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
/
Neural activity alterations with learning in MBNL2 KO mice
(USC Thesis Other)
Neural activity alterations with learning in MBNL2 KO mice
PDF
Download
Share
Open document
Flip pages
Contact Us
Contact Us
Copy asset link
Request this asset
Transcript (if available)
Content
NEURAL ACTIVITY ALTERATIONS WITH LEARNING
IN MBNL2 KO MICE
SHAGUN WAZIR
A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)
AUGUST 2018
Copyright 2018 Shagun Wazir
2
ACKNOWLEDGMENT
It gives me immense pleasure to express a deep sense of gratitude to my mentor
and PI, Dr. Sita Reddy. Her inquisitive nature, meticulous scrutiny, scientific advice
and timely suggestions were solely the reason behind the successful completion of
this dissertation.
I would like to extend my heartfelt gratitude to my committee members, Dr. Lucio
Comai, Dr. Hongwei Dong and Dr. Marcelo Coba for their time, prompt advice and
guidance through this journey.
It is my privilege to thank Dr. Jonkyu Choi for his utmost patience, and dedicated
approach to teaching me the basics of scientific bench-work and guiding me
through all the nooks and loopholes of laboratory protocols.
Last, but not the least I would like thank my fellow budding scientists Evan Feeley,
Abhinash Khandelwal, Allen Lee, Melody Gregarosian for their continuous support
and co-operation. Their kindness, constant encouragement and enthusiasm has been
extremely beneficial to my work.
3
CONTENTS
Acknowledgment ………………………………………………………………...2
Content …………………………………………………………………………...3
List of Figures …………………………………………………………………….4
List of Tables ……………………………………………………………………..5
Abstract ………………………………………………………………………….6
1. Introduction ……………………………………………………………………..8
1.1 Myotonic Dystrophy …………….……………………...............8
1.2 Pathogenesis ……………………………………………………8
1.3 RNA-Binding proteins ………………………………………...10
1.4 Muscleblind-like Proteins ……………………………………..10
1.5 HYPOTHESIS ………………………………………………...11
1.6 Novel object Recognition ……………………………………...11
1.7 Neural Circuits ………………………………………………...14
1.8 Role of MBNL Proteins ……………………………………….15
2. Materials and Methods ………………………………………………………..18
2.1 Materials ………………………………………………………18
2.2 Methods ……………………………………………………….20
2.2.1 Novel Object Recognition Test ………………………20
2.2.2 Immunohistochemistry ……………………………….21
2.2.3 Splice Analysis ……………………………………….24
2.2.4 Cell culture and Immunocytochemistry ……………...30
2.2.5 Polysome profiling and Western Blot ………………..32
3. Results and Conclusion ………………………………………………………..39
3.1 Mouse generation ……………………………………………..39
3.2 Novel Object Recognition Test ……………………………….39
3.3 Immunohistochemistry to test for cFos expression …………...40
3.4 Alternative splicing of GABRG2 and TMEM16B ……………65
3.5 Immunocytochemistry to test for presence of RNA granules ....67
3.6 Polysome profiling and Immunoblotting to test for mRNA
transport ……………………………………………………….68
4. Discussion ………………………………………………………………………70
5. References ……………………………………………………………………...74
4
LIST OF FIGURES
Fig 1.1: Schematic representation of the design of experiments followed for this
thesis
Fig 1.2: A neural circuit is made up of Pre-synaptic and post-synaptic neurons
Fig 2.1: The novel object recognition test
Fig 3.1: MBNL2 KO mouse generation
Fig 3.2: Mouse groups analyzed for cFos expression
Fig 3.3: Dorsal Hippocampus cFos stains of HC WT and TR WT
Fig 3.4: Ventral Hippocampus cFos stains of HC WT and TR WT
Fig 3.5: Amygdala cFos stains of HC WT and TR WT
Fig 3.6: Middle cortex cFos stains of HC WT and TR WT
Fig 3.7: Bottom cortex cFos stains of HC WT and TR WT
Fig 3.8: Top cortex cFos stains of HC WT and TR WT
Fig 3.9: Dorsal hippocampus cFos stains of TR WT and TR KO
Fig 3.10: Ventral hippocampus cFos stains of TR WT and TR KO
Fig 3.11: Amygdala cFos stains of TR WT and TR KO
Fig 3.12: Middle cortex cFos stains of TR WT and TR KO
Fig 3.13: Bottom cortex cFos stains of TR WT and TR KO
Fig 3.14: Top cortex cFos stains of TR WT and TR KO
Fig 3.15: Dorsal hippocampus cFos stains of HC WT and HC KO
Fig 3.16: Ventral hippocampus cFos stains of HC WT and HC KO
Fig 3.17: Amygdala cFos stains of HC WT and HC KO
Fig 3.18: Middle cortex cFos stains of HC WT and HC KO
Fig 3.19: Bottom cortex cFos stains of HC WT and HC KO
Fig 3.20: top cortex cFos stains of HC WT and HC KO
Fig 3.21: Dorsal hippocampus cFos stains of HC KO and TR KO
Fig 3.22: Ventral hippocampus cFos stains of HC KO and TR KO
Fig 3.23: Amygdala cFos stains of HC KO and TR KO
Fig 3.24: Middle cortex cFos stains of HC KO and TR KO
Fig 3.25: Bottom cortex cFos stains of HC KO and TR KO
Fig 3.26: Top cortex cFos stains of HC KO and TR KO
Fig 3.27: Splice pattern of GABRG2 in WT and MBNL2KO Cortex
Fig 3.28: Splice pattern of GABRG2 in WT and MBNL2KO Hippocampus
Fig 3.29: Splice pattern of TMEM16B in WT and MBNL2KO Cortex
Fig 3.30: Splice pattern of TMEM16B in WT and MBNL2KO Hippocampus
Fig 3.31: HT22 cells stained with SYTO14 to detect RNA granules
Fig 3.32: RNA profile (Krichevsky & Kosik, 2001)
Fig 3.33: RNA profile with presence of RNA granule fraction
5
LIST OF TABLES
Table 2.1: Blocking buffer dilution table for cFos staining
Table 2.2: Primary antibody dilution table for cFos staining
Table 2.3: Triton X-100 dilution table for cFos staining
Table 2.4: Secondary antibody dilution table for cFos staining
Table 2.5: Lysis buffer and LSG for polysome fractionation
Table 2.6: Stacking and running gels for western blot
Table 2.7: TBST preparation table for western blots
Table 3.1: Total cFos counts of HC WT and TR WT Hippocampus
Table 3.2: Total cFos counts of HC WT and TR WT Amygdala
Table 3.3: Total cFos counts of HC WT and TR WT Middle Cortex
Table 3.4: Total cFos counts of HC WT and TR WT Bottom Cortex
Table 3.5: Total cFos counts of TR WT and TR KO Hippocampus
Table 3.6: Total cFos counts of TR WT and TR KO Amygdala
Table 3.7: Total cFos counts of TR WT and TR KO Middle Cortex
Table 3.8: Total cFos counts of TR WT and TR KO Bottom Cortex
Table 3.9: Total cFos counts of HC WT and HC KO Hippocampus
Table 3.10: Total cFos counts of HC WT and HC KO Amygdala
Table 3.11: Total cFos counts of HC WT and HC KO Middle cortex
Table 3.12: Total cFos counts of HC WT and HC KO Bottom Cortex
Table 3.13: Total cFos counts of HC KO and TR KO Hippocampus
Table 3.14: Total cFos counts of HC KO and TR KO Amygdala
Table 3.15: Total cFos counts of HC KO and TR KO Middle cortex
Table 3.16: Total cFos counts of HC KO and TR KO Bottom cortex
6
ABSTRACT
Myotonic dystrophy (DM1) is an autosomal dominant disease that is caused by a
CTG trinucleotide repeat expansion in the 3’ untranslated region of the DM1
protein kinase (DMPK) gene. The DM1 model of the disease proposes that mutant
transcripts expressing expanded CUG repeats sequester a family of double stranded
RNA binding factors, the Muscleblind-like proteins, which in humans are encoded
by three genes MBNL1, MBNL2 and MBNL3. DM1 patients manifest a
compromised Central Nervous System. They are known to display cognitive and
behavioral abnormalities including learning and memory deficits. Of the 3 types of
MBNL proteins, the MBNL2 protein is abundantly present in the brain. I
hypothesized that deficits in MBNL2 function drive cognitive impairment in DM1
patients. Consistent with the CNS dysfunction observed in DM1 patients, MBNL2
KO mice perform poorly in a novel object recognition test (NOR) for learning and
memory compared to the WT mice. In order to capture this observed dysfunction
as a measurable biochemical outcome, I studied neuronal activity in WT and KO
mice, before and after training. cFos expression, which is used as a marker for early
neuronal activity, was used to assess CNS function in WT and MBNL2 KO mice.
The WT mice were first stained for cFos. These mice displayed an expression of
cFos in the Hippocampus, Cortex and Amygdala, the regions of the brain that have
been associated with learning and memory. Thus, establishing a system to study
neuronal activity in these mice. Subsequently the KO mice that performed poorly
in the NOR test, exhibited an increase in the expression of cFos compared to the
WT mice in the hippocampus, amygdala and certain parts of the cortex.
7
Interestingly, with training the fold increase in c-fos expression was lower in
MBNL2 KO mice when compared to WT animals. This suggested that the increase
in cFos expression in the KO mice could be an effect of an increase in excitation or
decrease in inhibition in neural circuitry. As prior studies in MBNL2 KO mice have
demonstrated diminished EPSP, taken together these results I propose that MBNL2
could play a regulatory role in inhibitory neural circuitry. As MBNL2 is known to
regulate alternative splicing and mRNA transport, I hypothesized that MBNL2
target genes that are associated with brain inhibitory circuits are alternatively
spliced in MBNL2 KO mice. I curated a list of genes from previously published
data and selected GABAbrg2, and TMEM16B as prime candidates for the splicing
assay. The results of this assay did not show a difference in the splice patterns of
these genes between the WT and KO cortex and hippocampus regions. I further
proposed that abnormality in inhibitory neural circuitry in the KO mice could be
the result of an early developmental phenomena. A set of immunostaining
experiments have been designed to study synapse formation and connectivity in
WT and MBNL2KO mice before training. In a parallel set of experiments, I’m
studying mRNA transport by MBNL2. Based on earlier records, I have performed
preliminary experiments to confirm the presence and composition of mRNA
granules that are known to be involved in mRNA transport in neurons.
8
1. INTRODUCTION
1.1 Myotonic dystrophy
Myotonic dystrophy (dystrophia myotonica, DM) is an autosomal dominant, multi-
systemic disease and is the most dominant form of inherited muscular dystrophy in
adults. It displays a wide range of symptoms, such as myotonia, progressive muscle
loss, cataracts, cardiac conduction defects, insulin resistance and cognitive
impairments (Nakamura et al., 2016). It affects roughly 1 in 8000 people worldwide
(Cho & Tapscott, 2007). DM is amongst the microsatellite expansion disorders in
which a trinucleotide repeat sequence within a specific gene, typically containing a
variable number of repeats within the population, expands to a pathogenic range
(Lee & Cooper, 2009). There are two forms of myotonic dystrophy, DM1 and DM2,
that have been studied to date. In DM1, disease symptoms result from an abnormal
expansion of a CTG trinucleotide repeat sequence in the 3′ untranslated region
(UTR) of myotonic dystrophy protein kinase (DMPK) gene on chromosome 19
(Nakamura et al., 2016). DM2 is caused by an expansion of a CCTG tetra-
nucleotide repeat in intron 1 of the CCHC-type zinc finger, nucleic acid binding
protein (CNBP) gene on chromosome 3 (Nakamura et al., 2016). DM1 is the more
common of the two forms in the United States (Lee & Cooper, 2009).
1.2 Pathogenesis
In 1992, the DM1 mutation was found to have expanded (up to >4000) CTG repeats
located within the 3′-UTR of the DMPK gene in exon 15 (Lee & Cooper, 2009).
With an increase in repeat size, the severity of the symptoms increased while the
9
age of onset decreased. Several hypotheses had been proposed to explain the
pathogenesis of this disease. One of them was that, aberrant expansion of repeats
inhibit DMPK mRNA or protein production, resulting in DMPK haplo-
insufficiency (Lee & Cooper, 2009). This hypothesis was demonstrated by
decreased expression of DMPK mRNA and protein in DM1 muscle. Surprisingly,
DMPK KO mice displayed mild myopathy and cardiac conduction defects in older
animals (Lee & Cooper, 2009). The mice did not exhibit myotonia which is one of
the characteristic symptoms of DM1. Therefore, the multi-systemic features of
DM1 could not be explained solely by DMPK haplo-insufficiency (Lee & Cooper,
2009). The RNA gain-of-function hypothesis proposed that the mutant RNA
transcribed from aberrant expansion of DMPK is sufficient to induce symptoms of
the disease (Lee & Cooper, 2009). ‘This was suggested by several observations: (i)
loss of function of DMPK or surrounding genes did not reproduce major features
of DM1, (ii) the expanded CTG repeats, transcribed into CUG repeats that
accumulate in discrete nuclear foci, (iii) expression of only the DMPK 3′- UTR
with 200 CTG repeats is sufficient to inhibit myogenesis’ (Lee & Cooper, 2009).
The HSA
LR
(human skeletal α-actin gene) mouse model expressing 250 CTG
repeats in the 3’ UTR not only developed myotonia but also exhibited muscle
histology similar to that of DM1 (Lee & Cooper, 2009). This suggested that CUG
repeats alone, regardless of the gene content, were sufficient to induce pathogenic
features of DM1 (Lee & Cooper, 2009). Six years later, another type of myotonic
dystrophy that wasn’t caused by the DM1 mutation was identified and termed DM2
(Lee & Cooper, 2009). Unlike DM1, there was no congenital form of DM2
10
reported. DM2 resulted from a CCTG tetra-nucleotide repeat expansion within
intron 1 of the ZNF9 (zinc finger 9) gene, and exhibited symptoms similar to that
of DM1 (Lee & Cooper, 2009). ‘The fact that two repeat sequences located in
entirely different genes can cause such similar disease features implied a common
pathogenic mechanism by RNA gain-of-function. One explanation of how repeat-
containing RNA could cause disease symptoms was through interaction with RNA-
binding proteins’ (Lee & Cooper, 2009).
1.3 RNA binding proteins (RBP) play a key role in the regulation and
coordination of gene expression in cells. All stages of the RNA life cycle are
regulated by a number of RNA binding proteins (Jazurek, Ciesiolka, Starega-
Roslan, Bilinska, & Krzyzosiak, 2016). Any shortcoming in RBP expression and
function or a mutation in target RNA molecules can disrupt protein-RNA networks
and result in human diseases, such as cancer, autoimmune pathologies, metabolic
and neurological diseases (Jazurek et al., 2016).
1.4 Muscleblind-like proteins (MBNL) are tissue-specific RNA metabolism
regulators, encoded by three genes MBNL1, MBNL2 and MBNL3, in mammals
(Konieczny, Stepniak-Konieczna, & Sobczak, 2014). The three members of the
family are structurally similar, consisting of four zinc finger (ZnF) domains critical
for recognizing a common consensus sequence in pre-mRNA and mRNA targets
(Konieczny et al., 2014). They differ widely in their distribution pattern. MBNL1
serves the primary role in most tissues, with the exception of the brain where
MBNL2 is predominantly expressed (Konieczny et al., 2014). On the other hand,
MBNL3 expression is much more restricted, with some functions reported in
11
muscle cell differentiation and regeneration. ‘One of the most important cellular
tasks of MBNLs is tissue-specific alternative splicing regulation, in which MBNL1
and 2 have largely compensatory roles’ (Konieczny et al., 2014). In addition to
alternative splicing, MBNLs also regulate gene expression by mediating cellular
mRNA transport and stability (Konieczny et al., 2014). The loss of MBNL function
in myotonic dystrophy, seems to be a secondary effect to the expression of
pathological CUG and CCUG expansions (in DM1) that sequester much of the
available pool of MBNL proteins in the nuclei (Konieczny et al., 2014).
Recap: Adults with Myotonic dystrophy type 1 manifest symptoms of cognitive
and behavioural impairment including learning and memory deficits. Among
the 3 types of MBNL proteins, MBNL2 is predominantly expressed in the
brain.
1.5 Based on existing data I hypothesized that, ‘Deficits in MBNL2 functions
drive cognitive impairment in DM1 patients’
1.6 Novel Object Recognition Test (NOR)
Since the beginning of the 20
th
century, animal models of memory have been
considered as the subject of many scientific publications (Antunes & Biala, 2012).
In animals, cognitive function is accessed through different kind of behaviors by
several specific, experimental models of memory and learning (Antunes & Biala,
2012). An MBNL2 KO mouse model was created using the cre/loxP recombinase
system. The mice were transported to the UCLA behaviour core facility in Los
Angeles. These mice were subjected to a novel object recognition test for learning
12
and memory. This test can be evaluated by the differences in the exploration time
of novel and familiar objects, between control and experimental mice (Antunes &
Biala, 2012). It is a simple behavioral assay of memory that relies mainly on a
rodent’s innate exploratory behavior in the absence of externally applied rules or
reinforcement (Antunes & Biala, 2012). The NOR task is particularly attractive
because no external motivation, reward, or punishment but only a little training
or habituation is required, and it can be completed in a relatively short period of
time (Antunes & Biala, 2012).
Consistent with the characteristics of DM1 patients, MBNL2 KO mice
performed poorly in the Novel Object Recognition test.
A series of experiments were designed and exhibited in a systematic manner to
understand the molecular mechanism by which MBNL2 regulates learning and
memory.
Fig 1.1: Schematic representation of the design of experiments followed for
this thesis
13
Goal 1: To establish a system where the observed dysfunction in MBNL2 KO mice
could be recapitulated as a measurable biological phenomena based on neural
activity.
Immediately after the NOR test, the brain sections were stained for a neural
activity protein marker called cFos. cFos is an immediate early gene that belongs
to family of proto-oncogenes which are activated rapidly due to a wide set of
stimuli. Expression of cFos is a marker of neuronal activity because it is expressed
when neurons fire action potentials.
The WT mice were first stained for cFos. These mice displayed an expression of
cFos in the Hippocampus, Cortex and Amygdala, the regions of the brain that have
been associated with learning and memory. Thus, establishing a system to study
neuronal activity in mice.
Goal 2: To study the difference in neuronal activity in WT and MBNL2 KO mice
before and after training.
The KO mice that performed poorly in the NOR test, interestingly exhibited an
increase in the expression of cFos compared to the WT mice in the hippocampus,
amygdala and certain parts of the cortex. With training the fold increase in c-fos
expression was lower in MBNL2 KO mice when compared to WT animals.
Before examining this interesting observation and the reason for the increase in
neural activity in MBNL2 KO mice, I would like to introduce the concept of neural
circuitry.
14
1.7 Neural Circuits: A general Idea
Fig 1.2: A neural circuit is made up of Pre-synaptic and post-synaptic neurons
Neurons fire action potentials from a pre-synaptic neuron to one or more post-
synaptic neurons forming a neural circuit. Neural circuits are inhibitory or
excitatory in nature depending on the type of signal received by the post-synaptic
neuron. In the pre-synaptic neuron a gene in the cell body is transcribed into
mRNA, this RNA molecule is then carried to a specific location (axon to the
synapse), where it receives developmental signals and is translated into a protein.
These proteins present in the synapse work in different ways to control the flow of
neurotransmitters, that are released into the synaptic cleft and absorbed by post-
synaptic neurons to initiate inhibition or excitation of that neuron.
The increase in cFos expression in the KO mice could be an effect of an
increase in excitation or decrease in inhibition in neural circuitry.
15
MBNL2 knockouts exhibited impaired spatial memory on a hippocampal-
dependent task studied previously. This lab performed electrophysiological
recordings on hippocampal slices to evaluate the effects of Mbnl2 loss on NMDA
receptor (NMDAR)-mediated synaptic transmission and synaptic plasticity.
NMDAR is a glutamate receptor that is studied in response to excitation in neurons.
Loss of MBNL2 expression resulted in decreased synaptic NMDAR activity, and
learning and memory deficits (Charizanis et al., 2012).
As MBNL2 KO mice demonstrated diminished EPSP, taken together these
results I propose that MBNL2 could play a regulatory role in inhibitory neural
circuitry.
1.8 Role of MBNL2 in neurons
mRNA Splicing- Alternative pre-mRNA splicing has an important role in the
control of neuronal gene expression. MBNL2 regulates the splicing of many
neuronally expressed genes.
Since, Inhibitory neural circuits are regulated by chloride channels, I hypothesized
that MBNL2 target genes that are associated with brain inhibitory circuits are
alternatively spliced in MBNL2 KO mice. Based on previously published data I
selected GABAbrg2, and TMEM16B as prime candidates for the splicing assay.
The results of this assay did not show a difference in the splice patterns of these
genes between the WT and KO cortex and hippocampus regions. I further proposed
that abnormality in inhibitory neural circuitry in the KO mice could be the result of
an early developmental phenomena. A set of immunostaining experiments have
16
been designed to study synapse formation and connectivity in WT and MBNL2KO
mice before training.
mRNA Transport- The enrichment of RNA in a specific subcellular region, is a
mechanism for the establishment and maintenance of cellular polarity in a variety
of systems (Gagnon & Mowry, 2011). This spatially restricts gene expression and
is an important post-transcriptional regulatory machinery required for specific
protein synthesis and function. ‘In neuronal cells, mRNA are transported from the
nucleus to the dendrites and along the axon to the synapse to maintain cellular
polarity and synaptic plasticity’ (Gagnon & Mowry, 2011). While the consequences
of RNA localization are well appreciated, mechanisms that are responsible for
carrying out mRNA transport remain arguable (Gagnon & Mowry, 2011). The
presence of cytoplasmic granules that contain translationally repressed mRNAs in
germ cells, embryos and neurons is known since a long time. These
macromolecular complexes are collectively called RNA granules. The term defines
a broad spectrum of granules, ranging from neuronal RNA transport granules to
specific structures for the storage of maternal mRNAs (Kaphingst, Persky, &
Lachance, 2010).
In a parallel set of experiments, I examined the presence and localization of
endogenous RNA complexes in living neurons. To visualize RNA, I used the
membrane-permeable dye SYTO 14, which fluoresces on binding to nucleic acids.
SYTO 14 (Molecular Probes) is a cell-permeant dye with fluorescent enhancement
on binding to nucleic acids and at least a 50% greater quantum yield for RNA versus
DNA (Knowles et al., 1996). Based on earlier studies I deduced an RNA profile
17
from mouse whole brains by sucrose gradient polysome fractionation. The presence
of a heavy fraction at the end of the RNA profile suggests a specific mechanism of
motor directed mRNA transport as opposed to mRNA diffusion (Krichevsky &
Kosik, 2001). To establish the presence of RNA granules in the heavy fraction, and
MBNL2 within these granules, I immunoblotted the fractions for granules specific
marker proteins like Kif5a and MBNL2 respectively (Krichevsky & Kosik, 2001).
18
2. MATERIALS AND METHODS
2.1 Materials
Preliminary.129sv/B6 MBNL2 KO mice were generated in our lab using the
cre/loxP recombinase system. The mice were genotyped and 10 MBNL2 WT and
KO mice were sent to the UCLA behavioral core facility, where 5 WT and KO mice
were subjected to the novel object recognition test. The other 5 WT and KO mice
were used as house controls. The mice were immediately perfused with 4% PFA
and their brains were dissected and embedded in agarose until sectioned using a
microtome. The sections were 33um thick and were stored in an anti-freeze solution
in 24 well plates at -20C until used.
Immunohistochemistry. The sections were stained using cFos (1:1000) as the
primary antibody to measure neural activity and Anti-rabbit Alexa 594 (1:500) as
the secondary antibody. The cfos expression was analyzed using the Keyence
fluorescence microscope. The cFos foci were counted using ImageJ and the
statistical analysis was carried out using Graph-pad Prism.
Splice analysis. Two MBNL2 WT and KO brains were analyzed for splice variants
of Gabbrg2 and Tmem16B. The brains were dissected for RNA extraction (Trizol)
and cDNA prep (buffers and reagents mentioned in Methods). RT PCR. Primers
were used for Gabrg2 and Tmem16B, chloride channel genes (PCR protocol
mentioned in Methods). Cell culture and Immunocytochemistry. HT22 cells
(hippocampal cell line) were cultured in DMEM (1x) growth media with 10% FBS
and 1% Pen/strep. The cells were differentiated in Differentiation media and stained
with SYTO 14 (50nM/1000cells). The stained HT22 cells were analyzed under the
19
Keyence microscope. Polysome profiling and Western blots. MBNL2 WT brains
were lysed (buffers used are mentioned in Methods) and the cytoplasmic
component was subjected to polysome profiling to generate an RNA profile. The
fractions collected from the profile were immunoblotted to detect the presence of
KiF5A and MBNL2KO.
PRIMER NAME PRIMER SEQUENCE
TMEM16B-E1-F CAAGGGGCTTGGCATCTGG
TMEM16B-E3-R GGCTCATTGGCATCCAGGTA
TMEM16B-E3-F CGCATGCACTTTCACGACAA
TMEM16B-E8-R ATCATGAAGCGGGTAGGCAG
TMEM16B-E8-F CTGCCTACCCGCTTCATGAT
TMEM16B-E15-R TCATATTCAGGCCGAGAGTGT
TMEM16B-E15-F AAGGAGAGTGGCAAGTCAGC
TMEM16B-E19-R CTTCCAGGTCTGCCCACAAA
TMEM16B-E19-F GGGCAGACCTGGAAGCTATG
TMEM16B-E23-R TGATGACGGAGAACTTGCCA
TMEM16B-E23-F TGGCAAGTTCTCCGTCATCA
TMEM16B-E25-R AGACGGGCAGACAAAACAGA
TMEM16B-E25-F TCTGTTTTGTCTGCCCGTCT
TMEM16B-E26-R AGGAGCTGCAGACAAACCTG
GABRG2-E1-F TCCTGCTATCGCTCTACCCA
GABRG2-E3-R ACTGGACCAATGCTGTTCACA
GABRG2-E3-F GCATTGGTCCAGTGAATGCT
GABRG2-E5-R GGGCAGGAGTGTTCATCCAT
GABRG2-E5-F ATGGATGAACACTCCTGCCC
GABRG2-E8-R AGGAGACCTTGGGCAGAGAT
GABRG2-E6-F TCCTTTGTTGGATTGAGGAATACA
GABRG2-E9-R GGTTGCTGATCTGGGACGAA
GABRG2-E7-F TCCAGACTTACATTCCCTGCAC
GABRG2-E10-R ACTACGTTGAGACAGAGAAACACA
20
2.2 Methods
2.2.1 NOR
Fig 2.1: The novel object recognition test
‘Habituation, familiarization, and test phase are the three phases of the task
procedure. In the habituation phase, each animal is allowed to freely explore the
arena in the absence of objects’(Antunes & Biala, 2012). The animal is then
removed and placed in its holding cage. In the familiarization phase, one animal
is placed in the open arena containing two identical objects, for some time
(Antunes & Biala, 2012). The experimental context is not very different during
the familiarization and the test phase. After a retention time, during the test phase,
the animal is returned to the open arena where one of the objects is replaced by a
novel object (Antunes & Biala, 2012).
‘During both the familiarization and the test phase, objects are located in opposite
and symmetrical corners of the arena and location of novel versus familiar object
is counterbalanced (Antunes & Biala, 2012). Normal rats spend more time
exploring the novel object during the first few minutes of the test phase, and when
this bias is observed, the animal could remember the sample object’ (Antunes &
21
Biala, 2012). ‘The strongest novel object preference scores tend to occur early in
the test phase; while the novel object is still relatively novel, since in the course
of time, the novel object became familiar’ (Antunes & Biala, 2012). ‘Despite
animals spent more time exploring the novel object, the recognition performance
varies according to the delay between the two phases, and the time the mice take
to explore the sample during the familiarization phase. This is the foundation of
the NOR test’ (Antunes & Biala, 2012).
2.2.2 Immunohistochemistry
This protocol was followed for 3 House control brain sections and 3 trained brain
sections. (this protocol is in accordance to the Dong lab protocol, ZNI)
Take two wells of brain sections (~9 sections/well) from each plate.
• Use well 5 as a negative control- No primary antibody used, follow all the other
steps.
• Use well 6 as the test sample
1) After separating brain tissue from agarose, rinse 3 times with 1x KPBS,
10mins/rinse for both the wells.
2) Non-specific antibody binding sites are blocked with blocking buffer for 1hour,
(shaking) for both the wells.
22
Table 2.1: Blocking buffer dilution table for cFos staining
Volume needed
(ml)
5% NGS from
100% stock (ul)
0.3% tritonX from
10% stock (ul)
Volume of 1x
KPBS (ml)
5 250 150 Upto 5ml
10 500 300 10
20 1000 600 20
25 1250 750 25
3) Rinse section 2 times in 1x KPBS, 3mins/rinse
4) Incubate sections in Primary antibody for 72hours at 4C, (shaking), for WELL 6
ONLY
Table 2.2: Primary antibody dilution table for cFos staining
Volume needed
(ml)
2% NGS from
100% stock (ul)
Primary antibody
C-FOS (1:1000)
(ul)
Volume of 1x
KPBS (ml)
5 100 5 Upto 5ml
10 200 10 10
20 400 20 20
25 500 25 25
While performing the primary antibody step for well 6 sections, incubate well 5
sections in IN 2% NGS in 1x KPBS for 72hours.
AFTER 72 HOURS:
5) Rinse sections 2 times in 1x KPBS, 10mins/rinse for WELL 6 ONLY.
6) Rinse sections with 0.1% tritonX (from 10% stock) for 10mins
23
Table 2.3: Triton X-100 dilution table for cFos staining
7) Wash sections with 1x KPBS 2times for 3mins each
8) Incubate sections with secondary antibody for 3 hours, (shaking) for BOTH THE
WELLS.
Table 2.4: Secondary antibody dilution table for cFos staining
9) Rinse tissue 2 times in 1x KPBS, 10mins/rinse.
Mounting sections:
With a brush, take the brain sections out and mount the sections from each well on
a slide (1 well per slide)
Add 2-3 drops of mounting solution (40% glycerol in ddH2O)
Place coverslip on the slide
Volume needed
(ml)
0.1% tritonX from
10% stock (ul)
Volume of 1x KPBS (ml)
5 50 Upto 5ml
10 100 10
20 200 20
25 250 25
Volume needed
(ml)
2% NGS from
100% stock (ul)
Secondary antibody
(anti-rabbit- alexa
594) (1:500) (ul)
Volume of 1x
KPBS (ml)
5 100 10 Upto 5ml
10 200 20 10
20 400 40 20
25 500 50 25
24
Analysis and quantification:
The brain sections were analysed under the keyence microscope at 10x, 20x, and
40x magnitudes. Images at 40x were taken and the cfos spots were quantified using
the ImageJ software.
Based on previously published data, I selected two chloride channel genes that
were MBNL2 targets, and played a functional role in the Brain- Gabrg2,
Tmem16B
2.2.3 Splice analysis
Dissection.
1) Keep liquid nitrogen, PBS and the dissection area and tools clean and ready.
2) Mice were then asphyxiated with CO2, and a cervical dislocation was carried out.
3) Next, the mouse was place on the dissection board (stomach facing down) and a
cut was made around the neck with surgical thin scissors.
4) The cut was the used to make a slit with the same scissors across the head upto the
nose carefully.
5) A tiny hole was located at the back end of the skull and was used to fix one end of
the scissor, that would then help you cut open the skull.
6) Small scissors were used to break open the rest of the skull.
7) The skull was separated with forceps until the entire brain along with the olfactory
bulbs right at the top can be seen.
8) Lift it up gently using the tools that work best and clean the brain with PBS.
25
9) Dissect the cortex and the hippocampus after cutting the brain into two halves from
each half, and quick freeze it with liquid nitrogen.
10) Stored until further used for splice analysis.
RNA extraction.
Prep for experiment
1) Label tubes and keep in ice
2) Use phenol bag for all trizol disposal
3) Because brain tisuue is soft, no grinding of tissue needed, directly proceed to
homogenizing
4) Add tissue to tubes…
Homogenizing sample
1) Determine your sample type and volume and homogenize sample at room
temperature using Trizol reagent.
Add 500ul of Trizol per 50-100mg of tissue sample, mush the tissue first, and
homogenize sample using glass teflon or power homogenizer.
Add 500ul of trizol to the tubes after this, and homogenizing by pipetting up and
down at least 10 times.
*If samples look cloudy- Tissues or cells with high fat, protein or extra-cellular
content should be centrifuged at 11,200rpm for 10 mins at 4C following this
homogenizing step. The supernatant contains RNA, this can be used for further
processing.
2) Homogenized sample can be stored at -60 to -80C for at least a month. If not frozen,
the sample should be processed immediately.
26
Phase separation
1) Thaw samples, in the meanwhile get another set of tubes ready
2) Make tubes for 75% ethanol, 100% isopropanol, and chloroform.
*if there are too many samples, place half of them back on ice and work on them
later.
3) Homogenize samples after thawing by pipetting 6-7 times.
4) Incubate the homogenized sample for 5 mins at room temperature (time this)
5) Add 0.2ml of chloroform per 1ml of Trizol used for homogenization.
*add chloroform quickly, cause its heavy and can sink to the bottom
6) Shut the lids tight, and shake vigorously by hand for 15 seconds (time this)
7) Incubate for 2-3 mins at RT
8) Centrifuge the sample at 11,200 rpm for 15mins at 4C.
9) Draw out the topmost colorless aqueous phase, and put that into a new vial.
10) Proceed with RNA isolation from the separated aqueous phase.
*The other 2 phases can be stored for DNA and protein isolation if required-
although not an efficient technique.
RNA isolation
*if the sample is small (<10mg) add 5-10ug of glycogen to the sample. Glycogen
acts as an aqueous phase carrier of the RNA.
1) 0.5ml of 100% isopropanol to the aqueous phase per 1ml of Trizol-sample solution.
2) Incubate at -80C for 30mins/or until used. It can also be incubated at room
temperature for 10mins (smaller the tissue amount, incubation should be longer,
generally at -80C)
27
*at this point you can work on the rest of the samples that were originally kept on
ice
3) Take samples out, let them thaw
4) Centrifuge at 11,200 rpm for 15mins at 4C (RNA is often invisible, and forms a
gel-like pellet at the side and bottom of the tube)
5) Remove the supernatant and wash the pellet with 1ml of 75% ethanol
*the sample can now be stored at -20C for a year or 4C for a week, if not
immediately used for further processing, OR
6) Vortex briefly and centrifuge the tube at 8900 rpm for 7mins at 4C. Discard the
wash.
7) Remove supernatant carefully – at first 950ul, then 50ul with p-20,p-200.
8) Allow pellet to air dry for 10 mins- until white pellet turns translucent. Do not allow
pellet to dry completely.
9) Proceed for RNA resuspension.
RNA resuspension
1) Add about 15-30ul of NFW, depending on the amount of RNA sample you have as
the pellet. You do not want to over dilute it.
2) Keep on ice until you finish adding water to all tubes.
3) Tap tubes to dissolve the pellet (switch the nano-drop on at this point)
4) Transfer tubes to heat-block for 10mins. Keep tapping the tubes, to mix for atleast
2-3 mins.
*the RNA has now been extracted and can be stored at -80C for further use, OR
5) Spin down the sample- for 40sec.
28
6) Check RNA concentration on nano-drop, using NFW as the Blank (load oly about
1-2ul of sample to check conc)
cDNA synthesis and gel electrophoresis.
1) Remove RT kit vials from -20C, and leave to thaw.
• Random primer
• Buffer RT
• Water
• dNTP mix
2) Vortex these tubes after thawing
3) Calculate volume of RNA to be added (after measuring concentration), keeping in
mind the amount wanted.
Eg: 2ug of RNA template= 2/concRNA
Also calculate remaining amount of water to make up 15ul.
4) Make master mix – 1ul RP, 1ul dNTP, 5ul of water- 7ul for each sample.
5) Label PCR tubes as per samples.
6) Add samples to tubes, then proceed to add remaining amount of water to make 15ul
(calculated when calculating volume of RNA), then add master mix-7ul to each
sample.
7) Centrifuge PCR tubes for 30sec.
8) Put into PCR machine at 65C for 5mins (RNA denaturing tab- RUN)
9) Prepare master enzyme mix in a pre-cooled tube; 5x RT buffer- 4ul, enzyme- 1ul=
5ul per sample.
10) Add 5ul to each sample after incubation at 65C
29
11) Tap tubes- just to mix a little
12) Centrifuge tubes for 10-20sec.
13) Close tubes tight, put into PCR machine
25C- 10mins
50C- 30mins
85C- 5mins
10C- ∞
14) Store at -80 for further use.
Splicing assay
1) cDNA- 2ul (50ng)
2) NFW- 4.3ul
3) 2x PCR mix- 7.5ul
4) Primer (F+R)- 1.2ul
Total reaction volume 15ul per pcr tube (no. of samples and exon pairs being tested
are considered and master mixes are made to make work easier)
Add all of the above to labeled pcr tubes and spin down for 20s.
Run splicing assay PCR protocol
Store tubes at 4C until needed to run gels.
Gels.
Protocol for making 2% gel (1 gel- 10-12wells). Volumes are changed for making
2 gels at a time.
1) Pour 50ml (1X) TBE buffer in a conical flask
2) Weigh 1g of agarose, and add it to the buffer
30
3) Mix and warm the mixture in an oven for 1-2mins, or until the agarose has
completely dissolved and you see a clear solution
4) Add 5ul of EtBr to the solution
5) Mix the solution and pour into the gel plates
6) Place the comb in the plate, and let the gel harden (20-30mins)
7) Place the gel in the electrophoretic unit
8) Load the ladder and sample, and run the gel for 25mins at 100V
2.2.4 Cell culture and Immunocytochemistry
1) Make growth medium using DMEM (1x) using FBS and Pen/strep.
2) Pour 7ml of this medium into 10ml vial (cell culture tubes)
3) Add the cell line (stored in 0.5ml pcr tubes) into the vial with 7ml medium
4) Centrifuge for 2-3minutes
5) Decant supernatant obtained after centrifugation
6) Suspend the cell pellet with 1ml media, and mix to make sure it dissolves
7) Add 7ml medium to this suspension, and mix gently
8) Pour this mix into cell culture plates (generally 10cm plates are used)
9) Tilt the plate (sideways) to mix and distribute the mix uniformly on the plate
10) Store the plate in an incubator (34°C- warm temp), for cells to grow and multiply,
for further procedures.
Sub- culture
1) After taking the cell culture plates out of the incubator, decant the media
2) Add 5ml PBS for 1-2mins and tilt the plates to clean all the cells on the plate
31
3) Decant PBS
4) Add 3ml trypsin to the plate, and incubate for 2-3mins (time for incubation depends
on how long your cells take to detach from the plate)
5) Add 5ml of your medium to the detached cells
6) Collect the entire mix in 10ml vials and centrifuge for 2-3mins
7) Decant the supernatant obtained
8) Suspend the pellet in 1ml media, and mix until dissolved
9) Add 2ml more of the media (depends on how many plates you want to sub culture
into. In this case its 1 into 3 plates, so total volume is 3ml) and mix gently
10) Pour out 1ml each into 3 cell culture plates.
11) Add 7ml media into each of these plates
12) Tilt the plates to mix and spread the cells uniformly
13) Store again, in the incubator (34°C), for further sub-culturing or harvesting.
The following procedure is carried out in a biosafety cabinet, for maintaining
aseptic conditions.
**HT22 cells have a short dividing time, thus they can be sub-
cultured/differentiated after 24 hours. HT22 cells should be washed with HBSS or
DMEM, so as to prevent loss of cells before staining (avoid using PBS)
1) Culture HT22 cells- subculture until you obtain well-structured HT22 cells
(DMEM- growth medium)
2) Transfer cells to chamber slides, allow them to attach to the slide (overnight, in
growth medium)
32
3) Differentiate HT22 cells- number of hours depends on the number cells. For eg:
20,000 cells (24 hours), works…but must try different number of cells and different
time slots for better results), 10,000 cells/5000 cells (36 hours)- specifically for
chamber slides.
For live cell staining (healthy cells for 2hours)
4) -add 50nM/1000 cells of SYTO 14 to the media (with N2 supplements)
5) -incubate the cells at 37C for 20mins
6) -wash twice with DMEM before evaluation
7) Place the chamber slide in the fluorescent microscope (dismantle the chamber slide,
and add mounting medium) and analyze the images.
For fixed cell staining of granules (this step requires, 4% paraformaldehyde
treatment, right after differentiation, and before staining)
8) -add 500nM of SYTO 14 to the media (with N2 supplements)
9) -incubate the cells at 37C for 10
10) -wash twice with DMEM before evaluation
The protocol for SYTO staining was taken from (Knowles et al., 1996)
**For this experiment, live cell staining was performed
2.2.5 Polysome Profiling and Western Blots
1) Whole brains were harvested from wild type and KO mbnl2 mice. Each brain was
pulverized under liquid nitrogen using a pestle and a mortar and the powder was
transferred to a micro-centrifuge tube.
33
2) The ground brain was lysed in 1 mL of lysis buffer, homogenizing with a
homogenizer (20strokes)
Table 2.5: Lysis buffer and LSG for polysome fractionation
1.0 M TrisHCL (7.5)
20mM 25mM
5.0 M NaCl
10mM
(add 160mM to
170mM later)
25mM
1.0 M MgCl2
3mM
(add 10mM to make
13mM later)
5mM
RNasin
1mM
1M Dithiotreitol
1mM
Triton X-100 (1%)
0.3%
1 M Sucrose 0.05M 15% and 45%
sucrose made with
buffer.
**cycloheximide was also added (stock- 10mg/ml; working stock- 0.1mg/ml)
**Rnasin and DTT volumes are added to the 1ml lysis buffer after its put into the
micro- centrifuge tubes with the brain powder.
**TritonX- 100 was added after homogenizing
3) After lysing the cells the nuclei were removed by centrifugation (4600rpm, 10 min,
at 4°C), (I added another round of centrifugation at 5500 rpm because I did not have
enough pellet)
4) The supernatant obtained is the cytoplasmic fraction with cell debris.
Lysis Buffer
Linear sucrose
gradient buffer
(15%- 45%)
34
5) Centrifuge at 11200 rpm, in the cold room for 10mins to remove the cell debris.
6) The cytoplasmic extracts in the supernatant is then supplemented with NaCl and
MgCl2 to obtain 170, 13 mM concentrations of salts respectively.
7) The supernatant (about 1ml) was layered onto a 10 mL linear sucrose gradient
(15%–45% sucrose), that was made with the gradient buffer.
8) The gradient was then Centrifuged for 120 min at 36,000 rpm at 4°C, with brake
off in Beckman centrifuge.
Fractions of about 600-700ul were collected by continuous monitoring at 254nm
(For making the gradient I followed the polysome profiling protocol (PPP)
prepared by Cyntia Neben, Dr. Amy Merills lab)
Table 2.6: Stacking and running gels for western blot
Separating
gel 10mL
Stacking
gel 5mL
H2O 4.6 3.4
30% acrylamide 2.7 0.83
10% SDS 0.1 0.05
10% APS 0.1 0.05
TEMED 0.006 0.005
Tris HCl 1.5 M
pH 8.8
2.5
Tris HCl 1M pH 6.8 0.63
**the 10% APS only goes in right before you are ready to load the gel because
that’s what causes the gel to bind up
35
Once the gel has formed, dismantle the gel from the casting chamber, gently remove
the combs, and wash the wells with ddH2O. Label the wells with a sharpie.
Denaturing Gel electrophoresis:
Dismount the gel from the casting chamber and fix it in the gel doc
1) Fill the lower buffer chamber with running buffer, so that the glass plates will be
submerged 2-3 cm with buffer. Fill the upper buffer chamber up to the top of the
gel with running buffer.
2) By now, you must have your samples prepared (25-30ul for 0.5mm combs), heat
denatured (on a heat block, at 90-100C) and centrifuged.
3) 25ul of sample was loaded from a total of 30ul (5ul of 5x sample buffer and 25ul
of sample)
4) Attach the lid of the gel system and plug in the cables to a high voltage power
supply.
5) Let half the gel run with a voltage of 100V, you can then increase it to about 130-
150V.
Semi-dry Transfer: You will require 2blotting pads, 2filter papers, 1NC membrane,
and the transfer buffer ready for the transfer.
Once the gel is ready for transfer, dismount the gel from the gel apparatus, and
crack the gel case open. Scrape the gel off the glass and place the gel in transfer
buffer.
36
1) In the meanwhile, dip the the other materials in the transfer membrane and place
them over each other in the form of a sandwich.
2) The sequence of the sandwich is;
Blot pad1
Filter paper1
NC membrane
Gel
Filter paper2
Blot pad2
3) Place the sandwich in the semi-dry transfer apparatus
4) Run the transfer at 24V for 30mins (length of transfer depends on size of protein of
interest)
Western Blot:
1) After transfer, open the apparatus, and cut the membrane according to the MW of
the antibodies, of the proteins to be detected (Biorad protein standards)
2) Place the membrane strips in different compartments of a plastic vessel and label
the compartments with the PA names.
3) Add 5ml of 5% skimmed milk to each compartment (volume depends on the
compartment size) and shake for 1 hour.
5% skimmed milk (can be changed according to antibody visibility)
1X TBST- (100mM salt)
Salt conditions can be changed according to different antibodies for clearer
backgrounds.
37
Table 2.7: TBST preparation table for western blots
Reagent Volume
1M Tris- HCl, pH
7.5, Autoclaved
10ml
5M NaCl 20ml
10% tween20 5ml
ddH2O 965ml
Add 5g of Non-fat dried milk to 100ml TBST, to make 5% skimmed milk as a
blocking buffer.
Primary antibody:
1) Use skimmed milk (5ml) to dilute primary antibodies as per their dilution ranges.
Different antibodies have different dilution ratios (refer to the company’s
recommendations)
2) Add primary antibodies, cover and incubate overnight at 4C
Secondary antibody:
1) Next day, decant PA, and wash with TBST (3 times for 10mins each)
2) Add secondary antibody (diluted n skimmed milk- specific dilution for mouse,
rabbit, and goat) and incubate for 1 hour, at room temperature.
3) Decant secondary antibody, and wash with TBST 4 times for 10mins each.
Antibody protocol for this experiment:
MBNL2
Primary antibody: 1:200- 25ul in 5ml of 5% skimmed milk- 24 hours incubation
38
Washing step after PA incubation: 3 times (10mins each)
Secondary antibody (anti-mouse): 1:10000- 0.5ul in 5ml of 5% skimmed milk- 1
hour incubation
Washing step after SA incubation: 4 times (10mins each)
KiF5A
Primary antibody: 1:1000- 5ul in 5ml of 5% skimmed milk- 24 hours incubation
Washing step after PA incubation: 3 times (10mins each)
Secondary antibody (anti-rabbit): 1:10000- 0.5ul in 5ml of 5% skimmed milk- 1
hour incubation
Washing step after SA incubation: 4 times (10mins each)
Imaging: Can use the fuji film (for digital imaging) or perform simple X-ray
imaging.
1) Add ECL, substrate (1.5-2ml per membrane)
ECL (25ul solution B IN 1ml solution A)
3) Let the ECL act for about 1-2mins
4) After that, dry the membrane using chem wipes, remove excess ECL.
5) Place the membrane on a black background (I used a black base for digital imaging)
6) Immediately take the membranes to the Fuji film, set the parameters to high
resolution, precision, and auto exposure, and image the blots.
39
3. RESULTS AND CONCLUSION
3.1 Mouse generation
Fig 3.1: MBNL2 KO mouse generation
Result: MBNL2 KO mice were created using the cre/loxP recombinase system.
Conclusion: These mice were used to study learning and memory by the NOR test.
3.2 Novel Object Recognition Test
Result: The NOR test showed that MBNL2 KO exhibit learning and memory
deficits compared to the WT mice.
40
Conclusion: The MBNL2 KO mouse model proved to be a good model to study
cognitive impairment, since the mice manifested similar deficits when subjected to
training as those seen in DM1 patients.
3.3 Test: Immunohistochemistry to test for cFos expression
3 WT and KO mouse brains were sectioned and stained for cFos, neural activity.
Images were captured in accordance to specific regions of the brain involved in
learning and memory. The images shown, depict the average neural activity
proportional to the expression of cFos between WT and MBNL2 KO Hippocampus,
Amygdala and Cortex of the 6 brains (3 WT and 3 KO)
Fig 3.2: Mouse groups analyzed for cFos expression
41
• Change in Neural activity in WT mice before and After training in the
Hippocampus, Amygdala, and Cortex
1) HIPPOCAMPUS- The table displays cumulative counts of both sections of the
hippocampus (Dorsal and Ventral)
Table 3.1: Total cFos counts of HC WT and TR WT Hippocampus
P value – 0.0010
Mean for HC WT – 9.167
Mean for TR WT – 38.17
There is significant difference in the expression of cFos between HC WT and TR
WT
HC WT TR WT
12 59
11 32
5 25
8 56
8 25
11 32
42
Dorsal section
Fig 3.3: Dorsal Hippocampus cFos stains of HC WT and TR WT
Ventral section
Fig 3.4: Ventral Hippocampus cFos stains of HC WT and TR WT
43
2) AMYGDALA
Table 3.2: Total cFos counts of HC WT and TR WT Amygdala
P value – 0.0008
Mean for HC WT – 5.667
Mean for TR WT – 32
There is significant difference in the expression of cFos between HC WT and TR
WT
Fig 3.5: Amygdala cFos stains of HC WT and TR WT
HC WT TR WT
12 41
6 36
2 27
3 50
5 12
6 26
44
3) MIDDLE CORTEX- This region consists of somatosensory, auditory and
association cells of the cortex.
Table 3.3: Total cFos counts of HC WT and TR WT Middle Cortex
HC WT TR WT
24 75
27 151
3 69
27 114
20 51
24 39
P value – 0.0052
Mean for HC WT – 20.83
Mean for TR WT – 83.17
There is significant difference in the expression of cFos between HC WT and TR
WT
Fig 3.6: Middle cortex cFos stains of HC WT and TR WT
45
4) BOTTOM CORTEX- This region consists of the piriform cortex- that is receptive
of smell.
Table 3.4: Total cFos counts of HC WT and TR WT Bottom Cortex
HC WT TR WT
33 221
37 190
10 90
12 145
18 50
13 119
P value – 0.0014
Mean for HC WT – 20.50
Mean for TR WT – 135.8
There is significant difference in the expression of cFos between HC WT and TR
WT
Fig 3.7: Bottom cortex cFos stains of HC WT and TR WT
46
5) TOP CORTEX
Pvalue - 0.03
HC mean - 0
TR mean - 48
There is significant difference in the expression of cFos between HC WT and TR
WT
Fig 3.8: Top cortex cFos stains of HC WT and TR WT
Result: The WT mice showed an expression of cfos in the Hippocampus,
Amygdala, and some parts of the cortex. The WT mice after training, displayed a
higher expression of cFos in the same 3 regions of the brain when compared to the
house control.
Conclusion: cFos expression in WT mice subsequent to training is consistent with
the regions that are implicated in the novel object recognition test- Hippocampus,
Amygdala, Cortex. Therefore, examination of c-fos expression can be informative
47
in understanding why KO do not perform well in NOR. A system based on neural
activity was established.
• Change in Neural activity in KO mice After training in the Hippocampus,
Amygdala, and Cortex
1) HIPPOCAMPUS- This table displays total counts of both sections of the
Hippocampus (Dorsal and Ventral)
Table 3.5: Total cFos counts of TR WT and TR KO Hippocampus
TR WT TR KO
59 157
32 155
25 111
56 105
25 49
32 50
P value – 0.0089
Mean for TR WT – 38.17
Mean for TR KO – 104.5
There is significant difference in the expression of cFos between TR WT and TR
KO
48
Dorsal section
Fig 3.9: Dorsal hippocampus cFos stains of TR WT and TR KO
Ventral section
Fig 3.10: Ventral hippocampus cFos stains of TR WT and TR KO
49
2) AMYGDALA
Table 3.6: Total cFos counts of TR WT and TR KO Amygdala
TR WT TR KO
41 57
36 63
27 50
50 54
12 36
26 31
P value – 0.05
Mean for TR WT – 32
Mean for TR KO – 48.5
There is no significant difference in the expression of cFos between TR WT and
TR KO.
Fig 3.11: Amygdala cFos stains of TR WT and TR KO
50
3) MIDDLE CORTEX- This region consists of the somatosensory, auditory and
association cells of the cortex.
Table 3.7: Total cFos counts of TR WT and TR KO Middle Cortex
TR WT TR KO
75 262
151 268
69 168
114 188
51 26
39 136
P value – 0.047
Mean for TR WT – 83.17
Mean for TR KO – 174.7
There is significant difference in the expression of cFos between TR WT and TR
KO
Fig 3.12: Middle cortex cFos stains of TR WT and TR KO
51
4) BOTTOM CORTEX- This region consists of the piriform cortex- that is receptive
of smell.
Table 3.8: Total cFos counts of TR WT and TR KO Bottom Cortex
TR WT TR KO
221 324
190 362
90 190
145 260
50 121
119 155
P value – 0.06
Mean for TR WT – 135.8
Mean for TR KO – 235.3
There is no significant difference in the expression of cFos between TR WT and
TR KO
Fig 3.13: Bottom cortex cFos stains of TR WT and TR KO
52
5) TOP CORTEX
Pvalue= 0.6
HC mean= 44
TR mean= 56.8
There is no significant difference in the expression of cFos between TR WT and
TR KO
Fig 3.14: Top cortex cFos stains of TR WT and TR KO
Result: Trained KO mice showed an increase in cFos expression compared to the
trained WT mice in the Hippocampus, Amygdala, and the middle cortex.
Conclusion: This observation could be the result of abnormal inhibition or
excitation in neural circuitry.
53
• Change in Neural activity in KO mice at the basal level (before training)
1) HIPPOCAMPUS- The table displays total counts of both sections of the
hippocampus (Dorsal and Ventral)
Table 3.9: Total cFos counts of HC WT and HC KO Hippocampus
HC WT HC KO
12 6
11 11
5 32
8 24
8 68
11 68
P value – 0.044
Mean for HC WT – 9.16
Mean for HC KO – 34.83
There is significant difference in the expression of cFos between HC WT and HC
KO
54
Dorsal section
Fig 3.15: Dorsal hippocampus cFos stains of HC WT and HC KO
Ventral section
Fig 3.16: Ventral hippocampus cFos stains of HC WT and HC KO
55
2) AMYGDALA
Table 3.10: Total cFos counts of HC WT and HC KO Amygdala
P value – 0.024
Mean for HC WT – 5.66
Mean for HC KO – 14.83
There is significant difference in the expression of cFos between HC WT and HC
KO
Fig 3.17: Amygdala cFos stains of HC WT and HC KO
HC WT HC KO
12 18
6 12
2 8
3 10
5 29
6 12
56
3) MIDDLE CORTEX- This region consists of somatosensory, auditory and
association cells of the cortex.
Table 3.11: Total cFos counts of HC WT and HC KO Middle cortex
HC WT HC KO
24 42
27 55
3 44
27 29
20 96
24 41
P value – 0.014
Mean for HC WT – 20.83
Mean for HC KO – 51.17
There is significant difference in the expression of cFos between HC WT and HC
KO
Fig 3.18: Middle cortex cFos stains of HC WT and HC KO
57
4) BOTTOM CORTEX- This region consists of the piriform cortex
Table 3.12: Total cFos counts of HC WT and HC KO Bottom Cortex
P value – 0.03
Mean for HC WT – 20.50
Mean for HC KO – 50.67
There is significant difference in the expression of cFos between HC WT and HC
KO
Fig 3.19: Bottom cortex cFos stains of HC WT and HC KO
HC WT HC KO
33 48
37 34
10 25
12 42
18 102
13 53
58
5) TOP CORTEX
Pvalue= 0.008
HC mean= 0
TR mean= 6.5
There is significant difference in the expression of cFos between HC WT and HC
KO
Fig 3.20: top cortex cFos stains of HC WT and HC KO
Result: The untrained mice also show an increase in cFos expression in the KO
compared to the WT.
Conclusion: At the basal level, cFos expression shows the same trend as shown
after training.
59
• Change in Neural activity in KO mice before and after training in the
Hippocampus, Amygdala and Cortex
1) HIPPOCAMPUS- This table displays total counts of both sections of the
hippocampus.
Table 3.13: Total cFos counts of HC KO and TR KO Hippocampus
HC KO TR KO
6 157
11 155
32 111
24 105
68 49
68 50
P value – 0.011
Mean for HC KO – 34.83
Mean for TR KO – 104.5
There is significant difference in the expression of cFos between HC KO and TR
KO
60
Dorsal section
Fig 3.21: Dorsal hippocampus cFos stains of HC KO and TR KO
Ventral section
Fig 3.22: Ventral hippocampus cFos stains of HC KO and TR KO
61
2) AMYGDALA
Table 3.14: Total cFos counts of HC KO and TR KO Amygdala
HC KO TR KO
18 57
12 63
8 50
10 54
29 36
12 31
P value – 0.0002
Mean for HC KO – 14.83
Mean for TR KO – 148.50
There is significant difference in the expression of cFos between HC KO and TR
KO
Fig 3.23: Amygdala cFos stains of HC KO and TR KO
62
3) MIDDLE CORTEX- This region of consists of somatosensory, auditory and
association cells of the cortex.
Table 3.15: Total cFos counts of HC KO and TR KO Middle cortex
HC KO TR KO
42 262
55 268
44 168
29 188
96 26
41 136
P value – 0.0085
Mean for HC KO – 51.17
Mean for TR KO – 174.7
There is significant difference in the expression of cFos between HC KO and TR
KO
Fig 3.24: Middle cortex cFos stains of HC KO and TR KO
63
4) BOTTOM CORTEX- This region consists of the piriform cortex that is receptive
of smell.
Table 3.16: Total cFos counts of HC KO and TR KO Bottom cortex
HC KO TR KO
48 324
34 362
25 190
42 260
102 121
53 155
P value – 0.0011
Mean for HC KO – 50.67
Mean for TR KO – 235.3
There is significant difference in the expression of cFos between HC KO and TR
KO
Fig 3.25: Bottom cortex cFos stains of HC KO and TR KO
64
5) TOP CORTEX
Pvalue= 0.009
HC mean= 6.5
TR mean= 56.33
There is significant difference in the expression of cFos between HC KO and TR
KO
Fig 3.26: Top cortex cFos stains of HC KO and TR KO
Result: There is an increase in the expression of cFos in the MBNL2KO mice after
training compared to the MBNL2KO mice before training.
Conclusion: The fold change in cFos expression in the KO mice subsequent to
training is lower than that seen in WT mice. This could be a result of impaired
excitation, or the effect of a system overload.
65
3.4 Test: Alternative splicing of GABRG2 and TMEM16B
GABRG2 CORTEX
Fig 3.27: Splice pattern of GABRG2 in WT and MBNL2KO Cortex
GABRG2 HIPPOCAMPUS
Fig 3.28: Splice pattern of GABRG2 in WT and MBNL2KO Hippocampus
Result: GABRG2 does not show evidence of alternative splicing in the cortex and
hippocampus regions of MBNL2 KO mouse brains.
66
TMEM16B CORTEX
Fig 3.29: Splice pattern of TMEM16B in WT and MBNL2KO Cortex
TMEM16B HIPPOCAMPUS
Fig 3.30: Splice pattern of TMEM16B in WT and MBNL2KO Hippocampus
Result: TMEM16B splice pattern analysis showed a lot of non-specific binding,
probably due to inconsistency in primer designing.
These technical errors can be corrected by
1) Re-designing primers to check specificity of the primer
67
2) Testing larger exon pairs that overlap the exon pairs with non-specific
binding, to check for errors other than non-specificity of primers.
Conclusion: GABAbrg2 show similar splice patterns in the WT and MBNL2 KO
cortex and hippocampus regions, suggesting that MBNL2 does not regulate
alternative splicing of this gene in adult mice.
Tmem16B needs to be re-checked for splice patterns between WT and MBNL2
mice before we can draw any conclusions. As of now, this data remains
inconclusive.
3.5 Test: Immunocytochemistry to test for presence of RNA granules
To check for the presence of RNA granules in axons of HT22 cell line (Knowles et
al., 1996)
Magnification: 10x Magnification: 20x
Fig 3.31: HT22 cells stained with SYTO14 to detect RNA granules
68
Result: SYTO 14 stains nucleic acids. SYTO 14 stained complexes outside the cell
body, along axons in HT22 cells
Conclusion: Presence of RNA complexes along axons of HT22 (neuronal) cell line
(Knowles et al., 1996).
3.6 Test: Polysome Profiling and Immunoblotting to test mRNA transport
To check for the presence and composition of a heavy granule fraction
Fig 3.32: This figure has been taken from (Krichevsky & Kosik, 2001)
Fig 3.33: RNA profile with presence of RNA granule fraction Western blot of
WT RNA fractions with Kif5a and MBNL2
69
Result: RNA PROFILE
• A heavy fraction was seen after the polysome fraction in the RNA profile, much
like the graph from the Krichevsky experiment (Krichevsky & Kosik, 2001).
• The fractions when immunoblotted, show the presence of Kif5a and MBNL2.
• The heavy fraction, showed the presence of kif5a and MBNL2.
• There is also a difference in migration patterns between fractions 1-8 and fractions
9-15.
Conclusion: The previously proclaimed RNA granule fraction was seen in the RNA
profile The fraction shows the presence of a motor protein, Kif5a and MBNL2. This
suggests that MBNL2 could be involved in the transport of selected mRNA that are
translocated via RNA granules in neurons.
70
4. DISCUSSION
‘DM1 is one of the most complex diseases. Discovery of a CTG/CUG unstable
expansion in the 3' UTR of the DM1 gene prompted researchers to investigate the
biological effects of untranslated unstable elements on the structure of chromatin,
efficiency of gene transcription, RNA processing, and signal transduction
pathways’ (Timchenko et al.,). ‘The CTG triplet repeat expansion in the normal
populations consists of 5–37 units; however, in DM1 patients CTG expansion is
significantly increased up to thousands of repeats. The number of CTG repeats
within the DMPK gene positively correlates with the severity of the symptoms’
(Timchenko et al.,). Since, DMPK haploinsufficiency did not seem to be the
striking reason for DM1 symptoms, RNA processing mouse models were
established. The current pathogenic models suggest that the most common cause of
the disease is through sequestration of important RBP in the nucleus, making them
unavailable for translation and their subsequent protein functions (Goodwin et al.,
2016).
Disease is primarily RNA-mediated, due to transcription of these genes into non-
coding microsatellite expansion RNAs. Toxicity could be a result of enhanced
binding of proteins to CUG expansions and depletion from their normal cellular
targets. Compelling evidence of a RNA toxicity model has been provided by several
publications (Goodwin et al., 2016). Muscleblind-like proteins are amongst the
most targeted RBP that are sequestered in the pathogenic state. Their most
important functions are that of, mRNA splicing and transport.
During development, alternative splicing of pre- mRNAs plays a critical role in the
71
extensive remodelling of cells and tissues. When MBNLs are sequestered by these
toxic RNAs, it disrupts alternative splicing, resulting in the persistence of fetal
splicing patterns in adult tissues (Charizanis et al., 2012).
DM1 patients manifest a compromised Central Nervous System. They are known
to display cognitive and behavioral abnormalities including learning and memory
deficits. Of the 3 types of MBNL proteins, the MBNL2 protein is abundantly
present in the brain. To test the hypothesis that sequestration of MBNL proteins
contributes to DM pathogenesis leading to cognitive impairment, an MBNL2 KO
mouse model was generated. WT and MBNL2 KO mice were subjected to a novel
object recognition test to study their ability to learn and memorize. ‘MBNL2
knockout mice show several phenotypes consistent with abnormalities observed in
myotonic dystrophy’ (Charizanis et al., 2012). The results of this test demonstrated
symptoms of cognitive impairment in MBNL2 KO mice as observed in DM1
patients. It was important to establish a system based on neural activity to
recapitulate this observed dysfunction as a measurable biological quantity. The WT
mouse brain sections were stained with cFos, a neural activity marker, and prolific
expression of cfos was seen in the hippocampus, amygdala, and certain regions of
the cortex. Coincidently these regions of the brain are involved in learning and
memory, thus, a system was created.
The following stains were performed on, MBNL2 KO’s (the mice that exhibited
learning and memory deficits), that showed an increase in the expression of cFos,
compared to their WT counterparts before and after training. In order to study the
molecular mechanism of this increase in cFos, in the KOs, we conducted
72
experiments to test the downstream effects of MBNL2 KO. An increase in neural
activity in MBNL2 KO’s could mean that there is an increase excitation at
excitatory synapses or a decrease in inhibition at inhibitory synapses. Prior
publications suggested that MBNL2 KO in mice causes a decrease in EPSP in the
hippocampus (Charizanis et al., 2012).
A decrease in inhibition, meant that MBNL2 targeted certain chloride channel
transcripts that regulate inhibitory pathways. The candidate genes selected to test
for alternative splicing were GABRG2 and TMEM16B based on their functions in
the brain. Untrained WT and KO cortex and hippocampus was dissected and tested
for splice pattern changes between the WT and MBNL2 KO mice. Results obtained
from the tests did not provide very conclusive answers as we had hoped. WT and
KO adult mice did not show a difference in their splice patterns for GABRG2. This
could mean that abnormality in inhibitory neural circuitry could be the result of an
early developmental phenomena. Further, a set of immunostaining experiments will
be designed to study the formation and connectivity of inhibitory circuits in the
early developmental stages of mice.
Many neurological diseases are also associated with mis-regulation of mRNA
localization. Studies suggest that mRNA localization requires the presence of RBP,
motor proteins, and and adaptor proteins that link motor proteins to the RBP
(Taliaferro et al., 2017). Localization of mRNA would require mRNA to be
transported in complexes, consisting of these necessary proteins. mRNA transport
via RNA granules is another study that had received recognition over the past few
years (Krichevsky & Kosik, 2001). Based on earlier records, I performed
73
preliminary experiments to confirm the presence and composition of mRNA
granules that are known to be involved in mRNA transport in neurons. Our data
suggests that RNA granules are present along axons (Knowles et al., 1996) and the
heavy granule fraction observed by polysome fractionation is comprised of KIF5a,
a motor protein, and MBNL2 (Krichevsky & Kosik, 2001). The presence of
MBNL2 in this complex is compelling evidence to acknowledge a function of this
protein in mRNA transport. In the future, polysome fractionation using sucrose
gradients can be performed on WT and MBNL2KO mice (Krichevsky & Kosik,
2001). The heavy granule fraction of the WT and KO mice can be sequenced to
check for difference in mRNA content. An MBNL2 dependent localization gene
list obtained from this experiment can further be used to study distal ALE isoforms
of these genes and their role in MBNL2 dependent mRNA localization (Taliaferro
et al., 2017).
74
5. REFERENCES
Nakamura, T., Ohsawa-Yoshida, N., Zhao, Y., Koebis, M., Oana, K., Mitsuhashi,
H., & Ishiura, S. (2016). Splicing of human chloride channel 1. Biochemistry and
Biophysics Reports, 5, 63–69. https://doi.org/10.1016/j.bbrep.2015.11.006
Cho, D. H., & Tapscott, S. J. (2007). Myotonic dystrophy: Emerging mechanisms
for DM1 and DM2. Biochimica et Biophysica Acta - Molecular Basis of Disease,
1772(2), 195–204. https://doi.org/10.1016/j.bbadis.2006.05.013
Lee, J., & Cooper, T. (2009). Pathogenic mechanisms of myotonic dystrophy.
Biochemical Society Transactions, 37(6), 1281–1286.
https://doi.org/10.1042/BST0371281
Jazurek, M., Ciesiolka, A., Starega-Roslan, J., Bilinska, K., & Krzyzosiak, W. J.
(2016). Identifying proteins that bind to specific RNAs - Focus on simple repeat
expansion diseases. Nucleic Acids Research, 44(19), 9050–9070.
https://doi.org/10.1093/nar/gkw803
Konieczny, P., Stepniak-Konieczna, E., & Sobczak, K. (2014). MBNL proteins and
their target RNAs, interaction and splicing regulation. Nucleic Acids Research,
42(17), 10873–10887. https://doi.org/10.1093/nar/gku767
Antunes, M., & Biala, G. (2012). The novel object recognition memory:
Neurobiology, test procedure, and its modifications. Cognitive Processing, 13(2),
93–110. https://doi.org/10.1007/s10339-011-0430-z
Charizanis, K., Lee, K. Y., Batra, R., Goodwin, M., Zhang, C., Yuan, Y., …
Swanson, M. S. (2012). Muscleblind-like 2-Mediated Alternative Splicing in the
Developing Brain and Dysregulation in Myotonic Dystrophy. Neuron, 75(3), 437–
450. https://doi.org/10.1016/j.neuron.2012.05.029
Gagnon, J. A., & Mowry, K. L. (2011). Molecular motors: Directing traffic during
RNA localization. Critical Reviews in Biochemistry and Molecular Biology, 46(3),
229–239. https://doi.org/10.3109/10409238.2011.572861
Kaphingst, K. A., Persky, S., & Lachance, C. (2010). NIH Public Access, 14(4),
384–399. https://doi.org/10.1080/10810730902873927.Testing
Knowles, R. B., Sabry, J. H., Martone, M. E., Deerinck, T. J., Ellisman, M. H.,
Bassell, G. J., & Kosik, K. S. (1996). Translocation of RNA granules in living
neurons. The Journal of Neuroscience : The Official Journal of the Society for
Neuroscience, 16(24), 7812–7820.
75
Krichevsky, A. M., & Kosik, K. S. (2001). Neuronal RNA granules: A link between
RNA localization and stimulation-dependent translation. Neuron, 32(4), 683–696.
https://doi.org/10.1016/S0896-6273(01)00508-6
Timchenko LT, Tapscott SJ, Cooper TA, et al. Myotonic Dystrophy: Discussion of
Molecular Basis. In: Madame Curie Bioscience Database [Internet]. Austin (TX):
Landes Bioscience; 2000-2013. Available from:
https://www.ncbi.nlm.nih.gov/books/NBK6512/
Goodwin, M., Mohan, A., Batra, R., Lee, K., José, F., Gómez, F., … Swanson, M.
S. (2016). HHS Public Access, 12(7), 1159–1168.
https://doi.org/10.1016/j.celrep.2015.07.029.MBNL
Taliaferro, J. M., Vidaki, M., Oliveira, R., Olson, S., Zhan, L., Wang, E. T., …
Burge, C. B. (2017). HHS Public Access, 61(6), 821–833.
https://doi.org/10.1016/j.molcel.2016.01.020.Distal
Abstract (if available)
Abstract
Myotonic dystrophy (DM1) is an autosomal dominant disease that is caused by a CTG trinucleotide repeat expansion in the 3’ untranslated region of the DM1 protein kinase (DMPK) gene. The DM1 model of the disease proposes that mutant transcripts expressing expanded CUG repeats sequester a family of double stranded RNA binding factors, the Muscleblind-like proteins, which in humans are encoded by three genes MBNL1, MBNL2 and MBNL3. DM1 patients manifest a compromised Central Nervous System. They are known to display cognitive and behavioral abnormalities including learning and memory deficits. Of the 3 types of MBNL proteins, the MBNL2 protein is abundantly present in the brain. I hypothesized that deficits in MBNL2 function drive cognitive impairment in DM1 patients. Consistent with the CNS dysfunction observed in DM1 patients, MBNL2 KO mice perform poorly in a novel object recognition test (NOR) for learning and memory compared to the WT mice. In order to capture this observed dysfunction as a measurable biochemical outcome, I studied neuronal activity in WT and KO mice, before and after training. cFos expression, which is used as a marker for early neuronal activity, was used to assess CNS function in WT and MBNL2 KO mice. The WT mice were first stained for cFos. These mice displayed an expression of cFos in the Hippocampus, Cortex and Amygdala, the regions of the brain that have been associated with learning and memory. Thus, establishing a system to study neuronal activity in these mice. Subsequently the KO mice that performed poorly in the NOR test, exhibited an increase in the expression of cFos compared to the WT mice in the hippocampus, amygdala and certain parts of the cortex. Interestingly, with training the fold increase in cFos expression was lower in MBNL2 KO mice when compared to WT animals. This suggested that the increase in cFos expression in the KO mice could be an effect of an increase in excitation or decrease in inhibition in neural circuitry. As prior studies in MBNL2 KO mice have demonstrated diminished EPSP, taken together these results I propose that MBNL2 could play a regulatory role in inhibitory neural circuitry. As MBNL2 is known to regulate alternative splicing and mRNA transport, I hypothesized that MBNL2 target genes that are associated with brain inhibitory circuits are alternatively spliced in MBNL2 KO mice. I curated a list of genes from previously published data and selected GABAbrg2, and TMEM16B as prime candidates for the splicing assay. The results of this assay did not show a difference in the splice patterns of these genes between the WT and KO cortex and hippocampus regions. I further proposed that abnormality in inhibitory neural circuitry in the KO mice could be the result of an early developmental phenomena. A set of immunostaining experiments have been designed to study synapse formation and connectivity in WT and MBNL2KO mice before training. In a parallel set of experiments, I’m studying mRNA transport by MBNL2. Based on earlier records, I have performed preliminary experiments to confirm the presence and composition of mRNA granules that are known to be involved in mRNA transport in neurons.
Linked assets
University of Southern California Dissertations and Theses
Conceptually similar
PDF
Mbnl1 and Mbnl2 play roles in brain structural integrity
PDF
The possible aberrant function of MBNL1 with insulin receptor (IR-B) mRNA in myotonic dystrophy type I
PDF
Mechanism of Mbnl1/2-depletion mediated neural defects
PDF
Using ribosome footprinting to detect translational efficiency of Mbnl1x2 KO muscle cells
PDF
The splicing error of FOXP1 in type I myotonic dystrophy
PDF
Elucidation of MBNL1 function in the nervous system of myotonic dystrophy type 1
Asset Metadata
Creator
Wazir, Shagun
(author)
Core Title
Neural activity alterations with learning in MBNL2 KO mice
School
Keck School of Medicine
Degree
Master of Science
Degree Program
Biochemistry and Molecular Biology
Publication Date
08/03/2018
Defense Date
06/08/2018
Publisher
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
alternative splicing,cFos,cognitive impairment,MBNL2,mRNA transport,myotonic dystrophy type 1,neural activity,neural circuitry,NOR test,OAI-PMH Harvest
Format
application/pdf
(imt)
Language
English
Contributor
Electronically uploaded by the author
(provenance)
Advisor
Reddy, Sita (
committee chair
), Coba, Marcelo (
committee member
), Comai, Lucio (
committee member
), Dong, Hongwei (
committee member
)
Creator Email
wazir.shagun17@gmail.com,wazir@usc.edu
Permanent Link (DOI)
https://doi.org/10.25549/usctheses-c89-51789
Unique identifier
UC11672626
Identifier
etd-WazirShagu-6629.pdf (filename),usctheses-c89-51789 (legacy record id)
Legacy Identifier
etd-WazirShagu-6629.pdf
Dmrecord
51789
Document Type
Thesis
Format
application/pdf (imt)
Rights
Wazir, Shagun
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
alternative splicing
cFos
cognitive impairment
MBNL2
mRNA transport
myotonic dystrophy type 1
neural activity
neural circuitry
NOR test