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The NMDA Receptor and Disrupted Glutamate Signaling: Is a microRNA the Link?

4 March 2009. Disturbances in glutamate signaling caused by disruptions in the N-methyl-D-aspartate receptor (NMDA-R) pathway have been implicated in numerous psychiatric disorders, particularly schizophrenia (see Current Hypotheses by B. Moghaddam and D. Javitt). Though much is known about this pathway, especially the role of the calcium/calmodulin-dependent protein kinase II (CaMKII) in synaptic plasticity, the mechanisms linking NMDA-R dysfunction to the cognitive and behavioral phenotypes seen in schizophrenia are largely unknown. A new study led by Jannet Kocerha of The Scripps Research Institute in Florida and published online on 5 February in Proceedings of the National Academy of Sciences USA suggests that a microRNA (miRNA), miR-219, may be one such link.

miRNAs, snippets of RNA about 20 nucleotides long that block gene expression by binding to mRNA (which either gums up protein translation or targets the mRNA strand for destruction), are attractive candidates for neuropsychiatric research. As Joseph Coyle of Harvard Medical School points out in a commentary accompanying the new PNAS article, of the several hundred miRNAs identified in the 15 years since their discovery in the nematode C. elegans, “half . . . are expressed predominantly or exclusively in brain,” where they are believed to play crucial roles in orchestrating neural development and regulating plasticity (see SRF related news story).

Putting brakes on a CaM
After injecting mice with dizocilpine, an NMDA-R antagonist similar to phencyclidine (PCP) that produces schizophrenia-like behaviors in both rodents and humans, Kocerha’s team used microarrays to measure miRNA expression levels in the prefrontal cortex, and they found a significant reduction in miR-219, a brain-specific miRNA. Using another model of schizophrenia, mice carrying a hypomorphic mutation in Grin1, which codes for the NR1 subunit of the NMDA-R, the researchers found that miR-219 levels were similarly reduced in both prefrontal cortex and hippocampus.

The antipsychotic drugs haloperidol and clozapine are known to reverse the behavioral phenotypes seen in dizocilpine-treated or Grin1-mutant mice. When the scientists administered either drug, the hyperlocomotion and stereotypy characteristic of these mouse models was sharply reduced, and miR-219 expression was returned to baseline levels.

Having established that pharmacological or genetic disruption of the NMDA-R down-regulates miR-219, Kocerha and colleagues next used bioinformatic techniques to identify potential targets for miR-219. Out of six hybridization candidates for miR-219, the team settled on the gamma subunit of CaMKII (CaMKIIγ) since this kinase is involved in NMDA-R trafficking and rapid, transient expression of the receptor in neural dendrites to regulate plasticity. In vitro analyses with neuron-like cultured cells confirmed that miR-219 suppresses CaMKIIγ expression, an effect that was reversed when the cells were transfected with an antisense inhibitor of miR-219. These results were confirmed in cells from mouse prefrontal cortex. In complementary in vivo experiments, an antisense inhibitor of miR-219 was continually infused into the third ventricle of mice for seven days, and CaMKIIγ expression levels again increased. Moreover, infusion of the antisense inhibitor significantly attenuated the behavioral effects of dizocilpine. All told, the authors write, “these data support the hypothesis that miR-219 represents an integral component of NMDA-R signaling.”

Some caveats
In his commentary, Coyle identifies several “inconsistencies” in the data. First, the authors report that reductions in miR-219 levels in dizocilpine-treated mice were restricted to prefrontal cortex, but “the pathology of schizophrenia is widespread in the cortex,” writes Coyle (see also Coyle, 2006). Although miR-219 levels declined after acute administration of dizocilpine, this decline was not observed with chronic administration of the drug, which Coyle, citing the work of Mohn and colleagues (Mohn et al., 1999), says “is considered to be a better model of schizophrenia.” Moreover, in the Grin1-mutant mice—a genetic analogue to chronic dizocilpine administration—miR-219 was reduced in the hippocampus as well as the prefrontal cortex. Finally, though low miR-219 levels are implicated in the increased locomotion seen after acute dizocilpine treatment, the fact that this behavior was reduced when the actions of miR-219 were blocked with an antisense inhibitor is “counterintuitive,” says Coyle.

On the positive side of the ledger, Coyle notes that the gene coding for miR-219 is located at 6p21, which has been identified as a risk locus for schizophrenia (Roig et al., 2007). He also points out that miR-219 transcription is regulated by a complex including the circadian rhythm protein CLOCK, which has been implicated in bipolar disorder (see SRF related news story). "Thus, miR-219 provides a nexus for 2 risk pathways for serious mental illness: (i) psychosis via hypofunction of NMDA receptors through a downstream effect on CaMKIIγ, and (ii) mood instability by disruption of CLOCK–BMAL1 function, which has been implicated in bipolar disorder," he writes.—Pete Farley.

Reference:
Kocerha J, Faghihi MA, Lopez-Toledano MA, Huang J, Ramsey AJ, Caron MG, Sales N, Willoughby D, Elmen J, Hansen HF, Orum H, Kauppinen S, Kenny PJ, Wahlestedt C. MicroRNA-219 modulates NMDA receptor-mediated neurobehavioral dysfunction. Proc Natl Acad Sci USA. 2009 Feb 5. Abstract

Comments on Related News


Related News: Endogenous Clock and Bipolar Disorder—Simple Mutation Leads to Mouse Mania

Comment by:  Patricia Estani
Submitted 7 April 2007
Posted 8 April 2007
  I recommend the Primary Papers

Related News: 22q11 and Schizophrenia: New Role for microRNAs and More

Comment by:  Linda Brzustowicz
Submitted 21 May 2008
Posted 21 May 2008

While some have expressed frustration over the lack of clear reproducibility of linkage and association findings in schizophrenia, the importance of the chromosome 22q11 deletion syndrome (22q11DS) as a real and significant genetic risk factor for schizophrenia has often been overlooked. While the deletion syndrome is present in a minority of individuals with schizophrenia (estimates of approximately 1 percent), presence of the deletion increases risk of developing schizophrenia some 30-fold, making this one of the clearest known genetic risk factors for a psychiatric illness. As multiple genes are deleted in 22q11DS, it can be a challenge to determine which gene or genes are involved in specific phenotypic elements of this syndrome.

The May 11, 2008, paper by Stark et al. highlights the utility of engineered animals for dissecting the individual effects of multiple genes within a deletion region and provides an important clue into the mechanism likely responsible for at least some of the behavioral aspects of the phenotype. While some may argue about the full validity of animal models of complex human behavior disorders, these systems do have an advantage in manipulability that cannot be achieved in work with human subjects. A key feature of this paper is the comparison of the phenotype of mice engineered to contain a 1.3 Mb deletion of 27 genes with mice engineered to contain a disruption of only one gene in the region, DGCR8. The ability to place both of these alterations on the same genetic background and then do head-to-head comparisons on a number of behavioral, neuropathological, and gene expression assays allows a clear assessment of which components of the mouse phenotype may be attributed specifically to DGCR8 haploinsufficiency. Perhaps not surprisingly, DGCR8 seems to play a role in some, but not all, of the behavioral and neuropathological changes seen in the animals with the 1.3 Mb deletion. The fact that the DGCR8 disruption was able to recapitulate certain elements of the full deletion in the mice does raise its profile as an important candidate gene for some of the neurocognitive elements of 22q11DS, and makes it a potential candidate gene for contributing to schizophrenia risk in individuals without 22q11DS.

Also of great interest is the known function of DGCR8. While the gene name simply stands for DiGeorge syndrome Critical Region gene 8, it is now known that this gene plays an important role in the biogenesis of microRNAs, small non-coding RNAs that regulate gene expression by targeting mRNAs for translational repression or degradation. As miRNAs have been predicted to regulate over 90 percent of genes in the human genome (Miranda et al., 2006), a disruption in a key miRNA processing step could have profound regulatory impacts. Indeed, as reported in the Stark et al. paper and elsewhere (Wang et al., 2007), homozygous deletion of DGCR8 function is lethal in mice. What perhaps seems to be the most surprising result is that haploinsufficiency of DGCR8 function does not induce a more profound phenotype, given the large number of genes that would be expected to be affected if miRNA processing were globally impaired. The Stark et al. paper determined that while the pre-processed form of miRNAs may be elevated in haploinsufficient mice, perhaps only 10-20 percent of all mature miRNAs show altered levels, suggesting that some type of compensatory mechanism may be involved in regulating the final levels of the other miRNAs. Still, the 20-70 percent decrease in the abundance of these altered miRNAs could have a profound effect on multiple cellular processes, given the regulatory nature of miRNAs. In the context of the recent evidence for altered levels of some miRNA in postmortem samples from individuals with schizophrenia (Perkins et al., 2007), the Stark et al. paper adds further support for studying miRNAs as potential candidate genes in all individuals with schizophrenia, not just those with 22q11DS. This paper should serve as an important reminder of how careful analysis of a biological subtype of a disorder can reveal important insights that will be relevant to a much broader set of affected individuals.

References:

1. Stark KL, Xu B, Bagchi A, Lai WS, Liu H, Hsu R, Wan X, Pavlidis P, Mills AA, Karayiorgou M, Gogos JA. Altered brain microRNA biogenesis contributes to phenotypic deficits in a 22q11-deletion mouse model. Nat Genet. 2008 May 11; Abstract

2. Miranda KC, Huynh T, Tay Y, Ang YS, Tam WL, Thomson AM, Lim B, Rigoutsos I. A pattern-based method for the identification of MicroRNA binding sites and their corresponding heteroduplexes. Cell. 2006 Sep 22;126(6):1203-17. Abstract

3. Wang Y, Medvid R, Melton C, Jaenisch R, Blelloch R. DGCR8 is essential for microRNA biogenesis and silencing of embryonic stem cell self-renewal. Nat Genet. 2007 Mar 1;39(3):380-5. Abstract

4. Perkins DO, Jeffries CD, Jarskog LF, Thomson JM, Woods K, Newman MA, Parker JS, Jin J, Hammond SM. microRNA expression in the prefrontal cortex of individuals with schizophrenia and schizoaffective disorder. Genome Biol. 2007 Jan 1;8(2):R27. Abstract

View all comments by Linda Brzustowicz