Klotho increases antioxidant defenses in astrocytes and ubiquitin–proteasome activity in neurons

Klotho is an antiaging protein, and its levels decline with age and chronic stress. The exogenous administration of Klotho can enhance cognitive performance in mice and negatively modulate the Insulin/IGF1/PI3K/AKT pathway in terms of metabolism. In humans, insulin sensitivity is a hallmark of healthy longevity. Therefore, this study aimed to determine if exogenous Klotho, when added to neuronal and astrocytic cell cultures, could reduce the phosphorylation levels of certain insulin signaling effectors and enhance antioxidant strategies in these cells. Primary cell cultures of cortical astrocytes and neurons from mice were exposed to 1 nM Klotho for 24 h, with or without glucose. Klotho decreased pAKT and mTOR levels. However, in astrocytes, Klotho increased FOXO-3a activity and catalase levels, shielding them from intermediate oxidative stress. In neurons, Klotho did not alter FOXO-3 phosphorylation levels but increased proteasome activity, maintaining lower levels of PFKFB3. This study offers new insights into the roles of Klotho in regulating energy metabolism and the redox state in the brain.


Animals and ethics.
All procedures adhered to the ARRIVE guidelines (https:// arriv eguid elines.org), which are grounded in the Ethical Principle in Animal Research endorsed by the Brazilian College of Animal Experimentation (CONCEA).These procedures received approval from the Ethical Committee for Animal Research (CEUA) at the Biomedical Sciences Institute of the University of São Paulo, São Paulo, São Paulo State, Brazil.The protocol was officially registered under the number 12/2016 CEUA for animals utilized in experimentation.
Primary culture of cortical glial cells.Primary cortical glial cell cultures were derived from postnatal (P1-P3) mice of both sexes 42 .The meninges were carefully removed, and the cortices were dissected in cold Hank's Balanced Salt Solution (HBSS).The tissue was then dissociated using 0.25% trypsin at 37 °C for 5 min, after which Dulbecco's Modified Eagle Medium (DMEM) (supplemented with 4 mM glutamine, 10% Hyclone FetalOne III serum [GE Healthcare, USA], and 1% penicillin/streptomycin) was added.The cells were further dissociated using a Pasteur pipette, filtered, and plated at a density of 1.5 × 10 4 cells/cm 2 in a T75 flask.The medium was refreshed every 3 days.The cultures were incubated until they achieved confluency, typically within 10-14 days.The cells were then digested with 0.25% trypsin at 37 °C for 10 min to facilitate detachment from the flask.Digestion was halted by adding DMEM with 10% fetal bovine serum (FBS), after which the cells were centrifuged for 2 min at 2000 rpm.The resulting pellet was resuspended in DMEM 10% FBS and counted for plating.Each well in a 6-well plate received 1 × 10 6 cells.For a 24-well plate, cells were seeded at a density of 1 × 10 4 per well.The flasks were then incubated in an orbital shaker at 37 °C and 180 r/min for 15 h, following a previously described method to separate astrocytes from other glial cells 35 .The resulting culture, used for the experiments, was deemed an astroglial-enriched culture, as it contained 91.2% glial fibrillary acidic protein-labeled cells.Representative photomicrographs of the culture phenotype are provided in the supplementary material (see Supplementary Fig. S1 online).
Primary mouse (embryonic) cortical neuronal culture.Primary cortical neuronal cultures were prepared from both male and female C57BL/6 mouse embryos on gestational days 16th-17th (E16-17).The meninges were excised from the brain, and the cortices were sectioned into small fragments and incubated in a 2 mg/mL trypsin solution for 20 min at 37 °C in a 5% CO 2 incubator.Following the removal of the trypsin solution, the tissue was rinsed twice with HBSS.The tissue was then dissociated in HBSS containing 0.1 mg/mL DNAse through mechanical trituration using a glass pipette.The cells were counted and seeded at a density of 1 × 10 5 cells/cm 2 on dishes pre-coated with polyethyleneimine (Sigma-Aldrich).The neurons were maintained for 2 weeks in Neurobasal medium (GIBCO) supplemented with B27 (GIBCO), 2 mM L-glutamine, 100 U/mL penicillin, 100 mg/ mL streptomycin, and 0.25 mg/mL amphotericin B. A representative photomicrograph of the culture phenotype is presented in Supplementary Fig. S2, available in the supplementary material.On average, the primary culture comprised 80% neurons.
Transfection with FOXO reporter plasmid.The activity of the FOXO transcription factor was assessed using the pGL-3 × DBE plasmid (Promega) 43 , which incorporates three iterations of FOXO-responsive promoters and encodes firefly luciferase.Concurrently, the pRL-TK plasmid (Promega), expressing renilla luciferase constitutively, was transfected as an internal control.Astrocytes were seeded in 60 mm diameter plates (1 × 10 6 cells/ plate) and transfected after 48 h (approximately 60% confluence) using Fugene 6 (Promega) with 1 µg DBE DNA, 0.2 µg pRL-TK DNA, and a DNA: Fugene ratio of 1:6.Following 48 h of transfection, the cells were treated by washing twice with phosphate buffered saline (PBS) and incubating for 24 h in DMEM containing either 4.5 g/L or 1 g/L glucose concentration, with or without 1 nM Klotho.Post-treatment, the cells were lysed as per the Dual-Luciferase ® Reporter Assay system (Promega) guidelines.Equal sample volumes were used, and the luminescence was developed and read according to the manufacturer's instructions (Promega) on opaque white plates using a Synergy H1 Hybrid Multi-Mode plate reader (BioTek).The results are presented as the ratio of firefly luciferase fluorescence to renilla luciferase, normalized to the mean of the control group.
Cell viability assay: color formazan reduction (MTT).The MTT assays were conducted as previously described 44,45 .This method relies on the ability of viable cells to convert tetrazolium salt (MTT) into formazan, a colored compound.Cells were pretreated with either PBS or Klotho for a duration of 24 h, followed by a 30-min exposure to H 2 O 2 .Cell viability was ascertained by incubating cells with MTT (12 mM) in the cell culture medium for 2 h at 37 °C, 24 h post the final treatment.The dark crystals that formed were solubilized with DMSO, and the absorbance was measured at a wavelength of 570 nm using a microplate reader.The MTT (%) was calculated using the formula: (absorbance of the sample − absorbance of DMSO)/(absorbance of the control − absorbance of DMSO) × 100.
Immunoprecipitation. To assess protein phosphorylation and ubiquitination levels, astrocyte and neuron samples were examined through immunoprecipitation.These samples were obtained from pools of six wells from six-well plates, ensuring sufficient protein content (an average of 6 × 10 6 cells).The cells were gathered and homogenized in non-denaturing lysis buffer (20 mM Tris-HCl, 137 mM NaCl, 10% glycerol, 1% NP-40, and 2 mM EDTA, supplemented with 30 mM NaF phosphatase inhibitors, 20 mM sodium pyrophosphate, 5 mM β-glycerophosphate, 2 µg/ml leupeptin protease inhibitors, and 2 µg/mL antipain) using ultrasonication on ice.A total of 100 µg of protein (diluted in 400 µL of buffer) was incubated with 2 µg of antibody against phosphotyrosine (clone 4g10, Millipore) or against mono-and polyubiquitin K 29 , K 48 , K 63 (clone FK2, Enzo Life Sciences).For their respective negative controls, 2 µg of rabbit IgG or mouse IgG were used.The samples were incubated overnight with light orbital shaking.Subsequently, 20 µL of the suspension containing 50% agarose microspheres with protein A/G covalently immobilized was added, followed by incubation under light agitation for 3 h.The samples were then centrifuged and washed five times with ice-cold PBS.The final precipitate was resuspended in 20 µL of three-fold concentrated SDS sample buffer, homogenized, and centrifuged again, followed by heating at 70 °C for 10 min.After an additional centrifugation, the samples were loaded onto an SDS PAGE gel and analyzed by western blotting against PFKB3 (1:1000, Abcam #Ab96699) for FK2 and PKM2 (1:1000, Cell Signaling #3198).This assay was conducted on three independent cell cultures.

Klotho decreases AKT and mTOR phosphorylation in neurons.
In a prior study conducted by our group, we observed that treating astrocytes with 1 nM Klotho led to a rapid increase in aerobic glycolysis and lactate release via ERK activation, coupled with a simultaneous reduction in AKT phosphorylation 35 .Consequently, to ascertain whether 1 nM Klotho could modulate the Insulin/AKT/mTOR and FOXO3a signaling pathways in neurons, we treated primary cortical neurons with 1 nM Klotho for 24 h and subsequently analyzed them using western blotting.For the purpose of a positive control, cells were exposed to 100 nM insulin for the same duration.This assay involved the treatment and analysis of four independent cultures.As anticipated, insulin treatment increased AKT phosphorylation at Ser473 (Fig. 1A) and mTOR phosphorylation at Ser2448 (Fig. 1B).In contrast, Klotho treatment induced a reverse response, diminishing AKT and mTOR phosphorylation (p-AKT/AKT: control = 1.00 ± 0.05487; insulin = 1.346 ± 0.046; Klotho = 0.6568 ± 0.08439; p-mTOR/mTOR: control = 1.00 ± 0.03109; insulin = 1.308 ± 0.7588; Klotho = 0.7588 ± 0.0756; n = 4).The subsequent step involved verifying whether Klotho modulates FOXO3a phosphorylation.A western blot assay indicated that after a 24-h treatment period, no significant difference was detected in S253 phosphorylation levels (Fig. 1C).Each sample used in the western blot analysis contained a minimum of 2 × 10 6 cells.Each dot represents an independent cell culture.

Klotho promotes antioxidant defense through FOXO3a activity in astrocytes.
Following 24 h of treatment with either 1 nM Klotho or 100 nM insulin, FOXO3a phosphorylation levels in astrocytes were assessed using western blot analysis.Lysates were obtained from either a single well or two wells of a 6-well plate, yielding a total of 2 × 10 6 cells.Each data point in the graphs corresponds to a lysate derived from an independent cell culture.
To evaluate the transcriptional activity of the FOXO transcription factor directly, astrocytes were transfected with a plasmid housing copies of (i) the FOXO-responsive sequence (pGL-3xDBE), serving as the promoter unit of a gene encoding firefly luciferase and (ii) plasmids carrying a gene encoding promoter-regulated renilla luciferase constitutive thymidine kinase (pRL-TK), which served as an internal control for the transfection rate.Following transfection, astrocytes were maintained for 24 h under varying culture conditions (in media with high or low glucose concentrations), with or without 1 nM Klotho.The relative luminescence was then measured to determine the transcription rate of the luciferase gene and, by extension, the activity of the FOXO transcription factor.The results indicated that a lower glucose concentration stimulated FOXO activity.Interestingly, Klotho augmented transcription factor activity in both high and low glucose concentrations, implying a potential role for Klotho in modulating FOXO activity in astrocytes (high glucose control = 1.00 ± 0.09658; low glucose control = 12.35 ± 0.8978; high glucose klotho = 26.26± 1.316; low glucose klotho = 25.06 ± 2.942; n = 5) (Fig. 2B).The influence of FOXO on genes associated with antioxidant defense, such as manganese superoxide dismutase (MnSOD or SOD2) and catalase, has been previously described 46 .To explore the capacity of Klotho to stimulate catalase expression in astrocytes, a western blot analysis was conducted following a 24-h treatment period with varying concentrations of Klotho.The analysis indicated that catalase expression was induced by Klotho treatment across all tested concentrations, demonstrating a dose-independent response (Fig. 2C).This evidence implies that Klotho may activate antioxidant defense mechanisms within astrocytes, potentially via its regulation of FOXO transcriptional activity (control = 1.00 ± 0.02975; Klotho 0.2 nM = 1.80 ± 0.1034; 0.5 nM = 1.88 ± 0.103; 1.0 nM = 1.84 ± 0.07988; 5.0 nM = 1.87 ± 0.1039; n = 4).

Klotho protects astrocytes but not neurons against oxidative insult.
We conducted an investigation into the potential protective role of Klotho against oxidative damage.The viability of astrocytes and neurons exposed to varying concentrations of H 2 O 2 was assessed using the MTT assay.Our results indicated a significant reduction in astrocyte viability across all tested H 2 O 2 concentrations (Fig. 3A).To further evaluate the protective effect of Klotho against H 2 O 2 -induced damage, we selected a concentration that represented an intermediate level of insult, resulting in slightly less than 50% cell death.For this purpose, we opted for a concentration of 500 µM H 2 O 2. Astrocytes were pretreated with either recombinant Klotho or a vehicle (PBS) at varying concentrations for 24 h prior to a 30-min exposure to H 2 O 2 .Our findings demonstrated that all tested Klotho concentrations effectively mitigated astrocyte death induced by H 2 O 2 (Fig. 3B).This suggests that Klotho may play a protective role against intermediate oxidative damage in astrocytes (control = 1.00 ± 0.04143; Klotho 5 nM = 0.9354 ± 0.1413; H 2 O 2 500 µM = 0.4999 ± 0.04248; H 2 O 2 + Klotho 0.2 nM = 0.7802 ± 0.04736; All tested concentrations of H 2 O 2 significantly decreased cell viability in neurons compared to the control group.In neuronal culture, the same H 2 O 2 concentration that was used in astrocytes constituted a severe oxidative insult, killing nearly 75% of the neurons (Fig. 3C).Our lab previously demonstrated that 1 nM Klotho for 24 h could protect neurons from death following exposure to LPS-activated microglia-conditioned medium 47 .Consequently, we sought to determine if the same concentration could protect neurons from a severe oxidative insult.However, pretreatment with 1 nM Klotho did not offer neuroprotection against cell death induced by 30 min of exposure to 500 µM H 2 O 2 (Fig. 3D).

Klotho induces PFKFB3 ubiquitination and proteasome activity in neurons.
The literature has previously indicated that Klotho suppresses PFKFB3 activity in adipocytes 39 .Maintaining low PFKFB3 levels in neurons could potentially facilitate the PPP and augment NADPH levels, which are crucial for oxidative damage repair.To explore the potential role of Klotho in modulating PFKFB3 expression, primary astrocyte cultures and hippocampal cortical neurons were exposed to a 24-h treatment with 1 nM Klotho.Subsequently, PFKFB3 levels were evaluated using western blotting (Fig. 4).All original blots can be found in Supplementary Fig. S6.
Based on the transcriptome results from a neuronal microarray 35 , we decided to assess both the proteasome activity and the levels of poly-ubiquitinated proteins in neurons.These neurons were either treated with 1 nM Klotho for 24 h or maintained without insulin for the same duration.To measure proteasomal activity, we included a negative control group, which consisted of cells treated with MG 132 (50 µM) for 2 h.Each dot on the graph represents the average of values measured in 22-24 wells of a 96-well plate from a single cell culture.We conducted this analysis of proteasome activity across three independent cell cultures.

Discussion
The brain requires substantial amounts of oxygen and glucose to sustain high levels of neuronal activity 48 .Neurons possess a limited capacity to metabolize energy substrates other than glucose, which accounts for the abundance of mitochondria and the intense oxidative phosphorylation observed 49 .Given that optimal neuronal function and, consequently therefore, healthy cognitive performance necessitate proper metabolic cooperation between astrocytes and neurons, this study aimed to determine whether exogenous Klotho could inhibit insulin/IGF-1 signaling in neurons and activate FOXO-3a.As previously noted, FOXOs enhance the expression of catalase, which detoxifies ROS from hydrogen peroxide.Additionally, we aimed to investigate whether Klotho could modulate PFKFB3 levels in the CNS.PFKFB3, a crucial regulator of cellular metabolism 40 , should be maintained at low levels in neurons to improve antioxidant defenses.
The beneficial impact of Klotho on the activity of FOXO transcription factors is well-documented 18,50 .Klotho can mitigate the suppressive effect of AKT on FOXO transcription factors by negatively modulating the PI3K/ AKT pathway.This allows the transcription factors to relocate to the nucleus and become active.Consistent with our previous findings from astrocytes, Klotho has also been observed to reduce AKT and mTOR phosphorylation levels in cortical neurons (Fig. 1A), which is in agreement with our previous results from astrocytes 35 .
The subsequent step involved verifying the effects of Klotho on FOXO3a activity.Initially, we assessed its phosphorylation levels following a 24-h treatment with either 1 nM Klotho or 100 nM insulin in neurons.However, no discernible difference was noted in the FOXO3a phosphorylation levels within neurons (Fig. 1B).In contrast, within astrocytes, Klotho treatment impeded the inhibitory phosphorylation of the FOXO3a transcription factor.As anticipated, insulin demonstrated its inhibitory effect on this protein (Fig. 2A).This disparity in phosphorylation patterns suggests that FOXO3a activity in neurons and astrocytes is subject to distinct regulatory mechanisms.Indeed, Du et al. have demonstrated that insulin treatment does not impact the subcellular localization of FOXO3a 51 .Unlike astrocytes, FOXO3a activity in neurons responded to DNA damage or astrogliosis 52 .Interestingly, a region-specific pattern of FOXO3a regulation was observed, with a decrease in FOXO3a activity in the cortex, but not in the hippocampus of aged mice 50 .Conversely, another study found that when primary hippocampal neurons were treated with 4 µg/mL Klotho for 24 h, there was a sustained increase in AKT activation and inhibitory FOXO3a phosphorylation 19 .
We conducted a gene reporter assay to measure FOXO3a activity.The luciferase assay results, which align with those from the western blot analysis, indicate that Klotho amplifies FOXO3a activity under both low and high glucose conditions (Fig. 2B).FOXO3a, upon activation, produces several antioxidant proteins that play a www.nature.com/scientificreports/crucial role in the cellular response to oxidative stress 53 .Importantly, we noted a dose-dependent increase in catalase expression subsequent to Klotho treatment (Fig. 2C).
Aging correlates with diminished FOXO3a expression, which subsequently results in abnormal astrocyte activation, inflammation, and metabolic disturbances 51 .Klotho, an antiaging protein, may potentially enhance FOXO-3a activity and catalase expression in astrocytes, thereby suggesting a possible protective mechanism.
In the subsequent phase of our research, we explored the potential of Klotho to protect neurons and astrocytes from cell death triggered by an oxidative insult, specifically H 2 O 2 .In astrocytes, Klotho pretreatment significantly mitigated H 2 O 2 -induced cell death (Fig. 3A,B).For glial cells, a concentration of 500 µM H 2 O 2 constituted a moderate insult, while for neurons, the same concentration posed a severe insult (Fig. 3C).Consequently, pretreatment with an identical concentration of Klotho failed to safeguard neurons from H 2 O 2 -induced cell death (Fig. 3D).
Prior literature indicates that a 24-h pretreatment with Klotho can protect retinal pigment epithelial cells from H 2 O 2 -induced cell death.This protection is achieved by reducing cleaved-caspase 3 and Bax levels, and enhancing antioxidant activity, such as SOD2 and CAT 54 .These findings suggest that Klotho's protective role against oxidative stress could be attributed to a decrease in AKT-mTOR signaling, which subsequently leads to an increase in FOXO3a activity and catalase levels.Our research further supports the hypothesis that Klotho functions as an anti-aging protein with antioxidant properties 54 .
The dependence on mitochondrial oxidative phosphorylation (OXPHOS) for survival significantly distinguishes astrocytic metabolism from neuronal metabolism.In astrocytes, OXPHOS inhibition triggers glycolysis, a response not mirrored to the same extent 55 .Neurons rely on OXPHOS to prevent apoptosis resulting from energy failure 56 .This reliance is partially attributed to the diminished levels of a key glycolytic-promoting enzyme, PFKFB3 57 .
PFKFB3, a crucial enzyme, plays a significant role in managing energy metabolism and antioxidant defenses in neurons.This enzyme belongs to the PFKFB family, which is tasked with controlling the concentration of fructose-2,6-bisphosphate (F2,6BP) within cells.F2,6BP serves as a powerful activator for the glycolytic enzyme PFK-1, the primary enzyme in the glycolytic pathway responsible for the conversion of glucose into pyruvate.
The levels of PFKFB3 messenger RNA in neurons and astrocytes are identical.However, owing to ubiquitination leading to proteasomal degradation, the protein level of PFKFB3 in neurons is virtually null due to its ubiquitination and consequent proteasomal degradation 41 .Maintaining low PFKFB3 levels in neurons can promote the PPP, a crucial process for the production of NADPH and ribose-5-phosphate.These compounds are essential for nucleotide and fatty acid synthesis, as well as for antioxidant defenses 58 .
Conversely, glutamate-induced excitotoxicity stabilizes neuronal PFKFB3.This stabilization subsequently diminishes the activity of the PPP and augments glycolysis, leading to oxidative damage and, ultimately, neuronal death.These detrimental effects can be mitigated by the overexpression of glucose-6-phosphate dehydrogenase, the enzyme that catalyzes the rate-limiting step in the PPP 59 .A reduction in PFKFB3 levels within neurons can enhance PPP activity and NADPH production 59,60 .In adipocytes, PFKFB3 activity is decreased by Klotho 39 .
In this study, we demonstrated that Klotho can diminish PFKFB3 levels in neurons (Fig. 4B) by promoting an upsurge in ubiquitination and proteasome activity (Fig. 5).This discovery is particularly significant as the accumulation of misfolded and damaged proteins is a characteristic feature of aging and is associated with neurodegenerative diseases 61 .
In conclusion, our research offers novel insights into the functions of Klotho in controlling energy metabolism and the redox state within the brain.The data we have gathered indicates that Klotho inhibits insulin/IGF-1 signaling in neurons and functions as an antioxidant in astrocytes by increasing FOXO activity and promoting catalase expression.Additionally, Klotho reduces PFKFB3 content in neurons, a crucial factor in the regulation of NADPH levels and the overall cellular reducing potential.Our study highlights the interaction between energy metabolism and redox balance, proposing that Klotho could be instrumental in preserving this equilibrium (Fig. 6). https://doi.org/10.1038/s41598-023-41166-6 13:15080 | https://doi.org/10.1038/s41598-023-41166-6

Figure 2 .
Figure 2. Klotho affects FOXO-3a activity and catalase activity in astrocytes.(A) Western blot assay was performed to measure phosphorylation levels of FOXO-3a.Statistical analysis suggested that Klotho treatment decreased the FOXO-3a inhibitory phosphorylation.One way ANOVA followed by Tukey's multiple comparisons tests; [F = 23.86;P = 0.0003; R squared = 0.8413].Below the graph is the representative western blot digital images of p-FOXO-3a, FOXO-3a, and β-actin.(B) FOXO gene reporter activity.After 48 h of plasmid transfections, treatments were performed by incubating cells for 24 h with DMEM containing high (4.5 g/L) or low (1 g/L) concentrations of glucose, with or without 1 nM Klotho.Results represent the ratio of fluorescence of firefly luciferase to renilla luciferase, normalized to the mean of the control group (DMEM high glucose).One-way ANOVA statistical analysis followed by Tukey's multiple comparison tests.FOXO-3a activity was increased in all groups: CTL High vs. CTL Low (mean difference: − 11.35; q = 6.782),CTL High versus Klotho High (mean difference: − 25.26; q = 15.09),CTL High versus Klotho Low (mean difference: − 24.06; q = 14.38),CTL Low versus Klotho High (mean difference: − 13.91; q = 8.313), and CTL Low versus Klotho Low (mean difference: − 12.71; q = 7.598).No difference was observed between Klotho high and Klotho low glucose (mean difference: 1.196; q: 0.7148).P < 0.001, F = 50.66,and R squared = 0.9048.(C) Catalase relative activity.Statistical analysis suggested that all klotho concentrations increased catalase expression compared to that of the control group.[One-way ANOVA followed by Tukey's multiple comparison test.F = 18.38;R squared 0.8386; P < 0.0001].*P < 0.05; ***P < 0.0001.The original autoradiographs are available in Supplementary Figs.S4 and S5.Lysates were obtained from two or three wells of a 6-well plate to reach 2 × 10 6 cells.Each point in the graphs represents a lysate obtained from independent cell cultures, and four independent cell cultures were analyzed in these assays.

Figure 3 .
Figure 3. Viability of astrocytes and neurons after oxidative insult by different concentrations of H 2 O 2 and the protective effects of Klotho.Neurons and astrocytes were pretreated with PBS or Klotho with different concentrations.After 24 h, the cells were exposed to a 30-min insult with H 2 O 2 at different concentrations.Cellular viability was measured by MTT.(A) Astrocytes were challenged with different concentrations of H 2 O 2 .All concentrations tested were sufficient to reduce their viability compared to the control group.Oneway ANOVA followed by Tukey's multiple comparisons tests [F = 110.5;R squared = 0.8962; P < 0.0001, n = 12].A concentration of 500 µM was selected.(B) Astrocytes were pretreated with different concentrations of Klotho and challenged with 500 µM H 2 O 2 .Results suggest that 1 nM Klotho alone was not able to alter cell viability, although 500 µM H 2 O 2 decreased cell viability, as expected.The pretreatment with all different Klotho concentrations protected astrocytes from death.One-way ANOVA followed by Tukey's multiple comparisons tests [F = 11.83;R squared = 0.5221; P < 0.0001, n = 4-12].(C) Neurons were challenged with different concentrations of H 2 O 2 .All concentrations tested were sufficient to reduce their viability compared to the control group.One-way ANOVA followed by Tukey's multiple comparisons tests [F = 20.36;R squared = 0.8498; P < 0.0001, n = 4].(d) Neurons were pretreated with 1 nM Klotho for 24 h and challenged with 500 µM H 2 O 2 for 30 min.Compared to the control group, as expected, H 2 O 2 altered neuronal viability but Klotho did not.However, pretreatment with Klotho did not avoid cell death caused by oxidative insult.Therefore, compared to the control group, both groups (H 2 O 2 and H 2 O 2 + Klotho) had a decreased viability.One-way ANOVA followed by Tukey's multiple comparisons tests [F = 59.01;R squared = 0.9568; P < 0.0001, n = 3].*P < 0.05; ***P < 0.0001.

Figure 4 .
Figure 4. PFKFB3 levels measured by western blot in neurons and astrocytes, normalized by the control.Primary cultures of astrocytes and neurons were treated with 1 nM Klotho for 24 h in the presence or absence of insulin.(A) In astrocytes, the level of PFKFB3 was not altered by insulin or Klotho treatment within 24 h.(B) Tukey's multiple comparisons test indicated that in neurons, the cells that were deprived of insulin had reduced PFKFB3 levels compared to those of the control group [mean difference 0.4455, q = 7.641, P = 0.004].Similarly, the cell treated with Klotho had reduced PFKFB3 levels compared to the those of the control group [mean difference: 0.3563, q = 6.111,P = 0.0118].One-way ANOVA [F = 16.35;R squared = 0.8449; P = 0.0037].Below the graphs are representative images of a western blot for PFKFB3 and β-actin.N = 3-4 independent cultures.The original autoradiographs are available in Supplementary Fig. S6. https://doi.org/10.1038/s41598-023-41166-6

Figure 6 .
Figure 6.Schematic representation of the effects of Klotho in astrocytes and cortical neurons.The AKT inhibition by Klotho treatment induces transcriptional activity of FOXO transcription factors and promotes antioxidant defense in astrocytes by inducing catalase expression.In addition, Klotho treatment induced PFKFB3 ubiquitination and proteasome activity in neurons.Klotho is an important player in the adaptive defense response in astrocytes, and it increases proteasomal activity in neurons, which are both protective actions involving coupling between neurons and astrocytes against neurodegenerative processes.