KLF2 Regulates Neural Differentiation of Dental Pulp-derived Stem Cells by Modulating Autophagy and Mitophagy

Transplantation of stem cells for treating neurodegenerative disorders is a promising future therapeutic approach. However, the molecular mechanism underlying the neuronal differentiation of dental pulp-derived stem cells (DPSC) remains inadequately explored. The current study aims to define the regulatory role of KLF2 (Kruppel-like factor 2) during the neural differentiation (ND) of DPSC. We first investigated the transcriptional and translational expression of KLF2, autophagy, and mitophagy-associated markers during the ND of DPSC by using quantitative RT-PCR and western blot methods. After that, we applied the chemical-mediated loss- and gain-of-function approaches using KLF2 inhibitor, GGPP (geranylgeranyl pyrophosphate), and KLF2 activator, GGTI-298 (geranylgeranyl transferase inhibitor-298) to delineate the role of KLF2 during ND of DPSC. The western blot, qRT-PCR, and immunocytochemistry were performed to determine the molecular changes during ND after KLF2 deficiency and KLF2 sufficiency. We also analyzed the oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) using the Seahorse XFe24 analyzer. Our study demonstrated that the expression level of KLF2, autophagy, and mitophagy-associated markers were significantly elevated during the ND of DPSC. Next, we found that the KLF2 inhibitor, GGPP significantly reduced the ND of DPSC. Inversely, KLF2 overexpression accelerated the molecular phenomenon of DPSC’s commitment towards ND, indicating the crucial role of KLF2 in neurogenesis. Moreover, we found that the KLF2 positively regulated autophagy, mitophagy, and the Wnt5a signaling pathway during neurogenesis. Seahorse XFe24 analysis revealed that the ECAR and OCR parameters were significantly increased during ND, and inhibition of KLF2 marginally reversed them towards DPSC’s cellular bioenergetics. However, KLF2 overexpression shifted the cellular energy metabolism toward the quiescent stage. Collectively, our findings provide the first evidence that the KLF2 critically regulates the neurogenesis of DPSC by inducing autophagy and mitophagy.

aid in ameliorating continuous neuron degradation [4,5].Over the past few decades, extensive preclinical research has been done to prove the therapeutic efficacy of different stem cells, including mesenchymal stem cells (MSC), embryonic stem cells, and induced pluripotent stem cells for neurodegenerative disorders [6,7].
Human dental pulp stem cells (DPSC) exfoliate from the third molar teeth of humans and are of particular interest for clinical purposes because of their relative ease in accessibility, self-renewal, and multi-potency in bone, dental, and neural tissue regeneration [8].DPSC express mesenchymal markers, including CD73, CD90, CD105, CD146, and STRO-1 [9].Moreover, DPSC showed comparatively higher innate potential for neuronal differentiation due to their embryonic neural crest origin during development [10,11].Additionally, these cells expressed basal levels of neuronal markers such as PAX6, GBX2, and Nestin [12].Therefore, DPSC potentially cured neuronal loss in animal models of several damaged central nervous systems (CNS), such as spinal cord injury, stroke, Parkinson's disease, Alzheimer's disease, and retinal degeneration [13][14][15].Recent studies suggested that transplantation of pre-differentiated MSC exhibits a better therapeutic effect in the in vivo model system [16,17].Several studies established the in vitro model system to induce the neuronal differentiation of DPSC [18][19][20].However, a better understanding of the molecular signatures that occurred during neuronal differentiation of DPSC is imperative to enhance our in-depth knowledge of neurogenesis and develop more rational regenerative therapy for treating neurodegenerative diseases effectively.
Autophagy is a highly conserved catabolic pathway involved in self-renewal, differentiation of cells, and embryonic development, and acts as an intracellular caretaker by providing the nutrients for cellular functions and selectively eliminating the residual cytosolic wastes, damaged mitochondria, and toxic protein aggregates [21].The formation of autophagosomes, double membrane-bound vesicles, initiates autophagy, the fusion of autophagosomes with lysosomes forms autolysosomal vesicles, which degrades autophagic cargos and the subsequent recycling of macromolecules precursors.Selective elimination of damaged mitochondria through autophagy is known as mitophagy [22,23].Accumulating data suggest that autophagy and mitophagy play a critical role during the neural differentiation (ND) of stem cells to meet the desire for a high amount of energy and nutrition.Autophagy and mitophagy also regulate several other signaling pathways involved in adequate ND [24][25][26].Understanding the transcriptional regulators of such pathways will open new avenues in regenerative medicine.
Krüppel-like transcription factors (KLFs), a novel family of transcription factors, are implicated in regulating proinflammatory activation of myeloid cells [27], cell proliferation, and differentiation [28].KLF2, a principal member of the KLFs family, contains highly conserved Cys-2/ His-2 zinc-finger domains that bind to the GC-rich region and CACCC-elements of DNA sequences in the promoter of their downstream targets, acting as transcriptional activators or repressors [29].Previous studies reported that KLF2 underpins a broad range of biological functions such as neovascularization, endothelial and bone cell proliferation and function [30,31], maintenance of B cell homeostasis and plasma cell homing [32], mediation of the granulocyte inflammatory response [33], immune cell function [29], and promotion of inflammatory T cells.Previously, our lab demonstrated that KLF2 plays a critical role in maintaining bone homeostasis through the modulation of autophagy and mitochondrial functions [31,34].However, the physiological function of KLF2 on ND has not been explored yet.The current study is undertaken to understand how ND of DPSC affects the basal level of KLF2 and how altered expression of KLF2 regulates the basal level of autophagy and mitochondrial functions.Our findings might allow us to develop a strategy by manipulating the level of KLF2 during neurogenesis, which aids in existing approaches for regenerative therapies for neurodegenerative diseases.

KLF2 Sufficiency and KLF2 Deficiency
To overexpress the KLF2, DPSC were cultured in MEME with 2.5 μM of KLF2-inducer GGTI-298 (Sigma Aldrich, St. Louis, MO, USA, #G5169).After 24 h, the MEME medium was replaced by NDM with GGTI-298.The reduction of KLF2 was performed with 10 μM of KLF2 inhibitor Geranylgeranyl pyrophosphate (GGPP, Sigma Aldrich, St. Louis, MO, USA, #G6025).DPSC were cultured in NDM with GGPP for 7 days at 37 °C in a CO 2 incubator.The old medium was replaced by a fresh medium every third day.

qRT-PCR Analysis
The total RNA was extracted using TRIzol reagent, and the RNA concentration was detected by NanoDrop 8000 (Thermo Scientific).One μg of total RNA for each sample was reversed transcribed with a cDNA kit (Thermo Fischer Scientific, Waltham, MA, USA, #4,387,406) following the manufacturer's instruction.Then, cDNA was amplified with SYBR Green PCR (Thermo Fischer Scientific, Waltham, MA, USA, #4,309,155) by a thermal cycler PCR instrument (CFX96 Real-Time System, Bio-Rad Laboratories, Hercules, USA).The cycling parameters were as follows: initial hold at 95 °C for 10s, 40 cycles at 95 °C for 15s, 60 °C for the 60s, then 65 °C for 15s and 95 °C for 50s.Relative target transcripts expression was analyzed by normalizing the calculated Ct values (threshold cycle) of target genes with internal control, GAPDH.All primer sets of target genes used in this study are included in Table S1.

Western Blot Analysis
Each group of cells was lysed in pre-cooled RIPA buffer (Millipore Sigma Aldrich Corporation, Burlington, MA, USA, #20-188), supplemented with protease and phosphatase inhibitors (Thermo Scientific, Waltham, MA, USA #78,441) for 30 min on ice.The cell lysate was collected by centrifugation at 13,200×g for 15 min, and total cell protein concentration was quantified with a precision red advanced protein assay kit (Cytoskeleton Inc., Denver, CO, USA, #ADV02).An equal amount of protein (30 μg) for each sample and protein marker (Millipore Sigma Aldrich Corporation, Burlington, MA, USA, #GERPN800E) was loaded on 8-12% SDS-PAGE gels.Proteins were separated electrophoretically and then transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA, #1,620,115).The nitrocellulose membrane was then blocked with 5% non-fat skim milk or 5% bovine serum albumin (BSA, Sigma Aldrich Corporation, St. Louis, MO, USA, #A7906) for 2 h at room temperature (RT).After blocking, membranes were incubated overnight with primary antibody diluted in 5% BSA (dissolved in Tris-buffered saline with 0.1% Tween 20 detergent, TBST, Boston Bioproducts, Milford, MA, USA, #IBB-180) at 4 °C.The membrane was washed thrice with TBST for 10 min each and re-incubated in species-specific horseradish peroxidase (HRP) conjugated secondary antibodies for 1 h at RT. Afterward, the membrane was re-washed and exposed to an enhanced chemiluminescence substrate (Amersham Pharmacia Biotechnology, Amersham, UK, #RPN2232).The protein bands were visualized and band intensity was normalized to the internal control, GAPDH using Image J software (National we sequentially administered glucose (1 mM), oligomycin (1 μM), and 2-deoxyglucose (10 mM).ECAR parameters were calculated using Seahorse Wave software 2.6.1.

Mitochondrial Reactive Oxygen Species (ROS) Analysis
Mitochondrial ROS generation was estimated using the MitoSOX red compound (Thermo Scientific, Waltham, MA, USA #M36008).Non-fluorescence MitoSOX red oxidized with mitochondrial superoxide's free radicals and produce red fluorescence.In Brief, DPSC (2 × 10 3 ) were seeded on a six-well plate overnight.Next, the medium was replaced by the NDM and incubated for 7 days.After incubation, cells were washed with 1× PBS and incubated in a 5 μM working solution prepared in Hanks' balanced salt solution (HBSS) of the MitoSOX red for 15 min at 37 °C.After washing with 1× PBS, the images of cells were captured and analyzed using Leica Stellaris 8 Falcon STED confocal microscope at excitation and emission of 510/588 nm.

Cellular ROS Analysis
Intracellular ROS generation was evaluated by 2',7'-dichlorodihydrofluorescein diacetate (DCFDA, Sigma Aldrich, St. Louis, MO, USA, #4091-99-0).Intracellular ROS reacts with non-fluorescent DCFDA and converts it into fluorescence molecules.In short, DPSC (2 × 10 3 ) were seeded on a six-well plate for overnight.Next, the medium was replaced by NDM and incubated for 7 days.After incubation, cells were washed with 1× PBS and incubated in a 5 μM working solution (prepared in PBS of the DCFDA) for 20 min at 37 °C.After washing with 1× PBS, the images of cells were captured and analyzed using Leica Stellaris 8 Falcon STED confocal microscope at excitation of 495 nm and emission of 529 nm.

Statistical Analysis
Statistical analysis was performed using Graph Pad Prism 5.0 (Graph Pad Software, San Diego, CA, USA) by implementing the one-way analysis of variance (ANOVA).Tukey's multiple comparisons test and Dunnett's multiple comparison test were used for comparing multiple groups.All results are expressed as mean ± standard error of the mean (SEM).
Institutes of Health, Bethesda, MD, USA).Details of primary antibodies are mentioned in Table S2.

Immunocytochemistry
DPSC were plated at 2 × 10 3 cells/well in a 6-well plate and incubated for seven days in a CO 2 incubator at 37 °C.Afterward, the culture media was aspirated, and cells were washed three times with phosphate-buffered saline (PBS), followed by fixed with 4% PFA (Santa Cruz Biotechnology, ChemCruz, Dallas, TX, USA, #sc-281,692) for 20 min at RT.The cells were then permeabilized with 0.3% Triton X100 (Sigma Aldrich, St. Louis, MO, USA, #T9284) and 1% BSA in PBS (pH 7.4) for 10 min and probed with Primary antibodies (Details given in Table S2) overnight at 4 °C after blocking with 5% BSA and 0.3% Triton X100 in PBS for 1 h.The following day, the cells were washed thrice with PBS for 5 min each and incubated in the dark with goat anti-rabbit/mouse conjugated with Alexa 488/649 (1:1000) for 1 h at RT.The cells were re-washed and mounted with DAPI (Invitrogen, Thermo Scientific, Waltham, MA, USA #P36931).Images were captured and analyzed by using a DMI8 inverted super-resolution confocal microscope (Leica Stellaris 8 Falcon STED).

Cellular Bioenergetics Analysis
The oxygen consumption rate (OCR, mitochondrial respiration) and extracellular acidification (ECAR, glycolysis) were measured during ND of DPSC using an XF24 Extracellular Flux Analyzer (Seahorse Bioscience, Billerica, MA, USA).In brief, differentiated cells were dissociated and re-seeded in the XF 24-well cell culture microplate (Seahorse Bioscience, Copenhagen, Denmark) at the cell density of (3 × 10 4 ) for 24 h with 1 mL of respective media for each condition.At the same time, a sensor cartridge in Seahorse XF calibrant was hydrated at 37 °C in a non-CO 2 incubator overnight.On the day of Seahorse analysis, the culture was washed twice with XF base media supplemented with pyruvate, glutamine, and glucose, and all wells were filled with 500 μL of the medium/well.Then plates were incubated in a non-CO 2 incubator at 37 °C for 1 h.After that, Seahorse XF calibrant was supplemented with the ATP synthase inhibitor, oligomycin (1.5 μM), uncoupler carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP, 1 μM), complex III inhibitor rotenone, and antimycin A (0.5 μM) and then first calibrated the instrument using Seahorse Wave software 2.6.1.The calibrant plate was then replaced with an XF 24-well cell culture microplate, and oxygen consumption rate (OCR) was measured in terms of ATP turnover, maximal respiration, and reserve respiratory capacity.To perform the glycolysis stress test, (Fig. 1a).The results showed that the number of TUJ1positive cells was hiked after induction of ND.Almost all differentiated cells were positive for MAP2, SYN1, GFAP, and undifferentiated DPSC were negative for those markers.Real-time qPCR analysis showed that the mRNA expression of neural markers (MAP2 and SYN1) was significantly increased in a time-dependent manner during differentiation (Fig. 1b).We further confirmed the ND of DPSC using western blotting analysis and found that the neuron-specific class III β-tubulin or TUJ1 was increased when DPSC differentiated to neuron-like cells.MAP2, a more mature neural marker than TUJ1, was also gradually expressed during differentiation (Fig. 1c and Fig. S1a).To investigate the level of KLF2 during the ND of DPSC, we found that mRNA expression and protein levels of KLF2 were significantly increased during neurogenesis of DPSC.At the same time, the genes of autophagy, BECN1, ATG5, ATG7,

Effect of Neural Differentiation of DPSC on KLF2, Autophagy, Mitophagy, and Wnt Signaling Molecules
To investigate whether DPSC exhibit their efficacy to differentiate into neural lineages, we cultured DPSC with neural differentiation-specific media and investigated the morphological changes that occurred during ND of DPSC on day 7 using a bright field microscope.Undifferentiated DPSC exhibited spindle-shaped morphology while differentiated cells showed neural-like pyramidal shape with short dendrite projections and a longer projection similar to the axon (Fig. 1a).Next, we examined the expression of neural-specific marker molecules like MAP2, GFAP, SYN1, and TUJ1 after induction of ND using immunocytochemistry staining correlating to transcription data, was significantly decreased in the presence of GGPP compared to the ND group.TUJ1 expression was strikingly increased in the ND group and restored to the basal level of DPSC (Fig. 2c and d).Subsequently, the effect of KLF2 deficiency on the morphology of neural-like cell differentiation was assessed using bright field microscopy.We analyzed the soma area and neurite growth in the obtained images using ImageJ software.As shown in the graphs (Fig. 2e and f), an insignificant difference between groups was observed suggesting that partial KLF2 deficiency only suppresses the ND at the transcriptional and translational levels.
We next examined the effect of KLF2 deficiency on autophagy during ND.We observed that the mRNA expression level of autophagy markers (ATG7, BCEN1, and LC3B) was strongly decreased after partial KLF2 deficiency (Fig. 3a).In the western blot analysis, we found that the protein expression level of ATG5, LC3B, and LAMP1 was also decreased after the ND treated with GGPP (Fig. 3b and Fig. S3), indicating that KLF2 plays a vital role in the autophagy process.Immunofluorescences analysis of ATG5 and LC3B was strongly corroborating with western blot analysis.Likewise, mitophagy is essential for ND to eliminate the damaged mitochondria.So, we next wanted to investigate how the limited expression of KLF2 affects the transcriptional and translational of mitophagy-associated markers.The transcript level of PINK1 was marginally decreased in the ND with GGPP, while the gene expression of Parkin was strikingly decreased after the deficiency of KLF2 compared to ND (Fig. 3a).Corroborating to transcription data, the protein expression of Parkin in the ND with GGPP was remarkably decreased.In contrast, the protein expression of DRP1 was slightly downregulated in the GGPP-treated ND group (Fig. 3b and Fig. S3).Next, we performed immunocytochemistry to visualize the protein expression of ATG5, LC3B, PINK1, and Parkin.As anticipated, ATG5, LC3B, PINK1, and Parkin expression in DPSC and KLF2-deficient neural differentiated cells were approximately similar while increased during the ND (Fig. 3c and d).Our data indicate that KLF2 is essential to regulating mitophagy during ND of DPSC.We also determined whether KLF2 deficiency alters Wnt signaling during ND.We observed a significant transcriptional decrease in Wnt5a and DVL3 levels in KLF2deficient neural differentiated cells.Western blot analysis represents that a marginal reduction in protein expression of Wnt5a and DVL3 (Fig. 3a and Fig. S3).
Next, we investigated the intracellular ROS generation and mitochondrial superoxide generation by MitoSOX and DCFDA staining after NDs for 7 days because ROS acts as a signaling stimulator for cellular pathways.We found that differentiated neuron-like cells showed a significant decrease in ROS and mitochondria superoxide production and LC3B were also significantly increased after 7 and 10 days of ND (Fig. 1d).The western blot analysis revealed that after 7 and 10 days of induction, the ATG5, LC3B, and Beclin1 were highly expressed.LAMP1, a lysosomal membrane maker, facilitates the successful maturation of both autophagosomes and phagosomes.Like early autophagy markers, LAMP1 expression also gradually increased during ND (Fig. 1e and Fig. S1b).We further assessed the mitochondrial dynamics markers and found that the PINK1, Parkin, DRP1, and FIS1 significantly increased after 10 days of differentiation (Fig. 1f).We determined similar trends in protein expression of Parkin and DRP1, however, FIS1 did not show remarkable differences (Fig. 1g and Fig. S1c).These data suggest that ND in DPSC might require a transcriptional regulator, KLF2, along with autophagy and mitophagy-associated genes.To get more insight into the molecular mechanism underlying the ND of DPSC, we examined the Wnt pathway-specific markers (Wnt5a, LRP6, CTNNB1 and DVL3, GSK3B).RT-PCR analysis revealed that the gene level of Wnt5a/b and LRP6 was significantly increased during ND.However, β-catenin was significantly upregulated during only the initial days of ND (Fig. 1h).In the western blot analysis, WNT5a/b and DVL3 expression were increased at 7 and 10 days of differentiation.Correspondingly, protein expression of GSK3α/β declined during differentiation.However, mRNA expression of GSK3B was dramatically increased time-dependent manner (Fig. 1i and Fig. S1d).The difference in protein and gene expression may be due to post-translational modification.

Effect of KLF2 Deficiency on Autophagy, Mitophagy, Mitochondrial Dynamics, and Neural Differentiation of DPSC
To determine the role of KLF2 in ND, we cultured DPSC in the NDM without or with GGPP, an inhibitor of KLF2, and examined the consequences involved in neural-like cell differentiation of DPSC.We found the mRNA expression of KLF2 was marginally decreased in the presence of GGPP (ND + GGPP) compared to the neural differentiated control (ND), indicating that GGPP leads to KLF2 deficiency during ND.Concurrently, partial KLF2 deficiency led to a conspicuously decrease in the mRNA expression of neural markers, MAP2, SYN1, and NeuN, indicating that KLF2 is an essential transcription factor required for ND of DPSC (Fig. 2a).We next probed into the translational level changes of KLF2 and MAP2 in the DPSC during ND in the presence and absence of GGPP.As expected, the protein expression of KLF2 and MAP2 was decreased in ND + GGPP group compared to the ND group (Fig. 2b and Fig. S2).We also performed immunocytochemistry to detect the expression level of MAP2 and TUJ1.We found that MAP2 expression, respiration and spare respiration capacity during ND of DPSC was detected while GGPP slightly shifted such mitochondrial metabolism effect towards DPSC (Fig. 4c and Fig. S6a).We next sought to examine the extracellular acidification rate (ECAR) defining the glycolytic metabolism during ND.We found that the neural differentiating cells catabolize the glucose through the glycolysis pathway to fulfill energy demand in the absence of glutamine.The non-glycolytic acidification, glycolysis, glycolysis capacity, and glycolytic reserve were significantly hiked in neuronal-like differentiated cells, however, partial KLF2-deficient neural-like compared to DPSC.After the KLF2 deficiency, ROS and superoxide production was found to be shifted towards the basal level (Fig. 4a and b, Fig. S8).To investigate the mitochondrial functions, we examined the mitochondrial oxygen consumption rate (OCR) in basal respiration, mitochondrial respiration, spare respiration capacity conditions, and ATP production.The basal respiration and ATP production was significantly decreased in the ND control group compared to the undifferentiated control group.In contrast, an insignificant was observed between GGPP-treated ND and ND control group.A significant hike in maximum KLF2 overexpression accelerated ND rate (Fig. 5a).Western blot analysis showed that expression of KLF2 was increased when GGTI-298 was added to NDM.Consistently, the protein expression of MAP2 and TUJ1 was also marginally increased in KLF2 overexpressed group (Fig. 5b and Fig. S4).Next, we performed the immunostaining for MAP2 and TUJ1, and image intensity analysis revealed that overexpression of KLF2 induced neuronal differentiation rate compared to ND control (Fig. 5c and d).Subsequently, we observed that the overexpression of KLF2 increased marginally soma area and neurite growth compared to ND, indicating that KLF2 overexpression induces the rate of ND (Fig. 5e and f).
To further determine the function of KLF2 on autophagy during ND, we examined the alteration of transcriptional and translational in autophagy-associated markers after differentiated cells exhibited less non-glycolytic acidification and glycolysis (Fig. 4d and Fig. S6b).

Effect of KLF2 Overexpression on Autophagy, Mitophagy, Mitochondrial Dynamics, and Neural Differentiation of DPSC
Next, we focused on defining how KLF2 regulates neural differentiation; to do so, we stimulated DPSC with KLF2 inducer GGTI-298 and cultured it with NDM for 7 days.We found that the GGTI-298-treated DPSC were differentiated to neural-like cells, and showed higher KLF2 expression than cells differentiated in only the NDM.The mRNA expression of MAP2 and SYN1 was also markedly increased in the presence of GGTI-298.NeuN expression was increased only after the addition of GGTI-298, indicating in protein expression was detected in KLF2 overexpressed differentiated neural cells compared to neural differentiated cells (Fig. 6c and d).
Next, we wanted to examine the effect of KLF2 overexpression on intracellular ROS generation and mitochondrial superoxide production.An insignificant difference between the ND group and the ND + GGTI-298 group was observed in ROS production determined by MitoSOX and DCFDA staining (Fig. 7a and b, and Fig. S9).To investigate the mitochondrial functions, we analyzed that the addition of GGTI-298 in ND media strikingly reduces the basal respiration and ATP production of differentiated cells, indicating the neuronal-like differentiated cells may be shifted towards maturation (Fig. 7c and Fig. S7a).Similarly, in ECAR analysis, non-glycolytic acidification was reduced compared to neuronal-like differentiated cells.Though, glycolysis and glycolytic capacity were not significantly altered in ND + GGTI-298 group (Fig. 7d and Fig. S7b).KLF2 overexpression.A slight increase in the transcript level of ATG5, ATG7, and LC3B was determined in KLF2overexpressed ND cells compared to differentiated neural cells (Fig. 6a).Corroborating to mRNA level, western blot analysis for autophagosome markers (ATG5, LC3B, and Beclin1) and autolysosome marker (LAMP1) collectively revealed that KLF2 overexpression promotes marginal autophagy and facilitates neuronal differentiation (Fig. 6b and Fig. S5).We further wanted to investigate how KLF2 overexpression affects mitophagy during ND.Similar to autophagy, KLF2 overexpression slightly increased the mRNA expression of mitophagy-associated genes, PINK1, Parkin, FIS1, and DRP1, compared to normal ND (Fig. 6a).We further examined whether these effects were consistent with the translational level.We performed western blot analysis for Parkin and DRP1 and observed that Parkin expression was significantly increased compared to the ND group, while DRP1 expression persisted the same in both groups (Fig. 6b and Fig. S5).In the immunostaining analysis for ATG5, LC3B, PINK1, and Parkin, a dramatically increased DPSC possess inherent immunomodulatory and neuroprotective properties [4,5].Therefore, they have been subjected to clinical studies to cure neurodegenerative disorders more effectively.The molecular changes that occur during ND of DPSC remain unclear.Autophagy and mitophagy play a vital role during the proliferation and differentiation of stem cells.Autophagy-associated pathways cross-talk with KLF2, one of the kruppel-like transcription factors (KLFs) belonging to the zinc finger family, to maintain intracellular

Discussion
The replacement of degenerated neurons with pre-differentiated neural-like stem cells appears to be a promising stem cells-based therapy for neurodegenerative disorders [7].Dental-pulp-derived mesenchymal stem cells (DPSC) are highly prolific in differentiating towards multiple lineages, chondrocytes, adipocytes, neurons, and osteoblast cells in the presence of appropriate signals [8,10].Additionally, In this study, we first confirmed the neurogenic differentiation efficacy of DPSC using qRT-PCR, western blot analysis, and immunocytochemistry.Our findings showed that the expression of early and mature neural markers, MAP2, TUJ1, synapsin, and GFAP was gradually increased in a time-dependent manner during ND, indicating the supplemented signals in NDM efficiently induce the commitment of DPSC towards ND.Our findings showed that both autophagy and mitophagy increased during the ND of DPSC, as confirmed by the upregulated level of autophagy markers, LC3B, BECN1, ATG5, ATG7, and LAMP1, and mitophagy markers, PINK1, Parkin, DRP1, and FIS1 homeostasis [28].The molecular function and expression level of KLF2 under healthy physiological and pathological conditions differs.For instance, we previously showed that upregulated KLF2 positively regulates autophagy during osteoblastic differentiation.Conversely, we also demonstrated that KLF2 expression is reduced during the osteoclastogenic differentiation of myeloid cells and negatively regulates autophagy [31].A recent study reported that the KLF2 exhibits a protective role in neonatal hypoxicischemic brain injury in rats by increasing the expressions of the downstream signaling molecules IRF4 and HDAC7 [35].These findings create a curiosity about how KLF2 regulates the molecular changes during neurogenesis.ND of stem cells requires a much higher level of autophagy and mitophagy than the basal level of stem cells to meet the cellular nutrition and energy demands, as evident by previous studies [24].However, the expression level and role of that KLF2 deficiency may affect autophagy and mitophagy during ND of DPSC.Therefore, we are further motivated to investigate how KLF2 deficiency affects autophagy and mitophagy during the ND of DPSC.KLF2 deficiency exhibited a positive correlation in the reduction of autophagy markers (LC3B, ATG5, and LAMP1) and mitophagy markers (PINK1, Parkin, DRP1, FIS1) along with ND marker molecules (MAP2, TUJ1).This study corroborated previous studies in which siRNA-mediated knockdown of KLF2 reduces the autophagy and osteoblast differentiation of DPSC [38].Previous studies demonstrated that GGTI-298 inhibits the protein geranylgeranylation, and subsequently induces significantly the KLF2 expression [37].So, we next performed the experiment with KLF2 overexpression using indicating that the autophagy and mitophagy are essential for the ND.In consistent with our study, recent studies described that autophagy inducer, 5-Azacytidine enhances the ND of human placenta-derived mesenchymal stem cells [26].Interestingly, we found that KLF2 expression was increased during the ND of DPSC time-dependently.To further investigate the principal function of KLF2 on ND, we performed KLF2 deficiency, which slows down the efficacy of DPSC towards neuron-like cells.GGPP, an isoprenoid metabolite, regulates various cell-signaling pathways, including autophagy.Previous studies reported that deletion of GGPP triggers autophagy [36] and the addition of GGPP to cerivastatin-treated HUVEC significantly reduced elevated-KLF2 expression [37].These findings represent differentiated cells can switch between both cellular bioenergetics, glycolysis, and mitochondrial respiration.For instance, in the presence of glucose only, differentiating cells would fulfill their energy demands through glycolysis because the balance between glycolysis and mitochondrial respiration is essential for stem cell differentiation [47].GGPP-mediated KLF2 deficiency reduces extracellular acidification, which is consistent with our data.Taken together, our data established that KLF2 regulates autophagy, mitophagy pathways, and metabolic reprogramming during ND of DPSC.

Conclusion
In summary, we provide the first evidence of the induction of KLF2 during ND of DPSC, which positively regulates the autophagy and mitophagy pathways.We verified that KLF2-mediated autophagy and mitophagy control the ND of DPSC using chemical-mediated partial loss-of-function and gain-of-function approaches for the KLF2 gene.Our results show that KLF2 functions as a modulator of autophagy and mitophagy processes during neurogenesis.The current study unravels a strategy for targeting KLF2 that could be used to develop a new potential regenerative therapeutic approach for neurogenerative disorders.

Fig. 1
Fig. 1 KLF2, autophagy, and Wnt signaling molecules were induced during neural differentiation.a. Confocal microscopy images were shown for neural markers, MAP2 (green), GFAP (green), SYN1 (green) and TUJ1 (red), in differentiated neuronal-like cells (ND) and undifferentiated DPSC.b.The mRNA expression level of MAP2, GFAP, SYN1, and NeuN was determined by qRT-PCR at various time points of neuronal differentiation of DPSC.Statistical significance, p < 0.001 was shown (***) compared to DPSC controls.c.Western blot of MAP2 and TUJ1 on the 3, 7 and 10 days of DPSC' neuronal differentiation keeping GAPDH as an internal loading control.d.

Fig. 4
Fig. 4 Effect of KLF2 deficiency on redox signaling and cellular bioenergetics.a. MitoSOX-stained cells were shown after ND, ND + GGPP, and DPSC.Inset represents a zoom view of targeted cell.b.DCFDAstained cells were shown after ND, ND + GGPP, and DPSC.Inset represents a zoom view of targeted cell.c.Seahorse flux analysis of oxygen consumption rate (OCR) in cells after ND, ND + GGPP, and

Fig. 7
Fig. 7 Effect of KLF2 sufficiency on redox signaling and cellular bioenergetics.a. MitoSOX-stained cells were shown after ND, ND + GGTI-298, and DPSC.Inset represents a zoom view of targeted cell.b.DCFDA-stained cells were shown after ND, ND + GGTI-298, and DPSC.Inset represents a zoom view of targeted cell.c.Seahorse flux analysis of oxygen consumption rate (OCR) in cells after ND,