2.1 Cell culture
HEK293T cells are cultured in DMEM (Dulbecco’s modified Eagle’s medium) containing 10% fetal bovine serum, 2mM L-glutamine and 1% penicillin-streptomycin (Thermo Fisher), incubated in 37℃, 5% CO2.
2.2 HEK233T cell transfection
One day before transfection, seed 1*106 293T cells in 10cm dish to achieve ~70-80%. We used jetPRIME® (polyplus) to transfect plasmids into cells. The ratio between DNA and jetPRIME reagent is 1:2 or 1:3, total DNA is 10-15 μg/plate. Incubate the cells 24-48 hours post transfection.
2.3 Protein extraction and western blot
The total protein of 293T cells were extracted by RIPA lysis buffer (20mM Tris-HCl pH8.0, 137mM NaCl, 2mM EDTA, 1% NP-40) with 1% protease inhibitors (Roche) and 1% phosphatase inhibitor cocktail 2, 3 (Sigma), incubate on ice for 20 minutes. Protein lysate were clarified by centrifugation at 13,200 rpm for 25 minutes at 4℃, the supernatant was collected and stored in -80℃. Protein samples were separated on 10%
SDS-PAGE Electrophoresis system then transferred to PVDF membranes (Millipore).
The membrane was blocked in 5% bovine serum albumin (BSA) for 1 hour in room temperature, then incubated with primary antibodies anti-GFP (ab183734, Abcam, 1:10000), anti-FLAG (F3165, Sigma, 1:10000) amd anti-GAPDH (YIF-LF-PA0212, AbFrontier, 1:10000) diluted in blocking buffer, incubated overnight at 4℃. Target protein expression was detected using an ECL western blot detection system (PerkinElmer).
2.4 Immunoprecipitation
293T cells were transfected with 10μg pEGFP-NRIP or NRIP mutants, 1μg FLAG-AChR-α, p3XFLAG-CMV14 and pEGFP as internal control by jetPRIME transfection reagent (Polyplus). Cells were harvested 48 hours after transfection. Equal amount of protein lysates were incubated with adjust lysis buffer and antibody as mentioned at 4℃
overnight. 30μl Protein G Sepharose (GE Healthcare) beads were added to lysates and incubated at 4℃ for 2 hours. Wash the beads gently with lysis buffer (in the absent or present of 0.1% SDS) for 3 times. Targeting protein were eluted by 5X sample dye and following performed Western blot.
2.5 Immunofluorescence assay of cells
293T cells were seeding on cover-glass one day before transfection. Cells were
transfected with indicated plasmids by jetPRIME and incubated for 24 or 48 hours. The cells were rinsed with phosphate-buffered saline (PBS) three times. For immunofluorescence assay without antibody labeling, cells were fixed and permeabilized by ice cold methanol 5 minutes. After 3 times 10 minutes’ washes with PBS, the cover-glass were picked and mounted in DAPI Fluoromount-G (SounthernBiotech, Birmingham, AL, USA). For immunofluorescence assay that required antibodies labeling, cells were fixed in 2% para-formaldehyde (PFA) for 10~15 minutes, then washed with PBS for 3 times. Samples were then permeabilized by ice cold methanol 5 minutes, following washed with PBS for 3 times. Cells were blocked with 2% BSA in PBS for one hour at room temperature, then incubated with primary antibodies 4℃ overnight: anti-FLAG (F3165, Sigma), anti-GFP (sc-9996, Santa Cruz). After three 10-minuites washed with PBS, samples were stained with fluorescent secondary antibodies for 1 hour at room temperature and mounted in DAPI. For Wheat-germ agglutinin (WGA) staining for cell membrane, WGA were stained after 2% PFA fixed for 1 hour. Immunostained samples were analyzed by Carl Zeiss LSM880 confocal microscope.
2.6 AAV production
The gene fragment including EGFP-NRIP-C and EGFP-NRIP-CΔWD7 was amplified by PCR using pEGFP-NRIP-C and pEGFP-NRIP-CΔWD7 as template. The product was
then digested by BamHI and SalI and ligased into AAV-MCS vector under control of Human cytomegalovirus (CMV) promoter which is done by Szu-Wei Chang. For production of AAV-NRIP-C and AAV-C-ΔWD7, HEK293T cells were co-transfected with p AAV-NRIP-C or p AAV-C-ΔWD7, pAAV-DJ/8 and the adenovirus helper plasmid pHelper by calcium phosphate transfection and cultured for 72 hours. Cell lysates were harvested and lysed by freeze-thaw cycle, cause viruses to release to supernatant. The AAV particles were purified by CsCl density-gradient ultracentrifugation and dialyzed by dialysis cassette (Slide-A-Lyzer dialysis cassettes, Thermo; MWCO 10K) in dialysis buffer containing 350 mM NaCl with 5% sorbital in 1X PBS at 4℃. The viral titers were determined by dot blot assay. Virus were separated into small portions and stored at -80℃
until future used.
2.7 Muscle-specific NRIP knockout mice
The generation of muscle-specific NRIP knockout mice was described in previous study (Chen et al., 2015). Mouse NRIP genomic DNA (bMQ134h07) was obtained by screening a BAC library derived from mouse strain 129, which is a 19.2 kb mouse genomic DNA fraction inserted at the NotI-SpeI site of pL253, MC1-TK (thymidine kinase) gene that served as negative selection marker. The resulting construct was used as the backbone for subsequent insertion of loxP sequence from pL452 into intron 1. The final targeting
construct contained a homologous short 5’ arm of 4.3 kb and long 3’ arm of 10.6 kb. The targeting vector was linearized by DNA digestion with NotI (unique site) and electroporated into embryonic stem (ES) cells, and then G418-resistant clones were selected. The ES cells containing the floxed NRIP allele were injected into blastocytes of C57BL/6JNarl mice and implanted into pseudo-pregnant foster mothers. The chimeric offspring was back-crossed to the C57BL/6Jnarl mice (more than 10 generations) to generate the NRIP-LoxP heterozygous (NRIPflox/+) mice. To obtain muscle-specific knockout animals, NRIPflox/+ mice were crossed with MCK (muscle creatine kinase)-Cre heterozygous mice (Bruning et al., 1998), then the NRIPflox/+ ; MCK-Cre+ offspring were crossed with each other to generate NRIPflox/flox ; MCK-Cre+ mice (muscle-specific NRIP knockout mice). Other littermates were used as wild type (WT) controls.
2.8 In vivo AAV injection
Mice were anaesthetized by intraperitoneal injection of 2.5% avertin (200~300 μl). AAV virus were given by intramuscular injection into each bilateral gastrocnemius and tibialis anterior muscles at age 6 weeks. The therapeutic were analyzed for NMJ integrity, axonal denervation and motor neuron survival 10 weeks after gene therapy.
2.9 Frozen section preparation of mice spinal cord and gastrocnemius
Mice were anesthetized with 2.5% avertin and sacrificed by heart excision. For spinal cord, the vertebral column was carefully dissected from thoracic T1 segment to the base of the tail. The paravertebral muscles were removed, and spinal cord were flushed out from the vertebral fraction by PBS with the needle/syringe technique. Searched for the vertebral foramen at the lower part. When the opening is identified, the tip of the needle was inserted in the spinal canal and push the plunger of the syringe containing 1X PBS to flush out spinal cord at the thoracic end. Fresh lumbar segment (L3-L5) of spinal cord was incised and embedded into O.C.T. compound immediately. The O.C.T. block was frozen in isopentane pre-cooled with liquid nitrogen and stored at -80℃. The 30μm thickness serial frozen cross-sections of spinal cord from lumbar L5 segment were prepared by cryostat microtome for immunofluorescence assay. For gastrocnemius, the fresh gastrocnemius muscle dissected from mice was covered with 2% PFA for 2 hours at room temperature. Wash the muscles with 1X PBS 10 minutes for 3 times, then dehydrate with 30% sucrose in PBS and incubate at 4℃ overnight. Dehydrated tissues were then embedded in O.C.T. compound, then frozen in isopentane pre-cooled with liquid nitrogen and stored at -80℃. The 30μm thickness frozen cross-section of GAS were prepared by cryostat microtome for immunofluorescence assay.
2.10 Immunofluorescence assay of neuromuscular junction (NMJ)
The 30μm thickness frozen cross-sections of GAS were wash and then fixed by 2% para-formaldehyde. Wash the slide 10 minutes in 1X BS for three times and incubated with 0.1M glycine for 30 minutes to block residual aldehyde group. Sections were washed by 1XPBS 10 minutes three times and permeabilized in ice-clod methanol for 7 minutes, then 1X PBS washed for 10 minutes 2 times. Next, sections were blocked in blocking buffer containing 0.2% Triton-X-100 and 2% BSA in PBS for 1 hour at room temperature, following by incubating with anti-neurofilament (anti-NF, ab8135, rabbit-monoclonal, Abcam, 1:500) and anti-synaptophysin (anti-SYN, ab32127, rabbit-monoclonal, Abcam, 1:250 dilution) in blocking buffer at 4℃ overnight. After 1X PBS washing, the sections were incubated with secondary antibodies (488-conjugated goat anti-rabbit, 1:10000 dilution) and Alexa-594-conjugated α-bungarotoxin (α-BTX, B13423, Life technologies, 1:10000 dilution) for 2 hours in dark at room temperature. Last, sections were washed by 1X PBS 10 minutes for 3 times, then mounted with DAPI Fluoromount-G. The immunofluorescence images were visualized and pictured by Carl Zeiss LSM880 confocal microscope and Zeiss Zen black software (confocal pinhole 1.0 Airy unit; collect a z-stack of endplates with a 0.7-1.2μm interval between each optical slice). Α-BTX-stained structure that extend < 15μm in the z-dimension is considered as endplate and is imaged for analysis. The intact region of α-BTX, anti-NF and anti-SYN was defined as innervated endplate; while α-BTX only considered as axonal denervation.
2.11 Immunofluorescence assay of spinal cord
The frozen sections of spinal cord were rinsed by 1X PBS for 10 minutes to remove O.C.T.
and fixed in 2% PFA for 5=10 minutes. Sections were washed by 1X PBS for 10 minutes 3 times and permeabilized by 1% Triton X-100 in PBS for 3 minutes, then washed by 1X PBS for 10 minutes 3 times immediately. Next, sections were blocked by 5% BSA in PBS for 1.5 hours at room temperature, then incubated with anti-NeuN (MABN140, Millipore, 1:500 dilution) and anti-ChAT (AB144P, Millipore, 1:250 dilution) at 4℃ overnight.
After reaction with primary antibodies, sections were washed by 1X PBS for 10 minutes 3 times, then incubated with secondary antibodies (Cy3-conjugated goat anti-rabbit and 488-conjugated goat anti-goat, 1:10000 dilution, Jackson ImmunoResearch Laboratories) for 1 hour in dark at room temperature. Finally, sections were washed by 1X PBS for 10 minutes 3 times then mounted with DAPI Fluoromount-G. The immunofluorescence images were visualized and pictured by Zeiss Axioskop 40 Optical Microscope with AxioCam 702 camera and Zeiss Zen blue software. Ventral horn cells with NeuN and ChAT double positive signal with cross-section area (CSA) > 500μm2 were defined as α-motor neurons.
2.12 Statistical analysis
All statistical analyses and graphs were performed using GraphPad Prism software (GraphPad software Inc.). Results were analyzed by two-tailed student’s t test in this study.
Data were presented as mean ± standard error of the mean (SEM). P-value < 0.05 was considered as statistically significant.