Heme Mediates the Akt/mTOR Intracellular Signaling Pathway Through IGF1R in Microglia

Introduction:

Background: Alzheimer’s disease (AD) is one of the most prevalent forms of neurodegenerative disease, affecting over 30 million people worldwide. Meanwhile, dysregulated neuroinflammation consist of intracellular signaling pathways and microglial activation, has been identified as a core pathology for Alzheimer’s disease (AD). Despite the increasing advancements in phenotyping microglia and understanding molecular changes during AD pathogenesis, there remains an essential knowledge gap on the underlaying mechanisms driving dysregulated immune function. Previous studies have indicated the significance of insulin growth factor 1 receptor (IGF1R) induced Akt/mTOR signaling in AD pathogenesis, highlighting its downstream effect in mediating dysregulated brain immune signaling.
Heme, the oxygen carrier cofactor of hemoglobin, can act as an immunomodulatory molecule with elevated levels observed in AD patient brains. We have previously reported that heme suppresses astrocyte inflammatory activation in response to phagocytic capacity for Aβ clearance. Furthermore, we have revealed that heme mediates immune signaling in both Aβ-dependent and independent pathways. Given the established role of IGF1R-induced Akt/mTOR signaling in immune modulation, we investigated whether heme’s immunomodulatory ability is associated with this pathway. Moreover, being a relatively large receptor molecule, IGF1R has been shown to have multiple allosteric binding sites with the potential to elicit downstream Akt/mTOR phospho-signaling. Coupled with heme’s relatively small molecular size and high binding affinity, we hypothesized that heme mediates Akt/mTOR signaling via IGF1R in a manner distinct from IGF1 ligand in Alzheimer’s disease.

Materials and

Methods:

Methods: Mouse microglia cell (MMC) line was utilized for all cell culture experiments. MMCs were plated in 6-well plates, starved for 1.5 hours, and conditioned with combinations of heme (25uM) and IGF1 ligand (100ng/mL) for either 5- or 15- minutes followed by cell collection and protein extraction. For inhibition studies, cells were conditioned with IGF1R small molecule inhibitor BMS536924 (100ng/mL) for 1.5 hours before the 5- or 15/15-minute Heme/IGF1 ligand incubation, followed by cell collection and protein extraction. Protein concentrations were determined using a Pierce BCA
protein assay. Multiplexed phosphoprotein analysis was conducted for the Akt/mTOR pathway via Milliplex MAP Akt/ mTOR 11-Plex (p-Akt, p-GSK3/, p-IGF1R, p-IR, p-IRS1, p-mTOR, p-p70S6K, p-PTEN, p-RPS6, and p-TSC2).

Results, Conclusions, and Discussions: Results and Discussions:
Our result indicated that initially, 5min incubation with 25M heme resulted in elevated levels of Akt/mTOR pathway phosphorylation (Figure 1a). Although such heme-induced activation was less profound compared to IGF1 ligand-induced activation, the observed trends revealed heme’s effect in driving the Akt/mTOR phospho-signaling within minutes of incubation. Moreover, heme-induced Akt/mTOR phosphorylation was further amplified with 15-minute incubation (Figure 1b), suggesting a progressive, sustained effect of heme in regulating the Akt/mTOR signaling pathway. Notably, for both timepoints, heme-induced elevated signaling was normalized with incubation of IGF1R inhibitor BME536924, indicating the importance of IGF1R in supporting heme as an immunomodulator.
In addition, while IGF1 ligand initially served as a positive control for the Akt/mTOR phospho-signaling cascade, surprisingly, our data demonstrated that treatment with the combined treatment of IGF1 ligand and Heme led to attenuated effects compared to treatments with IGF1 ligand alone and heme alone in 5- and 15-minutes, respectively (Figure a-b). This phenomenon could result from competitive binding between the IGF1 ligand and heme, potentially exacerbated by heme-induced conformational changes in IGF1R, thereby highlighting heme’s regulatory role in the Akt/mTOR signaling pathway. Given the established significance of the IGF1R-induced Akt/mTOR phospho-signaling pathway and the known role of microglial activity in AD pathology, our findings suggested exciting heme-based therapeutic potentials targeting dysregulated inflammation for Alzheimer’s disease.

Conclusion: Together, our data demonstrated that in vitro, heme potentially exhibits immunomodulatory effects on the Akt/mTOR signaling pathway, initiating phosphorylation within 5min, and sustained to 15 minutes, while also attenuating IGF1 ligand-induced Akt/mTOR pathway activation. These findings highlight an entirely new mechanism of Akt/mTOR signaling in the control of microglial function. Given the established dysregulation of both this pathway and brain heme content in AD, these findings may lead to new heme-targeted therapies for promoting neuroprotective microglial function in this devastating disease.

Acknowledgements (Optional):