Targeting p75NTR activity alleviates the neurotoxic effect of high glucose on iPSC-derived dopaminergic neurons
Present Address:
KonstantinaChanoumidou1,2✉EmailKonstantina_chanoumidou@imbb.forth.gr
IoannaZota1,2
MariaAnnaPapadopoulou1,2
ChrystallaKonstantinou1,2
AlexandrosTsimpolis1,2
ElectraTsagliotis2
MariaTziortziou3
KaterinaNtarntani1
AnneGrünewald3
MatthieuDavidLavigne2
AchilleGravanis1,2
IoannisCharalampopoulos1,2✉Emailcharalampn@uoc.gr
1Department of Pharmacology, Medical SchoolUniversity of Crete71003HeraklionGreece
2Institute of Molecular Biology and BiotechnologyFoundation for Research and Technology-Hellas71003HeraklionGreece
3
A
Centre for Systems BiomedicineUniversity of LuxembourgEsch-sur-AlzetteL-4362Luxembourg, LuxembourgKonstantina Chanoumidou1,2*, Ioanna Zota1,2, Maria Anna Papadopoulou1,2, Chrystalla Konstantinou1,2, Alexandros Tsimpolis1,2, Electra Tsagliotis2, Maria Tziortziou3, Katerina Ntarntani1, Anne Grünewald3, Matthieu David Lavigne2, Achille Gravanis1,2, Ioannis Charalampopoulos1,2*
1. Department of Pharmacology, Medical School, University of Crete, Heraklion, 71003, Greece.
2. Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Heraklion, 71003, Greece.
3. Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Esch-sur-Alzette, L-4362, Luxembourg.
*Correspondence
Ioannis Charalampopoulos
charalampn@uoc.gr
Konstantina Chanoumidou
Konstantina_chanoumidou@imbb.forth.gr
Abstract
Background
Hyperglycemia, a hallmark of Diabetes Mellitus, is a metabolic condition that highly affects the nervous system. While evidence from epidemiological and animal studies links Diabetes to dopaminergic dysfunction and an increased risk of Parkinson’s disease, the underlying mechanisms remain unclear. Here, we examined the effects of high glucose on human iPSC-derived dopaminergic neurons and glial cells to better understand the pathogenic alterations that lead to neurotoxicity. Previous implication of neurotrophins in the neurological manifestations of Diabetes prompted us to focus on the role of p75NTR neurotrophin receptor (p75NTR) in dopaminergic neurodegeneration under hyperglycemic conditions.
Methods
iPSC-derived dopaminergic neurons, astrocytes and microglia were treated with high glucose (50mM, 100mM) for 48h to simulate hyperglycemia. Cytotoxicity assays, RNA sequencing and DNA damage assessments were employed to investigate the pathological alterations induced by high glucose exposure in neurons. Pharmacological targeting of p75NTR activity allowed investigation of its involvement in glucose neurotoxicity. Glial-mediated neurotoxicity was evaluated using conditioned media and inflammatory marker analysis.
Results
High glucose treatment led to DNA damage, activation of JNK signaling and cell death in neurons. Importantly, we observed upregulation of p75NTR and its pro-apoptotic ligand pro-NGF, suggesting activation of the pro-NGF/p75NTR axis in high glucose-treated neurons. Inhibition of p75NTR activity rescued neuronal cell death, identifying p75NTR as a central mediator of glucose neurotoxicity. Furthermore, glucose overload sensitized neurons to 6-hydroxydopamine (6-OHDA), increasing their vulnerability to neurotoxic insults—an effect reversed by p75NTR blockade. Treatment with BNN27, a synthetic NGF mimetic, prevented neuronal loss through p75NTR and TrkA receptors, suggesting neurotrophin signaling as a potential therapeutic target for combating high glucose-induced neuronal damage. Finally, we demonstrated the contribution of glial cells to neurodegeneration since high glucose treatment of iPSC-derived astrocytes and microglia enhanced their inflammatory potential and triggered the release of neurotoxic factors, causing pro-apoptotic effects on neurons.
Conclusions
Our findings show that high glucose impairs human dopaminergic neuron survival through activation of the pro-NGF/p75NTR axis and indirect glia-mediated mechanisms. Targeting p75NTR signaling may offer neuroprotective benefits in Diabetes-related neurodegeneration, particularly for patients at risk of Parkinson’s disease.
Keywords
iPSC
p75NTR
Neurotrophins
Dopaminergic Neurons
Glucotoxicity
Neurodegeneration
Neuroinflammation
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Introduction
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Hyperglycemia, a defining feature of Diabetes Mellitus (DM), is a metabolic condition that highly affects the nervous system accelerating neurodegeneration. Although the connection between DM and Alzheimer’s Disease is well documented (Biessels & Despa, 2018), its association with Parkinson’s Disease (PD) is still largely unexplored. Growing evidence links DM to dopaminergic neurodegeneration and increased risk of PD (Biosa et al., 2018; Chohan et al., 2021; Komici et al., 2021; Yang et al., 2017). Animal models and patients with DM show striatal dopaminergic dysfunction, altered dopamine neurotransmission and have increased risk for parkinsonian symptoms (D’Amelio et al., 2009; Kotagal et al., 2013; Pérez-Taboada et al., 2020). However, the mechanistic interlink between the two disorders remains unclear impairing the development of neuroprotective therapies.Glucose is the main source of energy for the brain and de-regulation of glucose levels associates with neuronal loss (Mergenthaler et al., 2013). Glucose neurotoxicity has been linked to mitochondrial dysfunction, oxidative stress and accumulation of AGEs (Tomlinson & Gardiner, 2008), mechanisms that are also common with many neurodegenerative disorders. Additionally, Diabetes leads to systemic inflammation, changes in Blood Brain Barrier (BBB) integrity and gliosis in the brain (Lee et al., 2024). Streptozotocin (STZ)-treated mice display increased brain sensitivity to peripheral LPS-induced inflammation (Lee et al., 2024) while STZ treatment in the rotenone model of PD activates microglia and eventually worsens neurodegeneration and motor symptoms (Zhang et al., 2024a).
Neurotrophins, a major class of endogenous neuroprotective molecules, have been associated with the neurological manifestations of Diabetes (He et al., 2024; Hellweg & Hartung, 1990). The pro-apoptotic NGF isoform, pro-NGF, is up-regulated in diabetic retinopathy (Elshaer et al., 2019a; Mysona et al., 2013), while BDNF protects hippocampal neurons from hyperglycemia-driven apoptosis (Zhong et al., 2019). The neuroregenerative properties of neurotrophin signaling indicate neurotrophin receptors as promising therapeutic targets for neuroprotection. The p75 neurotrophin receptor (p75NTR), a member of the TNF receptor superfamily, binds all mature and pro-neurotrophins. While best known for mediating pro-apoptotic signaling upon binding to pro-neurotrophins under pathological conditions (Knowles et al., 2009; Kraemer et al., 2014), p75NTR can also promote cell survival in a ligand- and context- dependent manner (Charalampopoulos et al., 2012; Meeker & Williams, 2015). Notably, p75NTR is up-regulated in peripheral diabetic neuropathy (Chilton et al., 2004; Humpert et al., 2007; Scarpini et al., 1996) while in STZ-treated rodents the pro-NGF/p75NTR signaling promotes Blood-Retina-Barrier disruption and neuroinflammation in retina (Elshaer et al., 2019b; Mysona et al., 2013). However, its role in brain pathology under diabetic conditions is yet unexplored.
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To date, all studies investigating the impact of hyperglycemia on the dopaminergic system have been conducted in animal models. Leveraging the technology of human induced Pluripotent Stem Cells (hiPSCs), we examined the effects of high glucose (HG) on hiPSC-derived dopaminergic (DA) neurons, astrocytes and microglia to gain deeper insights into the pathogenic mechanisms of hyperglycemia – induced neurodegeneration and the contribution of glial cells in this pathology in a human relevant model. hiPSC-derived cells enable recapitulation of disease relevant mechanism for molecular interrogation (Volpato et al., 2018). We focused on the role of p75NTR in glucose neurotoxicity and evaluated p75NTR as a potential target for intervention. Our findings show that HG primarily induces DNA damage and neuronal loss in a dose-dependent manner. RNA sequencing analysis confirmed the induction of numerous stress-related processes including p53 signaling, response to TNF and DNA damage repair. The accumulation of pro-NGF and p75NTR in HG-treated neurons suggests induction of the pro-apoptotic pro-NGF/p75NTR axis in hyperglycemic conditions. Notably, inhibition of p75NTR activity prevented neuronal cell death, revealing p75NTR receptor as a novel mediator of glucotoxicity in DA neurons. Glucose overload increased the vulnerability of DA neurons to 6-OHDA - induced cytotoxicity while inhibition of p75NTR activity significantly reduced this effect. We tested BNN27, a synthetic NGF mimetic which targets p75NTR and TrkA receptors, against HG-induced toxicity and we highlight its neuroprotective action in hyperglycemic condition. Finally, we showed that HG increases astrocyte and microglia responsiveness to pro-inflammatory stimuli and promotes the release of neurotoxic factors showcasing the critical involvement of glial cells in hyperglycemia-induced neurodegeneration.Materials and methods
Cell lines
Three healthy human iPSC lines (SFC856-03-04, SFC841-03-01, SBAD-03-01) were kindly provided by Dr Μ. Z. Cader and were reprogrammed as part of IMI StemBANCC (Morrison et al., 2015). iPSCs were reprogrammed from skin fibroblasts of three healthy control individuals. These cell lines were used for the generation of neural progenitor cells, dopaminergic neurons and astrocytes. Microglia were differentiated from a healthy iPSC line derived from a female donor provided by Anne Grünewald. All cell lines have been tested and were negative for mycoplasma contamination.
Generation of Neural Progenitor Cells (NPCs)
Neural progenitor cells were generated as previously described (Reinhardt et al., 2013). Briefly, iPSCs were cultured on mouse embryonic fibroblasts (MEFs) until they form dense colonies in hESC medium (DMEM-F12 medium (Gibco, 21331-020), 20% KO serum replacement (Gibco, 10828028), Non-essential amino acids (Gibco, 11-140-050), Pen/Strep (Gibco, 15140122), L-Glutamine (Gibco, A2916801), 2-Mercaptoethanol (Gibco, 31350010) supplemented with 5 ng/ml FGF2 (Peprotech, 100-18C)). Next, iPSC colonies were cut into small pieces and detached from MEFs via treatment with 2 mg/mL collagenase IV (Sigma, C1764) for 15-30min at 37oC. Cells were collected and resuspended in human embryonic stem cell (hESC) medium without FGF2 supplemented with 1 µM Dorsomorphin (Abcam, ab120843), 3 µM CHIR99021 (Sigma, SML1046), 10 µM SB-431542 (Stem Cell Technologies, 72232) and 0.5 µM Purmorphamine (Stem Cell Technologies, 72202). Embryoid bodies (EBs) were formed by culturing cells in non-adherent petri dishes (Corning) for six days. On the second day, medium was changed to N2B27 medium (1:1 Neurobasal (Gibco, 21103-049) and DMEM-F12 medium (Gibco, 21331-020), 1:100 B27 supplement lacking vitamin A (Gibco, 12587010), 1:200 N2 supplement (Gibco, 17502048), 1% penicillin/streptomycin (Gibco, 15140122)) supplemented with the same factors as on Day 0. On day 4, dorsomorphin and SB-431542 were removed, whereas 150µM L-Ascorbic acid (Sigma, A4544) was added to the medium. On day 6, EBs were partially broken into smaller pieces via pipetting and plated on Matrigel- (Matrigel Growth-factor-reduced, Corning, 354263) coated 12-well plates. When confluent, NPCs were passaged with Accutase (Sigma, A6964). After three passages, purmorphamine was replaced by 0.5 µM SAG (Abcam, ab142160). NPCs were expanded until passage 6 and then could be frozen or subcultured in NPC medium (N2B27 medium supplemented with 0.5µM SAG, 3µM CHIR, 150µM Ascorbic acid). NPC identity was evaluated with immunostaining for Nestin. NPCs were used for neuronal differentiation after passage 8.
Differentiation of NPCs towards dopaminergic neurons and glucose treatment
NPCs were seeded at density of 36.000 cells/cm2 on Matrigel-coated 12-well plate format in NPC medium (Day − 1). On Day 0, the medium was replaced by N2B27 medium containing 1 µm SAG, 75 µM AA, 2 ng/mL BDNF (Peprotech, 450-02) and 2 ng/mL GDNF (Peprotech, 450 − 10). On day 6, the medium was replaced by N2B27 medium containing 75 µM AA, 2 ng/mL BDNF, 2 ng/mL GDNF, 1 ng/mL TGF-β3 (Peprotech, 100-36E) and 100 µM dbcAMP (Sigma, D0627). 5 ng/mL Activin A (Stem Cell Technologies, 78001) was added to the medium from day 6 to day 10. Cells were split in ratio 1:3 on day 8 of differentiation. Neuronal identity was confirmed with immunostaining for the neuronal markers TUJ1, MAP2 and the dopaminergic marker TH on day 21. The percentage of TUJ1 + cells was > 90% at day 21. Neurons were cultured in N2B27 medium containing 20mM D-glucose. This glucose concentration was considered as the control condition. Neurons were exposed to 50mM or 100mM D-glucose (Sigma, G8769) for 48h (from day19 to day 21 of differentiation) to mimic hyperglycemia. To inhibit p75NTR activity, cells were treated with 2.5 ng/ml p75NTR inhibitor (neutralizing antibody) (Abcam, ab6172) for 48h as previously described (Papadopoulou et al., 2023).
Generation of astrocytes, stimulation and glucose treatment
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For the generation of human iPSC-derived astrocytes, we followed the protocol originally described by Perriot et al. (Perriot et al., 2021). On Day 0, iPSC colonies were cut and transferred to low-binding 6-well plates in NPC induction medium DMEM/F-12 (Gibco, 21331-020), Glutamax (Gibco, 3505006), Pen/Strep (Gibco, 15140122), 1x N2 (Gibco, 17502048), 1x B27 w/o vitamin A (Gibco, 12587010), 500ng/ml Noggin (Peprotech, 120-10c), 20µM SB-431542 (Stem Cell Technologies, 72232), 4ng/ml FGF-2 (Peprotech, 100-18C), 2µg/ml Laminin (Sigma-Aldrich, L2020) in order to form neural spheres. After 6-8h, the generated spheres were transferred to poly-L-ornithine (Sigma, P4957)/Laminin- (Sigma-Aldrich, L2020)-coated 6-well plates in order to attach and form neural rosettes. The medium was changed every other day. On day 10, the medium was switched to NPC expansion medium consisting of DMEM/F-12 (Gibco, 21331-020), Glutamax (Gibco, 3505006), N2 (Gibco, 17502048), B27 w/o vitamin A (Gibco, 12587010), 10ng/ml FGF-2 (Peprotech, 100-18C) and 10ng/ml EGF (R&D, 236-EG). Once neural rosettes were formed (around day13), the STEMdiff™ Neural Rosette Selection Reagent (Stem Cell Technologies, 05832) was used to isolate them. The rosettes were then transferred to poly-L-ornithine/Laminin-coated plates and were further cultured in NPC expansion medium. The cells were split using TrypLE (Gibco, 12605010). After 6–8 passages, homogenous SOX2 + and PAX6 + cell populations of NPCs were generated. For the astrocyte differentiation (50.000 cells/cm2), NPCs were plated on Matrigel in Astrocyte induction medium containing DMEM/F-12 (Gibco, 21331-020), Glutamax (Gibco, 3505006), N2 (Gibco, 17502048), B27 w/o vitamin A (Gibco, 12587010), 10ng/ml EGF (R&D, 236-EG) and 10ng/ml LIF (Peprotech, 300-05). Medium changes were performed every other day and the cells were passaged with TrypLE (Gibco, 12605010) when confluent. On Day 14, the medium was changed to Astrocyte medium containing DMEM/F-12 (Gibco, 21331-020), Glutamax (Gibco, 3505006), B27 w/o vitamin A (Gibco, 12587010) and 20ng/ml CNTF (Peprotech, 450 − 13). From Day 14 to Day 28, the cells were passaged when confluent in seeding density 40.000 cells/cm2 onto Matrigel-coated flasks. From Day 29 to Day 42, the cells were passaged when confluent in seeding density 25.000 cells/cm2. At Day 44, we obtained > 90% S100β + astrocytes. In this study, astrocytes of differentiation day 50 were used for experiments. Astrocyte medium contains 17.5mM D-glucose. This glucose concentration was considered as the control level. Astrocytes were exposed to high glucose 100mM D-glucose (Sigma, G8769) for 48h to generate hyperglycemic conditions. Cell exposure to 10ng/ml IL-1β beta (Peprotech, 200-01B) and 30ng/ml TNFa (Peprotech 300-01A) was used to activate astrocytes.Generation, stimulation of microglia and glucose treatment
Embryonic like macrophage precursors were generated from human iPSCs as previously described by (Haenseler et al., 2017; van Wilgenburg et al., 2013). Briefly, 4 × 106 iPSCs were seeded into an Aggrewell 800 well (STEMCELL Technologies, 34850) to form EBs, in EB medium containing mTESR (Stem Cell Technologies, 100–0276), 50 ng/mL BMP4 (Invitrogen, PHC9534), 50 ng/mL VEGF (Invitrogen, PHC9394), and 20 ng/mL SCF (Miltenyi, 130-096-695) supplemented with 10 µM Rock inhibitor (Abcam Biochemicals, Ab120129) on the day of plating. Medium was changed daily. After four days, EBs were harvested and transferred to a 6 well low attachment plate, where they were further cultured for 3 days in EB medium. Then, EBs were collected and transferred to a T175 flask in X-VIVO15 (Lonza, LZBE04-4418F) medium supplemented with 100 ng/mL M-CSF (Invitrogen, PHC9501), 25 ng/mL IL-3 (Invitrogen, PHC0033), 2 mM Glutamax (Gibco, 35050), 100 U/mL penicillin and 100 µg/mL streptomycin (Gibco, 15140122), and 0.055 mM β-mercaptoethanol (Gibco, 31350010). 10ml fresh medium was added weekly. After four weeks in culture, macrophage precursors were collected from the supernatant, were strained (40 µm, Corning) and plated (0.8 x 106 cells per well of a tissue culture 6-well plate) for terminal differentiation towards microglia. Cells were cultured in Microglia medium containing Advanced DMEM/F12 (Invitrogen), 0.055 mM β-mercaptoethanol (Gibco, 31350010), 10ng/ml GM-CSF (Peprotech, 300-03), 100ng/ml IL-34 (Peprotech, 200 − 34), 25ng/ml M-CSF (Invitrogen) and 50ng/ml TGFβ1 (Peprotech) for two weeks and medium was changed every other day. Microglia medium contains 17.5mM D-glucose. Microglia were treated with 100mM D-glucose (Sigma, G8769) for 48h to generate the hyperglycemic condition and 100ng/ml LPS (Invitrogen, 00497693) during the last 6h to activate cells.
Immunocytochemistry
Cells were fixed with 4% paraformaldehyde for 15min. Following three washes with 1xPBS (Gibco, 14190094), cells were treated with 0.1% Triton X-100 for 5 min. Next, the cells were incubated with blocking solution containing 10% serum, 0.3% Triton X-100 and 0.1% BSA (Gibco, 15260037) in 1× PBS for 1h at RT. Primary antibodies were added in a buffer containing 1% serum and 0.1% Triton-X in 1xPBS overnight at 4°C. Upon three washes with 1xPBS, the secondary antibodies were added in 1xPBS at RT for 1h. Cell nuclei were visualized with Hoechst (Invitrogen, H3570). Images were obtained using a Confocal Laser Scanning Microscope Leica TCS SP8. Primary antibodies are shown in Table 1.
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Antibody | Company | Cat. |
|---|---|---|
TUJ1 | Biolegend | 801201 |
MAP2 | Merck | MAB3418 |
TH | Abcam | Ab137721 |
S100β (E7C3A) | Cell Signaling | #90393 |
GFAP | Merck | ab5541 |
ΕΑΑΤ1 | Abcam | Ab416 |
IBA1 | Wako | 019–19,741 |
Synapsin I | Abcam | Ab254349 |
Synaptophysin | Merck | MAB5258 |
Phospho-Histone H2A.X (Ser139) | Merck | 05-636 |
TrkA | Merck | 06-574 |
TrkB | Merck | 07-225-I |
p75NTR | Biolegend | 839701 |
pJNK | Cell Signaling | 4668 |
tJNK | Cell Signaling | 9252 |
Beta Actin | Santa Cruz Biotechnology | sc-47778 |
Amyloid-β treatment
The Amyloid-β (1–42) peptide was purchased from AnaSpec (AS-20276, AnaSpec, Fremont, CA, USA) and oligomers were prepared according to the manufacturer’s instructions and previously described protocols (Kokkali et al., 2024; W. Li et al., 2011). Peptides were diluted in Neurobasal Medium (Gibco, 21103-049) at the indicated concentrations. Neurons were treated with 10µΜ Amyloid-β (1–42) for 48h.
6-OHDA treatment
6-Hydroxydopamine hydrochloride (6-OHDA) was dissolved according to manufacturer’s instructions (Sigma, H4381). Neurons were treated with 50µM 6-OHDA for 16h to induce oxidative stress.
BNN27 treatment
BNN27 was initially dissolved in DMSO to prepare a stock solution of 100mM. Next, cell culture medium was used to generate an intermediate dilution of 1mM. The solution was incubated for 10min at 37oC. Finally, neurons were treated with 1µM BNN27 for 48h and the effect on cell viability was evaluated with the Celltox assay (Promega, G8742).
Celltox Cytotoxicity Assay
Celltox assay (G8742, Promega Corporation, Maddison, WI, USA) was used to assess cell death according to the manufacturer's instruction. Celltox reagent was added to the cells 24h before imaging. Hoechst (1:10,000, H3570, Invitrogen, Waltham, MA, USA) was used for nuclei labeling. Cells were imaged with a Leica DM IL LED microscope.
Cell lysis and Western Blotting
Cells were solubilized with Pierce™ IP Lysis Buffer (87788, Thermo Fischer Scientific, Waltham, MA, USA) supplemented with protease inhibitors (539138, Calbiochem, Darmstadt, Germany) and phosphatase inhibitors (524629, Calbiochem, Darmstadt, Germany). Total proteins were separated by electrophoresis on SDS-PAGE gels and were transferred to nitrocellulose membranes (Cytiva, GE10600002). After blocking with 5% w/v bovine serum albumin (BSA) (Applichem, A1391) in 1xTBST, membranes were incubated overnight at 4οC with gentle shaking with the primary antibodies in 5% w/v BSA in 1xTBST. The membranes were incubated for 1h with HRP-conjugated secondary antibodies in 5% w/v BSA in 1xTBST at RT and immunoblots were developed using the ECL Western Blotting Kit (Thermo Fisher Scientific). Image analysis and quantification of band intensities were performed with ImageJ Software. Primary antibodies are shown in Table 1. Full Western Blot Images are provided in Figures S4,5.
Fig. 5
BNN27 mitigates the neurotoxic effect of high glucose via p75NTR and TrkA receptors. Celltox cytotoxicity assay. (a) Quantification and (b) representative images of dead neurons (green) upon treatment with HG (100mM), BNN27 (1µM), p75NTR inhibitor (2.5 ng/ml, anti-p75 Receptor antibody (MC-192) Abcam) and/or TrkA inhibitor (20µM, GW441756, G-190, Alomone labs, Jerusalem, Israel) for 48h. Scale bar 50µm. Data are shown as mean ± SEM of three biological replicates. Statistical significance was evaluated using a one-way ANOVA (P = 0.0001) and post hoc unpaired t-test (*P < 0.05, **&##P < 0.01).
RNA-seq library preparation and differential expression analysis
Isolation of RNA and 3′ RNA sequencing
Dopaminergic neurons were treated with 100mM D-glucose for 48h at day 19 of cell differentiation. We collected RNA from three independent cell differentiation experiments of one iPSC line (SFC856-03-04), considering them as three biological replicates. Total RNA was extracted using Trizol reagent (Thermo Scientific) as per the manufacturer’s protocol. The quantity and quality of extracted RNA samples were analyzed using RNA 6000 Nano kit on a bioanalyzer (Agilent). 500 ng of total RNA samples with RNA integrity number (RIN) > 7 were used for library construction using the 3′ mRNA-Seq Library Prep Kit FWD for Illumina (QuantSeq-LEXOGEN) as per the manufacturer’s instructions. Amplification was controlled by qPCR for obtaining optimal unbiased libraries across samples by assessing the number of cycles (15) required for amplification of the library. DNA High Sensitivity Kit for bioanalyzer was used to assess the quantity and quality of libraries, according to the manufacturer’s instructions (Agilent). Libraries were multiplexed and sequenced on an Illumina Nextseq 500 at the genomics facility of IMBB FORTH according to the manufacturer’s instructions.
Differential Expression Analysis (DEA) and Gene Ontology (GO) enrichment analysis
Essentially as detailed previously (Charou et al., 2024), the quality of the FASTQ files was assessed with the FastQC software., Reads were aligned to the human (hg38) genome with Hisat2 (hisat2 -p32 -x $REFERENCE_GENOME -q fastq/$FILE_ID.fastq -S $FILE_ID.sam --score-min L,0,-0.5 -k 2). Due to low quality of RNA and sequencing data generating poor alignment efficiency for one of the biological replicates of the High Glucose (100 mM D-glucose) condition, we kept only two replicates for this condition in the subsequent steps of the analysis. Htseq-counts was utilized to summarize reads at the gene level (htseq-count -f bam syesigene_idbam/$FILE_ID.bamdata/refs/Homo_sapiens/UCSC/hg38/Annotation/Genes/genes.gtf>$COUNTS_DIR/NGS$FILE_ID). Differentially expressed genes (DEGs) between the control (20mM) and High Glucose (100 mM) condition were identified by running EdgeR through SARTools R wrapper with batch (experiment date) effect correction. A significance threshold of an adjusted p-value < 0.05 was applied. Functional enrichment analysis was performed using Metascape. Volcano and bidirectional bar plots were created in R with custom in-house scripts (available upon request).
Quantitative RT-PCR
Total RNA was extracted from cells using Nucleozol (Macherey Nagel, 740404200). 1500ng RNA was reversely transcribed to cDNA using the High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific 4368814). qRT-PCR was carried out using the ΚΑPA SYBR FAST qPCR Master Mix (KAPA, KK4602) and gene expression was normalized to Beta-Actin. The primers are presented in Table 2.
Gene | Forward Primer | Reverse Primer |
|---|---|---|
IL-6 | 5’-ACTCACCTCTTCAGAACGAATTG-3’ | 5’-CCATCTTTGGAAGGTTCAGGTTG-3’ |
Cxcl10 | 5’-GTGGCATTCAAGGAGTACCTC-3’ | 5’-TGATGGCCTTCGATTCTGGATT-3’ |
IL-8 | 5’-GGTGCAGTTTTGCCAAGGAG-3’ | 5’-TTCCTTGGGGTCCAGACAGA-3’ |
Ccl5 | 5’-TCATTGCTACTGCCCTCTGC-3’ | 5’- TACTCCTTGATGTGGGCACG-3’ |
Beta-Actin | 5’-CCAACCGCGAGAAGATGAC-3’ | 5’-TAGCACAGCCTGGATAGCAA-3’ |
ELISA
Pro-NGF and pro-BDNF protein levels were quantified in the cell lysis and the supernatant of neurons cultured in 12 well plate format (one well per condition) by using the human pro-NGF and the human pro-BDNF Rapid ELISA kits (Biosensis, BEK-2226-1P/2P for pro-NGF and BEK-2237-2P for pro-BDNF), respectively. Briefly, the cell supernatant was collected, supplemented with protease inhibitors (539138, Calbiochem, Darmstadt, Germany) and centrifuged for 5min at 10.000xg to remove floating cells. Cell lysis and protein extraction was performed with the Pierce™ IP Lysis Buffer (87788, Thermo Fischer Scientific, Waltham, MA, USA). For the ELISA, the cell supernatant was used non-diluted, whereas the cell lysis was diluted 1:2 for the pro-NGF and 1:3 for the pro-BDNF with the Assay Diluent A. ELISA was performed according to the manufacturer’s instructions. Absorbance was measured at 450nm with the Apollo ELISA Reader - Berthold Technologies.
Statistics
Statistical analyses were conducted using GraphPad Prism 8. Details of the analyses are described within the figure legends. For the Celltox cytotoxicity assays and Western Blots we used neurons differentiated from three different human iPSC lines. At least three independent sets of experiments were performed for qPCR to ensure reproducibility, and representative data are shown. RNAseq differential expression analysis was conducted using SARTools. Genes were filtered for low counts using a cpmCutoff of 1, Trimmed Mean of M-values (TMM) was used for normalization and p-values were adjusted using the Benjamini-Hochberg (BH) correction to control the false discovery rate (FDR).
Results
High glucose induces DNA damage and cell death in DA neurons
We first generated neural progenitor cells (NPCs) from three human iPSC lines using small molecules as previously described (Reinhardt et al., 2013). NPCs were then differentiated towards DA neurons. The resulting cell population was characterized by immunostaining for the neuronal markers TUJ1 and MAP2 and the dopaminergic marker TH (Fig. 1a). Neurons were differentiated and maintained in basal medium containing 20 mM D-glucose. Upon achieving terminal differentiation, neurons were exposed to high glucose (HG) conditions by supplementing the medium with 50 mM or 100 mM D-glucose for 48 hours, thereby simulating hyperglycemic conditions.
Fig. 1
High glucose has a pro-apoptotic effect on iPSC-derived dopaminergic neurons. a) Schematic of cell differentiation and glucose treatment procedures. Dopaminergic neurons were differentiated from Neural Progenitor Cells (NPCs). At Day 19 of differentiation, neurons were exposed to HG (50mM or 100mM) for 48h. Cell identity was confirmed with immunostaining for the neuronal markers TUJ1 (green), MAP2 (green) and the dopaminergic marker TH (red). Scale bar 25µm. b) Celltox cytotoxicity assay. Representative images and quantification of dead neurons(green) after cell exposure to HG for 48h. Scale bar 50µm. c) Western blot analysis and quantification of the DNA damage marker Phospho-Histone H2A.X (Ser139) in Control and HG-treated neurons. Phospho-Histone H2A.X levels were normalized to β-Actin. d) Immunofluorescence staining showing neurons positive for Phospho-Histone H2A.X (Ser139) (green) after HG treatment for 48h. Scale bar 10µm. e) Western Blot for phospho-JNK and JNK in Control and HG-treated neurons. Graph showing the ratio of pJNK/JNK/β-Actin. f) Immunofluorescence analysis of Synapin I and Synaptophysin in Control and HG- treated neurons. Scale Bar 50µm. For b and e data are presented as mean ± SEM of three biological replicates and the statistical significance was evaluated using one-way ANOVA (b**P = 0.0045, e*P = 0.0115) and post hoc unpaired two-tailed t-test (* P < 0.05). For c data are presented as mean ± SEM of four biological replicates. Statistical significance was evaluated with unpaired t-test (*P = 0.02). For f data are presented as mean ± SEM of three biological replicates and statistical significance was evaluated using unpaired t test (*P < 0.05). Full-length blots are presented in Figure S4.
Assessment of cell viability using the Celltox cytotoxicity assay showed that HG treatment induced neuronal cell death in a dose-dependent manner. The fraction of dead cells increased from 2.09% ± 0.44% (Control) to 4.51% ± 0.53% and 11.9% ± 2.1% after treatment with 50mM and 100mM HG, respectively (Fig. 1b). Glucose overload is known to induce oxidative stress and to render cells more vulnerable to DNA damage (Gelaleti et al., 2015; Rahmoon et al., 2023). Indeed, immunocytochemistry and Western blot analyses of phosphorylated H2AX, a marker of double-stranded DNA breaks, confirmed that DA neurons accumulate DNA damage in response to HG (Fig. 1c,d). In accordance with the apoptotic phenotype, we found elevated phosphorylation levels of the JNK protein in HG-treated neurons suggesting activation of the JNK signaling (Fig. 1e). Finally, in agreement with studies reporting deregulation of synaptic proteins and synaptic transmission under hyperglycemia in hippocampal neurons (Ripoli et al., 2020; Zhong et al., 2019), we found that HG treatment significantly decreased the expression of Synapsin I and Synaptophysin in DA neurons implying disturbance of synaptic plasticity (Fig. 1f). Overall, high glucose has a pleiotropic toxic effect on DA neurons eventually leading to cell death in a dose-dependent manner.
Global Transcriptomic Analysis of high glucose-treated neurons shows induction of stress related processes
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To gain deeper insights into the molecular impact of hyperglycemia on DA neurons, we performed RNA sequencing to compare the global gene expression profiles between HG-treated and Control neurons. Glucose treatment induced transcriptional changes in neurons, with 81 genes up-regulated and 209 genes down-regulated compared to non-treated cells (p adj < 0.05) (Fig. 2a, b Additional File 1: Figure S1, Additional File 2: Table S1, Additional File 3: Table S2). Gene Ontology (GO) and Reactome Gene Set (RGS) enrichment analyses of the up-regulated genes indicated induction of processes related to cellular stress including DNA damage repair, negative regulation of secretion and cellular senescence (Fig. 2c, d, Additional File 4: Table S3). Notably, genes involved in cholesterol biosynthesis, such as MVK and FDFT1, were upregulated, consistent with previous findings linking hyperglycemia to enhanced cholesterol production (Fenton et al., 2023; Sun et al., 2014) (Fig. 2b,d). Interestingly, the most significantly enriched GO term was "cell cycle phase transition", despite DA neurons being post-mitotic. This suggests HG triggers compensatory expression of cell survival and cycle regulatory genes, including BIRC5 (a component of the chromosomal passenger complex) and CCNB1/2 (Additional File 3: Table S2, Additional File 4: Table S3, Additional File 6: Table S5). In line with the observed induction of DNA damage in HG-treated neurons, pathway analysis revealed induction of PLK1. PLK1 is part of the DNA damage response (DDR) mechanism that protects cells from entering the cell cycle with damaged DNA (Hyun et al., 2014) (Fig. 2e). Additionally, pro-apoptotic pathways including p53 and p73 were induced (Fig. 2e). In parallel, analysis of the down-regulated genes indicated changes in the post-synaptic cytoskeleton organization and affected ERA genomic pathway that associates with neuroprotection in DA neurons (Bains & Roberts, 2016; Bourque et al., 2015) (Fig. 2c, e, Additional File 5: Table S4, Additional File 6: Table S5). Overall, the RNA sequencing analysis corroborated the experimental evidence of DNA damage and cell death induction under HG.Fig. 2
Differential gene expression analysis by RNA-seq of neurons exposed to high glucose. a) Up- (81; red dots) and down- (209; blue dots) regulated differentially expressed genes (DEGs) (p adj < 0.05) were identified and plotted on a volcano plot to show genes with perturbed expression in HG-treated neurons (100mM D-glucose, 48h) compared to Control neurons. b) Heatmap showing averaged z-scores across replicates and Log2 Fold
Change (LFC) for all differentially expressed genes (DEGs) (padj <
0.05) identified between control neurons (blue) and HG-treated (100mM
D-glucose, 48h) neurons (purple). DEGs are sorted based on descending
LFC values. c, d, e) Functional enrichment analysis using Metascape (Enrichment P Cutoff = 0.05). Bidirectional bar plots showing a selection (see supplementary table for full list and details) of significant terms of GO Biological Processes, Reactome Gene Sets and Pathways, respectively. NOTE: Both directions show positive values of -Log(p-value) (significance of enrichment). Term bars are color coded based on the gene list of origin. Red bars, enriched terms using all up-regulated genes as input; blue bars, enriched terms using all down-regulated genes as input.
p75NTR is upregulated and participates in high glucose-driven cell death in DA neurons
Considering the pro-apoptotic potential of p75NTR receptor in different types of neurons in the CNS and its previous involvement in diabetic peripheral neuropathy (Chilton et al., 2004) we set out to investigate its role in high glucose-induced toxicity in dopaminergic neurons.
First, we analyzed the expression pattern of neurotrophin receptors. TrkA, TrkB and p75NTR receptors are expressed in human DA neurons (Fig. 3a,b, Additional File 1: Figure S2a). Although the expression of the neuroprotective Trk receptors remains unaffected (Fig. 3a), HG significantly upregulated the expression of the p75NTR receptor (Fig. 3b). p75NTR has no intrinsic catalytic activity but signals via interacting with effector proteins that engage to different signaling pathways (Charalampopoulos et al., 2012). To further investigate the role of p75NTR in glucose neurotoxicity, we used a p75NTR neutralizing antibody that binds the extracellular domain of the receptor serving as a selective inhibitor (Kokkali et al., 2024; Papadopoulou et al., 2023). Strikingly, inhibition of p75NTR activity effectively ameliorated the neurotoxic effect of high glucose indicating that p75NTR mediates glucose neurotoxicity in DA neurons (Fig. 3c). Inhibition of p75NTR activity decreased the number of dead cells from 4.67% ±0.39–3.21%±0.27% in 50mM HG and from 11.96% ± 2.1–4.9% ±1.02% in 100mM HG.
Fig. 3
Pro-NGF/p75NTR axis is up-regulated in glucose neurotoxicity in DA neurons. a) Western blot analysis and quantification of TRKA and TRKB in Control and HG- (100mM) treated neurons for 48h. Protein levels were normalized to β-Actin. b) Western blot analysis and quantification of p75NTR in Control and HG (50mM, 100mM) treated neurons for 48h. Protein levels were normalized to β-Actin. c) Celltox cytotoxicity assay. Representative photos and quantification of dead (green) neurons after treatment with HG (50mM, 100mM) and/or p75NTR inhibitor (2.5 ng/ml, anti-p75 Receptor antibody (MC-192) Abcam) for 48h. Scale bar 50µm. d,e) ELISA quantification of (d) pro-NGF protein levels in the cell lysate and (e) the secreted pro-NGF protein levels in the supernatant of DA neurons treated with HG (100mM) for 48h. f) Schematics of the up-regulated pro-NGF/p75NTR axis in DA neurons upon treatment with HG. For a-e data are presented as mean ± SEM of three biological replicates. For a and c statistical significance was evaluated with a two-way ANOVA (a-Interaction P = 0.41, c-Interaction P = 0.01) and post hoc unpaired two-tailed t-test (*P < 0.05). For b statistical significance was evaluated with a one-way ANOVA (P = 0.0046) and a post hoc unpaired t-test (*P < 0.05). For d and e statistical significance was evaluated with an unpaired t-test (*P < 0.05, d:P = 0.03; e:P = 0.01). Full-length blots are presented in Figure S5.
p75NTR can differentially regulate neuronal survival in a ligand- and context-dependent manner. It promotes neuronal survival when bound to mature neurotrophins in the presence of pro-survival Trk receptors, whereas it can induce pro-apoptotic signaling when bound to pro-neurotrophins (Meeker & Williams, 2015). Therefore, we next evaluated the expression and secretion levels of pro-apoptotic pro-neurotrophins, the pro-NGF and pro-BDNF, in DA neurons. ELISA measurement of pro-NGF showed a significant increase in the synthesis (Fig. 3d) and secretion (Fig. 3e) of pro-NGF in DA neurons in response to HG. In contrast, the levels of pro-BDNF remained unaffected (Additional File 1: Figure S2 b,c). Collectively, our data suggest the activation of an autocrine pro-apoptotic pro-NGF/p75NTR signaling in DA neurons, when exposed to high glucose (Fig. 3f) and demonstrate that modulation of p75NTR activity can alleviate glucose neurotoxicity.
High glucose increases neuronal vulnerability to 6-OHDA toxicity, an effect mitigated by p75NTR inhibition
Considering the increased risk of diabetic patients to develop dopaminergic dysfunction and PD, we next sought to determine whether high glucose increases DA neuron vulnerability to well established exogenous neurotoxic stimuli. We first treated neurons with the neurotoxin 6-hydroxydopamine (6-OHDA), which induces oxidative stress and impairs mitochondrial activity selectively in DA neurons (Iglesias González et al., 2019; Latchoumycandane et al., 2011). Neurons were exposed to HG for 48h with or without 50µM 6-OHDA co-administered during the last 16h (Fig. 4a). The analysis of the cytotoxicity assay showed that 6-OHDA had a toxic effect on DA neurons that was significantly exacerbated in hyperglycemic condition (Fig. 4b,c). Notably, inhibition of p75NTR activity significantly reduced the neurotoxic effect of 6-OHDA in HG-treated neurons suggesting its neuroprotective potential in a PD relevant model.
Fig. 4
p75NTR inhibition attenuates the high glucose- induced sensitization to 6-OHDA toxicity. Celltox cytotoxicity assay. a) Schematic overview of the experimental design. b,c) (b) Quantification and (c) representative images of dead neurons (green) upon exposure to HG (100mM) and p75NTR inhibitor (2.5 ng/ml, anti-p75 Receptor antibody (MC-192) Abcam) for 48 h and 6-OHDA (50µM) during the last 16h. Scale bar 50µm. For b data are shown as mean ± SEM of four biological replicates. Statistical significance was evaluated with one-way ANOVA (**P = 0.001) and post hoc unpaired t-test (*P < 0.05).
Next, we challenged DA neurons with Amyloid-β oligomers. Amyloid-β burden has been described in PD patients and has been associated with cognitive impairment in PD (Gomperts Stephen et al., 2013; Melzer et al., 2019). Additionally, although DA neurons are not the primarily affected neuronal cell population in Alzheimer’s Disease, preclinical and neuropathological evidence associates loss of DA neurons and low levels of dopamine with memory deficits in AD (Nobili et al., 2017). Therefore, we sought to determine the impact of high glucose on DA neuron susceptibility to the neurotoxic effect of Amyloid-β 1–42 oligomers, which are major components of amyloid plaques (Hampel et al., 2021). We treated DA neurons concomitantly with HG and 10µΜ Amyloid-β 1–42 oligomers for 48h. While Amyloid-β 1–42 treatment did not significantly affect the survival of DA neurons, HG resulted in a two-fold increase of dead cells compared to Amyloid-β treatment alone and ten-fold increase compared to non-treated cells (Additional File 1. Figure S3 a,b). These findings underline the influence of hyperglycemic events on the progression of neurodegeneration in PD patients with amyloid-β burden and suggest an interlink between DM, PD and cognitive dysfunction.
BNN27 exerts a neuroprotective effect in DA neurons via p75NTR and TrkA receptors
Based on the pro-apoptotic function of p75NTR, we next questioned whether neurotrophin signaling could be therapeutically targeted to alleviate HG-driven neurodegeneration. We tested a synthetic small-sized 17-spiro-steroid analog that acts as NGF mimetic, BNN27, which has gained interest as a therapeutic molecule for neuroprotection in diabetic retinopathy(Ibán-Arias et al., 2019) and Alzheimer’s Disease (Kokkali et al., 2024). Previous publications of our and other groups have shown that BNN27 acts as a selective activator of both TrkA and p75NTR receptors, promoting the survival of multiple types of neurons (Kokkali et al., 2024; Pediaditakis, Efstathopoulos, et al., 2016). Exposure of DA neurons to 1µM of BNN27 significantly alleviated high glucose-induced cell death, an effect that was abolished when using p75NTR or TrkA inhibitors (Fig. 5a, b). This effect became more pronounced with combinatorial administration of both inhibitors suggesting that BNN27 can protect DA neurons from glucose neurotoxicity acting via both NGF receptors, TrkA and p75NTR. Collectively, our findings highlight BNN27 as a promising neurotrophin-based therapeutic candidate capable of mitigating HG- induced dopaminergic neurodegeneration.
High glucose enhances the inflammatory potential of astrocytes and induces the secretion of neurotoxic factors
The current consensus in the literature is that neuroinflammation is a key interlink between DM and neurodegenerative diseases (Llorián-Salvador et al., 2024). Astrocytes are primary homeostatic cells in the brain but in response to stress stimuli, they display phenotypic changes that may increase the risk of neurodegeneration (Brandebura et al., 2023). In this context, we investigated the involvement of human astrocytes on dopaminergic neurodegeneration in hyperglycemic condition. We differentiated human iPSCs towards astrocytes as previously described (Perriot et al., 2021). Cell identity was characterized via immunostaining for the astrocyte markers GFAP, S100β and EAAT1 (Fig. 6a). Mature astrocytes were treated with HG (100mM D-glucose) for 48h to simulate hyperglycemia. Analysis of cell survival showed no apoptotic effect of HG on astrocytes indicating that astrocytes are more resistant to HG compared to neurons (Fig. 6b). To explore the influence of HG on astrocyte activation, we analyzed the expression of inflammatory markers in the presence or absence of exogenous pro-inflammatory stimuli. Astrocytes were treated with HG and/or 30ng/ml TNFa and 10ng/ml IL-1β to mimic the inflammatory microenvironment that exists in the diabetic brain due to increased BBB permeability and infiltration of immune cells. qPCR analysis showed increased gene expression of IL-6, IL-8 and Cxcl10 in HG-treated stimulated but not in unstimulated astrocytes (Figs. 6c,d) implying that HG was not adequate to activate these genes but exacerbates astrocyte responsiveness to pro-inflammatory stimuli. Finally, to evaluate how HG-treated astrocytes interfere with neuronal survival, we collected the Astrocyte Conditioned Medium (ACM) of both cytokine-stimulated and unstimulated astrocytes. DA neurons were exposed to ACM for 48h without additional glucose (Fig. 6e). Notably, ACM from hyperglycemic astrocytes—both with and without exogenous cytokine stimulation—caused significant neuronal death, with a stronger effect observed in the cytokine-stimulated group. Specifically, ACM from HG-treated astrocytes increased neuronal death by 1.6-fold compared to the control condition, while ACM from cytokine- and HG-treated astrocytes led to a 2.3-fold increase (Fig. 6f,g). Although cytokine expression levels were unchanged in non-stimulated cells, other secreted factors with toxic properties may account for this effect. Conclusively, our results indicate that HG enhances the inflammatory potential of astrocytes and induces the secretion of neurotoxic factors with a harmful effect on DA neurons.
Fig. 6
High glucose amplifies the pro-inflammatory response of astrocytes and indirectly contributes to neurodegeneration. a) Schematic of astrocyte differentiation and glucose treatment procedures. Astrocytes were differentiated and treated with HG for 48h prior to analysis. Representative photos showing S100β+, GFAP + and EAAT1 + astrocytes on differentiation day 50. Scale bar 50µm. b) Celltox cytotoxicity assay. Quantification of dead astrocytes upon treatment with HG (50mM and 100mM). c, d) mRNA levels of IL-6, IL-8 and Cxcl10 in astrocytes treated with (c) HG (100mM) in unstimulated conditions (Unstimulated) and (d) HG (100mM) stimulated with TNFa (30ng/ml) and IL-1β (10ng/ml) for 48h (TNFa- IL1β- stimulated). Gene expression was normalized to Beta-Actin. e) Schematic of Astrocyte Conditioned Medium (ACM) collection and its application to neuronal cultures for 48h. f) Celltox cytotoxicity assay in neurons treated with ACM from un- and stimulated astrocytes. +/- HG for 48h. Representative photos and quantification of dead neurons (green). Scale bar 50µm. For b, data are shown as mean ± SEM of four biological replicates. Statistical significance was evaluated with one-way ANOVA (P = 0.5) and post hoc unpaired t-test. For c, d, and g, data are shown as mean ± SEM of three biological replicates. For c and d, the statistical significance was evaluated with two-way ANOVA (c: Interaction P = 0.04, Column Factor P = 0.54; d: Interaction P = 0.39, Column Factor P = 0.0001) and post hoc paired t-test *P < 0.05. For g, the statistical significance was evaluated with two-way ANOVA (Column Factor P = 0.007) and post hoc t-test (*P < 0.05).
High glucose leads to neurotoxic microglia response
Microglia, the resident macrophages of the CNS, are the main mediators of neuroinflammation in the brain. Activated microglia may impair neuronal activity through the release of cytokines, chemokines and glutamate (Gao et al., 2023). To explore the impact of high glucose on human microglia and the secondary effect on DA neuropathology, we generated Iba1 + microglia from human iPSCs as previously described (Haenseler et al., 2017) (Fig. 7a). Microglia were exposed to 100mM D-glucose for 48h and/or 100ng/ml LPS during the last 6h to simulate a pro-inflammatory microenvironment. Alike astrocytes, qPCR analysis for IL-6, IL-8 and Ccl5 showed that microglia exposure to HG did not induce the expression of these inflammatory markers (Fig. 7b) though LPS stimulation enhanced the expression of IL-8 under HG (Fig. 7c). We next collected the microglia-conditioned media (MCM) for neuronal treatment (Fig. 7d). The MCM from HG-treated microglia, both un- and LPS-stimulated, increased neuronal cell death. The fraction of dead neurons was increased 1.9-fold and 1.5-fold after cell exposure to MCM from hyperglycemic un-stimulated and LPS-stimulated microglia, respectively, compared to Control (Fig. 7e,f). In summary, our data demonstrate that elevated glucose levels trigger microglia to secrete factors that impair DA neuron viability, a phenotype also observed in astrocytes. Our results point to an important role of glial cells in the neurological complications of diabetes and highlight neuron-glia crosstalk as a target for intervention in future therapeutic avenues.
Fig. 7
High glucose induces the release of neurotoxic factors by microglia. a) Schematic of iPSC differentiation to Iba1 + microglia (green) and glucose treatment. Microglia were treated with high glucose (100mM) for 48h. Scale bar 20µm. b, c) mRNA levels of IL-6, IL-8, and Ccl5 in (b) microglia treated with HG (100 mM) in unstimulated conditions (Unstimulated), and (c) microglia treated with HG in LPS (100 ng/mL) stimulated conditions (LPS-stimulated). Gene expression levels were normalized to Beta-Actin. d) Schematic of Microglia Conditioned Medium (MCM) collection. DA neurons were treated with 1:1 MCM/Neuronal differentiation medium for 48h. e,f) Celltox cytotoxicity assay in neurons treated with MCM. (e) Representative photos and (f) quantification of dead neurons upon treatment with MCM collected from unstimulated and LPS-stimulated microglia for 48h. Scale bar 50µm. For b and c, data are shown as mean ± SEM of five biological replicates. Statistical significance was evaluated with a two-way ANOVA (Column Factor c, d P = 0.06) and post hoc unpaired t-test (*P < 0.05). For f, data are shown as mean ± SEM of three biological replicates and statistical significance was evaluated with a two-way ANOVA (Column Factor P = 0.0013) and post hoc t-test (*P < 0.05).
Additional Files
Discussion
Hyperglycemia is a hallmark of DM but transient hyperglycemic events may arise also in other conditions, such as post Traumatic Brain Injury (Quintana-Pajaro et al., 2023), stress-hyperglycemia in patients with acute illness (Dungan et al., 2009) or gestational hyperglycemia (Rodolaki et al., 2023). Prospective cohort studies support that Type 2 DM increases the risk for PD and accelerates PD progression (L. Chen et al., 2025; Chohan et al., 2021; D’Amelio et al., 2009; Kotagal et al., 2013; Pérez-Taboada et al., 2020), while antidiabetic agents are being evaluated as modifiers of PD pathophysiology (Aguirre-Vidal et al., 2024; Wang et al., 2020). However, the underlying mechanisms of hyperglycemia-induced dopaminergic dysfunction remain poorly understood. To date, most evidence has been derived from animal models, limiting the translational relevance and clinical interpretation of the findings. Our study assessed the impact of HG on human iPSC-derived dopaminergic neurons and investigated their crosstalk with glial cells in order to better understand the pathological manifestations of hyperglycemia in the brain in an in vitro human-relevant model.
STZ-treated rodents exhibit decreased dopamine levels and dopaminergic neurodegeneration (Pérez-Taboada et al., 2020; Renaud et al., 2018). Accordingly, our analysis showed that exposure of human DA neuron to HG induces DNA damage, changes in synaptic protein expression and eventually cell death. Transcriptomic analysis confirmed the induction of DNA damage response and cell death-related pathways, like p53, in neurons when exposed to HG. Finally, we demonstrated that HG increases DA neuron susceptibility to 6-OHDA and Amyloid-β-induced cytotoxicity emphasizing the importance of glucose regulation in patients with PD or high Amyloid-β burden due to aging. Overall, our model recapitulates many glucotoxicity features described in diabetic animal models and can serve as a platform to identify mediators of glucose neurotoxicity in the human CNS.
Impaired neurotrophin signaling has previously been correlated with brain damage in diabetic patients. T2DM patients have lower serum BDNF (He et al., 2024), while up-regulation of BDNF reduces neuroinflammation (Han et al., 2021) and protects hippocampal neurons from hyperglycemia-driven apoptosis (Zhong et al., 2019). Furthermore, pro-NGF is up-regulated in the retina of STZ-treated rodents and promotes neuroinflammation (Mysona et al., 2013). Our study implicates for the first time the pan-neurotrophin receptor p75NTR in high glucose-induced neuronal loss in the brain. p75NTR is a transmembrane receptor that is up-regulated in the adult brain in response to injury and disease (Alder et al., 2016; Speidell et al., 2020). p75NTR can promote neuronal survival in conjunction with TrkA when bound to mature neurotrophins (Friedman, 2000), whereas the p75NTR-sortilin complex can induce apoptosis upon binding to pro-neurotrophins (Skeldal et al., 2012). Notably, p75NTR signaling has been already implicated in DA neuronal loss in the rotenone model of PD (L. W. Chen et al., 2008; Y. Chen et al., 2018) as well as in the neurological complications of DM in the PNS and retina (Barcelona et al., 2016; Chilton et al., 2004; Elshaer et al., 2019b; Humpert et al., 2007; Mysona et al., 2013; Scarpini et al., 1996). Here, we extend these findings by demonstrating its involvement in hyperglycemia-induced brain pathology. We show that p75NTR is up-regulated and displays a pro-apoptotic function in DA neurons in hyperglycemic conditions. HG treatment up-regulates p75NTR and its pro-apoptotic ligand pro-NGF eventually inducing an autocrine induction of the pro-NGF/p75NTR axis in DA neurons. p75NTR-mediated neuronal death has been associated with c-JUN phosphorylation and p53 activation (Aloyz et al., 1998; Costantini et al., 2005; Kraemer et al., 2014) in line with our findings of increased JNK phosphorylation and up-regulated p53 signaling in HG-treated neurons. Importantly, inhibition of p75NTR activity, by a commonly used neutralizing antibody that blocks its extracellular domain, protects neurons from HG-induced cell death indicating the therapeutic benefits of p75NTR modulatory strategies. Further analysis of pro-NGF and p75NTR levels in the CSF of diabetic patients is necessary to confirm their prognostic value in diabetic neurodegeneration.
Although the neuroprotective and anti-inflammatory properties of neurotrophins are well documented, they face therapeutic limitations due to low stability and poor BBB permeability. These challenges have been addressed by developing small synthetic DHEA-derived microneurotrophins, which mimic neurotrophin activity by targeting their receptors and show promise in neuroprotection and regeneration (Calogeropoulou et al., 2009; Gravanis et al., 2017). In this context, we tested the neuroprotective effect of an NGF synthetic mimetic, BNN27, against glucose neurotoxicity in DA neurons. BNN27 is a well characterized microneurotrophin able to penetrate the BBB that has gained interest as a neuroprotective and anti-inflammatory agent in the nervous system acting via both TrkA and p75NTR receptors (Ibán-Arias et al., 2019; Kokkali et al., 2024; Pediaditakis, Efstathopoulos, et al., 2016). Importantly, BNN27 has already been tested successfully against diabetic retinopathy acting as a neuroprotective and anti-inflammatory agent (Ibán-Arias et al., 2019). Here, we demonstrated that BNN27 can alleviate glucose neurotoxicity in DA neurons acting via p75NTR and TrkA receptors. BNN27-mediated neuroprotective actions have been shown to be dependent on the extracellular domain of p75NTR (Pediaditakis, Kourgiantaki, et al., 2016). We here observed that by blocking the extracellular domain of p75NTR, we could effectively protect neurons from high glucose-induced toxicity. We therefore propose that BNN27 confers neuroprotection by engaging the extracellular domain of p75NTR and preventing its activation by autocrine pro-NGF under hyperglycemic conditions. Additionally, BNN27 may alter the receptor's conformation or signaling capacity, shifting its activity away from apoptotic pathways toward a survival-promoting profile, inducing its ability to interact with the pro-survival TrkA receptor. Our results suggest that BNN27, which is currently under pre-clinical investigation against diabetic retinopathy, could be a lead molecule for further evaluation against cases of diabetic encephalopathy.
Accumulating evidence indicates that hyperglycemia-induced alterations in glial cells play a critical role in diabetes-induced neuropathology (Lee et al., 2024; Zhang et al., 2024). To explore changes in neuron-glia interaction in hyperglycemic conditions, we first examined the effect of HG on human iPSC-derived astrocytes and microglia. Our in vitro data indicate that, unlike neurons, human astrocyte survival is not affected by HG, likely due to their higher glycogen storage capacity(Staricha et al., 2020) and stronger antioxidant defenses (Y. Chen et al., 2020). Importantly, astrocyte exposure to HG significantly augmented the expression of pro-inflammatory mediators in the presence of inflammatory stimuli. Our results agree with findings from Bahniwal and colleagues who showed that HG induces the secretion of IL-6 and IL-8 in a human astrocytic cell line stimulated with IFNγ and IL1β but not in non-stimulated cells (Bahniwal et al., 2017). Similarly, HG treatment upregulated the expression of IL-8 in LPS-stimulated microglia suggesting that HG sensitizes glial cells to inflammatory stimuli and enhances their reactive response. The absence of a pronounced inflammatory response to HG in our human cell models—unlike the robust activation observed in animal studies in vivo and in vitro(Lee et al., 2024; Y. Li et al., 2021; Zhang et al., 2024b) – suggests that glial cells possess substantial glucose-buffering capacity, which is compromised under inflammatory conditions. Notably, while HG alone did not significantly upregulate classical pro-inflammatory markers in astrocytes or microglia, conditioned medium from HG-treated glial cells induced apoptosis in dopaminergic neurons. We propose that, under hyperglycemic conditions, glial cells may release neurotoxic factors independent of canonical inflammatory cytokines, like glutamate, nitric oxide and reactive oxygen species (Abdyeva et al., 2024; Quincozes-Santos et al., 2017), or that transient or post-transcriptionally regulated inflammatory signals were not captured at the time of analysis. The neurotoxic effects of the conditioned media were further enhanced when astrocytes and microglia were co-stimulated with additional inflammatory cues. Proteomic and metabolic characterization of the HG-treated glia will unravel the nature of the released molecules and will provide insights into the pathological alterations that contribute to neurodegeneration.
Conclusions
This study demonstrates that high glucose compromises human dopaminergic neuron survival by activating the pro-NGF/p75NTR signaling and engaging indirect, glia-mediated neurotoxic mechanisms. Our findings indicate that targeting p75NTR activity could alleviate dopaminergic neurodegeneration in patients experiencing hyperglycemic episodes and suggest the beneficial effect of the small molecule BNN27 against glucose neurotoxicity. Our research highlights the contribution of glia in neuronal loss in hyperglycemic conditions, suggesting the need for systematic neuroprotective and anti-inflammatory therapeutic strategies. Human iPSC-derived cellular models can be utilized as a platform for elucidating glucose-mediated toxicity parameters and evaluation of anti-diabetic drugs against dopaminergic neuropathology.
List of abbreviations
AD Alzheimer's disease
AGEs Advanced glycation end products
ACM Astrocyte conditioned medium
BBB Blood Brain Barrier
CNS Central Nervous System
CSF
Cerebrospinal fluid
DA Dopaminergic
DDR DNA damage response
DM Diabetes Mellitus
HG Hyperglycemia/High Glucose
iPSC induced Pluripotent Stem Cell
MCM Microglia Conditioned Medium
MEF Mouse Embryonic Fibroblast
NPC Neural Progenitor Cell
PD Parkinson’s Disease
PNS Peripheral Nervous System
P75NTR p75 neurotrophin receptor
T2DM Type 2 Diabetes Mellitus
Declarations
Ethics approval and consent to participate
The human iPSC lines provided by Dr Μ. Z. Cader were derived from human skin biopsy fibroblasts, following signed informed consent, with approval from the UK NHS Research Ethics Committee (REC: 13/SC/0179 and 10/H0505/71) and were derived as part of the IMI-EU sponsored StemBANCC consortium. The human iPSC line provided by Prof. Grünewald was derived from female donor fibroblasts, following research consent from the patient and “CNER” stands for Comité National d'Ethique de Recherche (CNER) (CNER N° 201411).
Consent for publication
Not applicable.
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Data Availability
The datasets supporting the conclusions of this article are available in the GEO repository, GSE291145 https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE291145. The following secure token has been created to allow review of record GSE291145 while it remains in private status: gzqjakwgrlgvlcr.
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Competing Interests
All authors, except Achille Gravanis, declare that they have not any competing financial interests in relation to the work described. Dr Achille Gravanis is the co-founder of spin-off Bionature EA LTD, proprietary of compound BNN27 (patented with the WO 2008/ 1555 34 A2 number at the World Intellectual Property Organization). Gravanis A is co-founder of Bionature E.A. Ltd. The BNN compounds are proprietary and patented by the Bionature E.A. Ltd (http://www.bionature.net) (Patent Number: WO2008/155534 A2).
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Funding
This research was funded by: (1) Bodossaki Postdoctoral Research Fellowship to K.C., (2) EMBO Scientific Exchange Grant STF_11143 to K.C., (3) the European Union HORIZON, under the European Innovation Council (EIC)-2022-PATHFINDEROPEN-01 program “SoftReach”, No 101099145 (4) the Institute of Pharmaceutical Research and Technology (IFET S.A.) under collaborative funding between FORTH and IFET S.A., (5) the framework of the Action ‘Flagship Research Projects in challenging interdisciplinary sectors with practical applications in Greek industry’, implemented through the National Recovery and Resilience Plan Greece 2.0 and funded by the European Union – NextGenerationEU (project code: TAEDR-0535850). An.G. was supported by the Luxemburg National Research Fund (FNR) within the framework of an ATTRACT career development grant (Model-IPD, FNR9631103). M.T. received funding from the FNR within the NextImmune2 DTU (PRIDE21/16749720).
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Author Contribution
K.C.: conception and design of the study, acquisition and interpretation of the data, drafting the text I.C.: design of the study, interpretation of data, drafting the manuscript, Ac.G.& An.G.: interpretation of data, revising the manuscript, I.Z.: acquisition of the data, revising the manuscript, M.P., A.T., M.T., C.K., K.N.: acquisition of the data, E.T. & M.L.: RNA-seq analysis
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Acknowledgement
We thank Dr Μ. Z. Cader for providing us three human iPSC lines. We thank the staff of the Genomics Facility of IMBB FORTH, Heraklion, Crete, Greece for RNA-seq library preparation, sequencing and preliminary analyses. We thank Prof. Joseph Papapmatheakis, Dr Androniki Kretsovali and As. Prof. Dimitrios Tzeranis for revising the manuscript.
Use of Artificial Intelligence
The authors declare that they have not use AI-generated work in this manuscript.
Electronic Supplementary Material
Below is the link to the electronic supplementary material
Supplementary Material 7
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Figures & Figure Legends
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Additional File 1
. pdf. Supplementary Material. Figure S1. Heatmap showing the expression profile of all differentially expressed genes (p adj < 0.05), identified between HG-treated neurons and control neurons. Figure S2. Neurotrophin receptors and pro-BDNF levels in DA neurons. Figure S3. High glucose triggers DA neuron susceptibility to Amyloid-β 1–42. Figure S4. Uncropped Western Blot images of Fig. 1. Figure S5. Uncropped Western Blot images of Fig. 3.
Additional File 2.
xls. Table S1. Annotation of Heatmap rows.
Additional File 3.
xls. Table S2. List of DEGs (p adj < 0.05).
Additional File 4.
xls. Table S3. Metascape enrichment analysis of the up-regulated genes from the DEG list.
Additional File 5.
xls. Table S4. Metascape enrichment analysis of the down-regulated genes from the DEG list.
Additional File 6.
xls. Table S5. Gene lists for selected metascape annotations.
Additional File 7.
png. Graphical Abstract
