The JAK/STAT Pathway Is Involved in Synaptic Plasticity

Summary The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway is involved in many cellular processes, including cell growth and differentiation, immune functions and cancer. It is activated by various cytokines, growth factors, and protein tyrosine kinases (PTKs) and regulates the transcription of many genes. Of the four JAK isoforms and seven STAT isoforms known, JAK2 and STAT3 are highly expressed in the brain where they are present in the postsynaptic density (PSD). Here, we demonstrate a new neuronal function for the JAK/STAT pathway. Using a variety of complementary approaches, we show that the JAK/STAT pathway plays an essential role in the induction of NMDA-receptor dependent long-term depression (NMDAR-LTD) in the hippocampus. Therefore, in addition to established roles in cytokine signaling, the JAK/STAT pathway is involved in synaptic plasticity in the brain.


Preparation of slices
The experiments on acute slices were performed on 400 µm thick parasagittal hippocampal slices obtained from juvenile (13 -17 day old) Wistar rats. Hippocampal organotypic slices were prepared from 8 day old Wistar rats, as described previously (Bortolotto et al., 2011;Jo et al., 2010). Procedures involving animals and their care were conducted in conformity with the institutional guidelines that are in compliance with national (UK animals (Scientific Procedures) Act 1986 andD.L.n.116, G.U., Suppl. 40, 1992) and international laws and policies (EEC Council Directive 86/609, OJ L 358, 1, 12 December 1987; Guide for the Care and Use of Laboratory Animals, U.S. National Council, 1996).
Individual responses were displayed and the average field EPSP amplitude of four successive responses were plotted on-line using WinLTP software (Anderson and Collingridge, 2007) and normalized to baseline. The NMDAR-LTD was induced by delivering 900 shocks at 1 Hz (LFS) and LTP was induced by delivering 100 shocks at 100 Hz, both at test intensity. KCl, 3; NaHCO 3 , 26; NaH 2 PO 4 , 1.4; CaCl 2 , 4; MgSO 4 , 4; glucose, 10; picrotoxin, 50µM and 2-chloroadenosine, 1 µM, bubbled with O 2 :CO 2 : 95:5%. Two stimulating electrodes (test and control input) were placed in the Schaffer collateral-commissural pathway and stimulated at 0.05 Hz to record AMPAR EPSCs (V h = -70 mV). To measure NMDAR EPSCs, neurons were held at +40 mV and the EPSC amplitude was measured 60 ms following the stimulus. NMDAR-LTD was induced using a pairing protocol (1 Hz for 6 min, V h = -40 mV). Access resistance was monitored constantly and neurons were discarded if this varied by more than 20% during the recording period.

Stimulation and western blot
For chemically-induced LTD, whole hippocampal slices (without CA3) were treated with either 20 µM NMDA for 3 min, 100 µM DHPG for 10 min or 50 µM carbachol for 10 min.
For LFS-induced LTD, hippocampal slices were stimulated (900 stimulations at 1 Hz) with two electrodes placed in the Schaffer collateral-commissural fibres. The stratum radiatum (SR) surrounding the stimulating electrodes, enriched in CA1 dendrites, and the stratum pyramidale (SP), enriched in CA1 cell bodies, were then microdissected within the next 10 min and washed in a cold buffer containing (mM): Tris, 20; NaCl, 150; EDTA, 5; EGTA, 1; NaF, 5 and Na 3 VO 4 , 1. The SR was lysed in the same buffer with 1% protease inhibitor (Roche Products, Welwyn Garden City, UK) and 1% Triton X-100 added. The SP was lysed in a sucrose buffer containing 11% sucrose, 10 mM HEPES, 5 mM NaF, 1% phosphatase inhibitor mixture 2 (Sigma-aldrich, St. Louis, MO) and 1% protease inhibitor. The lysates were homogenized with a pellet pestle and rotated for 30-40 min at 4°C. The SR was then centrifuged at 1,000 x g for 10 min and the supernatant was kept. The SP was centrifuged at 800 x g for 10 min and the pellet was washed once and passed through a 25G needle before a last centrifugation at 800 x g. Twenty µg of protein from these samples were denaturated at 95ºC for 5 min in a standard denaturating buffer, separated with SDS-PAGE and transferred onto a PVDF membrane The intensity of the bands was quantified using WCIF ImageJ (NIH) software. For each experiment, the ratio of phosphorylated protein over the total amount of protein was calculated in each condition and compared relative to the ratio obtained in control condition. Results are presented as mean ± SEM. A paired Student's t-test was then performed. A value of p < 0.05 was considered significant.

Subcellular fractionation
Rat hippocampi were Dounce homogenized with 0.32 M sucrose solution containing both a protease inhibitor cocktail (Roche) and a phosphatase inhibitor cocktail I, II (Sigma). The homogenized suspension was centrifuged twice at 1,400 x g for 10 min at 4 °C to remove intact cells and large cellular organelles. Note that the pellet from the first spin was resuspended in the sucrose buffer and pelleted again. The obtained supernatants were combined and centrifuged at 10,000 x g for 20 min at 4 °C resulting in a supernatant and a second pellet (P2). The P2 pellet was resuspended in 0.32 M sucrose solution and layered over a sucrose step gradient (0.85/1.0/1.2M). The tube was centrifuged at 82,500 x g for 2 h. The fraction between 1.0 M and the 1.2 M sucrose layers was collected and centrifuged at 17,000 x g to pellet the synaptosomal fraction.
The pellet (LP1 fraction) was then further solubilized in a modified RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 2 % NP-40 alternative (Sigma), 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA with a protease inhibitor cocktail) for 30 min rotating at 4 C and pelleted again to remove the remaining insoluble protein and lipid aggregates from the supernatant. Approximately 40 g of each sample was resolved by SDS-PAGE and the membrane probed for PSD-95 (Affinity BioReagents; 1:1,000) and JAK2 (1:500) overnight at 4°C. To test the efficiency of the shRNAs and for the experiments with Stattic, hippocampi were dissected and dissociated from 2 day old Wistar rats.

Hippocampal cell cultures
Transfection of the cells with the shRNAs was performed at DIV 4-6 using lipofectamine 2000 according to the manufacturer's protocol and the cells were fixed 2-3 days later.
Pharmacological treatment with D-Serine and NMDA was performed as described above, 4-8 days after dissociation, on cells incubated with either control vehicle DMSO or Stattic (50 µM) for 20-30 min. Cells were then washed and lysed in a standard lysis buffer as described previously.

JAK2 and STAT3 knockdown
Neurons were fixed with 4% PFA, washed with PBS and blocked with donkey serum as described previously. Neurons were then incubated with either STAT3 anbibody (as above) or JAK2 antibody (1:200, ab39636, abcam, Cambridge, MA) in 1% NDS and 0.1% triton. The secondary antibodies used were cyanine 3-conjugated donkey antirabbit (1:500; Jackson ImmunoResearch, West Grove, PA) or Alexa-594 donkey antimouse (1:1000, Invitrogen, Carlsbad, CA). Confocal images of transfected neurons were obtained with sequential acquisition on a Leica AOBS SP2 confocal imaging system attached to Leica DMIRE2 inverted microscope. Each image is a z-series of 10 images each averaged 4 times. The resulting z-stack was 'flattened' into a single image using maximum projection.

HEK cells and transfection
HEK293 cells were maintained in Dulbecco's Modified Eagle's medium supplemented 8 with 10% dialysed horse serum and 2 mM l-glutamine in a humidified incubator at 37 °C with 5% CO2. They were transfected with a pcDNA3-rJAK2(FL)-HA plasmid and the different shRNAs, using an Amaxa Nucleofector Kit V according to the manufacturer's instructions, after which 0.5 x 10 6 per well of transfected cells were plated on 6-well plates previously coated with poly-L-lysine and collagen. The cells were lysed in a standard lysis buffer, 72h after transfection, and the levels of JAK2 and GAPDH were analysed by western-blot.