Fibroblast Growth Factor Binding Research Articles

The tandem of Fibroblast Growth Factor (FGF21) and Adipocyte Fatty Acid Binding Protein (A-FABP) as a physiological macronutrient signal to prevent insulin resistance

The tandem of Fibroblast Growth Factor (FGF21) and Adipocyte Fatty Acid Binding Protein (A-FABP) as a physiological macronutrient signal to prevent insulin resistance

Abstract 4373: Molecular interactions of human receptor tyrosine kinase oncogenic fusions

Abstract Oncogenic fusions (OFs) play a critical role in cancer development. Despite the exponential increase in identifications, only a small number have been extensively characterized functionally. In this study, we are focused on receptor tyrosine kinase OFs (RTK-OFs) prevalent in aggressive cancers. Our primary objective is to elucidate the underlying molecular mechanisms of 182 in-frame RTK-OFs identified in The Cancer Genome Atlas. An automated high-throughput PCR technique will be used to clone individual gene fusions using the sequences obtained from RNA-Seq data sourced from the TCGA dataset. These clones will be used to establish an RTK-OF-expressing isogenic cell line library. Multiple Approaches Combined (MAC)-tag workflow combining Affinity Purification (AP) and proximity biotin-dependent (BioID) purification within a single construct will be used. Subsequent high-throughput EvoSep One Liquid Chromatography coupled to ultra-fast TimsTOF Pro 2 Mass Spectrometry sample processing enables sensitive and robust analysis. These methods enable the capture of stable protein complexes and transient interactors associated with RTK-OFs. In our proof of concept study, we have identified four in-frame variants of FGFR3::TACC3 oncogenic fusions.FGFR3, part of the fibroblast growth factor (FGF) RTK family, activates cellular proliferation and growth through FGF binding and receptor dimerization. In FGFR3-TACC3 OFs, diverse isoforms are observed, with prevalent rearrangements linking FGFR3 exon 17 to various TACC3 exons. TACC3 is vital for mitotic microtubule organization and stabilizes kinetochore fibers and the mitotic spindle through its conserved C-terminal coiled-coil domain (TACC domain). TACC3 contributes to the fusion with varying sizes of the C-terminal coiled-coil domain which all constitutively activate the FGFR3 tyrosine kinase. In our studies, we show that FGFR3::TACC3 variants have a different localization to their wild-type counterparts. While FGFR3 and TACC3 are found in the cytoplasm all the OF variants strongly localize around the nucleus. Our identified high-confidence interactors represent a comprehensive FGFR3::TACC3 interaction network and reveal central pathways and key interactors related to microtubule assembly and possible deregulation of the cell cycle. The resultant data from these advanced technologies enable the construction of comprehensive interaction networks, shedding light on critical signaling pathways and subnetworks relevant to RTK-OFs. Further in-depth analysis of 182 RTK-OFs may uncover actionable pathways and druggable interactors within these networks. Our multi-faceted research strategy holds significant promise for advancing targeted drug discovery, ultimately contributing to improved cancer treatment strategies. Citation Format: Giovanna Dashi, Markku Varjosalo. Molecular interactions of human receptor tyrosine kinase oncogenic fusions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4373.

B4 suppresses lymphoma progression by inhibiting fibroblast growth factor binding protein 1 through intrinsic apoptosis.

Lymphoma positions as the fifth most common cancer, in the world, reporting remarkable deaths every year. Several promising strategies to counter this disease recently include utilizing small molecules that specifically target the lymphoma cellular proteins to overwhelm its progression. FGFBP1 is a soluble intracellular protein that progresses cancer cell proliferation and is upregulated in several cancers. Therefore, inhibiting FGFBP1 could significantly slow down lymphoma progression through triggering apoptosis. Thus, in this study, a flavonoid B4, isolated from Cajanus cajan, has been investigated for its effects of B4 on lymphoma, specifically as an FGFBP1 inhibitor. B4 could selectively hinder the growth of lymphoma cells by inducing caspase-dependent intrinsic apoptosis through G1/S transition phase cell cycle arrest. RNA sequencing analysis revealed that B4 regulates the genes involved in B-cell proliferation and DNA replication by inhibiting FGFBP1 in vitro. B4 increases the survival rate of lymphoma mice. B4 also represses the growth of patient-derived primary lymphoma cells through FGFBP1 inhibition. Drug affinity responsive target stability experimentations authorize that B4 powerfully binds to FGFBP1. The overexpression of FGFBP1 raises the pharmacological sensitivity of B4, supplementing its specific action on lymphoma cells. This study pioneers the estimation of B4 as a possible anticancer agent for lymphoma treatment. These outcomes highlight its selective inhibitory effects on lymphoma cell growth by downregulating FGFBP1 expression through intrinsic apoptosis, causing mitochondrial and DNA damage, ultimately leading to the inhibition of lymphoma progression. These suggest B4 may be a novel FGFBP1 inhibitor for the lymphoma treatment.

KLF5 promotes esophageal squamous cell cancer through the transcriptional activation of FGFBP1

Krüpple-like factor 5 (KLF5) is a zinc-finger-containing transcription factor implicated in several human malignancies, but its potential regulatory mechanisms implicated in esophageal squamous cell carcinoma (ESCC) remain elusive. Here, we show that KLF5 is upregulated in ESCC, where its level was significantly associated with tumor differentiation and lymph node metastasis status. Upregulated KLF5 expression promoted the proliferation, migration, and invasion of ESCC cells. Reduced KLF5 showed the opposite effects. Mechanistically, KLF5 exerts its tumor promotion effect by up-regulating fibroblast growth factor binding protein 1 (FGF-BP1) and snail family transcriptional repressor 2 (SNAIL2). KLF5 binds to the promoter regions of FGF-BP1 and transcriptionally activates its expression. Our study indicated that KLF5 could promote esophageal squamous cell cancer proliferation, migration, and invasion by upregulating FGF-BP1/SNAIL2 signaling. Our work suggests that KLF5 might be a proto-oncogene in ESCC and implicated in ESCC metastasis.

Relationship between Carotid Intima-Media Thickness and Fibroblast Growth Factor Binding Protein-3 in Patients with Metabolic Syndrome

Relationship between Carotid Intima-Media Thickness and Fibroblast Growth Factor Binding Protein-3 in Patients with Metabolic Syndrome

Oncostatin M enhances osteoprotegerin synthesis but reduces macrophage colony‑stimulating factor synthesis in bFGF‑stimulated osteoblast‑like cells.

Bone remodeling is tightly controlled by various factors, including hormones, autacoids and cytokines. Among them, oncostatin M (OSM) is a multifunctional cytokine produced by osteal macrophages, which serves as an essential modulator of bone remodeling. Macrophage colony-stimulating factor (M-CSF) and osteoprotegerin are secreted by osteoblasts, and also have pivotal roles in the regulation of the bone remodeling process. The binding of basic fibroblast growth factor (bFGF), a key regulator of bone remodeling, to the corresponding receptor [fibroblast growth factor receptor (FGFR)] triggers the dimerization and activation of FGFRs, which causes the phosphorylation of FGFR substrates and subsequent activation of downstream effectors, including mitogen-activated protein kinases (MAPKs), via Grb2. bFGF can activate MAPKs, resulting in the synthesis of osteoprotegerin and vascular endothelial growth factor in osteoblast-like MC3T3-E1 cells. In the present study, the effects of OSM on bFGF-induced osteoblast activation were investigated in the synthesis of osteoprotegerin and M-CSF in osteoblasts. The release of osteoprotegerin and M-CSF were analyzed using ELISA. The mRNA expression levels of osteoprotegerin and M-CSF were analyzed using reverse transcription-quantitative PCR. Phosphorylation of p38 MAPK, stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and p44/p42 MAPK was assessed using western blotting. OSM enhanced bFGF-induced osteoprotegerin release and bFGF-stimulated mRNA expression of osteoprotegerin. By contrast, OSM suppressed the bFGF-induced release of M-CSF and bFGF-stimulated mRNA expression of M-CSF. SB203580, a p38 MAPK inhibitor, and SP600125, a SAPK/JNK inhibitor, suppressed the bFGF-stimulated M-CSF release, whereas PD98059, an upstream kinase inhibitor of p44/p42 MAPK, failed to suppress the M-CSF release stimulated by bFGF. Furthermore, OSM enhanced the bFGF-induced phosphorylation of p38 MAPK, but attenuated the bFGF-stimulated phosphorylation of SAPK/JNK. By contrast, OSM had little effect on the bFGF-induced phosphorylation of p44/p42 MAPK. SB203580 markedly reduced the amplification of bFGF-stimulated osteoprotegerin release enhanced by OSM. These results strongly suggested that OSM may possess divergent effects on bFGF-induced osteoblast activation, upregulation of p38 MAPK and downregulation of SAPK/JNK, leading to the amplification of osteoprotegerin synthesis and the attenuation of M-CSF synthesis.

TFCP2 is a transcriptional regulator of heparan sulfate assembly and melanoma cell growth

Heparan sulfate (HS) is a long, linear polysaccharide that is ubiquitously expressed in all animal cells and plays a key role in many cellular processes, including cell signaling and development. Dysregulation of HS assembly has been implicated in pathophysiological conditions, such as tumorigenesis and rare genetic disorders. HS biosynthesis occurs in a non-template-driven manner in the endoplasmic reticulum and Golgi through the activity of a large group of biosynthetic enzymes. While much is known about its biosynthesis, little is understood about the regulation of HS assembly across diverse tissue types and disease states. To address this gap in knowledge, we recently performed genome-wide CRISPR/Cas9 screens to identify novel regulatory factors of HS biosynthesis. From these screens, we identified the alpha globin transcription factor, TFCP2, as a top hit. To investigate the role of TFCP2 in HS assembly, we targeted TFCP2 expression in human melanoma cells using the CRISPR/Cas9 system. TFCP2 knockout cells exhibited decreased fibroblast growth factor binding to cell surface HS, alterations in HS composition, and slowed cell growth compared to wild-type cells. Additionally, RNA sequencing revealed that TFCP2 regulates the expression of multiple enzymes involved in HS assembly, including the secreted endosulfatase, SULF1. Pharmacological targeting of TFCP2 activity similarly reduced growth factor binding and increased SULF1 expression, and the knockdown of SULF1 expression in TFCP2 mutant cells restored melanoma cell growth. Overall, these studies identify TFCP2 as a novel transcriptional regulator of HS and highlight HS-protein interactions as a possible target to slow melanoma growth.

Dynamic Changes in Heparan Sulfate Nanostructure in Human Pluripotent Stem Cell Differentiation.

Heparan sulfate (HS) is a heterogeneous, cell-surface polysaccharide critical for transducing signals essential for mammalian development. Imaging of signaling proteins has revealed how their localization influences their information transfer. In contrast, the contribution of the spatial distribution and nanostructure of information-rich, signaling polysaccharides like HS is not known. Using expansion microscopy (ExM), we found striking changes in HS nanostructure occur as human pluripotent stem (hPS) cells differentiate, and these changes correlate with growth factor signaling. Our imaging studies show that undifferentiated hPS cells are densely coated with HS displayed as hair-like protrusions. This ultrastructure can recruit fibroblast growth factor for signaling. When the hPS cells differentiate into the ectoderm lineage, HS is localized into dispersed puncta. This striking change in HS distribution coincides with a decrease in fibroblast growth factor binding to neural cells. While developmental variations in HS sequence were thought to be the primary driver of alterations in HS-mediated growth factor signaling, our high-resolution images indicate a role for the HS nanostructure. Our study highlights the utility of high-resolution glycan imaging using ExM. In the case of HS, we found that changes in how the polysaccharide is displayed link to profound differences in growth factor binding.

Genetic diagnosis of microcephaly

Objective: To explore the diagnostic value of chromosome karyotype analysis, chromosomal microarray analysis (CMA) and whole exome sequencing (WES) in microcephaly. Methods: A total of 9 cases of microcephaly fetuses diagnosed by prenatal ultrasound or children with microcephaly diagnosed after birth were selected from the Sixth Affiliated Hospital of Guangzhou Medical University from January 2014 to August 2022.Karyotype analysis and/or CMA were used to detect. The cases with negative karyotype analysis and CMA results were further sequenced by trio-based WES (Trio-WES). Then the coding genes contained in the pathogenic copy number variation (CNV) fragments were analyzed by gene ontology (GO) enrichment. The genes related to the development of the central nervous system contained in the pathogenic CNV and the pathogenic genes found by Trio-WES were combined for gene interaction network analysis. Results: In this study, 9 cases of microcephaly were recruited, with the time of diagnosis ranged from 23 weeks of gestation to 7 years after birth, and the head circumference of fetus or children ranged from 18.3 to 42.5 cm (-7SD to -2SD). Karyotype analysis was detected in all 9 cases and no abnormality result was found. Eight cases were detected by CMA, and one abnormal was found. Five cases were detected by Trio-WES, and two cases were detected with likely pathogenic genes. The GO enrichment analysis of the coding gene in the 4p16.3 microdeletion (pathogenic CNV) region showed that: in biological process, it was mainly concentrated in phototransduction, visible light; in terms of molecular function, it was mainly concentrated in fibroblast growth factor binding; in terms of cell components, it was mainly concentrated in rough endoplasmic reticulum. Gene interaction network analysis suggested that CDC42 gene could interact with CTBP1, HTT and ASPM gene. Conclusions: CMA could be used as a first-line detection technique for microcephaly. When the results of chromosome karyotype analysis and/or CMA are negative, Trio-WES could improve the detection rate of pathogenicity of microcephaly.

MiR-4432 Targets FGFBP1 in Human Endothelial Cells.

MicroRNAs (miRs) are small non-coding RNAs that modulate the expression of several target genes. Fibroblast growth factor binding protein 1 (FGFBP1) has been associated with endothelial dysfunction at the level of the blood-brain barrier (BBB). However, the underlying mechanisms are mostly unknown and there are no studies investigating the relationship between miRs and FGFBP1. Thus, the overarching aim of the present study was to identify and validate which miR can specifically target FGFBP1 in human brain microvascular endothelial cells, which represent the best in vitro model of the BBB. We were able to identify and validate miR-4432 as a fundamental modulator of FGFBP1 and we demonstrated that miR-4432 significantly reduces mitochondrial oxidative stress, a well-established pathophysiological hallmark of hypertension.

Fibroblast growth factor-2 bound to specific dermal fibroblast-derived extracellular vesicles is protected from degradation

Fibroblast growth factor-2 (FGF2) has multiple roles in cutaneous wound healing but its natural low stability prevents the development of its use in skin repair therapies. Here we show that FGF2 binds the outer surface of dermal fibroblast (DF)-derived extracellular vesicles (EVs) and this association protects FGF2 from fast degradation. EVs isolated from DF cultured in the presence of FGF2 harbor FGF2 on their surface and FGF2 can bind purified EVs in absence of cells. Remarkably, FGF2 binding to EVs is restricted to a specific subpopulation of EVs, which do not express CD63 and CD81 markers. Treatment of DF with FGF2-EVs activated ERK and STAT signaling pathways and increased cell proliferation and migration. Local injection of FGF2-EVs improved wound healing in mice. We further demonstrated that binding to EVs protects FGF2 from both thermal and proteolytic degradation, thus maintaining FGF2 function. This suggests that EVs protect soluble factors from degradation and increase their stability and half-life. These results reveal a novel aspect of EV function and suggest EVs as a potential tool for delivering FGF2 in skin healing therapies.

Synthesis of a Systematic 64-Membered Heparan Sulfate Tetrasaccharide Library.

Heparan sulfate (HS) has multifaceted biological activities. To date, no libraries of HS oligosaccharides bearing systematically varied sulfation structures are available owing to the challenges in synthesizing a large number of HS oligosaccharides. To overcome the obstacles and expedite the synthesis, a divergent approach was designed, where 64 HS tetrasaccharides covering all possible structures of 2-O-, 6-O- and N-sulfation with the glucosamine-glucuronic acid-glucosamine-iduronic acid backbone were successfully produced from a single strategically protected tetrasaccharide intermediate. This extensive library helped identify the structural requirements for HS sequences to have strong fibroblast growth factor-2 binding but a weak affinity for platelet factor-4. Such a strategy to separate out these two interactions could lead to new HS-based potential therapeutics without the dangerous adverse effect of heparin-induced thrombocytopenia.

FGFBP1 as a potential biomarker predicting bacillus Calmette–Guérin response in bladder cancer

Accurate prediction of Bacillus Calmette–Guérin (BCG) response is essential to identify bladder cancer (BCa) patients most likely to respond sustainably, but no molecular marker predicting BCG response is available in clinical routine. Therefore, we first identified that fibroblast growth factor binding protein 1 (FGFBP1) was upregulated in failures of BCG therapy, and the increased FGFBP1 had a poor outcome for BCa patients in the E-MTAB-4321 and GSE19423 datasets. These different expression genes associated with FGFBP1 expression are mainly involved in neutrophil activation, neutrophil-mediated immunity, and tumor necrosis factor-mediated signal pathways in biological processes. A significant positive correlation was observed between FGFBP1 expression and regulatory T-cell (Treg) infiltration by the Spearman correlation test in the BCG cohort (r = 0.177) and The Cancer Genome Atlas (TCGA) cohort (r = 0.176), suggesting that FGFBP1 may influence the response of BCa patients to BCG immunotherapy through immune escape. Though FGFBP1 expression was positively correlated with the expressions of PD-L1, CTLA4, and PDCD1 in TCGA cohort, a strong association between FGFBP1 and PD-L1 expression was only detected in the BCG cohort (r = 0.750). Furthermore, elevated FGFBP1 was observed in BCa cell lines and tissues in comparison to corresponding normal controls by RT-qPCR, Western blotting, and immunohistochemical staining. Increased FGFBP1 was further detected in the failures than in the responders by immunohistochemical staining. Notably, FGFBP1 is positively associated with PD-L1 expression in BCa patients with BCG treatment. To sum up, FGFBP1 in BCa tissue could be identified as a promising biomarker for the accurate prediction of BCG response in BCa.

Synthetic Heparan Sulfate Hydrogels Regulate Neurotrophic Factor Signaling and Neuronal Network Activity.

Three-dimensional (3D) synthetic heparan sulfate (HS) constructs possess promising attributes for neural tissue engineering applications. However, their sulfation-dependent ability to facilitate molecular recognition and cell signaling has not yet been investigated. We hypothesized that fully sulfated synthetic HS constructs (bearing compound 1) that are functionalized with neural adhesion peptides will enhance fibroblast growth factor-2 (FGF2) binding and complexation with FGF receptor-1 (FGFR1) to promote the proliferation and neuronal differentiation of human neural stem cells (hNSCs) when compared to constructs with unsulfated controls (bearing compound 2). We tested this hypothesis in vitro using 2D and 3D substrates consisting of different combinations of HS tetrasaccharides (compounds 3 and 4) and an engineered integrin-binding chimeric peptide (CP), which were assembled using strain-promoted alkyne-azide cycloaddition (SPAAC) chemistry. Results indicated that the adhesion of hNSCs increased significantly when cultured on 2D glass substrates functionalized with chimeric peptide. hNSCs encapsulated in 1-CP hydrogels and cultured in media containing the mitogen FGF2 exhibited significantly higher neuronal differentiation when compared to hNSCs in 2-CP hydrogels. These observations were corroborated by Western blot analysis, which indicated the enhanced binding and retention of both FGF2 and FGFR1 by 1 as well as downstream phosphorylation of extracellular signal-regulated kinases (ERK1/2) and enhanced proliferation of hNSCs. Lastly, calcium activity imaging revealed that both 1 and 2 hydrogels supported the neuronal growth and activity of pre-differentiated human prefrontal cortex neurons. Collectively, these results demonstrate that synthetic HS hydrogels can be tailored to regulate growth factor signaling and neuronal fate and activity.

Semen Modulates Cell Proliferation and Differentiation-Related Transcripts in the Pig Peri-Ovulatory Endometrium.

Simple SummaryHomeostasis of the uterus after mating is crucial for the subsequent reproductive events. The post-mating inflammatory response is restricted to the uterus, but semen also modulates the expression of other genes involved in regulation along the female reproductive tract, including the oviduct. This study aims to determine if several ejaculate fractions of the pig may modulate cell proliferation and differentiation-related transcripts in different sections of the peri-ovulatory sow reproductive tract. Our data demonstrate that most of the mRNA expression changes of the 144 transcripts tested were induced by mating. Additionally, spermatozoa and seminal plasma also triggered differential expression of the transcripts tested. Finally, our data imply that spermatozoa, seminal plasma components, and the act of mating induce differential mechanisms in the peri-ovulatory female reproductive tract, which are essential for tissue repair.Uterine homeostasis is maintained after mating by eliminating pathogens, foreign cells, and proteins by a transient inflammation of the uterus. Such inflammation does not occur in the oviductal sperm reservoir (utero-tubal junction, UTJ), colonized by a population of potentially fertile spermatozoa before the inflammatory changes are triggered. Semen entry (spermatozoa and/or seminal plasma) modifies the expression of regulatory genes, including cell proliferation and differentiation-related transcripts. Considering pigs display a fractionated ejaculation, this study aims to determine whether different ejaculate fractions differentially modulate cell proliferation and differentiation-related transcripts in the sow reproductive tract during the peri-ovulatory stage. Using species-specific microarray analyses, the differential expression of 144 cell proliferation and differentiation-related transcripts was studied in specific segments: cervix (Cvx), distal and proximal uterus (DistUt, ProxUt), UTJ, isthmus (Isth), ampulla (Amp), and infundibulum (Inf) of the peri-ovulatory sow reproductive tract in response to semen and/or seminal plasma cervical deposition. Most mRNA expression changes were induced by mating. In addition, while mating upregulates the fibroblast growth factor 1 (FGF1, p-value DistUt = 0.0007; ProxUt = 0.0253) transcript in the endometrium, both its receptor, the fibroblast growth factor receptor 1 (FGFR1, p-value DistUt = 2.14 e−06; ProxUt = 0.0027; UTJ = 0.0458) transcript, and a potentiator of its biological effect, the fibroblast growth factor binding protein 1 (FGFBP1), were downregulated in the endometrium (p-value DistUt = 0.0068; ProxUt = 0.0011) and the UTJ (p-value UTJ = 0.0191). The FGFBP1 was downregulated in the whole oviduct after seminal depositions (p-value Isth = 0.0007; Amp = 0.0007; Inf = 6.87 e−05) and, interestingly, FGFR1 was downregulated in the endometrium in the absence of semen (p-value DistUt = 0.0097; ProxUt = 0.0456). In conclusion, the findings suggest that spermatozoa, seminal components, and the act of mating trigger, besides inflammation, differential mechanisms in the peri-ovulatory female reproductive tract, relevant for tissue repair.

Analysis of protein-heparin interactions using a portable SPR instrument

Optical biosensors such as those based on surface plasmon resonance (SPR) are a key analytical tool for understanding biomolecular interactions and function as well as the quantitative analysis of analytes in a wide variety of settings. The advent of portable SPR instruments enables analyses in the field. A critical step in method development is the passivation and functionalisation of the sensor surface. We describe the assembly of a surface of thiolated oleyl ethylene glycol/biotin oleyl ethylene glycol and its functionalisation with streptavidin and reducing end biotinylated heparin for a portable SPR instrument. Such surfaces can be batch prepared and stored. Two examples of the analysis of heparin-binding proteins are presented. The binding of fibroblast growth factor 2 and competition for the binding of a heparan sulfate sulfotransferase by a library of selectively modified heparins and suramin, which identify the selectivity of the enzyme for sulfated structures in the polysaccharide and demonstrate suramin as a competitor for the enzyme’s sugar acceptor site. Heparin functionalised surfaces should have a wide applicability, since this polysaccharide is a close structural analogue of the host cell surface polysaccharide, heparan sulfate, a receptor for many endogenous proteins and viruses.

KLF15 overexpression in myocytes fails to ameliorate ALS-related pathology or extend the lifespan of SOD1G93A mice

Amyotrophic Lateral Sclerosis (ALS) is a currently incurable disease that causes progressive motor neuron loss, paralysis and death. Skeletal muscle pathology occurs early during the course of ALS. It is characterized by impaired mitochondrial biogenesis, metabolic dysfunction and deterioration of the neuromuscular junction (NMJ), the synapse through which motor neurons communicate with muscles. Therefore, a better understanding of the molecules that underlie this pathology may lead to therapies that slow motor neuron loss and delay ALS progression. Kruppel Like Factor 15 (KLF15) has been identified as a transcription factor that activates alternative metabolic pathways and NMJ maintenance factors, including Fibroblast Growth Factor Binding Protein 1 (FGFBP1), in skeletal myocytes. In this capacity, KLF15 has been shown to play a protective role in Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA), however its role in ALS has not been evaluated. Here, we examined whether muscle-specific KLF15 overexpression promotes the health of skeletal muscles and NMJs in the SOD1G93A ALS mouse model. We show that muscle-specific KLF15 overexpression did not elicit a significant beneficial effect on skeletal muscle atrophy, NMJ health, motor function, or survival in SOD1G93A ALS mice. Our findings suggest that, unlike in mouse models of DMD and SMA, KLF15 overexpression has a minimal impact on ALS disease progression in SOD1G93A mice.

FGF binding protein 3 is required for spinal cord motor neuron development and regeneration in zebrafish

Fibroblast growth factor binding protein 3 (Fgfbp3) have been known to be crucial for the process of neural proliferation, differentiation, migration, and adhesion. However, the specific role and the molecular mechanisms of fgfbp3 in regulating the development of motor neurons remain unclear. In this study, we have investigated the function of fgfbp3 in morphogenesis and regeneration of motor neuron in zebrafish. Firstly, we found that fgfbp3 was localized in the motor neurons and loss of fgfbp3 caused the significant decrease of the length and branching number of the motor neuron axons, which could be partially rescued by fgfbp3 mRNA injection. Moreover, the fgfbp3 knockdown (KD) embryos demonstrated similar defects of motor neurons as identified in fgfbp3 knockout (KO) embryos. Furthermore, we revealed that the locomotion and startle response of fgfbp3 KO embryos were significantly restricted, which were partially rescued by the fgfbp3 overexpression. In addition, fgfbp3 KO remarkably compromised axonal regeneration of motor neurons after injury. Lastly, the malformation of motor neurons in fgfbp3 KO embryos was rescued by overexpressing drd1b or neurod6a, respectively, which were screened by transcriptome sequencing. Taken together, our results provide strong cellular and molecular evidence that fgfbp3 is crucial for the axonal morphogenesis and regeneration of motor neurons in zebrafish.

Impaired neural differentiation of MPS IIIA patient induced pluripotent stem cell-derived neural progenitor cells

Mucopolysaccharidosis type IIIA (MPS IIIA) is characterised by a progressive neurological decline leading to early death. It is caused by bi-allelic loss-of-function mutations in SGSH encoding sulphamidase, a lysosomal enzyme required for heparan sulphate glycosaminoglycan (HS GAG) degradation, that results in the progressive build-up of HS GAGs in multiple tissues most notably the central nervous system (CNS). Skin fibroblasts from two MPS IIIA patients who presented with an intermediate and a severe clinical phenotype, respectively, were reprogrammed into induced pluripotent stem cells (iPSCs). The intermediate MPS IIIA iPSCs were then differentiated into neural progenitor cells (NPCs) and subsequently neurons. The patient derived fibroblasts, iPSCs, NPCs and neurons all displayed hallmark biochemical characteristics of MPS IIIA including reduced sulphamidase activity and increased accumulation of an MPS IIIA HS GAG biomarker. Proliferation of MPS IIIA iPSC-derived NPCs was reduced compared to control, but could be partially rescued by reintroducing functional sulphamidase enzyme, or by doubling the concentration of the mitogen fibroblast growth factor 2 (FGF2). Whilst both control heparin, and MPS IIIA HS GAGs had a similar binding affinity for FGF2, only the latter inhibited FGF signalling, suggesting accumulated MPS IIIA HS GAGs disrupt the FGF2:FGF2 receptor:HS signalling complex. Neuronal differentiation of MPS IIIA iPSC-derived NPCs was associated with a reduction in the expression of neuronal cell marker genes βIII-TUBULIN, NF-H and NSE, revealing reduced neurogenesis compared to control. A similar result was achieved by adding MPS IIIA HS GAGs to the culture medium during neuronal differentiation of control iPSC-derived NPCs. This study demonstrates the generation of MPS IIIA iPSCs, and NPCs, the latter of which display reduced proliferation and neurogenic capacity. Reduced NPC proliferation can be explained by a model in which soluble MPS IIIA HS GAGs compete with cell surface HS for FGF2 binding. The mechanism driving reduced neurogenesis remains to be determined but appears downstream of MPS IIIA HS GAG accumulation.

Pilot Gene Expression and Histopathologic Analysis of Tracheal Resections in Tracheobronchomalacia

BackgroundThe airway structures and messenger RNA expression of genes that regulate airway inflammation and remodeling may be altered in the trachea of patients with tracheobronchomalacia (TBM). MethodsFourteen tracheal specimens obtained from 2005 to 2018 were used in this study. Surgical resection specimens from patients with TBM and tracheal stenosis (TS) were compared with control tracheal specimens obtained from autopsy cases. We investigated the messenger RNA expression of genes encoding fibroblast growth factor (FGF) binding protein 2 (FGFBP2), FGF receptor R3 (FGFR3), interleukin-1β (IL1β), tumor growth factor-β1 (TGFβ1), tissue inhibitor of metalloproteinases 1 (TIMP1), and intercellular adhesion molecule 1 (ICAM1) as well as established markers of airway inflammation including interferon-γ (IFNγ) and tumor necrosis factor (TNF). The relative expression of target transcripts was assessed by quantitative real-time polymerase chain reaction. A histologic examination of the same resected airway specimens was performed on formalin-fixed paraffin-embedded tissue sections. ResultsFGFBP2 and FGFR3 showed higher expression in TBM compared with TS and control groups (P < .05 and P < .01, respectively). Furthermore, both TGFβ1 and TIMP1 were elevated in TBM patients compared with control subjects (P < .05). Conversely ICAM1 was downregulated in TBM versus TS and control subjects (P < .05). IL1β, IFNγ, and TNF were increased in TBM, although it did not achieve statistical significance. Histologically compared with control airways both TBM and TS demonstrated submucosal fibrotic changes, with TBM additionally demonstrating alterations in elastin fiber quality and density in the posterior membrane. ConclusionsSignificant changes in gene expression are observed in the tracheal walls of patients with TBM and TS compared with control subjects.