Partial purification of [3H]mianserin binding sites

European Journal of Pharmacology', 87 (1983) 357-365

357

Elsevier BiomedicalPress

PARTIAL PURIFICATION O F I3HIMIANSERIN BINDING S I T E S B. CHAN and B.K. MADRAS * Psvchopharmacology Unit, Clarke Institute of Psychiatry, 250 College Street, Toronto, Ontario, Canada, and Department of Pharmacology', University of Toronto, Toronto, Ontario, Canada

Received 9 February 1982,revised MS received21 September 1982, accepted 16 November 1982

B. CHAN and B.K. MADRAS, Partial purification of [3H]mianserin binding sites, European J. Pharmacol. 87 (1983) 357-365. Frontal cortex membranes were solubilized with digitonin, prelabelled with 4 nM [3H]mianserin, and partially purified by isoelectric focussing. Bound [3H]mianserin separated from free [3H]mianserin as a single radioactive peak with a pI value of 5.03. A 14-fold purification was achieved. Focussing in the presence of 1 #M ketanserin (R 41468) or 1/~M chlorpyramine (a histamine Hrantagonist ) or 10/~M spiperone (an S2-antagonist ) completely abolished the peak of radioactivity at pH 5. Pharmacological characterization of the eluted radioactive peak revealed both histaminergic and serotonergic binding properties. The data suggest that the serotonergic and histaminergic components of [3H]mianserin binding to frontal cortex are not separable by solubilization, gel filtration or isoelectric focussing. Solubilized [3H]mianserin binding Purified receptors

Purified [3H]mianserin sites Soluble receptors

1. Introduction The neurochemical profile of mianserin, a tetracyclic antidepressant, is distinct from the tricyclic antidepressants (TCA)(Peet and Behagel, 1978). It is not an effective inhibitor of monoamine uptake (Goodlet et al., 1977; Raisman et al., 1979) and its binding to neurotransmitter receptors does not always parallel that of tricyclic derivatives (U'Prichard et al., 1978; Whitaker and Seeman, 1978; Raisman et al., 1979; Clements-Jewery and Robson, 1980; Diffley et al., 1980; Tang and Seeman, 1980; Peroutka and Snyder, 1980; Hall and Ogren, 1981). Further clarification of mianserin binding sites has emerged from the use of [3H]mianserin.

* To whom all correspondenceshould be addressed: PsychopharmacologyUnit, Clarke Institute of Psychiatry,250 College Street, Toronto, Ontario, Canada M5T 1R8. 0014-2999/83/0000-0000/$03.00 © 1983 ElsevierBiomedicalPress

[3H]Mianserin binding

Antidepressants

[3H]Mianserin labeling of cortical sites has a unique pharmacological profile, possible reflecting high affinity binding to predominately serotonergic ($2) and histaminergic (Hi) sites (Whitaker and Cross, 1980; Dumbrille-Ross et al., 1980, 1981; Peroutka and Snyder, 1981). The two sites have been differentiated by analysis of in vitro binding pharmacology and anatomical distribution of binding characteristics (Peroutka and Snyder, 1981). However, Scatchard analyses of [3n]mianserin saturation curves suggest a single population of binding sites with an apparent K D of 0.7-3 nM (Whitaker and Cross, 1980; Peroutka and Snyder, 1981; Dumbrille-Ross et al., 1980, 1981; Chan and Madras, 1982). Thus mianserin may bind with equi-potency primarily to S2- and H~-receptors. Isolation of the individual sites and comparison with similarly treated but differently labeled S2- and H~-sites would constitute significant evidence for the existence of two independent populations of mianserin binding sites, with similar drug affinities for each.

358 To pursue this goal, we have adapted techniques used to successfully solubilize and partially purify other monoamine receptors (Fraser and Venter, 1980; Madras et al., 1980, 1981; 1981; Lilly et al., 1981). The solubilization of [3H]mianserin binding sites has been described elsewhere (Chan and Madras, 1982). We now present a method for partial purification of binding sites by isoetectric focussing. Characterization studies indicate that serotonergic and histaminergic components of [3H]mianserin binding are not separable by digitonin solubilization, gel filtration, or isoelectfic focussing.

2. Materials and methods

1981, 1982) were gifts from Janssen Pharmaceutica (Beerse, Belgium). d-LSD was from the Health Protection Branch, Department of Health and Welfare, Ottawa, Canada. 2~2. Frontal cortex

Frozen canine brains from Pel-Freeze were defrosted at room temperature. Gray matter from the frontal cortex was gently scraped off with a scalpel. A combined mitochondrial (P2) and microsomal ( ~ ) fraction was prepared as described earlier (Madras et al., 1982; Chan and Madras, 1982). The final pellet was rehomogenized in 40 vol of 5 mM sodium phosphate, pH 7.2 at 4°C containing 1 mM magnesium sulfate. The pellet set aside for solubilization was resuspended in 10 vol phosphate buffer.

2.1. Materials

2.3. Solubilization procedure Frozen canine brains were purchased from PelFreeze Biologicals (Roger, AK). [3H]Mianserin (57.7-78.5 Ci/mmol) and scintillation fluor (Aquasol) were from New England Nuclear Co. (Boston, MA). Digitonin (lot 796678 and 703463), Tris (hydroxymethyl) aminomethane, L-ascorbic acid, disodium ethylenediaminetetraacetic acid (EDTA), ammonium sulfate, sodium dihydrogen phosphate, calcium chloride and magnesium sulfate were obtained from Fisher Chemical Co. (Toronto). Polyethylene glycol 6000, bovine gamma globulin (Cohn Fraction II), bovine albumin (Fraction V), serotonin creatinine sulfate, dopamine hydrochloride and histamine base were from Sigma Chemical Co. (St. Louis, MO). Sodium chloride, sodium hydrogen phosphate, sodium hydroxide were from J.T. Baker Chemical Co. (Phillipsburg, N.J.). Magnesium chloride was from BDH Chemical Co. (Toronto), Whatman G F / B glass fibre filters were from Whatman Co. Sephadex G-50 (fine), Sephacryl S-300 (superfine) and carrier ampholyte for isoelectfic focussing (Pharmalyte pH 3-10) were from Pharmacia Fine Chemicals. Sucrose (special enzyme grade) was from Schwarz/Mann. Polyacrylamide gel electrophoresis chemicals and protein microassay kit were from Bio-Rad Labs. Mianserin was a gift from Organon (Holland). Spiperone, Ketanserin and R 43448 (Leysen et aL,

[3H]Mianserin binding sites were solubilized with digitonin from a combined P2-P3 membrane fraction (4 mg protein/ml). The procedure for solubilization was identical to that used to extract dopamine D2-receptors from striata (Madras et al., 1982). Exposing the membrane fraction to digitonin for a period longer than 30 min resulted in no increase in receptor release. Approximately 41% of the protein in the P2-P3 fraction was solubilized into the clear supernatant and approximately 45% of the specific sites for [3H]mianserin were solubilized intact (Chan and Madras, 1982). 2.4. [3H]3#ianserin binding to membranes

The specific binding of [3H]mianserin to the P2-P3 membrane fractions were performed by minor modifications of standard methods (Hartley and Seeman, 1978) and are described elsewhere (Chan and Madras, 1982). The specific binding of [3H]mianserin was defined as the difference between the total and that occurring in the presence of 1/zM R 41468 (ketanserin) a novel antagonist at 5-HT2 receptors with high affinity for histamine HI-receptors (Leysen et al., t981, 1982). The amount of [3H]mianserin which absorbed to the filter ('background') in the absence of membrane

359 fraction was generally about 400 cpm. This background radioactivity was not displaceable by 1 # M ketanserin. Each assay was done in triplicate and repeated 3 times with a separate pool of tissue.

2.5. Incubation conditions and assays for solubilized receptors All solutions of 3H-ligand and drugs were prepared in phosphate buffer. Glass test tubes received in order: 0.1 ml buffer, 0.1 ml [3 H]mianserin (final concentration usually 2 nM) and 0.4 ml soluble material (containing about 600 btg protein). The contents were incubated at 22°C for 1 h to permit complete equilibrium. Aliquots of 0.3 ml were removed from the mixture and assayed by Sephadex G-50 chromatography (Madras et al., 1982) or a polyethyleneglycol assay (Chan and Madras, 1982). Briefly, after incubation at 22°C the samples were chilled on ice. Polyethylene glycol 6000 (PEG 0.2 ml of 50%) containing 250 mM sodium chloride in phosphate buffer at 4°C was added (final concentration of PEG was 12% and NaC1 100 mM). The tubes were thoroughly mixed and placed on ice for 10-15 min. The 0.5 mt suspension was then diluted with 4 ml of cold 12% polyethylene glycol 6000 containing 100 mM sodium chloride in 5 mM phosphate buffer. The entire contents (4.5 ml) was poured slowly, under vacuum, through Whatman G F / B glass fibre filters. The filters were then washed twice with 5.0 ml of PEG (12% in 5 mM phosphate buffer containing 100 mM NaCI at 4°C) and left overnight at 4°C in 9 ml of scintillation fluor. Radioactivity was determined in a Mark II liquid scintillation spectrometer at about 42% efficiency. The amount of [3H]mianserin which was absorbed to filters in the absence of soluble receptors was generally about 200 cpm at 2 nM [3H]mianserin. This background radioactivity was not displaceable by 1 # M ketanserin (R 41468) (baseline for non-specific binding) or 1/~M mianserin.

2.6. lsoetectric focussing of soluble frontal cortex Preparative isoelectric focussing was done using modifications of procedures previously described (Soiefer and Venter, 1980; Charlton et al., 1980;

Madras et al., 1981; Lilly et al., 1981). Soluble receptors (12 ml) were incubated with 0.58 ml [3H]mianserin (4 nM) and 0.128 ml buffer or buffer containing competing drugs (chlorpyramine, 1/~M; spiperone, 10/~M). After attaining equilibrium, 11 ml of the prelabelled soluble preparation (containing 15-18 mg protein) was diluted to 45 ml with 5 mM phosphate buffer, and carrier ampholytes (0.3 ml) for isoelectric focussing were added. Anodic solution (14 ml water, 12 g sucrose, 0.2 ml concentrated sulphuric acid; pH 0.5-1.25) was loaded first into a 100 ml column (LKB; Sweden). A dense solution of sucrose consisting of 28 g sucrose, 40 ml water and 1.9 ml carrier ampholyte was mixed with the receptor sample in a gradient mixer (LKB) and loaded into the isoelectric focussing column over a period of 30 min. The cathode (0.1 g sodium hydroxide per 10 ml water; p H 10-12) was layered on top of the sucrose gradient. The column was focussed with starting voltage of 600-900 V and current of 15-18 mA. After 5-6 h, the voltage was 1600-1800 V and the current down to 2-5 mA. It was left at this power setting for an additional 14-16 h. The column was kept cold (at 4°C) during the entire 19-22 h by a jacket of water connected to a circulating cooling pump. The contents of the column were fractionated into 45-50 fractions of 2.5-3.0 ml. The volume and pH of each fraction was measured. An aliquot of each fraction (0.2 ml) was placed in a scintillation vial with 9 ml scintillation fluor and counted for radioactivity. For protein determination, 0.5 ml from each fraction was dialyzed against 100 mM phosphate buffer (pH 7.1) to remove the carrier ampholyte that interferes with the assay. Each experiment was repeated 2-3 times with a separate pool of tissue.

2. 7. Characterization of the bound [3H]rnianserin eluted from the isoelectric focussing columns Preparative isoelectric focussing of soluble frontal cortex prelabeled with 4 nM [3 H]mianserin revealed the presence of 3 peaks of radioactivity (pI 4.90, 7.81, 9.8-10.2). To characterize the 4.90 peak, it was washed free of carrier ampholyte. The pI 4.90 fractions (4-5 ml) were concentrated to 1 ml using Amicon Centriflo membrane cones ('cut off'

360

molecular weight: 25 000), washed twice with 5-6 ml of phosphate buffer, and centrifuged in a Beckman table top centrifuge at 1 200-1 400 × g. The Amicon Centriflo membrane cones were soaked in water for 30 min and then in phosphate buffer for another 30 min prior to use. The washed receptor fraction was reconstituted to the original volume of 4-5 ml and used immediately. Glass test tubes received in order: 0.05 ml buffer or buffer containing competing drugs, 0.05 ml [3H]mianserin (final concentration 4 nM), 0.2 ml partially purified and washed receptor preparation (containing about 25 /~g protein). The contents were incubated for 1 h, then a 0.25 ml aliquot was assayed by Sephadex G-50 chromatography as described previously. The characterization studies were performed twice with partially purified material from separate isoelectric focussing procedures.

TABLE 1 lsoelectric focussing of soluble frontal cortex prelabelled with 4 n M [3H]mianserin. Detail experiment procedure is given in ' Methods'. Duration of isoelectric focussing

(h) Total [ 3 H]mianserin bound added to column (fmol) A m o u n t of protein added to column (rag) Radioactivity recovered in peak pl value Purification (fold)

5-6

16-20

1 880

1642

12.98 84% 5.03_+0.16 (n = 3_+ S.D.) 15

15.85 78% 4.90_+0.14 (n = 5-+S.D.) 14

2.8. Protein determination Membrane and soluble protein were assayed by a modified (Markwell et al., 1978) method of Lowry (Lowry et al., 1951). For determination of the small quantity of protein present in the fractions eluted from the isoelectric focussing column, a microassay (capable of measuring 1-20/~g protein) developed by Bio-Rad Laboratories was used. Bovine albumin was used as standard for all protein determinations.

3. Results

3.1. Isoelectric focussing of soluble frontal cortex Digitonin solubilized [3H]mianserin receptive sites were prelabelled with 4 nM [3H]mianserin and partially purified by isoelectric focussing. After focussing for 5-6 h, bound [3H]mianserin separated from unbound [3H]mianserin as a single peak with a pI value of 5.03 + 0.16 (n = 3). The presence of 1 /~M ketanserin (R 41468) during the prelabelling incubation markedly reduced the radioactivity in the pH 5 fraction (data not shown). Focussing for a longer period (16-20 h) did not significantly alter the pI value recovery of radioactivity, or the extent of purification (table 1).

Isoelectric focussing for 16-20 h revealed a peak of radioactivity at pH 7.81 which was not apparent when focussing was done for 5-6 h. The presence of an excess of spiperone (10/~M) during the prelabelling incubation, markedly reduced the radioactivity in the pH 5 fraction not in the pH 7.81 fraction (fig. 1). Identical results were obtained with chlorpyramine (1 #M). Isoelectric focussing in the absence of soluble frontal cortex resulted in the complete disappearance of the pH 5 peak but the pH 7.81 peak of radioactivity remained (fig. 2). If 0.3 ml of the pH 7.81 fraction was passed through a column of Sephadex G-50 (M.W. exclusion limit 30 000) no radioactivity was detectable in the void volume, while similar treatment of the pH 5 fraction resulted in a huge peak of radioactivity in the eluate of fraction 5, 6, 7 (fig. 3). Polyacrylamide gel electrophoresis of the pH 7.81 fraction showed no protein bands while that of pH 5 showed several bands. Thus, the p H 5 fraction was concluded to be the receptor while the peak of radioactivity at pH 7.81 was established to be an artifact of prolonged focussing. This artifact could be the result of binding of [3H]mianserin to carrier ampholyte or digitonin at pH 7.81. Another possible explanation for the artifact is that it may be a radioactive impurity which has a different isoelectric point from free

361

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Fig. 1. Representative profile of isoelectric focussing of soluble frontal cortex prelabelled with 4 n M [3H]mianserin (78.5 C i / m m o l ) for 16-20 h. The free [3H]mianserin peak (not shown here) appears at = pH 9.7. Each fraction was about 2.5-3.0 ml. Detailed experimental procedure is given in 'Methods'. • • Total binding; • . . . . . . • pH gradient; © . . . . . . © focussing in presence of l 0 / z M spiperone. Similar results were obtained with excess chlorpyramine or ketanserin.

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[3H]mianserin. The pH 7.81 peak was consistently 0.6-1% ( n = 7 ) of the free [3H]mianserin. The [3H]mianserin supplied by New England Nuclear Co. was specified to be 98-99% pure. Focussing for 16-20 h may have provided the time for the radioactive impurity to separate from the free [3H]mianserin binding in the pH 5 fraction. Protein assay of the fractions eluted from the isoelectric focussing columns indicated a 14-fold enrichment of [3H]mianserin binding in the pH 5 fraction. This is probably an underestimate because ampholytes which are mixtures of amino acids and small chain peptides cannot be completely dialyzed and may interfere with protein determination. The competition curves for the pH 5 fraction are shown in fig, 4. Upon purification, chlorpyramine, a histamine H~-antagonist, potently displaced [3H]mianserin with a K i value of 1.42 nM while the K i of histamine decreased from over 100000 nM in the soluble to 20000 nM (table 2). Although serotonin still showed biphasic displacement, the first phase which constituted about 45% of specific binding in the membrane preparation, now only constituted about 29% in the partially purified material. By comparing the histamine and serotonin competition curves of solubilized (Chan and Madras, 1982) and purified (fig. 4), it is apparent that isoelectric focussing has differentially

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Fig. 3. Isoelectric focussing of solubilized canine frontal cortex prelabelled with 4 n M [3H]mianserin for 16-20 h revealed the presence of 2 peaks of radioactivity at pH 5 and 7.81. Elution profile after passing 0.3 ml of the pH 5 and p H 7.81 fraction through a column of Sephadex G-50 is shown here. The exclusion limit of Sephadex G-50 for globular protein is about 30000 daltons. Thus, the p H 5 fraction contains molecules with molecular weight greater than 30000 daltons that bind [3H]mianserin: whereas the pH 7.81 fraction contains molecules with molecular weight less than 30000 which bind [ 3 H]mianserin and is eluted together with the free [ 3H]mianserin in fraction No. 8.

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Fig. 4. Drug inhibition of [3H]mianserin binding to canine frontal cortex after isoelectric focussing. Each point is the mean of two different experiments using individual dog brains and vertical bars represent the standard deviation. The baseline was set by 1/xM R 41468 (Ketanserin). The total binding [3H]mianserin (4 nM) was 2 100 dpm, and the non-specific binding was 1 050 dpm. The amount of protein per assay was about 25 #g.

363 TABLE 2 Drug competition for [3H]mianserin binding sites in membrane, solubilized and purified preparations. [3H]Mianserin binding was assayed in the presence of five concentrations of drugs. Each K i value is the mean of at least 2-3 experiments. K i values were determined from concentration causing 50% inhibition of [3H]mianserin (ICs 0 value) by the equation: K i = IC50/(1 + L / K D ) , where L = concentration of [3H]mianserin and K D is the dissociation constant. K i (nM)

Chlorpyramine Ketanserin Spiperone Serotonin Histamine

Membrane

Solubilized

Purified

4 30 165 3 700 100 000

2 34 615 13 000 100 000

1 37 107 20 000 20 000

enriched the histamine sites relative to serotonin sites. Purification was indicated by the appearance of fewer protein bands after polyacrylamide gel electrophoresis of the pH 5 fraction as compared to the recovery of binding and protein in the

soluble (fig. 5). It was also indicated by the tubes containing the radioactive peak as a percentage of specific binding applied. Solubilized sites bound 91.59 f m o l / m g protein while purified sites (pooled from peak fractions) bound 13.02 f m o l / m g protein. The purification factor was calculated to be 14-fold. 3.2. Gel filtration of soluble frontal cortex on sephacryl S-300 column

Gel Filtration of soluble frontal cortex prelabelled with 4 nM [3H]mianserin resulted in bound [3H]mianserin being separated from free [3H]mianserin as a single peak which was displacable by 1 /xM ketanserin (not shown). The fractionation range of Sephacryl S-300 is very wide, ranging from 10000 to 1-2 × 10 6 daltons. [3H]Mianserin binding sites eluted at a volume corresponding to a molecular weight of 1.1 × l 0 6 daltons. This is, however, an approximation as the [3H]mianserin sites are probably in a digitonin micelle which would result in over-estimation of the molecular weight. A 5-fold purification was achieved. This experiment was done once. In the future, gel filtration on Sephacryl S-300 and isoelectric focussing will be performed in sequence to attempt a higher purification than is possible with either technique alone.

4. Discussion

A

B

C

D

E

F

G

H

Fig. 5. Polyacrylamide gel electrophoresis in SDS of digitonin solubilized canine frontal cortex in non-denaturing conditions (B); denaturing conditions (C). Digitonin solubilized canine frontal cortex prelabelled with 4 nM [3H]mianserin was subjected to purification by isoelectric focussing. Polyacrylamide gel electrophoresis of the purified fraction (pI 5) is indicated by (E) (F) (G) and (H). (E) was not very well destained. (A) and (D) are standards. The protein used for calibration are from bottom to top- lactate dehydrogenase (36000), catalase (60000), albumin (67000), ferritin (half unit, 220000) and thyroglobulin (330 000).

[3H]Mianserin binding sites were partially purified by isoelectric focussing. Bound [3H]mianserin separated from free [3H]mianserin as a single peak of radioactivity with a pI value of 5 and 14-fold purification. This peak is probably heterogeneous and consists of membrane proteins which are usually acidic. Both the D2-receptor and the fl-adrenergic receptor have pI values similar to the [3 H]mianserin one (Madras et al., Davis et al., in preparation; Fraser and Venter, 1980). When this procedure was repeated in the presence of 1/~M ketanserin (R 41468; a drug which is active at both serotonin S2- and histamine Hi-receptors ), 1 /xM chlorpyramine (a histamine Hi-antagonist ), or 10 /~M spiperone (a serotonin S2-antagonist),

364 the radioactive peak at p H 5 was completely abolished. More detailed characterization indicated that the partially purified material was both histaminergic and serotonergic in nature. The affinity (K~) of histamine for partially purified sites (20/~M) was greater than its affinity for the membrane or soluble (100 /zM) preparations. It still m a y be underestimated because sodium decreases the affinity of histamine at Hi-receptors (Gavish et al., 1979; Tran et al., 1978; Toll et al., 1980; Chang and Snyder, 1980). The K~ values of histamine and serotonin are equal in the purified material, while serotonin was more potent than histamine in the membrane and solubilized preparations. Ketanserin which binds to both histamine Hi- and serotonin S2-receptors with high affinity, displayed the same K~ value in the membrane, solubilized and isoelectric focussing purified material. Spiperone, a serotonin S2antagonist, displaced [3H]mianserin with a shallow slope in both the membrane and soluble preparations but showed a sharp displacement curve with a Kj of 107 nM in the partially purified material. In fact, the K i of spiperone was lowest in the purified material compared with values detected in the membrane and solubilized preparation. The binding of chlorpyramine, a histamine H~antagonist, was undiminished in the purified preparation. Thus, either the isoelectric focussing procedure purified a single [3H]mianserin binding site or alternately, and more likely, the physiocochemical characteristics of the sites (S 2 and H~, Peroutka and Snyder, 1981) are so similar that they are inseparable with these techniques. If we assume that these sites co-purify, then isoelectric focussing probably differentially enriched the amount of histamine Hi-sites relative to serotonin S2-sites. The high affinity phase of the serotonin competition curve represented about 29% of the specific [3H]mianserin binding to purified sites while in the membrane it represented about 45% of the binding. This differential enrichment may be a consequence of greater H~-receptor stability a n d / o r retention of the [3H]mianserin label during isoelectric focussing. Other receptors have been partially purified by isoelectric focussing, including the fl-adrenergic (Fraser and Venter, 1980) and dopamine (D2-type) (Madras et al., 1981; Davis et

al., 1981; Lilly et al., 1981). The pI value for these receptors was in the range of 5, indicating that these neurotransmitter receptors are acidic. Isoelectric focussing thus constitutes a rapid method for partial enrichment (15-20 fold) of membrane receptors. Its value as a technique for separating individual receptors is yet unproven. The present work represents the first attempt at enriching simultaneously the S2- and H~-binding sites, both of which may be responsible for the antidepressant action of mianserin. The data here indicates that the serotonergic and histaminergic components of [3H]mianserin binding to canine frontal cortex are not separable by solubilization, isoelectric focussing or gel filtration on Sephacryl S-300. Preparation of monoclonal antibodies to the purified material is currently underway. Such antibodies will be useful for probing structural homology between histamine H~- and serotonin S2-receptors and other antidepressant binding sites.

Acknowledgements We thank Dr. S.W. Tang, Anne Dumbrille-Ross and Dr. P. Seeman for useful discussions. Ms. Betty Chan was a recipient of an MRC studentship. Supported by grants from the Banting Institute, Bickell Foundation and Ontario Mental Health Foundation.

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