1. Introduction
Parkinson’s disease (PD) is the second most common neurodegenerative disease and
is characterized by aggregation of alpha-synuclein (-Syn) in the brain,
principally in the substantia nigra pars compacta (SNPc) and striatal regions
[1, 2]. -Syn is the major component of Lewy bodies (LBs), which are one
of the characteristic pathological features of PD. Levodopa, a dopamine
precursor, is currently the drug of choice in the treatment of PD. However, its
long-term use is associated with decreased efficacy and side-effects such as
dyskinesia and motor dysfunction [3, 4]. Furthermore, the current therapeutic
regimens including carbidopa-levodopa, dopamine agonists, catechol
O-methyltransferase (COMT) inhibitors, and anticholinergics provide only limited
improvement in patient quality of life and survival due to their reduced efficacy
after long-term usage. Therefore, the identification of other potential disease
target(s) and the development of new therapeutic strategies are urgently needed
for PD.
The major causes of PD pathogenesis are mitochondrial dysfunction,
neuroinflammation, and -Syn aggregation [5, 6, 7]. Recently,
González-Rodríguez et al. [8] reported that loss or disruption
of mitochondrial complex I in the dopaminergic neurons of mice was sufficient to
cause progressive, human-like Parkinsonism. Earlier reports suggested that
cytosolic and mitochondrial deposition of aggregated -Syn promoted the
fragmentation of mitochondria, which was then followed by mitochondrial
dysfunction [5, 9, 10]. Hence, the overexpression of -Syn alters
mitochondrial fusion/fission dynamics, thereby contributing to mitochondrial
dysfunction and the pathogenesis of PD [11, 12, 13].
The brain renin-angiotensin system (RAS) plays a crucial role in the
pathogenesis of Parkinsonism. Angiotensin II (AII), the most important effector
peptide in RAS, exerts its effect via the AII type 1 receptor (AT1R) and the AII
type 2 receptor (AT2R) [14, 15]. In physiological conditions, activation of AT2R
counteracts the function of AT1R [16]. In PD, the upregulation of AT1R expression
contributes to neuroinflammation and apoptosis, leading to dopaminergic cell
death. In our earlier study using a mouse model of PD, we reported that blockade
of AT1R with a selective AT1R antagonist, Telmisartan (Tel), resulted in
neuroprotection [15]. An interesting report by Valenzuela et al. [17]
(2016) revealed the presence of AT1R and AT2R on the mitochondrial membrane of
dopaminergic neurons, thus demonstrating a link between RAS and mitochondrial
function. There is now accumulating evidence that PD is associated with
alterations in mitochondrial dynamics and in the biogenesis of proteins such as
mitofusin protein 1 (MFN1) and Peroxisome proliferator-activated receptor-gamma coactivator- (PGC1) [18]. Recently, we also
demonstrated that AT1R blockade by Tel protects the expression of
mitochondrial-specific genes (PINK1, Parkin, and
PARK7) in the brain tissue of a mouse PD model, as well as locomotor and
gait function [6]. However, the underlying mechanisms behind restoration of
mitochondrial proteins/functions in PD models are still poorly understood and
require further research. The aim of the current study was therefore to
investigate the signaling mechanisms by which Tel protects mitochondrial
function, gait activity, and neuronal cells from death in a mouse PD model. The
agent 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was used to induce PD,
since it is a well-established inhibitor of brain SNPc mitochondrial complex I in
animal models [15, 19]. The current study reveals the effects of Tel on key
proteins involved in the regulation of mitochondrial function.
2. Materials and Methodology
2.1 Chemicals and Reagents
Telmisartan (Product No. T2861) and Probenecid (Product No. P1975) were
purchased from Tokyo Chemical Industry (TCI) Private Limited (Tamil Nadu, India).
MPTP hydrochloride (Cat. No.: HY-15608) was obtained from MedChemExpress (Middlesex County, NJ,
USA). The specific primary antibodies used in this study were:
anti--actin [1:1000 dilution, Cat# 13E5, Cell
Signaling Technology (CST), Danvers, MA, USA], anti-GSK3 (1:1000, Cat#
ab131356, Abcam, Cambridge, United Kingdom), anti-Phospho-(Ser9)-GSK3
(1:1000, Cat #sc-373800, Santa Cruz Biotechnology, Inc., Dallas, TX, USA),
anti-iNOS (1:1000, Cat# D6B6S, CST), anti-Akt1 (1:1000, Cat #BT-AP00347,
Bioassay Technology Laboratory (BT-Lab) Birmingham, United Kingdom),
anti-Phospho-(S473)-Akt (1:1000, Cat #BT-PHS00006, BT-Lab),
anti--Synuclein (1:1000, Cat #sc-12767, Santa Cruz), anti-Mfn1
(1:1000, Cat #sc-166644, Santa Cruz), anti-AT1R (1:1000, Cat #sc-515884, Santa
Cruz), anti-AT2R (1:1000, Cat #sc-518054, Santa Cruz), anti-Bax (1:1000, Cat
#2772, CST) and anti-PGC1 (1:1000, Cat #NBP3-08971, Novus Bilogical part of Bio-Teche India Private Limnited, Pune, India). The secondary antibodies used
were anti-rabbit (1:2000, Cat #7074, CST) and anti-mouse (1:2000, Cat#
sc-516102, Santa Cruz). The Adenosine triphosphate Enzyme-linked Immunosorbent Assay (ATP ELISA) kit was purchased from Thermo Fischer
(Catalog number: A22066, Waltham, MA, USA). All other chemicals and reagents used
in this study were analytical grade.
2.2 Animal Husbandry and Ethics Approval
Young male C57BL/6J mice (18–22 g body weight) were procured from Adita
Bioscience (Tumkur, Karnataka, India). The animals were housed in polypropylene
cages in the good laboratory practice standard central animal facility at JSS Academy of Higher Education And Research (JSS
AHER, Mysuru, India). Animals were acclimatized for 7 days in an experimental
room at a controlled temperature of 22 3 °C, 40–65% relative
humidity, and 12-h light/12-h dark cycles. This study was approved by the
Institutional Animal Ethics Committee (IAEC) (JSS AHER, Mysuru, India) (approval
number: JSSAHER/CPT/IAEC/016/2020).
2.3 Experimental Design and Treatments
Following acclimatization, mice were trained for the beam walk test (Fig. 1).
The animals were divided into 5 groups (n = 8 per group). Group A (vehicle) was
administered with 0.5% carboxymethylcellulose (CMC) by oral gavage + saline i.p.
Group B was given 0.5% CMC (vehicle) by oral gavage + MPTP [intraperitoneally at
250 mg/kg b.wt. (in 10 divided injections, each 25 mg/kg b.wt. at 3.5 days
interval) + Probenecid 250 mg/kg i.p.)] MPTP; positive control, and Groups C and
D were given Tel at 3 and 10 mg/kg, by oral gavage, respectively, up to 35 days +
MPTP (Vehicle or Tel was administered 1 hr before the first injection of MPTP and
thereafter daily once for 35 days). Group E (probenecid control) received 250
mg/kg i.p. In the study Probenecid was used here to reduce renal excretion of
MPTP and its metabolites, because its usage is recommended for experimental
protocols of chronic PD [20]. A separate group for Tel alone was not included,
since our earlier study showed that 10 mg/kg Tel did not cause any toxicity [15].
Mice were evaluated for motor functions using a beam walk every 10th day. On the
35th day, mice were euthanized using excess CO, and mouse brains were
collected and quickly frozen. Brain tissue was kept at –80 °C until the
preparation of extracts for biochemical and Western blot analyses. SNPc regions
(Bregma –3.16 mm, interaural 0.64 mm) were identified using the Paxinos
and Franklin mouse brain atlas [21].
Fig. 1.
Experimental design. TEL, Telmisartan; MPTP,
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; ATP, Adenosine triphosphate.
2.4 Gait Function Analysis
Beam Walk Experiment
Prior to MPTP administration, mice were pre-trained for the beam walk test by
allowing them to traverse the narrow 100 cm length runway with a dark escape box
at the other end [22]. An aversive stimulus was created by using a bright light
(100 lux) placed above the beam to motivate the walk. Mice were allowed a maximum
of 60 seconds for the travel. The observer for the experiment was blinded to the
identity of the treatment groups.
2.5 Western Blotting
The SNPc region from either side of the mouse brain was isolated and used for
Western immunoblot analyses. The isolated brain tissues were homogenized using
Radioimmunoprecipitation Assay (RIPA) lysis buffer (Cat #786-490, G-Biosciences, St. Louis, MO, USA) and a
protease and phosphatase inhibitor cocktail (MP Biomedicals, Santa Ana, CA, USA), incubated for 30 min on ice, and
then centrifuged (15,000 g at 4 °C for 10 min) to obtain individual
tissue lysates. Tissue lysate supernatants were collected and used for measuring
the protein concentration with a BCA Protein Assay Kit (Cat #23225,
ThermoFisher, Rockford, IL, USA). Aliquots of 40 µg protein were mixed with sodium
dodecyl sulfate (SDS)-sample loading buffer containing bromophenol blue and
-mercaptoethanol [SDS (CAS RN: 151-21-3), B-mercaptoethanol (CAS RN: 60-24-2), Sample Loading Buffer (B6104), from Tokyo Chemical Industry (TCI) Private Limited (Tamil Nadu, India)]. Electrophoresis using 10% SDS-polyacrylamide gels was
performed to separate the proteins, which were then electrically transferred onto
polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA, USA).
Bovine serum albumin, (BSA, 3%; Bio-Rad Laboratories) was used to block
nonspecific binding sites, and the membranes were rinsed three times with
Tris-buffered saline for 5 min each. Each membrane was then incubated with a
specific primary antibody at 4 °C for 8–10 h and then rinsed 3 times
for 30 min each with TBST (1% Tween20 + Tris Buffered Saline). Next, each
membrane was incubated with the appropriate secondary antibody (HRP-conjugated
anti-mouse IgG or anti-rabbit IgG) at room temperature for 2 hours and then
rinsed 3 times with TBST for 30 min each. Visualization and imaging of the target
protein bands were conducted using Clarity Max Western ECL Substrate (Cat#
1705062, Bio-Rad) for 10 min. Band intensities were quantified by ImageJ software
(Version 1.54, National Institutes of Health, Bethesda, MD, USA).
2.6 ELISA Assay
The Adenosine triphosphate (ATP) content of SNPc tissues was measured using an
Enzyme-linked Immunosorbent Assay (ELISA) kit as recommended by the manufacturer
(Catalogue number: A22066, Invitrogen™, Waltham, MA, USA) and a luminometer
(TECAN/SPARK 10M, Männedorf, Switzerland).
2.7 Statistical Analysis
Experimental data (mean differences) were analyzed using one-way analysis of
variance (ANOVA) followed by Tukey’s multiple comparison test, with a
p-value 0.05 considered to represent statistical significance.
GraphPad Prism 6.1 software (San Diego, CA, USA) was used for statistical
analysis. Final values were expressed as the mean standard error of the
mean (SEM).
3. Results
3.1 Effect of Tel on Gait Function in PD mice
Chronic MPTP + probenecid administration caused motor dysfunction and decreased
the body weight of mice in the MPTP control group. However, treatment with Tel
protected the normal body weight of MPTP mice (Body weight data is given in
Supplementary Materials).
3.2 Beam Walk
Mice in the MPTP group took significantly longer to reach the goal (dark escape
box at the end of the runway) on all of the locomotor test days [11th day
(p 0.01); 22nd and 32nd days (each p 0.001)] compared
to vehicle-treated mice (Fig. 2). Tel treatment significantly decreased the time
taken to reach the goal on the 22nd and 32nd days of the experiment (p 0.05 and p 0.001).
Fig. 2.
Effect of Tel on the time taken to cross the runway
(sec). ** and *** indicate p 0.01and p 0.001,
respectively, compared to the vehicle control. , and
indicate p 0.05, p 0.01, and
p 0.001, respectively, compared to the MPTP group. MPTP,
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.
3.3 Effect of Tel on ATP Content in PD Mouse Brains
The ATP content in SNPc regions of the mouse brains was determined with an ELISA
kit. ATP concentration was significantly lower in the MPTP group (group B)
compared to the vehicle (group A) and Probenecid (group E) control groups.
However, mice treated with Tel (3 and 10 mg/kg) showed a significantly higher ATP
content compared to MPTP-treated mice (Fig. 3).
Fig. 3.
Quantification of ATP content using a commercial ELISA kit.
SNPc region homogenates were diluted 10-fold and processed as
per the kit instructions. Luminescence was read at ~560 nm using
a luminometer. One-way ANOVA and Tukey’s multiple comparison tests were applied.
*** denotes a p-value 0.001 compared to the vehicle control.
denotes a p-value 0.001 compared to the MPTP group.
SNPc, substantia nigra pars compacta; ATP, Adenosine triphosphate; ELISA,
Enzyme-linked Immunosorbent Assay; ANOVA, Analysis of Variance.
3.4 Effects of Tel on the Expression of Key Signaling Proteins in
the Mouse Brain
Protein-Protein Interactions
Protein-protein interactions (PPI) were analyzed using the
Search Tool for the Retrieval of Interacting Genes/Proteins
(STRING) database (https://string-db.org/). Ten nodes of predicted protein
interactions were built based on genomic context analysis. The PPI enrichment
value (p-value: 2.81 10) indicated the proteins were
biologically connected, as described previously [23] (Fig. 4). The expected
number of edges was 4 large domains, which can expand to a final number of 21
edges, with an average node degree of 4.2 and an average local clustering
coefficient of 0.715. A primary interaction was found between AT1R (AGTR1) and
Akt1, which was in turn connected with GSK3B (GSK3), iNOS, IBA1
(Ionized calcium binding adaptor molecule 1, activated glial cell marker), Bax (BCL2 Associated X Protein, an apoptosis marker),
-Syn (SNCA), TH (tyrosine hydroxylase as a marker of
PD), PGC1 (PPARGC1A, a mitochondrial biogenesis marker) and
mitochondrial fusion protein 1 (MFN1). Interactions of GSK3 with
-Syn, PGC1 and Bax were also observed (Fig. 4).
Fig. 4.
Protein-protein interaction networks
(STRING Database). The lines between the protein connections
represent the types of interactions with other proteins with different color.
These are based on experimentally proven or predicted results from other
available databases. STRING, Search Tool for the Retrieval of Interacting
Genes/Proteins; AGTR1 (AT1R), Angiotensin II Receptor Type 1; AKT1, AKT
Serine/Threonine Kinase 1; iNOS, inducible nitric oxide synthase; BAX, BCL2 Associated X Protein; GSK3B, Glycogen Synthase
Kinase 3 Beta; IBA1, Ionized calcium binding adaptor molecule 1; TH, Tyrosine Hydroxylase; SNCA, Synuclein Alpha; PGC1A, PPARG Coactivator 1 Alpha; MFN1, Mitofusin 1.
To further validate the PPI data, the expression levels of specific proteins
were analyzed by Western blot analysis. Chronic MPTP injections significantly
upregulated the expression of AT1R (p 0.05), -Syn
(p 0.01), iNOS (trend for increase), IBA-1 (p 0.05) and
Bax (p 0.001) in MPTP-treated mouse brains compared to the vehicle
control group (Fig. 5). On the other hand, MPTP treatment downregulated the
expression of AT2R (a trend for decrease), Tyrosine Hydroxylase (TH) (p 0.05), Mitofusin 1 (MFN1) (p 0.001), p-Akt/Akt (p
0.01), p-GSK3 (Ser-9)/GSK3 (p 0.001) and PPARG
Coactivator 1 Alpha (PGC1) (p 0.01) in the MPTP group
compared to the vehicle control group.
Fig. 5.
Effects of Tel treatment on the protein levels. (a) TH, (b)
-Syn, (c) AT1R, (d) AT2R, (e) p-Akt (Ser 473)/Akt, (f)
p-GSK3 (Ser 9)/GSK3, (g) MFN1, (h) iNOS, (i)
IBA1, (j) PGC1, (k) Bax, (l) Western Blot Images. Data are expressed as the mean
SEM, with n = 3 mice/group. One-way ANOVA followed by Tukey’s multiple comparison
test was used to compare mean differences between groups. *, ** and *** indicate
p-values of 0.05, 0.01 and 0.001, respectively, compared to the
vehicle control. , and indicate p-values of
0.05, 0.01 and 0.001, respectively, compared to the MPTP group. ns
represents not significant. SEM, standard error of the mean; -Syn, alpha-synuclein; Bax, BCL2 Associated X Protein; PGC1, Peroxisome proliferator-activated receptor-gamma coactivator-.
Tel significantly restored the levels of AT1R, -Syn, GSK3,
iNOS, IBA1 and Bax following MPTP treatment. In addition, Tel significantly
upregulated the expression of TH, p-Akt/Akt, p-GSK3
(Ser-9)/GSK3, MFN1, and PGC1- compared to MPTP-treated mice,
but not AT2R expression (non-significant decrease). The significance levels
between the experimental groups are shown in Fig. 5.
4. Discussion
The findings of this study suggest the mechanism by which Tel, a selective AT1R
blocker, can protect mitochondrial function, gait function, and neuronal
apoptosis in a mouse model of PD. Moreover, the current study validated the
STRING database PPI associated with the renin-angiotensin system in PD (Fig. 4).
MPTP treatment in the present study was observed to upregulate -Syn
expression, consistent with earlier reports [24, 25]. -Syn inhibits TH
by activating protein phosphatase-2A. In turn, the inactivation of TH results in
dopamine depletion and apoptosis of dopaminergic cells [26]. Consistent with our
previous results using an MPTP-treated mouse model, MPTP caused severe gait
impairment and loss of body weight compared to the vehicle control group [6, 27].
The present results also showed that Tel treatment inhibits -Syn
expression in the MPTP-exposed mouse model, as well as protecting mitochondrial
and gait functions. Earlier studies from various authors including ourselves
found a correlation between decreased -Syn expression and improved
motor and gait function, which was further linked to increased dopamine turnover
[15, 28, 29].
The current work found that Tel prevented the increase in expression of AT1R,
while at least partially preventing AT2R in MPTP-exposed mice. In a rat model of
insulin resistance, inhibition of AT1R was shown to cause neuroprotection via the
Akt-mediated pathway in dorsal root ganglion (DRG) neurons [30]. MPTP treatment
decreased the active form of Akt (phosphorylated at Ser-473) and downregulated
p-GSK3 (Ser-9, inactive form), which in turn triggered apoptosis [31].
In the present study, Tel significantly increased Ser-473 phosphorylation of Akt
(activation), and upregulated inactive p-GSK3 (Ser-9), both of which
correlated with decreased Bax expression [32]. Hence, we propose that
Tel-mediated anti-apoptosis in PD mouse brains is likely to be caused by
modulation of the Akt/GSK3 pathway.
Cytosolic GSK3 phosphorylates -Syn, leading to its
aggregation and neuronal accumulation in PD [33]. On the other hand, the increase
in non-phosphorylated GSK3 (active form) stimulates autophagic clearance
of aggregated -Syn in neurons [34]. Hence, the decrease in
-Syn in Tel-treated mice could be mediated via the Akt-GSK3
pathway [35, 36, 37]. In addition, GSK3 promotes the inflammatory response
by activating microglia [38], which was evidenced by the upregulation of
inflammatory markers such as IBA1 and iNOS (non-significantly) in MPTP-treated
mice [39, 40]. Pretreatment with Tel inhibited IBA1 and iNOS expression, which
confirms the functional link between AT1R and GSK in PD.
The Akt and GSK3 proteins located in the mitochondria are highly active
compared to the cytosolic counterparts [41]. Upregulation of GSK3
(active form) resulted in aberrant mitochondrial function, which has been
implicated in PD [42]. GSK3 is an important Ser/Thr kinase that
regulates the degradation of PGC1, a critical transcriptional
coactivator for mitochondrial biogenesis [43, 44]. Activated GSK3
phosphorylates PGC1, which subsequently stimulates degradation of
PGC1 via the ubiquitin-dependent proteasomal system [44]. Inhibition of
GSK3 has in fact been shown to promote mitochondrial biogenesis in a
mouse model of cerebral stroke by restoring PGC1 levels [45]. In the
present study, Tel upregulated PGC1 and MFN1 while increasing the ATP
content, thus reflecting the improved mitochondrial functions in MPTP-treated PD
mouse brains. Tel showed higher PGC1 expression at lower
concentrations. That might be due to its more effective dual role, selective AT1
blocker and partial activator Peroxisome proliferator-activated receptor gamma
(PPAR-) at lower concentration [46, 47]. In summary, we demonstrated
that treatment of an MPTP mouse model of PD with Tel protected mitochondrial
function and gait activity through activation of the
Akt-GSK3-PGC1 pathway.
5. Conclusion
In conclusion, the present study showed that Tel upregulates p-GSK3
(Ser 9, inactive form) and PGC1 to preserve mitochondrial biogenesis
and bioenergetics in MPTP-treated mice (Fig. 6). The current results are
consistent with our earlier findings. Moreover, they provide additional evidence
regarding the signaling mechanisms by which Tel protects neuronal cells through
the preservation of mitochondrial and motor functions. Therefore, the present
results suggest that Tel could be a potential candidate for improving the
management of PD, at least as an adjunct therapeutic agent.
Fig. 6.
Tel protects mitochondrial function in the brain of a mouse
model of PD. MPTP is converted to MPP+ in glial cells by MAO (monoamine
oxidase). AT1R and AT2R are present on the mitochondrial membrane surface. AT1R
performs the opposite function to AT2R, and its blockade is neuroprotective. The
expression of AT1R in PD is elevated compared to AT2R. AT1R activation inhibits
p-Akt, triggers the aggregation of -Syn, and stimulates apoptosis by
activating p-Akt/GSK3 located in the mitochondria. Additionally, AT1R
activation inhibits mitochondrial biogenesis by suppressing the expression of
PGC1. However, blockade of AT1R by Tel reverses all of these negative
effects by regulating the Akt/GSK3/PGC1 pathway. MAO,
monoamine oxidase; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; MPP+,
1-methyl-4-phenylpyridinium; PD, Parkinson’s disease; TEL, Telmisartan.
Abbreviations
-Syn, alpha-synuclein; CMC, Carboxymethylcellulose; COMT, Catechol
O-methyltransferase; IAEC, Institutional Animal Ethics Committee; MFN1, mitofusin
protein 1; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; PD, Parkinson’s
disease; RAS, renin-angiotensin system; SNPc, Substantia nigra pars compacta;
Tel, Telmisartan; TH, tyrosine hydroxylase.
Availability of Data and Materials
The datasets used and/or analyzed during the current study are available from
the corresponding author on reasonable request.
Author Contributions
BR: conceptualized and designed the study, Performed, Data acquisition,
analysis, and Manuscript writing; ST, PGN and AS: Data acquisition, analysis, and
Manuscript Writing; AMM, PP and BJS: Data analysis
and manuscript editing; SBC: conceptualized and designed the study, Manuscript
Editing, and supervision. All authors contributed to editorial changes in the
manuscript. All authors read and approved the final manuscript. All authors have
participated sufficiently in the work and agreed to be accountable for all
aspects of the work.
Ethics Approval and Consent to Participate
The current study was approved by the Institutional Animal Ethics Committee
(IAEC) (JSS AHER, Mysuru, India) (Approval number:
JSSAHER/CPT/IAEC/016/2020).
Acknowledgment
The authors are thankful to National Institute on Alcohol Abuse and Alcoholism (NIAAA) and other respective institutions for providing the facilities.
Funding
This study was partially supported by the Indian Council of Medical Research (ICMR) (No-45/3/2019-PHA/BMS/OL).
Conflict of Interest
The authors declare no conflict of interest.