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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. 

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. 

Abstract Background The pond snail, Lymnaea stagnalis ( L. stagnalis ), has served as a valuable model organism for neurobiology studies due to its simple and easily accessible central nervous system (CNS). L. stagnalis has been widely used to study neuronal networks and recently gained popularity for study of aging and neurodegenerative diseases. However, previous transcriptome studies of L. stagnalis CNS have been exclusively carried out on adult L. stagnalis only. As part of our ongoing effort studying L. stagnalis neuronal growth and connectivity at various developmental stages, we provide the first age-specific transcriptome analysis and gene annotation of young (3 months), adult (6 months), and old (18 months) L. stagnalis CNS. Results Using the above three age cohorts, our study generated 55–69 millions of 150 bp paired-end RNA sequencing reads using the Illumina NovaSeq 6000 platform. Of these reads, ~ 74% were successfully mapped to the reference genome of L. stagnalis . Our reference-based transcriptome assembly predicted 42,478 gene loci, of which 37,661 genes encode coding sequences (CDS) of at least 100 codons. In addition, we provide gene annotations using Blast2GO and functional annotations using Pfam for ~ 95% of these sequences, contributing to the largest number of annotated genes in L. stagnalis CNS so far. Moreover, among 242 previously cloned L. stagnalis genes, we were able to match ~ 87% of them in our transcriptome assembly, indicating a high percentage of gene coverage. The expressional differences for innexins, FMRFamide, and molluscan insulin peptide genes were validated by real-time qPCR. Lastly, our transcriptomic analyses revealed distinct, age-specific gene clusters, differentially expressed genes, and enriched pathways in young, adult, and old CNS. More specifically, our data show significant changes in expression of critical genes involved in transcription factors, metabolisms (e.g. cytochrome P450), extracellular matrix constituent, and signaling receptor and transduction (e.g. receptors for acetylcholine, N-Methyl-D-aspartic acid, and serotonin), as well as stress- and disease-related genes in young compared to either adult or old snails. Conclusions Together, these datasets are the largest and most updated L. stagnalis CNS transcriptomes, which will serve as a resource for future molecular studies and functional annotation of transcripts and genes in L. stagnalis .