001 Case Study - Submission Point_b9014243 Dylan Atkinson.docx

Dylan Atkinson Applied Biochemistry Coursework
1
A Review of ‘Inhibition of EHMT1/2 rescues synaptic
and cognitive functions for Alzheimer’s disease’ A
paper by Zheng et al, 2019
Content
Page 1: Title Page
Page 2: Background Information
Page 3: Confirming Raised H3K9me2
Page 6: Inhibition of EHMT1/2
Page 7: Loss of Glutamate Receptors Recovered by BIX
Page 8: EHMT1/2 Inhibition with shRNA
Page 9: H3K9me2 in the Hippocampus
Page 10: Genome wide effects of EHMT1/2 inhibition
Page 11: Future Work
Page 12: References

2
Background Information
Dementia is a devastating neurodegenerative condition symptomized by memory problems,
difficulty concentrating and confusion. Affecting 900,000 people (Wittenberg et al, 2019) and
costing over £10 billion a year in social care in the UK alone. Alzheimer’s disease (AD) is the
most common cause of Dementia, first affecting the hippocampus causing learning, memory
and reasoning deficiencies, Alzheimer’s can progress to severe symptoms such as
hallucinations, delusions and problems with moving and self-care tasks (NHS,2021). This
disease has a huge effect not only on the patient, but also their families, carers and social care
systems. AD is caused by the build-up of proteins in neurons, mainly amyloids, and a decrease
in neurotransmitters produced by the cell, eventually the death of neurons occurs followed
by shrinkage of the area of the brain affected. Even with severe effects caused by this disease
it is not known what causes this process to start and develop, the disease can be familial,
showing a link to genetics in some patients. However, roughly 82% of cases have no known
definitive cause and appear random (Bird, 2018) suggesting a complex range of pathogenesis
and risk factors, both genetic and environmental. In addition to this, only symptomatic
treatments have been developed, such as acetylcholinesterase inhibitors, which work to
extend the action of the neurotransmitter acetylcholine by inhibiting the enzyme
acetylcholinesterase from breaking the neurotransmitter down, treating memory and
cognitive symptoms. Other therapies work to improve the quality of life of the patient such
as antidepressants. (NHS, 2021)
The paper cites epigenetics as a
pathogenesis of AD, looking
specifically at methylation of
histones to enhance/decrease
transcription of genes and if this
affects AD progression in humans.
Histone methylation is a
modification of certain amino
acids in a histone protein by
addition of up to 3 methyl groups,
methylation changes the position
of the DNA wound around the
histone by loosening or tightening
the tails, allowing transcription
factors to access DNA or restricting
access respectively (Whetstine,
2010), figure 1 shows a diagram to
indicate the principles of histone
methylation. A link between
epigenetics and development of
AD has yet to be fully proven, with most studies using mouse models to gain information,
however human samples would have to be studied if treatment is developed. (Cao et al, 2022)

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The paper by Zheng et al (2019) focus is on H3K9me2 and methyltransferases EHMT1/2.
EHMT1 is known to cause demethylation of H3K9me2 reducing transcription of DNA,
deficiency of transcription is known to be involved in other cognitive diseases such as
Kleefstra disease (Benevento et al, 2016) and blocking this enzyme found an increase in the
transcription of the genes increasing synaptic scaling. Zheng et al hypothesises that EHMT1/2
elevation and the subsequent dimethylation and reduction in DNA transcription is an
important part of the development of Alzheimer’s and inhibiting these enzymes can help in
the treatment of Alzheimer’s by increasing the production of proteins, specifically glutamate
receptors, AMPA and NMDA. These two ionotropic glutamate receptors (GluR) are both
imperative for fast synaptic transmission between neurons. (Purves et al, 2001) Reduction of
AMPA has been linked to the cognitive impairment seen in Alzheimer’s (O’Connor et al, 2020,
Zhang et al, 2018) and hypofunction of NMDAR has been linked to cell death. (Wang and
Reddy, 2017)
As with many other papers studying AD, Zhang et al uses mouse models for most of the
research, these models do not fully replicate the human disease but can provide significant
contributions to the understanding of theory. (Elder, 2010) Mouse models provide a
particular problem when analysing AD because the long development time of AD cannot be
replicated in mice, this makes it especially hard to look at the early stages of development,
where treatment can be best.
Confirming Raised H3K9me2
Zheng at al first identified and tested the hypothesis that there is an increased H3K9me2 in
AD, to do this Western Blots, quantitative PCR and immunohistochemistry were all used.
Western blotting techniques analysed the abundance of proteins that where targeted with
antibodies, using a horseradish peroxide secondary antibody followed by enhanced
chemiluminescence substrate to provide a detection signal. The signal is semi-quantitative,
allowing good comparison of total protein levels. (Mahmood and yang, 2012) This method
can be used to show data in a clear manner to compare how much protein is being produced,
making it very useful in a paper such as this, however, due to the layout of the figures Zheng
et al have made their results more complicated and obscure than they need to be.
Real time PCR is the gold standard in measuring target mRNA production by a cell with correct
primers it is extremely precise, accurate, and highly sensitive, which is needed for a study like
this with limited tissue samples (Bustin, 2000). Immunohistochemistry (IHC) is used to identify
H3K9me2 location in the nucleus of PFC neuron slices. Immunohistochemistry is also a gold
standard in detection of protein location and when employing a two-antibody system,
meaning one antibody targets the antigen of interest, with excess washed away removing
nonspecific binding, before a secondary antigen bound with a dye binds to the primary
antibody and produces a detection signal, meaning the analysis is highly specific of the target
antigen. Other methods could be used, such as immunofluorescence, however, will see little
difference in results as the main change is staining methods.

4
Figure 2: Taken from Zheng et al (2019) discussing H3K9me2 and EHMT1/2 levels in mice and
human neurons.

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Figure 2 from Zheng et al indicates the complexity of which the figures have been built, with
7 subsections making this figure harder to follow than needed. Results collected from human
tissues could have been included as a separate figure to this one. Figure 2: A shows an
increase of H3K9me2 in FAD mice compared to wildtype (WT) di-methylation of H3K9, proving
there is an increase in H2K9me2 in mice with AD, while tri-methylation stays the same. B and
C work to prove that the raise in methylation seen in the Alzheimer’s model is caused by an
increase in enzymes EHMT1/2, in the PCR in 2:C and western blot shows an increase in both
enzymes in FAD mice, while histone 3 level stays the same, 1:B shows increase in transcription
of the coding regions for EHMT1/2, so increase in concentration levels of these enzymes is
caused by a greater production of the enzymes. The proof that there is an increase of these
enzymes provides a potential therapeutic pathway. 2:D shows immunohistochemistry, the
location of H2K9me2 in PFC neurons was compared to the enzyme CaMKII, an abundant
enzyme in the brain used in the regulation of glutamatergic synapses (Lisman et al, 2002).
CaMKII allows the immunohistochemistry to show the outline of neurons, and where GluR
are located. This proved that there was an increase in H2K9me2 inside the nucleus of PFC
neurons. However, there also appeared to be a significant decrease in CaMKII enzymes in the
enlarged FAD image compared to WT. This could be an artifact of the method, if this slide was
examined later as the fluorophore will fade or maybe caused by AD in the FAD model. 2: E is
a quantification of the increase in H3K9me2 in neurons of mice PFC with no change in CaMKII,
however it does not state which figure magnification was used to quantify this. All these
figures go to prove an increase in EHMT1/2 and an increase in H2K9me2 seen in FAD mice.
Figure 2: F/G are repeats of 2: B and A respectively, using human post-mortem PFC, with
EHMT1 mRNA considerably higher in AD compared to the control, this isn’t seen in mice to
the same level, meaning in humans there may be a difference between EHMT1 raised levels
in AD compared to FAD mice. EHMT2 mRNA was also raised in AD but not to the same levels
seen in FAD mice, with 4 patients having lower EHMT2 in AD than the control mean, however
even with this difference H3K9me2 is raised in AD showing potential therapeutic targets.
Immunohistochemistry could have been performed on human PFC tissue instead of mice PFC,
which would have given more data to indicate whether there is an increase in H3K9me2 in
human AD.
Next Zheng et al (2019) sought
to find out whether histone
modification is age related.
Figure 3 A: B show that histone
methylation and EHMT1/2
levels do not increase in young
FAD mice. This may impact the
usefulness of treatment in the
future, Zheng et al had this data
stored in supplementary data
whereas this should have been
included in the main paper.
Figure 3: Taken from Zheng et al 9 (2019), A, showing western blots
and quantitative analysis of H3K9me2 levels in PFC of young FAD
mice, B showing analysis PCR analysis of mRNA levels of EHMT1/2
in young FAD mice

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Inhibition of EHMT1/2
As EHMT1/2 appears raised in AD this provides a potential therapeutic pathway for AD using
the BIX01294 EHMT1/2 inhibitor, western blotting (figure 4: A) showed a decreased mean
H3K9me2 levels in FAD Bix treated compared to FAD control, closer to levels seen in WT.
However, 1 sample was above the mean of saline treated FAD meaning treatment may not
work every time depending on other factors. Additionally, there was 5 WT samples in the
WT+Sal, 4 in WT+BIX, 11 in FAD+SAL, 10 in FAD+BIX, questions have to be asked why the same
number was not tested for each, or if some of the data has been removed.
Figure 4: Taken from Zheng et al (2019) measuring histone methylation of glutamate
receptors gene premotor region in FAD mice and FAD mice treated with EHMT1/2 inhibitor
B.

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Immunohistochemistry (4:B) shows a reduction of H3K9me2 in the nucleus when using BIX
in both WT and FAD. To measure the transcription of glutamate receptors GluA2 and NR2B
Zheng et al used ChIP and PCR to measurements gene transcription effected by methylation
of H3K9me2. ChIP in figures 4:C/E show H3K9me2 detectable 1200 bases upstream of GluR
genes, with greater enrichment seen in FAD mice which is the cause of reduced GluR, (4:D/F)
which was reduced in BIX treated mice. There was no effects on other upstream genes shown
in 4:G-F.
Loss of Glutamate Receptors Recovered by BIX
Figure 5: Measurements of glutamate receptors transcription and expression using PCR and
western blotting techniques.
Excess methylation seen in the previous figure leads to downregulation of the genes coding
for GluR, western blot shown in 5:B shows reduction of GluR protein subunits, with a
reduction ~30% for each of the 4 subunits, other proteins showed little change, however in
some FAD models there was no, or very little, change in GluA2 or NR1 subunits, which could
affect treatment in the future. Figure 5:A shows reduction of mRNA of glutamate receptor
proteins, showing that reduction in the proteins is caused by reduction in transcription rather
than an alternative cause. Reduction in these receptors is linked to AD development and

8
symptoms. 5:C adds BIX treatment with PCR of WT, 5:D adding protein levels to show BIX
increases both mRNA production and protein levels of the GluR proteins, however the
number of samples differed between the two tests, and between controls, PCR only has 5
treated FAD samples, but western blot shows 8; this questions again has data been left out to
make the study appear favourable to the hypothesis?
EHMT1/2 Inhibition with shRNA
To ensure the inhibition of EHMT1/2 Zheng et al chose to use targeted shRNA knockdowns of
EHMT1/2. Lentiviruses are used as they infect and transduce non-dividing cells e.g., neurons
(Wollebo et al, 2013), the vector pLKO.3G contained an eGFR marker for identification of
transduction success. Other papers have used BIX01294 as a specific inhibitor of EHMT1/2(Lin
et al, 2019, and Kubicek et al, 2007) which could have aided in the simplicity of the laboratory
work; however, shRNA provides more accurate and specific knockdowns. Figure 6 shows
shRNA inhibition causes knockdown of EHMT1/2 with decreased levels of H3K9me2 and
recovery of AMPA and NMDA receptors, meaning that inhibition of these enzymes provides
a therapeutic target for AD.
Figure 6: A: Effects of shRNA on EHMT1/2 from PFC slices with scrambled shRNA as control B:
quantified values taken from A.

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H3K9me2 in the Hippocampus
Another area of the brain severely affected by Alzheimer’s is the hippocampus (Babcock et a,
2021) therefor Zheng et al examined whether H3K9me2 is increased in these cells, and if this
is involved in the development of Alzheimer’s disease in the hippocampus.
Figure 7: A: Levels of H3K9me2 in hippocampus cells with B showing immunohistochemistry
of H3K9me2 and CamKII in hippocampus cells, C the quantification of B. D shows PCR data of
mRNA for glutamate receptors with treatment with BIX in FAD hippocampus.
There is little increase in methylation seen in the hippocampus, with a 0.22x mean increase
seen in immunoblotting, suggesting that BIX treatment may not work in the hippocampus,
however 7:C shows an increase in methylation in the nucleus of the neurons in line with the
increase seen in PFC. There are 3 samples in the immunohistochemistry that do not show any
increase of methylation in the hippocampus which could cause the results in B. Decreased
mRNA levels of GluR proteins are seen in FAD mice, BIX caused a decrease in mRNA of GluA2
subunit of AMPA in WT which could cause problems if treatment is given without analysis,
however FAD BIX treatment caused an increase in all GluR so there are future development
pathways for treatment.

10
Genome wide effects of EHMT1/2 inhibition
To see if other genes are affected by the increased methylation seen in FAD mice, and to see
the effects of BIX on these mice ChIP-seq (figure 8: A) showed greater occupancy of start sites
by H3K9me2 in FAD mice compared to WT and BIX treated. 8: B shows a Venn diagram of
genes with increased methylation in FAD, FAD and BIX decrease, this figure is not clear in what
it represents, with the intersection being increase in H3K9me2 in FAD which was then reduced
with treatment. This shows the increase in H3K9me2 affects multiple transcription sites of
multiple genes, reversed with BIX, this means other interactions if BIX need to be studied to
see effects on other genes. ChIP-seq data was used to identify methylation effects on
transcription of GluR genes (7:D), ChiP-seq high resolution allows identification of how
H3K9me2 interacts with the genes coding for GluA2, NR2B and NR2A, especially as binding is
close to the start sites.
Figure 7: genome wide analysis of H3K9me2 genes using ChIP analysis, taken from Zheng et
al.

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Future Work
Zheng et al (2019) found elevated repressive histone methylation H3K9me2 in PFC and
hippocampus in mice models which leads to a reduction of Glutamate receptor proteins
therefor a reduction in synaptic transmission efficiency. Inhibition of methyl transferases
EHMT1/2 leads to recovery of GluR and cognitive damage in FAD mice models. Wang et al
(2021) completed a similar study using Tau mice and EHMT2 inhibitor UNC0642 and found
similar results. However, Zheng et al found that there was little increase of H3K9me2 in early
stages of FAD Alzheimer’s development, which is the ideal target for medication to delay or
stop the development of AD. Grinan-Ferre et al (2019) found treatment of early onset FAD
mice with UNC0642 decreased excess methylation seen in his FAD models, closer to that of
WT suggesting that EHMT2 is a potential target for therapeutic treatment in AD.
This sets out many potential routes for future research and drug development. The most
obvious of which is if BIX01294 can repeat results of this experiment in human PCF and
hippocampus, first brain samples would have to be collected from deceased Alzheimer
patients to see if there are the same increase in EHMT1/2 and H3K9me2 seen in mice models.
Taking brain matter from Alzheimer patients is ethically ambiguous, as they may not have the
mental capacity to understand what will happen to them after they die and give informed
consent. If there is an increase in these enzymes a BIX inhibitor-based medication could be
developed, as BIX is dangerous for human consumption at the moment (ThermoFisher, 2021)
with it being toxic if ingested, which would be a favoured treatment route for AD. Off target
effects of BIX would also have to be studied, we know there are many other effects that BIX
inhibitor can have on the human body, such as BIX causing apoptosis of human bladder cancer
cells (Cui et al, 2015). If this drug is developed through preclinical trails, it would then have to
go through clinical research stages, this also brings up ethical problems, can an Alzheimer’s
patients themselves accept a new potentially risky treatment, and also what happens with
the control groups. These groups would likely be left on the best current treatment.
Alternatively, research can focus on what causes the increase in H3K9me2, such as Calderon-
Garciduenas et al (2020) who examined increase in methylation caused by increase in air
pollution. Other research can be done into the effects of the other genes affected by excess
methylation of H3K9me2 such as SHANK2 which could provide other treatment pathways for
Alzheimer’s disease.

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Reference:
1. Babcock, K. R., Page, J. S., Fallon, J. R., & Webb, A. E. (2021). Adult Hippocampal
Neurogenesis in Aging and Alzheimer's Disease. Stem cell reports, 16(4), 681–693.
https://doi.org/10.1016/j.stemcr.2021.01.019
2. Benevento, M., Iacono, G., Selten, M., Ba, W., Oudakker, A., Frega, M., Keller, J.,
Mancini, R., Lewerissa, E., Kleefstra, T., Stunnenberg, H. G., Zhou, H., van Bokhoven,
H., & Nadif Kasri, N. (2016). Histone Methylation by the Kleefstra Syndrome Protein
EHMT1 Mediates Homeostatic Synaptic Scaling. Neuron, 91(2), 341–355.
https://doi.org/10.1016/j.neuron.2016.06.003
3. Bird, T. D. (2018). Alzheimer disease overview. GeneReviews®.
https://www.mdpi.com/2413-4155/3/1/16
4. Bustin S. A. (2000). Absolute quantification of mRNA using real-time reverse
transcription polymerase chain reaction assays. Journal of molecular
endocrinology, 25(2), 169–193. https://doi.org/10.1677/jme.0.0250169
5. Calderón-Garcidueñas, L., Herrera-Soto, A., Jury, N., Maher, B. A., González-Maciel,
A., Reynoso-Robles, R., Ruiz-Rudolph, P., van Zundert, B., & Varela-Nallar, L. (2020).
Reduced repressive epigenetic marks, increased DNA damage and Alzheimer's
disease hallmarks in the brain of humans and mice exposed to particulate urban air
pollution. Environmental research, 183, 109226.
https://doi.org/10.1016/j.envres.2020.109226
6. Cao, Q., Wang, W., & Yan, Z. (2022). Epigenetics-based treatment strategies for
Alzheimer's disease. Aging, 14(10), 4193–4194.
https://doi.org/10.18632/aging.204096
7. Cui, J., Sun, W., Hao, X., Wei, M., Su, X., Zhang, Y., Su, L., & Liu, X. (2015). EHMT2
inhibitor BIX-01294 induces apoptosis through PMAIP1-USP9X-MCL1 axis in human
bladder cancer cells. Cancer cell international, 15(1), 4.
https://doi.org/10.1186/s12935-014-0149-x
8. Elder, G.A., Gama Sosa, M.A. and De Gasperi, R. (2010), Transgenic Mouse Models of
Alzheimer's Disease. Mt Sinai J Med, 77: 69-81. https://doi.org/10.1002/msj.20159
9. Griñán-Ferré, C., Marsal-García, L., Bellver-Sanchis, A., Kondengaden, S. M., Turga, R.
C., Vázquez, S., & Pallàs, M. (2019). Pharmacological inhibition of G9a/GLP restores
cognition and reduces oxidative stress, neuroinflammation and β-Amyloid plaques in
an early-onset Alzheimer's disease mouse model. Aging, 11(23), 11591–11608.
https://doi.org/10.18632/aging.102558
10. Lin, L., Liu, A., Li, H., Feng, J., & Yan, Z. (2019). Inhibition of Histone
Methyltransferases EHMT1/2 Reverses Amyloid-β-Induced Loss of AMPAR Currents
in Human Stem Cell-Derived Cortical Neurons. Journal of Alzheimer's disease :
JAD, 70(4), 1175–1185. https://doi.org/10.3233/JAD-190190
11. Lisman, J., Schulman, H., & Cline, H. (2002). The molecular basis of CaMKII function in
synaptic and behavioural memory. Nature reviews. Neuroscience, 3(3), 175–190.
https://doi.org/10.1038/nrn753
12. Mahmood, T., & Yang, P. C. (2012). Western blot: technique, theory, and trouble
shooting. North American journal of medical sciences, 4(9), 429–434.
https://doi.org/10.4103/1947-2714.100998
13. National Institutes of Health (2018) A scientific illustration of how epigenetics
mechanisms can affect health. https://commonfund.nih.gov/epigenomics/figure

13
14. NHS (2021) Overview Alzheimer’s Disease.
https://www.nhs.uk/conditions/alzheimers-disease/
15. O'Connor, M., Shentu, Y. P., Wang, G., Hu, W. T., Xu, Z. D., Wang, X. C., Liu, R., &
Man, H. Y. (2020). Acetylation of AMPA Receptors Regulates Receptor Trafficking
and Rescues Memory Deficits in Alzheimer's Disease. iScience, 23(9), 101465.
Advance online publication. https://doi.org/10.1016/j.isci.2020.101465
16. Purves D, Augustine GJ, Fitzpatrick D, et al. (2001), editors. Neuroscience. 2nd
edition. Sunderland (MA): Sinauer Associates. Glutamate Receptors.
https://www.ncbi.nlm.nih.gov/books/NBK10802/
17. ThermoFisher Scientific (2021) Safety data sheet.
https://www.fishersci.com/store/msds?partNumber=AC467831000&productDescrip
tion=BIX-01294+100MG&vendorId=VN00032119&countryCode=US&language=en
18. Wang, R., & Reddy, P. H. (2017). Role of Glutamate and NMDA Receptors in
Alzheimer's Disease. Journal of Alzheimer's disease : JAD, 57(4), 1041–1048.
https://doi.org/10.3233/JAD-160763
19. Wang, W., Cao, Q., Tan, T., Yang, F., Williams, J. B., & Yan, Z. (2021). Epigenetic
treatment of behavioral and physiological deficits in a tauopathy mouse
model. Aging cell, 20(10), e13456. https://doi.org/10.1111/acel.13456
20. Whetstine J.R (2010) Histone methylation: Chemically Inert but Chromatin Dynamic
https://reader.elsevier.com/reader/sd/pii/B9780123741455002874?token=287D50E
C1CC6A108158DC8158233939FD9D03DAB6C2CA3EA3EB842DF4F17CAB2E1330F8BB
1ED56B32DBA011D37D62BA1&originRegion=eu-west-
1&originCreation=20221114165330
21. Wittenberg, R., Hu, B., Barraza-Araiza, L., & Rehill, A. (2019). Projections of older
people with dementia and costs of dementia care in the United Kingdom, 2019–
2040. London: London School of Economics.
https://www.yhscn.nhs.uk/media/PDFs/mhdn/Dementia/Bulletin/2019/December%
202019/cpec_report_november_2019.pdf
22. Wollebo, H. S., Woldemichaele, B., & White, M. K. (2013). Lentiviral transduction of
neuronal cells. Methods in molecular biology (Clifton, N.J.), 1078, 141–146.
https://doi.org/10.1007/978-1-62703-640-5_12
23. Zhang, Y., Guo, O., Huo, Y., Wang, G., & Man, H. Y. (2018). Amyloid-β Induces AMPA
Receptor Ubiquitination and Degradation in Primary Neurons and Human Brains of
Alzheimer's Disease. Journal of Alzheimer's disease : JAD, 62(4), 1789–1801.
https://doi.org/10.3233/JAD-170879
24. Zheng, Y., Liu, A., Wang, Z. J., Cao, Q., Wang, W., Lin, L., Ma, K., Zhang, F., Wei, J.,
Matas, E., Cheng, J., Chen, G. J., Wang, X., & Yan, Z. (2019). Inhibition of EHMT1/2
rescues synaptic and cognitive functions for Alzheimer's disease. Brain : a journal of
neurology, 142(3), 787–807. https://doi.org/10.1093/brain/awy354
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