Inhibition of Cysteine Proteases via Thiol-Michael Addition Explains the Anti-SARS-CoV-2 and Bioactive Properties of Arteannuin B.

Artemisia annua is the plant that produces artemisinin, an endoperoxide-containing sesquiterpenoid used for the treatment of malaria. A. annua extracts, which contain other bioactive compounds, have been used to treat other diseases, including cancer and COVID-19, the disease caused by the virus SARS-CoV-2. In this study, a methyl ester derivative of arteannuin B was isolated when A. annua leaves were extracted with a 1:1 mixture of methanol and dichloromethane. This methyl ester was thought to be formed from the reaction between arteannuin B and the extracting solvent, which was supported by the fact that arteannuin B underwent 1,2-addition when it was dissolved in deuteromethanol. In contrast, in the presence of N-acetylcysteine methyl ester, a 1,4-addition (thiol-Michael reaction) occurred. Arteannuin B hindered the activity of the SARS CoV-2 main protease (nonstructural protein 5, NSP5), a cysteine protease, through time-dependent inhibition. The active site cysteine residue of NSP5 (cysteine-145) formed a covalent bond with arteannuin B as determined by mass spectrometry. In order to determine whether cysteine adduction by arteannuin B can inhibit the development of cancer cells, similar experiments were performed with caspase-8, the cysteine protease enzyme overexpressed in glioblastoma. Time-dependent inhibition and cysteine adduction assays suggested arteannuin B inhibits caspase-8 and adducts to the active site cysteine residue (cysteine-360), respectively. Overall, these results enhance our understanding of how A. annua possesses antiviral and cytotoxic activities.

Defective fractalkine-CX3CR1 signaling aggravates neuroinflammation and affects recovery from cuprizone-induced demyelination.

Microglia have been implicated in multiple sclerosis (MS) pathogenesis. The fractalkine receptor CX3CR1 limits the activation of pathogenic microglia and the human polymorphic CX3CR1I249/M280 (hCX3CR1I249/M280 ) variant increases disease progression in models of MS. However, the role of hCX3CR1I249/M280 variant on microglial activation and central nervous system repair mechanisms remains unknown. Therefore, using transgenic mice expressing the hCX3CR1I249/M280 variant, we aimed to determine the contribution of defective CX3CR1 signaling to neuroinflammation and remyelination in the cuprizone model of focal demyelination. Here, we report that mice expressing hCX3CR1I249/M280 exhibit marked demyelination and microgliosis following acute cuprizone treatment. Nanostring gene expression analysis in demyelinated lesions showed that hCX3CR1I249/M280 but not CX3CR1-deficient mice up-regulated the cuprizone-induced gene profile linked to inflammatory, oxidative stress, and phagocytic pathways. Although CX3CR1-deficient (CX3CR1-KO) and fractalkine-deficient (FKN-KO) mice displayed a comparable demyelination and microglial activation phenotype to hCX3CR1I249/M280 mice, only CX3CR1-deficient and CX3CR1-WT mice showed significant myelin recovery 1 week from cuprizone withdrawal. Confocal microscopy showed that hCX3CR1I249/M280 variant inhibits the generation of cells involved in myelin repair. Our results show that defective fractalkine signaling contributes to regional differences in demyelination, and suggest that the CX3CR1 pathway activity may be a key mechanism for limiting toxic gene responses in neuroinflammation. Cover Image for this issue: https://doi.org/10.1111/jnc.15416.

Advancing neuro-oncology of glial tumors from big data and multidisciplinary studies.

INTRODUCTION: Multidisciplinary studies for glial tumors has produced an enormous amount of information including imaging, histology, and a large cohort of molecular data (i.e. genomics, epigenomics, metabolomics, proteomics, etc.). The big data resources are made possible through open access that offers great potential for new biomarker or therapeutic intervention via deep-learning and/or machine learning for integrated multi-omics analysis. An equally important effort to define the hallmarks of glial tumors will also advance precision neuro-oncology and inform patient-specific therapeutics. This review summarizes past studies regarding tumor classification, hallmarks of cancer, and hypothetical mechanisms. Leveraging on advanced big data approaches and ongoing cross-disciplinary endeavors, this review also discusses how to integrate multiple layers of big data toward the goal of precision medicine. RESULTS: In addition to basic research of cancer biology, the results from integrated multi-omics analysis will highlight biological processes and potential candidates as biomarkers or therapeutic targets. Ultimately, these collective resources built upon an armamentarium of accessible data can re-form clinical and molecular data to stratify patient-tailored therapy. CONCLUSION: We envision that a comprehensive understanding of the link between molecular signatures, tumor locations, and patients' history will identify a molecular taxonomy of glial tumors to advance the improvements in early diagnosis, prevention, and treatment.

Role of the histone methyltransferases Ezh2 and Suv4-20h1/Suv4-20h2 in neurogenesis.

Mechanisms regulating neurogenesis involve broad and complex processes that represent intriguing therapeutic targets in the field of regenerative medicine. One influential factor guiding neural stem cell proliferation and cellular differentiation during neurogenesis are epigenetic mechanisms. We present an overview of epigenetic mechanisms including chromatin structure and histone modifications; and discuss novel roles of two histone modifiers, Ezh2 and Suv4-20h1/Suv4-20h2 (collectively referred to as Suv4-20h), in neurodevelopment and neurogenesis. This review will focus on broadly reviewing epigenetic regulatory components, the roles of epigenetic components during neurogenesis, and potential applications in regenerative medicine.

The chromatin repressors EZH2 and Suv4-20h coregulate cell fate specification during hippocampal development.

The cell fate transition from radial glial-like (RGL) cells to neurons and astrocytes is crucial for development and pathological conditions. Two chromatin repressors-the enhancer of zeste homolog 2 and suppressor of variegation 4-20 homolog-are expressed in RGL cells in the hippocampus, implicating these epigenetic regulators in hippocampal cell fate commitment. Using a double knockout mouse model, we demonstrated that loss of both chromatin repressors in the RGL population leads to deficits in hippocampal development. Single-nuclei RNA-Seq revealed differential gene expression and provided mechanistic insight into how the two chromatin repressors are critical for the maintenance of cycling cells in the dentate gyrus as well as the balance of cell trajectories between neuronal and astroglial lineages.

Region specific knock-out reveals distinct roles of chromatin modifiers in adult neurogenic niches.

Histone methyltransferases (HMTs) are present in heterogeneous cell populations within the adult brain including neurogenic niches. Yet the question remains whether loss of HMTs and the resulting changes in histone methylation alter cell fate in a region-specific manner. We utilized stereotaxic injection of Cre recombinant protein into the adult neurogenic niches, the subventricular zone (SVZ) adjacent to the lateral ventricle and the subgranular zone (SGZ) of the dentate gyrus. We confirmed that Cre protein was enzymatically active in vivo and recombination events were restricted to the vicinity of injection areas. In this study, we focus on using Cre mediated recombination in mice harboring floxed HMT: enhancer of zeste homolog 2 (EZH2) or suppressor of variegation homolog (Suv4-20h). Injectable Cre protein successfully knocked out either EZH2 or Suv4-20h, allowing assessment of long-term effects in a region-specific fashion. We performed meso-scale imaging and flow cytometry for phenotype analysis and unbiased quantification. We demonstrated that regional loss of EZH2 affects the differentiation paradigm of neural stem progenitor cells as well as the maintenance of stem cell population. We further demonstrated that regional loss of Suv4-20h influences the cell cycle but does not affect stem cell differentiation patterns. Therefore, Cre protein mediated knock-out a given HMT unravel their distinguishable and important roles in adult neurogenic niches. This Cre protein-based approach offers tightly-controlled knockouts in multiple cell types simultaneously for studying diverse regulatory mechanisms and is optimal for region-specific manipulation within complex, heterogeneous brain architectures.

Comparative analyses identify molecular signature of MRI-classified SVZ-associated glioblastoma.

Glioblastoma (GBM) is a highly aggressive brain cancer with limited therapeutic options. While efforts to identify genes responsible for GBM have revealed mutations and aberrant gene expression associated with distinct types of GBM, patients with GBM are often diagnosed and classified based on MRI features. Therefore, we seek to identify molecular representatives in parallel with MRI classification for group I and group II primary GBM associated with the subventricular zone (SVZ). As group I and II GBM contain stem-like signature, we compared gene expression profiles between these 2 groups of primary GBM and endogenous neural stem progenitor cells to reveal dysregulation of cell cycle, chromatin status, cellular morphogenesis, and signaling pathways in these 2 types of MRI-classified GBM. In the absence of IDH mutation, several genes associated with metabolism are differentially expressed in these subtypes of primary GBM, implicating metabolic reprogramming occurs in tumor microenvironment. Furthermore, histone lysine methyltransferase EZH2 was upregulated while histone lysine demethylases KDM2 and KDM4 were downregulated in both group I and II primary GBM. Lastly, we identified 9 common genes across large data sets of gene expression profiles among MRI-classified group I/II GBM, a large cohort of GBM subtypes from TCGA, and glioma stem cells by unsupervised clustering comparison. These commonly upregulated genes have known functions in cell cycle, centromere assembly, chromosome segregation, and mitotic progression. Our findings highlight altered expression of genes important in chromosome integrity across all GBM, suggesting a common mechanism of disrupted fidelity of chromosome structure in GBM.

Cross-species Analyses Unravel the Complexity of H3K27me3 and H4K20me3 in the Context of Neural Stem Progenitor Cells.

Neural stem progenitor cells (NSPCs) in the human subventricular zone (SVZ) potentially contribute to life-long neurogenesis, yet subtypes of glioblastoma multiforme (GBM) contain NSPC signatures that highlight the importance of cell fate regulation. Among numerous regulatory mechanisms, the post-translational methylations onto histone tails are crucial regulator of cell fate. The work presented here focuses on the role of two repressive chromatin marks tri-methylations on histone H3 lysine 27 (H3K27me3) and histone H4 lysine 20 (H4K20me3) in the adult NSPC within the SVZ. To best model healthy human NSPCs as they exist in vivo for epigenetic profiling of H3K27me3 and H4K20me3, we utilized NSPCs isolated from the adult SVZ of baboon brain (Papio anubis) with brain structure and genomic level similar to human. The putative role of H3K27me3 in normal NSPCs predominantly falls into the regulation of gene expression, cell cycle, and differentiation, whereas H4K20me3 is involved in DNA replication/repair, metabolism, and cell cycle. Using conditional knock-out mouse models to diminish Ezh2 and Suv4-20h responsible for H3K27me3 and H4K20me3, respectively, we found that both repressive marks have irrefutable function for cell cycle regulation in the NSPC population. While both EZH2/H3K27me3 and Suv4-20h/H4K20me3 have implication in cancers, our comparative genomics approach between healthy NSPCs and human GBM specimens revealed that substantial sets of genes enriched with H3K27me3 and H4K20me3 in the NSPCs are altered in the human GBM. In sum, our integrated analyses across species highlight important roles of H3K27me3 and H4K20me3 in normal and disease conditions in the context of NSPC.

Molecular targets of chromatin repressive mark H3K9me3 in primate progenitor cells within adult neurogenic niches.

Histone 3 Lysine 9 (H3K9) methylation is known to be associated with pericentric heterochromatin and important in genomic stability. In this study, we show that trimethylation at H3K9 (H3K9me3) is enriched in an adult neural stem cell niche- the subventricular zone (SVZ) on the walls of the lateral ventricle in both rodent and non-human primate baboon brain. Previous studies have shown that there is significant correlation between baboon and human regarding genomic similarity and brain structure, suggesting that findings in baboon are relevant to human. To understand the function of H3K9me3 in this adult neurogenic niche, we performed genome-wide analyses using ChIP-Seq (chromatin immunoprecipitation and deep-sequencing) and RNA-Seq for in vivo SVZ cells purified from baboon brain. Through integrated analyses of ChIP-Seq and RNA-Seq, we found that H3K9me3-enriched genes associated with cellular maintenance, post-transcriptional and translational modifications, signaling pathways, and DNA replication are expressed, while genes involved in axon/neuron, hepatic stellate cell, or immune-response activation are not expressed. As neurogenesis progresses in the adult SVZ, cell fate restriction is essential to direct proper lineage commitment. Our findings highlight that H3K9me3 repression in undifferentiated SVZ cells is engaged in the maintenance of cell type integrity, implicating a role for H3K9me3 as an epigenetic mechanism to control cell fate transition within this adult germinal niche.

Epigenetic regulation by chromatin activation mark H3K4me3 in primate progenitor cells within adult neurogenic niche.

Histone 3 lysine 4 trimethylation (H3K4me3) is known to be associated with transcriptionally active or poised genes and required for postnatal neurogenesis within the subventricular zone (SVZ) in the rodent model. Previous comparisons have shown significant correlation between baboon (Papio anubis) and human brain. In this study, we demonstrate that chromatin activation mark H3K4me3 is present in undifferentiated progenitor cells within the SVZ of adult baboon brain. To identify the targets and regulatory role of H3K4me3 within the baboon SVZ, we developed a technique to purify undifferentiated SVZ cells while preserving the endogenous nature without introducing culture artifact to maintain the in vivo chromatin state for genome-wide studies (ChIP-Seq and RNA-Seq). Overall, H3K4me3 is significantly enriched for genes involved in cell cycle, metabolism, protein synthesis, signaling pathways, and cancer mechanisms. Additionally, we found elevated levels of H3K4me3 in the MRI-classified SVZ-associated Glioblastoma Multiforme (GBM), which has a transcriptional profile that reflects the H3K4me3 modifications in the undifferentiated progenitor cells of the baboon SVZ. Our findings highlight the importance of H3K4me3 in coordinating distinct networks and pathways for life-long neurogenesis, and suggest that subtypes of GBM could occur, at least in part, due to aberrant H3K4me3 epigenetic regulation.

Molecular Characteristics in MRI-Classified Group 1 Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is a clinically and pathologically heterogeneous brain tumor. Previous studies of transcriptional profiling have revealed biologically relevant GBM subtypes associated with specific mutations and dysregulated pathways. Here, we applied a modified proteome to uncover abnormal protein expression profile in a MRI-classified group I GBM (GBM1), which has a spatial relationship with one of the adult neural stem cell niches, subventricular zone (SVZ). Most importantly, we identified molecular characteristics in this type of GBM that include up-regulation of metabolic enzymes, ribosomal proteins, and heat shock proteins. As GBM1 often recurs at great distances from the initial lesion, the rewiring of metabolism, and ribosomal biogenesis may facilitate cancer cells' growth and survival during tumor progression. The intimate contact between GBM1 and the SVZ raises the possibility that tumor cells in GBM1 may be most related to SVZ cells. In support of this notion, we found that markers representing SVZ cells are highly expressed in GBM1. Emerged findings from our study provide a specific protein expression profile in GBM1 and offer better prediction or therapeutic implication for this multifocal GBM.