Karima Schwab

Effects of oxidized and reduced forms of methylthioninium in two transgenic mouse tauopathy models

Given the repeated failure of amyloid-based approaches in Alzheimer’s disease, there is increasing interest in tau-based therapeutics. Although methylthioninium (MT) treatment was found to be beneficial in tau transgenic models, the brain concentrations required to inhibit tau aggregation in vivo are unknown. The comparative efficacy of methylthioninium chloride (MTC) and leucomethylthioninium salts (LMTX; 5–75 mg/kg; oral administration for 3–8 weeks) was assessed in two novel transgenic tau mouse lines. Behavioural (spatial water maze, RotaRod motor performance) and histopathological (tau load per brain region) proxies were applied. Both MTC and LMTX dose-dependently rescued the learning impairment and restored behavioural flexibility in a spatial problem-solving water maze task in Line 1 (minimum effective dose: 35 mg MT/kg for MTC, 9 mg MT/kg for LMTX) and corrected motor learning in Line 66 (effective doses: 4 mg MT/kg). Simultaneously, both drugs reduced the number of tau-reactive neurons, particularly in the hippocampus and entorhinal cortex in Line 1 and in a more widespread manner in Line 66. MT levels in the brain followed a sigmoidal concentration–response relationship over a 10-fold range (0.13–1.38 μmol/l). These data establish that diaminophenothiazine compounds, like MT, can reverse both spatial and motor learning deficits and reduce the underlying tau pathology, and therefore offer the potential for treatment of tauopathies.

Different pathways of molecular pathophysiology underlie cognitive and motor tauopathy phenotypes in transgenic models for Alzheimer's disease and frontotemporal lobar degeneration.

A poorly understood feature of the tauopathies is their very different clinical presentations. The frontotemporal lobar degeneration (FTLD) spectrum is dominated by motor and emotional/psychiatric abnormalities, whereas cognitive and memory deficits are prominent in the early stages of Alzheimer’s disease (AD). We report two novel mouse models overexpressing different human tau protein constructs. One is a full-length tau carrying a double mutation [P301S/G335D; line 66 (L66)] and the second is a truncated 3-repeat tau fragment which constitutes the bulk of the PHF core in AD corresponding to residues 296–390 fused with a signal sequence targeting it to the endoplasmic reticulum membrane (line 1; L1). L66 has abundant tau pathology widely distributed throughout the brain, with particularly high counts of affected neurons in hippocampus and entorhinal cortex. The pathology is neuroanatomically static and declines with age. Behaviourally, the model is devoid of a higher cognitive phenotype but presents with sensorimotor impairments and motor learning phenotypes. L1 displays a much weaker histopathological phenotype, but shows evidence of neuroanatomical spread and amplification with age that resembles the Braak staging of AD. Behaviourally, the model has minimal motor deficits but shows severe cognitive impairments affecting particularly the rodent equivalent of episodic memory which progresses with advancing age. In both models, tau aggregation can be dissociated from abnormal phosphorylation. The two models make possible the demonstration of two distinct but nevertheless convergent pathways of tau molecular pathogenesis. L1 appears to be useful for modelling the cognitive impairment of AD, whereas L66 appears to be more useful for modelling the motor features of the FTLD spectrum. Differences in clinical presentation of AD-like and FTLD syndromes are therefore likely to be inherent to the respective underlying tauopathy, and are not dependent on presence or absence of concomitant APP pathology.

Endothelin-1 overexpression restores diastolic function in eNOS knockout mice.

Background The cardiac nitric oxide and endothelin-1 (ET-1) systems are closely linked and play a critical role in cardiac physiology. The balance between both systems is often disturbed in cardiovascular diseases. To define the cardiac effect of excessive ET-1 in a status of nitric oxide deficiency, we compared left ventricular function and morphology in wild-type mice, ET-1 transgenic (ET+/+) mice, endothelial nitric oxide synthase knockout (eNOS−/−) mice, and ET+/+eNOS−/− mice. Methods and results eNOS−/− and ET+/+eNOS−/− mice developed high blood pressure compared with wild-type and ET+/+ mice. Left ventricular catheterization showed that eNOS−/− mice, but not ET+/+eNOS−/−, developed diastolic dysfunction characterized by increased end-diastolic pressure and relaxation constant tau. To elucidate the causal molecular mechanisms driving the rescue of diastolic function in ET+/+eNOS−/− mice, the cardiac proteome was analyzed. Two-dimensional gel electrophoresis coupled to mass spectrometry offers an appropriate hypothesis-free approach. ET-1 overexpression on an eNOS−/− background led to an elevated abundance and change in posttranslational state of antioxidant enzymes (e.g., peroxiredoxin-6, glutathione S-transferase mu 2, and heat shock protein beta 7). In contrast to ET+/+eNOS−/− mice, eNOS−/− mice showed an elevated abundance of proteins responsible for sarcomere disassembly (e.g., cofilin-1 and cofilin-2). In ET+/+eNOS−/− mice, glycolysis was favored at the expense of fatty acid oxidation. Conclusion eNOS−/− mice developed diastolic dysfunction; this was rescued by ET-1 transgenic overexpression. This study furthermore suggests that cardiac ET-1 overexpression in case of eNOS deficiency causes specifically the regulation of proteins playing a role in oxidative stress, myocytes contractility, and energy metabolism.

Dietary phytoestrogen supplementation induces sex differences in the myocardial protein pattern of mice: A comparative proteomics study

Elevated cardiovascular risk in postmenopausal women and beneficial actions of estrogen replacement in animal models have been related to protective effects of estrogens. However, randomized trials of hormone replacement therapy with synthetic estrogens in humans failed confirmation and phytoestrogens, natural plant hormones with agonistic properties for estrogen receptors, could represent potential alternatives. The aim of the present study is to characterize an animal model for alternative hormone replacement with genistein as a natural estrogenic compound. We performed a 2‐DE/ESI‐LC‐MS approach in order to identify protein species varying with genistein receipt and sex in their relative abundance in the healthy murine heart (http://www.mpiib‐berlin.mpg.de/2D‐PAGE). Oral genistein treatment revealed a substantial effect on the relative abundance of both estrogen receptors. Several enzymes of the fatty acid metabolism and their transcriptional regulators varied differentially in male and in female animals, at the transcript and/or the protein species level. Increased levels of enzyme species involved in the oxidative phosphorylation and generation of ROS were accompanied by decreased amounts of antioxidants in male mice receiving genistein compared with control males, which have been previously associated with various pathological conditions. Exposure of female animals to genistein provoked an increased abundance of two species of LIM domain‐binding protein and one species of desmin. These proteins have been associated with cardiac hypertrophy and our data warrant caution for the use of them as molecular markers, since the animals did not exhibit any histological signs of cardiac hypertrophy.

Dual feeding strategy for the production of α-amylase by Bacillus caldolyticus using complex media

In this study, the objective was to investigate an exponential feeding strategy for fed-batch production of thermostable alpha-amylase (E.C. 3.2.1.1.) from the Bacillus caldolyticus (DSM405). The parameters for establishing compositions of feed media and feeding rate were obtained by statistical analysis of batch and continuous shake flask experiments. These parameters were casitone to starch ratio of 2.67g(casitone)g(starch)(-1), maintenance coefficient 0.174g(casitone)g(DW)(-1)h(-1), cell yield 0.62g(DW)g(casitone)(-1) and mu(opt)=0.2h(-1). The exponentially fed fermentation resulted in yield of 120Uml(-1) alpha-amylase that was thermostable up to 105 degrees C. Results of the exponentially fed fermentation have been discussed in the light of a feed-back controlled fed-batch fermentation reported earlier by the authors. A comparison of the temperature and pH effects on amylase produced by B. caldolyticus and on several other commercially available amylases has also been presented.

Adaptation of proteomic techniques for the identification and characterization of protein species from murine heart

Disturbed energy metabolism with impaired fatty acid oxidation, ATP synthesis and changing levels of contractile proteins has been observed during the development and manifestation of cardiovascular diseases, with the latter showing sexual differences in terms of onset, manifestation and progress. Estrogenic compounds, such as estrogens and phytoestrogens, are known to exert beneficial effects on several cardiovascular parameters. However, global studies implying the normal, non-failing myocardium are rare. Thus, identifying and characterizing protein patterns involved in the maintenance of normal heart physiology at the protein species level will help understanding disease conditions. In this study, we performed an adapted 2-DE/MS approach in order to identify and characterize post-translational modified and truncated protein species from murine heart. Female and male animals of different age were receiving the phytoestrogen genistein and comparative analyses were performed to identify sex and genistein treatment-related effects. Selected 2-DE spots that exposed varying abundance between animal groups and identified as identical proteins were subject to multi-protease cleavage to generate an elevated sequence coverage enabling characterization of post-translational modifications and truncation loci via high-resolution MS. Several truncated, phosphorylated and acetylated species were identified for mitochondrial ATP synthase, malate dehydrogenase and trifunctional enzyme subunit alpha. However, confirmation of several of these modifications by manual spectra interpretation failed. Thus, our results warrant caution for the blind trust in software output. For the regulatory light chain of myosin, we identified an N-terminal processed species, which so far has been related to ischemic conditions only. We tried to unravel the information content of protein species separated by high-resolution 2-DE as an alternative to high-throughput proteomics, which mainly is interested in lists of protein names, ignoring the protein species identity.

Endothelin-1 overexpression and endothelial nitric oxide synthase knock-out induce different pathological responses in the heart of male and female mice

AIMS The nitric oxide and endothelin systems are key components of a local paracrine hormone network in the heart. We previously reported that diastolic dysfunction observed in mice lacking the endothelial nitric oxide synthase (eNOS-/-) can be prevented by a genetic overexpression of ET-1. Sexual dimorphisms have been reported in both ET-1 and NO systems. Particularly, eNOS-/- mice present sex related phenotypic differences. MAIN METHODS We used the ET-1 transgenic (ET+/+), eNOS-/-, and crossbred ET+/+eNOS-/- mice, and wild type controls. We measured cardiac function by heart catheterization. Cardiac ventricles were collected for histological and molecular profiling. KEY FINDINGS We report here that (i) the level of ET-1 expression in eNOS-/- mice was elevated in males but not in females. (ii) Left ventricular end-diastolic blood pressure was higher in male eNOS-/- mice than in females. (ii) eNOS-/- males but not females developed cardiomyocyte hypertrophy. (iv) Perivascular fibrosis of intracardiac arteries developed in female ET+/+ and eNOS-/- mice but not in males. Additionally, (v) the cardiac expression of metalloprotease-9 was higher in eNOS-/- males compared to females. Finally, (vi) cardiac proteome analysis revealed that the protein abundance of the oxidative stress related enzyme superoxide dismutase presented with sexual dimorphism in eNOS-/- and ET+/+ mice. SIGNIFICANCE These results indicate that the cardiac phenotypes of ET-1 transgenic mice and eNOS knockout mice are sex specific. Since both systems are key players in the pathogenesis of cardiovascular diseases, our findings might be important in the context of gender differences in patients with such diseases.

Dietary genistein enhances phosphorylation of regulatory myosin light chain in the myocardium of ovariectomized mice

There is evidence that isoflavones, such as genistein, can directly or indirectly improve lipid profile and lower blood pressure and hence exert cardiovascular protection. It is further believed, that genistein attenuates vascular contraction and thus vascular tone and blood pressure through altering the phosphorylation of the regulatory myosin light chain (MLC) probably via the myosin light chain kinase (MLCK) or the RhoA pathway. However, the direct role of genistein in the myocardium is poorly reviewed. In this study, we investigated the impact of genistein on the cardiac proteome in ovariectomized female mice using a 2DE‐MS approach. Dietary genistein intake considerably changed the abundance of several cytoskeletal and contractile proteins and enhanced the phosphorylation of MLC. The MLC phosphorylation was mediated through increased abundance of MLCK and inhibition of myosin light chain phosphatase latest known to be inversely regulated by RhoA. Contrary to others, in our model genistein did neither inhibit the cardiac MLCK, nor the cardiac RhoA pathway in vivo.

Alpha-Synuclein transgenic mice, h-α-SynL62, display α-Syn aggregation and a dopaminergic phenotype reminiscent of Parkinson's disease

HIGHLIGHTSTransgenic mice overexpressing human &agr;‐Syn under the control of the mouse Thy1‐promotor.Three transgenic lines express similar levels of &agr;‐Syn mRNA but a different number of cells expressing human &agr;‐syn.L62 has greatest level of aggregated &agr;‐Syn protein throughout brain and spinal cord and most severe motor phenotype.L62 mice display dopaminergic transmission deficits and altered D1 receptor function.L62 presents a model to study motor changes associated with Parkinsons disease. ABSTRACT Alpha‐Synuclein (&agr;‐Syn) accumulation is considered a major risk factor for the development of synucleinopathies such as Parkinsons disease (PD) and dementia with Lewy bodies. We have generated mice overexpressing full‐length human &agr;‐Syn fused to a membrane‐targeting signal sequence under the control of the mouse Thy1‐promotor. Three separate lines (L56, L58 and L62) with similar gene expression levels, but considerably heightened protein accumulation in L58 and L62, were established. In L62, there was widespread labelling of &agr;‐Syn immunoreactivity in brain including spinal cord, basal forebrain, cortex and striatum. Interestingly, there was no detectable &agr;‐Syn expression in dopaminergic neurones of the substantia nigra, but strong human &agr;‐Syn reactivity in glutamatergic synapses. The human &agr;‐Syn accumulated during aging and formed PK‐resistant, thioflavin‐binding aggregates. Mice displayed early onset bradykinesia and age progressive motor deficits. Functional alterations within the striatum were confirmed: L62 showed normal basal dopamine levels, but impaired dopamine release (upon amphetamine challenge) in the dorsal striatum measured by in vivo brain dialysis at 9 months of age. This impairment was coincident with a reduced response to amphetamine in the activity test. L62 further displayed greater sensitivity to low doses of the dopamine receptor 1 (D1) agonist SKF81297 but reacted normally to the D2 agonist quinpirole in the open field. Since accumulation of &agr;‐Syn aggregates in neurones and synapses and alterations in the dopaminergic tone are characteristics of PD, phenotypes reported for L62 present a good opportunity to further our understanding of motor dysfunction in PD and Lewy body dementia.

A Protein Aggregation Inhibitor, Leuco-Methylthioninium Bis(Hydromethanesulfonate), Decreases α-Synuclein Inclusions in a Transgenic Mouse Model of Synucleinopathy

α-Synuclein (α-Syn) aggregation is a pathological feature of synucleinopathies, neurodegenerative disorders that include Parkinson’s disease (PD). We have tested whether N,N,N′,N′-tetramethyl-10H-phenothiazine-3,7-diaminium bis(hydromethanesulfonate) (leuco-methylthioninium bis(hydromethanesulfonate); LMTM), a tau aggregation inhibitor, affects α-Syn aggregation in vitro and in vivo. Both cellular and transgenic models in which the expression of full-length human α-Syn (h-α-Syn) fused with a signal sequence peptide to promote α-Syn aggregation were used. Aggregated α-Syn was observed following differentiation of N1E-115 neuroblastoma cells transfected with h-α-Syn. The appearance of aggregated α-Syn was inhibited by LMTM, with an EC50 of 1.1 μM, with minimal effect on h-α-Syn mRNA levels being observed. Two independent lines of mice (L58 and L62) transgenic for the same fusion protein accumulated neuronal h-α-Syn that, with aging, developed into fibrillary inclusions characterized by both resistance to proteinase K (PK)-cleavage and their ability to bind thiazin red. There was a significant decrease in α-Syn-positive neurons in multiple brain regions following oral treatment of male and female mice with LMTM administered daily for 6 weeks at 5 and 15 mg MT/kg. The early aggregates of α-Syn and the late-stage fibrillar inclusions were both susceptible to inhibition by LMTM, a treatment that also resulted in the rescue of movement and anxiety-related traits in these mice. The results suggest that LMTM may provide a potential disease modification therapy in PD and other synucleinopathies through the inhibition of α-Syn aggregation.