Association between chronic stress-induced structural abnormalities in Ranvier nodes and reduced oligodendrocyte activity in major depression

Abstract

Repeated stressful events are associated with the onset of major depressive disorder (MDD). We previously showed oligodendrocyte (OL)-specific activation of the serum/glucocorticoid-regulated kinase (SGK)1 cascade, increased expression of axon-myelin adhesion molecules, and elaboration of the oligodendrocytic arbor in the corpus callosum of chronically stressed mice. In the current study, we demonstrate that the nodes and paranodes of Ranvier in the corpus callosum were narrower in these mice. Chronic stress also led to diffuse redistribution of Caspr and Kv 1.1 and decreased the activity in white matter, suggesting a link between morphological changes in OLs and inhibition of axonal activity. OL primary cultures subjected to chronic stress resulted in SGK1 activation and translocation to the nucleus, where it inhibited the transcription of metabotropic glutamate receptors (mGluRs). Furthermore, the cAMP level and membrane potential of OLs were reduced by chronic stress exposure. We showed by diffusion tensor imaging that the corpus callosum of patients with MDD exhibited reduced fractional anisotropy, reflecting compromised white matter integrity possibly caused by axonal damage. Our findings suggest that chronic stress disrupts the organization of the nodes of Ranvier by suppressing mGluR activation in OLs, and that specific white matter abnormalities are closely associated with MDD onset.

Figure 1. Chronic stress exposure causes morphological alterations in nodes and paranodes of Ranvier of the corpus callosum.

(A) Axon size and myelin thickness measured by g ratio (the ratio of the inner axonal diameter to the total outer diameter). The distribution of the g ratio is plotted as a function of axonal diameter. The results are expressed as the mean of six images (Cont; 304 axons, Stress; 324 axon) obtained from three independent experiments. *P < 0.05, Student’s t test. Cont; non-stressed control mice data, Stress; chronic stress-exposed mice data. (B) The distribution of the axon diameters of the corpus callosum in the control and repeated WIRS-exposed mice was assessed. Results are expressed as the mean of six images (300 nodes of Ranvier) obtained from three independent experiments. The results are expressed as the mean ± SEM. *P < 0.05, Student’s t test. (C) Electron micrographs of nodes (N) and paranodes (Pn) of Ranvier in longitudinal sections of the corpus callosum from control (left panels) and chronically stressed (right panels) mice. Scale bars, 1 μm. (D) Measurement of node width and paranode length. Results are expressed as mean ± SEM of six measurements from three independent experiments. *P < 0.05, Student’s t test. (E,H) Immunohistochemical analyses of Caspr (E) and K _v1.1 (H) expression in longitudinal sections of the corpus callosum from control (upper panels) and chronically stressed (lower panels) mice. Scale bar, 20 μm. (F,I) Representative images of Caspr (F) and K _v1.1 (I) expression. Scale bar, 5 μm. (G,J) Measurement of node width and the length of Caspr-labeled regions at paranodes (G), and node and paranode width and length of K _v1.1-labeled regions at juxtaparanodes (J). Results are expressed as the mean of six images (300 nodes of Ranvier) obtained from three independent experiments. Cas, Caspr-labeled region; N, node region; K _v, K _v1.1-labeled region; N + Pn, node and paranode region. *P < 0.05, Student’s t test.

Figure 2. Increase in the length of Caspr- and K _v1.1-immunoreactive regions at paranodes and juxtaparanodes of the corpus callosum upon chronic stress exposure.

(A,C) Immunohistochemical analyses of Na_v and Caspr (A), and K _v1.1 and Caspr (C) expression in longitudinal sections of the corpus callosum from control (left panels) and chronically stressed (right panels) mice. Scale bar, 10 μm. (B,D) Enlargement of the areas enclosed by squares in (A,C). Scale bar, 5 μm. Arrows indicate Na_v- and Caspr-positive (B) and K _v1.1- and Caspr-positive (D) boundary regions.

Figure 3. Chronic stress exposure leads to upregulation of adhesion molecules at adjacent nodes of Ranvier, causes morphological changes in mature oligodendrocytes, and decreases axonal activity in the corpus callosum.

(A) Western blot analysis of the expression of adhesion molecules (Caspr, total neurofascin, contactin, TAG1) and channels (Na_v and K _v1.1) at adjacent nodes of Ranvier in the corpus callosum of control and chronically stressed mice. (B) Quantification of protein bands from (A). Data are expressed as mean ± SEM of at least three independent experiments. *P < 0.05, Student’s t test. (C) Representative images of the processes of oligodendrocytes in control (Cont) and chronic stress exposed (Stress) mice. Scale bar, 40 μm. (F) Process complexity and branching are indicated by Sholl analysis. Quantification of intersection numbers within each radius (1–6). Results are the mean of six images obtained from three independent experiments and are expressed as the mean ± SEM. *P < 0.05, Student’s t test. (E,F) Na+/K+ transporting ATPase levels (E) and Na+/K+-ATPase activity (F) in the corpus callosum of control and chronically stressed mice. Data are expressed as mean ± SEM of at least three independent experiments. *P < 0.05, Student’s t test. (G,H) Time course of membrane potential activities of OPCs and mature OLs primary cultures (G) and membrane potential activities of 12 h DEX administration primary neurons (H) after 100 μM (G) or 10 μM (H) dexamethasone (DEX) administration. Data are expressed as mean ± SEM of at least three independent experiments. *P < 0.05, Student’s t test.

Figure 4. Chronic stress suppresses mGluR3 and 5 expressions in primary oligodendrocytes.

(A) mGluR1 to 5 mRNA expression in oligodendrocytes. Data are expressed as mean ± SEM of at least three independent experiments. *P < 0.05, Student’s t test. (B) Time course of Sgk1 mRNA expression in oligodendrocytes after 100 μM DEX administration. Data are expressed as mean ± SEM of at least three independent experiments. *P < 0.05, Student’s t test. (C–F) Real-time PCR analysis of Sgk1 (C), mGluR5 (D), and mGluR3 (E,F) mRNA expression in primary oligodendrocytes. Data are expressed as mean ± SEM of at least three independent experiments. *P < 0.05, Student’s t test.

Figure 5. Chronic stress induces nuclear translocation of phospho-SGK1.

(A) Western blot analysis of total and phosphorylated SGK1 protein levels in cytoplasmic (F1), membrane (F2), nuclear (F3), and cytoskeletal (F4) fractions of primary oligodendrocytes after treatment with 100 μM DEX. (B) Quantification of protein bands from (A). Data are expressed as mean ± SEM of at least three independent experiments. *P < 0.05, Student’s t test.

Figure 6. Chronic stress decreases mGluR3 and 5 expressions in a co-culture system and in the corpus callosum, and cAMP level in the corpus callosum.

(A) Representative images of immunocytochemical analysis of Neurofilament (NF) and MBP in oligodendrocyte and DRG-neuron co-culture system after treatment with (Cont) or without (DEX stimulations) 10 μM DEX for 12 hrs. Scale bar = 20 μm. (B) Sgk1, mGluR3, and -5 mRNA expression in co-culture system. Data are expressed as mean ± SEM of at least three independent experiments. *P < 0.05, Student’s t test. (C) Western blot analysis of mGluR3 and phosphorylated NDRG1 (oligodendrocyte stress marker) in the corpus callosum in control (Cont) and chronic stress-exposed (Stress) mice. (D) Quantification of protein bands from (C). Data are expressed as mean ± SEM of at least three independent experiments. *P < 0.05, Student’s t test. (E) cAMP levels in the corpus callosum of control (Cont) and chronically stressed (Stress) mice.

Figure 7. Lower fractional anisotropy (FA) values are observed in patients with MDD compared to healthy control subjects by DTI.

(A) Spherical voxels of interest (VOIs) placed on the anterior genu of the corpus callosum and (B) scatter plots of FA and axial diffusivity values in this region are shown for patients with MDD and controls. *Significantly lower FA and axial diffusivity values were observed in patients than in controls. Values were adjusted to the mean values of age and gender.