Cytoskeletal Genes Research Articles

Histopathologic Marks of Tongue in a Mouse Model of Oculopharyngeal Muscular Dystrophy Suggest Biomechanical Defects.

The tongue facilitates vital activities such as swallowing. Difficulty swallowing (dysphagia) is common in the elderly population and in patients with adult-onset neuromuscular disease. In oculopharyngeal muscular dystrophy (OPMD), dysphagia is often the first symptom. OPMD is an autosomal-dominant myopathy caused by a trinucleotide-expansion mutation in the gene encoding nuclear poly(A)-binding protein (PABPN)-1. Expanded-mutant PABPN1 forms insoluble nuclear aggregates that reduce the levels of the soluble form. Clinical tongue involvement in OPMD has been documented but is poorly understood. Histopathologic analysis of the tongue in an OPMD mouse model was done by light and electron microscopy combined with RNA sequencing. PABPN1 nuclear aggregates were found at moderate levels, whereas deposition of insoluble PABPN1 in blood vessels was prominent already at age 4 months. Muscle wasting of the tongue was age associated. RNA signatures of the OPMD tongue were enriched for mitochondrial and cytoskeletal genes. Electron microscopy revealed abnormalities in sarcomere and mitochondria organization in A17/+ mice, suggesting an energy and contractile deficit in OPMD tongue. This detailed analysis of the histopathology of the tongue in the A17/+ mouse model opens new avenues for understanding the mechanisms of dysphagia.

Single-molecule analysis reveals that IPMK enhances the DNA-binding activity of the transcription factor SRF.

Serum response factor (SRF) is a master transcription factor that regulates immediate early genes and cytoskeletal remodeling genes. Despite its importance, the mechanisms through which SRF stably associates with its cognate promoter remain unknown. Our biochemical and protein-induced fluorescence enhancement analyses showed that the binding of SRF to serum response elementwas significantly increased by inositol polyphosphate multikinase (IPMK), an SRF cofactor. Moreover, real-time tracking of SRF loci in live cell nuclei demonstrated that the chromatin residence time of SRF was reduced by IPMK depletion in fibroblasts. Conversely, elevated IPMK levels extended the SRF-chromatin association. We identified that IPMK binds to the intrinsically disordered region of SRF, which is required for the IPMK-induced stable interaction of SRF with DNA. IPMK-mediated conformational changes in SRF were observed by single-molecule fluorescence resonance energy transfer assays. Therefore, our findings demonstrate that IPMK is a critical factor for promoting high-affinity SRF-chromatin association and provide insights into the mechanisms of SRF-dependent transcription control via chaperone-like activity.

Substrate curvature influences cytoskeletal rearrangement and modulates macrophage phenotype.

Inflammation is a vital immune response, tightly orchestrated through both biochemical and biophysical cues. Dysregulated inflammation contributes to chronic diseases, highlighting the need for novel therapies that modulate immune responses with minimal side effects. While several biochemical pathways of inflammation are well understood, the influence of physical properties such as substrate curvature on immune cell behavior remains underexplored. This study investigates how substrate curvature impacts macrophage cytoskeletal dynamics, gene expression, and immunophenotype through mechanosensitive pathways. Gelatin-based microgels with tunable surface curvatures were fabricated via water-in-oil emulsification and crosslinked with genipin. Microgels were sorted into three size ranges, yielding high (40-50 µm), intermediate (150-250 µm), and low (350-400 µm) curvature profiles. Macrophages were seeded onto these microgels, and cytoskeletal dynamics were examined using confocal microscopy, SEM, and actin-specific staining. Gene expression of pro- and anti-inflammatory markers was quantified using qPCR. The role of actin polymerization was assessed using Latrunculin-A (Lat-A) treatment. Macrophages adhered effectively to both high- and low-curvature microgels, displaying curvature-dependent morphological changes. Confocal imaging revealed that macrophages on low-curvature microgels exhibited significantly higher F-actin density than those on high-curvature microgels. Correspondingly, qPCR analysis showed upregulation of pro-inflammatory markers (e.g., Tnf, Nos2) in high-curvature conditions, while anti-inflammatory markers (e.g., Arg1) were elevated in low-curvature conditions. Lat-A treatment reduced F-actin density and modulated gene expression patterns, confirming the cytoskeletal regulation of macrophage phenotype. These findings demonstrate that substrate curvature influences macrophage behavior by modulating cytoskeletal dynamics and associated immunophenotypic markers through actin-mediated transcriptional pathways. By controlling curvature, therapeutic biomaterials may direct immune responses, offering a new avenue for treating inflammatory diseases. This mechanobiological approach presents a promising strategy for precision immunomodulation in regenerative medicine.

Characterization of Cytoskeletal Profilin Genes in Plasticity Elongation of Mesocotyl and Coleoptile of Maize Under Diverse Abiotic Stresses.

The plasticity elongation of mesocotyl (MES) and coleoptile (COL) largely determines the morphology of maize seedlings under abiotic stresses. The profilin (PRF) proteins play a pivotal role in cytoskeleton dynamics and plant development via regulating actin polymerization. However, little is known about whether and how the expression of the ZmPRF gene family regulates MES and COL elongation in maize under adverse abiotic stresses. Here, a total of eight ZmPRF gene members were identified in the maize genome. They were mainly located in the cytoplasm, chloroplast, and mitochondrion, and clearly divided into four classes, based on phylogenetic analysis. Segmental duplication was the main driver for the expansion of ZmPRF genes. Ka/Ks analysis indicated that most ZmPRF genes were intensely purified and selected. Promoter cis-element analysis suggested their potential roles in response to growth and development, stress adaption, hormone response, and light response. The protein-protein interaction network and two independent RNA-sequencing analyses revealed that eight ZmPRF genes and their thirty-seven interacting genes showed varied expression patterns in MES and COL of three maize genotypes under different sowing depths, 24-epibrassinolide application, and light spectral-quality treatments, of which ZmPRF3.3 was a potential core conserved gene for breeding application. Moreover, the quantitative real-time PCR (qRT-PCR) verified that the relative expression levels of most ZmPRF genes in MES and COL under above treatments were significantly correlated with the plasticity elongation of MES and COL in maize. Therefore, these results perform a comprehensive overview of the ZmPRF family and will provide valuable information for the validation of the function of ZmPRF genes in maize development under diverse abiotic stress.

Maternal exposure to 4-tert-octylphenol causes alterations in the morphology and function of microglia in the offspring mouse brain

4-tert-Octylphenol (OP), an endocrine disrupting chemical is widely used in the production of industrial products. Prenatal exposure to endocrine-disrupting chemicals negatively affects the brain. However, the influence of OP exposure during neurodevelopment in adult offspring remains unclear. Thus, in the present study, we investigated the effects of maternal OP exposure on brain development in adult offspring by analyzing primary glial cell cultures and mice. Our findings revealed that OP exposure led to a specific increase in the mRNA expression of the ionized calcium-binding adapter molecule 1 (Iba-1) and the proportion of amoeboid microglia in the primary glial cell culture and adult offspring mice. Exposure to OP increased the transcriptional activation of Iba-1 and estrogen response element, which were counteracted by estrogen receptor antagonists ICI 182,780. Moreover, OP exposure increased the nuclear localization of the estrogen receptor. Remarkably, OP exposure decreased the mRNA expression levels of proinflammatory cytokines and genes associated with immune response in the brains of the offspring. OP exposure upregulated actin filament-related genes and altered cytoskeletal gene expression, as demonstrated by microarray analysis. The morphological changes in microglia did not result in an inflammatory response following lipopolysaccharide treatment. Taken together, the effects of OP exposure during neurodevelopment persist into adulthood, resulting in microglial dysfunction mediated by estrogen receptor signaling pathways in the brains of adult offspring mice.

RNA sequencing uncovers key players of cartilage calcification: potential implications for osteoarthritis pathogenesis.

Osteoarthritis (OA) is a joint disease linked with pathologic cartilage calcification, caused by the deposition of calcium-containing crystals by chondrocytes. Despite its clinical significance, the precise mechanisms driving calcification remain elusive. This study aimed to identify crucial players in cartilage calcification, offering insights for future targeted interventions against OA. Primary murine chondrocytes were stimulated with secondary calciprotein particles (CPP2) or left untreated (NT) for 6 h. Calcification was assessed by alizarin red staining. RNA was analyzed by Bulk RNA sequencing. Differentially expressed (DE) genes were identified (cutoff: abs(LogFC)>1 and adj.p-val < 0.05), and top 50 DE genes were cross-referenced with human OA datasets from previous studies (ie healthy vs OA cartilage, or undamaged vs damaged cartilage). RNA from NT and CPP2-stimulated primary human OA chondrocytes were used to validate genes by qPCR. CPP2 induced crystal formation by chondrocytes and significantly modulated 1466 genes. Out of the top 50 DE genes in CPP2, 27 were confirmed in published OA cartilage datasets. Of those genes, some are described in calcification and/or OA (Errfi1, Ngf, Inhba, Col9a1). Two additional ones (Rcan1, Tnfrsf12a) appear novel and interesting in the context of calcification and OA. We validated modulation of these six genes in calcifying human chondrocytes from 5 patients. Ultimately, we unveiled two distinct gene families modulated by CPP2: the first comprised cytoskeletal genes (Actb, Tpm1, Cfl1, Tagln2, Lmna), while the second encompassed extracellular matrix genes (Fmod, Sparc, Col9a1, Cnmd). CPP2 modulates genes in chondrocytes that could represent new targets for therapeutic interventions in OA.

IL-2 delivery to CD8+ T cells during infection requires MRTF/SRF-dependent gene expression and cytoskeletal dynamics

Paracrine IL-2 signalling drives the CD8 + T cell expansion and differentiation that allow protection against viral infections, but the underlying molecular events are incompletely understood. Here we show that the transcription factor SRF, a master regulator of cytoskeletal gene expression, is required for effective IL-2 signalling during L. monocytogenes infection. Acting cell-autonomously with its actin-regulated cofactors MRTF-A and MRTF-B, SRF is dispensible for initial TCR-mediated CD8+ T cell proliferation, but is required for sustained IL-2 dependent CD8+ effector T cell expansion, and persistence of memory cells. Following TCR activation, Mrtfab-null CD8+ T cells produce IL-2 normally, but homotypic clustering is impaired both in vitro and in vivo. Expression of cytoskeletal structural and regulatory genes, most notably actins, is defective in Mrtfab-null CD8+ T cells. Activation-induced cell clustering in vitro requires F-actin assembly, and Mrtfab-null cell clusters are small, contain less F-actin, and defective in IL-2 retention. Clustering of Mrtfab-null cells can be partially restored by exogenous actin expression. IL-2 mediated CD8+ T cell proliferation during infection thus depends on the control of cytoskeletal dynamics and actin gene expression by MRTF-SRF signalling.

Cardiac Atrophy, Dysfunction, and Metabolic Impairments: A Cancer-Induced Cardiomyopathy Phenotype

Muscle atrophy and weakness are prevalent features of cancer. Although extensive research has characterized skeletal muscle wasting in cancer cachexia, limited studies have investigated how cardiac structure and function are affected by therapy-naive cancer. Herein, orthotopic, syngeneic models of epithelial ovarian cancer and pancreatic ductal adenocarcinoma, and a patient-derived pancreatic xenograft model, were used to define the impact of malignancy on cardiac structure, function, and metabolism. Tumor-bearing mice developed cardiac atrophy and intrinsic systolic and diastolic dysfunction, with arterial hypotension and exercise intolerance. In hearts of ovarian tumor-bearing mice, fatty acid-supported mitochondrial respiration decreased, and carbohydrate-supported respiration increased-showcasing a substrate shift in cardiac metabolism that is characteristic of heart failure. Epithelial ovarian cancer decreased cytoskeletal and cardioprotective gene expression, which was paralleled by down-regulation of transcription factors that regulate cardiomyocyte size and function. Patient-derived pancreatic xenograft tumor-bearing mice show altered myosin heavy chain isoform expression-also a molecular phenotype of heart failure. Markers of autophagy and ubiquitin-proteasome system were upregulated by cancer, providing evidence of catabolic signaling that promotes cardiac wasting. Together, two cancer types were used to cross-validate evidence of the structural, functional, and metabolic cancer-induced cardiomyopathy, thus providing translational evidence that could impact future medical management strategies for improved cancer recovery in patients.

An integrated QTL and RNA-seq analysis revealed new petal morphology loci in Brassica napus L.

BackgroundRapeseed (Brassica napus L.) is one of the most important oil crops and a wildly cultivated horticultural crop. The petals of B. napus serve to protect the reproductive organs and attract pollinators and tourists. Understanding the genetic basis of petal morphology regulation is necessary for B. napus breeding.ResultsIn the present study, the quantitative trait locus (QTL) analysis for six B. napus petal morphology parameters in a double haploid (DH) population was conducted across six microenvironments. A total of 243 QTLs and five QTL hotspots were observed, including 232 novel QTLs and three novel QTL hotspots. The spatiotemporal transcriptomic analysis of the diversiform petals was also conducted, which indicated that the expression of plant hormone metabolic and cytoskeletal binding protein genes was variant among diversiform petals.ConclusionsThe integration of QTL and RNA-seq analysis revealed that plant hormones (including cytokinin, auxin, and gibberellin) and cytoskeleton were key regulators of the petal morphology. Subsequently, 61 high-confidence candidate genes of petal morphology regulation were identified, including Bn.SAUR10, Bn.ARF18, Bn.KIR1, Bn.NGA2, Bn.PRF1, and Bn.VLN4. The current study provided novel QTLs and candidate genes for further breeding B. napus varieties with diversiform petals.

TPPP-BRD9 fusion-related gallbladder carcinomas are frequently associated with intracholecystic neoplasia, neuroendocrine carcinoma, and a distinctive small tubular-type adenocarcinoma commonly accompanied with a syringomatous pattern

A fusion between tubulin polymerization-promoting protein (TPPP), a regulatory cytoskeletal gene, and the chromatin remodeling factor, bromodomain-containing protein 9 (BRD9), TPPP-BRD9 fusion has been found in rare cancer cases, including lung and gallbladder cancers (GBC). In this study, we investigated the histopathological features of 16 GBCs previously shown by RNA sequencing to harbor the TPPP-BRD9 fusion. Findings in the fusion-positive GBCs were compared with 645 GBC cases from the authors’ database. Among the 16 TPPP-BRD9 fusion-positive GBC cases, most were females (F:M = 7:1) of Chinese ethnicity (12/16), whereas the remaining cases were from Chile. The histopathological examination showed the following findings: 1) Intracholecystic neoplasm (ICN) in 7/15 (47% vs. 7% 645 reference GBCs, p < 0.001), all with gastro-pancreatobiliary phenotype, often with clear cell change, and in the background of pyloric gland metaplasia and extensive high-grade dysplasia. 2) Neuroendocrine carcinoma (NEC) morphology: 3 cases (27% vs. 4.6% in the reference database, p = 0.001) showed a sheet-like and nested/trabecular growth pattern of monotonous cells with salt-and-pepper chromatin characteristic of NECs. Two were large cell type, one had prominent clear cell features, a rare finding in GBNECs; the other one had relatively bland, well-differentiated morphology, and the remaining case was small cell type. 3) Adenocarcinoma identified in 8 cases had a distinctive pattern characterized by widely separated small, round tubular units with relatively uniform nuclei in a fashion seen in mesonephric adenocarcinomas, including hobnail-like arrangement and apical snouts, reminiscent of tubular carcinomas of the breast in many areas. In some foci, the epithelium was attenuated, and glands were elongated, some with comma shapes, which along with the mucinous/necrotic intraluminal debris created a “syringoid” appearance. 4) Other occasional patterns included the cribriform, glomeruloid patterns, and metaplastic tubular-spindle cell pattern accompanied by hemorrhage. In conclusion, TPPP-BRD9 fusion-positive GBCs often develop through intracholecystic neoplasms (adenoma-carcinoma sequence) of gastro-pancreatobiliary lineage, appear more prone to form NEC morphology and have a propensity to display clear cell change. Invasive adenocarcinomas arising in this setting often seem to display a distinctive appearance that we tentatively propose as the TPPP-BRD9 fusion-positive pattern of GBC.

CCG-1423-derived compounds reduce global RNA synthesis and inhibit transcriptional responses.

Myocardin-related transcription factors (MRTFs) are coactivators of serum response factor (SRF), and thereby regulate cytoskeletal gene expression in response to actin dynamics. MRTFs have also been implicated in transcription of heat shock protein (HSP)-encoding genes in fly ovaries, but the mechanisms remain unclear. Here, we demonstrate that, in mammalian cells, MRTFs are dispensable for gene induction of HSP-encoding genes. However, the widely used small-molecule inhibitors of the MRTF-SRF transcription pathway, derived from CCG-1423, also efficiently inhibit gene transcription of HSP-encoding genes in both fly and mammalian cells in the absence of MRTFs. Quantifying RNA synthesis and RNA polymerase distribution demonstrates that CCG-1423-derived compounds have a genome-wide effect on transcription. Indeed, tracking nascent transcription at nucleotide resolution reveals that CCG-1423-derived compounds reduce RNA polymerase II elongation, and severely dampen the transcriptional response to heat shock. The effects of CCG-1423-derived compounds therefore extend beyond the MRTF-SRF pathway into nascent transcription, opening novel opportunities for their use in transcription research.

Abstract 131: Local Pcsk6 Inhibition Attenuates VSMC Response In Vascular Injury And Restenosis

Intro: Vascular smooth muscle cell (VSMC) proliferation following endovascular interventions leads to restenosis, treated with drug-eluting stents. However, current drugs completely abolish vascular healing, cause thrombosis and clinical events. PCSK6 is a key protease in vascular remodeling and VSMCs activation upon vessel injury, via regulation of MMP2/14 activity. Here, we investigate whether local PCSK6 inhibition reduces VSMC proliferation and intimal hyperplasia. Methods: PCSK6 peptide inhibitor was tested in vitro on VSMCs migration, proliferation, apoptosis in comparison to rapamycin and paclitaxel. Internalization of a FITC labelled inhibitor was studied by flow cytometry and immunofluorescence. In vivo , PCSK6 inhibitor was administered at 3mg/kg IP using cationic microbubbles, followed by ultrasound-mediated local delivery in mice developing intimal hyperplasia after carotid ligation (n=8 vs 11). Carotid ligation in Tagln Cre+ /Pcsk6 fl/fl mice investigated the effect of PCSK6 specific ablation in VSMCs on intimal hyperplasia (n=7 vs 9). Carotids were collected from these and constitutive Pcsk6 -/- mice vs. controls for transcriptomic and histological analyses. Results: In vitro , PCSK6 inhibitor was internalized and inhibited both VSMC migration and proliferation (p&lt;0.0001), reducing the expression of PCSK6, MMP2 and GDF15 . In comparison, paclitaxel showed severe VSMC toxicity, whereas rapamycin inhibited more strongly VSMC migration and proliferation than the PCSK6 inhibitor. In vivo, the inhibitor reduced intimal hyperplasia (p=0.026), again, by repressing PCSK6 and MMP2 expression in treated vs. control mice, with a favorable safety profile. Likewise, conditional VSMC Pcsk6 knockouts showed less intimal hyperplasia (p=0.053). In carotids from Pcsk6 -/- mice, cytoskeletal (Synm) and peptidase (Pgpep1l, Klk1) genes were strongly downregulated, showing its importance for vascular matrix composition. Conclusion: Our proof-of-concept translational study shows that local application of a PCSK6 inhibitor is a viable method for specifically modulating VSMC activation in intimal hyperplasia following vascular injury

Abstract 3137: The Role Of Rna Binding Protein Fxr1 In Regulating Methylated Mrna Transcript Stability And Vascular Smooth Muscle Cell Contractility

Introduction/Background: A dysregulation in vascular smooth muscle cell (VSMC) cytoskeletal components may contribute to impaired contraction, leading to vascular disease. Post-transcriptional mechanisms of mRNA stability is an understudied, yet key mechanism in the regulation of VSMC contractility. m6A mRNA methylation is the most prevalent post-transcriptional modification and recognized to regulate mRNA stability. We recently showed that inducible expression of RNA binding protein FXR1 in injured arteries and stimulated hVSMCs regulates VSMC cytoskeletal gene mRNA stability, and FXR1 smc/smc mice have decreased VSMC contractility and blood pressure. Research Questions/Hypothesis: FXR1 functions as an m6A mRNA reader and regulates the stability of m6A methylated transcripts, altering VSMC contractility. Goals/Aims: To determine if the m6A methylation of mRNA transcripts by m6A methylases METTL3 and METTL14 play an important role in regulating cytoskeletal mRNA abundance and VSMC contractility and if transcript recognition by FXR1 is modified by transcript methylation state. Methods/Approach: m6A profiling was performed to investigate methylation status of hVSMC mRNA transcripts. Contraction assay following knockdown of METTL3 was performed to assess VSMC contraction. RNA immunoprecipitation experiments were performed to identify FXR1- interacting cytoskeletal transcripts following METTL3 knockdown. Biotinylated pulldown experiments were performed to assess FXR1’s interaction with methylated RNA probes. Results/Data: Cytoskeletal-related mRNA are the most highly methylated category of methylated transcripts. FXR1 interacts with methylated rather than unmodified target probes. Knockdown of the m6A methylase, METTL3 resulted in significantly decreased contraction of hVSMC. METTL3 knockdown significantly reduced FXR1-cytoskeletal mRNA interactions. Conclusions: These data support our hypothesis that FXR1 is an m6A methylated mRNA reader which regulates cytoskeletal mRNA stability and VSMC contractility.

Macrophage-enriched novel functional long noncoding RNAs LRRC75A-AS1 and GAPLINC regulate polarization and innate immune responses.

Macrophages (Mφs) are functionally dynamic immune cells that bridge innate and adaptive immune responses; however, the underlying epigenetic mechanisms that control Mφ plasticity and innate immune functions are not well elucidated. To identify novel functions of macrophage-enriched lncRNAs in regulating polarization and innate immune responses. Total RNA isolated from differentiating monocyte-derived M1 and M2 Mφs was profiled for lncRNAs expression using RNAseq. Impact of LRRC75A-AS1, GAPLINC and AL139099.5 knockdown was examined on macrophage differentiation,polarization markers, phagocytosis, and antigen processing by flow cytometry and florescence microscopy. Cytokine profiles were examined by multiplex bead array and cytoskeletal signaling pathway genes were quantified by PCR-based array. Gingival biopsies were collected from periodontally healthy and diseased subjects to examine lncRNAs,M1/M2marker expression. Transcriptome profiling of M1 and M2 Mφs identified thousands of differentially expressed known and novel lncRNAs. We characterized three Mφ-enriched lncRNAs LRRC75A-AS1, GAPLINC and AL139099.5 in polarization and innate immunity. Knockdown of LRRC75A-AS1 and GAPLINC downregulated the Mφ differentiation markers and skewed Mφ polarization by decreasing M1 markers without a significant impact on M2 markers. LRRC75A-AS1 and GAPLINC knockdown also attenuated bacterial phagocytosis, antigen processing and inflammatory cytokine secretion in Mφs, supporting their functional role in potentiating innate immune functions. Mechanistically, LRRC75A-AS1 and GAPLINC knockdown impaired Mφ migration by downregulating the expression of multiple cytoskeletal signaling pathways suggesting their critical role in regulating Mφ migration. Finally, we showed that LRRC75A-AS1 and GAPLINC were upregulated in periodontitis and their expression correlates with higher M1 markers suggesting their role in macrophage polarizationin vivo. Our results show that polarized Mφs acquire a unique lncRNA repertoire and identified many previously unknown lncRNA sequences. LRRC75A-AS1 and GAPLINC, which are induced in periodontitis, regulate Mφ polarization and innate immune functions supporting their critical role in inflammation.

Histone H1.0 couples cellular mechanical behaviors to chromatin structure

Tuning of genome structure and function is accomplished by chromatin-binding proteins, which determine the transcriptome and phenotype of the cell. Here we investigate how communication between extracellular stress and chromatin structure may regulate cellular mechanical behaviors. We demonstrate that histone H1.0, which compacts nucleosomes into higher-order chromatin fibers, controls genome organization and cellular stress response. We show that histone H1.0 has privileged expression in fibroblasts across tissue types and that its expression is necessary and sufficient to induce myofibroblast activation. Depletion of histone H1.0 prevents cytokine-induced fibroblast contraction, proliferation and migration via inhibition of a transcriptome comprising extracellular matrix, cytoskeletal and contractile genes, through a process that involves locus-specific H3K27 acetylation. Transient depletion of histone H1.0 in vivo prevents fibrosis in cardiac muscle. These findings identify an unexpected role of linker histones to orchestrate cellular mechanical behaviors, directly coupling force generation, nuclear organization and gene transcription.

Evolutionary dynamics of the cytoskeletal profilin gene family in Brassica juncea L. reveal its roles in silique development and stress resilience

Essential to plant adaptation, cell wall (CW) integrity is maintained by CW-biosynthesis genes. Cytoskeletal actin-(de)polymerizing, phospholipid-binding profilin (PRF) proteins play important roles in maintaining cellular homeostasis across kingdoms. However, evolutionary selection of PRF genes and their systematic characterization in family Brassicaceae, especially in Brassica juncea remain unexplored. Here, a comprehensive analysis of genome-wide identification of BjPRFs, their phylogenetic association, genomic localization, gene structure, and transcriptional profiling were performed in an evolutionary framework. Identification of 23 BjPRFs in B. juncea indicated an evolutionary conservation within Brassicaceae. The BjPRFs evolved through paralogous and orthologous gene formation in Brassica genomes. Evolutionary divergence of BjPRFs indicated purifying selection, with nonsynonymous (dN)/synonymous (dS) value of 0.090 for orthologous gene-pairs. Hybrid homology-modeling identified evolutionary distinct and conserved domains in BjPRFs which suggested that these proteins evolved following the divergence of monocot and eudicot plants. RNA-seq profiles of BjPRFs revealed their functional evolution in spatiotemporal manner during plant-development and stress-conditions in diploid/amphidiploid Brassica species. Real-Time PCR experiments in seedling, vegetative, floral and silique tissues of B. juncea suggested their essential roles in systematic plant development. These observations underscore the expansion of BjPRFs in B. juncea, and offer valuable evolutionary insights for exploring cellular mechanisms, and stress resilience.

Spatial transcriptomics reveals molecular dysfunction associated with cortical Lewy pathology

A key hallmark of Parkinson’s disease (PD) is Lewy pathology. Composed of α-synuclein, Lewy pathology is found both in dopaminergic neurons that modulate motor function, and cortical regions that control cognitive function. Recent work has established the molecular identity of dopaminergic neurons susceptible to death, but little is known about cortical neurons susceptible to Lewy pathology or molecular changes induced by aggregates. In the current study, we use spatial transcriptomics to capture whole transcriptome signatures from cortical neurons with α-synuclein pathology compared to neurons without pathology. We find, both in PD and related PD dementia, dementia with Lewy bodies and in the pre-formed fibril α-synucleinopathy mouse model, that specific classes of excitatory neurons are vulnerable to developing Lewy pathology. Further, we identify conserved gene expression changes in aggregate-bearing neurons that we designate the Lewy-associated molecular dysfunction from aggregates (LAMDA) signature. Neurons with aggregates downregulate synaptic, mitochondrial, ubiquitin-proteasome, endo-lysosomal, and cytoskeletal genes and upregulate DNA repair and complement/cytokine genes. Our results identify neurons vulnerable to Lewy pathology in the PD cortex and describe a conserved signature of molecular dysfunction in both mice and humans.

SRF transcriptionally regulates the oligodendrocyte cytoskeleton during CNS myelination

Myelination of neuronal axons is essential for nervous system development. Myelination requires dramatic cytoskeletal dynamics in oligodendrocytes, but how actin is regulated during myelination is poorly understood. We recently identified serum response factor (SRF)-a transcription factor known to regulate expression of actin and actin regulators in other cell types-as a critical driver of myelination in the aged brain. Yet, a major gap remains in understanding the mechanistic role of SRF in oligodendrocyte lineage cells. Here, we show that SRF is required cell autonomously in oligodendrocytes for myelination during development. Combining ChIP-seq with RNA-seq identifies SRF-target genes in oligodendrocyte precursor cells and oligodendrocytes that include actin and other key cytoskeletal genes. Accordingly, SRF knockout oligodendrocytes exhibit dramatically reduced actin filament levels early in differentiation, consistent with its role in actin-dependent myelin sheath initiation. Surprisingly, oligodendrocyte-restricted loss of SRF results in upregulation of gene signatures associated with aging and neurodegenerative diseases. Together, our findings identify SRF as a transcriptional regulator that controls the expression of cytoskeletal genes required in oligodendrocytes for myelination. This study identifies an essential pathway regulating oligodendrocyte biology with high relevance to brain development, aging, and disease.

Circadian disruption dysregulates lung gene expression associated with inflammatory lung injury.

Circadian systems drive the expression of multiple genes in nearly all cells and coordinate cellular-, tissue-, and system-level processes that are critical to innate immunity regulation. We examined the effects of circadian rhythm disorganization, produced by light shift exposure, on innate immunity-mediated inflammatory lung responses including vascular permeability and gene expression in a C57BL/6J murine model of inflammatory lung injury. A total of 32 C57BL/6J mice were assigned to circadian phase shifting (CPS) with intratracheal phosphate-buffered saline (PBS), CPS with intratracheal lipopolysaccharide (LPS), control (normal lighting) condition with intratracheal PBS, and control condition with intratracheal LPS. Bronchoalveolar lavage (BAL) protein, cell counts, tissue immunostaining, and differentially expressed genes (DEGs) were measured in lung tissues at 2 and 10 weeks. In mice exposed to both CPS and intratracheal LPS, both BAL protein and cell counts were increased at both 2 and 10 weeks compared to mice exposed to LPS alone. Multiple DEGs were identified in CPS-LPS-exposed lung tissues compared to LPS alone and were involved in transcriptional pathways associated with circadian rhythm disruption, regulation of lung permeability, inflammation with Rap1 signaling, and regulation of actin cytoskeleton. The most dysregulated pathways included myosin light chain kinase, MAP kinase, profilin 2, fibroblast growth factor receptor, integrin b4, and p21-activated kinase. Circadian rhythm disruption results in exacerbated immune response and dysregulated expression of cytoskeletal genes involved in the regulation of epithelial and vascular barrier integrity-the mechanistic underpinnings of acute lung injury. Further studies need to explore circadian disorganization as a druggable target.

The ctenophore Mnemiopsis leidyi deploys a rapid injury response dating back to the last common animal ancestor

Regenerative potential is widespread but unevenly distributed across animals. However, our understanding of the molecular mechanisms underlying regenerative processes is limited to a handful of model organisms, restricting robust comparative analyses. Here, we conduct a time course of RNA-seq during whole body regeneration in Mnemiopsis leidyi (Ctenophora) to uncover gene expression changes that correspond with key events during the regenerative timeline of this species. We identified several genes highly enriched in this dataset beginning as early as 10 minutes after surgical bisection including transcription factors in the early timepoints, peptidases in the middle timepoints, and cytoskeletal genes in the later timepoints. We validated the expression of early response transcription factors by whole mount in situ hybridization, showing that these genes exhibited high expression in tissues surrounding the wound site. These genes exhibit a pattern of transient upregulation as seen in a variety of other organisms, suggesting that they may be initiators of an ancient gene regulatory network linking wound healing to the initiation of a regenerative response.