SH-SY5Y Naïve and 293HEK cells were maintained in DMEM medium (Gibco) with 10% FBS (Gibco) and penicillin streptomycin. SH-SY5Y APP-Gal-4 cells were maintained in the same media supplemented with 200 mg/ml of G418 (Gibco).

We also obtained stable cell populations expressing farnesylated GFP (GFP-F), TrkB T and TrkB FL both GFP tagged. Farnesylation on the GFP targets the GFP to the cell membrane making it a better control for surface receptors. Transfected cells were selected with 400 mg/ ml G418 from 48 hours after transfection up to one month. Liquid nitrogen stocks were obtained. Expression of the constructs was verified by fluorescence microscopy and Western blot.

For ADAM activity assays, cells were treated with 10 mM in 0.5% DMSO of the ADAM10 agonist PMA (phorbol 12-myristate 13-acetate, Sigma) or DMSO for 10 hours. We also treated cells with 20 mM of all transretinoic acid (ATRA or RA, Sigma) in 0.5% DMSO or 0.5% DMSO alone, for 48 hours prior to collection.

Cell lysates were collected in extraction buffer provided with the R&D Systems ADAM activity kit and centrifuged at 4˚C. The supernatant was saved and combined with reaction buffer and substrate in a 96-well plate. The plate was incubated at 37˚C in the dark for 2 hours and then placed in a Tecan Infinite 200 plate reader controlled by the commercial software Magellan. An excitation wavelength of 355 nm and an absorption wavelength of 510 nm were used, the gain was automatically optimized and the integration time was set at 80 msec. Each well was read only once. As a positive control we used lysates from PMA treated cells. Each lysate was run in duplicates and readings were averaged. Background readings from wells containing reagents and substrate were divided by the experimental values and positive controls so that the ADAM10 activity is expressed in fold increase over background. The experiment was repeated twice independently for the cell lines and three times independently for the stable population of HEK293 cells transfected with TrkB constructs or GFP-F controls. Statistical analysis was conducted using the student t-test Bonferroni corrected for multiple comparison when applicable.

Cells were collected 48 hours after all transfection procedures. Whole cell lysates were prepared by lysing cells in the plate with ice-cold radio immuno-precipitation buffer (150 mM NaCl, 1% NP40, 0.5% DOC, 1% SDS, 50 mM Tris, pH 8.0) supplemented with Halt cocktail of protease and phosphatase inhibitors (ThermoFisher). Cell lysates were sonicated in an ice-cold water bath sonicator for 6 minutes then centrifuged 20 minutes at 4˚C at 14,000 rpm. The resulting supernatants were collected and protein concentration measured with the BCA protein concentration kit (Pierce) according to manufacturer’s instructions. Western blot samples were prepared at a final concentration of 1 - 2 mg/ml in 4X reducing loading buffer (InVitrogen) and heated at 70˚C for 10 minutes. 15 - 25 mg of total protein/well from the cell lysates were separated on 4% - 12% Tris-Glycine midi gels (InVitrogen) in MES-SDS running buffer (InVitrogen) and run at 190 mVolts for 45 minutes. The separated proteins were transferred to PVDF FL membranes (Millipore) using a Semi-Dry transfer apparatus (AA Hoefer TE77X) for 3 hours at 125 mAmp per gel. Membranes were blocked one hour at room temperature using Licor blocking buffer then probed overnight with primary antibodies diluted in Licor blocking buffer at 4˚C - 25˚C. Membranes were then washed for 5 minutes 4 times with 0.1% Tween (Sigma) in PBS. After washing, membranes were incubated in the dark with the appropriate seconddary antibody IRDye (Licor) diluted in Licor blocking buffer for one hour. Again membranes were washed as above and finally rinsed with PBS. Membranes were scanned on an Odyssey InfraRed scanner (Licor) at appropriate intensities and images acquired at 159 mm resolution. Band intensities were quantified with the provided built-in software (Licor) and always normalized to the actin loading control.

APP was detected using a rabbit antibody A8717 (Sigma) at 1:2000 dilution; TrkB GFP tagged proteins were detected using a mouse GFP antibody (Clontech) at 1:500 dilution; TrkB was also detected with a pan-TrkB rabbit antibody (Santa Cruz Biotechnology); BACE1 was detected using a rabbit antibody ab2077 (Abcam) at 1:1000 dilution; ADAM10 was detected using a rabbit antibody (Chemicon); actin was detected using a mouse antibody (Sigma) at 1:15000 dilution. Secondary IRDye antibodies (anti rabbit IR800 and anti mouse IR700) were purchased from Licor and used at 1:15000 dilution.

Cells were transfected at approximately 50% - 60% confluency in 96-well plates using Arrest-In transfection reagent (ThermoScientific) according to manufacturer’s protocol. For the reporter experiments TrkB plasmids, the GFP-F (negative control, Clontech) and the NFAT3- GFP plasmids (positive control, a gift of Dr. Chris Norris) were co-transfected with the NFAT luciferase reporter (Promega) or the pGL3 luciferase reporter (Promega). Experimental or control plasmids were transfected in 1:1 molar ratio with the luciferase reporter plasmids. In all transfections a renilla luciferase reporter (pRLSV40 or pRLTK, Promega) was used as a transfection normalizetion control at a molar ratio of 1:40 to the luciferase plasmid.

Cells were collected in Dual Glo Lysis Buffer (E2920, Promega) 48 hours post transfection and the assay was carried out as per manufacturer’s instructions. Plates were read in a plate reader. Luciferase signal was normalized to renilla luciferase levels and data was analyzed using t-tests with Bonferroni correction for multiple comparisons.

TrkB FL has been reported to increase transcription of the APP promoter via IP3K and Akt mediated pathways in SH-SY5Y cells [19]. These cells express basal levels of the TrkB receptors and BDNF, which is the natural ligand for TrkB, and therefore can activate the receptor through the canonical pathway. It is known that TrkB receptors can also autoactivate in absence of BDNF [21, 22]. Thus, we hypothesized that, in both HEK293 naive cells, which do not express BDNF or TrkB and SHSY5Y naive cells, TrkB FL transfection increases APP transcription. In fact, the signaling pathways triggered by TrkB FL engage molecules that are ubiquitously expressed in all cell lines.

We transiently transfected plasmids encoding farnesylated GFP (GFP-F), TrkB T and TrkB FL into HEK293 cells and measured APP levels 48 hours post-transfection.

We found that TrkB FL transfection significantly increased APP levels compared to GFP-F or TrkB T transfection (Figures 1(a) and (c)).

The extent of the increase, though, was modest. We then measured APP in a stable population of cells that had been selected for TrkB or GFP-F expression with antibiotic treatment for at least one month. We again found that cells expressing TrkB FL displayed higher levels of APP compared to GFP-F and TrkB T expressing cells (Figure 1(b)).

We transiently transfected TrkB T, TrkB FL and the GFP-F plasmid control in SH-SY5Y naïve cells and found similar results (Figures 2(a) and (b)).

We conclude that TrkB FL expression increases endogenous, full-length APP levels independent of BDNF while TrkB T does not.

TrkB FL has been shown to increase APP promoter transcription but also to increase sAPP-a levels in the media of SH-SY5Y cells [16]. We hypothesized that the increase in sAPP-a might be due to TrkB FL mediated PLC-g activation, since PLC-g produces diacylglycerol (DAG), an ADAM activity stimulator. Therefore we employed an ADAM activity assay to measure ADAM activity in cells that had been transfected with TrkB T or FL.

We initially checked for the assay reliability and the baseline ADAM activity of three different cell lines. A neuroblastoma cell line SH-SY5Y and the same cell line stably expressing APP fused to Gal4. We also used HEK293 cells. We collected lysates of cells treated with vehicle dimethyl sulfoxide (DMSO) or phorbol myristate acetate (PMA), a phorbol ester that mimics DAG activity.

SH-SY5Y cells showed a higher ADAM basal activity compared to HEK293 cells. PMA was able to increase the ADAM activity signal in both cell lines (Figure 3 (a)).

SH-SY5Y cells have been shown to display accumulation of sAPP-a when treated with retinoic acid [16]. The ADAM10 promoter contains retinoic acid responsive elements (RARE), so we hypothesized that this increase sAPP-a release is mediated by an increased transcription and maybe activity of ADAM10 in this cell line. To verify this, we treated SH-SY5Y cells for 48 hours with 20 mM RA or DMSO and measured ADAM activity in the cell lysates. We also treated cells for 10 hours with PMA as a positive control. Both RA and PMA treatments resulted in a detectable increase in ADAM10 activity, the increase induced by PMA was higher (Figure 3(b)). To verify that the increase in ADAM activity observed after RA treatment, was due to an increase in ADAM levels, we performed a Western blot analysis on the same cell lysates used for the ADAM activity assay. We found that there was an increase in ADAM10 levels in the cells trea-

ted with RA compared to the cells treated with vehicle only (DMSO) (Figures 3(c) and (d)).

These experiments show that the ADAM assay is able to detect differences in ADAM activity and validate its use in our in vitro system. They also show that RA causes increased ADAM activity by up-regulating the protein levels.

We then applied this assay to test the hypothesis that TrkB FL activates ADAM independently of BDNF. We obtained populations of HEK293 cells stably transfected with a GFP-F, TrkB T or TrkB FL. We measured ADAM activity of the cells stably transfected with the different constructs and compared them (Figure 4). There was no statistical difference between the ADAM activity levels detected in TrkB FL or TrkB T transfected cells compared to the GFP-F transfected cells (Figure 4).

We did not observe altered ADAM activity when transfecting TrkB FL or TrkB T in HEK293 cells. ADAMs are responsible for a-secretase cleavage of APP. Because we observed an increased APP processing upon TrkB FL transfection [20], we hypothesized that BACE1, responsible for b-secretase cleavage of APP, levels might be

affected upon TrkB transfection. To test this hypothesis, we measured BACE1 levels in cells transfected with the TrkB isoforms. We also employed TrkB FL mutants to identify the important functional domain mediating an effect on BACE1 (Figure 5(a)).

We found that TrkB FL significantly increased BACE1 levels compared to TrkB T. Transfection of the TrkB SHC isoform yielded BACE1 levels similar to TrkB FL. TrkB FL Y515F, mutated on the SHC binding site, decreased BACE1 levels compared to TrkB FL, suggesting a role of SHC binding to TrkB in mediating the effect. The tyrosine kinase inactive mutant, K571M, did not alter BACE1 levels compared to TrkB FL (Figure 5 (b)) suggesting that receptor phosphorylation is not required.

We observed an increase in BACE1 levels upon TrkB FL transfection. BACE1 levels can be modulated by the Ca²⁺ activated transcription factor NFAT1 [25]. Because TrkB FL can activate NFAT3, the neuronal specific form of NFAT, we hypothesized that TrkB FL might mediate activation of NFAT3 and thus increase BACE1 transcription. To test whether TrkB FL transfection was able to activate NFAT3, we transfected a NFAT luciferase reporter construct together with TrkB FL or TrkB T. We used a GFP tagged NFAT3 over-expression construct as a positive control and a renilla luciferase reporter to normalize transfection efficiency. In both SH-SY5Y naïve and HEK293 naïve cells, we found that TrkB FL transfection increased NFAT3 mediated luciferase activity compared to TrkB T and GFP-F control but not compared to the NFAT3 over-expression construct (Figures 6 (a) and (b)).

TrkB FL can activate signaling pathways that increase release of Ca²⁺ from intracellular storage, therefore the

effects that we observe might be due to Ca²⁺ mediated unspecific increase in transcription. To rule out this possibility we co-transfected the TrkB constructs, the control constructs and the NFAT3 construct together with a pGL3 luciferase reporter that does not respond to NFAT specific activation but would be subjected to general increases in transcription activity within the cells. We did not observe any differences in luciferase activity between TrkB T and TrkB FL when utilizing pGL3 as a reporter construct (Figures 6(c) and (d)) in both cell lines. This demonstrates that TrkB transcriptional effects are specific to NFAT. The increase in luciferase activity observed when transfecting NFAT3 and pGL3 luciferase is due to NFAT binding to the SV40 promoter, enhancing its transcription [26].