Gene expression profiling in cells with enhanced gamma-secretase activity

BACKGROUND: Processing by gamma-secretase of many type-I membrane protein substrates triggers signaling cascades by releasing intracellular domains (ICDs) that, following nuclear translocation, modulate the transcription of different genes regulating a diverse array of cellular and biological processes. Because the list of gamma-secretase substrates is growing quickly and this enzyme is a cancer and Alzheimer's disease therapeutic target, the mapping of gamma-secretase activity susceptible gene transcription is important for sharpening our view of specific affected genes, molecular functions and biological pathways. METHODOLOGY/PRINCIPAL FINDINGS: To identify genes and molecular functions transcriptionally affected by gamma-secretase activity, the cellular transcriptomes of Chinese hamster ovary (CHO) cells with enhanced and inhibited gamma-secretase activity were analyzed and compared by cDNA microarray. The functional clustering by FatiGO of the 1,981 identified genes revealed over- and under-represented groups with multiple activities and functions. Single genes with the most pronounced transcriptional susceptibility to gamma-secretase activity were evaluated by real-time PCR. Among the 21 validated genes, the strikingly decreased transcription of PTPRG and AMN1 and increased transcription of UPP1 potentially support data on cell cycle disturbances relevant to cancer, stem cell and neurodegenerative diseases' research. The mapping of interactions of proteins encoded by the validated genes exclusively relied on evidence-based data and revealed broad effects on Wnt pathway members, including WNT3A and DVL3. Intriguingly, the transcription of TERA, a gene of unknown function, is affected by gamma-secretase activity and was significantly altered in the analyzed human Alzheimer's disease brain cortices. CONCLUSIONS/SIGNIFICANCE: Investigating the effects of gamma-secretase activity on gene transcription has revealed several affected clusters of molecular functions and, more specifically, 21 genes that hold significant potential for a better understanding of the biology of gamma-secretase and its roles in cancer and Alzheimer's disease pathology.

Gamma-Secretase Dependent Gene Expression

Alexandra Marie-Isabelle Karoline Brigitte Magold

The causes of sporadic Alzheimer's Disease (AD), the most common form of dementia at old age, are still unknown. Familial early onset AD (FAD) is mainly caused by mutations of Presenilins (PS), the active subunits of γ-secretase. Processing of its substrate proteins by γ-secretase generates shorter fragments. In the case of the Amyloid Precursor Protein (APP) these are Aβ species, of which the ratio of Aβ40, which is 40 amino acids (aa) long and Aβ42, 42 aa in length respectively, has been connected with their aggregation and AD plaque formation. Another cleavage product of many γ-secretase substrate proteins are intracellular domains (ICDs), which enter the nucleus and alter gene transcription. Be it via statins, the levels of cAMP, the generation of which is also catalyzed by cilia specific Adenylyl-cyclase III (ACIII) or Wnt pathways, cilia can be connected with various AD relevant proteins, pathways and functions, but their physiological and thus pathological cerebral roles are not understood. We hypothesized that γ-secretase activity alterations impact transcription levels of specific genes and potentially signaling and functional pathways as well as cilia genes in ways relevant to AD. We determined the transcriptional impact of enhanced γ-secretase activity in Chinese hamster ovary (CHO) cells with a cDNA microarray experiment. Gene ontology cluster analysis of the 1981 genes reported by our microarray gave insights into functional groups represented. Matching of known physical protein-protein interactions as reported by the string 8.0 database for 21 genes, which were confirmed to be transcriptionally altered with γ-secretase activity, registered various members of three different Wnt pathways. Consistent with an hypothesized role of cilia, the array also reported two dozen of cilia-related genes. The most decreased transcript levels were of Anti-mitotic exit network homolog 1 (AMN1), a gene of predicted expression in the cilia of sensory olfactory neurons and for which protein data was available in yeast only. Our immunohistochemistry results confirm Amn1 protein expression in mammalian cells, specifically in murine and human brains. We showed that Amn1 protein is localized in cortical neurons and in sharply outlined intracellular structures close to motile cilia and the opposing cell pole of monocilia. Aged APPPS1 transgenic mice, which over express human APP and PS1 with FAD mutations, did neither show differences in lateral ventricular cilia range, cilia density, the percentage of ciliated hippocampal neurons nor ACIII protein levels. Consistently with previous reports of increased cAMP levels in the cerebrospinal fluid of AD patients, we detected significantly increased levels of ACIII protein in human AD cortex samples (AD n=11, non demented n=12). These results support a new focus on the role of cilia-related proteins in AD. They also suggest some limitations of the APPPS1 transgenic animal model. In recent years the impact and role of cilia-related proteins in various neurodegenerative diseases was demonstrated. This PhD thesis provides evidence that AD should be included in the list of neurodegenerative diseases which are being investigated for the impact and role of cilia-related proteins in their pathologies.

EPFL2011

Quantitative analysis of gene expression and transcriptional memory in mouse embryonic stem cells

Aleksandra Mandic

Quantitative studies of gene expression in have shown widespread variability in transcript and protein product abundance at the single-cell level. Such fluctuations in gene expression have been shown to lead to heterogeneous cellular responses and phenotypes, acting in a range of systems, from immunity, cancer to development. To better understand heterogeneity in cellular populations, we developed a novel method for the precise quantification of gene expression in single living cells. Furthermore, we focused on whether and to which extent transcriptional expression profiles are transmitted during cell division. In the first section, we described a new approach for quantitative measurements of gene expression, based on internal tagging of endogenous proteins with a reporter allowing luminescence and fluorescence time-lapse imaging. Using the currently brightest luminescent reporter, fluctuations of protein expression levels could be monitored in single living cells with high sensitivity and temporal resolution over extended time periods. Moreover, our system allowed measurement of single-cell protein degradation rates in the absence of protein synthesis inhibitors, as well as calculation of absolute synthesis rates over the cell cycle. Finally, the internal tag could be excised by inducible expression of Cre recombinase, which enabled estimation of endogenous mRNA half-lives by monitoring the decay of luminescent signal. Second, to monitor transcriptional activity and quantify memory across multiple endogenous genes in mouse embryonic stem cells, we generated cell lines in which a short-lived luciferase reporter allowed sensitive monitoring of transcriptional kinetics by luminescence imaging at high time resolution over long periods of time. By coupling live single-cell monitoring of transcriptional activity of 9 different promoters with mathematical modelling, we found that different levels of expression fluctuations shape transcriptional memory. Long fluctuations (1-10 generations) and short fluctuations (1-3 h), as well as their amplitudes, vary widely between across different genes and together shape transcriptional memory. Surprisingly, we have shown that there was a large level of gene-specific cell-to-cell synchronicity in transcriptional profiles. Overall, we uncovered that noise and timescales of slow and fast fluctuations, as well as their synchronisation over the cell cycle, defined how transcriptional dynamics were correlated within a lineage of related cells. In conclusion, this thesis described an innovative method that allows measurement of absolute protein expression levels, protein turnover, as well as mRNA half-lives for endogenously expressed genes. Our approach opens new avenues in quantitative studies of gene expression in single living cells. Furthermore, we revealed different levels of transcriptional fluctuations that shape gene specific transcriptional memory and its timescales in mouse embryonic stem cells.

EPFL2018

An epigenetic blockade of cognitive functions in the neurodegenerating brain

Johannes Gräff

Cognitive decline is a debilitating feature of most neurodegenerative diseases of the central nervous system, including Alzheimer's disease. The causes leading to such impairment are only poorly understood and effective treatments are slow to emerge. Here we show that cognitive capacities in the neurodegenerating brain are constrained by an epigenetic blockade of gene transcription that is potentially reversible. This blockade is mediated by histone deacetylase 2, which is increased by Alzheimer's-disease-related neurotoxic insults in vitro, in two mouse models of neurodegeneration and in patients with Alzheimer's disease. Histone deacetylase 2 associates with and reduces the histone acetylation of genes important for learning and memory, which show a concomitant decrease in expression. Importantly, reversing the build-up of histone deacetylase 2 by short-hairpin-RNA-mediated knockdown unlocks the repression of these genes, reinstates structural and synaptic plasticity, and abolishes neurodegeneration-associated memory impairments. These findings advocate for the development of selective inhibitors of histone deacetylase 2 and suggest that cognitive capacities following neurodegeneration are not entirely lost, but merely impaired by this epigenetic blockade.