Rabies Virus Hijacks and accelerates the p75NTR retrograde axonal transport machinery.

Animals and Dorsal Root Ganglion explants

ICR mice were bred and maintained at the Tel Aviv University animal care facility until the time of sacrifice. Spinal cords were dissected from E12.5–13.5 mice, followed by separation of Dorsal Root Ganglia (DRG) from meninges and additional spinal cord. The Institutional Animal Care Committee at the Tel Aviv University approved all the animal protocols in this work.

Microfluidic chamber preparation

Microfluidic chambers were fabricated using methods previously described in detail [56] . All microfluidic chambers were replica molded using PDMS (#41201841 Dow Corning) from masters that were patterned using the photosensitive epoxy SU-8 (Microchem). All masters consisted of two permanent SU-8 layers on a 3″ silicon wafer and were made in the clean room facility in Tel-Aviv University. The first layer of SU-8 (3 µm depth) contained the microgrooves, which were patterned by photolithography using a high-resolution chromium mask (5 µm minimum feature size; Advance Reproduction Corp.). The second layer of SU-8 (100 µm depth) contained the compartments, which were patterned by photolithography using a 20,000 dpi printed transparency mask (CAD/Art Services, Inc.). Chamber dimensions: channels: length 8.25 mm, width 1.5 mm; grooves: length 400 µm, width 15 µm, height 5 µm ( Fig. 1B ). A single 7 mm well was punctured into the “proximal” or explant channel, into which a “cave” was carved using a scalpel, to prevent explants from floating, 2 additional 1.2 mm wells were punctured into the channel on either side of the “explant” well to allow flow. Two 7 mm wells were punctured into both ends of the “distal” or axons channel to allow control over the distal channel. Microfluidic devices were cleaned of surface particles using adhesive tape and sterilized in 70% high-grade ethanol for 1 h. Devices were allowed to completely air dry under sterile conditions, attached to sterile 50 mm glass bottom dishes (FD5040-100, WPI) using gentle pressure and heated to 70°C for 20′ to improve adhesion to glass. Chambers were coated using 150 µl of 1.5 ng/ml Polyornithine (P-8638, Sigma) in PBS for 24 hours, which was replaced with 150 µl Laminin (L-2020, Sigma) 1∶333 in DDW for 24 hours. Laminin was replaced with culture medium until plating (1–3 days). Assembly and maintenance of DRG explants At the day of plating, media were removed from all wells, and a single DRG was inserted to each explant “cave” using a 20 µl tip. Following 1 hour of incubation at 37°C, 150 µl of medium were added to each well. Basic culture medium consisted of Neurobasal medium (Life Technologies) supplemented with 2% B-27 (Life Technologies), 1% penicillin-streptomycin (Biological Industries, Israel) and 1% Glutamax (Life Technologies). A gradient of murine NGF was created in order to encourage axons to cross the grooves to the distal axon compartment, by adding 100 ng/ml and 62.5 ng/ml murine NGF (Alomone labs) to the distal and proximal wells, respectively. Cultures were maintained at 37°C and 5% CO 2 , and media were refreshed every 2 days. Transport assays were initiated after axons had crossed the grooves and established an axonal network in the distal compartment, 3–5 days from plating ( Fig. 1D ). For TIRF imaging assays, 4–5 DRG were placed on 35 mm glass bottom dishes (FD35-100, WPI) with 20 µl each of DRG culture medium supplemented with 62.5 ng/ml NGF. DRG were incubated in 37°C for 4–5 hours to allow adhesion to glass, then supplemented with 2 ml DRG culture medium supplemented with 62.5 ng/ml NGF. Cultures were maintained at 37°c and 5% CO 2 for 48 hours before imaging. Fluorescent EGFP/mCherry rabies virus production rRABV EGFP-P ΔG/rRABV mCherry-P ΔG are recombinant rabies viruses in which EGFP or mCherry fluorescent reporters were fused to the phosphoprotein P and in which the glycoprotein G gene was deleted. Preparation and amplification was performed as previously described [14] , [57] . In brief, MG-136 cells, expressing the RABV matrix and glycoproteins after induction with doxycycline, were infected with rRABV EGFP-P ΔG. Cells were split, after which media were twice replaced with fresh medium supplemented with 1 mg/ml Doxycycline, to be collected 48 hours later. rRABV EGFP-P ΔG was concentrated from cell culture supernatants using the PEG virus precipitation kit (ab102538, Abcam). EGFP/sh-RNA-p75-EGFP lentivirus production HEK293t cells were transfected using calcium-phosphate precipitation with the viral vector pLL-EGFP and the helper plasmids pVSVG and pGag-PolGpt (kind gift from Eran Bacharach) or mix of 4 shRNA plasmids against murine p75, produced from Mouse GIPZ lentiviral target gene shRNAmir glycerol set (GE healthcare RMM4532-EG18053). Viral particles were collected 48 and 72 hours after transfection, filtered and concentrated using the PEG virus precipitation kit (ab102538, Abcam). DRG cultures, shRNA-p75 and RABV infection DRG were collected from E12.5–13.5 mice, trypsinized with Trypsin-EDTA solution B (Biological industries 03-052-1B) for 5 minutes in 37°C, and washed with complete F12 medium. Cells were dissociated by pipetting, counted and plated on PLO/Laminin coated 96 well plates, in a density of 15K cells per well. Cells were maintained with DRG culture medium supplemented with 62.5 ng/ml NGF. On day of plating, cells were infected with either LV-EGFP or a mix of 4 LV-shRNA-p75-EGFP (MOI≈5 and MOI≈10, respectively, due to high infectivity of LV-EGFP). 3–4 days from plating, cells were infected with ≈120K mCherry RABV particles for 0′, 30′ and 120′ in duplicates after which they were washed ×3 with DRG culture medium. 48 hours later, neurons showing obvious mCherry foci were counted in 6–9 fields per well, and divided by total number of counted neurons. Although cultures varied in axon network, non-neuronal cell populations and LV-infection levels, infection rates were lower in sh-RNA-p75-EGFP groups when compared to GFP control in all experiments. Due to differences in absolute infection rates, these were normalized to the rate obtained at the LV-EGFP control group. Infection of DRG explants in microfluidic chambers were performed on the day of plating, with ≈1×10 6 LV-EGFP particles, or a mix of 4×≈1–2×10 6 LV-sh-RNA-p75-EGFP particles. To estimate knockdown of p75, a DRG culture plated on a glass bottom dish was infected with sh-RNA-p75-EGFP as described above. At 4DIV, culture was treated with 1∶100 anti-p75-550 for 15′ and washed ×3 prior to imaging using spinning disc confocal. Fluorescent beads, markers and antibodies Qdot-NGF was prepared by mixing biotinylated murine-NGF 10 µg/ml (#N-240-B, Alomone Labs) with Quantum-Dot 605 streptavidin conjugate 1 µM ( Q10101 , Molecular Probes) in a molar ratio of 3∶1, respectively. Acidic compartments were delineated using Lysotracker Red DND-99 (Life Technologies) in a final concentration of 50 nM. Mitotracker Deep Red FM (Life Technologies) was used to denote mitochondria, in a final concentration of 100 nM. In order to track the co-transport of RABV with its receptors, the fluorescent extracellular antibodies Anti-p75NTR-ATTO-550, Anti-p75-ATTO-488 and Anti-TrkA-ATTO-633 (ANT-007-AO, ANT-007-AG and ANT-0180-FR, respectively, Alomone Labs) were used in a 1∶100 dilution. Live imaging of axonal transport Live imaging was performed on DRG explants grown in compartmental chambers, 3–5 DIV, upon forming an axonal network at the distal axon compartment. Prior to imaging, media from each well were replaced with 150 µl Neurobasal medium supplemented with 1% penicillin-streptomycin and 1% Glutamax (poor medium). Following two hours of starvation, 30 µl of poor medium were added to the proximal well, to induce compartmental separation. 2 µl of concentrated EGFP-RABV (≈120K particles), Qdot-NGF or both were added to one distal well, with additional 5 µl medium, to encourage flow through the distal axon channel ( Fig. 1B,C ). Chambers were incubated for 1–2 hours in 37°C and 5% CO 2 prior their placement on the microscope stage. Time lapse images of axons in the groove area ( Fig. 1B,C ), were acquired at 37°C and CO 2 controlled environment, using Nikon Eclipse Ti microscope equipped with Yokogawa CSU X-1 spinning disc confocal, controlled via iQ software (Andor). Multi-channel time lapses were captured using 60× lens, NA = 1.4 with 2000 msec intervals, with an approximate lag of 100–400 ms between channels. Digital images were taken with Andor iXon DU-897 EM-CCD camera.

Axonal transport image analysis

Time-lapse image analysis was carried out using Fiji, following subtraction of average intensity z-projection to exclude completely stationary fluorescent artifacts. XY coordinates of tracks were registered from 9 or more grooves (which contains typically 2–5 axons) in each experiment, using Manual Tracking plugin, while distances, velocities and MSD's were computed using MATLAB implementation for Fiji Trackmate plugin [58] . Tracks with run lengths