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Identification of disease modulating compounds in juvenile neuronal ceroid lipofuscinosis (JNCL)
(2016)
Mutationen im CLN3 Gen verursachen die neurodegenerative Erkrankung juvenile neuronale Zeroidlipofuszinose (JNCL). Bei dieser Erkrankung sind die Autophagie, der lysosomale pH Wert und der mitochondriale Metabolismus beeinträchtigt. Störungen dieser Prozesse führen zu einer erhöhten Verletzlichkeit neuronaler Zellen gegenüber alters- und umweltbedingten Schäden, einer Anhäufung von Autophagosomen und lysosomalem Speichermaterial, Zelltod und Neurodegeneration. Um die JNCL zu erforschen bedienen wir uns eines Zellmodels aus der Maus, welches die häufigste krankheitsauslösende CLN3 Mutation im Menschen, die Deletion der Exons 7 und 8, nachbildet. Die aus dem Kleinhirn dieser Mäuse stammenden cerebellaren Körnerstammzellen werden als CbCln3Δex7/8/Δex7/8 Zellen, solche aus wild-typ Mäusen als CbCln3+/+ Zellen bezeichnet. Die JNCL ist nicht heilbar und die Entwicklung von Wirkstoffen steht noch am Anfang.
Die vorliegende Arbeit befasst sich mit der Durchführung eines Hochdursatzscreenings um Wirkstoffe zu identifizieren, welche eine Anhäufung von Autophagosomen in CbCln3Δex7/8/Δex7/8 Zellen verhindern können. Unter 1750 verschiedenen untersuchten Wirkstoffen konnten wir 28 aktive „Hits“ identifizieren und stellten fest, dass Kalziumkanalblocker, Östrogene und HMG-CoA-Reduktase Inhibitoren gehäuft vertreten waren. Eine sorgfältige Untersuchung die möglichen Interaktionen der aktiven Wirkstoffe mit zellulären Signalwegen und die Analyse ihrer Dosis-Wirkungskurven unterstützte uns bei der Auswahl von Verapamil, Nicardipin und Fluspirilen zur näheren Untersuchung. Diese Wirkstoffe sind Kalziumkanalblocker und Fluspirilen blockt auch D2 Dopaminrezeptoren.
Außerdem untersuchten und quantifizierten wir mitochondriale Phänotypen in CbCln3Δex7/8/Δex7/8 Zellen. Unsere Untersuchungen ergaben, dass Mitochondrien in CbCln3Δex7/8/Δex7/8 Zellen einer signifikanten Hyperfusion unterliegen und ein schwächeres Membranpotenzial aufweisen. Weiterhin fanden wir eine Verringerung der maximalen der mitochondrialen Elektronentransportkapazität und eine verringerte Aktivität des Enzyms Zitratsynthase, welches die Effizienz des Zitratzyklus bestimmt.
Fluspirilen, Verapamil und, in geringerem Ausmaß, Nicardipin, verbesserten einige krankheitsbedingte lysosomale und mitochondriale Phänotypen. Des Weiteren konnten Verapamil und Nicardipin, nicht aber Fluspirilen, den erhöhten zellulären Kalziumspiegel in CbCln3Δex7/8/Δex7/8 Zellen absenken. Erniedrigungen im Kalziumgehalt können durch die Inhibition der kalziumabhängigen Protease Calpain 1 zu einer Induktion der Autophagie führen. Wir untersuchten, ob eine chemische Inhibition der Calpain 1-Protease die Anzahl der Autophagosomen in CbCln3Δex7/8/Δex7/8 Zellen senkt, und stellten fest, dass dies nicht der Fall ist. Eine Inhibition von Calpain 1 führte lediglich zu einem Anstieg der Zahl zellulärer Autophagosomen. Als Nächstes untersuchten wir die Auswirkung der Wirkstoffbehandlung auf den Autophagiefluss. Verapamil und Nicardipin hatten keinen Einfluss auf den Autophagiefluss in der getesteten Konzentration in CbCln3Δex7/8/Δex7/8 Zellen während Fluspirilen die Autophagie induzierte. Gleichzeitig stellten wir fest, dass hohe Dosen von Nicardipin und Verapamil teilweise vor einem Verlust des lysosomalen pH-Werts durch eine Behandlung mit Bafilomycin A1 schützen konnten. Da Fluspirilen auch ein Dopaminrezeptorblocker ist, untersuchten wir die Auswirkung einer erhöhten Dosis von Dopamin auf die Zahl der Autophagosomen. Wir fanden, dass eine mittlere Dosierung von Dopamin einen Trend zu einer leichten Verringerung von Autophagosomen in CbCln3Δex7/8/Δex7/8 Zellen zur Folge hat.
Wir vermuten, dass die Kalziumkanalblocker Verapamil und Nicardipin und der Dopaminrezeptorblocker Fluspirilen unterschiedliche zelluläre Signalwege benutzen, aber letztendlich um ähnliche Botenstoffe verwenden, um die Funktion der Lysosomen in CbCln3Δex7/8/Δex7/8 Zellen zu verbessern. Die Verringerung des intrazellulären Kalziumgehalts durch Verapamil und Nicardipin führt zu einer Aktivierung von Adenylatzyklasen, welche eine Erhöhung des intrazellulären cAMP Spiegels herbeiführen. Fluspirilen inhibiert Dopaminrezeptoren vom Typ D2 (D2DR), was zu einer selektiven Aktivierung von Dopaminrezeptoren des Typs D5 (D5DR) führen könnte. Im Gegensatz zu D2 führen D5D Rezeptoren zu einer Aktivierung von Adenylatzyklasen und einer Erhöhung des cAMP Spiegels. cAMP aktiviert die Protein Kinase A (PKA), welche durch eine Proteinphosphorylierung von lysosomalen Chloridkanälen und Protonenpumpen die lysosomale Aktivität erhöht. Dies führt zu einer Verbesserung des Abbaus von Autophagosomen und lysosomalem Speichermaterial und zu einer verbesserten Zellgesundheit in CbCln3Δex7/8/Δex7/8 Zellen.
Eine Verbesserung der lysosomalen Funktion in der JNCL kann einen wirksamen Therapieansatz ergeben. Wir hoffen, dass die hier vorgestellten Methoden und Ergebnisse einen ersten Schritt in diese Richtung darstellen.
Juvenile neuronal ceroid-lipofuscinosis (JNCL) is a rare lysosomal storage disease in children with lethal outcome and no therapy. The origin of JNCL has been traced to autosomal recessive mutations in the CLN3 gene, and ~85% of the JNCL patients harbor a 1.02 kb deletion that removes the exons 7 and 8 and the surrounding intronic DNA (CLN3Δex7/8). So far, structure, function and localization of the CLN3 protein remain elusive. However, there is strong evidence that CLN3 modulates a process or condition that is essential in many cellular pathways. Lipid metabolism and antero-/retrograde transport, two mechanisms CLN3 was previously implicated in, fulfill these requirements. Notably, also a bioactive group of glycosphingolipids referred to as gangliosides is tightly interrelated with these functions. Furthermore, a-series gangliosides have been shown to be involved in the development and sustenance of the brain, where they are essential for neurite outgrowth and cell survival. Defects in ganglioside metabolism were shown to play a crucial role in many lysosomal storage disorders. However, the contribution of gangliosides to NCL pathology is largely unknown.
The present study analyzed central enzymes and metabolites of the a-series ganglioside pathway in a JNCL cell model. The core finding was, thereby, the reduced amount of the neuroprotective ganglioside GM1 in homozygous CbCln3Δex7/8 cells. This was caused by the enhanced action of the GM1-degrading multimeric enzyme complex and in particular, by the upregulation of protein levels and increased enzyme activity of β-galactosidase (Glb1).
Improved binding of Glb1 to substrate-carrying membranes was provided by an increase in LBPA levels. In combination with other smaller alterations in the ganglioside pattern, a shift towards less complex gangliosides became present. The resulting loss of neuroprotection may be the reason for the multifocal pathology in homozygous CbCln3Δex7/8 cells.
The second part of the present study investigated the cellular mechanisms behind the altered ganglioside profile with regard to the potential role of CLN3. Here, the anterograde transport of GM1 to the plasma membrane presented a positive correlation with the amount of full-length CLN3. In case of the truncated protein this correlation was missing, resulting in reduced PM staining with CTxB-FITC. However, transfection of full-length CLN3 in these cells restored the CTxB-FITC intensity. Based on the neuroprotective role of GM1, the corresponding increase in GM1 levels may be the cause for the restoration effects observed in previous studies using full-length CLN3. Hence, administration of GM1 was expected to improve cell viability of homozygous CbCln3Δex7/8 cells and beyond that to rescue potentially some disease phenotypes. However, no effect could be observed. The reason for this may be reduced caveolar uptake and the mislocalization of ganglioside GM1 to the trans-Golgi network (TGN) and redirection towards degradative compartments.
Both are in line with the idea of an impaired endocytic flux in CLN3 deficiency. The observed localization of CLN3 in the TGN suggests a potential role for CLN3 in the lipid sorting machinery, subsequently altering membrane composition and its regulatory functions. The resulting imbalance may affect many of the cellular processes impaired in JNCL.
Batten disease refers to neuronal ceroid lipofuscinoses (NCLs), which are inherited lysosomal storage diseases with diverse ages of onset and cause progressive neurodegeneration. The most common NCL is Juvenile NCL (JNCL), which begins in early childhood and is characterized by lysosomal accumulation of subunit c of the mitochondrial ATP synthase (subunit c). JNCL is caused by mutations in the gene CLN3. This gene encodes the CLN3 protein, a transmembrane protein of unknown structure. Localization of CLN3 is ambiguous, and its exact cellular function is not known. Thereby, it is unclear what mechanisms lead to neurodegeneration in JNCL. Models of JNCL present disturbed membrane bound organelles and cytoskeleton as well as impaired autophagy and lysosomal function. The JNCL gene defect that most patients harbor is deletion of the exons 7 and 8 of CLN3. In the Cln3Δex7/8/Δex7/8 mouse model of JNCL, this deletion has been introduced to the mouse Cln3 gene.
The actin cytoskeleton consists of filaments formed through polymerization of actin and provides a framework which defines cellular morphology and also facilitates cell motility, cytokinesis, and cell surface remodeling. Rho GTPases are signaling proteins which regulate the assembly and dynamics of the actin cytoskeleton and play an important role in neuronal morphology. Rho GTPases need to be membrane-anchored in order to become active and initiate a signaling cascade. Their membrane anchorage is achieved through their geranylgeranyl tails, which they acquire through prenylation. Protein prenylation refers to the attachment of a geranylgeranyl or farnesyl group to the C-terminus of a protein. The enzyme geranylgeranyl transferase (GGTase) catalyzes geranylgeranylation, whereas geranylgeranyl pyrophosphate (GGPP) is the donor of the geranylgeranyl group. Cells produce GGPP as well as cholesterol and other lipids through the mevalonate pathway (MVA pathway).
The aim of this study was to analyze how the JNCL gene defect affects cellular morphology, especially the actin cytoskeleton and Rho GTPases, and the MVA pathway which is connected with Rho GTPase activation. These important cellular components play crucial roles in neurons and are implicated in other neurodegenerative diseases, but have received little attention in JNCL. The immortalized CbCln3Δex7/8/Δex7/8 cerebellar precursor cell line from Cln3Δex7/8/Δex7/8 mice was used for the experiments and provides a genetically accurate, neuronal cell model of JNCL. CbCln3Δex7/8/Δex7/8 cells present subunit c accumulation only when aged at confluency, but sub-confluent cells display other phenotypes. The experiments of this study were performed both with confluency-aged and sub-confluent cells. Filamentous actin was visualized, and protein levels as well as membrane localization of several small Rho GTPases was analyzed biochemically. Also the protein levels of GGTase and the key enzymes of the mevalonate pathway were determined.
Staining pattern of filamentous actin was disturbed in confluency-aged CbCln3Δex7/8/Δex7/8 cells. Additionally it was found out that these cells did not grow to wild-type size and exhibited an elongated peroxisomal morphology. Rho GTPases had reduced total levels and showed a tendency of decreased membrane localization. Levels of GGTase and the MVA pathway enzymes were altered. Results of sub-confluent CbCln3Δex7/8/Δex7/8 cells were similar with the exception of HMG-CoA reductase, which is the rate-limiting enzyme of the MVA pathway: while its level in confluency-aged CbCln3Δex7/8/Δex7/8 cells was increased, at sub-confluency it showed a reduced level. Also, in contrast with the confluency-aged cells, Rho GTPases presented a tendency of increased membrane localization.
The results of this study reveal that the accurate JNCL gene defect alters cellular morphology and the activity of the MVA pathway in neuronal cells. Small cell size and disrupted architecture of the actin cytoskeleton are confirmed as neuronal JNCL phenotypes, and the peroxisome is introduced as a novel cellular component affected in JNCL. Through defects in endocytosis, autophagy, lysosomal and mitochondrial function, and cytoskeleton, the JNCL gene defect may prevent cells from growing to wild-type size. The JNCL gene defect may attenuate the MVA pathway via mitochondrial dysfunction and/or upregulation of degradative processes. Attenuation of the MVA pathway may contribute to impaired membrane rafts, which are an established phenotype of JNCL cells. As indicated by reduced GGTase level and supported by downregulation of lipid production through the MVA pathway, the JNCL gene defect might also decrease prenylation of proteins.
Alzheimer’s disease (AD) is a common, age associated neurodegenerative disease that manifests as progressive dementia and is characterized by accumulation of the amyloid beta (Aβ) peptide which is a processing product of a transmembrane protein termed Alzheimer Amyloid Precursor Protein (APP). The Aβ peptide is generated by a sequential proteolytic processing of APP by two distinct proteases that are termed β- and γ-secretase. The β-secretase, also called BACE-1 or memapsin 2, belongs to the family of aspartyl proteases. BACE-1 evidently cleaves APP in an acidic endosomal compartment after endocytosis of APP, thereby facilitating Aβ peptide generation.
Sorting of transmembrane proteins is generally controlled by sorting signals in the cytoplasmic domains of the cargo proteins. The short cytoplasmic tail of BACE-1 with 23 amino acids contains a sorting signal of the acidic cluster, di-leucine (ACDL) type. The two Leu residues in this determinant are important for the clathrin mediated endocytosis of BACE-1, whereas the acidic residues together with the Leu are required for the endosomal sorting and recycling of BACE-1 back to the plasma membrane. The ACDL motif binds to the members of the GGA (Golgi-localized γ ear-containg ARF- binding proteins) family (GGA1-GGA3) that are involved in the sorting of BACE-1.
One of the major aims of this study was to address the role of flotillins in the intracellular sorting of BACE-1. This study shows that flotillin-1 directly binds to the di-leucine motif in the cytoplasmic tail of BACE-1, whereas flotillin-2 only shows an association mediated by flotillin-1. Flotillin-1 competes with GGA2 for the binding to BACE-1 tail, and thus influences the endosomal sorting of BACE-1. Importantly, depletion of flotillins results in an altered localization of the wildtype BACE-1, whereas the plasma membrane resident Leu to Ala (LLAA) mutant is not affected. Flotillin knockdown results in an accumulation of BACE-1, implicating reduced degradation and enhanced stability of this protease. Thus, flotillins appear to be important for the cellular targeting of BACE-1 and also influence the amyloidogenic processing of APP, as demonstrated by an increase in the amyloidogenic C-99 processing fragments.
When flotillin depleted cells were subjected to apoptotic stresses including Aβ25-35 synthetic peptide (inducer of the extrinsic apoptosis pathway) or several chemotherapeutic agents (staurosporine, brefeldin A, doxorubicin, carboplatin and paclitaxel: intrinsic apoptosis pathway) and cytotoxicity was determined, various apoptotic markers were activated in flotillin depleted cells. Caspase-3 and GGA3 are well accepted apoptosis markers and an enhanced caspase-3 cleavage was detected upon STS induced apoptosis in SH-SY5Y, HeLa, and HaCaT cell lines and increased GGA3 cleavage was observed in MCF7 cell line.
One of the major reasons for the apoptotic sensitivity in the absence of flotillins was a PI3K/Akt signaling defect. Neuroblastoma cells depleted of flotillins showed diminished levels of total Akt, phospho-Akt and phospho-ERK upon STS induced apoptosis. Since PI3K/Akt was the primary survival pathway affected upon STS induced apoptosis, ectopic expression of Akt in neuroblastoma cell line reduced caspase-3 cleavage and retarded apoptosis.
The direct downstream target of Akt is FOXO3a, whose localization was investigated in flotillin depleted cells. A major proportion of FOXO3a was localized in the nucleus of flotillin knockdown cells, implicating that FOXOs are active in these cells and subsequently trigger the transcription of death genes. Strikingly, an essential anti-apoptotic molecule and a major cancer target, Mcl-1, was inherently downregulated in flotillin knockdown cells. Mcl-1 is a chief member of the Bcl-2 family as it plays a pivotal role in cell survival and it is a critical protein in cancer therapeutics as suppression of Mcl-1 protein can curtail the survival and growth of tumorous cells.
Neuroblastoma cells were rescued from undergoing permanent damage due to STS induced apoptosis by overexpression of anti-apoptotic Bcl-2. Phorbol esters are well known PKC activators, and pre-treatment of neuroblastoma cells with phorbol esters along with staurosporine reduced caspase-3 cleavage.
These results demonstrate that absence of flotillins can sensitize cellular systems to apoptosis induction. The two main characteristics of cancer cells include resistance to apoptosis and unresponsiveness to chemotherapeutic agents. It is a well established fact that impaired apoptosis is central to tumour development. This study implicates that the downregulation of flotillin function can trigger cellular susceptibility and enhances apoptosis in response to conventional chemotherapeutic agents. Therefore, flotillins can serve as vital regulators in providing a more rational approach in molecular-targeted therapies for receding cancer growth and survival.
Nikotinische Acetylcholin Rezeptoren (nAChR) sind ligandengesteuerte Ionenkanäle der pentameren Cys-Loop Familie, welche nach Bindung des Neurotransmitters Acetylcholin exzitatorische Signale in Muskeln und Neuronen vermitteln. Während die Funktion der Rezeptoren an der synaptischen Membran relativ gut untersucht wurde, gibt es bis heute kaum Erkenntnisse über die intrazellulären Prozesse und Proteine, die der selektiven Assemblierung von homologen Untereinheiten zu funktionalen Rezeptorpentameren zugrundeliegen.
Das C. elegans Genom kodiert für mehr als 29 nAChR Untereinheiten-Gene und besitzt damit die größte Anzahl bekannter Homologe innerhalb der untersuchten Arten. An der neuromuskulären Synapse (NMJ) des Nematoden sind zwei Typen von nAChR bekannt: der heteromere Levamisolrezeptor (L-AChR) und der homomere Nikotinrezeptor (N-AChR). Innerhalb dieser Arbeit wurde der funktionale Zusammenhang zwischen den nikotinischen Rezeptoren der NMJ von C. elegans und einem neuen rezeptorassoziierten ER-Proteinkomplex der Proteine NRA-2 und NRA-4 untersucht. Ihre vertebraten Homologe Nicalin und Nomo wurden zuerst im ER vom Zebrafisch im Zusammenhang mit dem TGF-β Signalweg beschrieben. Mutation der Proteine hat einen Agonist-spezifischen Einfluss auf die Aktivität von L-AChR und N-AChR. Die subzellulären Lokalisationsstudien demonstrierten, dass die beiden Proteine im ER von Muskelzellen wirken und dort mit Rezeptoruntereinheiten co-lokalisieren. Weiterhin ließ sich nachweisen, dass die relative Menge einzelner L-AChR-Untereinheiten an der synaptischen Oberfläche reduziert bzw. erhöht ist. Da die Rezeptoraktivität in Zusammenhang mit der Untereinheiten Komposition steht, wurde die Rolle von zusätzlichen Untereinheiten wie ACR-8 untersucht. Dies zeigte, dass die zusätzliche Mutation der Untereinheit acr-8 in nra-2 Mutanten den Einfluss der nra-2 Einzelmutation auf die Aktivität des L-AChR revertiert. Basierend auf diesen Ergebnissen lässt sich die Hypothese formulieren, dass der NRA-2/NRA-4 Komplex im ER von C. elegans als Kontrollinstanz fungiert welche dafür sorgt, dass nur die jeweils „korrekten“ Untereinheiten in funktionale Rezeptoren eingebaut bzw. andere vom Einbau in das Pentamer abgehalten werden. Durch Fehlen des aktiven Komplexes in Mutanten können nicht vorgesehene -Untereinheiten (z. B. ACR-8) in funktionale Pentamere mit veränderter Funktionalität eingebaut werden.
The long sought molecular function of membrane raft-associated flotillin proteins is slowly becoming resolved, partially owing to the increasing knowledge about their interaction partners. Being ubiquitously expressed and evolutionarily highly conserved, flotillins carry out important cellular functions, one of which is the regulation of signal transduction pathways. This study shows that the signaling adaptor protein fibroblast growth factor receptor substrate 2 (FRS2) directly interacts both in vivo and in vitro with flotillin-1 (flot-1). FRS2 is an important docking protein of many receptor tyrosine kinases. It regulates downstream signaling by forming molecular complexes with other adaptor proteins and tyrosine phosphatases, and seems to be a critical mediator of sustained extracellular signal regulated kinase (ERK) activity. Flot-1 has also been implicated in the regulation of ERK activity upon EGF and FGF stimuli. Furthermore, flot-1 forms signalosomes with EGFR and the downstream components of the MAP kinase pathway. The newly discovered interaction between FRS2 and flot-1 was shown to be mediated by the phosphotyrosine binding (PTB) domain and, to a lesser extent, the C-terminus (CT) of FRS2 and by the C-terminus of flot-1. Flot-1 coprecipitated together with FRS2 from murine tissues and cell lysates, demonstrating that this interaction also takes place in vivo. Interestingly, flot-2, which shows a high homology to flot-1 and forms stable oligomeric complexes with it, does not appear to directly interact with FRS2. Novel insights into the functional role of the interaction between flot-1 and FRS2 were provided by the results showing that depletion of flot-1 affects the cellular localization of FRS2. In hepatocytes stably depleted of flot-1, FRS2 appeared to be more soluble. Furthermore, upon pervanadate stimulation of the cells, a small fraction of FRS2 was recruited into detergent resistant membranes, but the recruitment did not take place in the absence of flot-1. Triggered by the same stimulus, a fraction of FRS2 was translocated to the nucleus independently of flot-1. Overexpression of FRS2 has previously been shown to result in increased ERK activation. However, in cells depleted of flot-1, FRS2 was not able to compensate for the compromised ERK activation after EGF or FGF stimulation. This might imply that FRS2 and flot-1 are functionally interconnected and that FRS2 resides upstream of flot-1. Taken together, the results presented here indicate that this complex may be involved in the control of signaling downstream of receptor tyrosine kinases and is important for ensuring a proper signaling response. In the absence of flot-1, increased Tyr phosphorylation of FRS2 was observed. It is known that Tyr and Thr phosphorylation of FRS2 are reciprocally regulated. Since ERK is a known executor of the FRS2 Thr phosphorylation, and ERK activity was shown to be severely diminished upon flot-1 depletion, the increased Tyr phosphorylation of FRS2 was in agreement with this and might be a direct consequence of a decreased ERK activity upon flot-1 depletion. FRS2 owes its name to the major and the first described function of this protein as a substrate for FGFR. PTB domain of FRS2 was published to constitutively bind the juxtamembrane domain of FGFR. In this study, the PTB domain was mapped to be involved in the constitutive interaction with flot-1 and the competition was shown to exist between flot-1 and FGFR1 for binding to FRS2. Another novel interaction partner of FRS2 was discovered in the present study. Cbl-associated protein (CAP) is an adaptor protein with three SH3 domains and it plays a role during insulin signaling by recruiting the signaling complex to lipid rafts. CAP was previously shown to interact with flot-1 via the SoHo domain, and this interaction was found to be crucial for the lipid raft recruitment of other signaling components. Both the PTB domain and CT of FRS2 were found to mediate the interaction with CAP, whereas in CAP, the SoHo domain, together with the third SH3 domain, seems to bind to FRS2. SH3 domains mediate the assembly of specific protein complexes by binding to proline rich sequences, several of which are present in FRS2. Due to overlapping interaction domains, FRS2 and flot-1 competed for the binding to CAP. However, the interaction with neither CAP nor flot-1 was necessary for the observed nuclear translocation of FRS2. Since CAP is expressed as several tissue- and developmental stage-specific isoforms, a further aim of this study was to analyze the expression of its isoforms in mouse embryonic fibroblasts (MEFs). Many new isoforms were discovered here which have not been described in the literature so far. They all contain the SoHo domain and three SH3 domains, but differ among themselves by the presence and length of a proline-rich region that preceeds the SoHo domain and by a novel 20-amino acid (AA) stretch between the second and the third SH3 domain. The length of the proline-rich region turned out to be an important factor determining the strength of the interaction with FRS2. The interaction was found to be weakened by the increasing length of this region. The new isoforms possessing the 20-AA stretch are specifically expressed in murine muscular tissues, with the highest level in the heart. During adipogenesis, we observed a shift in the abundance of the isoforms, in that only the isoforms without the insertion were shown to be upregulated on mRNA level. However, during myogenesis, preferentially expressed isoforms were those with the insertion. The collected data implicate that isoforms with the 20-AA insertion might be more ubiquitous in nondifferentiated/embryonic cells and that the observed "isoform-switch" might be dependent on the cell fate and differentiation state.
The brain is characterized by its immune privileged state. However, recent studies suggest an extended contribution of hematopoietic cells to the brain. After transplantation of genetically labeled bone marrow into bone marrow depleted mice, not only labeled blood cells but also labeled neurons and other non-hematopoietic cells can be observed. Initially interpreted as transdifferentiated hematopoietic stem cells, this contribution later was identified as cell fusion of hematopoietic cells and neurons. Our lab previously addressed the question whether these fusion events also occur under non-invasive conditions. A Cre-LoxP based transgenic mouse line was used to irreversibly label all hematopoietic cells. In these mice, Cre expression is controlled by a hematopoietic promoter, thus causing recombination and subsequent marker gene expression restricted to blood cells. Interestingly, contribution of these hematopoietic cells to non-hematopoietic tissues was observed, but fusion could be excluded as the underlying mechanism. The Cre mRNA or protein seems to reach the non-hematopoietic cells from an external source. Extracellular vesicles, specifically exosomes, are increasingly recognized as a vehicle for the intercellular transfer of cellular components such as proteins or mRNAs. However, if they contribute to signaling between tissues in vivo is completely unknown and would represent a major paradigm shift for intercellular communication. Therefore, the aim of this PhD study is to investigate whether an exosomal transfer between the hematopoietic system and the brain exists. To confirm the previous results, a second Cre-LoxP mouse line that expresses the Cre recombinase under a different hematopoietic promoter is used additionally. Both mouse lines are screened for recombination and show comparable numbers and types of different non-hematopoietic cells. Besides hepatocytes and cells in lung and intestine, recombined Purkinje neurons in the cerebellum are detectable. To assess the influence of inflammation on these recombination events, different lesions such as peripheral tumors or peritonitis are applied to the mice. Inflammatory stimuli strongly increase the numbers of recombined Purkinje neurons. These neurons remain mononuclear, indicating that fusion does not occur. Also in human cerebellar material, no evidence for inflammation induced cell fusion is detectable. To screen for Cre recombinase containing exosomes, exosome purification protocols such as differential ultracentrifugation and sucrose gradient fractioning, are applied. The exosomal content is analyzed with nested PCR and western blot. Hematopoietically expressed Cre mRNA is detectable in blood plasma and hematopoietic cell culture conditioned medium. Further analysis reveals that this Cre mRNA but no Cre protein is contained in exosomes. The exosomal ability to induce recombination is investigated by injections into Cre reporter mice. After direct cerebellar injection, exosomes are sufficient to induce recombination of Purkinje neurons. Brain tissue of mice that received an inflammation is analyzed further to reveal other recombined cell types. The main immune cells of the brain, microglia, are not recombined. Mainly neuronal cell types are recombined in different areas of the brain. The observations made in this study are consistent with the hypothesis that a previously unrecognized way to communicate RNA based signals between the immune system and the brain exists. Specifically neurons are target cells for the uptake of hematopoietic exosomes and seem able to translate exosomal mRNA into functional protein. Microglial cells are neither involved as target cells, nor do they release Cre containing exosomes. By using the Cre-LoxP system, in vivo tracing of exosomes could be achieved for the first time. With this knowledge, other exosomal routes can be uncovered in future. The discovery of the exosomal transfer between the blood and the brain enables further research about the relevance of this signaling pathway. It will be important to investigate its role especially in the context of neural malfunctions and further studies might help to find new therapeutical approaches.
BMPs control postnatal dendrite growth and complexity in sympathetic neurons / von Afsaneh Majdazari
(2012)
The vertebrate nervous system is a complex network of billions of neurons connected by dendrites and axons, integrated to functional circuits and areas/organs in the central and peripheral nervous system. The cells of the nervous system origin from common progenitors, which take on different cell fates based on intrinsic and extrinsic factors. These factors determine general neuronal traits, but also the morphology and the type of connections made to other cells. Mechanisms underlying axonal and dendritic growth are well described in contrast to the initiation of neurite growth, which remains to be fully elucidated, especially concerning dendrite formation. Recently BMPs have been identified as candidate dendrite inducing factors in sympathetic, cortical and hippocampal neurons. Here we focus on the in vivo role of BMPs on dendrite growth in sympathetic neurons as their development and differentiation processes have been analyzed in detail.
Im Rahmen dieser Arbeit konnte die Bindeeigenschaft des synaptischen Vesikelproteins SV31 zu den divalenten Metallionen Zn2+, Ni2+ sowie Cu2+ nachgewiesen und reproduziert werden. Die Bindung an Zn2+ wurde dabei sowohl in vitro an der Sepharosesäule als auch in vivo in NGF-differenzierten PC12-Zellen bestätigt (3.2.1 - 3.2.3). In einer Kollaboration mit dem Max-Planck-Institut für Biophysik wurde des Weiteren eine mögliche Zinktransportfunktion von SV31 untersucht. Dafür wurde die Ladungstranslokation durch myc-SV31-enthaltene CHO-Zellmembranen nach Zinkzugabe gemessen (3.2.5). Weiterhin konnte durch subzelluläre Fraktionierung von PC12-Zellen ein Verteilungsmuster des neuen Proteins in Mikrosomen unterschiedlicher Dichte dokumentiert werden. Durch die andauernde Expression von SV31-RFP in stabil transfizierten PC12-Zellen kommt es außerdem zur Beeinflussung des Expressionsmusters zahlreicher Markerproteine und damit einhergehend zu einer Dichteverschiebung somatischer Organellen (3.3.1 - 3.3.3). Kolokalisationsstudien von SV31 mit Markerproteinen zahlreicher Zellorganellen ergaben partielle Fluoreszenzüberlagerungen mit synaptischen Vesikelproteinen sowie eine Anreicherung von SV31 in Nähe der Plasmamembran. In diesem Zusammenhang zeigt sich ebenfalls eine Übereinstimmung der Lokalisation von SV31 mit den SNAREProteinen SNAP25 und Syntaxin1A (3.4.1 - 3.4.3). Die Ergebnisse der vorliegenden Arbeit erweitern nicht nur das Wissen um die funktionellen Eigenschaften von SV31, sie geben auch Anlass zum Nachdenken über mögliche Interaktionspartner des neuen Vesikelproteins. Die Fähigkeit zur Zinkbindung und -akkumulation auf präsynaptischer Seite rückt SV31, im Hinblick auf neurodegenerative Erkrankungen wie Alzheimer und Parkinson, auch in einen medizinisch relevanten Kontext. Durch Deduktion der hier aufgezeigten Ergebnisse entsteht ein erweitertes Verständnis der Relevanz von SV31 als funktionelle, zinkbindende Einheit im Rahmen der synaptischen Transmission.
Die noradrenergen Neurone der sympathischen Ganglien und die cholinergen Neurone der parasympathischen Ziliarganglien gehen aus den NLZ hervor. BMP-Signale induzieren die Differenzierung beider Neuronentypen, die mit der Expression von Ascl1 und Phox2a/b beginnt. Im Fall der sympathischen Ganglien werden dann Hand2 und GATA2/3 exprimiert, was wiederum zur Expression der noradrenergen Marker TH und DBH führt, die auch in differenzierten Neuronen weiterhin vorhanden sind. Im Gegensatz dazu werden während der Entwicklung der parasympathischen Ziliarneurone sowohl Hand2 als auch TH/DBH nur transient exprimiert, die differenzierten Neurone besitzen zum Großteil einen cholinergen Phänotyp (Goridis und Rohrer, 2002; Müller und Rohrer, 2002).
Thema dieser Arbeit war die Untersuchung der Rolle der Hox-Gene bei der Differenzierung des PNS. 14 der analysierten Hox-Gene werden in den sympathischen Ganglien exprimiert, wobei wir uns bei der näheren Analyse auf das HoxB-Cluster beschränkt haben. HoxB5, HoxB6, HoxB7, HoxB8 und HoxB9 werden zwischen E4 und E7 in den sympathischen und sensorischen Ganglien exprimiert, wobei nur HoxB8 und HoxB9 eine deutliche Expression in den sympathischen Ganglien zeigen. Die HoxB-Gene könnten dem Expressionsmuster nach also eine Rolle bei der frühen Entwicklung und auch bei der Aufrechterhaltung des noradrenergen Phänotyps der sympathischen Ganglien spielen.
Die differenzielle Expression der HoxB-Gene in den sympathischen Neuronen und den Ziliarneuronen und ihre mögliche Beteiligung bei der Aufrechterhaltung des noradrenergen Charakters waren Ausgangspunkt für die ektopische Expression eines Vertreters des HoxB-Clusters, HoxB8, in den Ziliarganglien. In der Normalentwicklung wird die Expression von Hand2, TH und DBH nach E4 in den Ziliarneuronen stark reduziert (Abb. 22A). Wird HoxB8 in den Vorläuferzellen der Ziliarneurone in vivo überexprimiert, wird die Hand2-, TH- und DBH-Expression weit über E4 hinaus, bis mindestens E8 auf einem signifikant höheren Niveau gehalten (Abb. 22B). HoxB8 kann diesen Effekt allerdings nur ausüben, wenn es in den noch undifferenzierten Vorläuferzellen exprimiert wird. Die HoxB8-Überexpression in Primärkulturen von Ziliarneuronen an E5 oder E8 führt nur noch zu einem Anstieg der Hand2-Expression, hat aber keinen Einfluss mehr auf die noradrenerge Genexpression (Abb. 22B).
HoxB8 zeigt zusätzlich im Vergleich mit den anderen analysierten Hox-Genen einen spezifischen Effekt auf die Hand2-, TH- und DBH-Expression, denn sowohl das paraloge Hox-Gen HoxC8 als auch das anterior-exprimierte HoxB-Gen HoxB1 erreichen nur an E5 eine signifikante Expression der drei Gene. Weder HoxC8 noch HoxB1 können die Expression von Hand2 und TH/DBH über E5 hinaus aufrechterhalten (Abb. 22C), während HoxB8 deren Expression auch noch an E8 auf einem hohen Niveau halten kann.
Die HoxB8-vermittelte Aufrechterhaltung der TH- und DBH-Expression in den Ziliarneuronen konnte allerdings nicht in einen direkten Zusammenhang mit der erhöhten Hand2-Expression gebracht werden, da die Überexpression von Hand2 nicht zu einer Aufrechterhaltung von TH und DBH an E5 und E6 führt (Abb. 22C).
Die Effekte von HoxB8 auf die Entwicklung der Ziliarneurone, die durch HoxB8 z.T. noradrenerge, sympathische Eigenschaften annehmen, unterstützen die Vorstellung, dass HoxB8 bei der Differenzierung und Ausbildung des noradrenergen Phänotyps in sympathischen Ganglien eine Rolle spielt. Es konnte also erstmals einem Vertreter der Hox-Gen-Familie eine mögliche Funktion bei der Differenzierung autonomer Neurone zugeordnet werden.