Mitochondrial fission and altered Complex II expression and function are hallmarks of Huntington’s (HD) and Alzheimer’s (AD) diseases. Notably, peroxisomal fission protein, PEX11β, works in coordination with mitochondrial fission proteins and exhibits a unique co-expression pattern with respiratory chain complexes. However, whether a PEX11β-mediated shared mechanism drives mitochondrial fragmentation across different neurodegenerative conditions remains unclear. Using patient transcriptomic data and in vitro cellular models, we establish a novel connection between disease-associated downregulation of PEX11β, mitochondrial fragmentation, and impaired expression of fusion and Complex II genes. Restoration of PEX11β alleviates motor and cognitive deficits, and reinstates mitochondrial gene expression in the striatum of 3-NP-induced HD mice. In silico analysis and ChIP-PCR reveal Yin Yang 1 is a critical transcription factor regulating co-expression of PEX11β and Complex II genes. Our findings uncover PEX11β-mediated regulation of mitochondrial fission-fusion dynamics, linking mitochondrial integrity to behavioral outcomes, and offer a novel insight into amyloid-associated neuropathology.
Alternative splicing of the CSNK1A1 gene generates multiple CK1α isoforms with distinct subcellular localizations and regulatory properties. While cytoplasmic CK1α is a well-established negative regulator of canonical WNT signaling, the role of nuclear CK1α remains poorly understood. Here, we identify L-insert-containing nuclear CK1α isoforms as functional components of the β-catenin/TCF transcriptional complex in gastrointestinal cancer cells. Using isoform-specific assays, we demonstrate that CK1α isoforms containing the L-insert exhibit greater nuclear accumulation than isoforms lacking the L-insert and support WNT-driven transcription. Knockdown of L-insert-containing CK1α isoforms in esophageal adenocarcinoma (EAC) and colorectal cancer (CRC) models reduces β-catenin recruitment to TCF-bound chromatin and attenuates WNT target gene expression. Kinase-inactive CK1α mutants retained their association with the β-catenin/TCF complex but showed reduced WNT reporter activity, indicating that CK1α catalytic activity is dispensable for complex association but is required for full transcriptional activation. Consistent with the canonical negative regulatory function of CK1α, pharmacological activation of CK1α with SSTC3 increased β-catenin Ser45 phosphorylation, reduced active β-catenin abundance, and limited its nuclear availability and binding to TCF sites, thereby attenuating WNT transcriptional activity and reducing tumor-initiating cell (TIC)-enriched populations in gastrointestinal tumors. These effects translate into reduced tumorigenic potential and delayed tumor regrowth in vivo. Together, our findings identify L-insert-containing nuclear CK1α isoforms as positive regulators of WNT transcription, in contrast to the negative regulatory function of cytoplasmic CK1α within the β-catenin destruction complex, and provide a rationale for pharmacological CK1α activation as a strategy to target WNT-driven TICs.
P-glycoprotein (P-gp/MDR1) overexpression in macrophages contributes to tuberculosis (TB) treatment failure by reducing intracellular drug accumulation. The molecular mechanisms by which host macrophages upregulate P-gp remain poorly defined. The present study investigated whether CK2α functions as an upstream kinase regulating P-gp expression in macrophages and examined its therapeutic relevance in TB. P-gp regulation was characterized in PMA-differentiated THP-1 macrophages by immunoblotting, RT-PCR, qRT-PCR, immunofluorescence, co-immunoprecipitation, and subcellular fractionation, and in U-937 macrophages by immunoblotting. Signaling pathways were dissected using pharmacological inhibitors, siRNA knockdown, and overexpression. P-gp efflux function was assessed by rhodamine-123 accumulation assay. Flow cytometry was used to evaluate CK2α-dependent P-gp regulation in PBMC-derived primary macrophages. P-gp expression and TB pathology were examined in Mycobacterium tuberculosis H37Rv-infected BALB/c mice. During monocyte-to-macrophage differentiation, CK2α activated AKT at Ser129, leading to NF-κB-dependent P-gp induction. In mature macrophages, rifampin induced P-gp through CK2α-mediated Hsp90β phosphorylation and subsequent PXR stabilization. CK2 inhibition or CSNK2A1 knockdown suppressed P-gp expression through both pathways and increased intracellular rhodamine-123 accumulation. In Mtb-infected mice, co-administration of the CK2 inhibitor DRB with rifampin suppressed lung P-gp expression, reduced pulmonary bacterial burden compared to rifampin monotherapy, and attenuated lung inflammation. PBMC-derived primary macrophages showed the same CK2α-dependent dual pathway regulation. The present study identifies CK2α as a common upstream kinase for both NF-κB- and PXR-dependent P-gp induction in macrophages. CK2 inhibitors, for which clinical safety data are available from oncology trials, may warrant evaluation as adjunctive agents against P-gp-mediated drug resistance in TB treatment.
Endosomal dysfunction is increasingly recognised as an early event in Alzheimer’s disease. Hyperactivation of the early endosomal regulator Rab5 has been implicated in early AD-associated pathology, but its downstream signalling consequences and therapeutic relevance remain incompletely defined. We investigated whether Rab5-associated endo-lysosomal abnormalities are linked to oxytocin receptor trafficking defects and impaired receptor-associated signalling in early Alzheimer’s disease-relevant settings. Young APP/PS1 mice were used to assess endogenous endo-lysosomal abnormalities in the hippocampus. A hippocampus-targeted Rab5 overactivation model was established by stereotaxic delivery of AAV-hSyn-EGFP-Rab5Q79L to test whether sustained Rab5 activation was sufficient to induce similar alterations. Oxytocin receptor localisation, endosomal marker colocalisation, and OTR-associated downstream signalling readouts were evaluated by confocal immunofluorescence and western blotting. Receptor internalisation/recycling assays, membrane protein western blotting, OT rechallenge, and OTR antagonist experiments were performed in HEK293T cells to assess plasma-membrane OTR recovery and OT-responsive ERK/CREB phosphorylation. RNA sequencing, synaptic marker analysis, resting-state functional magnetic resonance imaging, structural magnetic resonance imaging, and behavioural testing were used to assess downstream molecular, circuit-level, and functional consequences. Group comparisons were analysed using one-way analysis of variance followed by Tukey’s post hoc test. Young APP/PS1 mice exhibited enlarged Rab5-positive early endosomal structures, increased overlap of Rab5 with Rab7 and LAMP1, abnormal intracellular redistribution of the oxytocin receptor, and reduced phosphorylation of ERK and CREB in the hippocampus. Rab5 overactivation reproduced Rab5-associated endosomal abnormalities and was accompanied by altered OTR endosomal association, impaired plasma-membrane OTR recovery, and reduced OT-evoked ERK/CREB phosphorylation. These receptor-level and signalling changes were accompanied by disruption of neuronal signalling-related transcriptional programmes, loss of excitatory and inhibitory synapses, reduced spontaneous brain activity, weakened hippocampal-medial prefrontal cortex functional connectivity, hippocampal atrophy, and cognitive impairment. Across the two models, MW150 treatment was associated with partial improvement in endosomal abnormalities, OTR-associated signalling readouts, and behavioural measures; additional synaptic and imaging improvements were observed in the Rab5-overactivation model. Rab5-associated endo-lysosomal trafficking abnormalities are accompanied by altered oxytocin receptor localisation, reduced OTR-associated signalling readouts, and multi-level early AD-relevant phenotypes. The partial improvement in molecular, synaptic, network, and behavioral abnormalities by MW150 supports the modifiability of the p38α–Rab5/endosomal axis. At the same time, further work is needed to establish whether OTR trafficking directly mediates these downstream phenotypes.
Tumor-associated macrophages (TAMs) are the most abundant infiltrating immune cells in tumors and contribute to the formation of an immunosuppressive niche. Ginsenoside Rg3, a triterpenoid glycoside derived from Panax ginseng, has been extensively documented to exert anti-tumor activities. However, whether the therapeutic efficacy of Rg3 against colorectal cancer (CRC) is closely associated with TAM-mediated immune suppression remains largely elusive. We investigated the immunomodulatory effects of Rg3 on TAM-mediated functions using murine CRC and human 3D organoid models. MS-DARTS technology was employed to identify the potential molecular targets of Rg3, which were further validated through CETSA, SPR, and molecular dynamics simulations. Metabolomic, proteomic, and transcriptomic analyses were performed to explore the metabolic and molecular mechanisms underlying Rg3-mediated tumor microenvironment (TME) remodeling. A macrophage-specific Glut1 knockout mouse model was established to assess the effects of Glut1 deficiency on TAM polarization and the therapeutic efficacy of Rg3. The treatment of Rg3 plus anti-PD-1 was evaluated to determine whether Rg3 enhances the anti-CRC effect of anti-PD-1. Rg3 inhibited CRC progression by modulating the immunosuppressive polarization of TAMs in a host immune-dependent manner. Rg3 directly binds to Glut1 on macrophages, thereby inhibiting p300/CBP-mediated acetylation and triggering metabolic reprogramming. This cascade reduces itaconate levels, leading to enhanced phosphorylation of JAK1 and subsequent activation of the STAT1 signaling pathway. By promoting STAT1 nuclear translocation, Rg3 increases the secretion of CXCL10/11, thereby facilitating the infiltration of CD8+ T cells into tumors. Rg3 enhances the therapeutic effect of anti-PD-1 against CRC progression, showing efficacy even in microsatellite stable CRC models. Rg3 is a promising candidate for targeting TAM metabolism to reshape the immunosuppressive TME and combat CRC development.
