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An Investigation of the Insulin Pathway and TDP-43 Based
Amyotrophic Lateral Sclerosis
Item type text; Electronic Thesis
Authors Podolsky, Taylor Catherine
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AN INVESTIGATION OF THE INSULIN PATHWAY AND TDP-43 BASED
AMYOTROPHIC LATERAL SCLEROSIS
Taylor Catherine Podolsky A Thesis Submitted to The Honors College In Partial Fulfillment of the Bachelors degree With Honors in Biology - Biomedical Studies
THE UNIVERSITY OF ARIZONAMAY 2013
Dr. Daniela C Zarnescu Department of Molecular and Cellular Biology The University of Arizona Electronic Theses and Dissertations Reproduction and Distribution Rights Form The UA Campus Repository supports the dissemination and preservation of scholarship produced by University of Arizona faculty, researchers, and students. The University Library, in collaboration with the Honors College, has established a collection in the UA Campus Repository to share, archive, and preserve undergraduate Honors theses.
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Abstract: The discovery of novel pathways and therapies in disease pathology is especially compelling in fatal neurodegenerative diseases like Amyotrophic Lateral Sclerosis (ALS), also known as Lou Gehrig’s Disease. We have developed a Drosophila model for ALS based on the overexpression of five variants of humanTDP-43, an RNA-binding protein that has been linked to ALS and other neurodegenerative diseases. In our fly model we can reproduce many of the ALS human pathologies including diminished locomotor function, and shortened life span. A primary screen of 1200 FDA approved compounds was performed, which led to the identification of compounds which rescue lethality, including six different antidiabetic drugs.
These drugs fell into three different categories, namely sulfonylureas, biguanides, and thiazolidinediones. Follow-up validations studies have shown locomotor function defects caused by TDP-43 toxicity. These findings suggest that antidiabetic drugs may have therapeutic potential for ALS.
Podolsky (May 2013) 3
Introduction to Amyotrophic Lateral Sclerosis:
Amyotrophic Lateral Sclerosis, from now on abbreviated ALS, is a neurodegenerative disease that affects motor neurons. The disease is characterized by death of motor neurons of several areas including: the brain, spinal cord, motor cortex and other areas of the central nervous system. This pathology leads to loss of motor function, paralysis, in-coordination, and respiratory failure. Some forms of ALS have also been linked to forms of dementia and dystonia, leading to many questions on how the disease manifests. Often ALS is diagnosed through ruling out other motor and neurological diseases. Most patients diagnosed with this disease die within three to five years of diagnosis, usually due to respiratory failure. (Rowland and Shnider 2001, Jawaid et al 2010, Clark 2005, Boillee et al. 2006). This disease was first characterized in 1874 by French neurobiologist and practitioner, Jean-Martin Charcot. ALS was left a medical mystery for many decades, leaving the how and why this disease occurred left to be answered. The name came through common observation of the degeneration of both upper and lower degeneration of motor neurons. ALS was often times used to describe other disease such as progressive muscular atrophy, and bulbar palsy; all of which are now distinguishable from each other (Boillee et al., 2006, Clark 2005).
ALS consists of two major types: familial (fALS) and sporadic (sALS). Approximately ninety percent of ALS cases are sporadic, and the other ten percent are familial. There is also sex bias with this disease, which affects men more frequently than women; ratio of incident being 1.4:1.0. It is estimated that five to eight thousand US citizens are diagnosed with ALS each year, some researchers and physicians argue that this is a relatively low estimate (Clark 2005, Boillee
Although there have been many discoveries and insight into the many causes and progression of ALS, still much is needed to be learned about this disease and what can be done for the patients and families affected by it.
TDP-43 based ALS Pathology:
Several loci have been linked to fALS. Mutations in superoxide dismutase, or SOD1, were among the first found in fALS. SOD1 is linked to around twenty percent of all fALS cases, or around two percent of all ALS diagnoses. Other loci have also been recently identified, one of them being TAR DNA-binding protein, or TDP-43 (Kabashi et al. 2010).
TAR DNA-binding protein (TDP-43) has a molecular weight of 43kD and contains 414 residues. TDP-43 has two RNA recognition motifs (RRM1 and RRM2) and a glycine rich domain in the C terminus. Mutations linked to ALS are mostly single amino acid substitutions and lie within the glycine rich C terminus with the exception of a single mutation found in the RNA recognition motif, RRM1. Figure 1 below from C. Lagier-Tourenne and D. W. Cleveland (2009) shows where several mutations lie within the protein sequence. These mutations are thought to increase TDP-43’s ability to phosphorylate and its targeting for degradation by the proteasome. This protein has been demonstrated to prepress transcription, regulate RNA splicing, mRNA shuttling, stability, and translation (Banks et al, 2008). TDP-43 is expressed in several tissues including the heart, liver, brain, kidney, and muscle (Neumann et al. 2006). TDPis normally localized in the nucleus but is associates with cytoplasmic inclusions in disease samples. Similar inclusions have been found in Drosophila expressing the human form of TDP
al. 2011). Aggregates or stress granules have also been shown in the C. elegans model that formed in response to environmental stressors. (Lagier-Tourenne and Cleveland 2009).
Figure 1: The various point mutations identified within TDP-43’s RRM1, RRM2, and Glycinerich C-terminus. Most of which are found within the C-terminus with the exception of D169G.
The red colored mutations signify sporadic mutations, and the black mutations signify the familial cases. Figure from Lagier-Tourenne and Cleveland 2009
Modeling ALS in Drosophila melanogaster:
The ability to model human disease in vivo in model organisms can provide valuable insights into complex pathological processes. In order to study the specific disease related pathologies, it is crucial to be able to control for gene expression within that particular tissue of interest. Many animal models have systems that allow a particular protein or gene to be expressed in isolated tissue types, allowing for a spatial and temporal way to test various assays.
The GAL-4 UAS system is a useful tool when driving expression of particular genes in different tissues in the Drosophila model. GAL4 is a yeast transcription activator, which regulars that transcription of a gene of interest via its binding site referred to as the Upstream Activating Sequences (UAS). GAL4 expressing fly lines casually referred to as drivers, can exert regulatory
specificity. With this system one can drive expression of fluorescently tagged proteins, which is useful in assays where visualizing regions or cells is crucial (Duffy 2002).
The Insulin Pathway and ALS:
The insulin pathway has been a target for many disease, pharmaceutical, and evolutionary investigations. Not only has the insulin pathway been linked to the disease diabetes mellitus but has other implications in ALS and neuronal function (Jawaid et al. 2010, Vaccaro et al. 2012, Bernardo and Minghetti 2008, and Kiaei 2008).
Peroxisome proliferator-activated receptor-γ (PPAR- γ) has a role in ALS and possibly glial cell functions. M. Kiaei (2008) showed that a small molecule PPAR- γ agonist (pioglitazone) had neuroprotective properties. This study compared findings of a SOD1
was involved in these regulatory processes of ALS pathology. Their results showed that by regulating DAF-16 transcriptional activity, the animals had decreased stress granules and extended lifespan. This process is thought to involve DAF-16 translocation to the nucleus where it activates numerous genes that are linked to survivorship and augmented stress resistance.
This connection to the insulin pathway and ALS is not limited to the model systems, but has also been shown in human patients. Jawaid et al. (2010) showed that patients with diabetes mellitus type 2 (DM) had later onset of ALS symptoms. This study compared 175 patients with ALS and DM to 2196 patients with just ALS. Their results showed that on average the age of onset for patients with ALS and DM was around 60.3 years compared to the non-diabetic group which age of onset was around 56.3 years, giving them a four year difference. They also tested the rate of progression and survival between these two groups finding that the trend continued where the diabetic group and a slower rate of progression and longer survival rates but the data failed to show statistical significance.
Materials and Methods:
Primary Screen Design:
Survivorship Assay: Male TDP-43 expressing transgenic flies was crossed with female D42 expressing flies. Three D42 females were crossed to two TDP-43 males. Specific variants include: wt2 1L, D169G3, G298S4, A315T9, and N345K3. These flies are crossed and brooded on 1ml of food and drug mixture at either a 50 µM or 30 µM concentrations for seven days.
After seven days the TDP-43 and D42 parents are removed. After several days the progeny are screened multiple times. Observations are recorded based on developmental progress, and
ganglion. Below Figure 2 is a schematic of the survivorship assay screening from food making
Figure 2: This shows the screen flowchart from using the drugs in the Prestwick collection to mixing them in food (see glass vials with 1ml of food and drug mixture). Crosses are set up in these vials and screened for developmental stages and rescues.
Preparing the Drug and Food Mixture: Fifty milliliter bottles of stock food are created by a designated trained lab assistant. These bottles are melted down in a microwave until the food has formed a consistent liquid mixture. Once melted these bottles are placed in a hot water bath set at 55 degrees Celsius. The food is aliquoted into six milliliter amounts into glass vials, placed back into hot water bath, and capped. A glass vial containing the six milliliters of food is removed and dried. Drugs are added to a final concentration of 1, 5, 10, 25, or 50 µM to the melted food along with 100 µL of 1% bromophenol blue dye. 0.99-1.0 mL of the food and drug are pipetted into glass vials.
Antidiabetic Compound Survival Screen Design: Similar design to primary screen design but performed at 1, 5, 10, 25, or 50 µM concentrations, for a period of 25-30 days. At day 25 the pupae were counted and the number recorded.
Larval Turning Design: After the designated flies are removed from the drug and food mixture, as mentioned in the Antidiabetic Drug Larval Turning Food Design section, the larvae are raised until they reach late third instar larval stage of development, as denoted by vertically climbing up vial and leaving food. These larvae are removed and YFP confirmed on a grape juice agar plate.
Once confirmed one larva at a time is flipped using a paint brush until it is ventral side up. The timer is started at this point and the larva is timed until it has returned to dorsal side up and has made one forward progression movement. Thirty larvae per genotype are tested.
Transgenics: Human TDP-43 YFP constructs were obtained from Aaron Gitler (Standford University) and cloned into the pUAST vector. After sequence verification plasmids were sent to Genetics Services for germline transformation. Approximately ten transformants per construct were screed and balanced in the laboratory.
Statistic analysis: The student’s T-Test was performed using Microsoft Excel to determine statistical significance.
Intervention of small molecules helps to rescue lethality caused by human TDP-43
overexpression in Drosophila:
When raised at 25 degrees Celsius, progeny of the GAL-4 D42 x UAS TDP-43 cross do