An orally active apelin receptor agonist improves Alzheimer’s disease pathology and cognitive functions by restoring microglial metabolism

Technical Summary: An Orally Active Apelin Receptor Agonist Improves Alzheimer's Disease Pathology and Cognitive Functions by Restoring Microglial Metabolism

Problem Statement
Alzheimer's disease (AD) is characterized by amyloid-β (Aβ) accumulation, chronic neuroinflammation, and cerebral glucose hypometabolism. While microglial phagocytosis is essential for Aβ clearance, this function is compromised in AD due to metabolic dysfunction. Specifically, prolonged exposure to Aβ and inflammatory stimuli drives microglia toward a state of metabolic exhaustion, characterized by impaired oxidative phosphorylation (OXPHOS) and defective glucose utilization. Current therapeutic strategies targeting microglial metabolism are limited. Furthermore, while the apelin peptide (APLN) and its receptor (APJ) regulate glucose metabolism and mitochondrial function, endogenous apelin has a short half-life, and continuous peptide stimulation leads to receptor desensitization and internalization, limiting its therapeutic utility.

Methodology
The study utilized the 5xFAD transgenic mouse model (carrying five familial AD mutations) and in vitro models (HT22 neurons and BV2 microglia).

• Pharmacokinetics: The blood-brain barrier (BBB) penetration of the synthetic biased apelin receptor agonist, CMF-019, was assessed via liquid chromatography-mass spectrometry (LC-MS/MS) in plasma and brain tissue following oral gavage.
• In Vivo Treatment: 9-month-old 5xFAD mice received chronic oral administration of CMF-019 (10 mg/kg) for 3 months.
• Behavioral Assessment: Cognitive function was evaluated using the Morris Water Maze (spatial learning/memory), Novel Object Recognition (episodic memory), and Open Field Test (anxiety/motor function).
• Metabolic and Molecular Analysis:
  • Glucose Uptake: A novel in vivo method (NBDGcyto) was developed involving stereotaxic injection of the fluorescent glucose analogue 2-NBDG into the hippocampus, followed by flow cytometry to quantify cell-specific uptake (neurons vs. microglia).
  • Mitochondrial Function: Seahorse extracellular flux assays measured Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR) in BV2 microglia exposed to LPS or Aβ oligomers.
  • Morphology: Mitochondrial mass and morphology were assessed via TOMM20 immunofluorescence and Mitotracker staining.
  • Phagocytosis: In vitro phagocytosis was measured using fluorescent latex beads; in vivo Aβ clearance was assessed via colocalization of Iba1 (microglia) and Thioflavin S (Aβ).
  • Signaling: Western blotting analyzed the activation of the PI3K/Akt/mTOR and MAPK/Erk pathways.

Key Results

1. BBB Penetration and Insulin Sensitivity: CMF-019 successfully crossed the BBB, reaching peak brain concentrations within 0.5 hours and remaining detectable for 8 hours. In vitro, CMF-019 restored insulin sensitivity in insulin-resistant HT22 neurons by significantly increasing Akt phosphorylation (pAktS473) without affecting the Erk pathway.
2. Cognitive Improvement: Chronic oral treatment with CMF-019 significantly improved spatial learning and memory in 5xFAD mice, evidenced by reduced escape latency in the Morris Water Maze and increased time spent in the target quadrant during probe tests. It also improved episodic memory in the Novel Object Recognition test and reduced anxiety-like behavior.
3. Pathology Reduction: CMF-019 treatment reduced Aβ plaque burden (both total loading and plaque number) in the cortex and hippocampus. It specifically reduced soluble Aβ42 levels and shifted the plaque size distribution toward smaller, less toxic plaques. Furthermore, it significantly reduced neuroinflammation, evidenced by decreased astrogliosis (GFAP), microgliosis (Iba1), and levels of pro-inflammatory cytokines (TNF-α and IL-1β).
4. Restoration of Microglial Metabolism:
   • Glucose Uptake: While total hippocampal glucose uptake was impaired in 5xFAD mice, CMF-019 specifically restored glucose uptake in CD11b+ microglia, not in neurons.
   • Mitochondrial Function: In BV2 microglia exposed to chronic LPS or Aβ, CMF-019 restored basal and maximal oxygen consumption rates (OCR) and ATP production, counteracting the shift toward glycolysis. It also restored mitochondrial mass (TOMM20 intensity) and morphology (aspect ratio and form factor), reversing Aβ-induced fragmentation.
   • Signaling Mechanism: The metabolic rescue was associated with the activation of the Akt-mTOR pathway and the inhibition of GSK3β (via Ser9 phosphorylation).
5. Enhanced Phagocytosis: By restoring metabolic fitness, CMF-019 enhanced the phagocytic capacity of microglia both in vitro (bead uptake) and in vivo (increased colocalization of microglia with Aβ fibrils).

Significance and Claims
The authors claim that this study identifies apelin receptor activation via the orally active agonist CMF-019 as a novel therapeutic strategy for AD. The central finding is that CMF-019 rescues microglial metabolic dysfunction—specifically by enhancing glucose utilization and mitochondrial oxidative phosphorylation via the Akt-mTOR signaling axis. This metabolic reprogramming restores the energetic capacity of microglia required for effective Aβ phagocytosis. Consequently, the treatment leads to reduced amyloid burden, diminished neuroinflammation, and improved cognitive function in the 5xFAD mouse model. The paper positions apelin receptor agonism as a promising approach to target immunometabolism in AD, offering a potential solution to the limitations of peptide-based therapies and the lack of current treatments targeting microglial metabolism.