Restoring mGluR-LTD Failure with the Induction of Arc Protein in

the CA1 Region of APP/PS1 Mice

Yansheng Zheng

1,†

and Lumeng Sun

2,†

Dalhousie University, Truro, Nova Scotia, B2N 5E3, Canada

Stephen Perse Foundation, Cambridge, CB2 1NA, U.K.

†

They are both first author

Keywords: Alzheimer’s Disease, mGluR-LTD, Arc Protein.

Abstract. Alzheimer’s Disease (AD) has become a severe problem across the world. Besides the major two hypotheses

(β-amyloid protein and tau protein), recent studies have also found that metabotropic glutamate receptor 5-

dependent LTD (mGluR-LTD) failure is also one significant symptom of AD in APP/PS1 mice (AD mice

model). Suppression of the inhibitor of de novo protein synthesis during the mGluR-LTD has successfully

reversed the mGluR-LTD failure. Arc protein is an essential protein that coordinates the mGluR, and the level

of Arc protein in the CA1 region of APP/PS1 mice is reduced. Therefore, an experiment is designed to increase

the Arc level in APP/PS1 exogenously and endogenously to restore the mGluR-LTD by measuring post-

synaptic responses.

1 INTRODUCTION

1.1 Background

Scientists have long been working to find a possible

explanation for how Alzheimer’s Disease (AD) is

developed and methods for curing or mitigating the

AD symptoms. There are several hypotheses about

how it originated. The first one is the Tau and

Amyloid-beta hypothesis. It asserts that tau, which

accounts for the neuron tangles, and amyloid-beta,

which clumps into plaques, act together to cause

synaptic failure, resulting in AD (Förstl, Kurz 1999).

The second one is about vascular problems, which

may lead to a malfunction of the blood barrier. This

failure inhibits glucose from reaching the brain as

well as preventing the clearing away of toxic beta-

amyloid and tau proteins (Förstl, Kurz 1999).

Scientists have also studied microglia cells, a form of

glial cells, as they fail to remove waste and beta-

amyloid plaques in AD (Förstl, Kurz 1999). The

initial symptoms of AD are sometimes mistaken as

aging or stress, with the most serious deficit being the

loss of short-term memory, along with other subtle

vestigial deficits in executive functions of

attractiveness, planning, flexibility, and abstract

thinking (Bäckman, Jones, Berger, Laukka, Small

2004, Waldemar, Dubois, Emre, Georges, McKeith,

Rossor, Scheltens, Tariska, Winblad 2007). With the

development of AD, the early stages of AD are

characterized by linguistic difficulties and impaired

executive functions, deteriorating perception

(agnosia), or degenerating execution of movements

(apraxia) other than memory impairments (Förstl,

Kurz 1999). Patients at the middle stages of AD show

degeneration in speech, reading and writing skills;

more importantly, the long-term memory starts to be

impaired and behavioral and neuropsychiatric change

become more prevalent (Förstl, Kurz 1999, Frank

1994). During the final stage of AD, patients

completely lose speech ability, show extreme apathy

and exhaustion, and eventually die of external factors

like infection of pressure ulcers or pneumonia (Förstl,

Kurz 1999, Frank 1994). The occurrence of AD

greatly influences the society, such as the life of

patients’ family and high financial cost (Abreu,

Forlenza, Barros, de. 2005). Moreover, during covid-

19, the number of AD patients has increased by 16

percent, and one-third of the over-85 population is

estimated to suffer from it by 2031((Bäckman, Jones,

Berger, Laukka, Small 2004). As no one has ever

been cured from AD, finding a therapy to avoid

becoming its victim is essential.

Beside β-amyloid hypothesis and tau protein

hypothesis being accepted as 2 major possible causes

Zheng, Y. and Sun, L.

Restoring mGluR-LTD Failure with the Induction of Arc Protein in the CA1 Region of APP/PS1 Mice.

DOI: 10.5220/0011294500003443

In Proceedings of the 4th International Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 753-759

ISBN: 978-989-758-595-1

753

of Alzheimer’s disease. Yang and co-workers found

that in APP/PS1 mice (AD mice model), mGluR-

LTD (Long-term Depression) failure occurs, and

assumed this phenomenon as a potential cause of AD

(Yang, Zhou, Zimmermann, Cavener, Klann, Ma

2016). Therefore, they tried to restore mGluR-LTD to

mitigate the progress of AD in APP/PS1 mice. PERK

is one of the four kinases of elF2 (eukaryotic initiative

factor 2), and it is triggered to phosphorylate eIF2 on

the α subunit which inhibits general synthesis of

protein and impairs memory if the reduction in

protein synthesis is persistent (Ma, Klann 2014,

Trinh, Klann 2013, Wek, Jiang, Anthony 2006, Wek,

Cavener, 2007). Yang and co-workers

pharmacologically and genetically repressed the

expression of PERK (Yang, Zhou, Zimmermann,

Cavener, Klann, Ma 2016), in order to restore the de

novo protein synthesis during the mGluR-LTD, and

they successfully reversed the failure of mGluR-LTD

in APP/PS1 mice. Another research by Yang and co-

workers demonstrated that via pharmacological and

genetic suppression of eEf2K (a kinase for elongation

factor 2) (Yang, Zhou, Ryazanov, Ma. 2021), the

hippocampal mGluR-LTD impairments in APP/PS1

mice had been alleviated.

1.2 Project Design

It has been known that mGluR-LTD failure occurred

in APP/PS1 mice (Yang, Zhou, Zimmermann,

Cavener, Klann, Ma 2016), and unregulated LTD is

associated with dementia and AD (Wilkerson, Julia,

Albanesi, Huber 2018). Based on this, it is assumed

that if mGluR-LTD failure in APP/PS1 mice is

reversed, the mice’s AD symptoms will be improved.

Both these 2 studies focus on the restoration of the

de novo protein synthesis, which is involved in

process of mGluR-LTD, to alleviate the impairments

of mGluR-LTD. Therefore, since Arc protein

synthesis is included in de novo protein synthesis

during mGluR-LTD, this project aims to investigate

whether a great amount of Arc expression will

reverse mGluR-LTD failure, in order to mitigate AD

symptoms.

2 EXPERIMENTAL APPROACH

Whether the induction of a great amount of Arc

protein will reverse mGluR-LTD impairments in the

APP/PS1 mice.

3 METHODS AND MATERIALS

3.1 Mice

All mice were kept in a barrier rearing system

committed to transgenic mice, which accords with the

standards and policies of the US Department of

Agriculture’s Animal Welfare Information Center

and the NIH Guide for Care and Use of Laboratory

Animals. A 12-hour-light/dark cycle is maintained in

the system, with a regular feeding and cage-cleaning

schedule. male and female mice are equally selected

for this research. APP/PS1 transgenic mice (APPswe

+PSEN1/Δ9) and wild type mice were purchased.

FMR1 hippocampus conditional knock-out (FMR1

cKO) were bred, and the method will be described

below. The genotype of all mice was verified using

PCR. Mice around 12-15 months old were used for

this experiment.

3.2 Hippocampal Slice Preparation and

Electrophysiology

The hippocampi were removed from the brains of

mice of 12-15 months, then vibratome was used to

make hippocampal slices at 400 μm(Hu, Serrano,

Oury, Klann 2006). Slices were kept at room

temperature and submerged in artificial CSF (ACSF),

which contains the following (in mM:125 NaCl, 2.5

KCl, 1.25 NaH2PO4, 25 NaHCO3, 25 D-glucose, 2

CaCl2, and 1 MgCl2), and incubated for at least 2 h

to be removed for electrophysiology.

For electrophysiology, slices were transferred to

recording chambers (preheated at 32°C) which were

superfused with oxygenated ACSF. Extracellular

field and whole-cell patch-clamp recording in CA1

stratum radiatum using pipettes are performed

(Schmeisser et al 2012). fEPSPs (field Excitatory

PostSynaptic Potentials) and fEPSP slopes are

recorded using the same pipettes. Long-term

potentiation will be induced by a single tetanus of 100

pulses at 100 Hz (Malenka, Bear 2004). For long-

term depression, CA1 is isolated by a microcut set at

the edge of CA2/CA3 before recording synaptic

response, and LTD is induced by 15 min paired-pulse

stimulation at 1 Hz with 50 ms between single pulses

in the presence of 1ml gabazine (Weiler et al 1997).

For NMDA/AMPA ratio, NMDA and AMPA are

separately recorded by compound EPSC (Excitatory

PostSynaptic Currents) of -60mv and +40mv, and ten

consecutive EPSCs for each holding potential will be

averaged.

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3.3 Drug Treatment

DHPG (Abcam, Cambridge, MA) will be prepared

within a week and diluted before the experiment.

3.4 Endogenous Application

The Cre-LoxP system will be used to breed

conditional knock-out mice suitable for endogenous

expression of Arc protein. The translation of Arc

protein is strongly related to a gene named FMR1.

FMR1 gene codes Fragile X Retardation Protein

(FMRP), which serves as a negative regulator of

translation and binds to the mRNA (Weiler et al

1997). As shown in Figure 1, The Arc protein

translation is induced by the mGluR activated by

DHPG (mGluR agonist). As the FMRP binding to the

mRNA. FMRP inhibits the process of de novo protein

synthesis, so that the amount of protein translated is

maintained at a normal level, as well as the intensity

of mGluR-LTD (Hou, et al 2006, Laggerbauer 2001).

Therefore, the FMR1 gene will be conditionally

knocked out in the CA1 region of APP/PS1 mice, so

that the Arc protein level in that region will increase,

and the mGluR-LTD is likely to be restored. shows

the mechanism of FMR1 inhibiting the translation of

Arc protein. The occurrence of mGluR-LTD is

closely related to the FMRP inhibition.

First, after finding a tissue specific promoter in

the CA1 region of APP/PS1 mice, and then a Cre-

recombinase gene will be introduced downstream

after the promoter (Ray, Fagan, Brunicardi 2000).

Following the treatment, the FMR1 gene in CA1

region of APP/PS1 will be genetically modified with

2 sets of LoxP sites. As shown in Figure 2, each set

of LoxP sites contains 2 loxP sites flanking the

FMR1, and one set of Loxp site will react with the

Cre-recombinase, so that the FMR1 gene can be

inverted and shut down. Afterwards, those 2 types of

genetically modified mice (APP/PS1 Cre mice and

APP/PS1 lox P) will mate with each other. After

mating, their offspring will be APP/PS1/FMR1_

(CA1 region conditional knock-out) mice that can be

used for further treatments.

Figure 1: Mechanism of how FMR1 regulates the interaction between Arc mRNA and mGluR-LTD.

Restoring mGluR-LTD Failure with the Induction of Arc Protein in the CA1 Region of APP/PS1 Mice

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Figure 2: A sketch of how FMR1 conditional knock-out in CA1 region of APP/PS1 mice.

3.5 Exogenous Application

3.5.1 Peptide

A type of peptide called the Arg9 peptide will be

used, which is the most efficient peptide (Ray, Fagan,

Brunicardi 2000). The peptide gets into the cell in a

non-endocytosis transmembrane process. This

eliminates the need of entering the cell by

endocytosis of proteins, which will send the

endosome to the lysosome for degradation. However,

if it’s a non-endocytosis transmembrane process, it

means that the peptide will first attach to the cell

membrane and make a hole on the membrane. The

hole allows the peptide to go into the cell. However,

scientists are still investigating the detailed process of

how peptide gets into the cell as the peptide can repair

the hole so swiftly that scientists can’t observe it

(Jiawan, 2015).

3.5.2 Create Fusion Protein

The Arg9 peptide and arc protein will be fused

together to get the Arg9-Arc protein gene through

genetic engineering.

3.5.3 Construct Prokaryotic Expression

Plasmid

After the fusion protein gene is obtained, it will be

loaded into the plasmid for better transfecting cells.

This recombinant plasmid includes both the target

gene and the plasmid vector.

To get the target gene, the primers are designed

first. The primers will allow DNA to be inserted into

the plasmid. The design can be done by using Primer

Blast or Primer Premier 5.0. The 5 primer’s end will

contain the his-tag, which allows for easy purification

and detection of the recombinant protein. The 3

primer’s end will contain the green fluorescence

protein, which is useful for identifying the protein

localization and analyzing the gene expression. Then

the primers will clone the Arg9-Arc fragment, which

is the target gene fragment wanted. Restriction

enzyme digestion is performed to get this clonal

fragment and the cohesive ends of the plasmid vector.

The recombinant plasmid is then obtained via

enzyme-linking of the fragment and the plasmid

vector. This prokaryotic expression plasmid will then

be sent to the sequence analysis company to confirm

that it is constructed successfully.

3.5.4 Transfection of the Plasmid

A. Expression of the recombinant fusion protein

After DNA is loaded into the plasmid, it will be

transfected into Escherichia coli (E. coli). In E. coli,

the recombinant fusion protein will be expressed. As

adding the inducer will increase the amount of

expression, an inducer will be added when the

bacterial growth reaches the exponential phase of

growth. Herein, the amount of the inducer is being

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added and the time it’s going to induce will need to

be taken into consideration. To optimize the

expression conditions, tests will be set up to

investigate under which conditions the protein will

express the most.

Investigation of time for inducing

(1) Take bacterial fluid which has been induced 0

hour (h), 1h, 2h, 3h, 6h, 8h respectively.

(2) Extract lysates and run SDS-PAGE gel.

(3) Stain the protein gel and elute protein.

(4) Observe the protein band that is at the

expected molecular mass of Arg9-Arc fusion protein.

(5) With time of inducing increases, the

concentration of the target band increases, which

means the brightness of the band increases. Observe

after which hour, the brightness stops increasing. This

means that with this hour’s time of inducing, protein

expression amount is maximized.

B. Purification of the recombinant fusion protein

Then the recombinant fusion protein will be purified

by using the His-tag Ni purification system. This

system can result in the protein of high concentration.

The SDS-PAGE gel electrophoresis is the used to

assess the protein purity. If the target protein band at

the expected molecular mass of Arg9-Arc is distinct,

it means that the purity of protein is high, and the

protein is prepared to be sent into the cells.

4 GROUP SET-UP

1. 10 APP/PS1 mice + DHPG vs. 10 Wild type mice

+ DHPG (LTP, LTD and NMDA/AMPA

measurements)

2. 10 APP/PS1 mice + exogenous Arc protein in

dendrites + DHPG (in CA1 Region) vs. 1(LTP, LTD

and NMDA/AMPA measurements)

3. 10 APP/PS1 FMR1 cKO mice + DHPG (in

CA1 region) vs. 1 (LTP, LTD and NMDA/AMPA

measurements)

5 DATA ANALYSIS

Data are presented as mean ± SEM. For comparison

of the 2 groups, if the data are normally distributed,

then a 2-tailed student t-test was used, otherwise, a

Mann-Whitney test was used. For comparison

between multiple groups, if the data are normally

distributed, then an ANOVA was used, followed by

individual post hoc tests when applicable, otherwise,

a Kruskal-Wallis test was used.

6 DISCUSSIONS

Figure 3: Expected result from electrophysiology showing

LTP and LTD (Leff 2002).

If the results are in accord with the hypothesis, then

the LTP and LTD is likely to be observed in the

recorded diagram similar to Figure 3 below:

For AMPA ratio, a significant reduction of

AMPARs on the synaptic surface will be detected.

Based on these two phenomena, it can be concluded

that the induction of Arc protein reverses the mGluR-

LTD failure, and future study can be performed to

investigate whether the AD symptoms have been

mitigated with the induction of Arc protein.

7 CONCLUSIONS

In the experiment, the mGluR-LTD invalidation in

APP/PS1 mice is reversed through inducing Arc

protein through two approaches and observe whether

the cognitive deficits in the mice have been mitigated.

If the cognitive deficits are improved when mGluR-

LTD failure is reversed, it means that the part of

cognitive abilities is restored.

Meanwhile, if a significant reversal of mGluR-

LTD invalidation in APP/PS1 mice can be observed,

then the inducement of Arc protein can be a potential

Restoring mGluR-LTD Failure with the Induction of Arc Protein in the CA1 Region of APP/PS1 Mice

757

cure for AD. Although, future investigation of Arc

protein inducing mGluR-LTD failure is still required

to ensure the multi-relationship among Arc protein,

mGluR-LTD and the exact does of Ar protein that

needs to maximize the efficiency.

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