AAV-NRIP gene therapy ameliorates motor neuron degeneration and muscle atrophy in ALS model mice

Background Amyotrophic lateral sclerosis (ALS) is characterized by progressive motor neuron (MN) degeneration, leading to neuromuscular junction (NMJ) dismantling and severe muscle atrophy. The nuclear receptor interaction protein (NRIP) functions as a multifunctional protein. It directly interacts with calmodulin or α-actinin 2, serving as a calcium sensor for muscle contraction and maintaining sarcomere integrity. Additionally, NRIP binds with the acetylcholine receptor (AChR) for NMJ stabilization. Loss of NRIP in muscles results in progressive motor neuron degeneration with abnormal NMJ architecture, resembling ALS phenotypes. Therefore, we hypothesize that NRIP could be a therapeutic factor for ALS. Methods We used SOD1 G93A mice, expressing human SOD1 with the ALS-linked G93A mutation, as an ALS model. An adeno-associated virus vector encoding the human NRIP gene (AAV-NRIP) was generated and injected into the muscles of SOD1 G93A mice at 60 days of age, before disease onset. Pathological and behavioral changes were measured to evaluate the therapeutic effects of AAV-NRIP on the disease progression of SOD1 G93A mice. Results SOD1 G93A mice exhibited lower NRIP expression than wild-type mice in both the spinal cord and skeletal muscle tissues. Forced NRIP expression through AAV-NRIP intramuscular injection was observed in skeletal muscles and retrogradely transduced into the spinal cord. AAV-NRIP gene therapy enhanced movement distance and rearing frequencies in SOD1 G93A mice. Moreover, AAV-NRIP increased myofiber size and slow myosin expression, ameliorated NMJ degeneration and axon terminal denervation at NMJ, and increased the number of α-motor neurons (α-MNs) and compound muscle action potential (CMAP) in SOD1 G93A mice. Conclusions AAV-NRIP gene therapy ameliorates muscle atrophy, motor neuron degeneration, and axon terminal denervation at NMJ, leading to increased NMJ transmission and improved motor functions in SOD1 G93A mice. Collectively, AAV-NRIP could be a potential therapeutic drug for ALS. Supplementary Information The online version contains supplementary material available at 10.1186/s13395-024-00349-z.


Fig. S1
Fig. S1 NRIP expression in the spinal cord and skeletal muscles at 4 and 6 weeks of age in WT and SOD1 G93A mice.(A) NRIP expression in the spinal cord at 4 weeks of age in WT and SOD1 G93A mice.Total proteins from L3-L5 spinal cord were subjected to Western blot (WB) analysis for NRIP expression, with GAPDH serving as the loading control.The right panel shows the quantification of NRIP expression conducted through densitometry analysis.(B) NRIP expression in the gastrocnemius (GAS) muscles at 4 weeks of age in WT and SOD1 G93A mice.The right panel illustrates the quantification.(C) NRIP expression in the tibialis anterior (TA) muscles at 4 weeks of age in WT and SOD1 G93A mice.The right panel displays the quantification.(D) NRIP expression in the spinal cord at 6 weeks of age in WT and SOD1 G93A mice.The right panel represents the quantification of NRIP expression.(E) NRIP expression in the gastrocnemius (GAS) muscles at 6 weeks of age in WT and SOD1 G93A mice.The right panel illustrates the quantification.(F) NRIP expression in the tibialis anterior (TA) muscles at 6 weeks of age in WT and SOD1 G93A mice.The right panel shows the quantification.Each group consisted of N = 3 mice for both WT and SOD1 G93A.Data are presented as mean ± SEM.Statistical analysis was conducted using the Student t-test.*P < 0.05 and **P < 0.01.

Fig. S2
Fig. S2 Immunofluorescence analysis of AAV-GFP expression in the spinal cord of WT mice via intramuscular injection.The analysis was conducted on both control WT mice (without AAV-GFP treatment) and WT mice administered with AAV-GFP through intramuscular injection.The spinal cord was dissected into cervical, thoracic, and lumbar segments.Each segment was co-stained with anti-GFP (green, indicating GFP expression) and anti-NeuN (red, a neuron marker) antibodies.DAPI was used as the nuclear stain.Scale bars: 100 μm.

Fig. S3
Fig. S3 Transgene copy number determination in SOD1 G93A mice treated with AAV-NRIP and AAV-GFP.The transgene copy number in SOD1 G93A mice treated with AAV-NRIP and AAV-GFP was determined by real-time PCR.The ΔCt (cycle threshold) of transgene copy number was assessed by comparing a reference gene (mouse apob) with the transgene (human SOD1).The AAV-GFP group comprised 3 mice, and the AAV-NRIP group comprised 4 mice.Data are presented as mean ± SEM.Statistical analysis was performed using the Student t-test.ns, no significance.

Fig. S4
Fig. S4 Total distance moved and rearing frequency of SOD1 G93A mice with AAV-GFP or AAV-NRIP treatment in males and females.(A) The total distance moved by SOD1 G93A mice with AAV-GFP or AAV-NRIP treatment was recorded from 100 to 147 days using EthoVision Video Tracking Software (Noldus) during a 10-minute test.The total distance traveled in the box was measured.In the male group, data are presented as mean ± SEM. *P =0.045 at the age of 126 days; ns, no significance; oneway ANOVA with Tukey's post hoc test.In the female group, data are presented as mean ± SEM. *P =0.0496 at the age of 120 days; ns, no significance; one-way ANOVA with Tukey's post hoc test.(B) Rearing frequency, indicating the frequency of mice standing on their hindlimbs in the box, was recorded from 100 to 147 days.In the male group, data are presented as mean ± SEM. *P =0.0166 at the age of 126 days; ns, no significance; one-way ANOVA with Tukey's post hoc test.In the female group, data are presented as mean ± SEM. *P =0.0381 at the age of 120 days; *P =0.0429 at the age of 133 days; ns, no significance; one-way ANOVA with Tukey's post hoc test.

Fig. S5
Fig. S5 Therapeutic effects of AAV-NRIP on rotarod performance and grip force via intramuscular injection in SOD1 G93A mice.(A) Evaluation of AAV-NRIP efficacy in rotarod performance of SOD1 G93A mice at the age of 120 days.Mice were placed on a rotating rod set at 20 rpm, and the time until falling was recorded.N=6 for each group.(B) Analysis of grip force.Forelimb strength was measured five times for each mouse at the age of 120 days.N=6 for each group.Data are presented as mean ± SEM.Statistical analysis was conducted using the Student t-test.ns, no significance.(C) Kaplan-Meier survival curves comparing AAV-NRIP-treated and AAV-GFP-treated SOD1 G93A mice.Statistical differences were analyzed using the Logrank test.ns, no significance.