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Walker. Neuroimmunol Neuroinflammation 2020;7:194-214 I http://dx.doi.org/10.20517/2347-8659.2020.09 Page 205

Increased levels of CD33 in AD brains might function to restrict inflammatory activation as well as
inhibiting phagocytic receptors, but blocking CD33 activation could result in enhanced neuroinflammation.
A clear demonstration of the interaction of CD33 and TREM2 signaling occurred in experimental mouse
[80]
models . The loss of CD33 in these models resulted in increased levels of key proinflammatory cytokines.
Significant information on whether these proteins interact in AD brains could be obtained if high-
resolution immunohistochemistry could show microglia staining for both proteins in vivo. The phenotypes
of CD33 or TREM2 positive microglia have not been rigorously investigated to determine if they primarily
express pro-inflammatory or reparative/homeostatic markers.

Progranulin
Increased expression of progranulin by microglia has also been observed in AD brains [81-83] . This protein
[84]
has multiple functions including neurotrophic, anti-inflammatory and lysosomal function regulation .
Progranulin expression is not restricted to myeloid cells with abundant neuronal expression having been
characterized. Mutations in GRN (progranulin gene) can cause some forms of frontotemporal dementia
(FTD), a neurodegenerative disease associated with neurodegeneration in the frontal and temporal cortex.
The mechanism for this degeneration has been associated with enhanced microglial inflammation caused
[85]
by partial loss of progranulin protein and its associated activity . This protein appears to be present
in most brain microglia, colocalizing with lysosomal proteins in brain sections, with increased levels in
[83]
plaque-associated microglia . As increased levels of progranulin can be protective, upregulated expression
in AD would be suggestive of a reparative stress-associated response to neurodegenerative changes. As a
marker to define microglial phenotypes, progranulin has limited utility, but its continued expression by
different types of microglia would suggest it is having an anti-inflammatory effect in AD affected tissue.
A recent gene profiling study of middle aged compared to old brain-derived microglia showed that GRN
[49]
mRNA expression was significantly higher in older microglia .

Toll-like receptors
Toll-like receptors (TLR) are a class of ten pattern recognition receptors associated with identifying
ligands from bacteria, viruses and fungi. However, they have also been identified to have a large range of
cellular ligands. Due to their demonstrated interactions with aggregated Aβ and α-synuclein, TLR2 and
TLR4 have been implicated in AD and PD, though immunohistochemistry for TLR4 has demonstrated
neuronal, not microglial, localization [86-88] . Activation of TLR9 by its ligand unmethylated double-stranded
DNA caused increased microglial phagocytosis of Aβ in experimental AD models ; however, there have
[89]
been no demonstrations of its localization in microglia in human brains. In a recent study focused on
[90]
TLR3, native ligand double-stranded RNA and the neuronal protein stathmin , we demonstrated distinct
microglial expression of TLR3 in human brains with increased expression in plaque-associated microglia,
and in endothelial cells, but not neurons or astrocytes . These results were different from previous studies
[91]
and dependent on the antibody used for immunohistochemistry. TLR3 had previously been defined as a
[92]
specific marker for dendritic myeloid cells including microglia .

Colony stimulating factor-1 receptor
The survival and proliferation of microglia is primarily dependent on the action of colony stimulating
[93]
factor-1 (CSF1) and IL34, ligands for CSF1R . Binding of these growth factors to CSF1R results in
microglial proliferation. These growth factors have distinct structures and though their expression is
regulated differently, they have overlapping properties. CSF1R expression in the brain is mainly restricted to
microglia. Studies using different CSF1R antagonist administered to mice resulted in knockout of microglia
from tissue with a variety of mostly therapeutic effects, although it can also be detrimental in some
circumstances depending on the disease model [94-99] . These studies have defined the significance of CSF1R
to microglial function. There has only been a single definitive study demonstrating localization of CSF1R
in microglia in human brains [100] . This study showed that all microglia constitutively expressed CSF1R