Methionine Sources Differently Affect Production of Reactive Oxygen Species, Mitochondrial Bioenergetics, and Growth of Murine and Quail Myoblasts In Vitro.

An optimal supply of L-methionine (L-Met) improves muscle growth, whereas over-supplementation exerts adverse effects. To understand the underlying mechanisms, this study aims at exploring effects on the growth, viability, ROS production, and mitochondrial bioenergetics of C2C12 (mouse) and QM7 (quail) myoblasts additionally supplemented (100 or 1000 µM) with L-Met, DL-methionine (DL-Met), or DL-2-hydroxy-4-(methylthio)butanoic acid (DL-HMTBA). In both cell lines, all the supplements stimulated cell growth. However, in contrast to DL-Met, 1000 µM of L-Met (C2C12 cells only) or DL-HMTBA started to retard growth. This negative effect was stronger with DL-HMTBA and was accompanied by significantly elevated levels of extracellular H2O2, an indicator for OS, in both cell types. In addition, oversupplementation with DL-HMTBA (1000 µM) induced adaptive responses in mitochondrial bioenergetics, including reductions in basal (C2C12 and QM7) and ATP-synthase-linked (C2C12) oxygen consumption, maximal respiration rate, and reserve capacity (QM7). Only QM7 cells switched to nonmitochondrial aerobic glycolysis to reduce ROS production. In conclusion, we found a general negative effect of methionine oversupplementation on cell proliferation. However, only DL-HMTBA-induced growth retardation was associated with OS and adaptive, species-specific alterations in mitochondrial functionality. OS could be better compensated by quail cells, highlighting the role of species differences in the ability to cope with methionine oversupplementation.

Stroke target identification guided by astrocyte transcriptome analysis.

Astrocytes support normal brain function, but may also contribute to neurodegeneration when they become reactive under pathological conditions such as stroke. However, the molecular underpinnings of this context-dependent interplay between beneficial and detrimental properties in reactive astrogliosis have remained incompletely understood. Therefore, using the RiboTag technique, we immunopurified translating mRNAs specifically from astrocytes 72 hr after transient middle cerebral artery occlusion in mice (tMCAO), thereby generating a stroke-specific astroglial translatome database. We found that compared to control brains, reactive astrocytes after tMCAO show an enrichment of transcripts linked to the A2 phenotype, which has been associated with neuroprotection. However, we found that astrocytes also upregulate a large number of potentially neurotoxic genes. In total, we identified the differential expression of 1,003 genes and 38 transcription factors, of which Stat3, Sp1, and Spi1 were the most prominent. To further explore the effects of Stat3-mediated pathways on stroke pathogenesis, we subjected mice with an astrocyte-specific conditional deletion of Stat3 to tMCAO, and found that these mice have reduced stroke volume and improved motor outcome 72 hr after focal ischemia. Taken together, our study extends the emerging database of novel astrocyte-specific targets for stroke therapy, and supports the role of astrocytes as critical safeguards of brain function in health and disease.

ABC Transport Proteins in Cardiovascular Disease-A Brief Summary.

Adenosine triphosphate (ATP)-binding cassette (ABC) transporters may play an important role in the pathogenesis of atherosclerotic vascular diseases due to their involvement in cholesterol homeostasis, blood pressure regulation, endothelial function, vascular inflammation, as well as platelet production and aggregation. In this regard, ABC transporters, such as ABCA1, ABCG5 and ABCG8, were initially found to be responsible for genetically-inherited syndromes like Tangier diseases and sitosterolemia. These findings led to the understanding of those transporter's function in cellular cholesterol efflux and thereby also linked them to atherosclerosis and cardiovascular diseases (CVD). Subsequently, further ABC transporters, i.e., ABCG1, ABCG4, ABCB6, ABCC1, ABCC6 or ABCC9, have been shown to directly or indirectly affect cellular cholesterol efflux, the inflammatory response in macrophages, megakaryocyte proliferation and thrombus formation, as well as vascular function and blood pressure, and may thereby contribute to the pathogenesis of CVD and its complications. Furthermore, ABC transporters, such as ABCB1, ABCC2 or ABCG2, may affect the safety and efficacy of several drug classes currently in use for CVD treatment. This review will give a brief overview of ABC transporters involved in the process of atherogenesis and CVD pathology. It also aims to briefly summarize the role of ABC transporters in the pharmacokinetics and disposition of drugs frequently used to treat CVD and CVD-related complications.

Accumulation of murine amyloid-ß mimics early Alzheimer's disease.

Amyloidosis mouse models of Alzheimer's disease are generally established by transgenic approaches leading to an overexpression of mutated human genes that are known to be involved in the generation of amyloid-ß in Alzheimer's families. Although these models made substantial contributions to the current knowledge about the 'amyloid hypothesis' of Alzheimer's disease, the overproduction of amyloid-ß peptides mimics only inherited (familiar) Alzheimer's disease, which accounts for <1% of all patients with Alzheimer's disease. The inherited form is even regarded a 'rare' disease according to the regulations for funding of the European Union (www.erare.eu). Here, we show that mice that are double-deficient for neprilysin (encoded by Mme), one major amyloid-ß-degrading enzyme, and the ABC transporter ABCC1, a major contributor to amyloid-ß clearance from the brain, develop various aspects of sporadic Alzheimer's disease mimicking the clinical stage of mild cognitive impairment. Using behavioural tests, electrophysiology and morphological analyses, we compared different ABC transporter-deficient animals and found that alterations are most prominent in neprilysin × ABCC1 double-deficient mice. We show that these mice have a reduced probability to survive, show increased anxiety in new environments, and have a reduced working memory performance. Furthermore, we detected morphological changes in the hippocampus and amygdala, e.g. astrogliosis and reduced numbers of synapses, leading to defective long-term potentiation in functional measurements. Compared to human, murine amyloid-ß is poorly aggregating, due to changes in three amino acids at N-terminal positions 5, 10, and 13. Interestingly, our findings account for the action of early occurring amyloid-ß species/aggregates, i.e. monomers and small amyloid-ß oligomers. Thus, neprilysin × ABCC1 double-deficient mice present a new model for early effects of amyloid-ß-related mild cognitive impairment that allows investigations without artificial overexpression of inherited Alzheimer's disease genes.

Homer 1 genotype AA variant relates to congenital splay leg syndrome in piglets by repressing Pax7 in myogenic progenitors.

Introduction: Porcine congenital splay leg syndrome (PCS) is a major birth defect in piglets, resulting in lameness and high mortality rates. The multifactorial pathogenesis of PSC is not well understood but includes a polygenic inheritance. Methods: Here, in addition to morphological investigations, we characterized the expression of myogenic genes and functional (proliferation and differentiation) properties of myogenic precursor/satellite cells (SATCs) in 1 day-old PCS piglets, non-affected littermates (LCs), and piglets from PCS-free healthy litters (HCs). In addition, PCS phenotypes were related to the SNP Homer1_rs325197091 within the Homer1 locus, which has been identified as a potential hereditary cause of PCS. Results and discussion: Samples from musculus semitendinosus (ST) of PCS piglets had a higher proportion of type II fibers, reflecting myofiber immaturity. In addition, myofiber atrophy, a lower number of myonuclei per fiber (ST), and a higher apoptotic activity (in ST and longissimus dorsi muscle; LD) were found in the PCS group. A higher proportion of cycling committed myoblasts (Pax7+/Ki67+ cells) occurred in samples from PCS-affected piglets, and on the other hand, the mRNA expression of genes involved in differentiation (muscle differentiation 1; MyoD, myogenin; MyoG) was repressed compared with HCs. Cultured SATCs from PCS-affected animals showed a temporal shift in peak expression of Pax7, MyoD, and MyoG toward days 3 and 4 of their 7 days differentiation regime. In vitro experiments with isolated SATCs confirmed the lower differentiation potential and the delayed progression of the myogenic processes in cells from piglets with PCS phenotype. In addition, Pax7 and desmin were differently expressed in Homer1_rs325197091 genotype variants (GG, GA, and AA). Both genes showed the lowest expression in the homozygous AA-variant, which was most frequently found in PCS-affected animals. The homozygous AA-variant was also associated with lower expression of the truncated Homer1-subtype 205. Thus, we hypothesize that in PCS, the balance between Homer1 proteins and its signaling functions is changed in a way detrimental to the myogenic differentiation program. Our results demonstrated direct negative effects of the Homer1 AA genotype on Pax7 expression, but the exact mode of action still needs to be elucidated.

Congenital Splay Leg Syndrome in Piglets-Current Knowledge and a New Approach to Etiology.

The porcine congenital splay leg syndrome (PCS), even though being of transient nature, is still one of the most important causes for piglet losses due to its high incidence and mortality. Although, described decades ago, the pathogenetic mechanism is still elusive. Numerous, mostly descriptive studies characterized the syndrome at clinical, histological and cellular levels but resulted in a highly diverse picture of the syndrome. Broad variability in phenotypical expression and, in case of proper care, the rapid recovery of affected animals complicated a systematical analysis of the underlying pathogenesis. Although, several environmental factors were discussed as potential causes of PCS, most of the evidence points to a hereditary basis of PCS. Nevertheless, only few of the suggested candidate genes from transcriptome and mapping analyses, like F-box protein 32 (FBXO32), could be confirmed so far. Only recently, a genome wide association study revealed genomic regions on five porcine chromosomes and named a number of potential candidate genes, among them homer scaffold protein 1 (HOMER1). This new candidate-a cellular scaffold protein-plays a role in a plethora of cellular signaling cascades, and is not only involved in skeletal muscle differentiation but also critical for muscular function. In this review, we critically elucidate the current state of knowledge in the field and evaluate current achievements in the identification of the pathogenetic mechanism for the syndrome.

DNp73-induced degradation of tyrosinase links depigmentation with EMT-driven melanoma progression.

Melanoma is an aggressive cancer with poor prognosis, requiring personalized management of advanced stages and establishment of molecular markers. Melanomas derive from melanocytes, which specifically express tyrosinase, the rate-limiting enzyme of melanin-synthesis. We demonstrate that melanomas with high levels of DNp73, a cancer-specific variant of the p53 family member p73 and driver of melanoma progression show, in contrast to their less-aggressive low-DNp73 counterparts, hypopigmentation in vivo. Mechanistically, reduced melanin-synthesis is mediated by a DNp73-activated IGF1R/PI3K/AKT axis leading to tyrosinase ER-arrest and proteasomal degradation. Tyrosinase loss triggers reactivation of the EMT signaling cascade, a mesenchymal-like cell phenotype and increased invasiveness. DNp73-induced depigmentation, Slug increase and changes in cell motility are recapitulated in neural crest-derived melanophores of Xenopus embryos, underscoring a previously unnoticed physiological role of tyrosinase as EMT inhibitor. This data provides a mechanism of hypopigmentation accompanying cancer progression, which can be exploited in precision diagnosis of patients with melanoma-associated hypopigmentation (MAH), currently seen as a favorable prognostic factor. The DNp73/IGF1R/Slug signature in colorless lesions might aid to clinically discriminate between patients with MAH-associated metastatic disease and those, where MAH is indeed a sign of regression.

Drug Repositioning Inferred from E2F1-Coregulator Interactions Studies for the Prevention and Treatment of Metastatic Cancers.

Metastasis management remains a long-standing challenge. High abundance of E2F1 triggers tumor progression by developing protein-protein interactions (PPI) with coregulators that enhance its potential to activate a network of prometastatic transcriptional targets. Methods: To identify E2F1-coregulators, we integrated high-throughput Co-immunoprecipitation (IP)/mass spectometry, GST-pull-down assays, and structure modeling. Potential inhibitors of PPI discovered were found by bioinformatics-based pharmacophore modeling, and transcriptome profiling was conducted to screen for coregulated downstream targets. Expression and target gene regulation was validated using qRT-PCR, immunoblotting, chromatin IP, and luciferase assays. Finally, the impact of the E2F1-coregulator complex and its inhibiting drug on metastasis was investigated in vitro in different cancer entities and two mouse metastasis models. Results: We unveiled that E2F1 forms coactivator complexes with metastasis-associated protein 1 (MTA1) which, in turn, is directly upregulated by E2F1. The E2F1:MTA1 complex potentiates hyaluronan synthase 2 (HAS2) expression, increases hyaluronan production and promotes cell motility. Disruption of this prometastatic E2F1:MTA1 interaction reduces hyaluronan synthesis and infiltration of tumor-associated macrophages in the tumor microenvironment, thereby suppressing metastasis. We further demonstrate that E2F1:MTA1 assembly is abrogated by small-molecule, FDA-approved drugs. Treatment of E2F1/MTA1-positive, highly aggressive, circulating melanoma cells and orthotopic pancreatic tumors with argatroban prevents metastasis and cancer relapses in vivo through perturbation of the E2F1:MTA1/HAS2 axis. Conclusion: Our results propose argatroban as an innovative, E2F-coregulator-based, antimetastatic drug. Cancer patients with the infaust E2F1/MTA1/HAS2 signature will likely benefit from drug repositioning.

Sideritis spp. Extracts Enhance Memory and Learning in Alzheimer's ß-Amyloidosis Mouse Models and Aged C57Bl/6 Mice.

Nowadays, Alzheimer's disease is the most prevalent epiphenomenon of the aging population. Although soluble amyloid-ß (Aß) species (monomers, oligomers) are recognized triggers of the disease, no therapeutic approach is able to stop it. Herbal medicines are used to treat different diseases in many regions of the world. On the Balkan Peninsula, at the eastern Mediterranean Sea, and adjacent regions, Sideritis species are used as traditional medicine to prevent age-related problems in elderly. To evaluate this traditional knowledge in controlled experiments, we tested extracts of two commonly used Sideritis species, Sideritis euboea and Sideritis scardica, with regard to their effects on cognition in APP-transgenic and aged, non-transgenic C57Bl/6 mice. Additionally, histomorphological and biochemical changes associated with Aß deposition and treatment were assessed. We found that daily oral treatment with Sideritis spp. extracts highly enhanced cognition in aged, non-transgenic as well as in APP-transgenic mice, an effect that was even more pronounced when extracts of both species were applied in combination. The treatment strongly reduced Aß42 load in APP-transgenic mice, accompanied by increased phagocytic activity of microglia, and increased expression of the α-secretase ADAM10. Moreover, the treatment was able to fully rescue neuronal loss of APP-transgenic mice to normal levels as seen in non-transgenic controls. Having the traditional knowledge in mind, our results imply that treatment with Sideritis spp. extracts might be a potent, well-tolerated option for treating symptoms of cognitive impairment in elderly and with regard to Alzheimer's disease by affecting its most prominent hallmarks: Aß pathology and cognitive decline.

Astroglial NF-kB contributes to white matter damage and cognitive impairment in a mouse model of vascular dementia.

Vascular cognitive impairment is the second most common form of dementia. The pathogenic pathways leading to vascular cognitive impairment remain unclear but clinical and experimental data have shown that chronic reactive astrogliosis occurs within white matter lesions, indicating that a sustained pro-inflammatory environment affecting the white matter may contribute towards disease progression. To model vascular cognitive impairment, we induced prolonged mild cerebral hypoperfusion in mice by bilateral common carotid artery stenosis. This chronic hypoperfusion resulted in reactive gliosis of astrocytes and microglia within white matter tracts, demyelination and axonal degeneration, consecutive spatial memory deficits, and loss of white matter integrity, as measured by ultra high-field magnetic resonance diffusion tensor imaging. White matter astrogliosis was accompanied by activation of the pro-inflammatory transcription factor nuclear factor (NF)-kB in reactive astrocytes. Using mice expressing a dominant negative inhibitor of NF-kB under the control of the astrocyte-specific glial fibrillary acid protein (GFAP) promoter (GFAP-IkBα-dn), we found that transgenic inhibition of astroglial NF-kB signaling ameliorated gliosis and axonal loss, maintained white matter structural integrity, and preserved memory function. Collectively, our results imply that pro-inflammatory changes in white matter astrocytes may represent an important detrimental component in the pathogenesis of vascular cognitive impairment, and that targeting these pathways may lead to novel therapeutic strategies.

Metabotropic P2Y1 receptor signalling mediates astrocytic hyperactivity in vivo in an Alzheimer's disease mouse model.

Astrocytic network alterations have been reported in Alzheimer's disease (AD), but the underlying pathways have remained undefined. Here we measure astrocytic calcium, cerebral blood flow and amyloid-ß plaques in vivo in a mouse model of AD using multiphoton microscopy. We find that astrocytic hyperactivity, consisting of single-cell transients and calcium waves, is most pronounced in reactive astrogliosis around plaques and is sometimes associated with local blood flow changes. We show that astroglial hyperactivity is reduced after P2 purinoreceptor blockade or nucleotide release through connexin hemichannels, but is augmented by increasing cortical ADP concentration. P2X receptor blockade has no effect, but inhibition of P2Y1 receptors, which are strongly expressed by reactive astrocytes surrounding plaques, completely normalizes astrocytic hyperactivity. Our data suggest that astroglial network dysfunction is mediated by purinergic signalling in reactive astrocytes, and that intervention aimed at P2Y1 receptors or hemichannel-mediated nucleotide release may help ameliorate network dysfunction in AD.

Impaired mitochondrial energy production and ABC transporter function-A crucial interconnection in dementing proteopathies of the brain.

Ageing is the main risk factor for the development of dementing neurodegenerative diseases (NDs) and it is accompanied by the accumulation of variations in mitochondrial DNA. The resulting tissue-specific alterations in ATP production and availability cause deteriorations of cerebral clearance mechanisms that are important for the removal of toxic peptides and its aggregates. ABC transporters were shown to be the most important exporter superfamily for toxic peptides, e.g. ß-amyloid and α-synuclein. Their activity is highly dependent on the availability of ATP and forms a directed energy-exporter network, linking decreased mitochondrial function with highly impaired ABC transporter activity and disease progression. In this paper, we describe a network based on interactions between ageing, energy metabolism, regeneration, accumulation of toxic peptides and the development of proteopathies of the brain with a focus on Alzheimer's disease (AD). Additionally, we provide new experimental evidence for interactions within this network in regenerative processes in AD.

Reduced Alzheimer's disease pathology by St. John's Wort treatment is independent of hyperforin and facilitated by ABCC1 and microglia activation in mice.

Soluble ß-amyloid peptides (Aß) and small Aß oligomers represent the most toxic peptide moieties recognized in brains affected by Alzheimer's disease (AD). Here we provide the first evidence that specific St. John's wort (SJW) extracts both attenuate Aß-induced histopathology and alleviate memory impairments in APP-transgenic mice. Importantly, these effects are attained independently of hyperforin. Specifically, two extracts characterized by low hyperforin content (i) significantly decrease intracerebral Aß42 levels, (ii) decrease the number and size of amyloid plaques, (iii) rescue neocortical neurons, (iv) restore cognition to normal levels, and (iv) activate microglia in vitro and in vivo. Mechanistically, we reveal that the reduction of soluble Aß42 species is the consequence of a highly increased export activity in the bloodbrain barrier ABCC1transporter, which was found to play a fundamental role in Aß excretion into the bloodstream. These data (i) support the significant beneficial potential of SJW extracts on AD proteopathy, and (ii) demonstrate for the first time that hyperforin concentration does not necessarily correlate with their therapeutic effects. Hence, by activating ABC transporters, specific extracts of SJW may be used to treat AD and other diseases involving peptide accumulation and cognition impairment. We propose that the anti-depressant and anti-dementia effects of these hyperforin-reduced phytoextracts could be combined for treatment of the elderly, with a concomitant reduction in deleterious hyperforin-related side effects.

ABC transporters B1, C1 and G2 differentially regulate neuroregeneration in mice.

BACKGROUND: ATP-binding cassette (ABC) transporters are essential regulators of organismic homeostasis, and are particularly important in protecting the body from potentially harmful exogenous substances. Recently, an increasing number of in vitro observations have indicated a functional role of ABC transporters in the differentiation and maintenance of stem cells. Therefore, we sought to determine brain-related phenotypic changes in animals lacking the expression of distinct ABC transporters (ABCB1, ABCG2 or ABCC1). METHODOLOGY AND PRINCIPAL FINDINGS: Analyzing adult neurogenesis in ABC transporter-deficient animals in vivo and neuronal stem/progenitor cells in vitro resulted in complex findings. In vivo, the differentiation of neuronal progenitors was hindered in ABC transporter-deficient mice (ABCB1(0/0)) as evidenced by lowered numbers of doublecortin(+) (-36%) and calretinin(+) (-37%) cells. In vitro, we confirmed that this finding is not connected to the functional loss of single neural stem/progenitor cells (NSPCs). Furthermore, assessment of activity, exploratory behavior, and anxiety levels revealed behavioral alterations in ABCB1(0/0) and ABCC1(0/0) mice, whereas ABCG2(0/0) mice were mostly unaffected. CONCLUSION AND SIGNIFICANCE: Our data show that single ABC transporter-deficiency does not necessarily impair neuronal progenitor homeostasis on the single NSPC level, as suggested by previous studies. However, loss of distinct ABC transporters impacts global brain homeostasis with far ranging consequences, leading to impaired neurogenic functions in vivo and even to distinct behavioral phenotypes. In addition to the known role of ABC transporters in proteopathies such as Parkinson's disease and Alzheimer's disease, our data highlight the importance of understanding the general function of ABC transporters for the brain's homeostasis and the regeneration potential.

Determination of spatial and temporal distribution of microglia by 230nm-high-resolution, high-throughput automated analysis reveals different amyloid plaque populations in an APP/PS1 mouse model of Alzheimer's disease.

One early and prominent pathologic feature of Alzheimer's disease (AD) is the appearance of activated microglia in the vicinity of developing ß-amyloid deposits. However, the precise role of microglia during the course of AD is still under discussion. Microglia have been reported to degrade and clear ß-amyloid, but they also can exert deleterious effects due to overwhelming inflammatory reactions. Here, we demonstrate the occurrence of developing plaque populations with distinct amounts of associated microglia using time-dependent analyses of plaque morphology and the spatial distribution of microglia in an APP/PS1 mouse model. In addition to a population of larger plaques (>700µm(2)) that are occupied by a moderate contingent of microglial cells across the course of aging, a second type of small ß-amyloid deposits develops (≤400µm(2)) in which the plaque core is enveloped by a relatively large number of microglia. Our analyses indicate that microglia are strongly activated early in the emergence of senile plaques, but that activation is diminished in the later stages of plaque evolution (>150 days). These findings support the view that microglia serve to restrict the growth of senile plaques, and do so in a way that minimizes local inflammatory damage to other components of the brain.

Cerebral amyloid-ß proteostasis is regulated by the membrane transport protein ABCC1 in mice.

In Alzheimer disease (AD), the intracerebral accumulation of amyloid-ß (Aß) peptides is a critical yet poorly understood process. Aß clearance via the blood-brain barrier is reduced by approximately 30% in AD patients, but the underlying mechanisms remain elusive. ABC transporters have been implicated in the regulation of Aß levels in the brain. Using a mouse model of AD in which the animals were further genetically modified to lack specific ABC transporters, here we have shown that the transporter ABCC1 has an important role in cerebral Aß clearance and accumulation. Deficiency of ABCC1 substantially increased cerebral Aß levels without altering the expression of most enzymes that would favor the production of Aß from the Aß precursor protein. In contrast, activation of ABCC1 using thiethylperazine (a drug approved by the FDA to relieve nausea and vomiting) markedly reduced Aß load in a mouse model of AD expressing ABCC1 but not in such mice lacking ABCC1. Thus, by altering the temporal aggregation profile of Aß, pharmacological activation of ABC transporters could impede the neurodegenerative cascade that culminates in the dementia of AD.