Bhaskar Roy

Nanotopography directs mesenchymal stem cells to osteoblast lineage through regulation of microRNA-SMAD-BMP-2 circuit.

The aim of this study was to investigate if chemically produced nanotopography on titanium (Ti) surface induces osteoblast differentiation of cultured human bone marrow mesenchymal stem cells (hMSCs) by regulating the expression of microRNAs (miRs). It was demonstrated that Ti with nanotopography induces osteoblast differentiation of hMSCs as evidenced by upregulation of osteoblast specific markers compared with untreated (control) Ti at day 4. At this time‐point, miR‐sequencing analysis revealed that 20 miRs were upregulated (>twofold) while 20 miRs were downregulated (>threefold) in hMSCs grown on Ti with nanotopography compared with control Ti. Three miRs, namely miR‐4448, ‐4708, and ‐4773, which were significantly downregulated (>fivefold) by Ti with nanotopography affect osteoblast differentiation of hMSCs. These miRs directly target SMAD1 and SMAD4, both key transducers of the bone morphogenetic protein 2 (BMP‐2) osteogenic signal, which were upregulated by Ti with nanotopography. Overexpression of miR‐4448, ‐4708, and 4773 in MC3T3‐E1 pre‐osteoblasts noticeably inhibited gene and protein expression of SMAD1 and SMAD4 and therefore repressed the gene expression of key bone markers. Additionally, it was observed that the treatment with BMP‐2 displayed a higher osteogenic effect on MC3T3‐E1 cells grown on Ti with nanotopography compared with control Ti, suggesting that the BMP‐2 signaling pathway was more effective on this surface. Taken together, these results indicate that a complex regulatory network involving a miR‐SMAD‐BMP‐2 circuit governs the osteoblast differentiation induced by Ti with nanotopography. J. Cell. Physiol. 229: 1690–1696, 2014.

Identification of MicroRNA-124-3p as a Putative Epigenetic Signature of Major Depressive Disorder

Major depressive disorder (MDD) is predicted to be the second leading cause of global disease burden by 2030. A large number of MDD patients do not respond to the currently available medication because of its poorly understood etiology. Recently, studies of microRNAs (miRNAs), which act as a molecular switch of gene expression, have shown promise in identifying a molecular network that could provide significant clues to various psychiatric illnesses. Using an in vitro system, a rodent depression model, and a human postmortem brain, we investigated the role of a brain-enriched, neuron-specific miRNA, miR-124-3p, whose expression is highly dysregulated in stressed rodents, and identified a set of target genes involved in stress response and neural plasticity. We also found that miR-124-3p is epigenetically regulated and its interaction with the RNA-induced silencing complex (RISC) is compromised in MDD. Using blood serum, we found similar dysregulation of miR-124-3p in antidepressant-free MDD subjects. Altogether, our study demonstrates potential contribution of miR-124-3p in the pathophysiology of MDD and suggests that this miRNA may serve as a novel target for drug development and a biomarker for MDD pathogenesis.

Understanding epigenetic architecture of suicide neurobiology: A critical perspective

Current understanding of environmental cross-talk with genetic makeup is found to be mediated through an epigenetic interface which is associated with prominent reversible and heritable changes at gene expression level. Recent emergence of epigenetic modulation in shaping the genetic information has become a key regulatory factor in answering the underlying complexities associated with several mental disorders. A comprehensive understanding of the pertinent changes in the epigenetic makeup of suicide phenotype exhibits a characteristic signature with the possibility of using it as a biomarker to help predict the risk factors associated with suicide. Within the scope of this current review, the most sought after epigenetic changes of DNA methylation and histone modification are thoroughly scrutinized to understand their close functional association with the broad spectrum of suicide phenotype.

The Life Span Model of Suicide and Its Neurobiological Foundation

The very incomprehensibility of the suicidal act has been occupying the minds of researchers and health professionals for a long time. Several theories of suicide have been proposed since the beginning of the past century, and a myriad of neurobiological studies have been conducted over the past two decades in order to elucidate its pathophysiology. Both neurobiology and psychological theories tend to work in parallel lines that need behavioral and empirical data respectively, to confirm their hypotheses. In this review, we are proposing a “Life Span Model of Suicide” with an attempt to integrate the “Stress-Diathesis Model” and the “Interpersonal Model of Suicide” into a neurobiological narrative and support it by providing a thorough compilation of related genetic, epigenetic, and gene expression findings. This proposed model comprises three layers, forming the capability of suicide: genetic factors as the predisposing Diathesis on one side and Stress, characterized by epigenetic marks on the other side, and in between gene expression and gene function which are thought to be influenced by Diathesis and Stress components. The empirical evidence of this model is yet to be confirmed and further research, specifically epigenetic studies in particular, are needed to support the presence of a life-long, evolving capability of suicide and identify its neurobiological correlates.

Unfolded protein response and associated alterations in toll-like receptor expression and interaction in the hippocampus of restraint rats

Recent evidence suggests that the cellular response to stress often elicits the unfolded protein response (UPR), which has an active role in major depression in emotionally relevant regions of the brain, such as the hippocampus. Much of the UPR activity has been found to be coalesced with the pro-inflammatory environment of the depressed brain. Specifically, downstream transcriptions of pro-inflammatory cytokines and increased regulation of candidate inflammatory mediators, such as toll-like receptors (TLRs), are promoted by the UPR. The present study examined the hippocampus associated expression profile of Tlr genes and their interaction with the UPR chaperone GRP94 in stress-induced rodent model of depression (restraint stress model). Also, the expression status of UPR related genes was evaluated in hippocampus using the same model. mRNA and protein levels of Tlr and UPR associated genes were examined by qRT-PCR and Western blot, respectively. Co-immunoprecipitation (Co-IP) method was used to determine the direct interaction between TLRs with GRP94 in depressed rat brain. The results showed that both UPR (Xbp-1, its spliced variant sXbp-1, Atf-6, Chop, and Grp94) and Tlr (2, 3, 4, 7 and 9) genes were significantly upregulated in the hippocampi of rats who were exposed to restraint stress. Similar upregulation was observed in the protein levels of the above-mentioned TLRs and the UPR chaperone protein GRP94 as well as total and phosphorylated forms of sensor proteins IRE1α and PERK. Further, a significantly increased interaction was observed between GRP94 and the activated TLR proteins. Since, increased inflammatory activity in vulnerable areas like hippocampus is coherently associated with depressed brain; our present data suggest that the UPR may be an integral part of increased activity of inflammatory regulations in depression.

Altered miRNA expression network in locus coeruleus of depressed suicide subjects

Norepinephrine (NE) is produced primarily by neurons in the locus coeruleus (LC). Retrograde and ultrastructural examinations reveal that the core of the LC and its surrounding region receives afferent projections from several brain areas which provide multiple neurochemical inputs to the LC with changes in LC neuronal firing, making it a highly coordinated event. Although NE and mediated signaling systems have been studied in relation to suicide and psychiatric disorders that increase the risk of suicide including depression, less is known about the corresponding changes in molecular network within LC. In this study, we examined miRNA networks in the LC of depressed suicide completers and healthy controls. Expression array revealed differential regulation of 13 miRNAs. Interaction between altered miRNAs and target genes showed dense interconnected molecular network. Functional clustering of predicated target genes yielded stress induced disorders that collectively showed the complex nature of suicidal behavior. In addition, 25 miRNAs were pairwise correlated specifically in the depressed suicide group, but not in the control group. Altogether, our study revealed for the first time the involvement of LC based dysregulated miRNA network in disrupting cellular pathways associated with suicidal behavior.

MicroRNA 665 Regulates Dentinogenesis through MicroRNA-Mediated Silencing and Epigenetic Mechanisms

ABSTRACT Studies of proteins involved in microRNA (miRNA) processing, maturation, and silencing have indicated the importance of miRNAs in skeletogenesis, but the specific miRNAs involved in this process are incompletely defined. Here, we identified miRNA 665 (miR-665) as a potential repressor of odontoblast maturation. Studies with cultured cell lines and primary embryonic cells showed that miR-665 represses the expression of early and late odontoblast marker genes and stage-specific proteases involved in dentin maturation. Notably, miR-665 directly targeted Dlx3 mRNA and decreased Dlx3 expression. Furthermore, RNA-induced silencing complex (RISC) immunoprecipitation and biotin-labeled miR-665 pulldown studies identified Kat6a as another potential target of miR-665. KAT6A interacted physically and functionally with RUNX2, activating tissue-specific promoter activity and prompting odontoblast differentiation. Overexpression of miR-665 reduced the recruitment of KAT6A to Dspp and Dmp1 promoters and prevented KAT6A-induced chromatin remodeling, repressing gene transcription. Taken together, our results provide novel molecular evidence that miR-665 functions in an miRNA-epigenetic regulatory network to control dentinogenesis.

Understanding the Neuroepigenetic Constituents of Suicide Brain

Stressful life incidents often cause a predisposition for developing mental disorders such as major depressive disorder (MDD). Impaired neurocognitive and neuro-vegetative functions of the central nervous system are the hallmarks of this mental illness. Blunted responses from emotionally salient regions of the brain including cortex, hippocampus, and amygdala have been associated with MDD-related behavioral changes. Moreover, improper signal processing and neuronal atrophy were held responsible for the overall dysfunctionality of these vulnerable regions in the MDD brain. The prevalence of genetic susceptibility along with adverse environmental stimuli often makes the situation worse for MDD patients, leading to an increased risk of suicidal behavior and eventually death by suicide. Despite considerable efforts to understand the complex neurobiology associated with MDD and suicidal behavior, their pathological determinants remain mostly elusive. Recent research, however, has shown that epigenetic perturbations have a formidable impact on the etiopathogenesis of MDD. Understanding the neuroepigenetic nature of this mental disorder may provide opportunities to devise more effective treatment strategies. Moreover, this can potentially lead to identifying predictive biomarkers associated with suicide risk. The present chapter critically reviews studies pertaining to epigenetic signatures of MDD and suicide brain.

Role of HPA and the HPG Axis Interaction in Testosterone-Mediated Learned Helpless Behavior

Affective disorders show sex-specific differences in prevalence, symptoms, and complications. One hypothesis for this discrepancy is the interaction between the hypothalamic-pituitary-adrenal (HPA) axis and hypothalamic-pituitary-gonadal (HPG) axis. The present study investigates the influence of androgen on the behavioral phenotype and explores how it interacts with HPA axis genes. Gonadectomized (GDX) and GDX rats treated with testosterone propionate (T) were tested for learned helplessness (LH) behavior and compared with tested controls (TC). Prefrontal cortex was used for analyses of HPG- axis-related genes (androgen receptor, (Ar); estrogen receptor-β (Er-β)) and HPA axis-related genes (corticotropin-releasing hormone, (Crh); glucocorticoid receptor, (Nr3c1); corticotropin-releasing hormone receptor 1, (Crhr1); corticotropin-releasing hormone receptor 2, (Crhr2); FK506 binding protein 5, (Fkbp5)). Promoter-specific CpG methylation in the Crh gene was determined by bisulfite sequencing. Chromatin immunoprecipitation (ChIP) assay was used for determining ER-β binding on the proximal promoter region of Crh gene. Serum testosterone levels confirmed a testosterone-depleted GDX group, a group with supraphysiological levels of testosterone (T) and another group with physiological levels of testosterone (control (C)). Unlike GDX rats, T group exhibited significantly higher LH score when compared with any other group. Crh and Fkbp5 genes were significantly upregulated in GDX group compared with controls, whereas Er-β showed a significant downregulation in the same group. Methylation analysis showed no significant differences in-between groups. ChIP assay was unable to determine a significant change in ER-β binding but revealed a notable contrast in Crh promoter occupancy between T and GDX groups. Altogether, the present study reveals an increased susceptibility to depression-like behavior due to chronic supraphysiological level of androgen via HPA axis inhibition.

Long Noncoding RNA-Associated Transcriptomic Changes in Resiliency or Susceptibility to Depression and Response to Antidepressant Treatment

Abstract Background Recent emergence of long noncoding RNAs in regulating gene expression and thereby modulating physiological functions in brain has manifested their possible role in psychiatric disorders. In this study, the roles of long noncoding RNAs in susceptibility and resiliency to develop stress-induced depression and their response to antidepressant treatment were examined. Methods Microarray-based transcriptome-wide changes in long noncoding RNAs were determined in hippocampus of male Holtzman rats who showed susceptibility (learned helplessness) or resiliency (nonlearned helplessness) to develop depression. Changes in long noncoding RNA expression were also ascertained after subchronic administration of fluoxetine to learned helplessness rats. Bioinformatic and target prediction analyses (cis- and trans-acting) and qPCR-based assays were performed to decipher the functional role of altered long noncoding RNAs. Results Group-wise comparison showed an overrepresented class of long noncoding RNAs that were uniquely associated with nonlearned helplessness or learned helplessness behavior. Chromosomal mapping within the 5-kbp flank region of the top 20 dysregulated long noncoding RNAs in the learned helplessness group showed several target genes that were regulated through cis- or trans-actions, including Zbtb20 and Zfp385b from zinc finger binding protein family. Genomic context of differentially expressed long noncoding RNAs showed an overall blunted response in the learned helplessness group regardless of the long noncoding RNA classes analyzed. Gene ontology exhibited the functional clustering for anatomical structure development, cellular architecture modulation, protein metabolism, and cellular communications. Fluoxetine treatment reversed learned helplessness-induced changes in many long noncoding RNAs and target genes. Conclusions The involvement of specific classes of long noncoding RNAs with distinctive roles in modulating target gene expression could confer the role of long noncoding RNAs in resiliency or susceptibility to develop depression with a reciprocal response to antidepressant treatment.