PCD within the OB
Nucleus
Anti-IBA1
Hippocampus
Olfactory Bulb
Tissue Sections
30 m
shown in red and Iba-IR is shown in green.
Dying Neurons
Tissue Sections
30 μm
Huntington's
disease mice
at 11 weekss
immuno-reactivity (IR). Fractalkine-IR is
Utilizing an antibody to a specific protein marker for microglia (Iba-1, Wako Chemicals)
and a fractalkine-specific antibody (R & D Systems), we will evaluate the expression of
fractalkine by OB neurons. Dying cells will be identified using antibody for activated
caspase-3 (Cell Signaling Tech.) expressed by apoptotic cells.
Olfactory
Bulb
Try additional fractalkine antibodies to see if the staining pattern is consistent
Look for changes in OB fractalkine expression under conditions of increased cell
death: mouse model of Huntington’s disease shows increased granule cell death
cell layer showing patches of fractalkine
Experimental Design
Female
C57B16
6
Fractalkine (Membrane-bound form)
Fractalkine (Secreted form)
Presence of fractalkine within microglia
Microglia:
Express fractalkine receptor (CX3CR1) [5]
Immune system of the brain [2]
Macrophage lineage [2]
Participate in programmed cell death [2,7]
Fractalkine is present in both the hippocampus and the olfactory bulb (Fig. 1-3)
Fractalkine is expressed throughout the bulb granule cell layer (Fig. 2-3)
There may be an interaction between fractalkine and microglia in the olfactory
bulb, since microglia express the fractalkine receptor, CX3CR1, and some
microglia had fractalkine-IR associated with them, as shown in Figure 3
The antibody used for fractalkine does not show a specific association between
fractalkine and activated caspase-3 (Fig. 4)
In contrast to a prior study using a different antibody, fractalkine is expressed
throughout the olfactory bulb granule cell layer and hippocampal dentate gyrus
and is not limited to the small number of cells that would be undergoing
programed cell death (as shown in Figures 1-4)
Although it is associated with some microglia (Fig. 2-3), fractalkine is not likely to
be a specific signal to the microglia, if this pattern of antibody staining is
accurately showing fractalkine expression and distribution
Acknowledgements
We would like to thank Dr. Evelyn Frazier, Dr. John R. Nambu, Ramon Garcia-Areas, fellow
honors peers, and fellow lab mates.