Analysis of the post-mortem brain tissue from two regions of the cerebral cortex in schizophrenia revealed a substantial alteration of the post-transcriptional regulatory environment, characterized by a global increase in miRNA expression. This change could have profound implications for the development and ongoing pathophysiology of the disorder, as each miRNA has the capacity to regulate the expression of hundreds of target genes. Studies in model systems have highlighted the significance of miRNA expression in brain development and a host of more specific neurobiological functions, such as regulation of the left–right asymmetry, long-term potentiation and establishment or maintenance of dendrites.17, 18, 19, 20, 21
In this work, we focused on the implications of miRNAs in the miR-15 family and closely related miR-107 as these functionally convergent miRNAs were consistently upregulated in both regions of the cerebral cortex in schizophrenia, and could collectively contribute a significant biological influence.
The miR-15 family of miRNAs has already been shown to have an important role in chronic lymphocytic leukemia, with a well-characterized association between a reduction of miR-15a/miR-16 concentration and increased expression of the anti-apoptosis gene BCL2
The relationship between miR-15 family expression and this gene may also have the opposite implications in schizophrenia, which has been associated with a downregulation of BCL2
expression. Reduced BCL2
expression in schizophrenia, perhaps in response to increased miR-15 family expression, is thought to contribute to elevated cortical apoptosis, cerebral atrophy and even a reduction in the risk of some forms of cancer.23, 24, 25
Pathway analysis of predicted target genes suggested that there are probably many other ways of influence of this group of miRNAs that are of significance to schizophrenia, such as axon guidance, long-term potentiation, WNT, ErbB and MAP kinase signaling (Supplementary Table 4). Many of these predicted target genes, such as RGS4, GRM7, GRIN3A, HTR2A, RELN, VSNL1, DLG4, DRD1
, have been shown to be associated with schizophrenia.26, 27, 28, 29, 30, 31, 32, 33
In relation to the current study, RGS4 and VSNL1
were reported to be downregulated in the same STG tissue;8, 34
however, the expression of these and other candidate genes has not been analyzed at the protein level in these cohorts. To further examine the potential for a functional relationship between MREs in these candidate genes and the miR-15-related miRNAs, we established luciferase reporter constructs and measured the degree of silencing from individual miRNA. Regulation of 3′-UTR elements from the metabotropic glutamate receptor GRM7
and the N-methyl--aspartic acid (NMDA) receptor subunit GRIN3A
was particularly strong and, along with DLG4
(PSD95; scaffold protein that supports these and other receptors in the post-synaptic density), provides a post-transcriptional mechanism that could underlie the many accounts of schizophrenia-associated glutamatergic hypofunction.35
It may also explain the apparent conflict between the schizophrenia-associated reduction of region-specific protein expression in the absence of change or even paradoxical increase in corresponding mRNA.36
Another target gene element that showed a consistent response to miR-107 and the miR-15 family miRNAs was one derived from the Reelin (RELN
) 3′-UTR. RELN
is a secreted glycoprotein involved in neuronal migration and synaptogenesis during development. It is also important for the establishment of long-term memory in the adult brain because of its role in the modulation of synaptic activity and dendritic spine development.37 RELN
is a highly plausible candidate gene and its expression has been shown to be altered in schizophrenia.30, 38
Although this alteration has been associated with epigenetic regulation though promoter hypermethylation,39, 40
it is now conceivable that post-transcriptional gene silencing is also contributing to RELN
dysregulation in schizophrenia.
Collectively, these experiments were broadly supportive of a role for this group of miRNAs in the regulation of schizophrenia-associated target genes; however, the response was quite variable for the individual miRNAs, with miR-107 showing the most consistent activity, whereas miR-195 appeared to have the least activity against the elements tested here. In contrast, a recent study has found that miR-195 (among others) was capable of regulating BDNF
expression in vitro
The temporal and spatial expression pattern of this miR-15 family member in the DLPFC was inversely correlated with BDNF
and may be important for the developmental regulation of this schizophrenia candidate gene.
Experiments in animal systems may also provide important insight into the behavioral consequences of altered cortical miRNA expression. In a recent study, mice treated with the NMDA receptor antagonist MK801 and hypomorphic GRIN1
(NR1) mutants showed a marked decrease in miR-219 expression.42
CaMKIIγ, a predicted target gene for this miRNA involved in NMDA signaling, was shown to be sensitive to miR-219 concentration in vitro
. Moreover, suppression of miR-219 expression via intraventricular delivery of the corresponding LNA-modified anti-miR restored MK801 induced neurobehavioral dysfunction back to levels approaching that of the controls.42
Interestingly, in our study miR-219 was the most highly upregulated miRNA in the DLPFC and, in addition to the miR-15 family-related miRNA, could also be mediating a schizophrenia-associated reduction in NMDA signaling. These observations add support to the idea that these altered miRNAs are influential in the regulation of schizophrenia-associated genes and provide the basis of a model for the influence of disease-related miRNAs on genes involved in synaptic structure and function ().
Figure 5 Model for miRNA-associated dysregulation of synaptic structure and function in schizophrenia. The microprocessor activity is elevated in cortical nuclei as a consequence of a schizophrenia-associated increase in DGCR8 expression. The increase in pri-miRNA (more ...)
Although the examples of gene–miRNA interactions mentioned above and modeled in provide a conceptual framework for the mechanisms that may take place in the context of cortical miRNA dysregulation, they may only touch the surface of the broader ramifications for gene regulation in this altered environment. In this regard, it is worth noting that gene expression profiles in the same STG cohort (albeit smaller than the one examined in this study) showed more than twice as many downregulated genes in schizophrenia compared with those upregulated.5
This observation at the mRNA level has been observed in other studies as well,4, 43, 44
and may be reflective of an elevation in global gene silencing mediated by increased miRNA expression in these tissues.
The question of why such an extensive and consistent change in cortical miRNA expression was seen in the schizophrenia group led to the consideration of key components of the miRNA biogenesis pathway. Significantly, we identified a corresponding upregulation of the microprocessor component DGCR8
mRNA in both the STG and DLPFC. This alteration was consistent with an increase of both mature miRNA and precursor forms of miR-181b and miR-26b in the absence of a change in their level of transcription. Although the mechanism behind this apparent increase in DGCR8
expression at the mRNA level is currently unknown, the gene is situated within a region of the genome that is prone to spontaneous structural variation associated with schizophrenia and other neurodevelopmental disorders.45, 46, 47
Microdeletion at this locus (22q11.2) is responsible for the DiGeorge/Velocardiofacial syndrome, which is also strongly associated with schizophrenia.48
In a neurodevelopmental model, mice with a specific deficiency in DGCR8
and miRNA biogenesis showed similar behavioral deficits to the larger hemizygous deletion of a larger region of chromosome 16 syntenic to that of the 22q11 locus in humans.49
Although the deletion model and human syndrome, involving haploinsufficiency, does not accord with the increased DGCR8 activity observed in this study, the low copy repeats that give rise to deletion can also induce microduplication. Interestingly, the syndrome associated with duplication at this locus also appears to be associated with behavioral abnormalities and cognitive deficits akin to those seen in the deletion syndrome, but, like schizophrenia, shows fewer dysmorphic features.50
As a consequence of this more subtle phenotype, the frequency of this poorly characterized syndrome may be underrepresented through misdiagnosis.50
Microduplications are also more difficult to identify by classical cytogenetic approaches than microdeletions, which may have historically masked their relative abundance. In theory, however, they are just as abundant as microdeletions, a view supported in a recent molecular analysis of copy number variation, which found that microduplications outnumbered microdeletions and were more highly associated with schizophrenia.47
In view of these observations, it is possible that changes in DGCR8
expression in some individuals could be due to increased gene dosage through chromosomal microduplication.
Alternatively, the increase in DGCR8
may be due to transcriptional or post-transcriptional dysregulation. A recent study has shown that DGCR8
is post-transcriptionally autoregulated by its own microprocessor complex.51
Contrary to expectation, this is probably not miRNA-mediated as the 3′-UTR for DGCR8
mRNA is almost devoid of predicted MREs, an exception being an MRE for the microprocessor-independent miRNA-intron or mirtron hsa-miR-1227.52
The mechanism is instead related to the presence of primary miRNA-like hairpin structures in the DGCR8
mRNA, which are themselves substrates for cleavage by the microprocessor. Cleavage results in destabilization of the mRNA and reduction in DGCR8
Polymorphisms with a capacity to destabilize these secondary structures in the mRNA could hinder feedback inhibition and result in DGCR8
In conclusion, our data suggest that schizophrenia is associated with a global increase in miRNA biogenesis and expression in the cerebral cortex. This could have profound neurodevelopmental and broader neurological implications in the context of schizophrenia by influencing genes involved in cortical structure and neural plasticity. It also has significance for our understanding of the mechanism underlying patterns of cortical gene expression associated with the disorder.