Ashutosh Dharap

Dysregulation of target genes of nonsense-mediated decay (NMD) in the brain remains sparsely known in tauopathies. PS19 transgenic mice expressing human mutant P301S tau were evaluated for levels of lncRNA Gas5, a target of NMD. The results show Gas5 decreased in the brains of PS19 mice as they aged. We evaluated the consequences of blocking the NMD-mediated turnover of Gas5 using a small molecule administered intranasally to PS19 mice. The results show NPC86 disassociated Upf1 and Gas5, thereby hindering NMD. NPC86 treatment increased Gas5 levels in the cortex of male PS19 mice concurrent with a highly significant decrease in pTau S214 and neuroinflammatory genes while increasing insulin signaling. Consequently, digital spatial profiling identified Gas5-regulated genes and pathways. NPC86 treatment enhanced neuronal homeostasis, synaptic vesicle transport and mitochondrial function and downregulated neuroinflammatory pathways. The nodal genes in Parkinson’s signaling pathway, the multiple sclerosis signaling pathway, the Gα(s) signaling pathway, the neuroinflammation signaling pathway, and the interferon gamma signaling pathway were decreased in response to NPC86. The study demonstrates the potential of selectively stabilizing NMD-target lncRNA Gas5 levels to alleviate early neurodegenerative pathology in tauopathy.

BACKGROUND: Recent advances in high-throughput transcriptomics have revealed extensive gene expression changes during cerebral ischemia. However, the changes in 3-dimensional chromatin architecture and long-range chromatin looping between genes and distal regulatory elements that drive these gene expression changes remain virtually unexplored. In this study, we map the landscape of the dynamic alterations in topologically associating domains and chromatin loops in the ischemic cortex and evaluate their contributions to poststroke transcriptional changes. METHODS: We used high-throughput chromatin conformation capture (Hi-C) to profile genome-wide changes of the 3-dimensional chromatin architecture in the cerebral cortex of adult mice following a 1-hour middle cerebral artery occlusion and 6 hours of reperfusion or sham treatment. We conducted RNA sequencing to identify the differentially expressed genes that are concomitantly altered in association with the stroke-induced topologically associating domains and loops. RESULTS: We identified 293 altered topologically associating domains following stroke, harboring a total of 60 upregulated differentially expressed genes and 106 downregulated differentially expressed genes. Chromatin looping analysis identified 4323 differentially altered loops in response to stroke, of which 1583 had enriched interactions and 2741 had diminished interactions. These loci included previously unknown enhancer and silencer elements, which were linked to a total of 88 upregulated and 96 downregulated genes. Upregulated genes associated with altered topologically associating domains and loops were linked to endothelial and immune cell functions, suggesting a role for dynamic chromatin remodeling in the poststroke cerebrovascular and neuroimmune response. Conversely, downregulated genes were associated with neuronal and synaptic functions, suggesting a role in poststroke neuronal fate. CONCLUSIONS: This study is the first to map changes in the 3-dimensional chromatin of the adult cerebral cortex following ischemic stroke. Our findings reveal novel regulatory elements directly associated with stroke-altered genes that may be involved in the regulation of these genes via dynamic changes in chromatin architecture and looping characteristics to fine-tune their expression.

Noncoding DNA undergoes widespread context-dependent transcription to produce noncoding RNAs. In recent decades, tremendous advances in genomics and transcriptomics have revealed important regulatory roles for noncoding DNA elements and the RNAs that they produce. Enhancers are one such element that are well-established drivers of gene expression changes in response to a variety of factors such as external stimuli, cellular responses, developmental cues, and disease states. They are known to act at long distances, interact with multiple target gene loci simultaneously, synergize with other enhancers, and associate with dynamic chromatin architectures to form a complex regulatory network. Recent advances in enhancer biology have revealed that upon activation, enhancers transcribe long noncoding RNAs, known as enhancer RNAs (eRNAs), that have been shown to play important roles in enhancer-mediated gene regulation and chromatin-modifying activities. In the brain, enhancer dysregulation and eRNA transcription has been reported in numerous disorders from acute injuries to chronic neurodegeneration. Because this is an emerging area, a comprehensive understanding of eRNA function has not yet been achieved in brain disorders; however, the findings to date have illuminated a role for eRNAs in activity-driven gene expression and phenotypic outcomes. In this review, we highlight the breadth of the current literature on eRNA biology in brain health and disease and discuss the challenges as well as focus areas and strategies for future in-depth research on eRNAs in brain health and disease.

Recent studies have reported widespread stimulus-dependent transcription of mammalian enhancers into noncoding enhancer RNAs (eRNAs), some of which have central roles in the enhancer-mediated induction of target genes and modulation of phenotypic outcomes during development and disease. In cerebral ischemia, the expression and functions of eRNAs are virtually unknown. Here, we applied genome-wide H3K27ac ChIP-seq and genome-wide RNA-seq to identify enhancer elements and stroke-induced eRNAs, respectively, in the mouse cerebral cortex during transient focal ischemia. Following a 1-h middle cerebral artery occlusion (MCAO) and 6 h of reperfusion, we identified 77 eRNAs that were significantly upregulated in stroke as compared to sham, of which 55 were exclusively expressed in stroke. The knockdown of two stroke-induced eRNAs in the mouse brain resulted in significantly larger infarct volumes as compared to controls, suggesting that these eRNAs are involved in the post-stroke neuroprotective response. A preliminary comparison of eRNA expression in the male versus female cortices revealed sex-dependent patterns that may underlie the physiological differences in response to stroke between the two sexes. Together, this study is the first to illuminate the eRNA landscape in the post-stroke cortex and demonstrate the significance of an eRNA in modulating post-stroke cortical brain damage.

Ischemic stroke is an acute brain injury with high mortality and disability rates worldwide. The pathophysiological effects of ischemic stroke are driven by a multitude of complex molecular and cellular interactions that ultimately result in brain damage and neurological dysfunction. The Human Genome Project revealed that the vast majority of the human genome (and mammalian genome in general) is transcribed into noncoding RNAs. These RNAs have several important roles in the molecular biology of the cell. Of these, the long noncoding RNAs are gaining particular importance in stroke biology. High-throughput analysis of gene expression using methodologies such as RNA-seq and microarrays have identified a number of aberrantly expressed lncRNAs in the post-stroke brain and blood in experimental models as well as in clinical samples. These expression changes exhibited distinct temporal and cell-type-dependent patterns. Many of these lncRNAs were shown to modulate molecular pathways that resulted in deleterious as well as neuroprotective outcomes in the post-stroke brain. In this review, we consolidate the latest data from the literature that elucidate the roles and functions of lncRNAs in ischemic stroke. We also summarize clinical studies identifying differential lncRNA expression changes between stroke patients and healthy individuals, and genetic variations in lncRNA loci that are correlated with an increased risk of stroke development.

Gene expression in cerebral ischemia has been a subject of intense investigations for several years. Studies utilizing probe-based high-throughput methodologies such as microarrays have contributed significantly to our existing knowledge but lacked the capacity to dissect the transcriptome in detail. Genome-wide RNA-sequencing (RNA-seq) enables comprehensive examinations of transcriptomes for attributes such as strandedness, alternative splicing, alternative transcription start/stop sites, and sequence composition, thus providing a very detailed account of gene expression. Leveraging this capability, we conducted an in-depth, genome-wide evaluation of the protein-coding transcriptome of the adult mouse cortex after transient focal ischemia at 6, 12, or 24 h of reperfusion using RNA-seq. We identified a total of 1007 transcripts at 6 h, 1878 transcripts at 12 h, and 1618 transcripts at 24 h of reperfusion that were significantly altered as compared to sham controls. With isoform-level resolution, we identified 23 splice variants arising from 23 genes that were novel mRNA isoforms. For a subset of genes, we detected reperfusion time-point-dependent splice isoform switching, indicating an expression and/or functional switch for these genes. Finally, for 286 genes across all three reperfusion time-points, we discovered multiple, distinct, simultaneously expressed and differentially altered isoforms per gene that were generated via alternative transcription start/stop sites. Of these, 165 isoforms derived from 109 genes were novel mRNAs. Together, our data unravel the protein-coding transcriptome of the cerebral cortex at an unprecedented depth to provide several new insights into the flexibility and complexity of stroke-related gene transcription and transcript organization.