Prolonged activation of microglia leads to excessive release of proinflammatory mediators, which are detrimental to brain health. Therefore, there are significant efforts to identify pathways mediating microglial activation. Recent studies have demonstrated that fatty acid-binding protein 4 (FABP4), a lipid binding protein, is a critical player in macrophage–mediated inflammation. Given that we have previously identified FABP4 in microglia, the aim of this study was to assess whether FABP4 activity contributed to inflammation, metabolism and immune function (i.e. immunometabolism) in immortalised mouse microglia (BV-2 cells) using the proinflammatory stimulus lipopolysaccharide (LPS) to induce general microglial activation. Microglial FABP4 expression was significantly increased following exposure to LPS, an outcome associated with a significant increase in microglial proliferation rate. LPS-stimulated BV-2 microglia demonstrated a significant increase in the production of reactive oxygen species (ROS) and tumour necrosis factor-alpha (TNF-α), phosphorylation of c-Jun N-terminal kinase (JNK), increased expression of Toll-like receptor 4 (TLR4), and reduced expression of uncoupling protein 2 (UCP2), all of which were reversed following FABP4 genetic silencing and chemical inhibition with BMS309403. The oxidation rate of ³H-oleic acid and microglial uptake of ³H-2-deoxy-D-glucose were modulated with LPS activation, processes which were restored with genetic and chemical inhibition of FABP4. This is the first study to report on the critical role of FABP4 in mediating the deleterious effects of LPS on microglial immunometabolism, suggesting that FABP4 may present as a novel therapeutic target to alleviate microglia-mediated neuroinflammation, a commonly reported factor in multiple neurodegenerative diseases. Graphical Abstract

P‐glycoprotein (P‐gp) is an efflux transporter at the blood–brain barrier (BBB) that hinders brain access of substrate drugs and clears endogenous molecules such as amyloid beta (Aβ) from the brain. As biometals such as copper (Cu) modulate many neuronal signalling pathways linked to P‐gp regulation, it was hypothesised that the bis(thiosemicarbazone) (BTSC) Cu‐releasing complex, copper II glyoxal bis(4‐methyl‐3‐thiosemicarbazone) (Cu^II[GTSM]), would enhance P‐gp expression and function at the BBB, while copper II diacetyl bis(4‐methyl‐3‐thiosemicarbazone) (Cu^II[ATSM]), which only releases Cu under hypoxic conditions, would not modulate P‐gp expression. Following treatment with 25–250 nM Cu^II(BTSC)s for 8–48 h, expression of P‐gp mRNA and protein in human brain endothelial (hCMEC/D3) cells was assessed by RT‐qPCR and Western blot, respectively. P‐gp function was assessed by measuring accumulation of the fluorescent P‐gp substrate, rhodamine 123 and intracellular Cu levels were quantified by inductively coupled plasma mass spectrometry. Interestingly, Cu^II(ATSM) significantly enhanced P‐gp expression and function 2‐fold and 1.3‐fold, respectively, whereas Cu^II(GTSM) reduced P‐gp expression 0.5‐fold and function by 200%. As both compounds increased intracellular Cu levels, the effect of different BTSC backbones, independent of Cu, on P‐gp expression was assessed. However, only the Cu‐ATSM complex enhanced P‐gp expression and this was mediated partly through activation (1.4‐fold) of the extracellular signal‐regulated kinase 1 and 2, an outcome that was significantly attenuated in the presence of an inhibitor of the mitogen‐activated protein kinase regulatory pathway. Our findings suggest that Cu^II(ATSM) and Cu^II(GTSM) have the potential to modulate the expression and function of P‐gp at the BBB to impact brain drug delivery and clearance of Aβ. image

P-glycoprotein (P-gp), expressed at the blood-brain barrier (BBB), is critical in preventing brain access to substrate drugs and effluxing amyloid beta (Aβ), a contributor to Alzheimer’s disease (AD). Strategies to regulate P-gp expression therefore may impact central nervous system (CNS) drug delivery and brain Aβ levels. As we have demonstrated that the copper complex copper diacetyl bis(4-methyl-3-thiosemicarbazone) (Cu(ATSM)) increases P-gp expression and function in human brain endothelial cells, the present study assessed the impact of Cu(ATSM) on expression and function of P-gp in mouse brain endothelial cells (mBECs) and capillaries in vivo, as well as in peripheral organs. Isolated mBECs treated with Cu(ATSM) (100 nM for 24 h) exhibited a 1.6-fold increase in P-gp expression and a 20% reduction in accumulation of the P-gp substrate rhodamine 123. Oral administration of Cu(ATSM) (30 mg/kg/day) for 28 days led to a 1.5 & 1.3-fold increase in brain microvascular and hepatic expression of P-gp, respectively, and a 20% reduction in BBB transport of [3H]-digoxin. A metallomic analysis showed a 3.5 and 19.9-fold increase in Cu levels in brain microvessels and livers of Cu(ATSM)-treated mice. Our findings demonstrate that Cu(ATSM) increases P-gp expression and function at the BBB in vivo, with implications for CNS drug delivery and clearance of Aβ in AD.

Exercise improves cognitive function in Alzheimer’s disease (AD) via mechanism that are not fully clear. Here, we first examined the effect of voluntary exercise training (VET) on energy metabolism and cognitive function in the APP/PS1 transgenic mouse (Tg) model of familial AD. Next, we profiled extracellular vesicles (EVs) and examined whether they may play a role in the protective effects of VET via intranasal administration of EVs, purified from the blood of sedentary (sEV) and/or acutely exercised (eEV) donor wild-type mice into APP/PS1Tg mice. We show that VET reduced resting energy expenditure (REE) and improved cognition in APP/PS1 Tg mice. Administration of eEV, but not sEV, also reduced REE, but had no effect on cognition. Taken together, these data show that exercise is effective intervention to improve symptoms of AD in APP/PS1Tg mice. In addition, eEVs mediate some of these effects, implicating EVs in the treatment of age-related neurodegenerative diseases.

Background: Iron overload is implicated in many neurodegenerative diseases, where there is also blood–brain barrier (BBB) dysfunction. As there is a growing interest in the role of iron in modulating key BBB proteins, this study assessed the effect of iron on the expression and function of P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and claudin-5 in primary mouse brain endothelial cells (MBECs) and their abundance in mouse brain microvessel-enriched membrane fractions (MVEFs). Methods: Following a 48 h treatment with ferric ammonium citrate (FAC, 250 µM), MBEC protein abundance (P-gp, BCRP and claudin-5) and mRNA (abcb1a, abcg2, and cldn5) were assessed by western blotting and RT-qPCR, respectively. Protein function was evaluated by assessing transport of substrates ³H-digoxin (P-gp), ³H-prazosin (BCRP) and ¹⁴C-sucrose (paracellular permeability). C57BL/6 mice received iron dextran (100 mg/kg, intraperitoneally) over 4 weeks, and MVEF protein abundance and iron levels (in MVEFs and plasma) were quantified via western blotting and inductively coupled plasma-mass spectrometry (ICP-MS), respectively. Results: FAC treatment reduced P-gp protein by 50% and abcb1a mRNA by 43%, without affecting ³H-digoxin transport. FAC did not alter BCRP protein or function, but decreased abcg2 mRNA by 59%. FAC reduced claudin-5 protein and cldn5 mRNA by 65% and 70%, respectively, resulting in a 200% increase in ¹⁴C-sucrose permeability. In vivo, iron dextran treatment significantly elevated plasma iron levels (2.2-fold) but did not affect brain MVEF iron content or alter P-gp, BCRP or claudin-5 protein abundance. Conclusions: Iron overload modulates BBB transporters and junction proteins in vitro, highlighting potential implications for CNS drug delivery in neurodegenerative diseases.