Take a look at the Recent articles

Abstract

Microglia categorized into macrophage is considered to be a major cell for immunity in central nervous system. Activated microglia release nitric oxide (NO) and proinflammatory cytokines to damage neurons, and also produce neurotrophins to protect neurons. In addition, activated microglia enhance endocytotic activity to engulf invading microorganisms and scavenge cell debris and damaged cells. Engulfment of neurons by activated microglia may play roles in the pathogenesis of neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD). Tissue-type transglutaminase (transglutaminase 2; TG2) is a cross-linking enzyme, which is activated in AD, PD and Huntington’s diseases. TG2 is involved in connection to phagocytes with apoptotic cells. In the present review, we explained in microglia the endocytosis mechanisms and the involvement of TG2 in endocytosis to be associated with neurodegenerative diseases.

Key words

transglutaminase, microglia, endocytosis, neurodegenerative diseases

Introduction

In pathological mechanism of various neurodegenerative diseases, it is well known that the activation of glial cells play important roles for both exacerbation and recovery [1-3]. Therefore, the elucidation of the mechanism of glial activation is essential for overcome such diseases. Astrocytes play various important roles in central nervous system (CNS), such as maintenance of blood brain barrier, scavenging some neurotransmitters, control of ionic balance in brain parenchyma. These functions of astrocytes serve the maintenance of brain homeostasis [4-6]. Functional changes of astrocytes are involved in neurodegenerative processes in various CNS diseases including Alzheimer’s diseases (AD).

Microglia categorized into macrophage is considered to be a major cell for immunity in CNS. Activated microglia release nitric oxide (NO) and proinflammatory cytokines to damage neurons, and also produce neurotrophins to protect neurons. In addition, they engulf invading microorganisms and scavenge cell debris and damaged cells [1,2,7,8]. Microglial phagocytosis contributes to maintaining of homeostasis of CNS and to developing neural network physiologically. However, it is reported that NO production and phagocytosis by hyper-activated microglia cause neuronal death in neurodegenerative diseases [3].

Tissue-type transglutaminase (TG2) is ubiquitously expressed in various cells and shows Ca²⁺ concentration-dependent enzyme activity catalyzing to form e-(g-glutamyl)lysine isopeptide bond crosslinking between glutamine and lysine residues of proteins [9,10]. TG2 is involved in cell adhesion and construct of cytoskeleton [11]. In addition, extracellular TG2 protein has binding domains to integrin and fibronectin in a Ca²⁺-independent manner [9,10,12]. These functions contribute to extracellular matrix formation, tissue structures stabilization and epithelia barrier. Moreover, TG2 is reported to play also as a G-protein, protein disulfide isomerase and protein kinase; participating in various intracellular signaling [9,10,13,14]. These various functions of TG2 contribute to proliferation, apoptosis, migration, inflammation, phagocytosis, and so on [9,10,15]. In CNS, several studies show that TG2 is over-expressed in the brains of AD, Parkinson’s disease (PD) and Huntington’s disease (HD) [9,10]; indicating that TG2 might play important roles in the CNS diseases.

When phagocytes engulf dead/apoptotic cells, they need to interact with “eat me” signal, such as phosphatidylserine (PS), which is exposed on surface of target cells [16,17]. It was reported in peritoneal macrophage that TG2 can bind integrin b₃ and milk fat globule EGF factor 8 protein (MFG-E8), and that TG2 protein is involved in recognition of apoptotic cells; suggesting that TG2 mediates the binding between vitronectin receptor (VR) and MFG-E8 in TG enzyme activity-independent manner [15,18]. TG2 might be associated with microglial phagocytosis as well as peritoneal macrophage. In the present review, we described especially in microglia that TG2 might be involved in the mechanisms of endocytosis, and that the endocytosis might be associated with neurodegenerative diseases.

Transglutaminases

Transglutaminase (TG), a crosslinking enzyme consisting eight isozymes identified, catalyzes Ca²⁺-dependently to form e-(g-glutamyl)lysine isopeptide bond crosslinking between glutamine and lysine residues in proteins or incorporating primary amines at selected peptide-bound glutamine residues [9]. Each TG in mammals is generally localized in a specific tissue and a type of cells, and plays important roles physiologically including skin cornification, blood clotting and wound healing [9]. Among them, TG2 is ubiquitously expressed in various tissues and has various functions as described above contributing to proliferation, apoptosis, migration, inflammation, phagocytosis, and so on [9,10,15]. TG2 is activated in pathological conditions including neurodegeneration, autoimmune diseases, and inflammatory diseases [9,19]. Blood coagulation factor XIII-A (FXIII-A), another TG, is reported to be expressed mainly in microglia in AD patient’s postmortem brain [20] and the mutation of FXIII might be associated with AD [21]. In monocyte/macrophage, TG2 and FXIII-A are reported to be involved in phagocytosis of apoptotic cells [15,18,22].

Phagocytosis

Endocytosis is generally divided into two categories, pinocytosis and phagocytosis, with size of particle up-taken; pinocytosis is defined as uptake of particles smaller than 1–2 µm and phagocytosis is as uptake over 2 µm [23-25]. In case of scavenging apoptotic cells, phagocytotic cells need to contact with “eat me” signal of target apoptotic cells by their receptors [16,17]. Phosphatidylserine (PS) is known to be a major “eat me” signal, most of which normally exist in inner leaflet of cell membrane; however, when cells are undergone apoptosis, PS is exposed on surface of outer leaflet. Recently, it was reported that PS exposure on cell surface was occurred not only in apoptotic cells but also in activated cells by several stimulation such as intracellular Ca²⁺ elevation [26]. It might cause phagocytosis of viable cells.

An adaptor protein, MFG-E8 consists of four domains, E1 and E2 homologous regions to epidermal growth factor (EGF) and C1 and C2 homologous regions to coagulation factor 8, and proline/threonine rich domain is additionally inserted between them resulting to be called a long-form of MFG-E8 [27]. C2 region of MFG-E8 binds to PS and RGD motif in E2 region of MFG-E8 binds to a VR of macrophage formed by integrin a_vb_3/5 dimer; thereby, macrophage can recognize and uptake the apoptotic cells [28,29]. In peripheral tissues, MFG-E8 protein is mainly released from macrophages and used for phagocytosis [28].

It is reported that microglia also release and utilize MFG-E8 to recognize neurons when stimulated by lipopolysaccharide (LPS) or amyloid b (Ab) [3,30,31]. It was reported that microglia derived from MFG-E8 knockout mice could endocytose neurons using MFG-E8 containing in the conditioned medium of astrocytes derived from wild-type mice [31]. Therefore, microglia might use MFG-E8 protein derived from both of microglia and astrocytes. In co-culture of neurons and microglia, it is reported that LPS-stimulated NO production from microglia causes exposure of PS on neurons and that microglia recognize neuronal PS as “eat me” signal, resulting to uptake the neurons [30]. The report suggests that excessive NO production and/or facilitation of phagocytosis are thought to be involved in neuronal death.

Involvement of TGs in phagocytosis

It has been reported in astrocytes and microglia that TG2 might participate in inflammatory responses including inducible NO synthase (iNOS) expression and NO production [19,32,33]. Exposure to LPS has been believed to stimulate intracellular signaling pathway through nuclear factor-kappa B (NF-kB) activation [34,35]. In addition, NF-κB binding site is identified on the TG2 promoter region [36]. It was reported that TG enzyme activity is increased by LPS exposure and that activated NF-κB might be involved in TG2 expression [19]. Moreover, Lee et al. reported that inhibitors of TG enzyme activity reduced LPS-induced NO production and that TG2 polymerized IκB (inhibitor of NF-kB) to activate NF-κB; indicating that TG2 augments the NF-kB activation pathway in a self-enhancing manner [19,32].

On the other hand, it has been reported in peritoneal macrophage that TG2 protein is involved in recognition of apoptotic cells [15,18]. It was reported that the phagocytosing activity was decreased in peritoneal macrophage derived from TG2 knockout mice and that TG2 protein interacted with adaptor proteins of PS independently of TG enzyme activity [15]. TG2 was reported to bind each of MFG-E8 and VR which are necessary proteins in the mechanism of phagocytosis [37].

Microglial endocytosis is known to be up-regulated by LPS-stimulation [38] and LPS-simulation in mouse microglial cell line BV-2 increased TG2 expression [39]. In the previous study, we reported in activated microglia that TG2 might be involved in endocytosis of fluorescent beads and dead cells, because of inhibition of the endocytosis by cystamine, an inhibitor of TG enzyme activity [39,40]. Cystamine also inhibited LPS-increased TG2 expression, iNOS expression, and NO production [39]. Therefore, TG2 expression and TG enzyme activity might be closely associated with both NO production and endocytosis in activated microglia.

Phagocytosis in neurodegenerative diseases

It has been reported the association between TGs and neurodegeneration [41-43]. On the other hand, microglial phagocytosis has been reported to be involved in neuronal death in neurodegenerative diseases [44,45]. As described above, in co-culture of neurons and microglia, it is reported that NO derived from LPS-stimulated microglia causes exposure of PS on neurons and that microglia recognize neuronal PS to uptake the neurons [30]. In AD brain, it is known to occur Ab accumulation and aggregation resulting to senile plaques. It was reported that uptake of soluble Ab monomer would be observed in resting microglia and the soluble Ab uptake would not activate microglia [46]; on the other hand, uptake of aggregated Ab was reported to activate microglia [47]. In neuronal/glial mixed culture, nanomolar Ab, which was not directly toxic to neurons but activated microglia, induced neuronal death by microglial phagocytosis [3]. It has been reported also in PD model that microglial phagocytosis might be involved in neuronal loss [48-50]. Taken together, the control of microglial phagocytosis might be important for neuronal survival in neurodegenerative diseases and the regulations of TG2 expression and TG enzyme activity might be targets of the mechanism to control microglial phagocytosis.

Conclusion

Engulfment of neurons by activated microglia may play roles in the pathogenesis of neurodegenerative diseases such as AD and PD. TG2 is a cross-linking enzyme, which is activated in AD, PD and HD. In monocyte/macrophage, TG2 is reported to involve in phagocytosis of apoptotic cells. Also in microglia, TG2 might be involved in endocytosis of invading microorganisms, aggregated proteins, cell debris and damaged cells. Regulations of TG2 expression and TG enzyme activity in microglia might play critical roles in neuronal survival in various CNS diseases through the regulation of endocytosis activity as well as inflammation.

References

1. Kreutzberg GW (1996) Microglia: a sensor for pathological events in the CNS. Trends Neurosci 19: 312-318. [Crossref]
2. Nakamura Y (2002) Regulating factors for microglial activation. Biol Pharm Bull 25: 945-953. [Crossref]
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: 39904-39913. [Crossref]
4. Anderson CM, Swanson RA (2000) Astrocyte glutamate transport: review of properties, regulation, and physiological functions. Glia 32: 1-14. [Crossref]
5. Abbott NJ (2002) Astrocyte-endothelial interactions and blood-brain barrier permeability. J Anat 200: 629-638. [Crossref]
6. Correale J, Villa A (2009) Cellular elements of the blood-brain barrier. Neurochem Res 34: 2067-2077. [Crossref]
7. 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: 95-100. [Crossref]
8. Motoyoshi A, Nakajima H, Takano K, Moriyama M, Kannan Y, et al. (2008) Effects of amphotericin B on the expression of neurotoxic and neurotrophic factors in cultured microglia. Neurochem Int 52: 1290-1296. [Crossref]
9. Griffin M, Casadio R, Bergamini CM (2002) Transglutaminases: nature's biological glues. Biochem J 368: 377-396. [Crossref]
10. Grosso H, Mouradian MM (2012) Transglutaminase 2: Biology, relevance to neurodegenerative diseases and therapeutic implications. Pharmacol Ther 133: 392-410. [Crossref]
11. Basso M, Berlin J, Xia L, Sleiman SF, Ko B, et al. (2012) Transglutaminase inhibition protects against oxidative stress-induced neuronal death downstream of pathological ERK activation. J Neurosci 32: 6561-6569. [Crossref]
12. Belkin AM (2011) Extracellular TG2: emerging functions and regulation. FEBS J 278: 4704-4716. [Crossref]
13. Nakaoka H, Perez DM, Baek KJ, Das T, Husain A, et al. (1994) Gh: A GTP-binding protein with transglutaminase activity and receptor signaling function. Science 264: 1593-1596. [Crossref]
14. Mishra S, Melino G, Murphy LJ (2007) Transglutaminase 2 kinase activity facilitates protein kinase A-induced phosphorylation of retinoblastoma protein. J Biol Chem 282: 18108-18115. [Crossref]
15. Tóth B, Garabuczi É, Sarang Z, Vereb G, Vámosi G, Aeschimann D, et al. (2009) Transglutaminase 2 is needed for the formation of an efficient phagocyte portal in macrophage engulfing apoptosis cells. J Immunol 182: 2084-2092. [Crossref]
16. Fadok VA, Chimini G (2001) The phagocytosis of apoptotic cells. Semin Immunol 13: 365-372. [Crossref]
17. Ravichandran KS (2011) Beginnings of a good apoptotic meal: the find-me and eat-me signaling pathways. Immunity 35: 445-455. [Crossref]
18. Szondy Z, Sarang Z, Molnár P, Németh T, Piacentini M, et al. (2003) Tranglutaminase 2-/- mice reveal a phagocytosis-associated crosstalk between macrophages and apoptotic cells. Proc Natl Acad Sci USA 100: 7812-7817. [Crossref]
19. Park KC, Chung KC, Kim YS, Lee J, Joh TH, et al. (2004) Transglutaminase 2 induces nitric oxide synthesis in BV-2 microglia. Biochem Biophys Res Commun 323: 1055-1062. [Crossref]
20. Akiyama H, Kondo H, Ikeda K, Arai T, Kato M, et al. (1995) Immunohistochemical detection of coagulation factor XIIIa in postmortem human brain tissue. Neurosci Lett 202: 29-32. [Crossref]
21. Gerardino L, Papaleo P, Flex A, Gaetani E, Fioroni G, et al. (2006) Coagulation factor XIII Val34Leu gene polymorphism and Alzheimer's disease. Neurol Res 28: 807-809. [Crossref]
22. Sárváry A, Szucs S, Balogh I, Becsky A, Bárdos H, et al. (2004) Possible role of factor XIII subunit A in Fcgamma and complement receptor-mediated phagocytosis. Cell Immunol 228: 81-90. [Crossref]
23. 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: 307-313. [Crossref]
24. Shanbhag AS, Jacobs JJ, Black J, Galante JO, Glant TT (1994) Macrophage/particle interactions: effect of size, composition and surface area. J Biomed Mater Res 28: 81-90. [Crossref]
25. 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: 50-57.
26. Segawa K, Nagata S (2015) An Apoptotic 'Eat Me' Signal: Phosphatidylserine Exposure. Trends Cell Biol 25: 639-650. [Crossref]
27. 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: 1209-1218. [Crossref]
28. Hanayama R, Tanaka M, Miwa K, Shinohara A, Iwamatsu A, et al. (2002) Identification of a factor that links apoptotic cells to phagocytes. Nature 417: 182-187.
29. Hanayama R, Nagata S (2005) Impaired involution of mammary glands in the absence of milk fat globule EGF factor 8. Proc Natl Acad Sci U S A 102: 16886-16891. [Crossref]
30. Neher JJ, Neniskyte U, Zhao J, Bal-Price A, Tolkovsky AM, et al. (2011) Inhibition of microglial phagocytosis is sufficient to prevent inflammatory neuronal death. J Immunol 186: 4973-4983.
31. Fricker M, Neher JJ, Zhao JW, Théry C, Tolkovsky AM, et al. (2012) MFG-E8 mediates primary phagocytosis of viable neurons during neuroinflammation. J Neurosci 32: 2657-2666.
32. Lee J, Kim YS, Choi DH, Bang MS, Han TR, et al. (2004) Transglutaminase 2 induces nuclear factor-kappaB activation via a novel pathway in BV-2 microglia. J Biol Chem 279: 53725-53735. [Crossref]
33. 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: 812-818.
34. Pålsson-McDermott EM, O'Neill LA (2004) Signal transduction by the lipopolysaccharide receptor, Toll-like receptor-4. Immunology 113: 153-162. [Crossref]
35. Lee JY, Lowell CA, Lemay DG, Youn HS, Rhee SH, et al. (2005) The regulation of the expression of inducible nitric oxide synthase by Src-family tyrosine kinase mediated through MyD88-independent signaling pathways of Toll-like receptor 4. Biochem Pharmacol 70: 1231-1240.
36. Caccamo D, Campisi A, Currò M, Aguennouz M, Li Volti G, et al. (2005) Nuclear factor-?B activation is associated with glutamate-evoked tissue transglutaminase up-regulation in primary astrocyte cultures. J Neurosci Res 82: 858–865.
37. 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 ?5?1 integrin co-signaling. J Biol Chem 285: 40212-40229.
38. Hughes MM, Field RH, Perry VH, Murray CL, Cunningham C (2010) Microglia in the degenerating brain are capable of phagocytosis of beads and of apoptotic cells, but do not efficiently remove PrPSc, even upon LPS stimulation. Glia 58: 2017-2030.
39. Kawabe K, Takano K, Moriyama M, Nakamura Y (2015) Lipopolysaccharide-stimulated transglutaminase 2 expression enhances endocytosis activity in the mouse microglial cell line BV-2. Neuroimmunomodulation 22: 243-249.
40. 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: 1488-1495.
41. Beck KE, De Girolamo LA, Griffin M, Billett EE (2006) The role of tissue transglutaminase in 1-methyl-4-phenylpyridium (MPP+)-induced toxicity in differentiated human SH-SY5Y neuroblastoma cells. Neurosci Lett 11: 46-51.
42. Jeitner TM, Muma NA, Battaile KP, Cooper AJ (2009) Transglutaminase activation in neurodegenerative diseases. Future Neurol 4: 449-467. [Crossref]
43. Jeitner TM, Pinto JT, Krasnikov BF, Horswill M, Cooper AJ (2009) Transglutaminases and neurodegeneration. J Neurochem 109 Suppl 1: 160-166. [Crossref]
44. Brown GC, Neher JJ (2014) Microglial phagocytosis of live neurons. Nat Rev Neurosci 15: 209-216. [Crossref]
45. Neniskyte U, Vilalta A, Brown GC (2014) Tumour necrosis factor alpha-induced neuronal loss is mediated by microglial phagocytosis. FEBS Lett 588: 2952-2956. [Crossref]
46. Akiyama H, Mori M, Saido T (1999) Occurrence of the diffuse amyloid ß-protein (Aß) deposits with numerous Aß-containing glial cells in the cerebral cortex of patients with Alzheimer's disease. Glia 25: 324-331.
47. Mitrasinovic OM, Murphy GM Jr (2003) Microglial overexpression of the M-CSF receptor augments phagocytosis of opsonized Abeta. Neurobiol Aging 24: 807-815.
48. Marinova-Mutafchieva L, Sadeghian M, Broom L, Davis J, Medhurst A, et al. (2009) Relationship between microglial activation and dopaminergic neuronal loss in the substantia nigra: a time course study in a 6-hydroxydomanine model of Parkinson’s disease. J Neurochem 110: 966-975.
49. Zhang W, Phillips K, Wielgus A, Liu J, Albertini A, et al. (2011) Neuromelanin activates microglia and induces degeneration of dopaminergic neurons; implications for progression of Parkinson’s disease. Neurotox Res 19: 63-72.
50. Emmrich JV, Hornik TC, Neher JJ, Brown GC (2013) Rotenone induces neuronal death by microglial phagocytosis of neurons. FEBS J 280: 5030-5038. [Crossref]