Human neural progenitor cultures

Human neural progenitors (NP) (#ax0015) were obtained from Axol Bioscience (Cambridge, UK) and cultured following the procedures previously reported [37, 38]. According to the customer suggestions, NP were passaged maximum three times before using for the experiments. Shortly, NP were plated in precoated wells as well as slides using Geltrex coating solution (ThermoFisher, Milan, IT), and differentiated in Neurobasal supplemented with B27 media (ThermoFisher, Milan, IT). As controls for our experiments, some key results were confirmed also in #ax0016 (Axol Bioscience, Cambridge, UK).

For RNA silencing, 2 days after plating, NP (2,500,000 cell/well, diameter/well 35 mm) were incubated with a siRNA mix containing Sema 3A (Ambion, Milan, IT, #S20284) or Npn 1 (Ambion, Milan, IT, #107,267) or Plxn A2 siRNA (5 pmol, Ambion, Milan, IT, #S10700), 1 µl Lipofectamine Messenger Max mRNA Transfection Reagent (Invitrogen, Milan, IT) and 100 µl Opti-MEM medium (ThermoFisher, Milan, IT) and left in Neurobasal supplemented with B27 media (ThermoFisher, Milan, IT) for additional 48 h. In preliminary experiments to set working conditions, we used 5 pmol Silencer GAPDH siRNA (Ambion, Milan, IT, #AM4624) as positive control and Silencer Negative Control siRNA (Ambion, Milan, IT, #AM4611) as the negative control following supplier’s suggestions (data not shown).

For DNA transfection, 10 µg/ml of Sema 3A-GFP (OriGene Technologies Inc., Rockville, MD, USA, #RG213681) or GFP empty vector (OriGene Technologies Inc., Rockville, MD, USA #PS100010) were incubated in 1 µl Lipofectamine Stem Transfection Reagent (Invitrogen, Milan, IT) for 20 min and then the mix was transferred to NP that were left in Neurobasal supplemented with B27 media for 48 h. After 48 h, the medium was refreshed, and cells were cultured for additional 24 h.

In siRNA and DNA co-transfection experiments, NP were incubated with 10 µg/ml Sema 3A-GFP or GFP-empty vector and 5 pmol siNpn 1 (Ambion, Milan, IT, #107,267), siSema 3A (Ambion, Milan, IT, #S20284) or siPlxn A2 (Ambion, Milan, IT #S10700) in 1 µl Lipofectamine Stem Transfection Reagent (Invitrogen, Milan, IT) and left in Neurobasal supplemented with B27 media for 48 h.

Human primary microglia

Human fetal brain-derived primary cultures of microglia (HMC3, 37,089–01), purchased from Celprogen Inc. (Benelux, NL), were cultured in Essential medium 8 (ThermoFisher, Milan, IT), that is routinely used for stem cells grow and expansion and we previously used to keep in culture NP [37, 39]. DNA transfection was performed by incubating 10 µg/ml of Sema 3A with 15 µl of Lipofectamine 2000 (Invitrogen, ThermoFisher, Milan, IT), according to the manufacturer protocols. After 20 min, fresh media was added, and cells were left in culture for 48 h. 10 µg/ml of GFP-empty vector was used as transfection positive control.

Media from Sema 3A, GFP or non-transfected microglia was collected 48 h after transfection and transferred to NP culture. In order to minimize events related to changes in growth conditions, NP were cultured in Essential medium 8 (ThermoFisher, Milan, IT) at least 48 h before the experiment.

Western blot analysis (WB)

For protein isolation, cells were collected and homogenized in RIPA buffer (ThermoFisher, Milan, IT) supplemented with protease inhibitors (Sigma-Aldrich, Darmstadt, DE). After 60 min incubation on ice, the homogenates were centrifuged (14,000 rpm, 4 °C, 20 min) and soluble protein samples were stored at -80 °C until use. Protein concentration was determined with the Bradford assay. Equal amounts (30 μg) of proteins were separated on 4–15% precast polyacrylamide gel (Biorad Laboratoires, Milan, IT) under reducing conditions, transferred into PVDF membranes (Abcam, Cambridge, UK). Membranes were blocked with 5% Bovine Serum Albumin (BSA, Sigma-Aldrich, Milan, IT) in Tris-Buffered Saline-Tween (TBS-T, Biorad Laboratoires, Milan, IT) and incubated overnight with the appropriate primary antibody. Anti-mouse or anti-rabbit secondary antibodies (Santa Cruz Biotechnology, Dallas, TX, USA) were used to detect the primary antibody. The detection of the protein of interest is achieved using chemiluminescent method utilizing Clarity Western ECL Substrate (Biorad Laboratoires, Milan, IT). For digital quantification, densitometric analysis of the immunoreactive bands was performed using ImageLab 6.1.0 software (2020, Bio-Rad Laboratories, Milan, IT). The following primary antibodies were used (see also key resource Table): anti-β-actin (1:20,000, Sigma-Aldrich, #A3854), anti-iNOS (1:1000, Proteintech, #18,985–1-AP), anti-TNFα (1:1000, Proteintech #17,590–1-AP), anti-Sema 3A (1:1000, Invitrogen, #PA5-67,972), anti-Fyn (1:1000, Cell Signaling, #4023), anti-pFyn Tyr420 (1:1000, Cell Signaling, #2101S), anti-p35/CDK5 (1:1000, Cell signaling, #2506), anti-pCDK5 Tyr15 (1:1000, Cell Signaling, #94,254). The following secondary antibodies were used for immunoblotting: anti-rabbit IgG-HRP conjugated (1:5000, Santa Cruz, #sc-2357), anti-mouse mIgGk BP-HRP (1:5000, Santa Cruz, #sc-516102).

Enzyme-linked immunosorbent assay (ELISA)

Sema 3A protein levels in media from microglia overexpressing Sema 3A (Sema 3A media) or GFP (GFP media) or non-transfected (Ctrl media) were assessed using Elisa kit (ELISA, Cusabio, Houston, TX, USA #CSB-E15913h), according to the manufacturer instructions. Kit sensitivity was 0,156 ng/ml, according to the manufacturer information.

Immunofluorescence (IF)

NP were plated on precoated slides for approximately 2 days in Neurobasal supplemented with B27 media (250,000 cells/well; diameter/well 16 mm) and then transfected with siRNA or DNA or exposed to microglia conditioned media as described in each figure. NP were fixed in 4% PFA-methanol free solution (ThermoFisher, Milan, IT), washed with Dulbecco's Phosphate Buffered Saline (DPBS, ThermoFisher, Milan, IT) and permeabilized with 0.05% Triton X-100 (Bio-Rad Laboratories, Milan, IT) for 3–5 min at room temperature. Triton was eliminated and cells were rinsed with DPBS. Then, cells were processed for the staining and incubated with primary antibodies overnight at 4 °C. The primary antibodies used were: anti-Ankirin G (1:200, Invitrogen, #33–8800), anti-βIII Tubulin (1:1000, Abcam, #ab Ab18207), anti-CD68 (1:1000, Proteintech, #66,231–2-Ig) anti-CD86 (1:1000, Proteintech, #13,395–1-AP), anti-IBA1 (1:1000, Proteintech, #66,827–1-Ig), anti-iNOS (1:1000, Proteintech, #18,985–1-AP), anti-MAP-2 (1:1000, Invitrogen, #PA517646) anti-Npn 1 (1:1000, Abcam, #ab81321), anti-Plexin A2 (1:1000, Cell Signaling, #5658), anti-TMEM119 (1:1000, Proteintech, #66,948–1-Ig) anti-TNFα (1:1000, Cell Signaling, #3707). Fluorescent secondary antibodies conjugated to Alexa 488 (1:250, Invitrogen, #A-11029) or 594 (1:250, Invitrogen, #R37117) were used for primary antibodies’ detection for 45 min at room temperature. Nuclei staining was obtained with DAPI (Fluoroshield Mounting Medium with DAPI, Abcam, #AB104139) and imaged with Plan Apochromat 40x/1,3 Oil DIC M27 (Zeiss, Oberkochen, Germany) or EC Plan Neofluar 20x/0,50 M27 (Zeiss, Oberkochen, Germany) or EC Plan Neofluar 10x/0,30 (Zeiss, Oberkochen, Germany) objectives on a Zeiss LSM700 AxioObserver laser scanning confocal microscope equipped with a gallium arsenide phosphide photomultiplier tube (GaAsp-PMT) detector and controlled by a Zen black software (Zeiss, Oberkochen, Germany). Fluorescence images presented are representative of cells imaged in at least three independent experiments and were processed with Fiji, ImageJ2 software (National Institutes of Health, Bethesda, Marlan, USA). In order to analyze axonal length and dendrite organization, NP were plated onto glass slides coated in a 24-well plate at a density of 75,000 cells per well. Axonal length was assessed using NeuronJ plug-in (ImageJ); Sholl analysis and Strahler analysis were performed using Neuroanatomy plug-in (ImageJ). Representative skeleton masks in Figs. 1B and 3A were obtained using Synapse and Neurites Detector (SynD) software [40].

Fig. 1

Sema 3A overexpression induces axonal retraction and increased dendritic arborization in neurons after 4 days in culture. A IF analysis with Ank G (green) and β-III Tubulin (red) markers of neurons transfected with: Sema 3A-GFP (Sema 3A); Sema 3A siRNA (siSema 3A); Npn 1 siRNA (siNpn 1); and Sema 3A-GFP + siNpn 1 (Sema 3A + siNpn 1). As controls, we used neurons transfected with GFP empty vector (GFP) or non-transfected (Ctrl). Scale bar: 10 µm. 40 × objective. Skeleton mask is reported in B. C Axonal length was assessed by Neuron J plug-in. Data are the mean ± SEM of three independent experiments in triplicate (approximately 100 neurons). One-way ANOVA followed by Tukey’s test for multiple comparisons. *P < 0.05; **P < 0.01; ****P < 0.0001. D IF analysis with MAP-2 marker (red). Scale bar: 50 µm. 40 × objective. E Sholl analysis was performed by Neuroanatomy plug-in of ImageJ software on MAP-2-stained neurons. Data represent the number of dendritic branches at a given distance from the soma (1 µm-radius interval) and are the mean ± SEM of three independent experiments in triplicate. Two-way ANOVA followed by Tukey’s test for multiple comparisons. *P < 0.05 vs Ctrl and.#P < 0.05 vs Sema 3A + siNpn 1. See also Additional File 1: Table S2 for single value analysis. F Dendritic branches were clustered according to Strahler orders’ classification using Neuroanatomy plug-in of ImageJ software. At least 10 neurons were analysed in each slide. Experiments were replicated three times in triplicates. Two-way ANOVA followed by Tukey’s test for multiple comparison. **P < 0.01; ***P < 0.001; ****P < 0.0001

Pictures in Fig. 2F were acquired with the LMS980 confocal microscope (Zeiss, Oberkochen, Germany) equipped with a Plan Apochromat 63x/1,40 Oil DIC M27 objective (Zeiss, Oberkochen, Germany), using a GaAsP-PMT detector (Zeiss, Oberkochen, Germany) and a Zen Blue Software (Zeiss, Oberkochen, Germany). Quantitative data of microglia were obtained after acquisition of huge tile regions (at least 64 frames) with Celldiscoverer7 system (Zeiss, Oberkochen, Germany) and measured Fluorescence Mean Intensity (MFI) through analysis module of Zen 3.1 Software (Zeiss, Oberkochen, Germany) (Fig. 2E). For each marker and each experiment, a threshold for the MFI was established and all the cells with higher level of fluorescence were counted as positive for that specific marker. Percentage of positive cells was calculated on total number of nuclei in the field.

Fig. 2

Sema 3A activates the microglia proinflammatory M1 phenotype 48 h after transfection. A Representative immunofluorescence pictures of the percentage of microglia cells transfected with Sema 3A-GFP (Sema 3A) or GFP empty vector (GFP). As control, we used non-transfected microglia cells (Ctrl). Scale bar: 55 µm. 10 × objective. B ELISA assay on media from Sema 3A, GFP and Ctrl microglia 48 h after transfection. Sema 3A levels were expressed as ng/ml and normalized on the number of alive cells (mean ± SEM from 10 different fields) for each experimental point. Data are the mean ± SEM of three independent experiments performed in triplicate. ****P < 0.0001 vs Sema 3A media. C Extent of cell survival obtained by counting the number of DAPI positive nuclei before and after Sema 3A transfection as well as in GFP and Ctrl microglia. Data are the mean ± SEM of three independent experiments in quadruplicate. One-way ANOVA followed by Tukey’s test for multiple comparisons. ****P < 0.0001 vs Sema 3A. D Quantitative analysis of TMEM 119, Iba1, CD86, CD68, and E iNOS and TNFα positive cells. MFI was performed on the entire slide using Zeiss Celldiscoverer7. Values are normalized on the number of DAPI positive cells for each slide and expressed as % of Ctrl. Data are the mean ± SEM of three independent experiments performed in quadruplicate. One-way ANOVA followed by Tukey’s test for multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.001. Representative images of iNOS (red) and TNFα (red) and Sema 3A or GFP (green) staining are reported in F. Scale bar: 10 µm. 63 × objective

Live imaging

Live Imaging analysis was performed at CEINGE Advanced Light Microscopy Facility using the automated platform Celldiscoverer7 system (Zeiss, Oberkochen, Germany) equipped with a heated stage (37 °C and 5% CO2) and an Orca flash 4.0 camera (Hamamatsu). Briefly, timelapse of 6 well plates containing NP exposed to Sema 3A, GFP and Ctrl media were acquired using a Plan Apochromat 20x/0.7 objective and 1 × tubelens. Images (24 frames) were captured at 5 min intervals (Zeiss, Oberkochen, Germany) in phase gradient contrast. Axonal retraction was quantified by manual measuring of the distance between the neuronal soma and the axon edge, at time 0 and after 60 min of exposure to Sema 3A or control media, using the Zen 3.1 Software (Zeiss, Oberkochen, Germany).

All materials used for cell culture, WB, IF and live imaging experiments are reported in additional files (see Additional File 1: Table S1).

Statistical analysis

Data are expressed as mean ± SEM. All of the experiments were performed at least three times. The appropriate statistical test was selected using GraphPad Prism software version 9.0 for Windows (GraphPad Software, San Diego, CA, USA) and reported in the legend for each figure.

Data and code availability

Any additional information about this paper is available from the lead contact upon request.