High-fat diet (HFD)-induced comorbid cognitive and behavioural impairments are thought to be the result of persistent low-grade neuroinflammation. Metformin, a first-line medication for the treatment of type-2 diabetes, seems to ameliorate these comorbidities, but the underlying mechanism(s) are not clear. Pituitary adenylate cyclase-activating peptide (PACAP) and vasoactive intestinal peptide (VIP) are neuroprotective peptides endowed with anti-inflammatory properties. Alterations to the PACAP/VIP system could be pivotal during the development of HFD-induced neuroinflammation. To unveil the pathogenic mechanisms underlying HFD-induced neuroinflammation and assess metformin’s therapeutic activities, (1) we determined if HFD-induced proinflammatory activity was present in vulnerable brain regions associated with the development of comorbid behaviors, (2) investigated if the PACAP/VIP system is altered by HFD, and (3) assessed if metformin rescues such diet-induced neurochemical alterations. C57BL/6J male mice were divided into two groups to receive either standard chow (SC) or HFD for 16 weeks. A further HFD group received metformin (HFD + M) (300 mg/kg BW daily for 5 weeks) via oral gavage. Body weight, fasting glucose, and insulin levels were measured. After 16 weeks, the proinflammatory profile, glial activation markers, and changes within the PI3K/AKT intracellular pathway and the PACAP/VIP system were evaluated by real-time qPCR and/or Western blot in the hypothalamus, hippocampus, prefrontal cortex, and amygdala. Our data showed that HFD causes widespread low-grade neuroinflammation and gliosis, with regional-specific differences across brain regions. HFD also diminished phospho-AKT^(Ser473) expression and caused significant disruptions to the PACAP/VIP system. Treatment with metformin attenuated these neuroinflammatory signatures and reversed PI3K/AKT and PACAP/VIP alterations caused by HFD. Altogether, our findings demonstrate that metformin treatment rescues HFD-induced neuroinflammation in vulnerable brain regions, most likely by a mechanism involving the reinstatement of PACAP/VIP system homeostasis. Data also suggests that the PI3K/AKT pathway, at least in part, mediates some of metformin’s beneficial effects.

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.

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 soma (48.41% vs 31.36% in LPS-treated). 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.

Telomere biology disorders (TBDs) are a spectrum of multisystem inherited disorders characterized by bone marrow failure, resulting from mutations in the genes encoding telomerase or other proteins involved in maintaining telomere length and integrity. Pathogenicity of variants in these genes can be hard to evaluate, because TBD mutations show highly variable penetrance and genetic anticipation related to inheritance of shorter telomeres with each generation. Thus, detailed functional analysis of newly identified variants is often essential. Herein, we describe a patient with compound heterozygous variants in the TERT gene, which encodes the catalytic subunit of telomerase, hTERT. This patient had the extremely severe Hoyeraal-Hreidarsson form of TBD, although his heterozygous parents were clinically unaffected. Molecular dynamic modeling and detailed biochemical analyses demonstrate that one allele (L557P) affects association of hTERT with its cognate RNA component hTR, whereas the other (K1050E) affects the binding of telomerase to its DNA substrate and enzyme processivity. Unexpectedly, the data demonstrate a functional interaction between the proteins encoded by the two alleles, with wild-type hTERT rescuing the effect of K1050E on processivity, whereas L557P hTERT does not. These data contribute to the mechanistic understanding of telomerase, indicating that RNA binding in one hTERT molecule affects the processivity of telomere addition by the other molecule. This work emphasizes the importance of functional characterization of TERT variants to reach a definitive molecular diagnosis for patients with TBD, and, in particular, it illustrates the importance of analyzing the effects of compound heterozygous variants in combination, to reveal interallelic effects.