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vonderEmbse et al. Neuroimmunol Neuroinflammation 2020;7:345-59 I http://dx.doi.org/10.20517/2347-8659.2019.29 Page 355

This GxE interaction, with a double hit from environmental toxicant exposure and genetic proclivity to
AD, exacerbated and prolonged maladaptive microRNA expression profiles in response to an atypical
developing environment for each sex. Contrary to the observed upregulation of DAP12 expression over
time in females regardless of strain [Figure 1], postnatal Pb exposure significantly increased DAP12
immediately following cessation of exposure at PND 10, that then declined over time in both females
[Figure 2A and C] as well as Tg males [Figure 2D]. These results not only corroborate reports of sexually
[36]
dimorphic postnatal microglial development using DAP12 as a marker for immature, phagocytic
[20]
perinatal microglia , but also the heightened vulnerability of immature female microglia during this
temporally critical maturation. Also, these data further corroborate our model of GxE vulnerability in a
DOAD model of AD, as Pb-exposed Tg males also exhibited increased DAP12 expression [Figure 2D]
during a postnatal window in which expression does not temporally fluctuate in healthy male mice
[Figure 1]. Interpretation of these results is limited due to a lack of quantification of microglial number
and phagocytosis or neuronal apoptosis, and future studies are warranted to delineate early physiological
alterations to microglial-directed synaptic pruning resulting from atypical DAP12 expression, as well as the
ramifications for total microglia numbers in the adult brain. Likewise, without the addition of colocalized
staining with microglia-specific markers, cell-type-specific inferences cannot justifiably be made regarding
DAP12 expression. However, reports of its expression are limited to perinatal microglia and its critical role
in developmental phagocytosis of apoptotic neurons by microglia [19,49] lend credence to the interpretation
that DAP12-related neuroimmune development was significantly altered in a sex-biased manner in this
GxE model.

Importantly, this striking GxE effect on temporal DAP12 expression in Pb-exposed Tg females was
accompanied by equally striking effects on epigenetic changes related to neuroimmune function. Namely,
latent upregulation of miR-124 by Pb, indo, or both indo+Pb was observed in Tg females at PND 21
[Figure 4C]. The microRNA miR-124 critically regulates both microglial and neuronal development,
[27]
maturation, and function . Upregulation of miR-124 has been shown to promote microglial quiescence
and restrict proliferation through inhibition of CEBPα, the transcription factor PU.1, and its downstream
[29]
[28]
target CSF1R , while also promoting neuronal differentiation via repression of Sox9 . The female-
specific expansion of amoeboid microglia populations around PND 30, preceded by a highly proliferative
period to populate various brain regions , would suggest that a premature increase in miR-124, such
[36]
as that observed in postnatally exposed Tg females at PND 21 [Figure 4C], may not only prematurely
limit temporally-critical microglial maturation but also the proliferation and subsequent region-specific
populations in the adult brain. While the additional investigation is needed to confirm any long-term
changes to microglia and neuron population numbers, there is considerable evidence for a central role
of miR-124 in our experimental DOAD model of AD. Long-term neuronal changes have been reported
consequent to upregulation of miR-124, in which inhibition of REST by miR-124 delayed the maturation
of NMDA receptors by retaining the prevalence of highly excitable GluN2B subunits, the ectopic form
of which is associated with amyloid-β production [50,51] . Also, defects in glutamatergic synaptic function
[21]
along with reduced TrkB expression have been reported in DAP12-deficient mice . Given our previous
research demonstrating a compensatory increase in hippocampal TrkB expression in Pb-exposed Tg female
mice compared to WT at PND 120 that decreased with age and correlated with dysfunctional microglial
[4]
phenotypes , the decrease in hippocampal DAP12 expression [Figure 2] and upregulation of miR-124
[Figure 4] reported here substantiate the hypothesis that perturbation to neuroimmune development
promotes epigenetic changes for maladaptive microglia-neuron interactions involved in early AD
pathologies. Furthermore, increased miR-124 at PND 21 in Tg females regardless of exposure type [Figure 4C]
would suggest that this postnatal window represents a previously unseen window of vulnerability for
female microglia development to any type of immune-altering exogenous insult, be it proinflammatory (Pb)
or anti-inflammatory (indo).