Bromodomain and extraterminal domain (BET) proteins have emerged as therapeutic targets in multiple cancers, including the most common primary adult brain tumor glioblastoma (GBM). Although several ...BET inhibitors have entered clinical trials, few are brain penetrant. We have generated UM-002, a novel brain penetrant BET inhibitor that reduces GBM cell proliferation in vitro and in a human cerebral brain organoid model. Since UM-002 is more potent than other BET inhibitors, it could potentially be developed for GBM treatment. Furthermore, UM-002 treatment reduces the expression of cell-cycle related genes in vivo and reduces the expression of invasion related genes within the non-proliferative cells present in tumors as measured by single cell RNA-sequencing. These studies suggest that BET inhibition alters the transcriptional landscape of GBM tumors, which has implications for designing combination therapies. Importantly, they also provide an integrated dataset that combines in vitro and ex vivo studies with in vivo single-cell RNA-sequencing to characterize a novel BET inhibitor in GBM.
Apolipoprotein A1 (APOA1) is the major protein component of high-density lipoprotein (HDL) in plasma. We have identified an endogenously expressed long noncoding natural antisense transcript, ...APOA1-AS, which acts as a negative transcriptional regulator of APOA1 both in vitro and in vivo. Inhibition of APOA1-AS in cultured cells resulted in the increased expression of APOA1 and two neighboring genes in the APO cluster. Chromatin immunoprecipitation (ChIP) analyses of a ∼50 kb chromatin region flanking the APOA1 gene demonstrated that APOA1-AS can modulate distinct histone methylation patterns that mark active and/or inactive gene expression through the recruitment of histone-modifying enzymes. Targeting APOA1-AS with short antisense oligonucleotides also enhanced APOA1 expression in both human and monkey liver cells and induced an increase in hepatic RNA and protein expression in African green monkeys. Furthermore, the results presented here highlight the significant local modulatory effects of long noncoding antisense RNAs and demonstrate the therapeutic potential of manipulating the expression of these transcripts both in vitro and in vivo.
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•APOA1 locus has an endogenously expressed natural antisense transcript•APOA1-antisense (APOA1-AS) negatively regulates APOA1 transcription•APOA1-AS silences target gene expression, recruiting histone modifiers•Inhibiting APOA1-AS increases APOA1 expression in vitro and in vivo
Apolipoprotein A1 (APOA1) is the major protein component of high-density lipoprotein and mediates the reverse transport of cholesterol from tissues to the liver. Therefore, increasing circulating APOA1 levels may have therapeutic potential for the treatment of atherosclerosis. In this study, Wahlestedt and colleagues characterize a noncoding antisense RNA (APOA1-AS) that acts locally to suppress APOA1 expression. Promisingly, they found that blocking the activity of APOA1-AS led to an increase in APOA1 expression both in vitro and in vivo.
Schizophrenia is characterized by affective, cognitive, neuromorphological, and molecular abnormalities that may have a neurodevelopmental origin. MicroRNAs (miRNAs) are small noncoding RNA sequences ...critical to neurodevelopment and adult neuronal processes by coordinating the activity of multiple genes within biological networks. We examined the expression of 854 miRNAs in prefrontal cortical tissue from 100 control, schizophrenic, and bipolar subjects. The cyclic AMP-responsive element binding- and NMDA-regulated microRNA miR-132 was significantly down-regulated in both the schizophrenic discovery cohort and a second, independent set of schizophrenic subjects. Analysis of miR-132 target gene expression in schizophrenia gene-expression microarrays identified 26 genes upregulated in schizophrenia subjects. Consistent with NMDA-mediated hypofunction observed in schizophrenic subjects, administration of an NMDA antagonist to adult mice results in miR-132 down-regulation in the prefrontal cortex. Furthermore, miR-132 expression in the murine prefrontal cortex exhibits significant developmental regulation and overlaps with critical neurodevelopmental processes during adolescence. Adult prefrontal expression of miR-132 can be down-regulated by pharmacologie inhibition of NMDA receptor signaling during a brief postnatal period. Several key genes, including DNMT3A, GATA2, and DPYSL3, are regulated by miR-132 and exhibited altered expression either during normal neurodevelopment or in tissue from adult schizophrenic subjects. Our data suggest miR-132 dysregulation and subsequent abnormal expression of miR-132 target genes contribute to the neurodevelopmental and neuromorphological pathologies present in schizophrenia.
Glioblastoma (GBM) is a devastating adult brain cancer with high rates of recurrence and treatment resistance. Cellular heterogeneity and extensive invasion of surrounding brain tissues are ...characteristic features of GBM that contribute to its intractability. Current GBM model systems do not recapitulate some of the complex features of GBM and have not produced sufficiently-effective treatments. This has cast doubt on the effectiveness of current GBM models and drug discovery paradigms. In search of alternative pre-clinical GBM models, various 3D organoid-based GBM model systems have been developed using human cells. The scalability of these systems and potential to more accurately model characteristic features of GBM, provide promising new avenues for pre-clinical GBM research and drug discovery efforts. Here, we review the current suite of organoid-GBM models, their individual strengths and weaknesses, and discuss their future applications with an emphasis on compound screening.
A GGGGCC
hexanucleotide repeat expansion (HRE) in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), fatal neurodegenerative ...diseases with no cure or approved treatments that substantially slow disease progression or extend survival. Mechanistic underpinnings of neuronal death include C9ORF72 haploinsufficiency, sequestration of RNA-binding proteins in the nucleus, and production of dipeptide repeat proteins. Here, we used an adeno-associated viral vector system to deliver CRISPR/Cas9 gene-editing machineries to effectuate the removal of the HRE from the C9ORF72 genomic locus. We demonstrate successful excision of the HRE in primary cortical neurons and brains of three mouse models containing the expansion (500-600 repeats) as well as in patient-derived iPSC motor neurons and brain organoids (450 repeats). This resulted in a reduction of RNA foci, poly-dipeptides and haploinsufficiency, major hallmarks of C9-ALS/FTD, making this a promising therapeutic approach to these diseases.
The C9ORF72 hexanucleotide repeat expansion is the most common known genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two fatal age-related neurodegenerative ...diseases. The C9ORF72 expansion encodes five dipeptide repeat proteins (DPRs) that are produced through a non-canonical translation mechanism. Among the DPRs, proline-arginine (PR), glycine-arginine (GR), and glycine-alanine (GA) are the most neurotoxic and increase the frequency of DNA double strand breaks (DSBs). While the accumulation of these genotoxic lesions is increasingly recognized as a feature of disease, the mechanism(s) of DPR-mediated DNA damage are ill-defined and the effect of DPRs on the efficiency of each DNA DSB repair pathways has not been previously evaluated.
Using DNA DSB repair assays, we evaluated the efficiency of specific repair pathways, and found that PR, GR and GA decrease the efficiency of non-homologous end joining (NHEJ), single strand annealing (SSA), and microhomology-mediated end joining (MMEJ), but not homologous recombination (HR). We found that PR inhibits DNA DSB repair, in part, by binding to the nucleolar protein nucleophosmin (NPM1). Depletion of NPM1 inhibited NHEJ and SSA, suggesting that NPM1 loss-of-function in PR expressing cells leads to impediments of both non-homologous and homology-directed DNA DSB repair pathways. By deleting NPM1 sub-cellular localization signals, we found that PR binds NPM1 regardless of the cellular compartment to which NPM1 was directed. Deletion of the NPM1 acidic loop motif, known to engage other arginine-rich proteins, abrogated PR and NPM1 binding. Using confocal and super-resolution immunofluorescence microscopy, we found that levels of RAD52, a component of the SSA repair machinery, were significantly increased iPSC neurons relative to isogenic controls in which the C9ORF72 expansion had been deleted using CRISPR/Cas9 genome editing. Western analysis of post-mortem brain tissues confirmed that RAD52 immunoreactivity is significantly increased in C9ALS/FTD samples as compared to controls.
Collectively, we characterized the inhibitory effects of DPRs on key DNA DSB repair pathways, identified NPM1 as a facilitator of DNA repair that is inhibited by PR, and revealed deficits in homology-directed DNA DSB repair pathways as a novel feature of C9ORF72-related disease.
Genome instability is associated with myriad human diseases and is a well-known feature of both cancer and neurodegenerative disease. Until recently, the ability to assess DNA damage-the principal ...driver of genome instability-was limited to relatively imprecise methods or restricted to studying predefined genomic regions. Recently, new techniques for detecting DNA double strand breaks (DSBs) and single strand breaks (SSBs) with next-generation sequencing on a genome-wide scale with single nucleotide resolution have emerged. With these new tools, efforts are underway to define the "breakome" in normal aging and disease. Here, we compare the relative strengths and weaknesses of these technologies and their potential application to studying neurodegenerative diseases.
The recent discovery of vast non-coding RNA-based regulatory networks that can be easily modulated by nucleic acid-based drugs has opened numerous new therapeutic possibilities. Long non-coding RNA, ...and natural antisense transcripts (NATs) in particular, play a significant role in networks that involve a wide variety of disease-relevant biological mechanisms such as transcription, splicing, translation, mRNA degradation and others. Currently, significant efforts are dedicated to harnessing these newly emerging NAT-mediated biological mechanisms for therapeutic purposes. This review will highlight the recent clinical and pre-clinical developments in this field and survey the advances in nucleic acid-based drug technologies that make these developments possible.
With the capacity to modulate gene networks in an environmentally-sensitive manner, the role of epigenetic systems in mental disorders has come under intense investigation. Dysregulation of ...epigenetic effectors, including microRNAs and histone-modifying enzymes, may better explain the role of environmental risk factors and the observed heritability rate that cannot be fully attributed to known genetic risk alleles. Here, we aimed to identify novel epigenetic targets of the schizophrenia-associated microRNA 132 (miR-132).
Histone modifications were quantified by immunodetection in response to viral-mediated overexpression of miR-132 while a luminescent reporter system was used to validate targets of miR-132 in vitro. Genome-wide profiling, quantitative PCR and NanoSting were used to quantify gene expression in post-mortem human brains, neuronal cultures and prefrontal cortex (PFC) of mice chronically exposed to antipsychotics. Following viral-mediated depletion of Enhancer of Zeste 1 (EZH1) in the murine PFC, behaviors including sociability and motivation were assessed using a 3-chambered apparatus and forced-swim test, respectively.
Overexpression of miR-132 decreased global histone 3 lysine 27 tri-methylation (H3K27me3), a repressive epigenetic mark. Moreover, the polycomb-associated H3K27 methyltransferase, EZH1, is regulated by miR-132 and upregulated in the PFC of schizophrenics. Unlike its homolog EZH2, expression of EZH1 in the murine PFC decreased following chronic exposure to antipsychotics. Viral-mediated depletion of EZH1 in the mouse PFC attenuated sociability, enhanced motivational behaviors, and affected gene expression pathways related to neurotransmission and behavioral phenotypes.
EZH1 is dysregulated in schizophrenia, sensitive to antipsychotic medications, and a brain-enriched miR-132 target that controls neurobehavioral phenotypes.
•EZH1 is a novel brain-enriched target of miRNA-132.•EZH1 depletion leads to behavioral abnormalities associated with schizophrenia.•EZH1 expression is altered in schizophrenia and is sensitive to antipsychotic medications.
Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease with available treatments only marginally slowing progression or improving survival. A hexanucleotide repeat ...expansion mutation in the C9ORF72 gene is the most commonly known genetic cause of both sporadic and familial cases of ALS and frontotemporal dementia (FTD). The C9ORF72 expansion mutation produces five dipeptide repeat proteins (DPRs), and while the mechanistic determinants of DPR-mediated neurotoxicity remain incompletely understood, evidence suggests that disruption of nucleocytoplasmic transport and increased DNA damage contributes to pathology. Therefore, characterizing these disturbances and determining the relative contribution of different DPRs is needed to facilitate the development of novel therapeutics for C9ALS/FTD. To this end, we generated a series of nucleocytoplasmic transport “biosensors”, composed of the green fluorescent protein (GFP), fused to different classes of nuclear localization signals (NLSs) and nuclear export signals (NESs). Using these biosensors in conjunction with automated microscopy, we investigated the role of the three most neurotoxic DPRs (PR, GR, and GA) on seven nuclear import and two export pathways. In addition to other DPRs, we found that PR had pronounced inhibitory effects on the classical nuclear export pathway and several nuclear import pathways. To identify compounds capable of counteracting the effects of PR on nucleocytoplasmic transport, we developed a nucleocytoplasmic transport assay and screened several commercially available compound libraries, totaling 2714 compounds. In addition to restoring nucleocytoplasmic transport efficiencies, hits from the screen also counteract the cytotoxic effects of PR. Selected hits were subsequently tested for their ability to rescue another C9ALS/FTD phenotype—persistent DNA double strand breakage. Overall, we found that DPRs disrupt multiple nucleocytoplasmic transport pathways and we identified small molecules that counteract these effects—resulting in increased viability of PR-expressing cells and decreased DNA damage markers in patient-derived motor neurons. Several HDAC inhibitors were validated as hits, supporting previous studies that show that HDAC inhibitors confer therapeutic effects in neurodegenerative models.
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