β‐Lactolin Improves Mitochondrial Dysfunction in Aβ‐treated Mouse Hippocampal Neuronal Cells and a Human iPSC‐Derived Neuronal Cell Model of Alzheimer’s Disease

Abstract

AbstractBackgroundMitochondrial dysfunctions are a key hallmark of Alzheimer’s disease (AD). β‐Lactolin, a whey‐derived glycine–threonine–tryptophan–tyrosine tetrapeptide, has been previously reported to prevent AD‐like pathologies in an 5×FAD mouse model via regulation of microglial functions. However, the direct effect of β‐lactolin on neuronal cells and neuronal mitochondrial functions remains unknown. Here, we investigated the effects of β‐lactolin on mitochondrial dysfunctions in amyloid β (Aβ)‐treated mouse hippocampal neuronal HT22 cells and human induced‐pluripotent cell (hiPSC)‐derived AD model neurons.MethodMouse hippocampal neuronal HT22 cell line, and hiPSC‐derived AD model neuron (PSEN1 P117L mutation) and its wild‐type control were used in this study. HT22 cells were treated with β‐lactolin (1–100 nM) for 1 hr, and Aβ42 (1–10 μM) was added for the next 23 hrs. Oxygen consumption rate was measured using extracellular flux analyzer. Intracellular ATP level was determined by luciferase activity. Cells were stained with MitoTracker, JC‐1, or MitoSOX fluorescent dye, and then mitochondrial morphologies, membrane potential, and oxidative stress, respectively, were examined by high content image analysis. Cell viability was measured by MTT assay. Gene expression levels were evaluated by RT‐PCR. hiPSC‐derived wild‐type and AD neurons were cultured for 14 days under β‐lactolin treatment, and mitochondrial morphologies and membrane potentials were analyzed.ResultHT22 cells treated with both β‐lactolin and Aβ exhibited increased oxygen consumption rate and cellular ATP concentrations compared to cells treated with Aβ alone, suggesting that β‐lactolin improves mitochondrial respiration and energy production. In high content image analysis, β‐lactolin attenuated Aβ‐induced mitochondrial fragmentation, membrane potential decrease, and excess oxidative stress, eventually preventing neuronal cell death. Treatments with β‐lactolin increased gene expression of mitofusin‐2, which contribute to mitochondrial fusion events. Finally, β‐lactolin attenuated impairment in both mitochondrial morphologies and membrane potentials in hiPSC‐derived AD model neurons, suggesting β‐lactolin shows a suppressing effect on human AD pathologies.Conclusionβ‐lactolin improved AD‐related neuronal mitochondrial dysfunctions and suppressed neuronal cell death in both mouse and human AD model neuronal cells. The dual function of β‐lactolin on both neuron and microglia marks an advantage in AD prevention.