Biology subjects

Brown, A. D.

Publications and source records attributed to Brown, A. D..

11 recordsLinked to original sources

bioRxiv2025

The Integrated Stress Response Pathway Improves Aged Murine Muscle Stem Cell Activation and in vivo Regeneration.

For efficient regeneration, muscle stem cells (MuSCs) transition out of quiescence through a series of progressively more activated states. During MuSC aging, transition through the earliest steps is the slowest and delayed, with the molecular regulators that govern this transition not well characterized. By analyzing the dynamic changes of MuSCs at the molecular (scRNA-Seq and Cell Painting) and phenotypic (heteromotility) level at single cell resolution we found that the Integrated Stress Response (ISR) Pathway is a critical regulator of MuSC transition states. Aged MuSCs have increased baseline ISR activity in quiescence that does not increase during activation to levels observed in adult MuSCs. Rapid and transient pharmacological ISR activation in vitro was sufficient to increase aged MuSC activation rate and migratory behavior as well as alter the transcriptional states toward a younger phenotype. ISR activation also improved aged MuSC potency and aged mouse muscle regeneration in vivo. Therefore, pharmacological activation of the ISR has therapeutic potential to improve MuSC function and skeletal muscle repair during aging.

cell biology↗

bioRxiv2024

Unilateral auditory deprivation reveals brainstem origin of a sensitive period for spatial hearing

Early sensory experience can exert lasting perceptual consequences. For example, a brief period of auditory deprivation early in life can lead to persistent spatial hearing deficits. Some forms of hearing loss (i.e., conductive; CHL) can distort acoustical cues needed for spatial hearing, which depend on inputs from both ears. We hypothesize that asymmetric acoustic input during development disrupts auditory circuits that integrate binaural information. Here, we identify prolonged maturation of the binaural auditory brainstem in the guinea pig by tracking auditory evoked potentials across development. Using this age range, we induce a reversible unilateral CHL and ask whether behavioral and neural maturation are disrupted. We find that developmental CHL alters a brainstem readout of binaural function which is not observed when the CHL is induced in adulthood. Startle-based behavioral measures reveal poorer spatial resolution of sound sources, but only for high-frequency sound stimuli. Finally, single-unit recordings of auditory midbrain neurons reveal significantly poorer neural acuity to a sound location cue that largely depends on high-frequency sounds. Thus, these findings show that unilateral deprivation can disrupt developing auditory circuits that integrate binaural information and may give rise to lingering spatial hearing deficits.

neuroscience↗

bioRxiv2023

Exercise on cytokine responses in males and females: Effect of leucine, HMB and BCAA.

PurposeTo identify the effects of leucine, {beta}-hydroxy {beta}-methylbutyrate (HMB) and branched chain amino acid (BCAA) on post-exercise cytokine responses in females and males. MethodsMales (n=53) and females (n=37) completed 100 drop jumps and consumed either no supplement, leucine (3g/d), HMB (3g/d) or BCAA (4.5g/d) from 1d pre to 14d post-exercise. Muscle soreness, squat jumps, chair rises and creatine kinase (CK) were measured at pre, post, 24h, 48h, 7 and 14d. Blood lactate (pre, post), 10 cytokines (pre, 24h, 48h, 7d) and oestradiol (pre, 7d) were also measured. ResultsWithout supplementation post-exercise, soreness was induced in both males (6-fold) and females (5-fold). With supplementation, there were no increases in CK or oestradiol in females and no impact on muscle soreness, performance, or function in both sexes. In males, CK was elevated in untreated (48%) and leucine (69%) conditions vs baseline, but these were suppressed with HMB and BCAA. IL-7 was elevated in females vs males at baseline (6.3-fold), leucine increased IL-7 concentrations in females at 24h (17.0-fold), 48h (5.1-fold) vs males. With HMB, TNFr1- increased in females at 24h (2.2-fold), 48h (2.3-fold) and 7d (2.3-fold) vs males. In males with BCAA, TNFr1- decreased (P=0.06) from pre to 24h (6.8-fold), then increased (P<0.05) from 24 to 48h (8.0-fold). ConclusionAlthough supplements were without effect on soreness following exercise, the cytokine response was evoked by exercise and impacted significantly by leucine, HMB and BCAA in females vs males. This improved cytokine response in females could lead to improved resistance to damage.

physiology↗

bioRxiv2022

Macromolecular crowding is surprisingly unable to deform the structure of a model biomolecular condensate

The crowded interior of a living cell makes experiments on simpler in vitro systems attractive. Although these reveal interesting phenomena, their biological relevance can be questionable. A topical example is the phase separation of intrinsically-disordered proteins into biomolecular condensates, which is proposed to underlie the membraneless compartmentalisation of many cellular functions. How a cell reliably controls biochemical reactions in compartments open to the compositionally-varying cytoplasm is an important question for understanding cellular homeostasis. Computer simulations are often used to study the phase behaviour of model biomolecular condensates, but the number of relevant parameters explodes as the number of protein components increases. It is unfeasible to exhaustively simulate such models for all parameter combinations, although interesting phenomena are almost certainly hidden in the jungle of their high-dimensional parameter space. Here we have studied the phase behaviour of a model biomolecular condensate in the presence of a polymeric crowding agent. We used a novel compute framework to execute dozens of simultaneous simulations spanning the protein/crowder concentration space. We then combined the results into a graphical representation for human interpretation, which provided an efficient way to search the models high-dimensional parameter space. We found that steric repulsion from the crowder drives a near-critical system across the phase boundary, but the molecular arrangement within the resulting biomolecular condensate is rather insensitive to the crowder concentration and molecular weight. We propose that a cell may use the local cytoplasmic concentration to assist formation of biomolecular condensates, while relying on the dense phase reliably providing a stable, structured, fluid milieu for cellular biochemistry despite being open to its changing environment.

biophysics↗

bioRxiv2022

β-catenin interacts with the TAZ1 and TAZ2 domains of CBP/p300 to activate gene transcription

The transcriptional co-regulator {beta}-catenin is a critical effector of the canonical Wnt-signalling pathway, which plays a crucial role in regulating cell fate and maintaining tissue homeostasis. Deregulation of the Wnt/{beta}-catenin pathway is characteristic in the development of major types of cancer, where accumulation of {beta}-catenin promotes cancer cell proliferation and renewal. {beta}-catenin gene expression is facilitated through recruitment of co-activators such as histone acetyltransferases CBP/p300; however, the mechanism of their interaction is not fully understood. Here we investigate the interaction between the C-terminal transactivation domain of {beta}-catenin and CBP/p300. Using a combination of pulldown assays, isothermal titration calorimetry, and nuclear resonance spectroscopy we determine the disordered C-terminal region of {beta}-catenin binds promiscuously to the TAZ1 and TAZ2 domains of CBP/p300. We then map the interaction site of the C-terminal {beta}-catenin transactivation domain onto TAZ1 and TAZ2 using chemical-shift perturbation studies. Luciferase-based gene reporter assays indicate Asp750-Leu781 is critical to {beta}-catenin gene activation, and mutagenesis revealed that acidic and hydrophobic residues within this region are necessary to maintain TAZ1 binding. These results provide a mechanistic understanding of Wnt/{beta}-catenin gene regulation that underlies cell development and provide a framework to develop methods to block {beta}-catenin dependent signalling in the future.

biochemistry↗

bioRxiv2022

Structural basis of CBP/p300 recruitment by the microphthalmia-associated transcription factor

The microphthalmia-associated transcription factor (MITF) is a master regulator of the melanocyte cell lineage. Aberrant MITF activity can lead to multiple malignancies including skin cancer, where it modulates the proliferation and invasiveness of melanoma. MITF-dependent gene expression requires recruitment of transcriptional co-activators such as CBP/p300, but details of this process are not fully defined. Here, we investigate the structural and functional interaction between the MITF N-terminal transactivation domain (MITFTAD) and CBP/p300. A combination of pulldown assays and nuclear magnetic resonance spectroscopy determined that MITF binds both TAZ1 and TAZ2 domains of CBP/p300 with high affinity. The solution-state structure of the MITFTAD:TAZ2 complex reveals that MITF interacts with a hydrophobic surface of TAZ2, while remaining relatively dynamic. Peptide array and mutagenesis experiments determined that an acidic motif is integral to the MITFTAD:TAZ2 interaction and is necessary for transcriptional activity of MITF. Peptides that bind to the same surface of TAZ2 as MITFTAD, such as the adenoviral protein E1A, are capable of displacing MITF from TAZ2 and inhibiting transactivation. These results provide mechanistic insight into co-activator recruitment by MITF that are fundamental to our understanding of MITF targeted gene regulation and melanoma biology.

biochemistry↗

bioRxiv2021

Angiogenesis is critical for the regenerative effects of exercise.

Summary AbstractEnhancing axon regeneration is a major focus of nerve injury research, and the quality of the surgical nerve repair plays a large role in the aggregate success of nerve regeneration. Additionally, exercise is known to promote successful axon regeneration after surgical nerve repair. In this study, we asked how exercise-induced nerve regeneration is affected when a transected nerve is repaired with or without fibrin glue. Fibrin glue repaired nerves exhibited greater vasculature within the tissue bridge compared to nerves that were intrinsically repaired. Fibrin glue repaired nerves also exhibited more robust axon regeneration after exercise compared to nerves that were not repaired with fibrin glue. When angiogenesis of the tissue bridge was prevented, exercise was unable to enhance regeneration despite the presence of fibrin glue. These findings suggest that the biological properties of fibrin glue enhance angiogenesis within the repair site, and a vascularized bridge is required for enhanced axon elongation with exercise. The combination of fibrin glue repair and exercise resulted in notable differences in vascular growth, axon elongation, neuromuscular junction reinnervation, and functional recovery. Fibrin glue should be considered as an adjuvant for nerve repair to enhance the subsequent efficacy of activity- and physical therapy-based treatment interventions.

cell biology↗

bioRxiv2021

Degradation of ribosome and chaperone proteins is attenuated during the Differentiation of Replicatively Aged C2C12 Myoblasts

Age-related impairments in myoblast differentiation may contribute to reductions in muscle function in older adults, however, the underlying proteostasis processes are not well understood. Young (P6-10) and replicatively aged (P48-50) C2C12 myoblast cultures were investigated during early (0h-24h) and late (72h-96h) stages of differentiation using deuterium oxide (D2O) labelling and mass spectrometry. The absolute dynamic profiling technique for proteomics (Proteo-ADPT) was applied to quantify the absolute rates of abundance change, synthesis and degradation of individual proteins. Proteo-ADPT encompassed 116 proteins and 74 proteins exhibited significantly (P<0.05, FDR <5 %) different changes in abundance between young and aged cells at early and later periods of differentiation. Young cells exhibited a steady pattern of growth, protein accretion and fusion, whereas aged cells failed to gain protein mass or undergo fusion during later differentiation. Maturation of the proteome was retarded in aged myoblasts at the onset of differentiation, but their proteome appeared to catch up with the young cells during the early phase of the differentiation period. However, this catch up process in aged cells was not accomplished by higher levels of protein synthesis. Instead, a lower level of protein degradation in aged cells was responsible for the elevated gains in protein abundance. Our novel data point to a loss of proteome quality as a precursor to the lack of fusion of aged myoblasts and highlights dysregulation of protein degradation, particularly of ribosomal and chaperone proteins, as a key mechanism that may contribute to age-related declines in the capacity of myoblasts to undergo differentiation.

cell biology↗

bioRxiv2021

Fluorogenic EXO-Probe Aptamers for Imaging and Tracking Exosomal RNAs

Small extracellular vesicles (sEVs), or exosomes, play important roles in physiological and pathological cellular communication. sEVs contain both short and long non-coding RNAs that regulate gene expression and epigenetic processes. Studying the intricacies of sEV function and RNA-based communication requires tools capable of labeling sEV RNA. Here we developed a novel genetically encodable reporter system for tracking sEV RNAs comprising an sEV-loading RNA sequence, termed the EXO-Code, fused to a fluorogenic RNA Mango aptamer for RNA imaging. This fusion construct allowed the visualization and tracking of RNA puncta and colocalization with markers of multivesicular bodies; imaging RNA puncta within sEVs; and quantification of sEVs. This technology represents a useful and versatile tool to interrogate the role of sEVs in cellular communication via RNA trafficking to sEVs, cellular sorting decisions, and sEV RNA cargo transfer to recipient cells.

bioengineering↗

bioRxiv2020

Comparative analyses of two primate species diverged by more than 60 million years show different rates but similar distribution of genome-wide UV repair events

Nucleotide excision repair is the primary DNA repair mechanism that removes bulky DNA adducts such as UV-induced pyrimidine dimers. Correspondingly, genome-wide mapping of nucleotide excision repair with eXcision Repair sequencing (XR-seq), provides comprehensive profiling of DNA damage repair. A number of XR-seq experiments at a variety of conditions for different damage types revealed heterogenous repair in the human genome. Although human repair profiles were extensively studied, how repair maps vary between primates is yet to be investigated. Here, we characterized the genome-wide UV-induced damage repair in gray mouse lemur, Microcebus murinus, in comparison to human. Mouse lemurs are strictly nocturnal, are the worlds smallest living primates, and last shared a common ancestor with humans at least 60 million years ago. We derived fibroblast cell lines from mouse lemur, exposed them to UV irradiation. The following repair events were captured genome-wide through the XR-seq protocol. Mouse lemur repair profiles were analyzed in comparison to the equivalent human fibroblast datasets. We found that overall UV sensitivity, repair efficiency, and transcription-coupled repair levels differ between the two primates. Despite this, comparative analysis of human and mouse lemur fibroblasts revealed that genome-wide repair profiles of the homologous regions are highly correlated. This correlation is stronger for the highly expressed genes. With the inclusion of an additional XR-seq sample derived from another human cell line in the analysis, we found that fibroblasts of the two primates repair UV-induced DNA lesions in a more similar pattern than two distinct human cell lines do. Our results suggest that mouse lemurs and humans, and possibly primates in general, share a homologous repair mechanism as well as genomic variance distribution, albeit with their variable repair efficiency. This result also emphasizes the deep homologies of individual tissue types across the eukaryotic phylogeny.

genomics↗

bioRxiv2019

DNA methylation across the genome in aged human skeletal muscle tissue and muscle stem cells: The role of HOX genes and physical activity

Skeletal muscle tissue demonstrates global hypermethylation with aging. However, methylome changes across the time-course of differentiation in aged human muscle derived stem cells, and larger coverage arrays in aged muscle tissue have not been undertaken. Using 850K DNA methylation arrays we compared the methylomes of young (27 {+/-} 4.4 years) and aged (83 {+/-} 4 years) human skeletal muscle and that of young/aged muscle stem cells over several time points of differentiation (0, 72 hours, 7, 10 days). Aged muscle tissue was hypermethylated compared with young tissue, enriched for; pathways-in-cancer (including; focal adhesion, MAPK signaling, PI3K-Akt-mTOR signaling, p53 signaling, Jak-STAT signaling, TGF-beta and notch signaling), rap1-signaling, axon-guidance and hippo-signalling. Aged muscle stem cells also demonstrated a hypermethylated profile in pathways; axon-guidance, adherens-junction and calcium-signaling, particularly at later timepoints of myotube formation, corresponding with reduced morphological differentiation and reductions in MyoD/Myogenin gene expression compared with young cells. While young cells showed little alteration in DNA methylation during differentiation, aged cells demonstrated extensive and significantly altered DNA methylation, particularly at 7 days of differentiation and most notably in the focal adhesion and PI3K-AKT signalling pathways. While the methylomes were vastly different between muscle tissue and isolated muscle stem cells, we identified a small number of CpG sites showing a hypermethylated state with age, in both muscle and tissue and stem cells (on genes KIF15, DYRK2, FHL2, MRPS33, ABCA17P). Most notably, differential methylation analysis of chromosomal regions identified three locations containing enrichment of 6-8 CpGs in the HOX family of genes altered with age. With HOXD10, HOXD9, HOXD8, HOXA3, HOXC9, HOXB1, HOXB3, HOXC-AS2 and HOXC10 all hypermethylated in aged tissue. In aged cells the same HOX genes (and additionally HOXC-AS3) displayed the most variable methylation at 7 days of differentiation versus young cells, with HOXD8, HOXC9, HOXB1 and HOXC-AS3 hypermethylated and HOXC10 and HOXC-AS2 hypomethylated. We also determined that there was an inverse relationship between DNA methylation and gene expression for HOXB1, HOXA3 and HOXC-AS3. Finally, increased physical activity in young adults was associated with oppositely regulating HOXB1 and HOXA3 methylation compared with age. Overall, we demonstrate that a considerable number of HOX genes are differentially epigenetically regulated in aged human skeletal muscle and muscle stem cells and increased physical activity may help prevent age-related epigenetic changes in these HOX genes.

physiology↗