つくば生物ジャーナル
Tsukuba Journal of Biology
Vol.23 No.7 July 2024

www.biol.tsukuba.ac.jp/tjb

Abstracts of Graduation ResearchPresentations, July 2024
July 5th 2024

College of Biological Sciences
University of Tsukuba

表紙画

Cover art
平井

表紙画の解説唯子

Hirai Yuiko

Explanation of the cover art：

テーマ：「混」
水の中に数色の絵の具を落とすとそれぞれの色はやがて混ざり合い一様になります。一見するとそれは一色の色水ですが、複数の絵の具から構成されている
事実は変わりません。様々な分野の研究から形成される科学も似ているなぁと
最近感じます。
表紙画作成にあたりご尽力いただきました吉本賢一郎くんをはじめ、卒業研究発表会準備委員の皆様にこの場を借りて厚く御礼申し上げます。

Theme: "KON(Mixed)"
When several colors of paint are dropped into water, each color will
eventually mix and become uniform. At first glance, it appears to be one
color of water, but the fact remains that it is composed of multiple
colors of paint. I have recently felt that science, which is formed fromresearch in a variety of fields, is similar.
We would like to take this opportunity to thank Mr. Kenichiro Yoshimoto
and all the members of the Graduation Research Presentation Preparation
Committee for their efforts in creating the cover art.

July 5th 2024
11:35–11:50

Baby Bhavya Gummadi
Metabolic shift in gut microbiota is the potential route for cachexia prevention
1

11:50–12:05

GU YULIANG
The effect of phosphate deficiency on the relationship between host Arabidopsis
thaliana and facultative root hemiparasite Phtheirospermum japonicum2

12:05–12:20

川上 瞭
無腸類と藻類の共生関係の進化
3

12:20–12:35

Caecilia Sasikirana Basirin
Optimization of medium for enhanced biomass and lipid production in
Elliptochloris subsphaerica through mixotrophic cultivation
4

12:35-12:50

Maaya Marie Eguchi Grove
Impacts of future ocean acidification on the ecology of two sea urchin species
5

12:50-13:05

Kseniia Sevostianova
Loss of Usp15 leads to mitochondrial abnormalities6つくば生物ジャーナル

Tsukuba Journal of Biology (2024)

23(7), 1Ⓒ2024 筑波大学生物学類

Metabolic shift in gut microbiota is the potential route for cachexia prevention

Baby Bhavya Gummadi（筑波大学 生物学類） 指導教員：Yuya Sanaki（筑波大学 生存ダイナミクス研究センター）
Introduction
Cancer was traditionally studied as a genetic disease,
however, increasing evidence has revealed that it has a strong
impact on the host metabolism1. In the advanced stages ofcancer, a severe metabolic disorder known as cachexia willdevelop, which is characterized by strong muscle wasting or/and
fat mass loss. Currently, there is no established treatment forthis condition, and most cases are fatal. Using Drosophilamelanogaster as an in vivo cancer model, we found aremarkable gut dysbiosis in tumour-bearing flies, where thecommensal Acetobacter and Lactobacillus were replaced byEscherichia. Strikingly, inoculating the non-pathogenic
Escherichia coli K-12 strain was solely sufficient to induce
cachexia in the tumour-bearing flies. Moreover, we foundthat their heat-resistant metabolites mediated the cachexiapromoting effect of gut bacteria. To identify the causative
metabolites, we conducted a genetic screening targeting
metabolic pathways with an E. coli mutant library.

qualitatively by staining the culture with Dichloro-dihydrofluorescein diacetate (DCFH-DA, Dojindo).
Antioxidant capability was measured by 2,2-diphenyl-1picrylhydrazyl (DPPH, Dojindo) staining in LB broth following
the manufacturer’s protocol.
Results and Discussion
We found that inoculating tumour-bearing flies with E. coli
mutant strains deficient in nuoN, nuoL, pfkA, and ppc genes
resulted in prolonged host survival, suggesting that these genes
are required for cachexia development.
Log-rank p=0.04

Material & Methods• Genetic Screen
Wild-type Oregon-R flies were reared on a conventional food
(wheat flour, dried yeast, sugar, agar, propionic acid,methylparaben) at 25C. Unmated/virgin female flies weretreated with antibiotics-containing food (AmpicillinGentamicin-Levofloxacin) for at least 3 days to eliminate the gut
microbiota. The flies were injected with RasV12 tumour cells (500cells/fly) and then recovered in antibiotics containing food for 1day. E. coli strains from the Keio collection, with a single-gene
knock out2, were orally introduced to tumour-bearing flies.
And fly survival was assayed. A survival of more than 20 DAI(days after injection) was used as a criterion to consider thebacterial gene to be required for cachexia development (HITgene).
• Histological Assay
Muscle wasting was analysed by staining flight muscles withphalloidin (Thermo Fisher Scientific) and anti-actinin primary
antibody (DSHB). Fat body wasting was analysed by staining
abdominal and visceral fat with Nile red (Fujifilm-Wako).
Images were acquired by confocal microscope (Carl Zeiss,
LSM900). The length of a sarcomere was determined bymeasuring the distance between z-bands in continuous muscle
fibres. Fat mass loss was analysed as the density and size of lipid
droplets. All image analyses were performed on FIJI.
• Bacteria Characterization
The “HIT” mutants from the bacterial genetic screen wereanalysed for their growth in LB broth, on fly food surface, and in
the host gut. Reactive oxygen species (ROS) were detected

Histological assays revealed a clear loss in fat mass upon
inoculation with the control K-12 strain and the “HIT” strains.
However, muscle wasting was significantly rescued in the “HIT”
strains compared to those inoculated with the control K-12
strain, suggesting that muscle wasting plays a more critical rolein determining survival period. The nuo genes are the essential
components of oxidative phosphorylation, while pfkA and ppc
are involved in glycolysis; both of these metabolic pathways arepart of aerobic metabolism. As aerobic metabolism is theprimary source of reactive oxygen species (ROS) in cells, wehypothesized that the “HIT” strains produce less ROS than thestrains that induce cachexia. Furthermore, the “HIT” strains
may even have antioxidant properties resulting from theactivation of anaerobic pathways, allowing the host to bettercombat the tumour. ROS staining revealed that the “HIT”
strains generate less ROS than the wild-type strain. However,this trend is not limited to “HIT” strains; certain non-“HIT”
strains also exhibited low ROS levels. Additionally, the “HIT”
strains demonstrated a higher antioxidant capacity compared to
the non-“HIT” strains.
Hence, we propose that a metabolic shift in the gut microbiota
away from aerobic metabolism contributes to the attenuation ofcancer cachexia in the host, potentially through lower ROS
levels and increased antioxidant production.
References
1Gyamfi et al. (2022), Int. J Mol Sci. 23(3):1155.
2Baba et al. (2006), Molecular Systems Biology, 2(1)1

つくば生物ジャーナル

Tsukuba Journal of Biology (2024)

23(7), 2Ⓒ2024 筑波大学生物学類

The effect of phosphate deficiency on the relationship between host Arabidopsis thalianaand facultative root hemiparasite Phtheirospermum japonicum
GU YULIANG（筑波大学 生物学類）

指導教員：Louis Irving（筑波大学 生命環境系）

1.Introduction
Parasitic plants steal resources from a host plant, causing an
increase in parasite mass at the expense of the host plant. In aprevious study focusing on the effect of N supply on the alfalfa –Phtheirospermum japonicum (Pj) relationship, it was found thatN supply to host suppressed parasitism (Irving et al. 2019). In
this experiment, we aim to explore the effects of host andparasite P status on the parasitic relationship between a host
(Arabidopsis thaliana) and the parasite (P. japonicum).
2.Material & Methods
Plants were grown in root boxes as shown in Fig. 1. Half theboxes had plastic straw bridges between the host-only andinteraction sections, allowing host – parasite connection. Control
boxes did not have this bridge. The boxes were filled withvermiculite. Nutrient supply to the host and the parasite could
be controlled independently by feeding nutrients to the host-only
and parasite only sections, respectively. After plantestablishment, we provided + and -P Hoagland nutrient
solutions to the host-only and parasite only sections in a full
factorial design for four weeks. At harvest, each plant had the
dry mass measured. In experiment two, we quantified
parasite N and P status.

Fig.2. Biomass of P. japonicum at low and high phosphorus
levels, in the presence and absence of A. thaliana.
3.2 Experiment two
Attachment caused a decrease in host mass, and an increase in
P content, with the increase being larger in –P parasites.

Fig.3. P content of P. japonicum at low and high phosphorus
levels, in the presence and absence of A. thaliana.
4.Discussion
Our data suggests that -P parasites were more strongly
parasitic than +P parasites, presumably due to resource
abstraction. It is possible that the parasite had greater sinkstrength. Another possibility is that the -P parasites formed agreater number of haustorial connections, as found under -P
conditions in Rhinanthus minor by Davies and Graves (2000)

Fig.1. Split-root box design.
3. Results
3.1 Experiment one
Attachment caused an increase in Pj biomass in experiment one
but did not affect host mass. -P parasites achieved much higher
shoot mass than +P parasites.

In a future experiment, we would plant to use more replicates,
or increase the treatment duration to decrease variability.
Isotopic tracers may also be used to quantify host to parasite P
flux.
References
Irving LJ et al., 2019 Env Exp Bot 162 125 – 132
Davies and Graves, 2000 Oecologia 124:100–1062つくば生物ジャーナル

Tsukuba Journal of Biology (2024)

23(7), 3Ⓒ2024 筑波大学生物学類

無腸類と藻類の共生関係の進化川上瞭（筑波大学 生物学類）

指導教員：中野裕昭（筑波大学 生命環境系）

Amphidinium 属は全て Amphidinium gibbosum に同定された。
Introduction
無腸類とは、珍無腸動物門無腸目に属する、多くが海産の小型
無腸類は種ごとに特定の系統の藻類と共生していた。
無脊椎動物である。日本からは 30 種以上の無腸類が報告されているが、温帯域に生息している無腸類の報告は少ない。
無腸類最大の科であるConvolutidae科の多くの種は微細藻類と共生しており、緑藻のTetraselmis属、
渦鞭毛藻のAmphidinium
属およびSymbiodiniaceae科が共生藻として主に報告されている。
先行研究では、無腸類はそれぞれの種が特定の系統の藻類と共生することが示唆されている。しかし、共生藻を種まで同定した例
やその系統解析を行った例は少なく、また、研究に用いられた無腸類の分類群が網羅的でないため、無腸類と藻類の共生関係の進化は十分に考察されていない。
本研究は日本の温帯域における無腸類の生息状況を明らかにす
ること、そして無腸類と藻類の共生関係の進化についての考察を深めることを目的とし、静岡県下田市で採集した無腸類と共生藻図 1. 採集された無腸類。スケールバーは全て 500 µm。
の形態観察および分子系統解析を行った。
Discussion
本研究により 8 種の無腸類が採集され、海藻類の洗い出しが温Material & Methods
2024 年 4 月 11 日から 6 月 4 日の期間に 6 回、静岡県下田市の
帯性無腸類の採集に有効であることが示された。日本の温帯域に4 地点において海藻類の採集を行った。採集した海藻類を研究室おける無腸類の生息状況をより正確に把握するために、下田市にに持ち帰り、洗い出しによって無腸類を採集した。
おける継続調査と新たな地点の調査が必要である。
採集した無腸類はデジタルカメラを備えた双眼実体顕微鏡およ本研究でも、先行研究同様、無腸類が種ごとに特定の系統の藻
び光学顕微鏡で形態観察および撮影を行い、形態的特徴によって類と共生する結果が得られた。藻類と共生する無腸類の多くは幼８種に区別した。観察後の個体は 99.5%エタノールで固定した。
若体期に環境中から共生藻を獲得するが、この結果は無腸類が共固定した標本を用いて DNA 抽出および PCR を行い、配列を生藻を獲得する際に、特定の藻類の系統を選択するメカニズムが決定して分子系統解析に用いた。使用したプライマーは、本研究存在することを支持する。
で設計した無腸類と Amphidinium 属のそれぞれに特異的なセッ本研究で確認されたSymbiodiniaceae科の共生藻はこれまでにConvolutidae 科以外からも報告がある。Tetraselmis 属と共生す
ト 、 お よ び 先 行 研 究 で 用 い ら れ た Tetraselmis 属 とSymbiodiniaceae 科のそれぞれに特異的なセットである。
る無腸類は Convolutidae 科で広く見られるものの、同科以外か
らは報告がない。本研究でも系統的に離れた複数の Convolutidae
科無腸類から Tetraselmis 属が確認された。Amphidinium 属とResult本研究では形態的に区別される以下 8 種の無腸類が採集された。 共生する無腸類は、本研究を含めて Amphiscolops 属、Amphiscolops oni オニムチョウウズムシ (図 1-A): Te, Am
Heterochaerus 属および Waminoa 属の 3 属のみしか報告されておらず、これら 3 属は Convolutidae 科内で単系統群を形成する
Amphiscolops sp. 1 (図 1-B): Sy
ことが明らかになっている。
Amphiscolops sp. 2 (図 1-C): Te, Am
以上より、Convolutidae 科が他の科と分岐するよりも前にHeterochaerus sp. 1 (図 1-D): Am
Symbiodiniaceae 科と、Convolutidae 科の共通祖先においてHeterochaerus sp. 2 (図 1-E): ×
Convolutidae sp. 1 (図 1-F): Sy
Tetraselmis 属と、同科内の Amphiscolops 属、Heterochaerus 属
Convolutidae sp. 2 (図 1-G): Te
および Waminoa 属の 3 属の共通祖先において Amphidinium 属
Convolutidae sp. 3 (図 1-H): Syとの共生関係がそれぞれ獲得されたと考えられる。しかし、Convolutidae 科には 1 種類の藻類のみと共生する種や藻類と共各種の後ろの略語はそれぞれの種の共生藻を表す。Sy:
生しない種のように、この仮説から想定されるよりも共生藻の種
Symbiodiniaceae 科、
Te: Tetraselmis 属、
Am: Amphidinium 属。
類が少ない種も多い。このことから、共生関係の喪失も無腸類の分子系統解析の結果は、形態的特徴に基づく分類と矛盾しなか
進化過程において頻繁に生じたと考えられる。一方で、ある系統った。採集された全ての種が Convolutidae 科に含まれ、同科内のの共生藻が無腸類の進化過程において独立に複数回獲得された可系統関係は先行研究と概ね一致した。
能性も考えられ、無腸類で網羅的に本研究と同様な調査を行い、共生藻に関しては、Symbiodiniaceae 科は全て Symbiodinium
また、各種における共生メカニズムを比較する必要がある。
属に分類され、 Tetraselmis 属は複数の系統に分かれ、3

つくば生物ジャーナル

Tsukuba Journal of Biology (2024)

23(7), 4Ⓒ2024 筑波大学生物学類

Optimization of medium for enhanced biomass and lipid production in Elliptochloris subsphaerica
through mixotrophic cultivation
Caecilia Sasikirana Basirin（筑波大学 生物学類）

指導教員：Kenichiro Ishida（筑波大学 生命環境系）

Introduction
In response to the energy crisis driven by climate change,
research has shifted from fossil fuels to biofuels for theirrenewability. As a result, microalgae have emerged as apromising resource for biofuel due to their high efficiency of lipid
production. Among different cultivation methods, mixotrophic
cultivation, which integrates autotrophic and heterotrophic
growth, has demonstrated the ability to enhance biomass andlipid productivity, making it suitable for large-scale production.
Botryococcus braunii is one of the most researched species
for the development of biofuels, renowned for its highhydrocarbon production, reaching up to 86% of its weight(Borowitzka, 2018). However, its slow growth rate increases thecost of production. To address this, Elliptochloris subsphaerica,a green algal species closely related to B. braunii (Fučíková et al.,2014), is being explored for its potential.
E. subsphaerica is known to exist as a phycobiont in lichen(Masumoto, 2020) as well as free-living in various substrates(Veselá et al., 2024). This species has been observed to containsignificant lipid droplets within its cells. A previous study has
shown that E. subsphaerica exhibits rapid growth when
cultured in medium Y, achieving a dry cell weight (DCW) of 3.5
g/L in 14 days and lipid content 30% of its DCW (Yuzheng, 2021).
However, a following study has found that glucose concentration
is not significant for the growth of E. subsphaerica in medium Y(Polsilapa, 2023).
Thus, this experiment aims to determine the optimal
concentration of components X in medium Y to maximize
biomass and lipid accumulation in E. subsphaerica.

1. Growth characteristics
E. subsphaerica grown in
T4 medium showed thehighest growth (p<0.0001),
achieving a DCW of 10.07
g/L in 14 days with aproductivity rate of 0.68
g/L/day. Compared to theprevious experiment by
Yuzheng (2021), the DCW
in this study is nearly three Fig.1 Cell density of E.subsphaerica grown intimes higher than the different medium concentration in 14 days
mixotrophic culture using medium Y with glucose. These results
suggest that higher components X concentrations promote
greater growth by providing more nutrients, allowing algae toextend the growth period before reaching the stationary phase.
Further study is needed to determine the peak of growth byextending the experiment period and testing more treatments.
Additionally, the cell size in T4 medium reached the largest
diameter on day 14, measuring twice the diameter of cells on
day 0, with large lipid droplets.
2. Lipid contents
Fluorescent microscope observation of cells stained with Nile
Red showed bright stained lipid droplets of algae grown in all
media. Subsequent lipid extraction showed that algae grown in
T4 medium had the highest lipid content, with 37.6% of its DCW
as lipids (p<0.0001). This amount significantly surpasses theprevious study by Yuzheng (2021), which reported a lipid
content of 30% of DCW. These findings suggest that higher
components X concentrations not only enhance growth but alsosignificantly increase lipid accumulation in E. subsphaerica,
making it a viable candidate for sustainable oil production.
Further lipid content identification is needed in the future toenhance its potential as a valuable source of algal oil.

Material & Methods
1. Medium Preparation: Four media Y (T1, T2, T3, and T4) weremade with varying concentrations of components X.
2. Cultivation: Pre-culture cells were inoculated at an initial
density of 5𝑥𝑥106 cells/mL and maintained at 20°C with alight/dark cycle of 14/10 h, under continuous shaking at 100 rpm.
3. Cell growth and harvest: Cell density was monitored every
two days through a hemocytometer (C-chip) cell counting. Cells
were harvested on day 14 and stored at -80°C before being
freeze-dried for DCW.
4. Lipid staining and extraction: Cells were stained with Nile
Red to assess lipid content. Lipids were extracted from driedsamples using a chloroform/methanol (2:1) solution.
5. Statistical analysis: The experiments were conducted intriplicate. GraphPad Prism software was used to analyze thedata using ANOVA, followed by Tukey's post hoc test.
Results and Discussion

References
Borowitzka, M.A. (2018). Microalgae in health and disease
prevention, 23-72
Fučíková et al. (2014). Phycological Research, 62(4), 294-305
Masumoto, H. (2020). University of Tsukuba PHD Thesis
Polsilapa, T. (2023). Tsukuba Journal of Biology 2023, 22(7), 2
Veselá et al. (2024). Phycologia, 63(3), 317–338
Yuzheng, S. (2021). Tsukuba Journal of Biology 2021

4

つくば生物ジャーナル

Tsukuba Journal of Biology (2024)

23(7), 5Ⓒ2024 筑波大学生物学類

Impacts of future ocean acidification on the ecology of two sea urchin species

Maaya Marie Eguchi Grove（筑波大学 生物学類）

指導教員：Ben Harvey（筑波大学 生命環境系）

Introduction
Ocean acidification (OA) is a process where carbonate chemistry
is altered due to the increase in anthropogenic atmospheric CO2.
Calcifying organisms are generally negatively affected by OA,
with sea urchins particularly sensitive to the altered conditions
showing reduced growth and survival (Harvey et al. 2013). Sea
urchins are key benthic macroherbivores in contemporary
systems, playing an important role in the maintenance andcontrol of habitat structure and composition (Baggini et. al.,2015). Despite their importance, the effects of OA on theecological role of urchins are still not well-known. Here, we
assess how OA affects the ecology of two locally importanturchin species (Echinometra sp. and Diadema setosum) using a
CO2 seep in Shikine-jima (natural analogue for future OA).

and 1.5-fold increase in their GSI (p = 0.003), but no differences
between treatments for feeding rate or growth.
Discussion
Exposure to OA conditions resulted in different responses for theurchin Echinometra sp., with acute exposure leading toincreased feeding rates while metabolic rates remained stable,
and chronic exposure causing higher metabolic rates without acorresponding increase in feeding. These suggest that while
greater feeding rates may compensate for increased energy
demands under acute conditions, this was insufficient when
exposed to OA for extended (chronic) periods (termed anenergetic mismatch). D. setosum individuals also appeared tohave their feeding rate altered by exposure to OA, with theirfeeding rate suppressed when they were exposed to short-term
elevated CO2 conditions. This would suggest that unlike
Echinometra sp. they are unable to behaviorally compensate
even on the short-term, which may contribute towards their
particularly low abundance in the CO2 seep area. Reductions inavailable energy (due to increased metabolism or decreased
feeding rates) can reduce the ability of organisms to properly
maintain bodily functions, requiring them to alter their energy
allocations, with long-term implications for population survival.
Here, urchins exposed to (chronic) high-CO2 conditions were
smaller but had a significantly greater GSI. While this may
simply be a shift in reproductive timing between the sites, thismay also represent a terminal investment, where reproduction
is favoured over individual survival. Taken together, our results
suggest that while some species-specific responses may exist,
OA will generally cause energetic mismatches and alteredenergy allocations in urchins, leading to potential long-term
impacts on population survival. Given that urchins play a
crucial role in controlling habitat structure and composition,
declines in their feeding rates and/or population survival willhinder their ability to perform this role, potentially having
significant impacts on the future health of coastal ecosystems.

Material & Methods
The effects of OA on urchins were considered under two
temporal scales, acute exposure (1-month aquarium experiment
under OA compared to control conditions) and chronic exposure(lifetime residing in CO2 seep compared to adjacent bay undercontemporary conditions). For acute exposure, urchins wereindividually held for one-month under control- (pHT ~8.15), mid(pHT ~7.90), or high- (pHT ~ 7.70) CO2 conditions (n = 8 pertreatment). Both urchin species were tested separately. For
chronic exposure, individuals from the CO2 seep site (pHT ~7.80)
and adjacent bay (pHT ~8.15) were collected (n = 8 per site) andmaintained under their respective conditions whilemeasurements (described below) were taken. Only
Echinometra sp. were tested for chronic exposure due to theextremely low abundance of D. setosum in the CO2 seep area
(0.02 individuals m-2). At the end of all experiments, metabolism(O2 consumption), feeding rate, and the gonadal somatic index(GSI) via dissection were measured, as well as size, and growth(changes in wet weight).
Results
Under acute exposure, both Echinometra sp. and D. setosum
demonstrated similar metabolic rates, GSI and growth rates
under all treatments (control-, mid-, and high-CO2). Feeding
rates, however, did differ; for Echinometra sp., there was a twofold increase in mean feeding rates (albeit non-significantly, p =0.274) under high-CO2 conditions compared to control-CO2
conditions (with mid-CO2 similar to control). For D. setosum,
however, feeding rates were significantly suppressed (p = 0.017)
under high-CO2 conditions compared to control conditions.
When considering the longer-term (chronic) exposure toelevated CO2 conditions, Echinometra sp. individuals showed asignificant two-fold increase in their metabolic rate (p = 0.006)

References
Harvey, B.P., Gwynn-Jones, D. & Moore, P.J. (2013). Metaanalysis reveals complex marine biological responses to theinteractive effects of ocean acidification and warming. Ecology
and Evolution, 3, 1016–1030.
Baggini, C., Issaris, Y., Salomidi, M., & Hall-Spencer, J. (2015).
Herbivore diversity improves benthic community resilience toocean acidification. Journal of Experimental Marine Biology
and Ecology, 469, 98–104.

5つくば生物ジャーナル

Tsukuba Journal of Biology (2024)

23(7), 6Ⓒ 2024 筑波大学生物学類

Loss of Usp15 leads to mitochondrial abnormalities

Kseniia Sevostianova（筑波大学 生物学類）

指導教員：Fuminori Tsuruta（筑波大学 生命環境系）
using a confocal microscope and analysed by FIJI ImageJsoftware.
Rotenone treatment: Cells were incubated for 1 day after
transfection and treated with rotenone at the final
concentrations of 100, 250, 500, and 1000 nm for 4 or 8 hours
before fixation.

Introduction
The cerebral cortex is organised into 6 horizontal layersthat are characterised by varying cell populations and density.
Layer V (L5) consists of large pyramidal neurons. The longprojecting axons of these neurons travel to the spinal cord
forming the corticospinal tract, which is the main pathway forvoluntary motor control. In turn, axon degeneration is one of thefeatures of motor and neurodegenerative disorders. Recent
evidence suggests that the degeneration can be triggered by
mitochondrial dysfunction.
In this study, I assess the role of ubiquitin-specific protease
15 (USP15), a deubiquinating enzyme (DUB) from the USP
subfamily. USP15 is a relevant gene in several motor andneurodegenerative disorders. Prior research demonstrates thatthe knock-out (KO) of USP15 in mice leads to behavioural(impaired sociability and instincts) and physiologicalabnormalities (thinning of L5 in the cerebral cortex,
hippocampus shrinkage). However, the molecular mechanismsof these changes are unknown.
USP15 is a known antagonist of Parkin and a mitophagy
regulator, which makes USP15-mediated mitochondrial
regulation a potential factor in the progression ofneurodegeneration. I investigated the influence of USP15 on
mitochondria under basal and stressful conditions. I found thatthe mitochondria in USP15 KO MEFs have a distinct
phenotype and conduct clearance of damaged mitochondria
more efficiently.

Results
1. Loss of USP15 changes mitochondrial morphology in MEFs
To assess the morphology of mitochondria in WT and USP15
KO MEFs, I transfected pEYFP-Mito into MEFs and quantified
relevant mitochondrial parameters. The mean area of all
mitochondria in a cell was 19% lower, and the mean perimeterwas 15% lower in KO cells on average (p = 0.003). The
mitochondria in KO cells exhibited less branching. The mean
form factor and aspect ratio indicated more elongated andfibrous organelles in WT cells as opposed to more circular onesin KO cells. Overall, mitochondria in USP15 KO MEFs
exhibited predominantly round morphology.
2. Loss of USP15 promotes a more effective clearance ofdamaged mitochondria under stressful conditions
To investigate the influence of USP15 on mitophagy understressful conditions, I treated pEYFP-Mito transfected cells withrotenone, a respiration inhibitor. In both 4- and 8-hourtreatment groups, the mitochondria in KO cells displayed
significantly fewer fluorescent signals than in WT cells at
rotenone concentrations of 250 nm and above. To confirm therole of USP15 in regulating mitophagy, I conducted a rescue
experiment cotransfecting KO cells with pEYFP-Mito and USPMyc. After 4 hours of rotenone treatment, the mitochondrialarea was larger in cotransfected cells even at the 500 nm
rotenone concentration. These data suggest that loss of USP15
can enhance mitophagy in response to stressful conditions.

Material & Methods

Cell culture and transfection: Wild type (WT) and USP15 KO

mouse embryonic fibroblasts (MEFs) were cultured in
Dulbecco’s modified Eagle’s medium (high glucose)
supplemented with 10% fetal bovine serum and 1% penicillinstreptomycin-glutamine in a 37°C incubator with 5% CO2. Cells
were plated at the density of 3.0 x 104 in 3 ml per well andincubated for 1 day. Cells were transfected with the pEYFPMito and/or Myc-USP15 plasmids using appropriate amounts ofpolyethyleneimine MAX and Opti-MEM according to the
manufacturer’s instructions.
Immunocytochemistry: Cells were fixed 24 hours after
transfection with 4% paraformaldehyde in PBS, blocked in 0.4%
Triton X-100 in blocking solution (5% BSA in PBS), andincubated with primary antibodies overnight at 4°C. Afterwashing with PBS, cells were incubated with secondary
antibodies for 30 min at room temperature. The coverslips were
mounted onto slides, and fluorescence images were obtained

Discussion

USP15 KO MEFs have a distinct mitochondrial phenotype,possibly implying a shift in the fission/fusion balance towards
fission with mitochondria taking a shorter, more circular shape.
Enhanced mitophagy in KO cells induced by mitochondrial
stressors was also observed for other DUBs of the same family,such as USP30. Emerging evidence of functional differences of
mitochondria in axons gives more grounds for a claim of USP15– mitochondria interactions playing a substantial role in relateddisorders. Taken together, my results suggest that USP15mediated changes in mitochondria regulation make USP15 apotential therapeutical target.
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