Deconstruction of Neurotrypsin Reveals a Multi-factorially Regulated Activity Affecting Myotube Formation and Neuronal Excitability

Molecular Cloning

Recombinant constructs for NT-mini (Gly497-Leu875) and AGR-46 (Pro1635-Pro2068) agrin-like substrates were amplified using a Phusion DNA polymerase (Thermo Fisher Scientific) starting from a source synthetic gene (GeneWiz) corresponding the full-length human NT cDNA (UniProt P56730-1) and from a human agrin y0z0 cDNA (UniProt O00468-6) obtained from Source Biosciences, respectively, using the oligonucleotides listed in Sup. Table 1.

The resulting PCR products were subcloned into pCR4-TOPO vectors (Thermo Fisher Scientific) using BamHI and NotI restriction sites. These plasmids served as the basis for the generation of the inactivated Ser-825-Ala NT*-mini and of the various AGR-46 agrin splice variant combinations via site-directed mutagenesis. The corresponding PCR reactions were performed with Phusion DNA polymerase (Thermo Fisher Scientific) using the oligonucleotides listed in Sup. Table 1.

The longer agrin AGR-124 construct was designed as a codon-optimized cDNA synthetic gene (GeneWiz) for mammalian expression, spanning human agrin y4z19 (Uniprot O00468-1) FS9-LG3 domains (Ala893-Pro2068).

All constructs were verified using Sanger sequencing (Microsynth) and then were subcloned into modified pUPE.106.08 plasmids (U-Protein Express BV) for secreted protein expression in mammalian cell culture systems. For NT-mini, the final expression constructs contained an N-terminal 8xHis-SUMO tag used to stimulate expression and facilitate purification. For agrin constructs, the final expression constructs contained an N-terminal 6xHis-tag followed by a tobacco etch virus (TEV) protease cleavage site.

Recombinant Protein Production

All NT and agrin constructs were produced via transient transfection following the protocols described in [28], but using SFM-HEK293 (Expression Systems) cell suspension cultures adapted to grow in a serum-free medium (ESF, Expression Systems). Transfections were performed using linear polyethylenimine (PEI MAX, Polysciences) in a 5:1 (w:w) PEI:DNA ratio. Zymogen-to-enzyme activation of NT-mini constructs was performed during protein production by co-transfecting the NT-mini plasmid in a 1:5 ratio with a pUPE expression plasmid for mammalian expression (U-Protein Express BV) carrying the human cDNA of the pro-protein convertase Furin. Four hours post-transfection, the culture was supplemented with peptone supplement (Primatone RL, Sigma-Aldrich) at a final concentration of 0.6% (w/v). Transfected cultures were maintained for 6 days to allow recombinant protein expression and accumulation. On the 7th day, the growth media was harvested by centrifugation. The cells were discarded and the recombinant proteins were purified from the supernatant using liquid chromatography.

Recombinant Protein Purification

Cell media were adjusted with concentrated buffer to a final 200 mM NaCl, 25 mM HEPES/NaOH, and pH 8 and filtered through a 0.45-µm syringe filter (Sarstedt) using a peristaltic pump (Thermo Fisher Scientific). All chromatographic steps were carried out using an NGC chromatography system (Biorad), and followed by monitoring UV absorbance at 280 nm. Samples from each step were further analyzed with reducing and non-reducing SDS-PAGE. Purification of NT-mini or NT*-mini was obtained with a three-step Heparin/Ni-IMAC “mixed-mode” approach and completed by size exclusion chromatography (SEC). The filtered medium was supplemented with 5 mM CaCl2 (final concentration) and loaded, at room temp, on a 20-mL Heparin column (GE Healthcare) pre-conditioned with buffer NT.A (200 mM NaCl, 5 mM CaCl2, 25 mM HEPES/NaOH, pH 8). Unbound material was washed off the column with buffer NT.A, and weakly bound contaminants were removed by increasing the NaCl concentration to 250 mM. The protein of interest (NT-mini or NT*-mini) was then eluted into a 5-mL Ni-IMAC HisTrap Excel column (GE Healthcare) by further increasing the NaCl concentration to 600 mM (buffer NT.B). Unbound material was washed off the Ni-IMAC column with buffer NT.A, after which the recombinant protein was eluted using buffer NT.C (200 mM NaCl, 5 mM CaCl2, 250 mM imidazole, 25 mM HEPES/NaOH pH 8) into a 1-mL HiTrap Heparin HP column (GE Healthcare). This column was further washed with buffer NT.A, and the bound NT construct was de-tagged by loading the column with 10 mL of a 4 µg/mL SUMO protease solution. Tag cleavage was allowed to proceed in the column overnight at 4 °C, then the cleaved tag was washed off the column with buffer NT.A. At this point, NT-mini/NT*-mini was eluted from the heparin column using buffer NT.B. The obtained material was concentrated to reach a final volume of ≤ 0.5 mL and further purified by SEC on a Superdex 200 10/300 Increase column (GE Healthcare), equilibrated with GF buffer (200 mM NaCl, 25 mM HEPES/NaOH, pH 8). Finally, the purified NT-mini/NT*-mini samples were concentrated to 1 mg/mL with a Vivaspin Turbo 10 kDa MWCO concentrator (Sartorius), flash frozen in liquid nitrogen, and stored at − 80 °C. These materials served as stock solutions for all subsequent in vitro assays and cell culture experiments.

For agrin AGR-46 constructs, the filtered medium was loaded on a 5-mL HisTrap Excel column (GE Healthcare) equilibrated with buffer AG.A (500 mM NaCl, 25 mM HEPES/NaOH, pH 8). Non-specific contaminants were removed by washing the column with buffer AG.A supplemented with 25 mM imidazole, then the elution of recombinant agrin constructs was obtained using buffer AG.B (500 mM NaCl, 250 mM imidazole, 25 mM HEPES/NaOH, pH 8). Fractions were pooled and dialyzed overnight against buffer AG.A in the presence of 25 μg/mL His-tagged TEV protease. Tag and TEV protease removal was achieved by re-loading the dialyzed sample onto the 5-mL HisTrap Excel column (GE Healthcare) and collecting the unbound tagless sample. Fractions containing pure agrin constructs as assessed by SDS-PAGE analysis underwent concentration through a Vivaspin Turbo 30 kDa MWCO concentrator (Sartorius) to reach a final volume below 0.5 mL, and further purified by SEC on a Superdex 200 10/300 Increase column (GE Healthcare) equilibrated with buffer GF. All samples eluted as single peaks distant from the column void volume. Pooled fractions were concentrated to 3–10 mg/mL, flash frozen in liquid nitrogen, and stored at − 80 °C until usage.

The purification of the longer agrin AGR-124 substrate was carried out following the protocol described for the shorter AGR-46 constructs, with slight modifications in the steps following IMAC purification. Specifically, following the O/N tag-removal dialysis step, the protein was diluted 1:1 with 25 mM HEPES/NaOH pH 8, to reduce the [NaCl] to 250 mM, before applying the tagless material onto a 1-mL HiTrap Heparin HP column (GE Healthcare). The TEV protease and purification tag were then washed off the column with buffer AG.C (200 mM NaCl, 25 mM HEPES/NaOH, pH 8), while the bound AGR-124 substrate was eluted with buffer AG.A. This protein was further purified by SEC on a Superdex 200 10/300 Increase column (GE Healthcare) equilibrated with buffer AG.A. The recovered protein was concentrated (Vivaspin Turbo 30 kDa MWCO (Sartorius)) to 1.5 mg/mL and stored until usage as described previously.

Activity Assays with Peptide Substrates

NT-mini biochemical characterization was performed spectrophotometrically, at a fixed enzyme concentration of 1 µM, with p-nitroaniline (pNa)–bearing peptide substrates (Sup. Table 2) allowing to follow product generation as an increase in absorbance at 405 nm. Initial reaction velocities (V0) extrapolated from the linear regions of those plots were used to investigate reaction parameters, compare enzymatic activities, and determine kinetics. Time-course measurements were performed at 37 °C in a clear-bottom 386-well plate (Greiner) in GF buffer. For each measurement, the final reaction volume was 20 µL. Substrate and CaCl2 stock solutions were prepared, respectively, as 100 mM and 1 M stocks in GF buffer (200 mM NaCl, 25 mM HEPES/NaOH, pH 8) and diluted as necessary. Stocks of BaCl2 and ZnCl2 for the corresponding experiments were prepared in an identical fashion as those of CaCl2. Heparin stocks were prepared as a 1 mM solution by directly dissolving heparin sodium salt (Sigma #H3393) in reaction buffer. For experiments assessing activity in relation to ionic strength, the NaCl concentration of the reaction buffer was altered to cover a 100–500 mM range; reaction parameters were otherwise unchanged from reference conditions. Reference reactions were performed using 5 mM CaCl2 and 1 mM peptide substrate, respectively. Each reaction was prepared on ice, with 1 mg/mL NT-mini as the last component, and briefly mixed by pipetting before measurement. To prevent evaporation, each sample was then overlayed with a small drop (~ 7 µL) of paraffin oil. Triplicate kinetics measurements were performed using CLARIOstar (BMG LABTECH) or Glomax Discovery (Promega) plate readers over the span of 230 min monitoring light absorbance at wavelengths of 405 and 600 nm. Measurements at 600 nm were used to exclude possible aggregation phenomena and correct baselines. Absorbance values were converted to concentration curves by accounting for path length and using the pNa extinction coefficient at 405 nm (9500 M−1 cm−1). The linear region of these plots (50 points in the 10–50 min measurement interval) was used to extrapolate initial reaction velocities (V0) via linear regression. The V0 values were directly fit to the Michaelis–Menten equation using Prism 6.01 (GraphPad), which provided values of apparent kcat and Km along with their associated errors. Propagation of statistical error value during the calculation of kcat/Km values was carried out as described [29]. Absence of proteolytic activity in NT*-mini was assessed in the same manner. The determination of statistical significance for V0 comparisons in the presence of bivalent metal ions (Fig. 3b) or heparin (Fig. 4a) was performed with a one-way ANOVA coupled to a multiple comparison test (Bonferroni) in Prism 6.01 (GraphPad).

Assays investigating the effect of pH on peptide processing were carried out as described above, but with minor modifications in sample preparation. A series of 10 × reaction buffer stock solutions (2 M NaCl + 500 mM buffering agent) were prepared to cover a range of pH values (5.0–10.5). Sodium citrate was used to cover pH 5.0, Bis–Tris was used to cover pH 6.0–6.5–7.0, HEPES/NaOH was used to cover pH 7.5–8.0, tricine was used for pH 8.5, glycine was used for pH 9.0–9.5, and CAPS covered pH 10.0–10.5. Each experiment was prepared using 2 μL 10 × reaction buffer, 1 μL of 100 mM CaCl2, and 2 μL of 10 mM β-peptide substrate. Sample volume was brought up to 19 μL with water, and 1 μL NT-mini (final conc. 1 µM) was added as the last component. Substrate and CaCl2 stocks were prepared in water as opposed to the previously indicated buffer while NT-mini stocks remained unaltered. Mixes were prepared on ice, and kinetics measurements and V0 extrapolations were performed as described above.

Experiments investigating the role of Ca2+ in NT-mini activity were carried out using the 6-mer β-peptide. Substrate concentration was fixed at 10 mM and kinetics measurements were carried out with 1 µM NT-mini in the presence of increasing [Ca2+]. For these experiments, NT-mini was pre-diluted (1:1) with a 20 mM EDTA in GF buffer solution to allow for unambiguous evaluation of activity at low [Ca2+] (≤ 500 µM). CaCl2 solutions for these experiments were prepared as 10 × stocks (with respect to the desired assay [Ca2+]) adjusted to compensate for the EDTA supplemented to NT-mini. Reaction samples were prepared as described previously with slight modifications to keep NT-mini and substrate concentrations unaltered. Typical reaction mixes (20 µL) were composed as follows: 2 µL NT-mini/EDTA, 2 µL 10 × CaCl2, 2 µL 100 mM 6-mer β-peptide, 14 µL GF buffer. All reagents were prepared in GF buffer. Assays were monitored as described previously, and V0 measurements were used to generate a Michaelis–Menten plot for [Ca2+] which was used to derive the pertinent kinetic constants kcat and Km.

Evaluation of the Glycosylation of Recombinant Agrin Fragments

The amino acid sequences of AGR-124 and its y and z splice variants were subject to evaluation using the NetNGlyc [30] and NetOGlyc [31] servers for the in silico prediction of possible N- and O-linked glycosylation sites, respectively.

To experimentally probe the predictions, recombinant AGR-46 and AGR-124 constructs were subject to treatment with peptide N-glycosydase F (PNGase-F, New England BioLabs) for removal of N-linked glycosylation. Briefly, Glycoprotein Denaturing Buffer (New England BioLabs) was added to protein extracts to reach a 1 × concentration and the proteins were denatured at 95 °C for 10 min. After cooling, glycobuffer 2 (New England BioLabs), 1% NP-40, and 0.2 μL (100 Units) of recombinant PNGase-F were added and incubated at 37 °C for 1 h. The results were then analyzed using SDS-PAGE through comparison with non-treated samples.

O-linked glycosylations were assessed by determining the overall glycosylation content of AGR-124 using size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS). Briefly, samples were loaded onto a Protein KW.802.5 (Shodex) column mounted on a Prominence high-pressure liquid chromatography (HPLC) system (Shimadzu) connected to a miniDAWN MALS detector (Wyatt Technologies), a differential refractive index detector (Shimadzu RID-20A) for quantitation of the total mass and to a UV detector (Shimadzu SPD-20A) for evaluation of the sole protein content. SEC-MALS runs were carried out at a flow rate of 1 mL/min in 50 mM TRIS/HCl, 500 mM NaCl, and pH 8.0. Results were analyzed using the protein conjugate module of the Astra software (Wyatt Technologies), using an estimated dn/dc value of 0.185 ml/g for proteins and 0.140 ml/g for glycans, and an extinction coefficient of 71,500 M−1 cm−1 for the AGR-124 sample. The calibration of the instrument was verified by injection of 10 µl of 3 mg/l monomeric BSA (Sigma-Aldrich).

Time Course Digestions of Agrin-Like Substrates

Catalytic competence on native agrin-like substrates was assessed via time course assays using purified recombinant human agrin AGR-46 constructs. Digestions with NT-mini at 5 µg/mL were performed at a fixed starting substrate concentration of 0.5 mg/mL in GF buffer supplemented with 5 mM CaCl2. All necessary dilutions were performed with the reference reaction buffer. Typical reaction mixes were prepared on ice in a PCR tube (total reaction volume 110 μL) and were composed of 500 μg/mL agrin construct, 5 mM CaCl2, and 5 µg/mL NT-mini. NT-mini was added last, and then the solution was mixed by gentle pipetting. Reaction mixes were maintained at 37 °C in a PCR thermocycler (Eppendorf) and 10 µL samples were collected after 0, 5, 10, 15, 20, 25, 30, 60, 90, and 120 min. Each sample was immediately supplemented with 5 µL of SDS-PAGE reducing sample buffer, boiled for 10 min at 95 °C to block the reaction, and loaded on a 16% polyacrylamide gel. Protein bands were stained with colloidal Coomassie blue solution (0.02% Brilliant Blue G250 w/v, 20% ethanol v/v, 5% Al2(SO4)3w/v, 1.05 M phosphoric acid) overnight and carefully destained with 10% acetic acid v/v and 20% ethanol v/v. Images for analysis were taken with a ChemiDoc imager (BioRad) and substrate/product band densities were measured with ImageJ [32]. The intensity values for the substrate bands obtained at 0 min of digestion were set to represent starting substrate concentration (0.5 mg/mL or 10 µM), and the normalized intensities associated with subsequent time point bands were then plotted as a function of time. Prism6 (Graphpad) was used to plot a non-linear regression curve from which pseudo-first-order rate constants for substrate consumption (ks) and product generation (kCAF-22) were derived, along with their associated errors. The same procedure was followed for time-course digestions of the agrin-like AGR-124 substrate, and for experiments with NT*-mini. For AGR-124, the polyacrylamide gel composition (6%) was adjusted to maximize the resolution of high MW CAF-110 and CAF-90 product bands. Statistical significance was determined using a one-way ANOVA coupled with a multiple comparison test (Bonferroni) in Prism 6.01 (GraphPad) to compare ks values of AGR-46 y0z0 with AGR-46 y4z0, AGR-46 y0z0 with all y0z + variants, and AGR-46 y4z0 with all y4z + variants.

Thermal Denaturation Assays

The effects of Ca2+, Zn2+, Ba2+, heparin, EGTA, or EDTA on NT-mini’s stability were assayed directly using thermal denaturation coupled to nano-differential scanning fluorimetry (nano-DSF). For these experiments, the concentration of NT-mini was fixed at 2 µM (0.1 mg/mL), while bivalent metal ions (Ca2+, Zn2+, Ba2+) were assayed at 5 mM, chelators (EGTA, EDTA) were tested at 10 mM, and heparin was used at 1 mM. All chemicals (Sigma-Aldrich) were dissolved directly in GF buffer, to prepare stock solutions at high concentrations: CaCl2 1 M, BaCl2 1 M, ZnCl2 1 M, NT-mini 20 µM (≈ 1 mg/mL), heparin (Sigma #H3393) 4 mM (≈ 72 mg/mL), EGTA 1 M, and EDTA 1 M. The same buffer was also used for all necessary dilutions to reach the assay concentrations from those stocks.

For the evaluation of Ca2+-induced NT-mini stabilization, thermal denaturation nano-DSF experiments were conducted in the presence of increasing [Ca2+] covering the following concentrations: 1 µM, 10 µM, 100 µM, 1 mM, 10 mM, 100 mM, 1 M, 2 M. NT-mini was pre-treated with EDTA (10 mM) before being assayed at a final conc. of 2 µM. CaCl2 solutions were prepared as 10 × stocks (with respect to the desired assay conc.) adjusted to compensate for the EGTA supplemented with NT-mini. All stock solutions and necessary dilutions were prepared in GF buffer (200 mM NaCl, 25 mM HEPES/NaOH, pH 8).

Protein stability at differing pH values was tested at the same final 2 μM NT-mini concentration. Samples were prepared with the same 10 × reaction buffers used for the peptide digestion assays to cover the same pH range. Similarly, in these experiments, the necessary buffer dilutions were performed with water.

Protein unfolding was monitored in Tycho quartz capillaries (Nanotemper) using a Tycho NT.6 instrument (Nanotemper), which allowed for the determination of NT-mini’s unfolding temperatures (Ti) in the assayed conditions. The smoothed data were re-plotted using Prism 6.01 (Graphpad) for comparative purposes. Ti values for increasing Ca2+-induced stabilization were plotted as a function of [Ca2+] and used to extrapolate an EC50 via sigmoidal 4PL non-linear regression in Prism 6.01 (Graphpad) with default parameters.

SPR Determination of Heparin Binding and Affinity

Measurements of heparin binding were performed in a Biacore T200 instrument (GE Healthcare) using a heparin-coated chip (Xantec). All analyses were performed in tris-buffer saline (TBS, 150 mM NaCl, 50 mM Tris–HCl, pH 7.5) unless otherwise noted. After each sample injection, the chip was regenerated using a TBS solution supplemented with 1 M NaCl. For the identification of the heparin-binding domain, the murine SRCR3 (human SRCR4) domain and NT-mini were run individually at a concentration of 50 nM with a contact time of 60 s at a flow of 50 µL/min. For the estimation of NT-mini’s affinity for heparin, a decreasing series of 9 NT-mini concentrations (200–0.78 nM) were obtained by twofold serial dilution in TBS, starting from a protein stock of 22 µM. Steady-state binding was assayed using the single-cycle kinetics mode with a contact time of 60 s and a flow rate of 30 µL/min. Curves were analyzed using the kinetics fit of the Biacore evaluation software (GE Healthcare) with the Rmax parameters set to local fit. For visualization purposes, the SPR traces were exported and re-plotted using Prism 6.01 (Graphpad).

C2C12 Cultures

For maintenance, C2C12 myoblasts (kindly provided by DM Rossi, ICS Maugeri, Pavia) were grown in 75 cm2 T-flasks (VWR) at 37 °C in a humidified 5% CO2 atmosphere. Cells were maintained in a high-glucose DMEM (Thermo Fisher Scientific) medium supplemented with 10% (v/v) fetal bovine serum (FBS, Thermo Fisher Scientific) and 1% (v/v) non-essential amino acids (NEAA, Thermo Fisher Scientific). Growth medium was refreshed on average every 48 h, and cells were generally split 1/10 when at 60–70% confluence.

To perform differentiation experiments, cells were transferred to 6-well plates containing sterilized glass cover slips and seeded at ~ 50% confluence. Cultures were allowed to stabilize for 24 h before inducing differentiation. This was obtained using high-glucose DMEM medium with a reduced FBS content (1%) but still supplemented with 2% NEAA. Differentiation was generally protracted for 7 days before proceeding with immuno-staining. Growth media was refreshed every 48 h.

Treatments with NT-mini/NT*-mini (50 ng/mL) were performed throughout the 7-day differentiation period by supplementing the differentiation medium with the purified recombinant protein. All necessary stock dilutions were performed directly in differentiation medium.

C2C12 Staining and Analysis

Evaluation of myoblast-to-myotube differentiation was carried out using a live stain protocol adapted from the procedures described in Stanga et al. [33], McMorran et al. [34], and Harrison et al. [35]. Briefly, cellular boundaries were identified by staining cell-surface glycans with an Alexa645-wheat germ agglutinin (WGA) conjugate (Thermo Fisher Scientific), while nuclei were stained using Hoechst dye (Sigma-Aldrich). Acetylcholine receptor (AChR) clustering was evaluated using an Alexa594-bungarotoxin (Btx) conjugate (Thermo Fisher Scientific). Hoechst and Btx stains were added directly to the media in the 6-well plate, in a 1/2000 (v/v) and 1/1000 (v/v) ratio respectively, and incubated at 37 °C for 45 min. Staining with WGA was also performed directly in the culture media, but in a 1/250 (v/v) ratio and with a 15 min incubation time. After staining, the cells were washed once with PBS, fixed with a 4% (w/v) paraformaldehyde (PFA) in PBS solution at room temperature for 15 min, and washed again with PBS. Finally, the cover slips were mounted on microscopy slides with a drop of mounting media (Sigma-Aldrich). These mounts were wrapped in tin foil and allowed to set O/N at 4 °C before being imaged using a SP8 white-laser confocal microscope (Leica Microsystems). Filters were adjusted specifically for each stain to visualize nuclei (Hoechst, Ex 405 nm, Em 429 nm), AChRs (Btx, Ex 598 nm, Em 634 nm), and cell-surface glycans (WGA, Ex 653 nm, Em 692 nm). Z-stacks of 0.7-µm thickness were collected at 40 × magnification, and images were analyzed with ImageJ [32]. Myotube identification and assignment of nuclei were performed by manual slice-by-slice assessment of the Z-stacks. Fusion indexes were calculated as the fraction of total nuclei incorporated into myotubes. These data were normalized to the non-treated control conditions and analyzed on Prism 6.01 (GraphPad) to assess the statistical significance of the observed differences with a one-way ANOVA coupled with a multiple comparison test (Bonferroni). Violin-plots of analyzed data were generated with MATLAB [36] using the “Violinplot-Matlab” extension [37].

Electrophysiology in Acute Hippocampal Slices

Animal maintenance and experimental procedures were performed according to the international guidelines of the European Union Directive 2010/63/EU on the ethical use of animals and were approved by the local ethical committee of the University of Pavia (Italy) and by the Italian Ministry of Health (628/2017-PR). Acute 300-µm-thick brain slices were cut on the coronal plane from the hippocampus of 20–23-day-old C57BL/6 mice (either sex), as reported previously [38]. Briefly, mice were deeply anesthetized with halothane (Sigma-Aldrich) and killed by decapitation to remove the hippocampus (either left or right) for acute slice preparation using a vibratome (VT1200S, Leica Microsystems). The cutting procedure was performed in an ice-cold solution containing 87 mM NaCl, 2.5 mM KCl, 25 mM NaHCO3, 1.25 mM NaH2PO4, 75 mM sucrose, 25 mM glucose, 0.2 mM CaCl2, and 7 mM MgCl2 and bubbled with 95% O2 and 5% CO2 (pH 7.4). Slices were recovered at 32 °C for 1 h in Krebs solution containing 120 mM NaCl, 2 mM KCl, 1.2 mM MgSO4, 26 mM NaHCO3, 1.2 mM KH2PO4, 2 mM CaCl2, and 11 mM glucose, equilibrated with 95% O2–5% CO2 (pH 7.4). Subsets of slices were incubated for 90 min in Krebs solution containing either NT-mini or NT*-mini (100 ng/ml, in both cases). The slices were then transferred to the recording chamber of an upright microscope (Zeiss), perfused with oxygenated Krebs solution (2 ml/min), and maintained at 32 °C using a Peltier feedback device (Warner Instruments). Krebs solution contained NT-mini or NT*-mini (100 ng/ml, in both cases) in the subset of recordings on slices pre-incubated with the corresponding enzyme. Whole-cell patch-clamp recordings were performed on hippocampal CA1 pyramidal neurons using glass borosilicate patch pipettes (resistance 5.5–7 MΩ) filled with an intracellular solution containing (mM) 138 mM K-gluconate, 8 mM KCl, 10 mM HEPES, 0.5 mM EGTA, 4 mM Mg-ATP, and 0.3 mM Na-GTP adjusted to pH 7.3 with KOH. Signals were acquired using a Multiclamp 700B amplifier (Molecular Devices; cutoff frequency of 10 kHz) and digitized with a Digidata 1440A interface (Molecular Devices). After reaching the whole-cell configuration, voltage steps were used (from − 70 mV, 10 mV per step) to elicit the Na current and to evaluate passive properties. Neuronal intrinsic excitability was assessed in current-clamp mode from resting membrane potential (Vrest), by injecting 1 s current steps of increasing amplitude (from − 50 pA to 400 pA, with 25 pA increment). Membrane capacitance (Cm), input resistance (Ri), and series resistance (Rs) were calculated as reported previously [38, 39]. No significant difference was found in these parameters among the different conditions (controls, NT-mini-treated and NT*-mini treated neurons). Signals were analyzed offline using Clampfit 10 (pClamp suite, Molecular Devices). All drugs were obtained from Sigma-Aldrich (Merck). Data are reported as mean ± MSE (standard error of the mean) and compared for statistical significance using unpaired Student’s t test.

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