Presynaptic glycine receptors as a potential therapeutic target for hyperekplexia disease

Abstract

Although postsynaptic glycine receptors (GlyRs) as αβ heteromers attract considerable research attention, little is known about the role of presynaptic GlyRs, likely α homomers, in diseases. Here, we demonstrate that dehydroxylcannabidiol (DH-CBD), a nonpsychoactive cannabinoid, can rescue GlyR functional deficiency and exaggerated acoustic and tactile startle responses in mice bearing point mutations in α1 GlyRs that are responsible for a hereditary startle-hyperekplexia disease. The GlyRs expressed as α1 homomers either in HEK-293 cells or at presynaptic terminals of the calyceal synapses in the auditory brainstem are more vulnerable than heteromers to hyperekplexia mutation-induced impairment. Homomeric mutants are more sensitive to DH-CBD than are heteromers, suggesting presynaptic GlyRs as a primary target. Consistent with this idea, DH-CBD selectively rescues impaired presynaptic GlyR activity and diminished glycine release in the brainstem and spinal cord of hyperekplexic mutant mice. Thus, presynaptic α1 GlyRs emerge as a potential therapeutic target for dominant hyperekplexia disease and other diseases with GlyR deficiency.

Figure 1

The α1R271Q mutation impairs GlyR function and causes exaggerated startle behavior in mice. (a) Trace records of I_Gly in HEK-293 cells expressing WT and α1R271Q mutant receptors. (b) The Gly concentration-response curves for the WT (n=9) and α1R271Q (n=6) mutant receptors. The Gly EC₅₀ values are 60 ± 8 μM for the WT receptors and 21,000 ± 3,215 μM for the α1R271Q mutant receptors. (p=0.0006, t(10)=9.003, unpaired t-test). (c) Trace records of Gly sIPSCs in spinal dorsal horn neurons isolated from the WT and α1R271Q mutant mice. (d) The average values and data points of Gly sIPSC frequency and amplitude. (WT, n=23 from 6 mice; R271Q, n=11 from 5 mice) (Frequency, WT vs R271Q, * p=0.022, t(32)=2.4; Amplitude, WT vs R271Q, ** p=0.0031, t(32)=3.227; unpaired t-test) (e) The average values of startle response induced by white noise ranging from 80 to 120 decibel (dB) in WT (n=8) and α1R271Q (n=8) mice. Maximum startle amplitude (Vm) as a function of sound intensity. (f) The average values of startle responses induced by air puff in WT (n=5) and α1R271Q (n=10) mice.

Figure 2

DH-CBD rescues the α1R271Q mutation-induced GlyR deficiency and hyperreflexia in mice. (a) Chemical structure of CBD and DH-CBD. Trace records of I_Gly without and with DH-CBD (10 μM) in HEK-293 cells expressing WT and the α1R271Q GlyRs. (b) Gly concentration-response curves with or without DH-CBD (10 μM) in HEK-293 cells expressing α1R271Q GlyRs. (GlyEC₅₀ values: 21 ± 3.2 mM without DH-CBD (n=6) and 1.2 ± 0.1 mM with DH-CBD (n=5), p=0.0007, t(9)=5.05, unpaired t-test). (c) DH-CBD restoration of startle responses induced by different levels of acoustic sound in α1R271Q mutant mice (WT n=6, R271Q n=8, R271Q+DH-CBD, n=8). * p<0.05, ** p<0.01, F(1, 13)=5.3, F(1, 13)=14.74, Two-way ANOVA compared to vehicle injected group. (d) DH-CBD (50 mg/kg, i.p.) restoration of startle responses induced by different levels of tactile air-puff stimuli in α1R271Q mutant mice (1.5 PSI: WT n=6, R271Q n=10, R271Q+DH-CBD n=6; 5 PSI: WT n=6, R271Q n=6, R271Q+DH-CBD n=8). * p<0.05, *** p<0.001, F(5, 31)=25.6, One-way ANOVA followed by Tukey’s post hoc test. (e) Hind feet clenching behavior in the α1R271Q mutant mouse and the restoration of this behavior by DH-CBD (i.p. 30 mg/kg). (f) Photo images of DH-CBD restoration of righting reflex behavior. (g) Concentration dependence of DH-CBD restoration of prolonged righting reflex time in the α1R271Q mutant mice (WT n=8; R271Q+DH-CBD (0–30mg/kg), n=10). ** p<0.01, *** p<0.001, F(4, 39)= 27.1, one-way ANOVA followed by Tukey’s post hoc test.

Figure 3

Site-specific restoration of hyperekplexic GlyR dysfunction and startle response by DH-CBD. (a) The average EC₅₀ values of Gly concentration-response curves without and with DH-CBD (10 μM) in HEK-293 cells expressing various hyperekplexic mutant α1 subunits. * p<0.05, ** p<0.01, *** p<0.001, t(12–18)>2.2, unpaired t-test. (b) The average E_max values of Gly concentration-response curves without and with DH-CBD (10 μM) for various hyperekplexic mutant α1 subunits. * p<0.05, *** p<0.001, t(13–19)>2.2, unpaired t-test. (c) The average startle responses to acoustic stimuli in wild type (WT) littermates, α1M287L, α1R271Q, α1Q266I, and α1S267Q mutant mice injected with vehicle (blue) and injected with DH-CBD at 50 mg/kg, i.p. (red). * p<0.05, *** p<0.001, t(12–15)>2.2 between vehicle and DH-CBD injection in the mutant mice, unpaired t-test. (d) Correlation analysis of DH-induced percentage change of the Gly EC₅₀ values and the DH-CBD-induced percent changes of the startle response in mice carrying corresponding mutant GlyRs (p=0.0005, linear regression).

Figure 4

Differential sensitivity of homomeric and heteromeric GlyRs to hyperekplexic mutations and DH-CBD. (a) Trace records of Gly (1 mM)-activated current in HEK-293 cells expressing homomeric α1R271Q or heteromeric α1R271Q/β GlyRs. (b) Gly concentration-response curves of the WT or α1R271Q and β1 GlyR cDNAs transfected at different ratio (2 μg/ml WT (n=6) or α1R271Q cDNA plus 6 (n=8), 2 (n=6), 0.67 (n=6) or 0 (n=5) μg/ml β1 cDNA) in HEK-293 cells. (c) The EC₅₀ values of homomeric and heteromeric hyperekplexic mutant GlyRs (mutant α1 cDNA: β1 cDNA=1:3). (d) The Gly EC₅₀ values of heteromeric mutant α1β (1:3) subunits without and with DH-CBD (10 μM). (e) The Gly I_max values of homomeric and heteromeric hyperekplexic mutant GlyRs expressed in HEK-293 cells. (f) The Gly I_max values of heteromeric mutant α1β subunits without and with DH-CBD (10 μM). *p< 0.05, **p< 0.01, ***p<0.001, t(11–15)>2.4, unpaired t-test (c–f).

Figure 5

DH-CBD rescue of diminished glycine release in spinal slices from the α1R271Q mutant mice. (a) Trace records of Gly sIPSC in spinal slices from WT and α1R271Q mutant mice before and after DH-CBD (20 μM). The bar graphs representing the average frequency and amplitude of Gly sIPSC (WT, vehicle n=24 cells of 6 mice, DH-CBD n=12 cells of 4 mice; R271Q, vehicle n=14 cells of 5 mice, DH-CBD n=17 cells of 5 mice). * p<0.05, ** p<0.01, F(3, 60)=7.2, one-way ANOVA followed by Tukey’s post hoc test. (b) Trace records and average frequency and amplitude of the Gly mIPSCs before and after DH-CBD (20 μM). A significant difference in the Gly mIPSC frequency before and after DH-CBD (n=10 cells of 3 mice, * p=0.03, t(18)=2.37, unpaired t-test). (c) Cumulative plot analysis of the distribution of the inter-event interval and amplitude of Gly mIPSCs without and with DH-CBD. (d) Trace records and ratio of paired pulse responses recorded in spinal neurons before and after DH-CBD (20 μM) (n=7 cells of 3 mice, ** p=0.0072, t(12)=3.23, unpaired t-test). (e) PTX inhibition of I_Gly in HEK-293 cells expressing human α1R271Q GlyRs without (n=6) and with (n=6) β1 subunits. (f) The effect of PTX (30 μM) on Gly sIPSC frequency in the absence (n=12 cells of 4 mice) and presence (n=12 cells of 4 mice) of DH-CBD in the spinal slices of α1R271Q mutant mice. *p<0.05, **p<0.01, F(3, 41)=7.5, one-way ANOVA followed by Tukey’s post hoc test.

Figure 6

Differential sensitivity of presynaptic and postsynaptic GlyRs to hyperekplexic mutation and DH-CBD rescue. (a) Photo imaging of a calyx associated with a postsynaptic neuron. The scale bar (solid white) represents 10 μM. The schemes illustrate the recording configurations of calyceal terminal and MNTB principle neuron. I_Gly recorded from either presynaptic terminal (red trace) or postsynaptic membrane of MNTB principle neuron (blue trace). (b) The average maximal amplitudes of I_Gly recorded from presynaptic calyceal terminals (red, P12, n=4 cells of 3 mice; P14, n=7 cells of 3 mice; P16, n=4 cells of 3 mice; P18, n=3 cells of 2 mice) and postsynaptic MNTB neurons (blue, P12, n=6 cells of 3 mice; P14, n=4 cells of 2 mice; P16, n=5 cells of 4 mice; P18, n=5 cells of 2 mice) during development from P12 to P18. (c) Gly concentration-response curves recorded from calyceal terminals of wild type littermates (open squares, n=7 cells of 3 mice) and M287L homozygous mutant mice (solid squares, n=6 cells of 4 mice). (d) Gly concentration-response curves recorded from MNTB principle neurons of wild type littermates (open squares, n=7 cells of 3 mice) and M287L mutant mice (solid squares, n=7 cells of 3 mice). (e) The average amplitudes of I_Gly (Gly, 300 μM) from calyceal presynaptic terminals of WT (n=7 cells of 3 mice) and mutant mice in absence (n=9 cells of 5 mice) and presence (n=9 cells of 5 mice) of DH-CBD (* p=0.023, t(16)=3.62, unpaired t-test). (f) The average amplitudes of I_Gly (Gly, 100 μM) from MNTB principle neurons of WT (n=10 cells of 3 mice) and M287L mutant mice in the absence (n=6 cells of 3 mice) and presence (n=8 cells of 5 mice) of DH-CBD (p=0.98, t(12)=0.031, unpaired t-test).