CD38 is an enzyme that catalyzes the formation of cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate, both of which are involved in the mobilization of Ca2+ from intracellular stores. Recently, CD38 has been shown to regulate oxytocin release from hypothalamic neurons. Importantly, CD38 mutations are associated with autism spectrum disorders (ASD) and CD38 knockout (CD38−/−) mice display ASD-like behavioral phenotypes including deficient parental behavior and poor social recognition memory. Although ASD and learning deficits commonly co-occur, the role of CD38 in learning and memory has not been investigated. We report that CD38−/− mice show deficits in various learning and memory tasks such as the Morris water maze, contextual fear conditioning, and the object recognition test. However, either long-term potentiation or long-term depression is not impaired in the hippocampus of CD38−/− mice. Our results provide convincing evidence that CD38−/− mice show deficits in various learning and memory tasks including spatial and non-spatial memory tasks. Our data demonstrate that CD38 is critical for regulating hippocampus-dependent learning and memory without modulating synaptic plasticity.

Ursolic acid (UA) possesses various pharmacological activities, such as antitumorigenic and anti-inflammatory effects. In the present study, we investigated the mechanisms underlying the effects of UA against esophageal squamous cell carcinoma (ESCC) (TE-8 cells and TE-12 cells). The cell viability assay showed that UA decreased the viability of ESCC in a dose-dependent manner. In the soft agar colony formation assay, the colony numbers and size were reduced in a dose-dependent manner after UA treatment. UA caused the accumulation of vacuoles and LC3 puncta, a marker of autophagosome, in a dose-dependent manner. Autophagy induction was confirmed by measuring the expression levels of LC3 and p62 protein in ESCC cells. UA increased LC3-II protein levels and decreased p62 levels in ESCC cells. When autophagy was hampered using 3-methyladenine (3-MA), the effect of UA on cell viability was reversed. UA also significantly inhibited protein kinase B (Akt) activation and increased p-Akt expression in a dose-dependent manner in ESCC cells. Accumulated LC3 puncta by UA was reversed after wortmannin treatment. LC3-II protein levels were also decreased after treatment with Akt inhibitor and wortmannin. Moreover, UA treatment increased cellular reactive oxygen species (ROS) levels in ESCC in a time- and dose-dependent manner. Diphenyleneiodonium (an ROS production inhibitor) blocked the ROS and UA induced accumulation of LC3-II levels in ESCC cells, suggesting that UA-induced cell death and autophagy are mediated by ROS. Therefore, our data indicate that UA inhibits the growth of ESCC cells by inducing ROS-dependent autophagy.

Regulated IRE1-dependent decay (RIDD) is a critical cellular mechanism mediated by the endoplasmic reticulum (ER) stress sensor IRE1α, which cleaves a variety of RNA targets to regulate ER homeostasis. Current in vitro assays to study IRE1α activity largely rely on synthetic or in vitro transcribed RNA substrates, which may not fully replicate the physiological complexities of native RNA molecules. Here, we present a comprehensive protocol to assess IRE1α-dependent RNA cleavage activity using total RNA isolated directly from mouse tissues. This protocol provides a step-by-step guide for tissue collection, RNA isolation, an ex vivo RIDD assay, cDNA synthesis, and subsequent RT-PCR analysis of target mRNA cleavage products. Key reagents include active IRE1α protein, the RIDD-specific inhibitor 4μ8C, and target-specific primers for RIDD-regulated genes such as Bloc1s1 and Col6a1. Quantitative assessment is achieved using agarose gel electrophoresis and imaging software. This methodology enables the study of IRE1α's RNA cleavage activity under conditions that closely mimic in vivo environments, providing a more physiologically relevant approach to understanding the role of RIDD in cellular and tissue-specific contexts. Key features • Uses total RNA from mouse tissues instead of synthetic RNA to better reflect in vivo conditions. • Includes RIDD-specific controls such as IRE1α inhibitor (4μ8C) and RNase A to confirm targeted RNA cleavage. • Combines agarose gel electrophoresis and ImageJ quantification for both qualitative and statistical validation. • Allows comparative studies of IRE1α activity across multiple mouse tissues in different biological contexts.

The protein kinase R (PKR)-like endoplasmic reticulum (ER) kinase (PERK), a key ER stress sensor of the unfolded protein response (UPR), can confer beneficial effects by facilitating the removal of cytosolic aggregates through the autophagy-lysosome pathway (ALP). In neurodegenerative diseases, the ALP ameliorates the accumulation of intracellular protein aggregates in the brain. Transcription factor-EB (TFEB), a master regulator of the ALP, positively regulates key genes involved in the cellular degradative pathway. However, in neurons, the role of PERK activation in mitigating amyloidogenesis by ALP remains unclear. In this study, we found that SB202190 selectively activates PERK independently of its inhibition of p38 mitogen-activated protein kinase, but not inositol-requiring transmembrane kinase/endoribonuclease-1α (IRE1α) or activating transcription factor 6 (ATF6), in human neuroblastoma cells. PERK activation by SB202190 was dependent on mitochondrial ROS production and promoted Ca ²⁺ -calcineurin activation. The activation of the PERK-Ca ²⁺ -calcineurin axis by SB202190 positively affects TFEB activity to increase ALP in neuroblastoma cells. Collectively, our study reveals a novel physiological mechanism underlying ALP activation, dependent on PERK activation, for ameliorating amyloidogenesis in neurodegenerative diseases.