Master Transcriptional Regulator Research Articles

Identification and characterization of LuxR solo homolog PplR in pathogenic Pseudomonas plecoglossicida NB2011

Pseudomonas plecoglossicida is a causative agent of visceral granulomas in large yellow croaker (Larimichthys crocea). Quorum sensing (QS) is widely involved in imparting virulence to pathogenic bacteria; however, it has not been studied in P. plecoglossicida. In this study, we annotated a LuxR family transcriptional regulator in P. plecoglossicida NB2011 and designated as PplR. We aligned the protein sequence by BlastP and Clustal X2, monitored the N-acyl-homoserine lactone (AHL) signal production through cross-feeding bioassay and HC-MS/MS; investigated exogenous AHL signal binding by recombinant expression and thin layer chromatography; constructed a deletion mutant of the target gene by method of double homologous recombination; sequenced the transcript RNA and analyzed the data; additionally, characterized phenotypes of wild type and mutant strain. The LuxR homolog PplR was found to share high similarity with PpoR—the LuxR solo of Pseudomonas putida—without a cognate LuxI. The wild-type strain did not produce any AHL signals and the recombinant LuxR protein was found to bind C6-L-homoserine lactone (C6-HSL), C8-HSL, 3-oxo-C10-HSL, and 3-oxo-C12-HSL. RNA-seq analysis indicated 84 differentially expressed genes—5 upregulated and 79 downregulated—mainly enriched in gene ontology terms, such as flagella-dependent motility, integral component of membrane, DNA binding and transcription, and metal ion binding, suggesting that PplR is a master transcription regulator. The mutant strain showed attenuated biofilm-forming ability and stress resistance, and the data support a role for PplR in the regulation of these traits in P. plecoglossicida NB2011 independent of the presence of AHL signals. This is the first study to provide QS-related information on P. plecoglossicida.

Altruistic feeding and cell-cell signaling during bacterial differentiation actively enhance phenotypic heterogeneity.

Starvation triggers bacterial spore formation, a committed differentiation program that transforms a vegetative cell into a dormant spore. Cells in a population enter sporulation nonuniformly to secure against the possibility that favorable growth conditions, which put sporulation-committed cells at a disadvantage, may resume. This heterogeneous behavior is initiated by a passive mechanism: stochastic activation of a master transcriptional regulator. Here, we identify a cell-cell communication pathway containing the proteins ShfA (YabQ) and ShfP (YvnB) that actively promotes phenotypic heterogeneity, wherein Bacillus subtilis cells that start sporulating early use a calcineurin-like phosphoesterase to release glycerol, which simultaneously acts as a signaling molecule and a nutrient to delay nonsporulating cells from entering sporulation. This produced a more diverse population that was better poised to exploit a sudden influx of nutrients compared to those generating heterogeneity via stochastic gene expression alone. Although conflict systems are prevalent among microbes, genetically encoded cooperative behavior in unicellular organisms can evidently also boost inclusive fitness.

8805 Nucleostemin is a novel bifunctional transcriptional coregulator of theandrogenreceptorin prostate cancer cells

Abstract Disclosure: E. Cheung: None. C. Zhang: None. Androgen receptor (AR) is a master transcriptional regulator in prostate cancer, interacting with and recruiting distinct sets of coregulator proteins to mediate the expression of direct target genes. What are the coregulators of AR and how they function in AR transcriptional activity and prostate cancer biology are still unclear. Using chromatin-immunoprecipitation coupled with mass spectrometry, we identified a set of AR interacting proteins that are present in multiple prostate cancer cell lines. We provide biochemical, cell-based, and genomic evidence to show that Nucleostemin, also known as Guanine nucleotide-binding protein-like 3, is a novel AR coregulator. Specifically, we demonstrate Nucleostemin has dual coregulator activities, functioning as both a coactivator and a corepressor of AR-dependent transcription to regulate the expression of genes that are involved for prostate cancer progression and immune response. From cellular and animal studies, we found that inhibiting Nucleostemin sensitizes prostate cancer cells to Enzalutamide. Clinical analyses of Nucleostemin from prostate cancer patient cohorts revealed that it is expressed significantly higher in cancer than normal in tissues. Finally, we found Nucleostemin expression is an excellent predictor of disease-free survival. In conclusion, our work suggests that Nucleostemin is a novel AR coregulator and is a promising diagnostic and therapeutic target for prostate cancer treatment. Presentation: 6/2/2024

8464 A Unique SERM Reveals An Estrogen Receptor-Dependent Secretion Of DKK1 In Breast Cancer Cells

Abstract Disclosure: K.S. Young: None. G. Hancock: None. S. Kregel: None. S.W. Fanning: Grant Recipient; Self; Olema Oncology. Although Estrogen Receptor positive (ER+) breast cancer has been successfully targeted in primary tumors, the critical limitations of hormone therapies are ultimately revealed as many women often experience disease recurrence. A subset of these patients harbor a distinctive tyrosine to serine missense mutation at position 537 (Y537S) in ER leading to allele-specific transcriptional programs that enhance metastasis. ER is a ligand-dependent master transcriptional regulator. Probing unique ER structural features can reveal new transcriptional activities. We developed a structurally unconventional Selective Estrogen Receptor Modulator (SERM), T6I-29, to better understand these structure-transcriptional relationships. T6I-29 shows effective anti-tumoral activities in Y537S ESR1 breast cancer cells. To examine the anti-tumoral mechanism of T6I-29 action, we performed RNA-sequencing on ESR1 Y537S mutant breast cancer cells. Compared to other clinically relevant compounds, our drug uniquely and significantly downregulated DKK1, a tumor secreted protein known to modulate the Wnt/β-Catenin and PI3K/Akt pathways. Upregulation of DKK1 correlates with metastatic burden across multiple cancers but has not been evaluated in ESR1 mutant breast cancers. We find that DKK1 levels are significantly elevated in the plasma of 108 ER+ breast cancer patients compared to 100 matched healthy donors. Furthermore, in wild-type (WT) PIK3CA breast cancer cell lines, those with Y537S ESR1 mutation show constitutive expression and secretion of DKK1, while DKK1 expression and secretion is estrogen-dependent in WT breast cancer cells. Intriguingly, WT cells show significant surface bound DKK1, which is secreted into the media upon hormone stimulation and blocked by T6I-29. Understanding the ER-dependence and functional importance of DKK1 secretion offers a novel approach to potentially reduce breast cancer metastatic burden. Presentation: 6/3/2024

7993 Thienopyrimidine-Based Estrogen Receptor Modulators Adopt Unique Ligand Binding Poses to Elicit Anti-Proliferative Activities in ER+ Breast Cancer Cells

Abstract Disclosure: E.C. Fink: None. S.L. Mateus: None. N. Meganathan: None. V.K. Sammeta: None. J.D. Norris: None. D.P. McDonnell: None. T. Willson: None. S.W. Fanning: None. One in eight women will be diagnosed with breast cancer in their lifetimes. Estrogen receptor alpha (ER) drives breast cancer pathology and is expressed in approximately seventy percent of tumors. Hormone therapies are used to treat and prevent the metastasis of ER+ breast cancers and these patients have favorable 5-year survivals. However, most breast cancer patient deaths are ER+. While the next generation of antiestrogens have shown promise in the advanced setting, more work needs to be done to fully address these mortalities. ER is a ligand-dependent master transcriptional regulator, whereby ligand-specific ER conformational ensembles redirect multiple programs to produce oncogenic or therapeutic endpoints. To better understand ER structure-activity relationships, we have developed new antiestrogenic small molecules based on a thienopyrimidine scaffold. Comprehensive structural, ER activity, and cancer endpoint profiling shows that these new molecules adopt a unique ligand binding pose, which perturbs new structural elements within the orthosteric hormone binding pocket. These molecules downregulate ER target genes and halt the proliferation of ER+ breast cancer cells. Presentation: 6/3/2024

VEGF-dependent testicular vascularisation involves MEK1/2 signalling and the essential angiogenesis factors, SOX7 and SOX17

BackgroundAbnormalities of in utero testis development are strongly associated with reproductive health conditions, including male infertility and testis cancer. In mouse testes, SOX9 and FGF9 support Sertoli cell development, while VEGF signalling is essential for the establishment of vasculature. The mitogen-activated protein kinase (MAPK) pathway is a major signalling cascade, essential for cell proliferation, differentiation and activation of Sry during primary sex-determination, but little is known about its function during fetal testis morphogenesis. We explored potential functions of MAPK signalling immediately after the establishment of testis cords in embryonic day (E)12.5 Oct4-eGFP transgenic mouse testes cultured using a MEK1/2 inhibitor.ResultsRNA sequencing in isolated gonadal somatic cells identified 116 and 114 differentially expressed genes after 24 and 72 h of MEK1/2 inhibition, respectively. Ingenuity Pathway Analysis revealed an association of MEK1/2 signalling with biological functions such as angiogenesis, vasculogenesis and cell migration. This included a failure to upregulate the master transcriptional regulators of vascular development, Sox7 and Sox17, VEGF receptor genes, the cell adhesion factor gene Cd31 and a range of other endothelial cell markers such as Cdh5 (encoding VE-cadherin) and gap junction genes Gja4 and Gja5. In contrast, only a small number of Sertoli cell enriched genes were affected. Immunofluorescent analyses of control testes revealed that the MEK1/2 downstream target, ERK1/2 was phosphorylated in endothelial cells and Sertoli cells. Inhibition of MEK1/2 eliminated pERK1/2 in fetal testes, and CD31, VE-cadherin, SOX7 and SOX17 and endothelial cells were lost. Consistent with a role for VEGF in driving endothelial cell development in the testis, inhibition of VEGFR also abrogated pERK1/2 and SOX7 and SOX17 expressing endothelial cells. Moreover, while Sertoli cell proliferation and localisation to the testis cord basement membrane was disrupted by inhibition of MEK1/2, it was unaffected by VEGFR inhibition. Instead, inhibition of FGF signalling compromised Sertoli cell proliferation and localisation to the testis cord basement membrane.ConclusionsTogether, our data highlight an essential role for VEGF-dependent MEK1/2 signalling in promoting vasculature and indicate that FGF signalling through MEK1/2 regulates Sertoli cell organisation in the developing mouse testis.

The Nrf2-HO-1 system and inflammaging.

Nrf2 is a master transcriptional regulator of a number of genes involved in the adaptive response to oxidative stress. Among the genes upregulated by Nrf2, heme oxygenase-1 (HO-1) has received significant attention, given that the products of HO-1-induced heme catabolism have well established antioxidant and anti-inflammatory properties. This is evidenced in numerous models of inflammatory and autoimmune disease whereby induction of HO-1 expression or administration of tolerable amounts of HO-1 reaction products can ameliorate disease symptoms. Unsurprisingly, Nrf2 and HO-1 are now considered viable drug targets for a number of conditions. In recent years, the term 'inflammaging' has been used to describe the low-grade chronic inflammation observed in aging/aged cells. Increased oxidative stress is also a key factor associated with aging and there is convincing evidence that Nrf2, not only declines with age, but that Nrf2 and HO-1 can reduce cellular senescence and the senescence-associated secretory phenotype (SASP) which is now considered an underlying driver of age-related inflammatory disease. In this review, we describe the role of oxidative stress in 'inflammaging' and highlight the potential anti-aging properties of the Nrf2-HO-1 system. We also highlight established and newly emerging Nrf2 activators and their therapeutic application in age-related disease.

Development of DuoMYC: a synthetic cell penetrant miniprotein that efficiently inhibits the oncogenic transcription factor MYC.

The master regulator transcription factor MYC is implicated in numerous human cancers, and its targeting is a long-standing challenge in drug development. MYC is a typical 'undruggable' target, with no binding pockets on its DNA binding domain and extensive intrinsically disordered regions. Rather than trying to target MYC directly with classical modalities, here we engineer synthetic miniproteins that can bind to MYC's target DNA, the enhancer box (E-Box), and potently inhibit MYC-driven transcription. We crafted the miniproteins via structure-based design and a combination of solid phase peptide synthesis and site-specific crosslinking. Our lead variant, DuoMYC, binds to E-Box DNA with high affinity (KD ~ 0.1 µM) and is able to enter cells and inhibit MYC-driven transcription with submicromolar potency (IC50 = 464 nM) as shown by reporter gene assay and confirmed by RNA sequencing. Notably, DuoMYC surpasses the efficacy of several other recently developed MYC inhibitors. Our results highlight the potential of engineered synthetic protein therapeutics for addressing challenging intracellular targets.

Development of DuoMYC: a synthetic cell penetrant miniprotein that efficiently inhibits the oncogenic transcription factor MYC

The master regulator transcription factor MYC is implicated in numerous human cancers, and its targeting is a long‐standing challenge in drug development. MYC is a typical ‘undruggable’ target, with no binding pockets on its DNA binding domain and extensive intrinsically disordered regions. Rather than trying to target MYC directly with classical modalities, here we engineer synthetic miniproteins that can bind to MYC’s target DNA, the enhancer box (E‐Box), and potently inhibit MYC‐driven transcription. We crafted the miniproteins via structure‐based design and a combination of solid phase peptide synthesis and site‐specific crosslinking. Our lead variant, DuoMYC, binds to E‐Box DNA with high affinity (KD ~ 0.1 µM) and is able to enter cells and inhibit MYC‐driven transcription with submicromolar potency (IC50 = 464 nM) as shown by reporter gene assay and confirmed by RNA sequencing. Notably, DuoMYC surpasses the efficacy of several other recently developed MYC inhibitors. Our results highlight the potential of engineered synthetic protein therapeutics for addressing challenging intracellular targets.

OVO positively regulates essential maternal pathways by binding near the transcriptional start sites in the Drosophila female germline.

Differentiation of female germline stem cells into a mature oocyte includes the expression of RNAs and proteins that drive early embryonic development in Drosophila. We have little insight into what activates the expression of these maternal factors. One candidate is the zinc-finger protein OVO. OVO is required for female germline viability and has been shown to positively regulate its own expression, as well as a downstream target, ovarian tumor, by binding to the transcriptional start site (TSS). To find additional OVO targets in the female germline and further elucidate OVO's role in oocyte development, we performed ChIP-seq to determine genome-wide OVO occupancy, as well as RNA-seq comparing hypomorphic and wild type rescue ovo alleles. OVO preferentially binds in close proximity to target TSSs genome-wide, is associated with open chromatin, transcriptionally active histone marks, and OVO-dependent expression. Motif enrichment analysis on OVO ChIP peaks identified a 5'-TAACNGT-3' OVO DNA binding motif spatially enriched near TSSs. However, the OVO DNA binding motif does not exhibit precise motif spacing relative to the TSS characteristic of RNA polymerase II complex binding core promoter elements. Integrated genomics analysis showed that 525 genes that are bound and increase in expression downstream of OVO are known to be essential maternally expressed genes. These include genes involved in anterior/posterior/germ plasm specification (bcd, exu, swa, osk, nos, aub, pgc, gcl), egg activation (png, plu, gnu, wisp, C(3)g, mtrm), translational regulation (cup, orb, bru1, me31B), and vitelline membrane formation (fs(1)N, fs(1)M3, clos). This suggests that OVO is a master transcriptional regulator of oocyte development and is responsible for the expression of structural components of the egg as well as maternally provided RNAs that are required for early embryonic development.

OVO positively regulates essential maternal pathways by binding near the transcriptional start sites in the Drosophila female germline

Differentiation of female germline stem cells into a mature oocyte includes the expression of RNAs and proteins that drive early embryonic development in Drosophila. We have little insight into what activates the expression of these maternal factors. One candidate is the zinc-finger protein OVO. OVO is required for female germline viability and has been shown to positively regulate its own expression, as well as a downstream target, ovarian tumor, by binding to the transcriptional start site (TSS). To find additional OVO targets in the female germline and further elucidate OVO’s role in oocyte development, we performed ChIP-seq to determine genome-wide OVO occupancy, as well as RNA-seq comparing hypomorphic and wild type rescue ovo alleles. OVO preferentially binds in close proximity to target TSSs genome-wide, is associated with open chromatin, transcriptionally active histone marks, and OVO-dependent expression. Motif enrichment analysis on OVO ChIP peaks identified a 5’-TAACNGT-3’ OVO DNA binding motif spatially enriched near TSSs. However, the OVO DNA binding motif does not exhibit precise motif spacing relative to the TSS characteristic of RNA polymerase II complex binding core promoter elements. Integrated genomics analysis showed that 525 genes that are bound and increase in expression downstream of OVO are known to be essential maternally expressed genes. These include genes involved in anterior/posterior/germ plasm specification (bcd, exu, swa, osk, nos, aub, pgc, gcl), egg activation (png, plu, gnu, wisp, C(3)g, mtrm), translational regulation (cup, orb, bru1, me31B), and vitelline membrane formation (fs(1)N, fs(1)M3, clos). This suggests that OVO is a master transcriptional regulator of oocyte development and is responsible for the expression of structural components of the egg as well as maternally provided RNAs that are required for early embryonic development.

Integrating Blood Biomarkers and Marine Brown Algae-Derived Inhibitors in Parkinson's Disease: A Multi-scale Approach from Interactomics to Quantum Mechanics.

Parkinson's disease (PD) involves alpha-synuclein accumulation according to Braak's pattern, with diverse clinical progressions that complicate diagnosis and treatment. We aimed to correlate Braak's pattern with rapid progressive PD to identify blood-based biomarkers and therapeutic targets exploiting brown algae-derived bioactives for potential treatment. We implemented a systematic workflow of transcriptomic profiling, co-expression networks, cluster profiling, transcriptional regulator identification, molecular docking, quantum calculations, and dynamic simulations. The transcriptomic analyses exhibited highly expressed genes at each Braak's stage and in rapidly progressive PD. Co-expression networks for Braak's stages were built, and the top five clusters from each stage displayed significant overlap with differentially expressed genes in rapidly progressive PD, indicating shared biomarkers between the blood and the PD brain. Further investigation showed, NF-kappa-B p105 as the master transcriptional regulator of these biomarkers. Molecular docking screened phlorethopentafuhalol-A from brown algae, exhibiting a superior inhibitory effect with p105 (- 7.51kcal/mol) by outperforming PD drugs and anti-inflammatory compounds (- 5.73 to - 4.38kcal/mol). Quantum mechanics and molecular mechanics (QM/MM) calculations and dynamic simulations have confirmed the interactive stability of phlorethopentafuhalol-A with p105. Overall, our combined computational study shows that phlorethopentafuhalol-A derived from brown algae, may have healing properties that could help treat PD.

SERPINA3 is a marker of cartilage differentiation and is essential for the expression of extracellular matrix genes during early chondrogenesis

Serine proteinase inhibitors (serpins) are a family of structurally similar proteins which regulate many diverse biological processes from blood coagulation to extracellular matrix (ECM) remodelling. Chondrogenesis involves the condensation and differentiation of mesenchymal stem cells (MSCs) into chondrocytes which occurs during early development. Here, and for the first time, we demonstrate that one serpin, SERPINA3 (gene name SERPINA3, protein also known as alpha-1 antichymotrypsin), plays a critical role in chondrogenic differentiation. We observed that SERPINA3 expression was markedly induced at early time points during in vitro chondrogenesis. We examined the expression of SERPINA3 in human cartilage development, identifying significant enrichment of SERPINA3 in developing cartilage compared to total limb, which correlated with well-described markers of cartilage differentiation. When SERPINA3 was silenced using siRNA, cartilage pellets were smaller and contained lower proteoglycan as determined by dimethyl methylene blue assay (DMMB) and safranin-O staining. Consistent with this, RNA sequencing revealed significant downregulation of genes associated with cartilage ECM formation perturbing chondrogenesis. Conversely, SERPINA3 silencing had a negligible effect on the gene expression profile during osteogenesis suggesting the role of SERPINA3 is specific to chondrocyte differentiation. The global effect on cartilage formation led us to investigate the effect of SERPINA3 silencing on the master transcriptional regulator of chondrogenesis, SOX9. Indeed, we observed that SOX9 protein levels were markedly reduced at early time points suggesting a role for SERPINA3 in regulating SOX9 expression and activity. In summary, our data support a non-redundant role for SERPINA3 in enabling chondrogenesis via regulation of SOX9 levels.

Sustained cancer-relevant alternative RNA splicing events driven by PRMT5 in high-risk neuroblastoma.

Protein arginine methyltransferase 5 (PRMT5) is over-expressed in a wide variety of cancers and is implicated as having a key oncogenic role, achieved in part through its control of the master transcription regulator E2F1. We investigated the relevance of PRMT5 and E2F1 in neuroblastoma (NB) and found that elevated expression of PRMT5 and E2F1 occurs in poor prognosis high-risk disease and correlates with an amplified Myelocytomatosis viral-related oncogene, neuroblastoma-derived (MYCN) gene. Our results show that MYCN drives the expression of splicing factor genes that, together with PRMT5 and E2F1, lead to a deregulated alternative RNA splicing programme that impedes apoptosis. Pharmacological inhibition of PRMT5 or inactivation of E2F1 restores normal splicing and renders NB cells sensitive to apoptosis. Our findings suggest that a sustained cancer-relevant alternative RNA splicing programme desensitises NB cells to apoptosis, and identify PRMT5 as a potential therapeutic target for high-risk disease.

An HSF1–JMJD6–HSP feedback circuit promotes cell adaptation to proteotoxic stress

Heat Shock Factor 1 (HSF1) is best known as the master transcriptional regulator of the heat-shock response (HSR), a conserved adaptive mechanism critical for protein homeostasis (proteostasis). Combining a genome-wide RNAi library with an HSR reporter, we identified Jumonji domain-containing protein 6 (JMJD6) as an essential mediator of HSF1 activity. In follow-up studies, we found that JMJD6 is itself a noncanonical transcriptional target of HSF1 which acts as a critical regulator of proteostasis. In a positive feedback circuit, HSF1 binds and promotes JMJD6 expression, which in turn reduces heat shock protein 70 (HSP70) R469 monomethylation to disrupt HSP70-HSF1 repressive complexes resulting in enhanced HSF1 activation. Thus, JMJD6 is intricately wired into the proteostasis network where it plays a critical role in cellular adaptation to proteotoxic stress.

Nuclear factor erythroid 2-related factor 2 activation in streptozotocin-induced diabetic rats normalize renal hemodynamics and oxygen consumption.

Diabetic kidney disease is a major contributor to end stage renal disease. A change in kidney oxygen homeostasis leading to decreased tissue oxygen tension is an important factor initiating alterations in kidney function in diabetes. However, the mechanism contributing to changed oxygen homeostasis is still unclear. Hyperglycemia-induced production of reactive oxygen species and an altered response to them have previously been demonstrated. In the present study, chronic treatment with DL-sulforaphane to induce nuclear factor erythroid 2-related factor 2 (Nrf2) expression, a master transcriptional regulator binding to antioxidant response elements inducing increased protection against reactive oxygen species, is studied. Sprague-Dawley rats were made diabetic using streptozotocin and either left untreated or received daily subcutaneous injections of DL-sulforaphane for 4 weeks. Age-matched non-diabetic rats served as controls. After 4 weeks of treatment, rats were anesthetized using thiobutabarbital, and kidney functions were studied in terms of glomerular filtration rate (GFR), renal blood flow (RBF), sodium transport, kidney oxygen consumption, and kidney oxygen tension. Mitochondria was isolated from kidney cortical tissue and investigated using high-resolution respirometry. GFR was increased in diabetics but not RBF resulting in increased filtration fraction in diabetics. DL-sulforaphane treatment did not affect RBF and GFR in controls but decreased the same parameters in diabetics. Increased GFR resulted in increased sodium transport and oxygen consumption, hence decreased efficiency in diabetics compared to controls. Increased oxygen consumption in diabetics resulted in decreased cortical tissue oxygen tension. DL-sulforaphane treatment decreased oxygen consumption in diabetics, whereas transport efficiency was not significantly affected. DL-sulforaphane treatment increased cortical pO2 in diabetics. DL-sulforaphane treatment affects renal hemodynamics, improving cortical oxygen tension but not mitochondrial efficiency.

Th17 pathway‐related immune signatures in the pathobiology of myasthenia gravis: Integrating the roles of regulatory/effector cytokines and transcription factors

AbstractObjectivesMyasthenia gravis (MG) is an autoimmune disorder mediated by antibodies against the acetylcholine receptor (AChR), muscle specific kinase (MuSK), and other antigens in the neuromuscular junction. Antibody production is influenced by T lymphocytes. The T helper 17 (Th17) subset, an inflammatory lineage of Th cells, is associated with several autoimmune diseases. Functional interactions between T lymphocytes and pathogenic antibody responses including aberrant Th17 cell responses have been reported in MG. However, the precise mechanism(s) underlying the activation and/or pathogenic transformation of Th17 cells are not clearly known. The current study aimed at simultaneously exploring the roles of the inducers, master regulator transcription factors, and effectors of Th17 cells in patients with MG.MethodsIn this cross‐sectional study, quantification of gene expression of IL6, IL17A, STAT3, and RORC in the peripheral mononuclear cells by quantitative polymerase chain reaction (qPCR) as well as estimation of plasma levels of IL‐1β, IL‐6, and IL‐17A cytokines by multiplex suspension assay were carried out in 59 patients with MG and 61 healthy controls.ResultsGene expression levels of IL17A were significantly upregulated in patients as compared to healthy controls. The plasma levels of IL‐1β, IL‐6, and IL‐17A were significantly elevated in patients compared to healthy controls. IL17A as well as RORC gene expressions correlated with the clinical features. IL17A gene expression levels were higher in AChR‐MG patients.ConclusionThe present study supports the crucial role of the Th17 pathway in the pathobiology of MG, including its potential influence on disease severity.

Single-nucleus multi-omics of Parkinson’s disease reveals a glutamatergic neuronal subtype susceptible to gene dysregulation via alteration of transcriptional networks

The genetic architecture of Parkinson’s disease (PD) is complex and multiple brain cell subtypes are involved in the neuropathological progression of the disease. Here we aimed to advance our understanding of PD genetic complexity at a cell subtype precision level. Using parallel single-nucleus (sn)RNA-seq and snATAC-seq analyses we simultaneously profiled the transcriptomic and chromatin accessibility landscapes in temporal cortex tissues from 12 PD compared to 12 control subjects at a granular single cell resolution. An integrative bioinformatic pipeline was developed and applied for the analyses of these snMulti-omics datasets. The results identified a subpopulation of cortical glutamatergic excitatory neurons with remarkably altered gene expression in PD, including differentially-expressed genes within PD risk loci identified in genome-wide association studies (GWAS). This was the only neuronal subtype showing significant and robust overexpression of SNCA. Further characterization of this neuronal-subpopulation showed upregulation of specific pathways related to axon guidance, neurite outgrowth and post-synaptic structure, and downregulated pathways involved in presynaptic organization and calcium response. Additionally, we characterized the roles of three molecular mechanisms in governing PD-associated cell subtype-specific dysregulation of gene expression: (1) changes in cis-regulatory element accessibility to transcriptional machinery; (2) changes in the abundance of master transcriptional regulators, including YY1, SP3, and KLF16; (3) candidate regulatory variants in high linkage disequilibrium with PD-GWAS genomic variants impacting transcription factor binding affinities. To our knowledge, this study is the first and the most comprehensive interrogation of the multi-omics landscape of PD at a cell-subtype resolution. Our findings provide new insights into a precise glutamatergic neuronal cell subtype, causal genes, and non-coding regulatory variants underlying the neuropathological progression of PD, paving the way for the development of cell- and gene-targeted therapeutics to halt disease progression as well as genetic biomarkers for early preclinical diagnosis.

Nuclear factor Y, a key player in neuronal gene regulation.

Establishing a functional nervous system is a complex process requiring tightly controlled gene expression programs to achieve the correct differentiation of distinct neuronal subtypes. The molecular programs required for neurons to acquire neuron-type-specific, and core pan-neuronal features mostly rely on sequence-specific transcription factors (TFs), which recognize and bind to cis-regulatory motifs present in the promoters of target genes. Recently, we investigated the role and mode of action of the NF-Y complex, a ubiquitously expressed transcriptional master regulator, in the Caenorhabditis elegans nervous system. We found that NFYA-1 is a pervasive regulator of neuron-specific and pan-neuronal gene batteries that are essential for neuronal development and function. Furthermore, we concluded that NFYA-1 acts cell autonomously by either directly binding to conserved motifs in target gene promoter regions or indirectly by regulating other transcriptional regulators to fine-tune gene expression. However, further studies are required to fully define the impact of the NF-Y complex on nervous system regulatory networks and how NF-Y coordinates with other TFs in this regard.

Incomplete autophagy and increased cholesterol synthesis during neuronal cell death caused by a synthetic cannabinoid, CP-55,940

There is a propensity for synthetic cannabinoid abuse to spread worldwide. CP-55,940, a synthetic cannabinoid having the ability to activate both CB1 and CB2 receptors, has been shown to induce cell death in neurons as well as other cells. Here we investigate molecular events underling the adverse effects of CP-55,940 on neuronal cells. Exposure of mouse neuroblastoma Neuro2a cells to 10–50 µM CP-55,940 results in concentration-dependent cell death that is not accompanied by an induction of apoptosis. CP-55,940 also stimulates autophagy, but the stimulation is not followed by an increase in autophagic degradation. Transcriptome analysis using DNA microarray revealed the increased expression of genes for the cholesterol biosynthesis pathway that is associated with the activation of SREBP-2, the master transcriptional regulator of cholesterol biosynthesis. However, free cholesterol is localized mainly to cytoplasmic structures, although it is localized to the plasma membrane in healthy cells. Thus, cellular trafficking of cholesterol seems to be somewhat disrupted in CP-55,940 stimulated cells. These results show for the first time that CP-55,940 stimulates autophagy as well as cholesterol biosynthesis, although not all the processes involved in the cellular response to CP-55,940 seem to be complete in these cells.