02 Jul 2019

Some microglia are from Mars; others from Venus. Well, no. But in some scenarios, to which sex a microglial cell belongs appears to hold some sway over how it responds. Consider tau. At a joint Keystone symposia—Neurodegenerative Diseases: New Insights and Therapeutic Opportunities, and Neural Environment in Disease: Glial Responses and Neuroinflammation—Li Gan reported that female tauopathy mice expressing an AD risk variant of TREM2 exhibited a stepped-up microglial reaction and had worse memory. The same TREM2 mutation did no such thing in male mice.

In tau mice, the R47H-TREM2 mutation triggered neurotoxic inflammation only in females.
MicroRNAs disproportionately tweak gene expression in males.
Chimeric mice allow researchers to explore human microglial responses to Aβ and tau pathology.

Gan, who is now at Weill Cornell Medical College in New York, also uncovered profound differences in how microRNAs control gene expression in males versus females. Other researchers found that microglia dictate pain sensitivity in sex-specific ways. In all, the findings cast microglia as pulling the strings behind some sex differences. At least in mice.

Many presentations at Keystone focused on these resident immune cells and TREM2. To name a few, David Holtzman of Washington University, St. Louis, David Hansen, Genentech, and Kim Green, University of California, Irvine, implicated TREM2 and other microglial factors in shaping Aβ plaques, which influenced the subsequent development of tau pathology (see Part 1 of this series). But does TREM2 also affect tau independently of Aβ? On this question, Gan reported surprising results in the P301L model of tauopathy. Her lab made use of a menagerie of mouse lines, including TREM2 knock-ins in which one or both copies of the mouse gene were replaced by the human one. The knock-ins expressed either the common variant of human TREM2, or the R47H-TREM2 variant that boosts risk for AD, and the animals were generated on the wild-type or P301L background. In addition, Gan examined P301L mice on a TREM2 knockout or wild-type background. This is where sex differences became apparent.

Compared to 1-year-old wild-type mice expressing a copy of normal human TREM2, those carrying a copy of the R47H AD variant had a spatial-learning deficit. It was greater in female than male mice. This sex difference grew further when Gan crossed the TREM2 mice to a P301L background. In Keystone, she reported that P301L mice expressing one copy of R47H-TREM2 had the same burden of tau pathology as mice expressing normal mouse or human TREM2. That said, female P301L mice expressing the AD risk variant did more poorly at spatial learning, whereas the mutation appeared to slightly benefit performance of male P301L mice.

Gan pegged maladaptive microglia as a likely culprit behind the greater memory loss in female P301Ls. She reported that microglia in both male and female P301L mice expressed the disease-associated microglia (DAM) signature of genes. However, expression of certain DAM genes shifted only in female mice that expressed TREM2-R47H. In females, R47H dialed up expression of many pro-inflammatory cytokines of the DAM signature and lessened expression of a suite of neuronal genes. In male P301L mice, the R47H variant exerted no overt change on the DAM signature.

Single-nucleus RNA sequencing told a similar story, in that a population of microglia expressing DAM genes was overrepresented in R47H-TREM2 female P301L mice compared with mice expressing the common variant of the microglial receptor.

Gan concluded that the tau-related effects of the R47H TREM2 variant are not equivalent to those of TREM2 deficiency. At least in females. While TREM2 deficiency reportedly dampens the DAM signature in both male and female P301L mice, the R47H mutation ramped it up and shifted it toward a more pro-inflammatory, potentially damaging profile in females, she said. Gan attributed the worse memory loss in female R47H-TREM2s to this shift. She emphasized that this sexual dimorphism was not due to more tau pathology in female P301L mice, but to an altered microglial response to that burden.

The prevailing view in the field holds that R47H saps TREM2 function. Gan acknowledged that her data appear to go against that. Still, she said, the mutation might reduce TREM2’s response to Aβ, which is known to bind TREM2, yet have a different effect in the context of tau pathology alone.

When asked about the shifted microglial response to tau that Gan reported in female mice, Hansen hesitated. The gender difference could come down to timing, he noted. He reported that in his PS2APP TREM2 knockout model, females develop Aβ pathology sooner than males do, but ultimately the sexes even out. Gan’s observation of sex dimorphism—albeit in a tau only model—was made when the mice were 7 to 9 months old. Hansen agreed with Gan’s point that while TREM2 clearly mediates Aβ-instigated tau pathology, the receptor’s function in the context of tau pathology alone could be different.

Gan is exploring the source of this sexual dimorphism in microglial responses further. MicroRNAs are known to fine-tune gene expression—could they be at play here? By comparing microRNA expression profiles in microglia isolated from male and female mice, graduate student Lay Kodama noted a slew of differentially expressed microRNAs. Kodoma also compared the mRNA transcriptomes of microglia isolated from wild-type and dicer knockout mice, which cannot generate mature microRNA. Removing dicer altered the expression of nearly 1,000 genes in male microglia, but only about 100 in female microglia, suggesting that microRNA disproportionately affect gene expression in microglia from males. Indeed, deleting dicer in male P301Ls exacerbated tau pathology, but in female P301Ls it did not. Overall, Gan interpreted her data to suggest that male and female microglia use different biological mechanisms to maintain homeostasis.

“These results indicate that males and females likely respond to AD-like neurodegeneration differently,” commented Li-Huei Tsai of Massachusetts Institute of Technology. “It would be very interesting to compare the transcriptomes of men and women who carry R47H in multiple cell types to see if there are differences,” she added.

Using single-cell RNA sequencing of human prefrontal cortex samples, Tsai recently reported sex differences in neuron and oligodendrocyte responses to AD, to which she attributed sex differences in AD risk and in white-matter pathology (Jul 2018 conference news; May 2019 news). Her study recovered too few microglia from the human samples to properly compare the sexes. However, at Keystone, Tsai reported that single-cell RNA sequencing from hippocampal samples, which have higher numbers of microglia, is underway, and could support such an analysis.

Next, Gan wants to test whether the sex differences are intrinsic to the cells or are imparted by the brain environment, as well as whether they hold true in human microglia.

Both these questions might find an answer with human microglia chimeric mouse models, such as the one developed by Matthew Blurton-Jones at the University of California, Irvine. At Keystone, Blurton-Jones characterized his model, in which immunodeficient mice receive transplants of iPSC-derived human microglial progenitors. Blurton-Jones generated them in response to mounting data showing that human microglia are different from their mouse counterparts in ways that matter greatly to the study of neurodegenerative disease (May 2019 news).

Alzforum covered some of this work at the AD/PD conference in Lisbon, along with findings from a similar chimeric model developed by Bart De Strooper at the U.K. Dementia Research Institute and KU Leuven (Apr 2019 conference news). Blurton-Jones had reported that when the recipient mice were 5xFAD, transplanted human microglia surrounded plaques but expressed different genes than the DAM signature of mouse microglia. At Keystone, he added that when the human microglia expressed a copy of R47H-TREM2, they were less energetic at rallying around plaques. Blurton-Jones has started transplanting microglia into P301L mice, and his initial findings suggest that transplanted human microglia nibble away at tau-laden neurons.

Both Gan and Blurton-Jones intend to use chimeric models to find out what is behind the sex differences that Gan saw in microglial responses to tauopathy. For example, by transplanting iPSC-derived microglia from women into male mice, or from men into female mice, the researchers can perhaps distinguish relative contributions of the brain environs from microglia-intrinsic properties that might drive the sex differences.

A smattering of other presentations at Keystone highlighted sex-specific differences in how microglia respond to a variety of perturbations. For example, Sandra Siegert from the Institute of Science and Technology in Klosterneuburg, Austria, reported that, in response to treatment with anesthetics, female microglia transformed into phagocytic cells that attacked structures surrounding parvalbumin-positive interneurons. Male microglia ultimately did the same, but responded more slowly. The findings suggest that female microglia may act faster on inhibitory interneurons to counteract dips in neuronal activity.

Julia Kuhn, who was a postdoc at the University of California, San Francisco, and is now at Genentech, reported a finding on pain. In response to peripheral nerve injury, Kuhn found, sensory neurons in the spinal cord pump out CSF-1, which signals through CSF-1R on spinal microglia (Guan et al., 2016). Males, but not females, are known to become hypersensitive to pain following such injuries. At Keystone, Kuhn reported why: The rush of CSF-1 in the spinal cord recruits T cells, which dampen microglial activation. In males, no T cells are recruited, and microglia become overactivated, stoking pain.

Yajing Xu of University College London reported that differences in the way males and females respond to pain could come down to how microglia sculpt nerve fibers during development. During the first week of life, microglia from both sexes engulfed portions of nerve fibers that receive touch and pain signals in the dorsal horn of the spinal cord, Xu found. When Xu nicked the hind paw of the mice during this time period, microglia responded by crowding the nerve fibers that transmitted the injury signal. However, only male microglia stepped up their engulfment of nerve fibers, essentially hijacking this normal developmental process, Xu said. She is investigating whether these differences in microglial engulfment of nerve fibers shape pain sensitivity in adulthood.

Overall, researchers agreed that microglia respond differently between the sexes in some instances, but what drives those differential responses, and how they ultimately affect the course of disease, remain to be explored.—Jessica Shugart

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Down to Sex? Boy and Girl Microglia Respond Differently

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