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RNA in ALS
DISCOVERING PATIENT-SPECIFIC RNA EDITING EVENTS IN
SPORADIC ALS
Maria Teresa Chavez
July 2013
RNA in ALS
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Specific Aims
I hypothesize that RNA editing underlies pathogenesis of amyotrophic lateral
sclerosis (ALS). I propose a multi-disciplinary study with the goal of finding RNA
editing events characteristic of patients with ALS and determining the functional
impact they may have on the nervous system. The first aim consists of deriving
iPSCs from fibroblasts from patients with sporadic ALS and differentiating them into
neurons and glia and sequencing the patient’s genome from both cell types. The
second aim will focus on identifying patient-specific RNA editing events and
determining which, if any, RNA editing events are shared among several patients.
The last aim will target the expression of those RNA editing events in cell cultures.
I Specifically propose the following aims:
Specific Aim 1: Deriving neural cell populations from fibroblasts from ALS patients
and sequencing their genomes and transcriptomes.
Specific Aim 2: Identification of patient-specific RNA editing events
Specific Aim 3: Blocking the expression of aberrant RNA editing events in sALS iPSCs
neurons and glia in culture
RNA in ALS
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Background
Amyotrophic lateral sclerosis (ALS), was first described1 in 1869 by Dr. Jean Martin
Charcot. ALS is a neurodegenerative disorder
which causes injury and death of
upper motor neurons in the motor cortex, and of lower motor neurons in the
brainstem and spinal cord. This results in progressive muscle weakness, atrophy,
and spasticity culminating in death from respiratory failure. The average survival
rate from symptom onset is approximately 3 to 5 years, although a small proportion
of patients have a more favorable prognosis. It is estimated that about 285,200
people currently live with ALS and about 16 of them die each day.
In the last few years, remarkable advances have occurred in unraveling the
pathogenic processes and factors underlying ALS, although they are multifactorial
and, at present, not fully understood.
Also recently, defects in RNA editing have been implicated in common nervous
system disorders, including ALS. However the individual functional changes resulting
from editing are known for only a few sites.2 Since the changes that are observed
are often small in magnitude, it is vital to understand the functional impact (if any)
of small changes in transcript editing on the nervous system over life-long periods of
time.2
There are many genes that have been associated with ALS, the most common ones
being SOD1, TARDBP and FUS. One puzzle for understanding ALS is that the known
ALS-causing gene products have diverse physiological functions. Genes associated
with ALS so far include several categories: mRNA processing, Axonal Transport,
Endosomal Vesicle trafficking, and Ubiquitination among other functions. (10, 11)
Understanding the role of RNA in ALS is a relatively new research topic, with one of
the first relevant discoveries being TDP43 aggregates in ALS 16 in 2007.This subfield
has grown substantially, especially in the past 3 years.
Abnormal oxidation of mRNA has been reported in patients with ALS and SOD1
transgenic mice starting with in the pre-symptomatic stage.13 It has been
hypothesized that blocking RNA oxidation at the prodromal stage may prevent/slow
the disease progression. However, once symptoms of the disease are already
evident, the antioxidant treatment may be already too late, thus no significant
beneficial effect was observed.
Under normal conditions, TDP-43 proteins locate in the nucleus and are degraded
through the proteasome pathway. However, in ALS, aberrant modifications and
cleavage of TDP-43 generate abnormal 25 kDa fragments. Because the 25 kDa
fragments lack the nuclear localization signal, they aggregate in the cytoplasm.14
The RNA-binding ability of FUS is critical in driving cellular toxicity and mutating the
RNA-binding residues of FUS has been shown to be enough to abolish this process.
Cytoplasmic mislocalization of mutant FUS is dependent on its RNA-binding ability,
and cytoplasmic localization of mutant FUS is a critical step in ALS pathogenesis.
Wild-type (RNA binding-incompetent) FUS does not incorporate into the cytoplasmic
SGs but FUS carrying ALS mutations does under stress conditions.15.
Only two types of canonical RNA editing are known to exist; adenosine-to-inosine (Ato-I; I is recognized as G) editing and cytosine-to-uracil (C-to-U) editing. The use of
RNA in ALS
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next-generation sequencing technologies allows comparative analysis of RNA
sequencing (RNA-seq) and genome sequencing data from the same individual.18
A mismatch between a genomic DNA sequence and its RNA transcript can be
considered a candidate RNA editing event if a sequencing error can be ruled out.
Therefore, most approaches involve mapping the RNA-seq reads to a genome,
comparing DNA and RNA sequences and calling RNA editing events. However, close
attention must be paid to achieve accurate mapping of the reads and effective
removal of potential false positives that result from various artifacts or errors.8
Small interfering RNA (siRNA) is a class of double-stranded RNA molecule
interferes with the expression of genes. siRNA is antisense of its RNA target. It
introduced into the cell, recognized by DICER and the RISC complex where it
guides DICER to the RNA target and when that happens DICER cleaves the
target, ultimately destroying it.
that
gets
then
RNA
siRNA is the most commonly used method for RNA knockdown but the effect is often
unsatisfactory, primarily due to the fact that siRNA delivery is substantially reduced
as ~70% of the internalized siRNA undergoes exocytosis through egress of LNPs
from late endosomes/lysosomes.19
iPS Cells as a model for ALS
Induced pluripotent stem cells are a type of pluripotent stem cells derived from nonpluripotent cells by inducing the expression of specific genes. iPSCs were first
produced in 2006 from mouse cells and in 2007 from human cells by Shinya
Yamanaka. iPSC-derived cells have advantages over primary and immortalized cells
because they can provide inexhaustible, scalable, and genetically relevant sources
for cell-based drug screening.3
To date, the efficacy of experimental ALS therapies in human clinical trials has been
disappointing (21,22) and one of the biggest obstacles to the development of novel
therapies is the lack of translation from preclinical mouse models of
neurodegenerative diseases to humans. iPSC-derived neurons and astrocytes are the
most genetically precise model of ALS available to date, and therefore have the
potential to transform the current state of therapeutics development for this
disease(5, 6)
Cellular and molecular phenotypes associated with ALS have been identified using
ALS patient–specific iPSC–derived motor neurons.7 The first model of ALS using iPSCs
was successfully created in 2008, using cells from an 82 year old patient with
Familial ALS.20 According to that paper, “patient-specific iPS cells generated from
individuals with sporadic disease could carry the precise constellation of genetic
information associated with pathology in that person. This approach would allow
study of living motor neurons generated from ALS cases with unknown genetic
lesions, providing insight into their intrinsic survival properties, their interactions
with other cell types, and their susceptibility to the environmental conditions that are
considered to play an important role in ALS pathogenesis.”
Rationale and significance
The purpose of this study is finding RNA editing events from patients with sporadic
ALS. Some of the questions that are addressed in this proposal are: Are there RNA
RNA in ALS
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editing events in sporadic ALS neurons? Which, if any, of the patient-specific RNA
editing events are shared among several patients? What is the functional impact
individual RNA editing events have on the nervous system? and ultimately, is
blocking mutant RNA expression a promising therapeutic approach for ALS and other
neurodegenerative disorders?
My hypothesis is that RNA is in fact an important factor of disease progression in ALS
and that blocking RNA editing events will help delay disease progression by reducing
the toxicity inside the neuron and as a result delay, or stop, neuronal death.
By understanding the functional impact RNA has in the nervous system, we can
design a therapy for the disease. Also by studying what happens when you block the
mutations in mice we might be able to see if siRNA or other gene silencing
approaches are something worth pursuing. This will enable us to get a sense for the
challenges that lie ahead.
The use of small inhibitory RNA technologies to suppress the expression of mutant
RNA in transgenic mice is rapidly becoming a viable therapeutic target. In this, the
use of sequence specific RNA interference (RNAi) can result in the post
transcriptional inactivation of gene expression by promoting specific endonucleolytic
cleavage of mRNA targets. This is of specific relevance in that such techniques can
lead to silencing of mutant gene expression, while leaving expression of the wildtype gene intact. (8, 9,17)
RNA editing influences neural function but is not well understood whether changes in
neural activity can affect RNA editing to exert control over transcript diversity and
protein function. For some of the transcripts that undergo activity-dependent editing,
such as glutamate and serotonin receptor subunits, defects in editing have been
implicated in common nervous system disorders, including AD, PD and ALS; yet the
individual functional changes resulting from editing are known for only a few sites.
Since the changes that are observed are often small in magnitude, it is vital to
understand the functional impact of small changes in transcript editing on the
nervous system.8
Experimental Methods and design
Specific Aim 1
iPSCs will be derived from skin cells of patients with ALS and differentiated into both
motor neurons, as described in Bilican et. Al. (2012)5, and glia, as described in Serio
et. Al. (2013)6; these will be co-cultured. The DNA and RNA will be sequenced from
both the glia and neurons to look for patient-specific RNA editing events4 with
Illumina HiSeq 2500.
Because each iPSC line contains the same genetic material as every cell in the
patient who developed ALS, these cell lines are the most genetically precise models
of neurodegenerative disease currently available. However, the reprogramming
process introduces some variability into the cells. To get around this, we’ll examine
two cell lines from the same patient.
Specific Aim 2
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RNA in ALS
Specific Aim 3
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