Abstract
Mutations in GlyR α1 or β subunit genes in humans and rodents lead to severe startle disease characterized by rigidity, massive stiffness and excessive startle responses upon unexpected tactile or acoustic stimuli. The recently characterized startle disease mouse mutant shaky carries a missense mutation (Q177K) in the β8-β9 loop within the large extracellular N-terminal domain of the GlyR α1 subunit. This results in a disrupted hydrogen bond network around K177 and faster GlyR decay times. Symptoms in mice start at postnatal day 14 and increase until premature death of homozygous shaky mice around 4–6 weeks after birth. Here we investigate the in vivo functional effects of the Q177K mutation using behavioral analysis coupled to protein biochemistry and functional assays. Western blot analysis revealed GlyR α1 subunit expression in wild-type and shaky animals around postnatal day 7, a week before symptoms in mutant mice become obvious. Before 2 weeks of age, homozygous shaky mice appeared healthy and showed no changes in body weight. However, analysis of gait and hind-limb clasping revealed that motor coordination was already impaired. Motor coordination and the activity pattern at P28 improved significantly upon diazepam treatment, a pharmacotherapy used in human startle disease. To investigate whether functional deficits in glycinergic neurotransmission are present prior to phenotypic onset, we performed whole-cell recordings from hypoglossal motoneurons (HMs) in brain stem slices from wild-type and shaky mice at different postnatal stages. Shaky homozygotes showed a decline in mIPSC amplitude and frequency at P9-P13, progressing to significant reductions in mIPSC amplitude and decay time at P18-24 compared to wild-type littermates. Extrasynaptic GlyRs recorded by bath-application of glycine also revealed reduced current amplitudes in shaky mice compared to wild-type neurons, suggesting that presynaptic GlyR function is also impaired. Thus, a distinct, but behaviorally ineffective impairment of glycinergic synapses precedes the symptoms onset in shaky mice. These findings extend our current knowledge on startle disease in the shaky mouse model in that they demonstrate how the progression of GlyR dysfunction causes, with a delay of about 1 week, the appearance of disease symptoms.
Highlights
Glycinergic inhibition is prominent in brain stem and spinal cord, where it is involved in essential processes such as motor control (Lynch, 2004), inflammatory pain sensitization (Harvey et al, 2004) and rhythmic breathing (Manzke et al, 2010)
Using the spontaneous mouse Glycine receptors (GlyRs) α1 subunit mutant shaky, it has been demonstrated that the extracellular β8-β9 loop is a key structural and functional element for GlyR signaling that influences conformational changes including the M3-M4 domain involved in synaptic clustering and the formation of the glycine-binding pocket (Schaefer et al, 2017)
The shaky mouse model, harboring the GlyR α1 subunit mutation Q177K, is the first in vivo model revealing that the integrity of the β8-β9 loop in the extracellular domains (ECDs) is a key regulator of glycinergic signaling
Summary
Glycinergic inhibition is prominent in brain stem and spinal cord, where it is involved in essential processes such as motor control (Lynch, 2004), inflammatory pain sensitization (Harvey et al, 2004) and rhythmic breathing (Manzke et al, 2010). Defects in glycinergic transmission are the underlying cause of the neurological motor disorder hyperekplexia (OMIM 149400, startle disease, stiff baby syndrome). Human hyperekplexia is caused by mutations in the GLRA1, GLRB, or SLC6A5 genes, encoding GlyR α1 and β subunits and the glycine transporter GlyT2. Symptoms in human hyperekplexia range from exaggerated startle reactions - due to unexpected acoustic or tactile stimuli - to muscle stiffness, apnea, and loss of postural control. Individuals with startle disease are typically treated with low doses of the benzodiazepine clonazepam, a positive allosteric modulator of GABAA receptors (Christian et al, 2013)
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