Fig.1. NRIP is a membrane-bound protein.
(A)The plasmid (named nuclear receptor interaction protein (NRIP-EGFP) was transfected into HEK293T cells by jetPRIME. After 24 hours, immunofluorescence assay was performed to examine NRIP distribution. EGFP-NRIP (green) localized at cytosol, membrane, and nucleus. (B) Co-stained with Wheat Germ Agglutinin (WGA; red, for cell membrane) and anti-GFP (NRIP). For WGA immunofluorescence staining of cell membrane, cells were incubated with WGA after 2% paraformaldehyde fixed, before permeabilized. Green: NRIP; red: cell membrane. (C) Co-stained with WGA(red, for Golgi apparatus) and anti-GFP (NRIP). For WGA immunofluorescence staining of Golgi apparatus, cells were incubated with WGA after 2% paraformaldehyde fixed and permeabilized by ice-cold methanol. Green: NRIP, red: Golgi apparatus. (D) Co-stained with calnexin (red, for endoplasmic reticulum marker) and anti-GFP (NRIP). Green:
NRIP; red: endoplasmic reticulum (ER). Scale bar, 5μm.
Fig.2. NRIP can interact with AChR α, or β, or δ subunit.
(A) NRIP interacts with either AChR-α or β. HEK293T cells were transfected with EGFP-NRIP and Flag-AChR-α or Flag-AChR-β by jetPRIME. 48 hours after transfection, immunoprecipitation with anti-EGFP (NRIP) was performed from of each transfected lysates; and then western blot analysis by anti-Flag (AChR). Total 1mg lysates were performed immunoprecipitation assay and 50 μg (5% of immunoprecipitation lysates) as input. GAPDH as an internal control. Arrow head indicates the precipitated AChR by NRIP. (B) NRIP interacts with either AChR-α or δ. HEK293T cells were transfected with EGFP-NRIP and Flag-AChR-α or Flag-AChR-β by jetPRIME transfection.
Immunoprecipitation with anti-EGFP (NRIP) was performed from each transfected lysates; and then western blot analysis by anti-Flag (AChR). Total 1mg lysates were performed immunoprecipitation assay and 50 μg (5% of immunoprecipitation lysates) as input. GAPDH as an internal control. Arrow head indicates the precipitated AChR by NRIP, while star sign represents heavy chain.
Fig.3. NRIP colocalizes with AChR α, β, γ, δ.
Immunofluorescence assay with anti-GFP (green) and anti-Flag (red) in HEK293T cells co-transfected Flag-AChR α, β, γ, δ subunits together with EGFP-NRIP by jetPRIME for 48 hours. NRIP was colocalized with AChR 4 subunits in cells (merge). The AChR aggregation was counted by area >0.5 μm2 as AChR cluster). The AChR aggregates in cytoplasm were observed in cytoplasm when co-transfect EGFP-NRIP. Scale bar, 5 μm.
Fig.4. The WD40 domain 7 located at NRIP-C is responsible for for AChR-α binding.
(A) Schematic map of NRIP mutants. Green box represents EGFP, grey box as WD40 domains, and orange box as IQ domain. The mutants include: EGFP-tagged NRIP full length FL); EGFP-tagged N-terminal of NRIP, containing WD1 ~ WD5 (NRIP-N); EGFP-tagged C-terminal of NRIP, containing IQ domain and WD6~WD7 (NRIP-C);
EGFP-tagged NRIP full length truncated IQ domain (NRIP-ΔIQ), containing WD1 ~ WD7 domain and truncated IQ domain; EGFP-tagged NRIP C-terminal truncated WD7 domain (ΔWD7), containing IQ domain and WD6 domain; and EGFP-tagged C-terminal truncated WD6,7 domain (C-ΔWD67), containing IQ domain only (B) Mapping domain of NRIP for AChR-α binding. Immunoprecipitation assay for NRIP mutants-binding with AChR-α, HEK293T cells were transfected with Flag-AChR-α and EGFP-NRIP mutants by jetPRIME for 48 hours. Immunoprecipitation with anti-FLAG (AChR-α) was performed from each transfected lysates; and then western blot analysis by anti-GFP (NRIP). Total 1mg lysates were performed immunoprecipitation assay and 50 μg (5% of immunoprecipitation assay) as input. GAPDH as an internal control. Both NRIP-FL and NRIP-ΔIQ had interaction with AChR-α. EGFP-NRIP-C, containing WD6 and WD7 domain, had AChR binding ability; while C-ΔWD7 lost AChR-α binding;
indicating WD7 domain is responsible for AChR-α binding. Arrow head: the precipitated NRIP mutant protein by AChR-α.
Fig.5. The cluster formation of NRIP with AChR-α in 293T cells.
(A) HEK293T cells were co-transfected with mCherry-AChR-α and each EGFP-NRIP mutant by jetPRIME for 48 hours. The plasmid (pmCherry-AChR-α) was constructed by Hsin-Hsiung Chen (Supplementary Fig.S5.). The cells were fixed and then pictured by confocal microscopy without antibody labeling, that the green and red signals were self- fluorescence of EGFP and mCherry. Green: EGFP or NRIP mutants; red: AChR-α; merge:
combine signals of EGFP-NRIP mutant and AChR-α. The AChR aggregation area >0.5 μm are defined as AChR cluster. Scale bar, 5μm. (B) Quantification analysis of the cluster number was counted as the AChR aggregation size >0.5μm. NRIP-FL group contained more cluster formation than control (EGFP vector only) (47.95 vs. 0.3 per cell). The cluster number between NRIP-FL and NRIP-C have no significant difference (47.95 vs.
46.65); while C-ΔWD7 have decrease cluster number than NRIP-FL (11.55 vs. 46.65, P<0.0001). (C) Quantification analysis of % of NRIP mutants for cluster formation to NRIP-Fl. The average cluster number of NRIP-FL set as 100%, cluster number of each mutant was calculated to percentage to NRIP-FL. Set 25% of NRIP-FL cluster number as border line, cluster number which is more than 25% of NRIP-FL were defined as positive AChR cluster formation, such as NRIP-C and NRIP-ΔIQ (95.20% and 84.98%). Cluster number which is less than 25% NRIP-FL was defined as negative AChR cluster formation, such as NRIP-N and C-ΔWD7 (23.25% and 24.09%). (D) Quantification analysis of the cluster number locolized on cell membrane was counted as the AChR aggregation size
>0.5μm. NRIP-FL group contained cluster formation on membrane while control group (EGFP vector only) didn’t (19.7 vs. 0 per cell). The cluster number between NRIP-FL and NRIP-C have no significant difference (19.7 vs. 18.45); while C-ΔWD7 have decrease cluster number than NRIP-FL (11.55 vs. 4.6, P<0.0001). Total 20 cells per group were counted from 5 independent experiments. Data are mean ± SEM by two tailed Student‘s t-test. ****P<0.0001; ns: no significance.
Fig.6. Subcellular location of NRIP mutants in HEK293T cells.
(A) Fluorescent image of EGFP-NRIP (green) subcellular location at HEK293T cells. 24 hours after transfection; NRIP-full length and NRIP-ΔIQ expressed both at cytoplasm and nucleus, while NRIP-C expressed only in cytoplasm, and NRIP-N expressed in nucleus. (B) Summary of the subcellular location, AChR-α binding and cluster formation.
Both NRIP and NRIP-ΔIQ, which expressed in cytoplasm and nucleus, had AChR-α binding ability and cluster formation in HEK293T cells. NRIP-N, expressed only in nucleus, had weak AChR binding ability and cluster formation compared to NRIP. NRIP-C, expressed in cytoplasm, had strong AChR binding ability and cluster formation. C-ΔWD7 expressed in cytoplasm same as NRIP-C, but lost its binding ability and cluster formation due to loss of WD7; implying that WD7 domain of NRIP is responsible for AChR-α binding. Scale bar, 10μm.
Fig.7. Adeno-associated virus (AAV)-NRIP C and AAV-C-ΔWD7 generation.
The adeno-associated viruses (AAV) encoding NRIP-C or C-ΔWD7 were generated. Co-transfected pAAV-DJ/8, pHelper and pAAV-MCS-NRIP-C (or pAAV-MCS- CΔWD7) into HEK293T cells using calcium phosphate transfection for 72 hours; the cell lysates were harvested and cells were broken by several freeze/thaw cycles. Supernatant viruses were harvested and purified through CsCl density-gradient ultracentrifugation and dialysis. (A) Western blot analysis of the recombinant AAV-NRIP-C and AAV-C-ΔWD7 infection efficiency. The recombinant viruses were infected in HEK293T cells; 24 hours after infection, anti-EGFP was immunostained for NRIP-C and C-ΔWD7 expression in different dose of viruses; due to each construct contained EGFP tag. GAPDH as internal control. Left panel: arrow indicates NRIP-C’s band. Right panel: expression of C-ΔWD7 in different dose. (B) Immunofluorescence assay of anti-EGFP (green) for NRIP-C or C-ΔWD7) expression and DAPI (nucleus, blue) in frozen sections from gastrocnemius (GAS) in AAV-treated NRIP cKO mice. 6-weeks cKO mice were given intramuscular injection to bilateral GAS muscles of virus (AAV-NRIP-C: 2.7*109 vg in a total volume of 20 μl for each muscle; AAV- C-ΔWD7: 3.6*1010 vg in a total volume of 20 μl for each muscle.). GAS muscles were collected 1 week after virus injection. GAS muscles of WT mice without virus infection were collected as no virus control. Scale bar, 50μm.
Fig.8. Intramuscular NRIP-C gene therapy of NRIP cKO can rescue neuromuscular integrity.
(A) Schematic protocol for AAV-NRIP-C gene therapy. Given intramuscular (i.m.) injection of AAV-EGFP (control), AAV-NRIP-C or AAV-C-ΔWD7 into bilateral gastrocnemius and tibialis anterior muscles at age 6-week old NRIP cKO mice. 10 weeks after injection, the mice were sacrificed and analyzed NMJ integrity and α-motor neuron number. (B) Immunofluorescence assay of α-BTX [red, for acetylcholine receptors (AChRs)], anti-synaptosphysin (SYN) and anti-neurofiliment (NF) [green, for axonal terminals] and DAPI (blue) in frozen sections from gastrocnemius (GAS) in AAV-treated cKO mice 10 week after virus injection. Scale bar, 20 μm. (C) Quantitative analysis of neuromuscular junction area. The quantification of NMJ area was analyzed by ImageJ software. The NMJ area of AAV-NRIP-C-treated cKO mice was larger than control group (236.06 μm2 vs. 189.05 μm2, P<0.01) and AAV-C-ΔWD7 treated group (236.06 μm2 vs.
204.31 μm2, P<0.05). Control, N=3 mice; AAV-NRIP-C treatment, N=3 mice; AAV-C-ΔWD7 treatment, N=4 mice. The number inside each bar represents the number of
examined NMJ (at least 30 NMJs were counted each GAS). (D) Axon denervation analysis. Denervation was defined as no nerve terminal overlapped to AChR cluster.
White arrow indicates denervation endplates. The proportion of denervated endplates (measured by the percentage of denervated endplates to the total endplates on one GAS muscle frozen section), decreased in AAV-NRIP-C-treated cKO mice compared with control mice (6.58% vs. 11.36%); compared with AAV-C-ΔWD7 (6.58% vs. 10.70%).
Control, N=3 mice; AAV-NRIP-C treatment, N=3 mice; AAV-C-ΔWD7 treatment, N=4 mice. Data are mean ± SEM by two tailed Student‘s t-test. *P<0.05 and **P<0.01
Fig.9. Intramuscular NRIP-C gene therapy can rescue the survival of motor neurons of NRIP cKO mice.
(A) Immunofluorescence assay of NeuN (red), ChAT (green) and DAPI (blue) in frozen section of spinal cord L3-L5 regions from AAV-treated cKO mice was performed 10 weeks after virus injection. Yellow cells which showed double positive signal of NeuN and Chat with cross-section area (CSA) >500 μm2 would be counted as α-motor neurons.
Scale bar, 50 μm. (B) Quantification analysis of α-motor neuron number per anterior horn.
The α-motor neuron numbers of AAV-NRIP-C treated cKO mice were higher than control group (23.33 vs. 17.25, P<0.05). AAV-NRIP-C treated cKO mice also had higher α-motor neuron numbers than AAV-C-ΔWD7group (23.33 vs. 17.63, P<0.05). Control, n=3 mice;
AAV-NRIP-C treatment, n=2 mice; AAV-C-ΔWD7 treatment, n=3 mice. Data are mean
± SEM by two tailed Student‘s t-test. *P<0.05
Fig.10. Summary comparison of intramuscular AAV-NRIP mutants gene therapy in rescuing neuromuscular integrity and survival of motor neuron in NRIP cKO mice.
These data were in combination with Yun-Hsin’ data by normalized AAV-NRIP mutants data to AAV-EGFP group.
(A) Quantitative analysis of neuromuscular junction area. The quantification of NMJ area of NRIP mutants was analyzed, NRIP mutants were normalized to AAV-EGFP group (set as 1), then analyzed as fold change of each mutant to the corresponding AAV-EGFP control. The NMJ area of AAV-NRIP treated cKO mice was larger than control group (AAV-EGFP) (1 vs. 1.14, P<0.05). The NMJ area of AAV-NRIP-C-treated cKO mice was also significantly larger than control group (1 vs. 1.25, P<0.001). There was no significant difference between AAV-C-ΔWD7 treated group and control group (1 vs. 1.08). Control, N=9 mice; AAV-NRIP treatment, N=9; AAV-NRIP-C treatment, N=3 mice; AAV-C-ΔWD7 treatment, N=4 mice. (B) Axon denervation analysis. The proportion of denervated endplates of NRIP mutants was normalized to AAV-EGFP group, then analyzed as fold change (NRIP mutant/EGFP control). Compare to the control group,
The proportion of denervated endplates also decreased in AAV-NRIP-C-treated cKO mice compared with control mice (1 vs. 0.58, P<0.05). In contrast, AAV-C-ΔWD7 treated cKO mice didn’t have significantly decreased denervated endplates than control group (1 vs.
0.94). Control, N=9 mice; AAV-NRIP treatment, N=9 mice; AAV-NRIP-C treatment, N=3 mice; AAV-C-ΔWD7 treatment, N=4 mice. (C) Quantification analysis of α-motor neuron number per anterior horn. The α-motor neuron numbers were normalized to AAV-EGFP group, then analyzed as fold change. The number of α-motor neuron of AAV-NRIP treated cKO mice were higher than AAV-EGFP-control group (1 vs. 1.17, P<0.05). AAV-NRIP-C treated cKO mice were higher than AAV-EGFP -control group (1 vs. 1.35, P<0.01).
There was no significant difference between AAV-C-ΔWD7 treated group and control group (1 vs. 1.02). Control, n=9 mice; AAV-NRIP treatment, N=8 mice; AAV-NRIP-C treatment, n=3 mice; AAV-C-ΔWD7 treatment, n=2 mice. Data are mean ± SEM by two tailed Student‘s t-test. *P<0.05, **P<0.01 and ***P<0.001.