Molecular Psychiatry (2009) 14, 556–562 & 2009 Nature Publishing Group All rights reserved 1359-4184/09 $32.00 www.nature.com/mp LETTERS TO THE EDITOR DISC-1 Leu607Phe alleles differentially affect centrosomal PCM1 localization and neurotransmitter release

Molecular Psychiatry (2009) 14, 556–557; doi:10.1038/ values±s.d.: Phe607 DISC-1, 0.92±0.18, untrans- mp.2009.13 fected control, 0.86±0.13; Leu607 DISC-1, 0.90±0.22, untransfected control, 0.88±0.21). The The putative susceptibility Dis- Leu607Phe polymorphism also affected neurotrans- rupted-in-schizophrenia 1 (DISC-1), encodes a pleio- mitter release, with Phe607 DISC-1, but not Leu607 tropic with many subcellular localizations DISC-1, decreasing noradrenaline release at 5 mM and purported functions. One key role, synaptic [K þ ] (Figure 1c). Under these conditions, release is function, is thought to be, in part, due to effects on likely due to spontaneous vesicle fusion (see Supple- .1 In this context a recent study has mentary Material). Neither variant influenced nora- shown that DISC-1 regulates 1 drenaline release at 100 mM [K þ ]. Our results reveal (PCM1; another putative schizophrenia susceptibility that the Leu607Phe polymorphism affects two aspects gene2) localization to the ,3 an organelle of cellular function: centrosomal PCM1 localization that serves as the main organizing center, and spontaneous neurotransmitter release, but does and microtubule assembly is PCM1 dependent.4 The not alter microtubule dynamics. Leu607Phe substitution is one of the few DISC-1 What might be the molecular mechanism(s) under- polymorphisms to be associated with schizophrenia- lying the influence of the Leu607Phe polymorphism, related phenotypes that is coding,5,6 though direct and could the centrosomal and neurotransmitter evidence for its functionality is lacking. release changes be related? Our observation that Given the potential links between DISC-1, PCM1, DISC1 and PCM1 colocalization at the centrosome is the centrosome, microtubules and synapses, we less with Phe607 DISC-1 than with Leu607-DISC-1 wanted to determine whether the Leu607Phe poly- may provide a clue. Leu607 is the first residue of a morphism affects centrosomal PCM1 localization, putative leucine zipper motif,8 and it also resides microtubule dynamics or neurotransmitter release. within a domain, both of which are SH-SY5Y cells were chosen as they are human, have a important in regulating localization to the centro- neuronal phenotype, and have been utilized in some.9 The Leu607Phe substitution is predicted to neurotransmitter studies. Undifferentiated SH-SY5Y disrupt both the leucine zipper and the coiled cells were transfected with constructs containing V5- coil domain,10 which may explain our finding of tagged Leu607 or V5-tagged Phe607 DISC-1. Anti-V5 decreased colocalization of DISC-1 and the reduction and PCM1 double immunofluorescence was per- in centrosomal immunoreactive area of its binding formed to assess differences between the variants in partner, PCM1, after Phe607 DISC-1 transfection. Of centrosomal PCM1 immunoreactivity. Western blots note, the assembly of other centrosomal , and were utilized to quantify tyrosinated and detyrosi- microtubule anchorage and organization, are all nated a-tubulin, markers of dynamic and stable dependent on PCM1,3 suggesting that the Leu607Phe microtubules, respectively.7 For neurotransmitter re- polymorphism could, in this way, impinge upon lease studies, SH-SY5Y cells were loaded with [3H] microtubule function in the absence of changes on noradrenaline, and release was measured at two microtubule dynamics. [K þ ] concentrations (5 mM and 100 mM). Further As a key component of the cytoskeleton, micro- experimental details are available in Supplementary tubules are involved in many cellular processes, Material. including synaptic function and neurotransmitters Although both DISC-1 variants colocalized with release (see Supplementary Material). For example, PCM1 at the centrosome, the area of colocalization microtubules act as rails along which molecular seemed smaller in SH-SY5Y cells transfected with motors transport intracellular cargoes, including that Phe607 DISC-1 (Figure 1a). Quantitative assessments of precursors of synaptic vesicles, to axon terminals. confirmed that centrosomal PCM1 immunoreactivity Hence, altered neurotransmitter release observed after was significantly less after Phe607 DISC-1 transfec- Phe607 DISC-1 transfection could be due to PCM1- tion than after Leu607 DISC-1 transfection and in related changes in microtubule organization and its untransfected cells (Figure 1b). No changes in the resultant alteration in synaptic vesicle precursor ratio of tyrosinated to detyrosinated a-tubulin were transport. However, it is not possible from our data detected after transfection with either variant (mean to determine if decreased neurotransmitter release is Letters to the Editor 557 Figure 1 (a) The area of PCM1 immunoreactivity at the centrosome of untransfected SH-SY5Y cells (left panel) appeared larger than in cells transfected with Phe607 DISC- 1 (right panel). Arrows point to the centrosome. V5-tagged Phe607 DISC-1: green; PCM1: red; DISC-1 and PCM1 colocalization: yellow. Scale bar = 10 mm. (b) Quantitative analysis showed that centrosomal PCM1 immunoreactive

area differed between experimental groups (F2, 209 = 16.84, P < 0.001), being decreased in SH-SY5Y cells transfected with Phe607 DISC-1, as compared with untransfected cells and cells transfected with Leu706 DISC-1. No difference in PCM1 immunoreactivity at the centrosome was detected between untransfected cells and those transfected with Leu607 DISC-1 (P = 0.603). Each point represents centroso- mal PCM1 immunoreactive area for each cell measured divided by the average control (untransfected) value. (c) Transfection of SH-SY5Y cells with Phe607 DISC-1 resulted in decreased noradrenaline release at 5 mM [K þ ]in comparison to untransfected cells run in conjunction with this variant (*P = 0.032, paired sample t-test), but not after transfection with Leu607 DISC-1 (P = 0.207). Release at 100 mM [K þ ] did not change after transfection with either variant (both P > 0.200). See Supplementary Material for further information.

In summary, our data provide evidence that the DISC-1 Leu607Phe substitution is a functional variant, impacting on both centrosomal and synaptic function.

SL Eastwood1, CA Hodgkinson2 and PJ Harrison1 1Department of Psychiatry, University of Oxford, Warneford Hospital, Oxford OX3 7JX, UK and 2Section on Human Neurogenetics, National Institute of Alcohol Abuse and Alcoholism, Rockville, MD, USA E-mail: [email protected]

References

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