Cell Death & Disease

Orthodontic tooth movement relies on a tightly regulated pro-inflammatory and pro resorptive mechanoresponse of local periodontal ligament fibroblasts (PdLFs). Dysregulation is linked to complications such as root resorption and tooth loss. Hyperlipidemic conditions promote excessive PdL mechanoresponses, with growth differentiation factor 15 (GDF15) acting as potential regulator. This study examined the contribution of the inflammasome/pyroptosis pathway as underlying mechanism for dysregulated mechanoresponses. Human PdLFs were treated with palmitic acid (PA) or oleic acid (OA) for six days before 24 hours of compressive loading. PA increased CASP1, CASP4, and CASP3 activity, secretion of IL-1{beta}, IL-18, and HMGB1, and LDH release. Pharmacological blockade and siRNA-mediated knockdown of inflammasome- and pyroptosis-related targets revealed that NLRP3, CASP1, CASP4, and GSDMD partially contributed to monocyte and osteoclast overactivation. Silencing PA-increased GDF15, partially normalized the phenotype, at least in part by inflammasome/pyroptosis regulation. GDF15 acted through extracellular, and a nuclear signaling route, each accounting partially to this phenotype. Together, GDF15 partially regulates the PA-induced, pyroptosis-associated overactivated mechanoresponse alongside pyroptosis-independent mechanisms suggesting it as an interesting target for potential clinical interventions.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy characterized by high metastatic dissemination, therapy resistance, and poor clinical outcome. Inhibitor of differentiation 1 or ID1, is frequently overexpressed in PDAC and is associated with tumour progression and adverse clinical outcome. However, the mechanisms governing its post-transcriptional regulation remain insufficiently characterized. Here, we identify tumour-suppressive miR-615-5p as a regulator of ID1 expression in PDAC. Integrative in-silico target prediction prioritized miR-615-5p based on seed complementarity and thermodynamic stability with the ID1 3' -UTR. Expression analysis of available PDAC clinical datasets revealed reduced miR-615-5p expression associated with increased ID1 expression. Direct association was validated using luciferase reporter assays, where miR-615-5p suppressed 3' -UTR reporter activity of ID1 in a sequence dependent manner, while mutation of the predicted binding site attenuated this effect. Further biotinylated-RIP and AGO2-RIP assays demonstrated the co-enrichment of ID1 transcripts and miR-615-5p with AGO2 associated RISC complexes, while AntimiR mediated inhibition of miR-615-5p perturbs association between miR/ID1 to AGO2, supporting interaction specificity. Functionally, modulation of miR-615-5p altered ID1 expression and impacted PDAC cell migration in vitro. Mechanistic analyses further indicated that the miR-615-5p/ID1 axis influences autophagic flux where miR-615-5p mediated inhibition of autophagy suppresses ID1 dependent cellular migration. Collectively, these findings define a previously uncharacterized miRNA-dependent regulation of ID1 expression and link this axis to autophagy-associated migratory responses in PDAC cells. The study expands the post-transcriptional regulatory landscape of ID1 and provides a possible mechanism where suppression of miR-615-5p leads to ID1 overexpression and subsequent poor clinical outcome in PDAC cells.

ZNF217 is an established oncogenic transcription factor that promotes cancer progression and therapeutic resistance; however, the mechanisms regulating ZNF217 protein abundance remain poorly understood. Here, we identify ubiquitin-specific peptidase 15 (USP15) as a critical regulator of ZNF217 stability and define a reciprocal USP15-ZNF217 signaling loop that sustains malignant phenotypes in ovarian cancer. Stable overexpression of ZNF217 in OVCA420 ovarian cancer cells enhanced proliferation, epithelial-mesenchymal transition, migration, invasion, and extracellular matrix adhesion. Notably, ZNF217 overexpression increased USP15 protein abundance without altering USP15 mRNA levels, whereas ZNF217 depletion reduced USP15 protein levels, suggesting post-transcriptional regulation. Conversely, USP15 depletion markedly reduced ZNF217 protein abundance while increasing ZNF217 mRNA levels, indicating that USP15 regulates ZNF217 predominantly at the post-transcriptional level. Proteasome inhibition restored ZNF217 protein levels following USP15 depletion, further demonstrating that USP15 promotes ZNF217 protein stability. Functionally, USP15 depletion in ZNF217-overexpressing ovarian cancer cells suppressed proliferation and multiple metastatic phenotypes, including migration, invasion, extracellular matrix adhesion, anoikis resistance, and multicellular aggregate formation. In vivo, USP15 depletion significantly reduced tumor progression and metastatic burden and prolonged survival in mice bearing ZNF217-driven ovarian tumors. Furthermore, USP15 depletion enhanced the sensitivity of ZNF217-overexpressing cells to carboplatin, paclitaxel, and doxorubicin. Collectively, these findings identify USP15 as an upstream regulator of ZNF217 protein stability and reveal a positive-feedback loop between USP15 and ZNF217 that reinforces oncogenic signaling. Targeting USP15 may therefore represent an indirect therapeutic strategy for suppressing ZNF217-driven ovarian cancer, particularly given the challenges associated with directly targeting oncogenic transcription factors.

Endothelial senescence is increasingly recognized as a driver of vascular pathology, while immunoglobulin G (IgG) has recently been reported to accumulate in aging tissues and induce senescence in macrophages and microglia. In cerebral cavernous malformations (CCMs), IgG accumulation has been obviously observed in CCM lesions, but the contribution of IgG to endothelial injury remains unclear. Using multi-omic profiling, endothelial models, and CCM mice, we identified IgG-secreting plasma cells enriched in lesions associated with endothelial senescence, hemorrhage, and disease severity. CCM loss-associated mTOR activation impaired lysosomal acidification and IgG processing, promoting intracellular IgG accumulation. IgG, in turn, induced NF-kB-dependent endothelial senescence. In vivo, BCMA-mediated plasma cell depletion attenuated lesion progression, whereas IgG supplementation partially restored disease severity. Anti-CD38 treatment likewise reduced IgG accumulation, endothelial senescence, hemorrhage, and lesion progression. These findings identify lysosomal dysfunction-mediated IgG as a pathogenic trigger of endothelial senescence and support targeting the plasma cell-IgG axis in CCM.

MYCN amplification drives replication stress in high-risk neuroblastoma, yet how MYCN-amplified tumour cells tolerate this stress to sustain proliferation remains poorly understood. Here we show that FBXL12, an SCF ubiquitin ligase substrate receptor that targets the Fanconi anaemia protein FANCD2 for degradation at replication forks, as well as the broader Fanconi anaemia and replication stress transcriptional program are elevated in high-risk and MYCN-amplified neuroblastoma. High FBXL12 expression independently predicts poor survival across neuroblastoma patient cohorts. FBXL12 loss stabilizes FANCD2 on chromatin, elevates ATR-dependent replication stress signalling and DNA damage during S phase, and impairs proliferation of MYCN-amplified neuroblastoma cells in vitro and in vivo. Mechanistically, MYCN directly engages the FBXL12-FANCD2 complex and antagonises FBXL12-mediated degradation of FANCD2 at replication forks, revealing that the oncogenic driver of replication stress also actively preserves the chromatin-bound FANCD2 pool required to tolerate it. Beyond S phase, FBXL12 loss disrupts FANCD2-dependent mitotic DNA synthesis and transmits unresolved replication intermediates into daughter cells. FBXL12-deficient cells consequently show transcriptional activation of MYC target gene, ATR, and mTOR signalling programs, and this pathway-concordant state confers differential sensitivity to ATR, and mTOR-targeting compounds, nominating candidate therapeutic strategies for this disease subset. Together, these findings define a MYCN-FBXL12-FANCD2 axis as a clinically relevant vulnerability in high-risk neuroblastoma.

We report the generation and characterization of a human induced pluripotent stem cell (iPSC) line derived from dermal fibroblasts of a patient with Skogholt disease, a rare maternally inherited neurodegenerative syndrome associated with choroid plexus dysfunction and impaired cerebrospinal fluid (CSF) homeostasis. Patient fibroblasts were reprogrammed using the non-integrating Repro-OSKGM kit. The resulting iPSC line exhibited typical pluripotent morphology, expressed canonical pluripotency markers, maintained a normal karyotype, retained the disease-associated genetic variant, was mycoplasma-free, and demonstrated trilineage differentiation potential. We also made choroid plexus (ChP) like organoids from the generated iPSCs. This patient-specific iPSC line provides a valuable resource for generating choroid plexus organoids and neurons to investigate disease mechanisms and develop therapeutic strategies.

Myeloproliferative neoplasms (MPN) are chronic hematologic malignancies characterized by clonal myeloid expansion, inflammation, oxidative stress, and progressive bone marrow (BM) remodeling that may culminate in fibrosis and secondary acute leukemia. Here, we evaluated the therapeutic efficacy and the underlying mechanisms of melatonin (MT) and liposomal melatonin (nano-MT) in preclinical MPN models. MT selectively inhibited clonogenic growth of patient-derived peripheral blood mononuclear cells and induced pluripotent stem cell-derived CD34 hematopoietic stem and progenitor cells in comparison to healthy controls. This effect was associated with increased apoptosis, reduced reactive oxygen species (ROS), and decreased glucose uptake, independently of MT receptor signaling. Transcriptomic profiling of primary MPN CD34 cells revealed suppression of MYC targets, G2M checkpoint signaling, ROS, and glycolysis pathways. In co-culture models, MT reduced stromal -smooth muscle actin and phosphorylated SMAD2/3, indicating inhibition of TGF-{beta}-driven mesenchymal stromal cell-to-myofibroblast formation. In tamoxifen-inducible SclCreER;JAK2V617F mice, nano-MT achieved efficient spleen and BM targeting. Therapeutically, nano-MT reduced erythrocytosis, myeloid progenitor expansion, and BM IL-1{beta} levels. Longitudinal micro-computed tomography and histological analyses demonstrated normalization of BM architecture, reduced osteosclerotic remodeling and splenomegaly, decreased reticulin deposition and megakaryocyte numbers. In a dose-escalation study, nano-MT restored erythrocyte, hematocrit, and platelet counts and normalized megakaryocyte-erythroid progenitors. Combination treatment with ruxolitinib further reduced leukocytosis, neutrophilia, and monocytosis. Collectively, these findings demonstrate that (nano-)MT attenuates MPN and BM remodeling by targeting metabolic, inflammatory, and fibrotic pathways. This study provides the first evidence for a therapeutic benefit of nano-MT in MPN and establishes a rationale for further translational evaluation.

BCL2L13 is a mitochondrial BCL2 family protein linked to mitophagy and ceramide metabolism, but its role in NSCLC metastatic plasticity remains unclear. Human lung cancer Tissue Microarray and matched patient specimens showed subtype and site dependent BCL2L13 expression, with higher cytoplasmic granular staining in primary NSCLC and reduced, heterogeneous staining in lymph node metastases, most evident in adenocarcinoma and squamous cell carcinoma. Because Epithelial mesenchymal transition and anoikis resistance are central requirements for metastatic dissemination, this primary to node attenuation provided the rationale to test BCL2L13 knockdown and overexpression in metastasis relevant NSCLC models. In A549 and LLC cell lines. TGF beta 1 induced coordinated mitophagy and EMT with mitochondrial enrichment of BCL2L13. BCL2L13 knockdown impaired TGF beta 1 and carbonyl cyanide m chlorophenyl hydrazone associated mitophagy, reducing LC3 beta mitochondria colocalization, TOMM20, LAMP1 overlap and mitochondrial LC3 II, p62, TOMM20 turnover; BNIP3 and NIX redistribution did not compensate. BCL2L13 loss enhanced EMT marker switching and migration, whereas overexpression partially opposed these changes. During detachment, BCL2L13 knockdown reduced anoikis associated apoptosis despite preserved mitochondrial recruitment of BAX, BAK, BNIP3,NIX, altered BID processing, non parallel caspase activity and shifted FAK phosphorylation. Pharmacological autophagy modulation did not reverse this anoikis phenotype. Lipidomics identified adhesion state dependent ceramide synthases CerS2, CerS6 linked sphingolipid remodeling: BCL2L13 knockdown increased C24 linked sphingolipid species in attached cells but reduced C16, C24 ceramide related profiles during anoikis. These findings identify BCL2L13 downregulation as a metastasis associated mitochondrial-lipid state that limits mitophagic quality control while favoring EMT and detachment survival in NSCLC adenocarcinoma.

Keloids are benign fibroproliferative disorders majorly characterized by excessive extracellular matrix deposition, with recurrence rates exceeding 80% following conventional therapy. Although epigenetic dysregulation has been implicated in keloid pathogenesis, whether genome-wide DNA methylation actively drives pathological cellular reprogramming, and whether this state is therapeutically reversible, remains unclear. We performed genome-wide DNA methylation profiling on keloid tissues, matched primary keloid fibroblasts, and normal controls. Our analysis revealed a shared DNA hypermethylation pattern between keloid tissues and fibroblasts, which was validated by three independent public cohorts. By integrating DNA methylome and transcriptome, we demonstrated that DNA methylation-regulated genes were enriched in osteochondrogenesis-related pathways, such as cartilage and bone development pathways. Furthermore, pharmacologic inhibition of DNA hypermethylation by DNA demethylating agent decitabine reduced the expression of osteochondrogenic markers and inhibited collagen deposition and keloid growth in primary keloid fibroblasts and patient-derived xenograft (PDX) model, offering a potential therapeutic strategy of keloid.

Cholesterol efflux is an important determinant of cellular lipid homeostasis. However, how microglial excessive cholesterol accumulation affects neuronal synaptic integrity remains poorly understood, particularly in the context of Alzheimer's disease. Here, we utilized a conditional knockout mouse model targeting the cholesterol transporters ABCA1 and ABCG1 in microglia. The microglia-specific ABCA1/ABCG1 deficiency triggered marked cholesterol accumulation, microglial hypertrophy, downregulation of the homeostatic marker P2ry12, and upregulation of the reactivity-associated marker CD11b, indicating shift toward a reactive phenotype. This phenotype was accompanied by increased reactive oxygen species, consistent with enhanced oxidative stress in ABCA1/ABCG1-deficient microglia compared with control. Using organotypic hippocampal slice cultures, we investigated the downstream neuronal outcomes of microglial ABCA1/ABCG1 deficiency. Under basal conditions, microglial ABCA1/ABCG1 knockdown did not significantly alter dendritic spine density in CA1 pyramidal neurons. However, upon exposure to amyloid-beta (A{beta}) stress, microglial ABCA1/ABCG1 deficiency markedly exacerbated dendritic spine loss in CA1 pyramidal neurons. Taken together, our findings highlight an important role for ABCA1/ABCG1-dependent cholesterol efflux in maintaining microglial homeostasis and limiting neuronal synaptic vulnerability to A{beta}-associated stress. These results support further investigation of microglial cholesterol transport as a potential target for preserving synaptic resilience in Alzheimer's disease.

Lineage-restricted transcriptional programs establish cell identity and can create selective dependencies in cancer. Here, we identify POU2AF2, encoding the transcriptional co-activator OCA-T1, as a critical lineage-specific dependency in a subset of diffuse large B-cell lymphoma (DLBCL). Pan-cancer dependency analyses and patient cohorts reveal elevated POU2AF2 expression in genetically aggressive DLBCL, where its depletion markedly suppresses tumor growth in vitro and in vivo. Mechanistically, POU2AF2 cooperates with the B-cell lineage-defining transcription factor POU2F2 (OCT2) to activate lymphocyte activation gene programs through direct chromatin engagement, thereby sustaining malignant transcriptional networks. We further identified a key epigenetic regulatory axis composed of the lineage-specific transcription factor TCF3 and the histone methyltransferase SET1A-COMPASS that drives POU2AF2 expression downstream of B-cell receptor signaling. Single-cell transcriptomic analysis reveals that POU2AF2 marks and sustains an innate-like B1 B-cell population in vivo, a candidate cell of origin for lymphoma. Together, these findings define a lineage-restricted POU2AF2/POU2F2 transcriptional module, controlled by a TCF3/SET1A epigenetic network, that sustains both innate-like B-cell identity and malignant fitness in DLBCL. Our study uncovers a previously unrecognized lineage-specific transcriptional dependency and highlights POU2AF2 and its associated regulatory circuitry as potential therapeutic targets in aggressive B-cell malignancies.

Abstract Background: Cytolytic activity (CYT), a widely used transcriptomic surrogate of anti-tumor immune cytotoxicity derived from GZMA (granzyme A) and PRF1 (Perforin 1) expression, is associated with clinical outcomes across cancers. MicroRNAs (miRNAs) are key post-transcriptional regulators of tumor immunity, yet their pan-cancer roles in modulating cytolytic activity remain incompletely understood. Objective: This study aimed to identify conserved miRNA regulators of tumor cytolytic activity and their downstream gene-mediated networks across diverse cancer types, while evaluating their clinical and therapeutic relevance. Methods: Matched miRNA and mRNA expression profiles from 9,288 primary tumors across 31 TCGA cancer types were analyzed. A multi-stage framework was applied: per-cancer Spearman correlations (|{rho}| >= 0.30, FDR < 0.05) identified recurrent CYT-associated miRNAs (at least 3 cancer types); these were integrated with TargetScan-predicted targets and subjected to pan-cancer and cross-cancer triple filtering (miRNA-gene and gene-CYT associations). All associations underwent tumor purity adjustment using Consensus Purity Estimate (CPE), with LUMP (Leukocytes Unmethylation for Purity) as sensitivity analysis. Candidates were further prioritized by random forest modeling with bootstrap stability, cancer-type-adjusted Cox regression, mediation analysis, immune cell deconvolution, k-means molecular subtyping, pathway enrichment, and DGIdb-based drug-target prioritization. Results: The analysis converged on 38 high-confidence miRNA-gene-CYT regulatory triplets involving 9 conserved miRNAs and 31 target genes after stringent purity adjustment and multi-layer validation. All nine miRNAs exhibited complete bootstrap stability. Mediation analysis confirmed significant gene-level mediation in 37 of 38 triplets (FDR < 0.01), with mediated proportions up to 94%. The final miRNA signature defined two distinct pan-cancer immune subtypes (immune-hot vs. immune-cold) with significantly different cytolytic activity and overall survival (OS) (HR = 0.754, FDR = 1.12 x 10^-4). The network was enriched for T-cell activation and lymphocyte differentiation pathways and highlighted multiple druggable targets, including CTLA4 and CD274 (PD-L1), nominating 124 candidate compounds. Conclusions: In conclusion, this tumor purity-adjusted pan-cancer study defines a compact, reproducible, and clinically relevant miRNA network that regulates cytolytic activity across diverse malignancies. By linking miRNA biology to immune subtyping and actionable therapeutic targets, the present work provides a valuable foundation for advancing precision immuno-oncology.

Liver metastases (LM) remain a major cause of death from different cancer types, in particular malignancies of the gastrointestinal tract. Liver metastases predict a poor response to immunotherapy due, among others, to the immunotolerant microenvironment (ME) of the liver and loss of local and systemic cytotoxic T cells. Thus, strategies that can reprogram the immune ME of the liver and restore cytotoxic T cell reactivity are being sought. We previously reported that estrogen signaling blockade impedes the growth of LM by reducing MDSC accumulation and monocyte/macrophage polarization. The aim of this study was to elucidate the underlying mechanism(s) and assess whether estrogen signaling in the myeloid lineage was driving the immunotolerant ME of LM. To this end, we generated mice with conditional myeloid cell-specific deletions of estrogen receptors (ER) or ER{beta} and analyzed in these mice the effect of ER loss on the liver immune ME and the outgrowth of LM. In mice with ER, but not with ER{beta} deletion, we observed a marked reduction in the growth of murine colon carcinoma MC-38 liver metastases as compared to their respective controls. Flow cytometry and immunohistochemistry revealed a decrease in macrophages that were polarized to the pro-tumorigenic M2-like phenotype and a concomitant increase in activated CD8+ T and NK cells relative to controls. Bulk RNAseq analysis performed on hepatic immune cells infiltrating the liver revealed changes in the expression of key cytokines/chemokines mediating immune cell recruitment, activation and polarization, including Ccl5 (upregulated) and Csf1 (downregulated). Taken together, the data suggest that ER signaling in myeloid-derived cells programs the immune landscape and contributes to an immunosuppressive and metastases-growth permissive ME in the liver.

Despite the success of chimeric antigen receptor (CAR) T cell therapy for treatment of B cell acute lymphoblastic leukemia (B-ALL), its translation to acute myeloid leukemia (AML) has been hindered by limited efficacy and significant toxicity. Interferon-gamma (IFN{gamma}) blockade with emapalumab has recently emerged as a promising strategy to mitigate CAR T cell-related toxicities in B cell malignancies, based on evidence that IFN{gamma} is largely dispensable for optimal CAR T cell activity in B-ALL. Whether IFN{gamma} signaling is similarly non-essential in the AML context remains unclear. Here, we demonstrate that disruption of the IFN{gamma} axis impedes anti-AML CAR T cell function and prevents upregulation of target antigen CD123, the apoptotic mediator Fas, and the adhesion molecule ICAM-1 on AML cells. Conversely, exogenous IFN{gamma} enhances CAR T cell cytotoxicity and increases CAR T cell avidity for AML targets. These findings identify IFN{gamma} as a critical mediator of CAR T cell efficacy against AML by promoting increased target antigen expression, enhanced cytotoxicity, and stable CAR T/tumor interactions. Our results suggest that therapeutic IFN{gamma} blockade, including with emapalumab, may compromise CAR T cell responses in AML and should be approached with caution in this disease context.

Introduction: Gliomas integrate into neural circuits and heighten neuronal excitability, engaging in bidirectional communication whereby neuronal activity promotes tumor growth and proliferation. Aging reshapes the brain microenvironment through extracellular matrix changes, altered secretory factors, and immune dysfunction, creating conditions permissive to tumorigenesis and limiting immunotherapy efficacy in glioblastoma. However, its effect on neuronal excitability and signaling in glioblastoma remains poorly understood. Methods: We developed a novel classification system for glioblastoma by leveraging three classes of DNA methylation-based aging biomarkers: chronological, biological, and mitotic clocks. This approach stratified tumors into accelerated and decelerated epigenetic aging subtypes, which we then characterized at the molecular, functional, and clinical levels using multimodal analyses. Guided by these profiles, we evaluated the in vitro effects of the FDA-approved agents levetiracetam and riluzole, alone and in combination with temozolomide, on U87MG and A172 cell lines. Specifically, we assessed changes in cell viability, apoptosis, and the expression of marker genes related to stemness, neuronal hyperexcitability, and immunosuppression. Results: Tumors with decelerated epigenetic aging showed expression modules and CpG hypomethylation associated with neuronal activity and stemness, and carried significantly worse prognosis. Single-cell and spatial multi-omics analyses revealed enrichment for neurons and malignant neural stem-like cells in these tumors. They also displayed enhanced intercellular communication, driven predominantly by glutamate signaling across the malignant, neuronal, and immune compartments of the tumor microenvironment. In vitro pharmacological inhibition of glutamatergic signaling with levetiracetam and riluzole reduced cell viability, induced apoptosis, and suppressed expression of stemness, neuronal hyperexcitability, and immunosuppression markers. Both agents potentiated the cytotoxic and apoptotic effects of temozolomide, supporting glutamatergic inhibition as a strategy for improving chemosensitivity. Conclusion: By establishing a framework for decoding glioblastoma heterogeneity through epigenetic aging, we identified the glutamatergic pathway as a clinically actionable vulnerability. Our findings suggest that combining anti-glutamatergic therapies with temozolomide exerts synergistic antitumor effects while mitigating adverse chemotherapy-induced phenotypes, such as increased stemness, neuronal hyperexcitability, and immunosuppression, thereby laying the groundwork for novel therapeutic strategies.

Hepatocellular carcinoma (HCC) is particularly aggressive and difficult to treat. Due to the lack of early clinical diagnosis and the unsatisfactory clinical treatment effect, it is particularly important to identify novel markers that can predict tumor behavior in HCC. biogenesis of ribosomes BRX1 (BRIX1) is abundant in various tissues of the human body. However, the regulatory mechanisms and its role in various tissues are not fully understood. Here, we analyzed the expression pattern of BRIX1 in HCC from public gene expression databases and tissue samples from clinical HCC. We confirmed that BRIX1 was upregulated in both HCC cell lines and HCC paraffin section samples. BRIX1 depletion significantly dicreased the capacity of cells to grow and migrate in vitro, and knockdown BRIX1 suppressed tumor growth in xenograft tumor model. Mechanistically, BRIX1 depletion suppressed the MAPK/ERK pathway, as reflected by reduced phosphorylated ERK (p-ERK) levels. In summary, we provide a rational clue for the further investigation of BRIX1 as an invaluable biological marker for diagnosing and predicting prognosis of patients with HCC.

Although the brain was traditionally considered immune-privileged, recent studies show immune factors play key roles in brain function. Dysfunction of these factors is linked to neurodevelopmental disorders, but mechanisms remain unclear. Using a maternal immune activation (MIA) mouse model, we investigated immune-related genes in neurodevelopmental disorder pathogenesis. MIA mice showed increased locomotor activity and disrupted prepulse inhibition. RNA-seq and qPCR analyses revealed persistent increases in major histocompatibility complex class II (MHCII) expression and persistent decreases in GABAergic synapse-related gene expression, particularly glutamate decarboxylase (Gad) expression, in dopaminergic regions. These expressions were negatively correlated, and immunohistochemistry showed MHCII at postsynaptic GABAergic synapses on dopaminergic neurons. Patch-clamp recordings confirmed reduced mIPSC frequency in MIA mice. MHCII knockout mice showed opposite phenotypes, while MHCII overexpression in dopaminergic neurons decreased Gad expression. These results suggest MIA-induced MHCII upregulation enhances pruning of GABAergic synapses on dopaminergic neurons, leading to behavioral deficits.

Skin, the bodys largest mechanosensitive organ, relies on a tension gradient across epidermal layers to maintain structure and function, but how mechanical force contributes to epidermal development and disease pathogenesis is poorly understood. By anchoring intermediate filaments (IF) to the plasma membrane, desmosomes, the most abundant intercellular junctions in the epidermis, help create a supracellular scaffolding that provides mechanical resilience to the tissue. However, the contribution of the desmosome-IF network to the epidermal response to mechanical strain remains unknown. Here we show that the desmosome-IF connection is not only required to induce a proper cellular mechano-response but is actively strengthened in response to stretch through the PP2A-mediated phospho-regulation of the cytoskeletal linker protein desmoplakin (DP). Additionally, we show in human skin dephosphorylated DP localizes to high tension layers, suggesting this mechano-response mechanism is coordinated with the epidermal tension gradient. Furthermore, in models of Carvajal syndrome, a cardio-cutaneous disorder caused by truncating DP mutations, cells lose mechano-responsive behavior and exhibit abnormal morphology in high-tension epidermal layers. Together, these findings identify the DP-IF network as a key component of the response to mechanical strain and show that its disruption compromises epidermal homeostasis and contributes to disease pathogenesis.

Disseminated cancer cells (DCC) in non-metastatic carcinoma patient bone marrow (BM) are predictive of metastasis. Those detected by epithelial cytokeratin or EpCAM expression have poorly-characterized transcription profiles due to their extreme rarity: 1~2 cells per two million BM cells in every third non-metastatic patient. We here characterize the transcriptomes of DCCs. Single-cell RNA-sequencing (scRNA-seq) of 864 EpCAM-positive cells (from 1,151 cancer patients) in BM or lymph nodes (LN) revealed plasma, immune, myeloid, erythroid progenitor cells and two candidate DCC populations, termed M0-DCC and M1-DCC. M0-DCC, mostly from non-metastatic M0-stage patients, displayed the highest known adult stemness scores, and were transcriptomically reminiscent of human cleavage-stage, preimplantation embryos. M1-DCC represented cancer cells undergoing the epithelial-mesenchymal transition (EMT), corresponding to later, implanting and gastrulating embryos. Detection of early-embryo-like DCC categorised patients at highest risk for metastatic progression. Furthermore, high M0-DCC scores predicted the metastatic potential of human cell lines from the Cancer Cell Line Encyclopedia. M0-DCC gene expression profiles can be reversibly induced from M1-DCC-like cells in vitro. The close correspondence between gene expression profiles in immediate early embryonic development and metastatic founder cell candidates provides strong evidence that the onset of cancer and metastasis recruits mechanisms employed in fertilization.

Background. GBA2-associated hereditary spastic paraplegia (SPG46) is a rare autosomal recessive neurodegenerative disorder caused by loss-of-function mutations in GBA2, encoding the non-lysosomal glucocerebrosidase 2. GBA2 deficiency leads to glucosylceramide (GlcCer) accumulation and glucosylated cholesterol (GlcChol) depletion, causing cytoskeletal defects in immature neurons. However, the mechanisms linking lipid dysregulation to neuronal dysfunction remain poorly understood. Methods. We modelled GBA2 loss of function by chronic pharmacological inhibition in mouse cerebellar granule neurons (CGNs) and assessed neuronal morphology, synaptic organization, Ca2+ dynamics, mitochondrial function and actin cytoskeleton during maturation. Proteomic profiling was performed in GBA2-inhibited and GlcChol-supplemented neurons. Findings were validated in a zebrafish gba2 crispant model by evaluating motor behavior, cerebellar development, neuronal organization and mitochondrial function, and in patient-derived fibroblasts carrying a homozygous pathogenic GBA2 variant (NM_020944). The role of RAC1 was studied in both neurons and patients' cultured skin fibroblasts, and upon rac1 pharmacological inhibition in zebrafish crispants. Results. Chronic GBA2 inhibition impaired axonal outgrowth in immature CGNs but not neurite complexity in mature neurons, suggesting morphological compensation. Nevertheless, mature neurons displayed enlarged presynaptic terminals, impaired synaptic vesicle clustering and altered Ca2+ responses to potassium and glutamate, the latter associated with NMDA receptor redistribution without changes in total receptor levels. Mitochondrial alterations were observed in CGNs, patient fibroblasts and zebrafish, consistent with defective architecture of the mitochondrial network. Proteomics revealed convergent alterations in actin cytoskeleton, synaptic pathways and cellular metabolism following both GBA2 inhibition and GlcChol supplementation. GlcChol bidirectionally regulated RAC1 function, likely altering its spatial distribution rather than its global activation. Confocal imaging confirmed abnormal RAC1 and F-actin localization in patient fibroblasts. Zebrafish gba2 crispants recapitulated motor deficits, Purkinje cell loss, motor neuron disorganization and mitochondrial abnormalities. Pharmacological Rac1 inhibition rescued motor behavior and neuronal organization, linking cytoskeletal disorganization to the observed phenotype in the zebrafish model. Conclusions. Our findings identify a pathogenic GlcChol-RAC1-actin signalling axis linking lipid imbalance to synaptic disorganization, NMDA receptor redistribution and mitochondrial dysfunction in SPG46. The selective vulnerability of corticospinal neurons, cerebellar granule neurons and Purkinje cells may reflect their dependence on this pathway. Rac1 inhibition rescues disease phenotypes in vivo, highlighting this pathway as a promising therapeutic target.