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Title: APLP2 regulates neuronal stem cell differentiation during cortical development
Other Titles: APLP2 regulates neuronal stem cell differentiation during cortical development
Authors: Shariati, Seyed Ali Mohammad
Issue Date: 9-Dec-2013
Abstract: SummaryHuman amyloid precursor protein (APP) belongs to a gene family that codes for type I trans-membrane proteins in different species, including fruit flies, roundworms and mice. Amyloid Precursor-like Protein1 and 2 (APLP1 and APLP2) are the two other mammalian members of this gene family. Like APP, APLP1 and APLP2 are transmembrane proteins with a large extracellular N-terminal domain and short cytoplasmic C-terminal fragment. Another common feature of APP/APLPs is their processing by enzymatic activities called secretases. Unlike APLP1 and APLP2, APP processing can generate amyloid beta peptides (Abeta) which precipitate in the brain of Alzheimer patients. Therefore, it is not surprising that APP has been extensively studied in the context of Alzheimer disease.Nevertheless, APP and its two paralogues, APLP1 and APLP2, are expressed in early stages of brain development suggesting a physiological function for these proteins in early neurodevelopment. However, genetic knock-out and shRNA studies have led to contradictory conclusions about their role during embryonic brain development. In particular, down-regulation of APP in precursors and neurons of the developing cortex in vivo blocks the migration of neurons towards the cortical plate, while conversely, neurons in an APP/APLP1/APLP2 triple knock-out (ko) mouse over-migrate and accumulate ectopically in the marginal zone. The yet unexplained discrepancy between the over-migration effects in the triple ko and the blocked migration in the case of APP down-regulation shows that the role of the APP family members in the course of cortical development is still unclear.We hypothesized that APP and APLPs might regulate distinct processes in the developing cortex based on the differential mRNA expression profiles for APP gene family members. During cortical development: APP is found in the cortical plate (CP) and ventricular zone (VZ), APLP2 in the VZ and subventricular zone (SVZ) and APLP1 in the CP only. The restriction of APLP2 expression to the proliferative zones (VZ/SVZ) of the developing cortex suggests a specific function for APLP2 in the development and specification of cortical progenitors. Therefore we focused our attention on the involvement of APLP2 in cortical development.To this end, we interfered with APLP2 expression in developing cortices of wildtype (wt) mice using in utero electroporation. APLP2 down-regulation in wt cortices did not change cortical positioning of neurons. Next, we down-regulated APLP2 expression in APP/APLP1 dko mice reasoning that partial overlapping functions with APP and APLP1 might compensate for the absence of APLP2. While APP/APLP1 dko neurons migrated normally to the cortical plate, similar to their wt counterparts, further APLP2 down-regulation in APP/APLP1 dko cells blocked cells predominantly in the proliferative zones of the developing cortex, leading to altered cortical positioning. We will use the term “triple ko” in the rest of the thesis to refer to these APP/APLP1dko cells that express APLP2shRNA. Our analysis shows that arrested cells remain undifferentiated as demonstrated by the continuous expression of progenitor and mitotic markers. We find that the morphology of “APP/APLP1/APLP2 triple ko” migrating neurons and “triple ko” radial glia fibers, a major substrate for neuronal migration, is normal. Furthermore, the migration of triple ko neurons in vitro seems not affected. Further investigation of the properties of neuronal progenitors showed delayed neuronal differentiation and decreased cell cycle exit of the triple deficient cells. In summary, our data reveal a novel function of APLP2 in the regulation of proper cell cycle exit of neuronal progenitors.
Table of Contents: SUMMARY 3
SAMENVATTING 5
LIST OF FIGURES 7
ABBREVIATIONS 8
CHAPTER I: INTRODUCTION 9
1.1 AMYLOID PRECURSOR PROTEIN DOMAIN STRUCTURE AND FUNCTION 10
1.2 EVOLUTION OF THE AMYLOID PRECURSOR PROTEIN FAMILY 15
1.3 PROTEOLYTIC PROCESSING OF AMYLOID PRECURSOR PROTEINS 17
1.4 TEMPORALLY AND TISSUE SPECIFIC REGULATED EXPRESSION OF APP FAMILY PROTEINS 20
1.5 THE INTERACTION NETWORKS OF APP, APLP1 AND APLP2 SHOW SPECIFICITY 21
1.6 VARIOUS ROLES OF THE APP MEMBERS IN THE NERVOUS SYSTEM 22
1.6.1 The extracellular domain in development of C. elegans 22
1.6.2 D. melanogaster’s APPL in axonal wiring and synaptic function 23
1.6.3 The APP family in M. musculus 24
1.7 A ROLE OF APP AND APLPS IN CORTICAL DEVELOPMENT? 28
1.7.1 Cell cycle of neural stem cells 29
1.7.2 Radial migration and the role of APP proteins 31
CHAPTER II: RATIONALE, AIMS AND APPROACH 35
CHAPTER III: METHODS AND MATERIALS 37
3.1 DNA CONSTRUCTS 37
3.2 WESTERN BLOT 39
3.3 CELL CULTURES 39
3.4 MATRIGEL ASSAY FOR MIGRATION 39
3.5 KNOCK-OUT MICE 40
3.6 IN UTERO ELECTROPORATION 40
3.7 IMMUNOCYTOCHEMISTRY 40
3.8 CELL CYCLE EXIT 40
3.9 CONFOCAL IMAGING AND QUANTIFICATION 41
3.10 STATISTICS 41
CHAPTER IV: RESULTS 43
4.1 IDENTIFICATION OF FUNCTIONAL AND SPECIFIC SHRNAS TARGETING APLP2 44
4.2 DOWN-REGULATION OF APLP2 IN WT CORTICES DOES NOT CHANGE CORTICAL POSITIONING 45
4.3 DOWN-REGULATION OF APLP2 IN APP/APLP1 DKO CORTICES AFFECTS CORTICAL POSITIONING 47
4.4 MORPHOLOGY AND MIGRATORY BEHAVIOUR OF “TRIPLE KNOCK-OUT” CELLS REMAIN UNCHANGED 49
4.5 “TRIPLE KNOCK-OUT” CELLS REMAIN IN A PROLIFERATIVE PROGENITOR STATE 51
4.6 APLP2 HAS A PROGENITOR SPECIFIC FUNCTION AND IS DISPENSABLE FOR RADIAL MIGRATION 53
4.7 CELL CYCLE VARIABLES ARE REGULATED BY APLP2 56
CHAPTER V: GENERAL DISCUSSION AND PERSPECTIVES 61
5.1 HYPOTHESES ON APLP2 REGULATION OF NEURAL STEM CELL DIFFERENTIATION 61
5.2 IN UTERO ELECTROPORATION TO STUDY NEURODEVELOPMENTAL PROCESSES: THE EXAMPLE OF APP PROTEINS 62
5.3 CONTROVERSIES ON THE ROLE OF APP PROTEINS IN CORTICAL DEVELOPMENT 64
5.4 PERSPECTIVE ON BIOLOGICAL FUNCTION OF APP PROTEIN FAMILY DURING CORTICAL DEVELOPMENT. 65
REFERENCES: 69
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Laboratory for the Research of Neurodegenerative Diseases
Department of Human Genetics - miscellaneous

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