Myelin basic protein associates with AβPP, Aβ1-42, and amyloid plaques in cortex of Alzheimer's disease brain

Abstract

The goal of this study was to show that myelin and axons in cortical gray matter are damaged in Alzheimer's disease (AD) brain. Superior temporal gyrus gray matter of AD patients (9 male, 14 female) was compared to cognitively normal controls (8 male, 7 female). Myelin basic protein (MBP) and a degraded myelin basic protein complex (dMBP) were quantified by Western blot. Brain sections were immunostained for MBP, dMBP, axonal neurofilament protein (NF), autophagy marker microtubule-associated proteins 1A/B light chain 3B precursor (LC3B), amyloid-β protein precursor (AβPP), and amyloid markers amyloid β1-42 (Aβ1-42) and FSB. Co-immunoprecipitation and mass spectroscopy evaluated interaction of AβPP/Aβ1-42 with MBP/dMBP. Evidence of axonal injury in AD cortex included appearance of AβPP in NF stained axons, and NF at margins of amyloid plaques. Evidence of myelin injury in AD cortex included (1) increased dMBP in AD gray matter compared to control (p < 0.001); (2) dMBP in AD neurons; and (3) increased LC3B that co-localized with MBP. Evidence of interaction of AβPP/Aβ1-42 with myelin or axonal components included (1) greater binding of dMBP with AβPP in AD brain; (2) MBP at the margins of amyloid plaques; (3) dMBP co-localized with Aβ1-42 in the core of amyloid plaques in AD brains; and (4) interactions between Aβ1-42 and MBP/dMBP by co-immunoprecipitation and mass spectrometry. We conclude that damaged axons may be a source of AβPP. dMBP, MBP, and NF associate with amyloid plaques and dMBP associates with AβPP and Aβ1-42. These molecules could be involved in formation of amyloid plaques.

Keywords: Alzheimer's disease; amyloid-β; amyloid-β protein precursor; autophagy; axon damage; degraded myelin basic protein; myelin basic protein.

Fig. 1

Western blot analysis of MBP and dMBP in control (n = 10) and AD (n = 13) brains. Both intact MBP (~19 kDa) and degraded MBP (~37 kDa) were detected in control and AD brains. Note that intact MBP was markedly decreased in one AD case (A, upper panel, black arrow). In the same case, dMBP was markedly increased (A, upper panel, white arrow). Similarly, intact MBP was markedly decreased in one out of 10 control brains (A, lower panel, black arrow). In the same case, dMBP was markedly increased (A, lower panel, white arrow). The overall expression of MBP in AD was significantly increased compared to control (B, upper panel). The dMBP levels in AD were significantly increased compared to control as well (B, middle panel). The ratio of dMBP over MBP was significantly higher in AD compared to control (B, lower panel). MBP, myelin basic protein; dMBP, degraded myelin basic protein complex; AD, Alzheimer’s disease. *p < 0.05 versus control, ***p < 0.001 versus control. Error bars are standard errors of the mean.

Fig. 2

Immunohistochemistry of MBP and dMBP in the cortex of control and AD brains. In controls, MBP was predominantly stained in the myelin sheaths of cortex (A1). In contrast, soma of the pyramidal neurons showed accumulation of intensively stained MBP (B1 and B2, arrows) in AD brains compared to controls (A2, arrow). Using an antibody specifically against degraded MBP, we detected degraded myelin protein in the giant myelin sheaths (D1, arrows), soma and nuclei (D2, arrows) of pyramidal neurons of AD brains. In controls, dMBP was also detected in the nuclei of the pyramidal neurons (C1 and C2). Brown, positive staining; MBP, myelin basic protein; dMBP, degraded myelin basic protein complex; AD, Alzheimer’s disease. Bar = 25 μm.

Fig. 3

Immunohistochemistry of NF in the cortex of control and AD brains. In controls, NF was stained in axons (A1 and A2, arrows) of neurons. NF was also stained in the soma of the pyramidal neurons (B1 and B2, arrows) of AD brains. In addition, the proximal portions of axons were thickened in the AD brain (B2). Brown, positive staining; NF, neurofilament; AD, Alzheimer’s disease. Bar = 25 μm.

Fig. 4

Axonal injury associated with AβPP staining in AD brains. Axonal fragmentation was observed (B1, white arrows) in AD brain by immunofluorescence staining of an axonal marker, NF. AβPP was associated with these fragmented axons (B2 and B3, white arrows). In the areas where axonal fragmentation was severe or large areas of axons were lost (B1, yellow arrows), AβPP aggregates were observed (B2 and B3, yellow arrows). In control, NF was localized in axons or bundles of axons (A1) that did not stain for AβPP (A2 and A3). NF, neurofilament; AβPP, amyloid-β protein precursor; AD, Alzheimer’s disease. Bar = 25 μm.

Fig. 5

Immunostaining of myelin/axonal aggregates and amyloid aggregates in AD brains. Myelin aggregates (A, yellow arrows) were observed in plaque-like structures (A, blue arrow surrounded by red arrows). MBP aggregates were localized in the halo (A, surrounded by red arrows), but not the core (A, blue arrow) of the plaque. Using the axonal marker, NF, we detected axonal aggregates (B, red arrow). The axons were tangles in the center while their somata orientated to the periphery (B, yellow arrows). Aβ_1–42 was not stained in the pyramidal neurons (C, black arrows), but Aβ_1–42 did stain in the core (C, blue arrow surrounded by red arrows) of the plaque. In addition, short fragments of Aβ such as Aβ_1–16 (D) and Aβ_17–24 (E), as well as AβPP (F) were identified both in the plaques (D, E, F, red arrows) and pyramidal neurons (D, E, F, black arrows). Brown, positive staining; MBP, myelin basic protein; NF, neurofilament; AβPP, amyloid-β protein precursor; Aβ, amyloid-β; AD, Alzheimer’s disease. Bar = 25 μm.

Fig. 6

Myelin/axonal aggregates in the amyloid plaques of AD brains. In FSB positive amyloid plaque (A1 and A3, yellow arrows), myelin aggregates (A2 and A3, arrows) were observed. Note that some MBP aggregates (A2 and A3, yellow arrows) are located in the amyloid plaque whereas other MBP aggregates (A2 and A3, white arrows) were adjacent, but not within the FSB⁺ plaques. Similar NF⁺ aggregates (B2 and B3, arrows) were observed in AD brain as well. These NF⁺ aggregates were frequently located in the periphery of the FSB+ amyloid plaque (B1, arrows). In addition, dMBP aggregates (C2, arrows) were identified in Aβ_1–42⁺ plaques (C1 andC3, arrows) with dMBP and Aβ_1–42 being co-localized (C3). MBP, myelin basic protein; dMBP, degraded myelin basic protein complex; Aβ, amyloid-β; NF, neurofilament; FSB, (E, E)-1-fluoro-2,5-bis (3-hydroxycarbonyl-4-hydroxy) styrylbenzene; AD, Alzheimer’s disease. Bar = 25 μm.

Fig. 7

Co-IP of dMBP, AβPP, or Aβ_1–42 in control and AD brains. A) Immunoprecipitation (IP) was performed using the dMBP antibody in both control and AD brains. Immunoblots of the IP product with the AβPP antibody reveals the interaction of dMBP with AβPP as indicated by detecting two bands (a and b) of protein in both AD and control brains (A). The molecular weight (MW) of the two bands was about 32 kDa (band a) and 27 kDa (band b), respectively. Using IgG as a negative control, we detected a protein with MW around ~28 kDa (band c). It is likely that there is a specific binding of IgG with cleaved AβPP in aging brains since the binding was not detected when the brain sample was replaced by PBS (A). B) Quantification of the intensity of bands A and B. The intensity of the upper band A and lower band B was greater in AD compared to control brain (B). C) A reciprocal experiment of IP was performed using anti-AβPP or anti-Aβ_1–42 antibody to verify the findings in panel A. Immunoblots of the IP products with dMBP antibody reveals the interaction of AβPP or Aβ_1–42 with dMBP as indicated by detecting a protein in both AD and control brains (C). The MW of the protein was about 27 kDa. Comparing with IP result from panel A, it is likely that the protein with MW around 27 kDa is in the complex of dMBP-AβPP since this protein was detected in both IP products either with AβPP or dMBP. In addition, AβPP complexed with dMBP is likely containing Aβ_1–42 fragments since immunoblots with dMBP in IP product using Aβ_1–42 revealed same MW of protein as that in IP product using AβPP. AβPP, amyloid-β protein precursor; dMBP, degraded myelin basic protein complex; AD, Alzheimer’s disease. *p< 0.05 versus control, ***p<0.001 versus control. Error bars represent standard errors of the mean.