3Articles review and critique

http://dx.doi.org/10.14336/AD.2021.0404

*Correspondence should be addressed to: Dr. Jeremy M. Van Raamsdonk, McGill University, MeDiC and BRaIN Programs, McGill

University Health Centre, Quebec, Canada. Email:[email protected]. #these authors equally contributed this work.

Copyright: © 2021 Machiela E et al. This is an open-access article distributed under the terms of the Creative Commons Attribution

License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

ISSN: 2152-5250 1753

Original Article

Targeting Mitochondrial Network Disorganization is

Protective in C. elegans Models of Huntington’s Disease

Emily Machiela1,#, Paige D. Rudich2,3,#, Annika Traa2,3,#, Ulrich Anglas2,3, Sonja K. Soo2,3,

Megan M. Senchuk1, Jeremy M. Van Raamsdonk1,2,3,4,5,*

1 Laboratory of Aging and Neurodegenerative Disease, Center for Neurodegenerative Science, Van Andel

Research Institute, Grand Rapids MI 49503, USA 2 Department of Neurology and Neurosurgery, McGill University, Montreal, Quebec, H4A 3J1, Canada

3 Metabolic Disorders and Complications Program, and Brain Repair and Integrative Neuroscience Program,

Research Institute of the McGill University Health Centre, Montreal, Quebec, H4A 3J1, Canada 4 Division of Experimental Medicine, Department of Medicine, McGill University, Montreal, Quebec, Canada

5 Department of Genetics, Harvard Medical School, Boston MA 02115, USA

[Received January 2, 2021; Revised April 3, 2021; Accepted April 3, 2021]

ABSTRACT: Huntington’s disease (HD) is an adult-onset neurodegenerative disease caused by a trinucleotide

CAG repeat expansion in the HTT gene. While the pathogenesis of HD is incompletely understood, mitochondrial

dysfunction is thought to be a key contributor. In this work, we used C. elegans models to elucidate the role of

mitochondrial dynamics in HD. We found that expression of a disease-length polyglutamine tract in body wall

muscle, either with or without exon 1 of huntingtin, results in mitochondrial fragmentation and mitochondrial

network disorganization. While mitochondria in young HD worms form elongated tubular networks as in wild-

type worms, mitochondrial fragmentation occurs with age as expanded polyglutamine protein forms aggregates.

To correct the deficit in mitochondrial morphology, we reduced levels of DRP-1, the GTPase responsible for

mitochondrial fission. Surprisingly, we found that disrupting drp-1 can have detrimental effects, which are

dependent on how much expression is decreased. To avoid potential negative side effects of disrupting drp-1, we

examined whether decreasing mitochondrial fragmentation by targeting other genes could be beneficial. Through

this approach, we identified multiple genetic targets that rescue movement deficits in worm models of HD. Three

of these genetic targets, pgp-3, F25B5.6 and alh-12, increased movement in the HD worm model and restored

mitochondrial morphology to wild-type morphology. This work demonstrates that disrupting the mitochondrial

fission gene drp-1 can be detrimental in animal models of HD, but that decreasing mitochondrial fragmentation

by targeting other genes can be protective. Overall, this study identifies novel therapeutic targets for HD aimed

at improving mitochondrial health.

Key words: Huntington’s disease, mitochondria, mitochondrial dynamics, C. elegans, neuroprotection, genetics,

neuroprotection, neurodegeneration, aggregation, DRP1, animal model

Huntington’s disease (HD) is an autosomal dominant

neurodegenerative disease caused by an expansion of the

polyglutamine tract in the N-terminal of the huntingtin

(Htt) protein. The expression of the expanded

polyglutamine tract is both necessary and sufficient for

cellular toxicity [1], although loss of wild-type huntingtin

function might also contribute to disease pathogenesis [2].

HD is characterized by progressive cognitive decline,

neuropsychiatric abnormalities, and motor impairment

[3]. In unaffected individuals, the polyglutamine tract of

the Htt protein is polymorphic, containing from 9-34

glutamines. However, mutations in the HD gene leading

Volume 12, Number 7; 1753-1772, October 2021

Machiela E., et al Mitochondrial dynamics and HD

Aging and Disease • Volume 12, Number 7, October 2021 1754

to 35 or more glutamines have been shown to cause HD.

Within the disease range of 35 glutamines and above, age

of onset negatively correlates with the number of

glutamines present [4, 5]. Although Htt is expressed in

every cell of the body and pathology has been observed in

multiple tissues, cellular dysfunction and atrophy are most

severe in the GABAergic medium spiny neurons of the

striatum. The reasons for this selective vulnerability are

still unknown.

While the cause of cellular dysfunction in HD is still

incompletely understood, mitochondrial dysfunction is

thought to play a central role in disease pathogenesis [6,

7]. There is significant evidence for mitochondrial

dysfunction in HD patients and animal models including

decreased activity of complexes in the electron transport

chain [8], increased lactate production in the brain [9],

decreased levels of ATP production [10], decreased

mitochondrial membrane potential [11], and impaired

trafficking of mitochondria within the cell [12]. The

importance of mitochondrial dysfunction to HD

pathogenesis is also suggested by the fact that systemic

administration of 3-nitropropionic acid, a neurotoxin that

inhibits mitochondrial function, can reproduce symptoms

and neuropathological deficits that occur in HD [13, 14].

In addition, a genome-wide association study

investigating genetic modifiers for age of onset of HD

found pathways involving mitochondrial fission to

significantly modify age of onset of the disease [15].

Recent work has demonstrated that mitochondrial

dynamics are disrupted in HD. Mitochondria continually

change their shape in response to the needs of the cell, and

these changes impact both the function and distribution of

the mitochondria. Mitochondrial morphology is

determined by two opposing processes: fission and fusion.

Mitochondrial fission results in an increase in

mitochondrial fragmentation as new mitochondria are

pinched off of existing mitochondria or mitochondrial

networks. The fission process is mediated by dynamin-

related protein 1 (DRP-1/DRP1) with the help of

mitochondrial fission proteins (FIS-1/FIS-2/FIS1) and

mitochondrial fission factors (MFF-1/MFF-2/MFF1).

Conversely, mitochondrial fusion leads to decreased

mitochondrial fragmentation by joining individual

mitochondria together with other mitochondria to form

interconnected mitochondrial networks. The fusion

process requires merging of the inner mitochondrial

membrane by optic atrophy protein 1 (EAT-3/OPA1) and

the merging of the outer mitochondrial membrane by

mitofusin (FZO-1/MFN).

In HD cell lines [16-21], the 3-nitropropionic acid

neurotoxin model of HD [22], cells from HD mouse

models [23] and cells derived from HD patients [19, 23],

it has been found that mitochondria are more fragmented

than in unaffected controls. In addition, examination of

mitochondria by electron microscopy in brain sections

from R6/2 mice [20] and YAC128 mice [17] revealed the

presence of smaller mitochondria in HD mice compared

to controls, suggesting that increased mitochondrial

fragmentation also occurs in vivo. In these studies, it has

been shown that the expression of exon 1 fragments of

mutant Htt is sufficient to cause mitochondrial

fragmentation [16-18]. The increase in mitochondrial

fragmentation in HD could result from excess

mitochondrial fission, decreased mitochondrial fusion or

both. While the precise mechanism by which mutant Htt

causes mitochondrial fragmentation is still unclear,

contributing factors may include: alterations in expression

levels of fission and fusion proteins [20, 24, 25], an

increase in DRP-1 enzymatic activity resulting from

increased interaction with mutant Htt [17, 26], increased

S-nitrosylation of DRP-1 leading to increased fission

activity [18], increased levels of reactive oxygen species

[22, 27], decreased Nrf2 signaling [20], and increased

calcineurin activity [23].

Importantly, reducing mitochondrial fragmentation

has been shown to be beneficial in models of HD.

Decreasing the activity or expression of the mitochondrial

fission protein DRP-1 increases survival in cell models of

HD [16, 17, 19]. In addition, treating a worm model of

HD expressing exon 1 fragment of mutant Htt with 74

CAG repeats in body wall muscle with RNAi against drp-

1 improved the movement deficit present in these worms,

although the effect of this treatment on mitochondrial

morphology in these worms was not assessed [16].

Furthermore, treatment of the R6/2 mouse model of HD

with a DRP-1 inhibitor (P110-Tat) improved behavior,

survival and neuropathology in these mice, and resulted

in a significant increase in cristae area in electron

micrographs [19]. The P110-Tat DRP-1 inhibitor was also

able to ameliorate mitochondrial structural deficits in the

hearts of R6/2 mice, indicating that this inhibitor can also

be effective in muscle tissue [21]. Combined, these

results suggest that developing interventions that inhibit

DRP-1 may be beneficial in the treatment of HD.

In this work, we explore the role of mitochondrial

fragmentation in the pathogenesis of HD, and whether

targeting this deficit may be an effective strategy to treat

HD. To do this, we use C. elegans models, which permit

the visualization of mitochondrial morphology in a live

organism that exhibits quantifiable, disease-relevant

phenotypic deficits. We find that C. elegans models of HD

exhibit mitochondrial fragmentation, which is temporally

correlated with polyglutamine aggregation. We find that

decreasing levels of drp-1 fails to correct the deficit in

mitochondrial morphology and can be detrimental,

depending on the level of disruption. In contrast, treating

worms with other RNAi clones that decrease

Machiela E., et al Mitochondrial dynamics and HD

Aging and Disease • Volume 12, Number 7, October 2021 1755

mitochondrial fragmentation improved movement in

worm models of HD.

MATERIALS AND METHODS

Strains

The following strains were used in this study:

N2 (WT)

JVR240 syIs243[Pmyo-3::TOM20:RFP] referred to as

mitoRFP

MQ1699 Punc-54::Htt28Q:GFP referred to as BW-

Htt28Q

MQ1698 Punc-54::Htt74Q:GFP referred to as BW-

Htt74Q

AM138 rmIs120[Punc-54::24Q:YFP] referred to as

BW-24Q

AM141 rmIs133[Punc-54::40Q:YFP] referred to as

BW-40Q

MQ1753 drp-1 (tm1108)

JVR248 Punc-54::Htt28Q:GFP;syIs243[Pmyo-

3::TOM20:RFP]

JVR250 Punc-54::Htt74Q:GFP;syIs243[Pmyo-

3::TOM20:RFP]

JVR251 drp-1(tm1108);Punc-54::Htt74Q:GFP

JVR255 drp-1(tm1108);syIs243[Pmyo-3::TOM20:RFP]

JVR259 drp-1(tm1108);Punc-

54::Htt74Q:GFP;syIs243[Pmyo-3::TOM20:RFP]

JVR473 syIs243[Pmyo-3::TOM20:RFP];rol-6(su1006)

JVR474 rmIs120[Punc-54::24Q:YFP];syIs243[Pmyo-

3::TOM20:RFP];rol-6(su1006)

JVR475 rmIs133[Punc-54::40Q:YFP];syIs243[Pmyo-

3::TOM20:RFP];rol-6(su1006)

JVR463 Punc-54::Htt74Q:GFP;syIs243[Pmyo-

3::TOM20:RFP];rol-6(su1006)

JVR520 Punc-54::Htt28Q:GFP;syIs243[Pmyo-

3::TOM20:RFP]; rol-6(su1006)

JVR521 drp-1(tm1108);Punc-

54::Htt74Q:GFP;syIs243[Pmyo-3::TOM20:RFP]; rol-

6(su1006)

All strains were maintained at 20°C on NGM plates

seeded with OP50 bacteria. All crosses were confirmed by

genotyping using PCR and, where applicable, confirmed

by fluorescent microscopy.

Confocal imaging and quantification

Mitochondrial morphology was imaged and quantified

using worms that express mitochondrially-targeted RFP

specifically in body wall muscle (syIs243[Pmyo-

3::TOM20:RFP]). In order to facilitate imaging, these

worms were crossed into a rol-6 background. The rol-6

mutation results in animals moving in a twisting motion,

thus displaying a helix of muscle cells upon imaging

(Supplementary Fig. 1). This is beneficial when imaging

the mitochondria of body wall muscle cells in the

nematode as it ensures that several portions of both the

ventral and dorsal quadrants of muscle cells are within the

plane of view (facing the objective lens). This facilitates

mitochondrial imaging as the tubular mitochondrial

organization can be seen within much of the muscle.

Without the rol-6 mutation, the lateral side of the

nematode may face the objective lens with only the

longitudinal edges of the muscle being visible, thus

making it difficult to observe mitochondrial organization.

To image the mitochondria, approximately 20 young

adult worms were mounted on 2% agar pads and

immobilized using 10 µM levamisole. Worms were

imaged under a 63x objective lens on a Zeiss LSM 780 or

Nikon A1R Ti confocal microscope. All conditions were

kept the same for all images. For representative images, a

z-stack of images spaced 0.125-0.40 µm apart were

collected and a z-stack projection was created using either

Nikon Elements or ImageJ to compress stacks into a

single image.

For quantification, a single plane image taken in the

same body region for each worm was used to avoid the

complication of mitochondria being present in two planes.

This slice was made binary using the Nikon Elements

thresholding tool. A background subtraction of a constant

50 was applied. Next, a pixel picker was applied to

several control mitoRFP images to define the low and

high threshold levels. Once optimum threshold numbers

were defined for control images, these limits were applied

to all images to be quantified with the separate function

on. Size and circularity were not used to define thresholds.

Prior to creating the binary mask, images were manually

inspected for proper threshold parameters. In the event

that threshold parameters mislabeled mitochondria,

objects were manually included or excluded prior to

masking. Mitochondrial circularity, number, and area

were measured using the measure objects tool in Nikon

Elements AR after the threshold mask was applied. For

mitochondrial circularity, raw numbers were exported to

Microsoft Excel and averages were calculated prior to

statistical analysis in Graphpad Prism. All other

calculations were exported directly to Prism for analysis.

Oxygen consumption

Basal oxygen consumption rate was measured using a

Seahorse XFe96 analyzer (Seahorse bioscience Inc., North

Billerica, MA, USA)[28]. Synchronized worms at day 1

of adulthood were cleaned in M9 buffer (22 mM KH2PO4,

34 mM NA2HPO4, 86 mM NaCl, 1 mM MgSO4). Cleaned

nematodes were pipetted in calibrant (~50 worms per

well) into a Seahorse 96-well plate. Oxygen consumption

was measured six times and rates of respiration were

Machiela E., et al Mitochondrial dynamics and HD

Aging and Disease • Volume 12, Number 7, October 2021 1756

normalized to the number of worms in each individual

well. The plate readings were begun within 20 minutes of

introduction of the worms into the well. Reading from

each well were normalized relative to the number of

animals per well. Well probes were hydrated in a 175 µL

Seahorse calibrant overnight before this assay was begun.

We found it is important to turn off the heating incubator

to allow the Seahorse machine to reach room temperature

before placing nematodes inside the machine. For these

experiments, we chose to measure oxygen consumption

per worm so that we could compare the rate of oxidative

phosphorylation for the whole organism to the whole

organism phenotypes that we were measuring (e.g.

movement and lifespan). Also, we chose to use a Seahorse

extracellular flux analyzer to measure oxygen

consumption so that all of the strains being compared

could be measured at the same time so that the conditions

would be identical. With the low number of worms that

are used in each well, it would be difficult to accurately

measure protein content, especially given that worms will

often stick to pipet tips or the side of the dish during

transfer.

ATP production

ATP levels were measured using a luminescence-based

ATP kit [29]. Approximately 200 worms were age-

synchronized by a limited lay. Worms were collected in

de-ionized water, washed, and freeze-thawed three times.

The resulting pellet was sonicated in a Bioruptor

(Diagenode) with 30 cycles of 30 seconds on, 30 seconds

off. The pellet was boiled for 15 minutes to release ATP,

then spun at 4°C at 11,000  g for 10 minutes. The

supernatant was collected and measured using a

Molecular Probes ATP determination Kit (Life

Technologies). Luminescence was normalized to protein

content, which was measured with a Pierce BCA protein

determination kit (Thermo Scientific).

Rate of movement

For measuring the effects of drp-1, the rate of movement

was assessed by measuring thrashing rate in liquid using

video-tracking and computer analysis [30].

Approximately 50 pre-fertile day 1 young adult worms

were placed in M9 buffer on a clean NGM plate. Videos

were taken with an Allied Vision Tech Stingray F-145 B

Firewire Camera (Allied Vision, Exton, PA, USA) at

1024×768 resolution, 8-bit using the MATLAB image

acquisition toolbox. Analysis was performed using

wrMTrck plugin for ImageJ (publicly available at

www.phage.dk/plugins).

For screening the mitochondrial fragmentation genes,

the rate of movement was assessed by measuring absolute

crawling speed and thrashing rate using WormLab

2019.1.2 (MBF BioSciences). Experiments were done on

day 1 adults, and the animals were isolated as L4’s 24 hrs

before. Animals were exposed to RNAi using the parental

paradigm, or if the RNAi clone inhibited development, the

animals were grown on empty vector RNAi and placed on

the respective RNAi clone as L4’s (E04A4.4, C33A12.1,

abhd-11.1, iars-1, his-12 and acs-1). For the experiment,

worms were removed from their plates with M9 buffer,

washed twice with M9 buffer, and placed on clean 3-cm

NGM plates. A Kimwipe was used to remove excess

liquid and the worms were allowed to acclimate for 5

minutes. The plates were placed under a monochrome

digital camera (Basler acA2440 camera with an AF Micro

Nikkor 60 mm f/2.8 D lens) and tapped to stimulate

movement. 20-30 animals were normally in frame. The

worms were recorded using the WormLab software

(Version 2019.1.2), in 45 s long videos with a resolution

of 2456x2052 at a scale of 9.9 m/pixel. Crawling was

recorded at a frame rate of 7.5 frames/s. After recording

crawling, M9 buffer was added to the plate. Worms were

allowed to acclimate for 5 minutes and then recorded

while swimming at a frame rate of 14 frames/s. Worms

that were tracked for less than half of the video were

excluded from the analysis because the worms could have

left the field-of-view and then returned, causing double

counting. Crawling speed and thrashing rates were

analyzed using the Absolute Peristaltic Speed results and

Wave Initiation Rate results, respectfully, exported from

WormLab and processed using Microsoft Excel 2016

(Microsoft, Redmond, WA, USA). 3 replicates of ~20

worms were performed for each RNAi clone.

Imaging and quantification of aggregation

Experimental animals were day 1 adults and were isolated

at the L4 stage 24 hours before the experiments. Animals

were exposed to RNAi using the parental paradigm, or if

the RNAi clone inhibited development, the animals were

grown on empty vector RNAi and placed on the respective

RNAi clone as L4’s (E04A4.4, C33A12.1, abhd-11.1,

iars-1, his-12 and acs-1). 10 animals were mounted on 3%

agarose pads using 10mM levamisole for anesthesia and

imaged within 45 minutes of levamisole exposure. Images

were taken on a Nikon Eclipse Ti microscope with a

Nikon Plan Apo 20x/0.75 NA objective and a Zyla Andor

sCM05 camera. z-stacks of the animals were recorded

with a 2m step size. Analysis was performed in ImageJ

(Version 2.1.0/1.53c) by merging the z-stack using

Temporal-Color Code to color code the different z planes,

and then the aggregates were manually counted using the

Cell Counter plugin. At least 10 animals per clone were

Machiela E., et al Mitochondrial dynamics and HD

Aging and Disease • Volume 12, Number 7, October 2021 1757

quantified. Clones which caused a measurable decrease

were repeated for confirmation.

Lifespan

Lifespan was determined on nematode growth media

(NGM) agar plates with 25 μM 5-fluoro-2′-deoxyuridine

(FUdR) in order to reduce the development of progeny.

Plates with 25 μM FUdR do not completely prevent the

development of progeny to adulthood in the first

generation so animals were transferred to fresh agar plates

after 4 days [31]. After the initial transfer, worms were

moved to fresh plates weekly. Animal survival was

observed every 2 days by gentle prodding. Three

replicates of 30 animals each were completed.

Brood size

Brood size was determined by placing individual young

adult staged animals onto agar plates with daily transfers

to new plates until progeny production ceased. The

resulting progeny were allowed to develop to adulthood

before quantification. Three replicates of 5 animals each

were completed.

Post-embryonic development

Post-embryonic development (PED) was assessed by

moving eggs to agar plates. After 3 hours, newly hatched

L1 worms were transferred to a new plate. The hours from

hatching to the young adult transition was measured as the

PED time. Three replicates of 20 animals each were

completed.

Quantitative reverse-transcription PCR (qPCR)

mRNA was collected from pre-fertile young adult worms

using Trizol as previously described [32]. We collected

three biological replicates each for WT, BW-40Q and

BW-Htt74Q worms. The mRNA was converted to cDNA

using a High-Capacity cDNA Reverse Transcription kit

(Life Technologies/Invitrogen) according to the

manufacturer’s directions. qPCR was performed using a

FastStart Universal SYBR Green kit (Roche) in an AP

Biosystems real-time PCR machine [33, 34]. Primer

sequences utilized:

drp-1 L-GGTTTTCACAGACTTCGATGC

R-TA GGCTCCGAAGTAGCGAAA

fis-1 L-AGAAATTCTGGCGGCTCGT

R-GCG TGTGCAAGAGCAAGATA

fis-2 L-GGGAATCGTGTGTCTTGAGAAG

R-GG CCATCATGAGTCATTGC

mff-1 L-CCGCTCAATAGATTGATGAACA

R-T TGGGGACTTCCATTCTGAG

mff-2 L-TGGATAAACTTCCAACGGAAA

R-CC GGGCTGTGTCTAGCTCT

eat-3 L-GCGGCTAGAACGTGGTATGA

R-CGG GCTCTTTTACTGGAACA

fzo-1 L-GCTTTCTGCAGGTTGAAGGT

R-CGA CACCAGGGCTATCAAGT

gfp L-GACGACGGCAACTACAAGAC

R-TCC TTGAAGTCGATGCCCTT

Quantification of mitochondrial DNA

Mitochondrial:nuclear DNA ratios were calculated as

previously described with minor modifications [35].

Wild-type, BW-40Q and BW-Htt74Q worms were grown

on OP50 at 20°C. Animals were isolated at the L4

developmental stage and collected the following day as

day 1 adults, where three replicates of six worms for each

strain were isolated in 90 µl of worm lysis buffer and

lysed. The lysed worm samples were analyzed by

quantitative RT-PCR as previously described for C.

elegans [35], using established mitochondrial-DNA

specific nd-1 primers and nuclear-DNA specific cox-4

primers [35, 36]. Samples were collected three times, each

time with three biological replicates. We performed three

technical replicates on each of these samples. The primer

sequences used are as follows:

nd-1 L-AGCGTCATTTATTGGGAAGAAGAC

R-AAGCTTGTGCTAATCCCATAAATGT

cox-4 L-GCCGACTGGAAGAACTTGTC

R-GCG GAGATCACCTTCCAGTA

RNA interference (RNAi)

All RNAi clones were sequence verified. To knockdown

expression of genes, the RNAi clones were grown

approximately 12 hours in LB with 50 μg/ml carbenicillin.

Cultures were concentrated (5x) and seeded onto NGM

plates containing 5 mM IPTG and 50 μg/ml carbenicillin.

Plates were incubated to induce RNAi for 2 days at room

temperature. RNAi was performed at 20°C. For

experiments examining RNAi knockdown beginning in

the parental generation (L4 parental paradigm), L4 worms

were plated on RNAi plates, transferred to a new plate the

following day as gravid adults, and then removed after 24

hours. The progeny from these worms were used for

analysis.

Quantification of knockdown of drp-1 mRNA by drp-1

RNAi

Wild-type and BW-Htt74Q worms were grown on empty

vector (EV) and drp-1 RNAi following the RNAi parental

paradigm described in the RNAi methods. Levels of drp-

1 and act-3 were quantified through quantitative RT-PCR.

Machiela E., et al Mitochondrial dynamics and HD

Aging and Disease • Volume 12, Number 7, October 2021 1758

Primers were specifically designed to exclude the drp-1

RNA used to induce RNAi (drp-1: L-GAGATGTC

GCTATTATCGAACG, R-CTTTCGGCACACTATCC

TG). Three biological replicates each with three technical

replicates were performed.

Effect of RNAi clones on mitochondrial morphology

In order to examine the effect of RNAi clones on

mitochondrial morphology in mitoRFP;rol-6 and BW-

Htt74Q;mitoRFP;rol-6 worms, 30 L4 worms were picked

to plates seeded with EV, pgp-3, F25B5.6, and alh-12

RNAi-expressing bacteria. After 24 hours gravid adults

were picked to new RNAi plates. After another 24 hours

gravid adults were removed from the RNAi plates.

Progeny were allowed to develop to the young adult stage

before imaging.

Experimental design and statistical analysis

Experiments were performed such that the experimenter

was blinded to the genotype of the worms. Experimental

worms were randomly selected from maintenance plates.

For all experiments, we completed a minimum of three

biological replicates (independent population of worms

tested on a different day). Where possible (e.g.,

measurement of movement) assays were completed using

automated approaches with computer analysis to

eliminate any potential experimental bias. We did not

perform power calculations to determine the N required

for experiments as the Ns that are used in C. elegans

experiments are typically much greater than required to

identify a statistically significant difference. For

measurements of mitochondrial morphology, we used 8

biological replicates. For oxygen consumption we

performed at least 8 replicates with ~50 worms per

replicate. For ATP measurements, we performed 3

biological replicates with ~200 worms per replicate. For

mRNA measurements, we used 3 biological replicates of

a full 60 mm plate of worms. For the thrashing assays, we

quantified movement in at least 40 worms over 3

biological replicates. For lifespan assays, we completed

three biological replicates with at least 30 worms per

replicate. Brood size was measured in 6 worms

individually. Post-embryonic development time was

measured in 3 replicates of 25 worms per replicate.

Statistical significance of differences between groups was

determined by one-way, two-way or repeated measures

ANOVA using Graphpad Prism. Lifespan data were

graphed using a Kaplan-Meier survival plot and the

significance of differences between two plots was

determined using the Log-rank test. Error bars indicate

standard error of the mean. This study was not pre-

registered. No sample size calculations were performed.

This study did not include a pre-specified primary

endpoint.

RESULTS

Mitochondrial networks are disrupted in C. elegans

models of Huntington’s disease

In order to study the relationship between mitochondrial

fragmentation and disease pathogenesis, we first sought to

determine if mitochondrial dynamics are disrupted in

worm models of HD. To visualize the morphology of the

mitochondria, we crossed two different worm models of

HD to mitoRFP worms, which express the mitochondrial

targeting sequence of TOM-20 linked to RFP under the

body wall muscle myo-3 promoter (Pmyo-3::TOM-

20:RFP). The first is a worm model of HD that expresses

an exon 1 fragment of human huntingtin (Htt) connected

to either an unaffected-length 28Q or disease-length 74Q

repeats tagged with GFP in body wall muscle, which will

be referred to as BW-Htt28Q and BW-Htt74Q,

respectively. Both of these lines have been characterized

previously [16, 37].

To image these worms, we used confocal microscopy

in live, immobilized worms, as we and others have done

previously [28, 38-40]. While wild-type worms exhibit

parallel tracts of elongated mitochondria in their body

wall muscle cells, BW-Htt74Q worms, which express a

disease-length polyglutamine tract, exhibit mitochondrial

fragmentation (Fig. 1A) and disorganized mitochondrial

networks (Supplementary Fig. 2) at day 1 of adulthood. In

contrast, there was no change in mitochondrial structure

in BW-Htt28Q worms, which express an unaffected-

length polyglutamine tract (Fig. 1A; Supplementary Fig.

2). Quantification of mitochondrial morphology revealed

that BW-Htt74Q worms have a significantly increased

number of mitochondria (Fig. 1B) and decreased

mitochondrial area (Fig. 1C) compared to wild-type

mitochondria, but mitochondrial shape was unaffected

(Fig. 1D).

An increase in mitochondrial number could result

from mitochondrial biogenesis or mitochondrial

fragmentation. To distinguish between these two

possibilities, we measured mitochondrial DNA (mtDNA)

content. We found that mtDNA content in BW-Htt74Q

worms is the same as wild-type worms (Supplementary

Fig. 3). This suggests that the increase in mitochondrial

number results from fragmentation of existing

mitochondria, not increased mitochondrial biogenesis.

To determine how the disruption of mitochondrial

networks in BW-Htt74Q worms affects the function of the

mitochondria, we measured basal oxygen consumption

and ATP levels at day 1 of adulthood. Although

mitochondrial morphology is clearly disrupted in BW-

Machiela E., et al Mitochondrial dynamics and HD

Aging and Disease • Volume 12, Number 7, October 2021 1759

Htt74Q worms, both oxygen consumption (Fig. 1E) and

ATP levels (Fig. 1F) were equivalent to wild-type levels

in these worms. However, this result should be interpreted

cautiously as these are whole worm measurements in a

model in which the mutant Htt exon 1 fragment protein is

only expressed in the 95 body wall muscle cells out of 959

total cells in the worm.

Figure 1. Mitochondrial networks are disrupted in C. elegans models of Huntington’s disease. Worms expressing an

expanded, disease-length polyglutamine tract of 74Q in body wall muscle (BW-Htt74Q worms) exhibit mitochondrial

fragmentation and mitochondrial network disorganization (see Supplementary Fig. 2). In contrast, worms expressing a

shorter, unaffected-length polyglutamine tract of 28Q (BW-Htt28Q worms) have tubular mitochondria, similar to control

worms (mitoRFP worms) (A). Mitochondria are labelled with RFP (red), while Htt is labelled with GFP (green). mitoRFP

strain is syIs243[Pmyo-3::TOM20:RFP]. BW-Htt28Q and BW-Htt74Q worms also express syIs243[Pmyo-

3::TOM20:RFP] transgene. The images shown are from a single focal plane collected on a confocal microscope. Scale bars

indicate 15 µM. Quantification of mitochondrial morphology at day 1 of adulthood reveals that BW-Htt74Q worms have

an increased number of mitochondria (B) and decreased average mitochondrial area (C), both of which are consistent with

increased mitochondrial fragmentation. Mitochondrial shape is not significantly changed in BW-Htt74Q worms compared

to BW-Htt28Q and mitoRFP control worms (D). Despite the disruption of mitochondrial morphology, whole worm oxygen

consumption (E) and ATP levels (F) are unchanged in BW-Htt74Q worms. A minimum of three biological replicates were

performed. Bars indicate the mean value. One-way ANOVA was used to assess significance. Error bars indicate SEM. ROI

– region of interest. *p<0.05, **p<0.01, ***p<0.001.

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Figure 2. Mitochondrial networks are disrupted in BW-40Q worm model of Huntington’s disease. Worms expressing

an expanded, disease-length polyglutamine tract of 40Q in body wall muscle (BW-40Q worms) exhibit mitochondrial

fragmentation and disorganized mitochondrial networks (see Supplementary Fig. 4). In contrast, worms expressing a shorter,

unaffected-length polyglutamine tract of 24Q (BW-24Q worms) have tubular mitochondria, similar to control worms

(mitoRFP worms) (A). Mitochondria are labelled with RFP (red), while polyglutamine protein is labelled with YFP

(green/yellow). BW-24Q and BW-40Q worms express syIs243[Pmyo-3::TOM20:RFP] transgene. The images shown are

from a single focal plane collected on a confocal microscope. Scale bars indicate 15 µM. Quantification of mitochondrial

morphology at day 1 of adulthood reveals that BW-40Q worms have an increased number of mitochondria (B) and a trend

towards decreased average mitochondrial area (C). Mitochondrial circularity is significantly increased in BW-40Q worms

compared with wild-type worms (D). A minimum of three biological replicates were performed. Bars indicate the mean

value. One-way ANOVA was used to assess significance. Error bars indicate SEM. ROI – region of interest. *p<0.05.

To determine the extent to which the disruption of the

mitochondrial network is dependent on the presence of

Htt exon 1 fragment, we also examined mitochondrial

morphology in a model of HD that expresses a pure

polyglutamine tract. We examined worms that express

either an unaffected (24 glutamines) or disease-length (40

glutamines) polyglutamine tract tagged with YFP in body

wall muscle under the unc-54 promoter. These worms will

be referred to as BW-Q24 and BW-Q40 worms,

respectively. Both lines are integrated and previously

characterized [41].

As with BW-Htt74Q worms, we found that BW-40Q

worms (containing a disease-length polyglutamine tract)

exhibit mitochondrial fragmentation (Fig. 2A) and have

disrupted mitochondrial networks (Supplementary Fig.

4), while BW-24Q worms (containing an unaffected-

length polyglutamine tract) have elongated, tubular

mitochondria, similar to wild-type worms (Fig. 2A;

Supplementary Fig. 4). Like BW-Htt74Q worms, BW-

40Q worms have increased mitochondrial number (Fig.

2B) and decreased mitochondrial area (Fig. 2C) compared

to wild-type worms. The levels of mtDNA in BW-40Q

worms are equivalent to wild-type levels (Supplementary

Fig. 3), suggesting that the increase in mitochondrial

number results from mitochondrial fragmentation. In

addition, mitochondria in BW-40Q worms exhibit

increased circularity compared to those in wild-type

worms (Fig. 2D). This indicates that the expression of a

disease-length polyglutamine tract is sufficient to cause

mitochondrial fragmentation in body wall muscle

independent of any Htt protein sequence.

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Figure 3. Disruption of mitochondrial

network is associated with polyglutamine

aggregation. Images show neighboring body

wall muscle cells from worms expressing a

disease-length polyglutamine tract in body wall

muscles (BW-40Q worms) at the L4 stage of

development. One cell has diffuse expression of

the polyglutamine (PolyQ) protein and normal,

tubular mitochondrial networks, while the other

cell has a polyglutamine aggregate and a

disrupted mitochondrial network (A). Initially,

body wall muscle cells have tubular

mitochondria and diffuse polyglutamine protein.

Over time, an increasing number of body wall

muscle cells have fragmented mitochondria and

aggregated polyglutamine. Very few cells

exhibit mitochondrial fragmentation and diffuse

polyglutamine protein expression, or tubular

mitochondria with aggregated polyglutamine

protein (B). In BW-Htt-74Q worms,

mitochondrial morphology is similar to that in

wild-type worms during early development (C).

Polyglutamine protein aggregation increases

throughout development in BW-Htt74Q worms

(D). The images in panels A and C are

compressed z-stacks collected on a confocal

microscope. Scale bars indicate 25 µM. A

minimum of three biological replicates were

performed. Bars indicate the mean value. Error

bars indicate SEM.

Mitochondrial fragmentation is associated with

polyglutamine aggregation

In imaging mitochondrial morphology in worm models of

HD, we observed that, during development, neighboring

muscle cells could exhibit different mitochondrial

morphologies. While some cells exhibited parallel tracts

of elongated mitochondria in combination with diffuse

expression of polyglutamine protein, adjacent cells had

fragmented or disorganized mitochondrial networks and

aggregated polyglutamine protein (Fig. 3A). We rarely

observed the co-occurrence of disrupted mitochondrial

networks and diffuse polyglutamine localization.

To further explore this relationship, we performed a

time course examining mitochondrial morphology and

polyglutamine protein aggregation throughout

development in BW-40Q worms (Fig. 3B). Initially most

cells had tubular, elongated mitochondria and diffuse

polyglutamine protein. Over time, the number of cells

exhibiting this phenotype declined, while an increasing

number of cells had fragmented mitochondria and

aggregated polyglutamine protein. Throughout the time

course we observed few cells with fragmented

mitochondria and diffuse polyglutamine protein, or

tubular mitochondria and aggregated polyglutamine

protein. This suggests that polyglutamine aggregation and

mitochondrial fragmentation are temporally-linked

events.

To extend these findings to another model, we

examined aggregation and mitochondrial morphology

throughout development in BW-Htt74Q worms. We

found that during early development (developmental

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Aging and Disease • Volume 12, Number 7, October 2021 1762

stages from L1 to L4), mitochondria in BW-Htt74Q

worms are elongated and exist in parallel tracts (Fig. 3C),

similar to mitochondria in wild-type worms, but become

fragmented by the time worms reached adulthood (see

Fig. 1A). At the same time, BW-Htt74Q worms initially

have diffuse expression of the Htt exon 1 fragment protein

but show increased aggregation with age (Fig. 3D). Thus,

as in BW-40Q worms, both aggregation and

mitochondrial fragmentation increase with age, and both

tend to occur together in the same cell. Combined, this

indicates that mitochondrial fragmentation is strongly

associated with polyglutamine aggregation.

Figure 4. Inhibition of mitochondrial fission has detrimental effects in C. elegans models of Huntington’s disease

expressing expanded polyglutamine tracts in body wall muscle. To examine the effect of disrupting mitochondrial

fission in worm models of HD, a body wall muscle (BW-Htt74Q worms) model of HD was crossed to a drp-1 deletion

mutant. The drp-1 mutation significantly decreased movement (A) and lifespan (B) in BW-Htt74Q worms but had no

effect on wild-type worms (A, C). Loss of drp-1 resulted in decreased fertility (D) and slower post-embryonic development

(E) in both BW-Htt74Q and wild-type worms. While the drp-1 deletion did not affect oxygen consumption (F) in either

genotype, it resulted in a decreased levels of ATP (G). Deletion of drp-1 did not decrease the mitochondrial fragmentation

that is present in BW-Htt74Q worms (H), as indicated by quantification of mitochondrial number (I), mitochondrial area

(J) and mitochondrial shape (K). Note that we used wild-type worms as a control instead of BW-Htt28Q worms because

we observed the formation of aggregates in BW-Htt28Q worms, which could complicate the interpretation of the results.

The images in panel H are compressed z-stacks collected on a confocal microscope. Scale bars indicate 10 µM. A minimum

of three biological replicates were performed. Bars indicate the mean value. One-way ANOVA was used to assess

significance in H, I and J. Two-way ANOVA was used to assess significance in A, C, E, and F. Log-rank test was used to

assess significance in B. Repeated measures ANOVA was used to assess significance in D. Error bars indicate SEM. ROI

– region of interest. *p<0.05, **p<0.01, ***p<0.001.

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C. elegans models of Huntington’s disease have

increased expression of mitochondrial fission and

fusion genes

In order to explore the mechanism underlying the

disrupted mitochondrial networks observed in the HD

worm models, we used quantitative reverse transcription

PCR (qPCR) to measure expression of fission and fusion

genes in day 1 adult animals. We found that both HD

models in which the disease-length polyglutamine protein

is expressed in body wall muscle (BW-Htt74Q, BW-40Q)

showed significant changes in the expression of both

mitochondrial fission and fusion genes (Supplementary

Fig. 5). In both models, fis-1 and eat-3 mRNA levels were

elevated. It is unclear whether these changes in gene

expression contribute to the disruption of mitochondrial

networks observed in these strains, or whether these genes

are activated in an attempt to restore wild-type

mitochondrial morphology.

Disruption of mitochondrial fission can be detrimental

in a body wall muscle model of Huntington’s disease

Having shown that worm models of HD exhibit increased

mitochondrial fragmentation, we next sought to determine

if decreasing mitochondrial fission would help to restore

mitochondria morphology, and whether this would

ameliorate phenotypic deficits present in these worms. To

decrease mitochondrial fission, we crossed BW-Htt74Q

worms to a drp-1 deletion mutant (tm1108) [42]. In

worms, drp-1 is expressed highly in body wall muscle and

neurons [43], making it a good genetic target for the worm

models expressing an expanded polyglutamine tract in

body wall muscle. In these experiments, we used wild-

type worms as a control instead of BW-Htt28Q worms

because we observed the formation of polyglutamine

aggregates in BW-Htt28Q worms. Since this length of

polyglutamine tract does not aggregate in most HD

models, and is within the unaffected range in humans, the

aggregation in BW-Htt28Q worms could complicate the

interpretation of the results.

Surprisingly, we found that the drp-1 deletion

resulted in decreased movement, as measured by the rate

of thrashing in liquid of day 1 young adult animals (Fig.

4A), and decreased lifespan (Fig. 4B) in BW-Htt74Q

worms, but had no significant effect on these phenotypes

in wild-type worms (Fig. 4A, C). Although we and others

find that drp-1 mutants have a wild-type lifespan [44, 45],

it should be noted that one report has indicated that drp-1

mutants are short-lived compared to wild-type worms

[46]. The drp-1 deletion also decreased fertility, as

measured by self-brood size (Fig. 4D) and slowed

development (Fig. 4E) in both BW-Htt74Q worms and

wild-type worms. Examining the effect of the drp-1

deletion on mitochondrial function revealed no effect

on the rate of oxidative phosphorylation, as measured by

oxygen consumption (Fig. 4F), but caused a small

decrease in ATP levels (Fig. 4G). Finally, we found that

disruption of drp-1 did not decrease the mitochondrial

fragmentation present in BW-Htt74Q worms (Fig. 4H-K).

Taken together, these results suggest that inhibition of

mitochondrial fission can be detrimental in a body wall

muscle model of HD.

drp-1 deletion increases expression of polyglutamine

transgene

It was previously reported that RNAi against drp-1

increases expression of the polyglutamine transgene [16].

As increasing the levels of the polyglutamine protein

would be expected to increase toxicity, we sought to

determine whether drp-1 deletion also resulted in

increased transgene expression. Accordingly, we used

qPCR to measure the levels of polyglutamine transgene

expression in BW-Htt74Q worms compared to BW-

Htt74Q;drp-1 worms. We found that BW-Htt74Q;drp-1

worms showed a 92% increase in Htt74Q:GFP mRNA

compared to BW-Htt74Q worms (Supplementary Fig. 6).

This increase in polyglutamine expression could

contribute to the detrimental effects of drp-1 deletion in

this strain.

Decreasing mitochondrial fission through drp-1 RNAi

can be beneficial in a body wall muscle model of

Huntington’s disease

In contrast to our results obtained with a drp-1 deletion,

another group previously observed that RNAi against drp-

1 improved movement in BW-Htt74Q worms [16]. Since

a complete loss of DRP-1 can lead to a wide range of

abnormalities [47], we wondered if the drp-1 deletion has

detrimental effects that are independent of, or that

synergize with polyglutamine toxicity, which masked a

potential beneficial effect of decreasing drp-1 levels.

Accordingly, we investigated whether decreasing levels

of drp-1 by RNAi would be more beneficial in the HD

model. To do this, we fed BW-Htt74Q worms with RNAi

bacteria that directly targets drp-1. The RNAi was

administered using an L4 parental protocol, in which

RNAi knockdown is begun at the L4 stage of the parental

generation prior to testing their progeny (the experimental

generation). Using this paradigm, drp-1 RNAi-treated

worms exhibited drp-1 mRNA levels of approximately

30% of empty vector (EV) controls, and there was no

difference in the knockdown efficiency between wild-

type and BW-Htt74Q worms (Supplementary Fig. 7).

Unlike the drp-1 mutation, drp-1 RNAi did not

decrease the rate of movement in BW-Htt74Q worms

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Aging and Disease • Volume 12, Number 7, October 2021 1764

(Fig. 5A) and resulted in a small but significant increase

in lifespan in these worms (Fig. 5B, C). This suggests that

the detrimental effect of the drp-1 mutation on movement

in BW-Htt-74Q worms requires drp-1 levels to be reduced

beyond a specific threshold or be completely absent. Like

the drp-1 mutation, both wild-type and BW-Htt74Q

worms treated with drp-1 RNAi have markedly decreased

brood size compared to worms grown on EV RNAi (Fig.

5D).

To determine whether the mild improvement in

lifespan was associated with changes in mitochondrial

function, we quantified the effect of drp-1 RNAi on

mitochondrial form and function. While there was no

effect of drp-1 RNAi on oxygen consumption in wild-type

or BW-Htt74Q worms (Fig. 5E), it did result in decreased

ATP levels (Fig. 5F). As with the drp-1 mutation, the

disrupted mitochondrial networks present in BW-Htt74Q

worms were not rescued by drp-1 RNAi (Fig. 5G).

Mitochondrial number, area, and circularity were all

unchanged in BW-Htt74Q worms treated with drp-1

RNAi compared to EV (Fig. 5H-J).

Figure 5. Decreasing expression of mitochondrial fission protein DRP-1 through RNAi increases lifespan in

body wall muscle model of Huntington’s disease. The rate of movement in BW-Htt74Q worms is unchanged with

drp-1 RNAi (A). Knocking down drp-1 results in a small increase in lifespan in BW-Htt74Q worms (B), but has no

effect in wild-type worms (C). drp-1 RNAi decreases fertility in wild-type and BW-Htt74Q worms (D). While drp-

1 RNAi did not affect oxygen consumption in wild-type or BW-Htt74Q worms (E), knockdown of drp-1 reduced

ATP levels in both strains (F). Knocking down drp-1 expression using RNAi does not affect mitochondrial

morphology in control mitoRFP worms and does not restore tubular mitochondrial networks in BW-Htt74Q worms

(G). Quantification of mitochondrial morphology reveals no significant changes in mitochondrial number (H),

mitochondrial size (I) or mitochondrial shape (J) after treatment with drp-1 RNAi. The images in panel G are

compressed z-stacks collected on a confocal microscope. Scale bars indicate 10 µM. A minimum of three biological

replicates were performed. Bars indicate the mean value. Significance was assessed using two-way ANOVA

(A,C,D,E,G,H,I) or log-rank test (B,C). Error bars indicate SEM. ROI – region of interest. *p<0.05,

**p<0.01,***p<0.001.

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Figure 6. RNAi clones that decrease mitochondrial fragmentation improve movement in body wall

muscle model of Huntington’s disease. BW-Htt74Q and BW-Htt28Q control worms were treated with RNAi

against genes that were previously shown to decrease mitochondrial fragmentation when knocked down by

RNAi. Movement was then assessed by crawling and thrashing assays using an unbiased video-tracking

automated system. Ten of the 25 RNAi clones that decrease mitochondrial fragmentation were found to

increase the crawling rate in BW-Htt74Q worms (A). Of these 10 RNAi clones, two RNAi clones also

increased crawling speed in BW-Htt28Q control worms, indicating that 8 RNAi clones specifically improve

movement in the disease model (B). Five of the 25 RNAi clones that decrease mitochondrial fragmentation

increased the rate of movement in liquid (thrashing rate) of BW-Htt74Q worms (C). Only one of the RNAi

clones increased the thrashing rate of BW-Htt28Q worms (D). Three RNAi clones, F25B5.6, alh-12 and pgp-

3 increased both crawling speed and thrashing rate in BW-Htt74Q worms. Blue bars show BW-Htt28Q worms

treated with empty vector (EV). Grey bars show BW-Htt74Q worms treated with empty vector. Green bars

show RNAi clones that significantly increased movement. Red bars show RNAi clones that significantly

decreased movement. Blue dotted line shows rate of movement for BW-Htt28Q treated with EV. Grey dotted

line shows rate of movement for BW-Htt74Q treated with EV. Bars indicate the mean value. Significance was

assessed using one-way ANOVA. Error bars indicate SEM. *p<0.05, **p<0.01, ***p<0.001.

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Table 1. RNAi clones that improved movement in C. elegans models of Huntington’s disease.

Target

gene

Drosophila

homolog

Mammalian

homolog

Effect on

crawling

BW-Htt74Q

Effect of

crawling

BW-Htt28Q

Effect on

thrashing

BW-Htt74Q

Effect of

thrashing

BW-Htt28Q

Effect on

aggregation

drp-1 Drp1 DNM1L No effect No effect No effect No effect Decreased

sdha-2 SdhA SdhA Increased Increased No effect Decreased No effect

C34B2.8 ND-B16.6 NDUFA13 Increased No effect Decreased No effect No effect

gpd-4 Gapdh2 GAPDH Increased No effect No effect Decreased No effect

F25B5.6 Fpgs FPGS Increased No effect Increased No effect No effect

oatr-1 Oat OAT Increased Increased No effect No effect No effect

alh-12 Aldh ALDH9A1 Increased No effect Increased Decreased No effect

R10H10.6 CG2846 RFK Increased No effect No effect No effect No effect

dlat-2 muc DLAT Increased Decreased No effect Decreased No effect

pgp-3 Mdr49 ABCB4 Increased No effect Increased Decreased No effect

wht-1 w ABCG1 Increased No effect No effect No effect No effect

gpx-1 PHGPx GPX4 No effect No effect Increased No effect No effect

immt-2 Mitofilin IMMT No effect No effect Increased No effect No effect

Decreasing mitochondrial fragmentation through

multiple genetic targets rescues movement deficits in a

body wall muscle model of Huntington’s disease

Given that DRP-1 is the main protein required for

mitochondrial fission and we observed detrimental effects

of disrupting drp-1 in both wild-type worms and worm

models of HD, inhibiting DRP-1 might not be an ideal

therapeutic target for HD. Accordingly, we explored the

therapeutic potential of other genetic targets that decrease

mitochondrial fragmentation. A previous study performed

a targeted RNAi screen to examine the effect of knocking

down mitochondria-associated genes on mitochondrial

morphology [48]. In this study, they examined 719 genes

predicted to encode mitochondrial proteins and identified

25 RNAi clones that decrease mitochondrial

fragmentation in body wall muscle. We performed a

targeted RNAi screen to examine the effect of these 25

RNAi clones in BW-Htt74Q worms. Treatment with

RNAi was begun at the L4 stage of the parental generation

and the rate of movement was assessed in the progeny

(experimental generation). The rate of movement was

assessed by unbiased video-tracking of movement on

solid plates (crawling) and movement in liquid

(thrashing).

Figure 7. Most RNAi clones that decrease mitochondrial fragmentation do not cause a decrease in polyglutamine

aggregation. BW-40Q worms were treated with RNAi against genes which decrease mitochondrial fragmentation in

C. elegans when knocked down via RNAi. Animals were imaged as day 1 adults. Of the 26 RNAi clones tested, four

RNAi clones resulted in a small but significant decrease in total aggregates per worm: drp-1, abhd-11.1, E04A4.4, and

C33A12.1. Grey bars show BW-40Q worms treated with empty vector (EV). Green bars show RNAi clones that

significantly decreased aggregate number. None of the RNAi clones resulted in increased aggregation. Bars indicate

the mean value. Significance was assessed using one-way ANOVA. Error bars indicate SEM. *p<0.05, **p<0.01,

***p<0.001.

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We found that 10 of the 25 RNAi clones that decrease

mitochondrial fragmentation increase crawling speed in

BW-Htt74Q worms (Fig. 6A). Of these 10 RNAi clones,

two clones also increased movement in BW-Htt28Q

worms suggesting a non-specific beneficial effect on

movement (Fig. 6B). Thus, eight RNAi clones

specifically rescued movement deficits in worm models

of HD (Table 1). In examining the effect of these same 25

RNAi clones on movement in liquid, we found that five

of these clones significantly increased the thrashing rate

in BW-Htt74Q worms (Fig. 6C), while a separate clone

improved movement in BW-Htt28Q control worms (Fig.

6D). Of the five clones that improved thrashing, three of

them, F25B5.6, alh-12 and pgp-3, also improved crawling

speed (Table 1).

Decreasing mitochondrial fragmentation does not affect

polyglutamine aggregation

To better understand how the RNAi clones that suppress

mitochondrial fragmentation caused a decrease in motility

defects, we examined the effect of these clones on

polyglutamine aggregation. We found that drp-1 RNAi

caused a small but statistically significant decrease in the

number of aggregates, as did three of the other RNAi

clones (E04A4.4, C33A12.1, abhd-11.1). However, all of

these clones were also found to disrupt development and

may be suppressing aggregation by slowing development.

As none of these RNAi clones rescued motility deficits in

the HD worm model, this indicates that decreasing

aggregation is not sufficient to improve movement.

Importantly, we found that none of the RNAi clones

which did suppress motility defects caused a decrease in

aggregation levels (Fig. 7, Table 1). Thus, the ability of

these clones to rescue movement deficits is not through a

decrease in aggregation.

Figure 8. RNAi clones that improve movement correct mitochondrial fragmentation in worm model of

Huntington’s disease. BW-Htt74Q worms or mitoRFP control worms were treated with RNAi clones that were

shown to improve crawling speed on solid plates and thrashing rate. In every case, the RNAi clones decreased

mitochondrial fragmentation such that mitochondrial morphology in the treated worms was equivalent to mitoRFP

controls (A). Treatment with RNAi against pgp-3, F25B5.6 or alh-12 significantly decreased the number of

mitochondria in the region of interest (ROI) (B), significantly increased mitochondrial area (C), and significantly

decreased mitochondrial circularity (D). The images shown in panel A are a compressed z-stack collected on a

confocal microscope. Scale bar indicates 25 µM. Bar graphs indicate the mean value. Significance shown is the

difference from the respective EV RNAi control, and was assessed using one-way ANOVA. Segmentation of

mitochondria for quantification was performed using ImageJ’s segmentation and quantification of subcellular shapes

(SQUASSH) tool. Average number of mitochondria, average mitochondrial area and average mitochondrial

circularity were then measured using the analyze particles tool on ImageJ. Error bars indicate SEM. ***p<0.001.

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Amelioration of movement deficits is associated with

restoration of wild-type mitochondrial morphology

To further characterize the three RNAi clones that rescued

the movement deficits in BW-Htt74Q worms, we

examined the effect of these genes on mitochondrial

morphology and lifespan. We found that the mitochondria

of BW-Htt74Q worms treated with pgp-3, F25B5.6 or

alh-12 RNAi was indistinguishable from the

mitochondria from mitoRFP control worms (Fig. 8A).

The RNAi-treated BW-Htt74Q worms exhibited

elongated, tubular mitochondria with no signs of

mitochondrial fragmentation. In quantifying the effect of

these three RNAi clones on mitochondrial morphology in

BW-Htt74Q worms, we found that pgp-3, F25B5.6 or alh-

12 RNAi significantly decreased mitochondrial number

(Fig. 8B), increased average mitochondrial area (Fig. 8C),

and decreased mitochondrial circularity (Fig. 8D).

Finally, we examined the effect of pgp-3, F25B5.6 or

alh-12 RNAi on the lifespan of BW-Htt74Q worms. We

found that treatment with these three RNAi clones was

unable to restore BW-Htt74Q lifespan to wild-type length

(Supplementary Fig. 8). Combined, this suggests that

decreasing mitochondrial fragmentation in BW-Htt74Q

worms through treatment with RNAi against pgp-3,

F25B5.6 or alh-12 increases healthspan, as measured by

movement in liquid and on solid plates, but not overall

lifespan.

DISCUSSION

Expression of a disease-length polyglutamine tract in C.

elegans causes mitochondrial fragmentation

While the genetic cause of HD was identified in 1993,

there are still no treatments available for patients with this

devastating disorder that can alter disease progression

[49]. Based on several observations linking HD and

mitochondrial dysfunction, correcting mitochondrial

deficits has been the focus of much research on disease-

modifying therapies for HD [50]. Accumulating evidence

indicates that mitochondrial dynamics are disrupted in

HD [16-23]. We have extended these findings to show

that mitochondrial networks are also disrupted in two

different C. elegans models of HD in which a disease-

length polyglutamine tract is expressed in body wall

muscle. In both BW-Htt74Q and BW-40Q worms we

observed mitochondrial fragmentation and mitochondrial

network disorganization leading to an increased number

of smaller, more rounded mitochondria, with no increase

in mtDNA content. The increase in mitochondrial

fragmentation may be a precursor to mitophagy in order

to replace mitochondria that are damaged by

polyglutamine toxicity or could be a direct effect of the

expanded polyglutamine protein. The fact that we observe

increased expression of mitochondrial fission genes

suggests that the increase in mitochondrial fragmentation

might be at least partially due to an active process to

increase mitochondrial fission.

Importantly, our results indicate that polyglutamine

toxicity can cause mitochondrial fragmentation

independently of the huntingtin protein, as a worm model

expressing a pure polyglutamine tract linked to YFP also

exhibited disrupted mitochondrial networks (BW-40Q

worms). Consistent with this idea, mitochondrial

fragmentation has been observed in models of other

polyglutamine toxicity disorders including

Spinocerebellar ataxia 3 (SCA3), Spinocerebellar ataxia 7

(SCA7) and Spinal and bulbar muscular atrophy (SBMA)

[51-53]. Combined, this suggests that expression of an

expanded polyglutamine tract (or the presence of a CAG

repeat expansion in the DNA/RNA) may be sufficient to

cause mitochondrial fragmentation independently of the

surrounding protein context.

DRP1 may not be an ideal therapeutic target for

Huntington’s disease

Based on the observation of mitochondrial fragmentation

in HD patients and models, multiple groups have explored

the effect of decreasing the levels or activity of DRP-1 in

various models of HD [16, 17, 19, 21]. In each case,

decreasing the levels or activity of DRP-1 showed a

beneficial effect in HD models. However, in thinking

about developing a treatment for HD, DRP1 may not be

an ideal target. DRP1 is the main GTPase responsible for

mitochondrial fission, which is crucial for proper cellular

function. Consistent with this, a number of studies have

indicated that loss of DRP1 function can be detrimental

[46, 47, 54-56]. For example, loss-of-function mutations

in the gene that encodes DRP1 causes a wide range of

abnormalities in humans, including epilepsy and

encephalopathy [57]. Our results indicate that deletion of

drp-1 can be detrimental in wild-type worms (slow

development, decreased fertility) and exacerbate

phenotypic deficits in a body wall muscle model of HD

(decrease movement, shorten lifespan). Because of the

potential negative side effects of directly targeting DRP1,

it may be important to explore other approaches to

decrease mitochondrial fragmentation as potential

therapeutic strategies for HD.

Alternatively, it may be necessary to precisely control

the level of drp-1 disruption. While deletion of drp-1 had

detrimental effects in BW-Htt74Q worms, we found that

decreasing drp-1 levels through RNAi resulted in a small

but beneficial effect on lifespan. Similarly, a previous

study found that drp-1 RNAi can increase movement in

the same BW-Htt74Q worms [16]. This suggests that the

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Aging and Disease • Volume 12, Number 7, October 2021 1769

precise level of drp-1 depletion may need to be controlled

to observe a beneficial effect. The difference between drp-

1 deletion and drp-1 RNAi could also have resulted from

the fact that neurons in C. elegans have decreased

sensitivity to RNAi. In addition to identifying the optimal

level of drp-1 knockdown, it may be possible to minimize

detrimental side effects of disrupting drp-1 by knocking it

down in specific tissues, which could be tested using

tissue-specific RNAi strains, or by knocking it down

during specific periods of development or adulthood,

which could be done by administering the RNAi bacteria

targeting drp-1 during specific periods of time.

Novel therapeutic targets for Huntington’s disease

aimed at reducing mitochondrial fragmentation

Because of the potential detrimental effects of targeting

DRP-1, we examined 25 other genes which also suppress

mitochondrial fragmentation when knocked down using

RNAi. Eight of the RNAi clones improved crawling only

in the Htt-74Q strain, while five clones improved

thrashing only in the Htt-74Q strain. Combined, we

identified three RNAi clones (pgp-3, F25B5.6, and alh-

12) that improved motility in both assays. These three

RNAi clones were also able to completely restore

mitochondria morphology in BW-Htt74Q worms to wild-

type morphology. These genes represent novel

therapeutic targets for HD, which will be important to

validate in other models of HD.

The main trait shared between pgp-3, F25B5.6 and

alh-12 are that they cause elongated mitochondria when

knocked down by RNAi [48]. pgp-3 is a p-glycoprotein

and performs ATP-dependent export of toxins and

xenobiotics out of the cytoplasm. It is required for drug

resistance to colchicine and chloroquine and upregulated

when animals are exposed to heavy metals [58, 59]. pgp-

3 is conserved and has a human orthologue, ABCB4 (ATP

binding cassette subfamily B member 4), which is

upregulated in an R6/2 mouse model of HD [60].

F25B5.6 also has ATP-binding activity and is

predicted to be a folylpolyglutamate synthase. The human

homologue, FPGS, is a mitochondrial enzyme, which

maintains folylpolyglutamate concentrations in the

cytoplasm and mitochondria. It has not previously been

associated with HD or neurodegeneration.

alh-12 is an aldehyde dehydrogenase, which is

upregulated in long-lived C. elegans mutants [60]. The

human homologue, ALDH9A1, has not been

experimentally linked with HD, however a meta-analysis

of pathways affected in HD predicted that ALDH9A1 is

important for HD as it is involved in multiple metabolic

pathways that are affected in HD [61].

As all three of these genes have homologs in mice and

humans, a key next step will be to examine the effect of

these genes on mitochondrial morphology and

polyglutamine toxicity in mammalian models. These

validation steps could be performed in mammalian cells

or mouse models, which we and others have shown to

recapitulate many features of the human disease [62-65].

Polyglutamine aggregation is associated with

mitochondrial fragmentation, but can be experimentally

dissociated

While polyglutamine aggregation is associated with

toxicity, whether or not aggregation causes toxicity,

reduces toxicity or is an epiphenomenon is still debated.

Both polyglutamine aggregation and toxicity increase

with both age and the length of the glutamine repeat [41];

however, decreases in polyglutamine aggregation do not

always cause a decrease in polyglutamine toxicity [66],

and the formation of aggregates has been associated with

a decreased probability of death [67]. In our study, we

show that polyglutamine aggregation is associated with

mitochondrial fragmentation. In both the BW-Htt74Q

model and BW-40Q model, mitochondria are tubular at

hatching and polyglutamine proteins exhibit diffuse

localization. By the L4 stage of development,

mitochondria start to become fragmented, and this event

is temporally correlated with the formation of aggregates.

Once worms have reached young adulthood, essentially

all muscle cells have fragmented mitochondria and

aggregated polyglutamine protein.

Despite the tight correlation between aggregation and

mitochondrial fragmentation, our data show that these

phenotypes can be experimentally dissociated. Knocking

down the expression of pgp-3, F25B5.6 or alh-12

prevented mitochondrial fragmentation in BW-Htt74Q

worms (Fig. 8) but had no effect on aggregation. This

indicates that mitochondrial fragmentation is not required

for aggregation.

Similarly, while BW-Htt74Q worms and BW-40Q

worms have both movement deficits and polyglutamine

aggregation, our data indicates that these phenotypes can

be separated. None of the RNAi clones which improved

either crawling or thrashing defects in BW-Htt74Q worms

caused a decrease in the number of polyglutamine

aggregates, while RNAi clones that did decrease

aggregation in these worms did not have a beneficial

effect on movement. Combined, our results suggest that

polyglutamine aggregation is not responsible for the

movement deficits in this worm model of HD.

Conclusions

In this work, we show that C. elegans models of HD

exhibit mitochondrial fragmentation and disorganized

mitochondrial networks, which are associated with

Machiela E., et al Mitochondrial dynamics and HD

Aging and Disease • Volume 12, Number 7, October 2021 1770

polyglutamine aggregation. Our observation that

decreasing DRP-1 levels can have detrimental effects in a

body wall muscle model of HD, suggests that DRP1 may

not be an ideal therapeutic target for HD, or that great care

must be taken to ensure that DRP1 levels are only

decreased by a certain amount. As an alternative to

targeting DRP1, we identified three novel genetic targets

(pgp-3, F25B5.6, and alh-12) that improved both crawling

and swimming in a C. elegans model of HD. These

genetic targets also corrected deficits in mitochondrial

morphology, thereby demonstrating that mitochondrial

fragmentation can be prevented without disrupting the

mitochondrial fission machinery. These results suggest

that strategies aimed at correcting mitochondrial

fragmentation may be beneficial in the treatment of HD.

Acknowledgments

We would like to thank Rick Morimoto, Mervyn Monteiro

and Paul Sternberg for generating strains used in this

research. Some strains were provided by the CGC, which

is funded by NIH Office of Research Infrastructure

Programs (P40 OD010440). We would also like to

acknowledge the C. elegans knockout consortium and the

National Bioresource Project of Japan for providing

strains used in this research. This work was supported by

the Canadian Institutes of Health Research (CIHR; PI:

Van Raamsdonk); the Natural Sciences and Engineering

Research Council of Canada (NSERC; PI: Van

Raamsdonk); the National Institutes of General Medical

Sciences (Grant number R01GM121756; PI: Van

Raamsdonk); and the Van Andel Research Institute

(VARI). JVR received a salary award from Fonds de

Recherche du Quebec Santé (FRQS). AT received

scholarships from NSERC and FRQS. SKS received a

scholarship from FRQS. The funders had no role in study

design, data collection and analysis, decision to publish,

or preparation of the manuscript.

Conflict of Interest

The authors declare no financial or competing interests.

Supplementary Materials

The Supplemenantry data can be found online at:

www.aginganddisease.org/EN/10.14336/AD.2021.0404.

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