Multiple sclerosis (MS) is a devastating disease, and with the increasing number of cases each year, it is becoming a significant socioeconomic burden for the affected people and the entire community. The aetiology of MS is largely unknown, but genetic susceptibility, exposure to infections and/or environmental toxicants are recognised as risk factors. MS is characterised by the appearance of lesions/plaques in the central nervous system, caused by destruction of the myelin sheet by auto-reactive T cells. Symptoms range from mild impairment of daily motor functions to severe sensory and cognitive disabilities necessitating mobility assistance, medical and support from caregivers. Due to the progressive nature of the disease, MS is gaining more attention and research to better understand its multifaceted pathogenesis. In the present review, we focus on some of the latest research related to the neuroinflammatory component of the disease, since it appears to play a critical role in MS pathogenesis. The goal is to shed more light on this specific domain of MS, in an attempt to assist in the identification of novel treatment trajectories and management plans.

Following peripheral nerve injury, dysregulations of certain non-coding microRNAs (miRNAs) occur in Schwann cells. Whether these alterations are the result of local inflammation and/or correlate with perturbations in the expression profile of the protective vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase-activating polypeptide (PACAP) system is currently unknown. To address these issues, we aimed at profiling the expression of selected miRNAs in the rat RT4 Schwann cell line. Cells exposed to lipopolysaccharide (LPS), to mimic the local inflammatory milieu, were appraised by real-time qPCR, Western blot and ELISAs. We found that upon LPS treatment, levels of pro-inflammatory cytokines (IL-1β, -6, -18, -17A, MCP-1 and TNFα) increased in a time-dependent manner. Unexpectedly, the expression levels of VIP and PACAP were also increased. Conversely, levels of VPAC1 and VPAC2 receptors were reduced. Downregulated miRNAs included miR-181b, -145, -27a, -340 and -132 whereas upregulated ones were miR-21, -206, -146a, -34a, -155, -204 and -29a, respectively. Regression analyses revealed that a subset of the identified miRNAs inversely correlated with the expression of VPAC1 and VPAC2 receptors. In conclusion, these findings identified a novel subset of miRNAs that are dysregulated by immune challenge whose activities might elicit a regulatory function on the VIP/PACAP system.

Buspirone is an anxiolytic drug with robust serotonin receptor 1A (Htr1a) agonist activities. However, evidence has demonstrated that this drug also targets the dopamine D3 receptor (Drd3), where it acts as a potent antagonist. In vivo, Drd3 blockade is neuroprotective and reduces inflammation in models of Parkinson’s disease. To test if buspirone also elicited anti-inflammatory activities in vitro, we generated stable Drd3−/− and Htr1a−/− BV2 microglial cell lines using CRISPR-Cas9 technology and then tested the effects of buspirone after lipopolysaccharide (LPS) challenge. We found that LPS exposure had no effect on cell viability, except in Htr1a−/− cells, where viability was reduced (p < 0.001). Drug treatment reduced viability in Drd3−/− cells, but not in WT or Htr1a−/− cells. Buspirone counteracted LPS-induced NO release, NOS2, IL-1β and TNF-α gene expression in WT cells, whereas it exerted limited effects in Drd3−/− or Htr1a−/− microglia. In summary, our findings indicate that buspirone attenuates microglial polarization after LPS challenge. These results also highlight some major effects of Drd3 or Htr1a genetic ablation on microglial biology, raising important questions on the complex role of neurotransmitters in regulating microglia functions.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are two structurally related immunosuppressive peptides. However, the underlying mechanisms through which these peptides regulate microglial activity are not fully understood. Using lipopolysaccharide (LPS) to induce an inflammatory challenge, we tested whether PACAP or VIP differentially affected microglial activation, morphology and cell migration. We found that both peptides attenuated LPS-induced expression of the microglial activation markers Iba1 and iNOS (### p < 0.001), as well as the pro-inflammatory mediators IL-1β, IL-6, Itgam and CD68 (### p < 0.001). In contrast, treatment with PACAP or VIP exerted distinct effects on microglial morphology and migration. PACAP reversed LPS-induced soma enlargement and increased the percentage of small-sized, rounded cells (54.09% vs. 12.05% in LPS-treated cells), whereas VIP promoted a phenotypic shift towards cell subpopulations with mid-sized, spindle-shaped somata (48.41% vs. 31.36% in LPS-treated cells). Additionally, PACAP was more efficient than VIP in restoring LPS-induced impairment of cell migration and the expression of urokinase plasminogen activator (uPA) in BV2 cells compared with VIP. These results suggest that whilst both PACAP and VIP exert similar immunosuppressive effects in activated BV2 microglia, each peptide triggers distinctive shifts towards phenotypes of differing morphologies and with differing migration capacities.

to be reconsidered for publication in Neural Regeneration Research. The work has not been published elsewhere and is not under review with another journal. The authors have agreed to the resubmission of the revised version of the manuscript.In the revised work, we carefully considered all the recommendations given by the two expert reviewers and revised the methodology to reduce the percentage of similarity as reported by the Editorial office. We put all the efforts to correctly incorporate the suggested changes in the text. All the revisions in the paper were performed using the Microsoft Word tracked-changes option. Below you will find an itemised list with the responses to each reviewers' concerns. We are grateful to you and the reviewers for your support and hope that the revised submission, if accepted, will further contribute to the growth of the journal.