CFTR and PC2, partners in the primary cilia in autosomal dominant polycystic kidney disease.

Defects in the primary cilium are associated with autosomal dominant polycystic kidney disease (ADPKD). We used a combination of animal models, Western blotting, and confocal microscopy and discovered that CFTR and polycystin 2 (PC2) are both colocalized to the cilium in normal kidneys, with the levels of both being decreased in cystic epithelia. Cilia were longer in CFTR-null mice and in cystic cells in our ADPKD animal models. We examined septin 2, known to play a role in cilia length, to act as a diffusion barrier and to serve as an enhancer of proliferation. We found that septin 2 protein levels were upregulated and colocalized strongly with CFTR in cystic cells. Application of VX-809, the CFTR corrector, restored CFTR and PC2 toward normal in the cilia, decreased the protein levels of septin 2, and drastically reduced septin 2 colocalization with CFTR. Our data suggest that CFTR is present in the cilia and plays a role there, perhaps through its conductance of Cl-. We also postulate that septin 2 is important for localizing CFTR to the apical membrane in cystic epithelia.NEW & NOTEWORTHY CFTR is present in the primary cilia together with polycystin 2 (PC2). Ablation of CFTR makes cilia longer suggesting that CFTR plays a role there, perhaps through its conductance of Cl.

A new role for heat shock factor 27 in the pathophysiology of Clostridium difficile toxin B.

Clostridiumdifficile (CD) is a common pathogen that causes severe gastrointestinal inflammatory diarrhea in patients undergoing antibiotic therapy. Its virulence derives from two toxins, toxin CD, A and B (TcdA and TcdB) (Borriello et al. Rev Infect Dis 12, Suppl 2: S185-191, 1990). Among the prime candidates for CD colonization are patients with cystic fibrosis (CF), who are routinely treated with antibiotics and frequently hospitalized. Indeed, ~50% of patients with CF are colonized with virulent forms of CD but do not exhibit diarrhea (Bauer et al. Clin Microbiol Infect 20: O446-O449, 2014; Binkovitz et al. Am J Roentgenol 172: 517-521, 199; Zemljic et al. Anaerobe 16: 527-532, 2010). We found that TcdB has global effects on colonic cells, including reducing the steady-state levels of sodium-proton exchange regulatory factors, reducing the levels of heat shock protein (Hsp) 27, and increasing the fraction of total Hsp27 bound to the cystic fibrosis transmembrane conductance regulator (CFTR). Also, since some mutations in CFTR seem to be protective, we asked whether CFTR is a target of TcdB. We show here that TcdB increases the maturation of CFTR and transiently increases its function. These combined effects promote increased surface expression of CFTR, resulting in a transient increase in Cl- secretion. This increase is followed by a precipitous decline in both CFTR-dependent Cl- secretion and transepithelial resistance (TER), suggesting a breakdown in the epithelial cells' tight junctions. We also found that overexpressing Hsp27 reverses some of the deleterious effects of TcdB, in particular preserving TER and therefore likely the maintenance of barrier function. Thus, our data suggest that Hsp27 plays a role in the diarrhea generated by CD infection and is a potential therapeutic target for treating this diarrhea.NEW & NOTEWORTHYClostridium difficile (CD) is a common pathogen that causes severe gastrointestinal inflammatory diarrhea in patients undergoing antibiotic therapy. We provide new evidence that heat shock protein (Hsp) 27 is one of the key players in CD pathology and that increasing Hsp27 can prevent the decrease in transepithelial resistance induced by toxin CD B, pointing the way for pharmacologic therapies for patients with chronic CD infection that can increase Hsp27 as a means to mitigate the effects of CD on gastrointestinal pathology.

Restoration of F508-del Function by Transcomplementation: The Partners Meet in the Endoplasmic Reticulum.

BACKGROUND/AIMS: Because of the small size of adeno-associated virus, AAV, the cystic fibrosis conductance regulator, CFTR, cDNA is too large to fit within AAV and must be truncated. We report here on two truncated versions of CFTR, which, when inserted into AAV1 and used to infect airway cells, rescue F508-del CFTR via transcomplementation. The purpose of this study is to shed light on where in the cell transcomplementation occurs and how it results in close association between the endogenous F508-del and truncated CFTR. METHODS: We treated CF airway cells (CFBE41o-) with AAV2/1 (AAV2 inverted terminal repeats/AAV1 capsid) containing truncated forms of CFTR, ∆264 and ∆27-264 CFTR, who can restore the function of F508-del by transcomplementation. We addressed the aims of the study using a combination of confocal microscopy and short circuit currents measurements. For the latter, CF bronchial epithelial cells (CFBE) were grown on permeable supports. RESULTS: We show that both F508del and the truncation mutants colocalize in the ER and that both the rescued F508-del and the transcomplementing mutants reach the plasma membrane together. There was significant fluorescence resonance energy transfer (FRET) between F508-del and the transcomplementing mutants within the endoplasmic reticulum (ER), suggesting that transcomplementation occurs through a bimolecular interaction. We found that transcomplementation could increase the Isc in CFBE41o- cells stably expressing additional wt-CFTR or F508-del and in parental CFBE41o- cells expressing endogenous levels of F508-del. CONCLUSION: We conclude that the functional rescue of F508-del by transcomplementation occurs via a bimolecular interaction that most likely begins in the ER and continues at the plasma membrane. These results come at an opportune time for developing a gene therapy for CF and offer new treatment options for a wide range of CF patients.

Histone deacetylase 6 inhibition reduces cysts by decreasing cAMP and Ca2+ in knock-out mouse models of polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is associated with progressive enlargement of multiple renal cysts, often leading to renal failure that cannot be prevented by a current treatment. Two proteins encoded by two genes are associated with ADPKD: PC1 (pkd1), primarily a signaling molecule, and PC2 (pkd2), a Ca2+ channel. Dysregulation of cAMP signaling is central to ADPKD, but the molecular mechanism is unresolved. Here, we studied the role of histone deacetylase 6 (HDAC6) in regulating cyst growth to test the possibility that inhibiting HDAC6 might help manage ADPKD. Chemical inhibition of HDAC6 reduced cyst growth in PC1-knock-out mice. In proximal tubule-derived, PC1-knock-out cells, adenylyl cyclase 6 and 3 (AC6 and -3) are both expressed. AC6 protein expression was higher in cells lacking PC1, compared with control cells containing PC1. Intracellular Ca2+ was higher in PC1-knock-out cells than in control cells. HDAC inhibition caused a drop in intracellular Ca2+ and increased ATP-simulated Ca2+ release. HDAC6 inhibition reduced the release of Ca2+ from the endoplasmic reticulum induced by thapsigargin, an inhibitor of endoplasmic reticulum Ca2+-ATPase. HDAC6 inhibition and treatment of cells with the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) reduced cAMP levels in PC1-knock-out cells. Finally, the calmodulin inhibitors W-7 and W-13 reduced cAMP levels, and W-7 reduced cyst growth, suggesting that AC3 is involved in cyst growth regulated by HDAC6. We conclude that HDAC6 inhibition reduces cell growth primarily by reducing intracellular cAMP and Ca2+ levels. Our results provide potential therapeutic targets that may be useful as treatments for ADPKD.

Syntaxin 8 and the Endoplasmic Reticulum Processing of ΔF508-CFTR.

BACKGROUND/AIMS: Cystic fibrosis (CF) is a lethal recessive disorder caused by mutations in the CF transmembrane conductance regulator (CFTR). ΔF508, the most common mutation, is a misfolded protein that is retained in the endoplasmic reticulum and degraded, precluding delivery to the cell surface [1]. METHODS: Here we use a combination of western blotting, immunoprecipitation, and short circuit current techniques combined with confocal microscopy to address whether the SNARE attachment protein, STX8 plays a role in ΔF508's processing and movement out of the ER. RESULTS: Although the SNARE protein STX8 is thought to be functionally related and primarily localized to early endosomes, we show that silencing of STX8, particularly in the presence of the Vertex corrector molecule C18, rescues ΔF508-CFTR, allowing it to reach the cell surface and increasing CFTR-dependent chloride currents by approximately 2.5-fold over control values. STX8 silencing reduced the binding of quality control protein, Hsp 27, a protein that targets ΔF508-CFTR for sumoylation and subsequent degradation, to ΔF508-CFTR. STX8 silencing increased the levels of Hsp 60 a protein involving in early events in protein folding. CONCLUSION: STX8 knockdown creates an environment favorable for mature ΔF508 to reach the cell surface. The data also suggest that when present at normal levels, STX8 functions as part of the cell's quality control mechanism.

Combination of Correctors Rescue ΔF508-CFTR by Reducing Its Association with Hsp40 and Hsp27.

Correcting the processing of ΔF508-CFTR, the most common mutation in cystic fibrosis, is the major goal in the development of new therapies for this disease. Here, we determined whether ΔF508 could be rescued by a combination of small-molecule correctors, and identified the mechanism by which correctors rescue the trafficking mutant of cystic fibrosis transmembrane conductance regulator (CFTR). We transfected COS-7 cells with ΔF508, created HEK-293 stably expressing ΔF508, and utilized CFBE41o(-) cell lines stably transduced with ΔF508. As shown previously, ΔF508 expressed less protein, was unstable at physiological temperature, and rapidly degraded. When the cells were treated with the combination C18 + C4 the mature C-band was expressed at the cell surface. After treatment with C18 + C4, we saw a lower rate of protein disappearance after translation was stopped with cycloheximide. To understand how this rescue occurs, we evaluated the change in the binding of proteins involved in endoplasmic reticulum-associated degradation, such as Hsp27 (HspB1) and Hsp40 (DnaJ). We saw a dramatic reduction in binding to heat shock proteins 27 and 40 following combined corrector therapy. siRNA experiments confirmed that a reduction in Hsp27 or Hsp40 rescued CFTR in the ΔF508 mutant, but the rescue was not additive or synergistic with C4 + 18 treatment, indicating these correctors shared a common pathway for rescue involving a network of endoplasmic reticulum-associated degradation proteins.

Rescuing Trafficking Mutants of the ATP-binding Cassette Protein, ABCA4, with Small Molecule Correctors as a Treatment for Stargardt Eye Disease.

Stargardt disease is the most common form of early onset macular degeneration. Mutations in ABCA4, a member of the ATP-binding cassette (ABC) family, are associated with Stargardt disease. Here, we have examined two disease-causing mutations in the NBD1 region of ABCA4, R1108C, and R1129C, which occur within regions of high similarity with CFTR, another ABC transporter gene, which is associated with cystic fibrosis. We show that R1108C and R1129C are both temperature-sensitive processing mutants that engage the cellular quality control mechanism and show a strong interaction with the chaperone Hsp 27. Both mutant proteins also interact with HDCAC6 and are degraded in the aggresome. We also demonstrate that novel corrector compounds that are being tested as treatment for cystic fibrosis, such as VX-809, can rescue the processing of the ABCA4 mutants, particularly their expression at the cell surface, and can reduce their binding to HDAC6. Thus, our data suggest that VX-809 can potentially be developed as a new therapy for Stargardt disease, for which there is currently no treatment.

Inhibition of histone deacetylase 6 activity reduces cyst growth in polycystic kidney disease.

Abnormal proliferation of cyst-lining epithelium and increased intracystic fluid secretion via the cystic fibrosis transmembrane conductance regulator (CFTR) are thought to contribute to cyst growth in autosomal dominant polycystic kidney disease (ADPKD). Histone deacetylase 6 (HDAC6) expression and activity are increased in certain cancers, neurodegenerative diseases, and in Pkd1-mutant renal epithelial cells. Inhibition of HDAC6 activity with specific inhibitors slows cancer growth. Here we studied the effect of tubacin, a specific HDAC6 inhibitor, on cyst growth in polycystic kidney disease. Treatment with tubacin prevented cyst formation in MDCK cells, an in vitro model of cystogenesis. Cyclic AMP stimulates cell proliferation and activates intracystic CFTR-mediated chloride secretion in ADPKD. Treatment with tubacin downregulated cyclic AMP levels, inhibited cell proliferation, and inhibited cyclic AMP-activated CFTR chloride currents in MDCK cells. We also found that tubacin reduced cyst growth by inhibiting proliferation of cyst-lining epithelial cells, downregulated cyclic AMP levels, and improved renal function in a Pkd1-conditional mouse model of ADPKD. Thus, HDAC6 could play a role in cyst formation and could serve as a potential therapeutic target in ADPKD.

STIM1fl/fl Ksp-Cre Mouse has Impaired Renal Water Balance.

BACKGROUND/AIM: STIM1 is as an essential component in store operated Ca2+ entry. However give the paucity of information on the role of STIM1 in kidney, the aim was to study the function of STIM1 in the medulla of the kidney. METHODS: we crossed a Ksp-cre mouse with another mouse containing two loxP sites flanking Exon 6 of STIM1. The Ksp-cre mouse is based upon the Ksp-cadherin gene promoter which expresses cre recombinase in developing nephrons, collecting ducts (SD) and thick ascending limbs (TAL) of the loop of Henle. RESULTS: The offspring of these mice are viable without gross morphological changes, however, we noticed that the STIM1 Ksp-cre knockout mice produced more urine compared to control. To examine this more carefully, we fed mice low (LP) and high protein (HP) diets respectively. When mice were fed HP diet STIM1 ko mice had significantly increased urinary volume and lower specific gravity compared to wt mice. In STIM1 ko mice fed HP diet urine creatinine and urea were significantly lower compared to wt mice fed HP diet, however the fractional excretion was the same. CONCLUSION: These data support the idea that STIM1 ko mice have impaired urinary concentrating ability when challenged with HP diet is most likely caused by impaired Ca2+-dependent signal transduction through the vasopressin receptor cascade.

CFTR Controls the Activity of NF-κB by Enhancing the Degradation of TRADD.

BACKGROUND/AIMS: Chronic lung infection in cystic fibrosis leads to an inflammatory response that persists because of the chronic presence of bacteria and ultimately leads to a catastrophic failure of lung function. METHODS: We use a combination of biochemistry, cell and molecular biology to study the interaction of TRADD, a key adaptor molecule in TNFα signaling, with CFTR in the regulation of NFκB. RESULTS: We show that Wt CFTR binds to and colocalizes with TRADD. TRADD is a key signaling intermediate connecting TNFα with activation of NFκB. By contrast, ΔF508 CFTR does not bind to TRADD. NF-κB activation is higher in CFBE expressing ΔF508 CFTR than in cells expressing Wt CFTR. However, this differential effect is abolished when TRADD levels are knocked down. Transfecting Wt CFTR into CFBE cells reduces NF-κB activity. However the reduction is abolished by the CFTR chloride transport inhibitor-172. Consistently, transfecting in the correctly trafficked CFTR conduction mutants G551D or S341A also fail to reduce NFκB activity. Thus CFTR must be functional if it is to regulate NF-κB activity. We also found that TNFα produced a greater increase in NF-κB activity in CFBE cells than in the same cell when Wt CFTR-corrected. Consistently, the effect is also abolished when TRADD is knocked down by shRNA. Thus, Wt CFTR control of TRADD modulates the physiological activation of NF-κB by TNFα. Based on studies with proteosomal and lysosomal inhibitors, the mechanism by which Wt CFTR, but not ΔF508 CFTR, suppresses TRADD is by lysosomal degradation. CONCLUSION: We have uncovered a novel mechanism whereby Wt CFTR regulates TNFα signaling by enhancing TRADD degradation. Thus by reducing the levels of TRADD, Wt CFTR suppresses downstream proinflammatory NFκB signaling. By contrast, suppression of NF-κB activation fails in CF cells expressing ΔF508 CFTR.

Regulation of CFTR Expression and Arginine Vasopressin Activity Are Dependent on Polycystin-1 in Kidney-Derived Cells.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the development of multiple, progressive, fluid-filled renal cysts that distort the renal parenchyma, leading to end-stage renal failure, mainly after the fifth decade of life. ADPKD is caused by a mutation in the PKD1 or PKD2 genes that encode polycystin-1 (PC-1) and polycystin-2 (PC-2), respectively. PC-1 is an important regulator of several signaling pathways and PC-2 is a nonselective calcium channel. The CFTR chloride channel is responsible for driving net fluid secretion into the cysts, promoting cyst growth. Arginine vasopressin hormone (AVP), in turn, is capable of increasing cystic intracellular cAMP, contributing to cell proliferation, transepithelial fluid secretion, and therefore to disease progression. The aim of this study was to assess if AVP can modulate CFTR and whether PC-1 plays a role in this potential modulation. METHODS: M1 cells, derived from mouse cortical collecting duct, were used in the current work. The cells were treated with 10-7 M AVP hormone and divided into two main groups: transfected cells superexpressing PC-1 (Transf) and cells not transfected (Ctrl). CFTR expression was assessed by immunodetection, CFTR mRNA levels were quantified by quantitative reverse transcription-polymerase chain reaction, and CFTR net ion transport was measured using the Ussing chamber technique. RESULTS: AVP treatment increased the levels of CFTR protein and mRNA. CFTR short-circuit currents were also increased. However, when PC-1 was overexpressed in M1 cells, no increase in any of these parameters was detected. CONCLUSIONS: CFTR chloride channel expression is increased by AVP in M1 cells and PC-1 is capable of regulating this modulation.

Correctors Rescue CFTR Mutations in Nucleotide-Binding Domain 1 (NBD1) by Modulating Proteostasis.

We evaluated whether small molecule correctors could rescue four nucleotide-binding domain 1 (NBD1) mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene (A455E, S492F, ΔI507, and R560T). We first transfected Cos-7 cells (green monkey kidney cells) with A455E, S492F, ΔI507, or R560T and created HEK-293 (human embryonic kidney cells) cell lines stably expressing these CFTR mutations. The mutants showed lowered protein expression, instability at physiological temperature, and rapid degradation. After treatment with correctors CFFT-002, CFFT-003, C3, C4, and/or C18, the combination of C18+C4 showed the most correction and resulted in increased CFTR residing in the plasma membrane. We found a profound decrease in binding of CFTR to histone deacetylases (HDAC) 6 and 7 and heat shock proteins (Hsps) 27 and 40. Silencing Hsp27 or 40 rescued the mutants, but no additional amount of CFTR was rescued when both proteins were knocked down simultaneously. Thus, CFTR mutations in NBD1 can be rescued by a combination of correctors, and the treatment alters the interaction between mutated CFTR and the endoplasmic reticulum machinery.

Gout-causing Q141K mutation in ABCG2 leads to instability of the nucleotide-binding domain and can be corrected with small molecules.

The multidrug ATP-binding cassette, subfamily G, 2 (ABCG2) transporter was recently identified as an important human urate transporter, and a common mutation, a Gln to Lys substitution at position 141 (Q141K), was shown to cause hyperuricemia and gout. The nature of the Q141K defect, however, remains undefined. Here we explore the Q141K ABCG2 mutation using a comparative approach, contrasting it with another disease-causing mutation in an ABC transporter, the deletion of Phe-508 (ΔF508) in the cystic fibrosis transmembrane conductance regulator (CFTR). We found, much like in ΔF508 CFTR, that the Q141K mutation leads to instability in the nucleotide-binding domain (NBD), a defect that translates to significantly decreased protein expression. However, unlike the CFTR mutant, the Q141K mutation does not interfere with the nucleotide-binding domain/intracellular loop interactions. This investigation has also led to the identification of critical residues involved in the protein-protein interactions necessary for the dimerization of ABCG2: Lys-473 (K473) and Phe-142 (F142). Finally, we have demonstrated the utility of using small molecules to correct the Q141K defect in expression and function as a possible therapeutic approach for hyperuricemia and gout.

Mis-regulation of mammalian target of rapamycin (mTOR) complexes induced by albuminuria in proximal tubules.

High albumin concentrations in the proximal tubule of the kidney causes tubulointerstitial injury, but how this process occurs is not completely known. To address the signal transduction pathways mis-regulated in renal injury, we studied the modulation of mammalian target of rapamycin (mTOR) complexes by physiologic and pathophysiologic albumin concentrations in proximal tubule cells. Physiologic albumin concentrations activated the PI3K/mTORC2/PKB/mTORC1/S6 kinase (S6K) pathway, but pathophysiologically high albumin concentrations overactivated mTORC1 and inhibited mTORC2 activity. This control process involved the activation of ERK1/2, which promoted the inhibition of TSC2 and activation of S6K. Furthermore, S6K was crucial to promoting the over activation of mTORC1 and inhibition of mTORC2. Megalin expression at the luminal membrane is reduced by high concentrations of albumin. In addition, knockdown of megalin mimicked all the effects of pathophysiologic albumin concentrations, which disrupt normal signal transduction pathways and lead to an overactivation of mTORC1 and inhibition of mTORC2. These data provide new perspectives for understanding the molecular mechanisms behind the effects of albumin on the progression of renal disease.

Polycystin-1 negatively regulates Polycystin-2 expression via the aggresome/autophagosome pathway.

Mutations of the PKD1 and PKD2 genes, encoding polycystin-1 (PC1) and polycystin-2 (PC2), respectively, lead to autosomal dominant polycystic kidney disease. Interestingly, up-regulation or down-regulation of PKD1 or PKD2 leads to polycystic kidney disease in animal models, but their interrelations are not completely understood. We show here that full-length PC1 that interacts with PC2 via a C-terminal coiled-coil domain regulates PC2 expression in vivo and in vitro by down-regulating PC2 expression in a dose-dependent manner. Expression of the pathogenic mutant R4227X, which lacks the C-terminal coiled-coil domain, failed to down-regulate PC2 expression, suggesting that PC1-PC2 interaction is necessary for PC2 regulation. The proteasome and autophagy are two pathways that control protein degradation. Proteins that are not degraded by proteasomes precipitate in the cytoplasm and are transported via histone deacetylase 6 (HDAC6) toward the aggresomes. We found that HDAC6 binds to PC2 and that expression of full-length PC1 accelerates the transport of the HDAC6-PC2 complex toward aggresomes, whereas expression of the R4227X mutant fails to do so. Aggresomes are engulfed by autophagosomes, which then fuse with the lysosome for degradation; this process is also known as autophagy. We have now shown that PC1 overexpression leads to increased degradation of PC2 via autophagy. Interestingly, PC1 does not activate autophagy generally. Thus, we have now uncovered a new pathway suggesting that when PC1 is expressed, PC2 that is not bound to PC1 is directed to aggresomes and subsequently degraded via autophagy, a control mechanism that may play a role in autosomal dominant polycystic kidney disease pathogenesis.

Overcoming the cystic fibrosis sputum barrier to leading adeno-associated virus gene therapy vectors.

Gene therapy has not yet improved cystic fibrosis (CF) patient lung function in human trials, despite promising preclinical studies. In the human CF lung, inhaled gene vectors must penetrate the viscoelastic secretions coating the airways to reach target cells in the underlying epithelium. We investigated whether CF sputum acts as a barrier to leading adeno-associated virus (AAV) gene vectors, including AAV2, the only serotype tested in CF clinical trials, and AAV1, a leading candidate for future trials. Using multiple particle tracking, we found that sputum strongly impeded diffusion of AAV, regardless of serotype, by adhesive interactions and steric obstruction. Approximately 50% of AAV vectors diffused >1,000-fold more slowly in sputum than in water, with large patient-to-patient variation. We thus tested two strategies to improve AAV diffusion in sputum. We showed that an AAV2 mutant engineered to have reduced heparin binding diffused twice as fast as AAV2 on average, presumably because of reduced adhesion to sputum. We also discovered that the mucolytic N-acetylcysteine could markedly enhance AAV diffusion by altering the sputum microstructure. These studies underscore that sputum is a major barrier to CF gene delivery, and offer strategies for increasing AAV penetration through sputum to improve clinical outcomes.

Transcomplementation by a truncation mutant of cystic fibrosis transmembrane conductance regulator (CFTR) enhances ΔF508 processing through a biomolecular interaction.

We previously showed that a truncation mutant of CFTR missing the first four transmembrane segments of TMD1, Δ264 CFTR, binds to key elements in the ER quality control mechanism to increase the amounts of the mature C band of both wt and ΔF508 CFTR through transcomplementation. Here, we created a new construct, Δ27-264 CFTR. Even though Δ27-264 CFTR is rapidly degraded in the proteasome, steady state protein can be detected by Western blot. Δ27-264 CFTR can also increase the amounts of the mature C band of both wt and ΔF508 CFTR through transcomplementation. Electrophysiology experiments show that Δ27-264 CFTR can restore chloride channel currents. Further experiments with the conduction mutant S341A show conclusively that currents are indeed generated by rescued channel function of ΔF508 CFTR. Immunoprecipitation studies show that Δ27-264 binds to ΔF508-CFTR, suggesting a bimolecular interaction. Thus the adeno-associated viral vector, rAAV-Δ27-264 CFTR, is a highly promising CF gene therapy vector, because it increases the amount of mature band C protein both from wt and ΔF508 CFTR, and rescues channel activity of ΔF508 CFTR.

Future directions in early cystic fibrosis lung disease research: an NHLBI workshop report.

Since the 1989 discovery that mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF), there has been substantial progress toward understanding the molecular basis for CF lung disease, leading to the discovery and development of new therapeutic approaches. However, the earliest impact of the loss of CFTR function on airway physiology and structure and its relationship to initial infection and inflammation are poorly understood. Universal newborn screening for CF in the United States represents an unprecedented opportunity for investigating CF clinical manifestations very early in life. Recently developed animal models with pulmonary phenotypic manifestations also provide a window into the early consequences of this genetic disorder. For these reasons, the National Heart, Lung, and Blood Institute (NHLBI) convened a working group of extramural experts, entitled "Future Research Directions in Early CF Lung Disease" on September 21-22, 2010, to identify future research directions of great promise in CF. The priority areas identified included (1) exploring pathogenic mechanisms of early CF lung disease; (2) leveraging newborn screening to elucidate the natural history of early lung disease; (3) developing a spectrum of biomarkers of early lung disease that reflects CF pathophysiology, clinical outcome, and response to treatment; (4) exploring the role of genetics/genomics (e.g., modifier genes, gene-environmental interactions, and epigenetics) in early CF pathogenesis; (5) defining early microbiological events in CF lung disease; and (6) elucidating the initial airway inflammatory, remodeling, and repair mechanisms in CF lung disease.

Drosophila TRPA1 channel mediates chemical avoidance in gustatory receptor neurons.

Mammalian sweet, bitter, and umami taste is mediated by a single transduction pathway that includes a phospholipase C (PLC)beta and one cation channel, TRPM5. However, in insects such as the fruit fly, Drosophila melanogaster, it is unclear whether different tastants, such as bitter compounds, are sensed in gustatory receptor neurons (GRNs) through one or multiple ion channels, as the cation channels required in insect GRNs are unknown. Here, we set out to explore additional sensory roles for the Drosophila TRPA1 channel, which was known to function in thermosensation. We found that TRPA1 was expressed in GRNs that respond to aversive compounds. Elimination of TRPA1 had no impact on the responses to nearly all bitter compounds tested, including caffeine, quinine, and strychnine. Rather, we found that TRPA1 was required in a subset of avoidance GRNs for the behavioral and electrophysiological responses to aristolochic acid. TRPA1 did not appear to be activated or inhibited directly by aristolochic acid. We found that elimination of the same PLC that leads to activation of TRPA1 in thermosensory neurons was also required in the TRPA1-expressing GRNs for avoiding aristolochic acid. Given that mammalian TRPA1 is required for responding to noxious chemicals, many of which cause pain and injury, our analysis underscores the evolutionarily conserved role for TRPA1 channels in chemical avoidance.

Transduction of Ferret Surface and Basal Cells of Airways, Lung, Liver, and Pancreas via Intratracheal or Intravenous Delivery of Adeno-Associated Virus 1 or 6.

Cystic fibrosis (CF) is potentially treatable by gene therapy. Since the identification of the CF gene, preclinical and clinical trials have concentrated on achieving effective gene therapy targeting the lung. However, the lung has proven to be a formidable barrier to successful gene therapy especially for CF, and many clinical trials failed to achieve efficacy. Recent advances in vector design and adeno-associated virus (AAV) serotypes have increased the chances of success. Given that CF is a multi-organ disease, the goal of this study was to test whether a gene therapy approach involving AAV1 or AAV6 vector delivery via the systemic circulation would at the same time overcome the barrier of lung delivery and transduce organs commonly affected by CF. To accomplish this, we sprayed AAV1 containing green fluorescent protein (GFP) into the trachea or injected it intravenously (IV). We also tested AAV6 injected IV. No adverse events were noted. Ferrets were necropsied 30 days after vector delivery. AAV1 or AAV6 vector genomes, messenger RNA (mRNA) expression, and GFP were detected in all the tracheal and lung samples from the treated animals, whether AAV1 was sprayed into the trachea or injected IV or AAV6 was injected IV. Importantly, both surface epithelial and basal cells of the trachea and lung airways were successfully transduced, regardless of which route of delivery or vector serotype used for transduction. We detected also AAV1 and AAV6 vector genomes, mRNA expression, and GFP in the livers and pancreases, particularly in the acinar cells of the pancreatic duct. These data suggest that gene transfer is attainable in the airways, liver, and pancreas using either serotype, AAV1 or AAV6. Given that these same organs are affected in CF, systemic delivery of AAV may be the preferred route of delivery for a gene therapy for CF.