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