Mms 435p

Primary hippocampal neurons were isolated as described above or obtained from Life Technologies (A10840-01). Cells were plated in Neurobasal medium supplemented with 10% FBS, 1% glutamine, 2% G21, and Pen/strep. The following day, the medium is changed to serum-free Neurobasal. On day 6–7, cells were transduced with the indicated lentivirus (1 × 10 e9/ml) at 1:1000 dilution overnight, and the medium was replaced the next day. After allowing an additional day of recovery, cells were pulsed for 30 min with 100 µM forskolin (Sigma # F6886) prior to fixtation involving a 15 min pre-fix with 4% PFA in PBS added at a 1:1 ratio to the existing medium, followed by 10 min in 4% PFA solution only. Cells were then probed for p-CREB (i.e., cell signaling), BIII-tubulin (BioLegend MMS-435P) and the corresponding fluorescent secondary antibodies as indicated, as well as for DAPI. Cells were then imaged by fluorescent microscopy, and cell total corrected fluorescence of p-CREB in infected neurons was obtained using ImageJ. Statistical analyses were performed using Tukey’s multiple comparisons test under one-way ANOVA (GraphPad Prism6 software). Multiplicity adjusted p-values are reported.

Postnatal day 1 (P1) Ambra1^+/gt and Ambra1^+/+ littermates were used to isolate RGCs. The eyes were extracted and the retina isolated. The retina was dissociated using the Papain kit from Worthington (PDS LK003150l). Then 25,000 retinal cells were incubated in 96-well plates (Screenstar microplate, Greiner Bio-one, 655866), pre-incubated with poly-l-lysine and laminin, with neurobasal A (ThermoFisher, 21103049) medium containing 2% B27 (ThermoFisher, 17504044), 0.5% gentamicin (Gibco, 15710-049), 0.25% l-glutamine (Gibco, 25030-024). Cells were incubated up to 3 days. Light microscopic pictures were taken after 1 and 3 days in vitro (DIV) and axonal neurites were measured manually in masked pictures. Five images were taken per well (n = 15 per time point). DAPI (Sigma, D9542; 1:1000) staining was performed to determine the total number of cells as well as the number of apoptotic nuclei, which were assessed for each condition in a masked manner. Neuronal Class III Beta-Tubulin (TUJ1) (Biolegend, MMS-435P at 1:500) staining was performed to analyse the % of RGCs in the culture.

Primary antibodies included rabbit anti-hrGFP (Agilent Technologies, 240242, 1:200–1000), mouse anti-tubulin beta-3 chain (TUJ1 mouse, Covance/BioLegend, MMS-435P, 1:300), rabbit anti-tubulin beta-3 chain (TUJ1 rabbit, Covance/BioLegend, MRB-435P, 1:300 or Sigma T2200, 1:200), mouse anti-nestin (Millipore, MAB5326), rabbit anti-VGLUT1 (Synaptic Systems, 135 303, 4 μg/ml), rabbit anti-MAP-2 (Millipore, AB5622, 1:500), mouse anti-synaptophysin (SYP, BD Biosciences, 611880, 1:200), rabbit anti-CD45 (AbdSerotech, MCA1130 1:250), rat anti-CD11b (integrin alpha-M, BioRad, MCA74GA, 1:100), mouse anti-L1cam/MAC387 (BioRad, MCA874GT, 1:100), mouse anti-CD4 (BioRad, MCA749S, 1:100), rabbit anti-vimentin (Abcam, AB92547, 1:2000), and mouse anti-GFAP (Sigma, G3893, 1:2000).

Protein lysate was loaded on a NuPAGE 4–12% Bis-Tris Gel (Thermo Fisher Scientific). After electrophoresis, proteins were transferred onto a nitrocellulose membrane (Bio-Rad) and subsequently incubated with the following primary antibodies: mouse anti-FOXA2 (#sc374376, Santa Cruz, 1:500), rabbit anti-LMX1A (#ab10533, Millipore, 1:1,000), mouse anti-TUJ1 (#MMS-435P, Biolegend, 1:1,000), rabbit anti TH (#657012, Calbiochem, 1:1,000), mouse anti-GAPDH (#MAB374, Santa Cruz, 1:1,000). Image Lab (Bio-Rad) was used for densitometric analysis of the blots.

Immunocytochemical analyses were performed according to a standard protocol. Briefly, cells were fixed in 2% paraformaldehyde (Nacalai Tesque, Kyoto, Japan) for 1 h at room temperature, washed 3 times with PBS, and incubated in PBS containing 0.1% Triton X-100 for 10 min, as well as in 3% skim milk/PBS for 2 h at room temperature. The fixed cells were then incubated with the following primary antibodies diluted in 3% skim milk/PBS at 4 °C overnight: mouse anti-III-tubulin (MMS-435P, 1:2000, BioLegend, San Diego, CA, USA) or rabbit anti-MAP2 (AB5622, 1:1000, MilliporeSigma, Burlington, MA, USA). After washing three times with PBS, the cells were incubated with secondary antibodies diluted in 3% skim milk/PBS: Alexa Fluor 488 goat anti-mouse IgG (1:1000, Thermo Fisher Scientific, Waltham, MA, USA) or Alexa Fluor 594 goat anti-rabbit IgG (1:1000, Thermo Fisher Scientific, Waltham, MA, USA) for 2 h at room temperature. The DNA was stained with DAPI (D523, 1:1000; DOJINDO, Kumamoto, Japan) for 15 min. Images were analyzed using a fluorescence microscope (Axio Vert.A1,Carl Zeiss, Oberkochen, Germany).