CD4 T cells mediate brain inflammation and neurodegeneration in a mouse model of Parkinson's disease.

α-Synuclein, a key pathological component of Parkinson's disease, has been implicated in the activation of the innate and adaptive immune system. This immune activation includes microgliosis, increased inflammatory cytokines, and the infiltration of T cells into the CNS. More recently, peripherally circulating CD4 and CD8 T cells derived from individuals with Parkinson's disease have been shown to produce Th1/Th2 cytokines in response to α-synuclein, suggesting there may be a chronic memory T cell response present in Parkinson's disease. To understand the potential effects of these α-syn associated T cell responses we used an α-synuclein overexpression mouse model, T cell-deficient mice, and a combination of immunohistochemistry and flow cytometry. In this study, we found that α-synuclein overexpression in the midbrain of mice leads to the upregulation of the major histocompatibility complex II (MHCII) protein on CNS myeloid cells as well as the infiltration of IFNγ producing CD4 and CD8 T cells into the CNS. Interestingly, genetic deletion of TCRß or CD4, as well as the use of the immunosuppressive drug fingolimod, were able to reduce the CNS myeloid MHCII response to α-synuclein. Furthermore, we observed that CD4-deficient mice were protected from the dopaminergic cell loss observed due to α-syn overexpression. These results suggest that T cell responses associated with α-synuclein pathology may be damaging to key areas of the CNS in Parkinson's disease and that targeting these T cell responses could be an avenue for disease modifying treatments.

T cell infiltration in both human multiple system atrophy and a novel mouse model of the disease.

Multiple system atrophy (MSA) is a progressive neurodegenerative disorder characterized by abnormal accumulation of alpha-synuclein (α-syn) in oligodendrocytes accompanied by inflammation, demyelination, and subsequent synapse and neuronal loss. Little is known about the mechanisms of neurodegeneration in MSA. However, recent work has highlighted the important role of the immune system to the pathophysiology of other synuclein-related diseases such as Parkinson's disease. In this study, we investigated postmortem brain tissue from MSA patients and control subjects for evidence of immune activation in the brain. We found a significant increase of HLA-DR+ microglia in the putamen and substantia nigra of MSA patient tissue compared to controls, as well as significant increases in CD3+, CD4+, and CD8+ T cells in these same brain regions. To model MSA in vivo, we utilized a viral vector that selectively overexpresses α-syn in oligodendrocytes (Olig001-SYN) with > 95% tropism in the dorsal striatum of mice, resulting in demyelination and neuroinflammation similar to that observed in human MSA. Oligodendrocyte transduction with this vector resulted in a robust inflammatory response, which included increased MHCII expression on central nervous system (CNS) resident microglia, and infiltration of pro-inflammatory monocytes into the CNS. We also observed robust infiltration of CD4 T cells into the CNS and antigen-experienced CD4 T cells in the draining cervical lymph nodes. Importantly, genetic deletion of TCR-ß or CD4 T cells attenuated α-syn-induced inflammation and demyelination in vivo. These results suggest that T cell priming and infiltration into the CNS are key mechanisms of disease pathogenesis in MSA, and therapeutics targeting T cells may be disease modifying.

Targeting of the class II transactivator attenuates inflammation and neurodegeneration in an alpha-synuclein model of Parkinson's disease.

BACKGROUND: Parkinson's disease (PD) is characterized by intracellular alpha-synuclein (α-syn) inclusions, progressive death of dopaminergic neurons in the substantia nigra pars compacta (SNpc), and activation of the innate and adaptive immune systems. Disruption of immune signaling between the central nervous system (CNS) and periphery, such as through targeting the chemokine receptor type 2 (CCR2) or the major histocompatibility complex II (MHCII), is neuroprotective in rodent models of PD, suggesting a key role for innate and adaptive immunity in disease progression. The purpose of this study was to investigate whether genetic knockout or RNA silencing of the class II transactivator (CIITA), a transcriptional co-activator required for MHCII induction, is effective in reducing the neuroinflammation and neurodegeneration observed in an α-syn mouse model of PD. METHODS: In vitro, we utilized microglia cultures from WT or CIITA -/- mice treated with α-syn fibrils to investigate inflammatory iNOS expression and antigen processing via immunocytochemistry (ICC). In vivo, an adeno-associated virus (AAV) was used to overexpress α-syn in WT and CIITA -/- mice as a model for PD. Concurrently with AAV-mediated overexpression of α-syn, WT mice received CIITA-targeted shRNAs packaged in lentiviral constructs. Immunohistochemistry and flow cytometry were used to assess inflammation and peripheral cell infiltration at 4 weeks post transduction, and unbiased stereology was used 6 months post transduction to assess neurodegeneration. RESULTS: Using ICC and DQ-ovalbumin, we show that CIITA -/- microglial cultures failed to upregulate iNOS and MHCII expression, and had decreased antigen processing in response to α-syn fibrils when compared to WT microglia. In vivo, global knock-out of CIITA as well as local knockdown using lentiviral shRNAs targeting CIITA attenuated MHCII expression, peripheral immune cell infiltration, and α-syn-induced neurodegeneration. CONCLUSION: Our data provide evidence that CIITA is required for α-syn-induced MHCII induction and subsequent infiltration of peripheral immune cells in an α-syn mouse model of PD. Additionally, we demonstrate that CIITA in the CNS drives neuroinflammation and neurodegeneration. These data provide further support that the disruption or modulation of antigen processing and presentation via CIITA is a promising target for therapeutic development in preclinical animal models of PD.

α-Synuclein fibrils recruit peripheral immune cells in the rat brain prior to neurodegeneration.

Genetic variation in a major histocompatibility complex II (MHCII)-encoding gene (HLA-DR) increases risk for Parkinson disease (PD), and the accumulation of MHCII-expressing immune cells in the brain correlates with α-synuclein inclusions. However, the timing of MHCII-cell recruitment with respect to ongoing neurodegeneration, and the types of cells that express MHCII in the PD brain, has been difficult to understand. Recent studies show that the injection of short α-synuclein fibrils into the rat substantia nigra pars compacta (SNpc) induces progressive inclusion formation in SNpc neurons that eventually spread to spiny projection neurons in the striatum. Herein, we find that α-synuclein fibrils rapidly provoke a persistent MHCII response in the brain. In contrast, equivalent amounts of monomeric α-synuclein fail to induce MHCII or persistent microglial activation, consistent with our results in primary microglia. Flow cytometry and immunohistochemical analyses reveal that MHCII-expressing cells are composed of both resident microglia as well as cells from the periphery that include monocytes, macrophages, and lymphocytes. Over time, α-Synuclein fibril exposures in the SNpc causes both axon loss as well as monocyte recruitment in the striatum. While these monocytes in the striatum initially lack MHCII expression, α-synuclein inclusions later form in nearby spiny projection neurons and MHCII expression becomes robust. In summary, in the rat α-synuclein fibril model, peripheral immune cell recruitment occurs prior to neurodegeneration and microglia, monocytes and macrophages all contribute to MHCII expression.

The Metabotropic Glutamate Receptor 4 Positive Allosteric Modulator ADX88178 Inhibits Inflammatory Responses in Primary Microglia.

While the specific trigger of Parkinson Disease (PD) in most patients is unknown, considerable evidence suggests that the neuroinflammatory response makes an essential contribution to the neurodegenerative process. Drugs targeting metabotropic glutamate receptors (mGlu receptors), 7 Transmembrane (7TM) spanning/G protein coupled receptors that bind glutamate, are emerging as therapeutic targets for PD and may have anti-inflammatory properties. ADX88178 is novel potent, selective, and brain-penetrant positive allosteric modulator of the mGlu4 which is under evaluation for treatment of PD and other neurological disorders. We used microglia cultured from mouse brain to determine if ADX88178 had direct effects on the inflammatory responses of these cells. We studied both microglia from wild type and Grm4 knock out mice. We found that activation of mGlu4 with ADX88178 attenuated LPS-induced inflammation in primary microglia, leading to a decrease in the expression of TNFα, MHCII, and iNOS, markers of pro-inflammatory responses. These effects were absent in microglia from mice lacking mGlu4. These results demonstrate a cell-autonomous anti-inflammatory effect of ADX88178 mediated mGlu4 activation on microglia, and suggest that this drug or similar activators or potentiators of mGlu4 may have disease-modifying as well as symptomatic effects in PD and other brain disorders with an inflammatory component.

Upregulation of autophagy decreases chlorine-induced mitochondrial injury and lung inflammation.

The mechanisms of toxicity during exposure of the airways to chlorinated biomolecules generated during the course of inflammation and to chlorine (Cl2) gas are poorly understood. We hypothesized that lung epithelial cell mitochondria are damaged by Cl2 exposure and activation of autophagy mitigates this injury. To address this, NCI-H441 (human lung adenocarcinoma epithelial) cells were exposed to Cl2 (100 ppm/15 min) and bioenergetics were assessed. One hour after Cl2, cellular bioenergetic function and mitochondrial membrane potential were decreased. These changes were associated with increased MitoSOX signal, and treatment with the mitochondrial redox modulator MitoQ attenuated these bioenergetic defects. At 6h postexposure, there was significant increase in autophagy, which was associated with an improvement of mitochondrial function. Pretreatment of H441 cells with trehalose (an autophagy activator) improved bioenergetic function, whereas 3-methyladenine (an autophagy inhibitor) resulted in increased bioenergetic dysfunction 1h after Cl2 exposure. These data indicate that Cl2 induces bioenergetic dysfunction, and autophagy plays a protective role in vitro. Addition of trehalose (2 vol%) to the drinking water of C57BL/6 mice for 6 weeks, but not 1 week, before Cl2 (400 ppm/30 min) decreased white blood cells in the bronchoalveolar lavage fluid at 6h after Cl2 by 70%. Acute administration of trehalose delivered through inhalation 24 and 1h before the exposure decreased alveolar permeability but not cell infiltration. These data indicate that Cl2 induces bioenergetic dysfunction associated with lung inflammation and suggests that autophagy plays a protective role.

Postexposure aerosolized heparin reduces lung injury in chlorine-exposed mice.

Chlorine (Cl2) is a highly reactive oxidant gas that, when inhaled, may cause acute lung injury culminating in death from respiratory failure. In this study, we tested the hypothesis that exposure of mice to Cl2 causes intra-alveolar and systemic activation of the coagulation cascade that plays an important role in development of lung injury. C57Bl/6 mice were exposed to Cl2 (400 for 30 min or 600 ppm for 45 min) in environmental chambers and then returned to room air for 1 or 6 h. Native coagulation (NATEM) parameters such as blood clotting time and clot formation time were measured in whole blood by the viscoelastic technique. D-dimers and thrombin-anti-thrombin complexes were measured in both plasma and bronchoalveolar lavage fluid (BALF) by ELISA. Our results indicate that mice exposed to Cl2 gas had significantly increased clotting time, clot formation time, and D-dimers compared with controls. The thrombin-anti-thrombin complexes were also increased in the BALF of Cl2 exposed animals. To test whether increased coagulation contributed to the development of acute lung injury, mice exposed to Cl2 and returned to room air were treated with aerosolized heparin or vehicle for 20 min. Aerosolized heparin significantly reduced protein levels and the number of inflammatory cells in the BALF at 6 h postexposure. These findings highlight the importance of coagulation abnormities in the development of Cl2-induced lung injury.

Inter-α-inhibitor blocks epithelial sodium channel activation and decreases nasal potential differences in ΔF508 mice.

Increased activity of lung epithelial sodium channels (ENaCs) contributes to the pathophysiology of cystic fibrosis (CF) by increasing the rate of epithelial lining fluid reabsorption. Inter-α-inhibitor (IαI), a serum protease inhibitor, may decrease ENaC activity by preventing its cleavage by serine proteases. High concentrations of IαI were detected in the bronchoalveolar lavage fluid (BALF) of children with CF and lower airway diseases. IαI decreased amiloride-sensitive (IENaC) but not cAMP-activated Cl(-) currents across confluent monolayers of rat ATII, and mouse nasal epithelial cells grew in primary culture by 45 and 25%, respectively. Changes in IENaC by IαI in ATII cells were accompanied by increased levels of uncleaved (immature) surface α-ENaC. IαI increased airway surface liquid depth overlying murine nasal epithelial cells to the same extent as amiloride, consistent with ENaC inhibition. Incubation of lung slices from C57BL/6, those lacking phenylalanine at position 508 (∆F508), or CF transmembrane conductance regulator knockout mice with IαI for 3 hours decreased the open probability of their ENaC channels by 50%. ∆F508 mice had considerably higher levels the amiloride-sensitive fractions of ENaC nasal potential difference (ENaC-NPD) than wild-type littermates and only background levels of IαI in their BALF. A single intranasal instillation of IαI decreased their ENaC-NPD 24 hours later by 25%. In conclusion, we show that IαI is present in the BALF of children with CF, is an effective inhibitor of ENaC proteolysis, and decreases ENaC activity in lung epithelial cells of ∆F508 mice.

Influenza matrix protein 2 alters CFTR expression and function through its ion channel activity.

The human cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-activated chloride (Cl(-)) channel in the lung epithelium that helps regulate the thickness and composition of the lung epithelial lining fluid. We investigated whether influenza M2 protein, a pH-activated proton (H(+)) channel that traffics to the plasma membrane of infected cells, altered CFTR expression and function. M2 decreased CFTR activity in 1) Xenopus oocytes injected with human CFTR, 2) epithelial cells (HEK-293) stably transfected with CFTR, and 3) human bronchial epithelial cells (16HBE14o-) expressing native CFTR. This inhibition was partially reversed by an inhibitor of the ubiquitin-activating enzyme E1. Next we investigated whether the M2 inhibition of CFTR activity was due to an increase of secretory organelle pH by M2. Incubation of Xenopus oocytes expressing CFTR with ammonium chloride or concanamycin A, two agents that alkalinize the secretory pathway, inhibited CFTR activity in a dose-dependent manner. Treatment of M2- and CFTR-expressing oocytes with the M2 ion channel inhibitor amantadine prevented the loss in CFTR expression and activity; in addition, M2 mutants, lacking the ability to transport H(+), did not alter CFTR activity in Xenopus oocytes and HEK cells. Expression of an M2 mutant retained in the endoplasmic reticulum also failed to alter CFTR activity. In summary, our data show that M2 decreases CFTR activity by increasing secretory organelle pH, which targets CFTR for destruction by the ubiquitin system. Alteration of CFTR activity has important consequences for fluid regulation and may potentially modify the immune response to viral infection.

Nano-TiO2 particles impair adhesion of airway epithelial cells to fibronectin.

Titanium dioxide engineered nanoparticles (nano-TiO(2)) are widely used in the manufacturing of a number of products. Due to their size (<100 nm), when inhaled they may be deposited in the distal lung regions and damage Clara cells. We investigated the mechanisms by which short-term (1-h) incubation of human airway Clara-like (H441) cells to nano-TiO(2) (6 nm in diameter) alters the ability of H441 cells to adhere to extracellular matrix. Our results show that 1h post-incubation, there was a 3-fold increase of extracellular H(2)O(2), increased intracellular oxidative stress as demonstrated by 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) oxidation, and a 5-fold increase of phosphor-ERK1/2 as measured by Western blotting. These changes were accompanied by a 25% decrease of H441 adherence to fibronectin (p<0.05 compared to vehicle incubated H441 cells). Pretreatment with the ERK1/2 inhibitor U0126 for 3h, partially prevented this effect. In conclusion, short-term exposure of H441 cells to nano-TiO(2) appears to reduce adherence to fibronectin due to oxidative stress and activation of ERK1/2.

Comparison of ribavirin and oseltamivir in reducing mortality and lung injury in mice infected with mouse adapted A/California/04/2009 (H1N1).

AIM: To compare the efficacy of ribavirin and oseltamivir in reducing mortality, lung injury and cytokine response profile in pandemic influenza H1N1 (2009) infection. MAIN METHODS: We assessed survival, weight loss, lung viral load (by RT-PCR), lung injury (by protein content in bronchoalveolar lavage), and inflammation (cell counts, differentials and cytokines in the bronchoalveolar lavage) in BALB/c mice after infection with mouse-adapted pandemic influenza strain A/California/04/2009. KEY FINDINGS: Our results indicate that ribavirin (80 mg kg(-1)) and oseltamivir (50 mg kg(-1)) are equally effective in improving survival (100% vs. 0% in water treated controls), while ribavirin proved to be more effective in significantly preventing weight loss. Both drugs diminished the injury of the alveolar-capillary barrier by decreasing the protein detected in the BAL to baseline levels, and they were also equally effective in reduction lung viral loads by 100-fold. Administration of either drug did not decrease the amount of inflammatory infiltrate in the lung, but ribavirin significantly reduced the percentage comprised of lymphocytes. This study shows that these antivirals differentially regulate inflammatory cytokines and chemokines with ribavirin significantly reducing most of the cytokines/chemokines measured. Ribavirin treatment leads to a Th1 cytokine response while oseltamivir leads to a Th2 cytokine response with significant increase in the levels of the anti-inflammatory cytokine IL-10. SIGNIFICANCE: This study reveals new mechanistic insights in the way that ribavirin and oseltamivir exert their antiviral activity and supports the theory that ribavirin could potentially serve as an efficacious therapeutic alternative for oseltamivir resistant pandemic H1N1 strains.

Regulation of alveolar epithelial Na+ channels by ERK1/2 in chlorine-breathing mice.

The mechanisms by which the exposure of mice to Cl(2) decreases vectorial Na(+) transport and fluid clearance across their distal lung spaces have not been elucidated. We examined the biophysical, biochemical, and physiological changes of rodent lung epithelial Na(+) channels (ENaCs) after exposure to Cl(2), and identified the mechanisms involved. We measured amiloride-sensitive short-circuit currents (I(amil)) across isolated alveolar Type II (ATII) cell monolayers and ENaC single-channel properties by patching ATII and ATI cells in situ. α-ENaC, γ-ENaC, total and phosphorylated extracellular signal-related kinase (ERK)1/2, and advanced products of lipid peroxidation in ATII cells were measured by Western blot analysis. Concentrations of reactive intermediates were assessed by electron spin resonance (ESR). Amiloride-sensitive Na(+) channels with conductances of 4.5 and 18 pS were evident in ATI and ATII cells in situ of air-breathing mice. At 1 hour and 24 hours after exposure to Cl(2), the open probabilities of these two channels decreased. This effect was prevented by incubating lung slices with inhibitors of ERK1/2 or of proteasomes and lysosomes. The exposure of ATII cell monolayers to Cl(2) increased concentrations of reactive intermediates, leading to ERK1/2 phosphorylation and decreased I(amil) and α-ENaC concentrations at 1 hour and 24 hours after exposure. The administration of antioxidants to ATII cells before and after exposure to Cl(2) decreased concentrations of reactive intermediates and ERK1/2 activation, which mitigated the decrease in I(amil) and ENaC concentrations. The reactive intermediates formed during and after exposure to Cl(2) activated ERK1/2 in ATII cells in vitro and in vivo, leading to decreased ENaC concentrations and activity.

Enhancement of alveolar epithelial sodium channel activity with decreased cystic fibrosis transmembrane conductance regulator expression in mouse lung.

We sought to establish whether the cystic fibrosis transmembrane conductance regulator (CFTR) regulates the activity of amiloride-sensitive sodium channels (ENaC) in alveolar epithelial cells of wild-type, heterozygous (Cftr(+/-)), knockout (Cftr(-/-)), and ΔF508-expressing mice in situ. RT-PCR studies confirmed the presence of CFTR message in freshly isolated alveolar type II (ATII) cells from wild-type mice. We patched alveolar type I (ATI) and ATII cells in freshly prepared lung slices from these mice and demonstrated the presence of 4-pS ENaC channels with the following basal open probabilities (P(o)): wild-type=0.21 ± 0.015: Cftr(+/-)=0.4 ± 0.03; ΔF508=0.55 ± 0.01; and Cftr(-/-)=and 0.81 ± 0.016 (means ± SE; n ≥ 9). Forskolin (5 µM) or trypsin (2 µM), applied in the pipette solution, increased the P(o) and number of channels in ATII cells of wild-type, Cftr(+/-), and ΔF508, but not in Cftr(-/-) mice, suggesting that the latter were maximally activated. Western blot analysis showed that lungs of all groups of mice had similar levels of α-ENaC; however, lungs of Cftr(+/-) and Cftr(-/-) mice had significantly higher levels of an α-ENaC proteolytic fragment (65 kDa) that is associated with active ENaC channels. Our results indicate that ENaC activity is inversely correlated to predicted CFTR levels and that CFTR heterozygous and homozygous mice have higher levels of proteolytically processed ENaC fragments in their lungs. This is the first demonstration of functional ENaC-CFTR interactions in alveolar epithelial cells in situ.

Ascorbate and deferoxamine administration after chlorine exposure decrease mortality and lung injury in mice.

Chlorine (Cl(2)) gas exposure poses an environmental and occupational hazard that frequently results in acute lung injury. There is no effective treatment. We assessed the efficacy of antioxidants, administered after exposure, in decreasing mortality and lung injury in C57BL/6 mice exposed to 600 ppm of Cl(2) for 45 minutes and returned to room air. Ascorbate and deferoxamine were administered intramuscularly every 12 hours and by nose-only inhalation every 24 hours for 3 days starting after 1 hour after exposure. Control mice were exposed to Cl(2) and treated with vehicle (saline or water). Mortality was reduced fourfold in the treatment group compared with the control group (22 versus 78%; P = 0.007). Surviving animals in the treatment group had significantly lower protein concentrations, cell counts, and epithelial cells in their bronchoalveolar lavage (BAL). Lung tissue ascorbate correlated inversely with BAL protein as well as with the number of neutrophils and epithelial cells. In addition, lipid peroxidation was reduced threefold in the BAL of mice treated with ascorbate and deferoxamine when compared with the control group. Administration of ascorbate and deferoxamine reduces mortality and decreases lung injury through reduction of alveolar-capillary permeability, inflammation, and epithelial sloughing and lipid peroxidation.

Functional stability of rescued delta F508 cystic fibrosis transmembrane conductance regulator in airway epithelial cells.

The most common mutation in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene, Delta F508, results in the production of a misfolded protein that is rapidly degraded. The mutant protein is temperature sensitive, and prior studies indicate that the low-temperature-rescued channel is poorly responsive to physiological stimuli, and is rapidly degraded from the cell surface at 37 degrees C. In the present studies, we tested the effect of a recently characterized pharmacological corrector, 2-(5-chloro-2-methoxy-phenylamino)-4'-methyl-[4,5'bithiazolyl-2'-yl]-phenyl-methanone (corr-4a), on cell surface stability and function of the low-temperature-rescued Delta F508 CFTR. We demonstrate that corr-4a significantly enhanced the protein stability of rescued Delta F508 CFTR for up to 12 hours at 37 degrees C (P < 0.05). Using firefly luciferase-based reporters to investigate the mechanisms by which low temperature and corr-4a enhance rescue, we found that low-temperature treatment inhibited proteasomal function, whereas corr-4a treatment inhibited the E1-E3 ubiquitination pathway. Ussing chamber studies indicated that corr-4a increased the cAMP-mediated Delta F508 CFTR response by 61% at 6 hours (P < 0.05), but not at later time points. However, addition of the CFTR channel activator, 4-methyl-2-(5-phenyl-1H-pyrazol-3-yl)-phenol, significantly augmented cAMP-stimulated currents, revealing that the biochemically detectable cell surface Delta F508 CFTR could be stimulated under the right conditions. Our studies demonstrate that stabilizing rescued Delta F508 CFTR was not sufficient to obtain maximal Delta F508 CFTR function in airway epithelial cells. These results strongly support the idea that maximal correction of Delta F508 CFTR requires a chemical corrector that: (1) promotes folding and exit from the endoplasmic reticulum; (2) enhances surface stability; and (3) improves channel activity.

Activation of the unfolded protein response by deltaF508 CFTR.

Environmental insults and misfolded proteins cause endoplasmic reticulum (ER) stress and activate the unfolded protein response (UPR). The UPR decreases endogenous cystic fibrosis transmembrane conductance regulator (CFTR) mRNA levels and protein maturation efficiency. Herein, we investigated the effects of the folding-deficient deltaF508 CFTR on ER stress induction and UPR activation. For these studies, we developed and characterized stable clones of Calu3deltaF cells that express different levels of endogenous wild-type (WT) and recombinant deltaF508 CFTR. We also present a novel RT-PCR-based assay for differential quantification of wild-type CFTR mRNA in the presence of deltaF508 CFTR message. The assay is based on a TaqMan minor groove binding (MGB) probe that recognizes a specific TTT sequence (encoding phenylalanine at position 508 in human CFTR). The MGB probe is extremely specific and sensitive to changes in WT CFTR message levels. In RNA samples that contain both WT and deltaF508 CFTR mRNAs, measurement of WT CFTR mRNA levels (using the MGB probe) and total CFTR mRNA (using commercial primers) allowed us to calculate deltaF508 CFTR mRNA levels. The results indicate that overexpression of deltaF508 CFTR causes ER stress and activates the UPR. UPR activation precedes a marked decrease in endogenous WT CFTR mRNA expression. Furthermore, polarized airway epithelial cell lines are important tools in cystic fibrosis research, and herein we provide an airway epithelial model to study the biogenesis and function of WT and deltaF508 CFTR expressed within the same cell.

Enhanced cell-surface stability of rescued DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) by pharmacological chaperones.

Misfolded proteins destined for the cell surface are recognized and degraded by the ERAD [ER (endoplasmic reticulum) associated degradation] pathway. TS (temperature-sensitive) mutants at the permissive temperature escape ERAD and reach the cell surface. In this present paper, we examined a TS mutant of the CFTR [CF (cystic fibrosis) transmembrane conductance regulator], CFTR DeltaF508, and analysed its cell-surface trafficking after rescue [rDeltaF508 (rescued DeltaF508) CFTR]. We show that rDeltaF508 CFTR endocytosis is 6-fold more rapid (approximately 30% per 2.5 min) than WT (wild-type, approximately 5% per 2.5 min) CFTR at 37 degrees C in polarized airway epithelial cells (CFBE41o-). We also investigated rDeltaF508 CFTR endocytosis under two further conditions: in culture at the permissive temperature (27 degrees C) and following treatment with pharmacological chaperones. At low temperature, rDeltaF508 CFTR endocytosis slowed to WT rates (20% per 10 min), indicating that the cell-surface trafficking defect of rDeltaF508 CFTR is TS. Furthermore, rDeltaF508 CFTR is stabilized at the lower temperature; its half-life increases from <2 h at 37 degrees C to >8 h at 27 degrees C. Pharmacological chaperone treatment at 37 degrees C corrected the rDeltaF508 CFTR internalization defect, slowing endocytosis from approximately 30% per 2.5 min to approximately 5% per 2.5 min, and doubled DeltaF508 surface half-life from 2 to 4 h. These effects are DeltaF508 CFTR-specific, as pharmacological chaperones did not affect WT CFTR or transferrin receptor internalization rates. The results indicate that small molecular correctors may reproduce the effect of incubation at the permissive temperature, not only by rescuing DeltaF508 CFTR from ERAD, but also by enhancing its cell-surface stability.

Endoplasmic reticulum stress and the unfolded protein response regulate genomic cystic fibrosis transmembrane conductance regulator expression.

The unfolded protein response (UPR) is a cellular recovery mechanism activated by endoplasmic reticulum (ER) stress. The UPR is coordinated with the ER-associated degradation (ERAD) to regulate the protein load at the ER. In the present study, we tested how membrane protein biogenesis is regulated through the UPR in epithelia, using the cystic fibrosis transmembrane conductance regulator (CFTR) as a model. Pharmacological methods such as proteasome inhibition and treatment with brefeldin A and tunicamycin were used to induce ER stress and activate the UPR as monitored by increased levels of spliced XBP1 and BiP mRNA. The results indicate that activation of the UPR is followed by a significant decrease in genomic CFTR mRNA levels without significant changes in the mRNA levels of another membrane protein, the transferrin receptor. We also tested whether overexpression of a wild-type CFTR transgene in epithelia expressing endogenous wild-type CFTR activated the UPR. Although CFTR maturation is inefficient in this setting, the UPR was not activated. However, pharmacological induction of ER stress in these cells also led to decreased endogenous CFTR mRNA levels without affecting recombinant CFTR message levels. These results demonstrate that under ER stress conditions, endogenous CFTR biogenesis is regulated by the UPR through alterations in mRNA levels and posttranslationally by ERAD, whereas recombinant CFTR expression is regulated only by ERAD.

Mutations in the amino terminus of the cystic fibrosis transmembrane conductance regulator enhance endocytosis.

Efficient endocytosis of the cystic fibrosis transmembrane conductance regulator (CFTR) is mediated by a tyrosine-based internalization signal in the CFTR carboxyl-terminal tail 1424YDSI1427. In the present studies, two naturally occurring cystic fibrosis mutations in the amino terminus of CFTR, R31C, and R31L were examined. To determine the defect that these mutations cause, the Arg-31 mutants were expressed in COS-7 cells and their biogenesis and trafficking to the cell surface tested in metabolic pulse-chase and surface biotinylation assays, respectively. The results indicated that both Arg-31 mutants were processed to band C at approximately 50% the efficiency of the wild-type protein. However, once processed and delivered to the cell surface, their half-lives were the same as wild-type protein. Interestingly, indirect immunofluorescence and cell surface biotinylation indicated that the surface pool was much smaller than could be accounted for based on the biogenesis defect alone. Therefore, the Arg-31 mutants were tested in internalization assays and found to be internalized at 2x the rate of the wild-type protein. Patch clamp and 6-methoxy-N-(3-sulfopropyl)quinolinium analysis confirmed reduced amounts of functional Arg-31 channels at the cell surface. Together, the results suggest that both R31C and R31L mutations compromise biogenesis and enhance internalization of CFTR. These two additive effects contribute to the loss of surface expression and the associated defect in chloride conductance that is consistent with a disease phenotype.

Efficient intracellular processing of the endogenous cystic fibrosis transmembrane conductance regulator in epithelial cell lines.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent protein kinase A-activated chloride channel that resides on the apical surface of epithelial cells. One unusual feature of this protein is that during biogenesis, approximately 75% of wild type CFTR is degraded by the endoplasmic reticulum (ER)-associated degradative (ERAD) pathway. Examining the biogenesis and structural instability of the molecule has been technically challenging due to the limited amount of CFTR expressed in epithelia. Consequently, investigators have employed heterologous overexpression systems. Based on recent results that epithelial specific factors regulate both CFTR biogenesis and function, we hypothesized that CFTR biogenesis in endogenous CFTR expressing epithelial cells may be more efficient. To test this, we compared CFTR biogenesis in two epithelial cell lines endogenously expressing CFTR (Calu-3 and T84) with two heterologous expression systems (COS-7 and HeLa). Consistent with previous reports, 20 and 35% of the newly synthesized CFTR were converted to maturely glycosylated CFTR in COS-7 and HeLa cells, respectively. In contrast, CFTR maturation was virtually 100% efficient in Calu-3 and T84 cells. Furthermore, inhibition of the proteasome had no effect on CFTR biogenesis in Calu-3 cells, whereas it stabilized the immature form of CFTR in HeLa cells. Quantitative reverse transcriptase-PCR indicated that CFTR message levels are approximately 4-fold lower in Calu-3 than HeLa cells, yet steady-state protein levels are comparable. Our results question the structural instability model of wild type CFTR and indicate that epithelial cells endogenously expressing CFTR efficiently process this protein to post-Golgi compartments.