Epigenetic Changes in Alzheimer ' s disease
Relation to β -amyloid
Xianli Shen
Degree project in biology, Master of science (2 years), 2011 Examensarbete i biologi 45 hp till masterexamen, 2011
Biology Education Centre, Uppsala University, and Department of Neurobiology, Care Sciences and Society, Karolinska Institutet
Supervisor: Christina Unger Lithner, PhD
Summary
Alzheimer’s disease (AD) is the most common form of dementia leading to progressive impairment of episodic memory and cognitive decline, with clinical characterization in part by abnormal amyloid deposition. The amyloid β-protein (Aβ), which is the chief component of amyloid deposits, seems to play an essential role in the pathogenesis of AD. Epigenetic mechanisms cause heritable changes in gene expression primarily through reversible changes in DNA methylation and remodelling of chromatin structure. By influencing chromatin structure, dynamic modifications on histone H3 contribute to long-term changes in neuronal function. This project is focused on understanding the epigenetic changes in brain areas important for memory and learning, particularly studying how different forms and species of Aβ affect the epigenetic mechanisms in the brain. The aim is to understand the early effects of Aβ and how it affects gene regulation of neuronal cells during the disease process.
Viability assays were carried out to investigate the toxicity of different forms and species of Aβ. The results demonstrated that oligomeric forms of Aβ42 probably induced the most toxic effects in SH-SY5Y cells in vitro.Histones were extracted to study various modifications that occurred in H3. Quantitative analyses indicate that nanomolar concentrations of fibrillar and oligomeric forms of Aβ40 promote an increase in acetylation at H3K14 and phosphorylation at H3S10 in SH-SY5Y cells. Moreover, AD subjects showed increased acetylation at H3K14 and dimethylation at H3K9 associated with a low level of phosphorylation at H3S10. The results may provide insights into understanding the epigenetic effects of Aβ in brain areas. Future work will be aimed at utilizing a microarray approach to investigate increases in relevant mRNAs and thereby obtain specific candidate genes related to the different forms of Aβ.
Introduction
Alzheimer’s disease
In 1907, the physician Alois Alzheimer reported the first case of Alzheimer’s disease (AD) in Germany. In that case, Alzheimer described the clinical characteristics with increasing memory impairment and progressive cognitive disturbances (Alzheimer, 1907). AD was considered a rare disorder 100 years ago. However, nowadays it is recognized that AD is the most common form of dementia and mainly affects aged individuals (Ferri et al, 2005).
Several stages from normal to severe impairment are involved in AD. As the disease progresses, clinically common symptoms of AD appear including impaired short-term memory, cognitive decline and language deterioration. AD is associated with an immense social and economic burden (Wimo et al, 2010) that might become more serious due to the ageing of populations all over the world. Besides ageing, epidemiological studies of AD have indicated other risk factors like decreased reserve capacity of the brain in early or late life and vascular disease (reviewed by Mayeux, 2003). Environmental factors such as dietary intake of vitamins and exposure to toxins have been linked with AD as well (Luchsinger and Mayeux, 2004).
Pathology and beta-amyloid
The characteristic pathological hallmarks in AD include extracellular neuritic plaques and intracellular neurofibrillary tangles in the medial temporal lobe and cortex of the brain as well as loss of neurons and synapses in the basal forebrain and hippocampus (Nussbaum and Ellis, 2003). Moreover, reduced neurotransmitter levels have been suggested to cause the clinical symptoms of AD. The neurofibrillary tangle is mainly composed of tau which is a microtubule-associated protein. Abnormally hyperphosphorylated and glycosylated tau proteins aggregate and generate insoluble tangles inside neuronal cell, affecting synaptic function in many neurological diseases (Iqbal, et al, 2005). The primary component of neuritic plaque, also know as amyloid plaque, is beta-amyloid peptide (Aβ) with a length of 39-43 amino acids (Masters et al, 1985). Aβ is produced from sequential cleavage of amyloid precursor protein (APP) by β-secretase at the N-terminus of Aβ domain, and by γ-secretase at the intramembrane C-terminus (reviewed by Selkoe, 2001). APP can be alternatively cleaved by a non-amyloidogenic pathway, first by α-secretase within the Aβ domain, and then further by γ-secretase at the C-terminus. The pathogenic cleavage of APPs results in the liberation of Aβ peptides which form extracellular aggregates after secretion.
Though Aβ is produced constitutively under normal conditions, the peptide is degraded by the peptidases insulin-degrading enzyme and by endothelin-converting enzyme, and cleared from the brain in a process balanced by the efflux and the influx of Aβ across the blood-brain
barrier (Haass et al, 1992; Carson and Turner, 2002). The amyloid cascade hypothesis suggests that an imbalance between Aβ production and Aβ clearance is the central event leading to Aβ accumulation in brain, ultimately causing cell death and dementia (Hardy and Selkoe, 2002). Aβ oligomer appears to be more neurotoxic than fibrillar Aβ in nature (Walsh and Selkoe, 2007). The structure-toxicity correlation regarding oligomeric Aβ has been established, demonstrating that the rank order of nerotoxicity is: tetramer > trimer > dimmer >
monomer (Ono et al, 2009).
Epigenetics
Epigenetics refers to heritable changes in gene expression independent of alterations in DNA sequence (Holliday, 1987). Epigenetic mechanisms affect cellular function primarily through reversible changes in DNA methylation and remodelling of chromatin structure. DNA methyltrasferases (DNMTs) perform DNA methylation by transferring a methyl group from S-adenosyl methionine (SAM) to CpG units. Methylated CpG units, located in promoter regions, block the binding of transcription factors and recruit methyl-CpG-binding domain (MBD) proteins, giving rise to transcriptional repression. Repeating nucleosomes form eukaryotic chromatin. Each nucleosome is composed of two copies of four histones, H2A, H2B, H3 and H4, and DNA wrapping around the histones.
Histone modifications
The unstructured N-terminal tails of histones possess numerous residues which are susceptible to post-translational modification. To date, several types of histone modifications have been identified including acetylation, methylation of lysine residues and arginine redisues, phosphorylation, ubiquitination, sumoylation and ADP ribosylation (reviewed by Kouzarides, 2007). Modifications of histones are carried out by dynamically adding or removing chemical groups at specific sites of the histone tail. Transcriptional activation has been correlated with histone acetylation. Histone acetyltransferases (HATs) mediate acetylation of lysines, which can be reverted by histone deacetylases (HDACs) thereby inducing transcriptional repression. HDAC inhibitors were stated to improve synaptic deficits and cognition decline in various neurological diseases (Abel and Zukin, 2008). Histone methylation on lysines in promoter regions associates with transcriptional repression, but the same modification in a coding region actives transcription (Vakoc et al, 2005).
Aims
The research project focused on understanding the epigenetic changes in brain areas important for memory and learning, in particular how different forms and species of Aβ affect epigenetic mechanisms in neuronal cells. The aim was to understand the early effects of Aβ in the neuronal cells during the development of the disease.
Results
Thioflavin T assay
Thioflavin T (ThT) assays were performed to verify Aβ oligomer preparations, since Aβ oligomers are prone to aggregate to fibrils. In ThT assays, Aβ oligomers were diluted in the cell medium used in the viability assays. As the results show (Figure 1, A and B), under the experimental conditions, no increase of fluorescence was observed compared with the initial fluorescence. The ThT assays confirmed no fibrilization of oligomeric Aβ40 and Aβ42, with concentrations ranging from 1nM to1µM, occurred during 70 hours of incubation at 37°C
Figure 1. Aβ oligomers maintain stable oligomeric forms without fibrillization in conditions used in the viability assays. A) ThT assays of different concentrations of Aβ40. B) ThT assays of different concentrations of Aβ42. The ThT fluorescence intensity was divided by the initial intensity (F/F0).
Viability assay
The cell viability assays were carried out to investigate the toxicity of different species and forms of Aβ, and the viability was assessed by measuring MTT (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction. Human neuroblastoma SH-SY5Y cells were first treated with different species and forms of Aβ, and the MTT reduction in the cells was measured after incubation for 1 day, 3 days or 7days. The results (Figure 2A) indicate that SH-SY5Y cells treated with Aβ40 did not experience a significant decrease in viability. In contrast, Aβ42 obviously decreased the viability of cells, especially 100nM Aβ42 caused a 30% decrease in MTT reduction compared with the control. The neurotoxicity of Aβ42 observed was not dose-dependent, since lower MTT reduction levels were caused by
A B
Aβ42 with concentration higher than 100nM. The SH-SY5Y cells did not show any significant change of viability after exposure to Aβ for 1 day and 7 days (data not shown).
Mouse primary neuronal cells from the neocortex were treated with Aβ and incubated for 3 days before the viability assays. The primary cells retained viability after treatment with Aβ40 and oligomeric Aβ42 (Figure 2, B). A slight decrease of MTT reduction was observed in the cells exposed to 10nm or 1µM fibrillar Aβ42 (Figure 2, B).
0 5 0 1 0 0 1 5 0
- 9 - 8 - 7 - 6 - 5
Log C(M) Aβ
MTT reduction (% of control) Oligomeric Aβ40
Fibrillar Aβ40 Oligomeric Aβ42 Fibrillar Aβ42
0 50 100 150
-10 -9 -8 -7 -6
Log C(M) Aβ
MTT reduction (% of control) Oligomeric Aβ40
Fibrillar Aβ40 Oligomeric Aβ42 Fibrillar Aβ42
Figure 2. Viability assays of cells treated either with oligomeric and fibrillar Aβ40 and Aβ42.
A) MTT reduction in human neuroblastoma SH-SY5Y cells after 70 hours exposure to Aβ with concentrations ranging from 10-9M to 10-5M. B) MTT reduction in mouse primary neocortex cells exposed to Aβ with concentrations from 10-10M to 10-6M. The results are shown as mean ± SEM values from three independent experiments.
A
B
Western blot
Differential modifications on histones were investigated by western blotting with specific antibodies. SH-SY5Y cells were treated with oligomeric and fibrillar Aβ40 and Aβ42 for 70 hours before histone extraction. The software Image J (National Institutes of Health, US) was employed to quantify luminescence intensities of each band on films. The results (Figure 3, A and B) demonstrate that 1 nM fibrillar Aβ40 significantly induced H3 acetylation and phosphorylation and furthermore, that 10 nM oligomeric Aβ40 increased H3 acetylation and phosphorylation as well. In contrast, the two forms of Aβ42 did not lead to an increase of H3 acetylation or phosphorylation in the concentration range used. 10 nM Aβ40 oligomers and 100 nM fibrillar Aβ42 significantly induced dimethylation occurred on H3 while 1 nM fibrillar Aβ40 slightly increased H3 dimethylation.
0 500 1000 1500 2000 2500 3000
Acetylated H3/Total H3
Oligomeric Aβ40 Fibrillar Aβ40 Oligomeric Aβ42 Fibrillar Aβ42
Figure 3. The modifications on histone H3 from human neuroblastoma SH-SY5Y cells exposed to nanomolar and micromolar concentrations of Aβ. A) Acetylation on histone H3. B) Phosphoralation on histone H3. C) Dimethylation on histone H3. All results are expressed as modified histone H3/total histone H3 values.
0 1000 2000 3000 4000 5000 6000
Phosphoralated H3/Total H3
Oligomeric Aβ40 Fibrillar Aβ40 Oligomeric Aβ42 Fibrillar Aβ42
0 500 1000 1500 2000 2500 3000 3500
Dimethylated H3/Total H3
Oligomeric Aβ40 Fibrillar Aβ40 Oligomeric Aβ42 Fibrillar Aβ42
B
A
C
control 1 nM 10 nM 100 nM 1 µM
control 1 nM 10 nM 100 nM 1 µM control 1 nM 10 nM 100 nM 1 µM
Human post mortem brain tissues from 7 AD subjects and 3 control subjects were used to study the histone modifications. The results (Figure 4) suggest the AD subjects have higher levels of histone H3 acetylation and dimethylation plus lower phosphorylation on histone H3 compared with the healthy control subjects. Also, another independent experiment demonstrates the same comparison (data not shown).
Figure 4. The modifications on histone H3 in human post mortem brain tissues from the occipital cortex of 7 AD subjects and 3 healthy control subjects. The data is normalized as modified histone H3/total histone H3. The results are expressed as mean ± SEM values from 3 different samples (control) or 7 different samples (AD). *P<0.05.
0 0,5
1 1,5
2 2,5
3
Modified H3/Total H3
0 0,5 1 1,5 2 2,5 3
Modified H3/Total H3
0 0,5 1 1,5 2 2,5 3
Modified H3/Total H3
A) B) C)
*
Control AD
0 0,5
1 1,5
2 2,5
3
Modified H3/Total H3
0 0,5 1 1,5 2 2,5 3
Modified H3/Total H3
0 0,5 1 1,5 2 2,5 3
Modified H3/Total H3
A) B) C)
*
0 0,5
1 1,5
2 2,5
3
Modified H3/Total H3
0 0,5 1 1,5 2 2,5 3
Modified H3/Total H3
0 0,5 1 1,5 2 2,5 3
Modified H3/Total H3
A) B) C)
*
Control AD Control AD
Acetyl H3 Di-methyl H3 Phospho H3
Discussion
In this study, viability assays were utilized to investigate the neurotoxicity of different species and forms of Aβ. The Aβ preparation is verified by Thioflavin T assays that Aβ oligomers remained in the non-fibrillized state under the experimental conditions. The measurements of MTT reduction demonstrated that oligomeric and fibrillar forms of Aβ40 were not toxic to the neuroblastoma SH-SY5Y cells, whereas nanomolar concentrations of the oligomeric form of Aβ42 was more neurotoxic than the other Aβs tested. The results indicate that oigomeric form of Aβ42 may cause the most toxic effects in SH-SY5Y cells in vitro. It was reported that Aβ oligomers probably play an important role in synaptic impairment in AD (Walsh and Selkoe, 2007). However, Aβ42 oligomers did not show significant neurotoxicity to mouse primary neuronal cells in our experiments (Figure 2, B), but fibrillar Aβ42 slightly decreased the cell viability. It appears that both fibrillar and oligomeric Aβ42 are able to mediate neurotoxic effects.
Neuroblastoma SH-SY5Y cells were exposed to different species and forms of Aβ, in order to study how Aβ affects histone modification. The results suggest that nanomolar concentrations of fibrillar and oligomeric forms of Aβ40 contributed to the increase of acetylation at H3K14 and phosphorylation at H3S10. Fibrillar and oligomeric forms of Aβ42 did not induced H3 acetylation or phosphorylation. Moreover, nanomolar concentrations of Aβ40 oligomer and fibrilar Aβ42 greatly promoted dimethylation at H3K9. Human post mortem brain tissues were used to investigate differences in histone modification patterns between AD subjects and control subjects with the hope of better understanding how Aβ affects epigenetic modification in the brain in vivo. The brain tissues of AD subjects had developed a high level of acetylation at H3K14 and dimethylation at H3K9 but a lower level of phosphorylation at H3S10 relative to tissue from control subjects. These results might be of importance for understanding the epigenetic effects of Aβ in the brain area important for learning and memory.
To date, data regarding histone modifications in human brain tissue has been rarely reported. A case study indicated that increased trimethylation at H3K9 is associated with gene silencing in temporal cortex and hippocampus (Ryu et al, 2008). Histone acetylation is associated with activation of gene transcription. And histone methylation on lysines serves as a repressor or activator of transcription depending on where in the gene the modification occurs (Vakoc et al, 2005). In animal studies, the presence of Aβ in the prefrontal cortex of APPswe transgenic mice induced acetylation and phosphorylation on Histone H3 (Lithner et al, 2009). MAPK has been shown to regulate global histone acetylation and phosphorylation in the hippocampus (Chwang et al, 2006). Investigation of the mechanisms underlying Aβ effects on histone modification may provide insights into how Aβ affects cellular signal transduction in the central nervous system. Increased knowledge in this area will help us to understand the
plasticity of the brain as well as giving us an idea as to how to reverse disease progression and promote factors that stimulate regenerative processes.
Future work
mRNA of the neuronal cells would be extracted to investigate possible increased mRNA expression of the gene caused by certain forms of Aβ at low concentrations. The goal would be to utilize a micro-array (Chip) approach to investigate the increases in relevant mRNA species and thereby obtain specific candidate genes related to the different forms of Aβ.
Thereafter Chromatin Immunoprecipitation (ChIP) approaches would be applied to measure chromatin changes at specific loci of the genes of interest. Positive findings in these studies would reveal how different Aβ species and conformations affect cellular mechanisms in the central nervous system.
Materials and methods
Aβ preparation
All peptides of Aβ1-40 and Aβ1-42 were ordered from rPeptide Corporation. For preparation of fibrillar Aβ, peptides of Aβ1-40 and Aβ1-42 were dissolved in ddH2O and DMSO, respectively. The Aβ solutions were diluted in PBS and subsequently were shake for 72h in incubator at 37°C. To obtain homogenous soluble Aβ oligomers, HFIP treated Aβ1-40 and Aβ1-42 peptides were dissolved in DMSO, afterwards sonicated and filtered. The prepared Aβ solutions were divided into aliquots and were stored at -80°C.
Thioflavin T assay
The oligomeric Aβ aliquots were sonicated and diluted in the cell medium used in the viability assays, and thereafter were measured by thioflavin T fluorescence (ThT) assays.
Oligomeric Aβ40 and Aβ42 were added to RPMI-1640 medium and Neurobasal medium (Invitrogen) to final concentrations of 1nM, 10nM, 100nM and 1µM, respectively. Then thioflavin T and sodium azide were added to the cell medium to arrive concentrations of 1µM and 0.02%, respectively. Fluorescence intensity was measured for total 70 hours with intervals of 15 min at wavelength of 485nm at 37°C.
Cell culture and treatment Human neuroblastoma cell culture
Human neuroblastoma SH-SY5Y cells were cultured in RPMI-1640 medium (Invitrogen) supplemented with fetal bovine serum (10%) and antibiotic-antimycotic (1%). The cells were washed in phosphate buffered saline (PBS) and harvested and further plated on 96-well plates at a density of 5,000 cells/well. The cells were cultured for 3 days in a humidified incubator with 5% CO2 at 37°C before treatment with Aβ.
Mouse primary cell culture
The neocortices from 16 or 17days old mouse embryos were dissected, dissociated and plated on poly-D-lysine coated 96-well plates at a density of 20,000 cells/ml in Neurobasal medium (Invitrogen) supplemented with B27 (0.5%) and glutamate (1%). The cells were then maintained for 5 days in a humidified incubator with 5% CO2 at 37°C.
Aβ treatment
Oligomeric and fibrillar Aβ40 and Aβ42 were added to the different cell media used in the cell culture and viability assays. SH-SY5Y cells and primary mouse cortex cells were treated
with either oligomeric or fibrillar Aβ40 and Aβ42 with concentrations ranging from 10-9M to 10-5M.
Viability assay
After cell culture and treatment with Aβ, 10µl of Triton X-100 (10% dissolved in DPBS) was added into the wells as a blank control of 96-well plate. Thereafter, 20µl of One Solution Cell Proliferation Assay kit (Promega) was added into each well. The plates were incubated for 1 hour followed by optical density absorbance measurement at wavelength at 490nm.
Human brain tissue
Human post mortem brain tissues from the occipital cortex of 7 AD subjects and 3 control subjects were obtained from the Netherlands Brain Bank, Netherlands Institute for Brain Research. All brain tissues were used in compliance with ethical rules.
Histone extraction
All steps in this experimental operation were carried out on ice or in a centrifuge at 4°C.
Samples of treated cells or autopsy brain tissues were homogenized for 6 strokes using Dounce Homogenizer tubes. Homogenized samples were transferred into Eppendorf tubes and were centrifuged at 7,700g for 1 min. The supernatant was kept as a cytosolic fraction in tube on ice, while the pellet was resuspended in 0.5ml of 0.4N H2SO4 and was incubated for 30 min on ice. Samples were centrifuged for 10min at 14,000g. The pellet which contains the membrane fraction was stored on ice. And the supernatant was transferred to a new Eppendorf tube and 250µl of TCA with 4mg/ml deoxycholic acid was added into that tube. The proteins were immediately precipitated at that time point. The proteins were incubated for 30min followed by centrifugation for 30min at 14,000g. The supernatant was discarded and the tube was rinsed and incubated with 1ml cold acidified acetone (0.1% HCl in acetone) and 1ml cold acetone for 5 min in turn. The tube with the pellet was centrifuged for 5 min at 14,000g after each wash. The supernatant was discarded and the tube was allowed to air dry for 5min. In the end, the protein pellet was resuspended in 50µl 10mM Tris pH8.0 and stored at -80°C.
Protein assay
Ptotein concentrations were measured first by adding 25µl Solution A and 200µl Solution B (Bio-Rad Protein Assay kit) to 5µl sample and 5µl bovine serum albumin (BSA) with standard concentration ranging from 0 to 8 µg/µl. Then after the reaction for 15 min, optical density absorbance was measured at wavelength at 750nm. Protein concentrations were calculated based on the standard curve.
Western blot
Each sample was mixed with protein loading buffer (0.5M Tris-HCl pH6.8, 2% SDS, 10%
glycerol, 1.25% β-mercaptoethanol and 0.1% bromophenol blue). 2.5µg of histone from each sample was loaded on a pre-cast polyacrylamide gel (Bio-Rad) and electrophoresed for 2 hours at 100V in running buffer (0.25M glycine, 0.25M Tris and 3.5mM SDS). Proteins were transferred to polyvinylidene difluoride (PVDF) membranes using the Iblot Gel Transfer device (Invitrogen). After that, membranes were processed in MeOH for 15 seconds, dried using filter papers and then rehydrated in TBST (150mM NaCl, 20mM Tris-HCl pH7.4 and 0.1% Tween 20). Membranes were incubated with primary antibody overnight in at 4°C. The primary antibodies were prepared by dilution (1:1000) in 3% bovine serum albumin (BSA) in TBST. All of the primary antibodies were purchased from Millipore including anti-histone H3, anti-phospho-histone H3 (Ser 10), anti-acetyl-histone H3 (Lys 14) and anti-dimethyl-histone H3 (Lys 9). Thereafter membranes were washed in TBST for 5 min three times and incubated with secondary antibody for 1 hour at room temperature. The secondary antibody used in the experiments was goat anti-rabbit IgG conjugated with horseradish peroxidase (HRP) (Santa Cruz Biotechnology). After that, membranes were washed with TBST for 5 min and then incubated with ECL chemiluminescence kit (GE healthcare). Finally membranes were developed by CanoScan 5200F. The bands on membranes were stripped by washing in 0.2M NaOH for 20 min, and were subsequently blocked with 3% BSA in TBST for 45 min after washing. Then once again, membranes were incubated with new primary antibodies over night and this procedure was repeated as described above. Image J (National Institutes of Health, US) was used to quantify protein luminescence intensity of each band on membranes.
Acknowledgements
I would like to thank my supervisor Christina Unger Lithner, for giving me the opportunity to study epigenetics in Alzheimer’s disease. Thanks for her valuable discussions and practical helps. I would like to appreciate the journal club on every Tuesday morning for inspiring my mind. Thanks Professor Agneta Nordberg and all the other people in the division for creating the nice academic atmospheres.
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