IPLEX administration improves motor neuron survival and ameliorates motor functions in a severe mouse model of spinal muscular atrophy

Abstract

Spinal muscular atrophy (SMA) is an inherited neurodegenerative disorder and the first genetic cause of death in childhood. SMA is caused by low levels of survival motor neuron (SMN) protein that induce selective loss of α-motor neurons (MNs) in the spinal cord, resulting in progressive muscle atrophy and consequent respiratory failure. To date, no effective treatment is available to counteract the course of the disease. Among the different therapeutic strategies with potential clinical applications, the evaluation of trophic and/or protective agents able to antagonize MNs degeneration represents an attractive opportunity to develop valid therapies. Here we investigated the effects of IPLEX (recombinant human insulinlike growth factor 1 [rhIGF-1] complexed with recombinant human IGF-1 binding protein 3 [rhIGFBP-3]) on a severe mouse model of SMA. Interestingly, molecular and biochemical analyses of IGF-1 carried out in SMA mice before drug administration revealed marked reductions of IGF-1 circulating levels and hepatic mRNA expression. In this study, we found that perinatal administration of IPLEX, even if does not influence survival and body weight of mice, results in reduced degeneration of MNs, increased muscle fiber size and in amelioration of motor functions in SMA mice. Additionally, we show that phenotypic changes observed are not SMN-dependent, since no significant SMN modification was addressed in treated mice. Collectively, our data indicate IPLEX as a good therapeutic candidate to hinder the progression of the neurodegenerative process in SMA.

Figure 1

IGF-1 is strongly impaired in SMNΔ7 SMA mice. (A) IGF-1 circulating levels were analyzed by ELISA in P10 WT and SMA mouse sera and reported as absolute quantification. (B) RT-qPCR analyses of IGF-1, IGFBP-3 and Igfals mRNAs levels in the liver of P10 WT and SMA mice. Data are reported as mean ± SEM of 10 animals of each group. (***P < 0.001)

Figure 2

IPLEX treatment attenuates muscle wasting in a mouse model of SMA. Hematoxylin and eosin–stained cross-section histological analysis of TA muscles at P10 in WT (A), SMA vehicle (D) and SMA IPLEX (G). Panels (B), (E), (H) show magnifications of WT, SMA vehicle and SMA IPLEX respectively. Immunofluorescence staining of Laminin in TA cross-section of WT (C), SMA vehicle (F) and SMA IPLEX (I), scale bar = 100 μm. (J) Frequency distribution of fiber size in TA muscles of WT, SMA vehicle and SMA IPLEX mice, Mann-Whitney rank sum test: ***P < 0.001. (K) mRNA levels were analyzed by real time RT-PCR in quadriceps isolated from mice daily treated with vehicle or IPLEX (60 mg/kg) on P2–P10. Data are representative of 10 mice. Values represent mean ± SEM. *P < 0.05; **P < 0.01.

Figure 3

IPLEX does not affect the transcription, protein levels and activity of SMN in spinal cord and skeletal muscles of SMA mice. (A) and (B): Real time RT-PCR analyses of SMNΔ7 and full length mRNAs were carried out on mouse tissues. Values represent mean ± SEM. *P < 0.05. (C) and (D): Western blot analysis of equal amount of proteins from SMA mice, injected with vehicle or IPLEX. (E) and (F): Relative quantification of SMN protein levels by densitometric analysis. Total RNAs from the spinal cord (G) and quadriceps (H) of P10 WT and SMA mice (treated with vehicle or with IPLEX) were analyzed by real-time RT-PCR for quantifying the levels of specific snRNAs. Dashed gray line corresponds to snRNA levels of WT mice treated with vehicle, used as references. Data are representative of three independent biological replicates. Values represent mean ± SEM. *P < 0.05; n.s. = not significant.

Figure 4

Administration of IPLEX does not increase the survival and body weight of SMA mice, but it improves the motor behavior of SMA mice. (A) Kaplan-Meier survival curves of SMA mice treated with IPLEX (n = 22) or vehicle (n = 15). (B) Body weight time course analysis of SMA mice treated with vehicle and SMA mice treated with IPLEX. (C) Mice were assessed from d 7 to d 9 for their ability and inability to right themselves when placed on their backs on a level surface (Righting Reflex). Values represent mean ± SEM, comparing SMA IPLEX to vehicle treated mice; *P < 0.05. (D) P9 WT and SMA mice treated with vehicle or IPLEX were tested for their latency to complete the geotaxis test (Negative Geotaxis). Values represent mean ± SEM, comparing SMA IPLEX to SMA vehicle treated mice, *P < 0.05. SMA IPLEX versus SMA vehicle, Bonferroni post hoc test.

Figure 5

IPLEX prevents loss of MNs in the spinal cord of SMA mice. NF-H immunostaining in spinal cord ventral horn of WT (A, B, C), SMA vehicle (D, E, F) and SMA IPLEX (G, H, I) P10 mice. Figures 5B, E and H show Hoechst counterstaining. Figures 5C, F and I represent a particular of Figures 5B, E and H, respectively. (J) MNs mean number for section of WT, SMA vehicle and SMA IPLEX mice. Data were reported as mean ± SEM, Mann-Whitney rank sum test: *P < 0.02.