Schizophrenia, as a severe mental illness, the number of patients is far beyond imagination. Although its researches have gradually shifted from complete psychological-focused to the neurochemical level, the studies on treatments have not made much progression. In recent years, with the deepening of understanding of long non-coding RNA (lncRNAs), the co-expression pattern and the important roles of some dysregulated lncRNAs, such as HAR1F and DGCR5, in the development of schizophrenia have been revealed. Two potential treatments that target lncRNAs involved in schizophrenia were proposed, including targeted therapy via designed oligomers and genetic editing through CRISPR/Cas 9. These treatments only performed on lncRNAs associated with other diseases (cancer, thalassemia et al.), and there are still some limitations in both technology and ethical perspectives, but they provide possible research directions in the future.
Schizophrenia, a long-term and serious mental syndrome that mainly defined by the observations of symptoms of psychosis, was diagnosed over 21 million people among the whole world 1, 2. According to World Health Organization (WHO) 10-country study, the schizophrenia’s incidence rate is form 0.1 to 0.4 per thousand 3. It generally happens in the early adulthood, although there were cases that patients younger than 10 years old or older than 50 years old, they are very rare 4. Statistics indicate that the disease occurs more often at males (~12 million) than females (9 million); in addition, the average onset age of males (18 years old) is earlier than females (25 years old), but after 30 years old, the incidence rate of woman becomes higher 1, 4. Schizophrenia affects a wild range of conscious behaviors, language expression, emotional control, and views on things 1. Based on the Diagnostic and Statistical Manual of Mental Disorder-V (DMS-5), five common symptoms are determined, including delusion, hallucinations, disorganized speech, grossly disorganized or catatonic behavior and negative symptoms 5.
The number of people suffering from these severe symptoms is more than people think, and most patients are not well taken care of because of discrimination. Furthermore, because the time of onset is just when they first enter college or started to their own career, most patients have a hard life in the future. Thus, the study of pathogenesis and the corresponding treatments are even more important today. In the early studies, very few researchers paid attention to neurochemical reasons; however, this situation began to change after emergence of antipsychotic drugs, and neuropharmacology gradually became to mainstream 2. In the past decades, the explosive development of genetics sequencing techniques and the whole genome projects had started to reveal the roles of certain genes in the occurrence and development of schizophrenia. Evidence indicated genetic variants that are associated with early neurodevelopment, including neuronal growth and synapse formation, have effects on schizophrenia 2. Meng Q et al. investigated 10 lncRNAs in the schizophrenia risk-associated rare copy number variant (CNV) deletion regions, and they discovered a specific lncRNA DGCR5 in the chromosome 22q 11.2 CNV is able to regulate some schizophrenia-related genes 6.
This review will talk about lncRNAs, how they affect final outcome by interacting with macromolecules, and its contribution to the pathophysiology of schizophrenia. Two potential approaches to treat schizophrenia will also be discussed. Limitations and future direction will be featured.
Long non-coding RNAs
Based on the central dogma, RNA in eukaryotes acts as an intermediate that is transcripted from DNA and can be translated into protein, which influence the phenotype. However, human genome-wide studies revealed that only 2.94% RNAs are transcripted by protein coding regions of the genome and the rest of transcripted RNAs are called non-coding RNAs (ncRNA) 7, 8. These RNAs were previously regarded as junk with no proper function, but now scientists realize both the housekeeping ncRNAs and the regulatory ncRNAs play an essential role in regulating gene expression in central nervous system 7. According to the number of nucleotides in the RNA sequences, the regulatory ncRNAs are given different names, including short ncRNA (200 nucleotides) 7, 8.
How does lncRNA regulate gene expression?
LncRNA is able to affect phenotypic expression (ultimate outcomes) from varies levels, from DNA structure to the proteins 7. Figure 1 demonstrates that lncRNA can achieve this goal via transcriptional regulation, post-transcriptional interference and interaction with other molecules, including proteins and short ncRNAs 7. During transcription process, lncRNA can directly bind with DNA and acts as chromatin structure modifier to induce conformational change of chromatin; it also can modify the histone protein, which is a crucial molecule that helps fold linear DNA chain into solenoid, the 3° structure of DNA 7. As we know, proteins interact with regulatory region/ promotor of DNA to regulate gene transcription; lncRNA can be a coactivator of these proteins and affects their activities through binding 9. Furthermore, it targets certain proteins and leads them to another location/ genetic loci to alter transcription pattern 7, 9. LncRNAs not only bind to DNA but also interact with other RNAs, such as coding mRNA 7. LncRNA can change the splicing pattern of mRNA while binding to it, and also can degrade it. In addition, lncRNA is able to inhibit translation process because ribosome is hard to bind, as well as process the mRNA when they bind together 7.
Figure 1. Paradigms for how lncRNAs function. 7
LncRNAs involved in schizophrenia
A study compared the human genome with chimpanzee genome, and researchers found an lncRNA, HAR1F, is responsible for coordinating the formation of regional forebrain organization, because this lncRNA was specifically up-expressed during the critical period of the growth and differentiation of cortical neurons 7. Other reports also provided evidence that lncRNA had an irreplaceable effect on the development of the central nervous system. Schizophrenia, which is a disease that highly related to the central nervous system, several distinct lncRNAs expression patterns were discovered between normal functional population and schizophrenia population, this suggests abnormal expression/ dysregulated lncRNAs is one reason causes the disease 6, 8.
On the chromosome 22q 12.1, where tightly next to the schizophrenia candidate area, the chromosome 22q 11.2, lncRNA myocardial infarction associated transcript (MIAT) was confirmed to be involved in the occurrence of the illness 8. It had lower expression in patients’ brain; moreover, experimental result shows the reduced level of MIAT expression could alter some splice variation that contribute to the progression of schizophrenia 8. Except for MIAT, another lncRNA, DGCR5, which located in the same chromosome CNV region, was identified as potential regulator for multiple schizophrenia-associated genes in the recent study. First of all, Meng Q. et al. investigated the BrainGVEX and developmental Capstone datasets and discovered a significant high co-expression pattern between schizophrenia risk-associated genes and DGCR5 6. In addition, 601 schizophrenia RNA-seq samples indicated that DGCR5 has much lower level of expression in the patients’ postmortem brain than normal population’s brain samples 6. DGCR5 is the only down-regulated CNV- lncRNA that was determined in the postmortem brain samples 6. Furthermore, they induced overexpression, and knockdown of DGCR5, they found 13 schizophrenia related genes respond to the changes 6. Nevertheless, this study does have some limitations. Some genes that appeared co-expression patterns wit DGCR5 seemed not regulated by it, and the datasets they used to identify the role of CNV- lncRNAs was quite small.
LncRNA, the potential therapeutic target
As mentioned precedent, the treatments of schizophrenia was completely focused on the psychological level in 100 years ago, and researchers started to pay more attention to the treatments in hormone/ protein level after dopamine-releasing drugs emerged 2. Nowadays, researchers begin to consider the possibility of treating schizophrenia at the molecular/ genetic level. In targeted therapy for diseases, many targets that drugs aim are proteins or mRNAs; but the discovery of the importance of lncRNA, especially their potential regulatory function, leads lncRNA to a novel target group 10.
There are several advantages to use lncRNA as a target over protein and mRNA. First of all, over 30,000 particular lncRNAs were determined as the potential aims 11. Secondly, ~80% identified lncRNAs are unique cell/ tissue types, certain development period, and disease-state 10. Thus, inhibitors can directly against these lncRNAs to impede their functions without interrupting other normal functions or at least reducing side effects 10. Thirdly, for treating some diseases that are caused by abnormal down-regulated proteins, traditional targeted therapy has a hard time to find the proper target; however, targeting lncRNAs gives a hope to overcome this problem, because one function of lncRNAs is to suppress protein expression10. Fourthly, it is now possible to synthesize oligonucleotides (oligomers) for attacking mRNA, so the difficulty of developing oligomers for lncRNA will be relatively reduced 10.
Therapeutics based on oligomers
Oligomer refers to a small amount of monomers, usually 7 to 25 base-pairs (bp), which link together by phosphodiester linkages 10. The oligomers that used to target lncRNAs are artificially synthesized with specific backbone. Different oligomers’ backbones have distinct levels of bioability and binding affinity due to their compositions 10. For example, the phosphodiester can be replaced by sulfur 10. As the consequence, this modified linkage boosts stability of the oligomer without changing its binding affinity because this bond gives it the insensitive property to nuclease degradation 10. Designed oligomer is able to inhibit lncRNAs because it has ability to degrade lncRNAs through RNase-H mediation 10. RNase H-sensitive DNA fragment within the oligomer can trigger RNase H, and this specific degradation enzyme can identify the complex which is formed by lncRNA binding to oligomer 10. Other than degradation effect, oligomers can perform another interference strategy. It can induce conformational change of lncRNA by directly binding to it, for instance, oligomers modify 2° and 3° structure of lncRNA. Once lncRNA changes its shape, it is no longer able to associate with DNA, RNA and other macromolecules 10.
Although designed oligomers that in current clinical trials are only for cancer, hypercholemia and other diseases, it is still a promising research area for the treatment of schizophrenia 10.
Editing lncRNA using CRISPR/Cas 9: a novel potential approach to investigate and treat schizophrenia
In addition to use oligomers to inhibit lncRNAs, there is now a remarkable gene editing technique called CRISPR/Cas 9 that can targets lncRNA from the error source, genes.
The CRISPR/Cas 9 system contains an R-S structure that is composed of multiple repeats and non-repetitive spacers, and an operon which encode Cas protein 11. The R-S structure is capable of recognizing exogenous DNA, and it can be transcripted into crRNA precursor, and this transcription process is initiated by a promotor-like leader sequence 11. The Cas gene is responsible for encoding CRISP associated proteins that perform multiple functions, and it modifies crRNA precursors into mature crRNA via trans-activating crRNA 11.
Three steps mechanism allows CRISP/Cas 9 to work in bacteria. Firstly, gain the CRISP spacer sequence, secondly, transcription and modification of crRNA and thirdly, form complex by binding crRNA, trans-crRNA and Cas 9 together to edit exogenous DNA 12. Based on the mechanism, Wiedenheft et al. designed the CRISPR/Cas 9 gene editing technique 11. They constructed a sgRNA based on the dimer structure of crRNA and trans-crRNA, and then used this sgRNA to bind to Cas 9 protein 12. This Cas 9 is directed to bind to a specific gene locus and edit it 11, 12. Compare to previous editing techniques, CRISPR/Cas 9 has better ability to recognize targets, and it is simple to operate with low cost 11.
Now researchers have used this technique to edit lncRNAs, and there were cases that silenced two lncRNAs, UCA1 and MALAT1 from 100 to 3000bp 13. Ho et al. used a dual sgRNA that produced a larger deletion in two specific sites in human cells 13. Thus, if researchers knockdown a suspect schizophrenia risk-associated lncRNA, and compare the results with controls, the function/ role of that lncRNA can be determined 13.similarly, if scientists use CRISPR/Cas 9 to correct a defined schizophrenia-related lncRNA, the result may be pleasing.
Schizophrenia is a very complicate mental disorder that is caused by environmental factors and multiple aberrant regulated lncRNAs which are raised from CNVs, such as HAR1F and DGCR5. Even though lncRNAs do not translate into functional proteins, they can regulate ultimate outcomes in transcriptional level, post-transcriptional level to proteins by binding them in the specific regions. Several co-expression patterns between lncRNAs and schizophrenia related-genes were found, and their expression levels were down-regulated in patients’ brain tissues. This makes lncRNA as an ideal potential target for synthetic oligomers. Designed oligomers are capable to inhibit lncRNAs through degradation or interrupting their 2° and 3° conformations. This approach to treat schizophrenia is not perfect. First of all, an exact lncRNA with determined function is required for designing the oligomer. Secondly, there have no schizophrenia lncRNA targeted oligomer undergo clinical trials yet, so its actual effect remains to be evaluated. CRISPR/Cas 9 gene editing technology can directly act on genetic mutation regions, which allows it to investigate the role of suspect lncRNAs. According to the existing case that used CRISPR/Cas 9 to edit the pathogenic gene of thalassemia and some embryos successfully gain the correct sequence of that gene, hence using it to edit the problem CNV- lncRNA in schizophrenia seems promising. However, CRISPR/Cas 9 is not fully developed, and the consequences of off-target must be carefully considered. Besides, the genotoxicity of it remains unclear; there are also a great controversy of ethical issues in modifying human embryo genome.
In the future, more experiments based on animal models are necessary; besides, the experiments that edit schizophrenia CNV- lncRNAs based on CRISPR/Cas 9 may be able to use artificially manufactured human brain tissues, which are developed by neurogenesis/ tissue-genesis technology.
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