CD47 promotes peripheral T cell survival by preventing dendritic cell-mediated T cell necroptosis.

Conventional dendritic cells (cDCs) are required for peripheral T cell homeostasis in lymphoid organs, but the molecular mechanism underlying this requirement has remained unclear. We here show that T cell-specific CD47-deficient (Cd47 ΔT) mice have a markedly reduced number of T cells in peripheral tissues. Direct interaction of CD47-deficient T cells with cDCs resulted in activation of the latter cells, which in turn induced necroptosis of the former cells. The deficiency and cell death of T cells in Cd47 ΔT mice required expression of its receptor signal regulatory protein α on cDCs. The development of CD4+ T helper cell-dependent contact hypersensitivity and inhibition of tumor growth by cytotoxic CD8+ T cells were both markedly impaired in Cd47 ΔT mice. CD47 on T cells thus likely prevents their necroptotic cell death initiated by cDCs and thereby promotes T cell survival and function.

Targeting of SIRPα as a potential therapy for Langerhans cell histiocytosis.

Langerhans cell histiocytosis (LCH) is a rare neoplastic disorder characterized by inflammatory lesions arising from the anomalous accumulation of pathogenic CD1a+ CD207+ dendritic cells (DCs). SIRPα is a transmembrane protein highly expressed in myeloid cells such as DCs and macrophages. Here we show that SIRPα is a potential therapeutic target for LCH. We found that SIRPα is expressed in CD1a+ cells of human LCH lesions as well as in CD11c+ DCs in the spleen, liver, and lung of a mouse model of LCH (BRAFV600ECD11c mouse), in which an LCH-associated active form of human BRAF is expressed in a manner dependent on the mouse Cd11c promoter. BRAFV600ECD11c mice manifested markedly increased numbers of CD4+ T cells, regulatory T cells, and macrophages as well as of CD11c+ MHCII+ DCs in the spleen. Monotherapy with a mAb to SIRPα greatly reduced the percentage of CD11c+ MHCII+ DCs in peripheral blood, LCH-like lesion size in the liver, and the number of CD11c+ MHCII+ DCs in the spleen of the mutant mice. Moreover, this mAb promoted macrophage-mediated phagocytosis of CD11c+ DCs from BRAFV600ECD11c mice, whereas it had no effects on the viability or CCL19-dependent migration of such CD11c+ DCs or on their expression of the chemokine genes Ccl5, Ccl20, Cxcl11, and Cxcl12. Our results thus suggest that anti-SIRPα monotherapy is a promising approach to the treatment of LCH that is dependent in part on the promotion of the macrophage-mediated killing of LCH cells.

Preclinical evaluation of the efficacy of an antibody to human SIRPα for cancer immunotherapy in humanized mouse models.

Tumor-associated macrophages (TAMs) are abundant in the tumor microenvironment and are considered potential targets for cancer immunotherapy. To examine the antitumor effects of agents targeting human TAMs in vivo, we here established preclinical tumor xenograft models based on immunodeficient mice that express multiple human cytokines and have been reconstituted with a human immune system by transplantation of human CD34+ hematopoietic stem and progenitor cells (HIS-MITRG mice). HIS-MITRG mice supported the growth of both human cell line (Raji)- and patient-derived B cell lymphoma as well as the infiltration of human macrophages into their tumors. We examined the potential antitumor action of an antibody to human SIRPα (SE12C3) that inhibits the interaction of CD47 on tumor cells with SIRPα on human macrophages and thereby promotes Fcγ receptor-mediated phagocytosis of the former cells by the latter. Treatment with the combination of rituximab (antibody to human CD20) and SE12C3 inhibited Raji tumor growth in HIS-MITRG mice to a markedly greater extent than did rituximab monotherapy. This enhanced antitumor effect was dependent on human macrophages and attributable to enhanced rituximab-dependent phagocytosis of lymphoma cells by human macrophages. Treatment with rituximab and SE12C3 also induced reprogramming of human TAMs toward a proinflammatory phenotype. Furthermore, the combination treatment essentially prevented the growth of patient-derived diffuse large B cell lymphoma in HIS-MITRG mice. Our findings thus support the study of HIS-MITRG mice as a model for the preclinical evaluation in vivo of potential therapeutics, such as antibodies to human SIRPα, that target human TAMs.

Vomiting and hyperkalemia are novel clues for emergency room diagnosis of type 1 diabetic ketoacidosis: a retrospective comparison between diabetes types.

INTRODUCTION: Diabetic ketoacidosis (DKA) is an acute life-threatening complication in patients with type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM). Causes, underlying pathophysiology, and mortality differ significantly by diabetes type, which initial treatment is dependent on, but few reports on these differences are available. This study aimed to clarify differences in clinical characteristics between the diabetes types to extract important clinical clues for preventing DKA and ensuring appropriate initial treatment in the emergency room. METHODS: We retrospectively analyzed the clinical presentation of 24 T1DM patients and 13 T2DM patients admitted with DKA to Kobe City Medical Center West Hospital between April 2006 and December 2018. RESULTS: In T1DM, the main causes were insulin omission and new onset, and important factors were also misdiagnosis with consequent inappropriate insulin prescription and older age with dementia. In T2DM, the main causes were infection and excessive soft drink consumption. For all soft drink ketosis patients, this was the first presentation of diabetes. The main complaint differed between diabetes types. Vomiting was a characteristic symptom in T1DM DKA; most T2DM DKA patients presented with generalized malaise or decreased level of consciousness. On blood examination, serum potassium level was higher and HbA1c was lower in T1DM DKA. CONCLUSIONS: To prevent DKA, it is important to provide social support for elderly patients with T1DM DKA and lifestyle intervention for younger T2DM or obese patients. Vomiting and serum potassium levels contribute to the classification of diabetes type and subsequent initial treatment in the emergency room. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13340-021-00539-w.

GCN2 regulates pancreatic ß cell mass by sensing intracellular amino acid levels.

EIF2AK4, which encodes the amino acid deficiency-sensing protein GCN2, has been implicated as a susceptibility gene for type 2 diabetes in the Japanese population. However, the mechanism by which GCN2 affects glucose homeostasis is unclear. Here, we show that insulin secretion is reduced in individuals harboring the risk allele of EIF2AK4 and that maintenance of GCN2-deficient mice on a high-fat diet results in a loss of pancreatic ß cell mass. Our data suggest that GCN2 senses amino acid deficiency in ß cells and limits signaling by mechanistic target of rapamycin complex 1 to prevent ß cell failure during the consumption of a high-fat diet.

Variants in saposin D domain of prosaposin gene linked to Parkinson's disease.

Recently, the genetic variability in lysosomal storage disorders has been implicated in the pathogenesis of Parkinson's disease. Here, we found that variants in prosaposin (PSAP), a rare causative gene of various types of lysosomal storage disorders, are linked to Parkinson's disease. Genetic mutation screening revealed three pathogenic mutations in the saposin D domain of PSAP from three families with autosomal dominant Parkinson's disease. Whole-exome sequencing revealed no other variants in previously identified Parkinson's disease-causing or lysosomal storage disorder-causing genes. A case-control association study found two variants in the intronic regions of the PSAP saposin D domain (rs4747203 and rs885828) in sporadic Parkinson's disease had significantly higher allele frequencies in a combined cohort of Japan and Taiwan. We found the abnormal accumulation of autophagic vacuoles, impaired autophagic flux, altered intracellular localization of prosaposin, and an aggregation of α-synuclein in patient-derived skin fibroblasts or induced pluripotent stem cell-derived dopaminergic neurons. In mice, a Psap saposin D mutation caused progressive motor decline and dopaminergic neurodegeneration. Our data provide novel genetic evidence for the involvement of the PSAP saposin D domain in Parkinson's disease.

Early administration of dapagliflozin preserves pancreatic ß-cell mass through a legacy effect in a mouse model of type 2 diabetes.

AIMS/INTRODUCTION: The preservation of pancreatic ß-cell mass is an essential factor in the onset and development of type 2 diabetes mellitus. Recently, sodium-glucose cotransporter 2 inhibitors have been launched as antihyperglycemic agents, and their organ-protective effects are attracting attention. They are also reported to have favorable effects on the preservation of pancreatic ß-cell mass, but the appropriate timing for the administration of sodium-glucose cotransporter 2 inhibitors is obscure. MATERIALS AND METHODS: In the present study, we administered a sodium-glucose cotransporter 2 inhibitor, dapagliflozin, to an animal model of type 2 diabetes mellitus, db/db mice, and investigated the adequate timing and duration for its administration. We also carried out microarray analysis using pancreatic islets from db/db mice. RESULTS: We found that dapagliflozin preserved pancreatic ß-cell mass depending on the duration of administration and markedly improved blood glucose levels. If the duration was the same, the earlier administration of dapagliflozin was more effective in preserving pancreatic ß-cell mass, increasing serum insulin levels and improving blood glucose levels. From microarray analysis, we discovered that the expression of Agr2, Tff2 and Gkn3 was significantly upregulated after the early administration of dapagliflozin. This upregulated gene expression might provide a legacy effect for the preservation of pancreatic ß-cell mass. CONCLUSIONS: We expect that the early administration of dapagliflozin would provide a long-lasting effect in preserving pancreatic ß-cell mass.

Docosahexaenoic Acid Reduces Palmitic Acid-Induced Endoplasmic Reticulum Stress in Pancreatic Β Cells.

Endoplasmic reticulum (ER) stress leads to peripheral insulin resistance and the progression of pancreatic beta cell failure in type 2 diabetes. Although ER stress plays an important role in the pathogenesis of diabetes, it is indispensable for cellular activity. Therefore, when assessing the pathological significance of ER stress, it is important to monitor and quantify ER stress levels. Here, we have established a novel system to monitor ER stress levels quickly and sensitively, and using this method, we have clarified the effect of differences in glucose concentration and various fatty acids on the ER of pancreatic ß cells. First, we developed a cell system that secretes Gaussia luciferase in culture medium depending on the activation of the GRP78 promoter. This system could sensitively monitor ER stress levels that could not be detected with real-time RT-PCR and immunoblotting. This system revealed that hyperglycemia does not induce unfolded protein response (UPR) in a short period of time in MIN6 cells, a mouse pancreatic ß cell line. Physiological concentrations of palmitic acid, a saturated fatty acid, induced ER stress quickly, while physiological concentrations of oleic acid, an unsaturated fatty acid, did not. Docosahexaenoic acid, an n-3 unsaturated fatty acid, inhibited palmitic acid-induced ER stress. In this study, we have established a system that can sensitively detect ER stress levels of living cells in a short period of time. This system can be used to monitor the state of the ER in living cells and lead to the investigation of the significance of physiological or pathological ER stress levels.

Heme-dependent Inactivation of 5-Aminolevulinate Synthase from Caulobacter crescentus.

The biosynthesis of heme is strictly regulated, probably because of the toxic effects of excess heme and its biosynthetic precursors. In many organisms, heme biosynthesis starts with the production of 5-aminolevulinic acid (ALA) from glycine and succinyl-coenzyme A, a process catalyzed by a homodimeric enzyme, pyridoxal 5'-phosphate (PLP)-dependent 5-aminolevulinate synthase (ALAS). ALAS activity is negatively regulated by heme in various ways, such as the repression of ALAS gene expression, degradation of ALAS mRNA, and inhibition of mitochondrial translocation of the mammalian precursor protein. There has been no clear evidence, however, that heme directly binds to ALAS to negatively regulate its activity. We found that recombinant ALAS from Caulobacter crescentus was inactivated via a heme-mediated feedback manner, in which the essential coenzyme PLP was rel eased to form the inactive heme-bound enzyme. The spectroscopic properties of the heme-bound ALAS showed that a histidine-thiolate hexa-coordinated ferric heme bound to each subunit with a one-to-one stoichiometry. His340 and Cys398 were identified as the axial ligands of heme, and mutant ALASs lacking either of these ligands became resistant to heme-mediated inhibition. ALAS expressed in C. crescentus was also found to bind heme, suggesting that heme-mediated feedback inhibition of ALAS is physiologically relevant in C. crescentus.

Casein kinase 2 phosphorylates and stabilizes C/EBPß in pancreatic ß cells.

During the development of type 2 diabetes, endoplasmic reticulum (ER) stress leads to pancreatic ß cell failure. CCAAT/enhancer-binding protein (C/EBP) ß is highly induced by ER stress and AMP-activated protein kinase (AMPK) suppression in pancreatic ß cells, and its accumulation reduces pancreatic ß cell mass. We investigated the phosphorylation state of C/EBPß under these conditions. Casein kinase 2 (CK2) was found to co-localize with C/EBPß in MIN6 cells. It phosphorylated S222 of C/EBPß, a previously unidentified phosphorylation site. We found that C/EBPß is phosphorylated by CK2 under AMPK suppression and ER stress, which are important from the viewpoint of the worsening pathological condition of type 2 diabetes, such as decreased insulin secretion and apoptosis of pancreatic ß cells.

Histone deacetylase regulates insulin signaling via two pathways in pancreatic ß cells.

Recent studies demonstrated that insulin signaling plays important roles in the regulation of pancreatic ß cell mass, the reduction of which is known to be involved in the development of diabetes. However, the mechanism underlying the alteration of insulin signaling in pancreatic ß cells remains unclear. The involvement of epigenetic control in the onset of diabetes has also been reported. Thus, we analyzed the epigenetic control of insulin receptor substrate 2 (IRS2) expression in the MIN6 mouse insulinoma cell line. We found concomitant IRS2 up-regulation and enhanced insulin signaling in MIN6 cells, which resulted in an increase in cell proliferation. The H3K9 acetylation status of the Irs2 promoter was positively associated with IRS2 expression. Treatment of MIN6 cells with histone deacetylase inhibitors led to increased IRS2 expression, but this occurred in concert with low insulin signaling. We observed increased IRS2 lysine acetylation as a consequence of histone deacetylase inhibition, a modification that was coupled with a decrease in IRS2 tyrosine phosphorylation. These results suggest that insulin signaling in pancreatic ß cells is regulated by histone deacetylases through two novel pathways affecting IRS2: the epigenetic control of IRS2 expression by H3K9 promoter acetylation, and the regulation of IRS2 activity through protein modification. The identification of the histone deacetylase isoform(s) involved in these mechanisms would be a valuable approach for the treatment of type 2 diabetes.

The Presence of Venous Gas Does Not Affect the Prognosis in Emphysematous Cystitis.

Objective Emphysematous cystitis (EC) has a high mortality rate compared with urinary tract infection without emphysema. However, its prognostic factors have yet to be determined. The presence of venous gas is suspected to be a rare, adverse prognostic factor of EC. However, all four previously reported cases improved. We hypothesized that venous gas is not an adverse prognostic factor of EC and aimed to assess the effect of venous gas on the EC prognosis. Methods Medical records were reviewed retrospectively. Patients The patients diagnosed with EC at Yodogawa Christian Hospital between April 2004 and September 2014 were included. Results Venous gas was present in 15 of 23 patients with EC. There was no significant difference in the background or clinical presentation between patients with or without venous gas. All patients with venous gas survived without invasive measures, whereas 50% of patients without venous gas died. Conclusion There was no marked difference in the mortality rate due to EC between the patients with and without venous gas. Venous gas may be a more common and less worrying finding in EC than assumed. It does not reflect the severity of infection, and air embolisms have not been reported so far. Venous gas may not affect the prognosis. This may be due to the differences in the mechanism of venous gas production. Gas in EC may develop due to glucose fermentation and intravesical pressurization, in contrast to the necrotizing infection seen in other emphysematous infections. This is the first study to assess the effect of venous gas on EC prognosis.

Genome-wide identification and gene expression profiling of ubiquitin ligases for endoplasmic reticulum protein degradation.

Endoplasmic reticulum (ER)-associated degradation (ERAD) is a mechanism by which unfolded proteins that accumulate in the ER are transported to the cytosol for ubiquitin-proteasome-mediated degradation. Ubiquitin ligases (E3s) are a group of enzymes responsible for substrate selectivity and ubiquitin chain formation. The purpose of this study was to identify novel E3s involved in ERAD. Thirty-seven candidate genes were selected by searches for proteins with RING-finger motifs and transmembrane regions, which are the major features of ERAD E3s. We performed gene expression profiling for the identified E3s in human and mouse tissues. Several genes were specifically or selectively expressed in both tissues; the expression of four genes (RNFT1, RNF185, CGRRF1 and RNF19B) was significantly upregulated by ER stress. To determine the involvement of the ER stress-responsive genes in ERAD, we investigated their ER localisation, in vitro autoubiquitination activity and ER stress resistance. All were partially localised to the ER, whereas CGRRF1 did not possess E3 activity. RNFT1 and RNF185, but not CGRRF1 and RNF19B, exhibited significant resistance to ER stressor in an E3 activity-dependent manner. Thus, these genes are possible candidates for ERAD E3s.

Luman is involved in osteoclastogenesis through the regulation of DC-STAMP expression, stability and localization.

Luman (also known as CREB3) is a type-II transmembrane transcription factor belonging to the OASIS family that localizes to the endoplasmic reticulum (ER) membrane under normal conditions. In response to ER stress, OASIS-family members are subjected to regulated intramembrane proteolysis (RIP), following which the cleaved N-terminal fragments translocate to the nucleus. In this study, we show that treatment of bone marrow macrophages (BMMs) with cytokines - macrophage colony-stimulating factor (M-CSF) and RANKL (also known as TNFSF11) - causes a time-dependent increase in Luman expression, and that Luman undergoes RIP and becomes activated during osteoclast differentiation. Small hairpin (sh)RNA-mediated knockdown of Luman in BMMs prevented the formation of multinucleated osteoclasts, concomitant with the suppression of DC-STAMP, a protein that is essential for cell-cell fusion in osteoclastogenesis. The N-terminus of Luman facilitates promoter activity of DC-STAMP, resulting in upregulation of DC-STAMP expression. Furthermore, Luman interacts with DC-STAMP, and controls its stability and localization. These results suggest that Luman regulates the multinucleation of osteoclasts by promoting cell fusion of mononuclear osteoclasts through DC-STAMP induction and intracellular distribution during osteoclastogenesis.

Effects of early physiotherapy with respect to severity of pneumonia of elderly patients admitted to an intensive care unit: a single center study in Japan.

[Purpose] We performed early physiotherapy for elderly patients with pneumonia admitted to an intensive care unit (ICU), and examined the effects of this early physiotherapy on the severity of pneumonia. [Subjects and Methods] Patients for whom physiotherapy was started the day after admission to the ICU (acute phase) were assigned to the early intervention group and compared with patients in the standard intervention group. All patients were divided into three groups (Groups I, II, and III) based on the severity of pneumonia. We evaluated the ICU admission period, hospitalization period, and activities of daily living (ADL) before and after admission. [Results] With respect to the severity of pneumonia, Group II showed significant differences in the ICU admission period and rates of change in the operating range, cognitive domain, and Functional Independence Measure (FIM). Group III showed significant differences in the ICU admission period and rate of change in the cognitive domain (FIM item). The results were more favorable in the early intervention group than in the standard intervention group. [Conclusion] The ICU admission period was shorter and a reduction in the ADL level was prevented in Groups II, and III compared to Group I. This may have occurred because of the early rehabilitation.

Regulation of Pancreatic ß Cell Mass by Cross-Interaction between CCAAT Enhancer Binding Protein ß Induced by Endoplasmic Reticulum Stress and AMP-Activated Protein Kinase Activity.

During the development of type 2 diabetes, endoplasmic reticulum (ER) stress leads to not only insulin resistance but also to pancreatic beta cell failure. Conversely, cell function under various stressed conditions can be restored by reducing ER stress by activating AMP-activated protein kinase (AMPK). However, the details of this mechanism are still obscure. Therefore, the current study aims to elucidate the role of AMPK activity during ER stress-associated pancreatic beta cell failure. MIN6 cells were loaded with 5-amino-1-ß-D-ribofuranosyl-imidazole-4-carboxamide (AICAR) and metformin to assess the relationship between AMPK activity and CCAAT enhancer binding protein ß (C/EBPß) expression levels. The effect of C/EBPß phosphorylation on expression levels was also investigated. Vildagliptin and metformin were administered to pancreatic beta cell-specific C/EBPß transgenic mice to investigate the relationship between C/EBPß expression levels and AMPK activity in the pancreatic islets. When pancreatic beta cells are exposed to ER stress, the accumulation of the transcription factor C/EBPß lowers the AMP/ATP ratio, thereby decreasing AMPK activity. In an opposite manner, incubation of MIN6 cells with AICAR or metformin activated AMPK, which suppressed C/EBPß expression. In addition, administration of the dipeptidyl peptidase-4 inhibitor vildagliptin and metformin to pancreatic beta cell-specific C/EBPß transgenic mice decreased C/EBPß expression levels and enhanced pancreatic beta cell mass in proportion to the recovery of AMPK activity. Enhanced C/EBPß expression and decreased AMPK activity act synergistically to induce ER stress-associated pancreatic beta cell failure.

Promotion of Cancer Cell Proliferation by Cleaved and Secreted Luminal Domains of ER Stress Transducer BBF2H7.

BBF2H7 is an endoplasmic reticulum (ER)-resident transmembrane basic leucine zipper (bZIP) transcription factor that is cleaved at the transmembrane domain by regulated intramembrane proteolysis in response to ER stress. The cleaved cytoplasmic N-terminus containing transcription activation and bZIP domains translocates into the nucleus to promote the expression of target genes. In chondrocytes, the cleaved luminal C-terminus is extracellularly secreted and facilitates proliferation of neighboring cells through activation of Hedgehog signaling. In the present study, we found that Bbf2h7 expression levels significantly increased by 1.070-2.567-fold in several tumor types including glioblastoma compared with those in respective normal tissues, using the ONCOMINE Cancer Profiling Database. In some Hedgehog ligand-dependent cancer cell lines including glioblastoma U251MG cells, the BBF2H7 C-terminus was secreted from cells into the culture media and promoted cancer cell proliferation through activation of Hedgehog signaling. Knockdown of Bbf2h7 expression suppressed the proliferation of U251MG cells by downregulating Hedgehog signaling. The impaired cell proliferation and Hedgehog signaling were recovered by addition of BBF2H7 C-terminus to the culture medium of Bbf2h7-knockdown U251MG cells. These data suggest that the secreted luminal BBF2H7 C-terminus is involved in Hedgehog ligand-dependent cancer cell proliferation through activation of Hedgehog signaling. Thus, the BBF2H7 C-terminus may be a novel target for the development of anticancer drugs.

Difference in the Effect of Outpatient Pulmonary Rehabilitation Due to Variation in the Intervention Frequency: Intervention Centering on Home-based Exercise.

[Purpose] The purpose of this study was to investigate differences in effects caused by variation in the intervention frequency of outpatient pulmonary rehabilitation, in terms of the pulmonary function, lower-limb muscle strength, exercise tolerance, and quality of life (QOL). [Subjects and Methods] A total of 36 patients with mild to severe chronic obstructive pulmonary disease (COPD) were studied. These patients were all men over the age of 40 who did not require assistance for activities of daily living (ADL). Groups undergoing intervention once a month (M1 group) and once a week (W1 group) were compared in terms of the effects of outpatient pulmonary rehabilitation for a period of 12 weeks. Intervention during this time included supervised and home-based exercise. [Results] Comparison of before and after intervention revealed that the rate of change in the W1 group was significantly higher than that in the M1 group in terms of the QOL, lower-extremity muscle strength, and 6-minute walking distance. [Conclusion] Outpatient pulmonary rehabilitation programs yielded greater improvements in the W1 group than in the M1 group in terms of the QOL and exercise tolerance.

Master regulator for chondrogenesis, Sox9, regulates transcriptional activation of the endoplasmic reticulum stress transducer BBF2H7/CREB3L2 in chondrocytes.

The endoplasmic reticulum (ER) stress transducer, box B-binding factor 2 human homolog on chromosome 7 (BBF2H7), is a basic leucine zipper (bZIP) transmembrane transcription factor. This molecule is activated in response to ER stress during chondrogenesis. The activated BBF2H7 accelerates cartilage matrix protein secretion through the up-regulation of Sec23a, which is responsible for protein transport from the ER to the Golgi apparatus and is a target of BBF2H7. In the present study, we elucidated the mechanisms of the transcriptional activation of Bbf2h7 in chondrocytes. The transcription of Bbf2h7 is regulated by Sex determining region Y-related high-mobility group box 9 (Sox9), a critical factor for chondrocyte differentiation that facilitates the expression of one of the major cartilage matrix proteins Type II collagen (Col2), through binding to the Sox DNA-binding motif in the Bbf2h7 promoter. BBF2H7 is activated as a transcription factor in response to physiological ER stress caused by abundant synthesis of cartilage matrix proteins, and consequently regulates the secretion of cartilage matrix proteins. Taken together, our findings demonstrate novel regulatory mechanisms of Sox9 for controlling the secretion of cartilage matrix proteins through the activation of BBF2H7-Sec23a signaling during chondrogenesis.

A bicyclic 1-deoxygalactonojirimycin derivative as a novel pharmacological chaperone for GM1 gangliosidosis.

Lysosomal ß-galactosidase (ß-Gal) deficiency causes a group of disorders that include neuronopathic GM1 gangliosidosis and non-neuronopathic Morquio B disease. We have previously proposed the use of small molecule ligands of ß-Gal as pharmacological chaperones (PCs) for the treatment of GM1 gangliosidosis brain pathology. Although it is still under development, PC therapy has yielded promising preclinical results in several lysosomal diseases. In this study, we evaluated the effect of bicyclic 1-deoxygalactonojirimycin (DGJ) derivative of the sp(2)-iminosugar type, namely 5N,6S-(N'-butyliminomethylidene)-6-thio-1- deoxygalactonojirimycin (6S-NBI-DGJ), as a novel PC for human mutant ß-Gal. In vitro, 6S-NBI-DGJ had the ability to inhibit the activity of human ß-Gal in a competitive manner and was able to protect this enzyme from heat-induced degradation. Computational analysis supported that the rigid glycone bicyclic core of 6S-NBI-DGJ binds to the active site of the enzyme, with the aglycone N'-butyl substituent, in a precise E-orientation, located at a hydrophobic region nearby. Chaperone potential profiling indicated significant increases of enzyme activity in 24 of 88 ß-Gal mutants, including four common mutations. Finally, oral administration of 6S-NBI-DGJ ameliorated the brain pathology of GM1 gangliosidosis model mice. These results suggest that 6S-NBI-DGJ is a novel PC that may be effective on a broad range of ß-Gal mutants.