Increased App expression in a mouse model of Down's syndrome disrupts NGF transport and causes cholinergic neuron degeneration.

Degeneration of basal forebrain cholinergic neurons (BFCNs) contributes to cognitive dysfunction in Alzheimer's disease (AD) and Down's syndrome (DS). We used Ts65Dn and Ts1Cje mouse models of DS to show that the increased dose of the amyloid precursor protein gene, App, acts to markedly decrease NGF retrograde transport and cause degeneration of BFCNs. NGF transport was also decreased in mice expressing wild-type human APP or a familial AD-linked mutant APP; while significant, the decreases were less marked and there was no evident degeneration of BFCNs. Because of evidence suggesting that the NGF transport defect was intra-axonal, we explored within cholinergic axons the status of early endosomes (EEs). NGF-containing EEs were enlarged in Ts65Dn mice and their App content was increased. Our study thus provides evidence for a pathogenic mechanism for DS in which increased expression of App, in the context of trisomy, causes abnormal transport of NGF and cholinergic neurodegeneration.

Computerized image analysis for quantitative neuronal phenotyping in zebrafish.

An integrated microscope image analysis pipeline is developed for automatic analysis and quantification of phenotypes in zebrafish with altered expression of Alzheimer's disease (AD)-linked genes. We hypothesize that a slight impairment of neuronal integrity in a large number of zebrafish carrying the mutant genotype can be detected through the computerized image analysis method. Key functionalities of our zebrafish image processing pipeline include quantification of neuron loss in zebrafish embryos due to knockdown of AD-linked genes, automatic detection of defective somites, and quantitative measurement of gene expression levels in zebrafish with altered expression of AD-linked genes or treatment with a chemical compound. These quantitative measurements enable the archival of analyzed results and relevant meta-data. The structured database is organized for statistical analysis and data modeling to better understand neuronal integrity and phenotypic changes of zebrafish under different perturbations. Our results show that the computerized analysis is comparable to manual counting with equivalent accuracy and improved efficacy and consistency. Development of such an automated data analysis pipeline represents a significant step forward to achieve accurate and reproducible quantification of neuronal phenotypes in large scale or high-throughput zebrafish imaging studies.

Blunt traumatic aortic transection: the endovascular experience.

BACKGROUND: Thoracic aortic transection resulting from blunt trauma is usually fatal. It is almost always associated with multiple, complex, nonaortic injuries that could be adversely affected by standard surgical repair of the aorta. Endovascular stenting techniques offer these patients a less physiologically disruptive treatment option. We studied the feasibility and safety of endovascular stent graft placement for treatment of acute traumatic aortic transection. METHODS: Between 1994 and 2001, 9 patients were treated emergently for aortic transections with stent graft placement. The first patient had a custom-made prototype, and the other 8 patients had the Cook-Zenith thoracic stent graft implanted. All were polyester-covered Z-stent construction and deployed through a femoral 20- to 24-F delivery sheath. RESULTS: Stent graft placement successfully sealed the aorta in all patients. One patient died as a result of a cerebrovascular accident. One patient required a brachial thrombectomy to relieve arm ischemia. The remaining eight patients were alive and without complications during the follow-up period (mean 21 months). CONCLUSIONS: Endovascular repair for acute aortic transection is a safe, effective, and timely treatment option. It may be the treatment of choice in patients with extensive associated injuries.

MBM fuel feeding system design and evaluation for FBG pilot plant.

A biomass fuel feeding system has been designed, constructed and evaluated for a fluidized bed gasifier (FBG) pilot plant at the University of Saskatchewan (Saskatoon, SK, Canada). The system was designed for meat and bone meal (MBM) to be injected into the gasifier at a mass flow-rate range of 1-5 g/s. The designed system consists of two stages of screw conveyors, including a metering stage which controlled the flow-rate of fuel, a rotary airlock and an injection conveyor stage, which delivered that fuel at a consistent rate to the FBG. The rotary airlock which was placed between these conveyors, proved unable to maintain a pressure seal, thus the entire conveying system was sealed and pressurized. A pneumatic injection nozzle was also fabricated, tested and fitted to the end of the injection conveyor for direct injection and dispersal into the fluidized bed. The 150 mm metering screw conveyor was shown to effectively control the mass output rate of the system, across a fuel output range of 1-25 g/s, while the addition of the 50mm injection screw conveyor reduced the irregularity (error) of the system output rate from 47% to 15%. Although material plugging was found to be an issue in the inlet hopper to the injection conveyor, the addition of air sparging ports and a system to pulse air into those ports was found to successfully eliminate this issue. The addition of the pneumatic injection nozzle reduced the output irregularity further to 13%, with an air supply of 50 slpm as the minimum air supply to drive this injector. After commissioning of this final system to the FBG reactor, the injection nozzle was found to plug with char however, and was subsequently removed from the system. Final operation of the reactor continues satisfactorily with the two screw conveyors operating at matching pressure with the fluidized bed, with the output rate of the system estimated based on system characteristic equations, and confirmed by static weight measurements made before and after testing. The error rate by this method is reported to be approximately 10%, which is slightly better than the estimated error rate of 15% for the conveyor system. The reliability of this measurement prediction method relies upon the relative consistency of the physical properties of MBM with respect to its bulk density and feeding characteristics.

The cytosolic loop of the gamma-secretase component presenilin enhancer 2 protects zebrafish embryos from apoptosis.

The gamma-secretase complex, composed of presenilin, presenilin enhancer 2 (Pen-2), nicastrin, and Aph-1, catalyzes the final cleavage of amyloid precursor protein to generate the toxic amyloid beta protein, the major component of plaques in the brains of Alzheimer disease patients. To understand the in vivo function of Pen-2, we used morphant technology available in zebrafish and transiently knocked down the expression of endogenous Pen-2 by injecting the morpholino (MO) against Pen-2. Two truncated Pen-2 proteins lacking either the cytosolic or the C-terminal domain were expressed in MO-injected embryos. This deletion analysis demonstrated that the Pen-2 cytosolic loop is essential for protecting developing embryos from caspase-dependent apoptosis caused by the reduction of Pen-2. Twelve amino acids in the C terminus of Pen-2 were dispensable and could not rescue the Pen-2 knockdown-induced apoptotic phenotype. Surprisingly, double knockdown of Pen-2 and nuclear factor kappaB component p65 abrogated the single Pen-2 MO-induced caspase activation, indicating that a previously reported pro-apoptotic role of NF-kappaB in some cell types could be manifested in a whole animal and that knockdown of Pen-2 may trigger pro-apoptotic activation of NF-kappaB.

Zebrafish lacking Alzheimer presenilin enhancer 2 (Pen-2) demonstrate excessive p53-dependent apoptosis and neuronal loss.

Gamma-secretase cleavage, mediated by a complex of presenilin, presenilin enhancer (Pen-2), nicastrin, and Aph-1, is the final proteolytic step in generating amyloid beta protein found in brains of Alzheimer's disease patients and Notch intracellular domain critical for proper neuronal development. Here, we employ the zebrafish model to study the role of Pen-2 in neuronal survival. We found that (i) knockdown of Pen-2 using antisense morpholino led to a reduction of islet-1 positive neurons, (ii) Notch signaling was reduced in embryos lacking Pen-2 or other gamma-secretase components, (iii) neuronal loss in Pen-2 knockdown embryos is not as a result of a lack of neuronal precursor cells or cell proliferation, (iv) absence of Pen-2 caused massive apoptosis in the whole animal, which could be suppressed by simultaneous knockdown of the tumor suppressor p53, (v) loss of islet-1 or acetylated tubulin positive neurons in Pen-2 knockdown embryos could be partially rescued by knockdown of p53. Our results demonstrate that knockdown of Pen-2 directly induces a p53-dependent apoptotic pathway that contributes to neuronal loss and suggest that Pen-2 plays an important role in promoting neuronal cell survival and protecting from apoptosis in vivo.

Endoproteolysis of presenilin in vitro: inhibition by gamma-secretase inhibitors.

The final proteolytic step to generate the amyloid beta-protein (Abeta) of Alzheimer's disease (AD) from beta-amyloid precursor protein (APP) is achieved by presenilin (PS)-dependent gamma-secretase cleavage. AD-causing mutations in PS1 and PS2 result in a selective and significant increase in production of the more amyloidogenic Abeta42 peptide. PS1 and PS2 undergo endoproteolysis by an unknown enzyme termed presenilinase to generate the functional complex of N- and C-terminal fragments (NTF/CTF). To investigate the endoproteolytic activity that generates active PS, we used a mammalian cell-free system that allows de novo human PS NTF and CTF generation. PS NTF and CTF generation in vitro was observed in endoplasmic reticulum (ER)-enriched fractions of membrane vesicles and to a lesser extent in Golgi/trans-Golgi-network (TGN)-enriched fractions. AD-causing mutations in PS1 and PS2 did not alter de novo generation of PS fragments. Removal of peripheral membrane-associated and cytosolic proteins did not prevent de novo generation of fragments, indicating that presenilinase activity corresponds to an integral membrane protein. Among several general inhibitors of different protease classes that blocked the presenilinase activity, pepstatin A was the most potent inhibitor. Screening available transition state analogue gamma-secretase inhibitors led to the identification of two compounds that were able to prevent the de novo generation of PS fragments, with an expected inhibition of Abeta generation. Our studies provide a biochemical approach to characterize and identify this elusive presenilinase.

Presenilin endoproteolysis mediated by an aspartyl protease activity pharmacologically distinct from gamma-secretase.

Presenilin (PS)-dependent gamma-secretase cleavage is the final proteolytic step in generating amyloid beta protein (A beta), a key peptide involved in the pathogenesis of Alzheimer's disease. PS undergoes endoproteolysis by an unidentified 'presenilinase' to generate the functional N-terminal and C-terminal fragment heterodimers (NTF/CTF) that may harbor the gamma-secretase active site. To better understand the relationship between presenilinase and gamma-secretase, we characterized the biochemical properties of presenilinase and compared them with those of gamma-secretase. Similar to gamma-secretase, presenilinase was most active at acidic pH 6.3. Aspartyl protease inhibitor pepstatin A blocked presenilinase activity with an IC50 of approximately 1 microM. Difluoroketone aspartyl protease transition state analogue MW167 was relatively selective for presenilinase (IC50 < 1 microM) over gamma-secretase (IC50-16 microM). Importantly, removing the transition state mimicking moiety simultaneously abolished both presenilinase and gamma-secretase inhibition, suggesting that presenilinase, like gamma-secretase, is an aspartyl protease. Interestingly, several of the most potent gamma-secretase inhibitors (IC50 = 0.3 or 20 nM) failed to block presenilinase activity. Although de novo generation of PS1 fragments coincided with production of A beta in vitro, blocking presenilinase activity without reducing pre-existing fragment levels permitted normal de novo generation of A beta and amyloid intracellular domain. Therefore, presenilinase has characteristics of an aspartyl protease, but this activity is distinct from gamma-secretase.