Identification of a battery of tests for drug candidate evaluation in the SMNΔ7 neonate model of spinal muscular atrophy

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Abstract

Spinal muscular atrophy (SMA) is characterized by selective loss of α-motor neurons and is caused by homozygous loss or mutation in the survival motor neuron (SMN1) gene. Loss of SMN1 is partially compensated by the copy gene, SMN2. Currently, there are no specific treatments for SMA. Key features of SMA are modeled in mice by deletion of murine Smn, and insertion of both full length human SMN2 gene and the major aberrant splice isoform of the SMN2 gene (SMNΔ7; [Le, T.T., Pham, L.T., Butchbach, M.E., Zhang, H.L., Monani, U.R., Coovert, D.D., Gavrilina, T.O., Xing, L., Bassell, G.J., and Burghes, A.H. 2005. SMNDelta7, the major product of the centromeric survival motor neuron (SMN2) gene, extends survival in mice with spinal muscular atrophy and associates with full-length SMN. Hum Mol Genet 14: 845-857]). The present study identified moderate-throughput, quantitative behavioral tests in neonatal SMN2+/+;SMNΔ7+/+;Smn−/− mice. It also addresses methodological approaches and common interpretational challenges in a neonatal model with motor deficiencies and frequent deaths. Animals were assessed daily for body weight and survival, and every other day for neonatal well-being indices and tests of motor function such as performance on the hind-limb suspension test (a.k.a. tube test) and geotaxis. The tube test is a novel non-invasive motor function test specifically designed for neonatal rodents. We found progressive deterioration in SMA model mice for most measures studied particularly body weight, survival, body temperature and motor function with differences appearing as early as P3. Power analysis showed that body weight, survival, righting reflex, geotaxis and tube test had highest predictive power for drug efficacy studies. This multi-functional component battery of tests provides a rapid and efficient means to identify, evaluate and develop candidate therapies as a prelude to human clinical trials.

Introduction

Proximal spinal muscular atrophy (SMA) is a common autosomal recessive neurodegenerative disease of spinal cord α-motor neurons and brainstem nuclei, and is the leading genetic cause of death among infants and toddlers. Symptoms include weakness and atrophy of both proximal limb and respiratory muscles with restricted breathing, locomotion and postural abnormalities. The primary lesion in SMA is a deficiency of survival motor neuron (SMN) protein caused by loss or mutation in the SMN1 gene (Lefebvre et al., 1995). Humans also possess a second copy of the gene, SMN2, which differs from SMN1 chiefly by a C to T transition in exon 7. The majority of SMN2 transcripts lack exon 7 and encode a truncated, less-functional protein. As the copy number of SMN2 increases, the severity of SMA symptoms decreases and the age of onset is delayed (Wirth et al., 2006).

There are currently no therapeutic agents available for SMA that target the primary genomic lesion or pathological mechanisms, although strategies to increase SMN2 production have been evaluated (Andreassi et al., 2004, Andreassi et al., 2001, Brahe et al., 2005, Brichta et al., 2003, Jarecki et al., 2005, Lunn et al., 2004, Mercuri et al., 2004, Riessland et al., 2006, Singh et al., 2006, Sumner et al., 2003). Additional therapeutic approaches include neuroprotective agents, neurotrophic factors, myotrophic factors and gene replacement (Azzouz et al., 2004, Hayashi et al., 2002, Hirtz et al., 2005, Lesbordes et al., 2003). Increasingly, candidate therapeutics to treat SMA are becoming available through academic, government-sponsored and pharmaceutical industry research programs. A standardized, quantitative and moderate-throughput system to test and compare such therapeutics in relevant animal models would be of benefit in accelerating candidates to clinical studies (Hirtz et al., 2005). While in vitro systems that focus on a particular drug target have the advantages of high throughput and target specificity (Jarecki et al., 2005), they generally lack the complex cellular interactions and developmental processes that replicate the cascade of pathological mechanisms and the functional manifestations for direct comparison to the clinical course seen in SMA patients. For genetically caused diseases such as SMA, the recapitulation of genetic defects in mice offers both a model of disease pathogenesis and a system to evaluate candidate therapeutics. It is critical to identify quantitative tests adapted to the specifics of SMA disease mice for examining effects of candidate medications in a standardized protocol with a high capacity and statistical power. Ideally, these tests should evaluate parameters that have homologues in human clinical disease, and evaluate not only survival, but also other phenotypes that impact the quality of life.

Following the discovery that deletion of the mouse Smn gene is embryologically lethal (Schrank et al., 1997), Burghes et al. (Monani et al., 2000), and Li et al. (Hsieh-Li et al., 2000) demonstrated that insertion of the human SMN2 transgene into the background of the Smn knockout rescues the embryonic lethality. The mice described by Monani et al. died between time of birth and 6 days of age with reduced numbers of brainstem and spinal cord motor neurons (Monani et al., 2000), providing a model for severe SMA disease. As in humans (Wirth et al., 2006), the severity of the phenotype in these mice decreased as the number of SMN2 transgenes was increased; at high copy number the transgene restored mice to normal phenotype and lifespan (Monani et al., 2000).

Similarly, Le et al. (2005) found that a transgenic cDNA corresponding to the major aberrant isoform of the SMN2 gene (SMNΔ7) extended the lifespan of the severe strain to 13.3 days. Notably, survival was proportional to the expression level of brain SMNΔ7 mRNA. This prolonged lifespan extended into the period when the animal's behavioral phenotype appears more relevant to the clinical presentation of symptoms of Type-I/Type-II SMA and, importantly, it extends the period for quantitative assessment. Specifically, Le et al. (2005) reported that SMN2+/+;SMNΔ7+/+;Smn−/− mice were smaller in size and weight from at least postnatal day (P) 5 onwards (in the Le et al study, the day of birth was defined as P1). At P5, impairments in righting reflex were noted. By P9, but not P4, loss of lumbar spinal cord neurons was observed. By P10, SMN2+/+;SMNΔ7+/+;Smn−/− mice displayed abnormal gait, clumsiness and ‘shakiness’. Although no abnormalities in gastrocnemius muscle dystrophin staining or dystrophy were observed at P14, muscle fibers were small in size. Some neuromuscular junctions were not innervated and displayed disassembled acetylcholine receptors.

The purpose of the present study was thus to design and evaluate the utility of selected phenotypic tests to quantify key features in the SMN2+/+;SMNΔ7+/+;Smn−/− neonatal mouse model of SMA disease for future drug evaluation. While survival is an important outcome parameter, we also sought measures of muscle function to evaluate therapeutics that could potentially impact the quality of life. We assembled a battery of straightforward, easy-to-perform, rapid and moderate throughput tests of survival, motor function and indices of neonatal well-being (El-Khodor and Boksa, 1997). We then identified those tests having high statistical power to discriminate SMA disease mice from control mice for subsequent use in the quantitative and comparative evaluation of candidate therapeutic compounds for SMA. In addition, we have developed a novel non-intrusive behavioral test for the evaluation of neonatal motor function.

Section snippets

Animals

Smn gene was disrupted by employing a targeting vector encoding a neomycin cassette and a lacZ gene. The construct was introduced into embryonic stem and then injected into C57BL/6 blastocytes. The resulting chimeric animals were crossed to C57BL/6 for several generations (Schrank et al., 1997). The transgenic alleles were created as follows: a 35.5 kb BamHI genomic fragment encoding the human SMN2 promoter and gene was injected into fertilized FVB/N mouse oocytes and founder animal 89 was

Survival

The three animals that died at birth (2 WT and 1 KO) were excluded from the study and thus not represented in the survival curve. Such early fatalities are not uncommon and are generally not viewed as representative of SMA, nor as the result of the inclusion of the human SMN2 transgene (Jablonka et al., 2000). During the course of the observation period (from birth to P22) one female WT pup died at P7 and six HET pups died between P12 and P16 (one female at P12, two females and two males at P14

Discussion

Compared to the severe SMN2+/+;Smn−/− mouse model which rapidly succumbs to disease within 5 days of birth (Hsieh-Li et al., 2000), the inclusion of the SMNΔ7 transgene was only partially beneficial in attenuating the severity of symptoms; the SMN2+/+;SMNΔ7+/+;Smn−/− mice (KO mice) eventually succumbed with SMA-like disease within 18 days (Le et al., 2005). Nevertheless, this delay enables mice to survive long enough for key quantitative behavioral measures to be made for indices of neonatal

Acknowledgments

We would like to acknowledge the following SMA Foundation mouse steering committee members for their valuable scientific input: Drs. Arthur Burghes, Mathew Butchbach, Greg Cox, Christine DiDonato, Catherine Hall, Jill Heemskerk, Richard Paylor and Michael Sendtner. We thank Drs. Sylvie Ramboz, Emer Leahy, Karen Chen and Shoshana Shendelman for their critical reading of the manuscript. This work was supported in part by SMA Foundation and NIH/NINDS and PsychoGenics Inc.

References (34)

  • AvilaA.M. et al.

    Trichostatin A increases SMN expression and survival in a mouse model of spinal muscular atrophy

    J. Clin. Invest.

    (2007)
  • AzzouzM. et al.

    Lentivector-mediated SMN replacement in a mouse model of spinal muscular atrophy

    J. Clin. Invest.

    (2004)
  • BarrettA.C. et al.

    Effect of GABA agonists and GABA-A receptor modulators on cocaine- and food-maintained responding and cocaine discrimination in rats

    J. Pharmacol. Exp. Ther.

    (2005)
  • BraheC. et al.

    Phenylbutyrate increases SMN gene expression in spinal muscular atrophy patients

    Eur. J. Hum. Genet.

    (2005)
  • BrichtaL. et al.

    Valproic acid increases the SMN2 protein level: a well-known drug as a potential therapy for spinal muscular atrophy

    Hum. Mol. Genet.

    (2003)
  • CohenJ.

    Statistical Power Analysis for the Behavioral Sciences

    (1988)
  • DuchenL.W. et al.

    Clinical and pathological studies of an hereditary neuropathy in mice (dystonia musculorum)

    Brain

    (1964)
  • Cited by (0)

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