DDX19B Differentially Expressed in the Brains of Patients with Psychotic

1 DDX19B is differentially expressed in the brains of patients with psychotic disorders. 2 Shahan Mamoor1 3 [email protected] 4 East Islip, NY, USA

6 We used public and published microarray data1,2 to identify the most significant gene expression changes in the brains of patients with psychotic disorders. We DDX19B as 7 differentially expressed in the dorsolateral prefrontal cortex of patients with schizophrenia and 8 schizoaffective disorder. In neurons of the dorsolateral prefrontal cortex from patients with 9 psychotic disorders, DDX19B expression was significantly decreased.

23 Keywords: schizophrenia, psychotic disorders, systems biology of schizophrenia, psychosis, 24 DDX19B. 25

PAGE 1 OF 14 1 Psychotic disorders including schizophrenia and schizoaffective disorder are 2 characterized by the presence of psychosis, or an inability to engage in reality testing3,4. Patients 3

4 with psychotic disorders can experience positive as well as negative symptoms5. Positive

5 symptoms include hallucinations, visual and auditory, as well as delusions, or fixed false beliefs5. 6 Negative symptoms include anhedonia, or an inability to experience pleasure, as well as 7

8 avolition, or loss of motivation5. To understand how specific regions of the brain differ most

9 significantly in terms of gene expression in patients with schizophrenias, we used published and 10 public microarray data to compare global gene expression profiles of brain tissues from patients 11

12 with schizophrenia with that of non-affected, control subjects1,2. We found that DDX19B was 13 among the genes whose expression changed most significantly in patients with schizophrenia in 14

15 the deep pyramidal neurons in patients with schizophrenia and in the layer 3 and layer 5

16 pyramidal neurons of the dorsolateral pre-frontal cortex in patients with schizophrenia. 17 DDX19B may be relevant for future study of genes pertinent to the biology of schizophrenia. 18

19 Methods 20

21 We used microarray datasets GSE145554 (1) and GSE87610 (2) for this differential gene

22 expression analysis in conjunction with GEO2R. GSE145554 was generated using Affymetrix 23

24 Human Gene 1.0 ST Array technology; for this analysis we used n=11 deep pyramidal neurons

25 from anti-psychotic-treated patients with schizophrenia and n=11 deep pyramidal neurons from 26 control subjects, as well as n=2 deep pyramidal neurons from patients with schizophrenia (not 27

28 treated with anti-psychotic medications) and n=11 deep pyramidal neurons from control subjects.

PAGE 2 OF 14 1 GSE87610 was generated using Affymetrix Human Genome U219 with n=72 for control 2 subjects and n=65 for patients with schizophrenia, using L3 and L5 pyramidal neurons from the 3

4 dorsolateral prefrontal cortex. The Benjamini and Hochberg method of p-value adjustment was

5 used for ranking of differential expression but raw p-values were used for assessment of 6 statistical significance of global differential expression. Log-transformation of data was auto- 7

8 detected, and the NCBI generated category of platform annotation was used. A statistical test

9 was performed to evaluate whether DDX19B expression was significantly different when 10 comparing neurons from the DLPFC from control subjects and or from patients with 11

12 schizophrenia or schizophrenia and schizoaffective disorder, respectively, using a two-tailed, 13 unpaired t-test with Welch’s correction. We used PRISM for all statistical analyses (Version 14

15 8.4.0)(455).

17 Results

18 We mined published and public microarray datasets to identify differentially expressed 19 genes in the brains of patients with psychotic disorders. 20

21 DDX19B is differentially expressed in deep pyramidal neurons of patients with schizophrenia. 22

23 We identified DDX19B as among the genes whose expression changed most significantly 24 genome-wide when comparing the transcriptional profiles deep pyramidal neurons from control, 25

26 non-affected subjects and from patients with schizophrenia; all of these patients had been treated

27 with antipsychotic medications (Table 1). When sorting each of the transcripts identified by 28

PAGE 3 OF 14 1 microarray based on degree of change in expression between control subjects and patients with 2 schizophrenia, DDX19B ranked 215 out of 32321 total transcripts (Table 1). Differential 3

4 expression of DDX19B in the deep pyramidal neurons of patients with schizophrenia was

5 statistically significant (Table 1; p=0.028177). 6

7 DDX19B is differentially expressed in the L3 and L5 pyramidal neurons of the dorsolateral 8 prefrontal cortex in patients with schizophrenia.

9 We mined a second microarray dataset to determine if differential expression of DDX19B 10

11 in patients with psychotic disorders could be observed in a separate dataset. We found a

12 DDX19B/DDX19A transcript that was among the genes most differentially expressed when 13 comparing L3 and L5 pyramidal neurons from patients with schizophrenia to that of normal 14

15 subjects. When sorting each of the transcripts identified by microarray based on degree of

16 change in expression between control subjects and patients with schizophrenia, DDX19B/ 17 DDX19A ranked 6698 out of 49386 total transcripts (Table 2). Differential expression of the 18

19 DDX19B/DDX19A transcript in the L3 and L5 pyramidal neurons of the DLPFC in patients with

20 schizophrenia was statistically significant (Table 2; p=4.74E-02). 21

22 23 Differential expression of DDX19B in deep pyramidal neurons of patients is independent of anti- 24 psychotic medication usage.

25 We probed the same dataset where we identified differential expression of DDX19B in 26 the deep pyramidal neurons to determine if this could be observed in schizophrenia patients not 27

28 treated with anti-psychotic medications. We again found that DDX19B was among the genes

PAGE 4 OF 14 1 whose expression was most significantly different genome-wide when comparing the 2 transcriptional profiles of deep pyramidal neurons from control, non-affected subjects and from 3

4 patients with schizophrenia (Table 3). When sorting each of the transcripts identified by

5 microarray based on degree of change in expression between control subjects and patients with 6 schizophrenia not being treated with anti-psychotic medications, DDX19B ranked 36 out of 7

8 32321 total transcripts (Table 1). Differential expression of DDX19B in the deep pyramidal

9 neurons of patients with schizophrenia not being treated with anti-psychotic medications was 10 statistically significant (Table 3; p=0.00222084). 11

13 DDX19B is expression is significantly higher in the deep pyramidal neurons of patients with 14 schizophrenia. 15

16 We obtained exact mRNA expression levels for the differentially expressed DDX19B 17 transcript, from control subjects and from patients with schizophrenia, to directly compare 18

19 DDX19B expression levels in the deep pyramidal neurons. We also performed a statistical test

20 to determine if this difference in DDX19B expression was statistically significant. DDX19B was 21 expressed at higher levels in the deep pyramidal neurons in patients with schizophrenia, and this 22 23 difference was statistically significant (Figure 1; p=0.0142). We calculated a mean fold change 24 of 1.0690 ± 0.0729 in DDX19B expression when comparing deep pyramidal neurons from anti- 25

26 psychotic treated patients with schizophrenia to that of control subjects (Table 1).

27 We also obtained exact mRNA expression levels for the differentially expressed 28

PAGE 5 OF 14 1 DDX19B transcript, from control subjects and from patients with schizophrenia that were had 2 not been treated with anti-psychotic medications, to directly compare DDX19B expression levels 3

4 in the deep pyramidal neurons. We also performed a statistical test to determine if this difference

5 in DDX19B expression was statistically significant. DDX19B was expressed at higher levels in 6 the deep pyramidal neurons in patients with schizophrenia that had not been treated with anti- 7

8 psychotic medications; this difference was not statistically significant (Figure 2; p=0.3150). We

9 calculated a mean fold change of 1.1355 ± 0.1056 in DDX19B expression when comparing deep 10 pyramidal neurons from patients with schizophrenia not treated with anti-psychotic medications 11

12 to that of control subjects (Table 3); thus, while this fold increase is greater than observed in 13 patients treated with anti-psychotic medications, the change was not statistically significant. 14

15 Thus, we found that DDX19B was among the most differentially expressed genes in the

16 brains of patients with schizophrenia, both in deep pyramidal neurons and in L3 and L5 17 pyramidal neurons of the dorsolateral prefrontal cortex; we observed significantly increased 18

19 expression of DDX19B in the deep pyramidal neurons of patients with schizophrenia; this

20 increase was independent of history of anti-psychotic medication usage. 21

22 Discussion 23

24 The DEAD-box helicase DDX19B is the human homologue of yeast Dbp5 which has

25 central functions in the nuclear export of messenger RNAs (mRNAs) and possesses both RNA- 26 binding and ATPase activity6-8. 27

28 Human DDX19B localizes to the cytoplasmic face of the nuclear pore complex (NPC)

PAGE 6 OF 14 1 and interacts with the amino-terminus of nucleoporin Nup159p (vertebrate Nup214); this 2 interaction is important for association with the NPC6. DDX19B is activated through 3

4 interactions with Gle1 and the inositol hexakisphosphate InsP68. Microinjection of mutant

5 human DDX19B protein into Xenopus oocytes blocks the nuclear export of mRNA6. In yeast, 6 Dbp5 can facilitate rearrangement or remodeling of exported ribonucleoproteins (RNPs) by 7

8 displacing nuclear export receptor Mex67p (the yeast homologue of TAP) from exported

9 RNPs7,9. 10 DDX19B is also involved in the elongation and termination phases of translation. 11

12 DDX19B supports formation of the termination complex (TC) and functions during stop codon 13 recognition10. DDX19B also interacts with the pre-termination complex and supports association 14 10 15 of elongation factors eEF1 and eEF2, resulting in the stabilization of terminating ribosomes .

16 Deletion of DDX19B in zebrafish using morpholino oligonucleotides results in curvature of body 17 axis and development of small eyes11. 18

19 We found using published and public microarray data that DDX19B is among the genes 20

21 whose expression changes most significantly in the deep pyramidal neurons and in the layer 3

22 and layer 5 pyramidal neurons of the dorsolateral pre-frontal cortex brains of patients with 23 schizophrenia. Changes in DDX19B gene expression observed here are more significant than 24

25 the vast majority of gene expression changes in two neuronal cell types of patients with 26 schizophrenia; differential expression of DDX19B appears to be independent of anti-psychotic 27

28 medication usage.

PAGE 7 OF 14 1 References 2 1. GSE145554. Robert McCullumsmith, University of Toledo. Toledo, OH, USA. Wu 3 X, Shukla R, Alganem K, Depasquale E, Reigle J, Simmons M, Hahn C, Haroutunian 4 V, Meller J, Meador-Woodruff J, McCullumsmith R.

5 2. Arion, D., Huo, Z., Enwright, J.F., Corradi, J.P., Tseng, G. and Lewis, D.A., 2017. 6 Transcriptome alterations in prefrontal pyramidal cells distinguish schizophrenia from bipolar and major depressive disorders. Biological psychiatry, 82(8), pp.594-600. 7

8 3. Cannon, T.D., 2015. How schizophrenia develops: cognitive and brain mechanisms underlying 9 onset of psychosis. Trends in cognitive sciences, 19(12), pp.744-756.

10 4. Arciniegas, D.B., 2015. Psychosis. Continuum: Lifelong Learning in Neurology, 21(3 11 Behavioral Neurology and Neuropsychiatry), p.715.

12 5. Andreasen, N.C., Nopoulos, P., Schultz, S., Miller, D., Gupta, S., Swayze, V. and Flaum, M., 13 1994. Positive and negative symptoms of schizophrenia: past, present, and future. Acta Psychiatrica Scandinavica, 90, pp.51-59. 14 15 6. Schmitt, C., von Kobbe, C., Bachi, A., Pante, N., Rodrigues, J.P., Boscheron, C., Rigaut, G., 16 Wilm, M., Seraphin, B., Carmo-Fonseca, M. and Izaurralde, E., 1999. Dbp5, a DEAD-box protein required for mRNA export, is recruited to the cytoplasmic fibrils of nuclear pore 17 complex via a conserved interaction with CAN/Nup159p. The EMBO journal, 18(15), pp. 18 4332-4347.

19 7. Tran, E.J., Zhou, Y., Corbett, A.H. and Wente, S.R., 2007. The DEAD-box protein Dbp5 20 controls mRNA export by triggering specific RNA: protein remodeling events. Molecular cell, 28(5), pp.850-859. 21

22 8. Weirich, C.S., Erzberger, J.P., Flick, J.S., Berger, J.M., Thorner, J. and Weis, K., 2006. Activation of the DExD/H-box protein Dbp5 by the nuclear-pore protein Gle1 and its 23 coactivator InsP 6 is required for mRNA export. Nature cell biology, 8(7), pp.668-676. 24

25 9. Gross, T., Siepmann, A., Sturm, D., Windgassen, M., Scarcelli, J.J., Seedorf, M., Cole, C.N. and Krebber, H., 2007. The DEAD-box RNA helicase Dbp5 functions in translation 26 termination. Science, 315(5812), pp.646-649. 27 10.Chen, X., Liu, M., Lou, H., Lu, Y., Zhou, M.T., Ou, R., Xu, Y. and Tang, K.F., 2019. 28 Degradation of endogenous proteins and generation of a null-like phenotype in zebrafish using Trim-Away technology. Genome biology, 20(1), p.19.

PAGE 8 OF 14 1 11.Lund, M.K. and Guthrie, C., 2005. The DEAD-box protein Dbp5p is required to dissociate 2 Mex67p from exported mRNPs at the nuclear rim. Molecular cell, 20(4), pp.645-651.

PAGE 9 OF 14 1

2 Rank ID p-value t B FC Gene Gene name

3 215 7997048 0.028177 2.3636215 -3.7 1.0690 ± DDX19B DEAD-box helicase 0.0729 19B 4

5 Table 1: DDX19B is differentially expressed in deep pyramidal neurons in patients with schizophrenia. 6

7 The rank of differential expression, the probe/transcript ID, the p-value with respect to global

8 differential expression, t, a moderated t-statistic, B, the log-odds of differential expression between the two groups compared, the fold change of DDX19B in deep pyramidal neurons of 9 patients with schizophrenia when compared to deep pyramidal neurons from control subjects, the 10 gene and gene name are listed in this chart. 11

PAGE 10 OF 14 1 Rank ID p-value t B Gene

2 6698 11723656_s_at 4.74E-02 2.0007635 -4.12976 DDX19A /// DDX19B 3

4 Table 2: DDX19B is differentially expressed in L3 and L5 pyramidal neurons of the 5 dorsolateral prefrontal cortex in patients with schizophrenia.

6 The rank of differential expression, the probe/transcript ID, the p-value with respect to global 7 differential expression, t, a moderated t-statistic, B, the log-odds of differential expression 8 between the two groups compared, the fold change of DDX19B in neurons of the DLPFC when compared to DLPFC neurons from control subjects, the gene and gene name are listed in this 9 chart. 10

PAGE 11 OF 14 1

2 Rank ID p-value t B FC Gene Gene name

3 36 7997048 0.00222084 3.716986 -3.59 1.1355 ± DDX19B DEAD-box helicase 0.1056 19B 4

5 Table 3: Differential expression of DDX19B in the deep pyramidal neurons of patients with schizophrenia is independent of anti-psychotic medication usage. 6

7 The rank of differential expression, the probe/transcript ID, the p-value with respect to global

8 differential expression, t, a moderated t-statistic, B, the log-odds of differential expression between the two groups compared, the fold change of DDX19B in deep pyramidal neurons of 9 patients with schizophrenia not treated with antipsychotic medications when compared to deep 10 pyramidal neurons from control subjects, the gene and gene name are listed in this chart. 11

PAGE 12 OF 14 1 DDX19B 2 6.5 0.0142 3

4 6.0 5

6 5.5

7 mRNA expression AU (arbitrary units) 5.0 8

9 4.5 Control Schizophrenia 10

12 Figure 1: DDX19B is expressed at significantly higher levels in the deep pyramidal neurons 13 of patients with schizophrenia.

14 The mRNA expression of DDX19B in deep pyramidal neurons from control subjects (left) and 15 in deep pyramidal neurons from patients with schizophrenia (right) is represented with mean 16 mRNA expression level marked and the result of a statistical test evaluating the significance of difference in DDX19B expression between control deep pyramidal neurons and deep pyramidal 17 neurons from patients with schizophrenia, a p-value, listed above. 18

PAGE 13 OF 14 1 DDX19B 2 6.5 0.3150 3

4 6.0 5

6 5.5

7 mRNA expression AU (arbitrary units) 5.0 8

9 4.5 Control Schizophrenia 10

12 Figure 1: DDX19B is expressed at higher levels in the deep pyramidal neurons of patients with schizophrenia independent of anti-psychotic medication usage. 13

14 The mRNA expression of DDX19B in deep pyramidal neurons from control subjects (left) and

15 in deep pyramidal neurons from patients with schizophrenia not being treated with anti- psychotic medications (right) is represented with mean mRNA expression level marked and the 16 result of a statistical test evaluating the significance of difference in DDX19B expression 17 between control deep pyramidal neurons and deep pyramidal neurons from patients with 18 schizophrenia not being treated with anti-psychotic medications, a p-value, listed above.

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