Deconstructing sex: Strategies for undoing binary thinking in neuroendocrinology and behavior.

The scientific community widely recognizes that "sex" is a complex category composed of multiple physiologies. Yet in practice, basic scientific research often treats "sex" as a single, internally consistent, and often binary variable. This practice occludes important physiological factors and processes, and thus limits the scientific value of our findings. In human-oriented biomedical research, the use of simplistic (and often binary) models of sex ignores the existence of intersex, trans, non-binary, and gender non-conforming people and contributes to a medical paradigm that neglects their needs and interests. More broadly, our collective reliance on these models legitimizes a false paradigm of human biology that undergirds harmful medical practices and anti-trans political movements. Herein, we continue the conversations begun at the SBN 2022 Symposium on Hormones and Trans Health, providing guiding questions to help scientists deconstruct and rethink the use of "sex" across the stages of the scientific method. We offer these as a step toward a scientific paradigm that more accurately recognizes and represents sexed physiologies as multiple, interacting, variable, and unbounded by gendered preconceptions. We hope this paper will serve as a useful resource for scientists who seek a new paradigm for researching and understanding sexed physiologies that improves our science, widens the applicability of our findings, and deters the misuse of our research against marginalized groups.

Feeding neurons integrate metabolic and reproductive states in mice.

Balance between metabolic and reproductive processes is important for survival, particularly in mammals that gestate their young. How the nervous system coordinates this balance is an active area of study. Herein, we demonstrate that somatostatin (SST) neurons of the tuberal hypothalamus alter feeding in a manner sensitive to metabolic and reproductive states in mice. Whereas chemogenetic activation of SST neurons increased food intake across sexes, ablation decreased food intake only in female mice during proestrus. This ablation effect was only apparent in animals with low body mass. Fat transplantation and bioinformatics analysis of SST neuronal transcriptomes revealed white adipose as a key modulator of these effects. These studies indicate that SST hypothalamic neurons integrate metabolic and reproductive cues by responding to varying levels of circulating estrogens to modulate feeding differentially based on energy stores. Thus, gonadal steroid modulation of neuronal circuits can be context dependent and gated by metabolic status.

Feeding Neurons Integrate Metabolic and Reproductive States in Mice.

Trade-offs between metabolic and reproductive processes are important for survival, particularly in mammals that gestate their young. Puberty and reproduction, as energetically taxing life stages, are often gated by metabolic availability in animals with ovaries. How the nervous system coordinates these trade-offs is an active area of study. We identify somatostatin neurons of the tuberal nucleus (TNSST) as a node of the feeding circuit that alters feeding in a manner sensitive to metabolic and reproductive states in mice. Whereas chemogenetic activation of TNSST neurons increased food intake across sexes, selective ablation decreased food intake only in female mice during proestrus. Interestingly, this ablation effect was only apparent in animals with a low body mass. Fat transplantation and bioinformatics analysis of TNSST neuronal transcriptomes revealed white adipose as a key modulator of the effects of TNSST neurons on food intake. Together, these studies point to a mechanism whereby TNSST hypothalamic neurons modulate feeding by responding to varying levels of circulating estrogens differentially based on energy stores. This research provides insight into how neural circuits integrate reproductive and metabolic signals, and illustrates how gonadal steroid modulation of neuronal circuits can be context-dependent and gated by metabolic status.

Estrogen-sensitive medial preoptic area neurons coordinate torpor in mice.

Homeotherms maintain a stable internal body temperature despite changing environments. During energy deficiency, some species can cease to defend their body temperature and enter a hypothermic and hypometabolic state known as torpor. Recent advances have revealed the medial preoptic area (MPA) as a key site for the regulation of torpor in mice. The MPA is estrogen-sensitive and estrogens also have potent effects on both temperature and metabolism. Here, we demonstrate that estrogen-sensitive neurons in the MPA can coordinate hypothermia and hypometabolism in mice. Selectively activating estrogen-sensitive MPA neurons was sufficient to drive a coordinated depression of metabolic rate and body temperature similar to torpor, as measured by body temperature, physical activity, indirect calorimetry, heart rate, and brain activity. Inducing torpor with a prolonged fast revealed larger and more variable calcium transients from estrogen-sensitive MPA neurons during bouts of hypothermia. Finally, whereas selective ablation of estrogen-sensitive MPA neurons demonstrated that these neurons are required for the full expression of fasting-induced torpor in both female and male mice, their effects on thermoregulation and torpor bout initiation exhibit differences across sex. Together, these findings suggest a role for estrogen-sensitive MPA neurons in directing the thermoregulatory and metabolic responses to energy deficiency.

Hypothalamic estrogen receptor alpha establishes a sexually dimorphic regulatory node of energy expenditure.

Estrogen receptor a (ERa) signaling in the ventromedial hypothalamus (VMH) contributes to energy homeostasis by modulating physical activity and thermogenesis. However, the precise neuronal populations involved remain undefined. Here, we describe six neuronal populations in the mouse VMH by using single-cell RNA transcriptomics and in situ hybridization. ERa is enriched in populations showing sex biased expression of reprimo (Rprm), tachykinin 1 (Tac1), and prodynorphin (Pdyn). Female biased expression of Tac1 and Rprm is patterned by ERa-dependent repression during male development, whereas male biased expression of Pdyn is maintained by circulating testicular hormone in adulthood. Chemogenetic activation of ERa positive VMH neurons stimulates heat generation and movement in both sexes. However, silencing Rprm gene function increases core temperature selectively in females and ectopic Rprm expression in males is associated with reduced core temperature. Together these findings reveal a role for Rprm in temperature regulation and ERa in the masculinization of neuron populations that underlie energy expenditure.

Sexes on the brain: Sex as multiple biological variables in the neuronal control of feeding.

Neuronal interactions at the level of vagal, homeostatic, and hedonic circuitry work to regulate the neuronal control of feeding. This integrative system appears to vary across sex and gender in the animal and human worlds. Most feeding research investigating these variations across sex and gender focus on how the organizational and activational mechanisms of hormones contribute to these differences. However, in limited studies spanning both the central and peripheral nervous systems, sex differences in feeding have been shown to manifest not just at the level of the hormonal, but also at the chromosomal, epigenetic, cellular, and even circuitry levels to alter food intake. In this review, we provide a brief orientation to the current understanding of how these neuronal systems interact before dissecting selected studies from the recent literature to exemplify how feeding physiology at all levels can be affected by the various components of sex.

Propionic Acid Shapes the Multiple Sclerosis Disease Course by an Immunomodulatory Mechanism.

Short-chain fatty acids are processed from indigestible dietary fibers by gut bacteria and have immunomodulatory properties. Here, we investigate propionic acid (PA) in multiple sclerosis (MS), an autoimmune and neurodegenerative disease. Serum and feces of subjects with MS exhibited significantly reduced PA amounts compared with controls, particularly after the first relapse. In a proof-of-concept study, we supplemented PA to therapy-naive MS patients and as an add-on to MS immunotherapy. After 2 weeks of PA intake, we observed a significant and sustained increase of functionally competent regulatory T (Treg) cells, whereas Th1 and Th17 cells decreased significantly. Post-hoc analyses revealed a reduced annual relapse rate, disability stabilization, and reduced brain atrophy after 3 years of PA intake. Functional microbiome analysis revealed increased expression of Treg-cell-inducing genes in the intestine after PA intake. Furthermore, PA normalized Treg cell mitochondrial function and morphology in MS. Our findings suggest that PA can serve as a potent immunomodulatory supplement to MS drugs.

IDOL regulates systemic energy balance through control of neuronal VLDLR expression.

Liver X receptors limit cellular lipid uptake by stimulating the transcription of Inducible Degrader of the LDL Receptor (IDOL), an E3 ubiquitin ligase that targets lipoprotein receptors for degradation. The function of IDOL in systemic metabolism is incompletely understood. Here we show that loss of IDOL in mice protects against the development of diet-induced obesity and metabolic dysfunction by altering food intake and thermogenesis. Unexpectedly, analysis of tissue-specific knockout mice revealed that IDOL affects energy balance, not through its actions in peripheral metabolic tissues (liver, adipose, endothelium, intestine, skeletal muscle), but by controlling lipoprotein receptor abundance in neurons. Single-cell RNA sequencing of the hypothalamus demonstrated that IDOL deletion altered gene expression linked to control of metabolism. Finally, we identify VLDLR rather than LDLR as the primary mediator of IDOL effects on energy balance. These studies identify a role for the neuronal IDOL-VLDLR pathway in metabolic homeostasis and diet-induced obesity.

Testosterone Differentially Affects T Cells and Neurons in Murine and Human Models of Neuroinflammation and Neurodegeneration.

The high female-to-male sex ratio of multiple sclerosis (MS) prevalence has continuously confounded researchers, especially in light of male patients' accelerated disease course at later stages of MS. Although multiple studies have concentrated on estrogenic mechanisms of disease modulation, fairly little attention has been paid to androgenic effects in a female system, and even fewer studies have attempted to dissociate hormonal effects on the neurodegenerative and neuroinflammatory processes of MS. Herein, we demonstrate the differential effects of hormone treatment on the acute inflammatory and chronic neurodegenerative phases of murine experimental autoimmune encephalomyelitis. Although s.c. treatment with testosterone and aromatase inhibitor applied beginning on the day of immunization ameliorated initial course of disease, similar treatment administered therapeutically exacerbated chronic disease course. Spinal cord analyses of axonal densities reflected the clinical scores of the chronic phase. In vitro, testosterone treatment not only decreased Th1 and Th17 differentiation in an aromatase-independent fashion, but also exacerbated cell death in induced pluripotent stem cell-derived primary human neurons under oxidative stress conditions in an aromatase inhibitor-dependent manner. Thus, through the alleviation of inflammatory processes and the exacerbation of neurodegenerative processes, androgens may contribute to the epidemiologic sex differentials observed in MS prevalence and course.

The immunomodulatory effect of laquinimod in CNS autoimmunity is mediated by the aryl hydrocarbon receptor.

Though several functional properties of laquinimod have been identified, our understanding of the underlying mechanisms is still incomplete. Since the compound elicits similar immunomodulatory effects to ligands of the aryl hydrocarbon receptor (AhR), we compared the efficacy of laquinimod in experimental autoimmune encephalomyelitis (EAE)-afflicted wild-type and AhR-deficient mice. Laquinimod failed to ameliorate clinical symptoms and leukocyte infiltration in AhR-deficient mice; however, treatment exerted neuroprotection by elevation of brain-derived neurotrophic factor (BDNF) independent of genetic profile. Thus, our data identify the AhR pathway in these mutant mice as crucial for the immunomodulatory, but not neuroprotective, efficacy of laquinimod in EAE.

Multiple Sclerosis Patient-Specific Primary Neurons Differentiated from Urinary Renal Epithelial Cells via Induced Pluripotent Stem Cells.

As multiple sclerosis research progresses, it is pertinent to continue to develop suitable paradigms to allow for ever more sophisticated investigations. Animal models of multiple sclerosis, despite their continuing contributions to the field, may not be the most prudent for every experiment. Indeed, such may be either insufficient to reflect the functional impact of human genetic variations or unsuitable for drug screenings. Thus, we have established a cell- and patient-specific paradigm to provide an in vitro model within which to perform future genetic investigations. Renal proximal tubule epithelial cells were isolated from multiple sclerosis patients' urine and transfected with pluripotency-inducing episomal factors. Subsequent induced pluripotent stem cells were formed into embryoid bodies selective for ectodermal lineage, resulting in neural tube-like rosettes and eventually neural progenitor cells. Differentiation of these precursors into primary neurons was achieved through a regimen of neurotrophic and other factors. These patient-specific primary neurons displayed typical morphology and functionality, also staining positive for mature neuronal markers. The development of such a non-invasive procedure devoid of permanent genetic manipulation during the course of differentiation, in the context of multiple sclerosis, provides an avenue for studies with a greater cell- and human-specific focus, specifically in the context of genetic contributions to neurodegeneration and drug discovery.