Nakeirah Christie, Hannah Fay, Amy Lee, Sheana Algama, Jordan Grant, Fridien Tchoukoua, Paul Sands, Emily Javadi, David Spears, Jacob Zalewski, Emily Williams, John Peyton Bush

Abstract

Machado Joseph Disease (MJD) is a neurodegenerative disorder caused by an expansion of CAG (polyQ) repeats within the gene that codes for the ataxin-3 (AT3) protein. This expansion leads to protein aggregation and a toxic-gain of function, but understanding the mechanisms by which aggregated ataxin-3 affects cell function is not well understood. We utilize the model organism C. elegans to investigate the toxicity and aggregation of the ataxin-3 protein in different cell and tissue-types. Specifically, we are interested in how cellular protein homeostasis (“proteostasis”) impacts aggregation and toxicity of the mutated protein in different tissues. To address this, we characterized the aggregation and toxicity of a C-terminal fragment of ataxin-3 (AT3CT) with various polyQ tract lengths expressed in C. elegans body wall muscle cells or neurons. Toxicity was determined by performing motility assays and aggregation was determined by fluorescence microscopy. Because it has previously been shown that neurons control the organismal heat shock response, we wondered whether animals expressing a disease-associated, aggregation-prone variant of ataxin-3 in neurons would have an impaired HSR. To address this, we performed qRT-PCR of heat-inducible genes. Surprisingly, our data suggest that ataxin-3 expressed in neurons has little effect on the organismal heat shock response, despite a clear age-dependent increase in aggregation.

Age-Dependent Aggregation of AT3CT in Muscle Cells as Compared to Neuronal Cells

Fluorescence Micrographs of C. elegans expressing the C-terminus of the ataxin-3 protein (AT3CT) in either the body wall muscle cells (orange background) or neuronal cells (blue background). The AT3CT protein was tagged with YFP to allow for visualization. Representative individual animals were imaged from L4 stage until Day 11 of adulthood.

Motility was determined as a function of thrashing in liquid. Individual L4 larvae or animals at day 4 of adulthood were picked to a 10 µL drop of M9 on a microscope slide and were given a 30 s adjustment period before counting thrashing rate. Thrashes (defined as the head crossing the vertical midline of the body) were counted for 60 s. A minimal n-number of n = 30 was assayed for each genotype or time point indicated.

Characterizing the Aggregation and Toxicity of the MJD-Associated Ataxin-3 protein Expressed in Body Wall Muscle cells as Compared to Neuronal cells of C. elegans

qRT-PCR showing the relative expression levels of the endogenous F44E5.4 (Hsp70) mRNA before (-HS) and after (+HS) heat shock in wild (N2) animals as compared to animals expredssing AT3CT in body wall muscle cells or neurons.

Conclusions

• AT3CT aggregation and toxicity is polyQ-length dependent in body wall muscles cells.

• AT3CT aggregation and toxicity is polyQ-length dependent and modulated by aging.

Future Directions

• Develop an AT3CT intestinal line to continue comparing AT3CT toxicity and aggregation in various tissue types

• Use RNAi to knock the expression of proteostasis network genes to identify regulators of AT3CT aggregation and toxicity.

The polyQ-conAT3taining C-terminal domain (lacking the N-terminal 257 amino acids) of AT3 was fused to YFP and expressed in body wall muscle cells under the control of the unc-54 promoter.

YFP

unc-54

YFPAT3CT

Q45unc-54

YFPAT3CT

Q63unc-54

C. elegans were transformed with the following gene constructs

L4

AT3C

T Q

45AT

3CT

Q63

AT3C

T Q

14AT

3CT

Q75

Day 1 Day 2 Day 4 Day 8Day 5 Day 9 Day 11

GFP Phalloidin

N2

AT3CT(Q45)

AT3CT(Q63)

Expression of polyQ-expanded AT3CT in C. elegans body wall muscle cells leads to polyQ length-

dependent foci formation

Fluorescence micrographs showing fixed N2 (wild type), AT3CT (Q45)::YFP and AT3CT (Q63) animals imaged for YFP fluorescence (green) or phalloidin-stained actin filaments (red).

0 10 20 30 40 50 600

20

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60

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120

Rela

tive

Fluo

resc

ence

Inte

nsity

Time (s)bleach

AT3CT(Q63)

YFP

AT3CT(Q45) (foci)

AT3CT(Q45) (diffuse)

Q0

(YFP

)

AT3C

T Q

45-Y

FP

AT3C

T Q

63-Y

FP

YFP monomer

aom

PolyQ Length-Dependent Aggregation

Native gel showing the YFP-containing protein species that accumulate in lines expressing YFP alone, At3CT(Q45)::YFP, or AT3CT(Q63)::YFP. Aggregates (a), oligomers (o), and monomers (m) are indicated.

Fluorescence Recovery after Photobleaching (FRAP) for diffuse fluorescent protein in YFP or AT3CT(Q45)::YFP-expressing animals, or fluorescent foci in AT3CT(Q45)::YFP or AT3CT(Q63)-expressing animals.

462 proteostasis regulators were identified in genetic screens. Published gene lists were compared to identify unique or overlapping hits. Genes that overlapped between two of the three studies (21 total) appear as hybrid colors (green, orange, purple). Genes (8) that appeared in all three studies are white. Together, these genes represent the proteostasis network and may modulate AT3CT aggregation and toxicity.

The Proteostasis NetworkAT3CT Expression Does not Inhibit the

Heat Shock Response

Motility Assays of AT3CT-expressing C. elegans suggest

tissue-specific toxicity

Body

Wal

l Mus

cle

Cells

Neu

rona

l Cel

ls

Muscles Neurons

N2-HS N2 +HSAT3CT Q

45 -HSAT3CT Q

45 +HSAT3CT Q

63 -HS AT3CT Q

63 +HS AT3CT Q

14 -HSAT3CT Q

14 +HSAT3CT Q

75 -HSAT3CT Q

75 +HS

0500

1000150020002500300035004000

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tive

Hsp7

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xpre

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n

N2 AT3CTQ45(muscle)

AT3CTQ63(muscle)

AT3CTQ14(neurons)

AT3CTQ75(neurons)

N2 AT3CTQ45(muscle)

AT3CTQ63(muscle)

AT3CTQ14(neurons)

AT3CTQ75(neurons)

L4 Larval Stage Day 4 of Adulthood

Thra

shes

/min

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/min