Glitazone Treatment Rescues Phenotypic Deficits in a Fly Model of Gaucher/Parkinson’s Disease

Parkinson’s Disease (PD) is the most common movement disorder, and the strongest genetic risk factor for PD is mutations in the glucocerebrosidase gene (GBA). Mutations in GBA also lead to the development of Gaucher Disease (GD), the most common type of lysosomal storage disorder. Current therapeutic approaches fail to address neurological GD symptoms. Therefore, identifying therapeutic strategies that improve the phenotypic traits associated with GD/PD in animal models may provide an opportunity for treating neurological manifestations of GD/PD. Thiazolidinediones (TZDs, also called glitazones) are a class of compounds targeted for the treatment of type 2 diabetes, and have also shown promise for the treatment of neurodegenerative disease, including PD. Here, we tested the efficacy of glitazone administration during development in a fly GD model with deletions in the GBA homolog, dGBA1b (GBA1^ΔTT/ΔTT). We observed an optimal dose of pioglitazone (PGZ) at a concentration of 1 μM that reduced sleep deficits, locomotor impairments, climbing defects, and restoration of normal protein levels of Ref(2)P, a marker of autophagic flux, in GBA1^ΔTT/ΔTT mutant flies, compared to GBA1^+/+ control flies. These data suggest that PGZ may represent a potential compound with which to treat GD/PD by improving function of lysosomal-autophagy pathways, a cellular process that removes misfolded or aggregated proteins.     

GCase and LIMP2 Abnormalities in the Liver of Niemann Pick Type C Mice

The lysosomal storage disease Niemann–Pick type C (NPC) is caused by impaired cholesterol efflux from lysosomes, which is accompanied by secondary lysosomal accumulation of sphingomyelin and glucosylceramide (GlcCer). Similar to Gaucher disease (GD), patients deficient in glucocerebrosidase (GCase) degrading GlcCer, NPC patients show an elevated glucosylsphingosine and glucosylated cholesterol. In livers of mice lacking the lysosomal cholesterol efflux transporter NPC1, we investigated the expression of established biomarkers of lipid-laden macrophages of GD patients, their GCase status, and content on the cytosol facing glucosylceramidase GBA2 and lysosomal integral membrane protein type B (LIMP2), a transporter of newly formed GCase to lysosomes. Livers of 80-week-old Npc1^−/− mice showed a partially reduced GCase protein and enzymatic activity. In contrast, GBA2 levels tended to be reciprocally increased with the GCase deficiency. In Npc1^−/− liver, increased expression of lysosomal enzymes (cathepsin D, acid ceramidase) was observed as well as increased markers of lipid-stressed macrophages (GPNMB and galectin-3). Immunohistochemistry showed that the latter markers are expressed by lipid laden Kupffer cells. Earlier reported increase of LIMP2 in Npc1^−/− liver was confirmed. Unexpectedly, immunohistochemistry showed that LIMP2 is particularly overexpressed in the hepatocytes of the Npc1^−/− liver. LIMP2 in these hepatocytes seems not to only localize to (endo)lysosomes. The recent recognition that LIMP2 harbors a cholesterol channel prompts the speculation that LIMP2 in Npc1^−/− hepatocytes might mediate export of cholesterol into the bile and thus protects the hepatocytes.     

A Fluorescent sp2-iminosugar with pharmacological chaperone activity for gaucher disease: synthesis and intracellular distribution studies

Gaucher disease (GD) is the most prevalent lysosomal-storage disorder, it is caused by mutations of acid β-glucosidase (β-glucocerebrosidase; β-Glu). Recently, we found that bicyclic nojirimycin (NJ) derivatives of the sp(2)-iminosugar type, including the 6-thio-N'-octyl-(5N,6S)-octyliminomethylidene derivative (6S-NOI-NJ), behaved as very selective competitive inhibitors of the lysosomal β-Glu and exhibited remarkable chaperone activities for several GD mutations. To obtain information about the cellular uptake pathway and intracellular distribution of this family of chaperones, we have synthesized a fluorescent analogue that maintains the fused piperidine-thiazolidine bicyclic skeleton and incorporates a dansyl group in the N'-substituent, namely 6-thio-(5N,6S)-[4-(N'-dansylamino)butyliminomethylidene]nojirimycin (6S-NDI-NJ). This structural modification does not significantly modify the biological activity of the glycomimetic as a chemical chaperone. Our study showed that 6S-NDI-NJ is mainly located in lysosome-related organelles in both normal and GD fibroblasts, and the fluorescent intensity of 6S-NDI-NJ in the lysosome is related to the β-Glu concentration level. 6S-NDI-NJ also can enter cultured neuronal cells and act as a chaperone. Competitive inhibition studies of 6S-NDI-NJ uptake in fibroblasts showed that high concentrations of D-glucose have no effect on chaperone internalization, suggesting that it enters the cells through glucose-transporter-independent mechanisms.     

Synthesis and Evaluation of Hybrid Structures Composed of Two Glucosylceramide Synthase Inhibitors

Glucosylceramide metabolism and the enzymes involved have attracted significant interest in medicinal chemistry, because aberrations in the levels of glycolipids that are derived from glucosylceramide are causative in a range of human diseases including lysosomal storage disorders, type 2 diabetes, and neurodegenerative diseases. Selective modulation of one of the glycoprocessing enzymes involved in glucosylceramide metabolism—glucosylceramide synthase (GCS), acid glucosylceramidase (GBA1), or neutral glucosylceramidase (GBA2)—is therefore an attractive research objective. In this study we took two established GCS inhibitors, one based on deoxynojirimycin and the other a ceramide analogue, and merged characteristic features to obtain hybrid compounds. The resulting 39‐compound library does not contain new GCS inhibitors; however, a potent (200 nm ) GBA1 inhibitor was identified that has little activity toward GBA2 and might therefore serve as a lead for further biomedical development as a selective GBA1 modulator.     

Chaperone Activity of Bicyclic Nojirimycin Analogues for Gaucher Mutations in Comparison with N‐(n‐nonyl)Deoxynojirimycin

Gaucher disease (GD), the most prevalent lysosomal storage disorder, is caused by mutations of lysosomal β‐glucosidase (acid β‐Glu, β‐glucocerebrosidase); these mutations result in protein misfolding. Some inhibitors of this enzyme, such as the iminosugar glucomimetic N‐(n‐nonyl)‐1‐deoxynojirimycin (NN‐DNJ), are known to bind to the active site and stabilize the proper folding for the catalytic form, acting as “chemical chaperones” that facilitate transport and maturation of acid β‐Glu. Recently, bicyclic nojirimycin (NJ) analogues with structure of sp² iminosugars were found to behave as very selective, competitive inhibitors of the lysosomal β‐Glu. We have now evaluated the glycosidase inhibitory profile of a series of six compounds within this family, namely 5‐N,6‐O‐(N′‐octyliminomethylidene‐NJ (NOI‐NJ), the 6‐thio and 6‐amino‐6‐deoxy derivatives (6S‐NOI‐NJ and 6N‐NOI‐NJ) and the corresponding galactonojirimycin (GNJ) counterparts (NOI‐GNJ, 6S‐NOI‐GNJ and 6N‐NOI‐GNJ), against commercial as well as lysosomal glycosidases. The chaperone effects of four selected candidates (NOI‐NJ, 6S‐NOI‐NJ, 6N‐NOI‐NJ, and 6S‐NOI‐GNJ) were further evaluated in GD fibroblasts with various acid β‐Glu mutations. The compounds showed enzyme enhancement on human fibroblasts with N188S, G202R, F213I or N370S mutations. The chaperone effects of the sp² iminosugar were generally stronger than those observed for NN‐DNJ; this suggests that these compounds are promising candidates for clinical treatment of GD patients with a broad range of β‐Glu mutations, especially for neuronopathic forms of Gaucher disease.     

GBA Mutations Influence the Release and Pathological Effects of Small Extracellular Vesicles from Fibroblasts of Patients with Parkinson’s Disease

Heterozygous mutations in the GBA gene, encoding the lysosomal enzyme glucocerebrosidase (GCase), are the strongest known genetic risk factor for Parkinson’s disease (PD). The molecular mechanisms underlying the increased PD risk and the variable phenotypes observed in carriers of different GBA mutations are not yet fully elucidated. Extracellular vesicles (EVs) have gained increasing importance in neurodegenerative diseases since they can vehiculate pathological molecules potentially promoting disease propagation. Accumulating evidence showed that perturbations of the endosomal–lysosomal pathway can affect EV release and composition. Here, we investigate the impact of GCase deficiency on EV release and their effect in recipient cells. EVs were purified by ultracentrifugation from the supernatant of fibroblast cell lines derived from PD patients with or without GBA mutations and quantified by nanoparticle tracking analysis. SH-SY5Y cells over-expressing alpha-synuclein (α-syn) were used to assess the ability of patient-derived small EVs to affect α-syn expression. We observed that defective GCase activity promotes the release of EVs, independently of mutation severity. Moreover, small EVs released from PD fibroblasts carrying severe mutations increased the intra-cellular levels of phosphorylated α-syn. In summary, our work shows that the dysregulation of small EV trafficking and alpha-synuclein mishandling may play a role in GBA-associated PD.     

A Specific Activity‐Based Probe to Monitor Family GH59 Galactosylceramidase,the Enzyme Deficient in Krabbe Disease

Galactosylceramidase (GALC) is the lysosomal β‐galactosidase responsible for the hydrolysis of galactosylceramide. Inherited deficiency in GALC causes Krabbe disease, a devastating neurological disorder characterized by accumulation of galactosylceramide and its deacylated counterpart, the toxic sphingoid base galactosylsphingosine (psychosine). We report the design and application of a fluorescently tagged activity‐based probe (ABP) for the sensitive and specific labeling of active GALC molecules from various species. The probe consists of a β‐galactopyranose‐configured cyclophellitol‐epoxide core, conferring specificity for GALC, equipped with a BODIPY fluorophore at C6 that allows visualization of active enzyme in cells and tissues. Detection of residual GALC in patient fibroblasts holds great promise for laboratory diagnosis of Krabbe disease. We further describe a procedure for in situ imaging of active GALC in murine brain by intra‐cerebroventricular infusion of the ABP. In conclusion, this GALC‐specific ABP should find broad applications in diagnosis, drug development, and evaluation of therapy for Krabbe disease.     

Inter-residual Hydrogen Bonding in Carbohydrates Unraveled by NMR Spectroscopy and Molecular Dynamics Simulations

Carbohydrates, also known as glycans in biological systems, are omnipresent in nature where they as glycoconjugates occur as oligo- and polysaccharides linked to lipids and proteins. Their three-dimensional structure is defined by two or three torsion angles at each glycosidic linkage. In addition, transglycosidic hydrogen bonding between sugar residues may be important. Herein we investigate the presence of these inter-residue interactions by NMR spectroscopy in D₂O/[D₆]DMSO (70:30) or D₂O and by molecular dynamics (MD) simulations with explicit water as solvent for disaccharides with structural elements α-d -Manp-(1→2)-d -Manp, β-d -GlcpNAc-(1→2)-d -Manp, and α-d -Glcp-(1→4)-β-d -Glcp, all of which have been suggested to exhibit inter-residue hydrogen bonding. For the disaccharide β-d -GlcpNAc-(1→2)-β-d -Manp-OMe, the large extent of O5′ ⋅⋅⋅ HO3 hydrogen bonding as seen from the MD simulation is implicitly supported by the ¹H NMR chemical shift and ³J_HO3,H3 value of the hydroxy proton. In the case of α-d -Glcp-(1→4)-β-d -Glcp-OMe, the existence of a transglycosidic hydrogen bond O2′⋅⋅⋅HO3 was proven by the presence of a cross-peak in ¹H,¹³C HSQC-TOCSY experiments as a result of direct TOCSY transfer between HO3 of the reducing end residue and H2′ (detected at C2′) of the terminal residue. The occurrence of inter-residue hydrogen bonding, albeit transient, is judged important for the stabilization of three-dimensional structures, which may be essential in maintaining a conformational state for carbohydrate–protein interactions of glycans to take place in biologically important environments.     

Evaluation of the Correlation between Porphyrin Accumulation in Cancer Cells and Functional Porphyrin Positions of the Phenyl Group

Porphyrin selectively shows tumour accumulation and has attracted attention as a carrier molecule for drug delivery systems (DDS). Porphyrin has two functional sites termed the meso- and β-positions. In previous work, meso-porphyrin derivatives with an alkyl group were found to exhibit greater accumulation in human breast cancer cells (MCF-7). To identify the correlation between porphyrin accumulation and functional porphyrin positions of other functional groups, the accumulation of porphyrin derivatives with a phenyl group was investigated. The β-porphyrin derivative with a phenyl group showed higher accumulation in MCF-7 cells and greater affinity for albumin than the meso-porphyrin derivative. The results of density functional theory (DFT) calculations suggest that the β-porphyrin derivative with a phenyl group had higher planarity across the total structure than the meso-porphyrin derivative. It was concluded that the greater planarity of the β-porphyrin derivative with a phenyl group might lead to superior MCF-7 cell accumulation.     

Activity-based profiling of retaining β-glucosidases: a comparative study

Activity-based protein profiling (ABPP) is a versatile strategy to report on enzyme activity in vitro, in situ, and in vivo. The development and use of ABPP tools and techniques has met with considerable success in monitoring physiological processes involving esterases and proteases. Activity-based profiling of glycosidases, on the other hand, has proven more difficult, and to date no broad-spectrum glycosidase activity-based probes (ABPs) have been reported. In a comparative study, we investigated both 2-deoxy-2-fluoroglycosides and cyclitol epoxides for their utility as a starting point towards retaining β-glucosidase ABP. We also investigated the merits of direct labeling and two-step bio-orthogonal labeling in reporting on glucosidase activity under various conditions. Our results demonstrate that 1) in general cyclitol epoxides are the superior glucosidase ABPs, 2) that direct labeling is the more efficient approach but it hinges on the ability of the glucosidase to be accommodated in the active site of the reporter (BODIPY) entity, and 3) that two-step bio-orthogonal labeling can be achieved on isolated enzymes but translating this protocol to cell extracts requires more investigation.     

Accurate Molecular Diagnosis of Gaucher Disease Using Clinical Exome Sequencing as a First-Tier Test

Gaucher disease (GD) is an autosomal recessive lysosomal disorder due to beta-glucosidase gene (GBA) mutations. The molecular diagnosis of GD is complicated by the presence of recombinant alleles originating from a highly homologous pseudogene. Clinical exome sequencing (CES) is a rapid genetic approach for identifying disease-causing mutations. However, copy number variation and recombination events are poorly detected, and further investigations are required to avoid mis-genotyping. The aim of this work was to set-up an integrated strategy for GD patients genotyping using CES as a first-line test. Eight patients diagnosed with GD were analyzed by CES. Five patients were fully genotyped, while three were revealed to be homozygous for mutations that were not confirmed in the parents. Therefore, MLPA (multiplex ligation-dependent probe amplification) and specific long-range PCR were performed, and two recombinant alleles, one of them novel, and one large deletion were identified. Furthermore, an MLPA assay performed in one family resulted in the identification of an additional novel mutation (p.M124V) in a relative, in trans with the known p.N409S mutation. In conclusion, even though CES has become extensively used in clinical practice, our study emphasizes the importance of a comprehensive molecular strategy to provide proper GBA genotyping and genetic counseling.     

Improving Antibody-Tubulysin Conjugates through Linker Chemistry and Site-Specific Conjugation

Tubulysins have emerged in recent years as a compelling drug class for delivery to tumor cells via antibodies. The ability of this drug class to exert bystander activity while retaining potency against multidrug-resistant cell lines differentiates them from other microtubule-disrupting agents. Tubulysin M, a synthetic analogue, has proven to be active and well tolerated as an antibody-drug conjugate (ADC) payload, but has the liability of being susceptible to acetate hydrolysis at the C11 position, leading to attenuated potency. In this work, we examine the ability of the drug-linker and conjugation site to preserve acetate stability. Our findings show that, in contrast to a more conventional protease-cleavable dipeptide linker, the β-glucuronidase-cleavable glucuronide linker protects against acetate hydrolysis and improves ADC activity in vivo. In addition, site-specific conjugation can positively impact both acetate stability and in vivo activity. Together, these findings provide the basis for a highly optimized delivery strategy for tubulysin M.     

不同的β成核剂聚丙烯的结晶性能研究

本文报导了用偏光显微镜观察IPP切片中β球晶的生长过程和形态;借助DSC法、光学解偏振法和大角X衍射法研究含不同β成核剂的IPP在等温和非等温条件下的结晶能力。实验结果表明,对比三种不同的成核剂,发现其结晶速率为RPP>DC>GD,而在纺丝过程中由于成核速率占主导作用,因此卷绕丝中β晶含量也为RPP>DC>GD。研究结果还表明,提高结晶温度,降低冷却速率,有利于提高β晶聚丙烯中的β晶含量。     

Glucosylceramide Mimics: Highly Potent GCase Inhibitors and Selective Pharmacological Chaperones for Mutations Associated with Types 1 and 2 Gaucher Disease

A series of iminoxylitol derivatives carrying a C‐linked di‐O‐acyl or di‐O‐alkyl glyceryl substituent were prepared and characterized. All of these compounds, which were designed as glucosylceramide (GlcCer) mimics, were nanomolar inhibitors of lysosomal β‐glucosidase (glucocerebrosidase, GCase). Two of these pseudoglycolipids were further evaluated for their ability to enhance the activity of mutant GCase in human Gaucher cells. Although the di‐O‐hexyl ether was surprisingly devoid of chaperoning activity on both N370S and L444P GCases, the di‐O‐decanoyl ester was a potent chaperone of the L444P hydrolase, capable of increasing the residual activity of the enzyme by a factor of two at a very low concentration (50 nM ); such a significant effect on the L444P mutation in human fibroblasts has not yet been observed. In heat‐stress studies, the diether was found to be much more effective in stabilizing the wild‐type enzyme than the diester. Four representative pseudoglycolipids were also assayed as inhibitors of GlcCer synthase, because such compounds could find use in the substrate reduction therapy approach to treat lysosomal storage diseases, but these compounds revealed only moderate activity. As efficient pharmacological chaperones, new structures such as the di‐C₁₀‐ester constitute leads for the development of therapeutic agents for types 2 and 3 Gaucher disease, the most severe neuronopathic forms of this lysosomal disease.     

Comparison of Bioorthogonal β-Lactone Activity-Based Probes for Selective Labeling of Penicillin-Binding Proteins

Penicillin-binding proteins (PBPs) are a family of bacterial enzymes that are key components of cell-wall biosynthesis and the target of β-lactam antibiotics. Most microbial pathogens contain multiple structurally homologous PBP isoforms, making it difficult to target individual PBPs. To study the roles and regulation of specific PBP isoforms, a panel of bioorthogonal β-lactone probes was synthesized and compared. Fluorescent labeling confirmed selectivity, and PBPs were selectively enriched from Streptococcus pneumoniae lysates. Comparisons between fluorescent labeling of probes revealed that the accessibility of bioorthogonal reporter molecules to the bound probe in the native protein environment exerts a more significant effect on labeling intensity than the bioorthogonal reaction used, observations that are likely applicable beyond this class of probes or proteins. Selective, bioorthogonal activity-based probes for PBPs will facilitate the activity-based determination of the roles and regulation of specific PBP isoforms, a key gap in knowledge that has yet to be filled.     

Glucocerebrosidase Enhancers for Selected Gaucher Disease Genotypes by Modification of α‐1‐C‐Substituted Imino‐D‐xylitols (DIXs) by Click Chemistry

A series of hybrid analogues was designed by combination of the iminoxylitol scaffold of parent 1C9‐DIX with triazolylalkyl side chains. The resulting compounds were considered potential pharmacological chaperones in Gaucher disease. The DIX analogues reported here were synthesized by CuAAC click chemistry from scaffold 1 (α‐1‐C‐propargyl‐1,5‐dideoxy‐1,5‐imino‐D ‐xylitol) and screened as imiglucerase inhibitors. A set of selected compounds were tested as β‐glucocerebrosidase (GBA1) enhancers in fibroblasts from Gaucher patients bearing different genotypes. A number of these DIX compounds were revealed as potent GBA1 enhancers in genotypes containing the G202R mutation, particularly compound DIX‐28 (α‐1‐C‐[(1‐(3‐trimethylsilyl)propyl)‐1H‐1,2,3‐triazol‐4‐yl)methyl]‐1,5‐dideoxy‐1,5‐imino‐D ‐xylitol), bearing the 3‐trimethylsilylpropyl group as a new surrogate of a long alkyl chain, with approximately threefold activity enhancement at 10 nM . Despite their structural similarities with isofagomine and with our previously reported aminocyclitols, the present DIX compounds behaved as non‐competitive inhibitors, with the exception of the mixed‐type inhibitor DIX‐28.     

ACCELERATED LIGHT-INDUCED AGING OF α-, β-, AND γ-13C-ENRICHED CELL WALL-DEHYDROGENATION POLYMERS STUDIED WITH SOLID STATE 13C NMR SPECTROSCOPY

ABSTRACT

Light-induced aging of lignocellulosic materials has been studied with a new technique involving selectively α-, β-, and γ-¹³C-enriched cell wall-dehydrogenation polymers (CW-DHPs) and solid state ¹³C NMR spectroscopy. The results from cross-polarization magic angle spinning (CP/MAS) ¹³C NMR experiments of unirradiated and irradiated CW-DHP have revealed mainly a decrease in the amount of end-groups of both coniferaldehyde and coniferyl alcohol type. The results suggest that these end-groups become saturated and that the terminal functionalites, i.e., γ-aldehyde and γ-hydroxymethyl groups, at least to some extent, are retained. The results indicate further that no detectable cleavage of the β-O-4 bonds occurs in the examined lignocellulosic model. In terms of proposed mechanisms of yellowing, there is marginal evidence that up to 2% of the α-labeled sites are converted by irradiation to α-carbonyls (aldehyde or ketones); moreover, we cannot dismiss the possibility that the precursor structures giving rise to these few α-carbonyls are β-O-4 structures. The ¹³C-enriched CW-DHP was formed directly on spruce (Picea abies) wood tissue (differentiating xylem) by administering selectively ¹³C-labeled coniferin at pH 6.0 in the presence of glucose oxidase and β-glucosidase, i.e., no phenol-oxidizing enzyme was added and the wood cells’ own enzymes polymerized the precursor.     

C-X-C Motif Chemokine Ligand 9 and Its CXCR3 Receptor Are the Salt and Pepper for T Cells Trafficking in a Mouse Model of Gaucher Disease

Gaucher disease is a lysosomal storage disease, which happens due to mutations in GBA1/Gba1 that encodes the enzyme termed as lysosomal acid β-glucosidase. The major function of this enzyme is to catalyze glucosylceramide (GC) into glucose and ceramide. The deficiency of this enzyme and resultant abnormal accumulation of GC cause altered function of several of the innate and adaptive immune cells. For example, augmented infiltration of T cells contributes to the increased production of pro-inflammatory cytokines, (e.g., IFNγ, TNFα, IL6, IL12p40, IL12p70, IL23, and IL17A/F). This leads to tissue damage in a genetic mouse model (Gba1^9V/−) of Gaucher disease. The cellular mechanism(s) by which increased tissue infiltration of T cells occurs in this disease is not fully understood. Here, we delineate role of the CXCR3 receptor and its exogenous C-X-C motif chemokine ligand 9 (CXCL9) in induction of increased tissue recruitment of CD4⁺ T and CD8⁺ T cells in Gaucher disease. Intracellular FACS staining of macrophages (Mϕs) and dendritic cells (DCs) from Gba1^9V/− mice showed elevated production of CXCL9. Purified CD4⁺ T cells and the CD8⁺ T cells from Gba1^9V/− mice showed increased expression of CXCR3. Ex vivo and in vivo chemotaxis experiments showed CXCL9 involvement in the recruitment of Gba1^9V/− T cells. Furthermore, antibody blockade of the CXCL9 receptor (CXCR3) on T cells caused marked reduction in CXCL9- mediated chemotaxis of T cells in Gba1^9V/− mice. These data implicate abnormalities of the CXCL9-CXCR3 axis leading to enhanced tissue recruitment of T cells in Gaucher disease. Such results provide a rationale for blockade of the CXCL9/CXCR3 axis as potential new therapeutic targets for the treatment of inflammation in Gaucher disease.