Amyloid beta and impairment in multiple memory systems in older transgenic APP TgCRND8 mice.

The relationship between amyloid beta and cognitive dysfunction in mouse models of Alzheimer's disease has been evaluated predominantly with the spatial reference memory version of the water maze task. However, as Alzheimer's disease encompasses decline in multiple memory systems, it is important to also utilize non-spatial tasks to fully characterize the role of amyloid on behaviour in animal models. We used the TgCRND8 mouse model of Alzheimer's disease to evaluate the effect of amyloid on spatial reference memory, as well as on the non-spatial task of acquisition of conditioned taste aversion, and on the procedural task of swimming to a visible platform. We demonstrate that 8- to 12-month-old TgCRND8 mice are significantly impaired in all three tasks, and that the levels of soluble amyloid beta are significantly correlated with impairment in spatial reference memory, but not with impairment in conditioned taste aversion or swimming to a visible platform. Insoluble fractions of amyloid, which correspond closely to amyloid plaque burden in the brain, are not associated with any behavioural measure. Our study extends the characterization of the model to stages of advanced amyloid pathology and demonstrates that older TgCRND8 mice are impaired in multiple memory systems, including procedural tasks, which are spared at younger ages. The lack of association between amyloid plaques and memory decline supports clinical findings in Alzheimer's patients.

Abeta-degrading enzymes: modulators of Alzheimer's disease pathogenesis and targets for therapeutic intervention.

The accumulation of Abeta (amyloid beta-protein) peptides in the brain is a pathological hallmark of all forms of AD (Alzheimer's disease) and reducing Abeta levels can prevent or reverse cognitive deficits in mouse models of the disease. Abeta is produced continuously and its concentration is determined in part by the activities of several degradative enzymes, including NEP (neprilysin), IDE (insulin-degrading enzyme), ECE-1 (endothelin-converting enzyme 1) and ECE-2, and probably plasmin. Decreased activity of any of these enzymes due to genetic mutation, or age- or disease-related alterations in gene expression or proteolytic activity, may increase the risk for AD. Conversely, increased expression of these enzymes may confer a protective effect. Increasing Abeta degradation through gene therapy, transcriptional activation or even pharmacological activation of the Abeta-degrading enzymes represents a novel therapeutic strategy for the treatment of AD that is currently being evaluated in cell-culture and animal models. In this paper, we will review the roles of NEP, IDE, ECE and plasmin in determining endogenous Abeta concentration, highlighting recent results concerning the regulation of these enzymes and their potential as therapeutic targets.

High throughput screens for the identification of compounds that alter the accumulation of the Alzheimer's amyloid beta peptide (Abeta).

Evidence gathered over the last two decades suggests that beta amyloid (Abeta), the predominant proteinaceous component of senile plaques, plays an early and critical role in the etiology and pathogenesis of Alzheimer's disease (AD). Thus, it is reasonable to hypothesize that compounds capable of reducing the accumulation of Abeta may be of value therapeutically. Additionally, compounds that influence Abeta accumulation may be useful as tools to further dissect the cellular pathways that regulate Abeta production and accumulation. To screen for compounds that affect Abeta levels, we have established high throughput, cell-based assays capable of the sensitive and selective detection of Abeta40 in parallel with the more amyloidogenic form of the peptide, Abeta42. To validate the approach, we examined the effects of several compounds previously identified to influence Abeta accumulation. Analysis of peptide accumulation following treatment with these compounds showed results similar to those previously published. Currently, we are using this assay to screen drugs that have already received FDA approval for the treatment of other diseases and over-the-counter natural product extracts. If compounds such as these can be identified that lower Abeta in the brain, they may represent one of the fastest and most cost effective methods to therapy.

Degradation of the Alzheimer's amyloid beta peptide by endothelin-converting enzyme.

Deposition of beta-amyloid (Abeta) peptides in the brain is an early and invariant feature of all forms of Alzheimer's disease. As with any secreted protein, the extracellular concentration of Abeta is determined not only by its production but also by its catabolism. A major focus of Alzheimer's research has been the elucidation of the mechanisms responsible for the generation of Abeta. Much less, however, is known about the mechanisms responsible for Abeta removal in the brain. In this report, we describe the identification of endothelin-converting enzyme-1 (ECE-1) as a novel Abeta-degrading enzyme. We show that treatment of endogenous ECE-expressing cell lines with the metalloprotease inhibitor phosphoramidon causes a 2-3-fold elevation in extracellular Abeta concentration that appears to be due to inhibition of intracellular Abeta degradation. Furthermore, we show that overexpression of ECE-1 in Chinese hamster ovary cells, which lack endogenous ECE activity, reduces extracellular Abeta concentration by up to 90% and that this effect is completely reversed by treatment of the cells with phosphoramidon. Finally, we show that recombinant soluble ECE-1 is capable of hydrolyzing synthetic Abeta40 and Abeta42 in vitro at multiple sites.

Reduction of Abeta accumulation in the Tg2576 animal model of Alzheimer's disease after oral administration of the phosphatidyl-inositol kinase inhibitor wortmannin.

The abnormal accumulation of the amyloid beta protein (Abeta) has been implicated as an early and critical event in the etiology and pathogenesis of Alzheimer's disease (AD). Compounds that reduce Abeta accumulation may therefore be useful therapeutically. In cell-based screens we detected a significant reduction in Abeta concentration after treatment with the phosphatidylinositol kinase inhibitors wortmannin and LY294002. To determine the effect of this class of compounds on in vivo Abeta accumulation, we administered wortmannin to the Tg2576 mouse model of AD. Oral administration of wortmannin over four months resulted in a significant, non-overlapping 40%-50% reduction in the number of senile plaques, one of the pathological hallmarks of AD. Sandwich ELISA analysis of formic acid extractable Abeta in the brain of treated animals indicates that both Abeta40 and the longer, more amyloidogenic form of the peptide, Abeta42, were significantly reduced. These data provide the first direct evidence that compounds identified by their ability to reduce Abeta concentration in vitro can reduce Abeta accumulation and deposition in the brain, thus establishing a basic paradigm for the identification and evaluation of additional compounds that lower Abeta accumulation.

Transport of bifunctional proteins across respiratory epithelial cells via the polymeric immunoglobulin receptor.

Neutrophil elastase (NE) contributes to progression of the lung disease characteristic of cystic fibrosis (CF). We developed a strategy that permits the delivery of alpha(1)-antitrypsin (alpha(1)-AT) to inaccessible CF airways by targeting the respiratory epithelium via the polymeric immunoglobulin receptor (pIgR). A fusion protein consisting of a single-chain Fv directed against human secretory component (SC) and linked to human alpha(1)-AT was effectively transported in a basolateral-to-apical direction across in vitro model systems of polarized respiratory epithelium consisting of 16HBEo cells transfected with human pIgR complementary DNA, which overexpress the receptor, and human respiratory epithelial cells grown in primary culture at an air-liquid interface. When applied to the basolateral surface, the anti-SC Fv/alpha(1)-AT fusion protein penetrated the respiratory epithelia, with transcytosis of the fusion protein being related to the amount of SC detected at the apical surface. Significantly less fusion protein crossed the cells in the opposite direction. In addition, because the antihuman SC Fv/alpha(1)-AT fusion protein was transported vectorially and deposited into the small volume of apical surface fluid, the antiprotease component of this protein was concentrated atop the epithelium. Thus, in cell models, this system is capable of concentrating the antiprotease of the fusion protein, in the thin film of epithelial surface fluid to a level expected to be therapeutic in the airways of many patients with CF.

In vitro transport of active alpha(1)-antitrypsin to the apical surface of epithelia by targeting the polymeric immunoglobulin receptor.

In cystic fibrosis (CF), the intense host inflammatory response to chronic infection largely accounts for the progressive pulmonary disease, and ultimately death. Neutrophils are the prominent inflammatory cells in the lungs of patients with CF, and large amounts of neutrophil elastase (NE) are released during phagocytosis. Besides having direct effects on structural elastin, NE stimulates the release of proinflammatory mediators from the respiratory epithelium and is a potent secretogogue. Therapeutic use of elastase inhibitors in CF has been complicated by difficulties in delivery to the critical site in the airway-the surface of the epithelium. We describe a unique strategy to protect the respiratory epithelial cell surface directly by capitalizing on the nondegradative transcytotic pathway of the polymeric immunoglobulin receptor (pIgR). A recombinant fusion protein was constructed consisting of an antihuman pIgR single-chain Fv (scFv) antibody linked to human alpha(1)-antitrypsin (A1AT), an inhibitor of NE. The recombinant scFv-A1AT fusion protein bound specifically to the pIgR on the basolateral surface of an epithelial cell monolayer, and was transported and released into the apical medium where the A1AT domain was capable of forming an inactivation complex with NE. Thus, A1AT linked to an antihuman pIgR scFv was delivered in receptor-specific fashion from the basolateral to apical surface and was released as an active antiprotease, indicating that it is feasible to deliver therapeutic proteins to the apical surface of epithelia by targeting the pIgR.

Immunologic responses to gene transfer into mice via the polymeric immunoglobulin receptor.

The respiratory epithelium is the primary target tissue for gene therapy of cystic fibrosis, and several methods of gene transfer permit the introduction of the gene encoding the normal cystic fibrosis transmembrane conductance regulator into cells of the respiratory tract in animals. DNA complexes based on Fab antibodies to secretory component have been used to mediate the delivery and uptake of expression plasmids into the respiratory tract via the polymeric immunoglobulin receptor both in vitro and in vivo. We evaluated the efficacy of gene transfer after several administrations of the DNA complexes, and examined the immunogenicity and toxicity of repetitive administration of anti-secretory component Fab-based complexes. Mice received single or multiple injections of the DNA complexes containing the plasmid pGL2 every 21 days after the initial treatment, and lysates from the lung and liver were assayed for luciferase expression. Luciferase activity was detected in the lungs of mice that received a single injection of the DNA complexes, whereas transgene expression was significantly lower in the mice that received three injections of the DNA complexes (17338 +/- 5469 integrated light units/mg and 3771 +/- 1778 integrated light units/mg, respectively). Serum samples from animals that underwent single or multiple injections were analyzed for a serologic response against the conjugate-DNA complexes by ELISA. No anticomplex antibodies were detected in the mice after a single injection. An escalating antibody response was noted with increasing number of treatments with the conjugate-DNA complexes. This serologic response was directed exclusively against the rabbit-derived, anti-secretory component (anti-SC) Fab antibody, and not against either the plasmid DNA or poly-L-lysine. Single injection of the conjugate-DNA complexes did not result in the consumption of circulating complement. Using direct immunofluorescence, perivascular deposits of immunoglobulin G were found in the liver of animals that received three treatments; no such deposition was detected in the lungs or kidneys. No increase in inflammatory cell infiltrates was observed in tissues after single and repeated injections of the DNA complexes. Thus, we conclude that repeated injections of the anti-SC Fab-based complexes evoked a humoral immune response against the heterologous Fab portion of the complex that was associated with reduced efficiency of gene transfer.

Overexpression of R domain eliminates cAMP-stimulated Cl- secretion in 9/HTEo- cells in culture.

The intracellular hydrophilic region of the cystic fibrosis transmembrane conductance regulator (CFTR), the R domain, has been postulated to be a regulator of the Cl-channel. Under basal conditions R blocks the channel, but when phosphorylated, R undergoes conformational change to open the channel. Overexpression of R in 9/HTEo- cells, a human tracheal epithelial cell line with adenosine 3',5' -cyclic monophosphate (cAMP)-regulated Cl- conductance due to CFTR, caused reduced basal Cl- conductance and elimination of its response to isoproterenol, but ionomycin-stimulated Cl- efflux was preserved. Cells which overexpressed R showed no downregulation of endogenous CFTR mRNA and had normal cAMP production and protein kinase A (PKA) activity, so R did not act at these levels. Although the precise mechanism by which R affects CFTR conductance is undetermined, these cell lines could be useful in separating the cell biological consequences of impaired Cl- transport from those of mutant CFTR per se.

Dietary changes improve survival of CFTR S489X homozygous mutant mouse.

Over 90% of untreated CFTR S489X homozygous (CF) mutant mice reportedly die of intestinal obstruction by 40 days of age, significantly limiting their usefulness as a model for the human disease. Because the period of highest mortality is during the week after weaning, we hypothesized that providing a low-residue liquid diet would improve survival and growth. When 99 CF mice that survived to 10 days of age were fed Peptamen (Clintec Nutrition), an elemental liquid diet, and housed on corn-cob bedding, 88% of them survived to maturity (50 days). The diet causes only minor histologic and ion transport changes in the intestines of normal mice and does not reduce growth rate or size. CF mice raised on Peptamen continue to display severe pathological changes in the intestine and completely lack a adenosine 3',5'-cyclic monophosphate-inducible chloride current in the cecum. This combination of dietary and bedding changes provides a reliable method for keeping CF mice alive well into adulthood and will be useful for the evaluation of the effect and duration of potential therapies for CF.

Modification of chloride ion transport in human erythrocyte ghost membranes by photoaffinity labeling reagents based on the structure of 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB).

Using human red blood cell ghost membranes, we have evaluated 5-nitro-2-[N-3-(4-azidophenyl)-propylamino]-benzoic acid and 5-nitro-2-[N-3-(4-azido-2,3,5,6-tetrafluorophenyl)-propylamino]- benzoic acid (FAzNPPB) as photoaffinity labeling agents based on the structure of the widely important Cl- channel blocker 5-nitro-2-(3-phenylpropyl-amino)-benzoic acid (NPPB). The tetrafluoro-substituted aryl azide was found to be a more effective photoinactivating agent than the corresponding protio compound. Using a tritiated version ([3H]FAzNPPB), we demonstrated that photoinactivation of Cl- flux was accompanied by photolabeling of the band 3 protein and membrane lipids. Both processes were diminished in the presence of NPPB and the related arylanthranilate flufenamic acid. Photolabeling resulted in the incorporation of 1.0 +/- 0.2 mol 3H/mol protein in the band 3 integral membrane domain, whereas the cytoplasmic domain was essentially unlabeled. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, photolabeling was found to be the result of partial labeling of at least three different regions of the membrane domain. Based on trypsin proteolysis, reverse-phase high-performance liquid chromatography and electrospray ionization mass spectrometry analysis, it is proposed that one of the sites of photolabeling is the peptide lys-phe-lys (590-592). FAzNPPB is a successful polyfluoro aryl azide photoaffinity labeling agent which may be of further use in studying the diverse effects of arylanthranilates on biological membranes.

Gene transfer into the airway epithelium of animals by targeting the polymeric immunoglobulin receptor.

Genes of interest can be targeted specifically to respiratory epithelial cells in intact animals with high efficiency by exploiting the receptor-mediated endocytosis of the polymeric immunoglobulin receptor. A DNA carrier, consisting of the Fab portion of polyclonal antibodies raised against rat secretory component covalently linked to poly-L-lysine, was used to introduce plasmids containing different reporter genes into airway epithelial cells in vivo. We observed significant levels of luciferase enzyme activity in protein extracts from the liver and lung, achieving maximum values of 13,795 +/- 4,431 and 346,954 +/- 199,120 integrated light units (ILU) per milligram of protein extract, respectively. No luciferase activity was detected in spleen or heart, which do not express the receptor. Transfections using complexes consisting of an irrelevant plasmid (pCMV lacZ) bound to the bona fide carrier or the expression plasmid (pGEMluc) bound to a carrier based on an irrelevant Fab fragment resulted in background levels of luciferase activity in all tissues examined. Thus, only tissues that contain cells bearing the polymeric immunoglobulin receptor are transfected, and transfection cannot be attributed to the nonspecific uptake of an irrelevant carrier-DNA complex. Specific mRNA from the luciferase gene was also detected in the lungs of transfected animals. To determine which cells in the lungs are transfected by this method, DNA complexes were prepared containing expression plasmids with genes encoding the bacterial beta-galactosidase or the human interleukin 2 receptor. Expression of these genes was localized to the surface epithelium of the airways and the submucosal glands, and not the bronchioles and alveoli. Receptor-mediated endocytosis can be used to introduce functional genes into the respiratory epithelium of rats, and may be a useful technique for gene therapy targeting the lung.