title: Genome-wide analysis of C99-overexpressing human neuroblastoma cells : effects of C99 cleavage products on gene expression, signal transduction and phosphorylation status
creator: Uhrig, Markus
subject: ddc-570
subject: 570 Life sciences
description: The human genome comprises approximately 20,000-25,000 genes. The genes known to be involved in Alzheimer’s disease (AD) are the amyloid precursor protein (AbetaPP), presenilin 1 (PS1), presenilin 2 (PS2) and apolipoprotein E (APOE). However, which additional genes are involved in its etiology is a controversial topic. C99, the C-terminal cleavage product of AbetaPP is the direct, in vivo occurring, precursor of Abeta-peptides. It is proteolytically processed, resulting in the generation of several Abeta-peptides. In contrast to the form with 40 amino acids (Abeta40), which is regarded as the physiological form, the variant with 42 amino acids (Abeta42) is thought to be the pathogenic form triggering the pathophysiological cascade in AD. In order to produce different Abeta42 and Abeta40 levels, the Abeta precursors C99I45F and C99V50F (two C99 mutants, known to generate different amounts of Abeta42 and Abeta40) were overexpressed in human neuroblastoma cells. This resulted, due to varying intracellular cleavage by gamma-secretase, in different Abeta42 and Abeta40 levels accompanied by the generation of their respective APP intracellular domains (AICDs). The goal of this thesis was to obtain information about effects of the different C99 cleavage products. Therefore different C99 mutants were overexpressed in the human neuroblastoma cell line SH-SY5Y resulting in different Abeta42/Abeta40 levels and a genome-wide transcriptomic (Affymetrix) and proteomic response of these cells was monitored. As was expected, many genes previously reported to be associated with AD were found to be differentially expressed. Importantly, further genes, not yet linked to AD, have been identified and new cross-talk between genes/proteins has been suggested. The genes assumed to play a predominant role in response to altered C99 cleavage products are indicated in brackets and full gene names are to be found in Chapter 10, Abbreviations. In this thesis, it was demonstrated that an increased Abeta42/Abeta40 ratio, but not a decreased Abeta42/Abeta40 ratio, induced upregulation of the retinoic acid binding protein CRABP1 on the transcript level and on the protein level. This, in turn, reduced the responsiveness of SH-SY5Y cells to retinoic acid, which reduced their differentiation potential. Knockdown of the increased CRABP1 levels by siRNA rescued the differentiation potential of these cells. These findings, in conjunction with known functional properties of CRABP1, suggest that up-regulated CRABP1 might, possibly also in vivo, prevent the terminal differentiation of neural precursor cells into functional neurons (disturbed neurogenesis). Furthermore, the phosphorylation status of proteins was determined providing further insights into signal transduction pathways. The IGF2/IGF1R/PKC together with the PI3K/AKT survival pathway was found to be inversely regulated by an altered Abeta42/Abeta40 ratio (IGF2, IGFBP5, PKC, AKT1). The chromosomal locus 11p15.5 was identified as a hot spot: an increased Abeta42/Abeta40 ratio, in contrast to a decreased one, down-regulated the IGF2-H19 imprinted region (chr.11p15.5) indicating that imprinting may play a role in AD. In consequence of an increased Abeta42/Abeta40 ratio, GSK3beta was hyperphosphorylated on the activating site Y216, and tau441 showed stronger phosphorylation on S199/S202, two sites reported to turn tau, upon phosphorylation, into a protein with possible toxic properties. APLP2 was found to be up-regulated as a consequence of an increased Abeta42/Abeta40 ratio, whereas APLP1 was up-regulated in response to a decreased Abeta42/Abeta40 ratio, indicating inverse Abeta-dependent regulation of APLP1/2 expression. In response to an increased Abeta42/Abeta40 ratio, expression levels of a set of genes were altered in such a way that a tendency towards faster blood coagulation and reduced fibrinolysis could be recognized on the transcript level (PLAT, TFPI2, FGB, SERPINF1, SERPINE2). This view is supported by observations in AD patients who have a greater risk of strokes and diminished cerebral perfusion. As Abeta-inducible candidate kinases possibly involved in tau-phosphorylation, DYRK1, CDKL1, CDKL5, CDK6, DCAMKL1, ERK1 and PFKP were identified due to their altered expression levels (or phosphorylation status for ERK1). Indeed, previous studies demonstrated that Abeta42 induces tau hyperphosphorylation in animal models.  PFKP, which plays a key role in glycolysis, was found to be dysregulated in consequence of a changed Abeta42/Abeta40 ratio and could possibly reflect disturbed regulation of glucose and ATP metabolism in brains of AD patients. A potential function was assigned to the hitherto uncharacterized KIAA0125, namely to be a counter player of neurogenin 2, which was co-regulated with C99 and neuronatin. Furthermore, the following areas were distinctly affected by a changed Abeta42/Abeta40 ratio (for most genes determined on the transcript level and for some additionally on the protein level): Copper transport/metabolism with special regard to ATP7A, several (metallo) proteases (ADAMTS9, ADAMTS3, MMP8, PREP, ECEL1, CTSD, PRSS12), metalloprotease inhibitors (TIMP3, TIMP1), extracellular matrix proteins or related enzymes thereof (RELN, COL4A1, COL4A2, HS3ST2), cytoskeletal proteins (ACTA2, ACTN1), mitochondrial respiratory chain components (NDUFB9), cytochromes (b-561 and b-245), dopamine, serotonin and glutamate -metabolism with special regard to DDC, HMP19 and GAD1. Effects of BDNF (non-catalytic isoform of TRKB), membrane fusion of neurotransmitter containing vesicles (STX3A, SYN2), Ca2+ influx (AMPA2), acetylcholine receptors (CHRNA7), Notch signaling (DNER, TLE1, TLE2, JAG1, CUTL2), TGFbeta signaling (TGFB2, TGFBR2, BAMBI, BMP7), WNT signaling (DKK2, DKK4), G protein signaling (RGS4) and ERK/MEK signaling (ERK1, MEK1). Growth cone guidance, synaptogenesis and dendritic branching are expected to be impaired as consequence of an increased Abeta42/Abeta40 ratio but improved as a result of a decreased one (SEMA3A, SEMA3C, L1CAM, PTN, SLIT1). Further genes involved in the following areas were distinctly influenced by a changed Abeta42/Abeta40 ratio: Glutamate/ammonia metabolism (GLUL, GLS), urea cycle (ASS), cell cycle regulation (CCND1, CDKN1A), glucocorticoid regulated kinase (SGK), receptors (PTGER2, EGFR, AMPA2, AMPA3, GRM7, GRM8c, GLRB, GABRB3), long term potentiation (CREB1), axonal transport (DNCLI2), angiogenesis (HGF, VEGF), T-cell receptors and connected transcription factors (T-cell receptor alpha and delta locus, GATA3), lipoprotein associated proteins or adaptor proteins hereof (LRP4, PDZK1), vesicular transport of organelles and microtubule association (VMP), transcription factors (PBX1, SHOX2) and stress-related factors (ADRB1). Taken together, the identified genes, proteins and pathways have given new and deeper insight into the effects of different C99 cleavage products and they have provided new hypotheses for the pathological mechanisms of AD. Furthermore they are possible candidates for genetic risk factors and may be helpful in explaining the mechanisms of non-genetic risk factors. The gene expression pattern, specific for an increased Abeta42/Abeta40 ratio, could be useful, together with clinical data, for the diagnosis of AD. Finally, some of the identified transcripts, proteins and pathways might turn out to be suitable drug targets.
date: 2008
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/8496/1/Dissertation_Markus_Uhrig.pdf
identifier: DOI:10.11588/heidok.00008496
identifier: urn:nbn:de:bsz:16-opus-84965
identifier:   Uhrig, Markus  (2008) Genome-wide analysis of C99-overexpressing human neuroblastoma cells : effects of C99 cleavage products on gene expression, signal transduction and phosphorylation status.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/8496/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng