Ataxin-3 Expression Reduces HSR in C.elegans

By: David Pride,Gosife Okoye, and Neal Bartl

Overview

● Machado-Joseph disease (SCA-3)○ Poly-Q Expansion○ Expanded CAG repeats on Ataxin-3 gene○ Ataxin-3 protein aggregates result in degeneration of

cerebellum, cortex, pons, and basal ganglia (Chanu et al, 2013)

● How does the cell respond to this?○ The Heat Shock Response!

The Heat Shock Response

● When does it occur?○ Environmental Stress: Heat shock, oxidative

stress, heavy metals, and toxic chemicals○ Growth and Development: Growth factors,

differentiation, and the cell cycle○ Pathophysiology: Soft tissue injury, fever,

inflammation, and infection○ Protein Conformational diseases:

Neurodegenerative diseases (including SCA-3), cancer, metabolic diseases, and aging

What We Already Know

● Poly (Q) diseases have been found to form aggregates that sequester HSP’s and reduce their bioavailability (Chanu et al, 2013)

● Cummings et al (2001): Overexpression of inducible HSP-70 chaperone suppresses neuropathology and improves motor function in SCA1 mice.○ Molecular chaperones may prevent aggregation/misfolding of Poly(Q)

proteins

● Ataxin-3 Caenorhabditis Elegans knockout: Enhanced stress response/ overexpression of molecular chaperones in knockout animals (Rodrigues et al, 2011)○ Ataxin-3 toxicity decreases stress response

Scientific Questions

● What effects does ataxin-3 expression have on HSR?○ Protein misfolding such as those that result in ataxin-3 aggregation

induce HSR. But how does that ataxin-3 expression affect HSR Robustness:■ In Muscle cells?

■ In Neuronal cells?

● Does HSR as a biochemical process malfunction when ataxin-3 is

expressed in the muscles or neurons of C. elegans?

Hypotheses

● Ataxin-3 expression will diminish the robustness of HSR in both muscle and neuronal cells.

● Decrease in HSR robustness due to ataxin-3 expression will be more drastic in neuronal than in muscle cells (due to the neuro-degenerative nature of the resulting aggregation).

● Aggregation (number of PolyQ repeats) will be proportional to the decrease in HSR robustness

Approach: Overview of Materials and Methods

● C. elegans Cell lines:

o N2 (WT)

o AT3CT Q45 (mt muscle)

o AT3CT Q65 (mt muscle)

o 419 (mt neuronal)

o 422 (mt neuronal)

● ~ 20 L4, Day 1 & 2 adult worms per plate

Approach: Overview of Materials and Methods contd...

● Heat Shock Assay

o All Cell Lines

● RNA Isolation

o Trizol Method for RNA Extraction

● Removal of Genomic DNA

o DNA Free Protocol from Ambion

● cDNA Synthesis & RT-PCR

o iSCRIPT Kit for cDNA synthesis and iQ™SYBR® Green kit for RT-PCR

Approach: Heat Shock Assay

● All Cell Lines

● 15 minutes at 34℃

● Control

Approach: RNA Isolation & DNA Inactivation

● Trizol Methodo M9 Common lab stock

5.8g Na2HPO4•7H2O 3.0g KH2PO4 5.0g NaCl 0.25g MgSO4•7H2O ddH2O (up to 1 L)

o Trizolo Chloroformo 2-Propanolo 70% EtOH

● DNA Free Protocolo Ambion Kit

Approach: cDNA Synthesis and RT-PCR

● iSCRIPT kit for cDNA synthesis from mRNA

● Reverse Transcriptase Polymerase Chain Reaction

o iQ™SYBR® Green kit

o Primers

Heat Shock Protein (HSP) 70● C12C8● F44E5.4

Actin Control

Results: Muscle cells exhibit greater heat shock robustness than neuronal cells with F44E5.4 HSP70 isotype

Figure 1: Muscle cells exhibit greater heat shock robustness than neuronal cells with F44E5.4 HSP isotype. Both Q45 and Q63 heat shocked muscle cell lines trend towards greater HSP expression than the 422 heat shocked neuronal cell line. (*) indicate statistical significance when compared to wild type positive control. (+) indicates heat shock induction while (-) indicates no heat shock induction.

Results contd...: Neuronal cells exhibit decreased stress response in comparison to muscle cells in C12C8 HSP70 isotype.

Figure 2: Neuronal cells exhibit decreased stress response as opposed to muscle cells in C12C8 HSP isotype. Q63+ heat shocked muscle cells trend towards increased HSP expression while 422+ heat shocked neuronal cells trend towards decreased HSP expression in comparison to the wild type positive control (N2+). (*) indicate statistical significance when compared to wild type positive control. (+) indicates heat shock induction while (-) indicates no heat shock induction.

CONCLUSION

● Expression of Ataxin-3 in the muscles or neurons of C.elegans decreases the robustness of HSR● The effect is more predominant in neuronal cells than in muscle cells.● The decrease in HSP70 expression is dependent on the number of PolyQ repeats● For our experiments, F44E5.4 HSP70 gives a more accurate representation of the result

● These results suggest that the expression of ataxin-3 in both muscular and neuronal cells of C.elegans results in significant decrease in the ability of the organismal cells to effectively launch a heat shock response. Overall, we conclude that the aggregation of ataxin-3 is able to limit the ability of the organism to effectively respond to the massive misfolding of ataxin-3 proteins that results in aggregation. In other words, ataxin-3 expression results in the malfunction of the organismal HSR.

● Future work might attempt to better understand the mechanism by which ataxin-3 expression reduces the expression of HSP70 thereby causing the malfunction of HSR.

References

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