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Functional study of Snord118 in microglila
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Functional study of Snord118 in microglila
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Content
Functional Study of Snord118 in Microglia
by
Yuanning Du
A Thesis Presented to the
FACULTY OF THE USC [KECK SCHOOL OF MEDICINE]
UNIVERSITY OF SOUTHERN CALIFORNIA
In partial Fulfilment of the
Requirements for the Degree
MASTER OF SCIENCE
(Biochemistry and Molecular Medicine)
August 2021
Copyright [2021] Yuanning Du
ii
ACKNOWLEDGEMENTS
I want to express my gratitude to my Principal Investigator, Dr. Jianfu Chen, who guided and
encouraged me conscientiously and responsibly in researching and writing this thesis during my
whole Master's life in these two years. I really would like to thank him for allowing me to join
his lab, work with the entire lab family, and get involved within the research. I would also like to
express my gratitude to other thesis committee members, Dr. Jian Xu and Dr. Zhipeng Lu. Their
professional comments and suggestions give me a lot of inspiration. I want to thank all lab
members Li Ma, Wei Zhang, Chen Liang, and Whan Supawadee for being an enthusiastic and
supportive team. I want to thank the USC CCMB for supporting me using all kinds of research
apparatus. And I would like to express my special thanks to my parents for supporting me in
completing my Master's studies. Thank you, everyone, for your help and support!
iii
TABLE OF CONTENTS
Acknowledgements..........................................................................................................................ii
List of Figures..................................................................................................................................v
Abstract ........................................................................................................................................viii
Introduction......................................................................................................................................1
LCC and Snord118......................................................................................................................1
Gliosis and microglia...................................................................................................................3
Blood-brain-barrier (BBB) ..........................................................................................................6
Materials and methods.....................................................................................................................9
Animals........................................................................................................................................9
Immunostaining............................................................................................................................9
Imaging and quantification.........................................................................................................10
Chapter 1: Generation of Snord118 flox; Cx3cr1-Cre mouse line ...............................................11
Chapter 2: Snord118 mutation contributes to the abnormal activation of microglia.....................13
Chapter 3: Snord118 flox induces the activation of microglia to change their morphology.........19
Chapter 4: The microglia activation induced by Snord118 might contribute to pericyte phenotype
in blood-brain-barrier.....................................................................................................................23
Chapter 5: The microglia activation induced by Snord118 might contribute to the change of
endothelial tight junctions in blood-brain-barrier..........................................................................29
iv
Chapter 6: Snord118 mutation though microglia – neurovascular unit cross-talk to affect blood-
brain-barrier functions...................................................................................................................33
Conclusion.....................................................................................................................................37
References......................................................................................................................................40
v
LIST OF FIGURES
1-1: Magnetic resonance imaging (MRI) of LCC brain..................................................................1
1-2: Two kinds of structure of snoRNAs.........................................................................................2
1-3: Model of 5' end of U8 snoRNA bind to the 5' end of 28S rRNA to interact ribosome
biogenesis.........................................................................................................................................3
1-4: Microglia morphology under both surveillant and activated conditions in septofimbrial
nucleus, hippocampus, and hypothalamus.......................................................................................5
1-5: Microglia activation is involved in these three common neurodegenerative diseases,
Alzheimer's disease, Parkinson's disease, and Frontotemporal dementia........................................6
1-6: Structure of the neurovascular unit...........................................................................................8
2-1: Gel electrophoresis of PCR genotyping results......................................................................12
2-2: Summary of the genotyping results........................................................................................12
3-1: Iba1 and CD68 co-staining in the cortex of control, Snord118 flox/+; Cx3cr1-Cre and
Snord118 flox/flox; Cx3cr1-Cre mice...........................................................................................14
3-2: The number of microglia in the striatum, cortex, corpus callosum, hippocampus, and
cerebellum......................................................................................................................................15
3-3: The area of the cell body in each microglia in the striatum, cortex, corpus callosum,
hippocampus, and cerebellum........................................................................................................16
3-4: The area of CD68 positive signal among each microglia cell body in the striatum, cortex,
corpus callosum, hippocampus, and cerebellum............................................................................17
vi
4-1: The morphology of microglia in the striatum, cortex, corpus callosum, hippocampus, and
cerebellum of control, Snord118 flox/+; Cx3cr1-Cre, and Snord118 flox/flox; Cx3cr1-Cre
group..............................................................................................................................................21
4-2: The quantification method we used to analyze the morphology change of microglia...........21
4-3: The number of end branches/number of primary branches in different brain regions of
control, Snord118 flox/+; Cx3cr1-Cre, and Snord118 flox/flox; Cx3cr1-Cre group....................22
5-1: There are SMA positive signals in deep brain regions of laminin KO mice, but no SMA
signals in the cortex or hippocampus.............................................................................................23
5-2: (A) CD31 positive – pericyte, lectin positive, (B) quantification of pericyte coverage,
pericyte coverage is defined as a percentage of CD13-positive surface area covering lectin-
positive surface area per field........................................................................................................24
5-3: CD31, PDGFR beta, SMA, and Hoechst co-staining in the striatum of control, Snord118
flox/+; Cx3cr1-Cre, and Snord118 flox/flox; Cx3cr1-Cre
group..............................................................................................................................................25
5-4: The percentage of SMA+; PDGFR beta+; CD31+ pericytes/PDGFR beta+ ; CD31 +
pericytes in striatum, cortex, and hippocampus.............................................................................26
5-5: Lectin and CD13 co-staining in the cortex of control, Snord118 flox/+; Cx3cr1-Cre, and
Snord118 flox/flox; Cx3cr1-Cre group..........................................................................................27
5-6: Quantification of the percentage of the area of CD13/Lectin in striatum, cortex, and
hippocampus of control, Snord118 flox/+; Cx3cr1-Cre, and Snord118 flox/flox; Cx3cr1-Cre
group..............................................................................................................................................28
vii
6-1: Co-staining of the endothelial-specific marker CD31 and Claudin5 in controls and after
VEGF inhibition.............................................................................................................................29
6-2: Claudin-5 and CD31 co-staining in the cortex of control, Snord118 flox/+; Cx3cr1-Cre, and
Snord118 flox/flox; Cx3cr1-Cre group..........................................................................................30
6-3: Quantification of the area of Claudin-5 in each unit area, the percentage of the area of
Claudin-5 within the area of CD31 in striatum, cortex, and hippocampus of control, Snord118
flox/+; Cx3cr1-Cre, and Snord118 flox/flox; Cx3cr1-Cre group..................................................31
7-1: Iba1 and AQP4 co-staining in the WT mice and mice with chronic systemic
inflammation..................................................................................................................................33
7-2: Iba1 and AQP4 co-staining microglia to see the phagocytosis of astrocyte endfeet..............34
7-3: (A) AQP4 and Iba1 co-staining in the cortex of control, Snord118 flox/+; Cx3cr1-Cre, and
Snord118 flox/flox; Cx3cr1-Cre group; (B) quantification of the percentage of microglia with
AQP4 inclusion..............................................................................................................................35
viii
ABSTRACT
The Snord118 mutation, which is also called U8, will cause leukoencephalopathy with
calcifications and cysts. In addition, it will result in neurological diseases. This mutation affects
the ribosome biogenesis progress to change the translational capacity, which could cause
abnormal cell growth. Microglia is a kind of immune cell located in the central neuron system.
The abnormal activity of microglia is always relevant to cell death and neuronal dysfunctions.
Recently, a study group has found a marked dissociation of microglia mRNA and protein
networks will be followed with innate immune activation. The ribosome-associated mRNAs will
not be translated, suggesting that the abnormal ribosome biogenesis would affect immune
response in microglia. We use the microglia-specific Snord118 gene mouse model to explore
microglia's role in this disease and discover the pathogenesis. We find that the microglia are
activated with Snord118 mutation, the number of microglia is significantly increased, and the
microglia morphology is changed in several different brain regions.
1
INTRODUCTION
LCC and Snord118:
Leukoencephalopathy with brain calcification and cysts(LCC) is a sporadic autosomal recessive
genetic disease. A previous study found that a biallelic mutation in the non-protein-coding gene
SNORD118 may be a fundamental reason for LCC (Emma M. Jenkinson et al., 2011). Patients
with LCC disease are often accompanied by symptoms, including nausea, vomiting, and ataxia.
Children also have delayed motor development. In all of these cases, their brain imaging
examinations revealed diffuse leukoencephalopathy and multiple cystic encephalopathies.
Histopathology also found that their brains will develop particular vascular diseases and
abundant Rosenthal fibers (K. Ummer et al., 2010).
Figure 1-1: Magnetic resonance imaging (MRI) of LCC brain: (b) A large right cerebellar cyst in
the right part of patient's brain; (d) brain with multiple calcifications (K. Ummer et al., 2010,)
Snord118, which is also called U8, is a kind of small nucleolar RNA (snoRNA). They will guide
the modifications of other RNAs. There are two standard classes of snoRNAs. The first one has a
conserved sequence motif C/D box (C (RUGAUGA) and D (CUGA)). They are associated with
2
methylation. Another one has H/ACA (H (ANANNA) and ACA) box, which is associated with
pseudouridylation (Tama's Kiss, 2002).
Figure 1-2: Two kinds of structure of snoRNAs (Tama' s Kiss, 2002)
Most of the time, rRNAs, tRNAs, and snRNAs need to be transcripted and modified precisely to
generate mature RNAs to realize their functions. Ribosome biogenesis is a complex biological
process in eukaryotes' development, which involved many different components in joining in and
steps to process. RNA polymerase (pol) I transcribe ribosome DNA to yield a single strand pre-
rRNA, which needs to be processed and modified to generate mature 18S, 5.8S, and 28S rRNAs.
The previous study has found that snoRNAs will affect 2′-O-methylation and pseudouridylation
on 18S, 5.8S, and 28S rRNAs (Kazimierz T. Tycowski et al., 1998).
U8 snoRNA has been proved to play an essential role in pre-rRNA processing, especially in
generating mature 28S and 5.8S rRNAs. The 5'end of U8 snoRNA can bind to the 5'end of 28S
rRNA to inhibit the interaction between 5'end of 28S and 3'end of 5.8S. This interaction should
appear in the normal mature ribosome. The function that U8 snoRNA can pair with 5'end of 28S
might alter the pre-rRNA folding pathway, which will affect the ribosome biogenesis (Brenda A.
Peculis, 1997). And then change the translational capacity to cause abnormalities in cell growth.
3
Figure 1-3: Model of 5' end of U8 snoRNA bind to the 5' end of 28S rRNA to interact ribosome
biogenesis (Brenda A. Peculis, 1997)
Gliosis and microglia:
One pathology of LCC is gliosis. Gliosis is a typical response of the central nervous
system(CNS) to neural injury, inflammation, or damage. Gliosis always presents in many kinds
of neural diseases, such as Alzheimer's disease, Korsakoff's syndrome, AIDS dementia complex,
Parkinson's disease, amyotrophic lateral sclerosis(ALS), etc. Under all of those cases, gliosis can
cause hypertrophy or proliferation in different glial cells, including astrocytes, microglia, and
oligodendrocytes. It will alter normal cellular activities and functions, generate widespread
effects on neurons and other cells. Among them, microglia are the most sensitive cells when
reacting to damage. They will give a rapid response, the microgliosis, or called activation of
microglia. And this initial microglia activity will regulate the astrogliosis. Some previous studies
have identified that the pro-inflammatory signals released by microglia can trigger astrogliosis.
After reducing microglia activation, the number of astrocytes also decreased (Dan Zhang et al.,
2013).
Microglia are the only vertical mesoderm cells that come from nerve tissue. Their nucleus is
small, about 5μm. They have different irregular shapes. They are located throughout the brain
4
and spinal cord, account for about 10–15% of all cells found within the brain. Microglia play a
significant immune role in the physiological processes of the central nervous system (CNS)
(Anthony J. Filiano et al., 2015). Microglia morphology has a high degree of plasticity, which is
closely related to its biological function status. In normal brain tissue, microglia are highly
ramified, within complicated structures, and the branches between cells rarely overlap.
Branched microglia are often called "resting/surveillant microglia." Resting microglia can make
contact directly with neuronal synapses to monitor the functional status of synapses. Under
normal circumstances, highly branched resting microglia provide a highly dynamic and efficient
monitoring system for the brain. When faced with inflammation, infection, or other neurological
diseases in the brain, microglia will be quickly activated and gain phagocytosis to clean the
damages. During the microglia activation, we can observe changes in their morphology. The
activated microglia cell body becomes larger, the branches become shorter, and the cell
morphology changes. When these activated microglia cells are further activated and adjusted, the
branches will disappear, the cell morphology will be amoebic, and they will obtain phagocytic
function. Therefore, we often define the activation state of microglia through their morphological
changes, and the activation state of microglia is usually closely related to the severity of the
damaged parts of the brain (Debasis Nayak et al., 2014). A previous study has observed that the
microglia morphology in different brain regions is not entirely the same. Only as for the resting
microglia, they have different morphologies in different areas. They are still various after the
morphology changes (Marí a del Mar Ferná ndez-Arjona et al., 2017).
5
Figure 1-4: Microglia morphology under both surveillant and activated conditions in
septofimbrial nucleus, hippocampus, and hypothalamus (Marí a del Mar Ferná ndez-Arjona et al.,
2017)
Microglia are involved in a series of neurodegenerative diseases in the human body. Nervous
system disorders can lead to inflammation, microglia activation, increased glial cells, and cause
phenotype changes. In acute neurodegenerative diseases, such as stroke, cerebral hypoxia, and
traumatic brain injury, the inflammatory mediators released by microglia are mainly cytokines
and chemokines (G. Jean Harry and Andrew D. Kraft, 2019). These acute inflammatory
reactions are usually beneficial to the survival of nerve cells, and they can reduce secondary
damage in the brain and repair damaged tissues. And we have known that chronic inflammation
mediated by microglia is involved in the pathological process of various chronic
neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Huntington's
disease, ALS, and so on (Hyuk Sung Kwon1 and Seong-Ho Koh, 2020). In the process of
6
chronic inflammation, microglia will be activated for a long time and then continue to release a
series of inflammatory mediators. It is generally believed that the chronic inflammatory response
mediated by microglia is harmful to the body and can cause nerve tissue damage.
Figure 1-5: Microglia activation is involved in these three common neurodegenerative diseases,
Alzheimer's disease, Parkinson's disease, and Frontotemporal dementia
Blood-brain-barrier (BBB):
The blood-brain-barrier refers to the barrier between blood plasma and brain cells. It is formed
by endothelial cells, pericytes, neurons, microglia, and astrocyte. These barriers can prevent a lot
of harmful substances from crossing the vessels and entering into CNS. It helps maintain the
essential stability of the internal environment of the brain tissue, which is vital for maintaining
the normal physiological status of the CNS significance. The selective permeability of blood-
brain-barrier restricts the movement of components from the systemic circulation to the brain. It
7
protects the brain from exposuring molecules harmless to peripheral organs but toxic to neurons
in the brain (Anna Carolina Carvalho da Fonseca et al., 2014). The neurovascular unit (NVU) is
a new concept representing the structure and function relationship between the brain and vessels.
It is constituted by the extensive network of endothelial cells together with neurons and glial
cells. It is responsible for regulating the integrity of the blood-brain-barrier and the function of
the CNS (Alexander H. Bell et al., 2020). The neurons communicate with astrocytes through the
astrocyte endfeet.
Pericytes are located between end feet and endothelial cells to form a stable vascular wall.
Endothelial cells contribute to tight junctions between adjacent cells. They are essential for
blood-brain-barrier integrity and function. Microglia also affect the operations of NVU.
Microglia play an essential role in the body's repair of blood-brain-barrier damage. When the
blood-brain barrier has abnormal permeability, nearby microglia will quickly gather and activate
to repair this damage. They will directly contact vascular endothelial cells and swallow the
astrocytes endfeet. Another research has found that the regulation effect of microglia could be
dual (Koichiro Haruwaka et al., 2019). Systemic inflammation induces the migration of
microglia in the brain to cerebral blood vessels. In the early stages of inflammation, vascular-
associated microglia most express the tight junction protein Claudin-5 to maintain the integrity
of the blood-brain-barrier and make physical contact with endothelial cells. When the
inflammation continues for a long time, microglia will swallow the endfeet of astrocytes and
damage the blood-brain-barrier function. This study of the dual effects of microglia on the blood-
8
brain-barrier is of great significance for elucidating how systemic immune activation affects
nerve function.
Figure 1-6: Structure of the neurovascular unit (Alexander H. Bell et al., 2020)
9
MATERIALS AND METHODS
Animals
Snord118 flox/flox and Cx3cr1-Cre mice were obtained from the Jackson Laboratory, and lines
were maintained by breeding with C57BL/6 mice. All animal studies were conducted under
protocols approved by the Institutional Animal Care and Use Committee at the University of
Southern California.
Immunostaining
Animals were deeply anesthetized and transcardially perfused with phosphate-buffered saline
(PBS) followed by 4% paraformaldehyde. Brains were post-fixed at 4 ̊C overnight in 4%
paraformaldehyde (PFA), then incubated in 30% sucrose/PBS solution for two days and
embedded in Tissue-Tek 394 OCT compound (Sakura). Coronal sections were sliced at 20 μm
using a cryostat (Leica CM1950). Sections were washed in PBS 3 times/5 min each and then
incubated with blocking buffer solution (5% normal goat serum, 1% BSA, 0.3% Triton X-100 in
PBS) for two h at room temperature. Sections were then incubated with rabbit anti-Iba1 (1:1000;
ab153696, Abcam), goat anti-Iba1 (1:500, Ab5076, Abcam), rat anti-CD68 (1:400;
MCA1957GA, Bio-rad), rabbit 594 anti-PDGFR beta (1:100, ab206872, Abcam), rabbit 488 α-
Smooth Muscle Actin (1:200, 34105S, CST), rat anti-mouse CD31 (1:200, 550274, BD
Pharmingen), Lectin 488 (1:200, L32470, ThermoFisher), goat anti-CD13 (1:100, AF2335,
Novus biologicals), mouse anti-Claudin-5 (1:200, VB298617, Invitrogen), rabbit anti-AQP4
(1:500, AB3594, Millipore) in blocking solution overnight at 4° C. After washing in PBS three
times/5 min each time, sections were incubated with species-specific fluorescently conjugated
secondary antibodies (1:400, Invitrogen) and Hoechst 33342 (1:200) in blocking buffer solution
10
for 1h at room temperature. After washing in PBS 3 times/5 min each, sections were mounted on
glass slides with mounting medium and coverslipped.
Imaging and quantification
Images of stained sections were acquired using a confocal microscope with a 20× or 60×
objective lens. All images were taken by using identical laser power, gain and offset values. In
addition, all images were acquired with a setting as z-series (stacks as three μm depth interval)
using 20X and 60X (1.4 NA) Leica oil immersion objective with identical parameters (laser
power, gain, and offset). The area or numbers are quantified by using ImageJ or manually.
11
Chapter 1
Generation of Snord118 flox; Cx3cr1-Cre mouse line
Cx3cr1-Cre mice express Cre recombinase under the direction of the Cx3cr1 promoter in the
mononuclear phagocyte system, making them useful for fate-mapping studies of microglia. We
use Snord118 flox/flox mice to mate with Cx3cr1-Cre mice to get wildtype (Snord118 +/+),
Snord118 flox/+; Cx3cr1-Cre and Snord118 flox/flox; Cx3cr1-Cre mice. The Snord118 flox/+;
Cx3cr1-Cre and Snord118 flox/flox; Cx3cr1-Cre mice, the Snord118 is knocked down in the
microglia. We get the targeted phenotypes in the microglia. The mice are euthanatized in one-
month-old.
Results:
We cut a little piece of mice's ears when they are fifteen-day-old to do the genotyping. When
running the PCR of the Cx3cr1-Cre gene, we only use the primer to detect the cre band, but not
the wildtype band. If a band is shown, it means this mouse has the Cx3cr1-Cre gene (Figure 2-1).
If not, it is a wildtype mouse. As for detecting the Snord118 flox gene, we use the primer, which
can catch the Snord118 flox band and wildtype band simultaneously. The Snord118 band is a
little longer than the wildtype band, which means the upper band represents the Snord118 flox
gene is existing in the mouse, the lower band is the wildtype band.
12
Figure 2-1: Gel electrophoresis of PCR genotyping results, (A) genotyping results of Cx3cr1-Cre
gene, (B) genotyping results of Snord118 flox gene.
Figure 2-2: Summary of the genotyping results in Figure 2-1
We picked up the mice whose PCR results do not have Cx3cr1-Cre band or only have Snord118
wildtpye band as wildtype/control group (#2, 4, 5, 8, 9, 11, 14, 15, 16, 18, 19), Snord118 flox/+;
Cx3cr1-Cre as heterozygote group (#10, 13, 20), Snord118 flox/flox; Cx3cr1-Cre as homozygote
group (#1, 3, 6, 7, 12,17). We generated many litters of mice like these to do the next
experiments.
A
B
13
Chapter 2
Snord118 mutation contributes to the abnormal activation of microglia
We can always observe microglia activation when the central nervous system gets infection,
inflammation, or damages. Iba1 antibody can label microglia, and CD68 antibody can label the
lysosome in microglia when activated (Barbara Klein et al., 2016). The number of microglia and
the area of CD68 positive signals should be both increased when microglia are activated. We
perform Iba1 and CD68 co-staining in the mouse brain sections to observe the activation status
of microglia in Snord118 flox mice. The brain has many different regions. The microglia in other
areas may have a different response when they face damages.
Results:
14
Figure 3-1: Iba1 (red) and CD68 (green) co-staining in the cortex of control, Snord118 flox/+;
Cx3cr1-Cre and Snord118 flox/flox; Cx3cr1-Cre mice
The area of the co-staining part (yellow) in the control group is tiny (Figure 3-1), and there is
even no CD68 positive signal in some microglia, which means these microglia are not activated
in the control mice. In the Snord118 flox/+; Cx3cr1-Cre tissue, every microglia has CD68
positive points in the cell body. As for the microglia in the Snord118 flox/flox; Cx3cr1-Cre
group, the CD68 positive dots are enormous in the cell body. They occupy a lot of part in the
microglia. The microglia are obviously activated in the cortex of Snord118 flox/flox; Cx3cr1-Cre
mice. We perform this kind of immunochemical staining in the striatum, cortex, corpus
15
callosum, hippocampus, and cerebellum. We also do a quantification to see this kind of change
of the area of CD68 positive signals.
Figure 3-2: The number of microglia in the (A) striatum, (B) cortex, (C) corpus callosum, (D)
hippocampus, and (E) cerebellum
16
Figure 3-3: The area of the cell body in each microglia in the (A) striatum, (B) cortex, (C) corpus
callosum, (D) hippocampus, and (E) cerebellum
17
Figure 3-4: The area of CD68 positive signal among each microglia cell body in the (A) striatum,
(B) cortex, (C) corpus callosum, (D) hippocampus, and (E) cerebellum
The number of microglia in the striatum increases significantly in the Snord118 flox/flox;
Cx3cr1-Cre group compared with the control group, nearly no change in the Snord118 flox/+;
Cx3cr1-Cre group (Figure 3-2 A). This condition is the same in the cortex of the brain (Figure 3-
2 B). But the number does not have any significant change in Snord118 flox/+; Cx3cr1-Cre or
Snord118 flox/flox; Cx3cr1-Cre group (Figure 3-2 C). As for in the hippocampus and
cerebellum, the number of microglia has a significant increase in Snord118 flox/+; Cx3cr1-Cre
group, and increases more in Snord118 flox/flox; Cx3cr1-Cre mutant mice (Figure 3-2 D, E).
The cell body of microglia has also increased in Snord118 flox mice. As we mentioned before, in
18
the process of microglia activation, the cell body of microglia will become huge. The cell body
area has a significant increase in Snord118 flox/+; Cx3cr1-Cre group, and becomes larger in the
Snord118 flox/flox; Cx3cr1-Cre group in the striatum (Figure 3-3 A). The increase also happens
in the corpus callosum and cerebellum (Figure 3-3 C, E). As for the cortex and hippocampus, we
observed a tiny increase in Snord118 flox/+; Cx3cr1-Cre group, but the change is not significant.
In the Snord118 flox/flox; Cx3cr1-Cre group, a significant increase of microglia cell body, is
compared with the control group (Figure 3-3 B, D).
The CD68 positive area slightly increases the Snord118 flox/+; Cx3cr1-Cre group in the
striatum, but this change is not significant. On the other hand, we can get a significant increase in
the Snord118 flox/flox; Cx3cr1-Cre group (Figure 3-4 A). This condition is also found in the
cortex and hippocampus (Figure 3-4 B, D). As for corpus callosum and cerebellum, in the
Snord118 flox/+; Cx3cr1-Cre mice, we can see a significant increase in CD68 positive signal
area, which is more considerable in the Snord118 flox/flox; Cx3cr1-Cre group (Figure 3-4 C, E).
The number of microglia is increasing, the area of the microglia cell body is becoming more
massive, and the size of CD68 positive signals is also growing. These analyses suggest that the
microglia in the almost whole brain is highly activated in the Snord118 flox mutant mice.
However, the level of activation may be different among different regions.
19
Chapter 3
Snord118 flox induces the activation of microglia to change their morphology
Another phenotype of microglia activation is the change of morphology. We use the Iba1
antibody to stain the microglia in different brain regions and use a confocal microscope with a
60× objective lens to get the pictures of each microglia to observe the changes.
Results:
20
21
Figure 4-1: The morphology of microglia in the striatum, cortex, corpus callosum, hippocampus,
and cerebellum of control, Snord118 flox/+; Cx3cr1-Cre, and Snord118 flox/flox; Cx3cr1-Cre
group
Just like the illustration in previous studies, the cell body is getting bigger in Snord118 flox/+;
Cx3cr1-Cre and Snord118 flox/flox; Cx3cr1-Cre group, the branches are shrinking, and nearly
wholly disappear in Snord118 flox/flox; Cx3cr1-Cre mice (Figure 4-1). The morphology of
microglia is different among every brain region. This kind of phenotype happened in other brain
regions. To quantify this morphology change, we use the number of end branches to divide each
microglia's primary branches (Figure 4-2). If the microglia is resting, there should be more end
branches, and the division results should be bigger. If the microglia are activated, the branches
disappear, decreasing the number of end branches.
Figure 4-2: The quantification method we used to analyze the morphology change of microglia
primary branch
end branch
22
Figure 4-3: The number of end branches/number of primary branches in different brain regions
of control, Snord118 flox/+; Cx3cr1-Cre, and Snord118 flox/flox; Cx3cr1-Cre group
In the striatum, corpus callosum, and cerebellum, the number of end branches/number of primary
branches have a significant decrease in the Snord118 flox/+; Cx3cr1-Cre group, and reduce more
in the Snord118 flox/flox; Cx3cr1-Cre group (Figure 4-3 A, C, E). The reduction means the end
branches are disappearing, the microglia are activated, the complexity of microglia morphology
is reducing. In the cortex and hippocampus, there is a minor decrease in the Snord118 flox/+;
Cx3cr1-Cre group, and has a significant reduction in the Snord118 flox/flox; Cx3cr1-Cre group
(Figure 4-3 B, D). Along with the activation process of microglia, this number keeps decreasing.
23
Chapter 4
The microglia activation induced by Snord118 might contribute to pericyte phenotype in
blood-brain-barrier
The neurodegeneration disease could affect the pericyte differentiation, and this kind of
influence could differ among different brain regions. After deleting laminin, the knock mice
express more smooth muscle actin (SMA) in deep brain regions (Yao Yao et al., 2013), which
means more smooth muscle actin appears, the pericyte is under a bad condition. But there is no
change in the cortex and hippocampus. It suggests that this kind of regulation is region-specific.
Based on the literature, we plan to use PDGFR beta and CD31 antibody to co-staining the total
pericyte, SMA antibody to stain the pericyte with a terrible condition.
Figure 5-1: There are SMA positive signals in deep brain regions of laminin KO mice, but no
SMA signals in the cortex or hippocampus (Yao Yao et al., 2013)
24
Pericyte coverage is also a standard parameter to determine the status of blood vessels. The loss
of pericyte happened in many diseases (Angeliki Maria Nikolakopoulou et al., 2017). Therefore,
we always use the CD13 antibody to stain the pericyte and the lectin antibody to stain the
endothelial cells to define the change of pericyte coverage.
Figure 5-2: (A) CD31 positive (purple) – pericyte, lectin positive (blue), (B) quantification of
pericyte coverage, pericyte coverage is defined as a percentage of CD13-positive surface area
covering lectin-positive surface area per field (Angeliki Maria Nikolakopoulou et al., 2017)
25
Results:
Figure 5-3: CD31 (red), PDGFR beta (yellow), SMA (green), and Hoechst (blue) co-staining in
the striatum of control, Snord118 flox/+; Cx3cr1-Cre, and Snord118 flox/flox; Cx3cr1-Cre group
We use the percentage of the area of SMA positive pericytes to the CD31 positive and PDGFR
beta positive pericytes to define the pericyte differentiation.
26
Figure 5-4: The percentage of SMA+; PDGFR beta+; CD31+ pericytes/PDGFR beta+ ; CD31 +
pericytes in striatum, cortex, and hippocampus
Although the percentage of SMA positive pericytes has a slight increase in Snord118 flox/+;
Cx3cr1-Cre, and Snord118 flox/flox; Cx3cr1-Cre group in striatum and hippocampus, there is
only a significant change in the Snord118 flox/flox; Cx3cr1-Cre in the striatum (Figure 5-4 A,
C). In the cortex, we do not observe any change (Figure 5-4 B). The pericyte differentiation is
affected by the microglia in the striatum and hippocampus but not in the cortex. Or the age of
experimental mice is not old enough to obtain the change of the pericyte differentiation. All
cerebral vessels are still in the growing period, and the differentiation will be impacted during
the whole period.
27
Figure 5-5: Lectin (green) and CD13 (red) co-staining in the cortex of control, Snord118 flox/+;
Cx3cr1-Cre, and Snord118 flox/flox; Cx3cr1-Cre group
We observe decreasing CD13 positive signals in each pericyte (Figure 5-5), which means the
microglia activation could affect the pericyte coverage. We quantify the area of CD13 positive
parts in Lectin positive pericyte parts to define the pericyte coverage.
28
Figure 5-6: Quantification of the percentage of the area of CD13/Lectin in striatum, cortex, and
hippocampus of control, Snord118 flox/+; Cx3cr1-Cre, and Snord118 flox/flox; Cx3cr1-Cre
group
There is no significant decrease of pericyte coverage between control and Snord118 flox/+;
Cx3cr1-Cre group (Figure A, B, C). As for the Snord118 flox/flox; Cx3cr1-Cre group, there is a
significant decrease in the pericyte coverage. Thus, we suggest that the microglia activation
induced by Snord118 could result in loss of pericyte coverage. Still, the activation induced by
Snord118 heterozygote might not be enough to contribute to the change of pericyte coverage.
29
Chapter 5
The microglia activation induced by Snord118 might contribute to the change of
endothelial tight junctions in blood-brain-barrier
Many studies have discussed that the BBB can selectively restrict the blood-to-brain diffusion of
molecular compounds, which is mandatory for cerebral neuronal function. And this property
depends on endothelial tight junctions (TJs) between adjacent cells (M. Rodewald et al., 2007).
The Claudin-5 antibody is used to stain the tight junction, and the CD31 antibody is used to stain
the endothelial cells. We also plan to perform this two antibody co-staining to observe the
change of the tight junction caused by microglia activation under Snord118 mutation.
Figure 6-1: Co-staining of the endothelial-specific marker CD31 and Claudin5 in controls and
after VEGF inhibition (M. Rodewald et al., 2007)
30
The literature used the area of Claudin-5 in each unit area and the area of Claudin-5 within the
endothelial cells to define the change of tight junctions.
Results:
Figure 6-2: Claudin-5 (green) and CD31 (red) co-staining in the cortex of control, Snord118
flox/+; Cx3cr1-Cre, and Snord118 flox/flox; Cx3cr1-Cre group
The area of Claudin-5 in Snord118 flox/+; Cx3cr1-Cre and Snord118 flox/flox; Cx3cr1-Cre
group has been decreased (Figure 6-2). We quantify this kind of change by using the same
methods in the reference paper.
31
Figure 6-3: Quantification of the area of Claudin-5 in each unit area (A, B, C), the percentage of
the area of Claudin-5 within the area of CD31 (D, E, F) in striatum, cortex, and hippocampus of
control, Snord118 flox/+; Cx3cr1-Cre, and Snord118 flox/flox; Cx3cr1-Cre group
We observe some decrease of the area of Claudin-5 in the unit area in the striatum and cortex,
although there is only a significant decrease in the Snord118 flox/flox; Cx3cr1-Cre group in the
cortex (Figure 6-3 A, B). And there is nearly no change of the Claudin-5 in the hippocampus
(Figure 6-3 C). The percentage of the area of Claudin-5 within the area of CD31 has a significant
decrease in Snord118 flox/+; Cx3cr1-Cre and Snord118 flox/flox; Cx3cr1-Cre group in the
striatum (Figure 6-3 D), a slight decrease in the Snord118 flox/+; Cx3cr1-Cre of the cortex
32
(Figure 6-3 E), a significant decrease in the Snord118 flox/flox; Cx3cr1-Cre group. In addition,
there is still hard to get the change in the hippocampus (Figure 6-3 F). The tight junctions are
affected to a certain extent by the microglia activation, especially in the Snord118 flox/flox;
Cx3cr1-Cre group of the striatum and cortex. In the hippocampus, the endothelial cell tight
junctions seem not to be affected by that abnormal microglia behavior.
33
Chapter 6
Snord118 mutation though microglia – neurovascular unit cross-talk to affect blood-brain-
barrier functions
The previous study has found that in the neurovascular unit, the neurons communicate with
astrocytes through the astrocyte endfeet (Koichiro Haruwaka et al., 2019). During the systemic
inflammation, the microglia will move forward to cerebral vessels, phagocytize the astrocyte
endfeet to affect the blood-brain-barrier functions. The article used aquaporin-4 (AQP4) antibody
to stain the astrocyte endfeet and use Iba1 to stain the microglia simultaneously to see the
movement of microglia and cross-talk between microglia and neurovascular unit.
Figure 7-1: Iba1 (green) and AQP4 (red) co-staining in the WT mice and mice with chronic
systemic inflammation (Koichiro Haruwaka et al., 2019)
During systemic inflammation, the percentage of vessel-associated microglia is increasing. Still,
the total density of microglia does not change, which means the microglia are moving forward to
the cerebral vessels from other parts of the brain.
34
Figure 7-2: Iba1 (green) and AQP4 (red) co-staining microglia to see the phagocytosis of
astrocyte endfeet (Koichiro Haruwaka et al., 2019)
The increasing percentage of microglia with AQP4 inclusion responses the phagocytosis
function of microglia is higher in systemic inflammation mice than normal mice. The astrocyte
endfeet is "swallowed" by the microglia to change the cross-talk ability between microglia and
the neurovascular unit. We perform the Iba1 and AQP4 antibody staining simultaneously to see
the phagocytosis of astrocyte endfeet by microglia under the microglia activation induced by
Snord118 mutation.
Results:
35
Figure 7-3: (A) AQP4 (green) and Iba1 (red) co-staining in the cortex of control, Snord118
flox/+; Cx3cr1-Cre, and Snord118 flox/flox; Cx3cr1-Cre group; (B) quantification of the
percentage of microglia with AQP4 inclusion
36
More microglia have attachment with cerebral vessels in Snord118 flox/+; Cx3cr1-Cre and
Snord118 flox/flox; Cx3cr1-Cre groups. However, no microglia connects with the wildtype
group vessels (Figure 7-3 A).
We quantify the percentage of microglia who has inclusion with cerebral vessels in a defined
unit area (Figure 7-3 B), there are almost 15% microglia connecting with blood vessels in
Snord118 flox/+; Cx3cr1-Cre and Snord118 flox/flox; Cx3cr1-Cre group, only around 5%
microglia have a connection with vessels in the control group. Thus, the microglia activation
induced by the Snord118 mutation contributes to the microglia moving forward to the cerebral
vessels and set up some microglia - neurovascular unit cross-talk to affect blood-brain-barrier
functions.
37
CONCLUSION
SNORD118 can cause neurological syndrome, leukoencephalopathy with calcifications and
cysts. The study of reporting cases found white matter with widespread calcifications,
vasculopathy with secondary ischemic lesions, and mineralization. Small vessels are resembling
vascular malformations and sporadic lymphocytic infiltration of vessel walls. The white matter is
also abnormal (Jenkinson EM et al., 2016). There is loss of myelin and axons, tissue rarefaction
with multifocal cystic degeneration, and the presence of foamy macrophages, secondary
calcifications, and gliosis (Guy Helman et al., 2020).
Microglia present in large numbers in all major brain regions. But there is a more than five-fold
variation in the density of microglia between different regions. For example, there are more
microglia in gray matter than white matter. The dense areas include the hippocampus, olfactory
telencephalon, basal ganglia, and substantia nigra. And the microglia morphology can be highly
complicated. There is wide variation in the length and complexity of branching of the processes.
And the systematic change in microglial morphology provides further evidence that these cells
are sensitive to the microenvironment. The microglia will activate them to perform phagocytize
functions to clear the damages (L. J. LAWSO et al., 1990).
The pericytes and endothelial cells in the neurovascular unit are critical regulator cells that
control interactions between neurons and the cerebral vessels. They are essential to maintain the
functions of blood-brain-barrier. They can regulate cerebral blood flow, support the blood-brain
barrier's stability, and control vascular development and angiogenesis (Lachlan Shaun Brown et
al., 2019). Blood–brain-barrier plays a vital role in the normal physiological and cognitive
functions of the brain. Any disruption within the brain or the systemic compartment triggers
38
microglia activation to induce BBB dysfunction and neuroinflammation. Both of them will result
in the pathogenesis of the vascular disease. This kind of blood-brain-barrier microglia cross-talk
is essential to affect the microglial response to neurovascular unit injury (HannahThurgur, 2019).
Our data observe that the microglia are obviously activated with Snord118 mutation. The cell
body of microglia is becoming huge, the complexity of branches is decreasing, and the CD68
positive lysosome signals are increasing. We capture the microglia morphology transformation.
These happened in one brain region and at least five brain regions (striatum, cortex, corpus
callosum, hippocampus, and cerebellum). The level of the change or activation is different
among every region, which means the regulation of Snord118 in microglia activation could be
region-specific.
We also find that the microglia activation contributed by the Snord118 may affect the pericytes
and endothelial cells functions to impact the blood-brain-barrier functions. The pericyte
differentiation is differently affected by the microglia activation. The pericyte coverage has some
decrease within the Snord118 mutation. And the endothelial cell tight junctions are also
decreased in the mutant mice. And all of these changes are still region-specific. The difference is
different among the striatum, cortex, and hippocampus. Previous literature has also found this
region-specific regulation of blood-brain-barrier functions (Roberto Villasenor et al., 2017). We
also detect that there should be some factors through microglia-neurovascular unit cross-talk to
affect the blood-brain-barrier function.
We use the mice of 30-day-old to perform all of these experiments. The disease might develop
with aging. And some studies have found that the regulation of brain-blood-barrier caused by
other factors could also be age-specific (Diana G. Bohannon et al., 2020). Next, we need to start
the same experiments by using aging mice to get more convincing data. We confirm some
39
impaction of pericytes and endothelial cells induced by microglia activation within Snord118
mutation, but whether the blood-brain-barrier has been affected or not, or how much it will affect
it, has not been studied. Next, we will pay attention to the effects on blood-brain-barrier
functions contributed by this kind of abnormal microglia activation.
40
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Abstract (if available)
Abstract
The Snord118 mutation, which is also called U8, will cause leukoencephalopathy with calcifications and cysts. In addition, it will result in neurological diseases. This mutation affects the ribosome biogenesis progress to change the translational capacity, which could cause abnormal cell growth. Microglia is a kind of immune cell located in the central neuron system. The abnormal activity of microglia is always relevant to cell death and neuronal dysfunctions. Recently, a study group has found a marked dissociation of microglia mRNA and protein networks will be followed with innate immune activation. The ribosome-associated mRNAs will not be translated, suggesting that the abnormal ribosome biogenesis would affect immune response in microglia. We use the microglia-specific Snord118 gene mouse model to explore microglia's role in this disease and discover the pathogenesis. We find that the microglia are activated with Snord118 mutation, the number of microglia is significantly increased, and the microglia morphology is changed in several different brain regions.
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Asset Metadata
Creator
Du, Yuanning
(author)
Core Title
Functional study of Snord118 in microglila
School
Keck School of Medicine
Degree
Master of Science
Degree Program
Biochemistry and Molecular Medicine
Degree Conferral Date
2021-08
Publication Date
07/28/2021
Defense Date
06/02/2021
Publisher
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blood-brain-barrier,microglia,microglia activation,neurological diseases,OAI-PMH Harvest,Snord118
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