Chronic hyperglycemia in type II diabetes results in impaired autophagy function, accumulation of protein aggregates, and neurodegeneration. However, little is known about how to preserve autophagy function under hyperglycemic conditions. In this study, we tested whether progranulin (PGRN), a neurotrophic factor required for proper lysosome function, can restore autophagy function in neurons under high-glucose stress. We cultured primary cortical neurons derived from E18 Sprague-Dawley rat pups to maturity at 10 days in vitro (DIV) before incubation in high glucose medium and PGRN for 24-72 h before testing for autophagy flux, protein turnover, and mitochondrial function. We found that although PGRN by itself did not upregulate autophagy, it attenuated impairments in autophagy seen under high-glucose conditions. Additionally, buildup of the autophagosome marker light chain 3B (LC3B) and lysosome marker lysosome-associated membrane protein 2A (LAMP2A) changed in both neurons and astrocytes, indicating a possible role for glia in autophagy flux. Protein turnover, assessed by remaining advanced glycation end-product levels after a 6-h incubation, was preserved with PGRN treatment. Mitochondrial activity differed by complex, although PGRN appeared to increase overall activity in high glucose. We also found that activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and glycogen synthase kinase 3β (GSK3β), kinases implicated in autophagy function, increased with PGRN treatment under stress. Together, our data suggest that PGRN prevents hyperglycemia-induced decreases in autophagy by increasing autophagy flux via increased ERK1/2 kinase activity in primary rat cortical neurons.

Estrogen plays fundamental roles in nervous system development and function. Traditional studies examining the effect of estrogen in the brain have focused on the nuclear estrogen receptors (ERs), ERα and ERβ. Studies related to the extranuclear, membrane-bound G proteincoupled estrogen receptor (GPER/GPR30) have revealed a neuroprotective role for GPER in mature neurons. In this study, we investigated the differential effects of GPER activation in primary rat embryonic (E18) hippocampal and cortical neurons. Microscopy imaging, multielectrode array (MEA), and Ca 2+ imaging experiments revealed that GPER activation with selective agonist, G-1, and non-selective agonist, 17β-estradiol (E2), increased neural growth, neural firing activity, and intracellular Ca 2+ more profoundly in hippocampal neurons than in cortical neurons. The GPER-mediated Ca 2+ rise in hippocampal neurons involve internal Ca 2+ store release via activation of phospholipase C and extracellular entry via Ca 2+ channels. 3Immunocytochemistry results revealed no observable difference in GPER expression/localization in neurons, yet RT-qPCR and western blot showed a higher GPER expression in the cortex than hippocampus, implying that GPER expression level may not fully account for its robust physiological effects in hippocampal neurons. We used RNA sequencing data to identify distinctly enriched pathways and significantly expressed genes in response to G-1 or E2 in cultured rat E18 hippocampal and cortical neurons. In summary, the identification of differential effects of GPER activation on hippocampal and cortical neurons in the brain and the determination of key genes and molecular pathways are instrumental toward an understanding of estrogen's action in early neuronal development. Significance StatementStudies of estrogen function via a non-nuclear G protein-coupled estrogen receptor (GPER/GPR30) in the brain have primarily focused on mature neurons and neuroprotective actions with little investigation into the role of GPER in early neural development. In this work, we discover differential effects of GPER on early neurite outgrowth, neuronal activity, and intracellular calcium signaling in primarily cultured rat embryonic (E18) hippocampal and cortical neurons. This study further highlights distinct, transcriptomic genes and pathways that are regulated by GPER agonists in early developing hippocampal and cortical neurons. These results advance our fundamental understanding of estrogen functions via GPER signaling in different (hippocampal versus cortical) neurons during early neuronal development. This knowledge is also instrumental for therapeutics for GPER-related neurodevelopmental disorders.

Type II diabetes affects over 530 million individuals worldwide and contributes to a host of neurological pathologies. Uncontrolled high blood glucose (hyperglycemia) is a major factor in diabetic pathology, and glucose regulation is a common goal for maintenance in patients. We have found that the neuronal growth factor progranulin protects against hyperglycemic stress in neurons, and although its mechanism of action is uncertain, our findings identified Glycogen Synthase Kinase 3β (GSK3β) as being potentially involved in its effects. In this study, we treated mouse primary cortical neurons exposed to high-glucose conditions with progranulin and a selective pharmacological inhibitor of GSK3β before assessing neuronal health and function. Whole-cell and mitochondrial viability were both improved by progranulin under high-glucose stress in a GSK3β—dependent manner. This extended to autophagy flux, indicated by the expressions of autophagosome marker Light Chain 3B (LC3B) and lysosome marker Lysosome-Associated Membrane Protein 2A (LAMP2A), which were affected by progranulin and showed heterogeneous changes from GSK3β inhibition. Lastly, GSK3β inhibition attenuated downstream calcium signaling and neuronal firing effects due to acute progranulin treatment. These data indicate that GSK3β plays an important role in progranulin’s neuroprotective effects under hyperglycemic stress and serves as a jumping-off point to explore progranulin’s protective capabilities in other neurodegenerative models.