TY - JOUR A1 - Müller, Thomas A1 - Mueller, Bernhard Klaus A1 - Riederer, Peter T1 - Perspective: Treatment for disease modification in chronic neurodegeneration JF - Cells N2 - Symptomatic treatments are available for Parkinson's disease and Alzheimer's disease. An unmet need is cure or disease modification. This review discusses possible reasons for negative clinical study outcomes on disease modification following promising positive findings from experimental research. It scrutinizes current research paradigms for disease modification with antibodies against pathological protein enrichment, such as α-synuclein, amyloid or tau, based on post mortem findings. Instead a more uniform regenerative and reparative therapeutic approach for chronic neurodegenerative disease entities is proposed with stimulation of an endogenously existing repair system, which acts independent of specific disease mechanisms. The repulsive guidance molecule A pathway is involved in the regulation of peripheral and central neuronal restoration. Therapeutic antagonism of repulsive guidance molecule A reverses neurodegeneration according to experimental outcomes in numerous disease models in rodents and monkeys. Antibodies against repulsive guidance molecule A exist. First clinical studies in neurological conditions with an acute onset are under way. Future clinical trials with these antibodies should initially focus on well characterized uniform cohorts of patients. The efficiency of repulsive guidance molecule A antagonism and associated stimulation of neurogenesis should be demonstrated with objective assessment tools to counteract dilution of therapeutic effects by subjectivity and heterogeneity of chronic disease entities. Such a research concept will hopefully enhance clinical test strategies and improve the future therapeutic armamentarium for chronic neurodegeneration. KW - neurodegeneration KW - repulsive guidance molecule A KW - neuroprotection KW - repair KW - oxidative stress KW - apoptosis KW - neurogenesis Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-236644 SN - 2073-4409 VL - 10 IS - 4 ER - TY - JOUR A1 - Voelker, Johannes A1 - Voelker, Christine A1 - Engert, Jonas A1 - Goemann, Nikolas A1 - Hagen, Rudolf A1 - Rak, Kristen T1 - Spontaneous Calcium Oscillations through Differentiation: A Calcium Imaging Analysis of Rat Cochlear Nucleus Neural Stem Cells JF - Cells N2 - Causal therapies for the auditory-pathway and inner-ear diseases are still not yet available for clinical application. Regenerative medicine approaches are discussed and examined as possible therapy options. Neural stem cells could play a role in the regeneration of the auditory pathway. In recent years, neural stem and progenitor cells have been identified in the cochlear nucleus, the second nucleus of the auditory pathway. The current investigation aimed to analyze cell maturation concerning cellular calcium activity. Cochlear nuclei from PND9 CD rats were microscopically dissected and propagated as neurospheres in free-floating cultures in stem-cell medium (Neurobasal, B27, GlutaMAX, EGF, bFGF). After 30 days, the dissociation and plating of these cells took place under withdrawal of the growth factors and the addition of retinoic acid, which induces neural cell differentiation. Calcium imaging analysis with BAPTA-1/Oregon Green was carried out at different times during the differentiation phase. In addition, the influence of different voltage-dependent calcium channels was analyzed through the targeted application of inhibitors of the L-, N-, R- and T-type calcium channels. For this purpose, comparative examinations were performed on CN NSCs, and primary CN neurons. As the cells differentiated, a significant increase in spontaneous neuronal calcium activity was demonstrated. In the differentiation stage, specific frequencies of the spontaneous calcium oscillations were measured in different regions of the individual cells. Initially, the highest frequency of spontaneous calcium oscillations was ascertainable in the maturing somata. Over time, these were overtaken by calcium oscillations in the axons and dendrites. Additionally, in the area of the growth cones, an increasing activity was determined. By inhibiting voltage-dependent calcium channels, their expression and function in the differentiation process were confirmed. A comparable pattern of maturation of these channels was found in CN NSCs and primary CN neurons. The present results show that neural stem cells of the rat cochlear nucleus differentiated not only morphologically but also functionally. Spontaneous calcium activities are of great relevance in terms of neurogenesis and integration into existing neuronal structures. These functional aspects of neurogenesis within the auditory pathway could serve as future targets for the exogenous control of neuronal regeneration. KW - neurogenesis KW - neural stem cells KW - neuronal oscillations KW - neuronal maturation KW - auditory pathway KW - regenerative capacity Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-248482 SN - 2073-4409 VL - 10 IS - 10 ER - TY - JOUR A1 - Fauser, Mareike A1 - Weselek, Grit A1 - Hauptmann, Christine A1 - Markert, Franz A1 - Gerlach, Manfred A1 - Hermann, Andreas A1 - Storch, Alexander T1 - Catecholaminergic Innervation of Periventricular Neurogenic Regions of the Developing Mouse Brain JF - Frontiers in Neuroanatomy N2 - The major catecholamines—dopamine (DA) and norepinephrine (NE)—are not only involved in synaptic communication but also act as important trophic factors and might ultimately be involved in mammalian brain development. The catecholaminergic innervation of neurogenic regions of the developing brain and its putative relationship to neurogenesis is thus of pivotal interest. We here determined DA and NE innervation around the ventricular/subventricular zone (VZ/SVZ) bordering the whole ventricular system of the developing mouse brain from embryonic day 14.5 (E14.5), E16.5, and E19.5 until postnatal day zero (P0) by histological evaluation and HPLC with electrochemical detection. We correlated these data with the proliferation capacity of the respective regions by quantification of MCM\(^{2+}\) cells. During development, VZ/SVZ catecholamine levels dramatically increased between E16.5 and P0 with DA levels increasing in forebrain VZ/SVZ bordering the lateral ventricles and NE levels raising in midbrain/hindbrain VZ/SVZ bordering the third ventricle, the aqueduct, and the fourth ventricle. Conversely, proliferating MCM\(^{2+}\) cell counts dropped between E16.5 and E19.5 with a special focus on all VZ/SVZs outside the lateral ventricles. We detected an inverse strong negative correlation of the proliferation capacity in the periventricular neurogenic regions (log-transformed MCM\(^{2+}\) cell counts) with their NE levels (r = −0.932; p < 0.001), but not their DA levels (r = 0.440; p = 0.051) suggesting putative inhibitory effects of NE on cell proliferation within the periventricular regions during mouse brain development. Our data provide the first framework for further demandable studies on the functional importance of catecholamines, particularly NE, in regulating neural stem/progenitor cell proliferation and differentiation during mammalian brain development. KW - brain development KW - ventricular zone KW - catecholamines KW - norepinephrine KW - dopamine KW - neurogenesis Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-212485 VL - 14 ER - TY - THES A1 - Ziegenhals, Thomas T1 - The role of the miR-26 family in neurogenesis T1 - Die Rolle der miR-26 Familie in der Neurogenese N2 - For the differentiation of a embryonic stem cells (ESCs) to neuronal cells (NCs) a complex and coordinated gene regulation program is needed. One important control element for neuronal differentiation is the repressor element 1 silencing transcription factor (REST) complex, which represses neuronal gene expression in non-neuronal cells. Crucial effector proteins of the REST complex are small phosphatases such as the CTDSPs (C-terminal domain small phosphatases) that regulate polymerase II activity by dephosphorylating the C-terminal domain of the polymerase, thereby repressing target genes. The stepwise inactivation of REST, including the CTDSPs, leads to the induction of a neuron-specific gene program, which ultimately induces the formation of neurons. The spatio-temporal control of REST and its effector components is therefore a crucial step for neurogenesis. In zebrafish it was shown that the REST-associated CTDSP2 is negatively regulated by the micro RNA (miR) -26b. Interestingly, the miR-26b is encoded in an intron of the primary transcript of CTDSP2. This gives the fundament of an intrinsic regulatory negative feedback loop, which is essential for the proceeding of neurogenesis. This feedback loop is active during neurogenesis, but inactive in non-neuronal cells. The reason for this is that the maturation of the precursor miR (pre-miR) to the mature miR-26 is arrested in non neuronal cells, but not in neurons. As only mature miRs are actively repressing genes, the regulation of miR-26 processing is an essential step in neurogenesis. In this study, the molecular basis of miR-26 processing regulation in the context of neurogenesis was addressed. The mature miR is processed from two larger precursors: First the primary transcript is cleaved by the enzyme DROSHA in the nucleus to form the pre-miR. The pre-miR is exported from the nucleus and processed further through the enzyme DICER to yield the mature miR. The mature miR can regulate gene expression in association with the RNA-induced silencing complex (RISC). Multiple different scenarios in which miR processing was regulated were proposed and experimentally tested. Microinjection studies using Xenopus leavis oocytes showed that slowdown or blockage of the nucleo-cytoplasmic transport are not the reason for delayed pre-miR-26 processing. Moreover, in vitro and in vivo miR-processing assays showed that maturation is most likely regulated through a in trans acting factor, which blocks processing in non neuronal cells. Through RNA affinity chromatographic assays using zebrafish and murine lysates I was able to isolate and identify proteins that interact specifically with pre-miR-26 and could by this influence its biogenesis. Potential candidates are FMRP/FXR1/2, ZNF346 and Eral1, whose functional characterisation in the context of miR-biogenesis could now be addressed. The second part of my thesis was executed in close colaboration with the laboratory of Prof. Albrecht Müller. The principal question was addressed how miR-26 influences neuronal gene expression and which genes are primarily affected. This research question could be addressed by using a cell culture model system, which mimics ex vivo the differentiation of ESCs to NCs via neuronal progenitor. For the functional analysis of miR-26 knock out cell lines were generated by the CRISPR/Cas9 technology. miR-26 deficient ESC keep their pluripotent state and are able to develop NPC, but show major impairment in differentiating to NCs. Through RNA deep sequencing the miR-26 induced transcriptome differences could be analysed. On the level of mRNAs it could be shown, that the expression of neuronal gene is downregulated in miR-26 deficient NCs. Interestingly, the deletion of miR-26 leads to selectively decreased levels of miRs, which on one hand regulate the REST complex and on the other hand are under transcriptional control by REST themself. This data and the discovery that induction of miR-26 leads to enrichment of other REST regulating miRs indicates that miR-26 initiates neurogenesis through stepwise inactivation of the REST complex. N2 - Für die Differenzierung von embryonalen Stammzellen (ESCs) zu Neuronen (NCs) bedarf es eines komplexen Genregulationsprogramms, welches sowohl zeitlich als auch räumlich reguliert werden muss. Ein wichtiger Faktor während der neuronalen Differenzierung ist der sogenannte „repressor element 1 silencing transcription factor“ (REST)-Komplex, welcher die Expression neuronaler Gene in nicht neuralen Zellen unterdrückt. Wichtige Effektorproteine im REST-Komplex sind kleine Phosphatasen (sog. CTDSPs), welche durch die Dephosphorylierung der C-terminalen Domäne der RNA-Polymerase II deren Aktivität an Zielgenen reprimiert. Die schrittweise Inaktivierung von REST, einschließlich der CTDSPs, führt hingegen zur Einleitung des neuronalen Genprogramms und damit zur Entwicklung von neuronalen Zellen. Die zeitliche Regulierung des REST-Komplexes und seiner assoziierten Effektor-Komponenten ist daher auf molekularer Ebene das entscheidende Ereignis in der Neurogenese. Studien in Zebrafisch haben gezeigt, dass die REST-assoziierte Phosphatase CTDSP2 von der micro-RNA (miR) -26b negativ reguliert wird, was zu einer reduzierten REST Aktivität führt. Interessanterweise liegt diese miR in einem Intron von CTDSP2, also dem Gen, welches sie selbst repremiert. Diese Konstellation stellt die Basis für eine intrinsische negative Rückkopplungsschleife dar und ist essentiell für das Voranschreiten der Neurogenese. Diese Schleife ist aktiv während der Neurogenese, jedoch inaktiv in neuronalen Stammzellen. Der Grund hierfür ist, dass die Reifung der miR-26b auf der Stufe der Prozessierung des miR-Vorläufers (pre-miR) zur reifen miR in Stammzellen, nicht aber in Neuronen, angehalten ist. Da nur reife miR funktionell aktiv sein können, ist die Regulation der miR-26 Reifung ein essentieller Schritt im Rahmen der Neurogenese. In dieser Dissertation sollte der Frage nach der molekularen Basis der regulierten Prozessierung der miR-26 im Rahmen der Neurogenese nachgegangen werden. Die Prozessierung von miR erfolgt über zwei Intermediate: Zunächst wird aus dem primären Transkript im Zellkern die pre-miR durch das Enzym DROSHA gebildet. Diese wird dann aus dem Kern exportiert und durch das Enzym DICER zur reifen miR weiterverarbeitet, die im Kontext des „RNA-Induced Silencing Complex“ (RISC) die post-transkriptionale Genexpression reguliert. Mirkoinjektionsstudien an Xenopus leavis Oocyten zeigten, dass eine Verlangsamung bzw. Blockade des nukleo-zytoplasmatischen Transports nicht Ursache für die verzögerte Prozessierung der pre-miR-26 sein kann. Stattdessen haben in vitro und in vivo Prozessierungsexperimente gezeigt, dass die Reifung der miR-26 sehr wahrscheinlich durch einen in trans agierenden Faktor gesteuert wird, der die Prozessierung in nicht-neuronalen Zellen blockiert. Durch RNA affinitätschromatographische Versuche gelang es, Proteine aus Maus- und Zebrafischlysaten zu isolieren und diese zu identifizieren, die spezifisch mit der pre-miR-26b interagieren und deren Biogenese beeinflussen könnten. Vielversprechende Kandidaten sind die Proteine FMRP/FXR1/2, ZNF346 und Eral1, deren funktionelle Charakterisierung im Kontext der miR-Biogenese nun möglich ist. In einer Kooperation mit der Arbeitsgruppe von Prof. Albrecht Müller wurde im zweiten Teil der Arbeit der prinzipiellen Frage nachgegangen, wie die miR-26 die neuronale Genexpression steuert und welche Gene hiervon primär betroffen sind. Diese Untersuchungen wurden durch die Etablierung eines Zellkultur-Protokolls ermöglicht, welches ex vivo die Differenzierung von ESC über neuronal Vorläufer Zellen (NPC) zu NCs erlaubte und so eine systematische Analyse dieses Prozess erlaubte. Für die Funktionsanalyse von miR-26 wurden über die CRISPR/Cas9 Technologie Zelllinien hergestellt, welche keine miR-26 mehr im Genom haben. miR-26 defiziente ESCs behalten ihren pluripotenten Status und ließen sich zu NPCs entwickeln. Die Weiterentwicklung von NPCs zu NCs war hingegen massiv eingeschränkt. Durch RNA Hochdurchsatzsequenzierung gelang es die miR-26 induzierten Genexpressionsunterschiede geanu zu identifizieren. Auf der Ebene der mRNA konnte gezeigt werden, dass die Expression von neuronalen Genen in miR-26 defizienten NCs herunterreguliert ist und dass unter diesen Bedingungen offensichtlich kein anderer Differenzierungsweg eingeschlagen werden konnte. Interessanterweise führte die Deletion von miR-26 zu einer selektiven Verminderung von miRs, die einerseits den REST Komplex regulieren, andererseits aber auch unter dessen transkriptionaler Kontrolle stehen. Diese Daten und die Entdeckung, dass die Induktion von miR-26 zur Anreicherung anderer REST regulierender miRs führt, lässt vermuten, dass miR-26 die Neurogenese durch die schrittweise Inaktivierung des REST Komplexe initiiert. KW - miRNS KW - Neurogenese KW - miR-26 KW - REST-Complex KW - miRNA Biogenesis KW - microrna KW - neurogenesis Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-156395 ER - TY - JOUR A1 - Kumar, Praveen A1 - Naumann, Ulrike A1 - Aigner, Ludwig A1 - Wischhusen, Joerg A1 - Beier, Christoph P A1 - Beier, Dagmar T1 - Impaired TGF-β induced growth inhibition contributes to the increased proliferation rate of neural stem cells harboring mutant p53 JF - American Journal of Cancer Research N2 - Gliomas have been classified according to their histological properties. However, their respective cells of origin are still unknown. Neural progenitor cells (NPC) from the subventricular zone (SVZ) can initiate tumors in murine models of glioma and are likely cells of origin in the human disease. In both, p53 signaling is often functionally impaired which may contribute to tumor formation. Also, TGF-beta, which under physiological conditions exerts a strong control on the proliferation of NPCs in the SVZ, is a potent mitogen on glioma cells. Here, we approach on the crosstalk between p53 and TGF-beta by loss of function experiments using NPCs derived from p53 mutant mice, as well as pharmacological inhibition of TGF-beta signaling using TGF-beta receptor inhibitors. NPC derived from p53 mutant mice showed increased clonogenicity and more rapid proliferation than their wildtype counterparts. Further, NPC derived from p53\(^{mut/mut}\) mice were insensitive to TGF-beta induced growth arrest. Still, the canonical TGF-beta signaling pathway remained functional in the absence of p53 signaling and expression of key proteins as well as phosphorylation and nuclear translocation of SMAD2 were unaltered. TGF-beta-induced p21 expression could, in contrast, only be detected in p53\(^{wt/wt}\) but not in p53\(^{mut/mut}\) NPC. Conversely, inhibition of TGF-beta signaling using SB431542 increased proliferation of p53\(^{wt/wt}\) but not of p53\(^{mut/mut}\) NPC. In conclusion, our data suggest that the TGF-beta induced growth arrest in NPC depends on functional p53. Mutational inactivation of p53 hence contributes to increased proliferation of NPC and likely to the formation of hyperplasia of the SVZ observed in p53 deficient mice in vivo. KW - mouse brain KW - tumors KW - cancer KW - TGF-beta KW - glioblastoma stem cell KW - pathways KW - expression KW - astrocytoma KW - glioblastoma KW - transforming growth factor-beta-1 KW - neurogenesis KW - gliomas KW - neural stem cell KW - p53 KW - subventricular zone KW - premalignant lesion Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-144262 VL - 5 IS - 11 ER -