Glycosylation of dentin matrix protein 1 is a novel key element for astrocyte maturation and BBB integrity

Abstract

The blood-brain barrier (BBB) is a tight boundary formed between endothelial cells and astrocytes, which separates and protects brain from most pathogens as well as neural toxins in circulation. However, detailed molecular players involved in formation of BBB are not completely known. Dentin matrix protein 1 (DMP1)-proteoglycan (PG), which is known to be involved in mineralization of bones and dentin, is also expressed in soft tissues including brain with unknown functions. In the present study, we reported that DMP1-PG was expressed in brain astrocytes and enriched in BBB units. The only glycosylation site of DMP1 is serine89 (S89) in the N-terminal domain of the protein in mouse. Mutant mice with DMP1 point mutations changing S89 to glycine (S89G), which completely eradicated glycosylation of the protein, demonstrated severe BBB disruption. Another breed of DMP1 mutant mice, which lacked the C-terminal domain of DMP1, manifested normal BBB function. The polarity of S89G-DMP1 astrocytes was disrupted and cell-cell adhesion was loosened. Through a battery of analyses, we found that DMP1 glycosylation was critically required for astrocyte maturation both in vitro and in vivo. S89G-DMP1 mutant astrocytes failed to express aquaporin 4 and had reduced laminin and ZO1 expression, which resulted in disruption of BBB. Interestingly, overexpression of wild-type DMP1-PG in mouse brain driven by the nestin promoter elevated laminin and ZO1 expression beyond wild type levels and could effectively resisted intravenous mannitol-induced BBB reversible opening. Taken together, our study not only revealed a novel element, i.e., DMP1-PG, that regulated BBB formation, but also assigned a new function to DMP1-PG.

Key words： blood-brain barrier; dentin matrix protein1; glycosylation; astrocyte; cell adhesion; proteoglycan

References

1 Alvarez JI (2011) The Hedgehog pathway promotes blood-brain barrier integrity and CNS immune quiescence. Science 334 (6063):1727–1731

2 Armulik A (2010) Pericytes regulate the blood-brain barrier. Nature 468(7323):557–561

3 Attwell D, Buchan AM, Charpak S, Lauritzen M, MacVicar BA, Newman EA (2010) Glial and neuronal control of brain blood flow. Nature 468(7321):232–243

4 Bell RD, Winkler EA, Singh I, Sagare AP, Deane R (2012) Apolipoprotein E controls cerebrovascular integrity via cyclophilin A. Nature 485:512–516

5 Bhattacharjee AK (2001) The effects of the Na+/Ca++ exchange blocker on osmotic blood–brain barrier disruption. Brain Res 900 (2):157–162

6 Dalkara T, Alarcon-Martinez L (2015) Cerebral microvascular pericytes and neurogliavas-cular signaling in health and disease. Brain Res 1623:3–17

7 Deane R, Sagare A, Hamm K, Parisi M, Lane S(2008) apoE isoform-specific disruption of amyloid beta peptide clearance from mouse brain. J Clin Investig 118:4002–4013

8 Drouin-Ouellet J, Sawiak SJ, Cisbani G, Lagacé M, Kuan WL (2015) Cerebrovascular and blood-brain barrier impairments in Huntington’s disease: potential implications for its pathophysiology. Ann Neurol 78:160–177

9 Feng JQ (2006) Loss of DMP1 causes rickets and osteomalacia and identifies a role for osteocytes in mineral metabolism. Nat Genet 38(11):1310–1315

10 George A, Sabsay B, Simonian PA, Veis A (1993) Characterization of a novel dentin matrix acidic phosphoprotein. Implications for induction of biomineralization. J Biol Chem 268(17):12624–12630

11 George A, Silberstein R, Veis A (1995) In situ hybridization shows Dmp1 (AG1) to be a developmentally regulated dentin-specific protein produced by mature odontoblasts. Connect Tissue Res 33(1–3):67–72

12 Halliday MR, Rege SV, Ma Q, Zhao Z, Miller CA (2016) Accelerated pericyte degeneration and blood–brain barrier breakdown in apolipoprotein E4 carriers with Alzheimer’s disease. J Cereb Blood Flow Metab 36:216–227

13 Huang YA, Zhou B, Wernig M, Südhof TC (2017) ApoE2, ApoE3, and ApoE4 differentially stimulate APP transcription and Aβ secretion. Cell 168:427–441

14 Knowland D (2014) Stepwise recruitment of transcellular and paracellular pathways underlies blood-brain barrier breakdown in stroke. Neuron 82(3):603–617

15 Korczyn AD (2015) Vascular parkinsonism–characteristics, pathogenesis and treatment. Nat Rev Neurol 11:319–326

16 Lorenz-Depiereux B (2006) DMP1 mutations in autosomal recessive hypophosphate-mia implicate a bone matrix protein in the regulation of phosphate homeostasis. Nat Genet 38 (11):1248–1250

17 Menezes MJ(2014) The extracellular matrix protein laminin α2 regulates the maturation and function of the blood-brain barrier. J Neurosci 34(46):15260–15280

18 Pan GY (2000) Intracarotid infusion of hypertonic mannitol changes permeability of blood brain barrier to methotrexate in rats. Acta Pharmacol Sin 21(7):613–616

19 Peng T (2008) Blocking of proteolytic processing and deletion of glycosaminoglycan side chain of mouse DMP1 by substituting critical amino acid residues. Cells Tissues Organs 189(1–4):192–197

20 Persidsky Y, Ramirez SH, Haorah J, Kanmogne GD (2006) Blood–brain barrier: structural components and function under physiologic and pathologic conditions. J Neuroimmune Pharmacol 1(3):223–236

21 Qin C (2006) A chondroitin sulfate chain attached to the bone dentin matrix protein 1 NH2-terminal fragment. J Biol Chem 281 (12):8034–8040

22 Romero IA (2003) Changes in cytoskeletal and tight junctional proteins correlate with decreased permeability induced by dexamethasone in cultured rat brain endothelial cells. Neurosci Lett 344:112–116

23 Rowe PSN (2000) MEPE, a new gene expressed in bone marrow and tumors causing osteomalacia. Genomics 67(1):54–68

24 Sun Y (2015) Glycosylation of Dentin Matrix Protein 1 is critical for osteogenesis. Sci Rep 5:17518

25 Sweeney MD, Sagare AP, Zlokovic BV (2015) Cerebrospinal fluid biomarkers of neurovascular dysfunction in mild dementia and Alzheimer’s disease. J Cereb Blood Flow Metab 35:1055–1068

26 Tarasoff-Conway JM, Carare RO, Osorio RS, Glodzik L, Butler T (2015) Clearance systems in the brain-implications for Alzheimer disease. Nat Rev Neurol 11:457–470

27 Terasawa M, Shimokawa R, Terashima T, Ohya K, Takagi Y, Shimokawa H (2004) Expression of dentin matrix protein 1 (DMP1) in nonmineralized tissues. J Bone Miner Metab 22 (5):430–438

28 Winkler EA, Sengillo JD, Sullivan JS, Henkel JS, Appel SH (2013) Blood-spinal cord barrier breakdown and pericyte reductions in amyotrophic lateral sclerosis. Acta Neuropathol 125:111–120

29 Wolburg H, Lippoldt A (2002) Tight junctions of the blood-brain barrier: development, composition and regulation. Vasc Pharmacol 38:323–337

30 Yao Y, Chen Z-L, Norris EH, Strickland S (2014) Astrocytic laminin regulates pericyte differentiation and maintains blood brain barrier integrity. Nat Commun 5:3413

31 Zhao Z, Nelson AR, Betsholtz C, Zlokovic BV (2015) Establishment and dysfunction of the blood-brain barrier. Cell 163:1064–1078

32 Zipser BD, Johanson CE, Gonzalez L, Berzin TM, Tavares R (2007) Microvascular injury and blood-brain barrier leakage in Alzheimer’s disease. Neurobiol Aging 28:977–986