LONP1 induction by SRT1720 attenuates mitochondrial dysfunction against high glucose induced neurotoxicity in PC12 cells

Journal Pre-proof LONP1 induction by SRT1720 attenuates mitochondrial dysfunction against high glucose induced neurotoxicity in PC12 cells

AnilKumar Kalvala, Veera Ganesh Yerra, Ashutosh Kumar PII:

S0887-2333(19)30730-1

DOI:

https://doi.org/10.1016/j.tiv.2019.104695

Reference:

TIV 104695

To appear in:

Toxicology in Vitro

Received date:

21 September 2019

Revised date:

17 October 2019

Accepted date:

17 October 2019

Please cite this article as: A. Kalvala, V.G. Yerra and A. Kumar, LONP1 induction by SRT1720 attenuates mitochondrial dysfunction against high glucose induced neurotoxicity in PC12 cells, Toxicology in Vitro(2018), https://doi.org/10.1016/j.tiv.2019.104695

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© 2018 Published by Elsevier.

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LONP1 Induction by SRT1720 Attenuates Mitochondrial Dysfunction Against High Glucose Induced Neurotoxicity in PC12 Cells Anil Kumar Kalvalaa, Veera Ganesh Yerrab, Ashutosh Kumara,* [email protected], [email protected], [email protected] a

Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER)-Hyderabad, Balanagar, India b

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Keenan Research Centre for Biomedical Science and Li Ka Shing Knowledge Institute, St. Michael’s Hospital, Toronto, ON, Canada * Corresponding author at: Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER)-Hyderabad, Balanagar, Hyderabad, TG500037 Abstract:

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Neuropathies caused by mitochondrial dysfunction are the most common and serious

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impediment of high glucose (HG)-induced toxicity. We have previously reported mitoprotective

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potency of Sirtuin1 (Sirt1) in diabetic neuropathy (DN) via targeting mitochondrial dysfunction but its nuclear control over mitochondrial bioenergetics remains unknown. Here, we studied the

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effect of SRT1720; a small molecule activator of Sirt1 in attenuating the HG mediated

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mitochondrial dysfunction in differentiated rat pheochromocytoma (PC12) cells and aiming to determine (1) whether SRT1720 can improve mitochondrial function in HG exposed PC12 cells

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(2) if yes then this effect is dependent or independent of mitochondrial Lon protease (LONP1) (3) and whether silencing of LONP1 affects the mitochondrial function or not. HG (30mM) exposed PC12 cells demonstrated reduced mitochondrial complex activities and oxygen consumption rate (OCR), decreased the expressions of Sirt1, peroxisome proliferator-activated receptor coactivator-1α (PGC1α), nuclear respiratory factor-2 (NRF2), LONP1 and ATP synthase c. SRT1720 treatment (4μM) significantly reversed these effects in hyperglycemia insulted PC12 cells but silencing the expression of LONP1 impeded this effect of SRT1720 on mitochondrial complex activities, OCR and mitochondrial membrane potential. Based on these

Journal Pre-proof findings, we inferred that SRT1720 might improve mitochondrial function in HG induced mitochondrial dysfunction in PC12 cells via stimulation of Sirt1-LONP1 axis

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Key Words: SRT1720, Sirt1, Mitochondrial Lon Protease, Mitochondrial Function

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1. Introduction

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Globally, mitochondrial research is gaining much importance in defining whole body

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metabolism, health and life span. Earlier mitochondrial dysfunction was expounded only in few disorders but now studies have been expanding and role of mitochondrial dysfunction has been

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illustrated in various disease affecting humans. Among the series of diseases and disorders, high glucose (HG) - induced mitochondrial dysfunction in neurons is the most studied ones in

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different experimental models, inspite of which, the mechanisms involved in the relationship

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between mitochondrial dysfunction and HG mediated stress in the neurons are not very clear. Decades of research involving eubacteria to eukaryotes revealed the crucial role of ATP dependent proteases in the regulation of mitochondrial homeostasis. Out of various ATP dependent mitochondrial matrix proteases; mitochondrial lon protease (LONP1) and caseinolytic peptidase (ClpP) unfold misfolded proteins and also degrade aggregated toxic proteins (Kalvala et al., 2019; Pryde et al., 2016). Among these, LONP1 is highly explored in improving mitochondrial function. Moreover, previous results of mitochondrial samples obtained from LONP1 silenced B16F10 melanoma cells subjected to proteomic analysis showed deeply

Journal Pre-proof compromised mitochondrial function (Gibellini et al., 2018). Additionally, LONP1 deficient cells compared to normal cells revealed compromised mitochondrial functionality with reduced electron transport chain (ETC) complex I, II and IV activities (Pinti et al., 2011). Furthermore, LONP1 also corrected protein misfolding, particularly misfolding caused by oxidativenitrosative stress, via interaction with heat shock proteins (HSPs) and elicited chaperone like activity (Quirós et al., 2014). Recently it was proved that nuclear respiratory factor-2 (NRF2)

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can act as master regulator of LONP1 expression based on the identification of NRF2 binding

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regions in the promoter region of LONP1 (Pinti et al., 2011).

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Several studies have reported the role of Sirtuin1 (Sirt1), a NAD+ dependent deacetylase, in

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alleviating neuropathic pain by improving mitochondrial function via deacetylating peroxisome

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proliferator-activated receptor coactivator (PGC1α)-NRF2 axis (Aquilano et al., 2010; Jia et al., 2016; Yerra et al., 2017). However, the relationship between Sirt1 and LONP1 with respect to

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mitochondrial function remains unclear. SRT1720 is studied by Sirtris pharmaceuticals and

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projected as a small molecule activator of Sirt1. Since its discovery, its specificity in activation of Sirt1 had been questioned and further defended (Dai et al., 2010; Pacholec et al., 2010). The

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administration of this drug in obese and diabetic animals improved insulin sensitivity, normalized glucose levels and also improved mitochondrial function (Milne et al., 2007). Interestingly, SRT1720 administered to the diabetic and obese mice enhanced life span by 18% when compared to untreated diabetic and obese mice (Mitchell et al., 2014). Therefore, this study was designed to explore the concurrent role of Sirt1 and LONP1 in the alleviation of HG-induced mitochondrial dysfunction using SRT1720; a small molecule activator of Sirt1 in PC12 cells.

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2. Materials and Methods 2.1. Materials: Unless specified all the chemicals are reagent grade and obtained from Sigma Aldrich, USA. SRT1720 was purchased from Selleckchem Ltd, Texas, USA. SMARTpool: Accell Lonp1 siRNA (E-092339-00-0005), Accell non targeting pool (D-001910-10-05) and

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Accell siRNA delivery media (B-005000-500) were purchased from Dharmacon, USA.

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2.2. Methods

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2.2.1. Experimental Design

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PC12 cell line was obtained from NCCS, Pune, India were cultured in RPMI 1640 media

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containing 5.5mM β-D glucose (Invitrogen) at 37°C temperature and 5% CO2. HG condition was

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simulated by exposing PC12 cells to 30mM β-D glucose (Saberi Firouzi et al., 2018) in RPMI 1640 media. The cells were divided into following groups: 1. NC: Normal control PC12 cells, 2.

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HG: Disease control group, 3. HG+SRT: HG PC12 cells treated with 4μM of SRT1720, 4.

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HG+SRT+LONP1-T: HG PC12 cells treated with 0.5μM of LONP1 siRNA plus 4μM SRT1720, 5. LONP-T: PC12 cells treated with 0.5μM of LONP1 siRNA and 6. siRNA negative control: PC12 cells exposed to non-targeting pool. Prior to the experiment, cells were differentiated into neurons by supplementing with 50ng/ml of nerve growth factor (NGF) in serum free media for 24h (Zhang et al., 2007), followed by transfecting PC12 cells with 0.5μM of LONP1 siRNA in siRNA delivery media with 5% FBS. After 24h, hyperglycemia was induced with 30mM β-D-glucose in RPMI 1640 media and then treated with 4μM SRT1720 for 24h followed by biochemical and molecular evaluations.

Journal Pre-proof 2.2.2. Cell Viability Assay PC12 cells were seeded in 96 well plates at the density of 5000 cells/well and incubated for 24h. After incubation, cells were treated with different concentrations of SRT1720 (0.39- 100μM) and co-treated with glucose (30mM) in RPMI 1640 media and incubated for 24h. Then MTT solution (5 mg/10 ml in RPMI) was added to the 96-well plate and incubated for 4h at 37°C. Medium was

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removed and crystals were solubilized using DMSO. Absorbance was measured at 570nm.

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2.2.3. Immunocytochemistry (ICC)

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Briefly, cells were washed with PBS for three times and fixed in 4% paraformaldehyde solution

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followed by washing with PBS. Then cells were blocked with 3% BSA solution and incubated with primary antibody; LONP1 (1:50) (cell signalling technology (CST), USA), PGC 1α (1:200)

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(Novus Biologicals, USA) and ATP synthase c (1:50) (Abcam, USA) in 3% BSA at 4°C

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temperature for 12h. Followed by incubation with secondary anti-rabbit antibody conjugated with rhodamine (Santa Cruz Biotechnology Inc., CA, USA) or FITC (Sigma) for 2h in dark at

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room temperature. After washing and mounting with Fluoroshield™ with DAPI histology

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mounting medium (Sigma), cells were subjected to confocal imaging using a confocal microscope (Leica TCS SP8 Laser Scanning Spectral Confocal) (Aquilano et al., 2010). 2.2.4. Western blotting PC12 cell lysates, prepared in RIPA buffer containing protease and phosphatase inhibitor (1:100), were resolved using SDS-PAGE and transferred on to the PVDF membrane followed by blocking with 5% non-fatty dried milk powder/3% BSA in TBST. The membranes were incubated with primary antibodies at 4°C for overnight; LONP1, SOD 2 (CST, USA), SIRT-1 (CST, USA), NRF2 (Santacruz biotechnologies, USA), ATP synthase, Aconitase 2 (CST, USA),

Journal Pre-proof HSP 27 (CST, USA) and PGC1α prepared at 1:1000 dilution in TBST. The membrane was incubated with HRP tagged secondary anti-rabbit and anti-mouse antibody for 2h at room temperature. Chemiluminescence signal was captured using a Fusion-FX imager (Vilber Lourmat, Germany) and relative band intensities were quantified by densitometry using Image-J software (version 1.48, NIH, USA) (Yerra et al., 2017).

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2.2.5. Oxyblot analysis

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Oxyblot analysis was performed in PC12 cell lysates using oxidized protein western blot assay

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protocol (ab178020, Abcam). The treated samples were subjected to Chemiluminescence wherein signal was captured using a Fusion-FX imager (Vilber Lourmat, Germany) and relative

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band densities were quantified by densitometry using Image-J software (version 1.48, NIH,

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USA).

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2.2.6. Analysis of mitochondrial potential (∆ψm) by JC-1 staining

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Briefly, cells were given allocated treatment and after 6h of treatment they were incubated for 15min with 5μM of JC-1. These cells were then PBS washed for three times and fluorescence

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images were captured at 20X magnification using Nikon eclipse Ti2 Fluorescence microscope, Japan. Additionally, 5μM of JC-1 treated cells were trypsinized, followed by centrifugation. The pellet was resuspended in PBS containing 5% FBS and subjected to fluorescence analysis using BD FACSVerse (BD Biosciences, CA, USA). 2.2.7. Measurement of mitochondrial respiration in neuronal rat pheochromocytoma cells Mitochondrial respiration was measured by using Seahorse XFp analyzer (Agilent technologies, USA). XFp analyzer designed with a transient 7μl chamber in a specialized 8 well microplates

Journal Pre-proof allowed measuring oxygen consumption rate (OCR) in real time (Roy Chowdhury et al., 2012). Briefly, neuronal cell density was maintained between 4000-5000 cells per well leaving A and H control wells. The cells were differentiated, transfected and treated according to the experimental design. One hour before measurement of OCR, RPMI 1640 media was changed with Dulbecco’s modified eagle medium with 1mM pyruvate and 2mM glutamate. Oligomycin (1μM); FCCP (Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone) (2 mM); and rotenone + antimycin A

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(1μM each) were injected sequentially through ports in the Sea- horse flux pak cartridges. Each

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loop was started with mixing for 3mins, delayed for 2mins and followed measuring OCR for

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3mins. This experiment allowed to measure the amount of oxygen consumption linked to ATP

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production, spare respiratory capacity, basal respiration and maximal respiration in respective wells. The data was imported into wave software and it allowed us to measure all

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aforementioned parameters after normalizing with the protein concentration of respective wells.

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The data was represented as OCR picomoles/min/mg/ml protein.

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2.2.8. Estimation of Aconitase-2 activity

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Aconitase-2 activity was measured by using aconitase activity assay kit (MAK051, Sigma Aldrich, USA) in the mitochondrial homogenates of PC12 cells. Absorbance was recorded at 450nm after addition of 10µl of developer solution to each well and results were reported in milliunit/ml/mg protein (Alvarez et al., 2017). 2.2.9. Measurement of mitochondrial complex activities The complex I (ab109721), complex II (ab109908), complex IV (ab109911) and complex V (ab109714) activities were estimated in isolated mitochondrial fractions of PC12 cells as per kit protocols (Abcam, UK) (Areti et al., 2017). The change in absorbance between the time points

Journal Pre-proof was calculated with normalized protein concentration (mOD/min/mg) and the values were reported as percentage of enzyme activity with respective to NC group. 3. Statistical Significance Data are represented as mean ± (Standard Error of Mean) SEM. The intergroup variation was measured by one-way analysis of variance (ANOVA) followed by "Bonferroni's multiple

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comparison post-hoc test" using the Graph Pad Prism. Results with p values <0.05 were

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considered to be statistically significant.

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4. Results

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Based on MTT results obtained, we have chosen 4μM of SRT1720 (IC50: 10.14µM) as a sub maximal dose to assess its neuroprotective potential in HG induced neurotoxicity in PC12 cells

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(Fig. 1).

4.1. Effect of SRT1720 on immunolocalization of PGC1α and expressions of Sirt1, NRF2 and PGC 1α in HG exposed and siRNA transfected PC12 cells Sirt1-PGC1α-NRF2 axis has an important role in regulating mitochondrial homeostasis. These protein expressions were significantly reduced; Sirt1 (P<0.01), PGC1α (P<0.001), and NRF2 (P<0.01) in HG exposed PC12 cells when compared to the normal cells. SRT1720 administration significantly (Sirt1 and NRF2 (P<0.01), PGC1α (P<0.005) v/s HG) attenuated this metabolic excess

mediated

protein

expression

levels

(Fig.2B&C).

Additionally,

PGC1α

immunolocalization was reduced in HG exposed PC12 cells and this effect reversed upon

Journal Pre-proof SRT1720 treatment (Fig.2A). However these protein expression levels were unaltered in LONP1 knocked down PC12 cells (Fig.2B&C) and LONP1 knockdown did not interfere with the effect of SRT1720 treatment in HG exposed PC12 cells. These results suggest that knockdown of LONP1 gene for 48 h has a neutral role in altering these protein expressions. 4.2. Effect of SRT1720 on LONP1 and protein carbonylation in high glucose exposed and

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LONP1 knockdown PC12 cells

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Interestingly, LONP1 expression was significantly (P<0.001) reduced in HG exposed

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(Fig.3A&C) and LONP1 gene silenced PC12 cells (P<0.001) when compared to normal control group (Fig. 3A&C). SRT1720 treatment to HG exposed PC12 cells significantly (P<0.001)

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improved LONP1 expression but same treatment failed to upregulate the LONP1 protein levels

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in LONP1 knocked down PC12 cells (Fig.3A&C). Indeed, Oxyblot analysis revealed the carbonylation of proteins was significantly (P<0.001) elevated in HG and LONP1 knocked down

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PC12 cells when compared to normal group (Fig.3B). However, SRT1720 treatment diminished

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the carbonylation of proteins in hyperglycemic PC12 but failed to alter in LONP1 knocked down

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PC12 cells (Fig.3B). Besides, Aco-2 being a specific substrate to LONP1, its protein expression (Fig.3C&D) and activity (Fig.3E) was significantly decreased in HG exposed PC12 cells and this effect is reversed by SRT1720 treatment. But, SRT1720 treatment was unable to recover the aco2 expression and activity in LONP1 silenced PC12 cells. Based on these results further we set out to determine the effect of SRT1720 and LONP1 transfection on mitochondrial biogenesis and function. 4.3. Effect of SRT1720 and siRNA mediated LONP1 transfection on mitochondrial biogenesis in HG exposed PC12 cells

Journal Pre-proof HG stimulus (30mM) to PC12 cells significantly (P<0.001) reduced ATP synthase c expression as evident from immunofluorescence and western blotting results (Fig.4A&B). SRT1720 cotreatment significantly (P<0.05) increased the expression (Fig.4A&B) of ATP synthase c in HG exposed PC12 cells (Fig.4A&B). While ATP synthase c expression was significantly (P<0.001) declined in siRNA transfected PC12 cells, to our surprise, the ATP synthase c expression levels were not reversed by SRT1720 treatment in siRNA transfected PC12 cells (Fig.4A&B). These

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results suggest that PGC 1α deacetylated by Sirt1 may induce ATP synthase c expression

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through LONP1 interface.

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4.4. Effect of SRT1720 administration and siRNA mediated LONP1 transfection on

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mitochondrial function in HG exposed PC12 cells

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Hyperglycemic stimulus (30mM) and siRNA transfection to PC12 cells significantly (P<0.001) resulted in mitochondrial membrane depolarization when compared to normal cells as seen in

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JC-1 assay (Fig.5G&H). SRT1720 co-treatment with high glucose exposure inhibited this

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mitochondrial depolarization significantly (P<0.05) when compared to hyperglycemic cells as

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seen in JC 1 results (Fig.5G&H). Additionally, mitochondrial complex activities; complex I, II, IV & V were significantly (P<0.001) compromised in HG exposed PC12 cells when compared to normal cells as shown in Fig.4C. SRT1720 treatment improved these complex activities in HG exposed PC12 cells as shown in Fig.4C. However, LONP1 transfection in PC12 cells significantly (P<0.001) displayed mitochondrial depolarization (Fig.5G&H) and reduced mitochondrial complex activities with or without HG/SRT1720 treatment. Further to corroborate these results we performed Mito stress assay using extra flux analyzer (Seahorse XF analyzer) (Fig.5A). HG stress significantly reduced basal (Fig.5B) and maximal respiration (P<0.001) (Fig. 5C), decreased ATP production (P<0.001) (Fig.5D) and spare respiratory capacity (P<0.001)

Journal Pre-proof (Fig.5E) when compared to normal cells. SRT1720 co treatment with HG stress reversed basal (P<0.01 v/s HG) and maximal respiration (P<0.05 v/s HG), restored ATP levels (P<0.05 v/s HG) and spare respiratory capacity (P<0.01 v/s HG). However, siRNA mediated LONP1 knockdown in PC12 cells with or without HG/SRT1720 treatment significantly (P<0.001) decreased minimal respiration, maximal respiration, ATP production and spare respiratory capacity when compared

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to the normal control group as shown in Fig. 5B,C,D&E.

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5. Discussion There is sufficient evidence that neurons demand high energy in the form of ATP derived from optimal glucose oxidation to drive vital processes of the cell (Berndt and Holzhütter, 2013). Previous studies suggested mitochondrial dysfunction as a key mechanism in HG-mediated neurotoxicity and further reported that Sirt1 activation improves mitochondrial function and

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biogenesis in experimental diabetic neuropathy (DN) (Cheng et al., 2016). However, it is still

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unclear as to how Sirt1 controls mitochondrial bioenergetics. In this study we explored the role

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of Sirt1 activation by SRT1720 treatment for controlling mitochondrial bioenergetics and also

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studied whether this effect is independent or dependent of LONP1 activation.

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The current study witnessed the downregulation of Sirt1, PGC 1α, NRF2, SOD2, LONP1, Aco-2 and HSP27 proteins in HG exposed PC12 cells and these protein expressions were upregulated

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by SRT1720 treatment. Moreover, mitochondrial function in terms of basal respiration, maximal

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respiration, spare respiratory capacity, ATP production, electron transport chain (ETC) complex

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activities and Aco-2 activity were improved by 4 μM SRT1720 treatment in HG exposed PC12 cells. Silencing of LONP1 gene using accel siRNA transfection assay kit furthered mitochondrial dysfunction in PC12 cells, interestingly SRT1720 treatment also failed to improve mitochondrial function of LONP1 deficient HG exposed PC12 cells. PGC 1α, nuclear factor erythroid 2 (NFE2)-related factor 2 (Nrf2) and Heat shock factor-1 (HSF1) deacetylated by Sirt1, immunolocalizes into the nucleus and activates several co-transcription factors (Hock and Kralli, 2009; Xue et al., 2016). Within the context of mitochondria, PGC1α activates nuclear respiratory factors, Nrf2 which inturn activates antioxidant enzymes and HSF-1

Journal Pre-proof activates HSPs to induce mitochondrial biogenesis and function (Choi et al., 2014; Westerheide et al., 2009). In line with previous reports, SRT1720 treatment increased the expressions of Sirt1, PGC 1α, NRF2, SOD2 and HSP 27 in HG exposed PC12 cells and thereby improved mitochondrial function and biogenesis. We also noticed the reduced expression of LONP1 in HG treated PC12 cells and SRT1720 treatment in HG exposed PC12 cells boosted the expression of LONP1. Nuclear- encoded LONP1 upregulation depends upon the transcriptional activity of

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NRF2 which is evident by identifying the presence of NRF2 consensus binding site at LONP1

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promoter region; - 623/+1 (Bahat et al., 2015; Pinti et al., 2011). With this, we could infer that

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LONP1 expression may be enhanced via Sirt1-PGC1α-NRF2 axis. Decades of research in

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exploring the role of LONP1, evidenced the loss and gain of mitochondrial function by conducting knock out and knock in of LONP1 gene respectively in various experimental models

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(Quirós et al., 2014; Strauss et al., 2015).

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Further, to understand the connection between Sirt1 and LONP1 in improving mitochondrial

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function, we convoked our experiments to find the effect of SIRT modulator, SRT1720 on mitochondrial function and biogenesis in presence and absence of LONP1. HG exposure and

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siRNA mediated knockdown of LONP1 in PC12 cells increased levels of carbonylated proteins, decreased aco-2 expression, diminished aco-2 activity, displayed depolarized mitochondria and compromised mitochondrial activity. As anticipated SRT1720 reversed these effects in HG insulted PC12 cells but failed to show similar effects in LONP1 gene transfected cells. These results suggest that SRT1720 may upregulate LONP1 expression which has resulted in bettering aco-2 expression and activity thereby, reducing the proteotoxic stress. LONP1 knockdown in mouse embryos have shown accumulation of large protein inclusion bodies with defective membrane potentials when compared to wild type embryos of same strain (Quirós et al., 2014)

Journal Pre-proof and have also demonstrated that oxidized aco-2 and oxidized mitochondrial transcription factor A (TFAM) are specific substrates that bind to LONP1 and get repaired into functional forms (Quirós, 2018; Strauss et al., 2015). These facts further corroborate our findings that LONP1 function is critical in protecting mitochondrial health in hyperglycemic stress. We also evaluated spare respiratory capacity (difference between ATP produced by oxidative

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phosphorylation at basal and at maximal respiration) which reflects the whole mitochondrial health in terms of ATP production. Hyperglycemic stress and knockdown of LONP1 gene also

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resulted in dipping of basal and maximal respiration, decreased spare respiratory capacity, ATP

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production and complex activities when compared to normal cells. Concurrently, SRT1720

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treatment to HG exposed PC12 cells significantly ameliorated these functional abnormalities of

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mitochondria but failed to produce a similar effect in LONP1 knockdown HG exposed PC12 cells. It can be inferred that, this dependency of ATP synthase c expression on LONP1 may be

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attributable to the transcriptional ability of LONP1 binding to mitochondrial DNA (Fu and

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Markovitz, 1998). But, it is still not apparent which mitochondrial proteins are controlled by transcriptional activity of LONP1 after its binding to mitochondrial DNA which has to be

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researched further. Based on these results, we anticipate that loss of transcriptional activity, chaperone activity and protease activity of LONP1 may be responsible for altered mitochondrial function and SRT1720 treatment increases LONP1 levels via stimulating Sirt1-PGC1α-NRF2 axis and may contribute in maintenance of mitochondrial quality in terms of mitochondrial function, biogenesis and proteostasis. In conclusion, our results hint towards a positive link between Sirt1 and LONP1 mediated through Sirt1-PGC1α-NRF2 in the regulation of mitochondrial bioenergetics during HG mediated mitochondrial dysfunction which may add a new dimension in developing therapeutics

Journal Pre-proof for hyperglycemia induced complications. SRT1720 administration offered mitoprotection against hyperglycemia induced mitochondrial damage to the cells and thus can further be explored for finding the exact patho-mechanisms responsible for the protective outcome against hyperglycemia induced stress.

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Acknowledgement

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Authors would like to acknowledge the financial support provided by the Department of

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Pharmaceuticals, Ministry of Chemicals and Fertilizers, Government of India for carrying out

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this work. Also, Authors sincerely thank to Dr. Eshvendar Reddy Kasala for proofreading of this

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manuscript.

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Conflicts of Interest: The authors have no conflicts of interest to declare

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Fig 1: Effect of SRT1720 on cell viability SRT1720 has found to produce significant neurotoxicity at concentrations greater than 6 µM (IC50: 10.14µM).Graphical representation of cell viability of PC12 cells treated with different concentrations (100-0.39 µM) of SRT1720. Values are expressed as mean ± SEM (n=3). **P<0.01 and ***P<0.001 Vs Normal PC12 cells. Fig 2: Effect of SRT1720 on expression levels of Sirt1, PGC 1α, NRF2 and SOD2 in hyperglycemic PC12 cells (A) Representative confocal microscopic pictures of rhodamine conjugated PGC 1α expression (B) Representative Immunoblots of Nerve growth factor differentiated PC12 cells and (C) Quantification of Sirt1, PGC 1α, NRF2 and SOD2 expressions in PC12 cells. Values

Journal Pre-proof are expressed as mean ± SEM (n=3). NC: normal PC12 cells, HG: Hyperglycemic PC12 cells, HG+SRT: SRT1720 treated cells, HG+SRT+LONP1-T: transfected cells exposed to glucose and SRT1720, LONP-T: Transfected PC12 cells and siRNA negative control. ***P<0.001, **P<0.01, *P<0.05 v/s HG and ^^^P<0.001, ^^P<0.01 v/s NC Fig 3: Effect of SRT1720 on expression of LONP1 in high glucose exposed PC12 cells (A) Representative photomicrographs of FITC conjugated LONP1 expression in hyperglycemic PC12 cells (B) Representative Oxyblot analysis of carbonylated proteins in

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differentiated PC12 cells, out of eleven lanes, the 1st,3rd,5th,7th,9th and 11th lane indicates

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the western blots obtained from derivatized protein samples with 1X DNPH solution and rest of the lanes indicates the western blots obtained from protein samples incubated with 1X

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derivatization control solution (C) Representative Immunoblots of NGF differentiated PC12 cells and (D) Quantification of LONP1, Aconitase-2, and HSP 27 expressions in PC12 cell

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lysates (E) Aconitase-2 activity in freshly prepared mitochondrial lysates of neuronal PC12

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cells. Values are expressed as mean ± SEM (n=3). NC: normal PC12 cells, HG: Hyperglycemic PC12 cells, HG+SRT: SRT1720 treated cells, HG+SRT+LONP1-T: transfected cells exposed to glucose and SRT1720, LONP-T: Transfected PC12 cells and

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^P<0.05 v/s NC

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siRNA negative control. ***P<0.001, **P<0.01, *P<0.05 v/s HG and ^^^P<0.001, ^^P<0.01,

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Fig 4: Effect of SRT1720 on ATP synthase expression and mitochondrial complex

(A) Representative confocal microscopic pictures of FITC conjugated ATP synthase c expression in hyperglycemic PC12 cells (B) Representative Immunoblots of NGF differentiated PC12 cells and Quantification of ATP synthase c expression in PC12 cells (C) Representing mitochondrial complex activities in freshly prepared mitochondrial lysates of neuronal PC12 cells. Values are expressed as mean ± SEM (n=3). NC: normal PC12 cells, HG: Hyperglycemic PC12 cells, HG+SRT: SRT1720 treated cells, HG+SRT+LONP1-T: transfected cells exposed to glucose and SRT1720, LONP-T: Transfected PC12 cells and siRNA negative control. ***P<0.001, *P<0.05 v/s HG and ^^^P<0.001, ^^P<0.01 v/s NC

Journal Pre-proof Fig 5: Measurement of mitochondrial function and mitochondrial membrane potential (ΔΨm) in cultured PC12 cells (A) Oxygen consumption rate (OCR) was measured at basal level with the subsequent and sequential addition of oligomycin (1µM), FCCP (2µM), and rotenone+antimycin A (1µM each) to the NC: normal PC12 cells, HG: Hyperglycemic PC12 cells, HG+SRT: SRT1720 treated cells, HG+SRT+LONP1-T: transfected cells exposed to glucose and SRT1720, LONP-T: Transfected PC12 cells and siRNA negative control. The levels of OCR are

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normalized with per mg total protein. Agilent wave software are used to measure the OCR data and the following histograms represents the quantification of (B) basal respiration, (C)

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maximal respiration, (D) ATP production and (E) spare respiratory capacity. (F) Representative fluorescence microscopic images showing JC-1 aggregates emitting red

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fluorescence (first lane), JC1 monomers emitting green fluorescence (second lane) and merge

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of red and green florescence (third lane). (G) Representative dot plotted graph of JC-1 by flow cytometer represents the scattered PC12 cells into red JC-1 and green population and

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(H) respective quantification of red: green fluorescence ratio indicating depolarized

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Graphical abstract

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mitochondria. Values are expressed as mean ± SEM (n=3)

SRT1720 treatment protected the differentiated PC12 cells from hyperglycemic stress by improving mitochondrial biogenesis and function. LONP1 is induced by SRT1720 treatment and the mitoprotective role of Sirt1 may depend upon the LONP1 activation.

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