SDC1-dependent TGM2 determines radiosensitivity in glioblastoma by coordinating EPG5-mediated fusion of autophagosomes with lysosomes.

Glioblastoma multiforme (GBM) is the most common brain malignancy insensitive to radiotherapy (RT). Although macroautophagy/autophagy was reported to be a fundamental factor prolonging the survival of tumors under radiotherapeutic stress, the autophagic biomarkers coordinated to radioresistance of GBM are still lacking in clinical practice. Here we established radioresistant GBM cells and identified their protein profiles using tandem mass tag (TMT) quantitative proteomic analysis. It was found that SDC1 and TGM2 proteins were overexpressed in radioresistant GBM cells and tissues and they contributed to the poor prognosis of RT. Knocking down SDC1 and TGM2 inhibited the fusion of autophagosomes with lysosomes and thus enhanced the radiosensitivity of GBM cells. After irradiation, TGM2 bound with SDC1 and transported it from the cell membrane to lysosomes, and then bound to LC3 through its two LC3-interacting regions (LIRs), coordinating the encounter between autophagosomes and lysosomes, which should be a prerequisite for lysosomal EPG5 to recognize LC3 and subsequently stabilize the STX17-SNAP29-VAMP8 QabcR SNARE complex assembly. Moreover, when combined with RT, cystamine dihydrochloride (a TGM2 inhibitor) extended the lifespan of GBM-bearing mice. Overall, our findings demonstrated the EPG5 tethering mode with SDC1 and TGM2 during the fusion of autophagosomes with lysosomes, providing new insights into the molecular mechanism and therapeutic target underlying radioresistant GBM.Abbreviations: BafA1: bafilomycin A1; CQ: chloroquine; Cys-D: cystamine dihydrochloride; EPG5: ectopic P-granules 5 autophagy tethering factor; GBM: glioblastoma multiforme; GFP: green fluorescent protein; LAMP2: lysosomal associated membrane protein 2; LIRs: LC3-interacting regions; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NC: negative control; RFP: red fluorescent protein; RT: radiotherapy; SDC1: syndecan 1; SNAP29: synaptosome associated protein 29; SQSTM1/p62: sequestosome 1; STX17: syntaxin 17; TGM2: transglutaminase 2; TMT: tandem mass tag; VAMP8: vesicle associated membrane protein 8; WT: wild type.

Use of pH-Active Catechol-Bearing Polymeric Nanogels with Glutathione-Responsive Dissociation to Codeliver Bortezomib and Doxorubicin for the Synergistic Therapy of Cancer.

Synergistic therapy holds promising potential in cancer treatment. Here, the inclusion of catechol moieties, a disulfide cross-linked structure, and pendent carboxyl into the network of polymeric nanogels with glutathione (GSH)-responsive dissociation and pH-sensitive release is first disclosed for the codelivery of doxorubicin (DOX) and bortezomib (BTZ) in synergistic cancer therapy. The pendent carboxyl groups and catechol moieties are exploited to absorb DOX through electrostatic interaction and conjugate BTZ through boronate ester, respectively. Both electrostatic interactions and boronate ester are stable at neutral or alkaline pH, while they are instable in an acidic environment to further recover the activities of BTZ and DOX. The polymeric nanogels possess a superior stability to prevent the premature leakage of drugs in a physiological environment, while their structure is destroyed in response to a typical endogenous stimulus (GSH) to unload drugs. The dissociation of the drug-loaded nanogels accelerates the intracellular release of DOX and BTZ and further enhances the therapeutic efficacy. In vitro and in vivo investigations revealed that the dual-drug loaded polymeric nanogels exhibited a strong ability to suppress tumor growth. This study thus proposes a new perspective on the production of multifunctional polymeric nanogels through the introduction of different functional monomers.

Overcoming multidrug resistance by intracellular drug release and inhibiting p-glycoprotein efflux in breast cancer.

Doxorubicin (DOX) is limited to use in clinical practice because of poor targeting, serious side effects and multidrug resistance (MDR). Vitamin E and its derivatives are currently considered as hydrophobic material that can reverse tumor MDR by suppressing the action of p-glycoprotein (p-gp). Therefore, reduction-sensitive amphiphilic heparosan polysaccharide-cystamine-vitamin E succinate (KSV) copolymers were designed to reverse breast cancer MDR cells. The spherical micelles (DOX/KSV) micelles which had suitable particle size presented redox-sensitive release character. Simultaneously, DOX-loaded reduction insensitive heparosan-adipic dihydrazide-vitamin E succinate (KV) micellar system was designed as a control. DOX/KSV and DOX/KV micelles had the higher capability to overcome tumor MDR than that free DOX. However, DOX/KSV had the highest amount of cellular uptake which might be caused by the synergistic intracellular drug release and inhibition of p-gp expression. The mechanism experiments revealed that DOX/KSV could be fast disassembled to release DOX after internalization into tumor cells. Moreover, DOX/KSV produced more ROS than free DOX and DOX/KV resulting in enhanced anticancer effect. In vivo tumor-bearing mice study suggested that DOX/KSV micelles could efficiently enhance antitumor effect by overcoming tumor MDR and reduce toxicity of DOX. The DOX/KSV micelles could synergistically increase the therapeutic effect of chemotherapeutic drug on tumor MDR cells.

Base-Sequence-Independent Efficient Redox Switching of Self-Assembled DNA Nanocages.

Stimuli responsivity has been extensively pursued in dynamic DNA nanotechnology, due to its incredible application potentials. Among diverse dynamic systems, redox-responsive DNA assembly holds great promise for broad applications, especially considering that redox processes widely exist in various physiological environments. However, only a few studies have been reported on redox-sensitive dynamic DNA assembly. Albeit ingenious, most of these studies are either dependent on the DNA sequence or involve chemical modification. Herein, a facile and universal mechanism to realize redox-responsive self-assembly of DNA nanocages (tetrahedron and cube) driven by the interconversion between cystamine and cysteamine toward dynamic DNA nanotechnology is reported.

Engineering a cleavable disulfide bond into a natural product siderophore using precursor-directed biosynthesis.

An analogue of the bacterial siderophore desferrioxamine B (DFOB) containing a disulfide motif in the backbone was produced from Streptomyces pilosus cultures supplemented with cystamine. Cystamine competed against native 1,5-diaminopentane during assembly. DFOB-(SS)1[001] and its complexes with Fe(iii) or Ga(iii) were cleaved upon incubation with dithiothreitol. Compounds such as DFOB-(SS)1[001] and its thiol-containing cleavage products could expand antibiotic strategies and Au-S-based nanotechnologies.

A Pharmacogenetic Discovery: Cystamine Protects Against Haloperidol-Induced Toxicity and Ischemic Brain Injury.

Haloperidol is an effective antipsychotic agent, but it causes Parkinsonian-like extrapyramidal symptoms in the majority of treated subjects. To address this treatment-limiting toxicity, we analyzed a murine genetic model of haloperidol-induced toxicity (HIT). Analysis of a panel of consomic strains indicated that a genetic factor on chromosome 10 had a significant effect on susceptibility to HIT. We analyzed a whole-genome SNP database to identify allelic variants that were uniquely present on chromosome 10 in the strain that was previously shown to exhibit the highest level of susceptibility to HIT. This analysis implicated allelic variation within pantetheinase genes (Vnn1 and Vnn3), which we propose impaired the biosynthesis of cysteamine, could affect susceptibility to HIT. We demonstrate that administration of cystamine, which is rapidly metabolized to cysteamine, could completely prevent HIT in the murine model. Many of the haloperidol-induced gene expression changes in the striatum of the susceptible strain were reversed by cystamine coadministration. Since cystamine administration has previously been shown to have other neuroprotective actions, we investigated whether cystamine administration could have a broader neuroprotective effect. Cystamine administration caused a 23% reduction in infarct volume after experimentally induced cerebral ischemia. Characterization of this novel pharmacogenetic factor for HIT has identified a new approach for preventing the treatment-limiting toxicity of an antipsychotic agent, which could also be used to reduce the extent of brain damage after stroke.

The 55 kDa tissue transglutaminase cross-linking active isoform TG induces cell death.

Transamidations are calcium-dependent reactions catalyzed by transglutaminase enzymes. Tissue transglutaminase (TG2 or TGC) is a multifunctional protein with a controversial role in apoptosis. The cross-linking function of transglutaminase enzymes has been shown to play a role in cell death. Human breast cancer cell lines (MCF7 and T47D), which express low endogenous levels of transglutaminase, were transiently transfected with the cross-linking 55 kDa active TG isoform or its precursor the 80 kDa full-length TGC. The increased frequency of apoptosis correlated with the increase in transglutaminase expression and the highest rates of apoptosis were found in cells transfected with the potent TG isoform as compared to the full length TGC. The calcium ionophores A231827 and maitotoxin, which are known to induce transamidation, were found to promote apoptosis, whereas cystamine, an active transglutaminase inhibitor, blocked apoptosis due to the over-expression of the active TG isoform. This is the first time that TG has been used in cellular transfections and the results presented show that TG is a potent inducer of cell death. This finding may help to clarify the conflicting functions of TG in the induction of cell death. The TG-dependent irreversible cross-linking of intracellular proteins, a function previously assigned to TGC, represents an important biochemical event in the induction of the structural changes present in cells during apoptosis.

Integrated electroosmotic perfusion of tissue with online microfluidic analysis to track the metabolism of cystamine, pantethine, and coenzyme A.

We have developed an approach that integrates electroosmotic perfusion of tissue with a substrate-containing solution and online microfluidic analysis of products, in this case thiols. Using this approach we have tracked the metabolism of cystamine, pantethine and CoA in the extracellular space of organotypic hippocampal slice cultures (OHSCs). Currently, little is known about coenzyme A (CoA) biodegradation and even less is known about the regulation and kinetic characteristics for this sequential multienzyme reaction. We found that the steady state percentage yields of cysteamine from cystamine and pantethine during the transit through OHSCs were 91% ± 4% (SEM) and 0.01%-0.03%, respectively. The large difference in the yields of cysteamine can be used to explain the drugs' different toxicities and clinical effectiveness against cystinosis. The kinetic parameters of the enzyme reaction catalyzed by the ectoenzyme pantetheinase are KM,C/α = 4.4 ± 1.1 mM and Vmax,C = 29 ± 3 nM/s, where α is the percentage yield of pantethine to pantetheine through disulfide exchange. We estimate that the percentage yield of pantethine to pantetheine through disulfide exchange is approximately 0.5%. Based on the formation rate of cysteamine in the OHSCs, we obtained the overall apparent Michaelis constant and maximum reaction rate for sequential, extracellular CoA degradation in an in situ environment, which are K'M = 16 ± 4 µM, V'max = 7.1 ± 0.5 nM/s. Kinetic parameters obtained in situ, although difficult to measure, are better representations of the biochemical flux in the living organism than those from isolated enzymes in vitro.

New mechanistic explanation for the localization of ulcers in the rat duodenum: role of iron and selective uptake of cysteamine.

Cysteamine, a coenzyme A metabolite, induces duodenal ulcers in rodents. Our recent studies showed that ulcer formation was aggravated by iron overload and diminished in iron deficiency. We hypothesized that cysteamine is selectively taken up in the duodenal mucosa, where iron absorption primarily occurs, and is transported by a carrier-mediated process. Here we report that cysteamine administration in rats leads to cysteamine accumulation in the proximal duodenum, where the highest concentration of iron in the gastrointestinal tract is found. In vitro, iron loading of intestinal epithelial cells (IEC-6) accelerated reactive oxygen species (ROS) production and increased [(14)C]cysteamine uptake. [(14)C]Cysteamine uptake by isolated gastrointestinal mucosal cells and by IEC-6 was pH-dependent and inhibited by unlabeled cysteamine. The uptake of [(14)C]cysteamine by IEC-6 was Na(+)-independent, saturable, inhibited by structural analogs, H(2)-histamine receptor antagonists, and organic cation transporter (OCT) inhibitors. OCT1 mRNA was markedly expressed in the rat duodenum and in IEC-6, and transfection of IEC-6 with OCT1 siRNA decreased OCT1 mRNA expression and inhibited [(14)C]cysteamine uptake. Cysteamine-induced duodenal ulcers were decreased in OCT1/2 knockout mice. These studies provide new insights into the mechanism of cysteamine absorption and demonstrate that intracellular iron plays a critical role in cysteamine uptake and in experimental duodenal ulcerogenesis.

Mechanism of putative neo-antigen formation from N-propionyl-4-S-cysteaminylphenol, a tyrosinase substrate, in melanoma models.

Metastatic melanoma is resistant to conventional therapies. N-propionyl-4-S-cysteaminylphenol (NPrCAP), an N-protected sulfur-amine analog of tyrosine, is a good substrate for tyrosinase and is selectively incorporated into melanoma cells, causing cytotoxicity in vitro and in vivo. We have recently shown that intratumoral injections of NPrCAP suppress not only the growth of primary B16F1 melanoma tumors but also of secondary, re-challenged tumors. The participation of CD8(+) T cells has been suggested for the NPrCAP-mediated anti-B16 melanoma immunity. In this study, the molecular mechanism of the NPrCAP cytotoxicity and immunogenicity was examined. The phenol NPrCAP was shown to be activated by mushroom tyrosinase to the ortho-quinone N-propionyl-4-S-cysteaminyl-1,2-benzoquinone (NPrCAQ), and the structure was confirmed by reducing it to the corresponding catechol. NPrCAQ reacted rapidly with biologically relevant sulfhydryl compounds such as cysteine, glutathione and bovine serum albumin. The NPrCAQ-thiol adduct formation was proven with a model thiol N-acetylcysteine by spectroscopic methods. The production and release of NPrCAQ-protein adducts was verified in B16F1 melanoma cells in vitro and in B16F1 melanoma-bearing mice in vivo through the detection of 5-S-cysteaminyl-3-S-cysteinylcatechol after acid hydrolysis of the protein fraction. These results suggest that the phenol NPrCAP, acting as a prohapten, can be activated in melanoma cells by tyrosinase to the quinone-hapten NPrCAQ, which binds to melanosomal proteins through their cysteine residues to form possible neo-antigens, thus triggering the immunological response. NPrCAP thus represents a potential new approach to immunotherapy against metastatic melanoma.

Mapping of O-GlcNAc sites of 20 S proteasome subunits and Hsp90 by a novel biotin-cystamine tag.

The post-translational modification of proteins with O-GlcNAc is involved in various cellular processes including signal transduction, transcription, translation, and nuclear transport. This transient protein modification enables cells or tissues to adapt to nutrient conditions or stress. O-Glycosylation of the 26 S proteasome ATPase subunit Rpt2 is known to influence the stability of proteins by reducing their proteasome-dependent degradation. In contrast, knowledge of the sites of O-GlcNAcylation on the subunits of the catalytic core of the 26 S proteasome, the 20 S proteasome, and the impact on proteasome activity is very limited. This is predominantly because O-GlcNAc modifications are often substoichiometric and because 20 S proteasomes represent a complex protein mixture of different subtypes. Therefore, identification of O-GlcNAcylation sites on proteasome subunits essentially requires effective enrichment strategies. Here we describe an adapted ß-elimination-based derivatization method of O-GlcNAc peptides using a novel biotin-cystamine tag. The specificity of the reaction was increased by differential isotopic labeling with either "light" biotin-cystamine or deuterated "heavy" biotin-cystamine. The enriched peptides were analyzed by LC-MALDI-TOF/TOF-MS and relatively quantified. The method was optimized using bovine α-crystallin and then applied to murine 20 S proteasomes isolated from spleen and brain and murine Hsp90 isolated from liver. Using this approach, we identified five novel and one known O-GlcNAc sites within the murine 20 S proteasome core complex that are located on five different subunits and in addition two novel O-GlcNAc sites on murine Hsp90ß, of which one corresponds to a previously described phosphorylation site.

Cystamine metabolism and brain transport properties: clinical implications for neurodegenerative diseases.

Cystamine has shown significant neuroprotective properties in preclinical studies of Parkinson's disease (PD) and Huntington's disease (HD). Cysteamine, its FDA-approved reduced form, is scheduled to be tested for clinical efficacy in HD patients. Here, we studied the key cystamine metabolites, namely cysteamine, hypotaurine and taurine, as well as cysteine, in order to identify which one is more distinctively responsible for the neuroprotective action of cystamine. After a single administration of cystamine (10, 50 or 200 mg/kg), naïve mice were perfused with phosphate-buffered saline (PBS) at 1, 3, 12, 24 or 48 h post-injection and brain and plasma samples were analyzed by two distinct HPLC methods. Although plasma levels remained under the detection threshold, significant increases in cysteamine brain levels were detected with the 50 and 200 mg/kg doses in mice perfused 1 and 3 h following cystamine injection. To further assess cysteamine as the candidate molecule for pre-clinical and clinical trials in PD, we evaluated its capacity to cross the blood brain barrier. Using an in situ cerebral perfusion technique, we determined that the brain transport coefficient (Clup) of cysteamine (259 µM) was 0.15 ± 0.02 µL/g/s and was increased up to 0.34 ± 0.07 µL/g/s when co-perfused in the presence of cysteine. Taken together, these results strongly suggest that cysteamine is the neuroactive metabolite of cystamine and may further support its therapeutic use in neurodegenerative diseases, particularly in HD and PD.

Relationship between three polymorphisms of methylenetetrahydrofolate reductase (MTHFR C677T, A1298C, and G1793A) gene and risk of prostate cancer: a case-control study.

BACKGROUND: We hypothesized that genetic polymorphisms in methylenetetrahydrofolate reductase (MTHFR) gene are associated with prostate cancer risk. METHODS: We genotyped three MTHFR polymorphisms (C677T, A1298C, and G1793A) and measured serum total homocysteine (tHcy), folate, and vitamin B12 levels in a case-control study of 174 cases and 348 normal healthy controls. The cancer-free controls were frequency matched to the cases by age (±2 years), educational level, occupational status, ethnicity, and smoking status. RESULTS: We found that the MTHFR 677TT and 1298CC genotypes were associated with an about 40% reduction in risk of prostate cancer (adjusted OR = 0.59, 95% CI = 0.41-0.94, and adjusted OR = 0.58, 95% CI = 0.32-0.91, respectively) compared to the 677CC, and 1298AA genotypes. The combined variant genotypes of 1298AC + 677CC were associated with a 30% reduction in risk of prostate cancer (OR = 0.70; 95% CI = 0.53-0.79). In contrast, the variant genotypes of 1793GA + 677CT were associated with slightly increased risk for prostate cancer (OR = 1.64; 95% CI = 0.86-2.15). Regarding prostate cancer aggressiveness, the 677TT genotype was associated with more than 50% decreased risk of high-grade prostate cancer (Gleason score >7) compared with the 677CC and 677CT genotypes (OR = 0.35, 95% CI = 0.24-0.64; P = 0.001). There was no significant difference in plasma levels of tHcy, folate, and vitamin B12 between the two groups with any genotypes. CONCLUSION: These data suggest that all three MTHFR polymorphisms may play a pivotal role in the developing prostate cancer. Larger studies in different ethnic populations and incorporating dietary folate intake are needed to replicate our findings.

Epoxy-amine synthesised hydrogel scaffolds for soft-tissue engineering.

Highly porous and biodegradable hydrogels based on poly(ethylene glycol) (PEG) and cystamine (Cys) were fabricated using epoxy-amine chemistry and investigated as scaffolds for soft-tissue engineering. Whereas the application of fused-salt templates provided a comprehensive interconnecting pore morphology, the incorporation of a specially designed poly(epsilon-caprolactone) (PCL) cross-linker provided enhanced mechanical function without adversely effecting the scaffolds positive biological interactions. The addition of only 1.2 wt% of the PCL cross-linker was sufficient to provide improvements in the ultimate stress of 30-40%. In vitro studies not only confirmed the non-cytotoxic nature of the scaffolds, but also their degradation products, which were isolated and characterised by nuclear magnetic resonance (NMR) and matrix-assisted laser desorption/ionisation time-of-flight mass spectroscopy (MALDI ToF MS). In vivo trials were conducted over a period of 8 weeks through implantation of the scaffolds into the dorsal region of rats. At both 2 and 8 week time points the explants revealed complete infiltration by the surrounding tissue and the development of a vascular network to support the newly generated tissue, without an excessive foreign-body response.

Measurement of sulfur-containing compounds involved in the metabolism and transport of cysteamine and cystamine. Regional differences in cerebral metabolism.

An HPLC method with coulometric detection is presented for the quantitation of cysteamine, cystamine, thialysine, glutathione, glutathione disulfide and an oxidized metabolite of thialysine [S-(2-aminoethyl)-L-cysteine ketimine decarboxylated dimer (AECK-DD)]. The advantage of coulometric detection is that derivatization is unnecessary if the analyte is redox sensitive. The method was used to quantitate several sulfur-containing compounds in plasma and brain following gavage feeding of cysteamine to rats. Cysteamine, cystamine, thialysine and AECK-DD were detected in the brains of these animals. Interestingly, cysteamine treatment resulted in greatly elevated levels of cerebral methionine, despite the fact that cysteamine is not a precursor of methionine.

N-propionyl-cysteaminylphenol-magnetite conjugate (NPrCAP/M) is a nanoparticle for the targeted growth suppression of melanoma cells.

A magnetite nanoparticle, NPrCAP/M, was produced for intracellular hyperthermia treatment of melanoma by conjugating N-propionyl-cysteaminylphenol (NPrCAP) with magnetite and used for the study of selective targeting and degradation of melanoma cells. NPrCAP/M, like NPrCAP, was integrated as a substrate in the oxidative reaction by mushroom tyrosinase. Melanoma, but not non-melanoma, cells incorporated larger amounts of iron than magnetite from NPrCAP/M. When mice bearing a B16F1 melanoma and a lymphoma on opposite flanks were given NPrCAP/M, iron was observed only in B16F1 melanoma cells and iron particles (NPrCAP/M) were identified within late-stage melanosomes by electron microscopy. When cells were treated with NPrCAP/M or magnetite and heated to 43 degrees C by an external alternating magnetic field (AMF), melanoma cells were degraded 1.7- to 5.4-fold more significantly by NPrCAP/M than by magnetite. Growth of transplanted B16 melanoma was suppressed effectively by NPrCAP/M-mediated hyperthermia, suggesting a clinical application of NPrCAP/M to lesional therapy for melanoma. Finally, melanoma cells treated with NPrCAP/M plus AMF showed little sub-G1 fraction and no caspase 3 activation, suggesting that the NPrCAP/M-mediated hyperthermia induced non-apoptotic cell death. These results suggest that NPrCAP/M may be useful in targeted therapy for melanoma by inducing non-apoptotic cell death after appropriate heating by the AMF.

The physiological role of the Gomori-positive glia: a new hypothesis.

On the basis of histological observations of the brains of intact animals and of those injected with a sulfur-containing material (cystamine) we propose that the main, if not unique, role of the Gomori-positive glia is to scavenge the brain for sulfur-containing material because such material when physiologically present (e.g. neurophysin) may give rise to free cysteine that is toxic to neurons, being a long-recognized neurotoxin.

Studies on the reducing systems for plant and animal thioredoxin-independent methionine sulfoxide reductases B.

Two distinct stereospecific methionine sulfoxide reductases (Msr), MsrA and MsrB reduce the oxidized methionine (Met), methionine sulfoxide [Met(O)], back to Met. In this report, we examined the reducing systems required for the activities of two chloroplastic MsrB enzymes (NtMsrB1 and NtMsrB2) from tobacco (Nicotiana tabacum). We found that NtMrsB1, but not NtMsrB2, could use dithiothreitol as an efficient hydrogen donor. In contrast Escherichia coli thioredoxin (Trx) could serve as a reducing agent for NtMsrB2, but not for NtMsrB1. Similar to previously reported human Trx-independent hMsrB2 and hMsrB3, NtMsrB1 could also use bovine liver thionein and selenocysteamine as reducing agents. Furthermore, the unique plant Trx-like protein CDSP32 was shown to reduce NtMsrB1, hMsrB2 and hMsrB3. All these tested Trx-independent MsrB enzymes lack an additional cysteine (resolving cysteine) that is capable of forming a disulfide bond on the enzyme during the catalytic reaction. Our results indicate that plant and animal MsrB enzymes lacking a resolving cysteine likely share a similar reaction mechanism.

Differential gene expression in mouse liver associated with the hepatoprotective effect of clofibrate.

Pretreatment of mice with the peroxisome proliferator clofibrate (CFB) protects against acetaminophen (APAP)-induced hepatotoxicity. Previous studies have shown that activation of the nuclear peroxisome proliferator activated receptor-alpha (PPARalpha) is required for this effect. The present study utilizes gene expression profile analysis to identify potential pathways contributing to PPARalpha-mediated hepatoprotection. Gene expression profiles were compared between wild type and PPARalpha-null mice pretreated with vehicle or CFB (500 mg/kg, i.p., daily for 10 days) and then challenged with APAP (400 mg/kg, p.o.). Total hepatic RNA was isolated 4 h after APAP treatment and hybridized to Affymetrix Mouse Genome MGU74 v2.0 GeneChips. Gene expression analysis was performed utilizing GeneSpring software. Our analysis identified 53 genes of interest including vanin-1, cell cycle regulators, lipid-metabolizing enzymes, and aldehyde dehydrogenase 2, an acetaminophen binding protein. Vanin-1 could be important for CFB-mediated hepatoprotection because this protein is involved in the synthesis of cysteamine and cystamine. These are potent antioxidants capable of ameliorating APAP toxicity in rodents and humans. HPLC-ESI/MS/MS analysis of liver extracts indicates that enhanced vanin-1 gene expression results in elevated cystamine levels, which could be mechanistically associated with CFB-mediated hepatoprotection.

Cystamine and cysteamine increase brain levels of BDNF in Huntington disease via HSJ1b and transglutaminase.

There is no treatment for the neurodegenerative disorder Huntington disease (HD). Cystamine is a candidate drug; however, the mechanisms by which it operates remain unclear. We show here that cystamine increases levels of the heat shock DnaJ-containing protein 1b (HSJ1b) that are low in HD patients. HSJ1b inhibits polyQ-huntingtin-induced death of striatal neurons and neuronal dysfunction in Caenorhabditis elegans. This neuroprotective effect involves stimulation of the secretory pathway through formation of clathrin-coated vesicles containing brain-derived neurotrophic factor (BDNF). Cystamine increases BDNF secretion from the Golgi region that is blocked by reducing HSJ1b levels or by overexpressing transglutaminase. We demonstrate that cysteamine, the FDA-approved reduced form of cystamine, is neuroprotective in HD mice by increasing BDNF levels in brain. Finally, cysteamine increases serum levels of BDNF in mouse and primate models of HD. Therefore, cysteamine is a potential treatment for HD, and serum BDNF levels can be used as a biomarker for drug efficacy.