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Microglia podosomes:
Characterization, Ca2+ regulation and potential role in migration
by
Tamjeed Ahmed Siddiqui
A thesis submitted in conformity with the requirements
Figure 16. Blocking CRAC channels results in loss of podosome (podonut) distribution
Figure 17. Immunolabeling of Orai1 show localization to podosome core in microglia
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Figure 18. Immunostaining of calmodulin (CaM) mostly shows localization to podosome
ring in microglia
Figure 19. Iba1 staining show localization to core in microglial podosomes
Figure 20. Immunolabeling of TRPM7 and podosome core in microglia
Figure 21. Updated schematic diagram showing podosome-associated molecules in
microglia based on findings in this thesis
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LIST OF TABLES
Table 1. Components that constitute podosomes, invadopodia and focal adhesions
Table 2. Summary of podosome components found in microglia and their corresponding
localization
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INTRODUCTION
Cell Migration
Cell migration is an important phenomenon that aids in various processes like
development of tissues, immune system function, angiogenesis and wound healing (Alberts
et al., 2008). It can also have a negative influence in pathological processes like tumour
metastasis. Cell migration is the result of a complex interplay between various intracellular
signalling pathways (e.g. Rho family GTPases, Cdc42, protein tyrosine kinases)(Ridley et al.,
2003; Alberts et al., 2008). Most of our understanding comes from studies based on different
cell types such as neutrophils, Dictyostelium, and fibroblasts (see reviews Lauffenburger and
Horwitz, 1996; Weiner, 2002; Le Clainche and Carlier, 2008; Vicente-Manzanares et al.,
2009). It is important to note that although cell migration is a well-studied phenomenon,
detailed mechanisms vary between different cell types and species (Becchetti and Arcangeli,
2010). Migration can be classified into two basic forms: random migration and directed
migration (Ridley et al., 2003; Gilbert, 2006). Random migration (chemokinesis) involves
intrinsic factors being activated that initiate migration without responding to any external
cues, such that cells migrate spontaneously in any direction. Directed migration, however,
adds more complexity to the intricate processes underlying cell migration. Migration
following an external cue, also known as chemotaxis, involves not only regulating the
migration machinery but also responding to the external stimuli via receptor signalling that
continually influences the migration machinery. In either case, cell migration is initiated by
inducing cell polarity and extension of the cell membrane and cytoplasm in the direction of
migration (Ridley et al., 2003). This establishes a leading edge at the cell front and a uropod
at the rear. The leading edge is the site of extensive assembly of cytoskeleton, mainly actin
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polymerization, protruding lamellipodia and a lamellum (Figure 1). Lamellipodia are sites of
rapid actin polymerization and protrusion that form membrane ruffles that have not yet
adhered to substrate. The lamellum, on the other hand, is located dorsal to lamellipodia, and
provides stability and traction by adhering to the extracellular matrix (ECM). The uropod in
the trailing end of migrating cells is a site of disassembly of cytoskeletal elements like
microtubules, and of retraction and detachment from ECM that allows cells to move forward.
In addition to signalling pathways that regulate cytoskeletal rearrangements,
regulation of intracellular Ca2+ is vital for cell migration (Evans and Falke, 2007). There is
evidence that many migrating cells maintain a descending Ca2+ gradient from rear-to-front of
the cell (Brundage et al., 1991; Laffafian and Hallett, 1995; Xu et al., 2004; Hoffmann et al.,
2009). Removal of extracellular Ca2+ impedes or stops cell migration, illustrating the
importance of local-, global- or spatiotemporally regulated Ca2+ concentrations during
migration (Brundage et al., 1991; Mandeville et al., 1995). Although it is believed that
migrating cells maintain a relatively low Ca2+ concentration at the cell front, Wei and
colleagues showed that directed migration is the result of localized Ca2+ rises in
microdomains (termed Ca2+ flickers) at cell front, which are short-lived and depend on
chemoattractant receptor signalling and membrane stretch (Wei et al., 2009). The authors
suggest that having a low Ca2+ background helps maintain a chemical driving force for Ca2+
entry through Ca2+ permeable channels at the cell front for subsequent initiation of Ca2+
signalling cascades necessary to steer the cells in a particular direction.
2
Figure 1. Schematic diagram of a migrating cell (Chhabra and Higgs, 2007).
Reprinted by permission from Macmillan Publishers Ltd: Nature Cell Biology. Chhabra ES, Higgs HN. The
many faces of actin: matching assembly factors with cellular structures. Copyright 2007
3
As important as regulation of signalling pathways and Ca2+ is to cell migration,
migrating cells also need to remodel the matrix in order to traverse through tissues (Alberts,
2009), which are made up of many cells that are held together by cell-cell interactions (e.g.
tight junctions) as well as cell-ECM contacts (Alberts, 2009). The ECM surrounding cells
serves not only as “glue” that allows cells to adhere for anchorage, but provides additional
support to keep the cells bound together. Characteristics of ECM in different tissues also
differ (Alberts, 2009); the components that make up the overall matrix are variable, leading
to differing rigidity, organization and fluidity. ECM typically consists of fibrillar collagens,
laminin and fibronectin, along with proteoglycans, glycosaminoglycans (GAGs) and
glycoproteins. The brain ECM, however, is very different from peripheral tissues, in that has
very little collagen and other commonly found ECM proteins (Bellail et al., 2004). Instead,
brain ECM primarily consists of glycosaminoglycan hyaluronan, proteoglycans, some of
which are specific for brain ECM (e.g. neurocan), and glycoproteins like tenascin-C. The
highly anionic hyaluronan, which is the major brain ECM component, binds hygroscopic
molecules (e.g., the cations, Na+ and Ca2+) and water (Bellail et al., 2004). The brain matrix
is water-rich and lacks rigid fibrillar collagens, making it softer than the ECM in other tissues
(Bellail et al., 2004). Taken together, this information means that cell migration through the
brain will require that cells degrade a specialized extracellular matrix and navigate through
an environment where cells are densely packed.
Invadosomes
Cell adhesion to the ECM substrate is also important for cell stability, anchorage and
traction during migration. Cell adhesion structures are sites where cells attach to the
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underlying substratum. Focal complexes and focal adhesions are well-documented and
extensively studied adhesion structures found in many cell types, which under some
circumstances provide traction for cell migration. Integrins at the cell-ECM contact sites are
adhesion molecules that mediate communication with the cell regarding its interaction with
the ECM, and influence many signalling cascades, including cytoskeletal rearrangements
(Caswell et al., 2009). In the 1980s, a new type of adhesion structure was discovered: the
‘podosome’ (David-Pfeuty and Singer, 1980; Wolosewick, 1984; Tarone et al., 1985; Linder
and Aepfelbacher, 2003), which was later proposed to play a role in tissue invasion.
Monocyte-derived cells (e.g. macrophages) (Messier et al., 1993), constitutively active Src-
transformed cell lines (David-Pfeuty and Singer, 1980; Tarone et al., 1985; Abram et al.,
2003), endothelial cells (Osiak et al., 2005), and vascular smooth muscle cells (Burgstaller
and Gimona, 2005) are now known to express podosomes. Invasive cancer cells (e.g. human
breast cancer) (Mandal et al., 2008) possess similar structures, which were named
‘invadopodia’ (the combined term, invadosomes, is often used for invadopodia and
podosomes) (Murphy and Courtneidge, 2011). Podosomes were recently discovered in vivo
in transforming growth factor beta (TGFβ)-stimulated endothelial cells in mice (Rottiers et
al., 2009) and also in 3D cultures of macrophages in vitro (Rottiers et al., 2009; Cougoule et
al., 2010) .
Because many components of invadosomes are also present in other cell-ECM
adhesion structures, it was initially difficult to distinguish invadosomes, and determine
whether they are indeed discrete adhesion structures. It is now clear that they exhibit some
morphological, protein composition, and functional similarities and differences (reviewed in
(Block et al., 2008). Both podosomes and invadopodia are F-actin rich structures that
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displays a unique ring and core structural arrangement (Figure 2) (Gimona, 2008). The ring
seems to have an adhesive nature and contains most of the integrins and adhesion-associated
molecules (e.g. talin, vinculin, paxillin); whereas, the core is rich in F-actin, and actin-
regulating molecules like Arp2/3 complex, WASP, and cortactin. Besides these cytoskeletal
regulators, other molecules have also been detected in invadosomes (Table 1) (Block et al.,
2008). Src tyrosine kinase signalling, a common signalling pathway involved in many
cellular processes including cell migration, is pivotal for the formation and regulation of
invadosomes (Linder and Aepfelbacher, 2003). Immunostaining for tyrosine phosphorylated
proteins shows localization to both the core and ring, suggesting their presence throughout
the structure. Unlike other adhesion structures, invadosomes can also degrade matrix due to
their association with matrix metalloproteinases (MMPs), and ADAMs (‘a disintegrin and
metalloproteinase’). There are several differences between the two subtypes of invadisomes.
Invadopodia have high degradative capability due to their slow turnover (>1 hr), whereas
podosomes are much more dynamic with turnover rates between 2 and 20 min, and shallower
matrix degradation. Invadopodia are larger structures (~5-8 µm in diameter) compared to
podosomes (~0.5-1 µm). Invadopodia are also sparsely distributed in cells (<10) while
podosomes are more compact and numerous (~20-100) (Murphy and Courtneidge, 2011).
Podosomes are protrusive adhesion structures primarily found at the ventral side of
polarised cells of the monocytic lineage, such as macrophages (Messier et al., 1993),
immature dendritic cells (Binks et al., 1998), and osteoclasts (Zambonin-Zallone et al., 1988;
Calle et al., 2006). Due to their adhesive properties, high turnover rates, and degradative
capacity, podosomes are well designed to aid highly migratory cells like macrophages in
traversing barriers like tissue layers (Carman et al., 2007). Indeed, when podosomes were
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discovered, the initial opinion was that they functioned in transcellular migration
(Wolosewick, 1984).
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Figure 2. (Top) Electron micrograph showing the distinct core and ring arrangement in a smooth muscle cell podosome (Gimona, 2008). (Bottom) Schematic diagram showing localization of common podosome-associated molecules (Linder and Aepfelbacher, 2003). Reprinted from The Lancet, Vol. 18, Gimona M, The microfilament system in the formation of invasive adhesions, 23-34, Copyright (2008), with permission from Elsevier Reprinted from The Lancet, Vol. 13, Linder S, Aepfelbacher M, Podosomes: adhesion hot-spots of invasive cells, 376-385, Copyright (2003), with permission from Elsevier
8
Table 1. Components that constitute podosomes, invadopodia and focal adhesions (Block et
al., 2008). Reprinted from The Lancet, Vol. 87, Block MR, Badowski C, Millon-Fremillon A, Bouvard D, Bouin AP,
Faurobert E, Gerber-Scokaert D, Planus E, Albiges-Rizo C, Podosome-type adhesions and focal adhesions, so
alike yet so different, 491-506, Copyright (2008), with permission from Elsevier
9
Introduction to monocytic immune cells
The immune system is a complex defensive array made up of several cell types with
different phenotypes (Alberts et al., 2008). All immune cells recognise and react to protect
the host from many pathophysiological circumstances, such as cancer, infection, and injury
(Kindt et al., 2007). Blood immune cells develop from a common hematopoietic stem cell,
through a process called hematopoiesis that can give rise to several blood cell types (Figure
3)(Kindt et al., 2007). One type of progenitor immune cell, the granulocyte-monocyte
progenitor cell (Kindt et al., 2007), can give rise to monocytes that can be released into the
blood circulation from the bone marrow. Circulating monocytes are undifferentiated immune
cells that can migrate into various tissues (a process called extravasation) to differentiate into
resident macrophages (as reviewed in Gordon and Taylor, 2005; Kindt et al., 2007). Gordon
and Taylor, in their review, highlight that these tissue macrophages are of heterogeneous
phenotypes, forming subpopulations with unique functions within their corresponding
microenvironments. For example, osteoclasts have the ability to remodel bone tissue,
alveolar macrophages in lungs remove debris and pathogens due to high expression of
pattern recognition receptors and scavenger receptors, and microglia in the adult central
nervous system (CNS) predominantly represent the innate immune system.
In this thesis, I investigated several physiological aspects of rat microglia in vitro.
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Figure 3. Schematic diagram illustrating development of immune cells in blood and CNS (Ransohoff and Cardona, 2010) Reprinted by permission from Macmillan Publishers Ltd: Nature. Ransohoff RM, Cardona AE. The myeloid cells of the central nervous system parenchyma. Copyright 2010
11
Microglia – Immune cells of the CNS Microglia are the resident sentinels of the CNS. Recently it has been shown that
microglia precursor cells of monocyte-lineage develop from mesodermal hematopoietic
cells–originating from the yolk sac– that enter and remain in the developing fetal brain (as
reviewed in Chan et al., 2007). It has been suggested that peri-natally and post-natally,
circulating monocytes in blood can also infiltrate into brain parenchyma (Figure 3)(reviews
Chan et al., 2007; Ransohoff and Cardona, 2010; Kettenmann et al., 2011). These cells then
proliferate, migrate to different regions of the brain and differentiate into microglial cells.
Microglia reside in an immune privileged environment, due to the blood brain barrier (BBB)
that separates the brain anatomically and physiologically, and strictly regulates substances
that cross the barrier (Alberts et al., 2008). Microglia are thought to form 5% to 20% of the
total glial cell population in the CNS (Kaur et al., 2010).
During post-natal development, microglia further differentiate within the brain, and
enter a quiescent state with a ramified morphology (Schlichter et al., 2010; Kettenmann et al.,
2011). In this non-activated state, microglia are non-migratory but have many processes
extending from the soma that are motile, constantly monitoring their environment through
pinocytosis, and interacting with other cells in CNS (Davalos et al., 2005; Nimmerjahn et al.,
2005; Kaur et al., 2010). Microglia respond first to perturbations of CNS homeostasis
(Miyake et al., 1988; Schilling et al., 2003). In response to a pathophysiological event,
microglia undergo a complex transformation process from the non-activated “resting” state to
“activated”. Once activated, microglia can exhibit one or more new phenotypes; e.g.,
increased proliferation, migration, phagocytosis, production of interleukins, cytokines and
chemokines (Hanisch and Kettenmann, 2007; Kaushal et al., 2007; Schlichter et al., 2010;
Kettenmann et al., 2011). The outcome of the microglial response is dependent on its
12
activation state, i.e., microglia react differently depending on the stimuli, factors released by
surrounding neuronal/glial tissue, and the pathophysiological context (Schwartz et al., 2006;
Carson et al., 2007; Colton, 2009; Kettenmann et al., 2011). Microglia are sometimes
referred to as brain macrophages, and can exhibit a wide range of immune functions that are
similar to peripheral macrophages; e.g., phagocytosis, free radical production, secretion of
chemokines and cytokines, and communication with rest of the immune system (Tambuyzer
et al., 2009). However, unlike macrophages, the response of activated microglia can be
immunologically silent, in which a system-wide immune reaction is not activated (Galea et
al., 2007). Along with the versatile nature of microglial activation, there is evidence of
microglial involvement in all neuropathologies (Kreutzberg, 1996; Streit et al., 2005; Block
et al., 2007; Hanisch and Kettenmann, 2007; Davoust et al., 2008; Colton and Wilcock, 2010;
Graeber and Streit, 2010; Kaur et al., 2010).
This thesis will focus on one of the reactive microglial phenotypes, which involves
cells migrating to sites of injury (e.g. to the lesion after intracerebral hemorrhage or ischemic
stroke) (Brockhaus et al., 1996; Zhang et al., 1997; Schlichter et al., 2010). Shortly after
stroke, our lab and other groups have shown that microglia/macrophages respond to the
injury by entering an activated state that includes a change in morphology from ramified to
amoeboid (Brockhaus et al., 1996; Zhang et al., 1997; Wasserman et al., 2008; Moxon-Emre
and Schlichter, 2010). Microglia/macrophages were then observed to progressively migrate
toward the damaged core from surrounding tissue, reaching maximal infiltration by 7 days
after both forms of stroke. Functionally, microglial migration can be either beneficial or
detrimental (Tambuyzer et al., 2009). For instance, beneficial effects include removal of
damaged cells and debris by phagocytosis, and secreting neurotrophic factors that aid in
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repair processes. It has even been suggested that activated microglia secrete soluble factors
that attract neural precursor cells to the site of injury where they can differentiate to produce
neurons and/or glial cells that aid in the repair process (Aarum et al., 2003). On the other
hand, microglial activation and migration can also be detrimental and cause secondary
damage after the initial insult; e.g., due to production of highly reactive oxygen species
(ROS) and nitric oxide-derived species like peroxynitrite (see review Boje and Arora, 1992;
Kettenmann et al., 2011). Furthermore, production of inflammatory factors, such as
interleukins and tissue necrosis factor (TNF-α), can cause additional neuro-inflammatory
damage that propagates from the site of injury to surrounding healthy tissue (see reviews
Chakraborty et al., 2010; Kettenmann et al., 2011).
Although microglial migration to the site of injury is a well-documented response that
is common to most of its reactive phenotypes, the mechanisms have not been studied
extensively. During development, microglia migrate to different regions of the brain,
(investigated mostly in quails), with migration tangentially at first, and then radially (Cuadros
et al., 1994; Cuadros et al., 1997; Marin-Teva et al., 1998; Rezaie and Male, 1999;
Navascues et al., 2000; Sanchez-Lopez et al., 2004). In their non-activated, quiescent,
ramified state, microglia motility is mainly seen as extension of the many processes arising
from their somata, without overall cell displacement (Davalos et al., 2005; Nimmerjahn et al.,
2005). However, factors like ATP released from necrotic cells in the damaged brain act as
chemo-attractants, activate microglia and alter their morphology to the amoeboid form
(Honda et al., 2001), which is associated with the migratory phenotype (Kettenmann et al.,
2011).
ATP allows Ca2+ entry through receptor signalling via metabotropic P2Y receptors or
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ionotropic P2X receptors that modulate intracellular pathways, such as PI3K/Akt signalling
(Ohsawa et al., 2007; Lalo et al., 2008). Some other physiological factors that activate and
attract microglia include ectonucleotidase-derived adenosine (Farber et al., 2008), glutamate
(Kim and Ko, 1998; Liu et al., 2009), chemokines (Biber et al., 2001; Liang et al., 2009),
bradykinin (Ifuku et al., 2007), growth factors (Bonifati and Kishore, 2007; Kettenmann et
al., 2011) and beta-amyloid (Stuart et al., 2007). There is evidence that signalling pathways
such as PI3K/Akt, Erk, non-receptor tyrosine kinases (e.g. Pyk2) are involved in microglial
migration (see review Kettenmann et al., 2011). Some ion channels proposed to be involved
in microglial migration are stretch-activated Ca2+ permeable channels, Ca2+-activated
potassium channels, the reversed mode of the Na+/Ca2+ exchanger (NCX), and Cl- channels
(see review Kettenmann et al., 2011). It is interesting that many of the molecules thought to
influence microglial migration are involved in Ca2+ signalling cascades; which suggests a
conserved, vital role for Ca2+ in migration and an important contribution of intracellular Ca2+
regulation (Evans and Falke, 2007), and a complex interplay between ligands, receptors, ion
channels, and the cytoskeleton. Microglia also need to migrate through the unique brain
ECM and tightly packed dense brain tissue without causing damage to normal cells. In this
context, there is very little known about migration mechanisms in microglia.
15
Podosomes in microglia
With Dr. Schlichter, a former graduate student, Catherine Vincent, discovered that
untreated microglia cells grown on glass cover slips show dot-like structures that are
abundant in F-actin, and enriched in the lamella region. Further investigation showed that
these structures possessed a ring pattern of talin staining and F-actin cores, and had
podosome-like dimensions, suggesting that microglia express podosomes in vitro (Figure 4).
Podosomes in macrophages also seem to be localized at the leading edge (Evans et al., 2003).
In addition, microglial podosomes often formed a larger structure that we called a ‘podonut’,
which is made up of many individual podosomes in an open ring formation. Functional
studies showed these structures have the ability to degrade fibronectin, a common non-brain
specific ECM substrate (Figure 5), which is a well-known function of podosomes (Chen et
al., 1985; Seals et al., 2005). Podonuts were observed after microglia were in culture for 20
hours (Figure 6) and formed at the cell-substrate attachment interface. It was also discovered
that SK3, a Ca2+ activated K+ channel, localized to the core of podosomes (Figure 5). In
preliminary studies (Vincent and Schlichter, 2010), blocking SK3 channel activity did not
affect podonut/podosome formation so its function in relation to these structures still requires
further investigation.
Activated microglia that are undergoing migration in the damaged CNS could benefit
from expression of these microscopic structures (podosomes), to provide the means for
localized degradation of brain ECM and to navigate through dense CNS tissue to reach the
site of injury.
16
A B
Figure 4. Primary rat microglia express podosomes. (A) Microglia immunolabeled for the
podosome core marker, Arp2 (red), and the podosome ring marker, talin (green), and the cell
nucleus (DAPI; blue) (B) Microglia immunolabeled for the podosome ring (talin; red), F-
actin in the core (phalloidin; green), and the nucleus (DAPI; blue). Boxes represent areas
chosen for higher magnification and colour separated on the right. Arrows show the
podosome ring and core structural arrangement. Scale bars = 5 µm. (Images from (Vincent
and Schlichter, 2010)
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Figure 5. Functional podosomes expressed in microglia, with SK3 localized in podosome
core. Microglia stained for SK3 (red) show that it co-localizes with the podosome core
marker, F-actin (phalloidin; blue). Images on the right are the boxed areas, magnified and
colour separated. F-actin dots co-localize with regions of degradation of FITC-labeled
fibronectin (green). Arrowheads show co-localization of SK3, F-actin and fibronectin-
degraded spots. Scale bar = 5 µm. (Images from (Vincent and Schlichter, 2010)
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Figure 6. Microglia in vitro express podonuts at 20 hr in culture. Images were acquired after
cells were in culture for the indicated number of hours; labelled for F-actin (phalloidin; red),
a podosome core marker. The large actin rings in the lamella region (podonuts) appeared at
the 20 hr time point. Scale bar = 20 µm. (Images from (Vincent and Schlichter, 2010)
19
Thesis Objectives
Podosome components are fairly conserved among different cell types; however,
some reports have shown apparent differences in their molecular make up. For example, in
smooth muscle cells, SM22α protein was shown to associate with the podosomal core
(Gimona et al., 2003). In dendritic cells, there is evidence that podosome assembly occurs in
a gelsolin-independent manner (Hammarfjord et al., 2011); contrary to the requirement for
gelsolin in osteoclasts (Ma et al., 2008; Crowley et al., 2009; Ma et al., 2010; Van Goethem
et al., 2010). With our lab first discovering podosomes in microglia (Vincent and Schlichter,
2010), and given that podosome components could vary in different cell types, I wanted to
further characterize podosomes in microglia using immunostaining and some functional
studies. In this thesis, I show that although some components are conserved between
podosomes in microglia and other cells; I have identified some molecules not previously
shown to associate with podosomes. Then, because Ca2+ plays a key role in cell migration,
and SK3 —a Ca2+ responsive channel— was found in the podosome core in microglia
(Vincent and Schlichter, 2010), I hypothesized that the regulation of podosomes is dependent
on Ca2+. Overall, the findings in this thesis contribute toward a better understanding of
podosomes, their features in microglia, and their Ca2+-dependence.
20
MATERIALS AND METHODS
Microglia cell cultures. Giulian and Baker (1986) were the first to develop
isolation and culturing protocols for microglia. The Schlichter lab published it’s first
microglia paper in 1996, and since then has refined the protocol such that after isolation,
neonatal rat microglia remain in a relatively resting state, as judged by very low expression
of many inflammatory mediators (Sivagnanam et al., 2010). Isolation of rat microglia was
done by Dr. Schlichter’s technician, Xiaoping Zhu, following standard protocols as described
previously (Kaushal et al., 2007; Ohana et al., 2009; Sivagnanam et al., 2010). Briefly,
primary microglia cultures were derived from 1 to 2 day old Sprague-Dawley rat pups. After
removing the meninges, the brain was dissected and mashed through a stainless steel sieve in
Minimum Essential Medium (MEM; Invitrogen, Burlington, Canada), and then centrifuged
at 1000g for 10 min. The supernatant was removed and the pellet was re-suspended in MEM
supplemented with 10% fetal bovine serum (FBS; Wisent, St-Bruno, Canada) and 0.05
mg/ml gentamycin (Invitrogen), and plated on flasks to maintain at 37ºC, 5% CO2
atmosphere. After 48 hr, cellular debris, non-adherent cells and supernatant were removed
and fresh medium was added. The mixed cell cultures were then maintained for another 5 to
6 days. Microglial suspensions were then obtained by shaking the mixed cultures on an
orbital shaker for 3-4 hours in 37ºC at 60 to 65 rpm. For my experiments, the suspension
containing microglial cells was then centrifuged for 10 min at 1000g and the cell pellet was
re-suspended in MEM supplemented with 2% FBS and 100 µM gentamycin. Cells were
plated at 60,000 cells per 15 mm diameter glass cover slip, incubated in the 2% FBS
supplemented MEM at 37ºC, 5% CO2, and used within 24 to 48 hr. The Schlichter lab has
repeatedly shown that primary microglia cultures obtained with this protocol are ≥99% pure,
21
based on labelling with several microglia markers: FITC-conjugated tomato lectin, or
antibodies against isolectin B4, ionized Ca2+ binding adaptor-1(Iba1) or CD11b (OX-42)
(Kaushal et al., 2007; Ohana et al., 2009; Sivagnanam et al., 2010).
Chemicals and antibodies. I used the channel blockers: 2-aminoethyl
diphenylborinate (2-APB; Sigma-Aldrich, Oakville, Canada), and spermine
tetrahydrochloride (Calbiochem, San Diego, CA). Several antibodies were used: rabbit
actinin), and adhesion-associated proteins (e.g. vinculin, paxillin, talin). PI3K activity in
podosomes would cease, preventing production of PIP2 at the membrane, which would
prevent Tks5 anchoring to the podosome membrane, allowing it to diffuse away. In turn,
Tks5 loss would diminish Nox1-mediated ROS generation in podosomes, and increase PTP
activity. When ER Ca2+ is replenished, the CRAC channel would close, and Ca2+ would be
removed from the nanodomain by buffering (by cytosolic proteins), uptake into organelles, or
61
pumping out due to plasma membrane Ca2+ pumps. The reduction in Ca2+ would close SK3
channels as apoCaM forms. Loss of the Ca2+ signal could influence other molecules; e.g.,
inactivating Pyk2 tyrosine kinase and other Ca2+ regulated molecules, such as Iba1.
Podosome association with the microtubule plus-end associated molecule, KF1C, might also
lead to disassembly of podosomes (Kopp et al., 2006).
62
Figure 21. Updated schematic showing podosome-associated molecules (modified from
(Linder and Aepfelbacher, 2003), with the molecules found in microglia in this thesis shown
in red boxes.
Adapted and reprinted from The Lancet, Vol. 13, Linder S, Aepfelbacher M, Podosomes: adhesion hot-spots of invasive cells, 376-385, Copyright (2003), with permission from Elsevier
63
FUTURE STUDIES
In this thesis, I conducted extensive immunostaining on primary cultures of rat
microglia, and identified both known and novel molecules in podosomes. Knowledge about
podosome components provides us with the foundation to explore some of the signalling
pathway(s) that might be involved in podosome formation and functions in microglia.
Continued characterization of the components of podosomes will help identify potential
pharmacological targets (e.g. SK3, Orai1, CaM, Iba1) for promoting or inhibiting podosome
formation. Selective drugs could then be used with immunocytochemistry and functional
assays to study podosome formation and roles. Both 2-D migration- and 3-D invasion assays
using brain-relevant ECM substrates would shed light on whether active podosomes are
needed for microglial migration or invasion. Because Ca2+ removal disrupted lamellipodia
formation, a structure required for microglia migration (Vincent and Schlichter, 2010), we
expect that microglia invasion through ECM-coated filters in Transwell™ chambers would
be inhibited in the absence of extracellular Ca2+, buffering of intracellular Ca2+, or blocking
specific Ca2+-entry and responding pathways (e.g., Orai1/CRAC and SK3 channels).
Unfortunately, primary microglia are extremely resistant to gene transfection, siRNA-
mediated knockdown or viral-mediated infection (Ohana et al., 2009); therefore, we have to
mainly rely on pharmacological approaches for these proposed studies.
I suggested in this thesis that microglial podosomes represent a nexus for Ca2+- and
ROS signalling pathways. Therefore, I would like to: (i) use Fura2-FF, an analogue of the
fluorescent Ca2+ indicator, Fura-2, to monitor and measure any rapid changes in local Ca2+ in
and around the tiny podosome structures; and (ii) monitor ROS levels with Rhodamine 123
in podonuts, and (iii) determine whether these two signals interact and are interdependent.
64
This would address the possible roles of Ca2+ and ROS as second messengers for podosome
regulation and function.
Our discovery of podosomes in microglia in vitro raises the possibility that migrating
microglia in the brain use these structures to locally degrade ECM and to navigate through
CNS tissue to reach the site of injury. Only a handful of studies have identified podosomes in
vivo, and never in microglia or the CNS. At this point, our in vitro studies have been limited
by using microglia grown in an artificial 2-dimensional environment. Ultimately, to study
podosomes in a 3-dimensional in vivo environment, we would like to use adult rats and our
ischemic and hemorrhagic stroke models, in which massive microglia migration is observed.
This would allow analysis with the naturally occurring complement of ECM molecules, and
factor in changes that occur after acute damage. For instance, Iba1, a microglia/macrophage-
specific marker that has been extensively used in our lab, is strongly up-regulated in
microglia/macrophages in rat brain after stroke. Thus, Iba1 could be used to identify these
cells and simultaneously label podosomes to assess their formation in the CNS in vivo.
In summary, there is evidence that microglia are involved in most neuropathological
processes (Tambuyzer et al., 2009), and microglial migration to the site of injury is a crucial
early response to acute CNS injury. Hence, determining the role of podosomes, and
mechanisms regulating their specific functions, will bring us an important step closer to
developing therapeutic measures that target microglia and brain inflammation.
65
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