As part of our coverage of DISC1 2010, held 3-6 September 2010 in Edinburgh, the United Kingdom, we bring you our final meeting missive, from Antonio Rampino, a postdoctoral fellow at the University of Edinburgh.
5 November 2010. Session 6, the first of two on the theme of Networks and Signaling, was opened by Chair Akira Sawa, Johns Hopkins University, Baltimore, Maryland, who introduced Kirsty Millar of Edinburgh University. Millar, whose talk was entitled, “DISC1 and Cyclic AMP Signaling: Modulation of the LIS1/NDE1/NDEL1 Complex and Links to NMDA Receptor Function,” started her presentation by reporting the already existing evidence on DISC1-cAMP signaling relationships (see SRF related news story). The first important link between DISC1 and cAMP signaling is represented by PDE4, a family of proteins primarily involved in cAMP degradation in the cell, some of which are known to be DISC1 interactors, and which are widely implicated in schizophrenia and other psychiatric conditions.
In the first series of experiments that Millar showed, she described how DISC1 modulates PDE4 response to cAMP levels in the cell, possibly through a mechanism of action where DISC1 determines conformational modifications in PDE4. This was demonstrated by overexpressing DISC1 and switching on cyclic amp signaling, then directly measuring the effect upon pde4 catalytic activity. Furthermore, data by Burgin et al. (Burgin et al., 2010) on PDE4 crystal structures demonstrate that phosphodiesterase 4 activity is regulated by controlling access to its active site, so it is possible that DISC1 stabilises the closed (not active) conformation of the enzyme through binding both its UCR2 and its catalytic domain.
DISC1-cAMP interaction is further complicated by the role of GSK3β, another DISC1 interactor whose activity is modulated by DISC1 itself (see SRF related news story). Data displayed by Millar show that PDE4 cAMP hydrolytic activity in SH-SY5Y cells is modulated by lithium chloride, an important mood stabilizer and GSK3β inhibitor. Moreover, PDE4 activity is modulated by specific pharmacological inhibition of GSK3β in the same cell line, according to data from Millar's colleague, Becky Carlyle. This evidence indicates that DISC1, PDE4, and GSK3β could have a coordinated action on cAMP signaling.
Another group of DISC1 interactors—namely LIS1, NDE1, and NDEL1—is possibly required to better understand the big picture of DISC1-cAMP signaling interaction. Partially anticipating Nick Bradshaw’s talk in Session 9, Millar illustrated that NDE1 is phosphorylated by PKA at the T131 site. This phosphorylation process (which is regulated via DISC1-PDE4 interaction) could be responsible for inhibition of NDE1 interaction with LIS1, while augmenting its interaction with NDEL1 (a prediction based on homology modeling). Based on this evidence, it is possible to hypothesize that DISC1/PDE4 determine the alternative interaction of NDE1 with LIS1 and NDEL1 in a cAMP-PKA dependent fashion. This would eventually result in influencing the neurodevelopmental processes regulated by the LIS1/NDE1/NDEL1 complex.
Further data presented by Millar show that PDE4 modulates cAMP production in response to NMDAR stimulation in mouse cortical neurons pharmacologically treated with an NMDAR agonist. At the same time, at the synapse, the Golgi apparatus and the centrosome, cAMP signaling is modulated by PKA activity through the intervention of a scaffolding protein called A kinase anchoring protein 9 (AKAP9). Millar, for the first time, reported the characterization of AKAP9-DISC1 complexes. In these data from her colleague Shaun Mackie, AKAP9 and
DISC1 colocalize in discrete compartments in hippocampal neurons, and endogenous PKA and PDE4D isoforms co-precipitate with DISC1 and AKAP9. The AKAP9 isoform involved here was the one known as Yotiao, previously proven to bind (and regulate) the NR1 subunit of the NMDAR and adenylate cyclase on the cytoplasmic side of the cell membrane (Lin et al., 1998; Westphal et al., 1999; Feliciello et al., 1999; Piggott et al., 2008; Terrenoire et al., 2009). Moreover, in vitro data from Mackie show that DISC1 and PDE4 associate with NR1. This NMDAR subunit can be phosphorylated by PKA at site S897, targeted mutations of which are associated in mouse with impaired AMPAR and NMDAR-mediated synaptic transmission and impaired LTP. Further in vitro experiments from the Edinburgh group show that NR1 phosphorylation at S897 is regulated by PDE4, Millar said.
These results allow us to imagine a complex molecular machinery where DISC1, PDE4, Yotiao, and NR1 interact with each other to regulate NMDAR activity, Millar concluded.
The second talk of this session was given by Li-Huei Tsai from the Massachusetts Institute of Technology, entitled “DISC1 and Wnt Signaling in Psychiatric Disease.” Opening her talk, Tsai described the already published data on Wnt signaling and DISC1. One of the key bits of evidence here is that DISC1 fine-tunes GSK3β/β-catenin signaling to regulate neural progenitor proliferation, and that DISC1 deficiency in adult mouse dentate gyrus causes hyperlocomotion and depression-like behaviors which can be rescued by GSK3β pharmacological inhibitors (see SRF related news story). The role of the DISC1-GSK3β-β-catenin pathway in neuronal development was further confirmed by the evidence that downregulation of DISC1 blunts β-catenin signaling in cortical neural progenitors in vitro and in utero, and that DISC1 silencing reduces progenitor proliferation in utero.
Tsai and collaborators have discovered a novel DISC1 interactor called Dixdc1 (see SRF related news story). This molecule, which is one of the three known molecules to posses a Dishevelled-Axin (DIX) domain, functions as a positive regulator of the Wnt/β-catenin pathway (Shiomi et al., 2003) and binds the C-terminus of DISC1. Tsai showed that the knockdown of Dixdc1 in the mouse embryonic neocortex results in a significant reduction in neural progenitors’ proliferation. More interestingly, this reduction leads to cell cycle exit and accelerates differentiation processes in neurons.
The effects of Dixdc1 or DISC1 knockdown were rescued by overexpression of the other gene. Furthermore, knockdown of Dixdc1 or DISC1 led to a reduction in Wnt3a-induced TCF/LEF reporter activity, a phenotype which could again be rescued by gene overexpression and overexpression of a Dixdc1 fragment that inhibits the interaction of Dixdc1 with DISC1. These data allowed Tsai to conclude that Dixdc1 and DISC1 modulate neuronal progenitors’ proliferation through the Wnt/β-catenin pathway.
Experiments on DISC1 and Dixdc1 knockdown also showed that both these expression manipulations were able to inhibit neuronal migration, but these knockdown phenotypes were not rescued by overexpression of the other gene or by expression of degradation-resistant β-catenin, bringing Tsai and colleagues to the hypothesis that Dixdc1 and DISC1 can regulate migration in a Wnt/β-catenin signaling independent fashion. Further experiments showed that Ndel1 (a well known DISC1 interactor) interacts with Dixdc1 both in vitro and in vivo. This interaction requires Dixdc1 phosphorylation at serine 250 by Cdk5 (while interaction between Dixdc1 and DISC1 is independent from Dixdc1 phosphorylation). Tsai and collaborators suggest that the phosphorylation of Dixdc1 can work as a switch for the involvement of DISC1 in progenitor cell proliferation and migration.
In the last part of her talk, Tsai described the results of a series of ongoing studies that her group is carrying out in collaboration with Pamela Sklar of the Stanley Centre for Psychiatric Research at the Broad Institute of MIT and Harvard. Tsai and Sklar have worked on the effects of structural and non-structural DISC1 variants on canonical Wnt signaling and brain development, showing how common DISC1 variants can actually be detrimental to protein function and interact with strong rare alleles to modulate disease presentation. In particular, Tsai presented data showing how common variants of DISC1 affect Wnt signaling and, consistently, neuronal proliferation.
As the third speaker of the session, Sawa invited on the stageJu Young Kim from Johns Hopkins University. Kim, whose talk was entitled, “AKT/mTOR Signaling Mediates DISC1 Function In Neuronal Development During Adult Neurogenesis,” briefly summarized the existing evidence supporting the role of DISC1 as a scaffold protein which regulates neurodevelopment. Studies by Duan et al. (see SRF related news story) and Faulker et al. (see SRF related news story) have shown how DISC1 suppression in proliferating neural progenitors leads to marked defects in a number of neurodevelopmental processes in adult hippocampus dentate gyrus. Kim and colleagues have recently shown that DISC1 effects on such processes is mediated via AKT/mTOR pathway through direct interaction of DISC1 with KIAA1212/Girdin complex (see SRF related news story). As further support to this evidence, Kim reported how DISC1 knockdown and AKT activation in adult-born neurons lead to similar developmental defects and similar defects in dendritic development in the adult brain. At the same time, rapamycin (an mTOR inhibitor) is able to rescue these phenotypes from DISC1 knockdown in both newborn neurons and in the adult brain. Following up on this evidence, Kim and collaborators recently found that further molecular mechanisms are potentially implicated in the DISC1-mTOR-AKT pathway, particularly the mTOR-Cyfip1-Elf4E pathway regulating adult neurogenesis.
The last contribution to Session 6 was by Talia Atkin from University College London, who talked about “The Effect of DISC1 on Mitochondrial Transport in Neurons." After a general introduction about the role of DISC1 in the regulation of brain function and in psychiatric diseases, Atkin focused her attention on mitochondria, the main topic of her presentation. A rich corpus of evidence shows that mitochondria are one of the main sources of energy in neurons, and that their correct functioning in the cell requires them to be transported from the cellular soma to the synaptic terminus, hence, the importance of better understanding the molecular machinery responsible for this transport process. DISC1, which colocalizes with mitochondria in neurons (Millar et al., 2005), has been implicated in this machinery through evidence that it interacts with kinesin-1 motors (Camargo et al., 2007). Using an RNAi approach and a real-time mitochondria movement assay, Atkin showed that RNAi to DISC1 inhibits mitochondrial movement in axons, and that DISC1 enhances mitochondria recruitment to the microtubular system. Based on this evidence, she concluded that DISC1 plays an important role in mitochondria transport and that some of the known variants of DISC1 gene could affect this process and lead to brain malfunction and psychiatric diseases.—Antonio Rampino.