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Psychiatric risk gene NT5C2 regulates protein translation in human neural progenitor cells

Rodrigo R.R. Duarte, Nathaniel D. Bachtel, Marie-Caroline Cotel, Sang H. Lee, Sashika Selvackadunco, Iain A. Watson, Gary A. Hovsepian, Claire Troakes, Gerome D. Breen, Douglas F. Nixon, Robin M. Murray, Nicholas J. Bray, Ioannis Eleftherianos, Anthony C. Vernon, Timothy R. Powell, Deepak P. Srivastava

Preprint posted on 12 November 2018 https://www.biorxiv.org/content/early/2018/11/12/468546.1

Article now published in Biological Psychiatry at http://dx.doi.org/10.1016/j.biopsych.2019.03.977

To in vitro and beyond! Establishing the function of a potential mental illness risk gene, NT5C2, and its role in regulating protein synthesis.

Selected by Joanna Cross

Background

According to the World Health Organization, 1 in 4 people will be affected by mental illness at some point in their lives, and currently around 450 million people suffer from these conditions.  Treatment options are improving but, for the majority of these disorders, the underlying mechanistic cause is unknown.  Schizophrenia, bipolar disorder and major depression are highly heritable, meaning that they are likely to be caused, at least in part, by genetic factors.  Genome-wide association studies (GWAS) are regularly used to identify areas of the genome that commonly contain mutations in patients, and genetic variants on chromosome 10q24 have been associated with combined risk for schizophrenia, bipolar disorder, major depression, autism and attention deficit hyperactivity disorder.  Although it is difficult to determine which gene located at this locus infers the most risk of these mental disorders, the cytosolic 5’-nucleotidase II (NT5C2) gene is suggested to be important.  NT5C2 functions to cleave or catalyze transfer of inorganic phosphate from purine and purine-derived nucleotides, such as adenosine, inosine and guanosine monophosphate.  As these compounds are important in many metabolic processes, for example gene transcription and protein synthesis, NT5C2 could be an important regulator of these processes.

 

Key Findings

In this preprint, Duarte et. al. aimed to show the functional effects of NT5C2 in mental disorders and the potential causal mechanisms.  They first wanted to see if expression of NT5C2 was altered in the brains of schizophrenia, bipolar disorder and major depression patients, using RNAseq data collected by the Stanley Neuropathology Consortium.  They found that NT5C2 is significantly reduced in schizophrenia and bipolar disorder, but not major depression, compared to controls. As both schizophrenia and bipolar disorder share psychotic manifestations, unlike major depression, this could hint at NT5C2’s particular role in risk for psychiatric disorders.

To further elucidate how reduced NT5C2 expression may influence pathogenesis, it is important to determine the specific cell type in which expression occurs.  The team achieved this using a wide array of techniques, including in silico modeling and sequencing from both mouse cortex and human neural precursor cells (hNPCs).  This showed that peak NT5C2 expression was present in neurons throughout development, although expression did persist into adulthood.

As NT5C2 appeared to be important during development, further studies were carried out in hNPCs.  In order to replicate the reduction in expression observed in schizophrenia and bipolar disorder, they used small interference RNA (siRNA) to knock down NT5C2 expression in hNPCs.  The transcriptomes of cells that had been exposed to this targeted treatment were then compared to those which had been treated with a scrambled, or non-target, siRNA; this revealed expression differences in 69 genes.  Using gene ontology software, they found that some of the most significantly downregulated genes were involved in protein translation. As AMPK and ribsosomal protein S6 (rpS6) have been previously linked to protein synthesis, the expression levels and extent of phosphorylation of these two proteins were studied.  It was found that with knock down of NT5C2, both the expression and phosphorylation state of AMPK increased, but only the phosphorylation state increased for rpS6. Therefore, they postulated that there is a link between NT5C2 and protein synthesis in hNPCs.

Although these in vitro functional studies supported the hypothesis that NT5C2 is important during development, they did not reveal the potential impact of reduced expression of NT5C2 at a systems level.  NT5C2 shares 60.5% sequence identity and 80.2% sequence similarity with the D. melanogaster homologue, CG32549, suggesting that these proteins may have the same or similar functionality.  Therefore, three fly lines were engineered, knocking down NT5C2 ubiquitously throughout the body, in the CNS or in the gut respectively.  No differences in survival were noted but there was a mean 20% reduction in climbing ability (a commonly used behavioural assay) in flies with ubiquitous and neuronal-specific knockdowns.  This effect was not observed in the flies with knockdown in the gut, showing that it is specific to neuronal populations, and there was no difference in climbing impairment between ubiquitous and neuronal-specific knock down lines. Decreased climbing ability has been related to lack of motivation in flies, perhaps indicating that NT5C2 is involved in the depressive states of mood disorders.

 

What I like about the preprint

Mental disorders affect a large portion of our society, including both the patients and their families. However, in order to administer the optimum treatment, the cause of these disorders needs to be found. Although this preprint does not establish the underlying cause of mental illness, it creates a great base for future study.  Using prior studies, the authors identified a potential candidate gene and implemented a wide range of techniques, both in vivo and in vitro, to establish functionality.  By understanding the function of the protein within the cell, mechanistic pathways can be developed and potential drug targets identified.

 

Questions for authors

Could the impairment in climbing ability in flies be related to NT5C2’s function in Parkinson’s disease and spastic paraplegia?

What changes happen in the transcriptome if knock-out hNPCs are treated with approved drugs for mental illness, e.g. lithium or anti-psychotics?

 

References

World Health Organization; http://www.who.int/whr/2001/media_centre/press_release/en/

Cross-Disorder Group of the Psychiatric Genomics Consortium, Lee SH, Ripke S, et al. Genetic relationship between five psychiatric disorders estimated from genome-wide SNPs. Nat Genet. 2013;45(9):984-94.

Ries, Ariane-Saskia et al. “Serotonin modulates a depression-like state in Drosophila responsive to lithium treatment” Nature communications vol. 8 15738. 6 Jun. 2017, doi:10.1038/ncomms15738

Tags: animal models, drosophila, mood disorders, neuroscience, stem cells

Posted on: 7 December 2018 , updated on: 11 December 2018

doi: https://doi.org/10.1242/prelights.6171

Read preprint (3 votes)

1 comment

4 years

Deepak Srivastava

Dear Joanna,

Thanks for providing this comprehensive summary of findings for our paper. Before answering your questions, we would like to highlight that, as you pointed out, the cause for major psychiatric disorders remains unknown to-date, so it is important for us to progress our understanding of the biological underpinnings for these conditions, as this has the potential to advance diagnostic and therapeutic options for patients. Furthermore, until ten years ago we did not know which regions of the genome were implicated in risk for mental illness, despite several studies corroborating a role for genetics in mediating susceptibility. In this context, genome-wide association studies (GWAS) enabled the psychiatric genetics field to move forward, but global analyses alone cannot elucidate with detail the biological processes impaired in these disorders. Our study modelled and characterised how genetic variants on chromosome 10q24 confer risk for psychiatric conditions. We previously found that risk is conferred via changes to NT5C2 expression, and now identified that this is associated with differential regulation of protein translation during early neurodevelopment, although this mechanism may persist during adult life. Ultimately, we believe that protein translation is one of the many important biological processes that are affected in patients at some point in life, and it’s possible that several psychiatric risk variants scattered through the genome alter the regulation of converging biological mechanisms, including protein translation, at specific developmental time points.

While we observed an alteration of NT5C2 expression associated with psychomotor abnormalities in Drosophila melanogaster, it’s difficult to compare motility behaviour in this model organism with psychomotor abnormalities experienced by patients, because there is obviously a substantial difference in the degree of complexity between these species. But by exploring the role of NT5C2 at a systems level using Drosophila as our model organism, our results raised the possibility that genetic variants affecting NT5C2 expression may be associated with motor disturbances observed in patients. This is corroborated by the fact that NT5C2 is also associated with spastic paraplegia and Parkinson’s Disease, as you pointed out. However, these findings raise other questions which we do not know the answer to at the moment, such as for example, does NT5C2 function mediate only psychomotor disturbances? Could it mediate other symptoms, such as psychosis, since it’s involved in protein translation, which is such a fundamental biological process? Could it regulate both, or additional symptoms?

Regarding the effect of drugs used in psychiatry on the transcriptome of knockdown hNPC cultures, it is very difficult to answer this question without empirical evidence. This could be explored using transcriptomic methods in our cell model supplemented with these drugs, or using in silico methods such as “connectivity mapping”, where the transcriptional effect of a drug is searched in a database of gene expression signatures associated with drugs applied to multiple cell lines. This could inform us that drugs used in psychiatry can revert the transcriptional changes associated with reduced NT5C2 expression… But would this be the case for every individual with schizophrenia? Could we remediate schizophrenia by exclusively altering NT5C2 function? The answer to these questions is “probably no”, but we believe that functional studies such as ours complement the body of evidence pertaining risk mechanisms associated with psychiatric disorders. The power combined of functional and pharmacological studies, studies using model organisms, clinical trials, and even analyses of larger cohorts of patients and unaffected controls using GWAS, will provide further clues about the biological processes impaired in psychiatric disorders, enabling us to develop our understanding of the biological mechanisms involved, and consequently fueling the discovery of novel biomarkers and drug targets for these conditions.

We appreciate your questions and interest in our work! Please feel free to message us if you have any questions or suggestions!

Best wishes,
Rodrigo and Deepak

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