Apoptosis signal-regulating kinase 1 (ASK1) is linked to neural stem cell differentiation after ischemic brain injury

Abstract

Neural stem cells (NSCs) have been suggested as a groundbreaking solution for stroke patients because they have the potential for self-renewal and differentiation into neurons. The differentiation of NSCs into neurons is integral for increasing the therapeutic efficiency of NSCs during inflammation. Apoptosis signal-regulating kinase 1 (ASK1) is preferentially activated by oxidative stress and inflammation, which is the fundamental pathology of brain damage in stroke. ASK1 may be involved in the early inflammation response after stroke and may be related to the differentiation of NSCs because of the relationship between ASK1 and the p38 mitogen-activated protein kinase pathway. Therefore, we investigated whether ASK1 is linked to the differentiation of NSCs under the context of inflammation. On the basis of the results of a microarray analysis, we performed the following experiments: western blot analysis to confirm ASK1, DCX, MAP2, phospho-p38 expression; fluorescence-activated cell sorting assay to estimate cell death; and immunocytochemistry to visualize and confirm the differentiation of cells in brain tissue. Neurosphere size and cell survival were highly maintained in ASK1-suppressed, lipopolysaccharide (LPS)-treated brains compared with only LPS-treated brains. The number of positive cells for MAP2, a neuronal marker, was lower in the ASK1-suppressed group than in the control group. According to our microarray data, phospho-p38 expression was inversely linked to ASK1 suppression, and our immunohistochemistry data showed that slight upregulation of ASK1 by LPS promoted the differentiation of endogenous, neuronal stem cells into neurons, but highly increased ASK1 levels after cerebral ischemic damage led to high levels of cell death. We conclude that ASK1 is regulated in response to the early inflammation phase and regulates the differentiation of NSCs after inflammatory-inducing events, such as ischemic stroke.

Figure 1

Upregulation of the levels of apoptosis signal-regulating kinase 1 (ASK1) and endogenous neural stem cells after cerebral ischemia. (a) Western blotting showed that the relative level of ASK1 was elevated from 8 to 24 h after cerebral ischemia in ischemic lesions. β-Actin was used as an internal control (mean±s.d., n=3–4). (b) Following cerebral ischemia for 7 days, SOX2-positive cells (red) were observed in the subventricular zone of the contralateral and ipsilateral hemispheres. Hoechst 33258 was used as a counterstain (blue). (c) The tables show significantly upregulated genes that were categorized as being related to inflammation or neurogenesis by assessing the microarrays. Scale bar=50 μm; *P<0.1 compared with normal group.

Figure 2

Relationship between cell death and differentiation in neural stem cells. The neurosphere size was measured by bright field microscopy, and cell death was measured by FACS analysis. (a) GAPDH gene silencing was used as a positive control and reduced the level of GAPDH protein. Downregulation was confirmed by western blot analysis (upper). Treatment with the ASK1-siRNA resulted in decreased ASK1 protein levels in NSCs (lower). (b) Neurosphere sizes were maintained more in the ASK1-siRNA treatment group than in the control group. (c) FACS analysis data show that the percentage of dead cells was lower in the ASK1-siRNA treatment group than in the control group after LPS treatment. *P<0.1. ASK1, apoptosis signal-regulating kinase 1; FACS, fluorescence-activated cell sorting; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; LPS, lipopolysaccharide; NS, not significant; NSC, neural stem cell; PI, propidium iodide; siRNA, small interfering RNA.

Figure 3

Photomicrographs of neural stem cells (NSCs) under apoptosis signal-regulating kinase 1 (ASK1) suppression conditions. Immunofluorescence images of a neural stem cell (DCX, green) and mature neuron (MAP2, red). The number of MAP2-positive cells increased after ASK1-small interfering RNA (siRNA) treatment in the lipopolysaccharide (LPS)-treated group compared with the LPS-only treatment group. The lane furthest to the right presents results without the primary antibody as a negative control. The bottom row shows the images after higher magnification.

Figure 4

Effect of apoptosis signal-regulating kinase 1 (ASK1) on neuronal differentiation through the p38 mitogen-activated protein kinase (MAPK) pathway. (a) DCX and MAP2 expression increased in the ASK1-small interfering RNA (siRNA) treatment group compared with the control group. (b) Phosphor-p38 expression decreased more in the ASK1-siRNA and the ASK1-siRNA with lipopolysaccharide (LPS)-treatment groups than in the LPS-only treatment group. *P<0.1.