BACKGROUND: Pituitary adenylate cyclase-activating peptide (PACAP) is a neuropeptide pivotal in migraine pathophysiology and is considered a promising new migraine drug target. Although intravenous PACAP triggers migraine attacks and a recent phase II trial with a PACAP-inhibiting antibody showed efficacy in migraine prevention, targeting the PACAP receptor PAC1 alone has been unsuccessful. The present study investigated the role of three PACAP receptors (PAC1, VPAC1 and VPAC2) in inducing migraine-relevant hypersensitivity in mice.
METHODS: Hindpaw hypersensitivity was induced by repeated PACAP38 injections. Tactile sensitivity responses were quantified using von Frey filaments in three knockout (KO) mouse strains, each lacking one of the PACAP-receptors (Ntotal = 160). Additionally, ex vivo wire myography was used to assess vasoactivity of the carotid artery, and gene expression of PACAP receptors was examined by qPCR.
RESULTS: PACAP38 induced hypersensitivity in WT controls (p < 0.01) that was diminished in VPAC1 and VPAC2 KO mice (p < 0.05). In contrast, PAC1 KO mice showed similar responses to WT controls (p > 0.05). Myograph experiments supported these findings showing diminished vasoactivity in VPAC1 and VPAC2 KO mice. We found no upregulation of the non-modified PACAP receptors in KO mice.
CONCLUSIONS: This study assessed all three PACAP receptors in a migraine mouse model and suggests a significant role of VPAC receptors in migraine pathophysiology. The lack of hypersensitivity reduction in PAC1 KO mice suggests the involvement of other PACAP receptors or compensatory mechanisms. The results indicate that targeting only individual PACAP receptors may not be an effective migraine treatment.

Dedicated assembly factors orchestrate stepwise production of many molecular machines, including the 28-subunit proteasome core particle (CP) that mediates protein degradation. Here, we report cryo-EM reconstructions of seven recombinant human subcomplexes that visualize all five chaperones and the three active site propeptides across a wide swath of the assembly pathway. Comparison of these chaperone-bound intermediates and a matching mature CP reveals molecular mechanisms determining the order of successive subunit additions, and how proteasome subcomplexes and assembly factors structurally adapt upon progressive subunit incorporation to stabilize intermediates, facilitate the formation of subsequent intermediates, and ultimately rearrange to coordinate proteolytic activation with gated access to active sites. The structural findings reported here explain many previous biochemical and genetic observations. This work establishes a methodologic approach for structural analysis of multiprotein complex assembly intermediates, illuminates specific functions of assembly factors, and reveals conceptual principles underlying human proteasome biogenesis.

Oral lichen planus (OLP) is a T cell-mediated autoimmune disease of oral mucosa concerning with the redox imbalance. Although glutamine uptake mediated by alanine-serine-cysteine transporter 2 (ASCT2) is critical to T cell differentiation, the exact mechanism remains ambiguous. Here, we elucidate a novel regulatory mechanism of ASCT2-mediated uptake in the differentiation and proliferation of T cells through maintaining redox balance in OLP. The results of immunohistochemistry (IHC) showed that both ASCT2 and glutaminase (GLS) were obviously upregulated compared to controls in OLP. Moreover, correlation analyses indicated that ASCT2 expression was significantly related to GLS level. Interestingly, the upregulation of glutamine metabolism in epithelial layer was consistent with that in lamina propria. Functional assays in vitro revealed the positive association between glutamine metabolism and lymphocytes infiltration. Additionally, multiplex immunohistochemistry (mIHC) uncovered a stronger colocalization among ASCT2 and CD4 and IFN-γ, which was further demonstrated by human Th1 differentiation assay in vitro. Mechanistically, targeting glutamine uptake through interference with ASCT2 using L-γ-Glutamyl-p-nitroanilide (GPNA) decreased the glutamine uptake of T cells and leaded to the accumulation of intracellular reactive oxygen species (ROS), which promoted dual specificity phosphatase 2 (DUSP2/PAC1) expression through activation of early growth response 1 (EGR1) to induce dephosphorylation of signal transducer and activator of transcription 3 (STAT3) and inhibit Th1 differentiation in turn. These results demonstrated that glutamine uptake mediated by ASCT2 induced Th1 differentiation by ROS-EGR1-PAC1 pathway, and restoring the redox dynamic balance through targeting ASCT2 may be a potential treatment for T cell-mediated autoimmune diseases.

The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) belongs to the glucagon/secretin family. PACAP interacts with the pituitary adenylate cyclase-activating polypeptide receptor type 1 (PAC1) and vasoactive intestinal peptide receptors 1 and 2 (VPAC1 and VPAC2), exhibiting functions in the immune, endocrine, and nervous systems. This peptide is upregulated in numerous instances of brain injury, acting as a neuroprotective agent. It can also suppress HIV-1 and SARS-CoV-2 viral replication in vitro. This work aimed to identify, in each peptide-receptor system, the most relevant residues for complex stability and interaction energy communication via Molecular Dynamics (MD), Free Energy calculations, and Protein-energy networks, thus revealing in detail the underlying mechanisms of activation of these receptors. Hydrogen bond formation, interaction energies, and computational alanine scanning between PACAP and its receptors showed that His1, Asp3, Arg12, Arg14, and Lys15 are crucial to the peptide\'s stability. Furthermore, several PACAP interactions with structurally conserved positions deemed necessary in GPCR B1 activation, including Arg2.60, Lys2.67, and Glu7.42, were significant for the peptide\'s stability within the receptors. According to the protein-energy network, the connection between Asp3 of PACAP and the receptors\' conserved Arg2.60 represents a critical energy communication hub in all complexes. Additionally, the ECDs of the receptors were also found to function as energy communication hubs for PACAP. Although the overall binding mode of PACAP in the three receptors was found to be highly conserved, Arg12 and Tyr13 of PACAP were more prominent in complex with PAC1, while Ser2 of PACAP was with VPAC2. The detailed analyses performed in this work pave the way for using PACAP and its receptors as therapeutic targets.Communicated by Ramaswamy H. Sarma.

The development of inflammatory pain seriously affects the activities and general functions of patients in daily life. At present, the research on the mechanism of pain relief is still insufficient. This study aimed to investigate the influence of PAC1 on the progression of inflammatory pain and its molecular mechanism. Lipopolysaccharide (LPS) was used to induce BV2 microglia activation to establish an inflammation model, and CFA injection was used to establish a mouse inflammatory pain model. The results showed that PAC1 was highly expressed in BV2 microglia induced by LPS. Knockdown of PAC1 significantly reduced LPS-induced inflammation and apoptosis in BV2 cells, and RAGE/TLR4/NF-κB signaling pathway was involved in the regulation of BV2 cells by PAC1. What\'s more, knockdown of PAC1 alleviated CFA-induced mechanical allodynia and thermal hyperalgesia in mice, as well as reduced the development of inflammatory pain to a certain extent. Therefore, Knockdown of PAC1 relieved inflammatory pain in mice by inhibiting the RAGE/TLR4/NF-κB signaling pathway. Targeting PAC1 may be a new direction for the treatment of inflammatory pain.

BACKGROUND: Resistance exercise induces thrombocytosis and increases platelet activation and function. These changes might be related to exercise variables including exercise intensity and type.
OBJECTIVE: We compared the effects of traditional resistance exercise (TRE) and circuit resistance exercise (CRE) on cellular markers of platelet activation and function.
METHODS: In this crossover study ten healthy male (mean±SD: age, 25.6±2.4 years) subjects performed TRE encompassed 3 sets of 10 repetitions at 100% of 10-RM (10 repetition maximum) for 6 exercises, and CRE protocols included 3 sets of 10 repetitions at 100% of 10-RM for all 6 exercises consecutively, in two separate weeks. To measure platelet indices, PAC1, CD41a, CD42b and CD62P three blood samples were taken before, immediately after exercise, and after 30 min recovery.
RESULTS: Lactate concentration, blood pressure, platelet count (PLT), and mean platelet volume (MPV) were significantly (p < 0.05) increased following both resistance exercise trials. Significant increases in PAC1, and CD62P; and significant reductions for CD42b and CD41a were detected following both REs (p < 0.05). However, changes in PAC1 and CD62P were significantly different between the two protocols (p < 0.05), with higher increases detected following CRE.
CONCLUSIONS: Acute RE increases platelet indices and platelet activation; and that CRE results in higher platelet activation than TRE, probably due to exercise-induced increases in shear stress.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are structurally related neuropeptides that are widely expressed in vertebrate tissues. The two neuropeptides are pleiotropic and have been associated with migraine pathology. Three PACAP and VIP receptors have been described: PAC1, VPAC1, and VPAC2. The localization of these receptors in relation to VIP and PACAP in migraine-relevant structures has not previously been shown in mice. In the present study, we used fluorescence immunohistochemistry, well-characterized antibodies, confocal microscopy, and three-dimensional reconstruction to visualize the distribution of PACAP, VIP, and their receptors in the basal blood vessels (circle of Willis), trigeminal ganglion, and brain stem spinal trigeminal nucleus (SP5) of the mouse CNS. We demonstrated a dense network of circularly oriented VIP fibers on the basal blood vessels. PACAP nerve fibers were fewer in numbers compared to VIP fibers and ran along the long axis of the blood vessels, colocalized with calcitonin gene-related peptide (CGRP). The nerve fibers expressing CGRP are believed to be sensorial, with neuronal somas localized in the trigeminal ganglion and PACAP was found in a subpopulation of these CGRP-neurons. Immunostaining of the receptors revealed that only the VPAC1 receptor was present in the basal blood vessels, localized on the surface cell membrane of vascular smooth muscle cells and innervated by VIP fibers. No staining was seen for the PAC1, VPAC1, or VPAC2 receptor in the trigeminal ganglion. However, distinct PAC1 immunoreactivity was found in neurons innervated by PACAP nerve terminals located in the spinal trigeminal nucleus. These findings indicate that the effect of VIP is mediated via the VPAC1 receptor in the basal arteries. The role of PACAP in cerebral arteries is less clear. The localization of PACAP in a subpopulation of CGRP-expressing neurons in the trigeminal ganglion points toward a primary sensory function although a dendritic release cannot be excluded which could stimulate the VPAC1 receptor or the PAC1 and VPAC2 receptors on immune cells in the meninges, initiating neurogenic inflammation relevant for migraine pathology.

Vasoactive Intestinal Peptide (VIP) and Pituitary Adenylate-Cyclase-Activating Peptide (PACAP) are anti-inflammatory neuropeptides that play important roles in human and rodent gut microbiota homeostasis and host immunity. Pharmacologically regulating these neuropeptides is expected to have significant health and feed efficiency benefits for agriculturally relevant animals. However, their expression profile in ruminant tissues is not well characterized. To this end, we screened for VIP and PACAP neuropeptides and their endogenous GPCRs using 15 different tissues from wethers and steers by RT-qPCR. Our results revealed relatively similar expression profiles for both VIP and PACAP neuropeptide ligands in the brain and intestinal tissue of both species. In contrast, the tissue expression profiles for VPAC1, VPAC2, and PAC1 were more widespread and disparate, with VPAC1 being the most diversely expressed receptor with mRNA detection in the brain and throughout the gastrointestinal tract. These data are an important first step to allow for future investigations regarding the VIP and PACAP signaling pathways in livestock ruminant species.

Homeostatic challenges may alter the drive for social interaction. The neural activity that prompts this motivation remains poorly understood. In the present study, we identify direct projections from the hypothalamic supraoptic nucleus to the cortico-amygdalar nucleus of the lateral olfactory tract (NLOT). Dual in situ hybridization with probes for pituitary adenylate cyclase-activating polypeptide (PACAP), as well as vesicular glutamate transporter (VGLUT)1, VGLUT2, V1a and V1b, revealed a population of vasopressin-receptive PACAPergic neurons in NLOT layer 2 (NLOT2). Water deprivation (48 h, WD48) increased sociability compared to euhydrated subjects, as assessed with the three-chamber social interaction test (3CST). Fos expression immunohistochemistry showed NLOT and its main efferent regions had further increases in rats subjected to WD48 + 3CST. These regions strongly expressed PAC1 mRNA. Microinjections of arginine vasopressin (AVP) into the NLOT produced similar changes in sociability to water deprivation, and these were reduced by co-injection of V1a or V1b antagonists along with AVP. We conclude that, during challenge to water homeostasis, there is a recruitment of a glutamatergic-multi-peptidergic cooperative circuit that promotes social behavior.

The sexually dimorphic bed nucleus of the stria terminalis (BNST) is comprised of several distinct regions, some of which act as a hub for stress-induced changes in neural circuitry and behavior. In rodents, the anterodorsal BNST is especially affected by chronic exposure to stress, which results in alterations to the corticotropin-releasing factor (CRF)-signaling pathway, including CRF receptors and upstream regulators. Stress increases cellular excitability in BNST CRF+ neurons by potentiating miniature excitatory postsynaptic current (mEPSC) amplitude, altering the resting membrane potential, and diminishing M-currents (a voltage-gated K+ current that stabilizes membrane potential). Rodent anterodorsal and anterolateral BNST neurons are also critical regulators of behavior, including avoidance of aversive contexts and fear learning (especially that of sustained threats). These rodent behaviors are historically associated with anxiety. Furthermore, BNST is implicated in stress-related mood disorders, including anxiety and Post-Traumatic Stress Disorders in humans, and may be linked to sex differences found in mood disorders.