Close
About
FAQ
Home
Collections
Login
USC Login
Register
0
Selected
Invert selection
Deselect all
Deselect all
Click here to refresh results
Click here to refresh results
USC
/
Digital Library
/
University of Southern California Dissertations and Theses
/
C9orf72 deficiency exacerbates motor deficits in C9ALS/FTD mouse models
(USC Thesis Other)
C9orf72 deficiency exacerbates motor deficits in C9ALS/FTD mouse models
PDF
Download
Share
Open document
Flip pages
Contact Us
Contact Us
Copy asset link
Request this asset
Transcript (if available)
Content
C9orf72 Deficiency Exacerbates Motor Deficits
in C9ALS/FTD Mouse Models
By
Qing Chang
A Thesis Presented to the
Faculty of USC Keck Medical School
In partial fulfillment of the
Requirements for the Degree
Master of Science
(Molecular Microbiology and Immunology)
May 2021
Copyright 2021 Qing Chang
ii
TABLE OF CONTENTS
List of Figures ............................................................................................................................ iii
Abstract ........................................................................................................................................ iv
Chapter1:Background ............................................................................................................ 1
1.1 C9orf72 and G4C2 repeats ...........................................................................1
1.2 C9ORF72 protein haploinsufficiency ...........................................................2
Chapter2:Methods .................................................................................................................. 4
2.1 Mouse models ...............................................................................................4
2.2 Rotarod Test .................................................................................................4
2.3 Grip Strength ..............................................................................................11
2.4 Three chamber tests ....................................................................................13
2.5 Novel Recognition Test ..............................................................................17
2.6 Five-Trial Social Memory Test ..................................................................20
Chapter3:Results ................................................................................................................... 21
3.1 C9orf72 protein levels decreased in crossed C9orf72+/- and C9BAC mice
..........................................................................................................................21
3.2 4-month-old Mutant mice performed no Anxiety issues ............................22
3.3 C9orf72 dose is critical for motor deficits in C9ALS/FTD mouse models 22
3.4 Motor dysfunction in C9ALS/FTD mice also shows age-dependent .........23
3.5 C9ALS/FTD mouse shows no social defects at 4 months old ...................26
Chapter4:Discussion ............................................................................................................ 29
4.1 The interaction of aging and loss- and gain-of-function in C9ALS/FTD
causes motor deficits phenotypes .....................................................................29
4.2 The evaluation of the gain-of-function FVB-C9BAC mouse model .........29
4.3 Potential mechanisms of pathological motor dysfunction in C9ALS/FTD
mice ..................................................................................................................30
Chapter5:Conclusion ........................................................................................................... 32
References .................................................................................................................................. 33
iii
List of Figures
Fig 1 Rotarod Equipment ...........................................................................................5
Fig 2 Grip Strength Equipment ................................................................................12
Fig 3 Grip Strength Equipment ................................................................................13
Fig 4 Novel Object Test ...........................................................................................20
Fig 5 Novel Object Test ...........................................................................................20
Fig 6 Motor deficits in 4 months old C9ALS/FTD mouse ......................................23
Fig 7 Exacerbate motor deficits in 10 months old C9ALS/FTD mouse ..................26
Fig 8 a, Five Trails Test; b,Novel Object Test ........................................................27
Fig 9 Three Chamber Test .......................................................................................27
Supplement Figure ..................................................................................................39
iv
Abstract
Background: The mos prominent hereditary amyotrophic lateral sclerosis (ALS) and
frontotemporal dementia (collectively, C9ALS / FTD) are triggered by hexanucleotide repeat
expansion in the C9ORF72 intron. The pathogenesis of C9ALS/FTD is due to C9ORF72
haploinsufficiency (loss of function) and toxicity benefit caused by toxic RNAs and dipeptide
repeat proteins (DPRs), modelled using human C9ORF72 bacterial artificial transgenic
chromosome mice (C9-BAC). It remains at best poorly known how loss-and gain-of-function
communicate and the biological relevance of their interactions.
Methods: In order to develop C9ALS/FTD mouse models (C9orf72+/-;C9-BAC), C9orf72+/-
(loss-of - function) mice were crossed with C9-BAC (gain-of-function) alleles in another mouse
strain, accompanied by cellular, neuropathological, and behavioral studies.
Results: By reducing the C9orf72 protein dose in the context of C9-BAC mice, we have
discovered that the C9orf72 deficiency promotes the age-dependent and dose-dependent motor
defects of C9-BAC mice.
Conclusions: These experiments presented the first in vivo proof of the relationship between
C9orf72 impairment and toxicity benefit resulting in motor activity defects by incorporating both
loss- and gain-of-function genetically engineered mice.
Keywords: C9orf72, gain of toxicity, axonal degeneration, motor behaviors, C9ALS/FTD
1
Chapter1:Background
1.1 C9orf72 and G4C2 repeats
The G4C2 hexanucleotide repeat expansion in the first intron of the chromosome 9 open
reading frame, 72 (C9ORF72), triggers the most common family amyotropic lateral sclerosis (ALS)
and fronto-temporal dementia (FTD). In patients under 60 years of age, frontotemporal dementia
(FTD) is the second most common dementia. (cite reference here) A common motor neuron (MN)
disorder that affects the upper and lower MNs and the corticospinal tract is amyotrophic lateral
sclerosis (ALS) [1,2]. Neurodegenerative diseases that arise at the same time in some patient
groups are frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) [5, 6]. Around
40 percent of familial ALS and approximately 5-10 percent of sporadic ALS cases are caused by
C9ORF72-associated ALS [7, 8].
FTD and ALS also share pathological hallmarks, including ubiquitin-TDP-43 neuronal
inclusions, in addition to clinical overlap [9,10]. C9ALS/FTD pathogenesis is known to be due to
C9ORF72 haploinsufficiency (loss of function) and toxic repeat transcription RNAs, as well as
repeat-associated non-AUG (RAN) translation (gain-of-function) dipeptide repeat proteins (DPRs)
[13-16]. Mice with intracerebroventricular injection of adeno-associated virus (AAV) encoding
G4C2 repeats and transgenic mice using C9ORF72 bacterial artificial chromosome (BAC) from
patient DNA have been developed to model gain of toxicity, including C9ALS/FTD gain-of-
function mice [17-22].
In patient tissues, RNA foci containing sense G4C2 and antisense G2C4 are identified and
can sequester RNA-binding proteins (RBPs), resulting in normal loss of function [23-25].
Different DPRs are produced by RNA translation, including poly-glycine-arginine (GA) and poly-
2
glycine-arginine (GR) from G4C2 sense transcripts, poly-proline-alanine (PA) and poly-proline-
arginine (PR) from G2C4 antisense, and poly-glycine-proline (GP) from repeat transcripts of both
sense and antisense. Poly (GA) accumulation has been associated with ER stress,
nucleocytoplasmic transport disruption, and proteasome dysregulation among different DPR
functions [26-28]. Several studies have shown that poly (GR)/(PR) is especially toxic and disrupts
various cellular functions, including the dynamics of stress granules, nucleocytoplasmic transport,
nucleoli, and control of translation [29-34].
1.2 C9ORF72 protein haploinsufficiency
C9ORF72 protein haploinsufficiency is a central potential disease mechanism that can work
in parallel with toxicity benefits from repeat transcription toxic RNAs and dipeptide repeat proteins
(DPRs) from repeat-associated non-AUG (RAN) translations [5, 6]. C9ORF72 expression is
decreased in C9ALS/FTD patient tissues, consistent with this loss-of-function notion [35-38];
C9ORF72 haploinsufficiency is known to lead to the degeneration of motor neurons (MNs)
induced pluripotent stem cells (iPSCs) [39] derived from ALS patients ; Therefore, from the
perspective of loss-of-function, studying C9orf72 biology can provide new insights into
C9ALS/FTD pathogenesis.
In comparison to toxicity gain, in vivo haploinsufficiency of C9ORF72 is comparatively less
well studied. In vitro studies have shown that C9orf72 with Smcr8 forms a protein complex and
regulates autophagy, a process dependent on lysosomes [40-46]. Autophagy is especially
important for terminally differentiated neurons. MNs with long axons are strongly polarized,
which remains a major challenge for the transport of degrading organelles from the distal tips to
the soma, where mature acid lysosomes containing degrading enzymes are located [48,49]. Despite
these advances, neither C9orf72 heterozygous nor homozygous knockout (KO) in neurons is
3
associated with neurodegeneration or motor defects in mice [50]. It is still important to determine
the in vivo significance of C9orf72 haploinsufficiency in C9ALS/FTD. It is poorly known whether
and how loss-of function and gain-of-function interact in vivo to facilitate behavioral deficits.
4
Chapter2:Methods
2.1 Mouse models
C9orf72 mutants (Cat#: 027068) and C9-BAC mice (Cat#: 029099) were purchased from
the Jackson Laboratory, Bar Harbor, Maine?. All animal studies were conducted under protocols
approved by the Institutional Animal Care and Use Committee (IACUC) at The University of
Southern California in Los Angeles, Callifornia.
2.2 Rotarod Test
2.2.1. Apparatus
To cushion any mice that fall off, a soft padded surface is placed at the base of the apparatus
for all tests. NB In the charts, this is not seen in order not to misrepresent any aspect of the
apparatus. We have never witnessed any injury caused by mice falling from such equipment, which
is normally 30-50 cm high, during the testing of hundreds of mice, both normal and mutant. Also,
we cleaned and sterilized the machinery between each tested mouse.
2.2.1.1 Rotarod
There are many commercial models of this system on the market, but some have drawbacks,
such as failing to accelerate to detect motor incoordination (rather than endurance) at an acceptable
speed. Another common flaw is that above the foundation, the rod is not high enough, leading to
mice tending to fall off or leap off.
The engineering sections of the Department of Pharmacology, University of Oxford, made
the rotarod shown in Figure 1 to the author's design. The rod has a diameter of 3 cm and is held 30
cm above the base of the apparatus. In a series of parallel ridges along the longitudinal axis, the
5
surface is knurled, allowing the mice to easily grip it (Figure 1). NB The depth of the ridges is a
crucial detail; the test would be much harder if the mouse does not get a good grip; if the grip is
too good. On the other hand, the mouse will "cartwheel" around the rod by passively holding on
to it. It might be useful to know that knurled wooden dowelling of a reasonable size for mice is
commercially available for 'amateur rotarod makers’ it does, of course, need to be treated with a
good waterproof varnish before use. The starting speed is set to 4 rpm and the rate of acceleration
to 20 rpm / min. The maximum velocity is 40 rpm.
The mouse is stopped from leaving the rod by two flanges. They have a diameter of 30 cm
(this will possibly be reduced to 20 cm). Their separation is set at 6 cm (maximum), but if they
appear to turn around on the rod, they might need to be adjusted lower for sub-adult mice.
Fig 1 Rotarod Equipment
6
2.2.1.2 Triple horizontal bars
The bars are made of brass, 38 cm high, supported at either end by a wooden support column
49 cm above the bench surface. The pillars are fastened to a heavy wooden base. There are three
available bar diameters: 2, 4 and 6 mm. The standard one we use is the 2 mm bar, but many mice
achieve maximum scores on this. Therefore, in an effort to refine the test, larger diameter bars
have been used, as mice do not handle these so well.
2.2.1.3 Static rods
Per 60 cm long, five wooden dowels or rods of varying thickness (35 (rod 1), 28, 22, 15 and
9 (rod 5) mm diameter) are fastened to the laboratory shelf by a G-clamp so that the rods protrude
horizontally into space. To show the finishing line, the end of the rod near the bench has a mark
of 10 cm from the end. 60 cm is the height of the rods above the floor.
Use only the biggest, smallest, and middle rods if a shorter and less thorough test is needed.
To test the development of Huntington's Disease in mice, Van Dellen et al.14 used only a single
rod.
2.2.1.4 Parallel bars
Two parallel steel bars with a length of 1 m and a diameter of 4 mm are fixed 30 mm (at the
center) apart from the wooden supporting columns at the ends. 60 cm above the floor are the bars.
2.2.2. Procedure
For all experiments, 5-20 min before testing, mice are carried to the experimental room to
ensure that they are fully awake. As a general rule, in order to allow muscle strength to recover
and return to normal levels of arousal, after each motor test, the mice rest by returning to their
home cage.
7
2.2.2.1 Rotarod
The rotarod is initially set to a starting speed of 4 rpm, 20 rpm/min acceleration rate.The tail
of the mouse is positioned on the spinning rod, facing away from the rotation direction, so that it
has to shift forward to remain upright. This is easier to do if the mouse is placed at an angle of 45 °
below horizontal towards the rod. Trying to lower the mouse from above would result in the
spread of its hind legs and the edges of the flanges being grasped. The mouse is released quickly
when the rod is almost touching, just ahead of and above the top dead center rods, allowing the
rod to be easily gripped. Using a thin (10-15 mm) dowel is a simpler and more reliable technique.
Th mouse is lowered to the dowel, parallel to the long axis, by keeping the mouse in one hand.
With the mouse 's head downwards, the dowel is tilted around 30 ° to allow it the mouse more time
to grasp the rotarod until it is released. The mouse is lowered and the dowel between the rotarod
flanges and the dowel is drawn downwards away from the mouse once the mouse is over the
spinning rod, and then the mousewill grab the rod and step forward.
Acceleration is started as long as the mouse faces forward. If the mouse is not facing forward,
the investigator waits until it faces forward before beginning acceleration at 10 seconds after the
mouse is put on the rod, and the investigator remembers the speed at which the mouse falls off.
Notice the time of fall if it falls off before 10 sec, and try again, up to three times in total, recording
the velocity at the first fall after the 10 sec mark. Falls before 5 sec, however, which are due to
poor placement by the experimenter, should not be registered. The mean velocity at fall is the data.
Instead of using the maximum velocity, this is accurate for the additional practice that the mouse
gets during the failed runs, which is thought will improve efficiency. Thus, a mouse that falls once
before 10 sec then remains on the second time scores (4 + 12)/2 = 8 rpm before 12 rpm.
8
A mouse that fell twice and continues on for scores of 10 rpm (4 + 4 + 10)/3=6 rpm. If a
cursor tries to grip it three times in 10 seconds, this gives it a score of 4 rpm.
One difficulty is that mice often avoid going forward and just grab the rod tightly, so they
passively somersault round, while most inevitably fall off. Therefore, it may be helpful to also
mention the velocity as the first such inversion happens. Using a rod with a greater diameter or
less prominent ridges may be a potential alternative.
2.2.2.2 Bars of horizontal bars
Since the thin 2 mm bar is simpler for mice to understand, it is advisable to test them on this
one first. Keeping the mouse by the tail, the investigator positions it in front of the unit on the
bench, slide it backwards about 20 cm quickly (this aligns it perpendicular to the bar), lifting it
rapidly and letting the mouse grab the horizontal bar with its forepaws only at the central point,
and drop the tail, beginning the stop clock at the same time. This may be challenging to do
effectively. Ccertain mice grip the horizontal bar easier if the tail is unexpectedly released The
mice can struggle to grip the horizontal bar if they believe they are still supported. The criteria
point is either a dropping from the bar until one of the bar's end columns is hit by the cursor, or the
time before a column is struck by one forepaw. The actual evaluation period is 30 seconds (cut-
off time).
If the mouse fails to adequately understand the bar for the first time, although this seems to
be due to the methodology of the experimenter rather than the mouse, the investigator attempts
again (after a short rest when checking another one or two mice) and does not report this dropping.
If the mouse dropped before 5 sec, and this apparently was not due to the experimenter's bad
positioning, then this experiment is repeated up to three times in an effort to get a score of > 5 sec.
9
If the second attempt is > 5 seconds, do not do a third experiment. In this case, take the date with
the highest ranking. A mouse that struggles to support itself continuously for > 5 sec scores just 1.
If the triple-bar variant is used, it will then be tested on thicker bars, if the mouse performs 5
on this first 2 mm bar, after a short rest time while another one or two mice are evaluated. The
rating scheme for the 4 and 6 mm bars is the same as for the 2 mm bars, and the combined sum is
the final result. Thus, a mouse that scores 5 on the 2 mm bar but declines after 13 seconds from
the 4 mm bar scores 5 + 3= 8.
(1) Scoring horizontal bars
Rating the horizontal bars: the first two periods are fewer than the last two since they are less
prone to collapse after the mice have initially learned the task: Dropping between 1-5 sec = 1
Falling between 6-10 sec = 2 Falling between 11-20 sec = 3 Falling between 21-30 sec = 4 Falling
after 30 sec = 5 Putting one forepaw without falling on a bar help = 5
As the optional usage of 4 and 6 mm bars is a recent addition to our protocol, we have no
detailed experience with them yet.
2.2.2.3 Rods Static
The mouse is placed at the very end of the longest rod (the length of one head from the end
is suitable for the nose tip). The investigator takes two measures: time of orientation (time taken
to orientate 180 ° from the starting point towards the shelf) and time of transit (time taken to fly to
the end of the shelf (nose from the shelf end of the rod above the 10 cm mark).
Orientation relies on the mouse being upright. If the mouse flips upside down and clings
under the rod, the overall orientation score of 120 sec is randomly given to it (for statistical
purposes). Do not measure it on smaller rods. Asa mouse that transits upright is clearly more
10
organized than one that transits when hanging upside down, the transit time becomes the highest
benefit.
If the mouse drops or exceeds the full test period (arbitrarily set at 120 seconds) after
orientation, then we do not test it on the smaller rods. For statistical reasons, 120 for that specific
rod and for subsequent ones are allocated to mice dropping, since it is presumed that they will fall
from a narrower rod again. If the mouse turns upside down, remember the case as well. The mouse
must remain upright on the rod for efficient transit as well as orientation. Otherwise, the highest
ranking is given to it. After it hits the end of the rod or sinks, the cursor is deleted.
The mouse is then returned to the home cage to relax after checking on one rod as you
examine another mouse. Then, the investigator puts the mouse on the rod of the next smaller
sized mouse and measures it again in the same manner. If the mouse slips off a rod after being ofor
longer than 5 seconds, the investigator avoids checking. The mouse is replaced, and another
attempt is made if the mouse ifalls off in fewer than 5 seconds (as dropping inside 5 seconds may
be attributed to faulty positioning by the experimenter), with a maximum of three attempts, and
then, the better outcome is used. .
2.2.2.4 Bars in parallel
With its longitudinal axis opposite to that of the bars, the mouse is positioned in the middle
of the two bars. All front paws should be on one bar and both hind paws on the other bar. Scoring
for the static rods is equal to that. Take two measures: the period taken before the start location is
oriented 90 ° by the mouse, and the duration until one of the end supports is eventually hit.
Remember this occurrence as well, if the cursor flips upside down. As for the static rods test,
without it flipping upside down, orientation and transit must be accomplished, otherwise the full
duration is allocated. It is uncommon for the mice to collapse in fewer than 5 seconds for relatively
11
stable mice. If this happens, it is possibly due to bad positioning of the mice. In such a case, the
outcome is discareded and the cursor is retested.
2.2.3. Evaluation
To test latency to decline, the sum of the three trials was used. The tail retained each mouse
and lowered it into the apparatus. They required the front paws to comprehend the assembly. The
mouse in the horizontal plane was then pushed backwards before the pull-bar was enabled. The
trial was performed four times, and reported the highest force created by pushing the animal away
from the wire mesh.
2.3 Grip Strength
2.3.1. Procedure Implementation
Using the Hand Dynometer Type 78010 Adapted from Lafayette Instruments illustrates Ithe
participant 's application of the methodology prior to assessment. The investigator should be in a
standing posture, not touching their body, arms by their sides. The investigator should hold the
elbows loosely curved. On the non-dominant side, the investigator should perform the examination.
(Exception: If hand grip may be performed from the protocol from the other side of the body, the
investigator should go ahead and do the calculation and report it as usual, but leave a marginal
note showing the side used, if it is later looked at by an observer as an outlier). The investigator
should then tell the individual to pinch the dynamometer with as much power as necessary , being
mindful to squeeze just once with each measurement. In order to prevent the symptoms of muscle
weakness, three trials can be performed with a delay of between 10-20 seconds between each
experiment. The investigator should record each trial 's outcome to the nearest pound or kilogram,
w hether the score gap is less than 6.6 lbs. The exam, or 3 kgs., is over.(This sentence is not clear;
please fix it). If the gap is more than 6.6 lbs in the two scales or 3 kgs., then the investigator
12
should repeat the test after a pause time, again. In your data report, use the strongest 3
measurements (ie. the largest three). The outlier (the lowest value )is marked off with your name
where a 4th measurement is taken with the hand grip (where all of the 3 measurements are 3 kg
apart) such that the 3 maximum measurements are specifically identified for data entry. The results
are compared to the age and sex-specific norms published. (Fig 2)
Fig 2 Grip Strength Equipment
2.3.2. Evaluation
Based on the data collected from broad numbers of individual mice, the following tables are
averages. Large differences may not generally be deemed uncommon or representative of an issue
from these statistics, a 6'2 "big, 30-year-old man weighing 210 lb, for instance." However, a 30-
year-old, 5'7 "man weighing 135 lb can have a stronger grip power than normal. The grip strength
could be weaker than normal. Power testers are particularly effective in explaining improvement
over time. For starters, assessing the strength of an individual during the process of an illness or
accident will measure lack of strength. Measuring at the outset and again at frequent periods will
determine whether the treatment had the expected effects for someone suffering from an illness or
accident who is doing physical therapy.
13
2.4 Three chamber tests
2.4.1 Space Set Up and Equipment: (Fig 3)
Fig 3 Grip Strength Equipment
1. A rectangular, three-chamber box comprises the equipment for Crawley's sociability and
choice for the social novelty examination. Each chamber measures 19 x 45 cm, and the dividing
walls are constructed of transparent plexiglass, with an open center portion enabling each chamber
to provide free entry for the mouse being tested.
2. An example using two identical containers with reversible lids, or wire cups, that are wide
enough to accommodate a single cursor. These are positioned vertically inside the apparatus, one
in each side chamber, and the naive /unfamiliar mouse would be enclosed. Every container consists
of metal wires that enable air to be transferred between the cylinder 's interior and outside but are
small enough to escape direct physical contact between an animal on the inside and one on the
outside.
3. Around 9:00am and 6:00 pm, behavioral testing can be done.
4. 650 lux is the general acceptable space illumination.
Three chamber test
Stranger1 Middle Empty
(S1) (M) (E)
Socialability
Stranger1 Middle Stranger2
(S1) (M) (S2)
Social Novelty/Memory
14
5. To track and report the observation and follow-up criteria referred to in section 4.3, use the
Observer 6.0 (Noldus) software (or a compatible alternative).
6. Both chambers should be cleaned with 70 % ethanol (between mice) after each trial and
then with Clidox 1:5:1 (between cages) to avoid olfactory cue bias and ensure adequate
disinfection, respectively, after each trial.
7. The person who makes the observation must be at least 2 meters away from the apparatus. .
2.4.2 Preparation for Animals:
1. Three to five mice should be housed per cage in a space with a 12 h light dark period
(lights on at 7:00 am) with access to food and water ad libitum, according to local Animal Care
Committee recommendations and procedures specifications and criteria.
2. Thirty minutes before the first test starts, the investigator should move all the cages holding
mice into the behavioral space.
3. For this experiment, there are two classes of mouse needed, one that serves as a control
animal, naive or 'unfamiliarized,' and one that is the focus of the research. Using a mouse with the
same history (usually C57BL6), age (usually 8-12 weeks), gender and weight, without any
previous interaction with the subject mouse, for the control mouse. Per experiment , two control
mice are needed, one is used for session I and another for session II. Between experiments, the
same control mice can be used.
2.4.3 Habitability (adaptation):
1. The investigator should use the separating Plexiglass walls to separate the right and left
compartments.
2. The investigator should place empty wire containment cups (one per site) in the center of
the right and left chambers.
15
3. For adaptation, the investigator should position the subject mouse at the center of the
middle chamber.
4. Habitat for five minutes.
2.4.4 Social affiliation Evaluation (session I) aspect:
1. In a wire containment cup that is placed in one of the side chambers, the investigator should
put one of the control mice ('Stranger 1') inside. The location of Stranger 1 is routinely altered
between trials on the left or right side of the room.
2. To permit free entry for the subject mouse to explore each of the three rooms, the
investigator should erase the walls between the compartments.
3. The investigator should start tracking and documenting the following parameters
immediately:
—Length and Time
Amount of direct (active) encounters, for each chamber separately, between the subject
mouse and the containment cup housing or not housing the Stranger 1 mouse. A clear touch
between the subject mouse and the containment cup, or a stretch of the subject mouse 's body 3-5
cm around the cup, shall be counted as an active contact;
—Length and Time
The amount of other topic mouse activities in each compartment, including wandering, self-
grooming, absence of more than 5 seconds of body motions ('freezing'), as well as odd actions,
such as hopping, repeated behavior, etc, should be reported.
—Length and Time
For each compartment, the investigator should record the number of entries. When the head
and four paws have reached the room, the mouse is deemed to be in the chamber.
16
Session I is 10 minutes in length.
2.4.5 Social Novelty / Test Session of Choice (Session II):
1. In the opposite side chamber (that had been empty during Session I), the investigator should
place a second control mouse ("Stranger 2") within an identical wire containment cup. The same
parameters mentioned in 4.3 are tracked, separating the activities between the subject mouse in the
presence of Stranger 1 and Stranger 2.
Session II has a length of 10 minutes.
2.4.6 Review of statistics:
—The investigator should analyze the following parameters using the Origin 6.0 program (or
a reasonable alternative), using the data recorded:
1. Complete number of connections and phone numbers
2. Total touch time between the experimental mouse and the empty storage cup vs. the cup
housing Stranger 1 (in session I) or the cup housing Stranger 1 vs. Stranger 2 (in session II);
3. Period average per contact;
4. Total amount and length (freezing, self-grooming, walking) of other activities
5. The cumulative time spent in each compartment by the subject mouse.
—For each of the above criteria, the investigator should evaluate the relevant discrepancies
by contrasting groups:
1. For a wild type (WT) subject, "Empty" containment cup vs "Stranger 1"
2. "For the experimental mouse," Empty "containment cup vs" Stranger 1 "(e.g. a knockout,
transgenic or drug-treated mouse, etc.).
3. "For a WT topic," Stranger 1 "vs" Stranger 2
17
4. "Stranger 1" versus "Stranger 2" (knockout, transgenic, or drug-treated, etc.) for the
laboratory mouse.
—Other criteria that help standardize this measure should also be taken into account, such as
previous expertise with distinguishing particular behavioral habits of the mouse, such as sniffing,
brushing, scratching, battling, mounting, which may be mostly based on subjective personal
observation. During the examination, the individual serving as the recorder of the behavioral
criteria should be oblivious to the genotype or circumstances of therapy and this knowledge should
be decoded afterwards.
2.5 Novel Recognition Test
Procedures (Fig 4)
2.5.1Habituation
Timing: 5 minutes
The main difference in the sum of habituation is between the three procedural differences
discussed here. Follow choice A if you are working out a long habit. Follow choice B if you are
carrying out a fast habit. If no 'habituation' is done (option C), then move to Phase 2 directly.
A. Long habit (repeat for three consecutive days)
1. The investigator should place the mouse facing the wall that is closest to the experimenter
in the empty open field and encourage it to explore the open field for 5 minutes.
2. The investigator should then send the mouse to its cage at home.
3. To eliminate olfactory signals, the investigator should disinfect the open field with 70
percent (vol / vol) ethanol.
4. Move 1A(i – iii) replicate 6 h later.
B. Short Habituation
18
1. The investigator should place the mouse facing the wall that is closest to the experimenter
in the empty open field and encourage it to explore the open field for 5 minutes.
2. Next, the investigator should send the mouse to its cage at home.
3. To eliminate olfactory signals, the investigator should then disinfect the open field with 70
percent (vol / vol) ethanol.
C. No Habituation
1. Skip to Phase 2 immediately.
2.5.2 Session on Familiarization
1. Timing per click < 10 min
2. The investigator should then place the two similar items in the open area, 5 cm away from
the walls (Fig . 2), twenty-four hours after Step 1.
3. In the open area, the investigatort should then position the cursor, with its head located
opposite the objects. The investigator should then start a stopwatch to measure the time taken for
complete discovery to meet the 20-s criterion.
4. The investigator should then allow the mouse to act openly. To monitor the time spent
exploring and object, the investigator should then use a second stopwatch before 20 s of cumulative
exploration time has been achieved. Two channels must be fitted with the second stopwatch, with
each channel being used by each item to monitor exploration time.
5. The investigator should then then stop the experiment after all items have been explored
for 20-s or when a 10-min duration (i.e. the cumulative session time) is over.
6. Notice the time spent on the first stopwatch to meet the criteria and the times provided by
the second stopwatch's two channels as the times spent exploring each item (total 20 s).
7.Send the mouse to its cage at home.
19
2.5.3 Session cleaning post-familiarization
2 min pacing
8. The investigator should then clean the items and the open field with 70 percent (vol / vol)
ethanol during the familiarization session to eliminate olfactory cues.
9. Before the next use, the investigator should then dry the artifacts and the open area.
2.5.4 Testing session
Timing per click < 10 min
10. The investigator should then replace the two common objects, one with a triplicate replica
(to guarantee that the previously utilized item has no residual olfactory indication) and the other
with a new one. Place them 5 cm away from the walls, at the same spot.
2.5.5 Phase Crucial
It is important to randomize the location of the novel object (left or right) between each mouse
and each checked category.
11. Steps 3-7 replicate.
2.5.6 Cleanup for post-test session
2 min pacing
12. The investigator should then clean the artifacts and the open field with 70 percent (vol /
vol) ethanol during the evaluation session to eliminate any olfactory clues.
13. Before the next use, the investigator should dry the artifacts and the open area.
10 min 5 min
20
Fig 4 Novel Object Test
2.6 Five-Trial Social Memory Test
In mouse models of CNS conditions, the Five-Trial Social Memory evaluation assesses
cognition, including the capacity to remember novel and common animals. Rodents will grow
conditioned to intruders through the span of many exposures and no longer find them as fascinating
as a brand fresh intruder. Four brief exposures to the same intruder in their home cage are offered
to the subject during training. The topic meets an entirely different attacker in the fifth trial. Each
route requires 1 minute.
For complete body examination, anogenital investigation, perioral investigation, body
investigation, grooming activity, sexual contact, and otherwise unknown interaction, all case test
trials are videotaped and subsequently examined. This examination is useful for the detection of
social amnesia in transgenic mice with autism symptoms or other deficiencies in social contact,
and for the identification of novel chemical entities influencing social memory. (Fig 5)
Fig 5 Five Trails Test
21
Chapter3:Results
3.1 C9orf72 protein levels decreased in crossed C9orf72+/- and
C9BAC mice
A C9ORF72 bacterial artificial chromosome (BAC) from C9ALS/FTD patient DNA was
used to generate re-center, gain-of-function mouse models under the influence of endogenous
regulatory elements. Interestingly, even at advanced ages, three out of four of these C9-BAC
transgenic mice did not develop motor activity deficits [51-53]. We hypothesized that C9orf72 has
neuroprotective effects against motor defects in C9-BAC mice because these C9-BAC mouse
models contain elevated C9orf72 proteins from the endogenous mouse gene.
We crossed C9orf72+/− mice with C9-BAC mice to test this hypothesis and investigate the
in vivo significance of C9orf72 haploinsufficiency and investigated the effects of C9orf72 protein
dose reduction (loss-of-function) in the context of C9-BAC (gain-of-function). We find that motor
behavior deficits are aggravated by C9orf72 loss and haploinsufficiency in a dose-dependent
manner. This occurs early in the course of pathogenesis (4 months of age). We selected the one
with motor deficits from among the four reported C9-BAC mouse models (we refer to this C9orf72
BAC Tg/+ model as the C9-BAC line here) [10]. For two generations, we crossed C9orf72+/− and
C9-BAC mice to decrease the levels of C9orf72 protein at distinct doses. Proteins from brain
tissues were isolated, and the predicted reduction in the dose of C9orf72 protein was confirmed
(Fig. 6a). The unchanged protein level of Atg101, which, based on our previous study [16], is
correlated with the C9orf72 / Smcr8 complex, indicates the specificity of reducing C9orf72
(Fig .6a).
22
3.2 4-month-old Mutant mice performed no Anxiety issues
We tracked a cohort of mice [20 WT (10 females + 10 males), 18 C9-BAC (11 females + 7
males), 26 C9orf72+/-;C9-BAC (14 females + 12 males), and 19 C9orf72-/-;C9-BAC (10 females
+ 9 males)] to study the impact of C9orf72 deficiency on the motor behaviors of C9-BAC mice.
For the following reasons, we omitted C9orf72+/- and C9orf72-/- mice: heterozygous and
homozygous C9orf72 KO mice displayed no neurodegeneration and motor defects based on
previous studies[8]; complete deletion of C9orf72, which does not occur in patients with C9ALS
/ FTD, resulted in autoimmune disorders and reduced mouse survival[1], which may complicate
large-scale behavior and survival studies. We found that when habits were measured, there were
no substantial variations between the four studied groups in their survival for about 4 months.
Related body weights were also exhibited, taking into account the sex of the mice (Additional file
1: Figure S1B-1C). We conducted an open field test [3] to evaluate their general anxiety levels. In
total distance traveled, distance traveled in the center, and time spent in the center, C9-BAC mice
with various C9orf72 levels behaved similarly (Additional file 1: Figure S1E-1 G).
3.3 C9orf72 dose is critical for motor deficits in C9ALS/FTD mouse
models
By means of an accelerating (4-40 rpm in 5 min) rotarod test, we next examined their motor
control and balance. Five trials a day were given to mice, with an inter-trial interval of 20min, for
4 consecutive days. In C9-BAC female mice (Fig. 6b), and in C9-BAC male mice on day 4 of the
rotarod assay (Fig. 6c), a C9orf72 dose-dependent decrease in latency to fall was observed (Fig.
6b). These findings indicate that in C9-BAC mice, motor control is responsive to C9orf72 protein
levels. In female mice, we further studied motor learning. Over the course of 4 consecutive days,
23
WT mice displayed an improvement in latency to dropping, suggesting successful motor learning
(Fig. 6d). C9-BAC mice had increased latency to fall on day 2 but decreased on days 3 and 4 (Fig.
6d). Importantly, in C9orf72+/-; C9-BAC and C9orf72-/-;C9-BAC animals, there was no increase
in latency to fall from day 1 to day 4 (Fig. 6d). These findings indicate that in a dose-dependent
way, C9orf72 deficiency inhibited motor control and motor learning of C9-BAC mice.
Fig 6 Motor deficits in 4 months old C9ALS/FTD mouse
a, Western blot analysis of C9orf72 and Atg101 protein levels in 2-month-old mouse cortex. β-Actin serves as the loading
control. b, c Accelerating rotarod test was performed on 4-month-old mice to examine the latency to fall of females (b)
and males (c). C9orf72 deficiency decreases the latency to fall of C9-BAC female mice in a dose-dependent manner. d A 4-
consecutive-day rotarod assay reveals defective motor learning in C9orf72
+/−
; C9-BAC and C9orf72
−/−
;C9-BAC female
mice in comparison to WT. e, f Grip strength test was performed to measure front paw strength in 4-month-old females
(e) and males (f). All data are presented as mean ± SEM using numbers (n) of mice as indicated. Statistical analyses were
performed with one-way ANOVA with Bonferroni’s post hoc test (*p < 0.05, **p < 0.01, ***p < 0.001, N.s represents no
significant difference detected in measurement of 2d, 3d, or 4d in comparison to that of 1d)
3.4 Motor dysfunction in C9ALS/FTD mice also shows age-
dependent
In an age- and dose-dependent way, C9orf72 dysfunction promotes motor defects of C9-BAC
mice. We previously stated that loss of C9orf72 induces the initiation of motor defects at the age
of 4 months in C9-BAC mice [44]. We speculated that when animals matured, these behavioral
defects will grow more serious. We based on female mice to evaluate this hypothesis, which
showed more robust and stable phenotypes than male mice [44]. In the history of C9-BAC mice,
distance traveled in the center, and time spent in the
center (Additional file 1: Figure S1E-1G).
We next examined their motor coordination and
balance using an accelerating (4–40rpm in 5min)
rotarod test. Mice were given five trials per day, with an
inter-trial interval of 20min, for 4 consecutive days. A
C9orf72 dose-dependent decrease in latency to fall was
detected in C9-BAC female mice (Fig. 1b), and in
C9-BAC male mice on day 4 of the rotarod assay
(Fig. 1c). These results suggest that motor coordination
is sensitive to C9orf72 protein levels in C9-BAC mice. We
further analyzed motor learning in female mice. WT mice
exhibited an increase in latency to fall over the course
of 4 consecutive days, indicating active motor learning
(Fig. 1d). Latency to fall of C9-BAC mice was increased
on day 2 but dropped on days 3 and 4 (Fig. 1d). Impor-
tantly, there was no increase in latency to fall from day 1
to day 4 in C9orf72
+/−
;C9-BAC and C9orf72
−/−
;C9-BAC
animals (Fig. 1d). These results suggest that C9orf72 defi-
ciency impaired motor coordination and motor learning
of C9-BAC mice in a dose-dependent manner.
To examine motor strength, we measured forearm
grip strength and found that it was significantly
reduced in both male and female C9orf72
−/−
;C9BAC
animals compared to other genotypes (Fig. 1e, f).
Lastly, we measured the maximal speed at which each
animal fell from the rotarod device. Results showed that
C9orf72 deficiency, in a dose-dependent manner,
decreased the maximum speed at which C9-BAC mice
fell (Additional file 1: Figure S1H, S1I), which is
consistent with the data on their latency to fall.
The rotarod assay revealed more evident motor impair-
ment in female mice than in male mice. This could be due
to toxic gain-of-function since C9-BAC female mice
exhibitedearlierand more pronouncedabnormalities than
male mice [10]. It will be important to examine using
similar cohorts of mice whether motor neurons (MNs)
degenerate or reduce in number in a C9orf72 dose-
dependent manner and whether these deficits correlate
with the observed motor behavior deficits. Future studies
should also investigate whether C9orf72 exhibits
dose-dependent effects in the three other C9-BAC mouse
models [7, 12, 13]. It will be informative to examine the
effects of C9orf72 deficiency in the background of
adeno-associated virus (AAV)-mediated G4C2 repeat
expression [2]. Our study indicates that C9orf72 haploin-
sufficiency contributes to disease onset in a mouse model
by exacerbating the pathogenic effects of RNA/DPR-me-
diated neurotoxicity. Together with a recent report on
patient iPSC-derived MNs [15], this study suggests indeed
that we should focus more on the combination of loss-
and toxic gain-of-function. Together, for the first time,
our mouse genetic studies showed that C9orf72 loss or
haploinsufficiency in a gain-of-function mouse model of
Fig. 1 C9orf72 dose is critical for motor deficits in C9ALS/FTD mouse models.a Western blot analysis of C9orf72 and Atg101 protein levels in 2-month-old
mouse cortex.β-Actin serves as the loading control. b, c Accelerating rotarod test was performed on 4-month-old mice to examine the latency to fall of
females (b)andmales(c). C9orf72 deficiency decreases the latency to fall of C9-BAC female mice in a dose-dependent manner. dA4-consecutive-day
rotarod assay reveals defective motor learning in C9orf72
+/−
;C9-BAC and C9orf72
−/−
;C9-BAC female mice in comparison to WT. e, f Grip strength test was
performed to measure front paw strength in 4-month-old females (e)andmales(f). All data are presented as mean±SEM using numbers (n) of mice as
indicated. Statistical analyses were performed with one-way ANOVA with Bonferroni’sposthoctest(*p<0.05, **p<0.01, ***p<0.001,N.srepresentsno
significant difference detected in measurement of 2d, 3d, or 4d in comparison to that of 1d)
Shao et al. Acta Neuropathologica Communications (2019) 7:32 Page 2 of 3
24
we removed proteins from spinal cord tissues and reported C9orf72 protein dose reduction (Fig.
6A). Atg101 is aligned with the C9orf72 / Smcr8 protein complex [34] and has not modified its
protein amount, showing the specificity of a decrease in the C9orf72 dose (Fig. 6A).
A 10-month cohort of female mice, including WT (n=12), C9-BAC (n=9), C9orf72+/-; C9-
BAC (n=16), and C9orf72-/-; C9-BAC (n=9), is tracked. Neurodegeneration and motor defects in
mice were not caused by C9orf72 heterozygous or homozygous mutation in neurons [43]. In mice,
total failure of activity of C9orf72, which does not occur in patients with C9ALS / FTD, has lead
to autoimmune disorders [45]. We omitted C9orf72+/- and C9orf72-/- mice from our behavior
experiments for these purposes and reduced overall animal usage. There were no major variations
in their longevity and body weights within these four classes at 10-month-old age, when motor
activities were performed. Using an accelerated (5-40 rpm in 5 min) rotarod test, we assessed their
motor control and balance. For four consecutive days with an inter-trial period of 20 minutes, mice
were given five trials a day. A C9orf72 dose-dependent decrease in latency was shown to decrease
in C9-BAC mice for four consecutive days (Fig. 7A). During the course of these 4 days, wild-type
(WT) and C9-BAC mice displayed an improvement in latency to decline, indicating their
successful learning processes (Fig. 7B). C9orf72+/-; C9-BAC or C9orf72-/-; C9-BAC mice, on
the other side, struggled to improve their drop latency (Fig. 7B), meaning that their motor learning
is hampered. These findings indicate that C9orf72 impairment interferes in a dose-dependent way
with the motor control and learning of C9-BAC mice.
Next, we tested the power of the forearm grip and observed a strict association between the
dose reduction of C9orf72 and the impairment of grip strength in 10-month-old mice (Fig. 7C).
On the other side, we only observed a substantial gap in Rotarod latency time between WT and
C9orf72-/-; C9-BAC mice at the age of 4 months. It has significant difference on the fourth day
25
testing (Fig. 7D), showing an age-dependent grip strength disturbance. We examined 4- and 10-
month age activity results to explore age-dependent motor deficits. C9orf72+/-; C9-BAC mice
reported more serious deficits in grip power and latency to collapse at 10 months of age relative to
4 months of age (Fig. 7D, 7E). Together, these observations indicate that C9orf72 dysfunction in
C9-BAC mice exacerbates motor activity defects in an age- and dose-dependent way.
a b
c d
26
e
Fig 7 Exacerbate motor deficits in 10 months old C9ALS/FTD mouse
a, the 10-month-old mice were exposed to accelerated rotarod test to test for female (b) latency. C9orf72 deficiency
reduces dose-dependent latency of C9-BAC female mice. b The C9orf72+/−, C9-BAC and C9orf72−/−; C9-BAC female
mice, relative to WT, displayed learning defects during the 4-day rotarod trial. c, Grip strength test in 10-month-old
females to assess front paw strength was conducted. d, Compare WT and C9+/-C9BAC groups on 4 months and 10
months old, there is a significant increase gap on the 4th day test. e, Relative to WT, for C9+/-C9BAC groups on 4 months
and 10 months old, there is a substantial gap on the grip strength test. All data is shown as mean ± SEM using mice (n) as
shown. A single way ANOVA statistical study with the examination of Bonferroni (* p < 0.05, * * * p < 0.01, * * * p <
0.001, N.s does not imply a major difference in 2d , 3d or 4d relative to the 1d)
3.5 C9ALS/FTD mouse shows no social defects at 4 months old
Since both familial ALS and FTD are associated with C9orf72 protein, we started
checking FTD phenotype on 4 months old mutant mice. For Five trail tests, between all groups,
there is no social memory defects (Fig. 8a). Meanwhile, the mice displayed normal object
recognition ability (Fig. 8b). In order to further examine, we also perform three chamber tests.
Unfortunately, there is no significant differences for all group mice which suggesting c9 dosage
doesn't affect 4 months old mice social ability like social memory (Fig. 9a) and social novelty
(Fig. 9b). Therefore, we believe c9 deficiency is not critical for C9 FTD pathogenesis at least in
the early stage of development.
d-4 months
d-10 months
-40
-20
0
20
40
60
Difference: 4 months - 10 months
✱✱✱✱
27
Fig 8 a, Five Trails Test; b, Novel Object Test
All data are presented as mean ± SEM using numbers (n) of mice as indicated. Statistical analyses were performed with
one-way ANOVA with Bonferroni’s post hoc test (*p < 0.05, **p < 0.01, ***p < 0.001, N.s represents no significant
difference detected in measurement of 2d, 3d, or 4d in comparison to that of 1d)
Fig 9 Three Chamber Test
All data are presented as mean ± SEM using numbers (n) of mice as indicated. Statistical analyses were performed with
one-way ANOVA with Bonferroni’s post hoc test (*p < 0.05, **p < 0.01, ***p < 0.001, N.s represents no significant
difference detected in measurement of 2d, 3d, or 4d in comparison to that of 1d)
28
29
Chapter4:Discussion
4.1 The interaction of aging and loss- and gain-of-function in
C9ALS/FTD causes motor deficits phenotypes
In an age- and dose-dependent way, C9orf72 dysfunction promotes motor defects of C9-BAC
mice. We also previously stated that C9orf72 deficiency induces the initiation of the motor deficit
of C9-BAC mice at the age of 4 months [54]. We found here that C9orf72 deficiency intensified
the development of the motor deficit of C9-BAC mice in an age- and dose-dependent way.
Compared to WT samples, only C9orf72-/-; C9-BAC mice exhibited decreased grip power at 4
months of age. In comparison, a strict C9orf72 dose-dependent effect on the grip power of C9-
BAC mice was observed in 10-month-old mice. At 10-, but not 4-, month-old age, C9orf72+/-;
C9-BAC mice exhibited a decline in grip power. Similarly, in the rotarod assay, C9orf72+/-; C9-
BAC mice had a more extreme decrease in latency to fall at 10- than 4-month-old era. Therefore,
aging, loss- and gain-of-function interact together in C9ALS / FTD mouse models to facilitate
motor activity deficits.
4.2 The evaluation of the gain-of-function FVB-C9BAC mouse
model
Why use the gain-of-function FVB-C9-BAC mouse model [19]? This is because motor
activity defects [20-21] were generated by this unique C9-BAC mouse model. We are mindful of
the mixed genetic history of our compound mutant mice and that, relative to C57BL/6 mice, the
FVB strain could be more vulnerable to excitotoxicity and show more severe neuronal death. For
30
more than two generations, all FVB-C9-BAC mice were crossed into C57BL/6J-C9orf72+/- mice
prior to our behavioral experiments, which minimized FVB context influences. After crossing with
C57BL/6J-C9orf72+/- all progenies of C9-BAC mice lived for 10 months, which differs with
~30%-35% mortality of FVB-C9-BAC mice between 20 and 40 weeks of age [19]. A mixed
genetic history in mouse models could be useful from the point of view of modeling illness. It has
been proposed, for example, to integrate genetic variation to boost Alzheimer's disease
translatability [55]. We used a broad cohort mouse, calculated at various time points, and centered
on female mice, to reduce the genetic history impact. Most notably, these trials have resulted in
statistically meaningful and reproducible motor dysfunction findings for C9-BAC mice with
various dose reductions for C9orf72. We are in the process of producing pure genetic C9orf72+/-;
C9-BAC mice.
4.3 Potential mechanisms of pathological motor dysfunction in
C9ALS/FTD mice
Interaction of loss and gain of function in the promotion of motor defects in C9ALS / FTD.
Motor defects were formed by the mixture of C9orf72+/- heterozygous and C9-BAC mice, not
separately. These findings pose the risk that, by exacerbating the pathogenic consequences of
toxicity gain, C9orf72 haploinsufficiency leads to disease. Alternatively, the two systems operate
on separate processes in tandem, both of which are necessary for the regulation of motor actions.
We carried out pathological experiments to shed light on this problem and noticed that C9orf72
deficiency facilitates the aggregation of RNA foci and DPRs in the CNS of C9-ABC mice, namely
poly (GA) and poly (GR). Poly (GA)/(GR) has been well-documented in different cell types and
disease models for their toxicity; poly (GA) ectopic expression contributes to motor defects in
31
mice [56]. Our observations thus confirm the idea that the mutation of C9orf72 exacerbates the
toxic RNA and DPRs, contributing to more serious motor defects in C9-BAC mice. How do
processes for failure and gain-of-function interact? It has been proposed that deficiency of C9orf72
facilitates poly (PR) clearance in cultured cells [58]. Alternatively, DPR protein biogenesis can be
increased by C9orf72 depletion. Stress and neuronal excitation [57-59] was stimulated by repeat-
associated non-AUG translation, which may be affected by C9orf72 deficiency-mediated
autophagy-lysosomal dysfunction accompanied by protein homeostasis disturbance [41,60]. To
overcome these problems, further mechanistic studies are required.
There are a few drawbacks to our research that can be clarified for the following purposes. In
postmortem patient brains, the number of inclusions for poly (PR) and poly (PA) is comparatively
rare [61,62]. We are unable to detect robust poly (PR)/(PA) signals in CNS tissues of 9-month-old
mice, consistent with clinical pathology evidence, which results in our relative narrow emphasis
on poly(GA)/(GR). In postmortem patient brains, relative to antisense RNA foci[63], more cells
produce sense RNA foci. We argued that comparing relatively low levels of antisense RNA foci
with C9orf72 dose reductions across different genotypes may be difficult. We concentrated on the
sensory RNA foci, thus. In C9-BAC mouse models, we observed that C9orf72 deficiency
increased the levels of sense RNA foci in the spinal cord, hippocampus, and cerebellum. In
addition to encouraging translation of DPRs, these findings show that C9orf72 depletion regulates
the transcription or turnover of RNA foci. We have not observed variations in hippocampus poly
(GA) abundance; among various genotypes, RNA foci appear identical in the cortex. This may be
induced in these CNS regions by comparatively less abundant staining of poly (GA) or RNA foci,
or the dose reduction-mediated modifications in C9orf72 are relatively subtle, rendering it
theoretically difficult to measure these variations. Alternatively, organisms are not old enough,
32
which is compatible with the relationships in the pathogenesis of C9ALS / FTD between ageing,
failure and gain-of-function.
In the NMJs of C9-BAC mice, C9orf72 deficiency disrupts axonal transmission of MNs and
facilitates axonal swelling. Autophagy-lysosomal functions [41,45] are controlled by C9orf72.
C9orf72 protein is also localized to the presynaptic terminals of MNs [64] in addition to its
interaction with lysosomes in cellular bodies. The experiments also posed the likelihood that
deficiency of C9orf72 impairs the autophagy-lysosomal degradation of synaptic proteins or
organelles, which in C9ALS / FTD mouse models leads to motor deficits. Future experiments
could decide how this axonal swelling deficit facilitates toxicity advantage.
Chapter5:Conclusion
Here we stated that in an age- and C9orf72 dose-dependent way, C9orf72 deficiency
promotes motor deficit progression of a C9ALS / FTD gain-of-function mouse model. For the
first time, these experiments presented in vivo proof of the relationship between processes of loss
and gain of function in fostering defects in motor activity in C9ALS/FTD.
33
References
1. Geser F, Lee VM-Y, Trojanowski JQ. Amyotrophic lateral sclerosis and frontotemporal lobar
degeneration: a spectrum of TDP-43 proteinopathies. Neuropathology. John Wiley & Sons, Ltd
(10.1111); 2010;30:103–12.
2. Ferrari R, Kapogiannis D, Huey ED, Momeni P. FTD and ALS: a tale of two diseases. Curr
Alzheimer Res. NIH Public Access; 2011;8:273–94.
3. Strong MJ. The syndromes of frontotemporal dysfunction in amyotrophic lateral sclerosis.
Amyotroph Lateral Scler. Taylor & Francis; 2008;9:323–38.
4. Lomen-Hoerth C, Anderson T, Miller B. The overlap of amyotrophic lateral sclerosis and
frontotemporal dementia. Neurology. 2002;59:1077–9.
5. Neumann M, Sampathu DM, Kwong LK, Truax AC, Micsenyi MC, Chou TT, et al.
Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis.
Science. American Association for the Advancement of Science; 2006;314:130–3.
6. Arai T, Hasegawa M, Akiyama H, Ikeda K, Nonaka T, Mori H, et al. TDP-43 is a component
of ubiquitin-positive tau-negative inclusions in frontotemporal lobar degeneration and
amyotrophic lateral sclerosis. Biochem. Biophys. Res. Commun. 2006;351:602–11.
7. Gijselinck I, Van Langenhove T, van der Zee J, Sleegers K, Philtjens S, Kleinberger G, et al. A
C9orf72 promoter repeat expansion in a Flanders-Belgian cohort with disorders of the
frontotemporal lobar degeneration-amyotrophic lateral sclerosis spectrum: a gene identification
study. Lancet Neurol. 2012;11:54–65.
8. Renton AE, Majounie E, Waite A, Simón-Sánchez J, Rollinson S, Gibbs JR, et al. A
hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-
FTD. Neuron. 2011;72:257–68.
9. DeJesus-Hernandez M, Mackenzie IR, Boeve BF, Boxer AL, Baker M, Rutherford NJ, et al.
Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes
chromosome 9p-linked FTD and ALS. Neuron. 2011;72:245–56.
10. Majounie E, Renton AE, Mok K, Dopper EGP, Waite A, Rollinson S, et al. Frequency of the
C9orf72 hexanucleotide repeat expansion in patients with amyotrophic lateral sclerosis and
frontotemporal dementia: a cross-sectional study. Lancet Neurol. 2012;11:323–30.
11. Gao F-B, Almeida S, Lopez-Gonzalez R. Dysregulated molecular pathways in amyotrophic
lateral sclerosis-frontotemporal dementia spectrum disorder. EMBO J. EMBO Press;
2017;36:2931–50.
12. Ling S-C, Polymenidou M, Cleveland DW. Converging mechanisms in ALS and FTD:
disrupted RNA and protein homeostasis. Neuron. 2013;79:416–38.
34
13. Taylor JP, Brown RH, Cleveland DW. Decoding ALS: from genes to mechanism. Nature.
Nature Publishing Group; 2016;539:197–206.
14. Gendron TF, Petrucelli L. Disease Mechanisms of C9ORF72 Repeat Expansions. Cold Spring
Harb Perspect Med. 2018;8:a024224.
15. Chew J, Gendron TF, Prudencio M, Sasaguri H, Zhang Y-J, Castanedes-Casey M, et al.
Neurodegeneration. C9ORF72 repeat expansions in mice cause TDP-43 pathology, neuronal
loss, and behavioral deficits. Science. 2015;348:1151–4.
16. Chew J, Cook C, Gendron TF, Jansen-West K, Del Rosso G, Daughrity LM, et al. Aberrant
deposition of stress granule-resident proteins linked to C9orf72-associated TDP-43
proteinopathy. Mol Neurodegener. BioMed Central; 2019;14:9.
17. Liu Y, Pattamatta A, Zu T, Reid T, Bardhi O, Borchelt DR, et al. C9orf72 BAC Mouse Model
with Motor Deficits and Neurodegenerative Features of ALS/FTD. Neuron. 2016;90:521–34.
18. O'Rourke JG, Bogdanik L, Muhammad AKMG, Gendron TF, Kim KJ, Austin A, et al. C9orf72
BAC Transgenic Mice Display Typical Pathologic Features of ALS/FTD. Neuron.
2015;88:892–901.
19. Peters OM, Cabrera GT, Tran H, Gendron TF, McKeon JE, Metterville J, et al. Human
C9ORF72 Hexanucleotide Expansion Reproduces RNA Foci and Dipeptide Repeat Proteins
but Not Neurodegeneration in BAC Transgenic Mice. Neuron. 2015;88:902–9.
20. Jiang J, Zhu Q, Gendron TF, Saberi S, McAlonis-Downes M, Seelman A, et al. Gain of
Toxicity from ALS/FTD-Linked Repeat Expansions in C9ORF72 Is Alleviated by Antisense
Oligonucleotides Targeting GGGGCC-Containing RNAs. Neuron. 2016;90:535–50.
21. Conlon EG, Lu L, Sharma A, Yamazaki T, Tang T, Shneider NA, et al. The C9ORF72
GGGGCC expansion forms RNA G-quadruplex inclusions and sequesters hnRNP H to disrupt
splicing in ALS brains. Elife. eLife Sciences Publications Limited; 2016;5:345.
22. Gendron TF, Bieniek KF, Zhang Y-J, Jansen-West K, Ash PEA, Caulfield T, et al. Antisense
transcripts of the expanded C9ORF72 hexanucleotide repeat form nuclear RNA foci and
undergo repeat-associated non-ATG translation in c9FTD/ALS. Acta Neuropathol. Springer
Berlin Heidelberg; 2013;126:829–44.
23. Haeusler AR, Donnelly CJ, Periz G, Simko EAJ, Shaw PG, Kim M-S, et al. C9orf72 nucleotide
repeat structures initiate molecular cascades of disease. Nature. Nature Publishing Group;
2014;507:195–200.
24. Zhang Y-J, Jansen-West K, Xu Y-F, Gendron TF, Bieniek KF, Lin W-L, et al. Aggregation-
prone c9FTD/ALS poly(GA) RAN-translated proteins cause neurotoxicity by inducing ER
stress. Acta Neuropathol. Springer Berlin Heidelberg; 2014;128:505–24.
35
25. Zhang Y-J, Gendron TF, Grima JC, Sasaguri H, Jansen-West K, Xu Y-F, et al. C9ORF72
poly(GA) aggregates sequester and impair HR23 and nucleocytoplasmic transport proteins.
Nature Neuroscience. Nature Publishing Group; 2016;19:668–77.
26. May S, Hornburg D, Schludi MH, Arzberger T, Rentzsch K, Schwenk BM, et al. C9orf72
FTLD/ALS-associated Gly-Ala dipeptide repeat proteins cause neuronal toxicity and Unc119
sequestration. Acta Neuropathol. 2014;128:485–503.
27. White MR, Mitrea DM, Zhang P, Stanley CB, Cassidy DE, Nourse A, et al. C9orf72 Poly(PR)
Dipeptide Repeats Disturb Biomolecular Phase Separation and Disrupt Nucleolar Function.
Molecular Cell. 2019.
28. Freibaum BD, Lu Y, Lopez-Gonzalez R, Kim NC, Almeida S, Lee K-H, et al. GGGGCC repeat
expansion in C9orf72 compromises nucleocytoplasmic transport. Nature. Nature Publishing
Group; 2015;525:129–33.
29. Zhang Y-J, Gendron TF, Ebbert MTW, O'Raw AD, Yue M, Jansen-West K, et al. Poly(GR)
impairs protein translation and stress granule dynamics in C9orf72-associated frontotemporal
dementia and amyotrophic lateral sclerosis. Nat. Med. Nature Publishing Group;
2018;24:1136–42.
30. Wen X, Tan W, Westergard T, Krishnamurthy K, Markandaiah SS, Shi Y, et al. Antisense
proline-arginine RAN dipeptides linked to C9ORF72-ALS/FTD form toxic nuclear aggregates
that initiate in vitro and in vivo neuronal death. Neuron. 2014;84:1213–25.
31. Mizielinska S, Grönke S, Niccoli T, Ridler CE, Clayton EL, Devoy A, et al. C9orf72 repeat
expansions cause neurodegeneration in Drosophila through arginine-rich proteins. Science.
American Association for the Advancement of Science; 2014;345:1192–4.
32. Jovičić A, Mertens J, Boeynaems S, Bogaert E, Chai N, Yamada SB, et al. Modifiers of
C9orf72 dipeptide repeat toxicity connect nucleocytoplasmic transport defects to FTD/ALS.
Nature Neuroscience. Nature Publishing Group; 2015;18:1226–9.
33. Jung J, Nayak A, Schaeffer V, Starzetz T, Kirsch AK, Müller S, et al. Multiplex image-based
autophagy RNAi screening identifies SMCR8 as ULK1 kinase activity and gene expression
regulator. Elife. eLife Sciences Publications Limited; 2017;6:2.
34. Yang M, Liang C, Swaminathan K, Herrlinger S, Lai F, Shiekhattar R, et al. A
C9ORF72/SMCR8-containing complex regulates ULK1 and plays a dual role in autophagy.
Sci Adv. American Association for the Advancement of Science; 2016;2:e1601167–7.
35. Amick J, Roczniak-Ferguson A, Ferguson SM. C9orf72 binds SMCR8, localizes to lysosomes,
and regulates mTORC1 signaling. Mol. Biol. Cell. American Society for Cell Biology;
2016;27:3040–51.
36. Sullivan PM, Zhou X, Robins AM, Paushter DH, Kim D, Smolka MB, et al. The ALS/FTLD
associated protein C9orf72 associates with SMCR8 and WDR41 to regulate the autophagy-
lysosome pathway. Acta Neuropathol Commun. BioMed Central; 2016;4:51.
36
37. Ugolino J, Ji YJ, Conchina K, Chu J, Nirujogi RS, Pandey A, et al. Loss of C9orf72 Enhances
Autophagic Activity via Deregulated mTOR and TFEB Signaling. PLoS Genet. Public Library
of Science; 2016;12:e1006443.
38. Sellier C, Campanari M-L, Julie Corbier C, Gaucherot A, Kolb-Cheynel I, Oulad-Abdelghani
M, et al. Loss of C9ORF72 impairs autophagy and synergizes with polyQ Ataxin-2 to induce
motor neuron dysfunction and cell death. EMBO J. 2016.
39. Webster CP, Smith EF, Bauer CS, Moller A, Hautbergue GM, Ferraiuolo L, et al. The C9orf72
protein interacts with Rab1a and the ULK1 complex to regulate initiation of autophagy. EMBO
J. EMBO Press; 2016;35:1656–76.
40. Wong E, Cuervo AM. Autophagy gone awry in neurodegenerative diseases. Nature
Neuroscience. 2010;13:805–11.
41. Kulkarni VV, Maday S. Compartment-specific dynamics and functions of autophagy in
neurons. Zhang H, Winckler B, Cai Q, editors. Dev Neurobiol. John Wiley & Sons, Ltd;
2018;78:298–310.
42. Sheng Z-H. The Interplay of Axonal Energy Homeostasis and Mitochondrial Trafficking and
Anchoring. Trends Cell Biol. 2017;27:403–16.
43. Koppers M, Blokhuis AM, Westeneng H-J, Terpstra ML, Zundel CAC, Vieira de Sá R, et al.
C9orf72 ablation in mice does not cause motor neuron degeneration or motor deficits. Ann.
Neurol. 2015;78:426–38.
44. Shao Q, Liang C, Chang Q, Zhang W, Yang M, Chen J-F. C9orf72 deficiency promotes motor
deficits of a C9ALS/FTD mouse model in a dose-dependent manner. Acta Neuropathol
Commun. BioMed Central; 2019;7:32.
45. Burberry A, Suzuki N, Wang J-Y, Moccia R, Mordes DA, Stewart MH, et al. Loss-of-function
mutations in the C9ORF72 mouse ortholog cause fatal autoimmune disease. Sci Transl Med.
2016;8:347ra93–3.
46. Maday S, Wallace KE, Holzbaur ELF. Autophagosomes initiate distally and mature during
transport toward the cell soma in primary neurons. J Cell Biol. Rockefeller University Press;
2012;196:407–17.
47. Cheng X-T, Zhou B, Lin M-Y, Cai Q, Sheng Z-H. Axonal autophagosomes recruit dynein for
retrograde transport through fusion with late endosomes. J Cell Biol. 2015;209:377–86.
48. Tammineni P, Ye X, Feng T, Aikal D, Cai Q. Impaired retrograde transport of axonal
autophagosomes contributes to autophagic stress in Alzheimer's disease neurons. Elife. eLife
Sciences Publications Limited; 2017;6:343.
49. Xie Y, Zhou B, Lin M-Y, Wang S, Foust KD, Sheng Z-H. Endolysosomal Deficits Augment
Mitochondria Pathology in Spinal Motor Neurons of Asymptomatic fALS Mice. Neuron.
2015;87:355–70.
37
50. Marangoni M, Adalbert R, Janeckova L, Patrick J, Kohli J, Coleman MP, et al. Age-related
axonal swellings precede other neuropathological hallmarks in a knock-in mouse model of
Huntington's disease. Neurobiol. Aging. 2014;35:2382–93.
51. Salvadores N, Sanhueza M, Manque P, Court FA. Axonal Degeneration during Aging and Its
Functional Role in Neurodegenerative Disorders. Front Neurosci. Frontiers; 2017;11:451.
52. Lüningschrör P, Binotti B, Dombert B, Heimann P, Perez-Lara A, Slotta C, et al. Plekhg5-
regulated autophagy of synaptic vesicles reveals a pathogenic mechanism in motoneuron
disease. Nat Commun. Nature Publishing Group; 2017;8:678.
53. Tankersley CG, Haenggeli C, Rothstein JD. Respiratory impairment in a mouse model of
amyotrophic lateral sclerosis. J. Appl. Physiol. 2007;102:926–32.
54. Mizielinska S, Lashley T, Norona FE, Clayton EL, Ridler CE, Fratta P, et al. C9orf72
frontotemporal lobar degeneration is characterised by frequent neuronal sense and antisense
RNA foci. Acta Neuropathol. Springer Berlin Heidelberg; 2013;126:845–57.
55. Lee Y-B, Chen H-J, Peres JN, Gomez-Deza J, Attig J, Stalekar M, et al. Hexanucleotide repeats
in ALS/FTD form length-dependent RNA foci, sequester RNA binding proteins, and are
neurotoxic. Cell Rep. 2013;5:1178–86.
55. Neuner SM, Heuer SE, Huentelman MJ, O'Connell KMS, Kaczorowski CC. Harnessing
Genetic Complexity to Enhance Translatability of Alzheimer's Disease Mouse Models: A Path
toward Precision Medicine. Neuron. 2019;101:399–411.e5.
56. Schludi MH, Becker L, Garrett L, Gendron TF, Zhou Q, Schreiber F, et al. Spinal poly-GA
inclusions in a C9orf72 mouse model trigger motor deficits and inflammation without neuron
loss. Acta Neuropathol. Springer Berlin Heidelberg; 2017;134:241–54.
57. Green KM, Glineburg MR, Kearse MG, Flores BN, Linsalata AE, Fedak SJ, et al. RAN
translation at C9orf72-associated repeat expansions is selectively enhanced by the integrated
stress response. Nat Commun. Nature Publishing Group; 2017;8:2005.
58. Cheng W, Wang S, Mestre AA, Fu C, Makarem A, Xian F, et al. C9ORF72 GGGGCC repeat-
associated non-AUG translation is upregulated by stress through eIF2α phosphorylation. Nat
Commun. Nature Publishing Group; 2018;9:51.
59. Westergard T, McAvoy K, Russell K, Wen X, Pang Y, Morris B, et al. Repeat-associated non-
AUG translation in C9orf72-ALS/FTD is driven by neuronal excitation and stress. EMBO Mol
Med. EMBO Press; 2019;11:e9423.
60. Corrionero A, Horvitz HR. A C9orf72 ALS/FTD Ortholog Acts in Endolysosomal Degradation
and Lysosomal Homeostasis. Curr Biol. 2018;28:1522–5.
61. Mackenzie IRA, Frick P, Grässer FA, Gendron TF, Petrucelli L, Cashman NR, et al.
Quantitative analysis and clinico-pathological correlations of different dipeptide repeat protein
38
pathologies in C9ORF72 mutation carriers. Acta Neuropathol. Springer Berlin Heidelberg;
2015;130:845–61.
62. Mackenzie IRA, Frick P, Neumann M. The neuropathology associated with repeat expansions
in the C9ORF72 gene. Acta Neuropathol. Springer Berlin Heidelberg; 2014;127:347–57.
63. DeJesus-Hernandez M, Finch NA, Wang X, Gendron TF, Bieniek KF, Heckman MG, et al.
In-depth clinico-pathological examination of RNA foci in a large cohort of C9ORF72
expansion carriers. Acta Neuropathol. Springer Berlin Heidelberg; 2017;134:255–69.
64. Frick P, Sellier C, Mackenzie IRA, Cheng C-Y, Tahraoui-Bories J, Martinat C, et al. Novel
antibodies reveal presynaptic localization of C9orf72 protein and reduced protein levels in
C9orf72 mutation carriers. Acta Neuropathol Commun. BioMed Central; 2018;6:72.
39
Supplement Figure 10
Fig. S1 4-month-old Mutant mice performed no Anxiety issues. (A, B) Body weight of female (A) and male (B) mice at
4 months of age. (C-E) Open field test was performed on 4-month-old mice to examine the total distance traveled (C),
distance traveled in the center (D), and percentage of time spent in the center (E). All data are presented as mean ± SEM
using numbers (n) of mice as indicated. Statistical analyses were performed with one-way ANOVA with Bonferroni’s post
hoc test (*p < 0.05, **p < 0.01, ***p < 0.001, n.s represents no significant difference detected).
Abstract (if available)
Abstract
Background: The most prominent hereditary amyotrophic lateral sclerosis and frontotemporal dementia (collectively, C9ALS/FTD) are triggered by hexanucleotide repeat expansion in C9ORF72 intron. The pathogenesis of C9ALS / FTD is due to C9ORF72 haploinsufficiency (loss of function) and toxicity benefit caused by toxic RNAs and dipeptide repeat proteins (DPRs), modelled using human C9ORF72 bacterial artificial transgenic chromosome mice (C9-BAC). It remains poorly known how loss- and gain-of-function communicate and the biological relevance of their interactions. ❧ Methods: In order to develop C9ALS/FTD mouse models (C9orf72+/-
Linked assets
University of Southern California Dissertations and Theses
Conceptually similar
PDF
Functional study of C9ORF72 and its implication in the pathogenesis of amyotrophic lateral sclerosis
PDF
The C9ORF72 repeat expansion impairs neurodevelopment
PDF
Modeling SynGAP1 truncating mutations in neurodevelopmental disease using iPSC-derived neurons
PDF
The splicing error of FOXP1 in type I myotonic dystrophy
Asset Metadata
Creator
Chang, Qing
(author)
Core Title
C9orf72 deficiency exacerbates motor deficits in C9ALS/FTD mouse models
School
Keck School of Medicine
Degree
Master of Science
Degree Program
Molecular Microbiology and Immunology
Publication Date
01/21/2021
Defense Date
11/12/2020
Publisher
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
axonal degeneration,C9ALS/FTD,C9ORF72,gain of toxicity,motor behaviors,OAI-PMH Harvest
Format
application/pdf
(imt)
Language
English
Contributor
Electronically uploaded by the author
(provenance)
Advisor
Landolph, Joseph (
committee chair
), Chen, JianFu (
committee member
), Schonthal, Axel (
committee member
), Zhen, Zhao (
committee member
)
Creator Email
qingc@usc.edu
Permanent Link (DOI)
https://doi.org/10.25549/usctheses-c89-416402
Unique identifier
UC11672269
Identifier
etd-ChangQing-9239.pdf (filename),usctheses-c89-416402 (legacy record id)
Legacy Identifier
etd-ChangQing-9239.pdf
Dmrecord
416402
Document Type
Thesis
Format
application/pdf (imt)
Rights
Chang, Qing
Type
texts
Source
University of Southern California
(contributing entity),
University of Southern California Dissertations and Theses
(collection)
Access Conditions
The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...
Repository Name
University of Southern California Digital Library
Repository Location
USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA
Tags
axonal degeneration
C9ALS/FTD
C9ORF72
gain of toxicity
motor behaviors