HDAC inhibition prevents white matter injury by modulating microglia/macrophage polarization through the GSK3β/PTEN/Akt axis

Abstract

Severe traumatic brain injury (TBI) elicits destruction of both gray and white matter, which is exacerbated by secondary proinflammatory responses. Although white matter injury (WMI) is strongly correlated with poor neurological status, the maintenance of white matter integrity is poorly understood, and no current therapies protect both gray and white matter. One candidate approach that may fulfill this role is inhibition of class I/II histone deacetylases (HDACs). Here we demonstrate that the HDAC inhibitor Scriptaid protects white matter up to 35 d after TBI, as shown by reductions in abnormally dephosphorylated neurofilament protein, increases in myelin basic protein, anatomic preservation of myelinated axons, and improved nerve conduction. Furthermore, Scriptaid shifted microglia/macrophage polarization toward the protective M2 phenotype and mitigated inflammation. In primary cocultures of microglia and oligodendrocytes, Scriptaid increased expression of microglial glycogen synthase kinase 3 beta (GSK3β), which phosphorylated and inactivated phosphatase and tensin homologue (PTEN), thereby enhancing phosphatidylinositide 3-kinases (PI3K)/Akt signaling and polarizing microglia toward M2. The increase in GSK3β in microglia and their phenotypic switch to M2 was associated with increased preservation of neighboring oligodendrocytes. These findings are consistent with recent findings that microglial phenotypic switching modulates white matter repair and axonal remyelination and highlight a previously unexplored role for HDAC activity in this process. Furthermore, the functions of GSK3β may be more subtle than previously thought, in that GSK3β can modulate microglial functions via the PTEN/PI3K/Akt signaling pathway and preserve white matter homeostasis. Thus, inhibition of HDACs in microglia is a potential future therapy in TBI and other neurological conditions with white matter destruction.

Keywords: inflammation; microglial polarization; myelination; oligodendrocyte; traumatic brain injury.

Fig. 1.

Proposed mechanism underlying the preservation of white matter by HDAC inhibition. Microglia/macrophages respond to TBI with proinflammatory changes that increase active, dephosphorylated PTEN. Active PTEN then converts phosphatidylinositol (3,5)-triphosphate (PIP3) to phosphatidyl (4,5)-biphosphate (PIP2), thereby inactivating the prosurvival kinase Akt and leading to microglial and oligodendroglial toxicity. HDAC inhibition counterbalances this inflammatory effect by increasing GSK3β, perhaps by relieving transcriptional repression of its mRNA. Active GSK3β then phosphorylates PTEN at Thr366, leading to a loss of PTEN activity. This loss of PTEN function results in activation of Akt, because PIP3 is no longer converted to PIP2. Activated Akt can now shift microglia from the destructive M1 phenotype toward the beneficial M2 phenotype and thereby elicit the protection of neighboring oligodendrocytes. Activated Akt also phosphorylates GSK3β, thereby disabling its pro-death effect. Knockdown of GSK3β or the PI3K/Akt inhibitor LY294002 each blocks the protective effects of HDAC inhibition, whereas the PTEN inhibitor bpV(pic) fails to provide any additional effect over that of HDAC inhibition.

Fig. 2.

Long-term preservation of white matter after TBI is promoted by Scriptaid. (A) Double immunofluorescent staining for dephosphorylated neurofilament protein (SMI-32) and MBP in the ipsilesional CC at 35 d after TBI or sham treatment. Nuclei are labeled with DAPI (blue). (Insets) High-power images. (B) Degree of WMI, expressed as the fold increases of SMI-32, fold decreases of MBP, and the ratio of SMI-32 to MBP. (C) Illustration of the CCI lesion (dashed line) and regions for immunohistochemistry and electron microscropy (red box). Stimulating and recording electrodes were positioned at the CC as shown to measure the evoked CAPs. (D) Representative traces of the evoked CAPs in the CC (stimulus, 0.3 mA; 0.48 mm lateral to the stimulating electrode) at 7 d post-TBI. (E) Signal conduction along nerve fibers, as measured by the amplitude of the N1 component of the CAPs in response to increasing stimulus strength (0.05–0.55 mA) at 7 d post-TBI. (F) N1 amplitude in response to a 0.3-mA stimulus at 3, 7, and 35 d post-TBI. Shown are the mean ± SEM values from six mice per group. ^#P ≤ 0.05; ^##P ≤ 0.01, ^###P ≤ 0.001 vs. sham; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 vs. TBI + vehicle.

Fig. 3.

HDAC inhibition indirectly reduces oligodendrocyte injury by modulating microglia/macrophages. (A) In vitro experiments using a CM transfer system or a Transwell system. Microglia cultures (M0) were incubated with Scriptaid (1 μM) or vehicle for 48 h; as positive controls, microglia were primed toward M1 or M2 using LPS (100 ng/mL) plus IFN-γ (20 ng/mL) or IL-4 (20 ng/mL), respectively, for 48 h (18). Cultured oligodendrocytes were exposed to 2-h OGD or control, non-OGD conditions and returned to normal medium. Twenty-four hours later, microglia CM was applied to oligodendrocytes for 24 h by pipetting or by a Transwell system. OLG, oligodendrocytes. (B) MBP and DAPI staining of oligodendrocytes exposed to OGD and cultured with CM from microglia exhibiting M0 (with or without Scriptaid), M1, or M2 phenotypes. (C–F) Oligodendrocyte survival and cell death were quantified by LDH release and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, respectively, after exposure to CM (C and D) or in Transwells (E and F). M0 phenotype in conjunction with Scriptaid or the M2 phenotype protected oligodendrocyte viability after OGD, whereas the M1 phenotype decreased oligodendrocyte viability. Shown are the mean ± SEM values from three independent experiments. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 vs. OGD + OLG; ^###P ≤ 0.001 vs. OGD + OLG + M0 microglia without Scriptaid; ^$$P ≤ 0.01, ^$$$P ≤ 0.001 vs. OGD + OLG + Scriptaid.

Fig. 4.

HDAC inhibition primes microglia toward the M2 phenotype in vitro and in vivo. (A) Double immunofluorescent staining for M1 marker CD16 or M2 marker CD206 with Iba1 marker for activated microglia in the ipsilesional CC at 7 d following TBI (vehicle and Scriptaid groups) or sham surgery (sham group). Shown is immunofluorescent staining for CD16 or CD206 (first or third column) and colabeling of the same sections for Iba1 and nuclei (DAPI; second and fourth columns). (B) Blinded cell counts of microglia immunolabeled for CD16 and CD206 from the same animals as shown in A. (C) Messenger RNA levels of CD16 and CD206 in the ipsilateral striatum at 7 d after TBI with or without Scriptaid. Scriptaid was injected at 3.5 mg/kg at 2 h after CCI, and this was repeated daily for the next 2 d (at 26 and 50 h after injury). (D) HDAC inhibitors Scriptaid, SAHA (2.5 µM), and VPA (4 μM) reduced LPS-induced production of TNF-α by microglia. (E) Microglial cultures were treated with LPS alone or in combination with Scriptaid, SAHA, or VPA for 48 h. Fluorescent microspheres were added for 3 h, and intramicroglial fluorescence intensity was measured. Scriptaid was more potent than SAHA or VPA in enhancing microsphere uptake by microglia. (F) Microglia were treated with LPS with or without Scriptaid as above and stained with phalloidin (green) to visualize F-actin. After a 3-h incubation, phagocytosed microspheres appeared red, and DAPI-stained nuclei appeared blue. Shown are the mean ± SEM values from four independent experiments. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 vs. LPS + vehicle.

Fig. 5.

HDAC inhibition modulates microglial polarization through the PI3K/Akt pathway. (A) LPS-induced HDAC activity in primary microglia cultures was reduced by Scriptaid. (B) Scriptaid prevented decreases in acetylated-histone 3 (H3) and acetylated H4 in primary microglia at 3 h after LPS, as measured by Western blot analysis. (C) Scriptaid prevented loss of p-PTEN (Ser380/Thr382/383) and p-Akt (Ser473) in LPS-challenged microglia, as measured by Western blot analysis. (D) Scriptaid largely prevented the decrease in p-mTOR (Ser2488) and p-GSK3β (Ser9) levels in LPS-treated microglia. (E) LPS reduced p-Akt levels, an effect largely blocked by the PTEN inhibitor dipotassium bisperoxo (picolinato) oxovanadate [bpV(pic)] (1 μM). Scriptaid partially preserved p-Akt levels in LPS-treated microglia alone or in the presence of bpV(pic), but not in the presence of the PI3K/Akt inhibitor LY294002 (10 µM). (F and G) LPS treatment of microglia increased TNF-α and NO production, an effect reduced by bpV(pic) and by Scriptaid. The effect or Scriptaid was reduced by LY294002, but not by bpV(pic). (H) Microsphere uptake was reduced by LPS alone, but increased by LPS + bpV(pic) or LPS + Scriptaid +/− bpV(pic); the effect of Scriptaid on LPS was blocked by LY294002. Thus, all LPS effects were opposed by Scriptaid, and the effects of Scriptaid were abolished by LY294002 and unchanged by [bpV(pic)]. Data are mean ± SEM values from four independent experiments. ***P ≤ 0.001 vs. LPS alone; ^##P ≤ 0.01, ^###P ≤ 0.001 vs. LPS + Scriptaid.

Fig. 6.

In vivo GSK3β knockdown attenuates Scriptaid-afforded protection against WMI. (A and C) Immunofluorescent staining for SMI-32 (upper row in A) and colabeling of the same sections for MBP and nuclei (DAPI) in the second row. Images were taken from the CC of animals at 7 d post-TBI. Lentiviral shRNA for GSK3β (shRNA-GSK3β) or scrambled shRNA (shRNA-Scr) was infused into the CC 7 d before TBI; Scriptaid was injected at 2, 26, and 50 h after TBI. WMI was expressed as the ratio of SMI-32 to MBP in the CC normalized to sham (C). (B and D) Immunofluorescent staining for GSK3β and Iba-1 in the CC at 7 d post-TBI as in A and C. Quantification of GSK3β⁺ cells is illustrated in D. Shown are the mean ± SEM values from six mice per group. ^#P ≤ 0.05, ^##P ≤ 0.01, ^###P ≤ 0.001 vs. sham; ***P ≤ 0.001 vs. vehicle; ^$$$P < 0.001 vs. Scriptaid.