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DISC1 Tied to Motivation, Oxidative Stress in Mice

July 16, 2013. Truncating both copies of the disrupted in schizophrenia 1 (DISC1) gene in mice impairs cognition and motivation, and leads to signs of oxidative stress in the brain, according to a study published July 8 in the Proceedings of the National Academy of Sciences. Led by Akira Sawa and Michela Gallagher, both at Johns Hopkins University in Baltimore, Maryland, the study delves into the effects of truncated copies of DISC1, which are proposed to interfere with normal DISC1 function. Mice homozygous for this putative dominant negative form (DN-DISC1) had problems adapting their behavior to changing conditions, were less likely to work for reward, and showed signs of oxidative stress in prefrontal cortex.

The results link DISC1 to the realms of motivation and behavioral flexibility, which are compromised in psychiatric disorders. Because DISC1 features as a player in neurodevelopment and synapse function (see SRF related news story), it may not be a surprise that it influences disparate domains of brain function. The same group, however, previously reported subtle phenotypes in mice heterozygous for DN-DISC1 (see SRF related news story), and so they set out to look at homozygotes. The DN-DISC1 model introduces a truncated form of the protein that mimics a product of the DISC1-disrupting chromosomal translocation found in a Scottish family beset by mental illness. Sawa and colleagues have proposed that this truncated DISC1 would bind normal DISC1 and prevent it from doing its job.

The study also suggests calming oxidative stress as a therapeutic approach for psychiatric diseases. Oxidative stress results when cells are overwhelmed by reactive oxygen molecules, and this condition can do some lasting damage in non-renewing neurons. Evidence for oxidative stress in schizophrenia comes from animal models (see SRF related news story) and from the decreased numbers of parvalbumin-containing interneurons found in schizophrenia. These fast-spiking, metabolically hungry neurons may be especially vulnerable to oxidative stress (see SRF related news story).

Fixated and unmotivated
First authors Alexander Johnson and Hanna Jaaro-Peled compared homozygous DN-DISC1 mice that resulted from breeding the heterozygous DN-DISC1 mice. Wild-type mice that were not littermates served as controls. As a first test of cognitive function, the researchers tested mice on reversal learning, which relies on the orbitofrontal cortex and measures how well the brain can change to a new set of rules. For example, the mice learned that poking their nose to the left would get them a sucrose reward, while poking it to the right would not. When the researchers switched the rules so that poking to the left gave no reward, but poking to the right did, control mice updated their behavior to the new rules, but DN-DISC1 mice initially did not.

Further testing suggested that this impairment stemmed from problems with connecting information about the value of a reward to behavior. For example, the researchers decreased the value of a food reward by letting the mice eat that food before the test—effectively ruining their appetite for that food. When they did this, the DN-DISC1 mice continued to press a lever associated with the food with gusto, as much as another lever that delivered a food they had not been fed ahead of time. In contrast, control mice scaled back their responses to the devalued food lever. This suggests a problem in getting the value of an outcome to shape behavior in DN-DISC1 mice.

Understanding how information about outcomes guides behavior also enters into the domain of motivation, and here the researchers also found differences in DN-DISC1 mice. When more effort was required to get a reward, this increased pleasure-related licking to that reward in control mice, but not in DN-DISC1 mice. This again suggests a disconnect between the value of something and its effect on behavior, which can be taken as a sign of reduced motivation. In another test, the researchers made it progressively harder for the mice to get their reward: Mice initially received sucrose after making 20 licks at an empty well, but the requirement gradually ratcheted up to 300 licks. Though the DN-DISC1 mice showed a normal preference for sucrose, they licked half the number of times controls did over the course of this test. Eventually, all DN-DISC1 mice (compared to half of the controls) quit licking altogether before the test was completed.

Prefrontal stress
The researchers then examined the prefrontal cortex for signs of oxidative stress. They found increased 8-oxo-dG staining, which marks spots of oxidative damage in DNA, in the orbitofrontal cortex in DN-DISC1 mice relative to controls. Another sign came in the form of the enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH): Under conditions of oxidative stress, GAPDH binds to a protein called Siah, and together they travel to the nucleus to spur a cascade of events. The researchers found a 10-fold increase in GAPDH-Siah binding in DN-DISC1 mice compared to controls. This increase occurred in prefrontal cortex, but not striatum, a brain area that interacts with prefrontal cortex. Although DN-DISC1 heterozygous mice have a reduced number of parvalbumin-containing neurons (see SRF related news story), the researchers did not report any pathology related to these neurons in DN-DISC1 homozygous mice.

The researchers mention they have developed inhibitors of the GAPDH cascade, but they do not test them in this study. If quelling oxidative stress normalizes cognition and motivation in these mice, this may pave the way for new therapeutics for psychiatric disorders. For now, it seems DISC1 has added a few more things to its bag of tricks, and future experiments will have to detail how DN-DISC1 expression leads to oxidative stress in prefrontal cortex.—Michele Solis.

Reference:
Johnson AW, Jaaro-Peled H, Shahani N, Sedlak TW, Zoubovsky S, Burruss D, Emiliani F, Sawa A, Gallagher M. Cognitive and motivational deficits together with prefrontal oxidative stress in a mouse model for neuropsychiatric illness. Proc Natl Acad Sci U S A. 2013 Jul 9. Abstract

Comments on Related News


Related News: Modeling Schizophrenia Phenotypes—DISC1 Transgenic Mouse Debuts

Comment by:  David J. Porteous, SRF AdvisorKirsty Millar
Submitted 2 August 2007
Posted 2 August 2007

Several genetic studies point to involvement of DISC1 in major psychiatric illness, including schizophrenia and bipolar disorder, but to date the only causal variant that has been definitively identified is the translocation between human chromosomes 1 and 11 that co-segregates with major mental illness in a large Scottish family and which directly disrupts the DISC1 gene (Millar at al., 2000). It has been speculated that a truncated form of DISC1 may be expressed from the translocated allele and, if so, that this could exert a dominant-negative effect, but there is no such evidence from studies of the translocation cases. Rather, the evidence from studies of lymphoblastoid cell lines carrying the translocation suggests that haploinsufficiency is the most likely disease mechanism in this family (Millar et al., 2005). The unresolvable caveat to this, of course, is that it has not been possible to determine whether this is true also for the brain. Moreover, it is far from certain that any productive product from the translocation chromosome would be a perfectly truncated protein encoded by all the remaining exons, as opposed to an exon-skip isoform, with or without a hybrid protein component borrowing sequence information from chromosome 11. What does seem likely from other human studies is that additional genetic mechanisms, including missense mutations, altered expression, and possibly also copy number variation, play a role in the generality of DISC1 as a risk factor.

The evidence in support of DISC1 as an important biological determinant across a spectrum of major mental illness has now received a further boost from the study in PNAS by Hikida et al. The Sawa lab describes a transgenic approach where a truncated human DISC1 protein is expressed from a CAMKII promoter. The truncation was designed to mimic the hypothetical truncation arising from the Scottish translocation. This forebrain-specific promoter confers preferential expression of the transgene at neonatal stages, as distinct from the expression of the endogenous protein, which is abundant from embryonic development into adulthood. This model therefore permits investigation of the effect of the truncated protein in the forebrain within a specific developmental window, against a background of endogenous mouse DISC1 expression. Since there is no evidence for production of a truncated protein from the translocated allele, the relevance of this model to psychiatric illness remains unclear. However, on the positive side and from a functional perspective, dominant-negative effects as a consequence of expressing the truncated protein have already been demonstrated in cultured cells, altering the subcellular distribution of DISC1 and interaction with DISC1 partner proteins. Moreover, expression of the truncated form of DISC1 mimics downregulation of DISC1 in vivo, inhibiting migration of neurons in the developing mouse cortex (Kamiya et al., 2005). Thus, this model has the genuine potential to enhance our understanding of the biology of DISC1.

This is, in fact, the third study describing mice expressing modified DISC1 alleles. In the first study, Gogos and colleagues (Kioke et al., 2006) studied the effects of a modified DISC1 allele carrying a spontaneous 25 bp deletion in exon 6 that is present in all 129 mouse strains (Koike et al., 2007; see SRF related news story). This allele additionally has an artificial stop codon in exon 8 and a downstream polyadenylation signal. After back-crossing this mutagenised version of the 129 allele onto a C57Bl6 background, they reported a deficit in an assay of working memory in both heterozygous and homozygous mutants, but other standard behavioral tests were unaltered or unreported, and there were no anatomical, electrophysiological, or pharmacological studies included. In the second study, one led by the Roder laboratory, Toronto, we described two mouse strains with missense mutations in exon 2, Q31L and L100P (Clapcote et al., 2007). Reductions in brain volume, deficits in a range of standard behavioral tests, and responses to pharmacological treatments were reported, which can be summarized as consistent with the 100P mutants displaying schizophrenia-like phenotypes and the 31L mutants, mood disorder-like phenotypes. There is a frustrating dearth of consistent biomarkers for schizophrenia, but one of the best replicated findings is by brain imaging of enlarged ventricles in schizophrenia (also, but less markedly, in bipolar disorder). Adding to the observations of Clapcote et al., arguably the most striking claim by Hikida et al. is for altered ventricular brain volume and reduced brain laterality following neonatal transgenic overexpression of truncated DISC1. Additionally, behavioral tests were reported that overlap in part with those reported earlier by Clapcote et al. That three studies all describe behavioral abnormalities consistent with modeling components of schizophrenia is reassuring. That there are clear differences, too, between the phenotypes should come as no surprise either, given the important differences in terms of genetic lesion and developmental expression. Other mouse models are in the pipeline and they, too, will be welcomed. Indeed, this is very much what is needed for the field to move forward. But we should do so with some caution, paying careful attention to the specific nature of the models, what they can and cannot tell us about DISC1 biology, and what they may or may not tell us about the human condition. Although none of these models relates directly to a known causal variant, it appears that the mouse models concur with the human genetic studies in suggesting that there are likely to be several routes by which DISC1 can perturb brain function leading to characteristics of human mental illness. What we need now is for the human genetic studies to catch up with the mouse so that defined pathognomic variants can be modeled.

View all comments by David J. Porteous
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Related News: Modeling Schizophrenia Phenotypes—DISC1 Transgenic Mouse Debuts

Comment by:  John Roder
Submitted 2 August 2007
Posted 2 August 2007

A new mouse model from the Sawa lab strengthens the evidence for the candidate gene DISC1 playing a role in psychosis and mood disorders. This important paper is the first to address one potential disease mechanism, that of a dominant-negative effect. Expression of the C-terminal deletion of human DISC1—which represented the original rearrangement found by the Porteous group in the Scottish families with schizophrenia and depression—in transgenic mice driven by the α CaMKII promoter, first described by Mark Mayford when a postdoctoral fellow in the Kandel lab, leads to mice showing behaviors consistent with schizophrenia and depression, with enlarged lateral ventricles. Since the Sawa group expressed the human C-terminal truncation in mouse with no change in mouse DISC1 levels, they feel this supports a dominant-negative mechanism. More direct experiments are required. For example, create a null mutant mouse for DISC1 and express the full-length and truncated human DISC1 under the influence of their own promoter in transgenic mice using human BACs. Full-length human DISC1 would, hopefully, rescue the null. If so, then a mixture of full-length and truncated DISC1 proteins could be tried. No rescue by the mixture of full-length and truncated proteins would suggest a dominant-negative mechanism.

The Porteous group has shown no detectable DISC1 protein in lymphoblasts from the patients, and put forward the following implicit model. The C-terminal truncation of DISC1 makes the protein unstable and sensitive to degradation, a plausible alternative notion. In my opinion both are likely in different schizophrenia patients with perturbations in DISC1, most of which are alterations other than the C-terminal truncation. Some have SNPs that lead to as yet uncharacterized disease. It has been shown by the Sawa lab that mice with a partial RNAi knockdown of DISC1 show perturbations in brain development, and if aged to 8-12 weeks these mice might have shown behavioral and neuropathological hallmarks of schizophrenia and depression. There is currently no null mutation in the mouse that would address this issue, although DISC1 is one of the genes being targeted in the NIH knockout mouse project. Fortunately, there are now several mouse models—the more the better. The Gogos lab has a 25bp deletion in exon 6 that removes some, but not all isoforms. The Roder lab used a reverse genetic screen of an ENU archive to generate two missense mutants in non-conserved amino acids. The phenotypes of all these lines are nicely summarized in the Sawa paper. This work represents a step forward in our understanding of the DISC1 gene.

View all comments by John Roder

Related News: Does Oxidative Stress Link NMDA and GABA Hypotheses of Schizophrenia?

Comment by:  John Krystal, SRF Advisor
Submitted 6 December 2007
Posted 9 December 2007

The paper by Behrens and colleagues provides exciting new data to suggest that NADPH oxidase plays an important role in the impact of the NMDA receptor antagonist, ketamine, upon parvalbumin-containing (PVC) fast-spiking GABA interneurons. The authors show that ketamine causes an activation of NADPH oxidase, resulting in increases in superoxide production. The elevation in free radicals, presumably toxic to these neurons, is associated with reduction in the expression of parvalbumin and GAD67. These effects of ketamine could be prevented by inhibition of NADPH oxidase.

These data were interpreted by the authors to help explain the schizophrenia-like effects of ketamine in healthy humans. I think that these data provide important insights into the impact of reductions in NMDA receptor function, and they may be relevant to schizophrenia. First, the data amplify the implications of the work of Kinney, Cunningham, and others who have shown that PVC interneurons express the NR2A subunit of the NMDA receptor and that deficits in NMDA receptor function may contribute to reduced GAD expression by these neurons. Since PVC deficits in GAD expression have been described in postmortem cortical tissue from people diagnosed with schizophrenia, the current data suggest that some of these findings may be attributable to activation of NADPH oxidase. It would be interesting to know whether there is an interaction between this consequence of deficits in NMDA receptor function, a feature associated with schizophrenia, and reductions in the cortical levels of glutathione, also associated with this disorder. Glutathione is a free radical scavenger. In other words, the emergence of GABA neuronal deficits may be an unfortunate consequence of the convergence of a disturbance in glutamatergic neurotransmission and a heritable abnormality in neural metabolism. These data highlight the potential importance of some very preliminary new data that suggest that N-acetyl-cysteine (NAC) may augment antipsychotic effects in treating schizophrenia. NAC raises intracellular glutathione and might be a treatment that targets the cellular process described by Behrens and colleagues.

The Behrens paper also highlights the importance of research studies exploring ketamine effects from a systems and cognitive neuroscience perspective. For example, it does not explain why ketamine effects produce symptoms and cognitive impairments associated with schizophrenia. It is likely that the work of scientists including H. Grunze, R. Greene, B. Moghaddam, R. Dingeldine, M. Cunningham, and others is important to consider. These investigators have shown that NMDA receptor antagonists reduce the recruitment of PVC interneurons in feed-forward inhibition pathways resulting in increased glutamatergic output. When NMDA receptors are blocked, the activity of these neurons produces dysfunctional effects, in that neural activity seems chaotic and the organized oscillatory activity of networks is disrupted. These disturbances in network function are paralleled by abnormal behaviors and cognitive impairments in animals and "schizophrenia-like" symptoms and cognitive deficits in humans. One potential solution to this problem would be to reduce glutamate release, a paradoxical suggestion for a disorder commonly thought of as "hypoglutamatergic" based on loss of cortical connectivity. Yet, in animals and humans, drugs that reduce glutamate release (lamotrigine, group II metabotropic glutamate receptor agonists) reduce the physiologic and behavioral effects of NMDA glutamate receptor antagonists. Further, there are now some encouraging clinical data that lamotrigine and, particularly, group II mGluR agonists, might have clinical efficacy in treating schizophrenia.

Overall, we seem to be working in a period where a wide variety of data from many sources is rapidly converging to capitalize on the insight that NMDA receptor antagonists, when administered to healthy people, transiently produce effects that resemble schizophrenia.

View all comments by John Krystal

Related News: Does Oxidative Stress Link NMDA and GABA Hypotheses of Schizophrenia?

Comment by:  Steven Siegel (Disclosure)
Submitted 6 December 2007
Posted 9 December 2007

The article by Behrens and colleagues provides evidence for a mechanistic link between NADPH oxidase and disruption of normal protein expression in some interneurons following the drug ketamine. Data presented demonstrate that addition of an NADPH oxidase inhibitor, given in the animal’s drinking water, blocked the effects of ketamine on a specific class of interneurons that contains parvalbumin. Several lines of research suggest that this population of cells is disrupted in schizophrenia, and that reductions of NMDA-type glutamate receptor activity may lead to that impairment. The important iterative advance in the current study links the reduction in NMDA receptor-mediated glutamate transmission to a specific intracellular mechanism and molecular pathway. Furthermore, the authors demonstrate that they can effectively block the cellular changes by inhibiting that pathway, suggesting a novel therapeutic target.

This leads to two major questions: 1) Could NADPH oxidase inhibitors, or similar mechanisms be used to avert the onset of schizophrenia if administered during a prodromal period? 2) Is the process of reduced parvalbumin expression reversible? Some studies have shown that drugs like ketamine, which reduce activity at NMDA receptors, actually lead to cell death, suggesting that only prevention would be possible. Alternatively, there is evidence that the parvalbumin-containing cells in schizophrenia may not be dead and gone, but rather have impaired function and loss of this particular protein. In this latter scenario, it is possible that the effects of the illness could be reversible. Given that ketamine also causes a variety of functional changes in animals, including electrical brain activity and behavior, the current work lays the groundwork for future studies to determine if co-administration of NADPH oxidase inhibitors can block the functional consequences of ketamine and, by extension, reduce NMDA receptor activity in general.

View all comments by Steven Siegel

Related News: Does Oxidative Stress Link NMDA and GABA Hypotheses of Schizophrenia?

Comment by:  Dan Javitt, SRF Advisor
Submitted 7 December 2007
Posted 10 December 2007

The study by Behrens and colleagues is an excellent illustration of how breaking with traditional paradigms can lead to identification of novel potential targets for intervention in schizophrenia. As detailed on the pages of Schizophrenia Research Forum (e.g., Interview with D. Lewis) and the cited articles from F. Benes, one of the most consistent findings in schizophrenia is the downregulation of PV and GAD67 expression in PV+ GABAergic interneurons. Dysfunction of these neurons, in turn, may be responsible for frontal neurocognitive and dopaminergic deficits. The underlying cause of the GABAergic interneuron changes, however, has only intermittently been investigated.

One of the leading potential mechanisms underlying reduced PV and GAD67 expression in brain in schizophrenia has always been NMDA dysfunction, given the strong expression of NMDA receptors on GABA interneurons, as described by Behrens and colleagues, and the well-known ability of NMDA antagonists to induce both symptoms and neurocognitive deficits closely resembling those of schizophrenia. Last year, Kinney and colleagues demonstrated that exposure to the NMDA antagonist ketamine reduced PV and GAD67 expression in GABAergic interneurons in vitro (Kinney et al., 2006). The present study builds upon this finding and demonstrates a similar phenomenon in vivo. Moreover, it builds upon this finding to demonstrate that these changes can be reversed by antagonists of NADPH oxidase, suggesting a potential target for intervention.

This study thus adds reduced GAD67 and PV expression in PV+ GABAergic interneurons to the long list of findings in schizophrenia that can be viewed as “downstream” of a more proximal deficit in NMDA-mediated neurotransmission, and supports interventions aimed specifically at frontal GABAergic interneurons, as well as more generally at reduced NMDA activity throughout brain. This preparation, moreover, may be appropriate to the testing of novel glutamatergic agents.

Behrens and colleagues' article, however, also leaves many questions unanswered. For example, loss of PV and GAD67 in schizophrenia is not confined to prefrontal cortex. It would be of interest to know, therefore, whether histological changes induced by ketamine are or are not confined to this region. As with all proposed new drug targets, it will also be important to know what other processes NADPH oxidase is involved with both inside and outside brain before proposing it too seriously as a drug target. It is one thing to reverse a specific deficit in a short-term treatment model, another to contemplate long-term treatment. At first glance, NADPH oxidase would seem to be a very general enzyme, which is being targeted to treat a very specific condition. Nevertheless, if NADPH oxidase activity can safely be blocked throughout the body long term, the present findings may point the way for new treatments for schizophrenia.

View all comments by Dan Javitt

Related News: Does Oxidative Stress Link NMDA and GABA Hypotheses of Schizophrenia?

Comment by:  Julie MarkhamJames I. Koenig
Submitted 10 December 2007
Posted 10 December 2007

The role of reactive oxygen species in the pathogenesis of schizophrenia is currently unclear. Several lines of evidence support a greater production of these reactive molecules in schizophrenia because of reduced levels of important buffers for superoxides, such as glutathione. Other research, however, suggests that antipsychotic drugs themselves increase the production of oxygen radicals. In this week’s issue of Science, Behrens and colleagues present data supporting the involvement of reactive oxygen species in the pathophysiology of schizophrenia. The authors have previously shown that administration of an NMDA receptor antagonist to primary cultures of cortical neurons results in the loss of GAD67 and parvalbumin (PV; a calcium-binding protein) from PV positive GABAergic interneurons (Kinney et al., 2006), similar to what has been observed in studies using postmortem tissue from patients with schizophrenia (e.g., Volk et al., 2000; Hashimoto et al., 2003). In this study, administration of the NMDA receptor antagonist ketamine was found to increase production of reactive molecules both in vitro (following bath application of the drug to cultured neurons) and in vivo (following two injections of the drug to mice). Moreover, inhibition of the enzyme NADPH oxidase prevented the reduction of both PV and GAD67 expression. The authors suggest that inhibition of NADPH oxidase may represent a novel treatment for both ketamine-induced psychosis and schizophrenia.

While the authors’ findings are undoubtedly exciting, some limitations of their approach need to be addressed before over-enthusiasm regarding NADPH oxidase inhibition as a treatment for schizophrenia is generated. Although the title advertises a “loss of phenotype of fast-spiking interneurons,” the reduction in PV and GAD67 expression from neurons that remain PV positive does not represent a loss of phenotype, and the ketamine-induced increase in superoxide production was not specific to interneurons (only 5-10 percent of primary cortical neuron cultures are PV positive, yet the effect was observed throughout sampled cells). Also, although their findings bear similarity to those observed in schizophrenia, there are notable differences. For instance, whereas the level of PV expression per cell is reduced in schizophrenia (Hashimoto et al., 2003), the level of GAD67 mRNA expression per cell does not differ between individuals with schizophrenia and controls; rather, it appears to be a reduction in the density of neurons that express the transcript. In contrast, Behrens and colleagues report a reduction in the expression per cell for both PV and GAD67. While this difference may simply be due to the fact that Behrens and colleagues examined levels of the proteins, the potential discrepancy should be recognized.

Perhaps the most important limitation to the work is the absence of a functional measure to determine whether the reduction in PV and GAD67 in cortical interneurons observed following ketamine administration results in any of the schizophrenia-associated endophenotypes which can be modeled in rodents. Animal models of schizophrenia employing developmental strategies have been very successful in this regard (reviewed in Carpenter and Koenig, in press), and it is unclear how functional outcomes from the acute pharmacological challenge in mature animals used in the present study might compare. Although the data as they stand are promising, they would be much more compelling if a functional deficit as a result of the treatment was observed and the authors could demonstrate that inhibition of NADPH oxidase prevented this deficit. Unfortunately, such a deficit is unlikely to be found following such a limited ketamine exposure. This is actually quite fortunate since ketamine is a popular general anesthetic in both human and veterinary medicine. Additionally, countless biomedical investigators routinely use ketamine as an anesthetic for survival surgeries; even in cases where the experimental design calls for multiple anesthetizations over the course of the study, no major functional disturbances in experimental animals have been reported. Our conclusion is that, while exposure to ketamine may induce features of neuropathology that bear some similarity to those observed in schizophrenia, the excitement about a treatment for ketamine-induced superoxide production should be tempered until it can be demonstrated that the treatment reverses a functional deficit that is relevant to schizophrenia.

References:

Carpenter WT, Koenig JI. The evolution of drug development in schizophrenia: past issues and future opportunities. Neuropsychopharmacology. (In press, 2007)

Hashimoto T, Volk DW, Eggan SM, Mirnics K, Pierri JN, Sun Z, Sampson AR, Lewis DA. Gene expression deficits in a subclass of GABA neurons in the prefrontal cortex of subjects with schizophrenia. J Neurosci. 2003 Jul 16;23(15):6315-26. Abstract

Kinney JW, Davis CN, Tabarean I, Conti B, Bartfai T, Behrens MM. A specific role for NR2A-containing NMDA receptors in the maintenance of parvalbumin and GAD67 immunoreactivity in cultured interneurons. J Neurosci. 2006 Feb 1;26(5):1604-15. Abstract

Volk DW, Austin MC, Pierri JN, Sampson AR, Lewis DA. Decreased glutamic acid decarboxylase67 messenger RNA expression in a subset of prefrontal cortical gamma-aminobutyric acid neurons in subjects with schizophrenia. Arch Gen Psychiatry. 2000 Mar;57(3):237-45. Abstract

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Related News: Does Oxidative Stress Link NMDA and GABA Hypotheses of Schizophrenia?

Comment by:  Gavin Reynolds
Submitted 10 December 2007
Posted 10 December 2007

For two decades, following the work by Benes and her colleagues, it has been increasingly apparent that there is a deficit in cortical GABAergic neurons in schizophrenia. Ten years ago we found that the parvalbumin (PV)-containing, but not calretinin-containing, subgroup of these neurons was selectively affected, and recently this specific deficit has been seen in animal models of the disease. Repeated administration of non-competitive NMDA receptor antagonists such as PCP, MK801, and ketamine can induce in rats some behaviors reminiscent of schizophrenia, as well as enduring deficits in PV expression.

Behrens and colleagues have identified some of the molecular mechanisms underlying this specific neurotoxicity of ketamine and, probably, other NMDA antagonists. That the effects of ketamine involve generation of reactive oxygen species (ROS) is not surprising, given the ubiquity of oxidative free radical production in neurotoxic processes. However, identifying the role of NADPH oxidase in producing ROS in response to ketamine, and demonstrating that this process determines the consequent toxic effects of ketamine on PV-containing and other neurons, are potentially important developments.

The importance of these findings to schizophrenia relies on the assumption that repeated administration of ketamine and, presumably, other NMDA antagonists not only models (some of) the pathophysiology of schizophrenia, it also mimics the process leading to this neuronal pathology. This is far from proven, although the NMDA receptor hypofunction hypothesis of Olney and Farber provides a useful model mechanism for this pathogenesis.

A useful proof of concept would be to move away from pharmacological approaches to other animal models of the disease. One such is the isolation rearing paradigm; in this model, induction of abnormal “schizophrenia-like” behaviors is also paralleled by a deficit in PV-containing neurons (Harte et al., 2007). A simple but very informative study here would be to determine whether inhibition of NADPH oxidase might protect against the development of these deficits. Of course, how the NMDA receptor-mediated deficits relate temporally to the natural history of schizophrenia is unclear; we do not know when the PV deficits occur in schizophrenia. There may be some hope for targeted treatment with, e.g., NADPH oxidase inhibitors if the neuronal pathology parallels a neurotoxic process that underlies the progressive cognitive disturbances as implied by Olney and Farber, but not if the PV deficits relate to an early and primary pathology of the disease.

References:

Harte M, Powell S, Swerdlow N, Geyer M, Reynolds GP (2007) Deficits in Parvalbumin and Calbindin Immunoreactive cells in the Hippocampus of Isolation Reared Rats. J Neural Transm 114, 893-898. Abstract

View all comments by Gavin Reynolds

Related News: Does Oxidative Stress Link NMDA and GABA Hypotheses of Schizophrenia?

Comment by:  Kenneth Johnson
Submitted 18 December 2007
Posted 18 December 2007

The recent study by Behrens and colleagues provides in vitro evidence that blockade of NMDA receptors by ketamine leads to a selective reduction in PV and GAD67 that appears to be due to the toxic effects of superoxide anion arising subsequent to the activation of NADPH oxidase. Blockade of the sublethal, toxic effects of ketamine in neuronal culture is consistent with our report demonstrating that the apoptotic effect of phencyclidine (PCP) on cortical neurons in vivo also could be prevented by the superoxide dismutase mimetic, M40403 (Wang et al., 2003). Though seemingly non-specific, superoxide dismutase mimetics may prove to be useful in the treatment of ketamine or PCP-induced psychosis because of the relative sparseness of critical life-promoting processes that require superoxide anion. Perhaps more importantly, a better understanding of the mechanisms underlying ketamine-induced loss of PV/GAD67 may lead to novel treatment modalities for schizophrenia.

While the primary focus of the report by Behrens and colleagues is on PV-expressing GABAergic interneurons, Fig. 1 clearly demonstrates that ketamine also affects a large population of non-PV neurons. This is consistent with our recent in vivo experiments in developing rats demonstrating that PCP administration on PN7 induces apoptosis of cortical PV-containing interneurons as well as principal neurons in layers II-IV of the cortex (Wang et al., 2007). Early postnatal administration of PCP also results in neuronal apoptosis in the hippocampus, striatum, and thalamus (Wang and Johnson, 2007. Thus, it may be premature to focus solely on this population of interneurons.

In thinking about the mechanism underlying the selective loss of PV interneurons following PCP, it is important to note that PV is not yet expressed on PN7, which is when PCP was administered in our paradigm (Wang et al., 2007). (The loss of PV-containing interneurons was measured at PN56, well after the time of PV expression on about PN10.) Interestingly, interneurons expressing calretinin and calbindin at the time of PCP administration were spared. These neurons showed no colocalization with cellular markers of apoptosis (terminal dUTP nick-end labeling [TUNEL] of broken DNA or cleaved caspase-3), indicating that calretinin- and calbindin-containing neurons were protected from the toxic effect of PCP and survived into adulthood (Wang et al., 2007). The mechanism underlying this selectivity for cortical PV-containing interneurons is unknown, but as Behrens and colleagues suggest, it could be because these neurons are dependent on a relatively large glutamatergic input for survival. It is also possible that the differing calcium buffering capacities of these interneurons play a role in the selective neurotoxic effect of NMDA receptor blockade. That is, since calcium binding proteins could also act to buffer decreases in intracellular Ca2+ levels caused by ketamine-induced blockade of NMDA receptors, it is possible that the lack of PV in these vulnerable interneurons reduces the ability of these cells to adequately buffer the ketamine-induced decrease in intracellular calcium. This is consistent with the lack of effect on other interneurons that express the calcium binding proteins calretinin and calbindin at the time of PCP administration. This suggests NMDA receptor blockade could cause the deletion of PV neurons because of a specific effect at a critical stage of development. However, cleaved caspase-3 (a hallmark of apoptosis) showed no colocalization with BrdU, a specific marker of S-phase proliferation (Wang et al., 2007). These data suggest that the loss of PV-containing neurons in this paradigm was not due to an effect of PCP on proliferating neurons, but rather an effect on postmitotic neurons.

We have reported recently that PCP in cortical neuronal culture causes neuronal apoptosis by interfering with the Akt-GSK-3β cascade, which is necessary for neuronal survival during development (Lei et al., 2007). Moreover, increasing synaptic strength by various means such as increasing calcium current via activation of L-type calcium channels completely blocks PCP-induced cell death by increasing Akt phosphorylation. It would be of great interest to determine whether PV-containing interneurons respond in a similar fashion.

In order to fully appreciate the significance of ketamine-induced loss of PV-containing neurons, it will be necessary to carefully compare the in vivo dose-related effects of ketamine or PCP that are truly selective for PV/GAD67-containing interneurons to those cortically mediated behaviors that have relevance to schizophrenia.

References:

Wang C, McInnis J, West JB, Bao J, Anastasio N, Guidry JA, Ye Y, Salvemini D, Johnson KM. Blockade of phencyclidine-induced cortical apoptosis and deficits in prepulse inhibition by M40403, a superoxide dismutase mimetic. J Pharmacol Exp Ther. 2003 Jan 1;304(1):266-71. Abstract

Wang, C.Z., Yang, S.F., Xia, Y. and Johnson, K.M. Induction of a selective cortical deficit of parvalbumin-containing interneurons by phencyclidine administration during postnatal brain development. Neuropsychopharmacology (In press, 2007).

Wang CZ, Johnson KM. The role of caspase-3 activation in phencyclidine-induced neuronal death in postnatal rats. Neuropsychopharmacology. 2007 May 1;32(5):1178-94. Abstract

Lei, G., Xia, Y. and Johnson, K.M. The role of Akt-GSK-3β signaling and synaptic strength in phencyclidine-induced neurodegeneration. Neuropsychopharmacology (In press, 2007).

View all comments by Kenneth Johnson

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Comment by:  Patricia Estani
Submitted 11 January 2008
Posted 13 January 2008
  I recommend the Primary Papers