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Dysregulation of miR-34a links neuronal development to genetic risk factors for bipolar disorder.

Bavamian S, Mellios N, Lalonde J, Fass DM, Wang J, Sheridan SD, Madison JM, Zhou F, Rueckert EH, Barker D, Perlis RH, Sur M, Haggarty SJ - Mol. Psychiatry (2015)

Bottom Line: Given their prominent role in brain function and disease, we hypothesized that microRNAs (miRNAs) might be of importance for BD.Of the predicted miR-34a targets, we validated the BD risk genes ankyrin-3 (ANK3) and voltage-dependent L-type calcium channel subunit beta-3 (CACNB3) as direct miR-34a targets.Using human iPSC-derived neuronal progenitor cells, we further show that enhancement of miR-34a expression impairs neuronal differentiation, expression of synaptic proteins and neuronal morphology, whereas reducing endogenous miR-34a expression enhances dendritic elaboration.

View Article: PubMed Central - PubMed

Affiliation: 1] Chemical Neurobiology Laboratory, Center for Human Genetics Research, Massachusetts General Hospital, Boston, MA, USA [2] Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, USA.

ABSTRACT
Bipolar disorder (BD) is a heritable neuropsychiatric disorder with largely unknown pathogenesis. Given their prominent role in brain function and disease, we hypothesized that microRNAs (miRNAs) might be of importance for BD. Here we show that levels of miR-34a, which is predicted to target multiple genes implicated as genetic risk factors for BD, are increased in postmortem cerebellar tissue from BD patients, as well as in BD patient-derived neuronal cultures generated by reprogramming of human fibroblasts into induced neurons or into induced pluripotent stem cells (iPSCs) subsequently differentiated into neurons. Of the predicted miR-34a targets, we validated the BD risk genes ankyrin-3 (ANK3) and voltage-dependent L-type calcium channel subunit beta-3 (CACNB3) as direct miR-34a targets. Using human iPSC-derived neuronal progenitor cells, we further show that enhancement of miR-34a expression impairs neuronal differentiation, expression of synaptic proteins and neuronal morphology, whereas reducing endogenous miR-34a expression enhances dendritic elaboration. Taken together, we propose that miR-34a serves as a critical link between multiple etiological factors for BD and its pathogenesis through the regulation of a molecular network essential for neuronal development and synaptogenesis.

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miR-34a expression is increased in the cerebellum of BD patients and in patient-derived cellular models of BDA) Mean ± SEM miR-34a levels depicted as fold change relative to controls. N = 34 control and 29 BD cerebellar samples. **p<0.01 compared to control conditions, based on a two-tailed, one-sample t-test. B) Mean ± SEM miR-34a levels in cerebellum of controls (N = 34) and BD patients segregated into medicated (N = 19) and non-medicated (N = 10). **p<0.01 based on ANOVA with Tukey's Multiple Comparison Test. C) Schematic of the neuronal differentiation process from iPSCs to eight weeks differentiated neurons. D) Immunostaining of Nestin in hNPCs and MAP2 in eight weeks differentiated neurons, scale bar = 50 µm. E) Graphs showing relative miR-34a expression in hNPCs and differentiated neurons of a healthy control individual (light grey circles) and a BD patient (dark grey circles). Data are shown as fold change relative to miR-34a level at hNPC stage. Stars over data depict statistical significance based on a two-tailed t-test (*p<0.05, **p<0.01, *p<0.001). F) Schematic of induced neurons (iNs) generation. G) miR-34a expression in iNs from three control and five BD subjects. The graph shows mean ± SEM miR-34a expression relative to control iNs. miR-34a expression was normalized to U6 and RNU44 expression, which control for the total amount of RNA present in each sample. *p<0.05 compared to control conditions, based on a two-tailed, one-sample t-test.
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Figure 1: miR-34a expression is increased in the cerebellum of BD patients and in patient-derived cellular models of BDA) Mean ± SEM miR-34a levels depicted as fold change relative to controls. N = 34 control and 29 BD cerebellar samples. **p<0.01 compared to control conditions, based on a two-tailed, one-sample t-test. B) Mean ± SEM miR-34a levels in cerebellum of controls (N = 34) and BD patients segregated into medicated (N = 19) and non-medicated (N = 10). **p<0.01 based on ANOVA with Tukey's Multiple Comparison Test. C) Schematic of the neuronal differentiation process from iPSCs to eight weeks differentiated neurons. D) Immunostaining of Nestin in hNPCs and MAP2 in eight weeks differentiated neurons, scale bar = 50 µm. E) Graphs showing relative miR-34a expression in hNPCs and differentiated neurons of a healthy control individual (light grey circles) and a BD patient (dark grey circles). Data are shown as fold change relative to miR-34a level at hNPC stage. Stars over data depict statistical significance based on a two-tailed t-test (*p<0.05, **p<0.01, *p<0.001). F) Schematic of induced neurons (iNs) generation. G) miR-34a expression in iNs from three control and five BD subjects. The graph shows mean ± SEM miR-34a expression relative to control iNs. miR-34a expression was normalized to U6 and RNU44 expression, which control for the total amount of RNA present in each sample. *p<0.05 compared to control conditions, based on a two-tailed, one-sample t-test.

Mentions: Given the known influences of mood stabilizers on miR-34a levels and its potential role in neuronal maturation and plasticity, we decided to investigate the link between miR-34a and BD. Interestingly, two recent studies43, 44, as well as unpublished data (S.J.H, manuscript in preparation), reported decreased expression of the two putative miR-34a targets ANK3 and CACNA1C in cerebellum of BD individuals. Based on these findings and emerging literature linking the cerebellum with BD43–52, we hypothesized that miR-34a could be dysregulated in the cerebellum of BD patients. To test this hypothesis, we measured miR-34a expression in postmortem human brain samples from cerebellum. We observed a significant increase in miR-34a levels in the cerebellum of BD patients (N = 29) relative to healthy controls (N = 34) (Figure 1A), whereas no differences were seen in prefrontal cortex (PFC) or anterior cingulate cortex (Figure S1A and S1B). In contrast to this BD and brain region-specific difference in miR-34a expression, based on the recent identification by GWAS of a new susceptibility locus within the first intron of ODZ4 where miR-708 is encoded16, which is predicted to target both ODZ4 itself and CACNA1C, the latter of which has shown multiple association with neuropsychiatric disorders53, we also measured miR-708 in the same cerebellum samples from BD individuals (Figure S1C), but did not see any differences compared to the healthy controls. Therefore, we did not pursue the investigation of miR-708 targets and focused our study on miR-34a.


Dysregulation of miR-34a links neuronal development to genetic risk factors for bipolar disorder.

Bavamian S, Mellios N, Lalonde J, Fass DM, Wang J, Sheridan SD, Madison JM, Zhou F, Rueckert EH, Barker D, Perlis RH, Sur M, Haggarty SJ - Mol. Psychiatry (2015)

miR-34a expression is increased in the cerebellum of BD patients and in patient-derived cellular models of BDA) Mean ± SEM miR-34a levels depicted as fold change relative to controls. N = 34 control and 29 BD cerebellar samples. **p<0.01 compared to control conditions, based on a two-tailed, one-sample t-test. B) Mean ± SEM miR-34a levels in cerebellum of controls (N = 34) and BD patients segregated into medicated (N = 19) and non-medicated (N = 10). **p<0.01 based on ANOVA with Tukey's Multiple Comparison Test. C) Schematic of the neuronal differentiation process from iPSCs to eight weeks differentiated neurons. D) Immunostaining of Nestin in hNPCs and MAP2 in eight weeks differentiated neurons, scale bar = 50 µm. E) Graphs showing relative miR-34a expression in hNPCs and differentiated neurons of a healthy control individual (light grey circles) and a BD patient (dark grey circles). Data are shown as fold change relative to miR-34a level at hNPC stage. Stars over data depict statistical significance based on a two-tailed t-test (*p<0.05, **p<0.01, *p<0.001). F) Schematic of induced neurons (iNs) generation. G) miR-34a expression in iNs from three control and five BD subjects. The graph shows mean ± SEM miR-34a expression relative to control iNs. miR-34a expression was normalized to U6 and RNU44 expression, which control for the total amount of RNA present in each sample. *p<0.05 compared to control conditions, based on a two-tailed, one-sample t-test.
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Figure 1: miR-34a expression is increased in the cerebellum of BD patients and in patient-derived cellular models of BDA) Mean ± SEM miR-34a levels depicted as fold change relative to controls. N = 34 control and 29 BD cerebellar samples. **p<0.01 compared to control conditions, based on a two-tailed, one-sample t-test. B) Mean ± SEM miR-34a levels in cerebellum of controls (N = 34) and BD patients segregated into medicated (N = 19) and non-medicated (N = 10). **p<0.01 based on ANOVA with Tukey's Multiple Comparison Test. C) Schematic of the neuronal differentiation process from iPSCs to eight weeks differentiated neurons. D) Immunostaining of Nestin in hNPCs and MAP2 in eight weeks differentiated neurons, scale bar = 50 µm. E) Graphs showing relative miR-34a expression in hNPCs and differentiated neurons of a healthy control individual (light grey circles) and a BD patient (dark grey circles). Data are shown as fold change relative to miR-34a level at hNPC stage. Stars over data depict statistical significance based on a two-tailed t-test (*p<0.05, **p<0.01, *p<0.001). F) Schematic of induced neurons (iNs) generation. G) miR-34a expression in iNs from three control and five BD subjects. The graph shows mean ± SEM miR-34a expression relative to control iNs. miR-34a expression was normalized to U6 and RNU44 expression, which control for the total amount of RNA present in each sample. *p<0.05 compared to control conditions, based on a two-tailed, one-sample t-test.
Mentions: Given the known influences of mood stabilizers on miR-34a levels and its potential role in neuronal maturation and plasticity, we decided to investigate the link between miR-34a and BD. Interestingly, two recent studies43, 44, as well as unpublished data (S.J.H, manuscript in preparation), reported decreased expression of the two putative miR-34a targets ANK3 and CACNA1C in cerebellum of BD individuals. Based on these findings and emerging literature linking the cerebellum with BD43–52, we hypothesized that miR-34a could be dysregulated in the cerebellum of BD patients. To test this hypothesis, we measured miR-34a expression in postmortem human brain samples from cerebellum. We observed a significant increase in miR-34a levels in the cerebellum of BD patients (N = 29) relative to healthy controls (N = 34) (Figure 1A), whereas no differences were seen in prefrontal cortex (PFC) or anterior cingulate cortex (Figure S1A and S1B). In contrast to this BD and brain region-specific difference in miR-34a expression, based on the recent identification by GWAS of a new susceptibility locus within the first intron of ODZ4 where miR-708 is encoded16, which is predicted to target both ODZ4 itself and CACNA1C, the latter of which has shown multiple association with neuropsychiatric disorders53, we also measured miR-708 in the same cerebellum samples from BD individuals (Figure S1C), but did not see any differences compared to the healthy controls. Therefore, we did not pursue the investigation of miR-708 targets and focused our study on miR-34a.

Bottom Line: Given their prominent role in brain function and disease, we hypothesized that microRNAs (miRNAs) might be of importance for BD.Of the predicted miR-34a targets, we validated the BD risk genes ankyrin-3 (ANK3) and voltage-dependent L-type calcium channel subunit beta-3 (CACNB3) as direct miR-34a targets.Using human iPSC-derived neuronal progenitor cells, we further show that enhancement of miR-34a expression impairs neuronal differentiation, expression of synaptic proteins and neuronal morphology, whereas reducing endogenous miR-34a expression enhances dendritic elaboration.

View Article: PubMed Central - PubMed

Affiliation: 1] Chemical Neurobiology Laboratory, Center for Human Genetics Research, Massachusetts General Hospital, Boston, MA, USA [2] Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, USA.

ABSTRACT
Bipolar disorder (BD) is a heritable neuropsychiatric disorder with largely unknown pathogenesis. Given their prominent role in brain function and disease, we hypothesized that microRNAs (miRNAs) might be of importance for BD. Here we show that levels of miR-34a, which is predicted to target multiple genes implicated as genetic risk factors for BD, are increased in postmortem cerebellar tissue from BD patients, as well as in BD patient-derived neuronal cultures generated by reprogramming of human fibroblasts into induced neurons or into induced pluripotent stem cells (iPSCs) subsequently differentiated into neurons. Of the predicted miR-34a targets, we validated the BD risk genes ankyrin-3 (ANK3) and voltage-dependent L-type calcium channel subunit beta-3 (CACNB3) as direct miR-34a targets. Using human iPSC-derived neuronal progenitor cells, we further show that enhancement of miR-34a expression impairs neuronal differentiation, expression of synaptic proteins and neuronal morphology, whereas reducing endogenous miR-34a expression enhances dendritic elaboration. Taken together, we propose that miR-34a serves as a critical link between multiple etiological factors for BD and its pathogenesis through the regulation of a molecular network essential for neuronal development and synaptogenesis.

Show MeSH
Related in: MedlinePlus