An epigenetic role for tip60 in APP mediated neuronal processes

Dynamic epigenetic regulation of neurons is emerging as a fundamental mechanism by which neurons adapt their transcriptional responses to specific developmental and environmental cues. Recent studies bolster the concept that age associated aberrant changes to the epigenetic modification code within the genome of the brain, specifically histone acetylation, cause gene misregulation that drive cognitive decline. Tip60 is a histone acetyltransferase (HATs) enzyme that is involved in the transcriptional regulation of genes enriched for neuronal function and control of synaptic plasticity. Accordingly, Tip60 has been implicated in the neurodegenerative disorder Alzheimer’s disease (AD) via transcriptional regulatory complex formation with the AD linked amyloid precursor protein (APP) intracellular domain (AICD). As such, misregulation of neuronal target genes by the Tip60/AICD complex is thought to lead to neurotoxicity associated with AD. However, a direct and causative epigenetic based role for Tip60 HAT activity in mediating such gene expression changes in vivo remains unclear and is thus the subject of this study. Chapter 1 reviews the role of specific HATs including Tip60 in mediating neuronal survival and high order brain functions like learning and memory. The pros and cons of using HDACi as therapeutic strategies and the beneficial effects of modulating the function of specific HATs in neurodegenerative diseases is also discussed. Chapter 2 demonstrates a functional interaction between Tip60 and APP during neurogenesis using a transgenic AD fly model that was uniquely adapted to induce varying levels of Tip60 HAT activity and describes the neuroprotective role Tip60 HAT activity exhibits towards AD neurodegenerative pathology. Chapter 3 demonstrates that Tip60 HAT activity in conjunction with APP mediates axonal growth of the Drosophila pacemaker cells, the sLNvs, and their production of the neuropeptide PDF to stabilize Drosophila sleep–wake cycles. The study provides novel insight into epigenetic-based regulation of sleep disturbances observed in neurodegenerative diseases like AD. Chapter 4 describes the effects Tip60 plays in mediating gene expression changes that underlie memory formation in Drosophila via the mushroom body encompassing neural circuit. Together, these studies add dTip60 to the growing list of HAT chromatin regulators critical for nervous system function.Ph.D., Biological Sciences -- Drexel University, 201

Targeting Specific HATs for Neurodegenerative Disease Treatment: Translating Basic Biology to Therapeutic Possibilities

Dynamic epigenetic regulation of neurons is emerging as a fundamental mechanism by which neurons adapt their transcriptional responses to specific developmental and environmental cues. While defects within the neural epigenome have traditionally been studied in the context of early developmental and heritable cognitive disorders, recent studies point to aberrant histone acetylation status as a key mechanism underlying acquired inappropriate alterations of genome structure and function in post-mitotic neurons during the aging process. Indeed, it is becoming increasingly evident that chromatin acetylation status can be impaired during the lifetime of neurons through mechanisms related to loss of function of histone acetyltransferase (HATs) activity. Several HATs have been shown to participate in vital neuronal functions such as regulation of neuronal plasticity and memory formation. As such, dysregulation of such HATs has been implicated in the pathogenesis associated with age-associated neurodegenerative diseases and cognitive decline. In order to counteract the loss of HAT function in neurodegenerative diseases, the current therapeutic strategies involve the use of small molecules called histone deacetylase (HDAC) inhibitors that antagonize HDAC activity and thus enhance acetylation levels. Although this strategy has displayed promising therapeutic effects, currently used HDAC inhibitors lack target specificity, raising concerns about their applicability. With rapidly evolving literature on HATs and their respective functions in mediating neuronal survival and higher order brain function such as learning and memory, modulating the function of specific HATs holds new promises as a therapeutic tool in neurodegenerative diseases. In this review, we focus on the recent progress in research regarding epigenetic histone acetylation mechanisms underlying neuronal activity and cognitive function. We discuss the current understanding of specific HDACs and H

Tip60 HAT activity mediates APP induced lethality and apoptotic cell death in the CNS of a Drosophila Alzheimer's disease model.

Histone acetylation of chromatin promotes dynamic transcriptional responses in neurons that influence neuroplasticity critical for cognitive ability. It has been demonstrated that Tip60 histone acetyltransferase (HAT) activity is involved in the transcriptional regulation of genes enriched for neuronal function as well as the control of synaptic plasticity. Accordingly, Tip60 has been implicated in the neurodegenerative disorder Alzheimer's disease (AD) via transcriptional regulatory complex formation with the AD linked amyloid precursor protein (APP) intracellular domain (AICD). As such, inappropriate complex formation may contribute to AD-linked neurodegeneration by misregulation of target genes involved in neurogenesis; however, a direct and causative epigenetic based role for Tip60 HAT activity in this process during neuronal development in vivo remains unclear. Here, we demonstrate that nervous system specific loss of Tip60 HAT activity enhances APP mediated lethality and neuronal apoptotic cell death in the central nervous system (CNS) of a transgenic AD fly model while remarkably, overexpression of Tip60 diminishes these defects. Notably, all of these effects are dependent upon the C-terminus of APP that is required for transcriptional regulatory complex formation with Tip60. Importantly, we show that the expression of certain AD linked Tip60 gene targets critical for regulating apoptotic pathways are modified in the presence of APP. Our results are the first to demonstrate a functional interaction between Tip60 and APP in mediating nervous system development and apoptotic neuronal cell death in the CNS of an AD fly model in vivo, and support a novel neuroprotective role for Tip60 HAT activity in AD neurodegenerative pathology

Gene expression changes of dTip60^E431Q misregulated target genes in the different transgenic lines.

a<p>Quantitative RT-PCR analysis was performed for the indicated target genes.</p>b<p>Staged second instar larvae ubiquitously expressing the indicated transgene(s) were used for cDNA preparation. Quantitative RT-PCR reactions were carried out in triplicate and the relative fold change was calculated using the 2−ΔΔCT method using RP49 as control.</p>§<p>Genes that were differentially regulated between flies expressing the Tip60 HAT mutant dTip60^E431Q alone and in conjunction with APP.</p> <p>Gene that were differentially regulated between flies overexpressing dTip60^WT alone or together with APP.</p

Generation and characterization of dTip60^E431Q containing APP or APP-dCT double transgenic flies.

<p>The dominant negative HAT defective lines dTip60^E431Q A or dTip60^E431Q B (Lorbeck <i>et al.</i>, 2011) were introduced into an APP or APP dCT background using standard genetic techniques. (<b>A</b>) Histogram depicting qPCR analysis of exogenous levels of dTip60^E431Q in staged F1 second instar larval progeny resulting from a cross between the ubiquitous driver 337 and either dTip60^E431Q (lines A and B), APP; dTip60^E431Q (lines A and B) or APP dCT; dTip60^E431Q (lines A and B). 337-Gal4 crossed to <i>w¹¹¹⁸</i> served as a control. Quantification of the exogenously expressed dTip60^E431Q mRNA levels relative to endogenously expressed dTip60 mRNA was done using the comparative CT method with RP49 as internal control as described in (Lorbeck <i>et al</i>, 2011). Asterisks (*) indicate significant fold change between the lines A and B for each genotype with values of p<0.05; n = 3. Error bars represent standard error of the mean. (<b>B</b>) Semiquantitative RT-PCR analysis of APP or APP dCT expression in the different transgenic lines to confirm APP transgene presence. cDNA was prepared as before from staged second instar larvae ubiquitously expressing dTip60^E431Q with APP or APP dCT (lines A or B in each case) and PCR amplified using primers that flank a 100 bp region in the N-terminal portion of APP. PCR products were visualized using 2% agarose gel containing ethidium bromide. Staged second instar larvae ubiquitously expressing APP or APP dCT were used as controls.</p

Generation and characterization of dTip60^WT containing APP or APP-dCT double transgenic flies.

<p>Flies expressing varying levels of wild type dTip60 (low, medium and high) were generated and then each introduced into APP or APP dCT background using standard genetic techniques. (<b>A</b>) The amount of wild type dTip60 that is exogenously induced relative to endogenous dTip60 was quantified by RT-PCR analysis of staged F1 second instar larvae resulting from the a cross between the ubiquitous driver 337 and either dTip60^WT (lines A, B and C), APP; dTip60^WT (lines A, B and C) or APP dCT; dTip60^WT (lines A, B and C). 337-Gal4 crossed to <i>w¹¹¹⁸</i> was used as control. The relative fold change in mRNA expression levels between exogenous and endogenous dTip60 was measured as described before using the comparative CT method with RP49 as the internal control, and these results are summarized in the histogram. The amount of exogenously induced wild type dTip60 levels is significantly different between lines A, B and C in each case with values of p<0.05; n = 3. Error bars represent standard error of the mean. (<b>B</b>) Semi-quantitative RT-PCR analysis of APP or APP dCT expression in the different dTip60^WT containing transgenic lines to confirm APP transgene presence. cDNA was prepared as before from staged second instar larvae ubiquitously expressing dTip60^WT with APP or APP dCT (lines A, B or C in each case) and PCR amplified using primers that flank a 100 bp region in the N-terminal portion of APP. PCR products were visualized using 2% agarose gel containing ethidium bromide. Staged second instar larvae ubiquitously expressing APP or APP dCT were used as controls.</p

Viability analysis indicates genetic interaction between Tip60 and APP in <i>Drosophila</i>.

<p>The indicated transgene was expressed ubiquitously in the fly using 337-Gal4 driver or pan-neuronally using 179 y-Gal4 driver. The number of F1 progeny that eclosed were counted daily. The percentage of eclosed flies was calculated relative to the wild type control (<i>w¹¹¹⁸</i>). All crosses were carried out in triplicate at 25°C. Overexpression of APP drastically reduced viability to <10% while no effect was observed due to expression of truncated version of APP lacking its C-terminal domain. Overexpression of varying levels of wild type dTip60 (dTip60^WT) also reduced viability in a dose independent manner. However, co-expression of dTip60^WT with APP partially rescued the lethal effects induced by APP expression in a dose dependent manner with the maximum effect observed with high levels of dTip60^WT. In the presence of APP lacking the C-terminus, overexpression of dTip60^WT had similar effects seen in flies that overexpressed dTip60^WT alone.</p

Transgenic fly lines used for this study.

a<p>The Tip60 P-element insertion is located on chromosome 3 and the APP P-element insertion is located on chromosome 2.</p>b<p>Indicates where the transgenic fly lines were generated.</p

Quantitative RT-PCR validation of selected apoptosis related genes identified by microarray analysis.

<p>(<b>A</b>) Histogram showing relative fold change in expression level of apoptosis related target genes in flies expressing dTip60^E431Q A. Staged second instar larvae were used for cDNA preparation. RT-PCR reactions were carried out in triplicate and the fold change was calculated using the 2−ΔΔCT method using RP49 as control. (<b>B</b>) List of selected apoptosis related target genes identified by microarray analysis and validated in the dTip60^E431Q A line using quantitative RT-PCR.</p