Article

ISSN 0006-2979, Biochemistry (Moscow), 2025, Vol. 90, No. 12, pp. 1999-2008 © The Author(s) 2025. This article is an open access publication.

Published in Russian in Biokhimiya, 2025, Vol. 90, No. 12, pp. 2108-2118.

1999

Mitochondrial Lipid Peroxidation Initiates Rapid

Accumulation of Lipofuscin in Cultured Cells

He Huan

, Alisa A. Panteleeva

, Ruben A. Simonyan

, Armine V. Avetisyan

Konstantin G. Lyamzaev

1,2,a

*, and Boris V. Chernyak

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University,

119991 Moscow, Russia

Russian Clinical Research Center of Gerontology, Pirogov Russian National Research Medical University,

Ministry of Health of the Russian Federation, 129226 Moscow, Russia

e-mail: lyamzaev@gmail.com

Received August 15, 2025

Revised September 12, 2025

Accepted September 16, 2025

Abstract— Activation of lipid peroxidation (LPO) in the mitochondria of rat H9c2 cardiomyoblasts and human

fibroblasts by the cystine transport inhibitor erastin or glutathione peroxidase 4 inhibitor RSL3 was accom-

panied by rapid (18 h) accumulation of lipofuscin. The mitochondria-targeted antioxidant SkQ1 and redox

mediator methylene blue, which prevents the formation of reactive oxygen species (ROS) in the mitochondrial

respiratory chain complex I, blocked both mitochondrial LPO and lipofuscin accumulation. These data indi-

cate that mitochondrial LPO serves as a driving force for the accelerated accumulation of lipofuscin in cells.

Rapid (24 h) lipofuscin formation was observed in isolated heart mitochondria in the presence of iron ions.

It was significantly accelerated by ROS generated in the respiratory chain complex I and blocked by SkQ1.

The question of whether oxidized components of mitochondria serve as a source for lipofuscin formation in

cells remains open. The results obtained suggest possible application of mitochondria-targeted compounds in

the treatment of diseases associated with excessive lipofuscin accumulation.

DOI: 10.1134/S0006297925602606

Keywords: lipid peroxidation, mitochondria, ferroptosis, lipofuscin

* To whom correspondence should be addressed.

INTRODUCTION

Lipofuscin (LF) is insoluble pigment aggregates

that accumulate in cells during aging. LF was first de-

scribed by the Danish physician and histologist Adolf

Hannover [1], although its association with aging had

not been recognized until the late nineteenth century.

LF is composed mainly of highly oxidized proteins,

with smaller amounts of lipids, carbohydrates, and

nucleic acids, and contains metal ions, including iron,

which determines its involvement in oxidative pro-

cesses [2]. The composition of LF varies considerably

across different types of cells; its analysis is complicat-

ed by extensive covalent cross-linking of constituent

macromolecules. In a body, LF primarily accumulates

in postmitotic cells, such as neurons and cardiomy-

ocytes. In actively phagocytizing cells, in particular,

macrophages [3] and retinal pigment epithelium

(RPE) cells [4], a substantial fraction of LF originates

from the endocytosed material. For example, in RPE

cells that phagocytize fragments of retinal-containing

photoreceptors, the major LF component is a retinal

dimer conjugated with ethanolamine (A2E). Accumu-

lation of LF in RPE cells has been implicated in the

development of age-related macular degeneration [5].

In cells, LF is found in the cytosol and endosomal

compartments. It is believed to be generated by oxida-

tion of various cellular constituents and delivered to

the lysosomes via autophagy [6]. Once inside the lyso-

somes, LF is mostly resistant to degradation but may

undergo modifications and even “grow” by reacting

with neighboring proteins through reactive surface

groups [6]. Inhibition of lysosomal proteolytic activity

in  vivo results in LF accumulation in multiple organs [7].

HUAN et al.2000

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

Mitochondria have been proposed as one of the prin-

cipal sources of LF [8]. Proteomic analysis of brain-

derived LF [9] identified several mitochondrial pro-

teins along with proteins from other cellular com-

partments, although the available data are insuffi-

cient to establish mitochondria as the primary site

of LF origin. Like other protein aggregates, LF can

inhibit the proteasomal activity, and its accumulation

in lysosomes may impair their function. However,

whether LF plays an active role in the age-related

cellular damage remains unknown.

The stimulation of autophagy in aged rats re-

duced the LF content, which correlated with a de-

crease in the proportion of cardiomyocytes displaying

senescence markers [10], although no causality could

be reliably established in such experiments. Exoge-

nously applied purified LF impaired the functioning

of cardiomyocytes [11] and RPE cells [12] and even

induced the death of fibroblasts [13]. The age-associ-

ated LF accumulation has been documented in var-

ious organs, including the heart [14]. Thus, smaller

short-lived species of primates were found to dis-

play much faster LF accumulation in the heart [15].

Examination of human cardiac tissue from individ-

uals aged 20 to 97 revealed that LF levels correlat-

ed with aging but did not depend on body weight

or cause of death [16]. LF accumulation during both

replicative and stress-induced senescence is a major

biomarker of aging in cultured cells and is widely

used in research [17].

Lipid peroxidation (LPO), in particular, oxidation

of polyunsaturated fatty acids in membrane phospho-

lipids, is a key driver of LF accumulation. Thus, iron

chelators capable of penetrating into cells and sup-

pressing LPO, prevented LF accumulation in  vitro [18]

and invivo [19]. Malondialdehyde, a major LPO prod-

uct, can react with one or two amino groups of mac-

romolecules with the formation of Schiff bases, which

undergo Maillard-type intramolecular rearrangements

and account for LF fluorescence at 450-470  nm (with

the excitation maximum at 360-390  nm) [20]. The for-

mation of aldehyde bridges is a critical mechanism

of protein–protein and protein–lipid cross-linking

during the generation and progressive “growth” of

LF aggregates.

Over the past decade, the studies of LPO have

been greatly stimulated by the discovery of ferropto-

sis, a form of regulated cell death that depends on

the iron-catalyzed LPO in cellular membranes [21].

It has been found that ferroptosis contributes to nu-

merous pathologies associated with oxidative stress,

including ischemic injury of the heart, brain, and

kidneys, as well as neurodegenerative disorders [22].

The interest in ferroptosis has been further fueled by

the ability of its inducers to act as anticancer agents

capable of overcoming tumor resistance linked to the

suppression of apoptosis [23]. The first identified spe-

cific inducer of ferroptosis was erastin, an inhibitor

of the cystine transporter SLC7A11 required for glu-

tathione biosynthesis [21]. A more selective and po-

tent inducer is the glutathione peroxidase  4 (GPX4)

inhibitor RSL3 [24], whose action does not depend on

glutathione levels and, therefore, is less constrained

by the cellular metabolic context compared to erastin

or inhibitors of glutathione biosynthesis.

Our recent work has shown that LPO in the in-

ner mitochondrial membrane (mitoLPO) is essential

for ferroptosis [25-28]. We found that erastin triggers

mitoLPO, which precedes ferroptotic cell death. The

mitochondria-targeted antioxidant SkQ1 [10-(6′-plas-

toquinonyl)decyltriphenylphosphonium bromide]

prevented both mitoLPO and erastin-induced cell

death. A similar effect was observed for methylene

blue (MB), a well-known redox mediator that allows

to bypass the electron transfer through the mito-

chondrial respiratory complex I, thereby suppressing

formation of reactive oxygen species (ROS) [29]. We

demonstrated that exogenous iron, supplied as am-

monium ferric citrate, induced rapid (within 24  h)

LF accumulation in rat H9c2 cardiomyoblasts, preced-

ing ferroptosis [27]. In this model, both LF accumu-

lation and mitochondrial LPO were blocked by SkQ1

and MB, highlighting the importance of mitoLPO in

the LF formation.

Here, we investigated LF accumulation in H9c2

cells and human fibroblasts treated with erastin and

RSL3. Our findings confirm that mitoLPO can drive

rapid LF accumulation in cells.

MATERIALS AND METHODS

Cell cultures. Rat H9c2 cardiomyoblasts (ECACC;

cat. no. 88092904) and primary human subcutaneous

fibroblasts (Biobank Shared Research Facility, Re-

search Centre for Medical Genetics, Moscow, Russia)

were cultured in Dulbecco’s Modified Eagle’s Medi-

um (DMEM) (Gibco, USA) supplemented with 2  mM

L-glutamine, 10% fetal bovine serum (FBS) (HyClone,

USA), 100  U/mL penicillin, and 100  U/mL streptomycin

(Gibco). Cell viability was assessed using the CellTit-

erBlue® assay (Promega, USA) according to the man-

ufacturer’s protocol. Optical density at 590  nm was

measured with a Fluoroskan Ascent FL microplate

reader (Thermo Labsystems, USA).

MitoLPO assay. MitoLPO was assessed using the

ratiometric fluorescent probe MitoCLox derived from

the well-established indicator C11-BODIPY581/591,

which specifically reacts with lipid peroxyl radicals,

resulting in the shift in its fluorescence spectrum [29].

The cells were incubated with 5  µM erastin or 100  nM

RSL3 for 18  h in the presence of 200  nM MitoCLox.

MITOCHONDRIAL LIPID PEROXIDATION DRIVES LIPOFUSCIN ACCUMULATION 2001

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

After incubation, the cells were detached using tryp-

sin/EDTA, centrifuged (900g, 5 min, 4°C), resuspended

in 50  µL PBS, and analyzed by imaging flow cytom-

etry using an Amnis FlowSight cytometer (Luminex

Corporation, USA) with excitation at 488  nm and

emission at 505-560  nm (Channel  2, Ch2) and 595-

642  nm (Channel  4, Ch4). Oxidation of MitoCLox was

quantified as the Ch2/Ch4 fluorescence intensity ra-

tio. The data were processed using the Amnis IDEAS®

software version 6.2 (Luminex). At least 4000 events

were recorded per sample. In cases where signal dis-

tribution deviated significantly from Gaussian, gat-

ing procedures were applied for accurate data eval-

uation.

Measurement of cellular LF content. The cells

were seeded in 12-well plates. After incubation for

24  h, 5  µM erastin or 100 nM RSL3 were added, ei-

ther alone or in combination with 100  nM  SkQ1,

250  nM  MB, 100  µM deferoxamine (DFO), or 200  µM

Trolox. LF autofluorescence was detected by flow

cytometry (excitation at 488  nm, emission at 505-

560  nm, Ch2) [30]. The data were analyzed with the

Amnis IDEAS® 6.2 software.

The LF content in H9c2 cells was additionally

assessed after seeding the cells in 6-well plates for

18  h, followed by treatment with 100  nM  RSL3, with

or without 100  nM  SkQ1, 250  nM  MB, 100  µM  DFO, or

200  µM  Trolox. After 18-h incubation, the cells were

lysed in 1% SDS, and fluorescence spectra were re-

corded at 500-650  nm (excitation at 405  nm) in a

FluoroMax-3 spectrofluorometer (Horiba Scientific,

Japan); fluorescence spectra of 1% SDS solution were

subtracted from the recorded spectra.

Measurement of cytosolic and mitochondrial

ROS. Cytosolic ROS levels were determined using

dichlorodihydrofluorescein diacetate (H

DCFDA) (In-

vitrogen Life Technologies, USA), while mitochon-

drial ROS were measured with MitoSOX (Invitrogen

Life Technologies). The cells were treated with 5  µM

erastin or 100  nM RSL3 for 18  h, followed by incu-

bation in fresh medium containing 1.8  µM H

DCF-DA

or 1  µM MitoSOX for 30 min. Fluorescence was mea-

sured using an Amnis FlowSight cytometer with exci-

tation at 488 nm and emission collected in Ch2.

LF content in isolated mitochondria. Mitochon-

dria were isolated from rat hearts in a buffer con-

taining 5  mM MOPS-KOH (pH  7.4), 250  mM sucrose,

1  mM EGTA, and 0.5  mg/mL bovine serum albumin.

Cardiac tissue was minced in ice-cold isolation buffer

(10  mL/g tissue) and homogenized in a Potter glass

homogenizer for 1-2  min. The homogenates were di-

luted to 20  mL/g tissue and centrifuged at 600g for

10  min. The supernatants were collected and centri-

fuged at 12,000g for 10  min. The pellets were resus-

pended in a minimal volume of the buffer, re-homog-

enized, diluted with 20  mL of the isolation buffer, and

centrifuged again (10 min, 12,000g). The final pellets

were resuspended and stored on ice.

Mitochondria were incubated in a medium con-

taining 50  µM FeSO

, 5  mM glutamate, 5  mM malate,

and 10  µM rotenone at 37°C with constant stirring[1].

Protein concentration was adjusted to 0.4  mg/mL (ac-

cording to Bradford assay). LF fluorescence was mea-

sured after the mitochondria were lysed in 1%  SDS

at 365-600  nm (excitation at 360  nm) with a Fluoro-

Max-3 spectrofluorometer. MitoCLox oxidation was

assessed as described previously [30].

Statistical data processing. The data are present-

ed as mean ± standard deviation (SD) from at least

three independent experiments and compared using

one-way ANOVA. Statistical significance was evaluat-

ed with the Prism 10.0 software (GraphPad Software,

USA). The differences were considered significant at

****  p  ≤  0.0001, ***  p  ≤  0.001, **  0.001  <  p  ≤  0.05, and

*  p  <  0.05.

RESULTS

Previously, we demonstrated that erastin induced

mitoLPO in SV40-transformed MRC5 human lung fi-

broblasts [25]. The data presented in Fig. 1 show that

mitoLPO (measured using MitoCLox) also occurred in

rat H9c2 cardiomyoblasts and non-transformed pri-

mary human dermal fibroblasts treated with either

erastin or RSL3. In all models employed, mitoLPO

preceded necrotic cell death. The mitochondria-tar-

geted antioxidant SkQ1 and the redox mediator MB

efficiently prevented both mitoLPO and cell death

(Fig. 1). Similarly, the cell-permeable iron chelator

DFO and the lipophilic antioxidant Trolox exerted

protective effects.

Measuring the levels of mitochondrial ROS with

the MitoSOX probe revealed that ROS accumulation

in our cellular models occurred concomitantly with

mitoLPO following the treatment with either erastin

or RSL3 (Fig.  2a). We also observed ROS accumula-

tion in the cytoplasm, as assessed with H

DCFDA

(Fig.  2b). In both compartments, ROS accumulation

was prevented by the same agents that inhibited

mitoLPO.

LF accumulation assessed by flow cytometry

based on its broad autofluorescence spectrum (505-

560  nm), was observed within the same 18-h time-

frame as mitoLPO and preceded cell death (Fig.  3).

SkQ1 and MB, as well as DFO and Trolox, completely

blocked LF accumulation. The measurements of flu-

orescence spectra in cell lysates following cell dis-

ruption with a detergent confirmed LF accumulation

(Fig.  3,d ande). Fluorescence microscopy revealed no

formation of LF granules or preferential LF accumu-

lation in the mitochondria.

HUAN et al.2002

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

Fig. 1. Erastin and RSL3 induced mitoLPO in rat H9c2 cardiomyoblasts (a) and primary human fibroblasts (b), as well

as cell death (c). Cells were incubated with 5 μM erastin or 100 nM RSL3, alone or in combination with 100  nM  SkQ1,

250  nM  MB, 100 μM DFO, or 200 μM Trolox for 18 h. MitoLPO was assessed by adding ratiometric fluorescent probe

MitoCLox to the medium. The ratio of green (Ch2) to red (Ch4) fluorescence was calculated using Amnis IDEAS® 6.2

software. Statistical significance: ****  p < 0.0001 and ***  p ≤ 0.001, differences between cells treated with erastin or RSL3

and all other treatment conditions (n = 4-6).

MITOCHONDRIAL LIPID PEROXIDATION DRIVES LIPOFUSCIN ACCUMULATION 2003

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

Fig. 2. Erastin and RSL3 induced the formation of (a) mitochondrial superoxide anion (cell staining with 1 μM MitoSOX

for 30 min) and (b) cytosolic ROS (cells staining with 1.8 μM H

DCFDA for 30 min) in H9c2 cardiomyoblasts and primary

human fibroblasts. In both cases, the cells were preincubated for 18 h with 5 μM erastin or 100 nM RSL3, alone or in

combination with 100 nM SkQ1, 250 nM MB, 100 μM DFO, or 200 μM Trolox. The data are shown as mean fluorescence

intensity (% of control). Statistical significance: ****p< 0.0001; ***p≤0.001; **0.001<p≤0.05; *p<0.05 vs. cells treated

with erastin or RSL3.

Interestingly, two distinct subpopulations differ-

ing markedly in the level of mitoLPO were observed

in the RSL3-treated fibroblasts (Fig. 1). A similar het-

erogeneity in mitoLPO had previously been detect-

ed in SV40-transformed MRC5 fibroblasts exposed

to erastin [25]. In H9c2 cells, the heterogeneity was

considerably less pronounced (Fig. 1), and the LF ac-

cumulation assay revealed population heterogeneity

only in fibroblasts (Fig. 3c). The nature of this het-

erogeneity remains unclear.

In our previous studies [25, 27, 28], we investi-

gated LPO in isolated rat heart mitochondria using

MitoCLox. It was found that LPO occurred only in

the presence of ferrous ions (Fe

), while addition of

the complex I inhibitor rotenone in the presence of

NAD-linked substrates (glutamate and malate) mark-

edly stimulated oxidation, consistent with the LPO in-

duction by ROS generated in complex I. Fluorescence

measurements after disruption of the mitochondria

with a detergent demonstrated detectable LF accumu-

lation within 24 h at 37°C, which further increased

after incubation for additional 24h (Fig. 4a). Notably,

under these conditions, LPO developed within 1-2  h

(Fig. 4b), substantially preceding LF accumulation.

No LF accumulation was observed in the absence of

ions. Rotenone promoted LF formation, whereas

SkQ1 blocked it, thus highlighting the role of com-

plex I-derived ROS (Fig. 4a).

Earlier studies [31] reported that the LF for-

mation in the mitochondria isolated from rat liver

occurred much faster than in the lysosomes or mi-

crosomes (membrane vesicles derived from disrupt-

ed endoplasmic reticulum). However, preparations of

liver mitochondria obtained by differential centrif-

ugation (as in [31]) are unavoidably contaminated

with peroxisomes, which contain lipolytic enzymes.

HUAN et al.2004

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

Fig. 3. Effects of erastin and RSL3 on the LF content in H9c2 cells(a) and primary human fibroblasts (b). Cells were incu-

bated for 18 h with 5 μM erastin or 100 nM RSL3, alone or in combination with 100  nM  SkQ1, 250  nM  MB, 100  μM  DFO,

or 200  μM  Trolox. LF fluorescence was recorded by flow cytometry at 505-560  nm (Ch2) (a, b, and c). LF accumulation

was further confirmed after cell lysis with 1% SDS, followed by spectrofluorimetry (d and e). Statistical significance:

****  p < 0.0001; ***  p ≤ 0.001; **  0.001 < p ≤ 0.05; * p < 0.05 vs. cells treated with erastin or RSL3.

Fig. 4. LF accumulation (a) and LPO rate (b) in isolated rat heart mitochondria. The incubation medium contained FeSO

(50 μM), glutamate (5 mM), malate (5 mM), rotenone (Rot, 10 μM), and SkQ1 (100 nM).

This contamination may explain the detection of LF

in the supernatant following mitochondrial sedimen-

tation at 12,000g [31]. The concerns about peroxiso-

mal contamination also extend to a later study [32],

which reported accelerated LF formation in liver mi-

tochondria upon heating or UV irradiation. In our ex-

periments on heart mitochondria, the risk of peroxiso-

mal contamination was minimal. The use of detergent

disruption in these experiments allowed LF quanti-

fication independently of its membrane association.

MITOCHONDRIAL LIPID PEROXIDATION DRIVES LIPOFUSCIN ACCUMULATION 2005

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

DISCUSSION

The data presented in Figs. 1 and 3 demonstrate

that the activation of mitoLPO by the cystine trans-

port inhibitor erastin or the GPX4 inhibitor RSL3

was accompanied by LF accumulation. The mito-

chondria-targeted antioxidant SkQ1 and the redox

mediator MB, which prevents ROS formation in com-

plexI of the mitochondrial respiratory chain, blocked

both mitoLPO and LF accumulation. Previously, we

showed [27] that the exogenous iron-induced LF ac-

cumulation in H9c2 cells was also inhibited by SkQ1

and MB. These findings indicate that mitoLPO acts as

a driving force of accelerated LF formation. Consis-

tent with this conclusion, another mitochondria-tar-

geted antioxidant, MitoTEMPO, was reported to sup-

press LF accumulation in fibroblasts chronically (for

10 days) treated with the prooxidant paraquat [33].

In the same model, the inhibitor of mitochondrial

fragmentation and mitophagy Mdivi-1 promoted LF

accumulation, further indicating the critical role of

mitochondria in LF biogenesis.

It should be noted that in our experiments, SkQ1

and MB suppressed ROS accumulation in both mito-

chondria and the cytoplasm (Fig.  2). Hence, we can-

not exclude the possibility that ROS generated in the

mitochondria contribute to the LF formation along-

side LPO products. At the same time, one of the LF

inducers used in our study was RSL3, which specifi-

cally blocks a defense mechanism against LPO. While

erastin can disrupt multiple antioxidant systems

through glutathione depletion, GPX4 inhibition selec-

tively promotes LPO without impairing other cellular

defenses. Further supporting the view that cellular

ROS accumulation results from LPO, DFO (an iron

chelator that selectively blocks LPO) prevented ROS

accumulation (Fig. 2).

Our data show an important role of mitochon-

dria in the LF formation but do not prove that mito-

chondria themselves constitute the primary substrate

for LF, as fluorescence microscopy revealed no de-

tectable LF accumulation in the mitochondria in our

models. However, it is well established that oxidized

mitochondria are targeted for mitophagy [34], so

LF accumulation in the mitochondria may be tran-

sient. Experiments with isolated rat heart mitochon-

dria (Fig.  4) demonstrated rapid LF formation that

was markedly accelerated by ROS generated in com-

plex I of the respiratory chain. Additional evidence

for the mitochondrial involvement in LF biogenesis

comes from the experiments where selective inhibi-

tion of the mitochondrial protease Lon in HeLa cells

promoted LF accumulation under chronic paraquat

treatment [33]. Nevertheless, it remains possible that

LPO and protein oxidation in mitochondria trigger LF

formation from other cellular components.

LF accumulation occurs not only during physi-

ological aging but also in various diseases, such as

age-related macular degeneration (AMD) [35], Star-

gardt disease (the most common form of inherited

juvenile macular degeneration) [36], Alzheimer’s dis-

ease [37], Parkinson’s disease [38], and other neuro-

degenerative disorders [39]. The pathogenic role of

LF has been best established in AMD [5] and Star-

gardt disease [40], in which LF-containing retinal-

dehyde dimers (A2E) contribute to the RPE damage

via photodynamic effects. Mitochondrial dysfunction

has been implicated in AMD pathogenesis [41], and

SkQ1 demonstrated therapeutic efficacy in this con-

text [42, 43]. Remofuscin (soraprazan), the first and

so far, only pharmacological inhibitor of LF accumu-

lation, has been proposed for the treatment of Star-

gardt disease [40]. Although the precise mechanism

of its action remains unclear, the studies in Caenor-

habditis elegans showed that remofuscin reduced

age-dependent LF accumulation and increased nema-

tode lifespan by 20% [44]. LF-loaded neurons appear

to be more susceptible to pathological changes in the

Alzheimer’s [39] and Parkinson’s [45] diseases. The

role of mitochondrial dysfunction in neurodegener-

ation is widely discussed [46]. SkQ1 significantly

attenuated progression of the Alzheimer’s [47] and

Parkinson’s [32] disease symptoms in various animal

models.

Taken together, our results suggest that mito-

chondria-targeted compounds can reduce LF accu-

mulation in cells, providing a rationale for their po-

tential therapeutic application in a broad spectrum

of conditions associated with excessive LF deposition.

Abbreviations

DFO deferoxamine

LF lipofuscin

MB methylene blue

mitoLPO mitochondrial lipid peroxidation

LPO lipid peroxidation

ROS reactive oxygen species

RPE retinal pigment epithelium

SkQ1

10-(6′-plastoquinonyl)decyltriphenyl-

phosphonium bromide

Acknowledgments

We thank the Moscow State University Development

Program PNR5 for access to the Amnis FlowSight cy-

tometer and Olympus IX83 microscope.

Contributions

B.V.Ch. and K.G.L. developed the concept and super-

vised the study; H.H., A.A.P., R.A.S., A.V.A., and K.G.L.

conducted the experiments; B.V.Ch. and K.G.L. an-

alyzed the data; B.V.Ch. and K.G.L. wrote the draft;

H.H. and K.G.L. edited the manuscript.

HUAN et al.2006

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

Funding

This work was supported by the Russian Science

Foundation (grant № 23-14-00061). The experimental

part of the work was supported by the Russian Sci-

ence Foundation (grant № 23-14-00061). Data analysis,

processing of results, and preparation of the article

text were carried out as part of a state assignment

from Lomonosov Moscow State University (topic No.

AAAA-A19-119031390114-5).

Ethics approval and consent to participate

All animal procedures and experiments were con-

ducted in accordance with international guidelines

for the care and use of laboratory animals and were

approved by the Institutional Ethics Committee of

the Belozersky Institute of Physico-Chemical Biology,

Moscow State University (protocol no. 012-5/05/2024,

March24, 2024).

Conflict of interest

The authors of this work declare that they have

noconflicts of interest.

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REFERENCES

1. Hannover, A. (1842) Mikroskopiske undersögelser af

nervesystemet, Kabernes Selkobs Naturv. Math. Afh.

Copenhagen, 10, 1-112.

2. Hohn, A., Jung, T., Grimm, S., and Grune, T. (2010)

Lipofuscin-bound iron is a major intracellular

source of oxidants: role in senescent cells, Free Rad-

ic. Biol. Med., 48, 1100-1108, https://doi.org/10.1016/

j.freeradbiomed.2010.01.030.

3. Lee, F.Y., Lee, T.S., Pan, C.C., Huang, A.L., and Chau,

L. Y. (1998) Colocalization of iron and ceroid in hu-

man atherosclerotic lesions, Atherosclerosis, 138, 281-

288, https://doi.org/10.1016/S0021-9150(98)00033-1.

4. Ablonczy, Z., Smith, N., Anderson, D. M., Grey, A. C.,

Spraggins, J., Koutalos, Y., Schey, K. L., and Crouch,

R. K. (2014) The utilization of fluorescence to iden-

tify the components of lipofuscin by imaging mass

spectrometry, Proteomics, 14, 936-944, https://

doi.org/10.1002/pmic.201300406.

5. Feldman, T. B., Dontsov, A. E., Yakovleva, M. A., and

Ostrovsky, M. A. (2022) Photobiology of lipofuscin

granules in the retinal pigment epithelium cells of

the eye: norm, pathology, age, Biophys. Rev., 14, 1051-

1065, https://doi.org/10.1007/s12551-022-00989-9.

6. Hohn, A., Sittig,A., Jung,T., Grimm, S., and Grune, T.

(2012) Lipofuscin is formed independently of macro-

autophagy and lysosomal activity in stress-induced

prematurely senescent human fibroblasts, Free Rad-

ic. Biol. Med., 53, 1760-1769, https://doi.org/10.1016/

j.freeradbiomed.2012.08.591.

7. Ivy, G. O., Kanai, S., Ohta, M., Smith, G., Sato, Y.,

Kobayashi, M., and Kitani, K. (1989) Lipofuscin-like

substances accumulate rapidly in brain, retina and in-

ternal organs with cysteine protease inhibition, Adv.

Exp. Med. Biol., 266, 31-45, https://doi.org/10.1007/

978-1-4899-5339-1_3.

8. Terman, A., Kurz, T., Navratil, M., Arriaga, E. A., and

Brunk, U.T. (2010) Mitochondrial turnover and aging

of long-lived postmitotic cells: the mitochondrial-lyso-

somal axis theory of aging, Antioxid. Redox Signal.,

12, 503-535, https://doi.org/10.1089/ars.2009.2598.

9. Ottis,P., Koppe,K., Onisko,B., Dynin,I., Arzberger,T.,

Kretzschmar, H., Requena, J. R., Silva, C. J., Huston,

J. P., and Korth, C. (2012) Human and rat brain lipo-

fuscin proteome, Proteomics, 12, 2445-2454, https://

doi.org/10.1002/pmic.201100668.

10. Li, W.W., Wang, H.J., Tan, Y.Z., Wang, Y.L., Yu, S.N.,

and Li, Z. H. (2021) Reducing lipofuscin accumula-

tion and cardiomyocytic senescence of aging heart

by enhancing autophagy, Exp. Cell Res., 403, 112585,

https://doi.org/10.1016/j.yexcr.2021.112585.

11. Walter, S., Haseli, S. P., Baumgarten, P., Deubel, S.,

Jung, T., Hohn, A., Ott, C., and Grune, T. (2025) Ox-

idized protein aggregate lipofuscin impairs cardio-

myocyte contractility via late-stage autophagy inhibi-

tion, Redox Biol., 81, 103559, https://doi.org/10.1016/

j.redox.2025.103559.

12. Davies, S., Elliott, M. H., Floor, E., Truscott, T. G.,

Zareba, M., Sarna, T., Shamsi, F. A., and Boulton,

M. E. (2001) Photocytotoxicity of lipofuscin in hu-

man retinal pigment epithelial cells, Free Radic.

Biol. Med., 31, 256-265, https://doi.org/10.1016/S0891-

5849(01)00582-2.

13. Baldensperger, T., Jung, T., Heinze, T., Schwerdtle, T.,

Hohn, A., and Grune, T. (2024) The age pigment li-

pofuscin causes oxidative stress, lysosomal dysfunc-

tion, and pyroptotic cell death, Free Radic. Biol. Med.,

225, 871-880, https://doi.org/10.1016/j.freeradbiomed.

2024.10.311.

MITOCHONDRIAL LIPID PEROXIDATION DRIVES LIPOFUSCIN ACCUMULATION 2007

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

14. Porta, E., Llesuy, S., Monserrat, A. J., Benavides, S.,

and Travacio, M. (1995) Changes in cathepsin B

and lipofuscin during development and aging in

rat brain and heart, Gerontology, 41, 81-93, https://

doi.org/10.1159/000213727.

15. Nakano,M., Oenzil,F., Mizuno,T., and Gotoh,S. (1995)

Age-related changes in the lipofuscin accumulation

of brain and heart, Gerontology, 41, 69-79, https://

doi.org/10.1159/000213726.

16. Kakimoto, Y., Okada, C., Kawabe, N., Sasaki, A.,

Tsukamoto, H., Nagao, R., and Osawa, M. (2019)

Myocardial lipofuscin accumulation in ageing and

sudden cardiac death, Sci. Rep., 9, 3304, https://

doi.org/10.1038/s41598-019-40250-0.

17. Faragher, R. G. A. (2021) Simple detection methods

for senescent cells: opportunities and challeng-

es, Front. Aging, 2, 686382, https://doi.org/10.3389/

fragi.2021.686382.

18. Barbouti, A., Lagopati, N., Veroutis, D., Goulas, V.,

Evangelou, K., Kanavaros, P., Gorgoulis, V. G., and

Galaris, D. (2021) Implication of dietary iron-chelat-

ing bioactive compounds in molecular mechanisms of

oxidative stress-induced cell ageing, Antioxidants (Ba-

sel), 10, 491, https://doi.org/10.3390/antiox10030491.

19. Naseri, N. N., Ergel, B., Kharel, P., Na, Y., Huang, Q.,

Huang, R., Dolzhanskaya, N., Burre, J., Velinov, M. T.,

and Sharma, M. (2020) Aggregation of mutant cyste-

ine string protein-alpha via Fe-S cluster binding is

mitigated by iron chelators, Nat. Struct. Mol. Biol.,

27, 192-201, https://doi.org/10.1038/s41594-020-0375-y.

20. Yin,D. (1996) Biochemical basis of lipofuscin, ceroid,

and age pigment-like fluorophores, Free Radic.

Biol. Med., 21, 871-888, https://doi.org/10.1016/0891-

5849(96)00175-X.

21. Dixon, S.J., Lemberg, K.M., Lamprecht, M.R., Skouta,R.,

Zaitsev, E. M., Gleason, C.E., Patel, D.N., Bauer, A.J.,

Cantley, A. M., Yang, W. S., Morrison, B., 3rd, and

Stockwell, B.R. (2012) Ferroptosis: an iron-dependent

form of nonapoptotic cell death, Cell, 149, 1060-1072,

https://doi.org/10.1016/j.cell.2012.03.042.

22. Berndt, C., Alborzinia, H., Amen, V. S., Ayton, S.,

Barayeu, U., Bartelt, A., Bayir, H., Bebber, C. M.,

Birsoy, K., Bottcher, J. P., Brabletz, S., Brabletz, T.,

Brown, A.R., Brune,B., Bulli,G., Bruneau,A., Chen,Q.,

DeNicola, G. M., Dick, T. P., Distefano, A., Dixon,

S. J., Engler, J. B., Esser-von Bieren, J., Fedorova, M.,

Friedmann Angeli, J. P., Friese, M. A., Fuhrmann,

D. C., Garcia-Saez, A. J., Garbowicz, K., Gotz, M.,

Gu, W., Hammerich, L., Hassannia, B., Jiang, X.,

Jeridi, A., Kang, Y. P., Kagan, V. E., Konrad, D. B.,

Kotschi, S., Lei, P., Le Tertre, M., Lev, S., Liang, D.,

Linkermann, A., Lohr, C., Lorenz, S., Luedde, T.,

Methner, A., Michalke, B., Milton, A. V., Min, J.,

Mishima, E., Muller, S., Motohashi, H., Muckenthaler,

M. U., Murakami, S., Olzmann, J. A., Pagnussat, G.,

Pan, Z., Papagiannakopoulos, T., Pedrera Puentes, L.,

Pratt, D. A., Proneth, B., Ramsauer, L., Rodriguez, R.,

Saito,Y., Schmidt,F., Schmitt,C., Schulze,A., Schwab,A.,

Schwantes, A., Soula, M., Spitzlberger, B., Stockwell,

B. R., Thewes, L., Thorn-Seshold, O., Toyokuni, S.,

Tonnus, W., Trumpp, A., Vandenabeele, P., Vanden

Berghe, T., Venkataramani, V., Vogel, F. C. E., von

Karstedt, S., Wang, F., Westermann, F., Wientjens, C.,

Wilhelm, C., Wolk, M., Wu, K., Yang, X., Yu, F.,

Zou, Y., and Conrad, M. (2024) Ferroptosis in

health and disease, Redox Biol., 75, 103211, https://

doi.org/10.1016/j.redox.2024.103211.

23. Stockwell, B. R. (2022) Ferroptosis turns 10: emerg-

ing mechanisms, physiological functions, and ther-

apeutic applications, Cell, 185, 2401-2421, https://

doi.org/10.1016/j.cell.2022.06.003.

24. Yang, W. S., SriRamaratnam, R., Welsch, M. E.,

Shimada, K., Skouta, R., Viswanathan, V. S., Cheah,

J.H., Clemons, P.A., Shamji, A.F., Clish, C.B., Brown,

L. M., Girotti, A. W., Cornish, V. W., Schreiber, S. L.,

and Stockwell, B. R. (2014) Regulation of ferroptot-

ic cancer cell death by GPX4, Cell, 156, 317-331,

https://doi.org/10.1016/j.cell.2013.12.010.

25. Lyamzaev, K. G., Panteleeva, A. A., Simonyan, R. A.,

Avetisyan, A.V., and Chernyak, B.V. (2023) Mitochon-

drial lipid peroxidation is responsible for ferroptosis,

Cells, 12, 611, https://doi.org/10.3390/cells12040611.

26. Huan, H., Lyamzaev, K. G., Panteleeva, A. A., and

Chernyak, B. V. (2024) Mitochondrial lipid peroxida-

tion is necessary but not sufficient for induction of

ferroptosis, Front. Cell Dev. Biol., 12, 1452824, https://

doi.org/10.3389/fcell.2024.1452824.

27. Lyamzaev, K. G., Huan, H., Panteleeva, A. A.,

Simonyan, R.A., Avetisyan, A.V., and Chernyak, B.V.

(2024) Exogenous iron induces mitochondrial lipid

peroxidation, lipofuscin accumulation, and ferropto-

sis in H9c2 cardiomyocytes, Biomolecules, 14, 730,

https://doi.org/10.3390/biom14060730.

28. Lyamzaev, K. G., Panteleeva, A. A., Simonyan, R. A.,

Avetisyan, A. V., and Chernyak, B.V. (2023) The criti-

cal role of mitochondrial lipid peroxidation in ferro-

ptosis: insights from recent studies, Biophys. Rev., 15,

875-885, https://doi.org/10.1007/s12551-023-01126-w.

29. Pap, E. H., Drummen, G. P., Winter, V. J., Kooij, T. W.,

Rijken, P., Wirtz, K. W., Op den Kamp, J. A., Hage,

W. J., and Post, J. A. (1999) Ratio-fluorescence mi-

croscopy of lipid oxidation in living cells using C11-

BODIPY(581/591), FEBS Lett., 453, 278-282, https://

doi.org/10.1016/S0014-5793(99)00696-1.

30. Malavolta, M., Giacconi, R., Piacenza, F., Strizzi, S.,

Cardelli, M., Bigossi, G., Marcozzi, S., Tiano, L.,

Marcheggiani, F., Matacchione, G., Giuliani, A.,

Olivieri, F., Crivellari, I., Beltrami, A. P., Serra, A.,

Demaria, M., and Provinciali, M. (2022) Simple de-

tection of unstained live senescent cells with imag-

ing flow cytometry, Cells, 11, 2506, https://doi.org/

10.3390/cells11162506.

HUAN et al.2008

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

31. Chio, K. S., Reiss, U., Fletcher, B., and Tappel, A. L.

(1969) Peroxidation of subcellular organelles: for-

mation of lipofuscinlike fluorescent pigments, Sci-

ence, 166, 1535-1536, https://doi.org/10.1126/science.

166.3912.1535.

32. Pavshintsev, V. V., Podshivalova, L. S., Frolova, O. Y.,

Belopolskaya, M. V., Averina, O. A., Kushnir, E. A.,

Marmiy, N. V., and Lovat, M. L. (2017) Effects of mi-

tochondrial antioxidant SkQ1 on biochemical and

behavioral parameters in a Parkinsonism model in

mice, Biochemistry (Moscow), 82, 1513-1520, https://

doi.org/10.1134/S0006297917120100.

33. Konig, J., Ott, C., Hugo, M., Jung, T., Bulteau, A. L.,

Grune, T., and Hohn, A. (2017) Mitochondrial contri-

bution to lipofuscin formation, Redox Biol., 11, 673-

681, https://doi.org/10.1016/j.redox.2017.01.017.

34. Picca, A., Faitg, J., Auwerx, J., Ferrucci, L., and

D’Amico, D. (2023) Mitophagy in human health, age-

ing and disease, Nat. Metab., 5, 2047-2061, https://

doi.org/10.1038/s42255-023-00930-8.

35. Pollreisz, A., Messinger, J. D., Sloan, K. R., Mitter-

mueller, T. J., Weinhandl, A. S., Benson, E. K., Kidd,

G. J., Schmidt-Erfurth, U., and Curcio, C. A. (2018)

Visualizing melanosomes, lipofuscin, and melanoli-

pofuscin in human retinal pigment epithelium using

serial block face scanning electron microscopy, Exp.

Eye Res., 166, 131-139, https://doi.org/10.1016/j.exer.

2017.10.018.

36. Huang, D., Heath Jeffery, R. C., Aung-Htut, M. T.,

McLenachan, S., Fletcher, S., Wilton, S. D., and Chen,

F. K. (2022) Stargardt disease and progress in thera-

peutic strategies, Ophthalmic Genet., 43, 1-26, https://

doi.org/10.1080/13816810.2021.1966053.

37. Stojanovic, A., Roher, A. E., and Ball, M. J. (1994)

Quantitative analysis of lipofuscin and neurofibril-

lary tangles in the hippocampal neurons of Alzhei-

mer disease brains, Dementia, 5, 229-233, https://

doi.org/10.1159/000106728.

38. Ulfig, N., Braak, E., and Braak, H. (1989) Changes

within the basal nucleus in Parkinson’s disease, Prog.

Clin. Biol. Res., 317, 493-500.

39. Moreno-Garcia,A., Kun,A., Calero,O., Medina,M., and

Calero, M. (2018) An overview of the role of lipofus-

cin in age-related neurodegeneration, Front. Neuros-

ci., 12, 464, https://doi.org/10.3389/fnins.2018.00464.

40. Fang,Y., Taubitz,T., Tschulakow, A.V., Heiduschka,P.,

Szewczyk, G., Burnet, M., Peters, T., Biesemeier, A.,

Sarna, T., Schraermeyer, U., and Julien-Schraer-

meyer,S. (2022) Removal of RPE lipofuscin results in

rescue from retinal degeneration in a mouse model

of advanced Stargardt disease: Role of reactive ox-

ygen species, Free Radic. Biol. Med., 182, 132-149,

https://doi.org/10.1016/j.freeradbiomed.2022.02.025.

41. Kaarniranta,K., Uusitalo,H., Blasiak,J., Felszeghy, S.,

Kannan, R., Kauppinen, A., Salminen, A., Sinha, D.,

and Ferrington,D. (2020) Mechanisms of mitochondri-

al dysfunction and their impact on age-related mac-

ular degeneration, Prog. Retin. Eye Res., 79, 100858,

https://doi.org/10.1016/j.preteyeres.2020.100858.

42. Muraleva, N. A., Kozhevnikova, O. S., Zhdankina,

A. A., Stefanova, N. A., Karamysheva, T. V., Fursova,

A. Z., and Kolosova, N. G. (2014) The mitochondria-

targeted antioxidant SkQ1 restores alphaB-crystallin

expression and protects against AMD-like retinopa-

thy in OXYS rats, Cell Cycle, 13, 3499-3505, https://

doi.org/10.4161/15384101.2014.958393.

43. Novikova, Y. P., Gancharova, O. S., Eichler, O. V.,

Philippov, P. P., and Grigoryan, E. N. (2014) Preven-

tive and therapeutic effects of SkQ1-containing Viso-

mitin eye drops against light-induced retinal degener-

ation, Biochemistry (Moscow), 79, 1101-1110, https://

doi.org/10.1134/S0006297914100113.

44. Oh, M., Yeom, J., Schraermeyer, U., Julien-Schraer-

meyer, S., and Lim, Y. H. (2022) Remofuscin induces

xenobiotic detoxification via a lysosome-to-nucle-

us signaling pathway to extend the Caenorhabditis

elegans lifespan, Sci. Rep., 12, 7161, https://doi.org/

10.1038/s41598-022-11325-2.

45. Braak, E., Sandmann-Keil, D., Rub, U., Gai, W. P.,

de Vos, R. A., Steur, E. N., Arai, K., and Braak, H.

(2001) alpha-synuclein immunopositive Parkinson’s

disease-related inclusion bodies in lower brain

stem nuclei, Acta Neuropathol., 101, 195-201, https://

doi.org/10.1007/s004010000247.

46. Klemmensen, M. M., Borrowman, S. H., Pearce, C.,

Pyles, B., and Chandra, B. (2024) Mitochondrial dys-

function in neurodegenerative disorders, Neurother-

apeutics, 21, e00292, https://doi.org/10.1016/j.neurot.

2023.10.002.

47. Stefanova, N. A., Muraleva, N. A., Skulachev, V. P.,

and Kolosova, N. G. (2014) Alzheimer’s disease-like

pathology in senescence-accelerated OXYS rats can

be partially retarded with mitochondria-targeted

antioxidant SkQ1, J. Alzheimers Dis., 38, 681-694,

https://doi.org/10.3233/JAD-131034.

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