Microglial cells, the resident immune cells of the central nervous system (CNS), are essential for maintaining CNS homeostasis. Dysregulation of microglial function is implicated in the pathogenesis of various neurodegenerative diseases. Vasoactive intestinal polypeptide receptors 1 and 2 (VPAC1 and VPAC2) are G-protein-coupled receptors (GPCRs) expressed by microglia, with their primary ligands being pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP). However, the specific roles of VPAC-type receptors in microglial regulation remain poorly understood. In this study, we generated VPAC1+/− and VPAC2+/− BV2 microglial cell lines using CRISPR-Cas9 gene editing and conducted a series of biological and molecular assays to elucidate the functions of these receptors. Our findings demonstrated that both mutant cell lines exhibited a polarized phenotype and increased migratory activity. VPAC1+/− cells showed enhanced survivability and baseline activation of the unfolded protein response (UPR), a protective mechanism triggered by endoplasmic reticulum (ER) stress, whereas this response appeared impaired in VPAC2+/− cells. In contrast, under lipopolysaccharide (LPS)-induced inflammatory conditions, UPR activation was impaired in VPAC1+/− cells but restored in VPAC2+/− cells, resulting in improved survival of VPAC2+/− cells, whereas VPAC1+/− cells exhibited reduced resilience. Overall, our findings suggest that VPAC1 and VPAC2 receptors play distinct yet complementary roles in BV2 microglia. VPAC2 is critical for regulating survival, ER stress responses, and polarization under basal conditions, while VPAC1 is essential for adaptive responses to inflammatory stimuli such as LPS. These insights advance our understanding of microglial receptor signaling and may inform therapeutic strategies targeting microglial dysfunction in neurodegenerative diseases.
Neuroinflammation within the central nervous system (CNS) is a process predominantly regulated by two types of resident glial cells: microglia and astrocytes. Although neuroinflammation is often perceived negatively and associated with CNS damage, its acute responses constitute a critical defensive mechanism designed to protect the brain from pathogens and/or injury while striving to restore homeostasis. In contrast, chronic CNS inflammation leads to the sustained release of neurotoxic inflammatory mediators, reactive oxygen species, and reactive nitrate species, all of which contribute to a deleterious microenvironment that impairs cellular function and CNS healing processes. Consequently, chronic neuroinflammation is widely recognized as a significant contributor to the pathogenesis of various neurodegenerative and traumatic neurological disorders, including multiple sclerosis (MS), Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis (ALS), and traumatic brain injury (TBI).
Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are neuropeptides extensively distributed within the CNS. These neuropeptides share structural similarities and exhibit a wide range of biological activities, including neuroprotection and anti-inflammatory effects. Their actions are mediated via three G-coupled receptors (GPCRs): the pituitary adenylate cyclase activating polypeptide receptor 1 (PAC1), vasoactive intestinal polypeptide receptor 1 (VPAC1), and vasoactive intestinal polypeptide receptor 2 (VPAC2), which mediate distinct cell-specific activities. Notably, PACAP has more than 100-fold greater affinity for the PAC1 receptor compared to VIP, yet both peptides bind with high affinity to VPAC1 and VPAC2 receptors. These differences suggest that PACAP/VIP receptor-specific biological effects largely depend on factors such as cell type, local receptor density, and localization.
VPAC1 and VPAC2 receptors are encoded by the VIPR1 and VIPR2 genes, respectively. These receptors are abundantly expressed in both the CNS and peripheral nervous system (PNS). Studies using VPAC1−/− mice subjected to experimental autoimmune encephalomyelitis (EAE)—a murine model of MS—demonstrated a delay in disease onset. These findings suggested that VIPR1 deficiency impairs CNS upregulation of chemokines and reduces the invasion of inflammatory cells from the periphery. In contrast, VPAC2−/− mice subjected to EAE displayed exacerbated clinical and histopathological features, accompanied by impaired regulatory T cell (Treg) function. These contrasting findings suggest that VPAC1 and VPAC2 receptors exert opposing functions during the effector phase of EAE, with VPAC1 primarily facilitating the onset of neuroinflammation, while VPAC2 acts as a molecular brake that restrains the inflammatory response. Similarly, in an experimental model of temporal lobe epilepsy, Serpa and colleagues reported differential regulation of VPAC1 and VPAC2 expression in the affected hippocampus, further supporting distinct roles for each receptor subtype.
Despite these advancements, the mechanisms underpinning the contrasting biological activities of VPAC receptors in microglia remain poorly understood. Given that PACAP and VIP are regulators of several key microglial functions—as evident from studies involving PACAP-deficient mice—a deeper understanding of the biological roles of VPAC1 and VPAC2 receptors in unstimulated and polarized microglia remains a critical unanswered question.
Importantly, understanding how altered VPAC signaling affects microglial behavior at baseline and under inflammatory stress may offer novel mechanistic insights into early microglial dysfunction during neurodegeneration. These in vitro findings could guide future experiments testing VPAC receptor modulation in animal models of MS or ALS, particularly by evaluating whether receptor-specific alterations impact disease onset, progression, or microglial activation profiles in vivo.
To address this gap, we used Clustered Regularly Interspaced Short Palindromic Repeats-Cas9 (CRISPR-Cas9) gene editing technology to generate murine BV2 microglial cell lines with heterozygous deletions of the VPAC1 or VPAC2 receptor genes (VPAC1+/− or VPAC2+/− microglia). These modified cell lines were subjected to a comprehensive battery of biological and molecular assays under resting conditions and after acute immune challenge with lipopolysaccharide (LPS). Our aim was to determine whether VPAC1 or VPAC2 haploinsufficiency would influence key microglial activities. A further goal was to elucidate any underlying intracellular adaptive mechanisms activated after partial gene ablation.
BV2 microglial cells were kindly provided by Dr Eryn Werry from the University of Sydney, Sydney, Australia. Cells were grown (37 °C, 5% CO 2) in Dulbecco’s Modified Eagle Medium (DMEM) mixture F-12 Ham (DMEM/F-12) supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS) and 1% Penicillin (100 IU/mL)/streptomycin (100 μg/mL) (Merck Life Science, Bayswater, VIC, Australia). To sensitize cells to the effect of an immune challenge, cells were serum-starved for 24 h prior to lipopolysaccharide (LPS) treatment (1 μg/mL; Merck Life Science, Bayswater, VIC, Australia) for 0, 6, 12, and 24 h under reduced serum conditions (DMEM/F12 nutrient mixture, 1% Fetal Bovine Serum, 1% Penicillin/streptomycin; Merck Life Science, Bayswater, VIC, Australia), as in prior work.
The all-in-one PX458 plasmid vector (Addgene, Watertown, MA, USA) was used for the generation of CRISPR-Cas9 expression plasmids containing VPAC1 or VPAC2 guide (g)-RNA sequences. gRNA primer sequences that were ligated to the CRISPR-Cas9 plasmid vector were designed using Benchling® and are listed in Table 1.
A detailed description of the protocol for the generation of our CRISPR-Cas9 expression vector and cell transfection is described in detail elsewhere. Cells were seeded and transfected with X-tremeGENE™ HP DNA Transfection Reagent (Merck Life Science, Bayswater, VIC, Australia) in Opti-MEM media (Thermo Fisher Scientific, Scoresby, VIC, Australia) supplemented with purified plasmid and P3000 reagent (Thermo Fisher Scientific, Scoresby, VIC, Australia). Reagent control cells underwent similar transfection; however, the plasmid was omitted. At 24 h post-transfection, green fluorescent protein (GFP)-positive cells were isolated and single-cell sorted using a BD FACSMelody™ cell sorter (BD Biosciences, Sydney, NSW, Australia) into a 96-well plate containing full growth media (FGM, DMEM/F12 nutrient mixture, 10% Fetal Bovine Serum, 1% Penicillin/streptomycin; Merck Life Science, Bayswater, VIC, Australia). Thereafter, single-cell clonal populations were expanded for 2–3 weeks and progressively sub-cultured into larger vessels (i.e., 24- and 6-well plates) until yielding sufficient cells for downstream biological and molecular applications.
To validate successful gene editing, genomic DNA was isolated from expanded clonal populations using the ISOLATE II Genomic DNA Kit (Millennium Science, Mulgrave, VIC, Australia) according to the manufacturer’s instructions. Purified DNA was then amplified by end-point polymerase chain reaction (PCR) using Phusion green Hot Start II High-Fidelity PCR Master Mix (Thermo Fisher Scientific, Scoresby, VIC, Australia) using PCR sequencing primers (shown in Table 2) and Sanger sequenced by the Australian Genomic Research Facility (AGRF, Westmead, NSW, Australia) using primer sets outlined in Table 3. To determine the successful targeting and efficiency of CRISPR-Cas9-induced gene deletion, sequencing results from VPAC1- and VPAC2-transfected clones were analyzed using
