Neurochemical Research

, Volume 43, Issue 1, pp 41–49 | Cite as

Microglia Endocytose Amyloid β Through the Binding of Transglutaminase 2 and Milk Fat Globule EGF Factor 8 Protein

  • Kenji Kawabe
  • Katsura TakanoEmail author
  • Mitsuaki Moriyama
  • Yoichi Nakamura
Original Paper


Activation of glial cells has been observed in neurodegenerative diseases including Alzheimer’s disease (AD). Aggregation of amyloid β (Aβ) is profusely observed as characteristic pathology in AD brain. In our previous study using microglial cell line BV-2, tissue-type transglutaminase (TG2) was found to be involved in phagocytosis (Kawabe et al., in Neuroimmunomodulation 22(4):243–249, 2015; Kawabe et al., Neurochem Res 2017). In the present study, we examined whether TG2 and milk fat globule EGF factor 8 protein (MFG-E8), an adaptor protein promotes macrophage to engulf apoptotic cells, were involved in Aβ endocytosis. When the neuronal/glial mixed culture was stimulated freshly prepared Aβ1−42 for 3 days, the incorporation of Aβ was observed by immunofluorescence staining technique in Iba-1-positive microglia. Cystamine, a broad competitive inhibitor of TGs, suppressed it. When aggregated Aβ was added to the mixed culture, the immunoreactivity of MFG-E8 surrounding Aβ was observed, and then followed by microglial endocytosis. Using western blotting technique, MFG-E8 was detected in cell lysate of astrocyte culture, and was also detected in the medium. When microglia culture was incubated with astrocyte conditioned medium, MFG-E8 levels in microglia tended to increase. It is likely that microglia might utilize MFG-E8 released from astrocytes as well as that expressed in themselves in order to endocytose Aβ aggregation. Furthermore, we confirmed that MFG-E8 could bind with TG2 in microglia culture by immunoprecipitate technique. These results suggest that microglia might uptake Aβ as a complex of aggregated Aβ/MFG-E8/TG2.


Amyloid β Microglia Astrocyte MFG-E8 Transglutaminase 2 


Amyloid beta


Astrocytes conditioned medium;


Alzheimer’s disease


Bovine serum albumin


Coomassie brilliant blue


Central nervous system


Dulbecco’s modified Eagle medium


Dimethyl sulfoxide


Epidermal growth factor


Fetal bovine serum


Fluorescein isothiocyanate


Glial fibrillary acidic protein


Horseradish peroxidase


Milk fat globule EGF factor 8 protein




Nitric oxide


Phosphate-buffered saline






Vitronectin receptor.



This work was supported in part by JSPS KAKENHI Grant No. JP15J12259 to K.K., JP26850209 to K.T., JP26450447 to M.M., and JP15K07768 to Y.N.


  1. 1.
    Kreutzberg GW (1996) Microglia: a sensor for pathological events in the CNS. Trends Neurosci 19:312–318CrossRefGoogle Scholar
  2. 2.
    Nakamura Y (2002) Regulating factors for microglia activation. Biol Pharm Bull 25:945–953CrossRefGoogle Scholar
  3. 3.
    Neniskyte U, Neher JJ, Brown GC (2011) Neuronal death induced by nanomolar amyloid β is mediated by primary phagocytosis of neurons by microglia. J Biol Chem 286(46):39904–39913CrossRefGoogle Scholar
  4. 4.
    Anderson CM, Swanson RA (2000) Astrocyte glutamate transport: review of properties, regulation, and physiological functions. Glia 32:1–14CrossRefGoogle Scholar
  5. 5.
    Abbott NJ (2002) Astrocyte-endothelial interactions and blood-brain barrier permeability. J Anat 200:629–638CrossRefGoogle Scholar
  6. 6.
    Correale J, Villa A (2009) Cellular elements of the blood-brain barrier. Neurochem Res 34:2067–2077CrossRefGoogle Scholar
  7. 7.
    Pratten MK, Lloyd JB (1986) Pinocytosis and phagocytosis: the effect of size of a particulate substrate on its mode of capture by rat peritoneal macrophages cultured in vitro. Biochim Biophys Acta 881:307–313CrossRefGoogle Scholar
  8. 8.
    Shanbhag AS, Jacobs JJ, Black J, Galante JO, Glant TT (1994) Macrophage/particle interactions: effect of size, composition and surface area. J Biomed Mat Res 28:81–90CrossRefGoogle Scholar
  9. 9.
    Aukunuru JV, Kompella UB (2002) In vitro delivery of nano- and micro-particles to human retinal pigment epithelial (ARPE-19) cells. Drug Del Technol 2:50–57Google Scholar
  10. 10.
    Fadok VA, Chimini G (2001) The phagocytosis of apoptotic cells. Semin Immunol 13:365–372CrossRefGoogle Scholar
  11. 11.
    Ravichandran KS (2011) Beginnings of a good apoptotic meal: the find-me and eat-me signaling pathways. Immunity 35(4):445–455CrossRefGoogle Scholar
  12. 12.
    Oshima K, Aoki N, Kato T, Kitajima K, Matsuda T (2002) Secretion of a peripheral membrane protein, MFG-E8, as a complex with membrane vesicles. Eur J Biochem 269:1209–1218CrossRefGoogle Scholar
  13. 13.
    Hanayama R, Tanaka M, Miwa K, Shinohara A, Iwamatsu A, Nagata S (2002) Identification of a factor that links apoptotic cells to phagocytes. Nature 417(6885):182–187CrossRefGoogle Scholar
  14. 14.
    Hanayama R, Nagata S (2005) Impaired involution of mammary glands in the absence milk fat globule EGF factor 8. Proc Natl Acad Sci USA 102(46):16886–16891CrossRefGoogle Scholar
  15. 15.
    Neher JJ, Neniskyte U, Zhao JW, Bal-Price A, Tolkovsky AM, Brown GC (2011) Inhibition of microglial phagocytosis is sufficient to prevent inflammatory neuronal death. J Immunol 186:4973–4983CrossRefGoogle Scholar
  16. 16.
    Fricker M, Neher JJ, Zhao JW, Théry C, Tolkovsky AM, Brown GC (2012) MFG-E8 mediates primary phagocytosis of viable neurons during neuroinflammation. J Neurosci 32(8):2657–2666CrossRefGoogle Scholar
  17. 17.
    Griffin M, Casadio R, Bergamini CM (2002) Transglutaminases: Nature’s biological glues. Biochem J 368:377–396CrossRefGoogle Scholar
  18. 18.
    Jeitner TM, Muma NA, Battaile KP, Cooper, AJL (2009) Transglutaminase activation in neurodegenerative diseases. Future Neurol 4:449–467CrossRefGoogle Scholar
  19. 19.
    Basso M, Berlin J, Xia L, Sleiman SF, Ko B, Haskew-Layton R, Kim E, Antonyak MA, Cerione RA, Iismaa S, Willis D, Cho S, Ratan RR (2012) Transglutaminase inhibition protects against oxidative stress-induced neuronal death downstream of pathological ERK activation. J Neurosci 32(19):6561–6569CrossRefGoogle Scholar
  20. 20.
    Belkin AM (2011) Extracellular TG2: emerging functions and regulation. FEBS J 278:4704–4716CrossRefGoogle Scholar
  21. 21.
    Grosso H, Mouradian MM (2012) Transglutaminase 2: biology, relevance to neurodegenerative diseases and therapeutic implications. Pharmacol Ther 133:392–410CrossRefGoogle Scholar
  22. 22.
    Szondy Z, Sarang Z, Molnár P, Németh T, Piacentini M, Mastroberardino PG, Falasca L, Aeschlimann D, Kovács J, Kiss I, Syegeydi E, Lakos G, Rajnavölgyi É, Birckbichler PJ, Melino G, Fésüs L (2003) Tranglutaminase 2-/-mice reveal a phagocytosis-associated crosstalk between macrophages and apoptotic cells. Proc Natl Acad Sci USA 100(13):7812–7817CrossRefGoogle Scholar
  23. 23.
    Tóth B, Garabuczi, É., Sarang Z, Vereb G, Vámosi G, Aeschimann D, Blaskó B, Bécsi B, Erdõdi B, Lacy-Hulbert A, Zhang A, Falasca L, Birge RB, Balajthy Z, Melino G, Fésüs L, Szondy Z (2009) Transglutaminase 2 is needed for the formation of an efficient phagocyte portal in macrophage engulfing apoptosis cells. J Immunol 182(4):2084–2092CrossRefGoogle Scholar
  24. 24.
    Kawabe K, Takano K, Moriyama M, Nakamura Y (2015) Lipopolysaccharide-stimulated transglutaminase 2 expression enhances endocytosis activity in mouse microglial cell line BV-2. Neuroimmunomodulation 22(4):243–249CrossRefGoogle Scholar
  25. 25.
    Kawabe K, Takano K, Moriyama M, Nakamura Y (2017) Amphotericin B increases transglutaminase 2 expression associated with upregulation of endocytotic activity in mouse microglial cell line BV-2. Neurochem Res 42:1488–1495CrossRefGoogle Scholar
  26. 26.
    Takano K, Shiraiwa M, Moriyama M, Nakamura Y (2010) Transglutaminase 2 expression induced by lipopolysaccharide stimulation together with NO synthase induction in cultured astrocytes. Neurochem Int 57(7):812–818CrossRefGoogle Scholar
  27. 27.
    Nakamura Y, Si QS, Kataoka K (1999) Lipopolysaccharide-induced microglial activation in culture: temporal profiles of morphological change and release of cytokines and nitric oxide. Neurosci Res 35:95–100CrossRefGoogle Scholar
  28. 28.
    Watanabe T, Totsuka R, Miyatani S, Kurata S, Sato S, Katoh I, Kobayashi S, Ikawa Y (2005) Production of the long and short forms of MFG-E8 by epidermal keratinocytes. Cell Tissue Res 321(2):185–193CrossRefGoogle Scholar
  29. 29.
    Akiyama H, Mori M, Saido T (1999) Occurrence of the diffuse amyloid β-protein (Aβ) deposits with numerous Aβ-containing glial cells in the cerebral cortex of patients with Alzheimer’s disease. Glia 25:324–331CrossRefGoogle Scholar
  30. 30.
    Mitrasinovic OM, Murphy GM Jr (2003) Microglial overexpression of the M-CSF receptor augments phagocytosis of opsonized Abeta. Neurobiol Aging 24(6):807–815CrossRefGoogle Scholar
  31. 31.
    Wang Z, Collighan RJ, Gross SR, Danen EH, Orend G, Telci D, Griffin M (2010) RGD-independent cell adhesion via a tissue transglutaminase-fibronectin matrix promotes fibronectin fibril deposition and requires syndecan-4/2 and α5β1 integrin co-signaling. J Biol Chem 285(51):40212–40229CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Kenji Kawabe
    • 1
  • Katsura Takano
    • 1
    Email author
  • Mitsuaki Moriyama
    • 1
  • Yoichi Nakamura
    • 1
  1. 1.Laboratory of Integrative Physiology in Veterinary SciencesOsaka Prefecture UniversityIzumisanoJapan

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