Profiling of microglia and neurons in a stem cell and murine model of anorexia nervosa

Anorexia Nervosa (AN) is a psychiatric disorder affecting 4% of females and 0.3% of males with high chronicity and mortality rates. Patients maintain a negative energy balance despite presence of evolutionary mechanisms that should prevent elective-starvation. Genome wide association studies (GWAS) have indicated dysregulation of metabolic signalling, and highlighted genetic variants associated with microglia. Mapping of variant genes highlight hypothalamic regions involved in energy homeostatic regulation, including the arcuate hypothalamus (ARC). Previously, our lab has identified microglial enlargement and branching in the ARC, alongside hypothalamic neuronal degeneration in an anorectic murine model, the anx/anx mouse. Thus, we propose that hypothalamic dysregulation involving microglia is underpinning negative energy balance in AN.

To investigate the molecular basis of starvation in AN, we obtained fibroblasts which were reprogrammed to induced pluripotent stem cells (iPSCs), from AN patients and healthy controls (HC). We differentiated iPSCs to microglia-like-cells (iMGs), alongside generating hypothalamic and cortical neurons. We extracted and fed synaptosomes from both neuronal populations to iMGs to assess synaptic engulfment, and performed RNA-sequencing to characterize the cells. Additionally, we stimulated the iMGs with glucagon-like-peptide-1 (GLP-1) to investigate response to peripheral appetite signalling peptides. Finally, we investigated hypothalamic microglia in the anx/anx mouse using spatial transcriptomics with differential gene expression and gene set enrichment analysis (GSEA). In addition, we performed further immunohistochemical (IHC) investigation of microglia in ARC and dorsal medial hypothalamus (DMH), performing quantification with IMARIS to assess synaptic pruning processes.

We identified that AN-derived iMGs had a reduced phagocytic capacity of synaptosomes compared to healthy controls, however this was specific to hypothalamic neurons as phagocytic capacity was not different from controls when fed with cortical synaptosomes. This indicates hypothalamic-specific reductions in synaptic engulfment by AN microglia. When stimulated with GLP-1, phagocytosis of hypothalamic synaptosomes was reduced in healthy control iMGs but remained the same in AN iMGs, indicating altered response to the peripheral appetite signalling hormone. In addition, we observed 81 differentially expressed genes (DEGs) in our AN iMGs RNAseq analysis compared to controls, including a downregulation of GLP1R. Together this indicates hypothalamic specific aberrances in microglial phagocytic processes. Moreover, we identified several DEGs in hypothalamic microglia of the anx/anx mice, with GSEA indicating involvement in synapse pruning processes. 3D quantifications of immunofluorescent staining of microglia and synapses in anx/anx mouse using IMARIS revealed increased microglial cell volume in the ARC and DMH of the anorectic mouse. In addition, this was associated with increased total synaptic engulfment in the mediobasal hypothalamus, however, interestingly synapse consumption was reduced when normalized to microglial volume, in line with our findings in iMGs.

Taken together, our data reveal aberrances in microglial synaptic engulfment of hypothalamic neurons and altered response to peripheral appetite signalling molecule GLP-1, strengthening the metabo-psychiatric view on AN suggested by large scale genetic studies.

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