29 research outputs found
Charakterisierung von drei Splicevarianten des humanen Rezeptors SorCS1
No full text1. Zusammenfassung
Die Familie der Wirbeltier Typ-I Transmembranrezeptoren, welche eine Vps10p-Domäne besitzen, beinhaltet fünf Mitglieder, Sortilin, SorLA/LR11 und SorCS1-3. Das Hauptmerkmal dieser Proteinfamilie ist die Vps10p-Domäne, welche am N-Terminus der luminalen/extrazellulären Region lokalisiert ist. Die Vps10p-Domäne wurde als erstes in dem gleichnamigen Rezeptor in der Hefe beschrieben. Dieser ist an Transport– und Sortierungsprozessen zwischen dem Trans-Golgi System und Endosomen beteiligt. Ein weiteres Kennzeichen dieser Rezeptorfamilie ist ein kurzer zytoplasmatischer Abschnitt, welcher in der gesamten Familie unterschiedliche Internalisierungs- und Sortierungssignale aufweist.
SorCS1 wird, genau wie die anderen Familienmitglieder, prominent, aber nicht ausschließlich, während der Entwicklung des Zentralen Nervensystems exprimiert und weist als einziges Spleißvarianten auf. Vom humanen SorCS1 Protein waren zu Beginn dieser Arbeit bereits zwei Isoformen bekannt, hSorCS1a und hSorCS1b. Im Rahmen dieser Arbeit wurde, eine dritte humane Spleißvariante, hSorCS1c, kloniert, und gemeinsam mit den zwei zuvor klonierten Isoformen charakterisiert.
Die drei Isoformen sind in ihrer luminalen/extrazellulären Domäne sowie der Transmembranregion identisch, unterscheiden sich aber vollständig in ihren zytoplasmatischen Abschnitten. Diese weisen verschiedene Bindemotive und Sortierungssignale auf. Es konnte zunächst gezeigt werden, dass die drei Spleißvarianten in unterschiedlichen Geweben differentiell exprimiert werden. Mittels stabil transfizierter Zelllinien wurde dann einerseits eine differentielle subzelluläre Verteilung der drei Isoformen nachgewiesen. Andererseits konnte gezeigt werden, dass die Spleißvarianten verschiedene Internalisierungsraten aufweisen. Während SorCS1b, für welches die stärkste Zelloberflächenlokalisation gezeigt wurde, gar nicht internalisiert wird, wird SorCS1a, das vor allem intrazellulär nachgewiesen wurde, rasch endozytiert. SorCS1c nimmt eine Zwischenstellung ein, wird aber eindeutig internalisiert. Somit sind zwei der drei Isoformen in der Lage die Internalisierung von Liganden zu vermitteln, während SorCS1b eine andere noch aufzuklärende Funktion besitzt. Somit kann SorCS1 als multifunktioneller Rezeptor betrachtet werden, der nach neueren Daten auch an der Entstehung von Typ 2 Diabetes verantwortlich sein soll
The functional role of the cell adhesion molecule L1: characterization of L1/687 and L1/858-863 transgenic mice
No full textThe cell adhesion molecule L1 has a key role during the development of the nervous system and is involved in different neuronal processes such as migration, adhesion, cell proliferation, neuritogenesis, synapse formation, and synaptic plasticity. Based on a previous work that showed the translocation of a fragment of L1, L1-70, into mitochondria, I showed in this thesis through flow cytometry that L1-70 is present on the surface of mitochondria and that interacts with different proteins involved in the mitophagy/mitochondrial quality control process. The interaction between L1 and mitophagy markers as LC3 and Parkin has been confirmed in vitro using proximity ligation assay on cerebellar granule cells, ELISA, and co-immunoprecipitation assays. Moreover, I showed an alteration in the levels of another mitophagy protein, BNIP3, in L1-deficient mice. To further determine the role of L1-70 in mitochondrial pathways, I used in this thesis two transgenic mice lines, expressing full-length L1, but no L1-70. To confirm the absence of L1-70 in the transgenic mice line, L1/687 and L1/858-863, proximity ligation assay was performed. The outcome of the experiment confirmed that the interaction of L1 with LC3 was due to the L1-70 fragment. Levels of LC3 were also detected by western blot on isolated mitochondria from L1/687 and L1/858-863, showing an alteration of the protein levels, but not its production. To understand the role of L1-70 in mitochondrial processes, different assays were performed. I showed that in cultured cerebellar neurons lacking L1-70 fragment, mitochondria move more retrogradely, exhibit reduced mitochondrial membrane potential and had lower ATP levels compared to wild-type littermates. These results suggest that L1-70 is important for mitochondrial homeostasis and its absence reflects on the mitochondrial quality and stability.
In addition, with staining of coronal brain sections from L1/858- 863 and L1/687, I showed that L1/858-863 brains, had diffuse cells and higher hippocampal area. The role of the L1-70 fragment was further analyzed with behavioral experiments in order to characterize L1/687 phenotype. Through open field, elevated plus maze, and marble burying test I showed that L1/687 mice did not display anxiety-like behavior compared to wild-type mice, but higher locomotor activity, expressed by the higher distance traveled in the arena. Besides, with the home cage activity test, I showed that L1/687 mice had a normal circadian rhythm, with L1/687 female mice being slightly more active compared to their wild-type littermates. In addition, with the social interaction test, I showed no alteration in the social behavior of transgenic mice that exhibited unaltered social memory and predilection for novel experiences. The use of motor tests as rotarod, beam walking and pole test highlighted that there was no physical impairment in L1/687 mice. Using the pole test and the beam walking, no differences were detected, suggesting that the absence of L1-70 does not lead to impairment in motor coordination, but more likely to a possible impairment in the learning circuit. Altogether, the new findings of this study could help reveals new aspects about the role and the importance of L1 and the L1-70 fragment in the murine central nervous system. L1-70 has in fact a role in mitochondrial dynamics and a possible function in mitochondrial quality control. These findings can be relevant in further understanding neurodegenerative diseases associated with mitochondrial impairments
Developmental expression of the estrogen receptor-related receptor γ in the nervous system during mouse embryogenesis
No full text
Identification of PNRC2 and TLE1 as activation function-1 cofactors of the orphan nuclear receptor ERRγ
No full textThe germ cell nuclear factor is required for retinoic acid signaling during Xenopus development
No full textThe germ cell nuclear factor (GCNF, NR6A1) is a nuclear orphan receptor that functions as a transcriptional repressor and is transiently expressed in mammalian carcinoma cells during retinoic acid (RA) induced neuronal differentiation. During Xenopus laevis development, the spatiotemporal expression pattern of embryonic GCNF (xEmGCNF) suggests a role in anteroposterior specification of the neuroectoderm. Here, we show that RA treatment of Xenopus embryos enhances xEmGCNF expression. Moreover, we present evidence for the relevance of this finding in the context of primary neurogenesis and hindbrain development. During early development of the central nervous system, RA signals promote posterior transformation of the neuroectoderm and increase the number of cells undergoing primary neurogenesis. Our loss-of-function analyses using a xEmGCNF-specific morpholino antisense oligonucleotide indicate that xEmGCNF is required for the effect of RA on primary neurogenesis. This may be caused by transcriptional regulation of the gene encoding the RA-degrading enzyme CYP26, since this gene is derepressed after depletion of xEmGCNF and an antimorph of xEmGCNF directly activates transcription of CYP26, also in absence of protein synthesis. The effect of xEmGCNF knockdown on hindbrain patterning is similar to conditions of reduced RA signaling, which may be caused by a reduction of RAR gamma expression specifically in the presumptive hindbrain
Transcriptional ERRγ2-mediated activation is regulated by sentrin-specific proteases
No full textInternational audienceModification with small ubiquitin-like modifiers (SUMOs) has emerged as an important means of regulating the activity of transcription factors, often by repressing their activity. The estrogen receptor-related receptor γ (ERRγ, ERR3, NR3B3) is a constitutively active orphan nuclear receptor. A phosphorylation-dependent sumoylation motif (PDSM) is located in close vicinity of the amino-terminally located ERRγ2-specific activation function 1 (AF-1). Its function can be substituted by a negatively charged amino acid-dependent sumoylation motif (NDSM). A mutational analysis reveals that ERRγ2-activity is modulated through sumoylation of a lysine residue at position 40 which in turn is regulated by phosphorylation. Phosphorylation in the +5-position relative to the sumoylation target is directly visualized by a high resolution electrophoretic mobility shift assay (EMSA). Sumoylation represses the activity of ERRγ both, with and without forced expression of the peroxisome proliferator-activated receptor gamma coactivator 1β (PGC-1β). Fusion proteins of a heterologous DNA binding domain with the ERRγ2 amino terminus demonstrate the function of the PDSM as the repression function 1 (RF-1) for the neighboring AF-1. Derepression is achieved by coexpression of Sentrin/SUMO-specific protease (SENP) family members. Together, our results demonstrate reversible phosphorylation-dependent sumoylation as a means to regulate the activity of an orphan nuclear receptor
Domains of ERRγ that mediate homodimerization and interaction with factors stimulating DNA binding
No full textDNA binding, protein interaction and differential expression of the human germ cell nuclear factor
No full text
