Upregulation of mesencephalic astrocyte-derived neurotrophic factor in glial cells is associated with ischemia-induced glial activation

Abstract

Background: Mesencephalic astrocyte-derived neurotrophic factor (MANF), a 20 kDa secreted protein, was originally derived from a rat mesencephalic type-1 astrocyte cell line. MANF belongs to a novel evolutionally conserved family of neurotrophic factors along with conserved dopamine neurotrophic factor. In recent years, ever-increasing evidence has shown that both of them play a remarkable protective role against various injuries to neurons in vivo or in vitro. However, the characteristics of MANF expression in the different types of glial cells, especially in astrocytes, remain unclear.

Methods: The model of focal cerebral ischemia was induced by rat middle cerebral artery occlusion. Double-labeled immunofluorescent staining was used to identify the types of neural cells expressing MANF. Primarily cultured glial cells were used to detect the response of glial cells to endoplasmic reticulum stress stimulation. Propidium iodide staining was used to determine dead cells. Reverse transcription PCR and western blotting were used to detect the levels of mRNA and proteins.

Results: We found that MANF was predominantly expressed in neurons in both normal and ischemic cortex. Despite its name, MANF was poorly expressed in glial cells, including astrocytes, in normal brain tissue. However, the expression of MANF was upregulated in the glial cells under focal cerebral ischemia, including the astrocytes. This expression was also induced by several endoplasmic reticulum stress inducers and nutrient deprivation in cultured primary glial cells. The most interesting phenomenon observed in this study was the pattern of MANF expression in the microglia. The expression of MANF was closely associated with the morphology and state of microglia, accompanied by the upregulation of BIP/Grp78.

Conclusions: These results indicate that MANF expression was upregulated in the activated glial cells, which may contribute to the mechanism of ischemia-induced neural injury.

Figure 1

Mesencephalic astrocyte-derived neurotrophic factor expression in the astrocytes of brain tissue. Brain samples were collected from the normal (A to C) and ischemic rat brain tissue (D to L). (D to F) and (J to L) Two-hour ischemia followed by 24-hour reperfusion; (G to I) 4-hour ischemia followed by 24-hour reperfusion. (A to I) Mesencephalic astrocyte-derived neurotrophic factor (MANF) expression in astrocytes was detected by double immunofluorescent staining for glial fibrillary acidic protein (GFAP) (red, A, D, and G) and MANF (green, B, E, and H) with anti-GFAP and anti-MANF, respectively. (J to L) MANF expression in neurons was detected with anti-MANF (red, K). The neurons were identified by anti-NeuN (green, J). Ischemia-induced MANF expression was found predominantly in neurons (K). A small number of astrocytes expressing MANF are indicated by arrows. Scale bar = 50 μm. (M to P) Specificity of the antibody against MANF in brain tissues was detected by immunohistochemistry and immunofluorescent staining. Brain sections were incubated with primary antibody (anti-MANF) in (N) and (P). Negative controls were performed by substituting the primary antibody with PBS (M and O). Scale bar = 50 μm.

Figure 2

Expression of mesencephalic astrocyte-derived neurotrophic factor in oligodendrocytes. Mesencephalic astrocyte-derived neurotrophic factor (MANF) was expressed in normal cerebral cortex (A to C) and ischemic cerebral cortex (D to J). The primary cultured oligodendrocytes were treated with vehicle (DMEM medium containing 5% serum) (K to N) or tunicamycin (1 μg/ml) (O to R). Twenty-four hours after treatment, immunofluorescent staining was performed. The oligodendrocytes were identified with anti-2-3-cyclic nucleotide 3-phosphodiesterase (anti-CNP) (red, B, E, I, M, and Q). MANF was detected with anti-MANF antibody (green, A, D, H, L, and P). Scale bar = 50 μm. (S) Percentage of MANF-positive cells in neural cells. The number of MANF-positive cells and the number of double positive cells co-expressing glial fibrillary acidic protein (GFAP), CD68, CNP, or NeuN were counted in five randomly selected fields under a high-power field (×400 magnification). The percentage of MANF expressing cells in the population of astrocytes, microglial, and oligodendrocytes was calculated.

Figure 3

Induction of mesencephalic astrocyte-derived neurotrophic factor expression in the primarily cultured astrocytes. Cells cultured in the DMEM medium containing 5% serum were used as controls (A to C). Glial cells were cultured as described in Materials and methods and treated with 1 μg/ml tunicamycin (D to F), 10 μM MG132 (G to I), and serum-free DMEM medium (J to L), respectively. Twenty-four hours after treatment, the astrocytes were identified with anti-glial fibrillary acidic protein (anti-GFAP) antibody (red, A, D, G, and J). Mesencephalic astrocyte-derived neurotrophic factor (MANF) expression was detected with monoclonal anti-MANF antibody (green, B, E, H, and K). Nuclei were stained with 4^′,6-diamidino-2-phenylindole (blue). The arrows show MANF immune-positive astrocytes after treatment. Scale bar = 50 μm.

Figure 4

Endoplasmic reticulum stress and mesencephalic astrocyte-derived neurotrophic factor expression in cultured primary glial cells. (A to D) Glial cells in the mixed culture were treated as indicated (serum+ control, DMEM medium containing 5% serum; Tm, tunicamycin, 1 μg/ml; MG132, 10 μM; serum–, serum-free DMEM medium). Twenty-four hours after treatment, the cells were collected and processed for RT-PCR and immunoblotting (IB). Levels of mRNAs (A) and proteins (C) were quantitated and normalized by actin. The quantitative data in (A) and (C) are shown in (B) and (D), respectively. Values expressed as mean ± standard error of the mean of three independent experiments. *P <0.05, **P <0.01, and ***P <0.001, compared with serum+ control. (E to P) Glial cells were treated with serum+ (E to G), Tm (H to J), MG132 (K to M), and serum– (N to P). Twenty-four hours after treatment, the cells were stained with 4^′,6-diamidino-2-phenylindole (F, I, L, and O) and propidium iodide (G, J, M, and P), and then observed with a microscope under bright field (E, H, K, and N) and fluorescence. Scale bar = 50 μm.

Figure 5

Differential expression of mesencephalic astrocyte-derived neurotrophic factor in the microglia in brain tissue. (A to D) A rod-shaped microglial cell within blood vessel in the normal cerebral cortex. (E to H) A ramified microglial cell under vascular endothelium in the normal cerebral cortex. (I to L) A rod-shaped microglial cell with weak mesencephalic astrocyte-derived neurotrophic factor (MANF)-positive immunoreaction in the ischemic cerebral cortex. (M to P) An amoeboid microglial cell with MANF-positive immunoreaction in the ischemic cerebral cortex. (Q to T) A round microglial cell with strong MANF-positive immunoreaction in the ischemic cerebral cortex. Microglial cells were identified with anti-CD68 antibody (green, A, E, I, M, and Q). MANF was detected with monoclonal anti-MANF (red, B, F, J, N, and R). Nuclei were stained by 4^′,6-diamidino-2-phenylindole (blue, C, G, K, O, and S). Scale bar = 50 μm.

Figure 6

BIP induction in the microglia in the ischemic cerebral cortex. (A to D) Ramified microglial cells in the ischemic cerebral cortex. (E to H) Round microglial cells in the ischemic cerebral cortex. Microglial cells were identified with anti-CD68 antibody (green, A and E). BIP was detected with rabbit anti-BIP (red, B and F). Nuclei were stained by 4^′,6-diamidino-2-phenylindole (blue, C and G). Scale bar = 50 μm.

Figure 7

Mesencephalic astrocyte-derived neurotrophic factor expression in cultured primary microglia. Glial cells were cultured as described in Materials and methods. The serum concentration in DMEM was lowered from 10% to 5% during treatment. Immunofluorescent staining was performed in vehicle controls (A to D) and 24 hours after treatment with 1 μg/ml tunicamycin (E to L). Microglia were identified with anti-CD68 antibody (green, A, E, and I). Mesencephalic astrocyte-derived neurotrophic factor (MANF) expression was detected with monoclonal anti-MANF antibody (red, B, F, and J). Nuclei were stained by 4^′,6-diamidino-2-phenylindole (blue, C, G, and K). Scale bar = 50 μm.