Human Neurons-hippocampal (HN-h)

High Glucose Promotes tau Hyperphosphorylation by Inhibiting the Expression of m⁶A Demethylase ALKBH5 in HN-h Cells

Elevated blood glucose in diabetes leads to tau hyperphosphorylation in hippocampal neurons, causing cognitive decline. m⁶A modifications affect cognitive functions and are linked with neurodegenerative conditions. ALKBH5, a m⁶A demethylase, is pivotal in neurodevelopment and potentially in neurodegeneration through its regulation of m⁶A marks. Qu's team aims to uncover the mechanism by which high glucose induces tau hyperphosphorylation via ALKBH5-mediated effects on Dgkh mRNA in hippocampal neurons, exploring potential therapeutic targets to reverse diabetic cognitive dysfunction.

Human Neurons-hippocampal (HN-h) cells were exposed to various high glucose (HG) concentrations (25, 33, 50, and 75 mM) to identify a condition that would not affect viability. A significant viability reduction was observed with 75 mM for 2 days and 50 mM for 3 days (Fig. 1A and B). Osmolarity control didn't significantly affect viability. Thus, 50 mM glucose for 2 days was selected for further experiments.The HG group showed significant decreases in ALKBH5 mRNA and protein levels compared to control samples (Fig. 1C and D). The levels of METTL3, METTL14, and FTO proteins remained unchanged. HG treatment significantly increased p-tau levels at T231 and PHF-1 epitopes (Fig. 1D), suggesting that HG induces tau hyperphosphorylation alongside reduced ALKBH5 expression in HN-h cells. To confirm ALKBH5's role in tau phosphorylation, HN-h cells were infected with a lentivirus carrying ALKBH5, reversing HG-induced tau hyperphosphorylation (Fig. 1E and F). The silencing of ALKBH5 using siRNA produced a slight elevation in p-tau levels at T231 and PHF-1 in comparison to control and showed more pronounced results than HG plus scrambled siRNA (Fig. 1G and H). Research demonstrates high glucose levels lead to reduced ALKBH5 expression which triggers tau protein hyperphosphorylation.

Fig. 1. ALKBH5 downregulation leads to tau hyperphosphorylation in HN-h cells (Qu M, Zuo L, et al., 2023).

Upregulation of miR-223-3p Expression is a Mechanism Involved in the Neuroprotective Effect of DEX Against H₂O₂ Induced-cytotoxicity

Dexmedetomidine (DEX) is an anesthetic known for its neuroprotective capabilities. It is used for sedation in medical procedures and has shown protection against various organ ischemia. Recent studies have linked its neuroprotection to modulation of specific pathways involving apoptotic and oxidative stress signaling, as well as inflammation. MicroRNAs (miRNAs) play roles in neuroprotection through regulation of cellular stress responses.

Wang's team investigated a potential implication of microRNA (miR)‑223‑3p in the DEX‑induced anti‑oxidative effect on neuronal cells. MTT assay indicated that DEX protects HN-h cells against H₂O₂-induced cytotoxicity. To investigate miR-223-3p's involvement in DEX protection against H₂O₂-based cell damage HN-H cells received miR-223-3p mimic or inhibitor treatment before H₂O₂ exposure. The RNA mimic resulted in higher miR-223-3p levels and the inhibitor led to lower miR-223-3p levels. MTT assays and flow cytometry outcomes displayed that the inhibitor increased both cellular death and apoptotic activity while the mimic showed protective effects against these outcomes (Fig. 2A and B). The activation of miR-223-3p through mimics reduced LDH leakage together with MDA, ROS, calcium ion levels and CAMII phosphorylation which indicates that miR-223-3p serves as a neuroprotective agent (Fig. 2C-H).

Fig. 2. Upregulation of miR-223-3p expression is a mechanism involved in the neuroprotective effect of DEX against H₂O₂ induced-cytotoxicity (Wang Q, Yu H, et al., 2018).