106 research outputs found
Transplantation reveals regional differences in oligodendrocyte differentiation in the adult brain
No full textTo examine the role of gray and white matter niches for oligodendrocyte differentiation, we used homo- and heterotopic transplantations into the adult mouse cerebral cortex. White matter-derived cells differentiated into mature oligodendrocytes in both niches with equal efficiency, whereas gray matter-derived cells did not. Thus, white matter promotes oligodendrocyte differentiation, and cells from this niche differentiate more easily, even in the less supportive gray matter environment
Multiple Splice Isoforms of Proteolipid M6B in Neurons and Oligodendrocytes
No full textProteolipids are abundant integral membrane proteins, initially described as structural proteins of CNS myelin. More recently, two neuronal proteins related to proteolipid protein (PLP), termed M6A and M6B, were identified, suggesting a common function of proteolipids in oligodendrocytes and neurons. We have analyzed the X-linked M6B gene and discovered an unexpected complexity of protein isoforms. Two promoters and alternative exons yield at least eight M6B proteins and polypeptides, differentially expressed in neurons and oligodendrocytes. Six isoforms are tetraspan membrane proteins that differ by highly conserved amino- and carboxy-terminal domains, termed α, β, ψ, and ω. In MDCK cells, the β-domain of M6B stabilizes tetraspan proteolipids at the cell surface, whereas non-β isoforms are more abundant in intracellular compartments. Cotransfection experiments suggest a physical interaction of M6B and mutant PLP, when retained in the endoplasmic reticulum, that may also contribute to oligodendrocyte dysfunction in Pelizaeus–Merzbacher disease
In vitro Studien zum Potenzial von Oligodendrozyten-Vorläuferzellen Nervenzellen zu ersetzen
No full textFragestellung: Nerve/glial antigen 2 (NG2)-Glia stellen im Zentralen Nervensystem eine mitotisch aktive, endogene Vorläuferpopulation dar. Aus ihr entstehen lebenslang Oligodendrozyten und während der Hirnentwicklung auch Astrozyten. Darüber hinaus werden NG2-Glia als Quelle für eine endogene neuronale Zellersatztherapie diskutiert. In vitro Experimente mit postnatalen NG2- Glia im Ratten- und Mausmodell sollen im Rahmen dieser Arbeit Aufschluss darüber geben, ob und unter welchen Bedingungen NG2-Glia zu Nervenzellen transdifferenzieren können. Die neurogenen, hochkonservierten Basic Helix Loop Helix Transkriptionsfaktoren Paired box 6 (Pax6) und Neurogenin 2 (Ngn2) wurden als geeignete Kandidaten identifiziert um das Zellschicksal von NG2-Glia zu beeinflussen.
Material und Methoden: Im Rahmen dieser Arbeit wird eine primäre Zellkultur von NG2-Glia aus der Ratte und aus der Maus etabliert. Zur Aufreinigung der Zellen werden verschiedene Methoden wie eine rein mechanische Separation, Magnet activated cell sorting und Immunopanning angewendet. Die angereicherten NG2-Glia werden in vitro mit Retroviren transduziert, um die Gene für die neurogenen Transkriptionsfaktoren Pax6 oder Ngn2 ektop zu exprimieren. Die transduzierten Zellen werden zu verschiedenen Zeitpunkten nach Transduktion mit einem Reporter-kodierenden Kontrollvirus, Pax6-Virus oder Ngn2-Virus mittels Immunzytochemie charakterisiert.
Ergebnisse: Die Transduktion mit dem Kontrollvirus hat keinen Einfluss auf das Zellschicksal. Im Vergleich zum Kontrollvirus ist der Anteil der transduzierten neuronalen Zellen in der Rattenkultur 10 Tage und 14 Tage nach Transduktion mit dem Pax6-Virus signifikant erhöht. Nach Transduktion mit dem Ngn2-Virus in der Rattenkultur ist der Anteil von β3Tubulin+-Zellen im Vergleich zum Kontrollvirus bereits nach 4 Tagen signifikant um das 10-fache erhöht. Zugleich ist der Anteil transduzierter O4+-Zellen im Vergleich zum Kontrollvirus signifikant geringer. Diese Ergebnisse lassen sich in der Mauskultur nach Transduktion mit dem Kontrollvirus, Pax6-Virus und Ngn2-Virus replizieren.
Diskussion: Da die forcierte Expression von Pax6 nach aktueller Datenlage keine neuronale Transdifferenzierung von Astrozyten induziert, ist der signifikante Anstieg neuronaler Zellen nach Transduktion auf das neurogene Potenzial von NG2-Glia zurückzuführen. Der signifikante Anstieg an transduzierten neuronalen Zellen nach Transduktion mit dem Ngn2-Virus bei gleichzeitig signifikanter Abnahme an transduzierten NG2-Glia stütz die Hypothese einer Transdifferenzierung von NG2-Glia zu Neuronen. Diese Beobachtung ist konsistent mit vorangegangenen in vitro- Studien zur Reprogrammierbarkeit von Astrozyten zu Neuronen nach Transduktion mit dem Ngn2-Virus. Zur abschließenden Beurteilung, in welchem Umfang die Neurogenese nach Transduktion mit dem Ngn2-Virus von NG2-Glia ausgeht, sind Abstammungsstudien z.B. im NG2-GFP-Mausmodell anzuschließen. Der beobachtete neurogene Effekt nach Transduktion mit dem Pax6- und dem Ngn2-Virus ermutigt weitere Reprogrammierungsstudien um NG2-Glia als endogene Stammzellpopulation zum Ziel einer neuronalen Zellersatztherapie zu machen
Role of GluA2-containing AMPARs in Oligodendrocyte Lineage Cells
No full textOligodendrocytes precursor cells (OPC), also called NG2 cells, proliferate or differentiate into myelin-producing oligodendrocytes throughout life. Myelination depends on neuronal activity. Although NG2 cells receive neuronal glutamatergic synaptic input throughout the CNS, factors mediating activity-dependent control of the oligodendrocyte lineage cell fate have not been resolved yet. Glutamatergic signalling in NG2 cells is mainly mediated by AMPARs. During the postnatal period, AMPARs are calcium impermeable due to the expression of the GluA2 subunit. Later in the adult period, NG2 cells downregulate the GluA2 subunit, therefore they express GluA2-lacking AMPARs, permeable to calcium. Thus, the decline of the proliferation rate of NG2 cells observed during the transition from the postnatal to the adult period is mirrored by an increase in the calcium-permeability of synaptic AMPA receptors. We hypothesized that the postnatally expressed GluA2 subunit enables the proliferation of NG2 cells by suppressing calcium entry during synaptic activity. Here, we assessed the impact of postnatal GluA2 subunit deletion on the oligodendrocyte lineage cell fate. In order to induce a conditional deletion of the GluA2 subunit in NG2 cells, we crossed the three following mouse lines. NG2CreER mouse line conditionally express Cre under the NG2 promotor. R26REYFP mouse line express the yellow fluorescent protein reporter after Cre expression. GluA2lox mouse line undergoes a deletion of Gria2 allele responsible for GluA2 expression. First, we followed the oligodendrocyte lineage cell fate with the BrdU and PCNA markers to examine NG2 cell proliferation, the cleaved caspase-3 marker to probe for cell death, and the CC1 marker to analyse differentiation of NG2 cells into oligodendrocytes. We found that GluA2 deletion in early postnatal NG2 cells increased BrdU uptake in NG2 cells without increasing cell density or cell death in the oligodendrocyte lineage cell population. Secondly, we investigated the role of GluA2 subunit on myelination establishment by measuring the internodes length during myelination onset and at a later timepoint during the postnatal phase. GluA2 deletion in NG2 cells shortened immature internodes during myelination onset. Thirdly, the role of the GluA2 subunit in NG2 cells was investigated in a motor learning task requiring newly formed oligodendrocytes. GluA2 deletion altered the motor learning performance in mice. Although it did not affect the myelination properties investigated or NG2 cells proliferation. Overall, the GluA2 subunit in postnatal NG2 cells regulates DNA synthesis in NG2 cells and myelination onset through the regulation of internode elongation. Our study highlights the prominence of glutamatergic synaptic input integration in postnatal NG2 cells to regulate oligodendrocyte lineage cell fate
GPR17 expressing NG2-Glia: Oligodendrocyte progenitors serving as a reserve pool after injury
No full textIn the adult brain NG2-glia continuously generate mature, myelinating oligodendrocytes. To which extent the differentiation process is common to all NG2-glia and whether distinct pools are recruited for repair under physiological and pathological conditions still needs clarification. Here, we aimed at investigating the differentiation potential of adult NG2-glia that specifically express the G-protein coupled receptor 17 (GPR17), a membrane receptor that regulates the differentiation of these cells at postnatal stages. To this aim, we generated the first BAC transgenic GPR17-iCreER(T2) mouse line for fate mapping studies. In these mice, under physiological conditions, GPR17(+) cells -in contrast to GPR17(-) NG2-glia- did not differentiate within 3 months, a peculiarity that was overcome after cerebral damage induced by acute injury or ischemia. After these insults, GPR17(+) NG2-glia rapidly reacted to the damage and underwent maturation, suggesting that they represent a 'reserve pool' of adult progenitors maintained for repair purposes. GLIA 2016;64:287-299
The reactions of glial cells in different mouse models of amyotrophic lateral sclerosis and Parkinson’s disease
No full textNeurodegenerative Erkrankungen wie beispielsweise Morbus Parkinson (PD) oder Amyotrophe Lateral Sklerose (ALS) werden in einer älter werdenden Gesellschaft zu einem zunehmenden Problem. Diese Krankheiten sind durch das Absterben bestimmter Neuronengruppen charakterisiert. Da das menschliche Gehirn aber nicht nur aus Neuronen, sondern auch aus Gliazellen besteht, wurde in dieser Arbeit untersucht, wie sich Gliazellen in verschiedenen Hirnregionen von Mäusen unterschiedlichen Alters mit bestimmten krankheitsauslösenden Mutationen verhalten. Hierfür wurde die Gliazellzahl analysiert und auch Proliferation und Differenzierung mittels BrdU näher betrachtet.
Zur genaueren Untersuchung von ALS wurde ein Mausmodell mit einem heterozygoten knock-out von TANK-binding kinase 1 (TBK1) und eine Mutation von TDP43 verwendet und der Motorcortex von 18 Monate alten Tieren näher betrachtet. Hier konnten vor allem Unterschiede in den Zahlen von Astrozyten und Mikroglia gezeigt werden, als auch in der Proliferation und Differenzierung von NG2-Glia.
Für PD wurden das Caudoputamen (cp), der Gyrus dentatus (dg) und der Motorcortex von Tieren mit einer α-synuclein Mutation betrachtet. Im cp reagierten hauptsächlich Astrozyten, wohingegen im dg vor allem NG2-Glia Unterschiede in ihrer Proliferation und Differenzierung zeigten. Im mc wiederum reagierten hauptsächlich Mikroglia sowie NG2-Glia.
Insgesamt konnte gezeigt werden, dass die Reaktionen von Gliazellen sehr heterogen sind und ihre Reaktionen unter anderem von der beobachteten Hirnregion, dem Alter und der Mutation abhängt.Neurodegenerative diseases such as Parkinson’s disease (PD) and amyotrophic lateral sclerosis (ALS) become a growing problem in an aging society. These diseases are characterized by the degeneration of specific neurons. However, since the brain does not only comprise of neurons but of glial cells as well, the behaviour of glial cells in several brain regions of mice with different pathologic mutations and of different ages was studied in this thesis. This was done by quantification of cell numbers of different glial cells and by fate mapping of proliferating cells via BrdU. For ALS, a heterozygous knockout of TANK-binding kinase 1 (TBK1) and a mutation in TDP43 in 18 months old mice were analysed, and in PD an α-synuclein knock in mouse model with 10 and 15 months old mice was studied. In ALS, quantification of glial cells was performed in the motor cortex (mc). Changes were seen in the numbers of reactive astrocytes and microglia, but also in the numbers of NG2-glial proliferation and differentiation.
In PD mice, the caudoputamen (cp), dentate gyrus (dg) and mc were analysed. In the cp, mainly astrocytes showed significant reactions, whereas in the dg mainly NG2-glia showed differences in proliferation and differentiation. In the mc, especially microglia numbers and NG2-glial differentiation and proliferation were altered.
In summary, it was shown that the reactions of glial cells are very heterogeneous and depend a lot on the brain region observed but also on the age and the mutation studied
Establishment of 3D human induced pluripotent stem cell-based culture systems to investigate myelination deficits in schizophrenia
No full textMyelination deficiency is a common finding in psychiatric diseases. Brain imaging studies show decreased myelin integrity in white matter tracts of schizophrenia patients. However, the molecular pathways behind the hypomyelination pathology are still unknown. The development of an in vitro platform to study the interaction of neurons and oligodendrocytes, in particular myelination, is essential to understand the cause of aberrant myelination. Three-dimensional (3D) cell culture systems are of great importance for disease modeling because they provide a more representative in vivo-like micro-environment to the cells. The aim of the thesis was the establishment of a physiologically relevant in vitro model to study oligodendrocyte lineage cell development and myelination which can be used for the identification and validation of potential targets related to schizophrenia.
3D scaffold-based in vitro platforms were developed for the generation of mixed cortical neurons and oligodendrocyte lineage cells from hiPSCs. These 3D in vitro platforms were used for the identification and validation of potential targets related to schizophrenia. To do this, hiPSCs derived from healthy controls and schizophrenia patients showing decreased white matter volume in the right prefrontal cortices, were utilized. hiPSC-derived mixed cortical neurons and oligodendrocyte lineage cells were investigated by transcriptomic and/or proteomic analysis to identify potential molecular targets that might be involved in processes leading to the hypomyelination phenotype. Two of the potential oligodendrocyte lineage cell-related targets were selected and validated in the 3D in vitro platform by external manipulation of their signaling pathways. Additionally, two controllable co-culture systems were established to investigate the interplay between neurons and oligodendrocyte lineage cells. These co-culture systems encompass both direct and indirect approaches. The direct co-culture system is tailored to explore the direct interaction of neurons and oligodendrocytes, specifically focusing on the myelination process. The indirect co-culture system was implemented to examine the impact of factors secreted by neurons on the development of oligodendrocyte lineage cells.
This thesis work provides an in vitro platform to study intrinsic and extrinsic factors of neurons and oligodendrocyte lineage cells in health and disease
Role of the G Protein-coupled Receptor 17 and the GPR17+NG2-glia in the adult brain under physiological conditions
No full textNG2-glia comprise a heterogeneous population of cycling cells that give rise to mature, myelinating oligodendrocytes. The mechanisms that regulate the process of differentiation from NG2-glia into oligodendrocytes are still not fully understood but over the last years the G Protein-coupled Receptor 17 (GPR17) has been on the spotlight as a possible key regulator. Interestingly, GPR17-expressing NG2-glia show under physiological conditions a slower and lower level of differentiation compared to NG2-glia without GPR17. In contrast, after a CNS insult these react with proliferation and differentiation in a high rate, pointing towards a role in repair processes. However, the role of GPR17+ NG2-glia under healthy conditions in adulthood has not been addressed yet. Therefore, I aimed here to characterize the GPR17-expressing NG2-glia.
Using transgenic mouse models, I showed restricted GPR17 expression in only some NG2-glia. Furthermore, I found that these cells constitute a distinct subset within the NG2-glia population, which shows a different gene expression profile and behavior when compared to the total NG2-glia population. Genetic depletion of GPR17+ cells showed that these are not contributing to the dynamic and continuous generation of new oligodendrocytes in the adult brain. Taken together, GPR17+ NG2-glia seem to play a distinct role under physiological conditions that goes beyond their classic differentiation control, that needs to be further elucidated.
These results open new avenues for using the GPR17 receptor as a target to change oligodendrogenesis under physiological and pathological conditions, highlighting the importance of further characterization of this protein for future pharmacological studies
Genetic manipulation of glial progenitors boosts oligodendrogenesis and myelination in the mammalian brain
No full textGlia, once considered as mere ‘glue’ for the central nervous system (CNS), have now emerged as active participants in almost every aspect of nervous system development, homeostasis, and even disease. Among these, oligodendroglia, comprising of oligodendrocyte progenitor cells (OPCs) and mature oligodendrocytes (OLs) are responsible for myelinating the CNS. Additionally, recent discoveries have implicated these cells in other processes including phagocytosis, synaptogenesis, ability to influence neural activity, and even animal behaviour. OPCs originate during embryogenesis from neural stem cells, establish a non-overlapping grid-like pattern across CNS, and persist throughout life. They are also one of the most proliferative cell types within the brain, which differentiate into OLs. Given their widespread presence and multifaceted functions, it is not surprising that oligodendroglia are implicated in the pathogenesis of diseases such as Multiple Sclerosis (MS). MS is a highly prevalent demyelinating disease, characterised by a severe loss of OLs, neuronal atrophy, and disrupted neural circuits. Furthermore, the endogenous mechanisms of repair and regeneration fail, leading to progressive deterioration, including motor deficits and cognitive decline. Current clinical therapies mainly focus on slowing disease progression and alleviating symptoms. Therefore, there is an urgent need for the development of novel and improved regenerative therapies. My doctoral research focused on OPCs as a therapeutic avenue due to their stem-cell-like properties. By leveraging established links between cell cycle regulation and proliferation, my study aimed to specifically target G1 phase shortening through Cdk4 and CyclinD1 (4D) overexpression. To first evaluate its effect under physiological conditions, I employed a sophisticated triple transgenic mouse line that allows for spatiotemporal control of 4D overexpression in oligodendroglia. This approach led to an increase in OPC proliferation in the white and grey matter of the brain, effectively enhancing oligodendrogenesis. Subsequently, I tested the efficacy of 4D in a preclinical model of MS using cuprizone-induced demyelination. While no significant improvements in learning and memory functions were evident, a comprehensive analysis of cellular and functional effects of 4D will shed light on its mechanisms of action. Additionally, it is plausible that 4D might have positive outcomes on other aspects of behaviour; however, this requires further investigation. Altogether, the findings presented in this thesis introduce a novel tool aimed at augmenting endogenous oligodendrogenesis under physiological conditions and represent a significant step toward developing innovative therapeutic strategies for demyelinating disorders.:Table of Contents
CHAPTER 1: INTRODUCTION
1.1. HISTORY OF OLIGODENDROGLIA 1
1.2. OLIGODENDROGLIA DURING DEVELOPMENT 4
1.3. OLIGODENDROGLIA IN ADULTHOOD 7
1.3.1. OPCS – DENSITY AND FUNCTIONS 7
1.3.2. OLS – DENSITY AND FUNCTIONS 8
1.4. OLIGODENDROGLIAL HETEROGENEITY 11
1.4.1. OPCS 11
1.4.2. OLS 12
1.5. OPC CELL CYCLE DYNAMICS 14
1.5.1. QUANTIFICATION OF OPC CELL CYCLE LENGTH 15
1.5.2. FACTORS INFLUENCING OPC CELL CYCLE 16
1.6. MYELIN AND MYELINATION 19
1.6.1. STRUCTURE AND COMPOSITION 19
1.6.2. FUNCTIONS 20
1.7. OLIGODENDROGENESIS AND BEHAVIOUR 21
1.7.1. LEARNING AND MEMORY 21
1.7.2. OTHERS 23
1.8. OLIGODENDROGLIA IN DISEASE AND REGENERATION 24
1.9. MS 26
1.9.1. MOUSE MODELS OF MS 28
1.10. CURRENT THERAPIES FOR DEMYELINATING DISEASES 31
1.11. AIM OF THE PROJECT 33
CHAPTER 2: MATERIALS AND METHODS
2.1. MATERIALS 36
2.1.1. MOUSE STRAINS 36
2.1.2. GENOTYPING PRIMERS 36
2.1.3. BUFFERS AND SOLUTIONS 37
2.1.4. CHEMICALS AND KITS 38
2.1.5. ANTIBODIES 39
2.2. METHODS 40
2.2.1. ANIMALS 40
2.2.2. GENOTYPING 40
2.2.3. DRUG TREATMENTS 40
2.2.4. BEHAVIOUR TESTS 41
2.2.4.1. OFT 41
2.2.4.2. EPM 42
2.2.4.3. ROTAROD 42
2.2.4.4. RW/CW 42
2.2.4.5. MWM 43
2.2.4.6. BM 44
2.2.5. IMMUNOHISTOCHEMISTRY 46
2.2.6. IMAGE ACQUISITION AND CELLULAR QUANTIFICATION 46
2.2.8. STATISTICS 47
CHAPTER 3: RESULTS - PART I
CELLULAR AND BEHAVIOURAL EFFECTS OF GENETIC MANIPULATION OF CELL CYCLE OF OLIGODENDROCYTE PROGENITORS VIA CDK4/CYCLIND1 (4D) OVEREXPRESSION
3.1. CHARACTERISATION OF 4D OVEREXPRESSION MEDIATED BY TRIPLE TRANSGENIC MICE 48
3.2. 4D OVEREXPRESSION IN ADULT MICE INCREASES OPC PROLIFERATION IN CC AND CTX 49
3.3. 4D-INDUCED INCREASE IN OPC PROLIFERATION IS AGE-DEPENDENT 51
3.4. 4D OVEREXPRESSION INCREASES DENSITY OF OLS AND MYELIN IN CC AND CTX 52
3.5. 4D-INDUCED INCREASE IN OPC PROLIFERATION IS TEMPORALLY CORRELATED TO ACTIVATION OF 4D 53
3.6. 4D OVEREXPRESSION DOES NOT AFFECT ANXIETY-LIKE BEHAVIOUR ON THE OPEN FIELD AND ELEVATED PLUS MAZE TEST 55
3.7. 4D OVEREXPRESSION LEADS TO IMPAIRED LEARNING ON THE MORRIS WATER MAZE TEST 57
3.8. 4D OVEREXPRESSION NEGATIVELY IMPACTS RUNNING SPEEDS ON THE RUNNING/COMPLEX WHEEL TEST 59
3.9. 4D OVEREXPRESSION HAS A LONG-TERM NEGATIVE EFFECT ON RUNNING SPEEDS ON THE RUNNING/COMPLEX WHEEL TEST 61
CHAPTER 4: RESULTS - PART II
CELLULAR AND BEHAVIOURAL CHARACTERISATION OF CUPRIZONE-INDUCED
DEMYELINATION MODEL OF MULTIPLE SCLEROSIS
4.1. CUPRIZONE DIET LEADS TO OLIGODENDROCYTE LOSS AND DEMYELINATION ACROSS BRAIN REGIONS 64
4.2. TERMINATION OF CUPRIZONE DIET TRIGGERS SPONTANEOUS REGENERATION ACROSS BRAIN REGIONS 66
4.3. CUPRIZONE-INDUCED DEMYELINATION IMPAIRS LEARNING ON THE MORRIS WATER MAZE TEST 68
4.4. CUPRIZONE-INDUCED DEMYELINATION ADVERSELY AFFECTS BODY WEIGHT AND PERFORMANCE ON THE RUNNING/COMPLEX WHEEL TEST 70
CHAPTER 5: RESULTS - PART III
BEHAVIOURAL EFFECT OF 4D-INDUCED OLIGODENDROGENESIS IN THE MODEL OF CUPRIZONE-INDUCED DEMYELINATION
5.1. 4D OVEREXPRESSION BEFORE THE ONSET OF CUPRIZONE-INDUCED DEMYELINATION DOES NOT RESCUE COGNITIVE PERFORMANCE ON BARNES MAZE 73
5.2. 4D OVEREXPRESSION BEFORE THE ONSET OF CUPRIZONE-INDUCED DEMYELINATION DOES NOT RESCUE MOTOR PERFORMANCE ON THE RUNNING/COMPLEX WHEEL TEST 75
5.3. SIMULTANEOUS 4D OVEREXPRESSION AND CUPRIZONE-INDUCED DEMYELINATION DOES NOT RESCUE MOTOR PERFORMANCE ON THE RUNNING/COMPLEX WHEEL TEST 78
CHAPTER 6: DISCUSSION
6.1. CELLULAR IMPLICATIONS OF 4D OVEREXPRESSION UNDER PHYSIOLOGICAL CONDITIONS 81
6.2. BEHAVIOURAL IMPLICATIONS OF 4D OVEREXPRESSION UNDER PHYSIOLOGICAL CONDITIONS 85
6.3. 4D AS A THERAPEUTIC TOOL 88
6.4. CONCLUSIONS AND OUTLOOK 90
REFERENCES 93
ACKNOWLEDGEMENTS 124
APPENDIX I 125
APPENDIX II 12
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