The CFTR Corrector, VX-809 (Lumacaftor), Rescues ABCA4 Trafficking Mutants: a Potential Treatment for Stargardt Disease.

BACKGROUND/AIMS: Mutations in ABCA4 cause Stargardt macular degeneration, which invariably ends in legal blindness. We studied two common mutants, A1038V (in NBD1) and G1961E (in NBD2), with the purpose of exploring how they interact with the cell's quality control mechanism. The study was designed to determine how these mutants can be rescued. METHODS: We expressed wt and mutant ABCA4 in HEK293 cells and studied the effect of the mutations on trafficking and processing and the ability of correctors to rescue them. We used a combination of western blotting, confocal microscopy and surface biotinylation coupled with pulldown of plasma membrane proteins. RESULTS: G1961E is sensitive to inhibitors of the aggresome, tubacin and the lysosome, bafilomycin A. Both mutants cause a reduction in heat shock protein, Hsp27. Incubation of HEK293 cells expressing the mutants with VX-809, an FDA approved drug for the treatment of cystic fibrosis, increased the levels of A1038V and G1961E by 2- to 3-fold. Importantly, VX-809 increased the levels of both mutants at the plasma membrane suggesting that trafficking had been restored. Transfecting additional Hsp27 to the cells also increased the steady state levels of both mutants. However, in combination with VX-809 the addition of Hsp27 caused a dramatic increase in the protein expression particularly in the G1961 mutant which increased approximately 5-fold. CONCLUSION: Our results provide a new mechanism for the rescue of ABCA4 trafficking mutants based on the restoration of Hsp27. Our results provide a pathway for the treatment of Stargardt disease.

Role of calcium in adult onset polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in genes encoding the polycystin (PC) 1 and 2 proteins. The goal of this study was to determine the role of calcium in regulating cyst growth. Stromal interaction molecule 1 (STIM1) protein expression was 15-fold higher in PC1-null proximal tubule cells (PN) than in heterozygote (PH) controls and 2-fold higher in an inducible, PC1 knockout, mouse model of ADPKD compared to a non-cystic match control. IP3 receptor protein expression was also higher in the cystic mice. Knocking down STIM1 with siRNA reduced cyst growth and lowered cAMP levels in PN cells. Fura2 measurements of intracellular Ca2+ showed higher levels of intracellular Ca2+, SOCE and thaspigargin-stimulated ER Ca2+ release in PN vs. PH cells. There was a dramatic reduction in thapsigargin-stimulated release of ER Ca2+ following STIM1 silencing or application of 2-APB, consistent with altered ER Ca2+ movement; the protein expression of the Ca2+-dependent adenylyl cyclases (AC) AC3 and AC6 was up- and down-regulated, respectively. Like STIM1 knockdown, application of the calmodulin inhibitor W7 lowered cAMP levels, further indicating that STIM1 regulates AC3 via Ca2+ We conclude that the high levels of STIM1 in ADPKD cells play a role in supporting cyst growth and promoting high cAMP levels and an increased release of Ca2+ from the ER. Thus, our results provide novel therapeutic targets for treating ADPKD.

Rescue of CFTR NBD2 mutants N1303K and S1235R is influenced by the functioning of the autophagosome.

The missing phenylalanine at position 508, located in nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane regulator (CFTR), is the most common cystic fibrosis mutation. Severe disease-causing mutations also occur in NBD2. To provide information on potential therapeutic strategies for mutations in NBD2, we used a combination of biochemical, cell biological and electrophysiological approaches and newly created cell lines to study two disease-causing NBD2 mutants, N1303K and S1235R. We observed that neither was sensitive to E64, a cysteine protease inhibitor. However, further investigation showed that when treated with a combination of correctors, C4 + C18, both mutants also responded to E64. Further exploration to assess aggresome throughput using the autophagy regulator LC3 as a marker showed that, in the absence of correctors, N1303K showed a stalled throughput of LC3-II to the aggresome. The throughput became active again after treatment with the corrector combination C4 + C18. Confocal microscopic studies showed that the N1303K and S1235R mutant proteins both co-localized with LC3, but this co-localization was abolished by the corrector combination and, to a lesser extent, by VX-809. Both the corrector combination and VX-809 increased the CFTR chloride channel function of both mutants. We conclude that correctors have a dual effect, particularly on N1303K: they improve trafficking and function at the plasma membrane and reduce the association with autophagosomes. After treatment with correctors persistent degradation by the autophagosome may limit restoration of function. Thus, mutations in NBD2 of CFTR, in contrast to ΔF508-CFTR, may require additional personalized strategies to rescue them.

A potential strategy for reducing cysts in autosomal dominant polycystic kidney disease with a CFTR corrector.

Autosomal dominant polycystic kidney disease (ADPKD) is associated with progressive enlargement of cysts, leading to a decline in function and renal failure that cannot be prevented by current treatments. Mutations in pkd1 and pkd2, encoding the polycystin 1 and 2 proteins, induce growth-related pathways, including heat shock proteins, as occurs in some cancers, raising the prospect that pharmacological interventions that target these pathways might alleviate or prevent ADPKD. Here, we demonstrate a role for VX-809, a corrector of cystic fibrosis transmembrane conductance regulator (CFTR), conventionally used to manage cystic fibrosis in reducing renal cyst growth. VX-809 reduced cyst growth in Pkd1-knockout mice and in proximal, tubule-derived, cultured Pkd1 knockout cells. VX-809 reduced both basal and forskolin-activated cAMP levels and also decreased the expression of the adenylyl cyclase AC3 but not of AC6. VX-809 also decreased resting levels of intracellular Ca2+ but did not affect ATP-stimulated Ca2+ release. Notably, VX-809 dramatically decreased thapsigargin-induced release of Ca2+ from the endoplasmic reticulum (ER). VX-809 also reduced the levels of heat shock proteins Hsp27, Hsp70, and Hsp90 in mice cystic kidneys, consistent with the restoration of cellular proteostasis. Moreover, VX-809 strongly decreased an ER stress marker, the GADD153 protein, and cell proliferation but had only a small effect on apoptosis. Given that administration of VX-809 is safe, this drug potentially offers a new way to treat patients with ADPKD.

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.

An inhibitor of histone deacetylase 6 activity, ACY-1215, reduces cAMP and cyst growth in polycystic kidney disease.

Adult-onset autosomal-dominant polycystic kidney disease (ADPKD) is caused by mutations in either the PKD1 or PKD2 gene, leading to malfunction of their gene products, polycystin 1 or 2. Histone deacetylase 6 (HDAC6) expression and activity are increased in PKD1 mutant renal epithelial cells. Here we studied the effect of ACY-1215, a specific HDAC6 inhibitor, on cyst growth in ADPKD. Treatment with ACY-1215 slowed cyst growth in a mouse model of ADPKD that forms massive cysts within 3 wk after knockout of polycystin 1 function. It also prevented cyst formation in MDCK.2 cells, an in vitro model of cystogenesis, and in an ADPKD cell line derived from the proximal tubules from a pkd1-/-.mouse (PN cells). In PN cells ACY-1215 also reduced the size of already established cysts. We found that ACY-1215 lowered cAMP levels and protein expression of adenylyl cyclase 6. Our results suggest that HDAC6 could potentially serve as a therapeutic target in ADPKD.

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.

Eag Domains Regulate LQT Mutant hERG Channels in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Human Ether á go-go Related Gene potassium channels form the rapid component of the delayed-rectifier (IKr) current in the heart. The N-terminal 'eag' domain, which is composed of a Per-Arnt-Sim (PAS) domain and a short PAS-cap region, is a critical regulator of hERG channel function. In previous studies, we showed that isolated eag (i-eag) domains rescued the dysfunction of long QT type-2 associated mutant hERG R56Q channels, by substituting for defective eag domains, when the channels were expressed in Xenopus oocytes or HEK 293 cells.Here, our goal was to determine whether the rescue of hERG R56Q channels by i-eag domains could be translated into the environment of cardiac myocytes. We expressed hERG R56Q channels in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and measured electrical properties of the cells with whole-cell patch-clamp recordings. We found that, like in non-myocyte cells, hERG R56Q had defective, fast closing (deactivation) kinetics when expressed in hiPSC-CMs. We report here that i-eag domains slowed the deactivation kinetics of hERG R56Q channels in hiPSC-CMs. hERG R56Q channels prolonged the AP of hiPSCs, and the AP was shortened by co-expression of i-eag domains and hERG R56Q channels. We measured robust Förster Resonance Energy Transfer (FRET) between i-eag domains tagged with Cyan fluorescent protein (CFP) and hERG R56Q channels tagged with Citrine fluorescent proteins (Citrine), indicating their close proximity at the cell membrane in live iPSC-CMs. Together, functional regulation and FRET spectroscopy measurements indicated that i-eag domains interacted directly with hERG R56Q channels in hiPSC-CMs. These results mean that the regulatory role of i-eag domains is conserved in the cellular environment of human cardiomyocytes, indicating that i-eag domains may be useful as a biological therapeutic.

Direct interaction of eag domains and cyclic nucleotide-binding homology domains regulate deactivation gating in hERG channels.

Human ether-á-go-go (eag)-related gene (hERG) potassium channels play a critical role in cardiac repolarization and are characterized by unusually slow closing (deactivation) kinetics. The N-terminal "eag" domain and a C-terminal C-linker/cyclic nucleotide-binding homology domain (CNBHD) are required for regulation of slow deactivation. The region between the S4 and S5 transmembrane domains (S4-S5 linker) is also implicated in this process, but the mechanism for regulation of slow deactivation is unclear. Here, using an eag domain-deleted channel (hERG Δeag) fused to Citrine fluorescent protein, we found that most channels bearing individual alanine mutations in the S4-S5 linker were directly regulated by recombinant eag domains fused to a cyan fluorescent protein (N-eag-CFP) and had robust Förster resonance energy transfer (FRET). Additionally, a channel bearing a group of eight alanine residues in the S4-S5 linker was not measurably regulated by N-eag-CFP domains, but robust FRET was measured. These findings demonstrate that the eag domain associated with all of the S4-S5 linker mutant channels. In contrast, channels that also lacked the CNBHD (hERG Δeag ΔCNBHD-Citrine) were not measurably regulated by N-eag-CFP nor was FRET detected, suggesting that the C-linker/CNBHD was required for eag domains to directly associate with the channel. In a FRET hybridization assay, N-eag-CFP had robust FRET with a C-linker/CNBHD-Citrine, suggesting a direct and specific interaction between the eag domain and the C-linker/CNBHD. Lastly, coexpression of a hERG subunit lacking the CNBHD and the distal C-terminal region (hERG ΔpCT-Citrine) with hERG Δeag-CFP subunits had FRET and partial restoration of slow deactivation. Collectively, these findings reveal that the C-linker/CNBHD, but not the S4-S5 linker, was necessary for the eag domain to associate with the channel, that the eag domain and the C-linker/CNBHD were sufficient for a direct interaction, and that an intersubunit interaction between the eag domain and the C-linker/CNBHD regulated slow deactivation in hERG channels at the plasma membrane.

Effect of daurisoline on HERG channel electrophysiological function and protein expression.

Daurisoline (1) is a bis-benzylisoquinoline alkaloid isolated from the rhizomes of Menispermum dauricum. The antiarrhythmic effect of 1 has been demonstrated in different experimental animals. In previous studies, daurisoline (1) prolonged action potential duration (APD) in a normal use-dependent manner. However, the electrophysiological mechanisms for 1-induced prolongation of APD have not been documented. In the present study, the direct effect of 1 was investigated on the hERG current and the expression of mRNA and protein in human embryonic kidney 293 (HEK293) cells stably expressing the hERG channel. It was shown that 1 inhibits hERG current in a concentration- and voltage-dependent manner. In the presence of 10 µM 1, steady-state inactivation of V(1/2) was shifted negatively by 15.9 mV, and 1 accelerated the onset of inactivation. Blockade of hERG channels was dependent on channel opening. The expression and function of hERG were unchanged by 1 at 1 and 10 µM, while hERG expression and the hERG current were decreased significantly by 1 at 30 µM. These results indicate that 1, at concentrations below 30 µM, exerts a blocking effect on hERG, but does not affect the expression and function of the hERG channel. This may explain the relatively lower risk of long QT syndrome after long-term usage.

Neuroprotective effect of dauricine after transient middle cerebral artery occlusion in rats: involvement of Bcl-2 family proteins.

Previous studies have shown that the bisbenzyl isoquinoline alkaloid dauricine can protect the brain against ischemic damage. We investigated here whether dauricine could inhibit neuronal apoptosis and modulate Bcl-2 family protein levels in a rat model of transient focal cerebral ischemia. Male Sprague-Dawley rats underwent a 60 min temporary occlusion of the middle cerebral artery (MCAO). Two doses of dauricine (5 and 10 mg/kg as low and high dose respectively) were administered intraperitoneally at 1 hour after MCAO. After neurological deficits were assessed at 3 hours and 24 hours of reperfusion, rats were killed and brain samples were collected. Apoptotic changes were evaluated by TUNEL method. The immunohistochemistry and Western blot were used to assess the protein expressions of Bcl-2 and Bax. RT-PCR was used to determine Bcl-2 and Bax mRNA expressions. Dauricine (5 and 10 mg/kg) treatment improved neurological deficits, diminished DNA fragmentation, increased Bcl-2 expression and reduced Bax expression in the penumbra. The infarct-reducing effects of dauricine may be due, in part, to the inhibition of apoptotic cell death via modulation Bcl-2 family protein in the penumbra.

Daurisoline suppressed early afterdepolarizations and inhibited L-type calcium current.

Our previous studies have shown that daurisoline (DS) exerted antiarrhythmic effects on various experimental arrhythmias. In this study, the effects of DS on early afterdepolarizations (EADs) and its possible mechanisms have been investigated. Cardiac hypertrophy was induced in rabbits by coarctating the abdominal aorta. The effects of DS on action potential duration (APD) and the incidences of EADs were studied in hypertrophied papillary muscles of rabbits in the conditions of low external K(+) concentration ([K(+)]o) and dofetilide (dof) by using standard microelectrode technique. The whole-cell patch clamp was used to record the L-type calcium current (I(Ca-L)) in isolated left ventricular cells of rabbits. The results showed that in hypertrophied papillary muscles of rabbits with low [K(+)]o ([K(+)]o = 2.7 mM), 1 microM dof prolonged APD(50) and APD(90) markedly and the incidence of EADs was 66.7% (4/6, p < 0.01); when 15 microM DS was applied, the incidence of EADs was 0% (0/4, p < 0.01) and the prolonged APD was shortened (p < 0.01). In a single myocyte, DS could also inhibit EADs induced by dof, low [K(+)]o and low external Mg(2+) concentration ([Mg(2+)]o) ([Mg(2+)](o) = 0.5 mM). DS could decrease the triangulation. In a single myocyte, DS could make the I-V curve upward, shift the steady-state activation curves to the right and the steady-state inactivation curves to the left and prolong the tau value of recovery curve obviously. These results suggested that DS could inhibit EADs which may be associated with its blockade effects on I(Ca-L).

Inhibitory effects of dauricine on early afterdepolarizations and L-type calcium current.

We have previously reported that dauricine exerted antiarrhythmic effects on various experimental arrhythmias. To further clarify its mechanism, the effects of dauricine on action potential duration (APD), early afterdepolarizations (EADs), triangulation, which is defined as the repolarization time from APD at 30% level (APD30) to APD at 90% level (APD90), and L-type calcium current (I(Ca-L)) were studied using standard microelectrode techniques on rabbit papillary muscles and whole-cell patch clamp techniques on single myocytes isolated from rabbits by enzymatic digestion, respectively. Cardiac hypertrophy was induced by coarctating the abdominal aorta of rabbits. The results showed that in papillary muscles of hypertrophied rabbits, 1 micromol/L dofetilide, a selective IKr blocker, prolonged APD50 and APD90 and induced EADs (4/6, p < 0.01) with hypokalemia ([K+]o = 2.7 mmol/L). Dauricine inhibited EADs (p < 0.01) and shortened the prolonged APD (p < 0.01). In single myocytes, dauricine also inhibited EADs induced by dofetilide, hypokalemia, and hypomagnesaemia. Dauricine decreased the triangulation and reduced the peak amplitude of I(Ca-L) at all potentials tested. Dauricine shifted the steady-state activation curves to the right and steady-state inactivation curves to the left and prolonged the tau value of the recovery curve. These results suggest that dauricine inhibits EADs and this effect may be associated with its blockade of I(Ca-L).

Inhibitory effect of dauricine on inflammatory process following focal cerebral ischemia/reperfusion in rats.

Our previous experimental studies showed that dauricine could protect the brain from ischemic damage, but the underlying mechanisms were unknown. In this study, we investigated the effect of dauricine on the changes of the inflammation process induced by ischemia/reperfusion (I/R). After I/R, the enzyme activity of MPO, the expression of ICAM-1 and the transcription of IL-1beta and TNF-alpha mRNA were all significantly increased (p < 0.01). And after treatment with dauricine, they were all significantly reduced compared to the vehicle-treated I/R group (p < 0.05 or p < 0.01). These results suggest that dauricin attenuates the inflammation process induced by I/R. The neuroprotective effect of dauricine may partly due to the inhibition acute inflammation induced by I/R.