12 January 2007. When both genetic and molecular studies link interacting proteins suspected of being involved in a specific disease, scientists can feel fairly confident that they have identified molecular machinations of pathological relevance. As such, DISC1 may have taken on increased importance in schizophrenia research. Three new studies, two molecular and one genetic, strengthen the case for the involvement of DISC1, and its partners, in the pathophysiology of this major psychiatric illness.
In the January 3 Journal of Neuroscience, Japanese researchers report that DISC1, or disrupted in schizophrenia, hitches a protein complex involved in neurodevelopment to the molecular motor that hauls cargo to the far ends of neuronal axons. In an early online publication on December 21, 2006, in Human Molecular Genetics, researchers in Finland report that in families affected by schizophrenia, there is a genetic association between DISC1 and a homolog of NudE-like (NUDEL), one member of the same protein complex. In a second Journal of Neuroscience paper, the Japanese group shows that DISC1 is also involved in shipping the protein Grb2, an intermediary between growth factor receptors and their downstream targets, out to axonal tips. Without DISC1, neurotrophin-3, one of only a few growth factors involved in neuronal survival, fails in one of its major roles—to induce elongation of axons. Together the three papers indicate that failures of axonal transport and hence poor maintenance of axonal tips, either during development or beyond, may be a key facet of schizophrenia.
DISCovered with Kinesin
Since disruption in the DISC1 gene was shown to strongly associate with schizophrenia and other major psychiatric illnesses, scientists have puzzled over the role of the protein product of the gene (see Millar et al., 2000). Some hints came when DISC1 was found to associate with a complex comprising NUDEL, lissencephaly-1 (LIS1), and 14-3-3ε, proteins that play key roles in neuronal development (see SRF related news story). Mutations in LIS1 cause lissencephaly, a rare neurodevelopmental disorder, while LIS1/NUDEL are known to co-migrate into axonal growth cones. The protein 14-3-3ε appears to regulate the transport process by keeping NUDEL chemically modified by phosphorylation. But how DISC1 fits into the picture has been unclear. Now, Kozo Kaibuchi and colleagues at Nagoya University, Osaka City University, and the Protein Research Network, Yokohama, show that DISC1 anchors the NUDEL/LIS1/14-3-3ε complex to kinesin-1, the motor protein that ferries cargo in an anterograde fashion, from the cell body toward the distal end of axons.
First author Shinichiro Taya and colleagues used protein purification methods to identify cytosolic molecules from rat brain that bind to DISC1. After passing extracts through an affinity column made up of the protein, they found that in addition to NUDEL, LIS1, and 14-3-3ε, the kinesin heavy chain protein, KIF5B, had also bound to the affinity matrix. Subsequently, they also detected other kinesin components, including the KIF5A heavy chain and the kinesin light chain, KLC1 (kinesin comprises a tetramer of two heavy and two light chains). A few other proteins also bound to the DISC1 column, but the authors chose to examine the kinesin interaction in depth, given its role in axonal transport.
Using test tube experiments and purified proteins, the researchers found that DISC1 specifically binds to KIF5A and KIF5B, brain-specific heavy chain isoforms. Then, using immunoprecipitation, they determined that DISC1 forms a ternary complex with KIF5A and NUDEL, and also with NUDEL and 14-3-3ε. Using deletion mutants of DISC1, they determined that the N-terminal of the protein was sufficient to bind KIF5A but not NUDEL, whereas the C-terminal alone could bind NUDEL but not KIF5A. Taken together, these findings suggest that DISC1 serves as a link between KIF5A and NUDEL with the N-terminal end binding the former and the C-terminal binding the latter.
Discovered with Kinesin
Microscopic analysis shows that DISC1 and the heavy chain of kinesin-1, KIF5A, colocalize in the growth cones of hippocampal neurons. [Copyright 2007 by the Society for Neuroscience.]
To test the physiological relevance of these experiments, Taya and colleagues examined protein-protein interactions in cells. Using PC12 cells, they found that DISC1 coimmunoprecipitates with KIF5A and the NUDEL/LIS1/14-3-3ε proteins, indicating that they all bind under normal cellular conditions. Fluorescence microscopy showed that DISC1 colocalizes with the other proteins at the distal part of axons in primary hippocampal neurons. Using high magnification, the researchers were able to pin this localization down to mainly the central part of the axonal growth cone, suggesting that the complex formed by the components may have a role in axonal elongation. In support of this idea, Taya and colleagues found that when they used RNAi to silence gene expression in these same neurons, the length of the axons was reduced in cells where either DISC1 or NUDEL/LIS1 was ablated. Knockdown of DISC1 also reduced the amount of NUDEL/LIS1/14-3-3ε in the distal axon but not in the cell body. All told, the data suggest that a major role for DISC1 is to transport the NUDEL complex from the soma to the axonal tips where it helps to facilitate axonal elongation; this function may be compromised in people with schizophrenia and other psychiatric disorders.
A New Mode of Transport
By coupling the LIS1/NUDEL/14-3-3ε complex or Grb2 to kinesin, DISC1 may ensure that the proteins are transported from cell bodies towards the plus end of microtubules and hence the distal ends of axons. [Model courtesy of Kozo Kaibuchi, Nagoya, Japan]
DISCerned Through Genetics
The genetic association study conducted by the Finnish group, led by Leena Peltonen of the National Public Health Institute, Helsinki, adds further support to the link between DISC1, the LIS1 complex, and schizophrenia. First author William Hennah and colleagues, in Finland and the U.S., analyzed 443 genetic markers in 458 Finnish families with hereditary links to the disease. The analysis expands on previous data sets used to study schizophrenia, culminating in one of the largest genetic association studies to date. Analysis of this large data set confirmed a statistically significant association of the DISC1 locus to the illness. A second locus, on chromosome 5, also emerged (at 5q12.3) and is within a region previously discovered to harbor a potential susceptibility gene in Icelandic, British, and Canadian families (see Sherrington et al., 1988 and Bassett et al., 1988). But it was in looking for genetic interactions that Hennah and colleagues found a link between DISC1 and NUDE, or nuclear distribution gene E homolog 1 (NDE1), which, like its cousin NUDEL, forms complexes with LIS1 and DISC1.
Because schizophrenia is now widely believed to be a polygenic disorder, affected by variation in many genes, the researchers asked what new genetic associations might emerge from their data if they analyzed only those samples that were positive for schizophrenia-linked DISC1 variations. Since the authors had previously identified a DISC1 haplotype (HEP3, comprising two single nucleotide polymorphisms) that associates with the illness, they split the samples into HEP3-positive and HEP3-negative pools and reanalyzed for genetic markers. This time a marker on chromosome 16 emerged as being significantly linked to schizophrenia in the HEP3-positive set. Because the marker, at chromosome 16p13, lies extremely close to the NDE1 locus, the researchers analyzed the gene in more detail.
Hennah and colleagues analyzed seven known single nucleotide polymorphisms (SNPs) that account for genetic variation in the NDE1 gene. They found that all seven were inherited together in the same haploblock, which could be “tagged,” or identified, by four of the seven SNPs (see SRF related news story on haplotype analysis). The researchers found that this tag-haplotype and all the individual SNPs, in fact, associate with schizophrenia, but only in females affected by the illness. Nevertheless, the findings suggest that the DISC1 and NDE1 genes may conspire together in the etiology of the disease and support the molecular data linking DISC1 to the NUDEL(NUDE)/LIS1/14-3-3ε complex. “This convergence of multiple lines of evidence starts to implicate not just DISC1 but a ‘DISC1 pathway’ that also incorporates NDE1 and PDE4B in the etiology of schizophrenia, potentially through underlying deficits in learning and memory,” write the authors. PDE4B, or phosphodiesterase 4B, is another protein that binds to DISC1 and has been tentatively linked to schizophrenia (see SRF related news story).
Linked to Signal Transduction
The phosphodiesterase angle also fits with Kaibuchi and colleagues’ other finding, that DISC1 helps transport the growth factor adaptor protein, Grb2. Since PDE4B regulates levels of cyclic AMP, a major signal transduction molecule, and Grb2 modulates signals coming through extracellular neurotrophins, such as neurotrophin-3 (NT-3), the evidence points to DISC1 as having modulatory effects on different cellular signals.
The Grb2 connection comes from a piece of scientific sleuthing similar to that reported in Kaibuchi and colleagues’ first paper. In their other paper in the same issue of the Journal of Neuroscience, first author Tomoyasu Shinoda and colleagues reported that DISC1 also anchors Grb2 to kinesin, facilitating its transport to axonal tips. Furthermore, using the same RNAi treatment described above to ablate DISC1, Shinoda and colleagues found that Grb2 levels at axonal tips were reduced, as was NT-3-induced activation of both ERK kinase and axonal elongation. The decreased axonal elongation and normal levels of Grb2 could only be restored by adding back full-length DISC1 but not an engineered protein that fails to bind Grb2. The data indicate that DISC1 is necessary to transport Grb2 to axonal tips where it helps transduce neurotrophin signals that induce axonal elongation and, perhaps, promote cell survival. The Grb2 connection also suggests a role for DISC1 in growth factor-mediated synaptic formation and maintenance, both in development and adulthood.
It is not clear at exactly what stage of neural development these DISC1 interactions might impact the biology that leads to schizophrenia, but researchers in the field now have some new and interesting biology that can be exploited in the search for potential treatments.—Tom Fagan.
Taya S, Shinoda T, Tsuboi D, Asaki J, Nagai K, Hikita T, Kuroda S, Kuroda K, Shimizu M, Hirotsune S, Iwamatsu A, Kaibuchi K. DISC1 regulates the transport of the NUDEL/LIS1/14-3-3e complex through kinesin-1. J. Neurosci. January 3, 2007;27:15-26. Abstract
Hennah W, Tomppo L, Hiekkalinna T, Palo OM, Kilpinen H, Ekelund J, Tuulio-Henriksson A, Silander K, Partonen T, Paunio T, Terwilliger JD, Lonnqvist J, Peltonen L. Families with the risk allele of DISC1 reveal a link between schizophrenia and another component of the same molecular pathway, NDE1. Hum. Mol. Genet. December 21, 2006. Advance Access Publication. Abstract
Shinoda T, Taya S, Tsuboi D, Hikita T, Matsuzawa R, Kuroda S, Iwamatsu A, Kaibuchi K. DISC1 regulates neurotrophin-induced axon elongation via interaction with Grb2. J. Neurosci. January 3, 2007;27:4-14. Abstract