👤 J Ham

Tinnitus is a complex neurological condition affecting 10-15% of adults worldwide, characterized by phantom auditory perception without external sound sources. While traditional investigations have focused on discrete auditory structures, emerging evidence suggests tinnitus involves broader alterations across central auditory regions. This study employed transcriptomic analysis to investigate molecular mechanisms underlying salicylate-induced tinnitus across multiple brain regions simultaneously. Male C57BL/6 N mice received daily intraperitoneal injections of sodium salicylate (350 mg/kg) for five consecutive days to induce tinnitus-like behavior, assessed using gap-prepulse inhibition of acoustic startle reflex. RNA sequencing was performed on auditory cortex, inferior colliculus, and cochlear nucleus tissues. Differential gene expression analysis, weighted gene co-expression network analysis, and functional annotation were conducted to identify shared molecular signatures and pathways across auditory centers. Principal component analysis revealed region-specific transcriptomic changes following salicylate treatment. Differential gene expression analysis identified Depp1 and Angptl4 as consistently upregulated genes across multiple brain regions, particularly within the inferior colliculus and cochlear nucleus. Weighted gene co-expression network analysis revealed a 215-gene module increased across all auditory regions in tinnitus mice, with functional annotation indicating enrichment for vasculature-related biological processes. Depp1 emerged as a central hub gene linking oxidative stress responses to autophagy mechanisms. This study shows that tinnitus pathology involves not only neuronal hyperactivity but also oxidative stress, neuroinflammation, and autophagy in the central auditory pathway. Depp1 acts as a molecular hub linking redox imbalance to cellular clearance, highlighting its potential as a therapeutic target and offering new insights for intervention. Show less

The glucagon-like peptide-1 receptor (GLP-1R) and glucose-dependent insulinotropic polypeptide receptor (GIPR) are important incretin receptors that are therapeutic targets for the treatment of type 2 diabetes and obesity. This study extensively characterised the metabolic phenotype of mice with global deletion of either the GLP-1R or GIPR side by side under identical conditions. Age-matched male wild-type (WT) C57Bl6NTac, GLP-1RKO or GIPRKO mice were placed on a high-fat or chow diet for 12 weeks, and a range of in vivo (weight gain, food intake, glucose tolerance, insulin tolerance, and whole-body energy metabolism) and ex vivo (white adipocyte lipolysis, brown adipose tissue and liver mitochondrial function, adipocyte and islet size, and hepatic steatosis) parameters were measured. While both WT and GLP-1RKO mice gained weight similarly on a HFD, obese high-fat-fed GLP-1RKO mice had altered glucose and insulin tolerance, and exhibited hepatic steatosis, highlighting the physiological importance of the GLP-1R in the regulation of blood glucose and lipid homoeostasis. In contrast, GIPRKO mice were partially resistant to diet-induced obesity compared to the WT mice, which was associated with a small reduction in food intake and intact epididymal and subcutaneous white adipocyte β-adrenoceptor-mediated lipolysis. Similarly, WT mice treated with a GIPR antagonist prevented weight gain due to a reduction in food intake on a HFD. These findings provide further support that the GLP-1R is important for normal glycaemic control, whereas the GIPR may play a role in the regulation of body weight. Show less

Bacteroides-centric gut dysbiosis reported to exacerbates liver cirrhosis via inflammation and fibrosis, therefore utilizing Bacteroides species as microbiome-based therapeutic logical to mitigate disease progression. Feces were collected from 52 Healthy and 144 Liver cirrhosis individuals for V3-V4 dependent 16rRNA-bsed comparative metagenomics analysis, followed a by microbiome depleted and non-depleted DDC mice model to explain the role of Bacteroidetes phylum classified microbial species P. plebeius in liver fibrosis pathophysiological pathways. Bacteroides presented cirrhosis-dependent decrease in human and animal microbiome, and negatively correlated to key molecular pattern associated with cirrhosis. P. plebeius significantly reduced in abundance and identified as a microbial biomarker for cirrhosis (AUC = 0.73) and treatment with P. plebeius significantly improved the levels of cirrhosis-related phenotypical and biochemical markers in the microbiome-depleted cirrhosis group. P. plebeius decrease the expression of S100a9, CCR1, ADAM8, TREM2, ITGAM, and MYO5A which are primarily responsible for inducing inflammation in liver cirrhosis. P. plebeius downregulated the fibrosis related genes expression including CD51, PLAT, ITGA3, CXCR4, and TGFBR1 and gene related to extracellular matrix formation including COL1A1, LTBP2, S100A6, and SMCO2. Additionally, P. plebeius treatment decreased the expression of hepatotoxicity-related genes including LPL, KRT18, ALDOA, and MCM10, and increased the expression of FABP1 and RDX. Additionally, P. plebeius normalized the expression of genes connected to two pathophysiological process including TIMP4, TGFB3, S100A8, PLSCR1, MMP8, CXCL4, and BMP. Our study revealed P. plebeius as a multifaceted bio-therapeutic candidate that normalized dysregulated gene expression and reversed hepatic inflammation, fibrogenesis, and hepatotoxicity. Show less

A homozygous loss-of-function (LoF) variant in POC5 was previously described in an individual with retinitis pigmentosa. We identified POC5 variants in 12 probands with a syndromic phenotype. We aim to define the phenotype spectrum and molecular mechanism associated with biallelic POC5 LoF variants. We studied a cohort of 12 families with bi-allelic LoF POC5 variants and performed detailed phenotype analysis. POC5 localization studies were performed in 3 proband-derived fibroblast cell lines. Detailed phenotyping of probands with POC5 variants expands the phenotype spectrum beyond ocular manifestations. This syndrome causes not only rod-cone dystrophy but also diabetes mellitus with severe insulin resistance and partial lipodystrophy, kidney disease, and muscle cramps. The POC5 protein plays an essential role during cell cycle and cilium formation. Interestingly, POC5 localization studies in 3 proband-derived fibroblast cell lines show aberrant localization suggesting a ciliary defect. The phenotypes of the 12 families in this study fit well within the ciliopathy phenotype spectrum, except for lipodystrophy, which is not common in ciliopathies. We describe a multiorgan syndrome caused by bi-allelic LoF variants in POC5. This underscores the pleiotropic effects of POC5 variants and highlights the significance of adipose tissue and metabolic dysfunction in ciliopathies. Show less

Osthole is active constituent of Cnidium monnieri (L.) Cuss. with various physiological functions including anti-inflammation and anti-lipedemic effects. However, the regulatory activity of osthole in colorectal cancer development, focusing on mitochondrial metabolism, is not well known. We hypothesized that osthole may suppress progression of colorectal cancer and aimed to determine the underlying mitochondrial metabolism and the autophagic flux. In this study, we elucidated the mechanism of action of osthole in colorectal cancer using an in vivo azoxymethane/dextran sodium sulfate (AOM/DSS) mouse model and an in vitro cell culture system. AOM/DSS mouse model was established and analyzed the effects of osthole on survival rate, diseases activity index, number of tumor and histopathology. Then, cell based assays including viability, cell cycle, reactive oxygen species (ROS), apoptosis, calcium efflux, and mitochondrial function were analyzed. Moreover, osthole-mediated signaling was demonstrated by western blot analyses. Osthole effectively suppressed the growth of colorectal tumors and alleviated AOM/DSS-induced intestinal injury. Osthole restored the function of goblet cells and impaired the expression of Claudin1 and Axin1 impaired by AOM/DSS. In addition, osthole specifically showed cytotoxicity in colorectal carcinoma cells, but not in normal colon cells. Osthole decreased the ASC/caspase-1/IL-1β inflammasome pathway and induced mitochondrial dysfunction in redox homeostasis, calcium homeostasis. Furthermore, osthole inhibited both oxidative phosphorylation (OXPHOS) and glycolysis, leading to the suppression of ATP production. Moreover, via combination treatment with chloroquine (CQ), we demonstrated that osthole impaired autophagic flux, leading to apoptosis of HCT116 and HT29 cells. Finally, we elucidated that the functional role of tiRNA These results suggest that osthole has the potential to manage progression of colorectal cancer by regulating autophagy- and mitochondria-mediated signal transduction. Show less

RPA is a critical factor for DNA replication and replication stress response. Surprisingly, we found that chromatin RPA stability is tightly regulated. We report that the GDP/GTP exchange factor DOCK7 acts as a critical replication stress regulator to promote RPA stability on chromatin. DOCK7 is phosphorylated by ATR and then recruited by MDC1 to the chromatin and replication fork during replication stress. DOCK7-mediated Rac1/Cdc42 activation leads to the activation of PAK1, which subsequently phosphorylates RPA1 at S135 and T180 to stabilize chromatin-loaded RPA1 and ensure proper replication stress response. Moreover, DOCK7 is overexpressed in ovarian cancer and depleting DOCK7 sensitizes cancer cells to camptothecin. Taken together, our results highlight a novel role for DOCK7 in regulation of the replication stress response and highlight potential therapeutic targets to overcome chemoresistance in cancer. Show less

The increasing pace of gene discovery in the last decade has brought a major change in the way the genetic causes of brain malformations are being diagnosed. Unbiased genomic screening has gained the first place in the diagnostic protocol of a child with congenital (brain) anomalies and the detected variants are matched with the phenotypic presentation afterwards. This process is defined as "reverse phenotyping". Screening of DNA, through copy number variant analysis of microarrays and analysis of exome data on different platforms, obtained from the index patient and both parents has become a routine approach in many centers worldwide. Clinicians are used to multidisciplinary team interaction in patient care and disease management and this explains why the majority of research that has led to the discovery of new genetic disorders nowadays proceeds from clinical observations to genomic analysis and to data exchange facilitated by open access sharing databases. However, the relevance of multidisciplinary team interaction has not been object of systematic research in the field of brain malformations. This review will illustrate some examples of how diagnostically driven questions through multidisciplinary interaction, among clinical and preclinical disciplines, can be successful in the discovery of new genes related to brain malformations. The first example illustrates the setting of interaction among neurologists, geneticists and neuro-radiologists. The second illustrates the importance of interaction among clinical dysmorphologists for pattern recognition of syndromes with multiple congenital anomalies. The third example shows how fruitful it can be to step out of the "clinical comfort zone", and interact with basic scientists in applying emerging technologies to solve the diagnostic puzzles. Show less

Bipolar disorder (BD) is one of the most heritable psychiatric disorders. A growing number of whole-exome sequencing (WES) studies for BD has been performed, however, no research has examined the association between single nucleotide variants (SNVs) from WES and structural magnetic resonance imaging (MRI) data. We sequenced whole-exomes in 53 patients with BD and 82 healthy control participants at an initial discovery stage and investigated the impacts of SNVs in risk genes from WES analysis on the cortical gray-matter thickness and integrity of white matter tracts and in the following stage. Cortical thickness and white matter integrity were investigated using the FreeSurfer and TRACULA (Tracts Constrained by UnderLying Anatomy). We identified 122 BD-related genes including KMT2C, AHNAK, CDH23, DCHS1, FRAS1, MACF1 and RYR3 and observed 27 recurrent copy number alteration regions including gain on 8p23.1 and loss on 15q11.1 - q11.2. Among them, single nucleotide polymorphism (SNP) rs4639425 in KMT2C gene, which regulates histone H3 lysine 4 (H3K4) methylation involved in chromatin remodeling, was associated with widespread alterations of white matter integrity including the cingulum, uncinate fasciculus, cortico-spinal tract, and superior longitudinal fasciculus. The small sample size of patients with BD in the genome data may cause our study to be underpowered when searching for putative rare mutations. This study first combined a WES approach and neuroimaging findings in psychiatric disorders. We postulate the rs4639425 may be associated with BD-related microstructural changes of white matter tracts. Show less

Human cohort studies have demonstrated a role for systemic metabolic dysfunction in osteoarthritis (OA) pathogenesis in obese patients. To explore the mechanisms underlying this metabolic phenotype of OA, we examined cartilage degradation in the knees of mice from different genetic backgrounds in which a metabolic phenotype was established by various dietary approaches. Wild-type C57BL/6J mice and genetically modified mice (hCRP, LDLr Metabolic phenotypes were confirmed in all studies as mice developed obesity, hypercholesteremia, glucose intolerance and/or insulin resistance. Aggravated cartilage degradation was only observed in two out of the twelve experimental setups, specifically in long-term studies in male hCRP and female ApoE*3Leiden.CETP mice. C57BL/6J and LDLr Long-term feeding of high-caloric diets consistently induced a metabolic phenotype in various C57BL/6J (-based) mouse strains. In contrast, the induction of articular cartilage degradation proved variable, which suggests that an additional trigger might be necessary to accelerate diet-induced OA progression. Gender and genetic modifications that result in a humanized pro-inflammatory state (human CRP) or lipoprotein metabolism (human-E3L.CETP) were identified as important contributing factors. Show less

Crosstalk between complement component 5a receptors (C5aRs) and TLRs in dendritic cells (DCs) occurs upon pathogen invasion; however, studies on C5aR and TLR crosstalk mainly focused on the modulating effect of C5a on TLR-induced cytokine production. To elucidate the breadth of C5aR and TLR4 crosstalk, the effect of simultaneous treatment with C5a and LPS was investigated in human monocyte-derived DCs (moDCs) 2 h after stimulation using whole transcriptome sequencing analysis. Although the effect of C5a on hallmark genes defining TLR4-induced DC maturation was limited at this time point, RNA sequencing analysis revealed a great variety of novel C5a targets, of which many interfere with TLR4-mediated immune activation. Analysis of functional relationships among these genes uncovered induction of a central immune regulatory network upon C5aR and TLR4 crosstalk, involving the transcription factors forkhead box (FOX)O1 and FOXO3 and the signaling molecules serum- and glucocorticoid-inducible kinase (SGK1), ribosomal S6 kinase 2 (RSK2), and PI3Kβ. C5aR and TLR crosstalk, furthermore, yielded down-regulation of mainly proinflammatory network branches, including IL-12B, IL-2Rα (IL-2RA), and jagged 1 (JAG1) and cooperative induction of predominantly anti-inflammatory network branches, including sphingosine kinase 1 (SPHK1), β2 adrenergic receptor (ADRB2), gastric inhibitory polypeptide receptor (GIPR), and four-and-a-half Lin11, Isl-1, and Mec-3 domains protein 2 (FHL2). Together, these data point toward induction of generalized immune regulation of DC function. Motif enrichment analysis indicate a prominent role for basic leucine zipper (bZIP) and IFN regulatory factor 4 (IRF4) transcription factors upon C5aR and TLR4 crosstalk. Additionally, differences were observed in the modulating capacity of C5a on DCs in the absence or presence of a pathogen (TLR stimulus). Our findings shed new light on the depth and complexity of C5aR and TLR4 crosstalk and provide new foci of research for future studies. Show less

Apolipoprotein A-I and A-IV are protein constituents of high-density lipoproteins although their functional difference in lipoprotein metabolism is still unclear. To compare anti-atherogenic properties between apoA-I and apoA-4, we characterized both proteins in lipid-free and lipid-bound state. In lipid-free state, apoA4 showed two distinct bands, around 78 and 67 Å on native gel electrophoresis, while apoA-I showed scattered band pattern less than 71 Å. In reconstituted HDL (rHDL) state, apoA-4 showed three major bands around 101 Å and 113 Å, while apoA-I-rHDL showed almost single band around 98 Å size. Lipid-free apoA-I showed 2.9-fold higher phospholipid binding ability than apoA-4. In lipid-free state, BS3-crosslinking revealed that apoA-4 showed less multimerization tendency upto dimer, while apoA-I showed pentamerization. In rHDL state (95:1), apoA-4 was existed as dimer as like as apoA-I. With higher phospholipid content (255:1), five apoA-I and three apoA-4 were required to the bigger rHDL formation. Regardless of particle size, apoA-I-rHDL showed superior LCAT activation ability than apoA-4-rHDL. Uptake of acetylated LDL was inhibited by apoA-I in both lipid-free and lipid-bound state, while apoA-4 inhibited it only lipid-free state. ApoA-4 showed less anti-atherogenic activity with more sensitivity to glycation. In conclusion, apoA-4 showed inferior physiological functions in lipid-bound state, compared with those of apoA-I, to induce more pro-atherosclerotic properties. Show less

Dual-specificity phosphatases (DUSPs) show distinct substrate preferences for specific MAPKs. DUSPs sharing a substrate preference for ERK1/2 may be classified as inducible or constitutive. In contrast to the inducible DUSPs which also dephosphorylate p38 MAPK and JNK in the major inflammatory pathways, constitutive DUSP6 and DUSP7 are specific to ERK1/2 and have not been studied in microglia and other immune cells to date. In the present study, we differentiated mRNA expression profiles of inducible and constitutive DUSPs that dephosphorylate ERK1/2 in microglia. Lipopolysaccharide (LPS) at 1 ng/ml induced prompt phosphorylation of ERK1/2 with peak induction at 30 min. LPS induced expression of DUSP1, DUSP2, and DUSP5 within 60 min, whereas DUSP4 expression was induced more slowly. DUSP6 and DUSP7 exhibited constitutive basal expression, which decreased immediately after LPS stimulation but subsequently returned to basal levels. The expression of DUSP6 and DUSP7 was regulated inverse to the phosphorylation of ERK1/2 in LPS-stimulated microglia. Therefore, we next investigated the correlation between DUSP6 and DUSP7 expression and ERK1/2 phosphorylation in resting and LPS-stimulated microglia. Inhibition of the ERK1/2 pathway by PD98059 and FR180204 resulted in a decrease in DUSP6 and DUSP7 expression, both in resting and LPS-stimulated microglia. These inhibitors partially blocked the LPS-induced expression of DUSP1, DUSP2, and DUSP4, but had no effect on DUSP5. Finally, we examined the role of DUSP6 activity in the downregulation of ERK1/2 phosphorylation. BCI, an inhibitor of DUSP6, increased the phosphorylation of ERK1/2. However, pretreatment with BCI inhibited the LPS-induced phosphorylation of ERK1/2. These results demonstrate that constitutive DUPS6 and DUSP7 expression was downregulated inverse to the expression of inducible DUSPs and the phosphorylation of ERK1/2 in LPS-stimulated microglia. The expression of DUPS6 and DUSP7 was mediated by ERK1/2 activity both in resting and LPS-stimulated microglia. In turn, DUSP6 suppressed the basal phosphorylation of ERK1/2, but exerted no suppressive effect on LPS-induced phosphorylation. Although DUSP6 is acknowledged as a negative regulator of the ERK1/2 pathway, such roles of DUSP6 need to be examined further in activated microglia. Show less

Multiple osteochondromas (MO) is a syndrome in which benign cartilage-capped neoplasms develop at the surface of the long bones. Most cases are caused by exonic changes in EXT1 or EXT2, but 15% are negative for these changes. Here we report for the first time a family of MO patients with germline genomic alterations at the EXT1 locus without detectable mutations or copy number alterations of EXT exonic sequences. Array-CGH showed an 80.7 kb deletion of Intron 1 of EXT1 and a 68.9 kb duplication proximal of EXT1. We identified a breakpoint between the distal end of the duplicated region and a sequence distal of the deleted region in the first intron. This breakpoint was absent in non-affected family members. The configuration of the breakpoint indicates a direct insertion of the duplicated region into the deletion. However, no other breakpoint was found, which suggests a more complex genomic rearrangement has occurred within the duplicated region. Our results reveal intronic deletion and duplication as a new causative mechanism for MO not detected by conventional diagnostic methods. Show less

Multiple osteochondromas (MO) is an autosomal dominant disorder caused by germline mutations in EXT1 and/or EXT2. In contrast, solitary osteochondroma (SO) is nonhereditary. Products of the EXT gene are involved in heparan sulfate (HS) biosynthesis. In this study, we investigated whether osteochondromas arise via either loss of heterozygosity (2 hits) or haploinsufficiency. An in vitro three-dimensional chondrogenic pellet model was used to compare heterozygous bone marrow-derived mesenchymal stem cells (MSCs EXT(wt/-)) of MO patients with normal MSCs and the corresponding tumor specimens (presumed EXT(-/-)). We demonstrated a second hit in EXT in five of eight osteochondromas. HS chain length and structure, in vitro chondrogenesis, and EXT expression levels were identical in both EXT(wt/-) and normal MSCs. Immunohistochemistry for HS, HS proteoglycans, and HS-dependent signaling pathways (eg, TGF-β/BMP, Wnt, and PTHLH) also showed no differences. The cartilaginous cap of osteochondroma contained a mixture of HS-positive and HS-negative cells. Because a heterozygous EXT mutation does not affect chondrogenesis, EXT, HS, or downstream signaling pathways in MSCs, our results refute the haploinsufficiency theory. We found a second hit in 63% of analyzed osteochondromas, supporting the hypothesis that osteochondromas arise via loss of heterozygosity. The detection of the second hit may depend on the ratio of HS-positive (normal) versus HS-negative (mutated) cells in the cartilaginous cap of the osteochondroma. Show less

The standard model of Wnt signaling specifies that after receipt of a Wnt ligand at the membranous receptor complex, downstream mediators inhibit a cytoplasmic destruction complex, allowing beta-catenin to accumulate in the cytosol and nucleus and co-activate Wnt target genes. Unexpectedly, shortly after Wnt treatment, we detected the dephosphorylated form of beta-catenin at the plasma membrane, where it displayed a discontinuous punctate labeling. This pool of beta-catenin could only be detected in E-cadherin(-/-) cells, because in E-cadherin(+/+) cells Wnt-induced, membranous beta-catenin was concealed by a constitutive junctional pool. Wnt-signaling-dependent dephosphorylated beta-catenin colocalized at the plasma membrane with two members of the destruction complex -- APC and axin -- and the activated Wnt co-receptor LRP6. beta-catenin induced through the Wnt receptor complex was significantly more competent transcriptionally than overexpressed beta-catenin, both in cultured cells and in early Xenopus embryos. Our data reveal a new step in the processing of the Wnt signal and suggest regulation of signaling output beyond the level of protein accumulation. Show less

In motile fibroblasts, stable microtubules (MTs) are oriented toward the leading edge of cells. How these polarized MT arrays are established and maintained, and the cellular processes they control, have been the subject of many investigations. Several MT "plus-end-tracking proteins," or +TIPs, have been proposed to regulate selective MT stabilization, including the CLASPs, a complex of CLIP-170, IQGAP1, activated Cdc42 or Rac1, a complex of APC, EB1, and mDia1, and the actin-MT crosslinking factor ACF7. By using mouse embryonic fibroblasts (MEFs) in a wound-healing assay, we show here that CLASP2 is required for the formation of a stable, polarized MT array but that CLIP-170 and an APC-EB1 interaction are not essential. Persistent motility is also hampered in CLASP2-deficient MEFs. We find that ACF7 regulates cortical CLASP localization in HeLa cells, indicating it acts upstream of CLASP2. Fluorescence-based approaches show that GFP-CLASP2 is immobilized in a bimodal manner in regions near cell edges. Our results suggest that the regional immobilization of CLASP2 allows MT stabilization and promotes directionally persistent motility in fibroblasts. Show less

Activation of the extracellular signal-regulated kinase 1 (ERK1) and ERK2 by neurotrophins, neuronal activity, or cAMP has been strongly implicated in differentiation, survival, and adaptive responses of neurons during development and in the adult brain. Recently, a new member of the mitogen-activated protein (MAP) kinase family, ERK5, was discovered. Like ERK1 and ERK2, ERK5 is expressed in neurons, and ERK5 stimulation by epidermal growth factor is blocked by the MAP kinase/ERK kinase 1 (MEK1) inhibitors PD98059 and U0126. This suggests the interesting possibility that some of the functions attributed to ERK1/2 may be mediated by ERK5. However, the regulatory properties of ERK5 in primary cultured neurons have not been reported. Here we examined the regulation of ERK5 signaling in primary cultured cortical neurons. Our data demonstrate that, similar to ERK1/2, ERK5 is activated by neurotrophins including brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and NT-4. BDNF stimulation of ERK5 required the activity of MEK5. Surprisingly, ERK5 was not stimulated by cAMP or neuronal activity induced by glutamate or membrane depolarization. In contrast to ERK1/2, ERK5 strongly activated the transcriptional activity of myocyte enhancer factor 2C (MEF2C) in pheochromocytoma 12 (PC12) cells and was required for neurotrophin stimulation of MEF2C transcription in both PC12 cells and cortical neurons. Furthermore, ERK1/2, but not ERK5, induced transcription from Elk1 and the cAMP/ Ca(2+) response element in PC12 cells. Our data suggest that mechanisms for regulation of ERK5 and downstream transcriptional pathways regulated by ERK5 are distinct from those of ERK1/2 in neurons. Furthermore, ERK5 is the first MAP kinase identified whose activity is stimulated by neurotrophins but not by neuronal activity. Show less