The Schizophrenia Susceptibility Gene dysbindin Controls Synaptic Homeostasis

At glutamatergic synapses of species as varied as Drosophila and humans, disruption of postsynaptic neurotransmitter receptor function can be precisely offset by an increase in presynaptic neurotransmitter release to homeostatically maintain normal postsynaptic excitation (1–3). The Drosophila neuromuscular junction (NMJ) is a glutamatergic synapse that is used as a model for this form of homeostatic signaling in the nervous system (1, 4, 5). Efficient homeostatic modulation of presynaptic release at the Drosophila NMJ can occur within 10 min after bath application of philanthotoxin-433 (PhTx), which persistently and specifically inhibits postsynaptic glutamate receptors (fig. S1) (4).

We systematically screened for mutations that block the rapid, PhTx-dependent induction of synaptic homeostasis (Fig. 1). Mutations in 276 genes were screened electrophysiologically (see supporting online text). For each mutant, we calculated an average value for the amplitude of both the spontaneous miniature excitatory junctional potential (mEJP) and evoked excitatory junctional potential (EJP) after treatment of the dissected neuromuscular preparation with PhTx for 10 min (4). We isolated 14 mutants with average EJP amplitudes more than two standard deviations smaller than the distribution mean (Fig. 1C, solid red bars). From these candidates we identified seven mutants that block synaptic homeostasis without an obvious effect on NMJ morphology or baseline synaptic transmission. We conclude that the molecular mechanisms of synaptic homeostasis can be genetically separated from the mechanisms responsible for normal neuromuscular development and baseline synaptic transmission.

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A fraction of the mutants we assayed (19.5%) are previously published genetic lesions. This allows us to rule out the involvement of numerous genes and associated biochemical processes. Mutations that disrupt RNA interference or microRNA processing, retrograde transsynaptic signaling, synaptic transmission, active zone assembly, synaptic vesicle endocytosis, and mitochondria all showed reliable homeostatic compensation (Fig. 1, D and E, and fig. S1). Therefore, synaptic homeostasis is a robust phenomenon unperturbed by a broad spectrum of synaptic mutations. In addition, significant homeostatic compensation in synaptojanin and endophilin mutants argues against the involvement of synaptic vesicle endocytosis and indicates that the size of the recycling synaptic vesicle pool is not a limiting factor for synaptic homeostasis. These data also underscore the importance and specificity of those mutations we identified that do block synaptic homeostasis. These include four ion channels, two of which are of unknown function, and two calcium-binding proteins of unknown function. Thus, homeostatic signaling at the NMJ may include previously unexplored mechanisms of synaptic modulation.

One mutation that was identified with a specific defect in homeostatic compensation is a transposon insertion that resides in the Drosophila homolog of dysbindin (CG6856; fig. S2). The dysbindin locus is linked with schizophrenia in humans (6–11). We identified a transposon insertion within Drosophila dysbindin (pBac^e01028, referred to as dysb¹; fig. S2) that showed a complete absence of homeostatic compensation after application of PhTx (Fig. 2, A and B). A similar effect was observed when dysb¹ was placed in trans to a deficiency that uncovers the dysb locus, indicating that the dysb¹ mutant was a strong loss-of-function or null mutation (Fig. 2, A and B). No significant change in baseline synaptic transmission in dysb¹ mutant larvae (0.5 mM extracellular calcium) was observed. Thus, under these recording conditions, this mutation disrupted synaptic homeostasis without altering baseline neurotransmission (Fig. 2B). As a control, synaptic homeostasis was normal in larvae in which the pBac^e01028 transposon was precisely excised (Fig. 2, C and D).

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The dysb gene is ubiquitously expressed in Drosophila embryos (fig. S2), consistent with widespread expression in vertebrates (12, 13). Therefore, we generated and expressed a dysbindin transgene in the dysb¹ mutant. Presynaptic expression of dysb fully restored homeostatic compensation in the dysb¹ mutant background, whereas muscle-specific expression of dysb did not (Fig. 2, C and D, and fig. S3). Thus, dysbindin is necessary presynaptically for the rapid induction of synaptic homeostasis.

We next asked whether dysbindin is also required for the sustained expression of synaptic homeostasis. We generated double-mutant flies harboring both the dysb¹ mutation and a mutation in a gene encoding a postsynaptic glutamate receptor (GluRIIA). GluRIIA mutant larvae normally show robust homeostatic compensation (14). However, homeostatic compensation was blocked in GluRIIA; dysb¹ double-mutant larvae (Fig. 2E). Thus, dysbindin was also necessary for the sustained expression of synaptic homeostasis over several days of larval development.

Next we demonstrated that synapse morphology was qualitatively normal in dysb mutants (Fig. 3A), taking into account the shape of the presynaptic nerve terminal as well as the levels, localization, and organization of synaptic markers including microtubules, synapsin, and synaptotagmin (fig. S4). Bouton number and active zone density are also normal in dysb mutants (Fig. 3, C to E) (15). Thus, the disruption of synaptic homeostasis in dysb¹ mutants is not a secondary consequence of altered or impaired NMJ development.

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In the vertebrate nervous system, dysbindin is associated with synaptic vesicles (16). We examined the localization of a Venus-tagged dysb transgene (ven-dysb) that rescues the dysb¹ mutant (Fig. 2, C and D). Ven-Dysb showed extensive overlap with synaptic vesicle–associated proteins when expressed in neurons (Fig. 3B). Thus, dysbindin functions presynaptically, potentially at or near the synaptic vesicle pool.

To further define the function of dysbindin, we investigated baseline synaptic transmission in the dysb mutant in greater detail. At 0.5 mM extracellular calcium, synaptic transmission in dysb¹ mutant larvae was indistinguishable from the wild type (Fig. 2, A and B). However, when extracellular calcium was reduced (0.2 to 0.4 mM Ca²⁺), baseline synaptic transmission was significantly impaired in dysb relative to the wild type (Fig. 4A), and this defect was rescued by presynaptic expression of dysb (fig. S5). Thus, there is an alteration of the calcium dependence of synaptic transmission in the dysb mutant. Indeed, at reduced extracellular calcium, both paired-pulse facilitation and facilitation that occurs during a prolonged stimulus train were increased in dysb mutants (Fig. 4B and fig. S6).

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In vertebrates, levels of dysb expression correlate with parallel changes in extracellular glutamate concentration (17). Therefore, we tested whether dysb overexpression might increase presynaptic release. In wild-type larvae overexpressing dysb in neurons, synaptic transmission was normal at low extracellular calcium (0.2 and 0.3 mM Ca²⁺) but was enhanced at relatively high extracellular calcium (0.5 mM Ca²⁺; Fig. 4C and fig. S5). The complementary effects of dysb loss of function and overexpression confirm that dysbindin has an important influence on calcium-dependent vesicle release.

The presynaptic Ca_V2.1 calcium channel, encoded by cacophony (cac), is required for synaptic vesicle release at the Drosophila NMJ (18). cac mutations decrease presynaptic calcium influx (19) and also block synaptic homeostasis (4). We therefore tested for a genetic interaction between dysb and cac during synaptic homeostasis. Because homozygous cac and dysb mutations individually block synaptic homeostasis, analysis of double-mutant combinations would not be informative. We resorted to an analysis of heterozygous mutant combinations and gene overexpression (5). Synaptic homeostasis was suppressed by a heterozygous mutation in cac (Fig. 4D). However, this suppression was not enhanced by the presence of a heterozygous mutation in dysb (Fig. 4 and fig. S7). In addition, neuronal overexpression of cac did not restore homeostatic compensation in dysb mutant larvae, and the enhancement of presynaptic release caused by neuronal dysb overexpression still occurred in a heterozygous cac mutant background (Fig. 4, E and F). Thus, dysbindin may function downstream or independently of Cac during synaptic homeostasis.

To further explore the relationship between dysbindin and Cac, we asked whether dysb mutations might directly influence presynaptic calcium influx. The spatially averaged calcium signal in dysb¹ was indistinguishable from the wild type, indicating no difference in presynaptic calcium influx (fig. S8). Thus, dysbindin appears to function downstream or independently of calcium influx to control synaptic homeostasis.

Through a systematic electrophysiological analysis of more than 250 mutants, we were able to rule out the involvement of numerous synaptic proteins and biochemical processes in the mechanisms of synaptic homeostasis and to show that this phenomenon is separable from the molecular mechanisms that specify structural and functional synapse development. We thus identify dysbindin as an essential presynaptic component within a homeostatic signaling system that regulates and stabilizes synaptic efficacy. Dysbindin functions downstream or independently of the presynaptic Ca_V2.1 calcium channel in the mechanisms of synaptic homeostasis.

Emerging lines of evidence suggest that glutamate hypofunction could be related to the etiology of schizophrenia (20–24). Likewise, reduced levels of dysbindin expression have been associated with schizophrenia (25, 26). The sandy mouse, which lacks dysbindin, has a decreased rate of vesicle release (~30% decrease), a correlated decrease in vesicle pool size, and an increased thickness of the postsynaptic density (27). We confirm a modest, facilitatory function for dysbindin during baseline transmission. However, numerous mutations with similar or more severe defects in baseline transmission show normal synaptic homeostasis (Fig. 1, D and E). By contrast, loss of dysbindin completely blocks the adaptive, homeostatic modulation of vesicle release, which suggests that the potential contribution of dysbindin mutations to schizophrenia may derive from altered homeostatic plasticity as opposed to decreased baseline glutamatergic transmission.