Temporal requirement for high SMN expression in SMA mice

Abstract

Spinal muscular atrophy (SMA) is caused by loss of the survival motor neuron 1 gene (SMN1) and retention of the SMN2 gene, resulting in reduced SMN. SMA mice can be rescued with high expression of SMN in neurons, but when is this high expression required? We have developed a SMA mouse with inducible expression of SMN to address the temporal requirement for high SMN expression. Both embryonic and early postnatal induction of SMN resulted in a dramatic increase in survival with some mice living greater than 200 days. The mice had no marked motor deficits and neuromuscular junction (NMJ) function was near normal thus it appears that induction of SMN in postnatal SMA mice rescues motor function. Early postnatal SMN induction, followed by a 1-month removal of induction at 28 days of age, resulted in no morphological or electrophysiological abnormalities at the NMJ and no overt motor phenotype. Upon removal of SMN induction, five mice survived for just over 1 month and two female mice have survived past 8 months of age. We suggest that there is a postnatal period of time when high SMN levels are required. Furthermore, two copies of SMN2 provide the minimal amount of SMN necessary to maintain survival during adulthood. Finally, in the course of SMA, early induction of SMN is most efficacious.

Figures

Figure 1.

Diagram of inducible Luciferase (Luci) Tetracycline response element (TRE) Survival motor neuron (SMN) transgenic construct and reverse tetracycline trans activator (rtTA) inserted into the ROSA26 locus with the floxed stop cassette removed (58). IRES, internal ribosome entry sequence; EGFP, enhanced green fluorescent protein; PminCMV, minimal cytomegalovirus promoter; Luc, Luciferase; SA, splice acceptor site; rtTA, reverse tetracycline trans activator; Dox, doxycycline.

Figure 2.

Analysis of SMN induction in neonatal mice. (A and B) Doxycycline was administered to the mother at PND0 (birth of the pups) and luciferase activity measured in the (A) brain and (B) spinal cord of the neonates at the indicated time after the start of induction. D indicates the number of days after the start of induction. (C and D) Western blots of (C) brain and (D) spinal cord tissue of the neonates for quantification of luciferase (Luci), SMN and actin. Non-I indicates mice of the same genotype as induced mice but without induction, ▵7 indicates ▵7 carrier mice (SMN2+/+;Smn+/−; SMN▵7+/+) without the inducible transgene. All induced mice are heterozygous for mouse Smn (Smn+/−) and SMN is detected with a human specific antibody. (E and F) Quantification of luciferase to actin ratio (grey bars) and SMN to actin ratio (white bars) from western blots of neonatal mice post-induction shown in (C) and (D). (n= 24 mice at day 3, n= 17 mice at day 17, n= 19 mice at day 10). (G and H) Real-time RT–PCR of full-length human SMN mRNA relative to cyclophilin mRNA after induction of SMN in (G) brain and (H) spinal cord tissue. (n= 4 mice for Non-I, n= 4 mice at day 3, n= 5 mice at day 5, n= 7 mice at day 10).

Figure 3.

Analysis of SMN induction in adult mice. (A and B) Doxycycline was administered to the mice at 28 days of age and then western blots of (A) brain and (B) spinal cord tissue of adult mice for quantification of luciferase (luci), SMN and actin performed. Non-I indicates mice of the same genotype as induced mice but without induction, ▵7 indicates ▵7 carrier mice (SMN2+/+; Smn+/−;SMN▵7+/+) without the inducible transgene. All induced mice are heterozygous for mouse Smn (Smn+/−) and SMN is detected with a human specific antibody. (C and D) Quantification of SMN to actin ratio (white bars), and luciferase to actin ratio (gray bars), from western blots of adult mice post-induction shown in (A) and (B). (n= 3 mice for brain and n= 3 mice for spinal cord). (E) Western blot of a series of tissues harvested 10 days after SMN induction. Induction of SMN and luciferase is observed in all tissues tested. (F–H) Immunostaining of human SMN in motor neurons of spinal cord sections from (F) high-copy SMN2 mice, (G) mice induced to express SMN for 28 days and (H) non-induced mice. Immunostaining is performed with a human specific SMN antibody and animals were sacrificed at 28 days of age. Arrows indicate gems, scale bar is 50 µm.

Figure 4.

Survival analysis of induced mice. (A) Survival curve of SMA mice induced for SMN at embryonic day 13 compared with non-induced mice of the same genotype. Non-induced SMA mice lived an average of 13 days and none lived past 18 days (n= 29 mice). Induced mice lived on average for 132 ± 32 days and some lived passed 200 days (n= 14 mice), Log Rank P ≤ 0.001 compared with non-induced mice. (B) Survival curve of SMA mice induced for SMN at PND0/PND01 compared with non-induced mice of the same genotype. The average survival of mice with PND0/PND01 induction was 86 days with a number of mice living past 200 days (n= 38 mice), Log Rank P ≤ 0.001 compared with non-induced mice. The non-induced SMA mice lived an average of 13 days and none lived past 18 days (n= 29). When induction was started at PND02 some mice did show extended survival (mean 25 ± 9, max 151 days) but a lower and less pronounced response was observed (n= 15 mice). (C and D) Western blot analysis showing SMA mice with SMN and luciferase induction (+Dox) and SMA mice that had SMN induced for 28 days and then doxycycline was removed for 10 days (−Dox) in (C) brain and (D) spinal cord tissue. Human specific SMN antibody was used (48). Non-I indicates animals of the same genotype apart from the mouse Smn locus (Smn+/−) that were not fed doxycycline. ▵7 indicates ▵7 SMA carrier mice. WT indicates wild-type mice. (E) Weight curve of carrier Smn+/− mice (n= 20 mice) and rescued Smn−/− SMA mice (n= 10 mice) with SMN induction. Notice the reduced weight of rescued mice although they do steadily increase in weight.

Figure 5.

Correction of neuromuscular phenotype in SMA mice with SMN induction. (A) Shown are the endplate current (EPC) evoked by nerve stimulation, the miniature endplate current (MEPC) thought to represent the post-synaptic response to acetylcholine in one synaptic vesicle and the response to repetitive stimulation of the nerve at 50 Hz for both a control endplate and an endplate from a mouse where SMN expression was rescued by the doxycycline-driven construct. There is no significant difference between the control and the SMN rescued NMJ in either EPC or MEPC amplitude. There is slightly more depression during a 50 Hz train of stimuli for the control endplate. (B–G) There is no significant difference between the control (B–D) and the SMN rescued (E–G) NMJ morphology, size or innervation pattern. (B and E) Anti-neurofilament 160, (C and F) alpha-bungarotoxin, (D and G) merged image. Scale bar is 20 µm.

Figure 6.

Decay of SMN induction upon doxycycline removal. (A and B) Doxycycline was removed and luciferase (Luci) activity measured at 2, 3, 5 and 10 days after removal of SMN induction in (A) brain (B) spinal cord. The mice where induced for 28 days starting at PND0. (n= 3 for brain, n= 3 spinal cord). (C and D) Western blot analysis of SMN, luciferase and actin protein after removal of SMN induction in (C) brain and (D) spinal cord using a human specific monoclonal antibody. –D: days after doxycycline removal. Non-I: non-induced mice and ▵7: ▵7 carrier mice that lack the inducible transgene. (E and F) Quantification of western blots showing the luciferase to actin ratio (gray bars) and the SMN to actin ratio (white bars) and at 2, 3, 5 and 10 days after removal of doxycycline in (E) brain (n= 3 mice) and (F) spinal cord (n= 3 mice). (G and H) Real-time RT–PCR of full-length human SMN mRNA relative to cyclophilin mRNA after removal of SMN induction for 15 days (–15D, n= 5 mice) compared with 28-day induction (I, n= 8 mice) and non-induced mice (Non-I, n= 6 mice) in (G) brain and (H) spinal cord tissue.

Figure 7.

No neuromuscular phenotype in SMA mice with removal of SMN induction. (A) Shown are the EPC, MEPC and the response to 50 Hz stimulation for both a control endplate and an endplate from a mouse where SMN expression was rescued by the doxycycline-driven construct, but doxycycline was removed 1 month earlier. (B–G) There is no significant difference between the control (B–D) and the SMN rescued (E–G) NMJ morphology, size or innervation pattern. (B and E) Anti-neurofilament 160, (C and F) alpha-bungarotoxin, (D and G) merged image. Scale bar is 20 µm.