Potential molecular mechanisms for decreased synaptic glutamate release in dysbindin-1 mutant mice

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Abstract

Behavioral genetic studies of humans have associated variation in the DTNBP1 gene with schizophrenia and its cognitive deficit phenotypes. The protein encoded by DTNBP1, dysbindin-1, is expressed in forebrain neurons where it interacts with proteins mediating vesicular trafficking and exocytosis. It has been shown that loss of dysbindin-1 results in a decrease in glutamate release in the prefrontal cortex; however the mechanisms underlying this decrease are not fully understood. In order to investigate this question, we evaluated dysbindin-1 null mutant mice, using electrophysiological recordings of prefrontal cortical neurons, imaging studies of vesicles, calcium dynamics and Western blot measures of synaptic proteins and Ca2 + channels.

Dysbindin-1 null mice showed a decrease in the ready releasable pool of synaptic vesicles, decreases in quantal size, decreases in the probability of release and deficits in the rate of endo- and exocytosis compared with wild-type controls. Moreover, the dysbindin-1 null mice show decreases in the [Ca2 +]i,expression of L- and N-type Ca2 +channels and several proteins involved in synaptic vesicle trafficking and priming. Our results provide new insights into the mechanisms of action of dysbindin-1.

Introduction

Schizophrenia is a relatively common neuropsychiatric disorder that often involves debilitating and treatment-refractory cognitive deficits that can significantly limit the psychosocial function of affected persons (Green et al., 2000). The disorder is highly heritable, and a number of candidate susceptibility genes have emerged recently (Gejman et al., 2010, Ayalew et al., 2012).

Of these putative risk genes, the gene encoding dystrobrevin-binding-protein-1 (i.e., dysbindin-1) — DTNBP1 is of particular interest. DTNBP1 lies within the chromosome 6p24-22 susceptibility locus (Straub et al., 1995), and multiple associations have been reported between variants of DTNBP1 and schizophrenia (Talbot et al., 2009, Maher et al., 2010). Beyond association between sequence variants and the disorder, a large proportion of schizophrenia patients exhibit lower dysbindin-1C protein in tissue from the PFC (Tang et al., 2009). There are three isoforms of dysbindin: dysbindin 1A, 1B and 1C. Dysbindin 1C is mainly found in postsynaptic sites in human tissue; however it has also been reported in presynaptic sites (Talbot et al., 2011). In mice, dysbindin 1C is the only form of protein found presynaptically (Talbot et al., 2011). Amongst its functions, dysbindin-1 is involved in the control of presynaptic release of glutamate. Recent studies (Chen et al., 2008, Jentsch et al., 2009) have reported that reduced expression of dysbindin-1 in mice dampened glutamate release in the PFC and hippocampus.

Dysbindin-1 is part of the Biogenesis of Lysosome-related Organelle Complex 1 (BLOC-1 complex) (Starcevic and Dell'Angelica, 2004) which is compromised by 8 proteins (dysbindin, snapin, muted, pallidin, cappuccino and BLOS 1–3). This complex has been related to multiple cellular functions including synaptic vesicle dynamics and stabilization of the t-SNARE complex (Larimore et al., 2011, Mullin et al., 2011, Newell-Litwa et al., 2009, Newell-Litwa et al., 2010). Interestingly, decreases in dysbindin-1 reduce the level of snapin (Feng et al., 2008) which, in turn, affects its association with SNAP25 and the interactions between SNAP25 and the calcium sensor synaptotagmin-1, thus impairing priming of vesicles. Moreover, changes in dysbindin-1 produce changes in synapsin 1 (Numakawa et al., 2004), which controls the movement of synaptic vesicles from the reserve pool to the ready-releasable pool (RRP) (Cesca et al., 2010), consequently facilitating synaptic vesicle trafficking following high frequency stimulation.

Here, we provide evidence that mice with loss dysbindin-1 expression exhibit a decrease in glutamate release that may be underlain by decreases in the expression of L- and N-type Ca2 + channels, resulting in deficits in [Ca2 +]i, abnormalities in synaptic vesicle priming and deficits in the replenishment of the ready releasable pool.

Section snippets

Animals

Studies were performed on mice carrying a large genomic deletion (exons 6–7; introns 5–7, Li et al., 2003) contained wholly within the DTNBP1 gene. We used mice that had been backcrossed to the C57Bl/6J background (Jackson Laboratories, Bar Harbor, Maine). All animals were genotyped as previously described (Jentsch et al., 2009). All the WT mice were littermates of the dys −/− mice. Male mice were used in the electrophysiological and molecular experiments described here; with the exception of

Loss of dysbindin-1 expression causes decreased release probability and numbers of vesicles

In order to investigate the origins of the lower glutamate release reported in dys −/− (Chen et al., 2008, Jentsch et al., 2009), we used high frequency stimulation (Dobrunz and Stevens, 1997, Schneggenburger et al., 1999, Taschenberger et al., 2002) to assess the number of ready releasable quanta (Nq). The method is based on the premise that high frequency stimulation-induced depression depends upon depletion of the ready releasable pool (RRP) of vesicles and that this can be estimated by

Discussion

Levels of the protein dysbindin-1 have been found to be reduced in synaptic tissue of schizophrenia patients, including the PFC (Talbot et al., 2004, Talbot et al., 2011, Tang et al., 2009). More importantly, dysbindin-1 has been suggested to play an important role in the negative symptoms and cognitive deficits present in this disorder (Maher et al., 2010, Tang et al., 2009, Talbot et al., 2004). Several authors have demonstrated that decreases in dysbindin-1 reduce glutamate release (Numakawa

Conclusion

By understanding the neurobiological functions for each of the proteins encoded by the genes involved in schizophrenia risk and the consequence of the functional mutations that associate with schizophrenia phenotypes, it is hoped that a convergent theory of cellular and network dysfunction in schizophrenia can be elucidated. Mechanistically, the data here provide a broader molecular explanation of the mechanisms of action of dysbindin. Decreased expression of dysbindin-1 has been shown to

Role of funding source

This research was funded by PHS grants MH-83269 (TC, JDJ, AL).

Contributors

Dr. Saggu performed all the Western blot studies.

Dr. Cannon and Dr. Jentsch reviewed and contributed to the manuscript.

Dr. Lavin designed the study and wrote the manuscript.

Conflict of interest

The authors do not have any conflict of interest to disclose.

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    This research was funded by PHS grants MH-83269 (TC, JDJ, AL).

    1

    Actual address: Department of Biology, Faculty of Sciences, University of Tabuk, Tabuk, Saudi Arabia.

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