Annexin A1 couples tanycyte nutrient sensing to microglial function and thermoregulation

Technical Summary: Annexin A1 couples tanycyte nutrient sensing to microglial function and thermoregulation

Problem Statement
The hypothalamus is a critical hub for integrating physiological signals to maintain energy homeostasis. While neuronal circuits are well-established as the primary regulators of this process, the role of glial cells in metabolic control is increasingly recognized. Tanycytes, specialized ependymal cells lining the third ventricle, are known to sense metabolic cues and communicate with neurons via metabolites and signaling molecules. However, the mechanisms by which tanycytes communicate with neighboring glial cells, specifically microglia, and how this interglial crosstalk influences systemic metabolic adaptation and thermoregulation, remain largely unexplored. Furthermore, the specific molecular mediators released by tanycytes that could modulate the hypothalamic immune environment are not well defined.

Methodology
The study employed a multidisciplinary approach combining in vivo mouse models, primary cell cultures, and advanced imaging and omics techniques:

• Ultrastructural Analysis: Semi-correlative electron microscopy was used to visualize tanycyte processes in vivo and in primary cultures, identifying multivesicular structures.
• Proteomics and Transcriptomics: Tanycyte-derived extracellular vesicles (EVs) were isolated via differential ultracentrifugation and subjected to mass spectrometry (LC-MS/MS) for proteomic profiling. Single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (Allen Brain Atlas) were utilized to map gene expression patterns of Anxa1 and its receptors (Fpr1, Fpr2) across hypothalamic cell types.
• Cell Culture Models: Primary tanycyte and microglia cultures were established to investigate nutrient-dependent trafficking of Annexin A1 (ANXA1) and its direct effects on microglial function. Glucose deprivation, refeeding, and calcium chelation experiments were conducted to dissect signaling pathways.
• Genetic and Pharmacological Manipulation:
  • Knockout: Tanycyte-specific Anxa1 knockout mice (TanAnxa1⁻/⁻) were generated using stereotaxic infusion of AAV1/2-Tshr-Cre into Anxa1^flox/flox mice.
  • Pharmacology: The ANXA1 mimetic peptide Ac2-26 was infused intracerebroventricularly (i.c.v.) to activate signaling. Lipopolysaccharide (LPS) was used to induce microglial inflammation, and the CSF1R inhibitor PLX5622 was used to deplete microglia.
• Physiological Assays: Mice were subjected to fasting-refeeding paradigms, high-fat diet (HFHS and 60HF) regimens, cold exposure tests, and β3-adrenergic receptor blockade. Metabolic parameters (body weight, glycemia, food intake, energy expenditure) and thermoregulatory responses (core and brown adipose tissue temperatures) were monitored.
• Immunohistochemistry and Molecular Biology: Techniques included RNAscope in situ hybridization, Western blotting, ELISA, qPCR, and confocal microscopy to assess protein localization, cytokine profiles, and gene expression.

Key Contributions and Results

1. Tanycytes Release Immune-Modulating EVs: The authors identified that tanycytes generate and release extracellular vesicles (EVs) containing a broad repertoire of immune-related proteins. Proteomic analysis revealed that Annexin A1 (ANXA1) is a highly enriched cargo within these tanycyte-derived EVs.
2. Metabolic Regulation of ANXA1 Secretion: ANXA1 expression and subcellular localization in tanycytes are dynamically regulated by nutrient availability.
   • Acute Regulation: Glucose exposure triggers a rapid, dose- and time-dependent redistribution of ANXA1 from the nucleus to CD9-positive multivesicular structures and the plasma membrane, facilitating its incorporation into EVs. This process is dependent on intracellular Ca²⁺ signaling and phosphorylation of ANXA1 at tyrosine 21.
   • Chronic Regulation: Chronic high-fat feeding leads to a reduction in hypothalamic ANXA1 protein levels, mimicking the phenotype observed in tanycyte-specific Anxa1 knockouts.
3. ANXA1 Modulates Microglial State: Tanycyte-derived ANXA1 acts on microglia via the formyl peptide receptor 1 (FPR1).
   • Resting State: Acute activation of ANXA1 signaling (via Ac2-26) promotes a homeostatic microglial morphology with increased ramification and reduced phagocytic activity.
   • Inflammatory State: ANXA1 signaling attenuates LPS-induced microglial activation, reducing the secretion of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) and enhancing anti-inflammatory IL-10 production. This effect is abolished when Fpr1 is silenced in microglia.
   • Loss of Signal: Deletion of Anxa1 specifically in tanycytes results in a reactive microglial phenotype characterized by enlarged soma, reduced process complexity, and increased density in the mediobasal hypothalamus.
4. Thermoregulatory Consequences: The disruption of the tanycyte-ANXA1-microglia axis impairs adaptive thermogenesis.
   • TanAnxa1⁻/⁻ mice exhibit elevated basal core body temperature and impaired cold tolerance.
   • Brown adipose tissue (BAT) in these mice shows signs of chronic activation (smaller lipid droplets, increased thermogenic gene expression) but fails to sustain thermogenic responses during acute cold challenges.
   • Crucially, microglial depletion in TanAnxa1⁻/⁻ mice rescues the thermoregulatory defects, restoring normal body temperature and cold-induced thermogenesis. This confirms that microglia are essential downstream effectors of tanycytic ANXA1 signaling in thermoregulation.

Significance
The paper establishes a previously unrecognized communication axis between tanycytes and microglia that links metabolic sensing to hypothalamic immune regulation and systemic thermoregulation. The authors propose that tanycytes act as immunomodulatory cells, releasing ANXA1 via EVs in a nutrient-dependent manner to maintain microglial homeostasis. When this signaling is disrupted (e.g., by chronic high-fat feeding or genetic deletion), microglia become activated, leading to impaired adaptive thermogenesis and metabolic dysfunction.

The study suggests that the decline in central ANXA1 signaling observed during chronic metabolic stress may contribute to hypothalamic neuroinflammation and the development of obesity-associated phenotypes. By defining the tanycyte-microglia axis, the work highlights glia-glia communication as a fundamental component of hypothalamic metabolic control and identifies ANXA1 as a potential therapeutic target for restoring hypothalamic homeostasis in metabolic diseases. The authors note that while the study was conducted in mice, the evolutionary conservation of tanycytes and the presence of ANXA1 in human cerebrospinal fluid suggest potential relevance to human metabolic disorders, though further investigation is required.