Biology subjects

Blum, J. E.

Publications and source records attributed to Blum, J. E..

6 recordsLinked to original sources

bioRxiv2024

Suppressing PDGFRβ Signaling Enhances Myocyte Fusion to Promote Skeletal Muscle Regeneration

Muscle cell fusion is critical for forming and maintaining multinucleated myotubes during skeletal muscle development and regeneration. However, the molecular mechanisms directing cell-cell fusion are not fully understood. Here, we identify platelet-derived growth factor receptor beta (PDGFR{beta}) signaling as a key modulator of myocyte fusion in adult muscle cells. Our findings demonstrate that genetic deletion of Pdgfr{beta} enhances muscle regeneration and increases myofiber size, whereas PDGFR{beta} activation impairs muscle repair. Inhibition of PDGFR{beta} activity promotes myonuclear accretion in both mouse and human myotubes, whereas PDGFR{beta} activation stalls myotube development by preventing cell spreading to limit fusion potential. Transcriptomics analysis show that PDGFR{beta} signaling cooperates with TGF{beta} signaling to direct myocyte size and fusion. Mechanistically, PDGFR{beta} signaling requires STAT1 activation, and blocking STAT1 phosphorylation enhances myofiber repair and size during regeneration. Collectively, PDGFR{beta} signaling acts as a regenerative checkpoint and represents a potential clinical target to rapidly boost skeletal muscle repair.

developmental biology↗

bioRxiv2024

Discovery and characterization of dietary antigens in oral tolerance

Food antigens elicit immune tolerance through the action of intestinal regulatory T (Treg) cells. Unlike food allergens, the proteins that mediate tolerance are mostly undescribed. Here, we found that epitopes derived from seed storage proteins are targets of murine intestinal Treg cells, with the most frequent response targeting the C-terminus of the maize protein alpha-zein. An MHC tetramer loaded with this antigen revealed that zein-specific T cells are predominantly intestinal Treg cells, develop concurrently with weaning, and constitute up to 2% of the peripheral Treg cell pool. Zein-responsive Treg cells repressed naive T cell proliferation ex vivo and prior dietary exposure resulted in a constrained response upon multiple inflammatory challenges in vivo, supporting a specific role for gut-resident Treg cells in suppressing systemic immune responses. Together, our work reveals the development, immune suppressive characteristics, and function of naturally occurring Treg cells that recognize dietary seed-storage proteins, a previously undescribed class of antigens in oral tolerance. One-sentence summaryImmunodominant epitopes from seed storage proteins are a target of intestinal regulatory T cells that modulate immune challenge to food.

immunology↗

bioRxiv2022

Impairments in SHMT2 expression or cellular folate availability reduce oxidative phosphorylation and pyruvate kinase activity

BackgroundSerine hydroxymethyltransferase 2 (SHMT2) catalyzes the reversible conversion of tetrahydrofolate (THF) and serine producing THF-conjugated one-carbon units and glycine in the mitochondria. Biallelic SHMT2 variants were identified in humans and suggested to alter the proteins active site, potentially disrupting enzymatic function. SHMT2 expression has also been shown to decrease with aging in human fibroblasts. Immortalized cells models of total SHMT2 loss or folate deficiency exhibit decreased oxidative capacity and impaired mitochondrial complex I assembly and protein levels, suggesting folate-mediated one-carbon metabolism (FOCM) and the oxidative phosphorylation system are functionally coordinated. This study examined the role of SHMT2 and folate availability in regulating mitochondrial function, energy metabolism, and cellular proliferative capacity in both heterozygous and homozygous cell models of reduced SHMT2 expression. MethodsPrimary mouse embryonic fibroblasts (MEF) were isolated from a C57Bl/6 dam crossed with a heterozygous Shmt2+/- male to generate Shmt2+/+ (wild-type) or Shmt2+/- (HET) MEF cells. In addition, haploid chronic myeloid leukemia cells (HAP1, wild-type) or HAP1 cells lacking SHMT2 expression ({Delta}SHMT2) were cultured for 4 doublings in either low-folate or folate-sufficient culture media. Cells were examined for proliferation, total folate levels, mtDNA content, protein levels of pyruvate kinase and PGC1, pyruvate kinase enzyme activity, mitochondrial membrane potential, and mitochondrial function. ResultsHomozygous loss of SHMT2 in HAP1 cells impaired cellular folate accumulation and altered mitochondrial DNA content, membrane potential, and basal respiration. Formate rescued proliferation in HAP1, but not {Delta}SHMT2, cells cultured in low-folate medium. Pyruvate kinase activity and protein levels were impaired in {Delta}SHMT2 cells and in MEF cells exposed to low-folate medium. Mitochondrial biogenesis protein levels were elevated in Shmt2+/- MEF cells, while mitochondrial mass was increased in both homozygous and heterozygous models of SHMT2 loss. ConclusionsThe results from this study indicate disrupted mitochondrial FOCM impairs mitochondrial folate accumulation and respiration, glycolytic activity, and cellular proliferation. These changes persist even after a potentially compensatory increase in mitochondrial biogenesis as a result of decreased SHMT2 levels.

biochemistry↗

bioRxiv2021

Reduced Shmt2 expression impairs mitochondrial folate accumulation and respiration, and leads to uracil accumulation in mouse mitochondrial DNA

BackgroundAdequate cellular thymidylate (dTMP) pools are essential for preservation of nuclear and mitochondrial genome stability. Previous studies have indicated that disruption in dTMP synthesis in the nucleus leads to increased uracil misincorporation into DNA affecting genome stability. To date, the effects of impaired mitochondrial dTMP synthesis in non- transformed tissues have been understudied. ObjectiveThis study aimed to determine the effects of decreased serine hydroxymethyltransferase 2 (Shmt2) expression and dietary folate deficiency on mitochondrial DNA integrity and mitochondrial function in mouse tissues. MethodsLiver mitochondrial DNA (mtDNA) content, and uracil content in liver mtDNA was measured in Shmt2+/- and Shmt2+/+ mice weaned onto either a folate-sufficient control diet (2 mg/kg folic acid, C) or a modified diet lacking folic acid (0 mg/kg folic acid, FD) for 7 wks. Shmt2+/- and Shmt2+/+ mouse embryonic fibroblasts (MEF cells) were cultured in defined culture medium containing either 0 or 25 nM folate to assess proliferative capacity and mitochondrial function. ResultsShmt2+/- mice exhibited 48-67% reduction in SHMT2 protein levels in tissues. Interestingly, Shmt2+/- mice consuming the folate-sufficient C diet exhibited a 25% reduction in total folate in liver mitochondria. There was also a >20-fold increase in uracil in liver mtDNA in Shmt2+/- mice consuming the C diet, and dietary folate deficiency also increased uracil content in mouse liver mtDNA from both Shmt2+/+ and Shmt2+/- mice. Furthermore, decreased Shmt2 expression in MEF cells reduced cell proliferation, mitochondrial membrane potential, and oxygen consumption rate. ConclusionsThis study demonstrates that Shmt2 heterozygosity and dietary folate deficiency impair mitochondrial dTMP synthesis, as evidenced by the increased uracil in mtDNA. In addition, Shmt2 heterozygosity impairs mitochondrial function in MEF cells. These findings suggest that elevated uracil in mtDNA may impair mitochondrial function.

biochemistry↗

bioRxiv2020

Lifelong Ulk1-mediated autophagy deficiency in muscle induces mitochondrial dysfunction and contractile weakness

The accumulation of damaged mitochondria due to insufficient autophagy has been implicated in the pathophysiology of sarcopenia resulting in reduced contractile and metabolic function. Ulk1 is an autophagy-related kinase that initiates autophagosome assembly and may also play a role in autophagosome degradation (i.e., autophagy flux), but the contribution of Ulk1 to healthy muscle aging is unclear. We found that Ulk1 phosphorylation declines in both human and mouse muscle tissue with age, therefore the purpose of this study was to investigate the role of Ulk1-mediated autophagy in skeletal muscle aging. At age 22 months (80% survival rate), muscle contractile and metabolic function were assessed using electrophysiology in muscle specific Ulk1 knockout mice (MKO) and their littermate controls (LM). Specific peak-isometric torque of the ankle dorsiflexors (normalized by tibialis anterior muscle cross-sectional area) and specific force of the fast-twitch extensor digitorum longus muscles were reduced in MKO mice compared to LM mice (p<0.03). Permeabilized muscle fibers from MKO mice had greater mitochondrial content, yet lower mitochondrial oxygen consumption and greater reactive oxygen species production compared to fibers from LM mice (p[≤] 0.04). Altered neuromuscular junction innervation patterns and changes in autophagosome numbers and/or flux in muscles from MKO may have contributed to decrements in contractile and metabolic function. Results from this study support an important role of Ulk1-mediated autophagy in skeletal muscle with age, reflecting Ulk1s dual role in maintaining mitochondrial integrity through autophagosome assembly and degradation. A lifetime of insufficient Ulk-1-mediated autophagy in skeletal muscle exacerbates age-related contractile and metabolic dysfunction.

physiology↗

bioRxiv2019

Serine and glycine are essential for human muscle progenitor cell population expansion

Skeletal muscle regeneration is reliant on a population of muscle specific adult stem cells (muscle progenitor cells; MPCs). During regeneration, the MPC population undergoes a transient and rapid period of population expansion, which is necessary to repair damaged myofibers and restore muscle homeostasis. Much research has focused on the age-related accumulation of negative regulators of regeneration, while the age-related decline of nutrient and metabolic determinants of the regenerative process needs examination. We hypothesized that older individuals, a population that is at risk for protein malnutrition, have diminished availability of amino acids that are necessary for MPC function. Here, we identified that levels of the non-essential amino acid serine are reduced in the skeletal muscle of healthy, older individuals. Furthermore, using stable-isotope tracing studies, we demonstrate that primary, human MPCs (hMPCs) exhibit a limited capacity for de novo biosynthesis of serine and the closely related amino acid glycine. We identified that serine and glycine are essential for hMPC proliferation and, therefore, population expansion. Serine and glycine were necessary to support synthesis of the intracellular antioxidant glutathione, and restriction of serine and glycine was sensed in an EIF2-dependent manner resulting in cell cycle arrest in G0/G1. In conclusion, we elucidate that, despite an absolute requirement of serine/glycine for hMPC proliferation, availability of serine in the skeletal muscle microenvironment is limited to the hMPCs of healthy older adults and is a likely underlying mechanism for impaired skeletal muscle regeneration with advancing age.\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=176 SRC=\"FIGDIR/small/833798v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (32K):\norg.highwire.dtl.DTLVardef@438298org.highwire.dtl.DTLVardef@32f66dorg.highwire.dtl.DTLVardef@1d08aeeorg.highwire.dtl.DTLVardef@511c7f_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

cell biology↗