22 June 2006. The DISC1 (disrupted in schizophrenia 1) gene and its protein product have been the subject of intense study since its identification as a putative risk factor for some cases of schizophrenia and other major mental disorders just six years ago. DISC1 protein plays an important role in brain development: perturbing expression of DISC1 in the organizing mouse brain leads to aberrant neuronal cell migration and neurite extension, which may underlie the neurodevelopmental origins of schizophrenia and bipolar disorder. But researchers have also begun to appreciate DISC1’s involvement in synaptic function in adults (see SRF related news story), an appreciation that should deepen upon viewing the first ultrastructural images of DISC1 localization in normal human neocortex, courtesy of Rosalinda Roberts and colleagues at the University of Maryland School of Medicine and Johns Hopkins Medical School.
Their study, published online May 30 in the Journal of Comparative Neurology, reveals that the protein can be found in pre- and/or postsynaptic structures, and in both excitatory and inhibitory synapses. DISC1 signal also lights up the nucleus and microtubules, consistent with the protein’s structure and known functions (see SRF related news story), though the authors failed to replicate mitochondrial localization from previous studies.
To determine DISC1 localization in adult parietal and frontal cortex, first author Brian Kirkpatrick and colleagues collected postmortem tissue from seven normal adults within hours of death. The DISC1 antibody they used (produced by coauthor Akira Sawa; see Ozeki et al., 2003) reacted with both pyramidal and nonpyramidal neuron cell bodies throughout most of the gray matter layers. The researchers saw prominent labeling in both cell bodies and apical dendrites. At light microscope-level resolution, they saw diffuse neuropil labeling and some glial labeling as well. These results agree with in situ hybridization and immunohistochemistry studies in both mice and monkeys, which found DISC1 in neurons across all cortical layers and, at least in monkeys, in some glia as well.
The researchers then hunted for DISC1 within neurons using electron microscopy. While those splotchy EM micrographs are not exactly cutting-edge technology for viewing cells in action, the "ultrastructuralists" have gleaned some interesting things over the years. Marc Colonnier first noted that synapses are either "asymmetrical," with thick, complex postsynaptic densities (PSDs), or "symmetrical," with thin PSDs more resembling the presynaptic membrane (Colonnier, 1968). It was later found that asymmetrical synapses are almost always excitatory, while symmetrical synapses are inhibitory or modulatory. In the neocortex, this means that asymmetrical synapses represent primarily communication between different cortical areas, or between the thalamus and cortex, while symmetrical synapses represent primarily local circuits.
Postsynaptic density labeling
In the photomicrograph above, postsynaptic density (PSD) labeling represents a novel finding. (Image courtesy of the authors, Journal of Comparative Neurology and John Wiley and Sons.)
Kirkpatrick and colleagues found DISC1 in nearly half of all synapses in their sections. The label was detected in axon terminals, PSDs, and dendritic spines, though not all these structures were necessarily labeled in a given synapse. The most common configuration was an unlabeled axon terminal making an asymmetric synapse with an immunoreactive spine. The finding of DISC1 signal in PSDs is novel, and in some cases only the PSD of a synapse, always asymmetric, was labeled. The authors note that the labeling of PSDs is known to be subject to artifacts, but they argue that the observation of DISC1 immunoreactivity in just a fraction of PSDs of each section suggests that the signal is genuine.
Classification of synapses as asymmetric or symmetric revealed dramatically different labeling patterns. Equal numbers of asymmetric versus symmetric synapses were DISC1-reactive, but most of the labeled asymmetric synapses (89 percent) had DISC1 localization in postsynaptic structures, whereas most labeled symmetric synapses (76 percent) showed DISC1 in the axon terminal. The presence of DISC1 in both asymmetric and symmetric synapses suggests it functions in corticocortical, thalamocortical, and local neocortical circuits. However, the fact that symmetric (inhibitory) synapses represent only a fraction (5-25 percent) of total cortical synapses (DeFelipe and Fariñas, 1992), yet accounted for half the labeled synapses in this study, raises the possibility that DISC1 has greater significance in inhibitory transmission.
In the cell body, the DISC1 antibody labeled ribosomes and some nuclear material. In dendritic shafts, the researchers detected labeling of microtubules, suggesting that DISC1 might be involved in transport in adult brain, consistent with its role in neuronal migration during development. No mitochondrial labeling was seen, in contrast to previous reports with human cells in culture and postmortem hippocampus (Brandon et al., 2005; James et al., 2004). The organelles of cellular protein excretion—Golgi apparatus, lysosomes, multivesicular bodies—were also found to be devoid of DISC1.
Immunolocalization studies are only as good as the antibodies used, and the authors present a panel of controls for antibody specificity. Like many DISC1 antibodies, the polyclonal serum recognized multiple species in immunoblots of human brain proteins. In addition to the full-length 105 kDa DISC1, the other bands consisted of a putative dimer, possibly phosphorylated DISC1 and a proteolytic fragment. Since all of the bands were completed with the original antigen (a peptide of DISC1), they were judged to be specific. Given the unknown nature of some of the reactive bands, it is reassuring that the immunolabeling results generally reinforce previous work on DISC1 function, and as such should serve as a guide to future work to unravel the multifaceted physiology of this protein.—Pat McCaffrey.
Kirkpatrick B, Xu L, Cascella N, Ozeki Y, Sawa A, Roberts RC. DISC1 immunoreactivity at the light and ultrastructural level in the human neocortex. J Comp Neurol. 2006 Jul 20;497(3):436-50. Abstract