Faculty Bibliography

BACKGROUND/UNASSIGNED:Bone regeneration following a fracture is dependent on multiple factors including skeletal stem cells (SSCs). Recruitment, proliferation, and differentiation of the SSCs is guided by the proteins and metabolites found within the fracture microenvironment. Understanding how intrinsic factors affect the fracture microenvironment has been a topic of ongoing investigation. This study sought to determine whether the levels of select proteins and metabolites within the fracture hematoma would be differentially expressed depending on the age of the patient. We hypothesized that a distinct set of proteins and metabolites found within the fracture hematoma microenvironment would be present at varying levels depending on patient age. METHODS/UNASSIGNED:The research study was reviewed and approved by an Institutional Review Board. Hematomas were collected from subjects aged 18 years old or older undergoing surgical intervention for a fracture. Hematoma samples were selected from the biorepository and assigned to one of two fracture groups including young ankle/hindfoot and aged ankle/hindfoot. Protein and metabolite levels within each hematoma were analyzed by liquid chromatography-mass spectrometry. RESULTS/UNASSIGNED:A total of seven hematomas were included in each the young ankle/hindfoot and aged ankle/hindfoot groups. From the global metabolomic analysis, creatine, 2-methylindoline, and acetyl-L-carnitine were identified as being differentially expressed between both groups. An untargeted metabolomic analysis of the two groups identified significant differences in the levels of an additional 66 metabolites. Proteomic analysis identified 34 proteins that were expressed at significantly different levels. CONCLUSIONS/UNASSIGNED:The level of metabolites and proteins found within the local fracture environment vary by patient age. Future investigations will focus on identifying a role for these proteins and metabolites in bone homeostasis and fracture healing. LEVEL OF EVIDENCE/UNASSIGNED:N/A, basic science investigation. SUPPLEMENTARY INFORMATION/UNASSIGNED:The online version contains supplementary material available at 10.1007/s43465-024-01284-3.

Mitochondrial dysfunction causes devastating disorders, including mitochondrial myopathy, but how muscle senses and adapts to mitochondrial dysfunction is not well understood. Here, we used diverse mouse models of mitochondrial myopathy to show that the signal for mitochondrial dysfunction originates within mitochondria. The mitochondrial proteins OMA1 and DELE1 sensed disruption of the inner mitochondrial membrane and, in response, activated the mitochondrial integrated stress response (mt-ISR) to increase the building blocks for protein synthesis. In the absence of the mt-ISR, protein synthesis in muscle was dysregulated causing protein misfolding, and mice with early-onset mitochondrial myopathy failed to grow and survive. The mt-ISR was similar following disruptions in mtDNA maintenance (Tfam knockout) and mitochondrial protein misfolding (CHCHD10 G58R and S59L knockin) but heterogenous among mitochondria-rich tissues, with broad gene expression changes observed in heart and skeletal muscle and limited changes observed in liver and brown adipose tissue. Taken together, our findings identify that the DELE1 mt-ISR mediates a similar response to diverse forms of mitochondrial stress and is critical for maintaining growth and survival in early-onset mitochondrial myopathy.

Understanding the molecular mechanisms underpinning diverse vaccination responses is critical for developing efficient vaccines. Molecular subtyping can offer insights into heterogeneous nature of responses and aid in vaccine design. We analyzed multi-omic data from 62 haemagglutinin seasonal influenza vaccine recipients (2019-2020), including transcriptomics, proteomics, glycomics, and metabolomics data collected pre-vaccination. We performed a subtyping analysis on the integrated data revealing five subtypes with distinct molecular signatures. These subtypes differed in the expression of pre-existing adaptive or innate immunity signatures, which were linked to significant variation in baseline immunoglobulin A (IgA) and hemagglutination inhibition (HAI) titer levels. It is worth noting that these differences persisted through day 28 post-vaccination, indicating the effect of initial immune state on vaccination response. These findings highlight the significance of interpersonal variation in baseline immune status as a crucial factor in determining the effectiveness of seasonal vaccines. Ultimately, incorporating molecular profiling could enable personalized vaccine optimization.

Forty percent of the US population and 1 in 6 individuals worldwide are obese, and the incidence of this disease is surging globally^1,2. Various dietary interventions, including carbohydrate and fat restriction, and more recently amino acid restriction, have been explored to combat this epidemic^3-6. We sought to investigate the impact of removing individual amino acids on the weight profiles of mice. Compared to essential amino acid restriction, induction of conditional cysteine restriction resulted in the most dramatic weight loss, amounting to 20% within 3 days and 30% within one week, which was readily reversed. This weight loss occurred despite the presence of substantial cysteine reserves stored in glutathione (GSH) across various tissues⁷. Further analysis demonstrated that the weight reduction primarily stemmed from an increase in the utilization of fat mass, while locomotion, circadian rhythm and histological appearance of multiple other tissues remained largely unaffected. Cysteine deficiency activated the integrated stress response (ISR) and NRF2-mediated oxidative stress response (OSR), which amplify each other, leading to the induction of GDF15 and FGF21, hormones associated with increased lipolysis, energy homeostasis and food aversion^8-10. We additionally observed rapid tissue coenzyme A (CoA) depletion, resulting in energetically inefficient anaerobic glycolysis and TCA cycle, with sustained urinary excretion of pyruvate, orotate, citrate, α-ketoglutarate, nitrogen rich compounds and amino acids. In summary, our investigation highlights that cysteine restriction, by depleting GSH and CoA, exerts a maximal impact on weight loss, metabolism, and stress signaling compared to other amino acid restrictions. These findings may pave the way for innovative strategies for addressing a range of metabolic diseases and the growing obesity crisis.

Dehydration and malnutrition are common and often underdiagnosed in hospital settings. Multidrug-resistant bacterial infections result in more than 35,000 deaths a year in nosocomial patients. The effect of temporal dietary and water restriction (DWR) on susceptibility to multidrug-resistant pathogens is unknown. We report that DWR markedly increased susceptibility to systemic infection by ESKAPE pathogens. Using a murine bloodstream model of methicillin-resistant Staphylococcus aureus infection, we show that DWR leads to significantly increased mortality and morbidity. DWR causes increased bacterial burden, severe pathology, and increased numbers of phagocytes in the kidney. DWR appears to alter the functionality of these phagocytes and is therefore unable to control infection. Mechanistically, we show that DWR impairs the ability of macrophages to phagocytose multiple bacterial pathogens and efferocytose apoptotic neutrophils. Together, this work highlights the crucial impact that diet and hydration play in protecting against infection.

The development of dendritic cells (DCs), including antigen-presenting conventional DCs (cDCs) and cytokine-producing plasmacytoid DCs (pDCs), is controlled by the growth factor Flt3 ligand (Flt3L) and its receptor Flt3. We genetically dissected Flt3L-driven DC differentiation using CRISPR-Cas9-based screening. Genome-wide screening identified multiple regulators of DC differentiation including subunits of TSC and GATOR1 complexes, which restricted progenitor growth but enabled DC differentiation by inhibiting mTOR signaling. An orthogonal screen identified the transcriptional repressor Trim33 (TIF-1γ) as a regulator of DC differentiation. Conditional targeting in vivo revealed an essential role of Trim33 in the development of all DCs, but not of monocytes or granulocytes. In particular, deletion of Trim33 caused rapid loss of DC progenitors, pDCs, and the cross-presenting cDC1 subset. Trim33-deficient Flt3⁺ progenitors up-regulated pro-inflammatory and macrophage-specific genes but failed to induce the DC differentiation program. Collectively, these data elucidate mechanisms that control Flt3L-driven differentiation of the entire DC lineage and identify Trim33 as its essential regulator.

Much is known about molecular mechanisms by which animals detect pathogenic microbes, but how animals sense beneficial microbes remains poorly understood. The roundworm Caenorhabditis elegans is a microbivore that must distinguish nutritive microbes from pathogens. We characterized a neural circuit used by C. elegans to rapidly discriminate between nutritive bacteria and pathogens. Distinct sensory neuron populations responded to chemical cues from nutritive Escherichia coli and pathogenic Enterococcus faecalis, and these neural signals are decoded by downstream AIB interneurons. The polyamine metabolites cadaverine, putrescine, and spermidine produced by E. coli activate this neural circuit and elicit positive chemotaxis. Our study shows how polyamine odorants can be sensed by animals as proxies for microbe identity and suggests that, hence, polyamines might have widespread roles brokering host-microbe interactions.

Mitochondrial dysfunction causes devastating disorders, including mitochondrial myopathy. Here, we identified that diverse mitochondrial myopathy models elicit a protective mitochondrial integrated stress response (mt-ISR), mediated by OMA1-DELE1 signaling. The response was similar following disruptions in mtDNA maintenance, from knockout of Tfam, and mitochondrial protein unfolding, from disease-causing mutations in CHCHD10 (G58R and S59L). The preponderance of the response was directed at upregulating pathways for aminoacyl-tRNA biosynthesis, the intermediates for protein synthesis, and was similar in heart and skeletal muscle but more limited in brown adipose challenged with cold stress. Strikingly, models with early DELE1 mt-ISR activation failed to grow and survive to adulthood in the absence of Dele1, accounting for some but not all of OMA1's protection. Notably, the DELE1 mt-ISR did not slow net protein synthesis in stressed striated muscle, but instead prevented loss of translation-associated proteostasis in muscle fibers. Together our findings identify that the DELE1 mt-ISR mediates a stereotyped response to diverse forms of mitochondrial stress and is particularly critical for maintaining growth and survival in early-onset mitochondrial myopathy.

Accumulation of advanced glycation end products (AGEs) on biopolymers accompanies cellular aging and drives poorly understood disease processes. Here, we studied how AGEs contribute to development of early onset Parkinson's Disease (PD) caused by loss-of-function of DJ1, a protein deglycase. In induced pluripotent stem cell (iPSC)-derived midbrain organoid models deficient for DJ1 activity, we find that lysosomal proteolysis is impaired, causing AGEs to accumulate, α-synuclein (α-syn) phosphorylation to increase, and proteins to aggregate. We demonstrated these processes are at least partly driven by astrocytes, as DJ1 loss reduces their capacity to provide metabolic support and triggers acquisition of a pro-inflammatory phenotype. Consistently, in co-cultures, we find that DJ1-expressing astrocytes are able to reverse the proteolysis deficits of DJ1 knockout midbrain neurons. In conclusion, astrocytes' capacity to clear toxic damaged proteins is critical to preserve neuronal function and their dysfunction contributes to the neurodegeneration observed in a DJ1 loss-of-function PD model.

Pancreatic ductal adenocarcinoma (PDAC) cells use glutamine (Gln) to support proliferation and redox balance. Early attempts to inhibit Gln metabolism using glutaminase inhibitors resulted in rapid metabolic reprogramming and therapeutic resistance. Here, we demonstrated that treating PDAC cells with a Gln antagonist, 6-diazo-5-oxo-L-norleucine (DON), led to a metabolic crisis in vitro. In addition, we observed a profound decrease in tumor growth in several in vivo models using sirpiglenastat (DRP-104), a pro-drug version of DON that was designed to circumvent DON-associated toxicity. We found that extracellular signal-regulated kinase (ERK) signaling is increased as a compensatory mechanism. Combinatorial treatment with DRP-104 and trametinib led to a significant increase in survival in a syngeneic model of PDAC. These proof-of-concept studies suggested that broadly targeting Gln metabolism could provide a therapeutic avenue for PDAC. The combination with an ERK signaling pathway inhibitor could further improve the therapeutic outcome.