Transient neuromotor phenotype in transgenic spastic mice expressing low levels of glycine receptor beta-subunit: an animal model of startle disease

Abstract

Startle disease or hereditary hyperekplexia has been shown to result from mutations in the alpha1-subunit gene of the inhibitory glycine receptor (GlyR). In hyperekplexia patients, neuromotor symptoms generally become apparent at birth, improve with age, and often disappear in adulthood. Loss-of-function mutations of GlyR alpha or beta-subunits in mice show rather severe neuromotor phenotypes. Here, we generated mutant mice with a transient neuromotor deficiency by introducing a GlyR beta transgene into the spastic mouse (spa/spa), a recessive mutant carrying a transposon insertion within the GlyR beta-subunit gene. In spa/spa TG456 mice, one of three strains generated with this construct, which expressed very low levels of GlyR beta transgene-dependent mRNA and protein, the spastic phenotype was found to depend upon the transgene copy number. Notably, mice carrying two copies of the transgene showed an age-dependent sensitivity to tremor induction, which peaked at approximately 3-4 weeks postnatally. This closely resembles the development of symptoms in human hyperekplexia patients, where motor coordination significantly improves after adolescence. The spa/spa TG456 line thus may serve as an animal model of human startle disease.

FIG. 1

Expression of the transgene in spa/spa mice hetero- and homozygous for the TG 456 insertion. (A) Northern analysis. Total brain RNA was probed with a 264-bp SspI–NcoI fragment encompassing exons 4 and 5 of the GlyR β cDNA. Control hybridizations of the same blot were performed using the GAPDH gene probe. wt, wild-type control mice. (B) Western analysis. Membrane proteins from spinal cord and brain stem were separated on SDS–PAGE gels, transferred to nitrocellulose, and specific bands were detected with the antibodies mAb 4a and anti-SVP against synaptophysin as a control. Immunoglobulins were visualized using horseradish peroxidase-coupled second antibodies and the ECL system (Amersham).

FIG. 2

Binding of ³[H] strychnine to spinal cord membranes. Genotypes: (○), wt; (□), spa/spa; and (▴), spa/spa-TG456/TG456. Data represent the means from two (wt, spa/spa) or three (spa/spa-TG456/TG456) animals, respectively. (A) Saturation binding curves and (B) Scatchard analyses of specific ³[H]-strychnine binding assays were performed in triplicate as described inBecker et al. (1993).

FIG. 3

Tremor inducibility in spa/spa, spa/spa-TG456 and spa/spa-TG456/TG456 animals. Animals of each genotype were tested at several time points for tremor inducibility by handling. Periods of tremor inducibility are indicated by solid lines. Note delayed and transient appearance of tremor symptoms in spa/spa-TG456/TG456 mice. Of the non-transgenic spa/spa animals, four were non-transgenic littermates from doubly heterozygous crosses, and three animals were derived from the original stock obtained from Jackson Laboratories.

FIG. 4

Electromechanical tracings of tremor-derived movement recorded from spa/spa, spa/spa-TG456/TG456 and wt animals. The amplitude is proportional to the strength of the movement recorded over time. For experimental details see text and Materials and methods.