The blood-brain barrier (BBB) is required for central nervous system (CNS) homeostasis across vertebrates and invertebrates, preventing paracellular diffusion from circulation while mediating transport of essential nutrients to the CNS. The mammalian BBB undergoes a decline in integrity with age, acting upstream of the age-associated onset of neurodegenerative diseases, such as Alzheimer’s disease (AD). Causes of age-related mammalian BBB decline remain poorly understood, and characterizing this process may offer a more comprehensive view of neurodegenerative onset. The BBB of Drosophila melanogaster shares homologous features to that of mammals and is highly amenable to genetic and physiologic BBB analysis. Despite these advantages as a BBB model, age-related changes to the Drosophila BBB are undescribed. Should a decrease in Drosophila BBB integrity occur with age, the range of available tools and assays could identify the causes of this decline and assess their homology in mammals. To evaluate the integrity of the aged Drosophila BBB, a brain penetration assay using tetramethylrhodamine dextran (TMRD) was tested. We report the identification of an abdominal region that, when injected with TMRD, permits fly survival and TMRD distribution throughout the hemolymph, showing promise as a BBB permeability assay. A possible cause of any potential age-related BBB integrity loss was assessed through immunofluorescence for proteins of the diffusion-resistant septate junctions (SJs). Two strong protein trap markers for the SPG SJs, Nrg::GFP and NrxIV::GFP, have been identified. With these protocols and tools, age-related changes to the Drosophila BBB can be evaluated.

Microglia are CNS immune cells involved in numerous important brain processes, spanning from early development to aging and neurodegeneration. Microglia are well-known for their phagocytic role in clearing the brain of synapses, pathogens, and protein aggregates, a process critical for protecting brain health and function. However, the mechanisms and stimuli that regulate microglial phagocytosis remain unknown along with potential differences in phagocytosis between regional microglial populations. Additionally, a connection between regional differences in microglial phagocytosis and long-term neuronal health remains to be elucidated. Despite this, there are few well-established methods that allow for the comparison of microglial phagocytosis in several brain regions at once. Here, we aim to develop an assay to accurately visualize and quantify the phagocytic abilities of microglia in acute brain sections of mice. The assay, modeled after a protocol by Dr. David Attwell’s lab, involves incubating acute brain sections with fluorescent beads, which act as phagocytic targets. During the optimization process, we refined the bead incubation chamber to best promote the health and stability of the slices, found that 45-minute and 1.5-hour periods were optimal for bead incubation, and used Imaris 3D-Reconstruction software to confirm microglial engulfment of beads. Bead incubation of slices on ice decreased bead phagocytosis, lending support for bonafide microglia-bead “interactions'' during normal assay conditions. Finally, two analysis approaches (bead-oriented vs. microglia-oriented) for future quantification were established. Through continued optimization, microglial phagocytosis will be effectively visualized and quantified, allowing for exploration of how microglial phagocytosis impacts brain health and function.

Achieving the ultimate promise of biomaterials to support tissue regeneration and therapeutic delivery depends on how host cells respond to them. Cells react to physical and chemical microenvironmental stimuli that influence their function and structure. Such responses can be instructed and assessed as a function of biomaterial properties. Antigen-presenting cell behavior is particularly important to investigate, as they are immune cells responsible for sampling antigens from a biomaterial that can direct an adaptive immune response, e.g. for vaccination. Here, we adjusted specific physical and chemical properties of a hydrogel biomaterial by incorporating adhesion peptides and using crosslinkers with different cleavability to evaluate how biomaterials and macrophages consequently interact. We created microporous annealed particle (MAP) gels with these different properties via microfluidic fabrication and measured the uptake of gel building blocks with fluorescent markers by the macrophages in vitro. The macrophages uptook significantly more fluorescent markers from gels without arginine-glycine-aspartic acid (RGD) peptides compared to gels with them. In addition, the cells uptook significantly more fluorescent markers from polyethylene glycol (PEG)-thiol crosslinked MAP gels compared to gels crosslinked with matrix metalloprotesase (MMP)-sensitive peptide linkers. Studying this interaction between cells and biomaterials will help lay the groundwork for advancing tunable biomaterials including the development of immunomodulatory platforms that can target specific cellular responses.

Adenosine 5’-triphosphate (ATP) plays a key role in storing and transferring energy in cells, allowing important biochemical reactions to occur in living things. Recent biochemical and molecular dynamics simulations have shown that ATP can effectively stabilize protein structure and inhibit aggregation. In a recent study, the interactions of ATPs were studied with three proteins: lysozyme, ubiquitin, and malate dehydrogenase, each found to contain ATP susceptible regions characterized by high flexibility and an abundance of charged residues. Another protein that interacts with ATP is cytochrome c (cytc), a soluble heme protein that plays a key role in the electron transport chain of mitochondria. Preliminary work in our group showed that bovine cytc can be stabilized by nonspecific ATP binding but also promoted the formation of a cytc dimer. Although previous studies reported in the literature have discussed mechanisms of cytc dimerization, none have discussed the role of ATP-induced dimerization. To better understand this phenomena, we employed mass spectrometry as a tool to characterize the dimerization of cytc in the presence of ATP. We tested if the binding of cytc to ATP was sequence dependent and probed the specificity of the cytc dimer by analyzing bovine, equine, and pigeon cytc. We found that the binding of cytc to ATP was not sequence dependent and that cytc dimerized in the presence of ATP across all three species. This study has the potential to explore if cytc dimerization is relevant in nature and better understand the interactions of ATP with proteins such as cytc.