Systematic interaction network filtering identifies CRMP1 as a novel suppressor of huntingtin misfolding and neurotoxicity

Assemblies of huntingtin (HTT) fragments with expanded polyglutamine (polyQ) tracts are a pathological hallmark of Huntington's disease (HD). The molecular mechanisms by which these structures are formed and cause neuronal dysfunction and toxicity are poorly understood. Here, we utilized available gene expression data sets of selected brain regions of HD patients and controls for systematic interaction network filtering in order to predict disease-relevant, brain region-specific HTT interaction partners. Starting from a large protein-protein interaction (PPI) data set, a step-by-step computational filtering strategy facilitated the generation of a focused PPI network that directly or indirectly connects 13 proteins potentially dysregulated in HD with the disease protein HTT. This network enabled the discovery of the neuron-specific protein CRMP1 that targets aggregation-prone, N-terminal HTT fragments and suppresses their spontaneous self-assembly into proteotoxic structures in various models of HD. Experimental validation indicates that our network filtering procedure provides a simple but powerful strategy to identify disease-relevant proteins that influence misfolding and aggregation of polyQ disease proteins.DFG [SFB740, 740/2-11, SFB618, 618/3-09, SFB/TRR43 A7]; BMBF(NGFN-Plus) [01GS08169-73, 01GS08150, 01GS08108]; HDSA Coalition for the Cure; EU (EuroSpin) [Health-F2-2009-241498, HEALTH-F2-2009-242167]; Helmholtz Association (MSBN, HelMA) [HA-215]; FCT [IF/00881/2013]info:eu-repo/semantics/publishedVersio

mTOR-dependent translation amplifies microglia priming in aging mice.

peer reviewedMicroglia maintain homeostasis in the brain. However, with age, they become primed and respond more strongly to inflammatory stimuli. We show here that microglia from aged mice had upregulated mTOR complex 1 signaling controlling translation, as well as protein levels of inflammatory mediators. Genetic ablation of mTOR signaling showed a dual yet contrasting effect on microglia priming: it caused an NF-κB-dependent upregulation of priming genes at the mRNA level; however, mice displayed reduced cytokine protein levels, diminished microglia activation, and milder sickness behavior. The effect on translation was dependent on reduced phosphorylation of 4EBP1, resulting in decreased binding of eIF4E to eIF4G. Similar changes were present in aged human microglia and in damage-associated microglia, indicating that upregulation of mTOR-dependent translation is an essential aspect of microglia priming in aging and neurodegeneration

Intrazellulärer Transport und Proteinprozessierung in einem nicht-viralen Transfektionsmodell

Saccharomyces cerevisiae wird allgemein als Modellsystem zur Untersuchung der Endocytose genutzt. Ergebnisse, die in Hefe gewonnen wurden, können in Bezug auf fast alle Aspekte auf das Säugersystem übertragen werden. Hier wurden definierte Deletionsstämme genutzt, um Schritte im endocytotischen Transport, in der endosomal/lysosomalen Ansäuerung und in dem intrazellulären Transport von Hydrolasen sowie Hydrolasen selbst zu identifizieren und zu lokalisieren, die die DNS Aufnahme und Freisetzung in der nicht-viralen Transfektion beeinflussen. Des Weiteren wurden in entsprechenden Deletionsmutanten Golgi-Proteine, Zellwandbestandteile und deren Transport zu ihren Wildtyp-Lokalisationen und teilweise auch der Einfluss von Aktin und seinen korrespondierenden Proteinen untersucht auf Effekte bezüglich der Transfektionseffizienz. Transportinhibierung in späteren endocytotischen Schritten, wie in der vps21 Mutante, erhöht die Transfektionseffizienz. Durch Quinacrine-Färbung-Untersuchungen unter Transfektionsbedingungen wurde gezeigt, dass eine Modifizierung des pH-Wertes im endosomal/vakuolären System, wie zum Beispiel in den Mutanten vph1 und stv1, zu einer erhöhten Transfektionseffizienz führt. Die Effizienz ist ebenfalls in spezifischen Fällen erhöht, wenn die Ionenzusammensetzung des endosomalen Systems beeinflusst wird (nhx1). Eine erhöhte Transfektionseffizienz wurde außerdem in Mutanten beobachtet, welche in dem CVT/Autophagie Prozess oder dem Transport von Hydrolasen zur Vakuole mutiert sind, wie in der Mutante vac8. Die Transfektionseffizienz kann des Weiteren erhöht werden durch Defekte an prozessierenden Proteinen des Golgi, wie in der kex2 Mutante, die defekt ist für eine wichtige prozessierende Golgi Protease, in der och1 Mutante, die defekt ist für ein Protein, dass in der frühen N-Glykosylierung des Golgi wichtig ist oder durch Beeinflussung der Wildtyp-Funktion dieser Proteine durch Deletion von PMR1, welches für den Golgi Mn2+/Ca2+-Import Transporter kodiert. Die Funktion von Kex2p ist abhängig von Ca2+-Ionen und die Funktion von Och1p von Mn2+-Ionen. Die natürliche Funktion von Kex2p könnte auch beeinflusst werden durch Disfunktionen in seinem Transport zwischen dem trans-Golgi-Netzwerk und den Endosomen. In dieser Promotionsarbeit wird ein Interaktionsnetzwerk der transfektions-beeinflussenden Faktoren präsentiert, welches Kex2p, Och1p, Pmr1p, Stv1p, Vph1p und Nhx1p beinhaltet. Zum Schluss kann noch gesagt werden, dass Einzeldefekte von Zellwandproteinen oder Proteinen, die involviert sind in den Aktin-Metabolismus, scheinbar nicht geeignet sind um die Transfektionseffizienz zu erhöhen. Zusammenfassend lässt sich sagen, dass die nicht-virale Transfektionseffizienz erhöht werden kann durch (i) die Inhibierung des Transportes endocytierten Materials, bevor es vakuoläre Bedingungen erreicht, (ii) die Erzeugung eines nicht natürlichen pH-Wertes in dem endosomal/vakuolären System, (iii) die Verlangsamung degradativer Prozesse durch Inhibierung vakuolärer Hydrolasen oder des Transports zwischen Golgi und dem späten Endosom/der Vakuole oder (iv) die Inhibierung/Unterbrechung der Prozessierung im Golgi.Saccharomyces cerevisiae is commonly used as a model system to study endocytosis. Results obtained with yeast can be transferred to the mammalian system in almost all aspects. Here, defined deletion mutants were utilized to identify and locate steps in endocytic transport, endosomal/lysosomal acidification and in intracellular transport of hydrolases, and hydrolases itself which influence DNA delivery in non-viral transfection. Furthermore, Golgi proteins, cell wall constituents and their transport to wild-type locations, and, partially, also the involvement of actin and its corresponding proteins were tested for effects on transfection efficiency in deletion mutants. Transport inhibition in later endocytic steps, such as in the vps21 mutant, enhances transfection efficiency. By applying transfection conditions in combination with quinacrine staining, it was demonstrated that modifying the pH-value of the endosomal/vacuolar system, for example in the vph1 and stv1 mutants, leads to enhanced transfection efficiency. Efficiency is also elevated in specific cases when the ion composition of the endosomal system is affected (nhx1). Enhanced transfection efficiency was further observed in mutants which are mutated in the CVT/autophagy pathway or hydrolase transport to the vacuole as in mutant vac8. Transfection efficiency can be further enhanced by deleting Golgi processing proteins as with the kex2 mutant deficient for an important Golgi processing protease, the och1 mutant deficient for an early Golgi participant in N-glycosylation, or by affecting these proteins' wild-type function by deleting PMR1 coding for the Golgi Mn2+/Ca2+-importing transporter. The function of Kex2p is dependent on Ca2+–ions and the function of Och1p on Mn2+–ions. Proper function of Kex2p might also be affected by malfunctions in the transport between the trans-Golgi network and endosomes. In this study, an interaction network affecting transfection is presented between Kex2p, Och1p, Pmr1p, Stv1p, Vph1p and Nhx1p. Finally, single defects affecting cell wall proteins or proteins involved in actin metabolism seem to be inappropriate to use to enhance transfection efficiency. In summary, non-viral transfection efficiency can be enhanced by (i) inhibiting the transport of endocytosed material before it meets vacuolar conditions, (ii) inducing a non-natural pH-value of the endosomal/vacuolar system, (iii) slowing down degradative processes by inhibiting vacuolar hydrolases or the transport between Golgi and late endosome/vacuole, or (iv) inhibiting/interrupting Golgi processing

Endocytic uptake of fluorescence labelled DNA in yeast.

After dispiriting results using viral vectors in gene therapy, by which a number of patients acquired cancer as a result of the use of retroviral vector constructs, the percentage of non-viral approaches has increased over recent years. To elucidate potential bottlenecks in the non-viral transfection process we here introduce a novel method to directly visualize endocytic non-viral DNA uptake in a transfection approach. This novel method allows for the first time to monitor the location of DNA which is taken up by endocytosis in yeast (Saccharomyces cerevisiae) wild type and mutant strains. More specifically it enables drawing conclusions about conditions favouring non-viral gene transfection

Defects in intracellular trafficking and endocytic/vacuolar acidification determine the efficiency of endocytotic DNA uptake in yeast.

The yeast Saccharomyces cerevisiae is a standard model system to study endocytosis. Here we describe the examination of a representative subset of deletion mutants to identify and locate steps in endocytic transport, endosomal/lysosomal acidification and in intracellular transport of hydrolases in non-viral transfection processes. When transport in late endocytosis is inhibited, transfection efficiency is significantly enhanced. Similarly, transfection efficiency is enhanced when the pH-value of the endosomal/vacuolar system is modified. Transfection efficiency is furthermore elevated when the N+/K+ transport in the endosomal system is disturbed. Finally, we observe enhanced transfection efficiency in mutants disturbed in the CVT/autophagy pathway and in hydrolase transport to the vacuole. In summary, non-viral transfection efficiency can be significantly increased by either (i) inhibiting the transport of endocytosed material before it enters the vacuole, or (ii) inducing a non-natural pH-value of the endosomal/vacuolar system, or (iii) slowing down degradative processes by inhibiting vacuolar hydrolases or the transport between Golgi and late endosome/vacuole

Absence of See1p, a widely conserved Saccharomyces cerevisiae protein, confers both deficient heterologous protein production and endocytosis.

The uncharacterized Saccharomyces cerevisiae open reading frame (ORF) YIL064w is predicted to encode a cytoplasmic 28 kDa protein, recognized by sequence similarity as a putative S-adenosyl-L-methionine methyltransferase. A micro-scale screening performed in our laboratory with the EUROSCARF S. cerevisiae BY4741 deletion mutant collection identified YIL064w deletion as negatively affecting secretory production of reporter alpha-amylase. The work presented here corroborates the later observations of the yil064w mutant in a larger-scale assay and shows that Yil064p is necessary for the efficient secretory production of two reporter proteins, murine alpha-amylase and fungal polygalacturonase. Further, we analysed endocytosis in the yil064w mutant strain and observed defects at both very early and later stages of endocytic transport in cells in the late logarithmic phase. The defects at very early stages may decisively account for the low transfection (DNA uptake by endocytosis) efficiency that we also observed in the yil064w mutant. These are the first in vivo data reporting a functional role for the protein encoded by ORF YIL064w and identify Yil064p, named here secretion and early endocytosis 1 protein (See1p), as a novel component of intracellular transport

Functional characterization of the recombinant N-methyltransferase domain from the multienzyme enniatin synthetase.

A 51 kDa fusion protein incorporating the N-methyltransferase domain of the multienzyme enniatin synthetase from Fusarium scirpi was expressed in Saccharomyces cerevisiae. The protein was purified and found to bind S-adenosyl methionine (AdoMet) as demonstrated by cross-linking experiments with (14)C-methyl-AdoMet under UV irradiation. Cofactor binding at equilibrium conditions was followed by saturation transfer difference (STD) NMR spectroscopy, and the native conformation of the methyltransferase was assigned. STD NMR spectroscopy yielded significant signals for H(2) and H(8) of the adenine moiety, H(1') of D-ribose, and S-CH(3) group of AdoMet. Methyl group transfer catalyzed by the enzyme was demonstrated by using aminoacyl-N-acetylcysteamine thioesters (aminoacyl-SNACs) of L-Val, L-Ile, and L-Leu, which mimic the natural substrate amino acids of enniatin synthetase presented by the enzyme bound 4'-phosphopantetheine arm. In these experiments the enzyme was incubated in the presence of the corresponding aminoacyl-SNAC and (14)C-methyl-AdoMet for various lengths of time, for up to 30 min. N-[(14)C-Methyl]-aminoacyl-SNAC products were extracted with EtOAc and separated by TLC. Acid hydrolysis of the isolated labeled compounds yielded the corresponding N-[(14)C-methyl] amino acids. Further proof for the formation of N-(14)C-methyl-aminoacyl-SNACs came from MALDI-TOF mass spectrometry which yielded 23 212 Da for N-methyl-valyl-SNAC, accompanied by the expected postsource decay (PSD) pattern. Interestingly, L-Phe, which is not a substrate amino acid of enniatin synthetase, also proved to be a methyl group acceptor. D-Val was not accepted as a substrate; this indicates selectivity for the L isomer

Neurons undergo pathogenic metabolic reprogramming in models of familial ALS.

OBJECTIVES: Normal cellular function requires a rate of ATP production sufficient to meet demand. In most neurodegenerative diseases (including Amyotrophic Lateral Sclerosis [ALS]), mitochondrial dysfunction is postulated raising the possibility of impaired ATP production and a need for compensatory maneuvers to sustain the ATP production/demand balance. We investigated intermediary metabolism of neurons expressing familial ALS (fALS) genes and interrogated the functional consequences of glycolysis genes in fitness assays and neuronal survival. METHODS: We created a pure neuronal model system for isotopologue investigations of fuel utilization. In a yeast platform we studied the functional contributions of glycolysis genes in a growth fitness assay iafter expressing of a fALS gene. RESULTS: We find in our rodent models of fALS, a reduction in neuronal lactate production with maintained or enhanced activity of the neuronal citric acid cycle. This rewiring of metabolism is associated with normal ATP levels, bioenergetics, and redox status, thus supporting the notion that gross mitochondrial function is not compromised in neurons soon after expressing fALS genes. Genetic loss-of-function manipulation of individual steps in the glycolysis and the pentose phosphate pathway blunt the negative phenotypes seen in various fALS models. CONCLUSIONS: We propose that neurons adjust fuel utilization in the setting of neurodegenerative disease-associated alteration in mitochondrial function in a baleful manner and targeting this process can be healthful