Quantitative Gait Analysis Using a Motorized Treadmill System Sensitively Detects Motor Abnormalities in Mice Expressing ATPase Defective Spastin - PubMed (original) (raw)

Quantitative Gait Analysis Using a Motorized Treadmill System Sensitively Detects Motor Abnormalities in Mice Expressing ATPase Defective Spastin

James W Connell et al. PLoS One. 2016.

Abstract

The hereditary spastic paraplegias (HSPs) are genetic conditions in which there is progressive axonal degeneration in the corticospinal tract. Autosomal dominant mutations, including nonsense, frameshift and missense changes, in the gene encoding the microtubule severing ATPase spastin are the most common cause of HSP in North America and northern Europe. In this study we report quantitative gait analysis using a motorized treadmill system, carried out on mice knocked-in for a disease-associated mutation affecting a critical residue in the Walker A motif of the spastin ATPase domain. At 4 months and at one year of age homozygous mutant mice had a number of abnormal gait parameters, including in stride length and stride duration, compared to heterozygous and wild-type littermates. Gait parameters in heterozygous animals did not differ from wild-type littermates. We conclude that quantitative gait analysis using the DigiGait system sensitively detects motor abnormalities in a hereditary spastic paraplegia model, and would be a useful method for analyzing the effects of pharmacological treatments for HSP.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1

Fig 1. SpastinN384K/N384K knock-in mouse generation and validation.

A) Schematic diagram of strategy used to generate spastinN384K allele. Numbering refers to spastin exons. Puro = puromycin resistance cassette, Neo = neomycin resistance cassette, Flp = flippase, FRT = flippase recognition target, UTR = untranslated region. B) DNA sequencing of PCR product generated using primers designed to amplify fragment of spastin exon 8 from genomic DNA of a wild-type mouse and spastinN384K/N384K littermate. C) Immunoblotting versus spastin of lysate or pellet fraction from foetal brain tissue (E17), from mice of the genotypes indicated. The histogram shows corresponding densitometry of the spastin bands in the lysate (n = 3 per genotype, p-values generated by paired t-tests). GAPDH immunoblotting or Coomassie staining serve to verify equal loading of lanes. Size bars indicate molecular weight in kD.

Fig 2

Fig 2. SpastinN384K/N384K mice develop axonal swellings.

A) Day 7 in vitro primary cortical neurons from animals with the genotypes indicated labeled with antibody to acetylated tubulin. DAPI staining (blue) was used to label nuclei. Examples of swellings seen in spastinN384K/N384K neurons are shown in the boxed areas, which are magnified in the right hand panels. B) Primary cortical neurons were labeled as in A) and the number of swellings per 250 nuclei was quantified in animals with the genotypes shown (n = 3 repeats per genotype). Mean number of swellings per 250 nuclei ± S.E.M.: spastwt/wt = 16.0 ± 1.53, spastN384K/wt = 16.3 ± 3.28 and for spastN384K/wt 62 ± 4.04. P-values were generated by 1-way ANOVA with Bonferroni post-test for comparison of individual pairs of genotypes. C) Day 7 in vitro primary cortical neurons from spastinN384K/N384K mice were labeled with the antibodies indicated. Swellings (arrowheads) were always MAP2 negative or tau positive, indicating that they were present in axons. Scale bars = 20μm.

Fig 3

Fig 3. Weight and key left hindlimb gait parameters in spastinN384K/N384K mice versus littermate controls at 1 year old.

Plots show values for each animal tested with the genotypes indicated. Squares = male animals, circles = females. A) weight in grammes, B) stride duration, C) swing phase duration, D) stance phase duration. E) Histogram showing the mean percentage of time that tested animals with the genotypes indicated spent in each phase of gait. F) Stride length in animals with the genotypes shown. Error bars = mean ± S.E.M. Numerical p-values for corresponding statistical tests using two-way ANOVA for the effect of gender and genotype on gait parameters are shown in Table 2. To facilitate interpretation, p-values for genotype are indicated on the plots by * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Fig 4

Fig 4. Key left hindlimb gait parameters in spastinN384K/N384K mice versus littermate controls at 4 months old.

Plots show values for each animal tested with the genotypes indicated. A) stride duration, B) swing phase duration, C) stance phase duration. D) Histogram showing the mean percentage of time that tested animals with the genotypes indicated spent in each phase of gait. E) Stride length in animals with the genotypes shown. Numerical values for corresponding statistical tests using one-way ANOVA for the effect of genotype on gait parameters are shown in Table 4. To facilitate interpretation, p-values for genotype are indicated on the plots by * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Error bars = mean ± S.E.M

Similar articles

Cited by

References

    1. Reid E. The hereditary spastic paraplegias. J Neurol. 1999;246(11):995–1003. Epub 2000/01/13. . - PubMed
    1. Fink JK. Hereditary spastic paraplegia. Neurol Clin. 2002;20(3):711–26. Epub 2002/11/16. . - PubMed
    1. Harding AE. The hereditary ataxias and related disorders. Edinburgh: Churchill Livingston; 1984.
    1. Blackstone C, O'Kane CJ, Reid E. Hereditary spastic paraplegias: membrane traffic and the motor pathway. Nat Rev Neurosci. 2011;12(1):31–42. Epub 2010/12/09. nrn2946 [pii] 10.1038/nrn2946 . - DOI - PMC - PubMed
    1. Finsterer J, Loscher W, Quasthoff S, Wanschitz J, Auer-Grumbach M, Stevanin G. Hereditary spastic paraplegias with autosomal dominant, recessive, X-linked, or maternal trait of inheritance. Journal of the neurological sciences. 2012;318(1–2):1–18. Epub 2012/05/05. 10.1016/j.jns.2012.03.025 . - DOI - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources