Molecular pathomechanisms of epilepsy associated gangliogliomas

Molecular pathomechanisms of epilepsyassociated gangliogliomas Dissertation zur Erlangung des Doktorgrades (Dr rer nat.) der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn vorgelegt von Eva Gembé aus Bonn Bonn 2013 Angefertigt mit der Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn Gutachter: Prof Dr Albert Becker Gutachter: Prof Dr Jörg Höhfeld Tag der Promotion: 09.12.2013 Erscheinungsjahr: 2014 Erklärung Diese Dissertation wurde im Sinne der Promotionsordnung vom 17.06.2011 am Institut für Neuropathologie des Universitätsklinikums Bonn unter der Leitung von Prof Albert Becker angefertigt Eidesstattliche Erklärung Hiermit versichere ich, dass die vorliegende Arbeit ohne unzulässige Hilfe Dritter und ohne die Benutzung anderer als der angegebenen Quellen angefertigt wurde Die aus fremden Quellen direkt oder indirekt übernommenen Gedanken sind gemäß der Promotionsordnung vom 17.06.2011 als solche kenntlich gemacht Bonn, Eva Gembé Table of contents Table of contents INTRODUCTION 1
1.1 Epilepsy 1
1.2 Focal epilepsies .3
1.3 Gangliogliomas 5
1.4 Molecular pathological aspects of gangliogliomas 8
1.5 LRP12 12
1.6 Cortical development 16
1.7 Aims of the study 19
MATERIALS 21
2.1 Equipment 21
2.2 Material and Reagents 24
2.2.1 Antibodies 24
2.2.2 Cell culture media 26
2.2.3 Chemicals 27
2.2.4 Diverse materials 28
2.2.5 Enzymes 29
2.2.6 Kits 29
I Table of contents 2.3 Oligonucleotides 31
2.3.1 Cloning 31
2.4 Vectors and vector construction 34
2.4.1 Multiple cloning sites (MCS) 34
2.4.2 Generated constructs 35
2.4.3 Plasmids obtained from other labs 36
2.5 Biological materials .37
2.5.1 Surgical specimens 37
2.5.2 Cell lines 39
2.6 Software 39
2.7 Bioinformatic analyses 40
METHODS 41
3.1 Molecular biological methods 41
3.1.1 mRNA isolation and cDNA synthesis 41
3.1.2 PCR protocols 42
3.1.3 Real-time PCR 44
3.1.4 Vector generation 44
3.1.5 Site-directed mutagenesis 45
3.1.6 Sequencing 45
3.2 Biochemical methods 46
3.2.1 Western blot analyses 46
3.2.2 Immunohistochemical stainings 46
II Table of contents 3.3 Cell culture .48
3.3.1 In vitro assays 49
3.3.1.1 Cell proliferation assays 49
3.3.1.2 Luciferase assays 49
3.3.2 rAAV virus production 50
3.3.3 Quantification of LRP12 mRNA/LRP12 knockdown efficiency in vitro 50
3.4 In utero electroporation studies 51
3.4.1 In utero electroporation 51
3.4.2 Analysis of PTZ induced seizure sensitivity 52
3.4.3 Quantification of LRP12 knockdown efficiency in vivo 52
3.5 Image analysis and quantification .53
3.6 Statistical analysis 54
RESULTS 55
4.1 Temporal and regional expression patterns of LRP12 and posttranslational modifications 55
4.1.1 Test for
-LRP12 antibody specificity 55
4.1.2 Expression of LRP12 in embryonic and adult mouse brain 56
4.1.3 Spatial expression of LRP12 in rodent brain 57
4.1.4 Posttranscriptional modifications of LRP12 59
4.2 Subcellular localization of LRP12 in neurons .60
4.2.1 LRP12 is localized in the soma and neurites of neurons 60
4.2.2 LRP12 is not localized at the Golgi apparatus 62
4.3 Frequent SNP in the LRP12 promoter region of ganglioglioma patients 64
III Table of contents 4.4 Biological relevance of rs9694676 .66
4.4.1 Bioinformatic analysis of the putative LRP12 promoter region 66
4.4.2 Luciferase assays of the LRP12 promoter region 69
4.5 Lack of LRP12 protein in neuronal and astroglial ganglioglioma components 70
4.6 Knockdown of LRP12 induces increased proliferation in cultured astroglial cells 74
4.6.1 In vitro testing of shRNA knockdown efficiency in HEK293T cells 74
4.6.2 In vitro cell proliferation assays 77
4.7 In vitro knockdown of LRP12 leads to substantially impaired arborization of developing neurons 79
4.7.1 In vitro LRP12 knockdown efficiency in neurons 79
4.7.2 In vitro arborization analyses 81
4.8 Embryonic knockdown of LRP12 by intraventricular in utero electroporation in mice leads to impaired migration of neurons 83
4.8.1 In vivo knockdown efficiency of LRP12 83
4.8.2 Embryonic knockdown of LRP12 leads to impaired migration of neurons 85
4.8.3 Positioning of electroporated neurons within cortical laminae 89
4.9 Increased propensity to PTZ-induced seizures in mice after LRP12 shRNA in utero electroporation 91
DISCUSSION 93
SUMMARY 107
IV Table of contents LIST OF FIGURES 109
LIST OF TABLES 113
ABBREVIATIONS 116
10 REFERENCES 126
11 ACKNOWLEDGEMENTS 142
V Introduction Introduction 1.1 Epilepsy Up to 10% of the population is affected by one seizure during lifetime Such onetime event does not necessarily lead to a manifestation of epilepsy Nevertheless, about 50 million people suffer from epilepsy, which is therefore regarded as one of the most common serious brain disorders worldwide Approximately 0.1% to 1.0% of the population is affected by epilepsy, often accompanied with e.g physical hazards due to unpredictability of seizure occurrence as well as social and economic burdens (1-4) The general public commonly associates epilepsy with the incidence of tonicclonic seizures, usually termed as ‘grand mal seizures’ In contrast to this, the clinical view of epilepsy is much more distinct In order to establish a common definition of epilepsy, the International League Against Epilepsy (ILAE) and the International Bureau for Epilepsy (IBE) accepted the definition of epileptic seizures as ‘a transient occurrence of signs and/or symptoms due to abnormal excessive or synchronous neuronal activity in the brain (2, 5) This somewhat vague definition of epilepsy implicates a diverse etiology and symptoms of epileptic seizures The ILAE Proposal for Revised Terminology for Organization of Seizure and Epilepsies 2010 distinguishes between generalized seizures, characterized by the arising and the propagation within bilaterally distributed networks, and focal seizures, which Introduction originate within networks limited to one hemisphere The appearance of generalized seizures is diverse, ranging from tonic-clonic over absence, clonic, tonic, atonic to myoclonic seizures For focal seizures, which were formally subdivided into simple partial (consciousness not impaired) and complex partial seizures (accompanied by impairment of consciousness), no specific classification is longer recommended Since 2010 the ILAE suggests to classify focal seizures by their semiologic features, such as by the occurrence and kind of auras, motorand autonomic impairment and the awareness and responsiveness of patients (5, 6) The underlying causes leading to epilepsy are characterized in the ILAE Proposal for Revised Terminology For Organization of Seizure and Epilepsies 2010 This report distinguishes between genetic, structural/ metabolic and ‘unknown causes’ for seizures In their terms genetically evoked epilepsies are a direct effect of a genetic defect, thereby seizures represent the core symptoms of the disorder This concept accounts for instance for channelopathies Furthermore, they concretize that the underlying cause of structural/ metabolic epilepsies are distinct disorders associated with a higher risk of developing epilepsy This includes on the one hand, acquired structural impairments such as stroke and trauma and on the other hand, lesions of genetic origin like tuberous sclerosis or cortical malformations Thereby, the disorder constitutes the link between genetic alteration and epilepsy In addition, epilepsies of ‘unknown causes’ are characterized as syndromes with yet unidentified nature of the underlying pathomechanisms (5, 6) During the last decades many antiepileptic drugs have been developed and in the majority, patients’ seizures (approximately 70%) can be controlled effectively with References 20 Blümcke I, Wiestler OD Gangliogliomas: an intriguing tumor entity associated with focal epilepsies J Neuropathol Exp Neurol 2002;61(7):575-584 21 Baumgartner C, Czech T, Serles W Aktueller Stand der prächirurgischen Epilepsydiagnostik Journal für Neurologie, Neurochirurgie und Psychiatrie 2001;2(2):21-35 22 Blümcke I, et al The CD34 epitope is expressed in neoplastic and malformative lesions associated with chronic, focal epilepsies Acta Neuropathol 1999;97(5):481-490 23 Aronica E, et al Ionotropic and metabotropic glutamate receptor protein expression in glioneuronal tumours from patients with intractable epilepsy Neuropathol Appl Neurobiol 2001;27(3):223-237 24 Wolf HK, Roos D, Blumcke I, 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D'Urso PI, De Tommasi C, Resta N, Ciappetta P Molecular genetic analysis in a case of ganglioglioma: identification of a new mutation Neurosurgery 2008;63(5):976-980 95 Grajkowska W, et al Ganglioglioma associated with alterations of NBN gene A case report Folia Neuropathol 2009;47(3):278-283 96 Markham K, Schuurmans C, Weiss S STAT5A/B activity is required in the developing forebrain and spinal cord Mol Cell Neurosci 2007;35(2):272282 139 References 97 De Bustos C, Smits A, Stromberg B, Collins VP, Nister M, Afink G A PDGFRA promoter polymorphism, which disrupts the binding of ZNF148, is associated with primitive neuroectodermal tumours and ependymomas J Med Genet 2005;42(1):31-37 98 Shastry BS SNP alleles in human disease and evolution J Hum Genet 2002;47(11):561-566 99 Garnis C, Coe BP, Zhang L, Rosin MP, Lam WL Overexpression of LRP12, a gene contained within an 8q22 amplicon identified by highresolution array CGH analysis of oral squamous cell carcinomas Oncogene 2004;23(14):2582-2586 100 Artegiani B, Calegari F Lentiviruses allow widespread and conditional manipulation of gene expression in the developing mouse brain Development 2013;140(13):2818-2822 101 De Vry J, et al In vivo electroporation of the central nervous system: a nonviral approach for targeted gene delivery Prog Neurobiol 2010;92(3):227244 102 Taniguchi Y, Young-Pearse T, Sawa A, Kamiya A In utero electroporation as a tool for genetic manipulation in vivo to study psychiatric disorders: from genes to circuits and behaviors Neuroscientist 2012;18(2):169-179 140 References 103 LoTurco JJ, Bai J The multipolar stage and disruptions in neuronal migration Trends Neurosci 2006;29(7):407-413 104 Binder DK, Steinhauser C Functional changes in astroglial cells in epilepsy Glia 2006;54(5):358-368 105 Heinemann U Basic mechanisms of partial epilepsies Curr Opin Neurol 2004;17(2):155-159 106 Wolf HK, Birkholz T, Wellmer J, Blumcke I, Pietsch T, Wiestler OD Neurochemical profile pharmacoresistant of focal glioneuronal epilepsies J lesions from Neuropathol patients Exp with Neurol 1995;54(5):689-697 107 Wong M Mechanisms of epileptogenesis in tuberous sclerosis complex and related malformations of cortical development with abnormal glioneuronal proliferation Epilepsia 2008;49(1):8-21 108 Manent JB, Represa A Neurotransmitters and brain maturation: early paracrine actions of GABA and glutamate modulate neuronal migration Neuroscientist 2007;13(3):268-279 141 Acknowledgements 11 Acknowledgements The realization of my Ph.D thesis would not have been possible without the help and support of others Therefore, I wish to express my gratitude to all people who contributed to this work I would like to express my gratitude to Prof Dr Albert Becker for giving me the opportunity to work on this interesting Ph.D thesis in his lab, for his willingness to help, his good advises and support and for the interesting discussions I cordially thank Prof Dr Jörg Höhfeld for his support and for being the coadvisor of my Ph.D thesis I would like to thank Prof Dr Susanne Schoch-McGovern for her great support, her enthusiasm and encouragement I wish to thank Prof Dr Torsten Pietsch for the opportunity to carry out this work at his institute I would like to thank all members and former members of the Becker and Schoch group I especially want to express my gratitude to Alex for carrying out the in utero electroporations and to Sabine for preparation of neurons and glial cells as well as for her encouraging words I thank Karen for the many answers to my questions Thank you, Katharina and Anne for all the cheerful lunches in the cafeteria and nice evenings with you and Mario Thank you to ‘Lady Pernhorst’ for ‘Mach et Otze’, ‘It flows off the tongue better’ and many, many other things Katrin and Leonie, I’m grateful for all these ‘early in the morning jogging tours’, 142 Acknowledgements conversations, support and proof reading my thesis I also like to thank Verena, Elisa, Tanja and Barbara for making the laboratory routine merrier! I thank Lioba and Daniela for ordering and assistance I am thankful for my friends Conny, Anja, Sobo and Marion who always lent me an ear and supported me Thank you so much for sharing the good and bad days during the last years! I am deeply grateful for my parents, my sister and my brother Thank you for your love, understanding and support in so many ways! 143 [...]... points to clusters of dysmorphic neurons located in the astroglial matrix) (F) So-called ‘satellite cells’ with expression of the oncofetal protein CD43 in the proximity of gangliogliomas point towards a developmentally compromized origin of gangliogliomas (CD34 IHC; arrows point to ‘satellite clusters’) Scale bar: A, B, E, F: 200
m; C, D: 50
m 1.4 Molecular pathological aspects of gangliogliomas Pathogenic... relies on the assumption of tumor -associated epileptic changes in adjacent non-tumorous brain tissue This idea is underlined by alterations of the composition of neuroactive proteins within perilesional brain areas and by the clinical observation of hyperexcitable tissue outside of the tumor entity itself (24, 25) To date, the molecular pathomechanisms leading to the manifestation of gangliogliomas still... region Nevertheless, SSCP and sequencing analyses of the LRP12 promoter area revealed a single nucleotide polymorphism (SNP) in the LRP12 promoter region of ganglioglioma patients Thus, the finding of low levels of LRP12 mRNA in gangliogliomas points to LRP12 as an interesting candidate gene potentially involved in the pathogenesis of epilepsy- associated gangliogliomas 1.5 LRP12 The low-density lipoprotein... Introduction 1.7 Aims of the study Molecular pathomechanisms leading to the formation of intractable epilepsyassociated gangliogliomas are still unclear Previous studies suggest these tumors to be highly complex and multifactorial diseases of cortical development (66-68) LRP12, a gene abundantly expressed during murine corticogenesis (37), combines distinct characteristics, supporting the hypothesis of LRP12 being... lesions, glial scars and encephalitic lesions In these cases the cause of epilepsy is symptomatic Merely a small proportion of temporal lobe epilepsy (7%) is of unknown pathology, as histomorphological analyses do not succeed to recognize pathological alterations (12, 13) A hallmark of temporal lobe epilepsy is the manifestation of seizures in adolescents or young adults Typical temporal lobe seizures... course of gangliogliomas point to an origin from developmentally 8 Introduction compromised precursor lesions (18, 19, 26) The presence of the stem cell marker CD34 supports the idea of a maldevelopmental origin of these neoplasms (18) Additionally, ongoing discussions deal with the issue whether the glial part, neuronal cells or both compartments of gangliogliomas constitute the neoplastic unit of these... characterizations of LRP12 expression levels during embryogenesis and in adult mice as well as analyses of post-translational modifications and LRP12’s subcellular localization are carried out Answering the questions posed above may contribute to a better understanding of the molecular pathomechanisms leading to the manifestation of gangliogliomas Hopefully, this study will aid the development of novel therapy... factor of gangliogliomas, in this particular case due to mutational events of the tumor suppressor gene TCS2 in glial cells, other investigations by Zhu et al (1997) suggest both glial and neuronal cells as neoplastic origin of gangliogliomas (27-30) Recently, two independent investigations by Schindler et al (2011) and Koelsche et al (2013) detected an increased rate of BRAF V600 mutations in gangliogliomas. .. neoplastic elements of gangliogliomas (30) Diagnosis of gangliogliomas can be challenging, as morphological appearances may be very similar to those observed in cortical infiltrating diffuse astrocytomas Quite recently, studies implicate that neuropathological differentiation of both tumors can be facilitated by immunohistochemical detection of IDH1 mutations, which are present in 75% of diffuse astrocytomas,... epileptic seizures originating from neocortical areas are termed as lateral temporal lobe epilepsy Benign brain tumors are most commonly identified as the 4 Introduction underlying cause of neocortical temporal lobe epilepsy The majority of these lowgrade neoplasms comprises gangliogliomas (17) 1.3 Gangliogliomas Gangliogliomas are benign brain tumors with only rare malignant progression Commonly, they ... astrocytes (Figure 1.6.2) (65) 18 Introduction 1.7 Aims of the study Molecular pathomechanisms leading to the formation of intractable epilepsyassociated gangliogliomas are still unclear Previous studies... clinical view of epilepsy is much more distinct In order to establish a common definition of epilepsy, the International League Against Epilepsy (ILAE) and the International Bureau for Epilepsy (IBE)... amounts of epithelial cells such as liver or kidney Cloning of a full-length human ST7 cDNA uncovered a protein of 859 amino acids with an estimated molecular weight of 92.8kDA Analysis of the