Polyamine (PA) spermine (SPM) (i) plays an essential role in the function of neurons, while (ii) accumulating predominantly in glial cells by an unknown mechanism. In addition, the translocation of SPM synthesis and redistribution in the developing and maturating retinas remains unclear. Therefore, the expression of the SPM-synthesizing enzyme, spermine synthase (SpmS), was compared in rat retinas on postnatal days 3, 21, and 120 using immunocytochemistry, Western blot (WB), and ImageJ analyses. The anti-glutamine synthetase (GS) antibody identified glial cells, and DAPI labeled the cell nuclei. At postnatal day 3 (P3), the neonatal retina shows widespread SpmS expression throughout most neuroblast cells, but absent in the developing synaptic layers and Müller cell (MCs) processes. By day 21 (P20), SpmS becomes strongly expressed in neurons, and not in glia. On day 120 (P120), SpmS was observed in synaptic areas, with significantly less presence in neuronal soma and still none in MCs. WBs showed a decrease in SpmS expression during maturation. Therefore, glial cells do not synthesize SPM, and the accumulation of SPM in MCs found earlier suggests that glial cells take up SPM via a hypothetical high-affinity SPM transporter. In glia, SPM regulates glial connexin (Cx43) and potassium (Kir4.1) channels, being a key player in CNS diseases and aging.

Hyperglycemia is linked to a higher risk of diabetes, epilepsy, and seizures, which contribute to increased mortality. Previous research has shown that hyperglycemia impairs the function of the polyamine-dependent Kir4.1 channels in cultured astrocytes, and a similar effect was observed in male astrocytes from diabetic (db/db) mice. However, whether there are sex differences in this effect remains unclear. This study aims to compare Kir4.1 channel function in 10–12 week old brains of female db/db mice with that in males and non-diabetic heterozygous (db/+). We performed mRNA analyses, Western blotting (WB), glucose level measurements, and patch-clamp recordings from hippocampal astrocytes in brain slices to assess Kir4.1 channel activity in db/db and db/+ mice of both sexes. Our results showed increased glucose levels in diabetic db/db mice, especially in males, along with (1) reduced synthesis of Kir4.1 channel protein, (2) reduced membrane potential, (3) decreased Kir currents, and (4) compromised potassium uptake. Female diabetic astrocytes exhibited significantly lower barium-blocked Kir4.1 currents compared to diabetic males. Additionally, barium-insensitive currents (leakage currents) were upregulated in db/db mice, likely as a compensatory response to hyperglycemia. In conclusion, diabetes impairs astrocyte function by downregulating Kir4.1 channels, with a more pronounced effect in male diabetic mice. This impairment may increase seizure risk by affecting the ability of astrocytes to maintain extracellular ion balance.

The effects of chronic hyperglycemia on CNS, glial homeostasis and Kir4.1 regulation is poorly understood. Elevated blood glucose levels affect polyamine-sensitive inwardly rectifying potassium channels, Kir4.1, in astrocytes and retinal Müller glial cells. These cells regulate extracellular potassium and clear glutamate, processes supported by Kir4.1. Dysregulation of Kir4.1 channels has been implicated in glutamate toxicity, epilepsy, ataxia, and diabetes-induced neurodegeneration. In this study, we used Tandem Mass Tag Proteomic analysis, Pyrosequencing, SDS-PAGE and Western blot analysis, in whole brain and astrocytic primary cultures in hyperglycemic conditions testing DNMT1 and AMPK activities with RG-108, compound C, and AICAR treatments to investigate how hyperglycemia alters Kir4.1 expression in astrocytes and explored two regulatory mechanisms: (i) DNA methylation mediated by DNA methyltransferase 1 (DNMT-1), which uses S-adenosylmethionine (SAM) as a methyl donor, and (ii) metabolic signaling via the AMPK/SIRT-1 cascade. We found that hyperglycemia (25 mM glucose) (i) increased DNMT-1 protein levels in astrocytes by 1.5–2-fold and (ii) decreased AMPK and (iii) SIRT-1 levels, accompanied by a reduction in Kir4.1 protein expression. Pharmacological inhibition of DNMT-1 with RG-108 decreased DNMT-1 levels and restored Kir4.1. Treatment with AICAR, a selective AMPK activator, rescued SIRT-1 and Kir4.1 expression. In summary, hyperglycemia engages dual regulatory pathways: (i) epigenetic via DNMT-1 and (ii) metabolic via AMPK/SIRT-1, which converge on Kir4.1 downregulation in astrocytes. Therefore, both DNMT-1 inhibition and AMPK activation rescued Kir4.1 expression, suggesting functional support for potassium and glutamate homeostasis and membrane potential stability in glia.