For immunocytochemistry, the primary antibodies used were a rabbit anti-enhanced green fluorescent protein (EGFP) from Invitrogen (Carlsbad, CA; used at a dilution of 1:500), a rat anti-hemagglutinin (HA) mAb and a mouse anti-Myc mAb (both used at a dilution of 1:400; Roche Applied Science, Mannheim, Germany). Western blots were probed with a mouse anti-RhoA (1:250; Santa Cruz Biotechnology, Santa Cruz, CA) and a mouse anti-PTP1B was used for immunoprecipitations (1:50; BD Transduction Laboratories, Lexington, KY). The goat anti-rabbit Cy2 (1:1000), goat anti-rat Cy3 (1:500), goat anti-mouse Cy3 (1:1000) and donkey anti-mouse-HRP (1:5000) secondary antibodies were all obtained from Jackson Immuno Research (West Grove, USA).
NGF from mouse salivary glands was obtained from Alomone Labs (Jerusalem, Israel), and it was used at a concentration of 100 ng/ml. Amyloid β (1-42) was obtained from NeoMPS (Strasbourg, France) and it was used at a concentration of 5 μM. This peptide was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol to obtain the oligomeric form of Amyloid β. After the solvent was allowed to evaporate for 2 hrs at room temperature, the peptide film was dissolved in DMSO, sonicated in a water bath for 10 min diluted to 100 μM in PBS and briefly vortexed, before it was incubated overnight at 4 °C. Aliquots were stored at -20°C. C3 ADP ribosyl transferase (Cytoskeleton Inc., Denver, CO), a cell permeable Rho inhibitor, was used at 500 ng/ml. CNFy (cytotoxic necrotizing factor from Yersinia pseudotuberculosis), a specific activator of RhoA, was produced as described previously  and used at 200 ng/ml. Raytide™EL, a general tyrosine kinase peptide substrate, and TAT-Pep5, a cell permeable p75NTR signalling inhibitor were used at 1.0 μM, each purchased from Calbiochem (Darmstadt, Germany). [γ-32P]ATP was obtained from Perkin-Elmer (Madrid, Spain).
The EGFP expressing vector (pEGFP-N1) was obtained from Clontech Laboratories, Inc. (Palo Alto, CA), while the pCDNA 3.1 Zeo encoding a HA-tagged form of wild type (wt) PTP1B was kindly provided by Carlos Arregui (Buenos Aires, Argentina) . The pRK5-Myc vector encoding a Myc-tagged dominant negative form of RhoA, RhoA N19 (Addgene plasmid 15901) , was obtained from Addgene (Cambridge, MA).
Primary hippocampal neuron cultures were prepared as described previously , with some minor modifications. Briefly, the hippocampus was removed from E17 CD1 mouse foetuses and dissociated into single cells following trypsin (Worthington, Lakewood, USA) and DNase I digestion (Roche Applied Science). Neurons were plated on glass coverslips or in plastic dishes coated with poly-L-lysine (Sigma-Aldrich, Madrid, Spain), and cultured in Neurobasal A supplemented with 2 mM GlutaMAX I, 100 units/ml penicillin and 100 μg/ml streptomycin (Gibco BRL, Crewe, UK). After 7 days in vitro (DIV) the neurons were exposed to Aβ, NGF, and/or the pharmacological agents indicated. TrkA-deficient PC12 cells, PC12nnr5 , were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% heat-inactivated foetal bovine serum (FBS), 10% heat-inactivated horse serum (Sigma-Aldrich), 2 mM GlutaMAX I, 100 units/ml penicillin and 100 μg/ml streptomycin (Gibco BRL). All cultures were kept at 37°C in a humidified atmosphere containing 5% CO2.
Cultured neurons were transfected at 7 DIV with different vectors using the Effectene Transfection Reagent (Qiagen GmbH, Hilden, Germany) according to a modified version of the manufacturer's instructions. Briefly, 0.6 μg of DNA was added to 120 μl of the EC buffer and 3.5 μl of the Enhancer for each 35 mm cell culture dish of hippocampal neurons. The solution was incubated for 5 min at room temperature before 10 μl of Effectene was added, and after a further 15 min incubation at room temperature, the final solution was added to hippocampal neurons. The medium was then changed after 3 h.
Immunocytochemistry, image acquisition and the morphometric analysis of labelled hippocampal neurons
At 16 h after transfection, the neurons were fixed for 30 min in 4% paraformaldehyde (PFA) prepared in PBS, they were then permeabilized for 15 min at room temperature with 0.5% Triton X-100 in PBS and blocked with 10% FBS in PBS containing 0.1% Triton X-100. The cells were incubated with the primary and secondary antibodies and the images of 10-20 neurons per coverslip were acquired digitally using a 63× oil immersion objective (Zeiss, Oberkochen, Germany). To analyze the dendrites, a region of interest (ROI) with a radius of 50 μm was projected onto EGFP-labelled neurons, its centre roughly coinciding with the centre of the soma. The dendrite length was expressed as the fraction of the dendritic tree that exceeds the limit of the ROI (fraction dendrites >50 μm). In co-transfection experiments, only double-labelled cells were analysed, which represented more than 90% of the single-labelled cells.
After treatment, the neurons were fixed for 30 min in 4% paraformaldehyde (PFA) in PBS and their nucleus was stained with the fluorescent dye, 4',6-diamidino-2-phenylindole (DAPI: Sigma-Aldrich). Non-viable neurons were recognized by nuclear condensation and/or the fragmentation of their chromatin. The number of viable neurons was counted in triplicate from ca. 50 fields in two independent experiments. In co-transfection experiments, only the nuclei of double-labelled cells were analysed.
To assay RhoA activation we followed a procedure described elsewhere . Briefly, stimulated PC12nnr5 cells were first lysed in buffer: 50 mM Tris [pH 7.5], 500 mM NaCl, 10 mM MgCl2, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS and a protease inhibitor cocktail (Roche Applied Science, Mannheim, Germany). The cleared lysates were incubated for 1 h at 4°C with Rhotekin-conjugated agarose beads (Cytoskeleton), and the beads were then collected by centrifugation and washed with the lysis buffer. Activated RhoA was detached from the beads by boiling for 5 minutes in Laemmli reducing buffer, after which it was immediately resolved by 12% SDS-PAGE and transferred to a nitrocellulose membrane. After blocking, the membranes were probed overnight at 4°C with a primary antibody directed against RhoA, which was detected with an HRP-conjugated secondary antibody that was visualised by enhanced chemiluminescence (GE-Healthcare, Piscataway, NJ). The intensity of the bands was evaluated by densitometry using ImageQuant software (GE-Healthcare).
Protein phosphatase 1B assay
The PTP1B assay was performed essentially as described previously , with minor modifications. Briefly, 7DIV cultured hippocampal neurons (1 × 106 cells) were collected and homogenized in RIPA buffer (50 mM Tris [pH7.5], 150 mM NaCl, 2 mM EDTA, 1.0% Triton X-100 and an anti-protease cocktail). Equal amounts of protein were incubated for 2 h at 4°C with a mouse anti-PTP1B mAb, and then 20 μl of protein G sepharose was added and incubated for additional 2 h with agitation. Immunoprecipitated complexes were washed twice in RIPA buffer, once with the assay buffer (25 mM imidazole [pH7.2], 0.1 mg/ml BSA, 10 mM DTT) and they were then resuspended in 25 μl of assay buffer. The phosphatase substrate Raytide was labelled at its unique tyrosine residue with [γ-32P]ATP as described previously . Assay mixtures (30 μl) containing the immunoprecipitated pellet and [32P]-labelled raytide (1 × 105 cpm) were incubated for 2 h at 30°C and the reaction was terminated by adding 750 μl of a charcoal mixture (0.9 M HCl, 90 mM sodium pyrophosphate, 2 mM NaH2PO4, 4% vol/vol Norit A). After centrifugation, the radioactivity in 400 μl of the supernatant was measured by scintillation counting. Blanks were determined by measuring the free [32P]phosphate in reactions where the immunoprecipitates were either boiled or omitted, and these values were subtracted from the reaction values.
Quantitative real time polymerase chain reaction (PCR)
After treatment, the total RNA was extracted from cultures at 7 DIV using the Illustra RNAspin Mini kit (GE-Healthcare) and first strand cDNA was prepared from the RNA using the First Strand Synthesis kit (Fermentas GmbH, St Leon-Rot, Germany). Quantitative PCR was performed using the ABI Prism 7000 Sequence Detector (Applied Biosystems, Weiterstadt, Germany) and TaqMan probes. Primers for Hes1 and the housekeeping gene GADPH (as a control) were selected as the Assay-on-Demand gene expression products (Applied Biosystems). All TaqMan probes were labelled with 6-carboxy fluorescein (FAM) and real time PCR was performed using the TaqMan Universal PCR Master Mix according to the manufacturers' instructions. Hes1 expression was normalized to the GAPDH expression.
Data are presented as the mean ± SEM and an unpaired Student's t-test was applied to determine the levels of significance, denoted as *p < 0.05, **p < 0.01, ***p < 0.001. All experiments were repeated at least twice.