Blockage of MLKL prevents myelin damage in experimental diabetic neuropathy

Abstract

SignificanceDiabetic neuropathy is a commonly occurring complication of diabetes that affects hundreds of millions of patients worldwide. Patients suffering from diabetic neuropathy experience abnormal sensations and have damage in their peripheral nerve axons as well as myelin, a tightly packed Schwann cell sheath that wraps around axons to provide insulation and increases electrical conductivity along the nerve fibers. The molecular events underlying myelin damage in diabetic neuropathy are largely unknown, and there is no efficacious treatment for the disease. The current study, using a diabetic mouse model and human patient nerve samples, uncovered a molecular mechanism underlying myelin sheath damage in diabetic neuropathy and provides a potential treatment strategy for the disease.

Keywords: MLKL; diabetic neuropathy; myelin.

Fig. 1.

STZ induces MLKL expression and causes sural nerve demyelination. (A) Twenty-four weeks after STZ injection, the ultrastructure of myelin in sural nerves was visualized by TEM. Whereas control mice had tightly packed myelin sheaths around their sural nerve, in STZ-treated mice, these myelin sheaths exhibited strong distal-to-proximal decompaction. (Scale bars, 1 µm.) (B) Immunoblotting with an antibody against MLKL in the distal (sural nerve [S]), middle (M), and proximal (P) segments of sciatic nerves in control and STZ-induced diabetic models; MLKL levels increased in sural nerves in diabetic mice. GAPDH was used as a loading control. (C) Representative immunofluorescence staining for MLKL (green) and the myelin sheath marker protein MBP (red) as well as their merged images on sural nerve sections from diabetic mice 8 wk after STZ injection. Note the high levels of colocalization between MLKL and MBP. (Scale bar, 10 µm.)

Fig. 2.

Whole body or Schwann cell–specific Mlkl deficiency prevents myelin decompaction and NCV decrease in diabetic mice. (A) Representative TEM images of the ultrastructures of the myelin sheaths of intact sural nerves and sural nerves from WT (n = 8) and Mlkl^−/− diabetic mice (n = 8). Left, low magnification. (Scale bars, 5 µm.) Right, high magnification of the boxed regions on the left. (Scale bars, 1 µm.) Note that the Mlkl^−/− mice exhibited significantly less severe myelin sheath decompaction than WT mice. (B) Quantification of intact myelin rings in WT and Mlkl^−/− diabetic mice (n = 150 myelin rings for each group). (C) At 24 wk post-STZ or control saline injection, the MNCVs and SNCVs of WT and Mlkl^−/− mice were measured. MLKL^−/− diabetic mice had significantly faster NCVs. (D) Immunoblotting of MLKL levels in Plp-Cre^ERT; Mlkl^fl/fl and Mlkl^fl/fl diabetic mice to assess Schwann cell–specific MLKL knockout; the distal (sural nerve), middle, and proximal segments of sciatic nerves were analyzed. MLKL levels were significantly lower in Plp-Cre^ERT; Mlkl^fl/fl mice. GAPDH was used as a loading control. (E) Representative TEM images of the ultrastructures of the myelin sheaths of sural nerves from Plp-Cre^ERT; Mlkl^fl/fl (n = 6) and Mlkl^fl/fl (n = 5) diabetic mice. Left, low magnification. (Scale bars, 5 µm.) Right, high magnification of the boxed regions on the left. (Scale bars, 1 µm.) (F) Quantification of E shows that Schwann cell–specific knockout of MLKL significantly blocked myelin sheath decompaction (n = 150 myelin rings for each group). (G) An NCV test showed that diabetic mice with Schwann cell–specific knockout of MLKL had significantly faster NCVs. Data are represented as mean ± SD.

Fig. 3.

Mlkl S441A–knockin mice are resistant to diabetes-induced myelin decompaction and NCV decrease. (A) Differential centrifugation of extracts from sural nerves in WT and S441A-knockin mice showed that WT MLKL was inserted into the lipid bilayer of the myelin sheath (in the same fraction as the PLP marker), but MLKL^S441A was only loosely associated with the myelin sheath (in the same fraction as MBP). (B) Representative TEM images of the ultrastructures of the myelin sheaths of sural nerves from WT (n = 8) and S441A-knockin (n = 6) diabetic mice. Left, low magnification. (Scale bars, 5 µm.) Right, high magnification of the boxed regions on the left. (Scale bars, 1 µm.) (C) Quantification of B shows that the S441A mutation of MLKL significantly blocked myelin sheath decompaction (n = 150 myelin rings for each group). (D) An NCV test indicated that S441A MLKL-knockin diabetic mice had significantly faster NCVs. Data are represented as mean ± SD.

Fig. 4.

An MLKL inhibitor blocks diabetes-induced myelin decompaction and NCV decrease. (A) Differential centrifugation of extracts from sural nerves in hMLKL-KI diabetic mice showed that MLKL was inserted into the lipid bilayer of the myelin sheath (in the same fraction as the PLP marker) but that MLKL was only loosely associated with the myelin sheath (in the same fraction as MBP) after TC013249 treatment. (B) Osmotic pumps, either containing vehicle or TC013249, were intraperitoneally implanted into mice (n = 5 for vehicle and n = 7 for TC013249) 4 wk post-STZ injection. Representative TEM images of the ultrastructures of the myelin sheaths of sural nerves from vehicle- and TC013249-treated hMLKL-KI diabetic mice. Left, low magnification. (Scale bars, 5 µm.) Right, high magnification of the boxed regions on the left. (Scale bars, 1 µm.) (C) Quantification of B shows that TC013249 treatment significantly blocked diabetes-induced myelin sheath decompaction (n = 150 myelin rings for each group). (D) An NCV test for vehicle- or TC013249- treated diabetic mice indicated that TC013249 treatment mitigated decreases in MNCV in diabetic mice. Data are represented as mean ± SD.

Fig. 5.

MLKL is activated in the myelin sheath of diabetic neuropathy patients. (A) Sural nerve sections from human patients with diabetic neuropathy were analyzed by immunofluorescence staining with antibodies against neurofilament (green) and MBP (red), DAPI (diamidino-2-phenylindole) was used to visualize nuclei (blue). Note the existence of a large axon without myelin wrapping (arrows). (Scale bar, 10 µm.) (B) Structured illumination microscopy fluorescence image of MBP from patients with diabetic neuropathy and healthy individuals. (Scale bars, 1.5 µm.) (C) Sural nerve sections from human patients with diabetic neuropathy and healthy individuals were analyzed by immunofluorescence staining with antibodies against MLKL (green) and MBP (red). DAPI was used to visualize nuclei (blue). Note the colocalization of MLKL and MBP in patients with diabetic neuropathy. Data are representative of three patients with diabetic neuropathy and three people from the control group. (Scale bars, 10 µm.)