In a recent study published in Neuron, researchers explored the biological mechanisms by which exercise-induced irisin attenuates amyloid-beta (Aβ) pathology in Alzheimer’s disease (AD).
Background
AD, a form of dementia, is characterized by progressive memory loss and severe cognitive impairment. Aβ protein deposition is a pathological hallmark of AD. Physical exercise has been shown to reduce Aβ deposition and neuroinflammation in AD mouse models. Irisin, an exercise-induced hormone, regulates lipid and glucose metabolism in adipose tissues and increases energy expenditure. Lower irisin levels have been reported in AD patients and AD murine models.
About the study
In the present study, researchers elucidated mechanisms underlying irisin-induced Aβ reduction in AD.
A three-dimensional (3D) human brain cell culture model of AD with robust Aβ generation was used, followed by a tau pathology (3D-AD) model. Irisin was administered to differentiated ReN cells that expressed familial AD-related APPSwedish/London amino acid substitutions (ReN-GA) or presenilin-1 and APPSwedish/London amino acid substitutions (ReN-mGAP) for two weeks at concentrations of 5.0 as well as 500 nanograms per mL. From two- or five-week differentiated three-dimensional cultures, samples of conditioned medium and cell pellets (3D gel) were obtained for analysis.
The enzyme lactate dehydrogenase was employed to detect cell death or cytotoxicity. Brain-derived neurotrophic factor (BDNF) expression was measured. Western blotting was done using 6E10 or G12A antibodies. Calcium stimulation and quick time-lapse imaging were used to measure the spontaneous activity of active cells in three-dimensional cultures after five weeks of differentiation.
The researchers investigated whether irisin may lower Aβ levels by increasing Aβ phagocytosis or enzymatic breakdown. In conditioned media comprising irisin-treated three-dimensional-AD cells, the researchers examined the levels of secreted neprilysin (secNEP) and insulin-degrading enzyme (secIDE), which are proteases released by astrocytes that can reduce Aβ levels.
The action of sacubitril, a neprilysin inhibitor, was examined in conditioned media comprising three-dimensional cultures. Human-induced pluripotent stem-cell-derived astrocyte (hiPSC-Astro) cells were treated with irisin to see if they were the source of the irisin-induced rise in secreted NEP.
Integrin αV and β5 subunit expression in ReN-mGAP and ReN-GA cells were validated using mass spectrometry (MS) and western blot analyses, respectively. Integrin β5 (ITGB5) activity was reduced genetically in ReN-GA and pharmacologically in ReN-mGAP cells to investigate whether integrin αV/β5 receptors were necessary for irisin to lower Aβ levels.
In five-week irisin-treated ReN-GA cells, the researchers examined signal transducer and activator of transcription 3 (STAT3) gene expression using specific astrocyte reactivity markers such as Cp, C3, glial fibrillary acidic protein (GFAP), cluster of differentiation 44 (CD44), adhesion molecule with Ig-like domain 2 (Amigo2), epithelial membrane protein 1 (Emp1), and serpin family G member 1 (Serping1).
In irisin-treated cells, the levels of astrocyte reactivity markers, S100 calcium-binding protein B (S100b), and GFAP were measured. Single-cell ribonucleic acid sequencing (scRNA-seq) was performed to examine irisin-induced cell-specific transcriptome alterations,
Results
The exercise-induced myokine, irisin, increased neprilysin production, which reduced A pathology in the AD culture. Integrin αV/β5 was the irisin receptor on astrocytes necessary to promote neprilysin secretion, which was achieved by downregulating the extracellular-signal-regulated kinase (ERK)-STAT3 signaling. At 500 nanograms per mL concentrations, irisin dramatically lowered Aβ-40 and 42 expressions in five-week ReN-GA cells and cellular pellets.
After five weeks of cellular differentiation, 500 nanograms per mL of irisin decreased Aβ-40 and 42 expressions in conditioned media and only Aβ42 expression in ReN-mGAP pellets, indicating more severe AD pathology than ReN-GA.
In five-week three-dimensional-AD cultures, irisin treatment did not enhance LDH release, showing that the irisin-induced Aβ reduction did not occur from cell death. Instead, it lowered LDH secretion at 500 nanograms per mL in five-week three-dimensional-AD cells at the time of endogenous loss of cells, showing neuroprotective benefits. Irisin showed no effect on BDNF expression in the five-week three-dimensional-AD cultures, demonstrating that the drop in Aβ caused by irisin was not BDNF-regulated.
Irisin quickly activated integrin receptors, as evidenced by increased phosphorylated focal adhesion kinase (p-FAK) and cyclic AMP response element-binding protein (CREB) phosphorylation in ReN-mGAP and hiPSC-Astro cultures. Irisin increased integrin signaling in the AD- and hiPSC-Astro cultures, based on the findings. Irisin lowered the expressions of nuclear factor kappa B (NF-kB) p65, complement C3 (C3), C3a receptor (C3aR), and apolipoprotein E (APOE) in the gels of five-week ReN-GA cells. Irisin treatment did not affect amyloid precursor protein (APP) processing.
In the AD cultures, irisin decreased the quantity and size of dystrophic neurites as well as cell hyperexcitability and boosted secNEP levels. Sacubitril inhibited secNEP activity in the three-dimensional culture medium.
Irisin and sacubitril co-treatment dramatically increased Aβ42 expression in AD cultures, indicating that irisin required increased secNEP activity to decrease Aβ42 expression. L-a-aminoadipate (L-AAA) therapy significantly reduced S100b and ITGB5 expression among ReN-mGAP cells, indicating that ITGB5 was mainly expressed in astrocytes in the 3D-AD model.
The capacity of irisin to lower STAT3, C3aR, and NF-kB p65 was abolished when combined with an integrin αV/β5 antibody. Irisin inhibited the expression of reactive astrocyte genes and proteins. Irisin decreased STAT3 levels by blocking interleukin-6 (IL-6)/ERK signaling, enhancing secNEP release. A1 inducer treatment decreased neprilysin expression in hiPSC-Astro cell lysates. Irisin also lowered the amount of phosphorylated tau protein in the body.
Conclusion
Overall, the study findings showed that irisin significantly reduced Aβ pathology by boosting the astrocytic release of the Aβ-degrading enzyme neprilysin, mediated via ERK-STAT3 signaling downregulation. The findings lend support to the development of irisin as a possible therapeutic target for Alzheimer’s disease.
