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Benusa et al. Neuroimmunol Neuroinflammation 2020;7:248-63 I http://dx.doi.org/10.20517/2347-8659.2020.03 Page 257

microglia from 3 week cuprizone treated mice had the greatest number of downregulated inflammatory
genes, and the top four uniquely upregulated genes associated with phagocytosis and oligodendrocyte
generation [guanine nucleotide-binding protein G(s) subunit, platelet derived growth factor alpha,
TYRO protein tyrosine kinase-binding protein (TyroBP), C-C chemokine receptor type]. Platelet derived
growth factor alpha is a mitogen that is critical for oligodendrocyte generation [134] . TyroBP is a microglial
transmembrane signaling polypeptide that forms phagocytosis active zones preparing microglia for
phagocytic activity [135] . Increased expression of TyroBP in the 3 week treated cuprizone mice, a treatment
time point that corresponds to early myelin loss, is consistent with findings from transcriptome microglial
analysis from demyelinating regions in other animal studies [136] and human tissue [137] . Thus, while microglia
from all three models exhibit pro-inflammatory (“M1”), reactive expression profiles, microglia maintained
a unique “fingerprint” for each model and these differences correspond with the integrity of the AIS,
suggesting that subtle changes in microglial phenotype may mediate either stability or disruption of
closely apposed neurons. It is still possible though that microglial phenotypes do not directly influence
AIS integrity. The direct association of microglia with the AIS suggests however, that this neuronal domain
may be particularly vulnerable to changes in microglial reactivity. These data support the growing body of
literature demonstrating that microglia exhibit a plethora of inflammatory expression profiles within an
“M1” phenotype despite having similar morphologies.

Transcriptomic defined subsets of microglia
Recently, several single cell RNA sequencing studies have begun to more clearly define subsets of microglia
[45]
in the developing,mature and healthy, and pathologic CNS [45,138,139] . Grabert and colleagues conducted
the first genome-wide comparison of RNA expression profiles from microglia isolated from specific brain
regions and across the adult life span. Their findings confirmed the presence of core profiles that distinguish
microglia from macrophages, underscoring their distinct origins. In addition, they observed three primary
RNA profiles that were regionally specific, demonstrating regional heterogeneity within the microglial
population. Although regional specific heterogeneity was observed, similarities persisted between the
cortex and the striatum, and between the cerebellum and hippocampus. With age, some of these differences
dissipated as the profile of hippocampal microglia appeared to converge with the profiles of microglia from
the cortex and striatum, while the profile of cerebellar microglia continued to diverge from the other three
regions to reveal region specific changes over time. Li et al. [138] reported that the majority of microglia in
mature, healthy CNS express similar profiles but significantly greater diversity was seen in postnatal CNS.
An interesting finding of Li et al. [138] was the similarity between a postnatal subset of microglia, termed
Proliferative-region Associated Microglia (PAM), and DAM, which demonstrates that genes expressed in
development are reactivated with aging and pathology. PAM appeared transiently in regions of developing
white matter, consistent with a role in phagocytosing the large numbers of oligodendrocytes that die during
myelination [140] . The authors further state that the complete chemokine and cytokine expression profile of
PAM supports additional roles including interacting with both neural and immune cells.

Using fluorescent assisted cell sorting gated by CD11b, CD45 and Cx3Cr1, Hammond et al. [139] defined nine
unique clusters of microglia in the whole brain based on expression profiles. The percent of cells in each
cluster changed across age and condition however. Canonical microglia genes were expressed by most cells
but only C1qa, Fcrls and Trem2 were expressed in all clusters. Interestingly, P2ry12, Cx3Cr1 and Tmem119,
which are frequently used as microglial identifiers [141-143] , were either expressed in very low levels, or
not at all in some clusters during development. Additionally, a novel subset of microglia, defined by the
expression of secreted phosphoprotein 1, similar to PAM described by Li et al. [138] , insulin like growth
factor 1 and immunomodulators from the galectin family and several lysosomal proteins, was observed
in the postnatal brain and associated with axonal tracts destined for myelination. Since these microglia
express lysosomal markers, it was proposed that these cells clear the way for continued axon outgrowth,
ultimately facilitating subsequent myelination. Other interesting findings include the lack of sex differences