The schizophrenia susceptibility gene dysbindin controls synaptic homeostasis

Abstract

The molecular mechanisms that achieve homeostatic stabilization of neural function remain largely unknown. To better understand how neural function is stabilized during development and throughout life, we used an electrophysiology-based forward genetic screen and assessed the function of more than 250 neuronally expressed genes for a role in the homeostatic modulation of synaptic transmission in Drosophila. This screen ruled out the involvement of numerous synaptic proteins and identified a critical function for dysbindin, a gene linked to schizophrenia in humans. We found that dysbindin is required presynaptically for the retrograde, homeostatic modulation of neurotransmission, and functions in a dose-dependent manner downstream or independently of calcium influx. Thus, dysbindin is essential for adaptive neural plasticity and may link altered homeostatic signaling with a complex neurological disease.

Figure 1. Electrophysiology-based screen for homeostatic mutations

(A) Flow diagram of screen strategy and outcome. (B) Histogram of average mEJP amplitude per genotype after PhTx application. Wild-type average mEJP (blue arrow) and wild-type average mEJP after PhTx application (black arrow) are indicated. (C) Histogram of average EJP amplitudes per genotype after PhTx application. Arrows as in (B). Red columns indicate values greater than 2 standard deviations from the mean. (D,E) Homeostatic increases in quantal content observed in published genetic mutations. Data are normalized to the same genotype without PhTx treatment in D, and to wild-type values in E. Full genotypes, n values, and references are shown in Tables S1,2.

Figure 2. Dysbindin is required presynaptically for synaptic homeostasis

(A) Mutations in dysbindin block the homeostatic increase in quantal content following PhTx application. Data are normalized to each genotype in the absence of PhTx. (B) Representative traces for data in (A). (C) Precise excision of the e01028 transposon (revertant) restores compensation. Neuronal-specific expression of dysb (with or without a venus tag; c155-GAL4/+; UAS-dysb/+; dysb¹) restores compensation. Muscle-specific expression (UAS-dysb/+; mhc-GAL4, dysb¹/dysb¹) does not. (D) Representative traces for data in (C). (E) Sustained homeostatic compensation is blocked in GluRIIA^SP16; dysb¹ double mutants.

Figure 3. NMJ morphology and Dysbindin localization

(A) Wild-type (w¹¹¹⁸) and dysb¹ mutant NMJs co-immunostained for anti-HRP (red; motoneuron membrane) and anti-DLG (green; postsynaptic muscle membrane). (B) ven-dysb expressed in neurons co-localizes with the synaptic vesicle associated protein synapsin (green). (C) Quantification of bouton number at muscles 4 and 6/7 comparing wild-type (n=19) and dysb (n=17) (p>0.41; Student’s t-test). (D) Quantification of nc82 puncta per NMJ comparing wild-type (n=15) and dysb (n=16) (p>0.97; Student’s t-test). (E) Density of nc82 puncta comparing wild-type (n=15) and dysb (n=16) (p>0.32; Student’s t-test). Scale bar is 5 microns.

Figure 4. Dysbindin modulates the calcium-dependence of vesicle release

(A) Quantal content as a function of extracellular calcium concentration. (B) Enhanced paired-pulse facilitation in dysb¹ mutants. (C) Quantal content is increased by neuronal overexpression of dysb (dysb OE; c155-GAL4/Y; UAS-dysb/+). (D) Homeostatic compensation is calculated as in Fig. 1E. Compensation is suppressed in cac^S/+ (n=15) compared with wild type (n=25; p<0.001; Student’s t-test) or dysb¹/+ (n=17; p<0.001; Student’s t-test) but is not further suppressed in cac^S/+; dysb¹/+ mutants (n=19; p>0.27; Student’s t-test). (E) Compensation remains blocked when cac is neuronally overexpressed in a dysb¹ mutant (n=9, dysb;cac OE; c155-GAL4/Y; UAS-cacGFP/+; dysb¹). (F) Quantal content is increased by neuronal overexpression of dysb (n=17, dysb OE; c155-GAL4/Y; UAS-dysb/+; p<0.0002; Student’s t-test) and when dysb is overexpressed in a cac^S mutant (n=19, cac^S/+; dysb OE; cac^S/c155-GAL4; UAS-dysb/+; p<0.006; Student’s t-test).