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Page 18 Yanguas-Casás. Neuroimmunol Neuroinflammation 2020;7:13-22 I http://dx.doi.org/10.20517/2347-8659.2019.31

specific inefficient responses. Sexual differentiation of the brain during early development likely underlies
the strong sex biases prevalent in many neurological conditions, as they acquire their sex specific identity
[30]
early in development, which persists during the injury response . Therefore, studying sex differences in
this context could shed some light on sex-specific disease mechanisms.

Beyond the neuroprotective effect of estrogens per se [96,97] , RNA-seq analysis revealed that female microglia
express more genes involved in cellular repair and inflammatory control than male microglia, which likely
[30]
contributes to a more favorable outcome in several injuries . Besides, recent studies have shown that
male microglia seem to be more reactive already under physiological conditions as well as have a shorter
[39]
lifespan . For example, female microglia show a higher mRNA expression of Shank 3, Fxyd1, Aqp1, or
Timp3 and a decreased mRNA expression of Akt1s1, Trem1, S100a9, or Cxcl2, as well as decreased NF-κB
activity levels, compared to male microglia .
[30]
Sex differences in microglia immunomodulatory response to lipopolysaccharide (LPS), a potent pro-
inflammatory agent, have been studied both in vitro and in vivo, and in both conditions male microglia
display a higher immune response of male microglia after LPS stimulation [90,98] , which is accompanied by
greater IL-1β mRNA and MHCI/II expression in male microglia, and decreased CD14 mRNA expression
in female microglia [39,98] .

Mouse models of forebrain or focal ischemia have shown that young adult female mice and rats sustain
lesser injury than males [99-101] . Moreover, female microglia display a neuroprotective phenotype in ischemic
[30]
stroke; indeed, when transplanted in male brains, they protect them from this disease .
Something important to keep in mind is that different subsets of microglia respond to various insults such
as aging or immune challenges differently. Microglia can be classified into gene expression clusters through
the lifespan of the individual. For example, on Postnatal Days 4 and 5, female microglia are enriched
for the genes Cd74, chemokine (C-C motif) ligand 24 (Ccl24), and Arg1 [102] . Interestingly, as the brain
ages, there is a progressive expansion of clusters that typically have few very cells in adolescent and adult
samples, which are enriched in inflammatory genes and are more responsive to interferon [102] . Combination
of deep single-cell transcriptome analysis, fate mapping, clonal analysis, in vivo imaging, and transgenic
mouse lines have allowed the identification of microglia subsets in different CNS compartments during
neuroinflammation [102-104] .

Single-cell sequencing of microglia in an Alzheimer’s disease mouse model revealed a unique AD-related
microglial phenotype, generated by a two-step process involving triggering receptor expressed on myeloid
cells 2 (Trem2). Activation is initiated in a TREM2-independent manner involving downregulation of
microglia checkpoints, followed by activation of a TREM2-dependent manner [104] . The relevance of sex in
these unique microglial subsets such as disease-associated microglia remains to be elucidated.

Interestingly, some genes have been linked with sex-specific phenotypes in the case of AD. One such gene
is Apoe, which codes apolipoprotein E (ApoE), a modulator of the CNS immune system that can have
differential outcomes on microglial function depending on the variant [105-107] . The ε4 variant of the gene,
which is expressed more strongly in females, has been linked with a higher risk of developing late-onset
AD in humans [56,108,109] . Microglia are a major source of plaque-associated ApoE, which is modulated by
TREM2 in AD mouse models [110] .

A sex-specific differential expression of Apoe in disease associated microglia has been found in a mouse
model of ALS [104,111,112] . Microglia isolated from female aged mice also have upregulation of ApoE transcripts
compared to males [113] . Overall, these findings suggest that ApoE is a gene that could partially explain the
sex differences found in AD and maybe other neurodegenerative disorders.