Supplemental Material:

WNT signaling in activated microglia is pro-inflammatory

Carina Sandbäck1, Jan Mulder2, Jenny Dahlström1, Ken Mackie3, Tibor Hortobagyi4, Heikki Tanila5,6, Lakshman Kumar Puli5, Katrin Färber7, Tibor Harkany2,8, Gunnar Schulte1,#

1Section for Receptor Biology & Signaling, Department of Physiology & Pharmacology, Karolinska Institutet, S-17177 Stockholm, Sweden; 2College of Life Sciences & Medicine, University of Aberdeen, Aberdeen, AB25 2ZD, United Kingdom; 3Department of Psychological and Brain Sciences, Indiana University, Bloomington, IN 47405, USA; 4Department of Clinical Neuroscience, Institute of Psychiatry, King's College London, London SE5 8AF, United Kingdom; 5A. I. Virtanen Institute, University of Eastern Finland, F-70210 Kuopio, Finland; 6Department of Neurology, Kuopio University Hospital, F-70211 Kuopio, Finland; 7Department of Cellular Neurosciences, Max Delbrück Center, D-13092 Berlin, Germany and 8Division of Molecular Neurobiology, Department of Medical Biochemistry & Biophysics, Karolinska Institutet, S-17177 Stockholm, Sweden.

#Address correspondence to: gunnar.schulte@ki.se

This file includes:

Human and mouse tissue preparation

Supplemental Table 1-5

Supplemental Figure 1-3

Preparation of human and mouse tissue for immunostaining

Human tissues.

Human brains (Table S1) were from subjects who had been consented for research and collected at the London Neurodegenerative Disease Brain Bank, Medical Research Council Centre for Neurodegeneration Research, Institute of Psychiatry, King’s College London, UK according to Local Research Ethics guidelines (#231/2001). Age-matched controls represent subjects with no clinical history of memory impairment. We examined β-catenin immunoreactivity on temporal lobe tissues encompassing the hippocampus and parahippocampal structures in patients with sporadic AD. All main stages of AD were studied such as transentorhinal, limbic, and isocortical; qualifying for ‘low’ (Braak I-II), ‘intermediate’ (Braak III-IV), and ‘high’ (Braak VI) likelihood of AD-related dementia according to the NIA-Reagan Institute diagnostic criteria (Hyman and Trojanowski, 1997) and for ‘possible’, ‘probable’ and ‘definite’ CERAD criteria (Mirra et al, 1991) (Table S1).

Animal tissues.

APdE9 mice and their wild-type littermates (10 and 13 months;n = 3/group) were anesthetized by isoflurane (5% in air; 1L/min flow rate), and transcardially perfused (100 ml/animal with 4 ml/min flow rate) with 4% paraformaldehyde in phosphate buffer (0.1M, pH7.4) that was preceded by a short pre-rinse with ice-cold physiological saline. Brains were post-fixed overnight. Tissue samples were cryoprotected in 30% sucrose in physiological saline for at least 48h. 40 μm coronal sections spanning the entire telencephalon were cut on a cryostat microtome. Animal experiments conformed to the European Communities Council Directive (86/609/EEC) and were approved by local ethical authorities (Stockholms Norra Djurförsöksetiska Nämnd, Stockholm, Sweden [N26/05]). Particular care was taken to minimize the number of animals and their suffering throughout the experiments.

Supplemental Table 1:

Table caption:	Clinicopathological characterization of post-mortem human tissue samples that were tested for their -catenin, CB2 cannabinoid receptor, GFAP and SNAP-25 levels.

Table legend:	Tissues from patients with Alzheimer’s disease (AD) and age-matched controls of both sexes were included in this study. Brain samples were obtained with written consent at the London Brain Bank of the Institute of Psychiatry, King’s College London, London, United Kingdom. Autopsy samples were coded to ensure anonymity. Pathological diagnosis including AD staging was performed by using routine histochemistry and immunocytochemistry protocols. The distribution and density of senile plaques, neurofibrillary tangles, neuronal atrophy, and cell loss was determined throughout. Sample material was classified by both the CERAD and Braak criteria, and subsequently grouped. Abbreviations: F, female; M, male; Braak, tangle stage according to (1); CERAD, likelihood of AD according to (2); PMD, post mortem delay (h); BW, brain weight (g); pH, pH of post-mortem brain tissue.

Number	Age (years)	Sex	Braak	CERAD	PMD	pH	BW	co-existent pathology
1 (A042/01)	52	F	0	control	44	6.37	n.a.
2 (A063/95)	82	F	0	control	48	6.63	1113
3 (A205/94)	71	F	0	control	30	6.3	1169
4 (A190/94)	26	F	0	control	10	5.9	1237
5 (A187/06)	71	F	1	possible AD	48	6.42	1292
6 (140/07)	81	F	1	possible AD	17	6.1	1132
7 (A033/94)	89	F	2	possible AD	96	6.12	n.a.	cerebrovascular disease
8 (A073/05)	93	M	2	possible AD	33	6.57	1324	old cerebellar infarcts
9 (A040/07)	82	F	2	possible AD	13	6.48	1218	argyrophilic grains
10 (A286/95)	105	F	3	possible AD	58	6.9	1369
11 (A307/98)	77	F	3	probable AD	43	6.63	n.a.	cerebrovascular disease
12 (A242/05)	82	M	4	probable AD	<24	6.78	1116
13 (A285/95)	87	F	4	probable AD	48	6.28	1156
14 (A093/97)	91	M	4	probable AD	62	6.31	1115
15 (A179/98)	88	F	4	probable AD	24	6.49	990
16 (A041/07)	87	F	4	probable AD	22	6.97	1188	motor neuron disease
17 (A188/07)	88	F	6	definite AD	19	6.35	900	mild small vessel dis.
18 (A219/07)	82	F	6	definite AD	5	6.45	1060
19 (A028/06)	81	F	6	definite AD	24	6.32	862
20 (A189/06)	79	F	6	definite AD	24	6.31	912	old cystic infarct
21 (A206/06)	71	F	6	definite AD	21	6.46	1270
22 (A195/06)	91	F	6	definite AD	23	6.36	954
23 (A230/06)	89	F	6	definite AD	26	6.5	1038

Supplemental Table 2:

Table caption:	Factors and their concentrations used in ligand microarrays to detect LRP phosphorylation and -catenin stabilization in immortalized N13 microglia-like cells (Fig. 5).

Table legend:	The final concentration of each factor is indicated. Control cultures were exposed to respective vehicles of the ligands listed. Methodological details are referred to in Materials and Methods. apomorphine, a dopamine receptor agonist; ATP and UTP, known to act at P2Y and P2X receptors; glutamate, AN excitatory neurotransmitter; interferon / and tumor necrosis factor , inflammatory cytokines ; isoproterenol, a -adrenergic receptor agonist; LiCl, GSK3 inhibitor; lipopolysaccaride (LPS), an inflammatory activator, bacterial cell wall component; NECA, an adenosine receptor agonist; thrombin, acting at protease-activated receptors.

Factor	Concentration
Apomorphine	10 µM
ATP	1 µM
Glutamate	100 µM
IFN α/β	10 U/ml
Insulin	50 ng/ml
Isoproterenol	100 µM
LiCl	20 nM
Lipopolysaccharide	100 ng/ml
NECA	100 µM
Thrombin	10 U/ml
TNFα	50 ng/ml
UTP	10 µM
WNT-3A	300 ng/ml

Supplemental Table 3:

Table caption:	Commercial antibodies used in this study.

Antigen	Species	Catalogue number	Company	Dilution
PHF-tau (AT8)	mouse	MN1020	Pierce/Thermo Scientific	1:1,000
-catenin	mouse	610154	BD Biosciences	1:1,000
-catenin	rabbit	Ab2365	Abcam	1:XXX
-actin	mouse	A5441	Sigma	1:10,000
Cd11b (OX-42)	rat	MCA711	Serotec	1:500
COX2	mouse	#610293	BD Biosciences	1:1,000
DVL1	mouse	sc-8025	Santa Cruz	1:1,000
DVL2	rabbit	sc-13974	Santa Cruz	1:1,000
DVL3	mouse	sc-8027	Santa Cruz	1:1,000
GFAP	mouse	MAB3202	Millipore	1:1,000
iNOS	rabbit	N7782	Sigma	1:5,000
IBA-1	goat	AB5076	Abcam	1:200
P-LRP6 (Ser1490)	rabbit	2568	Cell Signaling Technology	1:1,000

Supplemental Table 4:

Table caption:	Primer designs, their annealing temperatures and references.

Primer	Annealing temperature	Sequence (5’-3‘)	Reference
FZD1	60oC	Forward: GTC ATC GCC TGC TAC TTC TA	(3)
Reverse: AGC CTC GTG TAG AAC TTC CT
FZD2	61oC	Forward: TGC CGT CCT ATC TCA GCT ATA AGT TTC	(3)
Reverse: GTC TCC TCT TGC GAG AAG AAC AT
FZD3	55oC	Forward: CAG TCT GCT ACA TGA TGG TG	(3)
Reverse: CGC CAC TAA TAT TGT CAC CT
FZD4	59oC	Forward: GGG TTA TTC TGA CAC TCA CTT GGT T	(3)
Reverse: TGC GAT GTG GAA ATA AGA ACT GTG
FZD5	58oC	Forward: CCT GCT ACC AGC CGT CCT TC	(3)
Reverse: CAG TGA CAC ACA CAG GTA GCA C
FZD6	58oC	Forward: TGT TGG TAT CTC TGC GGT CTT CTG	RTprimer databaseID3699
Reverse: CTC GGC GGC TCT CAC TGA TG
FZD7	60oC	Forward: CTT CTC GGA CGA TGG CTA CC	(3)
Reverse: CCA TAC CGA AGA AGT AAA GCA CCA
FZD8	59oC	Forward: TTG GAA GTG ACC TCG CTC CTA G	(3)
Reverse: GGT TGG GCA TGT AAG TGT AGT TGT
FZD9	54oC	Forward: CTA CAA TGT CTA CTC CTT GGC	(4)
Reverse: CAG TAC CAG GTA GCA AGA TAG G
FZD10	60oC	Forward: ATG AAC AAT CAG ACC AAG ACA CCT G	(3)
Reverse: GGA CAC TTT GAC CAT GAA GAC CTC
LRP5	61oC	Forward: GAC ATC TAC AGC CGG ACA CTG TTC	this study
Reverse: TGG ACA TTG ATA GTG TTG GTG GC
LRP6	59oC	Forward: GCT ACA AAT GGC AAA GAG AAT GC	this study
Reverse: CAG TAT ACA AGC CAT GAC CAA ACA
ROR1	60oC	Forward: GGC AGC TCT TAC CAT GTT CTG AA	(3)
Reverse: TGG AAG GTA TCT CAT TCC AGC AC
ROR2	59oC	Forward: CCC CAA CAT CGT CTG TCT CCT A	this study
Reverse: CAT GGG AAC AGT AGC TGA AGA TCA
RYK	60oC	Forward: GGC TAT ATT AGC CTA GAG CCC TT	this study
Reverse: GCG AGG AAT AAA GGA AAT GAG TTA
WNT-1	61oC	Forward: CTT CGG CAA GAT CGT AAC C	(3)
Reverse: GCG AAG ATG AAC GCT GTT TCT
WNT-2	65oC	Forward: AAC GTC CCT CTC GGT GGA ATC	(3)
Reverse: TGT ACC ACC ATG AAG AGC TGA CC
WNT-3	65oC	Forward: CAG CGT AGC AGA AGG TGT GAA G	(3)
Reverse: ATG GCC AGG CTG TCA TCT ATG
WNT-3A	59oC	Forward: GAA CGC GAC CTG GTC TAC TAC G	(3)
Reverse: GTT AGG TTC GCA GAA GTT GGG T
WNT-7A	65oC	Forward: TGC GTG CCA GTC GAA ACA AG	(3)
Reverse: GAT ATA CAC CAG GTC AGT GTC CAT GG
actin	60oC	Forward: GCT CTG GCT CCT AGC ACC AT	(5)
Reverse: CCA CCG ATC CAC ACA GAG TAC

Supplemental Table 5:

Table caption:	Additional selection of genes involved in the pro-inflammatory response regulated by WNT-3A in primary microglia (FC, fold change).

Gene Name FC p value Nos2 nitric oxide synthase 2, inducible 44,72 0.047 Cd69 CD69 antigen 18,8 0.009 Mmp13 matrix metallopeptidase 13 14,649 0.035 Ptgs2 prostaglandin-endoperoxide synthase 2 14,454 0.0005 Mpa2l macrophage activation 2 like 7,522 0.008 Isg20 interferon-stimulated protein 7,474 0.021 Cd274 CD274 antigen 3,944 0.017 Cd86 CD86 antigen 3,499 0.040 Mmp14 matrix metallopeptidase 14 (membrane-inserted) 2,84 0.029

Supplemental Figure 1:

Figure caption:	Schematic summary of the WNT/-catenin signaling pathway.

Figure legend:	In the absence of WNTs, cytosolic -catenin levels are kept low by the constitutively active destruction complex. This is a multiprotein complex consisting of, among many other proteins, glycogen synthase kinase 3 (GSK-3), which phosphorylates -catenin resulting in its proteasomal degradation thus maintaining low cytosolic levels of -catenin. Upon WNT binding to both Frizzled (FZD) and LRP6, LRP becomes phosphorylated at multiple sites, such as Ser1490, which was assessed in the present study. In addition, the phosphoprotein disheveled (DVL) becomes activated by phosphorylation. Yet unknown mechanisms lead to an inactivation of the destruction complex through an increase in inhibitory phosphorylation of GSK-3. -Catenin can then translocate to the nucleus where it regulates T cell factor/lymphocyte enhancer factor (TCF/LEF)-mediated gene transcription leading to the induction of WNT-target genes.

Supplemental Figure 2:

Figure caption:	-catenin immuoreactivity is present in IBA-1+ microglia in human post-mortem brain from patients with Alzheimer's disease.

Figure legend:	(A) Three-dimensional rendering of an IBA-1+ microglia (a3 from Fig. 2) labeled with -catenin, the nuclear counterstain Hoechst33,342, and GFAP. Note the lack of -catenin in GFAP+ structures. In A1, the orthogonal Z-stack reveals cytosolic and perinuclear -catenin immunosignal in IBA-1+ microglia. (B) Spectral dye unmixing verified sharp peaks in the emission spectra at particular wavelenghts for Cy2 (2 and 3) and Cy3 (1). Scale bars in A, A1 = 15 µm; B = 5 µm.

Supplemental Figure 3:

Figure caption:	-catenin antibody in mouse, rat and human samples. Correlation between -catenin and SNAP-25.

Figure legend: (A) Western blot showing that an anti-β-catenin antibody (BD Biosciences) used throughout this study recognizes a band of the predicted molecular size for β-catenin in N13 microglia-like cells, primary microglia, and human brain. (B,B’, C) -catenin immunostaining in young adult rat brain confirms previous findings demonstrating predominant -catenin localization at hippocampal synapses. Arrows and asterisks point to unstained apical dendrites and perikarya of pyramidal cells, respectively, in the CA1 subfield of the rat hippocampus. Hoechst 33,342 nuclear counterstain appears in blue. (D, D1) Immunoblotting-based individual correlation analysis for b-catenin and the synaptic marker protein SNAP-25. Note the lack of expressional relationship between -catenin and SNAP-25 with disease progression despite a difference in SNAP-25 expression between control and mild AD. Scale bars in B = 20 µm, B1 = 10 µm, C = 15 µm.

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