Development of miRNA Types and Exosomes into Protective Agents against Huntington’s Disease

Name: Yejin Kong

School: Seoul Foreign School

City, Country: Seoul, South Korea

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Abstract
Huntington’s disease (HD) kills approximately 2.27 per million per population every year and slowly
makes everyday activities more difficult for those with the genetic mutation. Key symptoms of HD
include chorea, forgetfulness, impaired judgement, personality change, mood swings, and depression. HD
is clinically diagnosed by the HD mutation in the huntingtin gene: overly repeated C-A-G nucleotide
sequences. Abnormal sequencing in the gene causes abnormal protein synthesis in the huntingtin protein,
the impact of which compounds over time as the proteins create more clumps in the brain, which is why
HD is mostly diagnosed in middle-aged individuals - when the proteins have had enough time to cause
symptoms. HD is caused by prolonged damage to the striatum caused by the abnormal HD gene and
protein which makes brain cells eventually die. The link between miRNAs and HD have been studied
before but this study takes a step further by examining how this link can be exploited to create a cure.
Using three types of strategically selected miRNA types, this study showed that encapsulating the
miRNAs in exosomes for delivery into heavily damaged neurons increased cell proliferation. Mimicking
conditions of HD through the use of 6-hydroxydopamine (6-OHDA), this lab tested the impacts of the
three different miRNA types when delivered by exosomes into the damaged neuron cells and proved that
all three miRNAs did increase cell proliferation. Overall, this research focuses on the potential of miRNA
with exosome use in therapeutic treatment for blocking neuronal cell death of HD patients. As current
techniques evolve, this research can be used for the development of personalized therapy for HD patients.

Keywords: Molecular Biology, miRNA, Huntington Disease, 6-OHDA, Cell Death

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Table of Contents

Question 3

Hypothesis 3

1. Background Information 4
1.1 Exosomes 4
1.2 Relationship between miRNAs and Exosomes 4
1.3 Huntington’s Disease 4
1.4 A172 Glioblastoma Cell 5
1.5 Dopamine and Huntington’s Disease Relationship 5
1.6 Purpose and Hypothesis of this Experiment 6

2. Purpose and Significance of the Study 6

3. Materials List & Experimental Procedures 7

3.1 A172 Glioblastoma Cell 7
3.2 Retinoic Acid 7
3.3 6-OHDA (6-hydroxydopamine) 7
3.4 Isolated Exosomes and miRNA Insertion 7

4. Results Analysis and Discussions 9

Figure 1. Microscopic images of A172 Glioblastoma cells treated by varying levels of
retinoic acid (RA) for 24 hours. 9
Figure 2. Graphical representation of the IC50 level of the A172 Glioblastoma cell, a
quantitative measure of how much 6-OHDA is needed to inhibit 50% of the sample cell.
10
Figure 3. Results of the experiment testing the impact of three different miRNA types on
cells impacted by 6-OHDA observed up to 96 hours at 24 hours intervals. 11
Figure 4. Results of the experiment comparing the protective effects of the exosome +
reagent combination versus the effects of the exosome + reagent + miRNA combination
on cells impacted by 6-OHDA over 96 hours. 12

5. Conclusion 13

6. Research Impact 13

7. Possibilities for Future Studies 14

8. Reference 14
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Question

How does the insertion of potentially protective miRNA types encapsulated by isolated exosomes
positively or negatively impact brain cells dying from HD?

Hypothesis

The insertion of the chosen miRNA types will increase cell proliferation in previously dying brain cells.
Through research, several candidates of miRNA types were identified referring to previously published
papers that retrieved data from patients suffering from HD. By narrowing down these options to three
miRNA types that were clearly under or overregulated in patients, the degree of efficacy for this
experiment increased as the potential impact of the specific miRNA type was backed up by research.
Although the insertion of these miRNA types was not yet extensively researched, this experiment aims to
ameliorate this shortcoming to potentially develop a very efficient therapeutic method against HD.
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1. Background Information
1.1 Exosomes

Among many of its functions, such as maintaining the division amongst micro and
macromolecules, exosomes are also produced by almost every cell in the body and are shown to impact
distant cell signaling.1 Since they are so commonly produced and are mobile extracellular vesicles,
exosomes function as escape routes for different proteins and microRNAs (miRNAs) to move from one
cell to another even if it is far away. 2

Due to this property, however, the abnormal export of an exosome(s) just from one cell can cause
the death of key proteins and miRNAs, eventually causing misexpression. Recently, many studies have
been conducted to find a relationship between exosomes secreted from specific cells and their impact on
certain diseases. The many functions of exosomes leads to a variety of potential use cases, such as being
used as therapeutic tools in an array of different diseases, such as HD.

1.2 Relationship between miRNAs and Exosomes

MicroRNAs are small, non-coding strings of RNA with several functions that heavily impact
gene expression which is why their specific role in genetic diseases and the overall human anatomy are
being studied extensively. For example, one known function is how miRNAs regulate genes by creating
bonds with the three untranslated regions (UTR) of messenger RNAs (mRNAs) to cause the deregulation
of the target gene's expression, essentially giving them the power to change the expression of multiple
genes within a cell. This unique characteristic can be linked to potential positive or negative impacts the
lack or abundance of specific miRNAs can have in a genetic disease. 2

Some recent studies have even proposed links between miRNAs and exosomes as there has been
speculation around miRNA secretion being controlled by vesicular/exosomal controlled mechanisms.
Either way, exosomes do have a significant role within circulatory miRNA biology which is why
exosomes have been used as transportation methods for miRNAs in some studies in order to facilitate
gene exchange amongst different cells. Since miRNAs can't move on their own, scientists have found that
by being encapsulated by exosomes, the miRNAs can be protected when leaving or entering cells. 3

1.3 Huntington’s Disease

HD is caused by a genetic mutation in the DNA code of the Huntingtin gene (IT15) where the C-
A-G nucleotide sequence is overly repeated. This discovery has been named the "CAG repeat expansion"
where the general population has around 20 repeats in this gene while those with clinically diagnosed HD
have over 40 repeats. Everyone with this mutation will show symptoms at some point in their lives but the
reason why most patients are middle-aged when first diagnosed is because of the impact the genetic
mutation has on protein synthesis. Since genes are instructions for proteins, a huntingtin protein also
exists. The specifics of this protein’s function have not been found or verified but scientists assume that
the CAG repeat expansion causes an abnormally long chain of instructions making it difficult for the
protein to function properly. These proteins are consistently produced over the span of a lifetime and form
clumps in the brain which is why the brain cells of HD patients get damaged and eventually start to die,
causing both physical and neurological symptoms to worsen over time. 4
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Although this phenomenon affects the entire brain, the striatum is most heavily impacted. The
striatum is located deep within the brain and is in control of movement, mood, and behavior control. So,
prolonged damage to the striatum caused by the initial mutation in the huntingtin gene, and therefore the
huntingtin protein causes HD. HD does not have a cure at the moment partially because there simply is
not enough information on the specifics of what happens to those with the mutation. 5

1.4 A172 Glioblastoma Cell

The A172 Glioblastoma cell line originates from the brain tissue in homo sapiens. The specific
cell used in this experiment was derived from a 53 year old male with glioblastoma, meaning it will
constantly divide. The A172 cell is mono-layered, adherent to its surface, and epithelial meaning it has a
long, stretched morphology. This cell line is from a patient with glioblastoma which originates from glial
cells. Glial cells are non-neuronal cells located in the central and peripheral nervous systems meaning
they do not produce electrical impulses. They also maintain homeostasis, form myelin, and provide
support for neurons. 6

In order to maintain its viability, the A172 cell requires a cell culture media and buffer. To
provide the cell with its necessary nutrition for growth like glucose glutamate, this experiment will use
RPMI 1640. In addition, to check on the nutrition of the cell, phenol red will be used as it is able to
visually show when the pH levels have fallen under 7.5. These cells need to be resupplied with new
media every two to three days to maintain their vitality because otherwise the cell’s characteristics would
change, causing inaccurate results. However, the RPMI 1640 cell culture media is not enough to fulfill all
the cell’s nutritional needs, necessitating the use of a growth factor. For this cell, Fetal Bovine Serum
(FBS) was identified as the fitting growth factor (American Type Culture Collection [ATCC]). In an
effort to make the cell management process more safe, the 0.25% Trypsin-EDTA cell dissociation agent
will be used. The ATCC has recognized this as the appropriate dissociation solution as the trypsin
protease is able to cut proteins therefore helping the transportation of the cell when moving from one petri
dish to another. As the A172 cell is adherent to its surface through peptides, the trypsin protease is needed
to degrade the adherent protein, preventing cell damage from forceful detachment.

1.5 Dopamine and Huntington’s Disease Relationship

Dopamine is a neurotransmitter that sends signals between neuron cells and is an essential part in
controlling voluntary movements. An alteration of the dopamine balance in the striatum specifically leads
to pathological conditions like HD. Changes in the brain’s amount of dopamine and number of dopamine
receptors causes the abnormal movements and cognitive deficits HD brings. Evidence also shows that an
increased level of dopamine release induces chorea, a defining symptom of HD, while a reduction leads
to akinesia, the inability to make any voluntary action. So, the link between dopamine and HD is quite
clear. 7

Although the reason behind cell loss in HD is unclear it can involve too much glutamate being
released from cortical and thalamic terminals or an increased sensitivity of glutamate receptors (Cepeda
et.al). Glutamate is another neurotransmitter that helps nerve cells signal to one another. Glutamate
receptor function is controlled by the activation of dopamine receptors, and this relationship suggests that
an alteration in dopamine function and neurotransmission would significantly impact the motor and
cognitive symptoms of HD. 8
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Using this relationship, this experiment uses 6-hydroxydopamine (6-OHDA), a form of dopamine
that has been used in previous Parkinson’s studies to induce neuron damage. 6-OHDA causes massive
destruction of neurons and experts have been focusing on 6-OHDA’s “intracellular mechanisms at the
striatal level” 9. Therefore, 6-OHDA is a prime chemical to use in mimicking the effects of HD on neuron
cells and the central nervous system.

1.6 Purpose and Hypothesis of this Experiment

Despite the shortcoming in information on the link between HD to miRNAs and exosomes, this
study uses miRNA types that have been established to have a potential relationship with HD. For
example, the “MicroRNAs in CSF as prodromal biomarkers for Huntington Disease in the PREDICT-HD
study” tests those with HD and a control group without the genetic mutation to find six miRNA types that
“significantly increased in the prodromal HD gene expansion carriers”. Altered miRNA expression
caused the abnormal gene growth in HD so delivering downregulated suppressed miRNA types or
removing the excess can be an effective therapeutic method against HD. Using the advantages of
exosome-delivery such as the fact that they can encapsulate miRNAs to allow delivery, miRNAs can be
delivered to neurons damaged by HD as a recovery measure.

2. Purpose and Significance of the Study

Despite this method not being tested yet, it still has aspects that indicate its potential for success.
First, rather than using a completely new method without any scientific basis, this study proposes a
method that uses a pre-existing, standard protocol for transfection, a key part of the overall method itself.
When inserting the target miRNA into the derived exosomes, this method uses a common transfection
reagent, liposomes, to incubate the miRNA together so that the miRNA can penetrate into the exosome's
membrane which it cannot enter without the liposomes. Again, the miRNA + liposome substance gets
incubated with the exosome to transfect together and eventually enter a neuron cell to observe its effects.

Also, this study uses former knowledge regarding the CAG repeat expansions in HD to
manipulate neuron cells so they mimic this characteristic. Acquiring human cells with HD is not always
possible nor is animal testing which is why this method proposes the manipulation of healthy cells to
replicate that of HD-riddled ones in order to make the entire process more time and cost-efficient. To do
so, this study uses 6-hydroxydopamine (6-OHDA), a pro-oxidant neurotoxic compound that was used in
other studies to mimic HD. Scientists found that 6-OHDA increased mutated huntingtin (mHTT)
expression while suppressing HTT phosphorylation at Ser421, a modification that would have protected
against mHTT accumulation. 10 If the data gathered is significant enough, the method can later be used on
animals and potentially humans rather than on mimicked neuron cells but in its early stages, manipulating
isolated cells is a wiser course of action.

3. Materials List & Experimental Procedures

3.1 A172 Glioblastoma Cell

In this experiment, the A172 Glioblastoma cell was used for two main reasons: accessibility and
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manipulation. This cell line is very common and meets the two initial cell line requirements this
experiment had - first, that the cell originated from the homosapiens species and second, that it was from
brain tissue. In the early stages of this lab, the SH-SY5Y cell line was going to be used but because it did
not grow well and it was not a brain cell, the A172 cell was a more appropriate alternative. As evident
from figure 1, this cell was able to be manipulated to have neuron-like characteristics using retinoic acid,
a very conventional agent used for cell differentiation. Also, referring to section 1.4, this cell line also has
a very typical viability maintenance procedure consisting of a cell culture media and buffer. Considering
these benefits, the A172 cell was chosen as it would produce relevant results in an efficient manner.

3.2 Retinoic Acid

Retinoic acid is “a small lipophilic molecule derived from vitamin A” 11 that “plays key roles in
cell growth and differentiation” 12. In this experiment, it was used to differentiate the A172 Glioblastoma
cell to mimic a neuron cell. Normally, retinoic acid is used to differentiate embryonic stem cells into
motor neurons by “altering both encoding RNA and miRNA expression” 13 and this is the main objective
of retinoic acid use in this experiment as well. Cell differentiation is the process in which a cell
transforms into a more specialized type. This process changes a cell’s “shape, size, and energy
requirements” 14. Since HD is a neurodegenerative disease that is “characterized by the gradual and the
progressive loss of neurons” 15, an experiment aiming to find protective agents against the disease must
take place on neurons, the cell type that’s affected by the disease.

3.3 6-OHDA (6-hydroxydopamine)

This experiment uses 6-hydroxydopamine (6-OHDA), a form of dopamine that has been used in
previous Parkinson’s studies to induce neuron damage. 6-OHDA causes massive destruction of neurons
and experts have been focusing on 6-OHDA’s “intracellular mechanisms at the striatal level” 9. Therefore,
6-OHDA is a prime chemical to use in mimicking the effects of HD on neuron cells and the central
nervous system.

As previously established, 6-OHDA causes neuron damage and because HD is characterized by

the consistent neuron loss in the striatum, it makes sense to use 6-OHDA that causes neuron loss to mimic
HD conditions. 6-OHDA specifically was used because this model is already used as a valuable tool for
Parkinson’s disease, another neurodegenerative disease. Using its IC50 levels on the A172 Glioblastoma
cell line and observing the impact of different miRNA types and isolated exosomes on the cell’s viability
will help scientists come to a conclusion of whether or not a certain miRNA type helped or harmed the
cell’s proliferation.

3.4 Isolated Exosomes and miRNA Insertion

To isolate the exosomes from the A172 cell, the “Total Exosome Isolation (from cell culture
media) reagent”, produced by Invitrogen, and exosome depleted fetal bovine serum (FBS) was used,
following the guidelines provided by Invitrogen. This process needed to happen for several different
reasons. First, miRNAs cannot be transported into the cells by itself and exosome encapsulation has been
established as an effective method for transferring miRNAs into cells. This experiment tests the protective
abilities of miRNA types on HD and for this to effectively happen, the miRNAs need a method of
transportation which is exosomes in this experiment.
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4. Results Analysis and Discussions

Figure 1. Microscopic images of A172 Glioblastoma cells treated by varying levels of retinoic acid (RA)
for 24 hours.

This image shows several cell samples that have all been differentiated using retinoic acid.
Looking at the cell’s morphology, the sample treated with 0 μM of retinoic acid has several small pieces
with the occasional dark or white spot. The rest of the samples, treated with 10, 30, and 50 μM of retinoic
acid, all have a generally round shape that grows in size as the concentration of retinoic acid increases,
showing the progression of cell division. These samples seem to be epithelial with a long, stretched
morphology. All cell samples are attached to the bottom layer of the petri dish and the image was taken
from a bird’s eye view.
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Figure 2. Graphical representation of the IC50 level of the A172 Glioblastoma cell, a quantitative
measure of how much 6-OHDA is needed to inhibit 50% of the sample cell.

This graph compares the concentration of 6-OHDA to its impact on the cell absorbance of the
A172 Glioblastoma cell. Six data points were taken because there was not any change after 600 μM as the
last three data points showed very similar levels of absorbance. Absorbance levels show the amount of
light that can pass through a sample. If there are a lot of molecules in the cell, the light would have to hit
more molecules as it passes through meaning a high level of absorbance also indicates a high level of cell
viability, vice versa 16. When the cell was not treated with 6-OHDA, the cell’s absorbance was at
approximately 2.7 absorbance (ABS) and evidently started to decrease exponentially as soon as it got
treated with 6-OHDA, showing that the cell was getting killed by increasing the concentration of 6-
OHDA. The second and third data points did have some error bars but these do not impact the conclusion
that 6-OHDA kills the cell because the error bars do not intersect with another data point’s absorbance
levels. After the fitting curve was identified, IC50 was discovered as 49.73 μM, the concentration of 6-
OHDA that induces 50% of cell death. For the next experiment, we used 49.73 μM 6-OHDA to induce
cell death on A172.
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Figure 3. Results of the experiment testing the impact of three different miRNA types on cells impacted
by 6-OHDA observed up to 96 hours at 24 hours intervals.

These four bar graphs indicate the cell proliferation of the A172 Glioblastoma cell treated under
eight different conditions. Using the presto blue reagent to test for cell viability, two samples of cells in
different conditions were analyzed daily to allow for average and standard deviation calculations. Other
than the results after 24 hours, all three bar graphs showed a very low standard deviation, supporting this
experiment’s degree of accuracy. The overall levels of cell proliferation dropped in the 48 hours graph but
this sudden decrease is presumably due to human error as all eight conditions were impacted.

All four graphs show that 6-OHDA definitely decreased the cell proliferation of the sample cell
and its impact increased over time. Also, there was quite a significant increase in cell proliferation for the
three samples treated with the miRNA type and exosome combination without 6-OHDA when compared
to the no treatment control group. There was also an increase in cell proliferation between the cell sample
treated only with 6-OHDA and the three samples treated the combination of miRNA + exosome + 6-
OHDA showing there is a positive effect from this combination, whether this is from the miRNA or
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exosome. As time passed, the difference between the second bar graph compared to the last three
increased.

Figure 4. Results of the experiment comparing the protective effects of the exosome + reagent
combination versus the effects of the exosome + reagent + miRNA combination on cells impacted by 6-
OHDA over 96 hours.

Data from figure 4 shows there was a significant positive impact of the miRNA + exosome +
reagent on cell proliferation after they were treated with 6-OHDA. However, the positive effect on cell
proliferation may be induced either by exosomes with reagent or miRNA itself. To find out which factor
derives the protective effect against 6-OHDA induced cell death, two conditions were tested with the
addition of 6-OHDA: 1) exosomes with reagents 2) exosomes with reagents + miRNA.

Figure 4 shows that there is almost no difference in cell proliferation between cells treated only
with 6-OHDA versus those treated with the exosome + reagent combination while there’s a clear increase
in cell proliferation in samples treated with the miRNA types as well showing that the impact of the last
three bars is solely due to the addition of different miRNA types. Also, the error bars in the second and
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third bar graphs are almost none meaning this data is very reliable, again proving that the exosome +
reagent combination had no positive impact on cell proliferation. On the other hand, the error bars fourth
and last bars are quite significant but still show an increase in cell proliferation since the lower and upper
bounds of all error bars still have a higher level of cell proliferation when compared to the second, 6-
OHDA only bar. Therefore, the protective abilities of all three miRNA types against cells dying from 6-
OHDA has been proven.

5. Conclusion
Prior to the experiment, it was predicted that the insertion of certain miRNA types would increase
proliferation in cells dying from HD, mimicked using 6-OHDA. Since the miRNA types were chosen
based on pre-existing data about miRNA types impacted in the brains of HD patients, an experiment
targeted towards ameliorating this shortcoming should have positively impacted cell growth that was
already dying from HD. This hypothesis was supported as shown by figures 3 and 4 as they clearly show
that the miRNA types were able to cause an increase in cell proliferation.
While the change may seem trivial, considering that this experiment is exploring unknown topics
with a lot of room to grow, this initial data seems promising. Not only does it use 6-OHDA to mimic HD,
a relatively novel approach, but it also proves that miRNA types are effective in increasing cell
proliferation in damaged neuron cells. This study is one of the first to explore how miRNAs can be a
therapeutic method against HD and unlike some others that focus on one specific type, miR-124, this lab
explores the possibility of three types. One extension this research can take is to use combinations of the
three types tested above. Since all three miRNAs have shown a positive impact on cell proliferation,
theoretically, the combination should result in an exaggerated increase. If miRNAs prove to be an
effective therapeutic in fighting HD, this disease might have its first cure. Granted, not every patient is
under the same circumstances so there can not be just one solution but by finding protective miRNAs and
potentially using different combinations for each patient can be an effective way to at least slow down
disease progression.

6. Research Impact
In other studies that have used miRNAs and exosomes to discover more about HD, most have
used cells from another species to transfect into a miRNA vector. For example, a previous study used
genetic cloning technology to replicate their target miRNA into a plasmid to create miRNA vectors. They
then transfected HEK 293 human cells with the miRNA vector and the resulting cells were put into a
medium where scientists later chose cells that overexpressed the target miRNA. 17 The chosen cells were
cultured into an exosome-free medium where the exosomes in the cell were isolated and the level of
target miRNA left was quantified. To finish, the scientists injected the exosomes they harvested, that now
had their target miRNA, into transgenic mice using a syringe.
This experiment, on the other hand, has differences that improve the efficacy and accuracy of the
previously mentioned process. First, this experiment eliminates the need for vectors and cultured cells,
allowing for the target and origin cell for exosome isolation to be from the same subject. Much like the
idea behind organ transplants, it is much more preferable and beneficial if the original cell where an
exosome was derived has a similar genetic makeup as the target cell as there is a significantly lower
possibility of the target cell rejecting the exosome. However, this concept is not applied to the current,
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widespread method of miRNA delivery which is why this study proposes a method that theoretically
would work. Therefore, this study would be able to explore the possibility of a method that can produce
remarkably more accurate results thanks to the use of native cells.
As a whole, discovering effective miRNA types to recover and heal the neuron cells damaged
from HD will be the first plausible cure/management method for the disease. While the specific method
for injecting miRNAs into living human brains can be a future extension, this study shows the strong
possibility of this treatment method panning out theoretically.

7. Possibilities for Future Studies

Further testing such as manipulating the amount of miRNA or observing the samples for a longer
amount of time can address the shortcomings the current lab has. While there are thousands of potential
labs that can be conducted on this subject matter, at the beginning stages of exploration, it is much more
realistic to examine a given lab’s shortcomings then expand into broader possibilities like using different
miRNA types or isolating exosomes differently. In this research, for example, only 20 pmol of each
miRNA concentration was tested. While this was enough to indicate the relationship of miRNA +
exosome treatment on damaged cells, it did not cause a dramatic increase in cell proliferation. By testing
different amounts of the three miRNA types used here, an ideal quantity can be derived for these three
types. So, in future experiments, an optimal concentration of specific miRNA tpes should be analyzed to
maximize the protective effects on neuron cells from 6-OHDA induced cell death. Also, as evident from
the figures in section 4, all the samples were observed for only 96 hours. Again, this was still enough to
identify a relationship but observing the effects for a few weeks or even months can show how long the
protective qualities of the miRNA types need to fully take action but can also show how long a certain
concentration of miRNA lasts in the neuron cell before losing its protective qualities. Since the neuron
cells in patients with HD will constantly get damaged, being able to create a therapeutic that can adapt
and withstand these effects would be necessary. Therefore, a future study experimenting the different
time intervals miRNA injections may need to be tested.

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