Effects of Epigenetic Modification of PGC-1α by a Chemical Chaperon on Mitochondria Biogenesis and Visual Function in Retinitis Pigmentosa

Retinitis pigmentosa (RP) is a hereditary blinding disease characterized by gradual photoreceptor death, which lacks a definitive treatment. Here, we demonstrated the effect of 4-phenylbutyric acid (PBA), a chemical chaperon that can suppress endoplasmic reticulum (ER) stress, in P23H mutant rhodopsin knock-in RP models. In the RP models, constant PBA treatment led to the retention of a greater number of photoreceptors, preserving the inner segment (IS), a mitochondrial- and ER-rich part of the photoreceptors. Electroretinography showed that PBA treatment preserved photoreceptor function. At the early point, ER-associated degradation markers, xbp1s, vcp, and derl1, mitochondrial kinetic-related markers, fis1, lc3, and mfn1 and mfn2, as well as key mitochondrial regulators, pgc-1α and tfam, were upregulated in the retina of the models treated with PBA. In vitro analyses showed that PBA upregulated pgc-1α and tfam transcription, leading to an increase in the mitochondrial membrane potential, cytochrome c oxidase activity, and ATP levels. Histone acetylation of the PGC-1α promoter was increased by PBA, indicating that PBA affected the mitochondrial condition through epigenetic changes. Our findings constituted proof of concept for the treatment of ER stress-related RP using PBA and revealed PBA’s neuroprotective effects, paving the way for its future clinical application

The Neuroprotective Effect of Rapamycin as a Modulator of the mTOR-NF-κB Axis during Retinal Inflammation.

The determination of the molecular mechanism underlying retinal pathogenesis and visual dysfunction during innate inflammation, and the treatment effect of rapamycin thereon.The endotoxin-induced uveitis and retinitis mouse model was established by injecting lipopolysaccharide. The mice were subsequently treated with rapamycin, a mammalian target of rapamycin (mTOR) inhibitor. The rhodopsin mRNA and protein expression level in the retina and the photoreceptor outer segment (OS) length in immunohistochemical stainings were measured, and visual function was recorded by electroretinography. Inflammatory cytokines, their related molecules, mTOR, and LC3 levels were measured by real-time PCR and/or immunoblotting. Leukocyte adhesion during inflammation was analyzed using concanavalin A lectin.The post-transcriptional reduction in the visual pigment of rod photoreceptor cells, rhodopsin, OS shortening, and rod photoreceptor cell dysfunction during inflammation were suppressed by rapamycin. Activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and induction of inflammatory cytokines, such as interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1), and the activation of the downstream signaling protein, signal transducer and activator of transcription 3 (STAT3), which reduces rhodopsin in the retina during inflammation, were attenuated by rapamycin. Increased leukocyte adhesion was also attenuated by rapamycin. Interestingly, although mTOR activation was observed after NF-κB activation, mTOR inhibition suppressed NF-κB activation at the early phase, indicating that the basal level of activated mTOR was sufficient to activate NF-κB in the retina. In addition, the inhibition of NF-κB suppressed mTOR activation, suggesting a positive feedback loop of mTOR and NF-κB during inflammation. The ratio of LC3II to LC3I, which reflects autophagy induction, was not changed by inflammation but was increased by rapamycin.Our results propose the potential use of rapamycin as a neuroprotective therapy to suppress local activated mTOR levels, related inflammatory molecules, and the subsequent visual dysfunction during retinal inflammation

Chondroitin sulfate synthase-2 is necessary for chain extension of chondroitin sulfate but not critical for skeletal development.

Chondroitin sulfate (CS) is a linear polysaccharide consisting of repeating disaccharide units of N-acetyl-D-galactosamine and D-glucuronic acid residues, modified with sulfated residues at various positions. Based on its structural diversity in chain length and sulfation patterns, CS provides specific biological functions in cell adhesion, morphogenesis, neural network formation, and cell division. To date, six glycosyltransferases are known to be involved in the biosynthesis of chondroitin saccharide chains, and a hetero-oligomer complex of chondroitin sulfate synthase-1 (CSS1)/chondroitin synthase-1 and chondroitin sulfate synthase-2 (CSS2)/chondroitin polymerizing factor is known to have the strongest polymerizing activity. Here, we generated and analyzed CSS2(-/-) mice. Although they were viable and fertile, exhibiting no overt morphological abnormalities or osteoarthritis, their cartilage contained CS chains with a shorter length and at a similar number to wild type. Further analysis using CSS2(-/-) chondrocyte culture systems, together with siRNA of CSS1, revealed the presence of two CS chain species in length, suggesting two steps of CS chain polymerization; i.e., elongation from the linkage region up to Mr ∼10,000, and further extension. There, CSS2 mainly participated in the extension, whereas CSS1 participated in both the extension and the initiation. Our study demonstrates the distinct function of CSS1 and CSS2, providing a clue in the elucidation of the mechanism of CS biosynthesis

Cell Surface Heparan Sulfate Chains Regulate Local Reception of FGF Signaling in the Mouse Embryo

SummaryHeparan sulfate (HS) proteoglycans modulate the activity of multiple growth factors on the cell surface and extracellular matrix. However, it remains unclear how the HS chains control the movement and reception of growth factors into targeted receiving cells during mammalian morphogenetic processes. Here, we found that HS-deficient Ext2 null mutant mouse embryos fail to respond to fibroblast growth factor (FGF) signaling. Marker expression analyses revealed that cell surface-tethered HS chains are crucial for local retention of FGF4 and FGF8 ligands in the extraembryonic ectoderm. Fine chimeric studies with single-cell resolution and expression studies with specific inhibitors for HS movement demonstrated that proteolytic cleavage of HS chains can spread FGF signaling to adjacent cells within a short distance. Together, the results show that spatiotemporal expression of cell surface-tethered HS chains regulate the local reception of FGF-signaling activity during mammalian embryogenesis

GWAS-identified CCR1 and IL10 loci contribute to M1 macrophage-predominant inflammation in Behçet’s disease

Abstract Background Low C-C chemokine receptor 1 (CCR1) and interleukin (IL)-10 expression is associated with risk of Behçet’s disease (BD). The objective of the present study was to clarify the pathological roles of CCR1 and IL10 loci identified by previous BD genome-wide association studies (GWASs). Methods M1 and M2 macrophages (Mφ) were differentiated with granulocyte-macrophage colony-stimulating factor or macrophage colony-stimulating factor (M-CSF) from peripheral monocytes of healthy control subjects (HC) and patients with BD. Expression of CD68 and CD163 was evaluated to test for Mφ polarization. CCR1 and IL-10 messenger RNA (mRNA) and protein expression was compared according to CCR1 and IL10 single-nucleotide polymorphism (SNP) genotypes. The migratory ability of M1 and M2 Mφ toward CCR1 ligand macrophage inflammatory protein (MIP)-1α was compared. The ratio of M1 and M2 Mφ in skin lesions of BD and systemic sclerosis (SSc), which was reported to be M2 Mφ-dominant, was compared. To examine the plasticity of polarized Mφ, the differentiated cells were cultured with either the same or the other culture condition. Results Preferential expression of CD163, CCR1, and IL-10 was found in M2 Mφ compared with M1 Mφ. M2 Mφ migrated more sensitively to low concentrations of MIP-1α than M1 Mφ did. BD-derived M1 Mφ showed higher CCR1 surface expression than HC-derived M1 Mφ did. IL10 and CCR1 mRNA expression differences were observed by GWAS-identified SNP genotypes in polarized Mφ. BD skin lesions showed M1 Mφ predominance compared with SSc skin lesions. A plasticity assay revealed that M-CSF restored IL-10 synthesis and reduced IL-6 production by M1 Mφ. Conclusions The present study reveals that GWAS-identified SNPs contribute to M1 Mφ-predominant inflammation in BD. Our data also suggest that the skewed Mφ polarization is correctable by immunological intervention