Non-electrophilic TRPA1 modulator MNP-021 protects neuronal and glial function from diabetes-associated glucotoxicity and neuroinflammation

Diabetes-associated neurodegeneration is amplified by methylglyoxal (MGO)-driven dicarbonyl stress linking hyperglycemia to neuronal insulin resistance and maladaptive neuroinflammation. We tested the neuroprotective activity of MNP-021, a non-electrophilic transient receptor potential ankyrin 1 (TRPA1) modulator, in neurons and glial cells in vitro.

SH-SY5Y neurons were pretreated with MNP-021 and challenged with MGO, then profiled by high-content imaging, RNA-seq, Seahorse Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR), glycolytic stress assays, and immunoblotting for AKT, ERK, and cAMP response element-binding protein (CREB). In an additional sustained glucotoxicity paradigm, differentiated SH-SY5Y cells were exposed to high glucose for 7 days and viability was assessed by LIVE/DEAD™ high-content imaging. HMC3 glial cells were treated with MNP-021, exposed to Lipopolysaccharide (LPS), Tumor Necrosis Factor-α (TNF-α) or Aβ(25-35), and tested for viability and inflammatory markers by Enzyme-Linked Immunosorbent Assay (ELISA) and RT-qPCR.

MGO increased nucleus-to-cytoplasm area ratio by 49% and dysregulated glucose handling, increasing 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-NBDG) uptake by ~ 25%, with GLUT1/GLUT4 membrane redistribution; MNP-021 normalized morphology, glucose uptake, and transporter localization without cytotoxicity up to 10 µM. RNA-seq identified 754 MGO-deregulated genes, including ISR/metabolic nodes such as glucokinase (GCK), Sestrin-2 (SESN2), phosphoglycerate dehydrogenase (PHGDH), Phosphoserine Aminotransferase 1 (PSAT1), phosphoenolpyruvate carboxykinase 2 (PCK2); MNP-021 buffered stress-induced transcription with limited baseline effects, modulated mitochondrial morphology and redox-associated fluorescence while preserving overall respiratory flux and blunting stress-evoked compensatory glycolysis. MNP-021 restored pro-survival signaling (pAKT/pERK and nuclear pCREB), including insulin responsiveness during MGO exposure. In a sustained high-glucose model, MNP-021 reduced the high-glucose-associated increase in cell death, extending its protective effects beyond the acute MGO paradigm. MNP-021 reduced Interleukine-6 (IL-6)/TNF-α release while increasing Interleukine-10 (IL-10) and Arginase 1 (ARG1) (~ 1.9-fold vs. LPS/TNF-α) in HMC3 glial cells, shifting them toward a pro-resolving IL-10/ARG1 program with reduced amyloid β-peptide _25-35 (Aβ_(25-35))-evoked cytokine release and Glucagon-Like Peptide-1 (GLP-1) remaining very low (≤ 10 pg/mL) and not significantly increased in this system.

MNP-021 coordinates transcriptomic restraint, transporter-level glucose handling, mitochondrial resilience, and pro-survival/pro-resolving signaling in neuron-microglia cell models. These findings support further preclinical evaluation of MNP-021 in dicarbonyl-linked neuro-metabolic stress and provide pharmacological evidence consistent with a TRPA1-linked component of its activity.