The palpable observation in the sex bias of disease prevalence in the CNS has fascinated scientists for several generations. Brain sex dimorphism has been visualized by imaging and analytical tools at the tissue, cellular, and molecular levels. Recent work highlighted the specificity of such sex bias in the brain and its subregions, offering a unique lens through which disease pathogenesis can be investigated. The brain is the largest consumer of energy in the body and provides a unique metabolic environment for diverse lineages of cells. Immune cells are increasingly recognized as an integral part of brain physiology, and their function depends on metabolic homeostasis. This review focuses on metabolic sex dimorphism in brain tissue, resident, and infiltrating immune cells. In this context, we highlight the relevance of recent advances in metabolomics and RNA sequencing technologies at the single cell resolution and the development of novel computational approaches.
Among disorders of the CNS, females are more susceptible to multiple sclerosis (1), age-dependent Alzheimer’s disease (2), and major depressive disorders (3), whereas males are more likely to develop Parkinson’s disease (4), early onset schizophrenia (5), and autism (6) and have worse clinical outcome in multiple sclerosis (1) and glioma (7). In fact, sex dimorphism is observed among numerous other cognitive and emotional disorders where males are more affected by developmental onset disorders and females by a higher frequency of adult-onset disorders with some exceptions (8). Although not conclusive, sex difference can influence response to treatment in brain diseases (9, 10). Collectively, the clinical evidence points to the potential of leveraging temporal, regional, and cell-specific sex biases as an approach to investigate pathogenesis of CNS disorders and offer baby steps toward personalized medicine.
The exact mechanism of sex bias in disease is unknown but is likely induced by an interplay of complex traits, including hormonal, cellular, and regional brain differences, resulting in differential responses to stress, aging, and other disease-inducing processes. At the molecular level, gene expression differences across cell types and brain regions can be contributed by escapees of X chromosome inactivation, the Y chromosome and its transregulation of autosomal genes, among other mechanisms (11–13). Recent advances in RNA sequencing (RNA-seq) technology allowed comprehensive comparisons of sex bias at the level of gene expression in cells and tissue at increased resolution (13–22). These studies highlight brain subregion-specific sex bias as well as sex dimorphism of brain-resident immune cells. In the seminal work by Oliva et al. (21), genotype-tissue expression project data were leveraged to analyze 44 human tissues and a total of 16,245 RNA-seq samples. Of the 13 brain regions analyzed, cerebellum is the least sexually dimorphic (as measured by the number of differentially expressed genes), whereas spinal chord and hippocampus are at the other end of spectrum, with almost 3000 sex-biased genes in healthy individuals (21). Notably, ∼30% of the 13,294 sex-biased genes identified were found in only one or two tissues. In theory, the high tissue specificity makes it possible to leverage sex bias to investigate region-specific pathogenesis of different diseases.
Metabolism is a key regulator of brain function. The human brain is at 2% of the body weight but consumes 20% of its energy and is the largest consumer of glucose. Brain metabolism is intimately connected with neuron and glial cell function, and an age-dependent decline in glucose and oxidative metabolism is associated with pathogenesis of brain disorders (23–30). Is metabolism a component of brain sex dimorphism? Indeed, brain imaging and metabolomics approaches have alluded to metabolic sex bias. From RNA-seq studies of brain tissue (21) and microglia (31), we extrapolated differentially expressed genes of a metabolic nature (Fig. 1, metabolic enzymes and transporters as classified in Refs. 32, 33) and found that there are brain tissue and cell-specific sex biases in metabolic genes (between 1 and 2% of all differentially expressed genes). Overall, note that at the tissue level, the effector size of significant changes in sex-biased gene expression is either not reported or typically very small, in stark contrast to those obtained from a specific lineage of cells. This is likely contributed by the heterogeneity in cell lineage composition and the unique metabolic state in specific brain regions. These studies highlight the importance of evaluating metabolic changes at the cellular level in tissue. Advances in single-cell metabolomics and recent development of single-cell transcriptome-based computational approaches from our laboratory and those of others (34, 35) could be particularly beneficial in this context.
This review summarizes what we have learned in brain sex dimorphism by leveraging brain imaging, metabolomics, and transcriptome analyses of tissue, brain-resident, and infiltrating immune cells. We discuss how cell-intrinsic and cell-extrinsic factors such as glycolysis, mitochondrial substrate availability, epigenome regulation, IFN responses, and microbiome may regulate immune cell sex bias in metabolism and function contributing to CNS disorders. In this context, we discuss the opportunities and challenges we face to better cellular metabolism research by leveraging technological advances in the form of transcriptome-based and metabolome-based single-cell analyses.
Immune cells are sex dimorphic
Immune cells are important players in numerous brain disorders and are sex dimorphic. Sex bias has been reported for cells of both innate and adaptive immunity (reviewed in Ref. 36). For example, females have an increased response to TLR7 signaling, whereas males have increased responses to LPS at the level of macrophages, neutrophils, and dendritic cells (DCs) (36). Reports of sex bias in T cells appear to support increased activation and inflammatory potential of females (37–39). Overall, at steady state, macrophages, but not other immune cells, show the greatest sex differences at the transcriptome and epigenome levels, with further increases in response to external stimuli such as IFN signaling and aging (14, 15). Intriguingly, the opposite is true in human immune cells, where a greater magnitude of changes is seen in males with respect to declining naive T cells and an increase in monocytes and cytotoxic T cell function (20). Male immune cells also show earlier and stronger age-related epigenetic changes with unique alterations in B cell–specific loci (20). Therefore, it is important to understand sex bias in specific contexts. Next, we focus our discussion on sex dimorphism of relevant immune cells in brain disorders.
Brain-resident and infiltrating macrophages play a pivotal role in brain physiology and disease (40, 41). Microglia interact with neurons, oligodendrocytes, and their precursors and are actively involved in synaptic modeling, neuronal wiring, myelinogenesis, and vascularization of the developing and adult brain. In response to disease cues, microglia can also regulate astrocytes by secreting fragmented mitochondria, promoting astrogliosis and inflammatory neurodegeneration (42).
Essential to microglia function is their damage sensing and phagocytic capabilities, which are supported and regulated by metabolic homeostasis. A recent report suggests that under physiological conditions, microglia are flexible in mitochondrial fuel selection, and either glucose or glutamate is sufficient to maintain their role in parenchyma surveillance (43). When this flexibility is disrupted, however, such as by aging-induced glucose reliance or chronic amyloid β stimulation, macrophages/microglia have reduced ATP generation as a result of impaired glycolysis and glucose-dependent mitochondrial respiration (44, 45). In fact, restoring such metabolic dysfunction can reverse age-dependent cognitive decline, highlighting a dominant role of the metabolic state in macrophage function and brain physiology (44, 45).
Sex dimorphism has been described for microglia maturation, brain colonization, and involvement in pain perception (17, 18, 22, 46). In the acute response to traumatic brain injury, male mice show a more aggressive neuroinflammatory phenotype driven by massive infiltration of macrophages and sustained activation of microglia (47). In Alzheimer’s disease and its animal model, that is, APP/PS1 mice, female microglia are more activated, less phagocytic, and are associated with increased amyloidosis (48). Whereas the relative contributions of cell-intrinsic and cell-extrinsic factors in driving sex dimorphism of microglia are unclear, a clear role of specific disease context highlights the relevance of the tissue microenvironment.
Metabolically, microglia do not show sex bias in healthy aged wild-type mice, but they have increased glycolysis when isolated from female brain of APP/PS1 mice, consistent with their more activated phenotype (48). In aged APP/PS1 mice, female microglia have increased expression of most glycolytic enzymes and increased lactate production as estimated by extracellular acidification rate as well as direct measurement by metabolomics (48). Interestingly, intermediate glycolytic metabolites are largely not different between male and female microglia, highlighting the importance of measuring metabolic flux rather than steady-state metabolite levels. Collectively, these data support a notion that microglia sex bias in function and metabolic state is a consequence rather than a driver of sex bias in Alzheimer’s disease development. However, microglia-intrinsic sex bias can significantly contribute to disease progression, as suggested by successful prevention of cognitive decline through macrophage-specific interventions (44, 45).
Conventional and γδ T cells
Conventional CD4+ T cells, particularly Th17 cells, are potent inducers of neuroinflammation due to their ability to efficiently migrate through the blood-brain barrier through production of IL-17 and IL-22 (49). Naive male CD4+ T cells have a bias toward Th17 differentiation as reflected by increased IL-17 expression, a cell-intrinsic sex dimorphism dependent on androgen and PPARα (39). Once differentiated, male Th17 cells are also more pathogenic. Using a transfer model of experimental autoimmune encephalomyelitis (EAE), Doss et al. (50) showed that male but not female transgenic Th17 cells of the NOD background can transfer severe chronic EAE, regardless of the sex of the host. The increased pathogenicity of male Th17 cells is thought to depend on altered regulation of Jarid1C expression on the XY chromosome (50). This finding is consistent with the notion that Ag-specific Th17 cells, once generated, harbor intrinsic male bias in their ability to exacerbate disease outcome, and its connection to worsening clinical outcome of MS in male patients should be further investigated.
Despite cell-intrinsic male bias in Th17 cell differentiation and pathogenicity, the differentiation of Th17 cells is impaired in male SJL mice in response to EAE (39, 51), suggesting the importance of external factors. Zhang et al. (39) attributed the differential disease outcome to a poor ability of male CD4+ T cells to become Th1 cells. Alternatively, a recent report suggested that male SJL mice are protected against the onset of EAE due to the male-specific production of IL-33 by mast cells (51). Consequentially, an expansion of type 2 innate lymphoid cells in male mice induced the induction of a non-pathogenic Th2 cell response, which prevented EAE onset. It remains to be investigated whether the sex differences observed in murine CD4+ T cell differentiation and function are relevant in human MS, where women are more likely to develop MS (1).
CD8+ T cells can play both pathogenic and protective roles in neuroinflammation (52–54). Male CD8+ T cells show significantly higher brain infiltration in an ischemic stroke model (55). This infiltration is associated with a male bias in higher mortality and hemorrhagic transformation. Functionally, male CD8+ T cells produce higher levels of IFN-γ and are better suppressors of airway inflammation (56).
In addition to conventional T cells, recently, a meningeal resident population of γδ T cells were reported to play a significant role in regulating anxiety in naive mice due to their ability to make IL-17 (57). IL-17 is mostly studied in the context of autoimmune diseases and infections, but it is increasingly being recognized in other disease contexts. For example, in an Alzheimer’s disease model, female mice accumulate IL-17–producing cells, mainly γδ T cells, in brain and meninges, resulting in a memory deficit not observed in their male counterparts (58).
Few assessments of metabolic sex differences have been performed in T cells; however, one may expect novel discoveries in this arena in the coming years given extensive literature supporting metabolic pathways as key determinants of T cell fate and function. Overall, T cells demonstrate intrinsic sex dimorphism, but their differentiation and function are further regulated by the specific tissue microenvironment and disease context.
Metabolic sex dimorphism in brain tissue microenvironment
Evidence for brain sex bias in glycolysis
Sexual dimorphism in glycolysis could underlie sex difference in patient survival: higher glioma glycolysis is associated with decreased survival in males but better protection in females (7). Brain glucose metabolism can be visualized with brain imaging techniques at the level of brain structure, neuronal activity, and functional circuitry (59). Leveraging multitracer position-emission tomography (PET) scans of >200 cognitively normal individuals, aerobic glycolysis, estimated by the difference between total glucose use and oxygen consumption, was found to be higher in female brain as compared with males of the same age (26, 60, 61). Oxygen consumption can be contributed by other metabolic inputs of the tricarboxylic acid cycle such as glutamate and lipid, and thus it is important to note that alternative or complementary interpretations of this result is possible such as sex bias in mitochondrial fuel selection. The observed difference can also be contributed by sex bias in the frequencies of cell types that differentially use glycolysis.
At the molecular level in tissue, proteomic analysis of healthy rat hippocampal slices showed female bias in the expression of almost all glycolytic enzymes, glucose, and pyruvate transporters (62), a finding not recapitulated at the transcriptome level in human hippocampus (21). Curiously, both human and rat brain data suggest that there is a male bias in the expression of phosphofructokinase (PFK)-1, the rate-limiting enzyme that catalyzes the first committing step of glycolysis. As a gate keeper, PFK is highly regulated, and its activity is protected by cAMP (63, 64). cAMP degradation is controlled by phosphodiesterases, and two such enzymes (PDE4C and PDE7A) are preferentially expressed in multiple male human brain regions (21). We speculate that male brain may be less sensitive to cAMP-dependent activation of glycolysis and other metabolic pathways, and that regulation of PFK activity is a key molecular switch to sex bias in glycolysis. Notably, phosphodiesterases are drug development targets for many CNS diseases (65). The sex bias in phosphodiesterase expression highlights the relevance of these enzymes and calls for additional considerations of sex dimorphism in the context of related treatment. Further analyses, especially fluxomics such as that measured by high-field magnetic resonance spectroscopy (MRS) (66) and carbon tracing experiments, are needed to provide better insight into glycolytic sex bias in brain tissue.
Female brain has increased mitochondrial activity, relevance of pyruvate, glutamate, and branched-chain amino acids
Female brains show increased mitochondrial activity and maximal respiration (67, 68). In a pilot clinical study, imaged by 1H-magnetic resonance spectroscopy, healthy females were found to have the higher brain energy metabolite N-acetylaspartate in both white and gray matter-rich areas, consistent with higher mitochondrial function of PBMCs of the same donors, including elevated complex I, II, and IV activities and respiratory capacity (69). Rodent studies suggest that the female bias in mitochondrial respiration is not because of increased function of mitochondrial complexes (I–IV) per milligram of protein as measured by spectrophotometric assays (70), but is likely a result of increased numbers of mitochondria in female mouse brain (71). Interestingly, complex II, but not complex I, is thought to contribute to sex dimorphism in mitochondria respiration as measured by a high-resolution respiratory system in brain homogenate (71).
A female bias in overall mitochondrial activity, particularly complex II, is also found in other human tissues (72) and supported by gene and protein expression evidence in human and rodent brains (21, 62). Mitochondrial complex II, the succinate dehydrogenase complex, sits at the crossroad of the tricarboxylic acid (TCA) cycle and oxidative phosphorylation, and it uses succinate as a substrate (73). Although there is no evidence suggesting sex dimorphism in succinate dehydrogenase complex assembly or function (70), there is some evidence supporting sex bias in its substrate availability. Female human brains from several regions show elevated expression of oxoglutarate dehydrogenase (OGDH) and isocitrate dehydrogenase 2 (IDH2) (21), enzymes that convert isocitrate ultimately to succinyl-CoA, but not any of the downstream enzymes in the TCA cycle. In line with this observation, protein expression of most TCA cycle enzymes show male bias except for IDH2 and succinyl-CoA ligase (SUCLG), which are expressed at higher levels in female rat hippocampus slices (62). Thus, gene and protein expression patterns in pathways that regulate succinate generation may support a female bias in mitochondrial complex II activity.
What might contribute to substrate availability in the context of female mitochondrial bias? Pyruvate dehydrogenase complex isolated from mouse brain is thought to have a female bias in increased activity per milligram of protein (70). At the gene expression level, glutamate dehydrogenase 1 (GLUD1) is preferentially expressed in multiple brain regions from females (21). GLUD1 converts l-glutamate into α-ketoglutarate (aKG), an important intermediate of the TCA cycle, which can be subsequently converted into succinate. Branched-chain amino acids (BCAAs), such as isoleucine and valine, are another source of succinyl-CoA (74). BCAAs are first converted into branched chain α-keto acids, then subsequently irreversibly oxidized by branched-chain keto acid dehydrogenase (BCKDH) to generate succinyl-CoA. Metabolomic evidence suggests that valine has a male-biased accumulation in several mouse brain regions, which could be indicative of poor usage (75). Consistent with this view, both BCKDH E1 subunit α (BCKDHA), a component of BCKDH, and methylmalonyl-CoA mutase (MMUT), the last enzyme in valine catabolism that generates succinyl-CoA, are specifically elevated in human female brain regions (21). Furthermore, the elevated GLUD1 expression, when translated into female bias in activity, may promote branched chain α-keto acid activity favoring BCAA catabolism (74). Thus, a possible interpretation of these observations is that female brain might be better equipped for pyruvate, glutamate, and/or BCAA catabolism-fueled TCA and mitochondrial respiration, particularly that of complex II. Experiments aimed at direct measurement of sex bias in substrate fluxomics in brain tissue and resident cells, as well as subsequent functional analysis, are required to test these hypotheses.
In sum, there is significant sex bias in the brain metabolic environment, and it will undoubtedly alter immune cell function and in turn contribute to clinical outcome of relevant brain disorders.
Regulators of sex differences in brain immunometabolism
Tissue factors contributing to sex bias of immune cells
The CNS provides a niche-specific environment that can alter immune cell metabolic state and function. In the elegant work by Wu et al. (76), CD4+ T cells in inflamed CNS was shown to have reduced oxygen level as compared with those in small intestine or lymph nodes as measured by in vivo administration of pimonidazole (76). Consequentially, CNS Th17 cells rely heavily on glycolysis for energy generation, and perturbation of glucose-6-phosphate isomerase 1 (GPI1) niche selectively inhibited inflamed T cells in the CNS but not those in the intestine. Thus, the metabolic microenvironment can be a key regulator of immune cell metabolism and function.
Does sex bias in brain tissue metabolism further promote female microglia toward an altered metabolic state? Although this has not been formally investigated in the brain, the significance of tissue sex bias has been nicely demonstrated in the peritoneal environment, which showed functional relevance in protection against bacterial infection (77, 78). Leveraging sex-matched and mismatched, tissue-protected bone marrow chimeric mice, Bain et al. (77) showed that both male and female peritoneal macrophages have increased proliferative potential in male hosts, highlighting the significant contributions of tissue in addition to cell-intrinsic factors to sex dimorphism. In principle, the metabolic sex bias in brain tissue discussed earlier could all regulate microglia function. The female bias in increased reactive oxygen species in the aging brain may preferentially alter glycolysis (79). The proposed increase in BCAA catabolism in female brain can induce reactive oxygen species (80), reprogram macrophages (81), as well as promote inflammatory and restricting neuroprotective functions of microglia (82). The relevance of tissue sex bias is particularly interesting when considering brain-infiltrating macrophages. Peripheral macrophages are thought to have a male bias in glycolysis, distinct to microglia (14). Sex-matched and mismatched transfer experiments could help clarify the contributions of cell-intrinsic and cell-extrinsic properties of brain sex dimorphism on infiltrating immune cells.
In addition to the brain, the microbiome is a key regulator of microglia maturation and function (83). Mice that are germ-free, treated with broad-spectrum antibiotics, or even with reduced complexity of microbiota can induce global defects in microglia activation and response to viral infection. Intriguingly, a supplement of short chain fatty acids (butyric acid, acetic acid, and propionic acid), a bacterial fermentation product, can partially restore microglia maturation (83). A recent study showed a striking age-dependent sex difference in microglia at the level of gene expression, chromatin accessibility, and density in the absence of microbiome (31). The sex bias of adult microglia was largely dependent on the presence of microbiome in the developmental stage, as acute antibiotic treatment only induced mild sex dimorphism. The mechanisms underlying the gut-brain connection remain an intensively investigated area.
Molecular regulators of sex bias in immunometabolism
Sex hormones are key drivers of sex dimorphism and immune responses as reflected in the identification of a sex hormone response element in a large fraction of genes with sex bias in their expressions, expertly reviewed elsewhere (21, 36, 84, 85). In this review, we take a view of differentially expressed genes and discuss the relevance of histone 3 lysine 27 trimethylation (H3K27me3) and IFNs in regulating sex dimorphism in immune cell function and metabolic state.
H3K27me3, the canonically repressive epigenetic modification, is enriched in female placenta as compared with their male counterparts (86). In adult humans, of the differentially expressed genes between male and female brain regions, H3K27me3 targets are most significantly enriched, highlighting modifiers of H3K27 as key epigenetic regulators of sex bias in brain tissue and cells (21). For example, H3K27me3 can be modified by KDM6a, an X chromosome–linked, aKG-dependent histone demethylase preferentially expressed in female CD4+ T cells (87), female microglia (88), and multiple female human brain regions (21). H3K27me3 demethylation requires aKG and is regulated by its ratio to succinate (89, 90). The higher level of succinate observed in male microglia (Fig. 1) may further restrict KDM6A function.
Functionally, KDM6a modifies the repressive H3K27me3 mark by removing the methyl group, resulting in activation of genes in a cell- and tissue-specific manner (91, 92). Demethylation of H3K27me3 is an important process in CD4+ T cell activation (93), and the reduced male expression of KDM6a is consistent with previous findings that female T cells are more prone to activation (36). Furthermore, lineage-specific H3K27me3 islands have been identified for CD4+ T cell differentiation (94), and it is possible that differential expression of KDM6a in female versus male cells can result in sex bias in epigenome landscape that favors specific T cell fate. Indeed, genetic deletion or chemical inhibition of KDM6a in T cells restricts murine and human IL-17A production and increases resistance to EAE (87, 95). This observation is consistent with a previous report where IL-17 production was reduced in male cells, which has lower KDM6a expression, when isolated from SJL mice with EAE, but in contrast to the male bias in Th17 cell differentiation when naive murine CD4+ T cells or when human CD45+ naive cells were used (39). The divergent outcome of T cell cytokine production may be dependent on the expression of KDM6a and the distribution pattern of H3K27me3 in a specific cellular context such as naive, effector, or memory state.
Metabolically, reduction of H3K27me3 is important for activation of glycolytic genes and the subsequent increase in glycolysis (96), a critical promoter of T cell activation and Th17 cell differentiation. As well, IRF5, a target of H3K27me3 suppression, can increase M1 macrophage polarization and glycolysis in humans (97). Thus, the female bias in higher KDM6a expression at the cellular and brain tissue level is likely a key driver of sex dimorphism in the glycolytic state and activation of macrophage and T cells. The distribution patterns of H3K27me3 and sex-specific expressions of KDM6a and aKG collectively provide three layers of regulation where tissue- or disease-specific sex dimorphism can be achieved.
TLR7/Ddx3x/type I IFN
IFN response is a key regulator of anti-viral immunity (98–100) and also plays a significant role in neurologic disorders. Type I IFNs can be induced by amyloid β in microglia, which limit their phagocytic functions (101). IFN-β rescues neurodegeneration in models of Parkinson’s disease by regulating mitochondrial fission and dopaminergic neuron death (102). Sex dimorphism in IFN responses has been well characterized (15, 36). TLR7 is a critical inducer of type I IFN response and is best studied in the context of systemic lupus erythematosus, an example of sex dimorphism with a staggering 9-fold higher incidence rate in women. TLR7 is encoded on the X chromosome and can escape X chromosome inactivation in immune cells such as B cells, monocytes, and plasmacytoid DCs (103, 104). TLR7 signaling can enrich biallelic B cells, further promoting responsiveness to TLR7 ligand (104). The RNA helicase Ddx3x (DEAD-box helicase 3, X-linked) is another X chromosome–linked gene that can induce an IFN response against pathogens (105). Ddx3x deficiency in hematopoietic cells resulted in a reduced number of lymphocytes and impaired ability to combat Listeria monocytogenes in a sex-specific manner and cannot be replaced by Ddx3y (106).
Consistent with sex-dependent alterations in the expression of regulators of type I IFN, immune cells show sex bias in IFN responses (15, 103, 107, and E. Ricker, M. Manni, D. Flores-Castro, D. Jenkins, S. Gupta, J. Rivera-Correa, W. Meng, A.M. Rosenfeld, T. Pannellini, M. Bachu, et al., manuscript posted on bioRxiv, DOI: 10.1101/2021.01.20.427400). Human female plasmacytoid DCs have increased expression of IFN-α and IFN-β and their receptors (103, 108). IFN stimulation can significantly increase transcriptional and functional differences between male and female macrophages (15). IFNs are also emerging as key regulators of metabolic homeostasis. IFN signaling in DCs promotes aerobic glycolysis (109), alters mitochondrial fuel selection by promoting fatty acid oxidation, and restricts cholesterol biosynthesis in favor of cellular sterol lipid import (110–112). As such, it is conceivable that IFNs and their regulators provide the molecular basis for sex dimorphism in immunometabolism. Interestingly, IFNs were shown to promote trimethylation of H3K27me in human macrophages (113) and can be regulated by sex hormones (85), thus providing a potential link between the three-axis molecular regulator of sex dimorphism.
The future of brain metabolism research
Finally, we discuss recent advances in technologies that can be leveraged to integrate sex dimorphism at the gene, cell, and tissue level to inform mechanisms of disease.
Imaging techniques such as functional magnetic resonance imaging, PET, and high-field MRS are widely used in neuroscience and are among the least invasive techniques suitable for human brain research. PET and functional magnetic resonance imaging can both be leveraged to detect energy delivery and use, whereas MRS is suitable for measuring fluxomics in physiological and disease conditions. Signals detected by such imaging techniques are thought to be largely contributed by neurons; however, glial cells may play a role given the extensive metabolic crosstalk between such cell types (114).
Cell-specific imaging can be achieved leveraging radiolabeled markers with high cell specificity. TSPO, a mitochondrial membrane protein, is a widely used target for measuring microglia activation; M-CSF 1 receptor (CSF1R) is another target for microglia and myeloid cells; in contrast, monoamine oxidase B and type 2 imidazoline receptors are targeted for visualizing astrocytes (115, 116). These cell-specific markers, which typically label more than one lineage of cells, can be coupled with radiolabeled metabolite analogs (e.g., fluorodeoxyglucose) and enable visualization of cellular metabolism in disease (117). Recent development of a chemical exchange saturation transfer approach allows detection of hydroxyl, amine, and amide groups and has been leveraged for imaging of myo-inositol, glutamate, and glucose metabolism in preclinical models, adding to the growing list of metabolic molecules that can be detected (115).
Among methods to investigate brain metabolism, metabolomics remains a powerful tool to offer insights at the tissue and cellular level by measuring hundreds of metabolites. Metabolomics studies revealed the association of brain metabolism with physiological activities and disease conditions (118–122), and when combined with genome-wide association studies can be particularly useful in predicting specific disease-associated metabolites (123). Fluxomics, the measurement of flow of metabolites between different pathways, better enables investigation into the cellular metabolic state. Recent description of an in vivo approach to perform isotope tracing allows measurement of metabolic flux in physiological settings (124, 125). Spatial fluxomics, in contrast, can be leveraged to investigate subcellular metabolic profiles (126). One of the major obstacles of metabolomics is the large quantity of samples typically required. Advances in mass spectrometry–based approaches now offer single-cell metabolomics, enabling potential investigation of metabolic differences in a small number of cells present in tissue (127, 128). Notably, SpaceM, a tool that combines light microscopy and MALDI-imaging mass spectrometry, can provide high-throughput single-cell metabolomics measurements, yielding >100 metabolites for 1000 individual cells within an hour (35). These approaches, although still suffering from challenges, including intrinsic volatility of metabolites during sample processing and difficulty in pinpointing the pathogenesis of disease, offer an exciting toolbox for future brain metabolism research, especially when combined with other techniques.
Transcriptome and metabolic modeling
The ability to measure metabolites at a single-cell resolution will greatly advance our understanding of the heterogeneity of sex bias in brain-resident cells. Due to the scale and complexity of the metabolic network, however, changes observed in specific metabolites can be induced by dysfunction in a distant part of the network, especially because not all metabolites can be detected. Transcriptome-based analysis offers an alternative means to compare the expression of metabolic enzymes and transporters. When combined with computational approaches such as flux balance analysis, functional genomic data can be leveraged to contextualize metabolic flux (129). We recently validated a computational framework, Compass, that leveraged the statistical power of single-cell RNA-seq to investigate the metabolic flux of immune cells and found it a powerful tool to identify regulators of metabolic bifurcation and immune cell function (34). The growing bodies of single-cell RNA-seq data to map out human physiology (130) will provide the rich resources necessary to significantly advance our understanding of brain cellular metabolism.
The next challenge in brain metabolism research is to understand how brain-resident immune and non-immune cells cooperate to maintain metabolic homeostasis and support brain physiology in a sex-specific manner. It is apparent that sex-biased genes are distinct when comparing brain tissue and microglia. One might speculate that sex-specific metabolic adaptation may be needed to support the function of different cell lineages, resulting in distinct pathways to disease pathogenesis. Tools such as Compass (34) can be combined with spatial transcriptomics, high-throughput measurement of metabolic enzymatic activity (131), and single-cell metabolomics (35) to improve our understanding of metabolic sex bias at the cellular, spatial, and temporal levels and inform progression of CNS disorders.
This work was supported by National Multiple Sclerosis Society Grant TA-1605-08590.
Abbreviations used in this article
branched-chain amino acid
branched-chain keto acid dehydrogenase
experimental autoimmune encephalomyelitis
glutamate dehydrogenase 1
histone 3 lysine 27 trimethylation
magnetic resonance spectroscopy
tricarboxylic acid cycle
The authors have no financial conflicts of interest.
Chao Wang, Ph.D.
Assistant Professor, University of Toronto, Canada, 2020–present
Assistant Professor, Harvard Medical School, 2019–2020
Instructor, Harvard Medical School, 2016–2019
Post-doctoral Fellow, Harvard Medical School, 2012–2016
Post-doctoral Fellow, University of Toronto, Canada, 2011–2012
Ph.D., University of Toronto, Canada, 2011
B.Sc., Simon Fraser University, Canada, 1999
Raised by the lakes of mainland China, I was brought up with a strong sense of cultural identity. Much like how the immune system matures after finding its balance between the recognition of self and non-self, I was shaped by self-identity, as the cultural and ethnic diversity I was exposed to in Canada when I joined a United World College on the tip of Vancouver Island where students from 87 countries were present. The school motto was based on the quote from the late Canadian Prime Minister Lester B. Pearson: “How can there be peace without people understanding each other; and how can this be if they don’t know each other?” Although very much a geopolitical statement, my scientific career has benefited from it as I frequently seek different opinions and hypotheses by talking to fellow scientists and knowledge users with different cultural, ethnical, and educational backgrounds. I have also been extremely fortunate to have trained with a number of amazing scientific mentors that are inclusive and open-minded, which cultivated the inclination to seek diversity. In parallel, I often think the scientific truth lies deep within the diverse biological variables that are waiting to be organized into signatures and networks, and one must first appreciate the diversity of it before comprehension. In tribute to this issue, I contributed a review on sex dimorphism of immunometabolism in the brain which only begins to address the enormous diversity of the biological system which I so love.
Chao Wang, Ph.D.
Assistant Professor, University of Toronto, Canada