pharmacology toxicology and neuroscience seminar 2014
TRANSCRIPT
Sarah Lopez
Seminar 4/29/14
N
ALS Amyotrophic lateral sclerosis (ALS) Life expectancy of 2-5 years upon
diagnosis. Symptoms:
muscle weakness twitching (fasciculation) and
cramping impairment of the use of the arms and
legs "thick speech" and difficulty in
projecting the voice
Progress to difficulty in breathing/swallowing and paralysis
ALS.org
Cause of ALS Transactive response DNA-binding protein (TDP)-43
pathology estimated in 98% of cases, in both familial and sporadic ALS.
Work in yeast studies, primary neuronal cultures, and the rat models of disease in our lab have implicated the NMD factor upframeshift protein one (UPF1).
The aforementioned models use expression of the disease-related gene TDP-43.
Impact of ALS Most common degenerative disease of motor neurons
About 5000 people in the US will be diagnosed per year.
TDP-43 neuropathology is prevalent in:
amyotrophic lateral sclerosis
frontotemporal lobar degeneration
Alzheimer’s disease
traumatic brain injury
Overview: ALS Models and their NMD Assays
In vivo In vitro
• Model: Inject TDP43 and/or UPF1 AAV9 in temporal vein of neonatal rats
• NMD Analysis: mRNA levels of endogenous rat targets of NMD.
• Model: HEK293 cells transfected with TDP-43, UPF1, or pNW
• Advantages: - fraction of transduced cells - control of what is transfected • NMD Analysis: transfection
with plasmids coding either a normal mRNA sequence or one with a premature termination codon
Hypothesis
NMD plays a role in UPF1 protective effects for ALS
TDP-43 gene transfer leads to inhibition of NMD.
UPF1 gene delivery restores NMD function.
Rationale
TDP and UPF1 are both involved in RNA homeostasis and at least one isoform of TDP-43 is subject to NMD.
Goal
Measure markers of NMD after overexpressing TDP-43 or UPF1.
Introduction to NMD
NMD eliminates mRNA transcripts that contain premature termination codon (PTC).
Why? RNA surveillance
How?
Decapping leads to exonuclease susceptibility.
Chang, Y. et al. (2007). Annu. Rev. Biochem. 76:51–74.
Exon-junction complex (EJC)
Relation to stop codon determines its fate
Gre
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Methods – In vivo study
6 Sprague-Dawley rats (Harlan, Indianapolis, IN)
Injected with UPF1 AAV9 (N = 3) vs.
uninjected (N = 3)
Tissues were harvested for PCR at 12 weeks of age
RNA was isolated using STAT-60 and Qiagen Rneasy MinElute Kit
mRNA was converted to cDNA using iScript Reverse Transcriptase Supermix
Results – Rat cerebellum
• Endogenous
GPx levels
• Uninjected
trend is less NMD
-Trend for less
spliced mRNA
in UPF1 rats.
• N= 3, significant by one-tailed t-test (P = 0.10)
Methods
HEK293T cells
Co-transfected with DNA constructs
6 groups (UPF1, TDP, or pNW)
Reverse transcription-PCR
RNA extracted with chloroform/isopropanol
Converted to cDNA (iScript cDNA Synthesis Kit)
18 mRNA samples in total (N=3)
Recipe:
Norm
Ter
Norm
Ter
Norm
Ter
Purpose
pNW X X - - - - disease model TDP - - X X - -
UPF1 - - - - X X
GPx Norm
X - X - X - test plasmid
GPx Ter
- X - X - X
Gl Norm
X - X - X -
Gl Ter
- X - X - X
GFP
X X X X X X standard
pNW TDP UPF1
DNA: What is Norm and Ter?
Used as an index of NMD activity.
Expressed as a ratio of Ter form to Norm form.
How is NMD Assayed in Cell Culture?
(more of the PTC form) (less of the PTC form)
HEK293T cells
N = 1,
ongoing study
Y axis is Ratio of Ter/Norm
Ter means less NMD
Gl (p
erinu
clear)
GP
x (cytoso
lic)
Conclusion NMD’s role in disease model
Hypothesizing decrease in Ter mRNA when UPF1 expressed
Hypothesizing NMD inhibition by TDP-43
No cure for ALS, so we should explore new models and mechanisms
Future
Finish other mRNA samples
Try new cell line
Acknowledgements Kasey Jackson
Robert Dayton
Anu Sreedhar
Isaac Hardman
Elysse Orchard
Greg Petsko
Ronald Klein