Article

ISSN 0006-2979, Biochemistry (Moscow), 2024, Vol. 89, No. 7, pp. 1300-1312 © The Author(s) 2024. This article is an open access publication.

1300

Dose-Dependent Alterations of Lysosomal Activity

and Alpha-Synuclein in Peripheral Blood Monocyte-Derived

Macrophages and SH-SY5Y Neuroblastoma Cell Line

by upon Inhibition of MTOR Protein Kinase – Assessment

of the Prospects of Parkinson’s Disease Therapy

Anastasia I. Bezrukova

1,2,a

*, Katerina S. Basharova

, Galina V. Baydakova

Ekaterina Y. Zakharova

, Sofya N.Pchelina

1,2

, and Tatiana S. Usenko

1,2

Konstantinov Petersburg Nuclear Physics Institute, National Research Centre “Kurchatov Institute”,

188300 Gatchina, Leningrad Region, Russia

Pavlov First Saint Petersburg State Medical University, 197022 Saint Petersburg, Russia

Research Center for Medical Genetics, 115522 Moscow, Russia

e-mail: bezrukova_ai@pnpi.nrcki.ru

Received March 25, 2024

Revised May 22, 2024

Accepted June 9, 2024

Abstract— To date, the molecular mechanisms of the common neurodegenerative disorder Parkinson’s disease

(PD) are unknown and, as a result, there is no neuroprotective therapy that may stop or slow down the process

of neuronal cell death. The aim of the current study was to evaluate the prospects of using the mTOR molecule

as a potential target for PD therapy due to the dose-dependent effect of mTOR kinase activity inhibition on

cellular parameters associated with, PD pathogenesis. The study used peripheral blood monocyte-derived mac-

rophages and SH-SY5Y neuroblastoma cell line. As a result, we have for the first time showed that inhibition of

mTOR by Torin1 only at a concentration of 100nM affects the level of the lysosomal enzyme glucocerebrosidase

(GCase), encoded by the GBA1 gene. Mutations in GBA1 are considered a high-risk factor for PD development.

This concentration led a decrease in pathological phosphorylated alpha-synuclein (Ser129), an increase in its sta-

ble tetrameric form with no changes in the lysosomal enzyme activities and concentrations of lysosphingolipids.

Our findings suggest that inhibition of the mTOR protein kinase could be a promising approach for developing

therapies for PD, particularly for GBA1-associated PD.

DOI: 10.1134/S0006297924070113

Keywords: Parkinson’s disease, mTOR, Torin1, alpha-synuclein, glucocerebrosidase, autophagy, lysosomal enzyme

activity, lysosphingolipids

Abbreviations: ASMase,acid sphingomyelinase; GBA1-PD, PD associated with mutations in the GBA1 gene; GCase,glucoce-

rebrosidase; GLA,alpha-galactosidase; HexSph,hexosylsphingosine; LysoGb3,lyso-globotriaosylsphingosine; LysoSM,lyso-

sphingomyelin; PD,Parkinson’s disease; sPD,sporadic PD.

* To whom correspondence should be addressed.

INTRODUCTION

Parkinson’s disease(PD) is one of the most com-

mon neurodegenerative diseases characterized by

dopaminergic neuron death in the substantia nigra

as well as alpha-synuclein accumulation and aggrega-

tion[1]. Precise molecular mechanisms underlying PD

development remain unknown. Consequently, there

are no neuroprotective drugs capable of reversing or

slowing neurodegeneration. Recent studies suggest

that the processes such as neuroinflammation, mito-

chondrial dysfunction, disrupted lipid homeostasis,

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BIOCHEMISTRY (Moscow) Vol. 89 No. 7 2024

endoplasmic reticulum stress, and impaired au-

tophagolysosomal system may be involved in PD

molecular mechanisms [2, 3]. However, altered autoph-

agy, which accounts for about half of cellular alpha-sy-

nuclein degradation, is currently recognized as a key

factor underlying PD pathogenesis [4-6]. Thus, one of

the promising approaches for PD therapy may rely on

autophagy regulation primarily via the PI3K/AKT/mTOR

pathway [7, 8]. Previously, we and others revealed that

both sporadic PD (sPD) and PD caused by the GBA1

gene mutation (GBA1-PD), one of the most common

forms of PD, are associated with impaired PI3K/AKT/

mTOR signaling [9-14]. In this regard, transcriptome

analysis of GBA1-PD patient-specific cells and a mouse

model of parkinsonism triggered by lysosomal gluco-

cerebrosidase (GCase) dysfunction allowed us to iden-

tify altered expression of genes regulated by the PI3K/

AKT/mTOR cascade [9, 10]. Mutations in the GCase-en-

coding GBA1 gene represent a high genetic risk factor

for PD development resulting in downregulated GCase

activity and level in both homozygous and heterozy-

gous carriers [15, 16]. In turn, when mTOR inhibitors

targeted the PI3K/AKT/mTOR pathway, improved al-

pha-synuclein clearance was observed in both sPD

and GBA1-PD cells and mouse models [12, 17, 18].

However, it should be noted that both mTOR hyper-

activation and hypoactivation can result in lysosome

dysfunction, leading to cell death [19]. Hence, it is cru-

cial to maintain a balance between mTOR signaling

activation and lysosomal function. This study aimed

to assess to assess a dose-dependent effect of Torin 1-

mediated mTOR inhibition on cell parameters associ-

ated with PD, primarily activity of lysosomal enzymes

and lysosphingolipid levels, autophagy level, contents

of alpha-synuclein protein and GCase. The primary pe-

ripheral blood macrophages derived from neurologi-

cally healthy individuals and the SH-SY5Y neuroblasto-

ma cell line were used in this study because these cell

types are widely utilized by us and other researchers

for screening potential new drugs for neurodegenera-

tive diseases and for investigating disease-related mo-

lecular mechanisms, particularly those associated with

GBA1-PD [20-22]. Torin 1 was chosen as an inhibitor

of mTOR kinase activity. It has been previously proven

to effectively downregulate the level of phosphorylated

alpha-synuclein protein (Ser129) and restore the func-

tioning of the autophagolysosomal system in the pa-

tient-specific cells derived from the biallelic GBA1 gene

variant carriers (Gaucher disease) as well as patients

with GBA1-PD [12, 23].

MATERIALS AND METHODS

Characteristics of study participants. Six neu-

rologically healthy individuals (2 males, 4 females,

mean age – 30.3 ± 5.9 years) observed at the consul-

tative and diagnostic center of the Pavlov First Saint

Petersburg State Medical University were enrolled

in the study.

Primary peripheral blood macrophage culture.

Previously, we described the protocol used to obtain

a primary peripheral blood macrophage culture fol-

lowed by the isolation of a whole blood mononuclear

fraction from each participant [21, 24, 25]. On day 4 of

cultivation, a selective mTOR protein kinase inhibitor

Torin1 (Abcam, USA) was added to the primary mac-

rophage culture at various concentrations (25, 50, 100,

200nM) which were chosen based on pre-assessed lev-

els of cell survival. The cultures were then incubated

for 24h.

SH-SY5Y neuroblastoma cell line culture.

An SH-SY5Y neuroblastoma cell line, courtesy of

Dr. Sci. Biol. E. V. Kaznacheeva, Institute of Cytology of

the Russian Academy of Sciences, St. Petersburg, was

cultured in DMEM medium (Biolot, Russia) supple-

mented with 10% fetal bovine serum (Biolot) and 1%

gentamicin (Biolot), for 4 days at 37°C, 5% CO

. SH-SY5Y

neuroblastoma cell line used in the study underwent

not more than seven passages and was differentiat-

ed according to a previously described protocol [26].

On day 9 of cultivation a selective mTOR protein ki-

nase inhibitor Torin1 was added to SH-SY5Y neuroblas-

toma cell line at various concentrations (25, 50, 100,

200nM) chose based on the pre-assessed cell survival

levels. The cells were then incubated for 24h. Each ex-

periment was carried out in triplicate.

Survival of the mTOR protein kinase inhibitor-

exposed primary peripheral blood macrophage

culture and SH-SY5Y neuroblastoma cell line. The

macrophages were maintained for 5 days, while the

SH-SY5Y cells were cultured for 10 days, following

the protocol described earlier. Subsequently, both cell

types were exposed to Torin 1 at various concentra-

tions (25, 50, 100, 150, 200, 250, 300, 400nM) and culti-

vated for an additional 24 h under similar conditions.

Cell survival assessment was performed as previously

described [27]. For each Torin1 concentration, experi-

ments were performed in triplicate.

Immunofluorescence-assessed autophagy level.

A primary peripheral blood macrophage culture and

SH-SY5Y neuroblastoma cell line treated with Torin1

at various concentrations based on the pre-assessed

cell survival level left untreated, were incubated with

LysoTracker-Red DND-99 (Thermo Scientific, USA) for

30 min. Next, the cells were fixed using 4% parafor-

maldehyde (Sigma-Aldrich, USA) for 30 min, washed

with phosphate-buffered saline (Rosmedbio, Russia)

and incubated in a 1% bovine serum albumin (Biolot)

solution for 30 minutes. After that, the cells were

stained with primary anti-LC3B antibodies (ABclonal,

USA; A19665; 1:500) for 60min followed by staining

BEZRUKOVA et al.1302

BIOCHEMISTRY (Moscow) Vol. 89 No. 7 2024

with fluorescent Alexa Fluor 488-conjugated secondary

antibodies (Jackson ImmunoResearch Laboratories,

USA; 1: 400) for 60min. Finally, cells were analyzed

using a Leica TCS-SP5 confocal microscope (Leica Mi-

crosystem GmbH, Germany) and Fiji software (version

2.14.0/1.54f).

Protein levels of phosphorylated mTOR, GCase,

alpha-synuclein, and LC3B assessed by Western

blotting. Total protein level was quantified by using

a Pierce BCA Protein Assay kit (Thermo Scientific, Lith-

uania). Equal protein amounts were separated by elec-

trophoresis in a polyacrylamide gel (20% SDS-PAGE–

for LC3B protein, 12% SDS-PAGE– for other proteins)

and transferred to polyvinylidene fluoride membrane

(Bio-Rad, USA). Protein level of phosphorylated mTOR

(Ser2448), GCase, alpha-synuclein (phosphorylated

(Ser129), monomeric and tetrameric forms), LC3B

in primary peripheral blood macrophages derived

from neurologically healthy individuals and SH-SY5Y

neuroblastoma cell line treated with or without an

mTOR protein kinase inhibitor at different concen-

trations were quantified using specific primary an-

tibodies (1 : 1000): Phospho-mTOR-S2448, ABclonal,

cat. # AP0094, USA; Glucosylceramidase beta (GBA),

ABclonal, cat. # A8420; Phospho-α-Synuclein (Ser129)

(D1R1R), Cell Signal, cat. # 23706, USA; anti-alpha-sy-

nuclein oligomeric, Sigma, cat. # ABN2265, USA; LC3B,

ABclonal, cat. #A19665 and secondary peroxidase-con-

jugated antibodies (goat anti-rabbit HRP conjugate,

Abcam, cat. #ab6721, UK; 1 : 5000). After that, second-

ary antibodies were added and the formed complexes

were quantified using a Clarity Western ECL Blotting

Substrate detection system (Bio-Rad). Obtained pro-

tein quantities were normalized to GAPDH reference

protein (ABclonal, cat. # AC036; 1 : 15,000). For each

protein, experiments were performed in triplicate.

Western blot data were analyzed using Fiji software

(version 2.14.0/1.54f).

Lysosomal enzyme activity and lysosphingolip-

id concentrations. Enzymatic activity of lysosomal

enzymes (GCase, alpha-galactosidase (GLA), and sphin-

gomyelinase (ASMase)) as well as concentrations of the

relevant substrates (hexasylsphingosine (HexSph) –

a mixture of glycosylsphingosine and galactosylsphin-

gosine; lysosphingomyelin (LysoSM), and lysoglo-

botriaosylsphingosine (LysoGb3)) were assessed using

high-performance liquid chromatography with tan-

dem mass spectrometry in a primary peripheral blood

macrophage culture derived from the neurologically

healthy individuals and SH-SY5Y neuroblastoma cell

line treated with Torin1 at various concentrations or

untreated, following protocols previously described [24,

28-30]. All experiments were performed in triplicate.

Statistical analysis. Statistical data processing

was conducted using pre-installed R packages (ver-

sion 4.3.2) (https://cran.r-project.org/bin/windows/

base/). The normality of distribution in the obtained

data was assessed using the Shapiro–Wilk test.

Inter-group differences were evaluated using the

paired Wilcoxon test. A significance level of p < 0.05

was considered statistically significant. Clinical char-

acteristics of the study participants are presented as

mean ±standard deviation of the mean, experimental

values– as median (min-max).

RESULTS

Currently, therapeutic targets for PD treatment

are being extensively searched for worldwide. Here,

we investigated the dose-dependent effect of mTOR in-

hibitor on cell parameters known to be associated with

PDpathogenesis.

The degree of mTOR inhibition by Torin 1 was

evaluated in primary peripheral blood macrophage

cultures derived from neurologically healthy individ-

uals and in the SH-SY5Y neuroblastoma cell line. Cell

survival was evaluated in both the primary peripheral

blood macrophage culture and in the SH-SY5Y neuro-

blastoma cell line following treatment with the mTOR

protein kinase inhibitor Torin1, aiming to determine

its effective concentration range. Based on these as-

sessments, Torin1 concentrations (25, 50, 100, 200nM)

that reduced cell viability by no more than 80% in both

cell models were selected for subsequent experiments.

The dose-dependent inhibition of mTOR by To-

rin 1 was assessed by measuring the decrease in rela-

tive levels of phosphorylated mTOR protein (Ser2448)

in the primary peripheral blood macrophage culture

derived from neurologically healthy individuals and

in the SH-SY5Y neuroblastoma cell line was assessed

by analyzing decline in the relative level of phosphor-

ylated mTOR protein (Ser2448) (Fig. 1, a and b). In

the primary macrophage culture, Torin1 showed no

significant decrease in phosphorylated mTOR protein

levels at any of the tested concentrations compared

to untreated cells (Fig.1c). Conversely, in the SH-SY5Y

neuroblastoma cell line, Torin1 significantly reduced

phosphorylated mTOR protein levels at doses of 100

and 200 nM compared to the control (p < 0.01 and

p < 0.001, respectively; Fig.1d).

Torin 1-mediated dose-dependent protein ki-

nase mTOR inhibition affects autophagy, lysosom-

al activity, and GCase protein level in the prima-

ry peripheral blood macrophage culture derived

from the neurologically healthy individuals and in

SH-SY5Y neuroblastoma cell line. The level of auto-

phagy in the primary peripheral blood macrophage

culture derived from neurologically healthy individ-

uals and in SH-SY5Y neuroblastoma cell line was as-

sessed by analyzing relative level of the hallmark au-

tophagy marker LC3B-II protein along with fluorescent

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BIOCHEMISTRY (Moscow) Vol. 89 No. 7 2024

Fig. 1. Dose-dependent Torin1-mediated effect of mTOR inhibition on the protein level of phosphorylated mTOR (Ser2448).

a) Western blot analysis for mTOR phosphorylated (Ser2448) protein level in primary peripheral blood macrophages;

b)Western blot analysis for mTOR phosphorylated (Ser2448) protein level in tSH-SY5Y neuroblastoma cell line. c)Relative level

of mTOR phosphorylated (Ser2448) protein in primary peripheral blood macrophages (n=6, where n is the number of inde-

pendent samples); d)relative level of mTOR phosphorylated (Ser2448) protein level in SH-SY5Y neuroblastoma cell line (n=5,

where n is the number of independent cell line samples). **p<0.01; ***p<0.001; ****p<0.0001.

Fig. 2. Torin1-mediated effect of dose-dependent mTOR inhibition on the levels of LC3B-II and GCase proteins. a)Western blot

analysis for LC3B-II and GCase proteins in primary peripheral blood macrophages; b)Western blot analysis for LC3B-II and

GCase proteins in SH-SY5Y neuroblastoma cell line. c)Relative level of LC3B-II protein in primary peripheral blood macro-

phages (n=6, where n is the number of independent samples); d)relative level of GCase protein in primary peripheral blood

macrophages (n=6, where n is the number of independent samples); e)relative level of LC3B-II protein in SH-SY5Y neuroblas-

toma cell line (n=5, where n is the number of independent cell lines); f)relative level of GCase protein in SH-SY5Y neuroblas-

toma cell line (n=5, where n is the number of independent cell line samples). *p<0.05; **p<0.01; ***p<0.01.

staining of LC3B protein and lysosome assuming that

colocalization of LC3B with lysosomes could indi-

cate autophagosome-lysosome fusion (Fig.2,a andb;

Fig.3,a andb) [31].

In primary peripheral blood macrophages de-

rived from neurologically healthy individuals, treat-

ment with Torin 1 at a concentration of 200 nM re-

sulted in a significantly lower relative level of LC3B-II

protein compared to cultures exposed to 100 nM

Torin 1 and untreated controls (p < 0.05; Fig. 2c).

Conversely, exposure to lower doses of Torin 1 (25,

50, and 100 nM) tended to insignificantly elevate

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Fig. 3. Colocalization of LC3B with lysosomes upon Torin1-mediated dose-dependent mTOR inhibition. a)LC3B protein and

lysosome immunofluorescent staining in primary peripheral blood macrophages, 10µm; b)LC3B protein and lysosome im-

munofluorescent staining in SH-SY5Y neuroblastoma cell line, 10µm. c)colocalization of LC3B with lysosomes in primary pe-

ripheral blood macrophages (n=6, where n is the number of independent samples); d)colocalization of LC3B with lysosomes

in SH-SY5Y neuroblastoma cell line (n=5, where n is the number of independent cell line samples). *p<0.05; **p<0.01;

****p<0.0001; ns,not significant.

the LC3B-II level (p > 0.05; Fig. 2c). In contrast, in

SH-SY5Y neuroblastoma cell line treated with Torin 1

at any dose did not significantly alter the LC3B-II pro-

tein level (Fig.2e).

Furthermore, treatment of the primary periph-

eral blood macrophage culture derived from the

neurologically healthy individuals with Torin 1 at all

concentrations resulted in the higher degree of LC3B

colocalization with lysosomes compared to intact cells

(p < 0.05; Fig.3c). Similarly, in the SH-SY5Y neuroblas-

toma cell line, exposure to Torin1 at doses of 25 and

50 nM significantly increased LC3B colocalization

with lysosomes compared to control cells (p < 0.01 and

p < 0.0001, respectively; Fig.3d).

Activity of the lysosomal enzymes (GCase, GLA,

ASMase) and level of lysosphingolipids (HexSph,

LysoGb3, LysoSM) involved in ceramide metabolism

and associated with PD pathogenesis [28-30, 32, 33],

were also assessed in primary peripheral blood mac-

rophages derived from neurologically healthy individ-

uals and in SH-SY5Y neuroblastoma cell line treated

with autophagy inducer Torin 1 (Fig. 4). Interesting-

ly, only at the 100-nM Torin 1 dose was there no sig-

nificant effect on the lysosomal enzyme activity and

lysosphingolipid level compared to the untreated cells

(p > 0.05).

Moreover, it is worth mentioning that higher rel-

ative GCase protein level was discovered in primary

peripheral blood macrophages derived from the neu-

rologically healthy individuals (Fig. 2d) and in SH-

SY5Y neuroblastoma cell line (Fig. 2f) after exposure

to Torin1 at a concentration of 100nM (p > 0.05 and

p < 0.05, respectively) compared to the untreated cells.

Torin 1-mediated dose-dependent mTOR in-

hibition affects the levels alpha-synuclein forms

(monomeric, phosphorylated (Ser129), tetramer-

ic) in the SH-SY5Y neuroblastoma cell line. In this

study relative levels of different alpha-synuclein pro-

tein forms were assessed in SH-SY5Y neuroblastoma

cell line (Fig.5), but not in primary peripheral blood

macrophages due to limitations in the sensitivity of

the alpha-synuclein quantification assay. Treatment

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Fig. 4. Activity of lysosomal enzymes (GCase, GLA, ASMase) and levels of the relevant lysosphingolipid (HexSph, LysoGb3,

LysoSM) upon Torin1-mediated dose-dependent mTOR inhibition in primary peripheral blood macrophages (a and b; n=6,

where n is the number of independent samples) and in SH-SY5Y neuroblastoma cell line (c and d; n=5, where n is the number

of independent cell line samples). *p<0.05; **p<0.01; ns,not significant.

of SH-SY5Y neuroblastoma cell line with Torin 1 at

doses of 25, 100, and 200nM resulted in a significant

downmodulation of phosphorylated alpha-synuclein

protein (Ser129) compared to untreated cells (p<0.01;

Fig. 5, a, b). Additionally, for the first time, elevated

levels of tetrameric alpha-synuclein were detected in

this cell line after treated with Torin1 at concentra-

tions of 50, 100, and 200 nM compared to untreated

cells (p < 0.01; Fig. 5, a, d). In contrast, the level of

monomeric alpha-synuclein protein remained unal-

tered in SH-SY5Y neuroblastoma cell line treated with

Torin1 (p>0.05; Fig.5,a andc).

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Fig. 5. Torin1-mediated effect of dose-dependent mTOR inhibition on the levels of alpha-synuclein protein forms (monomeric,

phosphorylated (Ser129), tetrameric) in SH-SY5Y neuroblastoma cell line (n=5, where n is the number of independent cell line

samples). a)Western blot data for alpha-synuclein (monomeric, phosphorylated (Ser129), tetrameric) forms. b)Relative level

of phosphorylated (Ser129) alpha-synuclein protein; c)relative level of monomeric alpha-synuclein protein; d)relative level

oftetrameric alpha-synuclein protein; **p<0.01.

DISCUSSION

Currently, no neuroprotective therapy capable

of slowing down or stopping PD progression is avail-

able. The drugs used today only provide symptomat-

ic effects. Hence, there is an urgent need to identify

novel therapeutic targets for PD treatment, that may

downregulate alpha-synuclein protein accumulation

and reduce neuronal death. Recent evidence suggests a

key role of lysosomal dysfunction and autophagy in PD

pathogenesis [34, 35]. In this regard, proteins involved

in or regulating autophagy events may be among the

promising targets for developing PD therapy [36-38].

Here, we investigated the dose-dependent inhib-

itory effect of mTOR protein kinase essential for au-

tophagy regulation on cellular parameters associated

with PD pathogenesis Our findings revealed that mTOR

inhibition after exposure to varying Torin1 doses af-

fects activity of the lysosomal hydrolases and level of

the relevant substrates, lysosphingolipids, results in an

increase in the GCase protein level in the primary pe-

ripheral blood macrophage culture and SH-SY5Y neu-

roblastoma cell line, as well as downregulates the level

of phosphorylated alpha-synuclein (Ser129), while the

level of its tetrameric form in SH-SY5Y neuroblastoma

cell line exposed to autophagy inducer remains in-

creased.

mTOR is an intracellular serine-threonine pro-

tein kinase comprising a subunit of the multimolecu-

lar signaling complexes mTORC1 and mTORC2, which,

in turn, are components of PI3K/AKT/mTOR-axis. This

axis plays a crucial role in controlling signal transduc-

tion and various biological events, such as cell prolif-

eration, apoptosis, metabolism, angiogenesis, inflam-

mation, as well as maintaining lysosomal function and

autophagy [39, 40]. Alterations in the PI3K/AKT/mTOR

signaling cascade that affects autophagy may lead to

protein aggregate deposition and cell death in various

proteinopathies including PD [41, 42]. In particular,

changes in phosphorylated mTOR protein levels have

been observed in the substantia nigra of mice with

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-

induced parkinsonism as well as in SH-SY5Y neuro-

blastoma cell line carrying missense-mutation A53T

in the SNCA gene (the most common mutation in the

SNCA gene causing early-onset autosomal-dominant

PD. These changes increased mTOR/P70S6K signaling

and disrupted autophagy, promoting further A53T

alpha-synuclein aggregation [43-45]. Moreover, high-

er mTOR levels were found in the temporal cortex of

brain autopsies of the patients suffering from demen-

tia with Lewy bodies characterized by alpha-synuclein

protein accumulation similar to PD patients [46]. Pro-

teomic and Western blot analysis also showed elevated

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BIOCHEMISTRY (Moscow) Vol. 89 No. 7 2024

phosphorylated mTOR protein levels in neurons dif-

ferentiated from the induced pluripotent stem cells

(iPSCs) obtained from the patients with GBA1-PD [11,

12]. Thus, mTOR may be a promising target for devel-

oping GBA1-PD therapy.

However, the data on inhibited mTOR protein ki-

nase activity remain controversial, as both neuropro-

tective and neurotoxic effects were observed in various

PD models. This variability could be related to mTOR

imbalance, particularly due to the inhibitor doses used

to elicit cell death [8, 19]. Currently, inhibitors of mTOR

kinase activity are divided into four classes: antibiot-

ic allosteric inhibitors selectively inhibiting mTORC1

complex (first generation inhibitors, rapamycin and

its paralogs); ATP-competitive inhibitors capable of in-

hibiting both mTORC1 and mTORC2 complexes (second

generation inhibitors, Ku-0063794, WYE-3541, Torin1,

etc.); dual mTOR/PI3K inhibitors (second generation in-

hibitors, GNE477, NVP-BEZ235, etc.); other new inhibi-

tors (third generation inhibitors, P529, RapaLinks, etc.)

[47]. Currently, direct and indirect inhibitors of mTOR

kinase activity are undergoing clinical trials for assess-

ing therapeutic effectiveness in diverse neurodegener-

ative diseases and proteinopathies [PD (NCT05357989,

NCT05781711); Huntington’s disease (NCT04826692);

Alzheimer’s disease (NCT03748706, NCT04511416);

amyotrophic lateral sclerosis (NCT04577404)]. In our

study, we chose the direct mTOR inhibitor Torin 1,

which is not yet in clinical trials for neurodegenerative

diseases. However, studies using mouse models of in-

duced parkinsonism and cell lines from biallelic GBA1

gene variant carriers (Gaucher disease) and patients

with GBA1-PD have demonstrated that Torin 1 is effec-

tive with regard to the hallmark biochemical charac-

teristics related to PD pathogenesis. These include the

downregulation of phosphorylated alpha-synuclein

(Ser129), restoration of autophagolysosomal system

function and decrease of neurodegeneration [12, 23,

48], which allows considering it as a promising agent

for PD therapy particularly for GBA1-PD.

In our study, the dose-dependent effect of mTOR

kinase activity inhibition on lysosome functioning

was discovered in cell models. This was evidenced by

changes in level of essential autophagy marker LC3B-II,

GCase protein, as well as altered activity of lysosomal

hydrolases and levels of lysosphingolipids. Exposure

of the primary peripheral blood macrophage culture

to the mTOR inhibitor Torin1 at doses of 25, 50, and

100nM, and exposure of the SH-SY5Y neuroblastoma

cell line to Torin 1 at doses of 25, 50, 100, and 200nM,

resulted in a slight increase in the relative level of

LC3B-II protein. This was accompanied by an increased

degree of LC3B protein colocalization with lysosomes

in primary peripheral blood macrophage culture at all

tested doses of Torin1, and in SH-SY5Y neuroblasto-

ma cell line at Torin1 concentrations of 25 and 50nM.

Previously, it was shown that inhibition of the mTOR

kinase activity in mouse models of MPTP-induced par-

kinsonism and in the neuroblastoma cell line treated

with toxic 1-methyl-4-phenylpyridinium (MPP+) cations

results in altered LC3 protein level and upregulated

expression level of the main lysosome marker LAMP1,

both at mRNA and protein levels [43, 49]. Torin1-treat-

ed neurons derived from iPSC of GBA1-PD patients

were also shown to have elevated LC3B-II protein level

after exposure to chloroquine that inhibits autophago-

somal degradation [12].

Activity of lysosomal hydrolases was assessed

by analyzing the enzymes GCase, GLA, ASMase, as

well as the level of sphingolipids HexSph, LysoGb3,

LysoSM involved in ceramide metabolism, disruption

of which is associated with PD pathogenesis [28-30, 32,

33]. Thelysosomal enzymes GCase, GLA, ASMase are

encoded by the GBA1, GLA, SMPD1 genes, respective-

ly. Mutations in these genes result in lysosomal stor-

age diseases characterized by lower enzyme activity

and accumulation of lysosphingolipids and also are

considered as risk factors for PD development [50].

Wediscovered that activity of these enzymes decreas-

es at all Torin 1 doses in the primary peripheral blood

macrophage culture and SH-SY5Y neuroblastoma cell

line, except for the 100-nM concentration, when the

enzyme activities and substrate levels remained un-

changed similar to those in the cells without the au-

tophagy inducer. Hence, it suggests that exposure to

Torin1 at doses of 25, 50, and 200nM could cause im-

balance in cellular processes particularly affecting ly-

sosome functioning, which may eventually trigger cell

death [8, 19]. Apart from this, we assessed for the first

time the effect of Torin1 on the relative GCase enzyme

level, activity of which is decreased in GBA1-PD and

sPD [51-53]. We found that the relative GCase protein

level increased in the primary peripheral blood mac-

rophage culture and in the SH-SY5Y neuroblastoma

cell line after exposure to Torin 1 at concentrations

of 25 and 100 nM, and 50, 100, and 200 nM, respec-

tively. Previously, another inhibitor, RTB101, a dual

mTOR/PI3K inhibitor, was shown to decrease the lev-

el of glucosylceramide, an essential GCase substrate,

both in blood and cerebrospinal fluid of GBA1-PD

patients [54].

It is interesting to note that mTOR-dependent au-

tophagy inducers were also previously shown to af-

fect the level of alpha-synuclein protein that plays an

essential role in PD pathogenesis [12, 17, 18]. Various

alpha-synuclein forms exist in cells. The phosphory-

lated alpha-synuclein (Ser129) promotes its aggrega-

tion and exhibits the highest cell toxicity. This form is

most often found in pathological inclusions in PD [55].

In contrast, the tetrameric form of alpha-synuclein is

considered physiological and more stable, whereas

the monomeric form is prone to forming neurotoxic

BEZRUKOVA et al.1308

BIOCHEMISTRY (Moscow) Vol. 89 No. 7 2024

oligomers [56]. In this study, we observed decline

inthe level of phosphorylated alpha-synuclein protein

(Ser129) without changes in the monomeric form in

SH-SY5Y neuroblastoma cell line treated with Torin1

at doses of 25, 100, and 200nM compared to the intact

cells. Previously, a dose-dependent reduce in phosphor-

ylated (Ser129) and monomeric alpha-synuclein forms

was shown in cell and animal models overexpressing

alpha-synuclein after mTOR inhibition [17, 18, 57, 58].

In addition, neurons derived from iPSC-of GBA1-PD pa-

tients showed a slight downregulation of phosphory-

lated alpha-synuclein (Ser129) after Torin1 exposure

[12]. The mechanism underlying this decline in the

level of phosphorylated alpha-synuclein protein due to

Torin1-mediated mTOR inhibition remains unknown.

However, suppression mTOR activity by metformin,

asecond-generation inhibitor, like Torin1, was shown

to activate protein phosphatase 2A (PP2A) that may

dephosphorylate, alpha-synuclein, in primary murine

hippocampal neuron culture [18]. At the same time,

we were the first to demonstrate an increased level

of tetrameric alpha-synuclein form in SH-SY5Y neuro-

blastoma cell line upon exposure to Torin1 at all doses

examined compared to the control cells.

The current study has several limitations. Thepri-

mary peripheral blood macrophage culture was ob-

tained from neurologically healthy individuals and

SH-SY5Y neuroblastoma cell line was used without

induced parkinsonism and GCase enzyme dysfunc-

tion. Further studies with patient-specific cells derived

from patients with PD, particularly GBA1-PD, as well

as the cell lines that accurately model the disease,

are required.

CONCLUSION

Despite previous demonstrations of the neuro-

protective properties of Torin 1 in animal and cell

models, our study using primary peripheral blood

macrophage culture derived from the neurologically

healthy individuals and SH-SY5Y neuroblastoma cell

line provides deeper insights into the effect of mTOR

inhibitor Torin 1 on altering cell parameters, which

may be related to PD. In this regard, we demonstrat-

ed that exposure to Torin 1 at different doses may

decrease lysosomal hydrolase activities and elevate

lysosphingolipid concentrations, which could be fatal

to the cells. However, optimal doses of Torin 1 may

induce autophagy, increase GCase protein level, de-

crease phosphorylated alpha-synuclein (Ser129) level,

and increase its tetrameric form without significantly

affecting lysosomal hydrolase activities and lysosphin-

golipid levels. Torin1-mediated mTOR protein kinase

inhibition shows promise for developing therapies for

PD, particularly for the GBA1-PD. Determining thera-

peutic dosages of Torin1 will be crucial. Such studies

are very important for expanding our understanding

of the molecular mechanisms of various chemicals and

assessing their potential clinical applications.

Contributions. T.S.U. conceptualized and super-

vised the study; A.I.B., K.S.B., and G.V.B. conducted the

experiments; A.I.B., E.Y.Z., S.N.P., and T.S.U. discussed

the study data; A.I.B. wrote the manuscript; T.S.U. ed-

ited the manuscript.

Funding. The study was financially supported by

the Russian Science Foundation (grant no.24-25-00212).

Ethics declarations. All the procedures carried

out in the research with participation of humans

were in accordance with the ethical standards of the

National Research Ethics Committee and with the Hel-

sinki Declaration of 1964 and its subsequent chang-

es or with comparable ethics standards. Informed

voluntary consent was obtained from every partic-

ipant of the study. The study was approved by the

ethics committee of the Pavlov First Saint Petersburg

State Medical University (protocol no. 275 dated of

09/04/2023). The authors declare that they no conflicts

of interest.

Open access. This article is licensed under a Cre-

ative Commons Attribution 4.0 International License,

which permits use, sharing, adaptation, distribution,

and reproduction in any medium or format, as long

as you give appropriate credit to the original author(s)

and the source, provide a link to the Creative Commons

license, and indicate if changes were made. Theimages

or other third-party material in this article are includ-

ed in the article’s Creative Commons license, unless

indicated otherwise in a credit line to the material.

Ifmaterial is not included in the article’s Creative Com-

mons license and your intended use is not permitted

by statutory regulation or exceeds the permitted use,

you will need to obtain permission directly from the

http://creativecommons.org/licenses/by/4.0/.

REFERENCES

1. Surmeier, D.J. (2018) Determinants of dopaminergic

neuron loss in Parkinson’s disease, FEBSJ., 285, 3657-

3668, https://doi.org/10.1111/febs.14607.

2. Morris, H.R., Spillantini, M. G., Sue, C. M., and Wil-

liams-Gray, C. H. (2024) The pathogenesis of Par-

kinson’s disease, Lancet, 403, 293-304, https://

doi.org/10.1016/S0140-6736(23)01478-2.

3. Kouli, A, Torsney, K. M., and Kuan, W.L. (2018) Par-

kinson’s disease: etiology, neuropathology, and

pathogenesis, in Parkinson’s Disease: Pathogene-

sis and Clinical Aspects (Stoker, T. B., and Green-

land, J. C., eds) Brisbane (AU), Codon Publications,

mTOR FOR TARGETED THERAPY OF PARKINSON’S DISEASE 1309

BIOCHEMISTRY (Moscow) Vol. 89 No. 7 2024

Chap. 1, https://doi.org/10.15586/codonpublications.

parkinsonsdisease.2018.ch1.

4. Lynch-Day, M. A., Mao, K., Wang, K., Zhao, M., and

Klionsky, D. J. (2012) The role of autophagy in Par-

kinson’s disease, Cold Spring Harb. Perspect. Med., 2,

a009357, https://doi.org/10.1101/cshperspect.a009357.

5. Hou, X., Watzlawik, J. O., Fiesel, F. C., and Spring-

er, W. (2020) Autophagy in Parkinson’s disease,

J. Mol. Biol., 432, 2651-2672, https://doi.org/10.1016/

j.jmb.2020.01.037.

6. Nechushtai, L., Frenkel, D., and Pinkas-Kramar-

ski, R. (2023) Autophagy in Parkinson’s disease,

Biomolecules, 13, 1435, https://doi.org/10.3390/

biom13101435.

7. Khan, M. R., Yin, X., Kang, S.-U., Mitra,J., Wang, H.,

Ryu,T., Brahmachari,S., Karuppagounder, S.S., Kimu-

ra,Y., Jhaldiyal,A., Kim, H.H., Gu,H., Chen,R., Red-

ding-Ochoa,J., Troncoso,J., Na, C.H., Ha,T., Dawson,

V.L., and Dawson, T.M. (2024) Enhanced mTORC1 sig-

naling and protein synthesis in pathologic α-synucle-

in cellular and animal models of Parkinson’s disease,

Sci. Transl. Med., 15, eadd0499, https://doi.org/10.1126/

scitranslmed.add0499.

8. Lan,A., Chen,J., Zhao, Y., Chai,Z., and Hu, Y. (2017)

mTOR signaling in Parkinson’s disease, Neuro-

mol. Med., 19, 1-10, https://doi.org/10.1007/s12017-

016-8417-7.

9. Usenko,T., Bezrukova,A., Rudenok, M.M., Basharo-

va,K., Shadrina, M.I., Slominsky, P.A., Zakharova,E.,

and Pchelina,S. (2023) Whole transcriptome analysis

of substantia nigra in mice with MPTP-induced par-

kinsonism bearing defective glucocerebrosidase activ-

ity, Int.J. Mol. Sci., 24, 12164, https://doi.org/10.3390/

ijms241512164.

10. Usenko, T., Bezrukova, A., Basharova, K., Pantele-

eva, A., Nikolaev, M., Kopytova, A., Miliukhina, I.,

Emelyanov,A., Zakharova,E., and Pchelina,S. (2021)

Comparative transcriptome analysis in monocyte-de-

rived macrophages of asymptomatic GBA mutation

carriers and patients with GBA-associated Parkinson’s

disease, Genes (Basel), 12, 1545, https://doi.org/10.3390/

genes12101545.

11. Bogetofte, H., Ryan, B. J., Jensen, P., Schmidt, S. I.,

Vergoossen, D. L. E., Barnkob, M. B., Kiani, L. N.,

Chughtai, U., Heon-Roberts, R., Caiazza, M. C., Mc-

Guinness, W., Márquez-Gómez, R., Vowles, J., Bunn,

F.S., Brandes,J., Kilfeather,P., Connor, J.P., Fernandes,

H.J.R., Caffrey, T.M., Meyer,M., Cowley, S.A., Larsen,

M. R., and Wade-Martins, R. (2023) Post-translation-

al proteomics platform identifies neurite outgrowth

impairments in Parkinson’s disease GBA-N370S

dopamine neurons, Cell Rep., 42, 112180, https://

doi.org/10.1016/j.celrep.2023.112180.

12. Mubariz, F., Saadin, A., Lingenfelter, N., Sarkar, C.,

Banerjee, A., Lipinski, M. M., and Awad, O. (2023)

Deregulation of mTORC1–TFEB axis in human IPSC

model of GBA1-associated Parkinson’s disease,

Front. Neurosci., 17, 1152503, https://doi.org/10.3389/

fnins.2023.1152503.

13. Yu,L., Hu,X., Xu,R., Zhao,Y., Xiong,L., Ai,J., Wang,X.,

Chen, X., Ba, Y., Xing, Z., Guo,C., Mi, S., and Wu, X.

(2024) Piperine promotes PI3K/AKT/mTOR-mediated

gut-brain autophagy to degrade α-synuclein in Par-

kinson’s disease rats, J.Ethnopharmacol., 322, 117628,

https://doi.org/10.1016/j.jep.2023.117628.

14. Zhang, G., Yin, L., Luo, Z., Chen, X., He, Y., Yu, X.,

Wang,M., Tian,F., and Luo,H. (2021) Effects and po-

tential mechanisms of rapamycin on MPTP-induced

acute Parkinson’s disease in mice, Ann. Palliat. Med.,

10, 2889-2897, https://doi.org/10.21037/apm-20-1096.

15. Sidransky, E., and Lopez, G. (2012) The link be-

tween the GBA gene and parkinsonism, Lancet Neu-

rol., 11, 986-998, https://doi.org/10.1016/S1474-4422

(12)70190-4.

16. Emelyanov, A. K., Usenko, T. S., Tesson, C., Senkev-

ich, K. A., Nikolaev, M. A., Miliukhina, I. V., Kopy-

tova, A. E., Timofeeva, A. A., Yakimovsky, A. F., Les-

age,S., Brice,A., and Pchelina, S.N. (2018) Mutation

analysis of Parkinson’s disease genes in a Russian

data set, Neurobiol. Aging, 71, 267.e7-267.e10, https:/

/doi.org/10.1016/j.neurobiolaging.2018.06.027.

17. Xu,J., Ao, Y.-L., Huang,C., Song,X., Zhang,G., Cui,W.,

Wang, Y., Zhang, X. Q., and Zhang,Z. (2022) Harmol

promotes α-synuclein degradation and improves

motor impairment in Parkinson’s models via reg-

ulating autophagy-lysosome pathway, NPJ Parkin-

sons Dis., 8, 100, https://doi.org/10.1038/s41531-

022-00361-4.

18. Pérez-Revuelta, B. I., Hettich, M. M., Ciociaro, A.,

Rotermund,C., Kahle, P.J., Krauss,S., and Di Monte,

D.A. (2014) Metformin lowers Ser-129 phosphorylat-

ed α-synuclein levels via mTOR-dependent protein

phosphatase2A activation, Cell Death Dis., 5, e1209,

https://doi.org/10.1038/cddis.2014.175.

19. Zhu, Z., Yang, C., Iyaswamy, A., Krishnamoorthi, S.,

Sreenivasmurthy, S. G., Liu,J., Wang,Z., Tong, B.C.,

Song, J., Lu, J., Cheung, K. H., and Li, M. (2019) Bal-

ancing mTOR signaling and autophagy in the treat-

ment of Parkinson’s disease, Int.J. Mol. Sci., 20, 728,

https://doi.org/10.3390/ijms20030728.

20. Xicoy, H., Wieringa, B., and Martens, G. J. M. (2017)

TheSH-SY5Y cell line in Parkinson’s disease research:

a systematic review, Mol. Neurodegener., 12, 10,

https://doi.org/10.1186/s13024-017-0149-0.

21. Kopytova, A.E., Rychkov, G.N., Nikolaev, M.A., Bay-

dakova, G.V., Cheblokov, A.A., Senkevich, K.A., Bog-

danova, D.A., Bolshakova, O.I., Miliukhina, I.V., Bez-

rukikh, V.A., Salogub, G.N., Sarantseva, S.V., Usenko,

T.C., Zakharova, E.Y., Emelyanov, A.K., and Pchelina,

S. N. (2021) Ambroxol increases glucocerebrosidase

(GCase) activity and restores GCase translocation in

primary patient-derived macrophages in Gaucher dis-

BEZRUKOVA et al.1310

BIOCHEMISTRY (Moscow) Vol. 89 No. 7 2024

ease and parkinsonism, Parkinsonism Relat. Disord.,

84, 112-121, https://doi.org/10.1016/j.parkreldis.

2021.02.003.

22. Aflaki,E., Stubblefield, B.K., Maniwang,E., Lopez,G.,

Moaven, N., Goldin, E., Marugan, J., Patnaik, S.,

Dutra, A., Southall, N., Zheng, W., Tayebi, N., and

Sidransky, E. (2014) Macrophage models of Gauch-

er disease for evaluating disease pathogenesis and

candidate drugs, Sci. Transl. Med., 6, 240ra73, https://

doi.org/10.1126/scitranslmed.3008659.

23. Brown, R. A., Voit, A., Srikanth, M. P., Thayer, J. A.,

Kingsbury, T.J., Jacobson, M.A., Lipinski, M.M., Feld-

man, R.A., and Awad,O. (2019) mTOR hyperactivity

mediates lysosomal dysfunction in Gaucher’s disease

IPSC-neuronal cells, Dis. Model Mech., 12, dmm038596,

https://doi.org/10.1242/dmm.038596.

24. Usenko, T. S., Basharova, K. S., Bezrukova, A. I., Ni-

kolaev, M. A., Milyukhina, I. V., Baidakova, G. V.,

Zakharova, E.Yu., and Pchelina, S.N. (2022) Selective

inhibition of LRRK2 kinase activity for treatment of

Parkinson’s disease [in Russian], Meditsinskaya ge-

netika, 21, 26-29, https://doi.org/10.25557/2073-7998.

2022.12.26-29.

25. Kopytova, A.E., Rychkov, G.N., Cheblokov, A.A., Grig-

or’eva, E.V., Nikolaev, M.A., Yarkova, E.S., Sorogina,

D.A., Ibatullin, F. M., Baydakova, G.V., Izyumchen-

ko, A.D., Bogdanova, D.A., Boitsov, V. M., Rybakov,

A.V., Miliukhina, I.V., Bezrukikh, V.A., Salogub, G.N.,

Zakharova, E. Y., Pchelina, S. N., and Emelyanov,

A. K. (2023) Potential binding sites of pharmacolog-

ical chaperone NCGC00241607 on mutant beta-glu-

cocerebrosidase and its efficacy on patient-derived

cell cultures in Gaucher and Parkinson’s disease,

Int. J. Mol. Sci., 24, 9105, https://doi.org/10.3390/

ijms24109105.

26. Norradee,C., Khwanraj,K., Balit, T., and Dharmasa-

roja,P. (2023) Evaluation of the combination of met-

formin and rapamycin in an MPP

-treated SH-SY5Y

model of Parkinson’s disease, Adv. Pharmacol. Pharm.

Sci., https://doi.org/10.1155/2023/3830861.

27. Martínez, M.-A., Rodríguez, J.-L., Lopez-Torres, B.,

Martínez,M., Martínez-Larrañaga, M.-R., Maximilia-

no, J. E., Anadón, A., and Ares, I. (2020) Use of hu-

man neuroblastoma SH-SY5Y cells to evaluate gly-

phosate-induced effects on oxidative stress, neuro-

nal development and cell death signaling pathways,

Environ. Int., 135, 105414, https://doi.org/10.1016/

j.envint.2019.105414.

28. Pchelina, S., Baydakova, G., Nikolaev, M., Senkev-

ich, K., Emelyanov, A., Kopytova, A., Miliukhina, I.,

Yakimovskii,A., Timofeeva,A., Berkovich,O., Fedoto-

va,E., Illarioshkin,S., and Zakharova,E. (2018) Blood

lysosphingolipids accumulation in patients with Par-

kinson’s disease with glucocerebrosidase1 mutations,

Mov. Disord., 33, 1325-1330, https://doi.org/10.1002/

mds.27393.

29. Usenko, T.S., Senkevich, K.A., Bezrukova, A.I., Bayda-

kova, G.V., Basharova, K.S., Zhuravlev, A.S., Grache-

va, E.V., Kudrevatykh, A.V., Miliukhina, I.V., Krasa-

kov, I.V., Khublarova, L.A., Fursova, I.V., Zakharov,

D. V., Timofeeva, A. A., Irishina, Y. A., Palchikova,

E.I., Zalutskaya, N.M., Emelyanov, A.K., Zakharova,

E.Y., and Pchelina, S.N. (2022) Impaired sphingolip-

id hydrolase activities in dementia with Lewy bod-

ies and multiple system atrophy, Mol. Neurobiol., 59,

2277-2287, https://doi.org/10.1007/s12035-021-02688-0.

30. Usenko, T., Bezrukova, A., Basharova, K., Baydako-

va, G., Shagimardanova, E., Blatt, N., Rizvanov, A.,

Limankin, O., Novitskiy, M., Shnayder, N., Izyum-

chenko,A., Nikolaev,M., Zabotina,A., Lavrinova,A.,

Kulabukhova,D., Nasyrova,R., Palchikova,E., Zaluts-

kaya,N., Miliukhina, I., Barbitoff,Y., Glotov,O., Glo-

tov,A., Taraskina,A., Neznanov,N., Zakharova,E., and

Pchelina,S. (2024) Altered sphingolipid hydrolase ac-

tivities and alpha-synuclein level in late-onset schizo-

phrenia, Metabolites, 14, 30, https://doi.org/10.3390/

metabo14010030.

31. Tsukuba, T., Yanagawa, M., Kadowaki, T., Takii, R.,

Okamoto, Y., and Yamamoto, K. (2013) Cathepsin e

deficiency impairs autophagic proteolysis in macro-

phages, PLoS One, 8, e82415, https://doi.org/10.1371/

journal.pone.0082415.

32. Usenko, T.S., Senkevich, K.A., Basharova, K.S., Bezru-

kova, A.I., Baydakova, G.V., Tyurin, A.A., Beletskaya,

M.V., Kulabukhova, D.G., Grunina, M.N., Emelyanov,

A.K., Miliukhina, I. V., Timofeeva, A. A., Zakharova,

E. Y., and Pchelina, S. N. (2023) LRRK2 exonic vari-

ants are associated with lysosomal hydrolase activi-

ties and lysosphingolipid alterations in Parkinson’s

disease, Gene, 882, 147639, https://doi.org/10.1016/

j.gene.2023.147639.

33. Pchelina, S., Emelyanov, A., Baydakova, G., Ando-

skin, P., Senkevich, K., Nikolaev, M., Miliukhina, I.,

Yakimovskii,A., Timofeeva,A., Fedotova,E., Abramy-

cheva,N., Usenko,T., Kulabukhova,D., Lavrinova,A.,

Kopytova,A., Garaeva,L., Nuzhnyi,E., Illarioshkin,S.,

and Zakharova,E. (2017) Oligomeric α-synuclein and

glucocerebrosidase activity levels in GBA-associat-

ed Parkinson’s disease, Neurosci. Lett., 636, 70-76,

https://doi.org/10.1016/j.neulet.2016.10.039.

34. Navarro-Romero, A., Montpeyó, M., and Marti-

nez-Vicente, M. (2020) The emerging role of the ly-

sosome in Parkinson’s disease, Cells, 9, 2399, https://

doi.org/10.3390/cells9112399.

35. Lu,J., Wu,M., and Yue,Z. (2020) Autophagy and Par-

kinson’s disease, Adv. Exp. Med. Biol., 1207, 21-51,

https://doi.org/10.1007/978-981-15-4272-5_2.

36. Sanchez-Mirasierra, I., Ghimire, S., Hernandez-Di-

az, S., and Soukup, S. F. (2022) Targeting macroau-

tophagy as a therapeutic opportunity to treat Par-

kinson’s disease, Front. Cell Dev. Biol., 10, 921314,

https://doi.org/10.3389/fcell.2022.921314.

mTOR FOR TARGETED THERAPY OF PARKINSON’S DISEASE 1311

BIOCHEMISTRY (Moscow) Vol. 89 No. 7 2024

37. Moors, T.E., Hoozemans, J.J.M., Ingrassia,A., Becca-

ri,T., Parnetti,L., Chartier-Harlin, M.C., and van de

Berg, W. D. J. (2017) Therapeutic potential of auto-

phagy-enhancing agents in Parkinson’s disease, Mol.

Neurodegener., 12, 11, https://doi.org/10.1186/s13024-

017-0154-3.

38. Kinet, R., and Dehay, B. (2023) Pathogenic aspects

and therapeutic avenues of autophagy in Parkin-

son’s disease, Cells, 12, 621, https://doi.org/10.3390/

cells12040621.

39. Saxton, R. A., and Sabatini, D. M. (2017) mTOR sig-

naling in growth, metabolism, and disease, Cell, 168,

960-976, https://doi.org/10.1016/j.cell.2017.02.004.

40. Panwar,V., Singh,A., Bhatt,M., Tonk, R.K., Azizov,S.,

Raza, A. S., Sengupta, S., Kumar, D., and Garg, M.

(2023) Multifaceted role of mTOR (mammalian tar-

get of rapamycin) signaling pathway in human

health and disease, Signal Transduct. Target Ther., 8,

375, https://doi.org/10.1038/s41392-023-01608-z.

41. Chrienova, Z., Nepovimova, E., and Kuca, K. (2021)

Therole of mTOR in age-related diseases, J.Enzyme

Inhib. Med. Chem., 36, 1678-1692, https://doi.org/

10.1080/14756366.2021.1955873.

42. Perluigi, M., Di Domenico, F., and Butterfield, D. A.

(2015) mTOR signaling in aging and neurodegenera-

tion: at the crossroad between metabolism dysfunc-

tion and impairment of autophagy, Neurobiol. Dis.,

84, 39-49, https://doi.org/10.1016/j.nbd.2015.03.014.

43. Siracusa,R., Paterniti,I., Cordaro,M., Crupi,R., Brus-

chetta, G., Campolo, M., Cuzzocrea, S., and Esposi-

to,E. (2018) Neuroprotective effects of temsirolimus

in animal models of Parkinson’s disease, Mol. Neu-

robiol., 55, 2403-2419, https://doi.org/10.1007/s12035-

017-0496-4.

44. Jiang, T.F., Zhang, Y.J., Zhou, H.Y., Wang, H.M., Tian,

L.P., Liu,J., Ding, J.Q., and Chen, S.D. (2013) Curcum-

in ameliorates the neurodegenerative pathology in

A53T α-synuclein cell model of Parkinson’s disease

through the downregulation of mTOR/P70S6K sig-

naling and the recovery of macroautophagy, J.Neu-

roimmune Pharmacol., 8, 356-369, https://doi.org/

10.1007/s11481-012-9431-7.

45. Zhang, Z. N., Hui, Z., Chen, C., Liang, Y., Tang, L.L.,

Wang, S. L., Xu, C. C., Yang,H., Zhao, Y., and Zhang,

J. S. (2021) Mechanism of autophagy regulation

in MPTP-induced PD mice via the mTOR signal-

ing pathway by echinacoside, Neuropsychiatr. Dis.

Treat., 17, 1397-1411, https://doi.org/10.2147/NDT.

S299810.

46. Crews, L., Spencer,B., Desplats, P., Patrick,C., Pauli-

no,A., Rockenstein,E., Hansen,L., Adame,A., Galas-

ko,D., and Masliah,E. (2010) Selective molecular al-

terations in the autophagy pathway in patients with

Lewy body disease and in models of α-synucleinopa-

thy, PLoS One, 5, e9313, https://doi.org/10.1371/journal.

pone.0009313.

47. Chen, Y., and Zhou, X. (2020) Research progress of

mTOR inhibitors, Eur. J. Med. Chem., 208, 112820,

https://doi.org/10.1016/j.ejmech.2020.112820.

48. Zhuang, X.-X., Wang, S.-F., Tan,Y., Song, J.-X., Zhu,Z.,

Wang, Z. Y., Wu, M. Y., Cai, C. Z., Huang, Z. J., Tan,

J.Q., Su, H.X., Li,M., and Lu, J.H. (2020) Pharmaco-

logical enhancement of TFEB-mediated autophagy

alleviated neuronal death in oxidative stress-induced

Parkinson’s disease models, Cell Death Dis., 11, 128,

https://doi.org/10.1038/s41419-020-2322-6.

49. Dehay, B., Bové, J., Rodríguez-Muela, N., Perier, C.,

Recasens, A., Boya, P., and Vila, M. (2010) Patho-

genic lysosomal depletion in Parkinson’s disease,

J.Neurosci., 30, 12535-12544, https://doi.org/10.1523/

JNEUROSCI.1920-10.2010.

50. Robak, L.A., Jansen, I.E., van Rooij, J., Uitterlinden,

A.G., Kraaij,R., Jankovic,J., International Parkinson’s

Disease Genomics Consortium (IPDGC), Heutink, P.,

and Shulman, J.M. (2017) Excessive burden of lyso-

somal storage disorder gene variants in Parkinson’s

disease, Brain, 140, 3191-3203, https://doi.org/10.1093/

brain/awx285.

51. Murphy, K., Gysbers, A., Abbott,S., Tayebi,N., Kim,

W.S., Sidransky,E., Cooper, A., Garner,B., and Hall-

iday, G.M. (2014) Reduced glucocerebrosidase is as-

sociated with increased alpha-synuclein in sporad-

ic Parkinson’s disease, Brain, 137, 834-848, https://

doi.org/10.1093/brain/awt367.

52. Oftedal,L., Maple-Grødem,J., Dalen,I., Tysnes, O.-B.,

Pedersen, K.F., Alves,G., and Lange,J. (2023) Associa-

tion of CSF glucocerebrosidase activity with the risk of

incident dementia in patients with Parkinson disease,

Neurology, 100, e388-e395, https://doi.org/10.1212/

WNL.0000000000201418.

53. Chiasserini, D., Paciotti,S., Eusebi,P., Persichetti, E.,

Tasegian, A., Kurzawa-Akanbi, M., Chinnery, P. F.,

Morris, C. M., Calabresi, P., Parnetti, L., and Becca-

ri, T. (2015) Selective loss of glucocerebrosidase ac-

tivity in sporadic Parkinson’s disease and dementia

with Lewy bodies, Mol. Neurodegener., 10, 15, https://

doi.org/10.1186/s13024-015-0010-2.

54. Schneider, S. A., and Alcalay, R. N. (2020) Preci-

sion medicine in Parkinson’s disease: emerging

treatments for genetic Parkinson’s disease, J. Neu-

rol., 267, 860-869, https://doi.org/10.1007/s00415-

020-09705-7.

55. Anderson, J. P., Walker, D. E., Goldstein, J. M., De

Laat, R., Banducci, K., Caccavello, R. J., Barbour, R.,

Huang, J., Kling, K., Lee, M., Diep, L., Keim, P. S.,

Shen, X., Chataway, T., Schlossmacher, M. G., Seu-

bert, P., Schenk, D., Sinha, S., Gai, W. P., and Chil-

cote, T. J. (2006) Phosphorylation of Ser-129 is the

dominant pathological modification of α-synuclein

in familial and sporadic Lewy body disease, J. Biol.

Chem., 281, 29739-29752, https://doi.org/10.1074/jbc.

M600933200.

BEZRUKOVA et al.1312

BIOCHEMISTRY (Moscow) Vol. 89 No. 7 2024

56. Bartels,T., Choi, J.G., and Selkoe, D.J. (2011) α-Synu-

clein occurs physiologically as a helically folded te-

tramer that resists aggregation, Nature, 477, 107-111,

https://doi.org/10.1038/nature10324.

57. Decressac,M., Mattsson,B., Weikop,P., Lundblad,M.,

Jakobsson,J., and Björklund,A. (2013) TFEB-mediated

autophagy rescues midbrain dopamine neurons from

α-synuclein toxicity, Proc. Natl. Acad. Sci. USA, 110,

E1817-E1826, https://doi.org/10.1073/pnas.1305623110.

58. Webb, J. L., Ravikumar, B., Atkins, J., Skepper, J. N.,

and Rubinsztein, D.C. (2003) α-Synuclein is degrad-

ed by both autophagy and the proteasome, J. Biol.

Chem., 278, 25009-25013, https://doi.org/10.1074/jbc.

M300227200.

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