- Email: [email protected]
mortalin mRNA expression in the dorsolateral prefrontal cortex of human postmortem brain specimens
Schizophrenia Research 119 (2010) 228–231
Contents lists available at ScienceDirect
Schizophrenia Research j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / s c h r e s
Antipsychotic drug use is correlated with CRP40/mortalin mRNA expression in the dorsolateral prefrontal cortex of human postmortem brain specimens Joseph P. Gabriele a,⁎, Giuseppe F. Pontoriero a, Nancy Thomas a, Mark A. Ferro b, Geetha Mahadevan a, Duncan J. MacCrimmon a, Zdenek B. Pristupa a, Ram K. Mishra a a b
Department of Psychiatry and Behavioural Neurosciences, McMaster University, Hamilton, Ontario, Canada Department of Epidemiology and Biostatistics, University of Western Ontario, London, Ontario, Canada
a r t i c l e
i n f o
Article history: Received 15 July 2009 Received in revised form 21 December 2009 Accepted 29 December 2009 Available online 25 January 2010 Keywords: Schizophrenia Catecholamine regulated proteins Mortalin Prefrontal cortex Antipsychotic drugs
a b s t r a c t Heat shock proteins act as intracellular chaperones by assisting with proper protein folding in response to various cellular stresses. In doing so, these proteins protect the cell from unwanted protein aggregation, which in turn, plays an important role in the pathogenesis of numerous disorders. Previous reports from our laboratory have described a 40 kDa catecholamine regulated heat shock-like protein (CRP40), an alternate gene product of the 70 kDa mitochondrial heat shock protein, mortalin. CRP40 shares an intimate association with dopaminergic activity, specifically as it pertains to dopamine dysregulation in schizophrenia. This study investigates human CRP40/mortalin mRNA expression within dorsolateral prefrontal cortex postmortem specimens from normal control, schizophrenic and bipolar patients obtained from the Stanley Medical Research Institute. Real-time polymerase chain reaction was carried out for all patient samples (n = 105; n = 35 per group) in a blinded manner. No significant alterations in CRP40/mortalin mRNA expression levels were observed between control, bipolar and schizophrenic patients. However, multiple regression demonstrated a distinct positive correlation between CRP40/mortalin mRNA expression and lifetime use of antipsychotic drugs within the schizophrenic patient profile, after controlling for important confounding factors. Thus, the data suggest that human CRP40/mortalin is modulated by dopaminergic activity and may act to protect neurons from excess catecholamine activity in regions of the brain associated with psychosis. Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved.
1. Introduction The dopamine (DA) hypothesis, although its mechanisms of action are not fully understood, maintains that aberrations in DA signal transduction pathways remain one of the leading possibilities among the potential abnormalities implicated in psychosis (Seeman and Kapur, 2000). The psychotic and cognitive abnormalities associated with schizophrenia and bipolar disorder are responsive to antipsychotic drugs (ASDs); relief of partial or full dysfunctional symptoms has
⁎ Corresponding author. McMaster University, 1200 Main St. West, HSC Rm 4N81, Hamilton, Ontario, Canada L8N 3Z5. Tel.: +1 905 525 9140x22617. E-mail address: [email protected] (J.P. Gabriele).
implicated the dopaminergic pathways and DA receptors (Selemon and Rajkowska, 2003). Catecholamine regulated proteins (CRPs) have been previously shown to bind DA and other structurally related catecholamines (Ross et al., 1995, 1993). Our laboratory has identified a 40 kDa human brain CRP (CRP40), which has been shown to be primarily regulated by DA receptor agonists and antagonists (Nair and Mishra, 2001; Sharan et al., 2003). Corroborating this hypothesis, we have previously demonstrated that increased CRP40 protein expression is correlated with increased ASD treatment in schizophrenic human postmortem ventral striatal tissue samples (Gabriele et al., 2005). More recently, we have identified CRP40 to be an alternative gene product of HSPA9, the gene encoding the 70 kDa mitochondrial heat shock protein, mortalin, sharing
0920-9964/$ – see front matter. Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.schres.2009.12.039
J.P. Gabriele et al. / Schizophrenia Research 119 (2010) 228–231
identity to mortalin's carboxy-terminal domain (Gabriele et al., 2009). Studies have indicated that both mortalin and CRP40 share the ability to bind DA and regulate dopaminergic activity (Gabriele et al., 2009; Van Laar et al., 2009). The present study examined human CRP40/mortalin expression in control, schizophrenic and bipolar postmortem brain specimens. Because, human CRP40 is differentially expressed in the presence of DA and DA metabolites, this study focused on the effects of lifetime ASD use in schizophrenic postmortem specimens. CRP40/mortalin mRNA expression was assessed via real-time PCR in human dorsolateral PFC (DLPFC) brain samples. Our findings suggest that human CRP40/mortalin mRNA expression levels are related to lifetime ASD use and that human CRP40/mortalin may be dysfunctional in patients with schizophrenia. 2. Methods Coded postmortem DNase-treated RNA specimens of the DLPFC were obtained from the Stanley Medical Research Institute (SMRI). Real-time RT-PCR was conducted using the QuantiTect SYBR Green RT-PCR Kit (Qiagen Inc.) according to the manufacturer's protocols. Real-time RT-PCR reactions were performed in triplicate using 50 ng of total RNA and the following specific PCR primers (300 nM): forward primer, 5′TTGGCCGGCGATGTCACGGATGTG-3′ and reverse primer, 5′ACACACTTTAATTTCCACTTGCGT-3′. Real-time RT-PCR conditions were: 50 °C for 30 min (1 cycle), 95 °C for 15 s (1 cycle), followed by 40 cycles of 94 °C for 15 s, 60 °C for 30 s, and 72 °C for 45 s. Expression of the house-keeping gene, human cyclophilin, was also examined across groups and showed no significant alterations. A standard curve was constructed to calculate the absolute CRP40/mortalin mRNA copy number. Data were entered and analyzed with the Statistical Analysis System (SAS, Version 9.1.3). Associations between CRP40/mortalin mRNA expression and the other study variables were assessed using Pearson product moment correlation coefficients. The differences in CRP40/mortalin mRNA copy number between each patient group (control, bipolar disorder, schizophrenia) were determined using
229
analysis of variance (ANOVA). Backward, stepwise multiple regression was used to model the impact of lifetime ASD use on CRP40/mortalin mRNA expression within the schizophrenia patient profile, controlling for confounding factors. Diagnostic analyses for the regression analysis indicated that no outliers were influential and were thus retained in the model to maximize the statistical power. Some data was log transformed in order to maintain the assumption of normality. All statistical tests were two-tailed using a confidence level of α = 0.05. 3. Results Postmortem DLPFC RNA samples extracted from 35 control patients, 35 bipolar disorder patients, and 35 schizophrenic patients, were obtained from the SMRI. A summary of the relevant patient information is provided in Table 1 and is stratified according to patient diagnosis group (normal control, bipolar disorder, schizophrenia). Given the significant homology between CRP40 and mortalin, the primers utilized in this study amplified both the CRP40 and mortalin mRNA transcripts. 3.1. CRP40/mortalin mRNA expression is positively correlated with brain pH but no other variables in postmortem DLPFC tissue We first carried out Pearson correlation analyses at the P b 0.05 level to examine potential confounding factors on CRP40/mortalin expression. Some of these variables have previously been shown to negatively impact gene expression. Following logarithmic transformation of CRP40/mortalin expression, a positive correlation between brain pH and CRP40/mortalin expression was observed ( r = 0.26, P = 0.0086). No significant correlations were observed between any of the other clinical variables (age at death, postmortem interval, brain pH, RNA quality) and CRP40/ mortalin gene expression. Interestingly, examination of CRP40/mortalin gene expression across our three patient cohorts revealed no significant alterations in log transformed
Table 1 Descriptive subject statistics. Variable
Normal control (n = 35)
Bipolar disorder (n = 35)
Schizophrenia (n = 35)
Age (mean ± SD, years) Sex (M/F) Race (Caucasian/other) Age of onset (mean ± SD, years) Duration of illness (mean ± SD, years) Time in hospital (mean ± SD, years) Alcohol abuse at TOD (n) Drug abuse at TOD (n) Smoking at TOD (yes/no/unknown) Psychotic feature (yes/no/unknown) Lifetime antipsychotic use (FE ± SD, mg) Relative brain mass (mean ± SD, g) Right brain (n) Left brain (n) PMI (mean ± SD, h) Refrigerator interval (mean ± SD, h) Brain pH (mean ± SD) 28S:18S rRNA ratio (mean ± SD)
44.2 ± 7.6 (Range 31–60) 26/9 35/0 N/A N/A N/A 2 1 9/9/17 0/35/0 N/A 1444 ± 148.4 (Range 1120–1900) 19 16 29.4 ± 12.9 (Range 9–58) 3.6 ± 2.6 (Range 0–14) 6.61 ± 0.27 (Range 6.00–7.03) 2.18 ± 0.50 (Range 0.90–3.74)
45.3 ± 10.5 (Range 19–64) 17/18 33/2 25.1 ± 9.1 (Range 14–48) 20.1 ± 9.5 (Range 2–45) 0.5 ± 1.4 (Range 0–8) 11 10 16/6/13 21/12/2 10,035 ± 22,896 (Range 0–130,000) 1394 ± 139.1 (Range 1120–1670) 15 20 37.9 ± 18.4 (Range 12–81) 10.1 ± 10.4 (Range 1–54) 6.43 ± 0.30 (Range 5.76–6.97) 2.21 ± 0.75 (Range 0.49–4.18)
42.6 ± 8.5 (Range 19–59) 26/9 34/1 21.3 ± 6.1 (Range 9–34) 21.3 ± 10.2 (Range 1–45) 1.2 ± 2.3 (Range 0–12) 12 9 23/4/8 35/0/0 85,004 ± 100,335 (Range 50–400,000) 1442 ± 107.5 (Range 1170–1630) 18 17 31.4 ± 15.5 (Range 9–80) 6.0 ± 4.2 (Range 1–19) 6.48 ± 0.24 (Range 5.90 ± 6.93) 2.13 ± 0.55 (Range 1.18–3.81)
Abbreviations: M, male; F, female; N/A, not applicable; TOD, time of death; PMI, postmortem interval; FE, fluphenazine equivalents.
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sion was examined within our schizophrenia patient profile. Controlling for important confounding factors, multiple regression analysis revealed that lifetime ASD use was significantly associated with CRP40/mortalin mRNA expression levels (β = 0.07, P = 0.0125) in patients diagnosed with schizophrenia. Moreover, there was a positive correlation between these variables, whereby increased lifetime ASD use was associated with increased CRP40/mortalin gene expression within the DLPFC (Fig. 2). Interestingly, multiple regression analysis revealed no evidence to suggest a correlation between ASD use and mRNA expression in bipolar patients (data not shown). 4. Discussion Fig. 1. CRP40/mortalin mRNA expression within postmortem DLPFC brain specimens. Real-time PCR was performed on 105 patients samples obtained from the SMRI. Following ANOVA analysis, no significant alteration in CRP40/ mortalin mRNA expression was observed between either normal control (CTL) patient samples, or samples extracted from patients afflicted with bipolar disorder (BPD) and schizophrenia (SCZ). Data are presented as box plots of log transformed mean CRP40/mortalin mRNA expression.
CRP40/mortalin mRNA expression between control, bipolar or schizophrenic postmortem DLPFC tissue samples (F = 2.46, P = 0.0903) (Fig. 1). 3.2. CRP40/Mortalin mRNA expression is positively correlated with increased ASD use Our previous examination of CRP40 protein expression within the ventral striatum of human postmortem schizophrenic patients revealed a positive association between ASD use and CRP40 expression (Gabriele et al., 2005). As such, the relationship between ASD use and CRP40/mortalin expres-
This study examined the expression of mortalin and its 40 kDa alternative gene product CRP40, within postmortem DLPFC brain samples. Real-time RT-PCR suggested no significant alterations in CRP40/mortalin mRNA expression between all experimental cohorts as evidenced by the ANOVA analysis. However, after controlling for confounding variables, a direct correlation was observed between CRP40/mortalin mRNA copy number and the amount of lifetime schizophrenic ASD use; in the presence of DA modifying agents, CRP40/mortalin mRNA expression is significantly increased. Recently, our lab has shown reduced expression of CRP40 protein within the ventral striatum of schizophrenic patients (Gabriele et al., 2005). In contrast, our present study consisted of examination of human CRP40/mortalin mRNA expression within the DLPFC. Neuronal aberrations within the PFC have often been associated with diseases involving psychosis (Callicott et al., 2000; Glahn et al., 2007). Previous studies have demonstrated that there is a thinning of the PFC,
Fig. 2. A direct linear relationship between expression levels of CRP40/mortalin mRNA (expressed as copy number) and lifetime ASD use (expressed at fluphenazine equivalents (FE)). A solid line represents a fitted regression line, dotted lines represent the probability (0.95) that the ‘true’ fitted line (in the population) falls between the bands, while the blue region depicts the 95% confidence intervals.
J.P. Gabriele et al. / Schizophrenia Research 119 (2010) 228–231
specifically the inter-neurons of the PFC, which accounts for the decreased number of neurons in schizophrenic patients (Benes, 2000; Weinberger et al., 2001). Furthermore, studies have shown that abnormalities of synaptic circuitry, due to alterations in the neuronal architecture, may implicate the hypofrontality of DA activity in the PFC region (Benes, 2000; Weinberger, 1988). Corroborating this hypothesis, the medial PFC is known to be densely populated with dopaminergic afferents while also containing large amounts of mRNA encoding the DA D2 receptor (Pira et al., 2004). Current treatment options for the management of schizophrenia include the administration of ASDs, which bind with varying affinity to the DA D2 receptor. Our data in this study are correlational and therefore we cannot conclusively demonstrate causal relationships between ASD intake and CRP40/ mortalin expression. However, past reports from our lab have shown that the CRP40 protein is differentially regulated by ASDs, DA receptor agonists, and psychotropic drugs (Gabriele et al., 2002; Modi et al., 1996). Supporting these observations, the results from our current study indicate that patients administered higher doses of ASDs exhibited higher DLPFC CRP40/mortalin mRNA expression. This positive effect of ASD treatment on CRP40 expression was also observed in our recent examination of ventral striatal postmortem brain tissue (Gabriele et al., 2005). These findings correlate well with our working hypothesis that human CRP40 expression is directly related to the amount of ASD use, and subsequent increased DA release. Given their roles as chaperone proteins, misexpression of CRP40 and its parent protein, mortalin, may impart an inability to combat excess DA and associated oxidative metabolites within the PFC and other brain regions associated with schizophrenia. Supporting this notion, DA-mortalin conjugates were identified following exposure to DA quinone (Van Laar et al., 2009). It should be noted that given the 100% homology associated with CRP40 and mortalin, a major limitation with the current investigation was our inability to differentiate CRP40 from mortalin mRNA expression. However, we have examined the protein expression profiles for both mortalin and CRP40 in postmortem PFC samples obtained from the Human Brain and Spinal Fluid Resource Center (Los Angeles, California) and observed similar experimental findings (J. Gabriele, unpublished findings). In summary, human CRP40/mortalin is likely modulated by DA activity and may act as a molecular chaperone by protecting the cell from excess catecholamine and catecholamine metabolite activity in regions of the brain associated with psychosis. Furthermore, the results in this study suggest that ASDs have a significant influence on the CRP40 mRNA levels in patients with schizophrenia. In addition, future work should be aimed at examining whether reduced CRP40/ mortalin expression is either a cause or downstream effect of DA dysfunction within the PFC and related brain regions. Role of funding source Funding for this study was provided by the Canadian Institutes of Health Research (CIHR). The CIHR had no further role in study design, in the collection, analysis and interpretation of data, in the writing of the report, or in the decision to submit the paper for publication. Contributors All authors have made significant scientific contributions to this manuscript. Joseph P. Gabriele contributed to the conceptualization and
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implementation of the study, securing funding and contributed to the writing of the manuscript. Giuseppe F. Pontoriero contributed to the implementation of the study and to the writing of the manuscript. Nancy Thomas and Geetha Mahadevan conducted the real-time PCR analyses. Mark A. Ferro conducted the statistical analyses and contributed to the editing of the manuscript. Duncan J. MacCrimmon provided insight to our working hypothesis. Zdenek B. Pristupa designed all PCR primers and provided significant consultation. Ram K. Mishra contributed to the conceptualization of the study, securing funding and contributed to the editing of the manuscript. All authors have approved the final manuscript. Conflict of interest Joseph P. Gabriele, Zdenek B. Pristupa, and Ram K. Mishra jointly hold a patent on mortalin and its alternative gene product, CRP40. All other authors report no competing interests. Acknowledgements This work was funded by the Canadian Institutes of Health Research. Postmortem brain tissue was generously donated by the Stanley Medical Research Institute's brain collection courtesy of Drs. Michael B. Knable, E. Fuller Torrey, Maree J. Webster, and Robert H. Yolken.
References Benes, F.M., 2000. Emerging principles of altered neural circuitry in schizophrenia. Brain Res. Brain Res. Rev. 31 (2–3), 251–269. Callicott, J.H., Bertolino, A., Mattay, V.S., Langheim, F.J., Duyn, J., Coppola, R., Goldberg, T.E., Weinberger, D.R., 2000. Physiological dysfunction of the dorsolateral prefrontal cortex in schizophrenia revisited. Cereb. Cortex 10 (11), 1078–1092. Gabriele, J., Rajaram, M., Zhang, B., Sharma, S., Mishra, R.K., 2002. Modulation of a 40-kDa catecholamine-regulated protein following D-amphetamine treatment in discrete brain regions. Eur. J. Pharmacol. 453 (1), 13–19. Gabriele, J.P., Chong, V.Z., Pontoriero, G.F., Mishra, R.K., 2005. Decreased expression of a 40-kDa catecholamine-regulated protein in the ventral striatum of schizophrenic brain specimens from the Stanley Foundation Neuropathology Consortium. Schizophr. Res. 74 (1), 111–119. Gabriele, J., Pontoriero, G.F., Thomas, N., Thomson, C.A., Skoblenick, K., Pristupa, Z.B., Mishra, R.K., 2009. Cloning, characterization, and functional studies of a human 40-kDa catecholamine-regulated protein: implications in central nervous system disorders. Cell Stress Chaperones. Glahn, D.C., Bearden, C.E., Barguil, M., Barrett, J., Reichenberg, A., Bowden, C.L., Soares, J.C., Velligan, D.I., 2007. The neurocognitive signature of psychotic bipolar disorder. Biol. Psychiatry 62 (8), 910–916. Modi, P.I., Kashyap, A., Nair, V.D., Ross, G.M., Fu, M., Savelli, J.E., Marcotte, E.R., Barlas, C., Mishra, R.K., 1996. Modulation of brain catecholamine absorbing proteins by dopaminergic agents. Eur. J. Pharmacol. 299 (1–3), 213–220. Nair, V.D., Mishra, R.K., 2001. Molecular cloning, localization and characterization of a 40-kDa catecholamine-regulated protein. J. Neurochem. 76 (4), 1142–1152. Pira, L., Mongeau, R., Pani, L., 2004. The atypical antipsychotic quetiapine increases both noradrenaline and dopamine release in the rat prefrontal cortex. Eur. J. Pharmacol. 504 (1–2), 61–64. Ross, G.M., McCarry, B.E., Thakur, S., Mishra, R.K., 1993. Identification of novel catecholamine absorbing proteins in the central nervous system. J. Mol. Neurosci. 4 (3), 141–148. Ross, G.M., McCarry, B.E., Mishra, R.K., 1995. Covalent affinity labeling of brain catecholamine-absorbing proteins using a high-specific-activity substituted tetrahydronaphthalene. J. Neurochem. 65 (6), 2783–2789. Seeman, P., Kapur, S., 2000. Schizophrenia: more dopamine, more D2 receptors. Proc. Natl. Acad. Sci. U. S. A. 97 (14), 7673–7675. Selemon, L.D., Rajkowska, G., 2003. Cellular pathology in the dorsolateral prefrontal cortex distinguishes schizophrenia from bipolar disorder. Curr. Mol. Med. 3 (5), 427–436. Sharan, N., Chong, V.Z., Nair, V.D., Mishra, R.K., Hayes, R.J., Gardner, E.L., 2003. Cocaine treatment increases expression of a 40 kDa catecholamineregulated protein in discrete brain regions. Synapse 47 (1), 33–44. Van Laar, V.S., Mishizen, A.J., Cascio, M., Hastings, T.G., 2009. Proteomic identification of dopamine-conjugated proteins from isolated rat brain mitochondria and SH-SY5Y cells. Neurobiol. Dis. 34 (3), 487–500. Weinberger, D.R., 1988. Schizophrenia and the frontal lobe. Trends Neurosci. 11 (8), 367–370. Weinberger, D.R., Egan, M.F., Bertolino, A., Callicott, J.H., Mattay, V.S., Lipska, B.K., Berman, K.F., Goldberg, T.E., 2001. Prefrontal neurons and the genetics of schizophrenia. Biol. Psychiatry 50 (11), 825–844.
Contents lists available at ScienceDirect
Schizophrenia Research j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / s c h r e s
Antipsychotic drug use is correlated with CRP40/mortalin mRNA expression in the dorsolateral prefrontal cortex of human postmortem brain specimens Joseph P. Gabriele a,⁎, Giuseppe F. Pontoriero a, Nancy Thomas a, Mark A. Ferro b, Geetha Mahadevan a, Duncan J. MacCrimmon a, Zdenek B. Pristupa a, Ram K. Mishra a a b
Department of Psychiatry and Behavioural Neurosciences, McMaster University, Hamilton, Ontario, Canada Department of Epidemiology and Biostatistics, University of Western Ontario, London, Ontario, Canada
a r t i c l e
i n f o
Article history: Received 15 July 2009 Received in revised form 21 December 2009 Accepted 29 December 2009 Available online 25 January 2010 Keywords: Schizophrenia Catecholamine regulated proteins Mortalin Prefrontal cortex Antipsychotic drugs
a b s t r a c t Heat shock proteins act as intracellular chaperones by assisting with proper protein folding in response to various cellular stresses. In doing so, these proteins protect the cell from unwanted protein aggregation, which in turn, plays an important role in the pathogenesis of numerous disorders. Previous reports from our laboratory have described a 40 kDa catecholamine regulated heat shock-like protein (CRP40), an alternate gene product of the 70 kDa mitochondrial heat shock protein, mortalin. CRP40 shares an intimate association with dopaminergic activity, specifically as it pertains to dopamine dysregulation in schizophrenia. This study investigates human CRP40/mortalin mRNA expression within dorsolateral prefrontal cortex postmortem specimens from normal control, schizophrenic and bipolar patients obtained from the Stanley Medical Research Institute. Real-time polymerase chain reaction was carried out for all patient samples (n = 105; n = 35 per group) in a blinded manner. No significant alterations in CRP40/mortalin mRNA expression levels were observed between control, bipolar and schizophrenic patients. However, multiple regression demonstrated a distinct positive correlation between CRP40/mortalin mRNA expression and lifetime use of antipsychotic drugs within the schizophrenic patient profile, after controlling for important confounding factors. Thus, the data suggest that human CRP40/mortalin is modulated by dopaminergic activity and may act to protect neurons from excess catecholamine activity in regions of the brain associated with psychosis. Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved.
1. Introduction The dopamine (DA) hypothesis, although its mechanisms of action are not fully understood, maintains that aberrations in DA signal transduction pathways remain one of the leading possibilities among the potential abnormalities implicated in psychosis (Seeman and Kapur, 2000). The psychotic and cognitive abnormalities associated with schizophrenia and bipolar disorder are responsive to antipsychotic drugs (ASDs); relief of partial or full dysfunctional symptoms has
⁎ Corresponding author. McMaster University, 1200 Main St. West, HSC Rm 4N81, Hamilton, Ontario, Canada L8N 3Z5. Tel.: +1 905 525 9140x22617. E-mail address: [email protected] (J.P. Gabriele).
implicated the dopaminergic pathways and DA receptors (Selemon and Rajkowska, 2003). Catecholamine regulated proteins (CRPs) have been previously shown to bind DA and other structurally related catecholamines (Ross et al., 1995, 1993). Our laboratory has identified a 40 kDa human brain CRP (CRP40), which has been shown to be primarily regulated by DA receptor agonists and antagonists (Nair and Mishra, 2001; Sharan et al., 2003). Corroborating this hypothesis, we have previously demonstrated that increased CRP40 protein expression is correlated with increased ASD treatment in schizophrenic human postmortem ventral striatal tissue samples (Gabriele et al., 2005). More recently, we have identified CRP40 to be an alternative gene product of HSPA9, the gene encoding the 70 kDa mitochondrial heat shock protein, mortalin, sharing
0920-9964/$ – see front matter. Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.schres.2009.12.039
J.P. Gabriele et al. / Schizophrenia Research 119 (2010) 228–231
identity to mortalin's carboxy-terminal domain (Gabriele et al., 2009). Studies have indicated that both mortalin and CRP40 share the ability to bind DA and regulate dopaminergic activity (Gabriele et al., 2009; Van Laar et al., 2009). The present study examined human CRP40/mortalin expression in control, schizophrenic and bipolar postmortem brain specimens. Because, human CRP40 is differentially expressed in the presence of DA and DA metabolites, this study focused on the effects of lifetime ASD use in schizophrenic postmortem specimens. CRP40/mortalin mRNA expression was assessed via real-time PCR in human dorsolateral PFC (DLPFC) brain samples. Our findings suggest that human CRP40/mortalin mRNA expression levels are related to lifetime ASD use and that human CRP40/mortalin may be dysfunctional in patients with schizophrenia. 2. Methods Coded postmortem DNase-treated RNA specimens of the DLPFC were obtained from the Stanley Medical Research Institute (SMRI). Real-time RT-PCR was conducted using the QuantiTect SYBR Green RT-PCR Kit (Qiagen Inc.) according to the manufacturer's protocols. Real-time RT-PCR reactions were performed in triplicate using 50 ng of total RNA and the following specific PCR primers (300 nM): forward primer, 5′TTGGCCGGCGATGTCACGGATGTG-3′ and reverse primer, 5′ACACACTTTAATTTCCACTTGCGT-3′. Real-time RT-PCR conditions were: 50 °C for 30 min (1 cycle), 95 °C for 15 s (1 cycle), followed by 40 cycles of 94 °C for 15 s, 60 °C for 30 s, and 72 °C for 45 s. Expression of the house-keeping gene, human cyclophilin, was also examined across groups and showed no significant alterations. A standard curve was constructed to calculate the absolute CRP40/mortalin mRNA copy number. Data were entered and analyzed with the Statistical Analysis System (SAS, Version 9.1.3). Associations between CRP40/mortalin mRNA expression and the other study variables were assessed using Pearson product moment correlation coefficients. The differences in CRP40/mortalin mRNA copy number between each patient group (control, bipolar disorder, schizophrenia) were determined using
229
analysis of variance (ANOVA). Backward, stepwise multiple regression was used to model the impact of lifetime ASD use on CRP40/mortalin mRNA expression within the schizophrenia patient profile, controlling for confounding factors. Diagnostic analyses for the regression analysis indicated that no outliers were influential and were thus retained in the model to maximize the statistical power. Some data was log transformed in order to maintain the assumption of normality. All statistical tests were two-tailed using a confidence level of α = 0.05. 3. Results Postmortem DLPFC RNA samples extracted from 35 control patients, 35 bipolar disorder patients, and 35 schizophrenic patients, were obtained from the SMRI. A summary of the relevant patient information is provided in Table 1 and is stratified according to patient diagnosis group (normal control, bipolar disorder, schizophrenia). Given the significant homology between CRP40 and mortalin, the primers utilized in this study amplified both the CRP40 and mortalin mRNA transcripts. 3.1. CRP40/mortalin mRNA expression is positively correlated with brain pH but no other variables in postmortem DLPFC tissue We first carried out Pearson correlation analyses at the P b 0.05 level to examine potential confounding factors on CRP40/mortalin expression. Some of these variables have previously been shown to negatively impact gene expression. Following logarithmic transformation of CRP40/mortalin expression, a positive correlation between brain pH and CRP40/mortalin expression was observed ( r = 0.26, P = 0.0086). No significant correlations were observed between any of the other clinical variables (age at death, postmortem interval, brain pH, RNA quality) and CRP40/ mortalin gene expression. Interestingly, examination of CRP40/mortalin gene expression across our three patient cohorts revealed no significant alterations in log transformed
Table 1 Descriptive subject statistics. Variable
Normal control (n = 35)
Bipolar disorder (n = 35)
Schizophrenia (n = 35)
Age (mean ± SD, years) Sex (M/F) Race (Caucasian/other) Age of onset (mean ± SD, years) Duration of illness (mean ± SD, years) Time in hospital (mean ± SD, years) Alcohol abuse at TOD (n) Drug abuse at TOD (n) Smoking at TOD (yes/no/unknown) Psychotic feature (yes/no/unknown) Lifetime antipsychotic use (FE ± SD, mg) Relative brain mass (mean ± SD, g) Right brain (n) Left brain (n) PMI (mean ± SD, h) Refrigerator interval (mean ± SD, h) Brain pH (mean ± SD) 28S:18S rRNA ratio (mean ± SD)
44.2 ± 7.6 (Range 31–60) 26/9 35/0 N/A N/A N/A 2 1 9/9/17 0/35/0 N/A 1444 ± 148.4 (Range 1120–1900) 19 16 29.4 ± 12.9 (Range 9–58) 3.6 ± 2.6 (Range 0–14) 6.61 ± 0.27 (Range 6.00–7.03) 2.18 ± 0.50 (Range 0.90–3.74)
45.3 ± 10.5 (Range 19–64) 17/18 33/2 25.1 ± 9.1 (Range 14–48) 20.1 ± 9.5 (Range 2–45) 0.5 ± 1.4 (Range 0–8) 11 10 16/6/13 21/12/2 10,035 ± 22,896 (Range 0–130,000) 1394 ± 139.1 (Range 1120–1670) 15 20 37.9 ± 18.4 (Range 12–81) 10.1 ± 10.4 (Range 1–54) 6.43 ± 0.30 (Range 5.76–6.97) 2.21 ± 0.75 (Range 0.49–4.18)
42.6 ± 8.5 (Range 19–59) 26/9 34/1 21.3 ± 6.1 (Range 9–34) 21.3 ± 10.2 (Range 1–45) 1.2 ± 2.3 (Range 0–12) 12 9 23/4/8 35/0/0 85,004 ± 100,335 (Range 50–400,000) 1442 ± 107.5 (Range 1170–1630) 18 17 31.4 ± 15.5 (Range 9–80) 6.0 ± 4.2 (Range 1–19) 6.48 ± 0.24 (Range 5.90 ± 6.93) 2.13 ± 0.55 (Range 1.18–3.81)
Abbreviations: M, male; F, female; N/A, not applicable; TOD, time of death; PMI, postmortem interval; FE, fluphenazine equivalents.
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sion was examined within our schizophrenia patient profile. Controlling for important confounding factors, multiple regression analysis revealed that lifetime ASD use was significantly associated with CRP40/mortalin mRNA expression levels (β = 0.07, P = 0.0125) in patients diagnosed with schizophrenia. Moreover, there was a positive correlation between these variables, whereby increased lifetime ASD use was associated with increased CRP40/mortalin gene expression within the DLPFC (Fig. 2). Interestingly, multiple regression analysis revealed no evidence to suggest a correlation between ASD use and mRNA expression in bipolar patients (data not shown). 4. Discussion Fig. 1. CRP40/mortalin mRNA expression within postmortem DLPFC brain specimens. Real-time PCR was performed on 105 patients samples obtained from the SMRI. Following ANOVA analysis, no significant alteration in CRP40/ mortalin mRNA expression was observed between either normal control (CTL) patient samples, or samples extracted from patients afflicted with bipolar disorder (BPD) and schizophrenia (SCZ). Data are presented as box plots of log transformed mean CRP40/mortalin mRNA expression.
CRP40/mortalin mRNA expression between control, bipolar or schizophrenic postmortem DLPFC tissue samples (F = 2.46, P = 0.0903) (Fig. 1). 3.2. CRP40/Mortalin mRNA expression is positively correlated with increased ASD use Our previous examination of CRP40 protein expression within the ventral striatum of human postmortem schizophrenic patients revealed a positive association between ASD use and CRP40 expression (Gabriele et al., 2005). As such, the relationship between ASD use and CRP40/mortalin expres-
This study examined the expression of mortalin and its 40 kDa alternative gene product CRP40, within postmortem DLPFC brain samples. Real-time RT-PCR suggested no significant alterations in CRP40/mortalin mRNA expression between all experimental cohorts as evidenced by the ANOVA analysis. However, after controlling for confounding variables, a direct correlation was observed between CRP40/mortalin mRNA copy number and the amount of lifetime schizophrenic ASD use; in the presence of DA modifying agents, CRP40/mortalin mRNA expression is significantly increased. Recently, our lab has shown reduced expression of CRP40 protein within the ventral striatum of schizophrenic patients (Gabriele et al., 2005). In contrast, our present study consisted of examination of human CRP40/mortalin mRNA expression within the DLPFC. Neuronal aberrations within the PFC have often been associated with diseases involving psychosis (Callicott et al., 2000; Glahn et al., 2007). Previous studies have demonstrated that there is a thinning of the PFC,
Fig. 2. A direct linear relationship between expression levels of CRP40/mortalin mRNA (expressed as copy number) and lifetime ASD use (expressed at fluphenazine equivalents (FE)). A solid line represents a fitted regression line, dotted lines represent the probability (0.95) that the ‘true’ fitted line (in the population) falls between the bands, while the blue region depicts the 95% confidence intervals.
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specifically the inter-neurons of the PFC, which accounts for the decreased number of neurons in schizophrenic patients (Benes, 2000; Weinberger et al., 2001). Furthermore, studies have shown that abnormalities of synaptic circuitry, due to alterations in the neuronal architecture, may implicate the hypofrontality of DA activity in the PFC region (Benes, 2000; Weinberger, 1988). Corroborating this hypothesis, the medial PFC is known to be densely populated with dopaminergic afferents while also containing large amounts of mRNA encoding the DA D2 receptor (Pira et al., 2004). Current treatment options for the management of schizophrenia include the administration of ASDs, which bind with varying affinity to the DA D2 receptor. Our data in this study are correlational and therefore we cannot conclusively demonstrate causal relationships between ASD intake and CRP40/ mortalin expression. However, past reports from our lab have shown that the CRP40 protein is differentially regulated by ASDs, DA receptor agonists, and psychotropic drugs (Gabriele et al., 2002; Modi et al., 1996). Supporting these observations, the results from our current study indicate that patients administered higher doses of ASDs exhibited higher DLPFC CRP40/mortalin mRNA expression. This positive effect of ASD treatment on CRP40 expression was also observed in our recent examination of ventral striatal postmortem brain tissue (Gabriele et al., 2005). These findings correlate well with our working hypothesis that human CRP40 expression is directly related to the amount of ASD use, and subsequent increased DA release. Given their roles as chaperone proteins, misexpression of CRP40 and its parent protein, mortalin, may impart an inability to combat excess DA and associated oxidative metabolites within the PFC and other brain regions associated with schizophrenia. Supporting this notion, DA-mortalin conjugates were identified following exposure to DA quinone (Van Laar et al., 2009). It should be noted that given the 100% homology associated with CRP40 and mortalin, a major limitation with the current investigation was our inability to differentiate CRP40 from mortalin mRNA expression. However, we have examined the protein expression profiles for both mortalin and CRP40 in postmortem PFC samples obtained from the Human Brain and Spinal Fluid Resource Center (Los Angeles, California) and observed similar experimental findings (J. Gabriele, unpublished findings). In summary, human CRP40/mortalin is likely modulated by DA activity and may act as a molecular chaperone by protecting the cell from excess catecholamine and catecholamine metabolite activity in regions of the brain associated with psychosis. Furthermore, the results in this study suggest that ASDs have a significant influence on the CRP40 mRNA levels in patients with schizophrenia. In addition, future work should be aimed at examining whether reduced CRP40/ mortalin expression is either a cause or downstream effect of DA dysfunction within the PFC and related brain regions. Role of funding source Funding for this study was provided by the Canadian Institutes of Health Research (CIHR). The CIHR had no further role in study design, in the collection, analysis and interpretation of data, in the writing of the report, or in the decision to submit the paper for publication. Contributors All authors have made significant scientific contributions to this manuscript. Joseph P. Gabriele contributed to the conceptualization and
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implementation of the study, securing funding and contributed to the writing of the manuscript. Giuseppe F. Pontoriero contributed to the implementation of the study and to the writing of the manuscript. Nancy Thomas and Geetha Mahadevan conducted the real-time PCR analyses. Mark A. Ferro conducted the statistical analyses and contributed to the editing of the manuscript. Duncan J. MacCrimmon provided insight to our working hypothesis. Zdenek B. Pristupa designed all PCR primers and provided significant consultation. Ram K. Mishra contributed to the conceptualization of the study, securing funding and contributed to the editing of the manuscript. All authors have approved the final manuscript. Conflict of interest Joseph P. Gabriele, Zdenek B. Pristupa, and Ram K. Mishra jointly hold a patent on mortalin and its alternative gene product, CRP40. All other authors report no competing interests. Acknowledgements This work was funded by the Canadian Institutes of Health Research. Postmortem brain tissue was generously donated by the Stanley Medical Research Institute's brain collection courtesy of Drs. Michael B. Knable, E. Fuller Torrey, Maree J. Webster, and Robert H. Yolken.
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