Reagents

Retinoic acid (RA; R2625, Sigma-Aldrich, St. Louis, MO, USA) was prepared as a 10 mM stock solution in dimethyl sulfoxide (DMSO; Sigma-Aldrich). Phorbol 12-myristate 13-acetate (PMA; P1585), N6,2′-O-dibutyryladenosine 3′,5′-cyclic monophosphate sodium salt (dbcAMP; D0260), and brain-derived neurotrophic factor human (BDNF; B3795) were from Sigma-Aldrich. The γ-Eno peptide, corresponding to the last 30 amino acids of γ-enolase (AKYNQLMRIEEELGDEARFAGHNFRNPSVL), was obtained from GenScript (Piscataway, NJ, USA) and prepared as a 0.712 mM stock solution in Dulbecco’s phosphate-buffered saline (DPBS; D8537, Sigma-Aldrich). The irreversible inhibitor of cathepsin X, AMS36, was synthesized as reported by Pišlar et al. (Pišlar et al., 2014) and prepared as a 10 mM stock solution in DMSO.

Cell Cultures

The human neuroblastoma SH-SY5Y and murine neuroblastoma Neuro-2a cell lines were obtained from the American Type Culture Collection (CRL-2266 and CCL-131, respectively; Manassas, VA, USA). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F12 (D8437, Sigma-Aldrich) supplemented with 10% (v/v) fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin-streptomycin (P4333, Sigma-Aldrich). The human neuroblastoma LA-N-2 cell line was obtained from Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH (ACC 671; Braunschweig, Germany). Cells were cultured in DMEM/F12 (D8437, Sigma-Aldrich) supplemented with 20% (v/v) FBS (Thermo Fisher Scientific) and 2% penicillin-streptomycin (P4333, Sigma-Aldrich). Cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2 and grown to 80% confluence. Cells were grown in 75 cm2 flasks, passaged twice a week when approximately 80% confluent, and used only up to passage 30.

Cell Differentiation and Treatment

To differentiate the SH-SY5Y cell line into dopaminergic-, cholinergic-, and adrenergic-like neuronal cells, we adapted protocols from Kovalevich and Langford (Kovalevich & Langford, 2013), Shipley et al. (Shipley et al., 2016), Medeiros et al. (Medeiros et al., 2019), and Kume et al. (Kume et al., 2008). For differentiation experiments, plates were pre-coated with collagen (20 µg/mL; C3867; Sigma-Aldrich) in Dulbecco’s phosphate-buffered saline (DPBS; D8537, Sigma-Aldrich) for 2 h at 37 °C and washed twice with DPBS. SH-SY5Y cells were then seeded, and after 1 day, the cells were exposed to reduced-serum medium (2% FBS and 0.25% penicillin-streptomycin) containing 10 µM RA. Differentiation medium with the same proportion of DMSO was used as a negative control, representing non-differentiated SH-SY5Y cells. After 72 h, SH-SY5Y cell differentiation into the dopaminergic-like neuronal subtype was induced with reduced-serum medium (2% FBS and 0.25% penicillin-streptomycin) containing a combination of 10 µM RA and 80 nM PMA.

Cell differentiation into the cholinergic-like neuronal subtype was induced with reduced-serum medium (2% FBS and 0.25% penicillin-streptomycin) containing a combination of 10 µM RA and 50 ng/mL BDNF for an additional 72 h. To obtain the adrenergic-like neuronal subtype, the day after seeding, SH-SY5Y cells were exposed to reduced-serum medium containing 0.5 mM dbcAMP for 72 h. All control cells were treated with an equivalent volume of the solvent used for RA (DMSO) or dbcAMP (dH2O) and were characterized as non-differentiated SH-SY5Y cells.

To differentiate Neuro-2a cells into a dopaminergic-like neuronal subtype, we adapted the protocol from Tremblay et al. (Tremblay et al., 2010). Cells were seeded onto non-coated plates and the next day exposed to reduced-serum medium (2% FBS and 0.25% penicillin-streptomycin) containing 0.5 mM dbcAMP for 72 h. All control cells were treated with an equivalent volume of the solvent used for dbcAMP (dH2O) and were characterized as non-differentiated Neuro-2a cells.

To differentiate LA-N-2 cells into a cholinergic-like neuronal subtype, we adapted the protocol from Singh et al. (Singh et al., 1990). Cells were seeded onto non-coated plates and the next day exposed to reduced-serum medium (4% FBS and 0.5% penicillin-streptomycin) containing 10 µM RA for 72 h. All control cells were treated with an equivalent volume of the solvent used for RA (DMSO) and were characterized as non-differentiated LA-N-2 cells.

For γ-Eno peptide treatment, the peptide was added to the differentiation medium at a final concentration of 100 nM on days 1 and 4. Additionally, for cathepsin X inhibition, AMS36 at a final concentration of 10 µM was added to differentiation media on days 1 and 4. Non-treated control cells were treated with an equal volume of the solvent used for γ-Eno (DPBS) and AMS36 (DMSO).

Vector Construction

Human enolase 2 VersaClone cDNA (RDC3186; R&D Systems, Minneapolis, MN, USA) and the pcDNA3-GFP expression vector (13031; Addgene, Watertown, MA, USA) were used. The following primers were used for cloning: 5′-TTGTACAAGTCCATAGAGAAGATCTGG-3′ and 5′-AATCTAGATCACAGCACACTGG-3′ for enolase 2, and 5′-TTGTACAAGTCCATAGAGAAGATCTGG-3′ and 5′-AATCTAGATCAACTGGGATTACG-3′ for enolase 2 without the last two amino acids on its C-terminus (ΔC). PCR products were ligated into the XbaI and BsrGI sites of pcDNA3-GFP, placing enolase 2 in-frame downstream of GFP and resulting in N-terminal GFP fusions to enolase 2 or enolase 2-ΔC. Prepared vectors were multiplied in Escherichia coli TOP10. Enolase 2 and enolase 2-ΔC genes were verified by sequencing.

Cell Transfection

For the overexpression of γ-enolase, SH-SY5Y cells were seeded in growth medium without antibiotics into 6-well culture plates (1.5 × 105 cells/mL for the 7-day differentiation protocol and 3 × 105 cells/mL for the 4-day differentiation protocol). The next day, lipofection was performed using Lipofectamine 2000 (11668027; Thermo Fisher Scientific) according to the manufacturer’s instructions. Briefly, 2 µg of DNA plasmid in 100 µL of serum-free medium without antibiotics was mixed with an equal amount of medium containing 4 µL of Lipofectamine and incubated for 20 min at room temperature (RT). The DNA-lipid mixture was added to the cells dropwise and incubated at 37 °C and 5% CO2 for 5 h. For the 7-day cell differentiation protocol, cells were exposed to another transfection in the same manner on day 4. After transfection, the transfection medium was removed and replaced with differentiation medium as indicated in the Cell differentiation and treatment section. The plasmids used for γ-enolase overexpression were constructed using the pcDNA3-GFP vector, which contains a constitutively active cytomegalovirus (CMV) promoter. Consequently, the expression of GFP–γ-enolase fusion proteins is not under the control of differentiation agents such as RA, PMA, dbcAMP, or BDNF, as these treatments do not target any inducible promoter elements within the plasmid. To assess the efficiency of transfection, cells were visualized with fluorescence microscopy and the EVOS Cell Imaging System (Thermo Fisher Scientific) for qualitative assessment of protein expression and cell morphology and viability. Afterwards, cells were harvested for cell pellets, and the efficiency of γ-enolase transfection was evaluated by western blot analysis. Two specific mouse monoclonal antibodies were used: one raised against amino acids 416–433 of γ-enolase, which detects the C-terminal region and therefore recognizes only the full-length variant (1:250; sc-21738, Santa Cruz Biotechnology, Dallas, TX, USA), and another raised against amino acids 271–285, which detects both the full-length and C-terminally truncated (ΔC) variants, representing total γ-enolase expression (1:350; sc-21737, Santa Cruz Biotechnology).

For γ-enolase silencing, Neuro-2a and LA-N-2 cells were seeded in growth medium without antibiotics into 12-well culture plates (1 × 105 cells/mL). For mouse γ-enolase, Silencer® siRNA (s65514; Thermo Fisher Scientific) targeting the sequence 5′-GGACUUUGUCCGGAACUAUtt-3′ (sense) and 5′-AUAGUUCCGGACAAAGUCCtg-3′ (antisense) was used. For human γ-enolase, Stealth RNAi™ siRNA specific for ENO2 (HSS176535; Thermo Fisher Scientific) was applied, targeting the sequence 5′-CGACUAGGUGCAGAGGUCUACCAUA-3’. Cells were transfected with control siRNA (sc-37007; Santa Cruz Biotechnology) as control to siRNA of mRNA target. Cells were transiently transfected with Stealth RNAi/γ-Eno using Lipofectamine 2000 (Thermo Fisher Scientific), according to the manufacturer’s protocol. Briefly, Lipofectamine 2000 was gently mixed before use, diluted in serum-free medium without antibiotics, and left for 5 min at RT. The diluted Lipofectamine 2000 was then combined with the diluted siRNA oligomer in serum-free medium without antibiotics, gently mixed, and left for a further 20 min at RT. Next, 200 µL of the transfection complex was added to the cells to achieve a final siRNA concentration of 20 nM in the well and incubated at 37 °C in a humidified atmosphere of 5% CO2 for 5 h. The transfection medium was removed and replaced with differentiation medium as indicated in the Cell differentiation and treatment section. To assess the efficiency of transfection, cells were harvested for cell pellets, and the efficiency of γ-enolase transfection was evaluated by western blot analysis.

Evaluation of Cell Extensions

SH-SY5Y, Neuro-2a, and LA-N-2 cells were seeded in growth medium into 6-well culture plates in duplicate. The next day, cells were treated with differentiation medium as described above. Cell extensions were evaluated by morphological examination and captured using the EVOS Cell Imaging System (Thermo Fisher Scientific). ImageJ software was used to determine the number and length of cell extensions (in pixels) that were longer than the corresponding cell diameter. Approximately 100 cells per condition were measured in each experiment. The data are expressed relative to the control, i.e., non-differentiated cells.

Cell Proliferation Assay

SH-SY5Y, Neuro-2a, and LA-N-2 cells were seeded in growth media in 6-well culture plates in duplicate. The next day, cells were stained with CellTrace carboxyfluorescein succinimidyl ester (CFSE) reagent (1 µM; Thermo Fisher Scientific) according to the manufacturer’s protocol. Subsequently, the cells were differentiated as described above. Then, cells were collected, and their mean fluorescence intensities were measured in the BL-1 channel using a flow cytometer (Attune NxT, Thermo Fisher Scientific). The obtained data were analyzed in FlowJo software (Tree Star Inc., Ashland, OR, USA), and mean CFSE fluorescence intensities were normalized to the control cells. Three independent experiments with two replicates per differentiation condition were performed.

Cell Lysate Preparation

After 4 days of differentiation for Neuro-2a, LA-N-2, and dbcAMP-treated SH-SY5Y cells, or after 7 days of differentiation for SH-SY5Y cells treated with RA combined with PMA or BDNF, the cells were harvested. Cell lysis was completed with either of two lysis buffers: for assessing protein expression by western blotting and enzyme-linked immunosorbent assay (ELISA) (50 mM HEPES, pH 6.5, 150 mM NaCl, 1 mM EDTA, 0.25% Triton X-100) or for quantifying cathepsin X activity (50 mM Na-acetate, pH 5.5, 1 mM EDTA, 100 mM NaCl, 0.25% Triton X-100). After lysis, whole-cell lysates were incubated for 30 min on ice, preserved at − 80 °C, subjected to freeze-thaw cycles, sonicated, and centrifuged at 15,000 × g for 15 min at 4 °C. Protein concentrations in the obtained lysates were quantified using the DC Protein Assay (Bio-Rad, Hercules, CA, USA) and bovine serum albumin (BSA; Sigma-Aldrich) as a protein standard.

Western Blotting

Equal protein concentrations from whole-cell lysates (1 µg/µL) were denatured by the addition of SDS-PAGE buffer, heated (100 °C) for 5 min, and resolved by SDS-PAGE on 12% Tris-glycine gels. The proteins were then transferred to polyvinylidene difluoride membranes using iBlot (Thermo Fisher Scientific). Membranes were blocked in 5% (w/v) non-fat dried milk powder in Tris-buffered saline with Tween 20 (TBST; 20 mM Tris/HCl, pH 7.4, 137 mM NaCl, and 0.1% Tween 20) at RT for 1 h. Subsequently, the membranes were incubated overnight at 4 °C with primary antibodies in TBST containing 3% (w/v) BSA. The following primary antibodies and dilutions were used: mouse anti-tyrosine hydroxylase (1:250; sc-25269), mouse anti-acetylcholinesterase (1:500; sc-373901), mouse anti-α-enolase (1:500; sc-100812), mouse anti-γ-enolase raised against amino acids 416–433 of γ-enolase, determining the active form of γ-enolase (1:250; sc-21738, Santa Cruz Biotechnology), mouse anti-γ-enolase raised against amino acids 271–285 of γ-enolase, determining the total form of γ-enolase (1:350; sc-21737;Santa Cruz Biotechnology), rabbit anti-α−2 adrenergic receptors (ADRA2A) (1:300; 14266-1-AP), rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:10000; 10494-1-AP; both from Proteintech, Rosemont, IL, USA), mouse anti-β-tubulin (1:1000; T4026; Sigma-Aldrich), and goat anti-cathepsin X (1:500; AF934; R&D Systems, Minneapolis, MN, USA). The membrane for goat anti-cathepsin X antibody incubation was blocked with 1.5% (w/v) non-fat dried milk powder in TBST and diluted in TBST containing 1.5% non-fat dried milk powder and 1% (w/v) BSA. After washing, the following secondary horseradish-peroxidase-conjugated antibodies in TBST containing 5% (w/v) non-fat dried milk powder were added for 1 h: anti-mouse (1:5000; 111-035-068), anti-rabbit (1:5000; 111-035-045; both from Jackson ImmunoResearch, West Grove, PA, USA), and anti-goat (1:2000; sc-2354; Santa Cruz Biotechnology) antibodies. After washing, protein bands were visualized with enhanced chemiluminescence detection kits (Thermo Fisher Scientific) and recorded with a G: Box imager (Syngene, UK). When necessary, membranes were stripped with stripping buffer (62.5 mM Tris/HCl, pH 5.7, 100 mM 2-mercaptoethanol, and 2% SDS) for 1 h at 65 °C. Band intensities were quantified using Gene Tools software (Sygene). To determine the protein levels, values were expressed as a ratio to GAPDH and were normalized to the control sample.

ELISA

The protein levels of α-enolase and γ-enolase were detected with the ENO1 SimpleStep ELISA® (ab181417; Abcam) and NSE SimpleStep ELISA® (ab217778; Abcam) kits, respectively. Following the manufacturer’s instructions, standards and equal protein amounts of samples (50 µg) were added to the wells, followed by the antibody mix. After 1 h incubation, the wells were washed, and TMB Development Solution (Merck Millipore, MA, USA) was added. After 10 min, the reaction was stopped by adding Stop Solution. Protein amounts were determined by measuring the absorbance at 450 nm using a Tecan Safire spectrophotometer (Tecan Safire2), and protein levels were expressed relative to the control.

Cathepsin X protein levels were determined by ELISA. Microtiter plates were coated with equal aliquots of goat anti-cathepsin X antibody (1:250, AF934; R&D Systems) in 0.01 M carbonate/bicarbonate buffer, pH 9.6, and incubated at 4 °C overnight. After incubation with blocking buffer (2% BSA in PBS, pH 7.4) for 1 h at RT, samples containing equal protein amounts (25 µg) were added. Following a 2-h incubation at 37 °C, the wells were washed and filled with mouse anti-cathepsin X monoclonal antibody conjugated with horseradish peroxidase (1:3000; 3B10; produced in-house) in a blocking buffer. After an additional 1.5 h incubation at 37 °C, 100 µL of 3,3′,5,5′-tetramethylbenzidine substrate (Sigma-Aldrich) was added to each well. The reaction was stopped after 15 min by adding 50 µL of 2 M H2SO4 to each well. Protein levels were determined by measuring the absorbance at 450 nm using a Tecan Safire spectrophotometer (Tecan Safire2, Switzerland), and protein levels were expressed relative to the control.

Cathepsin X Activity Assay

Cell lysates were diluted to a protein concentration of 0.125 mg/mL in 50 mM acetate buffer, pH 5.5, with 5 mM dithiothreitol (43819; Sigma-Aldrich) and 1.5 mM EDTA (03690; Sigma-Aldrich) followed by incubation at 37 °C for 10 min. In duplicate, 95 µL of lysate was transferred into the wells of a black microtiter plate (Nunclon Delta Surface; Thermo Fisher Scientific) containing 5 µL of the previously added fluorogenic substrate Abz-FEK(Dnp)-OH (Puzer et al., 2005) at a final concentration of 5.9 µM. The subsequent degradation was monitored continuously at 320 ± 5 nm excitation and 420 ± 5 nm emission using a Tecan Safire spectrophotometer (Tecan Safire2, Switzerland). Kinetic measurements were analyzed with Magellan™ software (Magellan™, 2011), and the resulting data, expressed in relative fluorescence units, were normalized to the control sample.

Enolase Glycolytic Activity Assay

The glycolytic activity of enolase, i.e., the conversion of 2-phosphoglycerate to phosphoenolpyruvate, was assessed with the Enolase Assay Kit (ab241024; Abcam), according to the manufacturer’s instructions. Briefly, cell lysates were prepared using the Enolase buffer supplied in the Enolase Assay Kit, and protein concentrations were determined as described in the previous paragraph. Initially, standards and samples were prepared (1 µg/mL) and loaded in duplicate onto the plate, forming two separate sets of samples. Additionally, 100-fold and 1000-fold dilutions of the positive control were prepared and loaded onto the plate in duplicate. The reaction and a corresponding background control mixture (substituting substrate with buffer) were formulated and applied to the plate. Absorbance was measured at 570 nm using a Tecan Safire spectrophotometer in kinetic mode for 60 min at RT. Sample absorbances were corrected using their respective background controls. Results were expressed as the slope of the linear portion of the reaction curve over time, with data normalized to control cells.

Flow Cytometry Analysis of Protein Expression

Flow cytometry was used to analyze the protein expression of tyrosine hydroxylase (TH), choline acetyltransferase (ChAT), and ADRA2A in treated cells. For all experiments, cells were washed with PBS (pH 7.4), fixed with 4% paraformaldehyde (1510; Electron Microscopy Science, Hatfield, PA, USA) for 10 min at RT, and permeabilized with specific reagents. For TH detection, cells were permeabilized with 90% methanol at − 20 °C for 30 min, followed by incubation with anti-TH antibody (1:2500; ab209921, Abcam) at RT for 30 min. A rabbit monoclonal IgG phycoerythrin-conjugated isotype control (1:2500; ab209478, Abcam) was used under the same conditions. ChAT expression was assessed after permeabilization with 0.05% Triton X-100 in PBS for 15 min at RT, followed by incubation with anti-ChAT antibody (1:5000; ab224001, Abcam) for 30 min at RT. A rabbit monoclonal IgG allophycocyanin-conjugated isotype control (Abcam, ab232814) was included as a control. For ADRA2A detection, cells were permeabilized with 0.5% Tween 20 in PBS for 10 min at RT and incubated with anti-ADRA2A antibody (1:50; PA5-18475, Thermo Fisher Scientific) at 4 °C for 60 min, followed by Alexa Fluor 488-labeled secondary antibody (1:500; Thermo Fisher Scientific) for 30 min at RT. After antibody incubations, cells were washed and collected. Mean fluorescence intensities were measured using an Attune NxT flow cytometer (Thermo Fisher Scientific) in the BL-1 channel for TH and ADRA2A and the BL-2 channel for ChAT.

The relative TH, ChAT, and ADRA2A contents of the cells were evaluated using FlowJo software and were determined as the mean fluorescence of each sample relative to the fluorescence intensity of control cells.

Double Immunofluorescence Staining

Cells were seeded onto glass coverslips in growth medium in duplicate and subjected to the differentiation protocol. Afterwards, cells were fixed with 4% paraformaldehyde (1510; Electron Microscopy Science) in DPBS for 30 min at RT and then permeabilized with 0.5% Tween 20 (P9416; Sigma-Aldrich) in PBS for 10 min. Non-specific staining was blocked with 10% donkey serum (S-30; Sigma-Aldrich) in PBS with 0.05% Triton X-100 (T8787; Sigma-Aldrich) for 30 min at RT. Cells were then incubated with rabbit phycoerythrin (PE)-conjugated anti-TH (1:200; ab209921; Abcam), rabbit allophycocyanin (APC)-conjugated anti-ChAT (1:200; ab224001; Abcam), rabbit Alexa Fluor 555 anti-β-tubulin (1:200; ab202519; Abcam), goat anti-ADRA2A (1:50; PA5-18475; Thermo Fisher Scientific), rabbit anti-α-enolase (1:200; 11204-1-AP; Proteintech, Rosemont, IL, USA), mouse anti-γ-enolase (1:50; 66150-1-Ig; Proteintech), and goat anti-cathepsin X (1:75; AF934; R&D System) antibodies in blocking solution for 2 h at RT. Afterwards, the cells were washed with PBS, and those incubated with non-conjugated primary antibodies were further exposed to Alexa Fluor 488-, Alexa Fluor 555-, and Alexa Fluor 647-labeled secondary antibodies (1:1000; Thermo Fisher Scientific) for an additional 1.5 h. After washing with PBS, a ProLong Gold antifade reagent with DAPI (P36935; Thermo Fisher Scientific) was used for mounting the coverslips onto glass slides. Fluorescence microscopy was performed using a confocal microscope (LSM 710; Carl Zeiss, Oberkochen, Germany and AX; Nikon, Tokyo, Japan) with the ZEN 3.4 image software or NIS-Elements image software. Co-localization analysis was performed on immunofluorescence images using Carl Zeiss ZEN software. The degree of co-localization was quantified using the Pearson’s correlation coefficient (referred to as Correlation R). Relative co-localization areas were analyzed in at least 10 cells per condition.

Statistical Analysis

All statistical analyses were performed using parametric tests appropriate to the experimental design. Data are expressed as means with 95% confidence intervals (CI) from N independent biological replicates, with N specified in the corresponding figure legends. Differences between groups were assessed by one-way analysis of variance (ANOVA) followed, where appropriate, by Tukey’s post hoc multiple comparisons test. Analyses were conducted using GraphPad Prism version 10 (GraphPad Software, San Diego, CA, USA). Exact p-values are reported in the figure legends; for graphical representation, statistical significance is indicated as p < 0.05 (*), and p < 0.01 (**). Sample-size justification for key experiments was based on power analyses performed with G*Power 3.1, using effect sizes estimated from the data, an α-level of 0.05, and a target power of 0.80 (Supplementary Table S1).