The glymphatic-lymphatic system is increasingly recognized as fundamental for the homeostasis of the brain milieu since it defines cerebral spinal fluid flow in the brain parenchyma and eliminates metabolic waste. Animal and human studies have uncovered several important physiological factors regulating the glymphatic system including sleep, aquaporin-4, and hemodynamic factors. Yet, our understanding of the modulation of the glymphatic system is limited, which has hindered the development of glymphatic-based treatment for aging and neurodegenerative disorders. Here, we present the evidence from fluorescence tracing, two-photon recording, and dynamic contrast-enhanced magnetic resonance imaging analyses that 40 Hz light flickering enhanced glymphatic influx and efflux independently of anesthesia and sleep, an effect attributed to increased astrocytic aquaporin-4 polarization and enhanced vasomotion. Adenosine-A _2A receptor (A _2A R) signaling emerged as the neurochemical underpinning of 40 Hz flickering-induced enhancement of glymphatic flow, based on increased cerebrofluid adenosine levels, the abolishment of enhanced glymphatic flow by pharmacological or genetic inactivation of equilibrative nucleotide transporters-2 or of A _2A R, and by the physical and functional A _2A R–aquaporin-4 interaction in astrocytes. These findings establish 40 Hz light flickering as a novel non-invasive strategy of enhanced glymphatic flow, with translational potential to relieve brain disorders.

Major depressive disorder ranks as a major burden of disease worldwide, yet the current antidepressant medications are limited by frequent non-responsiveness and significant side effects. The lateral septum (LS) is thought to control of depression, however, the cellular and circuit substrates are largely unknown. Here, we identified a subpopulation of LS GABAergic adenosine A_2A receptors (A_2AR)-positive neurons mediating depressive symptoms via direct projects to the lateral habenula (LHb) and the dorsomedial hypothalamus (DMH). Activation of A_2AR in the LS augmented the spiking frequency of A_2AR-positive neurons leading to a decreased activation of surrounding neurons and the bi-directional manipulation of LS-A_2AR activity demonstrated that LS-A_2ARs are necessary and sufficient to trigger depressive phenotypes. Thus, the optogenetic modulation (stimulation or inhibition) of LS-A_2AR-positive neuronal activity or LS-A_2AR-positive neurons projection terminals to the LHb or DMH, phenocopied depressive behaviors. Moreover, A_2AR are upregulated in the LS in two male mouse models of repeated stress-induced depression. This identification that aberrantly increased A_2AR signaling in the LS is a critical upstream regulator of repeated stress-induced depressive-like behaviors provides a neurophysiological and circuit-based justification of the antidepressant potential of A_2AR antagonists, prompting their clinical translation.

Immune checkpoint blockade (ICB) therapy targeting the programmed death 1/programmed death-ligand 1 (PD-1/PD-L1) axis has achieved considerable success in treating a wide range of cancers. However, most patients with pancreatic cancer remain resistant to ICB. Moreover, there is a lack of optimal biomarkers for the prediction of response to this therapy. Palmitoylation is mediated by a family of 23 S-acyltransferases, termed zinc finger Asp-His-His-Cys-type palmitoyltransferases (ZDHHC), which precisely control various cancer-related protein functions and represent promising drug targets for cancer therapy. Here, we revealed that tumor cell-intrinsic ZDHHC9 was overexpressed in pancreatic cancer tissues and associated with impaired anti-tumor immunity. In syngeneic pancreatic tumor models, the knockdown of ZDHHC9 expression suppressed tumor progression and prolonged survival time of mice by modifying the immunosuppressive ('cold') to proinflammatory ('hot') tumor microenvironment. Furthermore, ZDHHC9 deficiency sensitized anti-PD-L1 immunotherapy mainly in a CD8⁺ T cell dependent manner. Lastly, we employed the ZDHHC9-siRNA nanoparticle system to efficiently silence ZDHHC9 in pancreatic tumors. Collectively, our findings indicate that ZDHHC9 overexpression in pancreatic tumors is a mechanism involved in the inhibition of host anti-tumor immunity and highlight the importance of inactivating ZDHHC9 as an effective immunotherapeutic strategy and booster for anti-PD-L1 therapy against pancreatic cancer.

Background: Multiple sclerosis (MS) is one of the most common autoimmune disorders characterized by the infiltration of immune cells into the brain and demyelination. The unwanted immunosuppressive side effect of therapeutically successful natalizumab led us to focus on the choroid plexus (CP), a key site for the first wave of immune cell infiltration in experimental autoimmune encephalomyelitis (EAE), for the control of immune cells trafficking. Adenosine A_2A receptor (A_2AR) is emerging as a potential pharmacological target to control EAE pathogenesis. However, the cellular basis for the A_2AR-mediated protection remains undetermined. Methods: In the EAE model, we assessed A_2AR expression and leukocyte trafficking determinants in the CP by immunohistochemistry and qPCR analyses. We determined the effect of the A_2AR antagonist KW6002 treatment at days 8–12 or 8–14 post-immunization on T cell infiltration across the CP and EAE pathology. We determined the critical role of the CP-A_2AR on T cell infiltration and EAE pathology by focal knock-down of CP-A_2AR via intracerebroventricular injection of CRE-TAT recombinase into the A_2AR^flox/flox mice. In the cultured CP epithelium, we also evaluated the effect of overexpression of A_2ARs or the A_2AR agonist CGS21680 treatment on the CP permeability and lymphocytes migration. Results: We found the specific upregulation of A_2AR in the CP associated with enhanced CP gateway activity peaked at day 12 post-immunization in EAE mice. Furthermore, the KW6002 treatment at days 8–12 or 8–14 post-immunization reduced T cell trafficking across the CP and attenuated EAE pathology. Importantly, focal CP-A_2AR knock-down attenuated the pathogenic infiltration of Th17⁺ cells across the CP via inhibiting the CCR6–CCL20 axis through NFκB/STAT3 pathway and protected against EAE pathology. Lastly, activation of A_2AR in the cultured epithelium by A_2AR overexpression or CGS21680 treatment increased the permeability of the CP epithelium and facilitated lymphocytes migration. Conclusion: These findings define the CP niche as one of the primary sites of A_2AR action, whereby A_2AR antagonists confer protection against EAE pathology. Thus, pharmacological targeting of the CP-A_2AR represents a novel therapeutic strategy for MS by controlling immune cell trafficking across CP.

The cluster of differentiation 38 (CD38) is a multifunctional transmembrane protein involved in numerous physiological and pathological processes including aging, neurodegenerative diseases, and tumorigenesis, hence is an attractive drug target. However, the mechanisms underlying the regulation of CD38 expression remain enigmatic. Herein, we report for the first time that CD38 is palmitoylated at Cys16, and that S-palmitoylation is required to maintain CD38 protein expression in tumor cells. Furthermore, we identify DHHC9 as the palmitoyl transferase and APT1 as the acylprotein thioesterase responsible for this crucial post-translational modification. Finally, we designed a competitive peptide of CD38 palmitoylation that decreases CD38 expression in tumor cells and suppresses tumor progression in vivo. These findings provide novel insight into CD38 regulation and highlight potential therapeutic strategies targeting CD38 palmitoylation for cancer treatment.

A new Cytolysin, termed as Gigantoxin-4, was isolated from the sea anemone Stichodactyla gigantea and found to be highly homologous with Cytolysin-3 (HMg III) from Heteractis magnifica, RTX-A from Radianthus macrodactylus, and Sticholysin-1 (St I) and Sticholysin-2 (St II) from Stichodactyla helianthus (homology 82%, 86%, 82% and 86% respectively). Its 20 N-terminal residues were identified and the full-length cDNA sequence was obtained by reverse transcription-polymerase chain reaction (RT-PCR). Multiple sequence alignments with other Cytolysins of the actinoporin family clearly indicated that Gigantoxin-4 belongs to this protein family. SDS-PAGE electrophoresis showed that this new actinoporin had a molecular mass of about 19 kDa, and possessed a high hemolytic activity to human erythrocytes (HA₅₀= 40 ng/ml), which was inhibited by pre-incubation with sphingomyelin (SM) or SM-cholesterol mixtures. Our in vivo experiments showed that Gigantoxin-4 had wide toxicity to the rat cardiovascular system and the respiratory system. A concentration of 30μg/kg Gigantoxin-4, i.v. produced a positive inotropic effect on the rat heart although final cardiovascular failure was inevitable, and 60μg/kg Gigantoxin-4 caused respiratory arrest rapidly resulting in rat death. HE staining indicated pathological changes in various organs and tissues after i.v. administration of Gigantoxin-4.