Bipolar disorder-iPSC derived neural progenitor cells exhibit dysregulation of store-operated Ca2+ entry and accelerated differentiation.

While most of the efforts to uncover mechanisms contributing to bipolar disorder (BD) focused on phenotypes at the mature neuron stage, little research has considered events that may occur during earlier timepoints of neurodevelopment. Further, although aberrant calcium (Ca2+) signaling has been implicated in the etiology of this condition, the possible contribution of store-operated Ca2+ entry (SOCE) is not well understood. Here, we report Ca2+ and developmental dysregulations related to SOCE in BD patient induced pluripotent stem cell (iPSC)-derived neural progenitor cells (BD-NPCs) and cortical-like glutamatergic neurons. First, using a Ca2+ re-addition assay we found that BD-NPCs and neurons had attenuated SOCE. Intrigued by this finding, we then performed RNA-sequencing and uncovered a unique transcriptome profile in BD-NPCs suggesting accelerated neurodifferentiation. Consistent with these results, we measured a slower rate of proliferation, increased neurite outgrowth, and decreased size in neurosphere formations with BD-NPCs. Also, we observed decreased subventricular areas in developing BD cerebral organoids. Finally, BD NPCs demonstrated high expression of the let-7 family while BD neurons had increased miR-34a, both being microRNAs previously implicated in neurodevelopmental deviations and BD etiology. In summary, we present evidence supporting an accelerated transition towards the neuronal stage in BD-NPCs that may be indicative of early pathophysiological features of the disorder.

Clozapine Increases Nestin Concentration in the Adult Male Rat Hippocampus: A Preliminary Study.

Patients with schizophrenia, and rodent models of the disease, both exhibit suppressed neurogenesis, with antipsychotics possibly enhancing neurogenesis in pre-clinical models. Nestin, a cytoskeletal protein, is implicated in neuronal differentiation and adult neurogenesis. We hypothesized that schizophrenia pathogenesis involves nestin downregulation; however, few studies have related nestin to schizophrenia. We assessed nestin protein concentration, prepulse inhibition (PPI), and social interaction in the MK-801 model of schizophrenia, with or without antipsychotic (clozapine) treatment. Adult male Sprague-Dawley rats were intraperitoneally administered saline or MK-801 (0.1 mg/kg) to produce a schizophrenia-like phenotype, with concomitant subcutaneous injections of vehicle or clozapine (5 mg/kg). PPI was assessed on days 1, 8, and 15, and social interaction was assessed on day 4. Hippocampus tissue samples were dissected for Western blotting of nestin concentration. MK-801 alone did not alter nestin concentration, while clozapine alone enhanced hippocampal nestin concentration; this effect was not apparent in animals with MK-801 and clozapine co-administration. MK-801 also produced schizophrenia-like PPI disruptions, some of which were reversed by clozapine. Social interaction deficits were not detected in this model. This is the first report of clozapine-induced enhancements of hippocampal nestin concentration that might be mediated by NMDA receptors. Future studies will explore the impact of neurodevelopmental nestin concentration on symptom onset and antipsychotic treatment.

A bidirectional competitive interaction between circHomer1 and Homer1b within the orbitofrontal cortex regulates reversal learning.

Although circular RNAs (circRNAs) are enriched in the brain, their relevance for brain function and psychiatric disorders is poorly understood. Here, we show that circHomer1 is inversely associated with relative HOMER1B mRNA isoform levels in both the orbitofrontal cortex (OFC) and stem-cell-derived neuronal cultures of subjects with psychiatric disorders. We further demonstrate that in vivo circHomer1 knockdown (KD) within the OFC can inhibit the synaptic expression of Homer1b mRNA. Furthermore, we show that circHomer1 directly binds to Homer1b mRNA and that Homer1b-specific KD increases synaptic circHomer1 levels and improves OFC-mediated behavioral flexibility. Importantly, double circHomer1 and Homer1b in vivo co-KD results in a complete rescue in circHomer1-associated alterations in both chance reversal learning and synaptic gene expression. Lastly, we uncover an RNA-binding protein that can directly bind to circHomer1 and promote its biogenesis. Taken together, our data provide mechanistic insights into the importance of circRNAs in brain function and disease.

Lithium inhibits oxidative stress-induced neuronal senescence through miR-34a.

Neuronal senescence, triggered by telomere shortening, oncogene activation, DNA damage, or oxidative stress, has been associated with neurodegenerative diseases' pathogenesis. Therefore, preventing neuronal senescence could be a novel treatment strategy for neurodegenerative diseases. Lithium (Li), the first-line treatment against bipolar disorder, has been shown to have neuroprotective effects in clinical, pre-clinical, and in vitro studies. Li can protect cells from senescence, and its effect on neuronal senescence was investigated in our study. Furthermore, we also investigated the effects of Li on the senescence-associated miR-34a/Sirt1/p53 pathway. In this study, hydrogen peroxide was used as an inducer for the "stress-induced premature senescence" model. In the senescence model, we have assessed Li's effects on senescence by analyzing ß-galactosidase activity, Sudan Black B, and senescence-associated heterochromatin foci (SAHF) stainings, and on cell cycle arrest by BrdU staining. Furthermore, expression levels of senescence and cell cycle arrest-related proteins (p53, p21, p16INK4a, and SIRT1) by western blotting. Finally, the effects of Li on senescence-associated miR-34a levels were measured by quantitative PCR. We show via Sudan Black B staining, ß-Gal activity assay, and by detecting SAHF, Li protects against senescence in neuronal cells. Then, Li's effect on signaling has also been determined on pathways involved in senescence and cell cycle arrest. Moreover, we have observed that Li has a modulatory effect on miR-34a expression. Therefore, we posit that Li suppresses senescence in neuronal cells and that this effect is mediated through miR-34a/Sirt1/p53 axis.

Adaptor Protein ShcD/SHC4 Interacts with Tie2 Receptor to Synergistically Promote Glioma Cell Invasion.

Gliomas are characterized by diffuse infiltration of tumor cells into surrounding brain tissue, and this highly invasive nature contributes to disease recurrence and poor patient outcomes. The molecular mechanisms underlying glioma cell invasion remain incompletely understood, limiting development of new targeted therapies. Here, we have identified phosphotyrosine adaptor protein ShcD as upregulated in malignant glioma and shown that it associates with receptor tyrosine kinase Tie2 to facilitate invasion. In human glioma cells, we find that expression of ShcD and Tie2 increases invasion, and this significant synergistic effect is disrupted with a ShcD mutant that cannot bind Tie2 or hyperphosphorylate the receptor. Expression of ShcD and/or Tie2 further increases invadopodia formation and matrix degradation in U87 glioma cells. In a coculture model, we show that U87-derived tumor spheroids expressing both ShcD and Tie2 display enhanced infiltration into cerebral organoids. Mechanistically, we identify changes in focal adhesion kinase phosphorylation in the presence of ShcD and/or Tie2 in U87 cells upon Tie2 activation. Finally, we identify a strong correlation between transcript levels of ShcD and Tie2 signaling components as well as N-cadherin in advanced gliomas and those with classical or mesenchymal subtypes, and we show that elevated expression of ShcD correlates with a significant reduction in patient survival in higher grade gliomas with mesenchymal signature. Altogether, our data highlight a novel Tie2-ShcD signaling axis in glioma cell invasion, which may be of clinical significance. IMPLICATIONS: ShcD cooperates with Tie2 to promote glioma cell invasion and its elevated expression correlates with poor patient outcome in advanced gliomas.

A psychiatric disease-related circular RNA controls synaptic gene expression and cognition.

Although circular RNAs (circRNAs) are enriched in the mammalian brain, very little is known about their potential involvement in brain function and psychiatric disease. Here, we show that circHomer1a, a neuronal-enriched circRNA abundantly expressed in the frontal cortex, derived from Homer protein homolog 1 (HOMER1), is significantly reduced in both the prefrontal cortex (PFC) and induced pluripotent stem cell-derived neuronal cultures from patients with schizophrenia (SCZ) and bipolar disorder (BD). Moreover, alterations in circHomer1a were positively associated with the age of onset of SCZ in both the dorsolateral prefrontal cortex (DLPFC) and orbitofrontal cortex (OFC). No correlations between the age of onset of SCZ and linear HOMER1 mRNA were observed, whose expression was mostly unaltered in BD and SCZ postmortem brain. Using in vivo circRNA-specific knockdown of circHomer1a in mouse PFC, we show that it modulates the expression of numerous alternative mRNA transcripts from genes involved in synaptic plasticity and psychiatric disease. Intriguingly, in vivo circHomer1a knockdown in mouse OFC resulted in specific deficits in OFC-mediated cognitive flexibility. Lastly, we demonstrate that the neuronal RNA-binding protein HuD binds to circHomer1a and can influence its synaptic expression in the frontal cortex. Collectively, our data uncover a novel psychiatric disease-associated circRNA that regulates synaptic gene expression and cognitive flexibility.

Activation of WNT and CREB signaling pathways in human neuronal cells in response to the Omega-3 fatty acid docosahexaenoic acid (DHA).

A subset of individuals with major depressive disorder (MDD) elects treatment with complementary and alternative medicines (CAMs), including the omega-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Previous studies in rodents suggest that DHA modulates neurodevelopmental processes, including adult neurogenesis and neuroplasticity, but the molecular and cellular mechanisms of DHA's potential therapeutic effect in the context of human neurobiology have not been well established. Here we sought to address this knowledge gap by investigating the effects of DHA using human iPSC-derived neural progenitor cells (NPCs) and post-mitotic neurons using pathway-selective reporter genes, multiplexed mRNA expression profiling, and a panel of metabolism-based viability assays. Finally, real-time, live-cell imaging was employed to monitor neurite outgrowth upon DHA treatment. Overall, these studies showed that DHA treatment (0-50 µM) significantly upregulated both WNT and CREB signaling pathways in human neuronal cells in a dose-dependent manner with 2- to 3-fold increases in pathway activation. Additionally, we observed that DHA treatment enhanced survival of iPSC-derived NPCs and differentiation of post-mitotic neurons with live-cell imaging, revealing increased neurite outgrowth with DHA treatment within 24 h. Taken together, this study provides evidence that DHA treatment activates critical pathways regulating neuroplasticity, which may contribute to enhanced neuronal cell viability and neuronal connectivity. The extent to which these pathways represent molecular mechanisms underlying the potential beneficial effects of omega-3 fatty acids in MDD and other brain disorders merits further investigation.

Chemogenomic analysis reveals key role for lysine acetylation in regulating Arc stability.

The role of Arc in synaptic plasticity and memory consolidation has been investigated for many years with recent evidence that defects in the expression or activity of this immediate-early gene may also contribute to the pathophysiology of brain disorders including schizophrenia and fragile X syndrome. These results bring forward the concept that reversing Arc abnormalities could provide an avenue to improve cognitive or neurological impairments in different disease contexts, but how to achieve this therapeutic objective has remained elusive. Here, we present results from a chemogenomic screen that probed a mechanistically diverse library of small molecules for modulators of BDNF-induced Arc expression in primary cortical neurons. This effort identified compounds with a range of influences on Arc, including promoting its acetylation-a previously uncharacterized post-translational modification of this protein. Together, our data provide insights into the control of Arc that could be targeted to harness neuroplasticity for clinical applications.

Erythropoietin Promotes Glioblastoma via miR-451 Suppression.

Erythropoietin (EPO) is an erythropoiesis stimulating growth factor and hormone. EPO has been widely used in the treatment of chronic renal failure, cancer, and chemotherapy-related anemia for three decades. However, many clinical trials showed that EPO treatment may be associated with tumorigenesis and cancer progression. EPO is able to cross blood-brain barriers, and this may lead to an increased possibility of central nervous system tumors such as glioblastoma. Indeed, EPO promotes glioblastoma growth and invasion in animal studies. Additionally, EPO increases glioblastoma cell survival, proliferation, migration, invasion, and chemoresistancy in vitro. However, the exact mechanisms of cancer progression induced by EPO treatment are not fully understood. Posttranscriptional gene regulation through microRNAs may contribute to EPO's cellular and biological effects in tumor progression. Here, we aimed to study whether tumor suppressive microRNA, miR-451, counteracts the positive effects of EPO on U87 human glioblastoma cell line. Migration and invasion were evaluated by scratch assay and transwell invasion assay, respectively. We found that EPO decreased basal miR-451 expression and increased cell proliferation, migration, invasion, and cisplatin chemoresistancy in vitro. miR-451 overexpression by transfection of its mimic significantly reversed these effects. Furthermore, ectopic expression of miR-451 inhibited expression of its own target genes, such as metalloproteinases-2 and -9, which are stimulated by EPO treatment and involved in carcinogenesis processes, especially invasion. These findings suggest that miR-451 mimic delivery may be useful as adjuvant therapy in addition to chemotherapy and anemia treatment by EPO and should be tested in experimental glioblastoma models.

Diagnostic and therapeutic potential of microRNAs in neuropsychiatric disorders: Past, present, and future.

Neuropsychiatric disorders are common health problems affecting approximately 1% of the population. Twin, adoption, and family studies have displayed a strong genetic component for many of these disorders; however, the underlying pathophysiological mechanisms and neural substrates remain largely unknown. Given the critical need for new diagnostic markers and disease-modifying treatments, expanding the focus of genomic studies of neuropsychiatric disorders to include the role of non-coding RNAs (ncRNAs) is of growing interest. Of known types of ncRNAs, microRNAs (miRNAs) are 20-25-nucleotide, single-stranded, molecules that regulate gene expression through post-transcriptional mechanisms and have the potential to coordinately regulate complex regulatory networks. In this review, we summarize the current knowledge on miRNA alteration/dysregulation in neuropsychiatric disorders, with a special emphasis on schizophrenia (SCZ), bipolar disorder (BD), and major depressive disorder (MDD). With an eye toward the future, we also discuss the diagnostic and prognostic potential of miRNAs for neuropsychiatric disorders in the context of personalized treatments and network medicine.

Cell division cycle 7-kinase inhibitor PHA-767491 hydrochloride suppresses glioblastoma growth and invasiveness.

BACKGROUND: Genomic instability is a hallmark of cancer cells, and this cellular phenomenon can emerge as a result of replicative stress. It is possible to take advantage of replicative stress, and enhance it in a targeted way to fight cancer cells. One of such strategies involves targeting the cell division cycle 7-related protein kinase (CDC7), a protein with key roles in regulation of initiation of DNA replication. CDC7 overexpression is present in different cancers, and small molecule inhibitors of the CDC7 have well-documented anti-tumor effects. Here, we aimed to test the potential of CDC7 inhibition as a new strategy for glioblastoma treatment. METHODS: PHA-767491 hydrochloride was used as the CDC7 inhibitor. Two glioblastoma cell lines (U87-MG and U251-MG) and a control cell line (3T3) were used to characterize the effects of CDC7 inhibition. The effect of CDC7 inhibition on cell viability, cell proliferation, apoptosis, migration, and invasion were analyzed. In addition, real-time PCR arrays were used to identify the differentially expressed genes in response to CDC7 inhibition. RESULTS: Our results showed that CDC7 inhibition reduces glioblastoma cell viability, suppresses cell proliferation, and triggers apoptosis in glioblastoma cell lines. In addition, we determined that CDC7 inhibition also suppresses glioblastoma cell migration and invasion. To identify molecular targets of CDC7 inhibition, we used real-time PCR arrays, which showed dysregulation of several mRNAs and miRNAs. CONCLUSIONS: Taken together, our findings suggest that CDC7 inhibition is a promising strategy for treatment of glioblastoma.

Lithium protects against paraquat neurotoxicity by NRF2 activation and miR-34a inhibition in SH-SY5Y cells.

Lithium is a mood stabilizing agent commonly used for the treatment of bipolar disorder. Here, we investigated the potential neuroprotective effect of lithium against paraquat toxicity and its underlying mechanisms in vitro. SH-SY5Y human neuroblastoma cells were treated with paraquat (PQ) 0.5 mM concentration after lithium pretreatment to test lithium's capability in preventing cell toxicity. Cell death was evaluated by LDH, WST-8, and tryphan blue assays. Apoptosis was analyzed using DNA fragmentation, Annexin V immunostaining, Sub G1 cell cycle analysis, and caspase-3 activity assays. BCL2, BAX, and NRF2 protein expression were evaluated by Western-blotting and the BDNF protein level was determined with ELISA. mRNA levels of BCL2, BAX, BDNF, and NRF2 target genes (HO-1, GCS, NQO1), as well as miR-34a expression were analyzed by qPCR assay. Functional experiments were done via transfection with NRF2 siRNA and miR-34a mimic. Lithium treatment prevented paraquat induced cell death and apoptosis. Lithium treated cells showed increased anti-apoptotic protein BCL2 and decreased pro-apoptotic protein BAX expression. Lithium exerted a neurotrophic effect by increasing BDNF protein expression. It also diminished reactive oxygen species production and activated the redox sensitive transcription factor NRF2 and increased its target genes expression. Knockdown of NRF2 abolished neuroprotective, anti-apoptotic, and anti-oxidant effects of lithium. Furthermore, lithium significantly decreased both basal and PQ-induced expression of miR-34a. Transfection of miR-34a specific mimic reversed neuroprotective, anti-apoptotic, and anti-oxidant effects of lithium against PQ-toxicity. Our results revealed two novel mechanisms of lithium neuroprotection, namely NRF2 activation and miR-34a suppression.

EPO Mediates Neurotrophic, Neuroprotective, Anti-Oxidant, and Anti-Apoptotic Effects via Downregulation of miR-451 and miR-885-5p in SH-SY5Y Neuron-Like Cells.

Erythropoietin (EPO) is a neuroprotective cytokine, which has been applied in several animal models presenting neurological disorders. One of the proposed modes of action resulting in neuroprotection is post-transcriptional gene expression regulation. This directly brings to mind microRNAs (miRNAs), which are small non-coding RNAs that regulate gene expression at the post-transcriptional level. It has not yet been evaluated whether miRNAs participate in the biological effects of EPO or whether it, inversely, modulates specific miRNAs in neuronal cells. In this study, we employed miRNA and mRNA arrays to identify how EPO exerts its biological function. Notably, miR-451 and miR-885-5p are downregulated in EPO-treated SH-SY5Y neuronal-like cells. Accordingly, target prediction and transcriptome analysis of cells treated with EPO revealed an alteration of the expression of genes involved in apoptosis, cell survival, proliferation, and migration. Low expression of miRNAs in SH-SY5Y was correlated with high expression of their target genes, vascular endothelial growth factor A, matrix metallo peptidase 9 (MMP9), cyclin-dependent kinase 2 (CDK2), erythropoietin receptor, Mini chromosome maintenance complex 5 (MCM5), B-cell lymphoma 2 (BCL2), and Galanin (GAL). Cell viability, apoptosis, proliferation, and migration assays were carried out for functional analysis after transfection with miRNA mimics, which inhibited some biological actions of EPO such as neuroprotection, anti-oxidation, anti-apoptosis, and migratory effects. In this study, we report for the first time that EPO downregulates the expression of miRNAs (miR-451 and miR-885-5p) in SH-SY5Y neuronal-like cells. The correlation between the over-expression of miRNAs and the decrease in EPO-mediated biological effects suggests that miR-451 and miR-885-5p may play a key role in the mediation of biological function.