Mechanoreceptor Piezo1 Is Downregulated in Multiple Sclerosis Brain and Is Involved in the Maturation and Migration of Oligodendrocytes in vitro

Abstract

Mechanical properties of the brain such as intracranial pressure or stiffness of the matrix play an important role in the brain's normal physiology and pathophysiology. The physical properties are sensed by the cells through mechanoreceptors and translated into ion currents which activate multiple biochemical cascades allowing the cells to adapt and respond to changes in their microenvironment. Piezo1 is one of the first identified mechanoreceptors. It modulates various central nervous system functions such as axonal growth or activation of astrocytes. Piezo1 signaling was also shown to play a role in the pathophysiology of Alzheimer's disease. Here, we explore the expression of the mechanoreceptor Piezo1 in human MO3.13 oligodendrocytes and human MS/non-MS patients' brains and investigate its putative effects on oligodendrocyte proliferation, maturation, and migration. We found that Piezo1 is expressed in human oligodendrocytes and oligodendrocyte progenitor cells in the human brain and that its inhibition with GsMTx4 leads to an increment in proliferation and migration of MO3.13 oligodendrocytes. Activation of Piezo1 with Yoda-1 induced opposite effects. Further, we observed that expression of Piezo1 decreased with MO3.13 maturation in vitro. Differences in expression were also observed between healthy and multiple sclerosis brains. Remarkably, the data showed significantly lower expression of Piezo1 in the white matter in multiple sclerosis brains compared to its expression in the white matter in healthy controls. There were no differences in Piezo1 expression between the white matter plaque and healthy-appearing white matter in the multiple sclerosis brain. Taken together, we here show that Piezo1-induced signaling can be used to modulate oligodendrocyte function and that it may be an important player in the pathophysiology of multiple sclerosis.

Keywords: MO3.13; mechanoreceptor; mechanosensation; mechanosensitive ion channel (MSC); migration; multiple sclerosis; oligodendrocyte (OLs); piezo1.

FIGURE 1

Piezo1 is expressed in oligodendrocyte linage cells in the human brain. (A) Piezo1 protein is present in OPCs (A2B5⁺), (B) in oligodendrocyte cell bodies (CC1⁺) and in (C) myelin (MBP⁺), as indicated by overlapping green (Piezo1) and red staining. Representative images of N = 3 human brains. Scale 20 μm.

FIGURE 2

Piezo1 agonism with Yoda-1 induces calcium signaling in MO3.13 oligodendrocytes. (A) Traces calculated from 50 s recordings show changes in Ca²⁺ levels overtime after stimulation with Piezo1 agonist Yoda-1 (20 μM) and no increase after HBSS (ctrl). Peak from t = 10 s to t = 24 s, AUC = 18.19 ± 1.49. Traces are shown as mean ± SEM of N = 4 independent experiments. (B) Corresponding bar graph shows increased Ca²⁺ signaling after receptor stimulation with agonist Yoda-1. Data presented as mean ± SEM, unpaired t-test, ****p < 0.0001, N = 4 independent experiments. (C) Representative images taken from microscope recordings show basal levels of Ca²⁺, followed by induction of Ca²⁺ after addition of Yoda-1 or HBSS (ctrl), and subsequent recovery. Scale 100 μm.

FIGURE 3

Inhibition of Piezo1 induces MO3.13 oligodendrocyte migration. (A) Representative images and a corresponding bar graph show increased oligodendrocyte migration after inhibition of Piezo1 signaling for 24 h with GsMTx4 (500 nm) (208.4 ± 8.21% vs. control). Agonism of Piezo1 with Yoda-1 (40 μm) induced opposite effects (58 ± 3.82% vs. control). Data presented as mean ± SEM, One-way analysis of variance and Tukey’s multiple comparisons test, ***p < 0.001, N = 8. Scale 25 μm. (B) The MTT cell proliferation assay showed significantly increased cell proliferation after 72 h of Piezo1 inhibition with GsMTx4 (224.8 ± 7.50% vs. control) with no differences compared to the 24 and 48 h controls. Activation of the receptor with Yoda-1 significantly reduced cell proliferation after 48 (110.8 ± 15.58% vs. control) and 72 h (139.0 ± 3.34% vs. control). Data presented as mean ± SEM, One-way analysis of variance and Sidak’s multiple comparisons test, *p < 0.05, **p < 0.01, ***p < 0.001, N = 4 independent experiments.

FIGURE 4

Expression of Piezo1 decreases in MO3.13 oligodendrocytes during maturation. (A) The mRNA expression of Piezo1 receptor in MO3.13 oligodendrocytes is downregulated already after 24 h of both, serum-free media (SFM) (28 ± 16% SFM 24 h vs. FBS) and SFM supplemented with PMA (8 ± 4% PMA 24 h vs. FBS). At 48 h of treatment with PMA (data not collected for 48 h SFM) Piezo1 mRNA briefly increases (45 ± 8% PMA 48 h vs. FBS, p = 0.0522) indicating a temporary upregulation of Piezo1 signaling during oligodendrocyte maturation. Data presented as mean ± SEM, One-way analysis of variance with Sidak’s multiple comparison’s test, p = 0.013, N = 3 independent experiments. **p < 0.01 (B) SFM (365 ± 20% SFM 72 h vs. FBS) and PMA (314 ± 46% OMA 72 h vs. FBS) treatment induce MO3.13 oligodendrocyty maturation. Data presented as mean ± SEM, One-way analysis of variance with Sidak’s multiple comparison’s test, p < 0.0001, N = 4 independent experiments. ***p < 0.001, ****p < 0.0001 (C) Representative WB image of OPC marker PDGFRα shows steadily decreasing levels of PDGFRα protein in MO3.13 oligodendrocytes stimulated with PMA or cultured in SFM. Data presented as mean ± SEM, One-way analysis of variance with Dunnett’s multiple comparison’s test, p < 0.0026, N = 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 5

Piezo1 expression is downregulated in MS brain. (A) Analysis of Piezo1 mRNA levels shows significantly reduced expression of Piezo1 in MS brain in white matter (WM) (32.7 ± 19.2% vs. ctrl). Unpaired Student’s t-tests, **p < 0.005, N = 2 ctrl and N = 5 MS brains. (B) No differences in Piezo1 expression were found in the MS brains between WM lesions (plaques) and lesion-free WM within the same MS brain (91.98 ± 19.77% plaque vs. WM). Unpaired Student’s t-tests, p > 0.05, N = 5 MS brains.