搜索结果：Knocked

Parkinson's disease (PD) is a neurodegenerative disorder characterized by alpha-synuclein (α-syn) aggregates termed Lewy bodies. To model PD pathology in vitro, preformed fibrils of α-syn (PFFs), which can be taken up by cells, provide a seed that drives misfolding and aggregation of endogenous α-syn, with new aggregates amplifying this process. External application of PFFs to dopaminergic neurons (DNs) increases aggregate formation, marked by α-syn phosphorylation at serine 129 (pS129-syn), a pathological PD marker. Building on this, we developed an automated synuclein seeding assay to quantify new α-syn aggregates in iPSC-derived DNs. Using pS129-syn as a readout, we show that PFFs elicit a time- and dose-dependent increase in pS129-syn aggregates. Our automated seeding assay further revealed that aggregate formation depends on endogenous α-syn levels. Treatment with PFFs produced a greater increase in pS129-syn aggregates in iPSC DNs derived from a PD patient with a triplication in the SNCA gene, which encodes the α-syn protein and which elevates total α-syn levels, relative to DNs from an isogenic iPSC line from the same individual, in which the SNCA gene mutation had been corrected by CRISPR/Cas9. In contrast, no pS129-syn signal was detected in neurons in which all copies of the SNCA gene had been knocked out (KO). This proof-of-principle automated high-content imaging workflow for synuclein seeding has been validated using isogenic cell lines with defined SNCA copy number variants and it offers a platform for assessing compounds and therapeutics that may impede α-syn aggregate formation.

Cholesterol plays a critical role in repairing white matter injury (WMI) following traumatic brain injury (TBI). The enzyme cholesterol 24-hydroxylase (CYP46A1) regulates the removal of cholesterol by converting it into 24(S)-hydroxycholesterol (24OHC). Although CYP46A1 has neuroprotective effects on various central nervous system disorders, its effect on WMI remains unclear. Adult male C57BL/6 mice underwent controlled cortical impact to model TBI. Experiments using the CYP46A1 activator efavirenz and CYP46A1-/- mice were utilized to elucidate the function of CYP46A1. Neurological function, WMI, and cholesterol metabolism were evaluated, and the mechanisms through which CYP46A1 affects these processes were explored. Efavirenz markedly improved outcomes and preserved white matter structure after TBI by increasing microglial phagocytic activity and myelin debris clearance, along with promoting oligodendrocyte precursor cell remyelination. Furthermore, efavirenz promoted cholesterol export by increasing 24OHC levels and activating liver X receptors (LXR). However, these neuroprotective effects of Efavirenz were partially diminished when CYP46A1 was knocked down or when LXR activity was blocked. Efavirenz administration promotes functional neurological recovery and sustains white matter integrity in TBI through the regulation of cholesterol homeostasis and the promotion of remyelination processes.

Abnormal glucose metabolism in the central nervous system is a major cause of sporadic Alzheimer's disease (SAD). We hypothesize that glycogen synthase kinase 3β (GSK3β) mediates cognitive impairment by inhibiting the Wnt/β-catenin pathway, which in turn induces abnormal glucose metabolism, synaptic damage, and mitochondrial dysfunction. To test this hypothesis, we injected streptozotocin bilaterally into the lateral ventricles of 100 male C57BL/6 J mice to establish in vivo models of SAD, and into HT22 cells to establish in vitro models of SAD. GSK3β expression was knocked down via adeno-associated virus (AAV) injection into the hippocampal CA1 region in vivo and via lentiviral transfection in vitro. We assessed cognitive function using the Morris water maze, Y-maze, and novel object recognition tests (n = 10). Glucose metabolism was evaluated by 18F-FDG PET imaging (n = 3), while synaptic and myelin sheath ultrastructure was examined using transmission electron microscopy (n = 6). Cell viability, mitochondrial function, and key protein expression were measured using CCK-8 assays, Seahorse analysis, and molecular biology techniques, respectively (n = 3, n = 6). In both in vivo and in vitro STZ-induced SAD models, GSK3β knockdown significantly reduced amyloid-β (1-42) deposition and tau hyperphosphorylation, activated the Wnt/β-catenin pathway, enhanced glucose metabolism, reversed glycolytic inhibition and mitochondrial dysfunction, and repaired synaptic and myelin sheath damage, ultimately improving cognitive deficits. Our findings demonstrate that GSK3β knockdown ameliorates STZ-induced SAD-like pathologies by restoring Wnt/β-catenin signaling and normalizing glucose metabolism, highlighting GSK3β as a potential therapeutic target for SAD.

Periodontitis involves dysregulated immunity where the NLRP3 inflammasome plays a key role, while the role of TRIM31, an E3 ubiquitin ligase, remains unknown in periodontitis. Human gingival fibroblasts (HGFs) and macrophages were stimulated with LPS and ATP; TRIM31 was overexpressed via AAV, and NLRP3 was knocked out via CRISPR; periodontitis was induced in WT and NLRP3-KO mice treated with AAV-TRIM31; bone loss, osteoclasts, and apoptosis were assessed. TRIM31 was downregulated in inflammation and correlated with M2 polarization. TRIM31 overexpression protected HGFs, promoted M2 polarization, and bound to NLRP3, thereby promoting K48-linked ubiquitination and degradation. In vivo, TRIM31 reduced bone loss, osteoclasts, and apoptosis; these effects were abolished in NLRP3-KO cells and mice. TRIM31 negatively regulates periodontal inflammation via ubiquitin-dependent NLRP3 degradation.

Phosphoprotein associated with glycosphingolipid-enriched microdomains 1 (PAG1) has been recognized as a biomarker for diabetic nephropathy (DN). This study aimed to elucidate the mechanism by which PAG1 influences DN. The expression and clinical significance of PAG1 in DN were assessed using bioinformatics analysis. PAG1 expression was validated in db/db mice and high glucose (HG)-treated human kidney-2 (HK-2) cells. Subsequently, PAG1 was knocked down in DN models to investigate its effects on DN progression, pyroptosis, and the transforming growth factorbeta 1 (TGF-β1)/Smads pathway. The TGF-β1/Smads pathway activator SRI-011381 was employed to further elucidate the role of PAG1 in DN regulation. Bioinformatics analysis revealed elevated PAG1 expression in DN samples, which correlated with various immune cells and pathways, such as chemokine pathway and cell cycle. This elevated expression was confirmed in animal and cell experiments. In db/db mice, PAG1 knockdown alleviated DN, reduced inflammatory factor levels, inhibited pyroptosis, and suppressed TGF-β1/Smads pathway. In vitro, PAG1 silencing promoted proliferation, inhibited apoptosis and fibrosis, reduced inflammatory and pyroptotic factor levels, and suppressed the TGF-β1/Smads pathway. These effects were reversed by SRI-011381 intervention. PAG1 promotes pyroptosis by upregulating the TGF-β1/Smads pathway, thereby exacerbating DN.

Astrocytes provide crucial metabolic support for neurons and undergo significant metabolic changes in Alzheimer's disease (AD). Aldolase C (ALDOC), an astrocyte-enriched glycolytic enzyme, may play a role in this process. This study aimed to investigate whether ALDOC modulates astrocytic metabolism to support neuronal energy supply in patients with AD and to assess its therapeutic potential. Hippocampal and cortical tissues from 6-month-old APP/PS1 and wild-type mice were subjected to western blotting, qPCR, and immunofluorescence staining for ALDOC and glycolytic proteins. An in vitro AD model was created using oligomeric β-amyloid (oAβ)-treated SVGp12 astrocytes. ALDOC was overexpressed or knocked down via plasmid or siRNA. Downstream effects on AMPK/mTOR/HIF-1α signaling and the expression of glycolytic markers (LDHA and PKM2) were evaluated by western blot and qPCR, as well as by lactate/ATP assays and extracellular acidification rate (ECAR) measurements. Neuron-astrocyte interactions were assessed in an SVGp12/SH-SY5Y coculture. Furthermore, the ability of magnesium ions to restore ALDOC expression was tested. ALDOC was specifically expressed in astrocytes but was downregulated in APP/PS1 mice, accompanied by reduced HIF-1α and LDHA levels, suggesting glycolytic impairment. Similar downregulation occurred in oAβ-treated SVGp12 cells. ALDOC overexpression was associated with altered AMPK/mTOR/HIF-1α signaling, enhanced glycolysis, and increased lactate and ATP production, whereas its knockdown had the opposite effects. These outcomes appeared to depend on HIF-1α, as suggested by the rescue experiments. In coculture, ALDOC overexpression in astrocytes supported neuronal metabolic function. Moreover, magnesium ions restored ALDOC activity and glycolysis in oAβ-treated astrocytes. These results suggest that ALDOC is downregulated in APP/PS1 mice and is associated with glycolytic impairment. In oAβ-treated astrocytes, ALDOC appears to regulate glycolysis through the AMPK/mTOR/HIF-1α axis and may support neuronal energy via the lactate shuttle. Magnesium ions appear to offer a potential strategy for addressing the metabolic deficits in AD.

Myocardial infarction is associated with massive cardiomyocyte loss, and embryonic stem cells (ESCs), owing to their cardiomyocyte differentiation ability, have emerged as a promising therapy. One potential factor involved in regulating ESC-to-cardiomyocyte differentiation is activating transcription factor 3 (Atf3), and this study clarifies its involvement. Atf3 was knocked out (ko) in mouse ESCs (mESCs) with CRISPR/Cas9. Both wild-type (WT) and Atf3-KO mESCs formed embryoid bodies (EBs) over 6 days, followed by adherent culture to induce cardiomyocyte differentiation from days 7-12; there, Atf3-KO had more beating EBs, at higher frequencies, from Day 8 of differentiation than WT. They also had increased expression of mesodermal markers on Days 3-6 (ex. T), cardiac progenitor markers on Days 6-9 (ex. Pdgfra), and cardiomyocyte differentiation marker on Days 9-12 (ex. cardiac troponin T), as measured by RT-qPCR and flow cytometry. Furthermore, cardiomyocyte differentiation-associated differentially-expressed genes were significantly upregulated in Atf3-KO EBs, compared to WT, under Gene Set Enrichment Analysis of RNA sequencing. All these effects in Atf3-KO mESCs were reversed upon "rescue", where Atf3 expression was restored in these cells. Therefore, Atf3-KO in mESCs promotes differentiation into mesodermal lineages, which further differentiate into cardiac progenitors, serving as a target for cell-based cardiac regeneration therapies.

As metabolic disorders associated with excessive lipid deposition in skeletal muscle rise, strategies to alleviate this accumulation have emerged as a major focus in metabolic research. Glycerol-3-phosphate acyltransferase 3 (GPAT3) is a key enzyme in triglyceride biosynthesis; however, its role in muscle lipid metabolism remains unclear. In this study, we overexpressed or knocked down GPAT3 in C2C12 myoblasts to evaluate its effects on muscle lipid metabolism and then validated the findings in GPAT3 knockout (KO) mice. GPAT3 deficiency significantly reduced lipid deposition in muscle cells. In C2C12 cells, changes in GPAT3 expression were accompanied by alterations in AMPK signaling activity. These results demonstrate that GPAT3 promotes lipid deposition in skeletal muscle. Our in vitro data reveal a correlation between GPAT3 and AMPK signaling; further in vivo validation and functional assays are required to clarify the role of AMPK in this process. This study highlights GPAT3 as a potential therapeutic target for metabolic diseases characterized by excessive intramuscular lipids.

Myocardial ischemia-reperfusion (MIR) injury remains the leading cause of adverse outcomes after myocardial infarction, and its prevention remains a major therapeutic challenge. The GSE160516 and GSE61592 datasets from the Gene Expression Omnibus database were analyzed to screen MIR-related differentially expressed genes. To simulate clinical MIR injury, male C57BL/6 mice were subjected to ligation of the left anterior descending coronary artery for 30 minutes, followed by reperfusion for 24 hours. In this study, the protein-protein interaction network was generated based on the 34 common upregulated genes, and among them, 11 genes were identified as hub genes, including solute carrier family 11 member 1 (Slc11a1), a gene associated with immune response. Herein, the upregulation of myocardial Slc11a1 was validated in mice with MIR injury. Mice subjected to MIR had improved cardiac function when Slc11a1 was knocked down via myocardial injection of Slc11a1 small interfering RNA (siRNA): the infarcted areas reduced from 38.43±4.13% to 22.18±3.45%; CK-MB levels decreased from 12.01±3.11 to 5.84±1.24. Slc11a1 silencing also reduced infiltration of F4/80 macrophages, deactivated the p65 proinflammatory pathway, and decreased the generation of IL-6 and TNF-α. Myocardial oxidative stress induced by IR challenge was attenuated in mice receiving Slc11a1 siRNA. Our study demonstrates that the abnormal elevation of Slc11a1 contributes to IR-related myocardial injury.

Groupers are economically significant fish in southern China and Southeast Asia, where viral diseases severely hinder the sustainable development of aquaculture. Singapore grouper iridovirus (SGIV) is a highly pathogenic virus in groupers that can cause mass mortality upon infection. NOD-like receptor thermal protein domain associated protein 3 (NLRP3), a crucial component of innate immunity, plays vital roles in various viral infections. However, whether SGIV infection induces NLRP3 inflammasome activation and the precise mechanisms involved remain to be elucidated. In this study, we investigated the mechanism using an in vitro SGIV infection model of GS cells. Our findings demonstrated that SGIV infection significantly upregulated the transcription of NLRP3 and IL-1β. Additionally, SGIV induced pronounced aggregation of NLRP3 and enhanced the interaction between NLRP3 and ASC. Further investigation revealed that SGIV infection markedly upregulated TLR9 transcription while promoting nuclear translocation of NF-κB and enhancing its promoter activity. Transfection with SGIV genomic DNA in vitro also significantly upregulated transcription of TLR9, NLRP3, and IL-1β, along with NF-κB promoter activity. When TLR9 expression was knocked down by siRNA, NF-κB promoter activity and transcription levels of NLRP3 and IL-1β were substantially reduced. Similarly, the NF-κB inhibitor JSH-23 significantly suppressed NLRP3 and IL-1β transcription. Moreover, SGIV DNA upregulated transcription of cGAS and STING, while STING overexpression enhanced Caspase-1 activity and promoted IL-1β activation. This study first elucidates that the genomic DNA of SGIV may upregulate the transcription of IL-1β through the TLR9/NF-κB and cGAS/STING pathways, thereby inducing the assembly of the NLRP3 inflammasome.

Drug-induced liver injury (DILI) is a global health issue with limited treatment options. The nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway plays a critical role in defending against DILI, making it a potential therapeutic target. This study aimed to investigate the hepatoprotective effect of salvianolic acid B (Sal B), a polyphenol derived from Salvia miltiorrhiza Bge., against DILI and to elucidate its underlying molecular mechanisms, particularly focusing on the Nrf2 pathway. Compounds screening was carried out to identify Sal B as a potent Nrf2 activator. Cellular thermal shift assay (CETSA), surface plasmon resonance (SPR), and microscale thermophoresis (MST) were employed to examine the direct interaction between Sal B and kelch-like ECH-associated protein-1 (Keap1), an Nrf2 inhibitor. Nrf2 knock-out (Nrf2-/-) mice and liver-specific Keap1 knockout (Hepcre-Keap1flox/flox) mice were used to assess the role of Nrf2 and Keap1 in Sal B-mediated hepatoprotection. Mass spectrometry-based chemoproteomic analysis and mutagenesis studies were performed to identify the specific cysteine residues modified by Sal B. Molecular dynamics simulations were used to analyze conformational changes in Keap1 following Sal B binding. Sal B strongly activated Nrf2, and its hepatoprotective effects were significantly diminished by Nrf2 knock-out. Sal B directly bound to Keap1. In Hepcre-Keap1flox/flox mice, DILI was attenuated, and Sal B's protective effects were weakened. Sal B covalently modified Cys395 and Cys434 within the Keap1 double-glycine repeat (DGR) domain. Mutagenesis of these residues impaired Sal B-Keap1 interaction and abolished Nrf2 activation. Sal B's modification altered the conformation of the Keap1 kelch domain, loosening the Nrf2 binding pocket and facilitating Nrf2 activation. Sal B exerts hepatoprotective effects against DILI by activating Nrf2 through covalent modification of Keap1 at Cys395 and Cys434. These findings provide mechanistic insights into the potential of Sal B as a novel therapeutic agent for DILI.

Abnormal DNA methylation occurs in Huntington's disease (HD, but the underlying mechanisms remain unclear. Using a knock-in pig model, we identify significant alterations in 5mC and 5hmC levels linked to neural genes. TET1, which converts 5mC to 5hmC, decreases prior to symptom onset in pigs-a change not observed in HD mice. TBP binding sites are abundant in the porcine and human TET1 promoter, but scarce in mice. Mutant huntingtin (mHTT) binds more strongly to TBP in HD pig brains, blocking TBP's access to the TET1 promoter, thereby reducing TET1 transcription and altering 5mC/5hmC patterns. Our findings reveal TET1 as a target of abnormal DNA methylation that contributes to selective neuronal vulnerability in HD pigs, highlighting the value of large mammalian models for studying disease pathogenesis.

Seaweeds, such as the fast-growing green alga Ulva prolifera, can be harnessed as valuable marine crops. The lack of scalable genome-editing tools hampers functional genomics to explore and elucidate algal molecular pathways with industrial importance. Here, we expanded precision genome modification in seaweeds by successfully demonstrating gene editing with a transgene-free AT-rich-targeting Cas system in U. prolifera. By evaluating various delivery buffers, comparing different Cas systems, and optimizing incubation temperatures, we determined suitable conditions for higher applicability of a novel Cas12a-aligned ST8 editor. We obtained more than 50 ST8-mediated knock-out mutants of a toxin-based endogenous marker gene, UpAPT, at 28℃ post-delivery incubations. Our work diversified the applicable genome editing tools in seaweeds, advancing algal functional genomics, and providing more strategies to precisely target unexplored seaweed resources.

SLC6A8 encodes the creatine transporter (CRT), which mediates creatine transport across the plasma membrane in the brain, including the blood-brain barrier and neurons. Creatine transporter deficiency (CTD), caused by pathogenic variants in SLC6A8, leads to cerebral creatine depletion and cognitive impairment. Here, we investigated the developmental molecular mechanisms underlying CTD using the pathogenic c.1681G>C (G561R) variant of Slc6a8, which corresponds to a variant identified in SLC6A8 in a patient with CTD. In vitro analyses using HEK293 cells expressing mutant mouse CRT carrying the G561R variant demonstrated impaired N-glycan maturation and plasma membrane localization of the transporter, resulting in markedly reduced creatine uptake, consistent with previous reports on the corresponding human CRT variant. To investigate the in vivo effects of this pathogenic variant, we generated CRT-G561R knock-in mice by introducing the c.1681G>C point mutation into the mouse Slc6a8 gene using the CRISPR/Cas9 system. These male mice exhibited severe reductions in brain creatine levels, postnatal growth retardation, and impaired spatial memory, despite preserved gross brain morphology. Quantitative proteomic analyses of the hippocampus and cerebral cortex during postnatal development revealed region-dependent protein alterations in CTD. The hippocampus showed pronounced early postnatal remodeling involving proteins related to actin cytoskeleton organization and vesicle-mediated membrane trafficking, whereas the cerebral cortex exhibited a more gradual response involving creatine biosynthesis-related enzymes and later-emerging mitochondrial pathways, including the mitochondrial translation machinery. These findings demonstrate stage- and region-dependent proteomic remodeling during postnatal brain development in CTD.Significance Statement Creatine transporter deficiency (CTD) causes cerebral creatine depletion and intellectual disability; however, the developmental mechanisms linking creatine loss to brain dysfunction remain unclear. We performed developmental proteomic profiling of the hippocampus and cerebral cortex using a mouse model carrying a pathogenic Slc6a8 variant identified in patients with CTD. Creatine transporter dysfunction induces distinct region- and stage-dependent molecular responses during postnatal brain maturation. The hippocampus shows early alterations in cytoskeleton-dependent membrane trafficking pathways, consistent with impaired synaptic and circuit maturation, whereas the cerebral cortex exhibits progressive metabolic and mitochondrial adaptations. These findings suggest that impaired creatine-dependent energy buffering disrupts distinct developmental programs across brain regions, potentially contributing to cognitive dysfunction by hindering early hippocampal circuit maturation.

Poorly permeable compounds in the beyond rule of 5 (bRo5) space often demonstrate acceptor well concentrations below the lower limit of quantification or limited resolution between apparent permeability (Papp) values. To address the acceptor well concentration limitation and better resolve differences between poorly permeable molecules, a straightforward solution was proposed, extend the incubation time from 2-h to 24-h to enable acceptor well concentrations to achieve quantifiable values. MDCK cells with knock out of the endogenous canine Mdr1 gene (MDCK-KO) were seeded on 96-Tanswell plates and grown to confluence over three days at which point the 24-h assay was initiated. The longer incubation time enabled receiver compartment concentrations to exceed limits of quantification enabling Papp determination in a test set of approximately 30 molecules. The approximately 10-fold increase in incubation time enabled Papp value determination of up to an order of magnitude lower compared to a 2-h incubation. The assay demonstrates good reproducibility and resolution between poorly permeable compounds. In summary, a straightforward modification of a traditional Transwell permeability assay has produced an assay suitable for the measurement and resolution of very poorly permeable compounds.

The requirement for the T-box transcription factors (TF) T-bet and Eomes in innate lymphoid cells (ILCs) beyond their development is not well understood. Here, we generate an inducible, NKp46-specific T-bet knock-out (KO) model and compare it to corresponding Eomes KO and combined T-bet Eomes double KO mice to define T-box TFs requirement in the homeostasis and function of mature NK cell and other NKp46+ ILC. Inducible T-bet deletion reduces stage IV NK cell numbers in the spleen and tissues, while preserving NK cells in the bone marrow and lymph nodes. Liver ILC1 and small intestine lamina propria NKp46+ ILC3 are also lost upon T-bet deletion, indicating the requirement for continuous T-bet expression in these ILC types. Combined T-bet and Eomes KO leads to a rapid loss of NK cells, markedly greater than with individual T-bet or Eomes KO. Direct comparison of inducible T-bet and Eomes KO models reveals that Eomes is critical for host protection against murine cytomegalovirus, whereas T-bet is dispensable, despite loss of ILC1. These findings establish the tissue-specific and non-redundant roles for T-box TFs in NKp46+ ILCs homeostasis and response to viral infection.

Microglial activation is a critical pathogenic mechanism of neurological damage in methylmalonic acidemia (MMA). The NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome is closely associated with neuroinflammatory processes. However, the mechanism of NLRP3 inflammasome activation in microglia by methylmalonic acid (MMAcid) remains unclear. Mouse microglia BV2 cells and hippocampal neuronal HT22 cells were stimulated with different concentrations of MMAcid. Neuronal cells were treated with conditioned medium derived from differently treated microglia. Microscopy was used to observe morphological changes of cells. Cell counting kit-8 (CCK-8) assay was performed to assess cell viability (VB). In addition, the mRNA and protein levels were detected via qRT-PCR, ELISA, and western blot. To further validate the mechanism, we used lentivirus infection and small interfering RNA (siRNA) transfection to overexpress (OE) and knockdown (KD) extracellular signal-regulated kinase (ERK) and analyzed the inflammatory pathway. Direct exposure of neuronal cells to MMAcid significantly reduced cell VB, accompanied by morphological features suggestive of pyroptosis. MMAcid activated microglia, which was accompanied by increased expression of inflammatory molecules, activation of the ERK/nuclear factor-κB (NF-κB)/NLRP3 signaling pathway, and elevated secretion of interleukin-1 beta (IL-1β) and tumor necrosis factor-α (TNF-α). Neuronal cell VB decreased when cocultured with the supernatant from MMAcid-treated microglia. Overexpression of ERK further enhanced microglial activation and potentiated MMA-induced neuronal pyroptosis-related signaling. Conversely, knocking down ERK expression in microglia reduced their activation by MMAcid and decreased cytokine secretion, but it did not completely reverse MMA-induced neuronal damage. MMAcid is associated with microglial activation, accompanied by alterations in the ERK/NF-κB/NLRP3 signaling pathway and increased release of inflammatory cytokines, which may contribute to neuronal pyroptosis-related changes.

The LRRK2 G2019S mutation is one of the most common genetic risk factors for Parkinson's disease (PD), yet LRRK2 G2019S knock-in (KI) mice rarely develop robust neurodegeneration under basal conditions, suggesting that additional environmental triggers are required for disease progression. Here, we established a clinically relevant gene-environment interaction mouse model of PD by subjecting LRRK2 G2019S KI mice to recurrent Citrobacter (C.) rodentium infection, a murine model of enteric bacterial inflammation. Repeated infection induced progressive PD-like phenotypes selectively in KI mice, including motor impairment, reduced locomotor activity, impaired motor coordination, selective nigrostriatal dopaminergic neurodegeneration, enhanced neuroinflammation, and pathological phosphorylated α-synuclein (p-αSyn) accumulation, whereas wild-type (WT) mice remained largely resistant. Mechanistically, infected KI mice developed markedly exacerbated colonic inflammation, epithelial barrier dysfunction, increased intestinal permeability, and enhanced inflammasome activation despite normal bacterial clearance, indicating that pathogenic LRRK2 signaling amplifies inflammatory responses rather than impairing antimicrobial defense. In parallel, recurrent infection induced pronounced intestinal p-αSyn accumulation and expansion of pathology beyond the epithelial layer in KI mice, supporting a gut-brain axis mechanism linking intestinal inflammation to neurodegeneration. Collectively, these findings demonstrate that the LRRK2 G2019S mutation functions as a sensitizing factor that cooperates with recurrent enteric inflammation to drive PD-related pathology. This study establishes a physiologically relevant LRRK2 G2019S gene-environment interaction mouse model that recapitulates key behavioral, neuropathological, and inflammatory features of PD.

Auxin conjugation represents a key metabolic mechanism in regulating auxin activity within plant cells. GRETCHEN HAGEN3 (GH3) enzymes conjugate the major naturally occurring auxin indole-3-acetic acid (IAA) with amino acids, thereby contributing to the maintenance of auxin homeostasis. Although the transcription of GH3 genes is auxin-regulated, the complexity of this regulation is still not fully understood. Therefore, in this study, we employed β-estradiol-inducible and CRISPR/Cas9-mediated knock-out transgenic tobacco (Nicotiana tabacum) cell lines with modified expression of representative genes from two GH3 subgroups with contrasting response to auxin, NtGH3.1a and NtGH3.6e. Using IAA metabolite profiling and bacterial enzyme assays, we show that NtGH3.1a preferentially catalyzes the formation of indole-3-acetyl-aspartate (IAA-Asp), while NtGH3.6e facilitates the production of the less-characterized conjugates indole-3-acetyl-glutamine (IAA-Gln) and 2-oxindole-3-acetyl-glutamine (oxIAA-Gln). We further validated these results by testing the Arabidopsis thaliana homologs of both GH3 subgroups, showing that AtGH3.1 favors aspartate, while both AtGH3.5/6 preferentially utilize glutamine. Finally, subcellular localization analyses using GFP-tagged NtGH3.1aT and NtGH3.6eT expressed under inducible promoters demonstrated that both enzymes localized to the nucleus and cytoplasm, independently of the presence of auxin. Moreover, we provide evidence of GH3 localization under native promoters, confirming their presence in both compartments. Collectively, our results suggest the evolutionary conservation of amino acid type-preferential IAA conjugation and underscore the functional divergence of GH3 isoforms in IAA metabolism.