Toxicity of Alzheimer's disease-associated Aβ peptide is ameliorated in a Drosophila model by tight control of zinc and copper availability

Amyloid plaques consisting of aggregated Aβ peptide are a hallmark of Alzheimer's disease. Among the different forms of Aβ, the one of 42aa length (Aβ42) is most aggregation-prone and also the most neurotoxic. We find that eye-specific expression of human Aβ42 in Drosophila results in a degeneration of eye structures that progresses with age. Dietary supplements of zinc or copper ions exacerbate eye damage. Positive effects are seen with zinc/copper chelators, or with elevated expression of MTF-1, a transcription factor with a key role in metal homeostasis and detoxification, or with human or fly transgenes encoding metallothioneins, metal scavenger proteins. These results show that a tight control of zinc and copper availability can minimize cellular damage associated with Aβ42 expressio

Bitopertin, a selective oral GLYT1 inhibitor, improves anemia in a mouse model of \u3b2-thalassemia

Anemia of \u3b2-thalassemia is caused by ineffective erythropoiesis and reduced red cell survival. Several lines of evidence indicate that iron/heme restriction is a potential therapeutic strategy for the disease. Glycine is a key initial substrate for heme and globin synthesis. We provide evidence that bitopertin, a glycine transport inhibitor administered orally, improves anemia, reduces hemolysis, diminishes ineffective erythropoiesis, and increases red cell survival in a mouse model of \u3b2-thalassemia (Hbbth3/+ mice). Bitopertin ameliorates erythroid oxidant damage, as indicated by a reduction in membrane-associated free \u3b1-globin chain aggregates, in reactive oxygen species cellular content, in membrane-bound hemichromes, and in heme-regulated inhibitor activation and eIF2\u3b1 phosphorylation. The improvement of \u3b2-thalassemic ineffective erythropoiesis is associated with diminished mTOR activation and Rab5, Lamp1, and p62 accumulation, indicating an improved autophagy. Bitopertin also upregulates liver hepcidin and diminishes liver iron overload. The hematologic improvements achieved by bitopertin are blunted by the concomitant administration of the iron chelator deferiprone, suggesting that an excessive restriction of iron availability might negate the beneficial effects of bitopertin. These data provide important and clinically relevant insights into glycine restriction and reduced heme synthesis strategies for the treatment of \u3b2-thalassemia

the role of the GxxxG-motif

Der Oligomerisierung des Aß42 Peptids wird eine ursächliche Rolle bei der Alzheimer Krankheit zugeschrieben. Bevor die Peptide zu Fibrillen aggregieren, wirken besonders niedere Oligomere neurotoxisch und könnten für den Verlust der kognitiven Fähigkeiten des Patienten verantwortlich sein. Da durch eine sequenzielle Prozessierung des APP Aß mit variierender Länge entstehen, war ein erster Schwerpunkt dieser Arbeit die Untersuchung der verschiedenen Peptide. Dabei konnte gezeigt werden, je kürzer die Peptide sind, desto weniger häufig werden höhere Oligomere ausbildet. Obwohl alle Peptide vergleichbar hohe Mengen an Tetrameren gebildet hatten und diesen Formen in der Literatur als potentiell toxisch beschrieben sind, konnte nur Aß42 ein toxischer Effekt zugeschrieben werden. Für das GxxxG-Dimerisierungsmotiv innerhalb der Aß Sequenz zeigte sich, dass dieses Motiv auch für die Aggregation und das pathologische Verhalten des A42 Peptids kritisch ist. Im Rahmen dieser Arbeit konnte erstmals der Einfluss eines Aminosäureaustausches innerhalb des Aß GxxxG-Motivs auf Aggregation, Struktur und Toxizität beschrieben werden. Strukturelle Untersuchungen zeigten, dass die Erhöhung der Hydrophobizität an Position Gly33 den Faltungskern des Peptides stabilisiert und die Oligomerisierung des Peptides forciert. Die Abschirmung der hydrophoben Oberflächen, führt zur Krümmung der Aggregate und lässt somit keine Fibrillenbildung mehr zu. Im Gegensatz dazu wird bei der Aß42 G29A Variante die tetramere Untereinheit durch Anlagerung gegenüberliegender Gly29 durch erhöhte Hydrophobizität stabilisiert, was in einer verlängerten Fibrillenbildung resultiert. Ein doppelter Austausch von Gly29 und Gly33 führt zu einer intermediären Oligomerisierung, wobei auch hier keine reifen Fibrillen gebildet wurden. Neben der Aggregation und Struktur wurde auch erstmals detailliert die funktionelle Rolle des GxxxG-Motives mittels Toxizitätstests und im Modellsystem für Lernen und Gedächtnis (LTP Messung) untersucht. Dabei stellte sich heraus, dass eine Substitution an Position Gly33 weder in vitro noch in vivo toxische Effekte hat, noch LTP inhibiert. Für das Aß42 wt und Aß42 G29A Peptid hingegen konnte gezeigt werden, dass weder Fibrillen, noch höhere Oligomere sondern ausschließlich niedere Oligomere, vor allem Tetramere, toxisch wirken. Diese Effekte konnten in reduzierter Form auch bei Aß42 G29/33A beobachtet werden, was das intermediäre Verhalten der Variante weiter unterstreicht. Dennoch konnte sowohl bei Aß42 wt als auch Aß42 G33I Peptid eine Reduktion der basalen synaptischen Transmission und eine Aufnahme des Peptids in die Zelle und den Zellkern ermittelt werden. Die Ergebnisse zeigen auf, dass das zentrale GxxxG-Motiv des Aß Peptids, und Gly33 im Besonderen, eine essentielle Rolle nicht nur im Aggregationsverhalten des Peptides spielt. In dieser Arbeit konnte erstmals gezeigt werden, dass vor allem Tetramere toxische Oligomerformen sind, jedoch der Aggregationszustand nicht grundsätzlich ausschlaggebend für die Toxizität ist. Besonders interessant ist, dass die strukturelle Veränderung des Peptides durch Austausch an Position Gly33 das pathologische Verhalten aufhebt. Diese Erkenntnisse eröffnen die Möglichkeit, mit dem Werkzeug neuer Aß42 Varianten, die genauen Mechanismen der toxischen Wirkungsweise von oligomeren Aß Formen zu erforschen. Daraus wiederum könnten weiterführend neue Therapieansätze entwickelt werden.The oligomerization of Aß42 peptides plays a critical role in Alzheimer’s disease. Before aggregating to fibrils, low-n oligomers are known to be neurotoxic and cause cognitive deficits. Based on the model of the sequential cleavage of APP there exist a heterogenous mixture of different Aß species of variying length. In the first part of this work, analysis of the oligomerization and toxicity of those shorter peptides were performed. It could be demonstrated that the shorter the peptide the fewer higher oligomers were generated. Although all peptides formed high amounts of tetramers, which were described in literature to be potentially toxic, only Aß42 cause toxic effects. Earlier studies could already underline the importance of the GxxxG- motivs within the Aß sequence in APP processing. Furthermore these motives were postulated to play a role in aggregation and pathological behavior of Aß42 peptides. In structural analyses it could be demonstrated that increased hydrophobicity at position Gly33 stabilizes the folding nucleus of the peptide and thereby accelerate the oligomerization. The shielding of the ß-sheets from the polar milieu leads to a bending of the peptide and thus a suppression of the fibril formation. In contrast, Aß42 G29A stabilizes the tetrameric subunit resulting in an elongated fibril formation. Even though double substitutions of Gly29 and Gly33 are resulting in an intermediate oligomerization but also do not form fibrils. Despite the aggregation and structure, a detailed functional role of the GxxxG-motifs was analyzed for the first time using toxicity assays and a system for learning and memory (LTP measurements). It was observed that Gly33 substitution neither in vitro nor in vivo had any toxic effects, or inhibited LTP. For Aß42 wt and Aß42 G29A peptides it could be demonstrated that neither fibrils nor higher oligomers influenced the cell viability. Only low-n oligomers, mainly tetramers dramatically reduced the number of cells. This effect could also be recorded in a reduced form for Aß42 G29/33A, underlining the intermediate behavior. Nevertheless, both Aß42 wt and Aß42 G33I peptides reduced the basal synaptic transmission and were taken up by the cells and transported into the nucleus. In conclusion these results demonstrate that the central GxxxG-Motiv of Aß and especially Gly33 plays a critical role in aggregational and toxic behavior of the peptide. Furthermore, the data reveal that even though tetramers are most toxic oligomers, toxicity does not depend on aggregation form, but the structural change. Interestingly, the structural changes caused by amino acid exchange at position Gly33 completely abolish the pathological behavior. These findings enable, with the new Aß42 variants as a powerful tool, the detailed analysis of the toxic mechanism of Aß42 oligomers and opens new possibilities for developing new therapeutic approaches

A Selective Oral GLYT1 Inhibitor, Improves Anemia and Red Cell Survival in a Mouse Model of Beta-Thalassemia

The unbalanced hemoglobin chain synthesis in beta-thalassemias leads to hemichrome-induced damage that contributes to ineffective erythropoiesis, hemolysis and reduced red cell survival. Iron overload related to ineffective erythropoiesis and abnormally low Hepcidin (Hamp), combined with the cytotoxic effects of free heme with free-alpha-chains play a key role in the increased generation of reactive oxygen species (ROS) in beta thalassemias. Here we used a specific and selective inhibitor of the plasma membrane expressed glycine transporter GlyT1 (Ro4917838). Use of Ro4917838 has been associated with a dose-dependent decrease in MCH, Hb, soluble transferrin receptor, and increase in absolute reticulocytes and RBC counts in several animal species, attributed to reduce glycine bioavailability in erythroblasts and decreased heme synthesis. In rats, Ro4917838 reduces heme synthesis, and down-regulates erythroid transferrin receptor, but does not interfere with hepcidin regulation and systemic iron homeostasis. We aimed to determine if reduced cellular availability of glycine in erythroblasts may reduce heme synthesis, and impact pathologic erythropoiesis in a mouse model for b-thalassemia. Wild-type control (WT) C57B6/2J, and beta-thalassemia Hbbth3/+ mice were treated with either vehicle or Ro4917838 at dosages of 3, 10, 30 mg/kg/d administered over 4 weeks once daily by gavage. RO4917838 administration was associated with significant improvements of central hallmarks of the b-thalassemia pathology. Reduced erythrocyte destruction was seen bydemonstrated a significant improvements in erythrocyte morphology and amelioration of hemoglobin reduction in reticulocytes. We also observed an impressively quick reduction of the circulating erythroblast count within 1 week of initiating treatment. This was also associated with decreased hemolysis biomarkers. Ro4917838 induced a significant reduction in extramedullary erythropoiesis and reduction in orthochromatic erythroblasts as well as insoluble alpha chain aggregates in circulating red cells. Red cell survival of b-thal mice treated with 30 mg/kg/day Ro4917838 significantly increased by more than 50%. CD71+ erythroid precursors significantly decreased in WT mice treated with Ro4917838 at 30 mg/kg and in b-thal mice at the dosage of 30 mg/kg/ d. These data suggest that Ro4917838 ameliorates anemia in a b-thalassemia mouse model and positively affects ineffective erythropoiesis and red cell survival in peripheral circulation. Ro4917838 may represent a novel therapeutic approach for the treatment of anemia in b-thalassemia patients

How female mice attract males:A urinary volatile amine activates a trace amine-associated receptor that indices male sexual interest

Individuals of many species rely on odors to communicate, find breeding partners, locate resources and sense dangers. In vertebrates, odorants are detected by chemosensory receptors of the olfactory system. One class of these receptors, the trace amine-associated receptors (TAARs), was recently suggested to mediate male sexual interest and mate choice. Here we tested this hypothesis in mice by generating a cluster deletion mouse (Taar2-9−/−) lacking all TAARs expressed in the olfactory epithelium, and evaluating transduction pathways from odorants to TAARs, neural activity and behaviors reflecting sexual interest. We found that a urinary volatile amine, isobutylamine (IBA), was a potent ligand for TAAR3 (but not TAAR1, 4, 5, and 6). When males were exposed to IBA, brain regions associated with sexual behaviors were less active in Taar2-9−/− than in wild type males. Accordingly, Taar2-9−/− males spent less time sniffing both the urine of females and pure IBA than wild type males. This is the first demonstration of a comprehensive transduction pathway linking odorants to TAARs and male sexual interest. Interestingly, the concentration of IBA in female urine varied across the estrus cycle with a peak during estrus. This variation in IBA concentration may represent a simple olfactory cue for males to recognize receptive females. Our results are consistent with the hypothesis that IBA and TAARs play an important role in the recognition of breeding partners and mate choice

Novel APP/Aβ mutation K16N produces highly toxic heteromeric Aβ oligomers

Here, we describe a novel missense mutation in the amyloid precursor protein (APP) causing a lysine-to-asparagine substitution at position 687 (APP770; herein, referred to as K16N according to amyloid-β (Aβ) numbering) resulting in an early onset dementia with an autosomal dominant inheritance pattern. The K16N mutation is located exactly at the α-secretase cleavage site and influences both APP and Aβ. First, due to the K16N mutation APP secretion is affected and a higher amount of Aβ peptides is being produced. Second, Aβ peptides carrying the K16N mutation are unique in that the peptide itself is not harmful to neuronal cells. Severe toxicity, however, is evident upon equimolar mixture of wt and mutant peptides, mimicking the heterozygous state of the subject. Furthermore, Aβ42 K16N inhibits fibril formation of Aβ42 wild-type. Even more, Aβ42 K16N peptides are protected against clearance activity by the major Aβ-degrading enzyme neprilysin. Thus the mutation characterized here harbours a combination of risk factors that synergistically may contribute to the development of early onset Alzheimer disease

Alzheimer Amyloid Peptide Aβ42 Regulates Gene Expression of Transcription and Growth Factors

The pathogenesis of Alzheimer's disease (AD) is characterized by the aggregation of amyloid-β (Aβ) peptides leading to deposition of senile plaques and a progressive decline of cognitive functions, which currently remains the main criterion for its diagnosis. Robust biomarkers for AD do not yet exist, although changes in the cerebrospinal fluid levels of tau and Aβ represent promising candidates in addition to brain imaging and genetic risk profiling. Although concentrations of soluble Aβ42 correlate with symptoms of AD, less is known about the biological activities of Aβ peptides which are generated from the amyloid-β protein precursor. An unbiased DNA microarray study showed that Aβ42, at sub-lethal concentrations, specifically increases expression of several genes in neuroblastoma cells, notably the insulin-like growth factor binding proteins 3 and 5 (IGFBP3/5), the transcription regulator inhibitor of DNA binding, and the transcription factor Lim only domain protein 4. Using qRT-PCR, we confirmed that mRNA levels of the identified candidate genes were exclusively increased by the potentially neurotoxic Aβ42 wild-type peptide, as both the less toxic Aβ40 and a non-toxic substitution peptide Aβ42 G33A did not affect mRNA levels. [...

Aβ42-oligomer Interacting Peptide (AIP) neutralizes toxic amyloid-β42 species and protects synaptic structure and function

The amyloid-β42 (Aβ42) peptide is believed to be the main culprit in the pathogenesis of Alzheimer disease (AD), impairing synaptic function and initiating neuronal degeneration. Soluble Aβ42 oligomers are highly toxic and contribute to progressive neuronal dysfunction, loss of synaptic spine density, and affect long-term potentiation (LTP). We have characterized a short, L-amino acid Aβ-oligomer Interacting Peptide (AIP) that targets a relatively well-defined population of low-n Aβ42 oligomers, rather than simply inhibiting the aggregation of Aβ monomers into oligomers. Our data show that AIP diminishes the loss of Aβ42-induced synaptic spine density and rescues LTP in organotypic hippocampal slice cultures. Notably, the AIP enantiomer (comprised of D-amino acids) attenuated the rough-eye phenotype in a transgenic Aβ42 fly model and significantly improved the function of photoreceptors of these flies in electroretinography tests. Overall, our results indicate that specifically “trapping” low-n oligomers provides a novel strategy for toxic Aβ42-oligomer recognition and removal

The cellular prion protein mediates neurotoxic signalling of β-sheet-rich conformers independent of prion replication

PrPC can interact with and mediate toxic signalling of misfolded β-sheet-rich oligomers of different origin

DNA-Encoded Library-Derived DDR1 Inhibitor Prevents Fibrosis and Renal Function Loss in a Genetic Mouse Model of Alport Syndrome

The importance of Discoidin Domain Receptor 1 (DDR1) in renal fibrosis has been shown via gene knockout and use of antisense oligonucleotides; however, these techniques act via a reduction of DDR1 protein, while we prove the therapeutic potential of inhibiting DDR1 phosphorylation with a small molecule. To date, efforts to generate a selective small-molecule to specifically modulate the activity of DDR1 in an in vivo model have been unsuccessful. We performed parallel DNA encoded library screens against DDR1 and DDR2, and discovered a chemical series that is highly selective for DDR1 over DDR2. Structure-guided optimization efforts yielded the potent DDR1 inhibitor 2.45, which possesses excellent kinome selectivity (including 64-fold selectivity over DDR2 in a biochemical assay), a clean in vitro safety profile, and favorable pharmacokinetic and physicochemical properties. As desired, compound 2.45 modulates DDR1 phosphorylation in vitro as well as prevents collagen-induced activation of renal epithelial cells expressing DDR1. Compound 2.45 preserves renal function and reduces tissue damage in Col4a3-/- mice (the preclinical mouse model of Alport syndrome) when employing a therapeutic dosing regime, indicating the real therapeutic value of selectively inhibiting DDR1 phosphorylation in vivo. Our results may have wider significance as Col4a3-/- mice also represent a model for chronic kidney disease, a disease which affects 10% of the global population