The protein of the developmentally regulated Disrupted-in-Schizophrenia-1 (DISC1) gene is involved in a wide variety of neurological processes in the developing mammalian brain. Mutations in DISC1 have been associated with a predisposition for schizophrenia (and other mood disorders), hence its name. DISC1 is expressed in discrete tissues in the mouse brain during 2 developmental phases. One occurs in utero, during early brain development; the second, at the onset of puberty.
We now have the ingredients necessary to explain a truly powerful finding involving DISC1, puberty, and the developing cortex of a mouse. By transiently altering the expression of DISC1, you can induce behaviors reminiscent of schizophrenia.
Using IUEP, GFP was bilaterally introduced into mouse lateral ventricles at day 14 postconception (called E14, for embryonic day). By day P56 (postnatal, 56th day), green cells were observed at many cortical sites, including the medial prefrontal cortex (mPFC), dorsolateral prefrontal cortex (dlPFC), orbitofrontal cortex (OFC), and anterior cingulate. This confirmed that with the introduction of foreign genes into the lateral ventricles during embryonic growth, expression could be observed in cortical regions long after birth.1 The constructs were found to be integrated into a lineage of pyramidal neurons.
What would happen if you performed an IUEP experiment at E14 with a green reporter gene, accompanied by an shRNA to DISC1? This would allow transient blocking of DISC1 expression at a time when the cells were forming critical cortical structures. This experiment was done, and preliminary data showed you could indeed get transient blocking of DISC1 expression in green cells using the technologies just described. Once again expressed primarily in pyramidal cells, the suppression of native DISC1 was shown not to be permanent. DISC1 was inhibited for 7 days postinjection but was found to be fully recovered, expressing in a normal pattern 3 weeks later.
This technique is very different from your standard KOAs. In fact, the technique is called “knockdown” to indicate this transient inhibition. What did the brain of these mice look like? How did the mice behave?
The researchers found some extraordinary things. The physical architecture of the cortex of these knockdowns was greatly affected, as were certain metabolic features. Dendritic cell abnormalities normally associated with apoptosis were found in cortical structures at P14 in the experimentals. Intriguingly, anomalies in the projection patterns of the mesocortical dopamine(Drug information on dopamine)rgic neurons to the mPFC were found in the knockdowns at P56, but not in the controls. There were decreases in the extracellular concentrations of dopamine in the mPFC, too, but not in other regions of the brain (the cerebellum and the nucleus accumbens had typical levels, for example). Levels of tyrosine hydroxylase, as measured by immunoreactivity, were inhibited as well. As you recall, tyrosine hydroxylase catalyzes the conversion of the amino acid tyrosine into a dopamine precursor. That development was disrupted by performing in utero gene transfer experiments affecting known, developmentally regulated genes in specific areas of the brain associated with cortical development.
Did any of these maturational anomalies translate into behavioral changes? Two appeared to, both presenting after the murine equivalent of puberty. One deficit occurred with memory, the other, with the ability to organize sensory information.
The memory test involved a standard behavioral protocol called a novel object recognition task (NORT). This protocol assesses mammalian visual working memory, measuring interactions between the hippocampus and cortex. No differences in NORT scores were observed in the prepuberty animals of either the knockdowns or the controls (P28). However, memory impairments were consistently observed in the knockdown animals but not in controls after puberty (P56). The same suite of findings was observed in response to a T-maze memory test. Intriguingly, cognitive scores could be greatly improved if clozapine(Drug information on clozapine) was administered to the P56 knockdowns.
Another behavioral measure involved an assessment termed the “prepulse inhibition test” (PPI). This test measures information processing abilities that directly involve the cortex (sensory gating). The same puberty-dependent patterns of behaviors observed in the NORT and maze tests were found in the PPI assessment. No differences were observed in the P28 animals of either the knockdowns or the controls, but great differences were observed in the P56 animals. Before, knockdown animals showed impairment, and the controls did not.
Both behavioral deficits are associated with behaviors commonly observed in schizophrenia. The changes are quite specific. In a forced-swim water maze test (an assay associated with depression in these animals), no differences were observed in any animal tested. Constructing a gene model using DISC1 with such specificity is quite an achievement. And it is not even the biggest one.
The real gold lies in the molecular elegance with which these data were obtained. Timed, transient ablation of gene expression was harnessed to create nonlethal animal models of a mental disease. So many developmental processes in the brain involve subtle, fleeting alterations in gene expression. Short of deploying the blunt, low-resolution instrument of KOAs, it is often difficult to conceive of experiments capable of ferreting out these more subtle secrets. Using IUEP in combination with RNAi assays in a knockdown paradigm can get at some of these issues, opening up an array of questions for ex-experimenters to consider; the potential to shed light on laundry lists of disorders is extraordinary.
We’ve come a long way since my first exposure to molecular manipulations involving whole animals. I have a feeling that schizophrenia is just the start.