High glucose upregulates myosin light chain kinase to induce microfilament cytoskeleton rearrangement in hippocampal neurons

Abstract

Chronic hyperglycemia leads to myosin light chain kinase (MLCK) upregulation and induces neuronal damage. However, the underlying molecular mechanism of neuronal damage in hyperglycemia has not yet been fully elucidated. In the present study, hippocampal neuronal cells were cultured and treated with a high glucose concentration (45 mmol/l). The results demonstrated that high glucose induced shrinking of the synapses, nuclear shape irregularity and microfilament damage. Filamentous actin (F‑actin) filaments were rearranged, cell apoptosis rate was increased and the protein expression of MLCK and phosphorylated (p)‑MLC was upregulated. The MLCK inhibitor ML‑7 largely reversed the alterations in the microfilament cytoskeleton, inhibited F‑actin depolymerization, reduced apoptosis and downregulated MLCK and p‑MLC protein expression. Overall, these results indicated that high glucose upregulated MLCK to promote F‑actin depolymerization, which induced microfilament cytoskeleton rearrangement in hippocampal neuronal cells.

Figure 1.

Morphology and identification of hippocampal neurons at different time points. (A) Cells exhibited a round, oval or tapered shape, and the cell bodies appeared bright and full, three-dimensional and with strong refraction; day 1, magnification ×200. (B) Volume of the cells was visibly increased, and the neurites are branched; day 4, magnification ×200. (C) Neurites were branched and overlapping; day 7, magnification ×200. (D) Hippocampal neuronal cells were identified following immunocytochemical staining with anti-neuron-specific enolase; magnification ×400.

Figure 2.

Ultrastructure of hippocampal neurons in culture. (A) In the control group, the morphology of the nucleus was regular, and the chromatins in the nucleus were abundant and homogeneously distributed. The cell organelles were abundant, the endoplasmic reticulum and mitochondria were normal in morphology, and there were large numbers of microfilaments in the cytoplasm (magnification, ×10,000). Black arrow, microfilament structural integrity. (B) In the high-glucose group, a large fragment of a relatively well-preserved perikaryon of the pyramidal neuron was observed. Certain mitochondria appeared to be enlarged. The nuclear membrane appeared to have shrunk (magnification, ×10,000). (C) In the high glucose+ML-7 group the nuclei were normal, round or oval, with abundant chromatin. The endoplasmic reticulum and Golgi complex were only partly edematous. The mitochondria were abundant and their morphology was normal (magnification, ×10,000). Red circles, abundant microfilament.

Figure 3.

Apoptosis rates in different groups. (A) Representative images of flow cytometry analysis of hippocampal neurons in different groups. (B) The bar graph presents the percentage of apoptotic cells. The experiments were performed in triplicate; the data are expressed as the mean ± standard deviation. **P<0.01.

Figure 4.

Structural alterations of F-actin in hippocampal neurons. (A) Representative images of immunofluorescent staining for F-actin (green) with DAPI nuclear staining (blue) in various groups (magnification, ×630). (B) Quantitative analysis of fluorescence intensity of F-actin. **P<0.01. F-actin, filamentous actin.

Figure 5.

MLCK and p-MLC protein expression in various groups. (A) Western blot analysis of MLCK and p-MLC protein expression in various groups, using β-actin as an endogenous control. (B) Quantitative analysis of MLCK and p-MLC protein expression levels; *P<0.05 and **P<0.01. MLCK, myosin light chain kinase; p-MLC, phosphorylated myosin light chain.