Faculty Bibliography

A balanced intake of macronutrients-protein, carbohydrate and fat-is essential for the well-being of organisms. An adequate calorific intake but with insufficient protein consumption can lead to several ailments, including kwashiorkor¹. Taste receptors (T1R1-T1R3)² can detect amino acids in the environment, and cellular sensors (Gcn2 and Tor)³ monitor the levels of amino acids in the cell. When deprived of dietary protein, animals select a food source that contains a greater proportion of protein or essential amino acids (EAAs)⁴. This suggests that food selection is geared towards achieving the target amount of a particular macronutrient with assistance of the EAA-specific hunger-driven response, which is poorly understood. Here we show in Drosophila that a microbiome-gut-brain axis detects a deficit of EAAs and stimulates a compensatory appetite for EAAs. We found that the neuropeptide CNMamide (CNMa)⁵ was highly induced in enterocytes of the anterior midgut during protein deprivation. Silencing of the CNMa-CNMa receptor axis blocked the EAA-specific hunger-driven response in deprived flies. Furthermore, gnotobiotic flies bearing an EAA-producing symbiotic microbiome exhibited a reduced appetite for EAAs. By contrast, gnotobiotic flies with a mutant microbiome that did not produce leucine or other EAAs showed higher expression of CNMa and a greater compensatory appetite for EAAs. We propose that gut enterocytes sense the levels of diet- and microbiome-derived EAAs and communicate the EAA-deprived condition to the brain through CNMa.

RATIONALE Coronavirus Disease-2019(COVID-19), caused by the severe acute respiratory syndrome coronavirus-2(SARS-CoV-2), causes multi-organ failure and death. Metabolic syndrome(MetSyn) characteristics are also risks for COVID-19. The Receptor for Advanced Glycation End-Products(RAGE) is a MetSyn mediator. SARS-CoV via its Spike protein binds ACE2 as its 1o-receptor, and may activate TLR2. Particulate matter(PM) similarly activates an innate immune response, partially via the RAGE receptor, and increases ACE2 expression. Excessive hyaluronan(HA) levels are found in lungs of COVID-19 patients. Reducing HA synthesis and stabilizing the HA shield surrounding cells may be therapeutic. A HA-binding peptide, P15-1, is anti-inflammatory and reduces HA. HA and its binding proteins may provide a link explaining synergistic ACE2 and RAGE signaling, reducing interaction of receptors with their ligands and ultimately inflammation-related changes in peripheral blood mononuclear cells(PBMCs), the severity of which correlate with patient outcome after SARS-CoV-2 exposure. Our focus is to develop novel therapeutic strategies for SARS-CoV-2 inflammation. To begin to explore our HYPOTHESIS that COVID-19 Spike protein and PM co-exposure synergistically induces an inflammatory phenotype and that phenotype can be mitigated by stabilizing the pericellular HA matrix and by inhibiting RAGE. METHODS. We performed in vitro exposure of PBMCs isolated from 9/11 World Trade Center(WTC) 1st- Responders to i. Media alone(MA); ii. WTC PM; iii. SARS-CoV-2 Spike RBD(C19); iv. C19 and PM; v. C19 and P15-1; vi. C19, PM and P15-1 vii. C19, PM and RAGE inhibitor(RAGEInh) FPS-ZM1; viii. LPS(positive control). Total mRNA levels for Cox-2, IL-1beta, IL-6 and MMP-13 24 hours after exposure were analyzed by real time PCR. Comparisons by Student's t- and Mann-Whitney U-tests. Correlations by Spearman's. Significance p<0.05. RESULTS COX-2, IL-1beta, IL-6 and MMP-13 mRNA levels were significantly increased 24-hrs after the administration of PM and C19. Co-exposure to PM and C19 yielded a synergistic increase in the mRNA of IL-beta, Figure 1B. P15-1 and RAGE inhibition significantly reduced mRNA levels of inflammatory markers in primary PBMCs exposed to C19, WTC PM, or a combination of the two, Figure 1. CONCLUSIONS Our work focuses on mitigating the COVID-19 inflammatory phenotype by stabilizing the pericellular HA matrix and by inhibiting RAGE. Preliminary data presented in this abstract will be further explored using PBMCs and cell lines in a multidisciplinary approach

Lysosomal trafficking and maturation in neurons remain poorly understood and are unstudied in vivo despite high disease relevance. We generated neuron-specific transgenic mice to track vesicular CTSD acquisition, acidification, and traffic within the autophagic-lysosomal pathway in vivo, revealing that mature lysosomes are restricted from axons. Moreover, TGN-derived transport carriers (TCs), not lysosomes, supply lysosomal components to axonal organelles. Ultrastructurally distinctive TCs containing TGN and lysosomal markers enter axons, engaging autophagic vacuoles and late endosomes. This process is markedly upregulated in dystrophic axons of Alzheimer models. In cultured neurons, most axonal LAMP1 vesicles are weakly acidic TCs that shuttle lysosomal components bidirectionally, conferring limited degradative capability to retrograde organelles before they mature fully to lysosomes within perikarya. The minor LAMP1 subpopulation attaining robust acidification are retrograde Rab7⁺ endosomes/amphisomes, not lysosomes. Restricted lysosome entry into axons explains the unique lysosome distribution in neurons and their vulnerability toward neuritic dystrophy in disease.

In order to understand the transmission and virulence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), it is necessary to understand the functions of each of the gene products encoded in the viral genome. One feature of the SARS-CoV-2 genome that is not present in related, common coronaviruses is ORF10, a putative 38-amino acid protein-coding gene. Proteomic studies found that ORF10 binds to an E3 ubiquitin ligase containing Cullin-2, Rbx1, Elongin B, Elongin C, and ZYG11B (CRL2^ZYG11B). Since CRL2^ZYG11B mediates protein degradation, one possible role for ORF10 is to "hijack" CRL2^ZYG11B in order to target cellular, antiviral proteins for ubiquitylation and subsequent proteasomal degradation. Here, we investigated whether ORF10 hijacks CRL2^ZYG11B or functions in other ways, for example, as an inhibitor or substrate of CRL2^ZYG11B While we confirm the ORF10-ZYG11B interaction and show that the N terminus of ORF10 is critical for it, we find no evidence that ORF10 is functioning to inhibit or hijack CRL2^ZYG11B Furthermore, ZYG11B and its paralog ZER1 are dispensable for SARS-CoV-2 infection in cultured cells. We conclude that the interaction between ORF10 and CRL2^ZYG11B is not relevant for SARS-CoV-2 infection in vitro.

Since alterations in the intestinal microbiota may induce systemic inflammation and polarization of macrophages to the M1 state, the microbiome role in atherosclerosis, an M1-driven disease, requires evaluation. We aimed to determine if antibiotic (Abx) induced alterations to the intestinal microbiota interferes with atherosclerotic plaque inflammation resolution after lipid-lowering in mice. Hyperlipidemic Apoe^-/- mice were fed a western diet to develop aortic atherosclerosis with aortas then transplanted into normolipidemic wild-type (WT) mice to model clinically aggressive lipid management and promote atherosclerosis inflammation resolution. Gut microbial composition pre and post-transplant was altered via an enteral antibiotic or not. Post aortic transplant, after Abx treatment, while plaque size did not differ, compared to Apoe^-/- mice, Abx^- WT recipient mice had a 32% reduction in CD68-expressing cells (p = 0.02) vs. a non-significant 12% reduction in Abx⁺ WT mice. A trend toward an M1 plaque CD68-expresing cell phenotype was noted in Abx⁺ mice. By 16S rRNA sequence analysis, the Abx⁺ mice had reduced alpha diversity and increased Firmicutes/Bacteroidetes relative abundance ratio with a correlation between gut Firmicutes abundance and plaque CD68-expressing cell content (p < 0.05). These results indicate that in a murine atherosclerotic plaque inflammation resolution model, antibiotic-induced microbiome perturbation may blunt the effectiveness of lipid-lowering to reduce the content of plaque inflammatory CD68-expressing cells.

Stem cells reside in specialized microenvironments or niches that balance stem cell proliferation and differentiation.¹

Mechanical stress during cell migration may be a previously unappreciated source of genome instability, but the extent to which this happens in any animal in vivo remains unknown. We consider an in vivo system where the adult stem cells of planarian flatworms are required to migrate to a distal wound site. We observe a relationship between adult stem cell migration and ongoing DNA damage and repair during tissue regeneration. Migrating planarian stem cells undergo changes in nuclear shape and exhibit increased levels of DNA damage. Increased DNA damage levels reduce once stem cells reach the wound site. Stem cells in which DNA damage is induced prior to wounding take longer to initiate migration and migrating stem cell populations are more sensitive to further DNA damage than stationary stem cells. RNAi-mediated knockdown of DNA repair pathway components blocks normal stem cell migration, confirming that active DNA repair pathways are required to allow successful migration to a distal wound site. Together these findings provide evidence that levels of migration-coupled-DNA-damage are significant in adult stem cells and that ongoing migration requires DNA repair mechanisms. Our findings reveal that migration of normal stem cells in vivo represents an unappreciated source of damage, which could be a significant source of mutations in animals during development or during long-term tissue homeostasis.

Skin scarring, the end result of adult wound healing, is detrimental to tissue form and function. Engrailed-1 lineage-positive fibroblasts (EPFs) are known to function in scarring, but Engrailed-1 lineage-negative fibroblasts (ENFs) remain poorly characterized. Using cell transplantation and transgenic mouse models, we identified a dermal ENF subpopulation that gives rise to postnatally derived EPFs by activating Engrailed-1 expression during adult wound healing. By studying ENF responses to substrate mechanics, we found that mechanical tension drives Engrailed-1 activation via canonical mechanotransduction signaling. Finally, we showed that blocking mechanotransduction signaling with either verteporfin, an inhibitor of Yes-associated protein (YAP), or fibroblast-specific transgenic YAP knockout prevents Engrailed-1 activation and promotes wound regeneration by ENFs, with recovery of skin appendages, ultrastructure, and mechanical strength. This finding suggests that there are two possible outcomes to postnatal wound healing: a fibrotic response (EPF-mediated) and a regenerative response (ENF-mediated).

Endothelial cells play a key role in the regulation of disease. Defective regulation of endothelial cell homeostasis may cause mesenchymal activation of other endothelial cells or neighboring cell types, and in both cases contributes to organ fibrosis. Regulatory control of endothelial cell homeostasis is not well studied. Diabetes accelerates renal fibrosis in mice lacking the endothelial glucocorticoid receptor (GR), compared to control mice. Hypercholesterolemia further enhances severe renal fibrosis. The fibrogenic phenotype in the kidneys of diabetic mice lacking endothelial GR is associated with aberrant cytokine and chemokine reprogramming, augmented Wnt signaling and suppression of fatty acid oxidation. Both neutralization of IL-6 and Wnt inhibition improve kidney fibrosis by mitigating mesenchymal transition. Conditioned media from endothelial cells from diabetic mice lacking endothelial GR stimulate Wnt signaling-dependent epithelial-to-mesenchymal transition in tubular epithelial cells from diabetic controls. These data demonstrate that endothelial GR is an essential antifibrotic molecule in diabetes.