Roy B. Dyer

Mutant Huntingtin Impairs Axonal Trafficking in Mammalian Neurons In Vivo and In Vitro

ABSTRACT Recent data in invertebrates demonstrated that huntingtin (htt) is essential for fast axonal trafficking. Here, we provide direct and functional evidence that htt is involved in fast axonal trafficking in mammals. Moreover, expression of full-length mutant htt (mhtt) impairs vesicular and mitochondrial trafficking in mammalian neurons in vitro and in whole animals in vivo. Particularly, mitochondria become progressively immobilized and stop more frequently in neurons from transgenic animals. These defects occurred early in development prior to the onset of measurable neurological or mitochondrial abnormalities. Consistent with a progressive loss of function, wild-type htt, trafficking motors, and mitochondrial components were selectively sequestered by mhtt in human Huntingtons disease-affected brain. Data provide a model for how loss of htt function causes toxicity; mhtt-mediated aggregation sequesters htt and components of trafficking machinery leading to loss of mitochondrial motility and eventual mitochondrial dysfunction.

Increased Bile Acid Biosynthesis Is Associated With Irritable Bowel Syndrome With Diarrhea

BACKGROUND & AIMS Variations in genes that regulate bile acid (BA) synthesis are associated with colonic transit in patients with irritable bowel syndrome (IBS). We investigated features of BA synthesis and excretion and genetic features of patients with different types of IBS. METHODS In 26 healthy volunteers, 26 patients with IBS and constipation (IBS-C), and 26 with IBS and diarrhea (IBS-D), we measured serum levels of 7α-hydroxy-4-cholesten-3-one (C4; a surrogate for BA synthesis) and fibroblast growth factor (FGF) 19 (an ileal hormone that downregulates BA synthesis). For stool samples, we measured concentration of BA, weight, and amount of fat when participants were given high-fat diets. Spearman correlations were used to explore relationships among factors. We analyzed 1 polymorphism in Klotho-β (KLB) and 3 in fibroblast growth factor receptor-4 (FGFR4) for all members of each group using analysis of covariance. RESULTS The concentration of BA in stool was associated with group (for a comparison of 3 groups; P = .057); it was higher in patients with IBS-D than IBS-C (P = .017). The serum level of C4 was higher in patients with IBS-D than IBS-C (P = .02) or healthy volunteers (P = .01); 38% of patients with IBS-D had increased serum levels of C4, compared with healthy volunteers. Serum level of C4 correlated with stool concentration of BA (rs = 0.606; P < .001), serum FGF19 (rs = -0.324; P = .007), and stool weight (rs = 0.366; P = .003). Stool concentration of BA correlated with weight (rs = 0.737; P < .001) and level of fat (rs = 0.528; P < .001). Body mass index correlated with serum level of C4 (rs = 0.423, P < .001) and stool concentration of BA (rs = 0.507, P < .001), and was higher in patients with IBS-D compared with other groups (overall P = .036). FGFR4 rs1966265 was associated with stool level of BA (P = .032). CONCLUSIONS Patients with IBS-D have greater body mass index and synthesize and excrete higher levels of BA than individuals with IBS-C or healthy volunteers. Serum levels of C4 might be used to identify patients with IBS-D who have BA malabsorption; studies are needed to determine if some patients have a genetic predisposition to this disorder.

Protection conferred by myocardial ATP-sensitive K+ channels in pressure overload-induced congestive heart failure revealed in KCNJ11 Kir6.2-null mutant

Ventricular load can precipitate development of the heart failure syndrome, yet the molecular components that control the cardiac adaptive response to imposed demand remain partly understood. Compromised ATP‐sensitive K+ (KATP) channel function renders the heart vulnerable to stress, implicating this metabolic sensor in the homeostatic response that would normally prevent progression of cardiac disease. Here, pressure overload was imposed on the left ventricle by transverse aortic constriction in the wild‐type and in mice lacking sarcolemmal KATP channels through Kir6.2 pore knockout (Kir6.2‐KO). Despite equivalent haemodynamic loads, within 30 min of aortic constriction, Kir6.2‐KO showed an aberrant prolongation of action potentials with intracellular calcium overload and ATP depletion, whereas wild‐type maintained ionic and energetic handling. On catheterization, constricted Kir6.2‐KO displayed compromised myocardial performance with elevated left ventricular end‐diastolic pressure, not seen in the wild‐type. Glyburide, a KATP channel inhibitor, reproduced the knockout phenotype in the wild‐type, whereas the calcium channel antagonist, verapamil, prevented abnormal outcome in Kir6.2‐KO. Within 48 h following aortic constriction, fulminant biventricular congestive heart failure, characterized by exercise intolerance, cardiac contractile dysfunction, hepatopulmonary congestion and ascites, halved the Kir6.2‐KO cohort, while no signs of organ failure or mortality were seen in wild‐type. Surviving Kir6.2‐KO developed premature and exaggerated fibrotic myocardial hypertrophy associated with nuclear up‐regulation of calcium‐dependent pro‐remodelling MEF2 and NF‐AT pathways, precipitating chamber dilatation within 3 weeks. Thus, KATP channels appear mandatory in acute and chronic cardiac adaptation to imposed haemodynamic load, protecting against congestive heart failure and death.

Mutant protein in Huntington disease is resistant to proteolysis in affected brain.

The cause of Huntington disease pathophysiology is unknown, but a major hypothesis suggests that toxicity arises from the cleavage and accumulation of amino-terminal fragments containing an expanded polyglutamine region. In evaluating huntingtin protein (HD) from human brain, transgenic animals and cells, we observed, unexpectedly, that mutant HD is more resistant to proteolysis than normal HD. The N-terminal cleavage fragments we observed arise from the processing of normal HD and are sequestered by full-length mutant HD. Our results support a model in which inhibition of proteolysis of mutant HD leads to aggregation and toxicity through the sequestering of important targets, including normal HD.

Microtubule destabilization and nuclear entry are sequential steps leading to toxicity in Huntington's disease

There has been a longstanding debate regarding the role of proteolysis in Huntingtons disease. The toxic peptide theory posits that N-terminal cleavage fragments of mutant Huntingtons disease protein [mutant huntingtin (mhtt)] enter the nucleus to cause transcriptional dysfunction. However, recent data suggest a second model in which proteolysis of full-length mhtt is inhibited. Importantly, the two competing theories differ with respect to subcellular distribution of mhtt at initiation of toxicity: nuclear if cleaved and cytoplasmic in the absence of cleavage. Using quantitative single-cell analysis and time-lapse imaging, we show here that transcriptional dysfunction is “downstream” of cytoplasmic dysfunction. Primary and reversible toxic events involve destabilization of microtubules mediated by full-length mhtt before cleavage. Restoration of microtubule structure by taxol inhibits nuclear entry and increases cell survival.

Effect of colesevelam on faecal bile acids and bowel functions in diarrhoea‐predominant irritable bowel syndrome

About one‐third of patients with IBS‐diarrhoea (irritable bowel syndrome‐D) have evidence of increased bile acid synthesis or excretion.

Candidate Serum Biomarkers for Prostate Adenocarcinoma Identified by mRNA Differences in Prostate Tissue and Verified with Protein Measurements in Tissue and Blood

BACKGROUND Improved tests are needed for detection and management of prostate cancer. We hypothesized that differential gene expression in prostate tissue could help identify candidate blood biomarkers for prostate cancer and that blood from men with advanced prostate disease could be used to verify the biomarkers presence in circulation. METHODS We identified candidate markers using mRNA expression patterns from laser-capture microdissected prostate tissue and confirmed tissue expression using immunohistochemistry (IHC) for the subset of candidates having commercial antisera. We analyzed tissue extracts with tandem mass spectrometry (MS/MS) and measured blood concentrations using immunoassays and MS/MS of trypsin-digested, immunoextracted peptides. RESULTS We selected 35 novel candidate prostate adenocarcinoma biomarkers. For all 13 markers having commercial antisera for IHC, tissue expression was confirmed; 6 showed statistical discrimination between nondiseased and malignant tissue, and only 5 were detected in tissue extracts by MS/MS. Sixteen of the 35 candidate markers were successfully assayed in blood. Four of 8 biomarkers measured by ELISA and 3 of 10 measured by targeted MS showed statistically significant increases in blood concentrations of advanced prostate cancer cases, compared with controls. CONCLUSIONS Seven novel biomarkers identified by gene expression profiles in prostate tissue were shown to have statistically significant increased concentrations in blood from men with advanced prostate adenocarcinoma compared with controls: apolipoprotein C1, asporin, cartilage oligomeric matrix protein, chemokine (C-X-C motif) ligand 11 (CXCL11), CXCL9, coagulation factor V, and proprotein convertase subtilisin/kexin 6.

FAVL elevation in human tumors disrupts Fanconi anemia pathway signaling and promotes genomic instability and tumor growth

Fanconi anemia (FA) is a rare human genetic disease caused by mutations in any one of 13 known genes that encode proteins functioning in one common signaling pathway, the FA pathway, or in unknown genes. One characteristic of FA is an extremely high incidence of cancer, indicating the importance of the FA pathway in tumor suppression. However, the role of this pathway in the development and progression of human cancers in individuals who do not have FA has not been clearly determined. Here, we report that elevated expression of what we believe to be a novel splice variant of FA complementation group L (FANCL), which we identified and named FAVL, can impair the FA pathway in non-FA human tumor cells and act as a tumor promoting factor. FAVL expression was elevated in half of the human carcinoma cell lines and carcinoma tissue samples tested. Expression of FAVL resulted in decreased FANCL expression by sequestering FANCL to the cytoplasm and enhancing its degradation. Importantly, this impairment of the FA pathway by FAVL elevation provided human cancer cells with a growth advantage, caused chromosomal instability in vitro, and promoted tumor development in a xenograft mouse model. These data indicate that FAVL impairment of the FA pathway likely contributes to the development of non-FA human cancers and therefore add a challenging layer of complexity to the pathogenesis of human cancer. We further believe that these data will prove useful for developing additional tools for fighting human cancer.

Role for SUR2A ED Domain in Allosteric Coupling within the KATP Channel Complex

Allosteric regulation of heteromultimeric ATP-sensitive potassium (KATP) channels is unique among protein systems as it implies transmission of ligand-induced structural adaptation at the regulatory SUR subunit, a member of ATP-binding cassette ABCC family, to the distinct pore-forming K+ (Kir6.x) channel module. Cooperative interaction between nucleotide binding domains (NBDs) of SUR is a prerequisite for KATP channel gating, yet pathways of allosteric intersubunit communication remain uncertain. Here, we analyzed the role of the ED domain, a stretch of 15 negatively charged aspartate/glutamate amino acid residues (948–962) of the SUR2A isoform, in the regulation of cardiac KATP channels. Disruption of the ED domain impeded cooperative NBDs interaction and interrupted the regulation of KATP channel complexes by MgADP, potassium channel openers, and sulfonylurea drugs. Thus, the ED domain is a structural component of the allosteric pathway within the KATP channel complex integrating transduction of diverse nucleotide-dependent states in the regulatory SUR subunit to the open/closed states of the K+-conducting channel pore.

Direct Evidence of Acetaminophen Interference with Subcutaneous Glucose Sensing in Humans: A Pilot Study.

BACKGROUND Recent advances in accuracy and reliability of continuous glucose monitoring (CGM) devices have focused renewed interest on the use of such technology for therapeutic dosing of insulin without the need for independent confirmatory blood glucose meter measurements. An important issue that remains is the susceptibility of CGM devices to erroneous readings in the presence of common pharmacologic interferences. We report on a new method of assessing CGM sensor error to pharmacologic interferences using the example of oral administration of acetaminophen. MATERIALS AND METHODS We examined the responses of several different Food and Drug Administration-approved and commercially available CGM systems (Dexcom [San Diego, CA] Seven(®) Plus™, Medtronic Diabetes [Northridge, CA] Guardian(®), and Dexcom G4(®) Platinum) to oral acetaminophen in 10 healthy volunteers without diabetes. Microdialysis catheters were placed in the abdominal subcutaneous tissue. Blood and microdialysate samples were collected periodically and analyzed for glucose and acetaminophen concentrations before and after oral ingestion of 1 g of acetaminophen. We compared the response of CGM sensors with the measured acetaminophen concentrations in the blood and interstitial fluid. RESULTS Although plasma glucose concentrations remained constant at approximately 90 mg/dL (approximately 5 mM) throughout the study, CGM glucose measurements varied between approximately 85 to 400 mg/dL (from approximately 5 to 22 mM) due to interference from the acetaminophen. The temporal profile of CGM interference followed acetaminophen concentrations measured in interstitial fluid (ISF). CONCLUSIONS This is the first direct measurement of ISF concentrations of putative CGM interferences with simultaneous measurements of CGM performance in the presence of the interferences. The observed interference with glucose measurements in the tested CGM devices coincided temporally with appearance of acetaminophen in the ISF. The method applied here can be used to determine the susceptibility of current and future CGM systems to interference from acetaminophen or other exogenous pharmacologic agents.Abstract Background: Recent advances in accuracy and reliability of continuous glucose monitoring (CGM) devices have focused renewed interest on the use of such technology for therapeutic dosing of insulin without the need for independent confirmatory blood glucose meter measurements. An important issue that remains is the susceptibility of CGM devices to erroneous readings in the presence of common pharmacologic interferences. We report on a new method of assessing CGM sensor error to pharmacologic interferences using the example of oral administration of acetaminophen. Materials and Methods: We examined the responses of several different Food and Drug Administration–approved and commercially available CGM systems (Dexcom [San Diego, CA] Seven® Plus™, Medtronic Diabetes [Northridge, CA] Guardian®, and Dexcom G4® Platinum) to oral acetaminophen in 10 healthy volunteers without diabetes. Microdialysis catheters were placed in the abdominal subcutaneous tissue. Blood and microdialysate samples were collecte...