Accumulating evidence suggests that the abnormal aggregation of amyloid- () peptide in Alzheimer's disease (AD) begins intraneuronally, within vesicles of the endosomal-lysosomal pathway where A is both generated and degraded. Metalloproteases, including endothelin-converting enzyme (ECE)-1 and -2, reside within these vesicles and normally limit the accumulation of intraneuronally produced A. In this study, we determined whether disruption of A catabolism could trigger A aggregation within neurons and increase the amount of A associated with exosomes, small extracellular vesicles derived from endosomal multivesicular bodies. Using cultured cell lines, primary neurons, and organotypic brain slices from an AD mouse model, we found that pharmacological inhibition of the ECE family of metalloproteases increased intracellular and extracellular A levels and promoted the intracellular formation of A oligomers, a process that did not require internalization of secreted A. In vivo, the accumulation of intraneuronal A aggregates was accompanied by increased levels of both extracellular and exosome-associated A, including oligomeric species. Neuronal exosomes were found to contain both ECE-1 and -2 activities, suggesting that multivesicular bodies are intracellular sites of A degradation by these enzymes. ECE dysfunction could lead to the accumulation of intraneuronal A aggregates and their subsequent release into the extracellular space via exosomes.Pacheco-Quinto, J., Clausen, D., Perez-Gonzalez, R., Peng, H., Meszaros, A., Eckman, C. B., Levy, E., Eckman, E. A. Intracellular metalloprotease activity controls intraneuronal A aggregation and limits secretion of A via exosomes.

Impaired clearance of amyloid-β peptide (Aβ) has been postulated to significantly contribute to the amyloid accumulation typical of Alzheimer's disease. Among the enzymes known to degrade Aβ in vivo are endothelin-converting enzyme (ECE)-1, ECE-2, and neprilysin (NEP), and evidence suggests that they regulate independent pools of Aβ that may be functionally significant. To better understand the differential regulation of Aβ concentration by its physiological degrading enzymes, we characterized the cell and region-specific expression pattern of ECE-1, ECE-2, and NEP by in situ hybridization and immunohistochemistry in brain areas relevant to Alzheimer's disease. In contrast to the broader distribution of ECE-1, ECE-2 and NEP were found enriched in GABAergic neurons. ECE-2 was majorly expressed by somatostatin-expressing interneurons and was active in isolated synaptosomes. NEP messenger RNA was found mainly in parvalbumin-expressing interneurons, with NEP protein localized to perisomatic parvalbuminergic synapses. The identification of somatostatinergic and parvalbuminergic synapses as hubs for Aβ degradation is consistent with the possibility that Aβ may have a physiological function related to the regulation of inhibitory signaling.

The Linac Coherent Light Source has added a self-seeding capability to the soft x-ray range using a grating monochromator system. We report the demonstration of soft x-ray self-seeding with a measured resolving power of 2000-5000, wavelength stability of 10(-4), and an increase in peak brightness by a factor of 2-5 across the photon energy range of 500-1000 eV. By avoiding the need for a monochromator at the experimental station, the self-seeded beam can deliver as much as 50-fold higher brightness to users.

BACE1 (β-secretase) and α-secretase cleave the Alzheimer's amyloid β protein (Aβ) precursor (APP) to C-terminal fragments of 99 aa (CTFβ) and 83 aa (CTFα), respectively, which are further cleaved by γ-secretase to eventually secrete Aβ and Aα (a.k.a. P3) that terminate predominantly at residues 40 and 42. A number of γ-secretase inhibitors (GSIs), such as N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT), have been developed with the goal of reducing Aβ to treat Alzheimer's disease (AD). Although most studies show that DAPT inhibits Aβ in a dose-dependent manner several studies have also detected a biphasic effect with an unexpected increase at low doses of DAPT in cell cultures, animal models and clinical trials. In this article, we confirm the increase in Aβ40 and Aβ42 in SH-SY5Y human neuroblastoma cells treated with low doses of DAPT and identify one of the mechanisms for this paradox. We studied the pathway by first demonstrating that stimulation of Aβ, a product of γ-secretase, was accompanied by a parallel increase of its substrate CTFβ, thereby demonstrating that the inhibitor was not anomalously stimulating enzyme activity at low levels. Secondly, we have demonstrated that inhibition of an Aβ degrading activity, endothelin converting enzyme (ECE), yielded more Aβ, but abolished the DAPT-induced stimulation. Finally, we have demonstrated that Aα, which is generated in the secretory pathway before endocytosis, is not subject to the DAPT-mediated stimulation. We therefore conclude that impairment of γ-secretase can paradoxically increase Aβ by transiently skirting Aβ degradation in the endosome. This study adds to the growing body of literature suggesting that preserving γ-secretase activity, rather than inhibiting it, is important for prevention of neurodegeneration.

The efficient clearance of amyloid-β (Aβ) is essential to modulate levels of the peptide in the brain and to prevent it from accumulating in senile plaques, a hallmark of Alzheimer's disease (AD) pathology.We and others have shown that failure in Aβ catabolism can produce elevations in Aβ concentration similar to those observed in familial forms of AD. Based on the available evidence, it remains plausible that in late-onset AD, disturbances in the activity of Aβ degrading enzymes could induce Aβ accumulation, and that this increase could result in AD pathology. The following review presents a historical perspective of the parallel discovery of three vasopeptidases (neprilysin and endothelin-converting enzymes-1 and -2) as important Aβ degrading enzymes. The recognition of the role of these vasopeptidases in Aβ degradation, beyond bringing to light a possible explanation of how cardiovascular risk factors may influence AD risk, highlights a possible risk of the use of inhibitors of these enzymes for other clinical indications such as hypertension. We will discuss in detail the experiments conducted to assess the impact of vasopeptidase deficiency (through pharmacological inhibition or genetic mutation) on Aβ accumulation, as well as the cooperative effect of multiple Aβ degrading enzymes to regulate the concentration of the peptide at multiple sites, both intracellular and extracellular, throughout the brain.

BACKGROUND:Orthotopic liver transplantation (OLT) models in rats have been investigated in many studies. The reconstruction of hepatic artery is required for reliable OLT and also requires advanced skills.METHODS:The hepatic artery reconstructions by a hand-suture technique and a new method using a micro T-tube were investigated in rats with a whole-liver syngeneic graft. Operative time and postoperative patency were compared between the hand-suture and micro T-tube techniques.RESULTS:Our technique using the micro T-tube shortened the operative time of recipient surgery compared with the hand-suture technique and prolonged the operative time for the donor. The patency ratio was maintained at 24h after OLT with hand suturing but was significantly reduced with the micro T-tube, which had a patency ratio of 0.83 only up to 6h after OLT.CONCLUSION:The micro T-tube technique may have potential usefulness in the rat OLT model but requires further modification.

AIM:To investigate the effect of matrix metalloproteinase-9 (MMP-9) on the remnant liver after massive hepatectomy in the mouse.METHODS:Age-matched, C57BL/6 wild-type (WT), MMP-9(-/-), and tissue inhibitors of metalloproteinases (TIMP)-1(-/-) mice were used. The mice received 80%-partial hepatectomy (PH). Samples were obtained at 6 h after 80%-PH, and we used histology, immunohistochemical staining, western blotting analysis and zymography to investigate the effect of PH on MMP-9. The role of MMP-9 after PH was investigated using a monoclonal antibody and MMP inhibitor.RESULTS:We examined the remnant liver 6 h after 80%-PH and found that MMP-9 deficiency attenuated the formation of hemorrhage and necrosis. There were significantly fewer and smaller hemorrhagic and necrotic lesions in MMP-9(-/-) remnant livers compared with WT and TIMP-1(-/-) livers (P < 0.01), with no difference between WT and TIMP-1(-/-) mice. Serum alanine aminotransaminase levels were significantly lower in MMP-9(-/-) mice compared with those in TIMP-1(-/-) mice (WT: 476 ± 83 IU/L, MMP-9(-/-): 392 ± 30 IU/L, TIMP-1(-/-): 673 ± 73 IU/L, P < 0.01). Western blotting and gelatin zymography demonstrated a lack of MMP-9 expression and activity in MMP-9(-/-) mice, which was in contrast to WT and TIMP-1(-/-) mice. No change in MMP-2 expression was observed in any of the study groups. Similar to MMP-9(-/-) mice, when WT mice were treated with MMP-9 monoclonal antibody or the synthetic inhibitor GM6001, hemorrhagic and necrotic lesions were significantly smaller and fewer than in control mice (P < 0.05). These results suggest that MMP-9 plays an important role in the development of parenchymal hemorrhage and necrosis in the small remnant liver.CONCLUSION:Successful MMP-9 inhibition attenuates the formation of hemorrhage and necrosis and might be a potential therapy to ameliorate liver injury after massive hepatectomy.

A screen of a library of synthetic drugs and natural product extracts identified a botanical extract that modulates the processing of amyloid precursor protein (APP) in cultured cells to produce a lowered ratio of amyloid-beta peptide (1-42) (Aβ42) relative to Aβ40. This profile is of interest as a potential treatment for Alzheimer's disease. The extract, from the black cohosh plant (Actaea racemosa), was subjected to bioassay guided fractionation to isolate active components. Using a combination of normal-phase and reverse-phase chromatography, a novel triterpene monoglycoside, 1, was isolated. This compound was found to have an IC(50) of 100 nM for selectively reducing the production of amyloidogenic Aβ42 while having a much smaller effect on the production of Aβ40 (IC(50) 6.3 μM) in cultured cells overexpressing APP. Using IP-MS methods, this compound was found to modulate the pool of total Aβ produced by reducing the proportion of Aβ42 while increasing the relative amounts of shorter and less amyloidogenic Aβ37 and Aβ39. Concentrations of 1 sufficient to lower levels of Aβ42 substantially (up to 10 μM) did not significantly affect the processing of Notch or other aspects of APP processing. When 1 (10 μg) was administered to CD-1 normal mice intracerebroventricularly, the level of Aβ42 in brain was reduced. Assays for off-target pharmacology and the absence of overt signs of toxicity in mice dosed with compound 1 suggest a comparatively selective pharmacology for this triterpenoid. Compound 1 represents a new lead for the development of potential treatments for Alzheimer's disease via modulation of gamma-secretase.

MicrosurgeryVolume 32, Issue 3 p. 253-254 Letter to the Editor Hepatic arterial reconstruction with a short-term patency by using micro T-tube in rat liver transplantation Tomohide Hori M.D., Ph.D., Corresponding Author Tomohide Hori M.D., Ph.D. hori.tomohide@mayo.edu Department of Neuroscience, Mayo Clinic Florida, Jacksonville, FL Divisions of Hepato-Pancreato-Biliary, Transplant and Pediatric Surgery, Department of Surgery, Kyoto University Hospital Kyoto, JapanDepartment of Neuroscience, Mayo Clinic in Florida, Birdsall Research Bldg., 3rd. Fl., Rm# 323, 4500 San Pablo Rd., Jacksonville, FL 32224Search for more papers by this authorShinji Uemoto M.D., Ph.D., Shinji Uemoto M.D., Ph.D. Divisions of Hepato-Pancreato-Biliary, Transplant and Pediatric Surgery, Department of Surgery, Kyoto University Hospital Kyoto, JapanSearch for more papers by this authorFeng Chen Ph.D., Feng Chen Ph.D. Department of Transplant Surgery, Mayo Clinic Florida Jacksonville, FLSearch for more papers by this authorAnn-Marie T. Baine B.S., Ann-Marie T. Baine B.S. Department of Transplant Surgery, Mayo Clinic Florida Jacksonville, FLSearch for more papers by this authorLindsay B. Gardner B.S., Lindsay B. Gardner B.S. Department of Transplant Surgery, Mayo Clinic Florida Jacksonville, FLSearch for more papers by this authorToshiyuki Hata M.D., Toshiyuki Hata M.D. Divisions of Hepato-Pancreato-Biliary, Transplant and Pediatric Surgery, Department of Surgery, Kyoto University Hospital Kyoto, JapanSearch for more papers by this authorAimee R. Herdt Ph.D., Aimee R. Herdt Ph.D. Department of Neuroscience, Mayo Clinic Florida, Jacksonville, FLSearch for more papers by this authorChristopher B. Eckman Ph.D., Christopher B. Eckman Ph.D. Department of Neuroscience, Mayo Clinic Florida, Jacksonville, FLSearch for more papers by this authorJustin H. Nguyen M.D., Justin H. Nguyen M.D. Department of Transplant Surgery, Mayo Clinic Florida Jacksonville, FLSearch for more papers by this author Tomohide Hori M.D., Ph.D., Corresponding Author Tomohide Hori M.D., Ph.D. hori.tomohide@mayo.edu Department of Neuroscience, Mayo Clinic Florida, Jacksonville, FL Divisions of Hepato-Pancreato-Biliary, Transplant and Pediatric Surgery, Department of Surgery, Kyoto University Hospital Kyoto, JapanDepartment of Neuroscience, Mayo Clinic in Florida, Birdsall Research Bldg., 3rd. Fl., Rm# 323, 4500 San Pablo Rd., Jacksonville, FL 32224Search for more papers by this authorShinji Uemoto M.D., Ph.D., Shinji Uemoto M.D., Ph.D. Divisions of Hepato-Pancreato-Biliary, Transplant and Pediatric Surgery, Department of Surgery, Kyoto University Hospital Kyoto, JapanSearch for more papers by this authorFeng Chen Ph.D., Feng Chen Ph.D. Department of Transplant Surgery, Mayo Clinic Florida Jacksonville, FLSearch for more papers by this authorAnn-Marie T. Baine B.S., Ann-Marie T. Baine B.S. Department of Transplant Surgery, Mayo Clinic Florida Jacksonville, FLSearch for more papers by this authorLindsay B. Gardner B.S., Lindsay B. Gardner B.S. Department of Transplant Surgery, Mayo Clinic Florida Jacksonville, FLSearch for more papers by this authorToshiyuki Hata M.D., Toshiyuki Hata M.D. Divisions of Hepato-Pancreato-Biliary, Transplant and Pediatric Surgery, Department of Surgery, Kyoto University Hospital Kyoto, JapanSearch for more papers by this authorAimee R. Herdt Ph.D., Aimee R. Herdt Ph.D. Department of Neuroscience, Mayo Clinic Florida, Jacksonville, FLSearch for more papers by this authorChristopher B. Eckman Ph.D., Christopher B. Eckman Ph.D. Department of Neuroscience, Mayo Clinic Florida, Jacksonville, FLSearch for more papers by this authorJustin H. Nguyen M.D., Justin H. Nguyen M.D. Department of Transplant Surgery, Mayo Clinic Florida Jacksonville, FLSearch for more papers by this author First published: 08 March 2012 https://doi.org/10.1002/micr.21946Citations: 2Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume32, Issue3March 2012Pages 253-254 RelatedInformation

BACKGROUND:Oxidative stress plays an important role in neuronal dysfunction and neuron loss in Alzheimer's brain. Previous studies have reported downregulation of CREB-mediated transcription by oxidative stress and Aβ. The promoter for CREB itself contains cyclic AMP response elements. Therefore, we examined the expression of CREB in the hippocampal neurons of Tg2576 mice, AD post-mortem brain and in cultured rat hippocampal neurons exposed to Aβ aggregates.RESULTS:Laser Capture Microdissection of hippocampal neurons from Tg2576 mouse brain revealed decreases in the mRNA levels of CREB and its target, BDNF. Immunohistochemical analysis of Tg2576 mouse brain showed decreases in CREB levels in hippocampus and cortex. Markers of oxidative stress were detected in transgenic mouse brain and decreased CREB staining was observed in regions showing abundance of astrocytes. There was also an inverse correlation between SDS-extracted Aβ and CREB protein levels in Alzheimer's post-mortem hippocampal samples. The levels of CREB-regulated BDNF and BIRC3, a caspase inhibitor, decreased and the active cleaved form of caspase-9, a marker for the intrinsic pathway of apoptosis, was elevated in these samples. Exposure of rat primary hippocampal neurons to Aβ fibrils decreased CREB promoter activity. Decrease in CREB mRNA levels in Aβ-treated neurons was reversed by the antioxidant, N-acetyl cysteine. Overexpression of CREB by adenoviral transduction led to significant protection against Aβ-induced neuronal apoptosis.CONCLUSIONS:Our findings suggest that chronic downregulation of CREB-mediated transcription results in decrease of CREB content in the hippocampal neurons of AD brain which may contribute to exacerbation of disease progression.

Mechanisms of brain edema in acute liver failure (ALF) are not completely understood. We recently demonstrated that matrix metalloproteinase 9 (MMP-9) induces significant alterations to occludin in brain endothelial cells in vitro and in brains of mice with experimental ALF (Hepatology 2009; 50: 1914). In this study we show that MMP-9-induced transactivation of epidermal growth factor receptor (EGFR) and p38 MAPK/NF kappa B (mitogen-activated protein kinase/nuclear factor-kappa B) signals participate in regulating brain endothelial occludin level. Mouse brain endothelial bEnd3 cells were exposed to MMP-9 or p38 MAPK up-regulation in the presence and absence of EGFR inhibitor, p38 MAPK inhibitor, NF kappa B inhibitor, and/or appropriate small interfering RNA. Reverse-transcription polymerase chain reaction (RT-PCR) and western blotting were used for messenger RNA and protein expression analyses. Immunohistochemical staining and confocal microscopy were used to demonstrate cellular EGFR activation. Intraperitoneal azoxymethane was use to induce ALF in mice. Brains of comatose ALF mice were processed for histological and biochemical analyses. When bEnd3 cells were exposed to MMP-9, EGFR was significantly transactivated, followed by p38 MAPK activation, I-kappa B alpha (I kappa B alpha) degradation, NF kappa B activation, and suppression of occludin synthesis and expression. Similar EGFR activation and p38 MAPK/NF kappa B activation were found in the brains of ALF mice, and these changes were attenuated with GM6001 treatment. Conclusion: EGFR activation with p38 MAPK/NF kappa B signaling contributes to the regulation of tight junction integrity in ALF. EGFR activation may thus play an important role in vasogenic brain edema in ALF. (HEPATOLOGY 2011;53:1294-1305)

Target-based drug discovery for Alzheimer's disease (AD) centered on modulation of the amyloid β peptide has met with limited success. Therefore, recent efforts have focused on targeting the microtubule-associated protein tau. Tau pathologically accumulates in more than 15 neurodegenerative diseases and is most closely linked with postsymptomatic progression in AD. We endeavored to identify compounds that decrease tau stability rather than prevent its aggregation. An extract from Myrica cerifera (bayberry/southern wax myrtle) potently reduced both endogenous and overexpressed tau protein levels in cells and murine brain slices. The bayberry flavonoids myricetin and myricitrin were confirmed to contribute to this potency, but a diarylheptanoid, myricanol, was the most effective anti-tau component in the extract, with potency approaching the best targeted lead therapies. (+)-aR,11S-Myricanol, isolated from M. cerifera and reported here for the first time as the naturally occurring aglycone, was significantly more potent than commercially available (±)-myricanol. Myricanol may represent a novel scaffold for drug development efforts targeting tau turnover in AD.

Background: Oxidative stress plays an important role in neuronal dysfunction and neuron loss in Alzheimer's brain. Previous studies have reported downregulation of CREB-mediated transcription by oxidative stress and A beta. The promoter for CREB itself contains cyclic AMP response elements. Therefore, we examined the expression of CREB in the hippocampal neurons of Tg2576 mice, AD post- mortem brain and in cultured rat hippocampal neurons exposed to A beta aggregates.Results: Laser Capture Microdissection of hippocampal neurons from Tg2576 mouse brain revealed decreases in the mRNA levels of CREB and its target, BDNF. Immunohistochemical analysis of Tg2576 mouse brain showed decreases in CREB levels in hippocampus and cortex. Markers of oxidative stress were detected in transgenic mouse brain and decreased CREB staining was observed in regions showing abundance of astrocytes. There was also an inverse correlation between SDS- extracted A beta and CREB protein levels in Alzheimer's post- mortem hippocampal samples. The levels of CREB-regulated BDNF and BIRC3, a caspase inhibitor, decreased and the active cleaved form of caspase-9, a marker for the intrinsic pathway of apoptosis, was elevated in these samples. Exposure of rat primary hippocampal neurons to A beta fibrils decreased CREB promoter activity. Decrease in CREB mRNA levels in A beta-treated neurons was reversed by the antioxidant, N-acetyl cysteine. Overexpression of CREB by adenoviral transduction led to significant protection against A beta-induced neuronal apoptosis.Conclusions: Our findings suggest that chronic downregulation of CREB-mediated transcription results in decrease of CREB content in the hippocampal neurons of AD brain which may contribute to exacerbation of disease progression.

AIM: To investigate our learning curves of orthotopic liver transplantation (OLT) in rats and the most important factor for successful surgery. METHODS: We describe the surgical procedures for our rat OLT model, and determined the operator learning curves. The various factors that contributed to successful surgery were determined. The most important surgical factors were evaluated between successful and unsuccessful surgeries. RESULTS: Learning curve data indicated that 50 cases were required for operator training to start a study. Operative time, blood loss, warm ischemic time, anhepatic phase, unstable systemic hemodynannic state, and body temperature after surgery significantly affected surgery success by univariate analysis, while the anhepatic phase was the most critical factor for success by multivariate analysis. CONCLUSION: OLT in rats is the only liver transplantation model that provides clinically relevant and reliable results. Shortened anhepatic phase is key to success in this model. (C) 2010 Baishideng. All rights reserved.

Globoid cell leukodystrophy (GLD) (Krabbe disease) is an autosomal recessive, degenerative, lysosomal storage disease caused by a severe loss of galactocerebrosidase (GALC) enzymatic activity. Of the >70 disease-causing mutations in the GALC gene, most are located outside of the catalytic domain of the enzyme. To determine how GALC mutations impair enzymatic activity, we investigated the impact of multiple disease-causing mutations on GALC processing, localization, and enzymatic activity. Studies in mammalian cells revealed dramatic decreases in GALC activity and a lack of appropriate protein processing into an N-terminal GALC fragment for each of the mutants examined. Consistent with this, we observed significantly less GALC localized to the lysosome and impairment in either the secretion or reuptake of mutant GALC. Notably, the D528N mutation was found to induce hyperglycosylation and protein misfolding. Reversal of these conditions resulted in an increase in proper processing and GALC activity, suggesting that glycosylation may play a critical role in the disease process in patients with this mutation. Recent studies have shown that enzyme inhibitors can sometimes “chaperone” misfolded polypeptides to their appropriate target organelle, bypassing the normal cellular quality control machinery and resulting in enhanced activity. To determine whether this may also work for GLD, we examined the effect of α-lobeline, an inhibitor of GALC, on D528N mutant cells. After treatment, GALC activity was significantly increased. This study suggests that mutations in GALC can cause GLD by impairing protein processing and/or folding and that pharmacological chaperones may be potential therapeutic agents for patients carrying certain mutations.