Prominent tauopathy and intracellular ß-amyloid accumulation triggered by genetic deletion of cathepsin D: implications for Alzheimer disease pathogenesis.

BACKGROUND: Cathepsin D (CatD) is a lysosomal protease that degrades both the amyloid-ß protein (Aß) and the microtubule-associated protein, tau, which accumulate pathognomonically in Alzheimer disease (AD), but few studies have examined the role of CatD in the development of Aß pathology and tauopathy in vivo. METHODS: CatD knockout (KO) mice were crossed to human amyloid precursor protein (hAPP) transgenic mice, and amyloid burden was quantified by ELISA and immunohistochemistry (IHC). Tauopathy in CatD-KO mice, as initially suggested by Gallyas silver staining, was further characterized by extensive IHC and biochemical analyses. Controls included human tau transgenic mice (JNPL3) and another mouse model of a disease (Krabbe A) characterized by pronounced lysosomal dysfunction. Additional experiments examined the effects of CatD inhibition on tau catabolism in vitro and in cultured neuroblastoma cells with inducible expression of human tau. RESULTS: Deletion of CatD in hAPP transgenic mice triggers large increases in cerebral Aß, manifesting as intense, exclusively intracellular aggregates; extracellular Aß deposition, by contrast, is neither triggered by CatD deletion, nor affected in older, haploinsufficient mice. Unexpectedly, CatD-KO mice were found to develop prominent tauopathy by just ∼ 3 weeks of age, accumulating sarkosyl-insoluble, hyperphosphorylated tau exceeding the pathology present in aged JNPL3 mice. CatD-KO mice exhibit pronounced perinuclear Gallyas silver staining reminiscent of mature neurofibrillary tangles in human AD, together with widespread phospho-tau immunoreactivity. Striking increases in sarkosyl-insoluble phospho-tau (∼ 1250%) are present in CatD-KO mice but notably absent from Krabbe A mice collected at an identical antemortem interval. In vitro and in cultured cells, we show that tau catabolism is slowed by blockade of CatD proteolytic activity, including via competitive inhibition by Aß42. CONCLUSIONS: Our findings support a major role for CatD in the proteostasis of both Aß and tau in vivo. To our knowledge, the CatD-KO mouse line is the only model to develop detectable Aß accumulation and profound tauopathy in the absence of overexpression of hAPP or human tau with disease-associated mutations. Given that tauopathy emerges from disruption of CatD, which can itself be potently inhibited by Aß42, our findings suggest that impaired CatD activity may represent a key mechanism linking amyloid accumulation and tauopathy in AD.

Prominent tauopathy and intracellular ß-amyloid accumulation triggered by genetic deletion of cathepsin D: Implications for Alzheimer disease pathogenesis.

Background: Cathepsin D (CatD) is a lysosomal protease that degrades both the amyloid-ß protein (Aß) and the microtubule-associated protein, tau, which accumulate pathognomonically in Alzheimer disease (AD), but few studies have examined the role of CatD in the development of Aß pathology and tauopathy in vivo. Methods: CatD knockout (KO) mice were crossed to human amyloid precursor protein (hAPP) transgenic mice, and amyloid burden was quantified by ELISA and immunohistochemistry (IHC). Tauopathy in CatD-KO mice, as initially suggested by Gallyas silver staining, was further characterized by extensive IHC and biochemical analyses. Controls included human tau transgenic mice (JNPL3) and another mouse model characterized by pronounced lysosomal dysfunction (Krabbe A). Additional experiments examined the effects of CatD inhibition on tau catabolism in vitro and in cultured neuroblastoma cells with inducible expression of human tau. Results: Deletion of CatD in hAPP transgenic mice triggers large increases in cerebral Aß, manifesting as intense, exclusively intracellular aggregates; extracellular Aß deposition, by contrast, is neither triggered by CatD deletion, nor affected in older, haploinsufficient mice. Unexpectedly, CatDKO mice were found to develop prominent tauopathy by just ~ 3 weeks of age, accumulating sarkosyl-insoluble, hyperphosphorylated tau exceeding the pathology in aged JNPL3 mice. CatDKO mice exhibit pronounced perinuclear Gallyas silver staining reminiscent of mature neurofibrillary tangles in human AD, together with widespread phospho-tau immunoreactivity. Striking increases in sarkosyl-insoluble phospho-tau (~ 1250%) are present in CatD-KO mice, but notably absent from Krabbe A mice collected at an identical antemortem interval. In vitro and in cultured cells, we show that tau catabolism is slowed by blockade of CatD proteolytic activity, including via competitive inhibition by Aß42. Conclusions: Our findings support a major role for CatD in the proteostasis of both Aß and tau in vivo. To our knowledge, CatD-KO mice are the only model to develop detectable Aß acumulation and profound tauopathy in the absence of overexpression of hAPP or human tau with disease-associated mutations. Given that tauopathy emerges from disruption of CatD, which can itself be potently inhibited by Aß42, our findings suggest that impaired CatD activity may represent a key mechanism linking amyloid accumulation and tauopathy in AD.

Vascular Involvements Are Common in the Branch Arteries of the Abdominal Aorta Rather Than in the Aorta in Vascular Ehlers-Danlos Syndrome.

Background: Vascular Ehlers-Danlos syndrome (vEDS) is a rare disorder with poor prognosis, owing to associated vascular complications. However, the most prevalent arterial problems in patients with vEDS are not well known. Methods: We retrospectively examined 20 consecutive patients diagnosed with vEDS and examined their clinical events, image findings, and therapies. Results: The age at first complication requiring admission was 29 ± 13 years. The observational period was 67 ± 30 months. Of the 20 patients, 17 took celiprolol at final assessment. At the final follow-up, the total number of complications relating to lesions and requiring admission was 16 for pulmonary lesions (8 patients), 16 for bowel lesions (8 patients), 5 for tendon/ligament lesions (2 patients), 18 for the branch arteries of the abdominal aorta (10 patients), 2 for the aorta (2 patients), and 7 for other arteries (6 patients). Of 54 arterial involvements (aneurysms, dissections, and ruptures), both with and without symptoms, 43 (80%) were in branches of the abdominal aorta (celiac artery and branches, 8; superior mesenteric artery, 4; renal arteries, 3; iliac arteries and branches, 28), 2 (4%) were in the aorta, and 9 were in other arteries. The diameter of the sinus of Valsalva was 29 ± 5 mm, within the normal range. During follow-up, 3 patients died due to suspected ruptures in a branch of the celiac artery, the superior mesenteric artery, and the aorta. Conclusion: Our findings indicate that lesions involving the branch arteries of the abdominal aorta, rather than aorta, were the most prevalent lesion type in patients with vEDS.

Contexte: Le syndrome d'Ehlers-Danlos vasculaire (SEDv) est une affection rare au pronostic sombre en raison des complications vasculaires qui y sont associées. Toutefois, les problèmes artériels les plus fréquents chez les patients atteints de SEDv sont mal connus. Méthodologie: Nous avons analysé de manière rétrospective les cas de 20 patients consécutifs ayant reçu un diagnostic de SEDv. Les données relatives aux manifestations cliniques, les résultats des examens d'imagerie et les traitements prescrits ont été examinés. Résultats: L'âge auquel la première complication nécessitant une hospitalisation est survenue était de 29 ± 13 ans. La période d'observation était de 67 ± 30 mois. Parmi ces 20 patients, 17 recevaient du céliprolol lors de l'évaluation finale. Lors de la dernière visite de suivi, le nombre total de complications associées aux lésions et ayant nécessité une hospitalisation comprenaient : 16 lésions pulmonaires (8 patients), 16 lésions intestinales (8 patients), 5 lésions tendineuses ou ligamentaires (2 patients), 18 complications touchant les ramifications (artères) de l'aorte abdominale (10 patients), 2 complications aortiques (2 patients) et 7 complications d'autres artères (6 patients). Sur les 54 atteintes artérielles (anévrismes, dissections et ruptures), qu'elles aient été symptomatiques ou pas, 43 (80 %) concernaient des ramifications de l'aorte abdominale (artère cœliaque et ses ramifications, 8; artère mésentérique supérieure, 4; artères rénales, 3; artère iliaque et ses ramifications, 28), 2 (4 %) concernaient l'aorte, et 9, d'autres artères. Le diamètre du sinus de Valsalva était de 29 ± 5 mm, soit dans les limites de la normale. Au cours de la période de suivi, 3 patients sont décédés en raison de ruptures suspectées d'une ramification de l'artère cœliaque, de l'artère mésentérique supérieure et de l'aorte. Conclusion: Les résultats de nos analyses indiquent que les lésions touchant des ramifications de l'aorte abdominale, plutôt que de l'aorte elle-même, étaient les types de lésions les plus fréquents chez les patients atteints de SEDv.

Outcomes of 8 Years of Noninvasive Prenatal Testing at Nippon Medical School Hospital.

BACKGROUND: Noninvasive prenatal testing (NIPT) is used to screen for fetal chromosomal abnormalities, such as fetal aneuploidy, and has been offered at our hospital since 2013. We analyzed data from our center to determine if NIPT screenees could be given more-accurate information on NIPT outcomes. METHODS: This retrospective observational study included 819 pregnant women who requested NIPT at Nippon Medical School Hospital from November 2013 to October 2021. We examined medical records for data on NIPT results and clinical outcomes. RESULTS: Of the 819 women, 764 (93.2%) underwent NIPT, and 55 (6.7%) did not. Of the 764 women who underwent NIPT, 17 received a positive result (2.2%), of whom 2 (11.8%), 4 (23.5%), and 11 (64.7%) received a positive result for trisomy 13, 18, and 21, respectively. The true-positive rates after definitive diagnoses of trisomy 13, 18, and 21 were 1 (50%), 3 (75%), and 11 (100%), respectively. Of the 17 positive results, there were two false-positive results (11.8%) (for trisomy 13 and trisomy 18). Eleven women with fetal aneuploidy terminated their pregnancies, and four cases resulted in intrauterine fetal death. Five neonates with negative NIPT results had congenital disease without chromosomal abnormality. Two patients had indeterminate results from the first blood sampling, possibly because of treatment with unfractionated heparin. The results of repeat testing after heparin cessation were negative. CONCLUSIONS: Our results were generally similar to nationwide data for Japan. NIPT providers can provide more detailed and individualized genetic counseling for each situation by understanding their own medical facility's data in detail.

A case of vascular Ehlers-Danlos syndrome with a ruptured hepatic artery after surgical treatment of peritonitis caused by the perforation of the colon.

BACKGROUND: Ehlers-Danlos syndrome (EDS) is an inherited disorder that causes connective tissue fragility. The vascular type of EDS (vEDS) caused by defective collagen type III production accounts for 5%-10% of all EDS cases. Patients can develop gastrointestinal or arterial ruptures, which cause poor prognosis. We report a case of a patient who experienced colonic rupture, which was immediately followed by arterial rupture. CASE PRESENTATION: A 40-year-old man who had been genetically diagnosed with vEDS 6 years previously was admitted to our hospital with ischemic colitis. After 3 days of conservative treatment, his abdominal pain worsened, and computed tomography (CT) revealed free air in the abdominal cavity. Pan-peritonitis due to perforation of the sigmoid colon was diagnosed. Intraperitoneal lavage and drainage and Hartmann's operation were urgently performed. Because the patient had confirmed vEDS, we performed the surgery in a protective manner. The postoperative course was initially good, and he was transferred to the general ward 3 days after surgery. However, 5 days after surgery, massive intra-abdominal hemorrhage suddenly occurred, and contrast-enhanced CT showed an aneurysm in the common hepatic artery that had ruptured; this aneurysm was not present before surgery and was far from the surgical field. Although we considered an emergency operation, the patient suddenly experienced cardiac arrest and was unresponsive to resuscitation. CONCLUSIONS: In cases of vEDS, vascular rupture can occur immediately after surgery for intestinal rupture. We recommend paying special attention to vascular complications in patients in their forties, as such complications are the most common causes of death.

Cathepsin D regulates cerebral Aß42/40 ratios via differential degradation of Aß42 and Aß40.

BACKGROUND: Cathepsin D (CatD) is a lysosomal protease that degrades both the amyloid ß-protein (Aß) and the microtubule-associated protein, tau, and has been genetically linked to late-onset Alzheimer disease (AD). Here, we sought to examine the consequences of genetic deletion of CatD on Aß proteostasis in vivo and to more completely characterize the degradation of Aß42 and Aß40 by CatD. METHODS: We quantified Aß degradation rates and levels of endogenous Aß42 and Aß40 in the brains of CatD-null (CatD-KO), heterozygous null (CatD-HET), and wild-type (WT) control mice. CatD-KO mice die by ~ 4 weeks of age, so tissues from younger mice, as well as embryonic neuronal cultures, were investigated. Enzymological assays and surface plasmon resonance were employed to quantify the kinetic parameters (KM, kcat) of CatD-mediated degradation of monomeric human Aß42 vs. Aß40, and the degradation of aggregated Aß42 species was also characterized. Competitive inhibition assays were used to interrogate the relative inhibition of full-length human and mouse Aß42 and Aß40, as well as corresponding p3 fragments. RESULTS: Genetic deletion of CatD resulted in 3- to 4-fold increases in insoluble, endogenous cerebral Aß42 and Aß40, exceeding the increases produced by deletion of an insulin-degrading enzyme, neprilysin or both, together with readily detectable intralysosomal deposits of endogenous Aß42-all by 3 weeks of age. Quite significantly, CatD-KO mice exhibited ~ 30% increases in Aß42/40 ratios, comparable to those induced by presenilin mutations. Mechanistically, the perturbed Aß42/40 ratios were attributable to pronounced differences in the kinetics of degradation of Aß42 vis-à-vis Aß40. Specifically, Aß42 shows a low-nanomolar affinity for CatD, along with an exceptionally slow turnover rate that, together, renders Aß42 a highly potent competitive inhibitor of CatD. Notably, the marked differences in the processing of Aß42 vs. Aß40 also extend to p3 fragments ending at positions 42 vs. 40. CONCLUSIONS: Our findings identify CatD as the principal intracellular Aß-degrading protease identified to date, one that regulates Aß42/40 ratios via differential degradation of Aß42 vs. Aß40. The finding that Aß42 is a potent competitive inhibitor of CatD suggests a possible mechanistic link between elevations in Aß42 and downstream pathological sequelae in AD.

Identification of BACE2 as an avid ß-amyloid-degrading protease.

BACKGROUND: Proteases that degrade the amyloid ß-protein (Aß) have emerged as key players in the etiology and potential treatment of Alzheimer's disease (AD), but it is unlikely that all such proteases have been identified. To discover new Aß-degrading proteases (AßDPs), we conducted an unbiased, genome-scale, functional cDNA screen designed to identify proteases capable of lowering net Aß levels produced by cells, which were subsequently characterized for Aß-degrading activity using an array of downstream assays. RESULTS: The top hit emerging from the screen was ß-site amyloid precursor protein-cleaving enzyme 2 (BACE2), a rather unexpected finding given the well-established role of its close homolog, BACE1, in the production of Aß. BACE2 is known to be capable of lowering Aß levels via non-amyloidogenic processing of APP. However, in vitro, BACE2 was also found to be a particularly avid AßDP, with a catalytic efficiency exceeding all known AßDPs except insulin-degrading enzyme (IDE). BACE1 was also found to degrade Aß, albeit ~150-fold less efficiently than BACE2. Aß is cleaved by BACE2 at three peptide bonds-Phe19-Phe20, Phe20-Ala21, and Leu34-Met35--with the latter cleavage site being the initial and principal one. BACE2 overexpression in cultured cells was found to lower net Aß levels to a greater extent than multiple, well-established AßDPs, including neprilysin (NEP) and endothelin-converting enzyme-1 (ECE1), while showing comparable effectiveness to IDE. CONCLUSIONS: This study identifies a new functional role for BACE2 as a potent AßDP. Based on its high catalytic efficiency, its ability to degrade Aß intracellularly, and other characteristics, BACE2 represents a particulary strong therapeutic candidate for the treatment or prevention of AD.

Comprehensive and innovative techniques for liver transplantation in rats: a surgical guide.

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 hemodynamic 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.

Designed inhibitors of insulin-degrading enzyme regulate the catabolism and activity of insulin.

BACKGROUND: Insulin is a vital peptide hormone that is a central regulator of glucose homeostasis, and impairments in insulin signaling cause diabetes mellitus. In principle, it should be possible to enhance the activity of insulin by inhibiting its catabolism, which is mediated primarily by insulin-degrading enzyme (IDE), a structurally and evolutionarily distinctive zinc-metalloprotease. Despite interest in pharmacological inhibition of IDE as an attractive anti-diabetic approach dating to the 1950s, potent and selective inhibitors of IDE have not yet emerged. METHODOLOGY/PRINCIPAL FINDINGS: We used a rational design approach based on analysis of combinatorial peptide mixtures and focused compound libraries to develop novel peptide hydroxamic acid inhibitors of IDE. The resulting compounds are approximately 10(6) times more potent than existing inhibitors, non-toxic, and surprisingly selective for IDE vis-à-vis conventional zinc-metalloproteases. Crystallographic analysis of an IDE-inhibitor complex reveals a novel mode of inhibition based on stabilization of IDE's "closed," inactive conformation. We show further that pharmacological inhibition of IDE potentiates insulin signaling by a mechanism involving reduced catabolism of internalized insulin. CONCLUSIONS/SIGNIFICANCE: The inhibitors we describe are the first to potently and selectively inhibit IDE or indeed any member of this atypical zinc-metalloprotease superfamily. The distinctive structure of IDE's active site, and the mode of action of our inhibitors, suggests that it may be possible to develop inhibitors that cross-react minimally with conventional zinc-metalloproteases. Significantly, our results reveal that insulin signaling is normally regulated by IDE activity not only extracellularly but also within cells, supporting the longstanding view that IDE inhibitors could hold therapeutic value for the treatment of diabetes.

Development of monoclonal antibodies and quantitative ELISAs targeting insulin-degrading enzyme.

BACKGROUND: Insulin-degrading enzyme (IDE) is a widely studied zinc-metalloprotease implicated in the pathogenesis of type 2 diabetes mellitus, Alzheimer disease (AD) and varicella zoster virus infection. Despite more than six decades of research on IDE, progress has been hampered by the lack of well-characterized reagents targeting this biomedically important protease. To address this important need, we generated and characterized new mouse monoclonal antibodies (mAbs) targeting natively folded human and rodent IDE. RESULTS: Eight monoclonal hybridoma cell lines were derived in house from mice immunized with full-length, natively folded, recombinant human IDE. The mAbs derived from these lines were shown to detect IDE selectively and sensitively by a wide range of methods. Two mAbs in particular-designated 6A1 and 6H9-proved especially selective for IDE in immunocytochemical and immunohistochemical applications. Using a variety of methods, we show that 6A1 selectively detects both human and rodent IDE, while 6H9 selectively detects human, but not rodent, IDE, with both mAbs showing essentially no cross reactivity with other proteins in these applications. Using these novel anti-IDE mAbs, we also developed sensitive and quantitative sandwich ELISAs capable of quantifying IDE levels present in human brain extracts. CONCLUSION: We succeeded in developing novel mAbs that selectively detect rodent and/or human IDE, which we have shown to be suitable for a wide range of applications, including western blotting, immunoprecipitation, immunocytochemistry, immunohistochemistry, and quantitative sandwich ELISAs. These novel anti-IDE mAbs and the assays derived from them constitute important new tools for addressing many unresolved questions about the basic biology of IDE and its role in multiple highly prevalent human diseases.

Colorectal cancer susceptibility associated with the hMLH1 V384D variant.

Lynch syndrome is an autosomal dominant colorectal cancer susceptibility syndrome caused by a dysfunction of DNA mismatch repair genes, including MLH1, MSH2, MSH6 and PMS2. However, the interpretation of certain changes in the mismatch repair genes is perplexing, as these changes do not necessarily affect the function of the protein. The pathogenicity of the hMLH1 1151T↷A variant, which results in an amino-acid substitution of valine for aspartic acid at codon 384 (V384D), is also controversial. This study was undertaken to assess the clinicopathological features of colorectal cancer patients harboring the hMLH1 V384D variant. Two independent Japanese cohorts, comprising 670 colorectal cancer patients and 332 cancer-free controls, respectively, were genotyped by polymerase chain reaction (PCR)-RFLP. The allele frequency of V384D was 0.75% in the control group and 3.1% in the colorectal cancer group (p<0.001). Thus, the V384D variant was associated with increased colorectal cancer susceptibility. However, only 5% of the colorectal cancer patients carrying the V384D variant had high micro-satellite instability; most had microsatellite-stable cancer. Additionally, these patients had no clear familial history of Lynch syndrome-related tumors. The combined results indicate that hMLH1 V384D allele frequency was 4.1-fold higher in the colorectal cancer group than in the control group. Thus, the hMLH1 V384D variant may contribute to the development of microsatellite-instable as well as -stable colorectal cancer.