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Epigenetic dysregulation of TGFβ signaling and other inflammatory genes in schizophrenia and bipolar disorder
neurology, psychiatry and brain research 20 (2014) 3–27
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/npbr
Epigenetic dysregulation of TGFb signaling and other inflammatory genes in schizophrenia and bipolar disorder Epigenetic dysregulation of TGFb signaling and other inflammatory genes in schizophrenia and bipolar disorder H.M. Abdolmaleky *, S. Thiagalingam Boston University School of Medicine, Boston, MA, USA *Corresponding author. Epidemiologic and genetic studies indicate that schizophrenia (SCZ) and bipolar disorder (BD) are complex and multi-factorial illnesses in origin, influenced by both genetic and environmental factors (e.g. birth in winter or early spring, and large cities, paternal age, famine, nutritional and perinatal complications) that may act through epigenetic mechanisms. Because of the poor success rate with >1000 genetic studies as well as large scale genome-wide association studies (GWAS) in finding the molecular basis in major mental diseases, efforts have been made by others and us in examining the roles of epigenetic aberrations in SCZ and BD pathogenesis. Our initial studies provided compelling evidence to support that the brain specific regulatory DNA methylation changes of genes such as RELN, MBCOMT, HTR2A and 5-HTT play important roles in SCZ and BD pathogenesis and similar alterations were also found in DNA from saliva of the patients.1 In order to generate a more comprehensive picture of the pathology of the diseases in SCZ & BD, we performed gene expression profiling using the Affymetrix U133 Plus 2.0 Human Transcriptome array for post-mortem brain samples from SCZ and BD patients vs. controls (each group 10) to identify driver genes in SCZ and BD pathogenesis. We identified >60 genes relate to inflammatory, cell-mediated immune response and inflammatory diseases which were up (TGFB2, ITGB5, FOXO3, MTOR, PERP, NQO1, AUTS2, BCL9 and MAP3K5) or down regulated (e.g. ITGB3, ITGAX, TNFRSF18, IFNA17, HSP90B1, KLF2, CD6, CD8B, CD9 and RUNX1) in SCZ and BD. Subsequent Ingenuity Pathway Analysis (IPA) identified TGFb2 as a highly likely gene that regulate the expression of other affected genes in SCZ and BD via the interconnected ERK, MAPK, PI3K and PKA signaling pathways. The top dysregulated genes linked to TGFb signaling pathway were: SMAD1, BMP7 and Wnt10A among others. Overlay of the affected pathways with the neuronal signaling pathways using IPA revealed that, these interlinked pathways are connected to dopamine, glutamate, GABA, glucocorticoid receptors, CREB, reelin and G protein coupled receptor signaling known to be affected in SCZ and BD. In confirmatory qRT-PCR analysis we found that TGFb2 expression is increased by 30% in SCZ [and to lesser extent in BD] vs. controls, in post-mortem brain samples donated by the Stanley Medical Research Institute (each group 35 samples, p = 0.02; two tailed t test). Subsequent DNA methylation profiling of selected samples used for the expression analysis, 0941-9500/$ – see front matter
by the Illumina 27k (for 12 samples) and 450k DNA methylation arrays (for another 12 samples) found 40% differences in the TGFb2 promoter DNA methylation levels in SCZ and BD vs. controls. Furthermore, almost half of the genes which correlated directly or inversely with the TGFb2 expression showed an aberrant promoter DNA methylation in SCZ and/or BD. Overall, our array data support that an epigenetically overactivated TGFb2 signaling along with an increased BMP7/ SMAD1 expression and epigenetic down-regulation of Wnt10A is linked to SCZ and BD pathogenesis (Abdolmaleky and Thiagalingam, unpublished). We have previously shown that an activated TGFb-Smad signaling pathway silences several genes by altering the binding capacity of DNMT1 to CpGs in gene promoters in breast cancer.2 It is likely that the increased TGFb2 expression in SCZ and BD could also have similar effects in the brain of affected individuals. The TGFb super family of cytokines is involved in the regulation of cellular processes, including cell division, differentiation, motility, adhesion and death. TGFb and BMPs signals by binding to the membrane receptors leading to transphosphorylation Smad1, Smad2, Smad3 and Smad5/8, which along with Smad4 translocate to the nucleus and form transcriptional complexes with DNA binding factors and co-activators/co-repressors. An increase in TGFb signaling and decrease in Wnt signaling can promote adult neuronal differentiation and migration, and inappropriate insertion into the neuronal network in SCZ.3 Consistent with our array data, a recent study reported an increase in the production of TGFb in psychotic patients and in First Episode Psychosis as well as in relapse phase of SCZ indicating that TGFb could be a marker for psychosis.4 A new meta-analysis also concluded that TGFb appears to be a state marker in SCZ. From the therapeutic point of view it is worthy to note that lithium increases the activity of cAMP/PKA signaling and inhibits Smad3/4-dependent TGFb signaling in neurons.5 Thus, antipsychotic drugs that block DRD2 receptor and increase cAMP level may have the same effect.
references 1. Abdolmaleky HM, Thiagalingam S. Can schizophrenia epigenome provide clues to the molecular basis of pathogenesis? Epigenomics 2011;3(6):679–83. 2. Papageorgis P, Lambert AW, Ozturk S, Gao F, Pan H, Manne U, et al. Smad signaling is required to maintain epigenetic silencing during breast cancer progression. Cancer Res 2010;70 (3):968–78. 3. Kalkman HO. Altered growth factor signaling pathways as the basis of aberrant stem cell maturation in schizophrenia. Pharmacol Ther 2009;121(1):115–22.
4
neurology, psychiatry and brain research 20 (2014) 3–27
4. Borovcanin M, Jovanovic I, Radosavljevic G, Djukic Dejanovic S, Bankovic D, Arsenijevic N, et al. Elevated serum level of type-2 cytokine and low IL-17 in first episode psychosis and schizophrenia in relapse. J Psychiatr Res 2012;46(11): 1421–6. 5. Liang MH, Wendland JR, Chuang DM. Lithium inhibits Smad3/ 4 transactivation via increased CREB activity induced by enhanced PKA and AKT signaling. Mol Cell Neurosci 2008;37 (3):440–53.
http://dx.doi.org/10.1016/j.npbr.2014.01.135 CSF outflow along spinal nerves – A neuroradiological documentation K. Bechter a,*, B. Schmitz b a
BKH Guenzburg/Ulm University, Department of Psychiatry II, Ludwig-Heilmeyer-Straße 2, D-89312 Guenzburg, Germany b BKH Guenzburg/Ulm University, Department of Neuroradiology, Ludwig-Heilmeyer-Straße 2, D-89312 Guenzburg, Germany *Corresponding author.
Quincke in 1872 demonstrated that CSF is flowing out from the subarachnoid spaces along spinal nerves into peripheral tissues. Outflow through the cribriform plate near olfactory nerves was extensively investigated whereas poorly at other sites along brain nerves and spinal nerves. From clinical observations during experimental treatments of therapy resistant depression with CSF filtration, it was hypothesized that CSF may interact with nerves all along the peripheral CSF outflow pathway (PCOP), and also at the wind up of the PCOP at the nerves ends and even with peripheral tissues.1 We recently demonstrated that leukaemia cells followed the PCOP along lumbar nerves into the periphery presumably in between epineurium and perineurium reaching even at subcutaneous tissues.2 Thus PCOP associated pathogenetic mechanisms via CSF signalling to respective tissues, although not proven yet, have the potential to better understand several unsolved pathogenetic aspects in neuroinflammatory disorders and also in a subgroup of severe psychiatric disorders, associated with low level neuroinflammation (LLNI). Indeed about 70% of patients with therapy resistant depression or schizophrenic spectrum disorders demonstrate some CSF abnormality, such findings compatible with the mild encephalitis hypothesis (compare3–6). In general in neuroinflammatory disorders, including fibromyalgic syndromes, one would expect the involvement of PCOP associated pathomechanisms. Here, we demonstrate in one human subject supposed to lumbar myelography for diagnostic reasons, that CSF was spontaneously flowing from the lumbar subarachnoid spaces down all neighbouring lumbar nerves into the periphery, making a distance of 50.8 mm in 30 min. These findings support the general plausibility of the PCOP hypothesis. For understanding of pathogenetic details much more research is required.
references
1. Bechter K. The peripheral cerebrospinal fluid outflow pathway – physiology and pathophysiology of CSF recirculation: a review and hypothesis. Neurol Psychiatry Brain Res 2011;17(3):51–66. 2. Schmitt M, Neubauer A, Greiner J, Xu X, Barth TF, Bechter K. Spreading of acute myeloid leukemia cells by trafficking along the peripheral outflow pathway of cerebrospinal fluid. Anticancer Res 2011;31(6):2343–5.
3. Maxeiner HG, Rojewski MT, Schmitt A, Tumani H, Bechter K, Schmitt M. Flow cytometric analysis of T cell subsets in paired samples of cerebrospinal fluid and peripheral blood from patients with neurological and psychiatric disorders. Brain Behav Immun 2009;23(1):134–42. 4. Bechter K, Reiber H, Herzog S, Fuchs D, Tumani H, Maxeiner HG. Cerebrospinal fluid analysis in affective and schizophrenic spectrum disorders: identification of subgroups with immune responses and blood-CSF barrier dysfunction. J Psychiatr Res 2010;44(5):321–30. 5. Kuehne LK, Reiber H, Bechter K, Hagberg L, Fuchs D. Cerebrospinal fluid neopterin is brain-derived and not associated with blood CSF barrier dysfunction. Acta Neurologica Scandinaciva 2013. 6. Bechter K. Updating the mild encephalitis hypothesis of schizophrenia. Prog Neuropsychopharmacol Biol Psychiatry 2013;42:71–91.
http://dx.doi.org/10.1016/j.npbr.2014.01.136 Brain-wide glymphatic waste drainage characterized by PET-MRI H. Benveniste a,b,*, M. Budassi c, S. David Smith a, M. Yu a, H. Lee a,b, M. Nedergaard c, P. Vaska d a
Departments of Anesthesiology and Radiology, Stony Brook University, NY, United States b Department of Physics, Brookhaven National Laboratory, Upton, NY, United States c Center for Translational Neuroscience, University of Rochester Medical Center, NY, United States d Department of Biomedical Engineering, Stony Brook University, NY, United States *Corresponding author. Introduction: According to theory, cerebrospinal fluid (CSF) is produced by the choroid plexus, then flows through the ventricles to the basal cisterns, subarachnoid space and into brain parenchyma via peri-arterial spaces, where it mixes with interstitial fluid (ISF) and eventually exits into large cerebral veins. CSF-ISF exchange is thought to act as a ‘sink’ for clearance of waste products in the brain. Recently, this concept was revisited when experiments revealed that CSF exchanges rapidly with ISF along a defined macroscopic pathway termed the ‘glymphatic’ pathway comprising a para-arterial influx route, a para-venous clearance route and an intracellular transastrocytic path, which connects the two para-vascular conduits.1 We recently developed a novel approach to map and quantify glymphatic pathway mass transport in the rodent brain using paramagnetic contrast agents in combination with MRI.2 We demonstrated that the small molecular weight (MW) molecule (Gd-DTPA, 938 Da) was able to access all components of the glymphatic pathway in the rodent brain whereas the larger contrast molecule GadoSpin (MW 200,000 Da) was restricted and limited to the most proximal part. This was in agreement with optical imaging studies showing that larger MW compounds are restricted from crossing the 20 nm gaps between overlapping astrocytic foot processes and therefore cannot access parenchymal ISF.1 However, when compared to small MW fluorescently tagged tracers which distributes rapidly within the brain-wide glymphatic network (30– 40 min),1 glymphatic transport of Gd-DTPA appears to be slower and does not reach the most central parts of the rodent brain within a circulation time of 2–3 h.2 We speculated that the slower transport of Gd-DTPA is due to the relative insensitivity (relative to fluorescence-based imaging) of T1weighted MRI to detect signal changes induced by low
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/npbr
Epigenetic dysregulation of TGFb signaling and other inflammatory genes in schizophrenia and bipolar disorder Epigenetic dysregulation of TGFb signaling and other inflammatory genes in schizophrenia and bipolar disorder H.M. Abdolmaleky *, S. Thiagalingam Boston University School of Medicine, Boston, MA, USA *Corresponding author. Epidemiologic and genetic studies indicate that schizophrenia (SCZ) and bipolar disorder (BD) are complex and multi-factorial illnesses in origin, influenced by both genetic and environmental factors (e.g. birth in winter or early spring, and large cities, paternal age, famine, nutritional and perinatal complications) that may act through epigenetic mechanisms. Because of the poor success rate with >1000 genetic studies as well as large scale genome-wide association studies (GWAS) in finding the molecular basis in major mental diseases, efforts have been made by others and us in examining the roles of epigenetic aberrations in SCZ and BD pathogenesis. Our initial studies provided compelling evidence to support that the brain specific regulatory DNA methylation changes of genes such as RELN, MBCOMT, HTR2A and 5-HTT play important roles in SCZ and BD pathogenesis and similar alterations were also found in DNA from saliva of the patients.1 In order to generate a more comprehensive picture of the pathology of the diseases in SCZ & BD, we performed gene expression profiling using the Affymetrix U133 Plus 2.0 Human Transcriptome array for post-mortem brain samples from SCZ and BD patients vs. controls (each group 10) to identify driver genes in SCZ and BD pathogenesis. We identified >60 genes relate to inflammatory, cell-mediated immune response and inflammatory diseases which were up (TGFB2, ITGB5, FOXO3, MTOR, PERP, NQO1, AUTS2, BCL9 and MAP3K5) or down regulated (e.g. ITGB3, ITGAX, TNFRSF18, IFNA17, HSP90B1, KLF2, CD6, CD8B, CD9 and RUNX1) in SCZ and BD. Subsequent Ingenuity Pathway Analysis (IPA) identified TGFb2 as a highly likely gene that regulate the expression of other affected genes in SCZ and BD via the interconnected ERK, MAPK, PI3K and PKA signaling pathways. The top dysregulated genes linked to TGFb signaling pathway were: SMAD1, BMP7 and Wnt10A among others. Overlay of the affected pathways with the neuronal signaling pathways using IPA revealed that, these interlinked pathways are connected to dopamine, glutamate, GABA, glucocorticoid receptors, CREB, reelin and G protein coupled receptor signaling known to be affected in SCZ and BD. In confirmatory qRT-PCR analysis we found that TGFb2 expression is increased by 30% in SCZ [and to lesser extent in BD] vs. controls, in post-mortem brain samples donated by the Stanley Medical Research Institute (each group 35 samples, p = 0.02; two tailed t test). Subsequent DNA methylation profiling of selected samples used for the expression analysis, 0941-9500/$ – see front matter
by the Illumina 27k (for 12 samples) and 450k DNA methylation arrays (for another 12 samples) found 40% differences in the TGFb2 promoter DNA methylation levels in SCZ and BD vs. controls. Furthermore, almost half of the genes which correlated directly or inversely with the TGFb2 expression showed an aberrant promoter DNA methylation in SCZ and/or BD. Overall, our array data support that an epigenetically overactivated TGFb2 signaling along with an increased BMP7/ SMAD1 expression and epigenetic down-regulation of Wnt10A is linked to SCZ and BD pathogenesis (Abdolmaleky and Thiagalingam, unpublished). We have previously shown that an activated TGFb-Smad signaling pathway silences several genes by altering the binding capacity of DNMT1 to CpGs in gene promoters in breast cancer.2 It is likely that the increased TGFb2 expression in SCZ and BD could also have similar effects in the brain of affected individuals. The TGFb super family of cytokines is involved in the regulation of cellular processes, including cell division, differentiation, motility, adhesion and death. TGFb and BMPs signals by binding to the membrane receptors leading to transphosphorylation Smad1, Smad2, Smad3 and Smad5/8, which along with Smad4 translocate to the nucleus and form transcriptional complexes with DNA binding factors and co-activators/co-repressors. An increase in TGFb signaling and decrease in Wnt signaling can promote adult neuronal differentiation and migration, and inappropriate insertion into the neuronal network in SCZ.3 Consistent with our array data, a recent study reported an increase in the production of TGFb in psychotic patients and in First Episode Psychosis as well as in relapse phase of SCZ indicating that TGFb could be a marker for psychosis.4 A new meta-analysis also concluded that TGFb appears to be a state marker in SCZ. From the therapeutic point of view it is worthy to note that lithium increases the activity of cAMP/PKA signaling and inhibits Smad3/4-dependent TGFb signaling in neurons.5 Thus, antipsychotic drugs that block DRD2 receptor and increase cAMP level may have the same effect.
references 1. Abdolmaleky HM, Thiagalingam S. Can schizophrenia epigenome provide clues to the molecular basis of pathogenesis? Epigenomics 2011;3(6):679–83. 2. Papageorgis P, Lambert AW, Ozturk S, Gao F, Pan H, Manne U, et al. Smad signaling is required to maintain epigenetic silencing during breast cancer progression. Cancer Res 2010;70 (3):968–78. 3. Kalkman HO. Altered growth factor signaling pathways as the basis of aberrant stem cell maturation in schizophrenia. Pharmacol Ther 2009;121(1):115–22.
4
neurology, psychiatry and brain research 20 (2014) 3–27
4. Borovcanin M, Jovanovic I, Radosavljevic G, Djukic Dejanovic S, Bankovic D, Arsenijevic N, et al. Elevated serum level of type-2 cytokine and low IL-17 in first episode psychosis and schizophrenia in relapse. J Psychiatr Res 2012;46(11): 1421–6. 5. Liang MH, Wendland JR, Chuang DM. Lithium inhibits Smad3/ 4 transactivation via increased CREB activity induced by enhanced PKA and AKT signaling. Mol Cell Neurosci 2008;37 (3):440–53.
http://dx.doi.org/10.1016/j.npbr.2014.01.135 CSF outflow along spinal nerves – A neuroradiological documentation K. Bechter a,*, B. Schmitz b a
BKH Guenzburg/Ulm University, Department of Psychiatry II, Ludwig-Heilmeyer-Straße 2, D-89312 Guenzburg, Germany b BKH Guenzburg/Ulm University, Department of Neuroradiology, Ludwig-Heilmeyer-Straße 2, D-89312 Guenzburg, Germany *Corresponding author.
Quincke in 1872 demonstrated that CSF is flowing out from the subarachnoid spaces along spinal nerves into peripheral tissues. Outflow through the cribriform plate near olfactory nerves was extensively investigated whereas poorly at other sites along brain nerves and spinal nerves. From clinical observations during experimental treatments of therapy resistant depression with CSF filtration, it was hypothesized that CSF may interact with nerves all along the peripheral CSF outflow pathway (PCOP), and also at the wind up of the PCOP at the nerves ends and even with peripheral tissues.1 We recently demonstrated that leukaemia cells followed the PCOP along lumbar nerves into the periphery presumably in between epineurium and perineurium reaching even at subcutaneous tissues.2 Thus PCOP associated pathogenetic mechanisms via CSF signalling to respective tissues, although not proven yet, have the potential to better understand several unsolved pathogenetic aspects in neuroinflammatory disorders and also in a subgroup of severe psychiatric disorders, associated with low level neuroinflammation (LLNI). Indeed about 70% of patients with therapy resistant depression or schizophrenic spectrum disorders demonstrate some CSF abnormality, such findings compatible with the mild encephalitis hypothesis (compare3–6). In general in neuroinflammatory disorders, including fibromyalgic syndromes, one would expect the involvement of PCOP associated pathomechanisms. Here, we demonstrate in one human subject supposed to lumbar myelography for diagnostic reasons, that CSF was spontaneously flowing from the lumbar subarachnoid spaces down all neighbouring lumbar nerves into the periphery, making a distance of 50.8 mm in 30 min. These findings support the general plausibility of the PCOP hypothesis. For understanding of pathogenetic details much more research is required.
references
1. Bechter K. The peripheral cerebrospinal fluid outflow pathway – physiology and pathophysiology of CSF recirculation: a review and hypothesis. Neurol Psychiatry Brain Res 2011;17(3):51–66. 2. Schmitt M, Neubauer A, Greiner J, Xu X, Barth TF, Bechter K. Spreading of acute myeloid leukemia cells by trafficking along the peripheral outflow pathway of cerebrospinal fluid. Anticancer Res 2011;31(6):2343–5.
3. Maxeiner HG, Rojewski MT, Schmitt A, Tumani H, Bechter K, Schmitt M. Flow cytometric analysis of T cell subsets in paired samples of cerebrospinal fluid and peripheral blood from patients with neurological and psychiatric disorders. Brain Behav Immun 2009;23(1):134–42. 4. Bechter K, Reiber H, Herzog S, Fuchs D, Tumani H, Maxeiner HG. Cerebrospinal fluid analysis in affective and schizophrenic spectrum disorders: identification of subgroups with immune responses and blood-CSF barrier dysfunction. J Psychiatr Res 2010;44(5):321–30. 5. Kuehne LK, Reiber H, Bechter K, Hagberg L, Fuchs D. Cerebrospinal fluid neopterin is brain-derived and not associated with blood CSF barrier dysfunction. Acta Neurologica Scandinaciva 2013. 6. Bechter K. Updating the mild encephalitis hypothesis of schizophrenia. Prog Neuropsychopharmacol Biol Psychiatry 2013;42:71–91.
http://dx.doi.org/10.1016/j.npbr.2014.01.136 Brain-wide glymphatic waste drainage characterized by PET-MRI H. Benveniste a,b,*, M. Budassi c, S. David Smith a, M. Yu a, H. Lee a,b, M. Nedergaard c, P. Vaska d a
Departments of Anesthesiology and Radiology, Stony Brook University, NY, United States b Department of Physics, Brookhaven National Laboratory, Upton, NY, United States c Center for Translational Neuroscience, University of Rochester Medical Center, NY, United States d Department of Biomedical Engineering, Stony Brook University, NY, United States *Corresponding author. Introduction: According to theory, cerebrospinal fluid (CSF) is produced by the choroid plexus, then flows through the ventricles to the basal cisterns, subarachnoid space and into brain parenchyma via peri-arterial spaces, where it mixes with interstitial fluid (ISF) and eventually exits into large cerebral veins. CSF-ISF exchange is thought to act as a ‘sink’ for clearance of waste products in the brain. Recently, this concept was revisited when experiments revealed that CSF exchanges rapidly with ISF along a defined macroscopic pathway termed the ‘glymphatic’ pathway comprising a para-arterial influx route, a para-venous clearance route and an intracellular transastrocytic path, which connects the two para-vascular conduits.1 We recently developed a novel approach to map and quantify glymphatic pathway mass transport in the rodent brain using paramagnetic contrast agents in combination with MRI.2 We demonstrated that the small molecular weight (MW) molecule (Gd-DTPA, 938 Da) was able to access all components of the glymphatic pathway in the rodent brain whereas the larger contrast molecule GadoSpin (MW 200,000 Da) was restricted and limited to the most proximal part. This was in agreement with optical imaging studies showing that larger MW compounds are restricted from crossing the 20 nm gaps between overlapping astrocytic foot processes and therefore cannot access parenchymal ISF.1 However, when compared to small MW fluorescently tagged tracers which distributes rapidly within the brain-wide glymphatic network (30– 40 min),1 glymphatic transport of Gd-DTPA appears to be slower and does not reach the most central parts of the rodent brain within a circulation time of 2–3 h.2 We speculated that the slower transport of Gd-DTPA is due to the relative insensitivity (relative to fluorescence-based imaging) of T1weighted MRI to detect signal changes induced by low