29 September 2008. The schizophrenia susceptibility gene candidate disrupted in schizophrenia 1 (DISC1) appears to be necessary for the proper targeting of axons and synapses in newly born neurons of the adult hippocampus, according to a study published in the September 16 issue of Proceedings of the National Academy of Sciences. A collaborative team, led by Edward Jones and Hwai-Jong Cheng at the University of California, Davis, and Hongjun Song at Johns Hopkins University, Baltimore, Maryland, reports that hippocampal mossy fibers from dentate granule cells generated in the adult mouse brain form synapses with CA3 pyramidal cells two weeks after their generation and that these synapses become mature within eight weeks. When DISC1 was knocked down, the researchers found defects in axonal path-finding and synapse formation.
Aberrant neurodevelopment may play a major role in schizophrenia. Since the disorder frequently emerges in late adolescence and adulthood, however, neurons may not start to go awry until these later stages of life. Traditional neuroscience theories once decreed that new neurons are only born in the neonate, but it is now well accepted that the adult brain can give life to new neurons and synapses. It seems clear, then, that it is possible for cells newly generated in adult brain to contribute to axonal miswiring in schizophrenia.
In this context, DISC1 is of tremendous interest to researchers. DISC1 is a key susceptibility gene candidate that has been associated with familial inheritance of schizophrenia and other major mental illnesses. Disruption of the protein causes abnormal cortical (see SRF related news story) and hippocampal development (see SRF related news story; SRF news story; SRF related news story), as well as reduced frontal cortical grey matter and impaired memory (Cannon et al., 2005). A study published last year by Song and colleagues found that knocking down DISC1 expression in adult mouse neural progenitors produced changes in neuronal position, morphology, firing, and integration into existing circuits for the mature dentate granule cells (see SRF related news story), strongly supporting the role that this gene plays in hippocampal connectivity, and possibly also in schizophrenia. In that study, Duan and colleagues focused on dendritic and somal effects of DISC1 disruption; in the current paper, first author Regina Faulkner of UC Davis and colleagues turn their attention to the output end of things, the axons and their synaptic terminals.
New cells, same old boutons
There are a number of lines of evidence supporting hippocampal involvement in schizophrenia neuropathology. These abnormalities are thought to contribute to neuropsychological impairment in schizophrenia, rather than psychotic symptoms (for review, see Harrison, 2004). In this new study, Faulkner and colleagues studied both the integration of newly generated dentate granule cells into adult brain circuitry, as well as how perturbation of DISC1 disturbs axonal and synaptic development in adult brain. As Kate Meyer and Jill Morris of Northwestern University, Chicago, have recently reported, DISC1 is expressed throughout the embryonic and adult hippocampus, including the proliferative zone (Meyer and Morris, 2008). Faulkner and colleagues transfected neurons in the dentate gyrus of adult mice using a retrovirus expressing green fluorescent protein (GFP). This method specifically labels proliferating cells and the progeny of those cells. They visualized the cells using confocal and quantitative immunoelectron microscopy, and three-dimensional reconstruction of GFP-labeled boutons, the axonal protuberances that house the presynaptic apparatus.
The researchers followed the migration of the mossy fiber axons that arise from these cells, and found that they reached a specific region of CA3 in the hippocampus—stratum lucidum—by one week following the dentate gyrus injection. The positively labeled (GFP+) axons began to form synaptic boutons by a week and a half following the dentate gyrus injection, and the GFP+ axons never traveled beyond the CA3 region. The mossy fibers of dentate granule cells generated in the adult therefore seemed to travel on the same path as pre-existing mossy fibers.
Because synaptic boutons formed, the authors concluded that synapses must be forming in CA3. Faulkner and colleagues further analyzed the characteristics of the boutons using electron microscopy. They found that at both eight and 16 weeks following the dentate gyrus injections, the GFP+ boutons appeared to be morphologically very similar to control boutons around them. They confirmed that the boutons established mature synaptic contacts at eight weeks by quantifying the number of invading dendritic spines and finding that GFP+ had a similar number of spine contacts as control.
A knockdown drag out synaptic fight
To see if DISC1 influenced this development of mossy fiber connections to CA3, the researchers knocked down DISC1 expression using retrovirus-mediated expression of a short hairpin RNA that interferes with expression of the mouse DISC1 gene (shRNA-D1). They labeled axons using green fluorescent protein as before. At one week following the injection of the shRNA-D1, the GFP+ mossy fiber axons were longer than control axons. After 1.5 weeks and at all times viewed thereafter, GFP+ mossy fiber axons extended beyond CA3 into the CA1 region of the hippocampus, something that never happened in control axons. Also, despite the axonal projections into CA1, boutons did not make clear synaptic contacts and in many cases did not appear to develop normally into mature boutons, as analyzed using electron microscopy. For boutons that did reach the CA3 region, bouton development actually appeared to be accelerated, with synaptic spine contact occurring sooner than in control animals.
The authors surmise that new neurons generated in adult dentate gyrus form normal mossy fiber contacts and become part of the regular brain circuitry. DISC1 seems to be important for regulating both the ability of axons to reach their appropriate target, as well as the rate at which normal synaptic contacts are made. Their findings are supported by studies showing that mossy fiber boutons observed in postmortem tissue taken from the brains of people with schizophrenia are different from mossy fibers observed in postmortem control brains, since they have contact with fewer dendritic spines and seem to make fewer synapses (Kolomeets et al., 2005; Kolomeets et al., 2007). Taken together with the report last year from Duan and colleagues, the present study suggests a pivotal role for DISC1 mutations in disrupting hippocampal circuitry in schizophrenia, although the exact mechanisms by which DISC1 affects adult neuro- and synaptogenesis remains to be understood.—Alisa Woods.
Faulkner RL, Jang MH, Liu XB, Duan X, Sailor KA, Kim JY, Ge S, Jones EG, Ming GL, Song H, Cheng HJ. Development of hippocampal mossy fiber synaptic outputs by new neurons in the adult brain. Proc Natl Acad Sci U S A. 2008 Sep 16;105(37):14157-62. Abstract