Distinct Aβ pathology in the olfactory bulb and olfactory deficits in a mouse model of Aβ and α‐syn co‐pathology

Several degenerative brain disorders such as Alzheimer''s disease (AD), Parkinson''s disease (PD) and Dementia with Lewy bodies (DLB) are characterized by the simultaneous appearance of amyloid‐β (Aβ) and α‐synuclein (α‐syn) pathologies and symptoms that are similar, making it difficult to differentiate between these diseases. Until now, an accurate diagnosis can only be made by postmortem analysis. Furthermore, the role of α‐syn in Aβ aggregation and the arising characteristic olfactory impairments observed during the progression of these diseases is still not well understood. Therefore, we assessed Aβ load in olfactory bulbs of APP‐transgenic mice expressing APP695 ^KM670/671NL and PSEN1 ^L166P under the control of the neuron‐specific Thy‐1 promoter (referred to here as APPPS1) and APPPS1 mice co‐expressing SNCA ^A30P (referred to here as APPPS1 × [A30P]aSYN). Furthermore, the olfactory capacity of these mice was evaluated in the buried food and olfactory avoidance test. Our results demonstrate an age‐dependent increase in Aβ load in the olfactory bulb of APP‐transgenic mice that go along with exacerbated olfactory performance. Our study provides clear evidence that the presence of α‐syn significantly diminished the endogenous and seed‐induced Aβ deposits and significantly ameliorated olfactory dysfunction in APPPS1 × [A30P]aSYN mice.     

VKORC1 −1639G>A and CYP2C9*3 are the major genetic predictors of phenprocoumon dose requirement

Purpose

Phenprocoumon, similar to other coumarin-derived anticoagulants, is associated with a large variation in the individual dose requirement to achieve stable anticoagulation. Polymorphisms in the vitamin K epoxide reductase complex subunit 1 (VKORC1) and the liver enzyme cytochrome P450 2C9 (CYP2C9) effectively account for the variability in warfarin and acenocoumarol response but are less well-defined pharmacogenetic predictors in phenprocoumon therapy.

Methods

A retrospective study was performed on 185 outpatients attending anticoagulation clinics in Austria and Germany. These patients were genotyped for the VKORC1 ?1639G>A and 3730G>A polymorphisms as well as for the CYP2C9 *2 and *3 polymorphisms using a reverse hybridisation-based teststrip assay.

Results

The VKORC1 ?1639A allele, which was present at a frequency of 41.4% in the study cohort, significantly reduced the mean weekly phenprocoumon dose by 3 mg (19%) in the heterozygous and by 6.7 mg (43%) in the homozygous state compared to wild-type carriers (15.5?±?6.8 mg, p?<?0.0001). A stepwise multiple regression analysis revealed that VKORC1 ?1639G>A, age and CYP2C9*3 were the major independent determinants of phenprocoumon dose, accounting for 14.2, 9.1 and 4.7% of its variability, respectively (p?≤?0.0007). The CYP2C9*2 polymorphism had a marginal influence (1.4%) and failed to reach statistical significance (p?=?0.062). The VKORC1 3730G>A genotype had no additional predictive power for individual dose variability.

Conclusion

Similar to warfarin and acenocoumarol, the VKORC1 ?1639G>A polymorphism had the highest impact on the maintenance dose of phenprocoumon. The factor age was the second most important predictor and explained a greater percentage of the variability than CYP2C9 genotype.     

Selective degeneration of septal and hippocampal GABAergic neurons in a mouse model of amyloidosis and tauopathy

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by brain accumulation of amyloid-β peptide and neurofibrillary tangles, which are believed to initiate a pathological cascade that results in progressive impairment of cognitive functions and eventual neuronal death. To obtain a mouse model displaying the typical AD histopathology of amyloidosis and tauopathy, we generated a triple-transgenic mouse line (TauPS2APP) by overexpressing human mutations of the amyloid precursor protein, presenilin2 and tau genes. Stereological analysis of TauPS2APP mice revealed significant neurodegeneration of GABAergic septo-hippocampal projection neurons as well as their target cells, the GABAergic hippocampal interneurons. In contrast, the cholinergic medial septum neurons remained unaffected. Moreover, the degeneration of hippocampal GABAergic interneurons was dependent on the hippocampal subfield and interneuronal subtype investigated, whereby the dentate gyrus and the NPY-positive interneurons, respectively, were most strongly affected. Neurodegeneration was also accompanied by a change in the mRNA expression of markers for inhibitory interneurons. In line with the loss of inhibitory neurons, we observed functional changes in TauPS2APP mice relative to WT mice, with strongly enhanced long-term potentiation in the medial-perforant pathway input to the dentate gyrus, and stereotypic hyperactivity. Our data indicate that inhibitory neurons are the targets of neurodegeneration in a mouse model of amyloidosis and tauopathy, thus pointing to a possible role of the inhibitory network in the pathophysiological and functional cascade of Alzheimer's disease.     

Comparison of Two Different Blood Sample Tubes for Platelet Function Analysis with the Multiplate® System

BACKGROUND: For whole blood aggregation on the Multiplate? system, the use of thrombin inhibitor as anticoagulant is recommended. So far sample tubes containing liquid lepirudin were provided (Dynabyte, Munich, Germany). They are not sterile and have to be stored refrigerated. For better handling now also sterile tubes with dried hirudin, storable at room temperature, are also provided (Dynabyte). The aim of this study was to compare the performance of these both sample tubes on the Multiplate system. PATIENTS AND METHODS: Blood was collected from 30 patients, treated with aspirin and/or clopidogrel, in tubes, and ASPItest, ADPtest and TRAPtest were performed. RESULTS: The correlation between both sample tubes was excellent with ASPItest (r(2) = 0.96), good with ADPtest (r(2) = 0.91) and also satisfying with TRAPtest (r(2) = 0.85). CONCLUSION: Both sample tubes are well qualified for platelet function analysis with the Multiplate system. The new sterile sample tube with dried hirudin may be preferred for better handling.     

First workshop for detection of heparin-induced antibodies: validation of the heparin-induced platelet-activation test (HIPA) in comparison with a PF4/heparin ELISA

BACKGROUND: No data exist regarding the inter-laboratory reproducibility of the heparin-induced-platelet-activation (HIPA) test, the most widely used functional assay in Germany for the detection of heparin-induced thrombocytopenia (HIT) antibodies. METHODS: Nine laboratories used an identical protocol to test eight different sera with the HIPA test. Five laboratories also tested the sera with a platelet factor 4 (PF4)/heparin-complex ELISA. Cross-reactivity with danaparoid-sodium was assessed using 0.2 aFXa units instead of heparin in the HIPA test. RESULTS: Two of nine laboratories had no discrepant HIPA test results. Four laboratories differed in one sample, one reported two discrepant results, and two laboratories reported more than two discrepant results. Cross-reactivity with danaparoid-sodium test results differed among laboratories. PF4/heparin ELISA results were identical in all five laboratories. CONCLUSION: The HIPA test requires strict quality control measures. Using both a sensitive functional assay (HIPA test) and a PF4/heparin ELISA will allow detection of antibodies directed to antigens other than PF4/heparin complexes as well as detection of IgM and IgA antibodies with PF4/heparin specificity.     

Epigenetic reprogramming by tumor-derived EZH2 gain-of-function mutations promotes aggressive 3D cell morphologies and enhances melanoma tumor growth

In addition to genetic alterations, cancer cells are characterized by myriad epigenetic changes. EZH2 is a histone methyltransferase that is over-expressed and mutated in cancer. The EZH2 gain-of-function (GOF) mutations first identified in lymphomas have recently been reported in melanoma (~2%) but remain uncharacterized. We expressed multiple EZH2 GOF mutations in the A375 metastatic skin melanoma cell line and observed both increased H3K27me3 and dramatic changes in 3D culture morphology. In these cells, prominent morphological changes were accompanied by a decrease in cell contractility and an increase in collective cell migration. At the molecular level, we observed significant alteration of the axonal guidance pathway, a pathway intricately involved in the regulation of cell shape and motility. Furthermore, the aggressive 3D morphology of EZH2 GOF-expressing melanoma cells (both endogenous and ectopic) was attenuated by EZH2 catalytic inhibition. Finally, A375 cells expressing exogenous EZH2 GOF mutants formed larger tumors than control cells in mouse xenograft studies. This study not only demonstrates the first functional characterization of EZH2 GOF mutants in non-hematopoietic cells, but also provides a rationale for EZH2 catalytic inhibition in melanoma.     

Fibrinogen Kaiserslautern (γ 380 Lys to Asn): a new glycosylated fibrinogen variant with delayed polymerization

An adult woman diagnosed with cerebral thrombosis following a caesarean section was found to have severely prolonged thrombin and reptilase times. Five other family members also had prolonged, but variable, thrombin and reptilase times. Analysis of purified fibrinogen on reducing SDS-PAGE revealed an additional band, in all family members, which migrated immediately below the normal Bβ band. Western blotting indicated that this band was a gamma chain and endoglycosidase-F digestion established that it contained an additional oligosaccharide side chain. Partial acid hydrolysis localized the new oligosaccharide to the C-terminus of the gamma chain. Amplification of this region by PCR and subsequent DNA sequencing demonstrated a single base substitution altering the normal 380 Lys (AAG) codon to Asn (AAT), producing a new Asn-Lys-Thr glycosylation site. The propositus and one other family member were homozygous for this mutation but the remaining four family members were heterozygous. The polymerization of purified fibrin monomers from the propositus was grossly abnormal; however, the polymerization curve was almost normalized by the removal of terminal sialic acid residues. This suggests that the polymerization defect was primarily caused by additional negatively charged sialic acid residues present on the new oligosaccharide. Further analysis of the D domain of purified fibrinogen established that calcium binding to the high affinity site remained unaffected by the bulky carbohydrate side chain or negatively charged sialic acid residues.     

Messungen des tubulären Harnstromes,seine Beziehungen zum Blutdruck und zur Inulin-Clearance

Zusammenfassung An Nembutal-narkotisierten Ratten wurde eine kontinuierliche Zunahme aller tubulären Passagezeiten von Lissamingrün (LG) bei arterieller Blutdrucksenkung (Entblutungshypotonie) gefunden. Bei einem Mitteldruck von 60 mm Hg hat die proximale Passagezeit um den dreifachen, die distale Passagezeit um den zehnfachen Betrag zugenommen.An jungen Katzen beträgt die Passagezeit von LG für den an der Nierenoberfläche sichtbaren Anteil des proximalen Konvolutes im Mittel 7,5 sec, die Schleifen-Passagezeit 35 sec und die distale Passagezeit 41 sec. Im Mitteldruckbereich unter 80 mm Hg sind bei Katzen wiederum alle Passagezeiten verlängert (distal > proximal), während die Passagezeit oberhalb dieses Wertes vom Druck unabhängig erscheint. Bei Blutdrucksenkungen sind neben der bekannten Einschränkung der Inulin-Clearance und der Diurese auch Abnahmen der proximalen Tubulusdurchmesser zu beobachten. Eine Abhängigkeit zwischen proximaler Stromstärke und arteriellem Mitteldruck findet sich nur im Druckbereich unter 80 mm Hg. Zwischen proximaler Stromstärke und Inulinclearance besteht eine fast lineare Korrelation. Aus direkten Strömungsgeschwindigkeits-Messungen und der proximalen Passagezeit wird die mittlere Länge des oberflächlich sichtbaren Anteiles eines proximalen Konvolutes abgeleitet, sowie der Nettowasserfluß für die innere Tubulusoberfläche berechnet.In der Diskussion wird das Problem der glomerulär-tubulären Balance erörtert. Unsere Ergebnisse scheinen die Hypothese von Gertz zu bestätigen. Ferner werden aus der Schleifen-Passagezeit und morphologischen Daten Strömungsgeschwindigkeiten in dem für die Harnkonzentrierung wichtigsten Bereich des Nierenmarkes berechnet und die Bedeutung von Strömungsbeschleunigungen für die Harnkonzentrierung diskutiert.

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Summary When the blood-pressure of rats is reduced by bleeding, we found a continous increase of all the tubular passage-times of Lissamingreen measured on the renal surface by incident-light microscopy. With a blood-pressure of 60 mm Hg the proximal passage-time of Lissamingreen was three-times the normal and the distal passage-time ten-times the normal.On young cats with normal blood-pressure Lissamingreen took an average of 7,5 sec to pass through the visible part of the proximal convolution; the average times taken to pass through the Henle-loops and the distal convolution were 35 sec and 41 sec respectively.As in the case of rats all passage-times increased when the mean blood-pressure was less than 80 mm Hg (the distal passage-time increased more than the proximal passage-time), whereas the proximal passage-time on higher values was not affected by pressure. This corresponds to the so-called autoregulation of the kidney. When the blood-pressure is reduced, a decrease of the diameter of the proximal tubules can be seen in addition to the known reduction in the inulin-clearance and diuresis. The correlation between the inulin-clearance and the diameter of the tubules is then at its most obvious. There is only a connection between the proximal tubular volume-flow and the mean blood-pressure when the pressure is below 80 mm Hg. However there is almost a linear correlation between the proximal tubular volume-flow and the inulin-clearance.The average length of the part of a proximal convolution visible on the surface is calculated from direct measurements of the flow-velocity and the passage-time at 2,6 mm. With those length an average proximal tubular volume-flow of 1.2±0.07 · 10^–4 mm³ · sec^–1 is to be found withCin>3.0 ml · min^–1 · kg^–1. OnCin 2.1 to 2.9 ml · min^–1 · kg^–1 the volume-flow is 8.9±0.7 · 10^–5 mm³ · sec^–1 and onCin<2.0 the volume-flow is 3.9±0.9 · 10^–5 mm³ · sec^–1.A net water reabsorption for internal tubular surface of approximately 5.0·10^–4 mm³ · mm^–2 is calculated from the decrease of the flow-velocity in the proximal convolution.The flow-velocity in the descending and ascending limb of the Henle-loop was calculated from the passage-times for the loops and from morphological data.The question of the glomerular-tubular balance was discussed and the conclusions tended to support the Gertz-theory.

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Mit 8 Textabbildungen

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Mit Unterstützung der Deutschen Forschungsgemeinschaft.

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Auszugsweise vorgetragen auf der 29. Tagung der Deutschen Physiologischen Gesellschaft, Tübingen 1964²⁹.

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Kir. klin. Sahlgrenska Sjukhuset, Universität Göteborg/Schweden.