were found with transgenic HA-tagged polyQ40 animals (Figures
S1A and S1B).
Effects of proteotoxicity on the UPRmtwere highly specific to
the length, neuronal expression, and form of proteotoxic stress
on the mitochondria. Transgenic animals expressing either
non-aggregative or severely neurotoxic expansions of polyQ
failed to elicit cell-non-autonomoushsp-6p driven GFP expres-
sion (Figure 1A). PolyQ40 expression in the body wall muscle,
rather than in the neurons, was also incapable of eliciting a
cell-non-autonomous mitochondrial response to proteotoxic
stress (Figure S1E). Moreover, neither neuronal expression of
the Alzheimer’s-related protein Ab1-42, nor mutant forms
of the ALS associated protein TDP-43, were sufficient to induce
the UPRmt(Figure S1F). Finally, neuronal polyQ40 expression
had a specific effect on mitochondrial stress response activa-
tion, as neither basal levels nor induction of chaperones and
stress-responsive genes, including hsp-16.2 (small hsp20/
alpha-B crystalline, responsive to heat shock) (Link et al.,
1999 ),hsp-4 (BiP/Hsp9a, responsive to ER stress) (Kapulkin
et al., 2005), orsod-3 (superoxide dismutase, responsive to
oxidative stress) (Honda and Honda, 1999) were affected by its
expression (Figure S1G). Collectively, these data suggest that
a cell-non-autonomous mitochondrial stress response is
invoked by polyQ40 protein expression in theC. elegansnervous
system.
We analyzed whether the effects of polyQ40 on UPRmtwere
conserved in mammalian cells. We expressed exon1 of the Htt
gene followed by polyglutamine expansions of lengths of 25Q,
78Q, 103Q, and 153Q in human primary fibroblasts. mtHSP70
levels increased with increasing lengths of polyglutamine expan-
sions and were significantly increased in cells expressing the
103Q and 153Q lengths (Figure 1F and 1G). This suggests that
the UPRmtinduction by polyglutamine toxicity is not specific to
C. elegans,but occurs in mammalian models of proteotoxicity
as well.
Distal hsp-6 Induction Requires Functional UPRmt
Components
Previous work inC. eleganshas identified multiple genetic com-
ponents required for the UPRmtstress response (Haynes et al.,
2007, 2010; Yoneda et al., 2004). The increased expression of
mitochondrial chaperones HSP-6 and HSP-60 requires the nu-
clear localization of transcription factors and co-regulators
DVE-1, UBL-5, and ATFS-1, while the protease CLPP-1 is
required for generating the mitochondrial-derived signal to acti-
vate the UPRmt. We predicted that the cell-non-autonomous in-
duction of mitochondrial chaperones observed with neuronal
polyQ40 expression requires one or more essential UPRmtfac-
tors. To test this hypothesis, we applied RNAi againstubl-5,
dve-1,atfs-1, andclpp-1, andcco-1to neuronal polyQ40 worms
expressinghsp-6p::GFP. RNAi againstdve-1,atfs-1, andclpp-1
blocked the induction of thehsp-6p::GFP reporter in peripheral
tissues, whilecco-1RNAi showed robust induction (Figures 2A
and 2B).ubl-5 knockdown also suppressed the UPRmt in
response to neuronal polyQ40, albeit to a lesser extent (Figures
2 A and 2B). These expression changes were also assessed us-
ing immunoblots for GRP75/HSP6 and GFP from animals har-
vested prior to reproduction to negate embryonic HSP-6 protein
levels (Figure S2A). RNAi-mediated knockdown ofatfs-1and
clpp-1resulted in a relative decrease in HSP6 expression and
a robust decrease in the GFP marker ofhsp-6reporter expres-
sion. However, RNAi towarddve-1andubl-5, while showing a
modest decrease in intestinal GFP fluorescence, showed little,
if any, reduction in whole-worm GRP75/HSP6 and GFP by
immunoblot analysis.
As an additional measure of UPRmtinduction in peripheral
cells, strains expressing adve-1p::DVE-1::GFP translational
fusion gene (Haynes et al., 2007) were used to assess the effects
of neuronal polyQ40 on the expression and localization of the
dve-1transcription factor in the intestine. In response to mito-
chondrial stress, DVE-1 accumulates in the nucleus, serving as
an additional proxy for induction of the UPRmt. We found
increased nuclear accumulation ofdve-1p::DVE-1::GFP in the in-
testinal cells of animals expressing the neuronal polyQ40 (Fig-
ure 2C and 2D). Taken together, cell-non-autonomous induction
of the UPRmtcaused by neuronal expressed PolyQ40 requires an
intact UPRmtsignaling pathway.Neuronally Expressed polyQ40 Physically Interacts with
Mitochondria and Affects Mitochondrial Function
Polyglutamine repeats within the Huntingtin (Htt) protein impairs
mitochondrial function and morphology through direct interac-
tion with the outer membrane of the mitochondria (Costa and
Scorrano, 2012; Panov et al., 2002). We hypothesized that
polyQ40 proteins may bind to and thus affect mitochondria in
the neurons ofC. elegans.To test this possibility, we isolated
mitochondrial and cytoplasmic fractions from wild-type and
neuronal polyQ40-expressing animals for the presence of polyQ
protein binding to the mitochondria. In these analyses, we
observed significant polyQ40 protein in both the mitochondrial
and cytoplasmic fractions (Figure 3A). Hyper-toxic polyQ67
was, in contrast, only slightly detectable in the mitochondrial
fraction (Figure 3A). We confirmed the specificity of our fraction-
ation using antibodies toward cytoplasmic and mitochondrial
proteins.
We repeated these fractionation experiments using Q19 as
well as a second cytoplasmic marker,b-actin. As expected,
Q19, which cannot induce the cell-non-autonomous response,
was not present in the mitochondrial fraction (Figure S3A). Using
strains in which Ab1-42 is expressed in the muscle we found that
the high molecular weight species of Ab1-42 were largely
excluded from the mitochondria (Figure S3B), while oligomers
were present in small amounts in the mitochondrial fraction. Of
note, Abexpressed in the muscle does not elicit a cell-non-
autonomous UPRmtresponse, and thus, localization may not
engage the same mitochondrial stress pathways as in the ner-
vous system. These results suggest that the interaction of
polyQ40 with mitochondria inC. elegansmay be responsible
for its capacity to invoke a cell-non-autonomous mitochondrial
stress response.
We tested if there was an overt metabolic consequence of the
physical interaction of polyQ40 and mitochondria and cell-non-
autonomous induction of the UPRmt. We examined the effects
of neuronal expression of polyQ40 on in vivo oxygen consump-
tion rates in whole animals. We found that the neuronal polyQ40
expressing strains had significantly lowered respiration inCell 166 , 1553–1563, September 8, 2016 1555