Biology subjects

Miller, B. F.

Publications and source records attributed to Miller, B. F..

At least 19 recordsLinked to original sources

bioRxiv2026

ULK1 and ULK2 Restrain Skeletal Myofiber Growth by Balancing Protein Synthesis and Degradation

Background Skeletal muscle is vital for mobility and metabolic regulation, impacting independence and overall health. Increases in skeletal muscle mass and contractile function during development, and their maintenance during adulthood and aging, rely on an intricate coordination between protein synthesis and degradation processes that remains incompletely understood. Here, we investigated a potential role for the autophagy-initiating kinases ULK1 and ULK2 in broadly modulating protein metabolism in skeletal muscle. Methods Studies were conducted in young (4-6 wk-old) and adult (7-10 mo.-old) mice with skeletal muscle-specific knockout of Ulk1 and Ulk2 (i.e., Ulk1/2skmDKO) and wild-type littermates (WT). Short-term deficiency of these proteins was achieved via electroporation of plasmids (encoding specific microRNAs targeting Ulk1 and Ulk2) into muscles of 4 mo.-old wild-type mice. Protein metabolism was assessed via deuterium oxide (D2O) labeling, whereas anabolic signaling was investigated under insulin and leucine administration. Results Lifelong Ulk1/2 deficiency markedly impaired autophagy flux (i.e., LC3-II accumulated with colchicine treatment only in wild-type mice, P<0.001), compromised muscle quality, as evidenced by an increase in centrally nucleated fibers (from 0.1% to 4.5% in females, and from 0.8% to 22.7% in males (P<0.001), primarily involving MyHC type 2b fibers) and impaired force of dorsiflexors and plantar flexors in males (20%, P<0.01), and plantar flexors in females (24%, P<0.01). Despite these deficits, Ulk1/2 deficiency promoted robust muscle hypertrophy, evidenced by increased diameters of all major MyHC fiber types in the tibialis anterior and soleus muscles (i.e., by 10-15% in males, and 14-20% in females, P<0.05). Short-term deficiency (up to 4 weeks) of Ulk1/2 in adult skeletal muscle, however, led to myofiber hypertrophy (13%, P<0.05) without impairments in force or changes in central nucleation of fibers, pointing to an initial period of muscle quality preservation. Mechanistically, Ulk1/2 deficiency led to elevated myofibrillar protein synthesis (23% higher Ksyn, P<0.05) and decreased mitochondrial and sarcoplasmic protein degradation (16% and 14% lower Kdeg, P=0.09 and P<0.05, respectively). Further mechanistic studies revealed that hypertrophy was accompanied by enhanced mTORC1 activity independent of AKT in Ulk1/2-deficient muscle. Conclusions These results indicate that ULK1 and ULK2 jointly sustain autophagy and limit mTORC1-driven protein synthesis to govern skeletal muscle protein metabolism, with lifelong deficiency increasing muscle size at the expense of quality and function, while short-term deficiency permits hypertrophy without impairment. These findings identify ULK1/2 as a novel node coordinating protein turnover in skeletal muscle, warranting investigation as a therapeutic strategy for atrophy and weakness.

cell biology↗

bioRxiv2026

Overactive PDGFRα and PDGFRβ promote distinct yet overlapping phenotypes of skeletal muscle fibrosis and stiffness, with PDGFRβ also driving drastic muscle growth

Background: Fibrosis accumulates in skeletal muscle over time and leads to greater muscle rigidity, stiffness, and increased risk of injuries. However, investigations of experimental models to study the mechanisms through which muscle fibrosis occurs are often confounded by injury or disease. The contribution of platelet-derived growth factor receptors alpha and beta (PDGFR or PDGFR{beta}) to muscle fibrosis is yet to be clarified. We hypothesized that both receptors would promote ECM deposition and fibrosis, causing muscle stiffening and weakness, with sex-specific differences arising due to hormonal influences on receptors. Methods: To test this hypothesis, we used a mouse model with inducible overactive PDGFR or PDGFR{beta} signaling and assessed various indicators of muscle function, metabolism, motor coordination, exercise capacity, collagen deposition, and muscle stiffness. Results: Overactive PDGFR led to more collagen deposition, increased collagen crosslinking, and higher AGE/LOX protein levels, all of which correlated with greater muscle stiffness compared to CON. Overactive PDGFR{beta} resulted in greater muscle mass and lower fat mass, and had higher collagen deposition in female mice compared to CON. There were also sex-specific differences with fibrotic remodeling, muscle stiffness, and muscle size in response to overactive PDGFR and PDGFR{beta} signaling. Conclusion: These findings establish PDGFR and PDGFR{beta} signaling as distinct regulators of muscle remodeling and establish overactive PDGFR as a mouse model to study skeletal muscle fibrosis in the absence of other confounding variables.

physiology↗

bioRxiv2025

HSF1 remodels mitochondrial biogenesis and function in cancer cells via TIMM17A

Mitochondria play critical roles in energy production and cellular metabolism. Despite the Warburg effect, mitochondria are crucial for the survival and proliferation of cancer cells. Heat Shock Factor 1 (HSF1), a key transcription factor in the cellular heat shock response, promotes malignancy and metastasis when aberrantly activated. To understand the multifaceted roles of HSF1 in cancer, we performed a genome-wide CRISPR screen to identify epistatic interactors of HSF1 in cancer cell proliferation. The verified interactors of HSF1 include those involved in DNA replication and repair, transcriptional and post-transcriptional gene expression, and mitochondrial functions. Specifically, we found that HSF1 promotes cell proliferation, mitochondrial biogenesis, respiration, and ATP production in a manner dependent on TIMM17A, a subunit of the inner membrane translocase. HSF1 upregulates the steady-state level of the short-lived TIMM17A protein via its direct target genes, HSPD1 and HSPE1, which encode subunits of the mitochondrial chaperonin complex and are responsible for protein refolding once imported into the matrix. The HSF1- HSPD1/HSPE1-TIMM17A axis remodels the mitochondrial proteome to promote mitochondrial translation and energy production, thereby supporting robust cell proliferation. Our work reveals a mechanism by which mitochondria adjust protein uptake according to the folding capacity in the matrix by altering TIM complex composition.

cell biology↗

bioRxiv2024

Combining in vivo 2-photon imaging with photoactivatable fluorescent labeling reveals strikingly low rates of mitochondrial dynamics in skeletal muscle.

Mitochondrial dynamics involve two distinct and opposing processes, fusion and fission. Traditionally we assess fusion and fission by snapshots of protein markers at distinct time points or in vitro models to infer outcomes in vivo. Recent technological advancements enable visualization of mitochondrial dynamics in vivo using fluorescent microscopy. Our study modified this technique to evaluate mitochondrial dynamics in skeletal muscle, comparing young (6mo) and old (24mo) mice in vivo and contrasting this to ex vivo and in vitro models. We hypothesized that in vitro and ex vivo models would have higher rates of dynamics than in vivo models and that young animals would have higher rates than old animals. We electroporated mitochondrial matrix-targeted photo-activatable GFP into the tibialis anterior (TA) of young and old C57Bl6 mice and imaged using multiphoton microscopy. We also measured rates of mitochondrial dynamics using single fibers isolated from the TA of the electroporated mice, as well as C2C12 myotubes transfected with the same plasmids. We found that the rates of dynamic events in vivo are slower than previously indicated, with the C2C12 myoblasts having the fastest rates of dynamic events across all models. We also observed that dynamic rates are slower in old animals compared to young animals. Finally, we found that rates of dynamic events were higher in old animals after an acute bout of exercise. Our data demonstrate it is possible to directly measure rates of mitochondrial dynamics in vivo. This technique provides a powerful tool to answer experimental questions about mitochondrial dynamics of skeletal muscle.

physiology↗

bioRxiv2024

Hyperactive mTORC1/4EBP1 Signaling Dysregulates Proteostasis and Accelerates Cardiac Aging.

The mechanistic target of rapamycin complex 1 (mTORC1) has a major impact on aging by regulation of proteostasis. It is well established that mTORC1 signaling is hyperactivated with aging and age-related diseases. Previous studies have shown that partial inhibition of mTOR signaling by rapamycin reverses the age-related decline in cardiac function and structure in old mice. However, the downstream signaling pathways involved in this protection against cardiac aging have not been established. TORC1 phosphorylates 4E-binding protein 1 (4EBP1) to promote the initiation of cap-dependent translation. The aim of this project is to examine the role of the mTORC1/4EBP1 axis in age-related cardiac dysfunction. We utilized a whole-body 4EBP1 KO mouse model, which mimics a hyperactive 4EBP1/eIF4E axis, to investigate the effects of hyperactive mTORC1/4EBP1 axis in cardiac aging. Echocardiographic measurements revealed that young 4EBP1 KO mice have no difference in cardiac function at baseline compared to WT mice. Interestingly, middle-aged (14-15-month-old) 4EBP1 KO mice show impaired diastolic function and myocardial performance compared to age-matched WT mice and their diastolic function and myocardial performance are at similar levels as 24-month-old WT mice, suggesting that 4EBP1 KO mice experience accelerated cardiac aging. Old 4EBP1 KO mice show further declines in systolic and diastolic function compared to middle-aged 4EBP1 KO mice and have worse systolic and diastolic function than age-matched old WT mice. Gene expression levels of heart failure markers are not different between 4EBP1 KO and WT mice at these advanced ages. However, ribosomal biogenesis and overall protein ubiquitination are significantly increased in 4EBP1 KO mice when compared to WT, which suggests dysregulated proteostasis. Together, these results show that a hyperactive 4EBP1/eIF4E axis accelerates cardiac aging, potentially by dysregulating proteostasis.

molecular biology↗

bioRxiv2024

Metformin treatment results in distinctive skeletal muscle mitochondrial remodeling in rats with different intrinsic aerobic capacities

The rationale for the use of metformin as a treatment to slow aging was largely based on data collected from metabolically unhealthy individuals. For healthspan extension metformin will also be used in periods of good health. To understand potential context specificity of metformin treatment on skeletal muscle, we used a rat model (HCR/LCR) with a divide in intrinsic aerobic capacity. Outcomes of metformin treatment differed based on baseline intrinsic mitochondrial function, oxidative capacity of the muscle (gastroc vs soleus), and the mitochondrial population (IMF vs SS). Metformin caused lower ADP-stimulated respiration in LCRs, with less of a change in HCRs. However, a washout of metformin resulted in an unexpected doubling of respiratory capacity in HCRs. These improvements in respiratory capacity were accompanied by mitochondrial remodeling that included increases in protein synthesis and changes in morphology. Our findings raise questions about whether the positive findings of metformin treatment are broadly applicable.

physiology↗

bioRxiv2024

SEraster: a rasterization preprocessing framework for scalable spatial omics data analysis

MotivationSpatial omics data demand computational analysis but many analysis tools have computational resource requirements that increase with the number of cells analyzed. This presents scalability challenges as researchers use spatial omics technologies to profile millions of cells. ResultsTo enhance the scalability of spatial omics data analysis, we developed a rasterization preprocessing framework called SEraster that aggregates cellular information into spatial pixels. We apply SEraster to both real and simulated spatial omics data prior to spatial variable gene expression analysis to demonstrate that such preprocessing can reduce resource requirements while maintaining high performance. We further integrate SEraster with existing analysis tools to characterize cell-type spatial cooccurrence. Finally, we apply SEraster to enable analysis of a mouse pup spatial omics dataset with over a million cells to identify tissue-level and cell-type-specific spatially variable genes as well as cooccurring cell-types that recapitulate expected organ structures. Availability and implementationSource code is available on GitHub (https://github.com/JEFworks-Lab/SEraster) with additional tutorials at https://JEF.works/SEraster.

bioinformatics↗

bioRxiv2023

Characterizing cell-type spatial relationships across length scales in spatially resolved omics data

Spatially resolved omics (SRO) technologies enable the identification of cell types while preserving their organization within tissues. Application of such technologies offers the opportunity to delineate cell-type spatial relationships, particularly across different length scales, and enhance our understanding of tissue organization and function. To quantify such multi-scale cell-type spatial relationships, we developed CRAWDAD, Cell-type Relationship Analysis Workflow Done Across Distances, as an open-source R package with source code and additional documentation at https://jef.works/CRAWDAD/. To demonstrate the utility of such multi-scale characterization, recapitulate expected cell-type spatial relationships, and evaluate against other cell-type spatial analyses, we applied CRAWDAD to various simulated and real SRO datasets of diverse tissues assayed by diverse SRO technologies. We further demonstrate how such multi-scale characterization enabled by CRAWDAD can be used to compare cell-type spatial relationships across multiple samples. Finally, we applied CRAWDAD to SRO datasets of the human spleen to identify consistent as well as patient and sample-specific cell-type spatial relationships. In general, we anticipate such multi-scale analysis of SRO data enabled by CRAWDAD will provide useful quantitative metrics to facilitate the identification, characterization, and comparison of cell-type spatial relationships across axes of interest.

bioinformatics↗

bioRxiv2023

Fibroblast growth factor-21 induces skeletal muscle atrophy and increases plasma amino acids in female mice: a potential role for glucocorticoids

BackgroundFibroblast growth factor-21 (FGF21) is an intercellular signaling molecule secreted by metabolic organs, including skeletal muscle, in response to intracellular stress. FGF21 crosses the blood brain barrier and acts via the nervous system to coordinate aspects of the adaptive starvation response, including increased lipolysis, gluconeogenesis, hepatic fatty acid oxidation, and activation of the hypothalamic-pituitary-adrenocortical (HPA) axis. Given its beneficial effects for hepatic lipid metabolism, pharmaceutical FGF21 analogues are in clinical trials treatment of fatty liver disease. We predicted pharmacologic treatment with FGF21 in-creases HPA axis activity and skeletal muscle glucocorticoid signaling and induces skeletal muscle atrophy in mice. MethodsWe treated male and female mice with FGF21 or saline, delivered either pe-ripherally or directly to the brain, to determine its effect on skeletal muscle. To identify metabolic pathways affected by FGF21, we analyzed untargeted primary metabolites measured in plasma by GCTOF-MS. To determine mechanisms underlying sex-and FGF21-dependent changes in muscle mass, we measured hormonal and molecular mediators of muscle protein synthesis and degradation. We performed stable isotope labeling with deuterium oxide to directly measure muscle protein synthesis. ResultsA short course of systemic FGF21 treatment decreased muscle protein synthe-sis (P < 0.001) and reduced tibialis anterior weight (P < 0.05); this was driven primarily by its effect in female mice (P < 0.05). Similarly, intracerebroventricular FGF21 reduced TA muscle fiber cross sectional area (P < 0.01); this was more apparent among female mice compared to male littermates (P < 0.05). In agreement with the reduced muscle mass, the topmost enriched meta-bolic pathways in FGF21-treated females were related to amino acid metabolism, and the relative abundance of plasma proteinogenic amino acids were increased up to three-fold (P < 0.05). FGF21 treatment increased hypothalamic Crh mRNA (P < 0.01), plasma corticosterone (P < 0.01), and adrenal weight (P < 0.05), and increased expression of glucocorticoid receptor target genes known to reduce muscle protein synthesis and/or promote degradation including Foxo1, Redd1, and Klf15 (P < 0.05). Again, these changes were driven primarily by effects of FGF21 in females (P < 0.05). ConclusionsFGF21 increased plasma amino acids and decreased skeletal muscle mass, together with activation of the HPA axis and glucocorticoid receptor target genes in skeletal muscle--and female mice were more sensitive to all these outcomes. Given the proposed use of FGF21 analogues for the treatment of metabolic disease, the study is both physiologically relevant and may have important clinical implications.

physiology↗

bioRxiv2023

17α-estradiol Alleviates High-Fat Diet-Induced Inflammatory and Metabolic Dysfunction in Skeletal Muscle of Male and Female Mice

Skeletal muscle has a central role in maintaining metabolic homeostasis. 17-estradiol (17-E2), a naturally-occurring non-feminizing diastereomer of 17{beta}-estradiol that demonstrates efficacy for improving metabolic outcomes in male, but not female, mice. Despite several lines of evidence showing that 17-E2 treatment improves metabolic parameters in middle-aged obese and old male mice through effects in brain, liver, and white adipose tissue little is known about how 17-E2 alters skeletal muscle metabolism, and what role this may play in mitigating metabolic declines. Therefore, this study aimed to determine if 17-E2 treatment improves metabolic outcomes in skeletal muscle from obese male and female mice following chronic high fat diet (HFD) administration. We hypothesized that male, but not female, mice, would benefit from 17-E2 treatment during HFD. To test this hypothesis, we used a multi-omics approach to determine changes in lipotoxic lipid intermediates, metabolites, and proteins related to metabolic homeostasis. In male mice, we show that 17-E2 alleviates HFD-induced metabolic detriments of skeletal muscle by reducing the accumulation of diacylglycerol (DAGs) and ceramides, inflammatory cytokine levels, and reduced the abundance of most of the proteins related to lipolysis and beta-oxidation. In contrast to males, 17-E2 treatment in female mice had little effect on the DAGs and ceramides content, muscle inflammatory cytokine levels, or changes to the relative abundance of proteins involved in beta-oxidation. These data support to the growing evidence that 17-E2 treatment could be beneficial for overall metabolic health in male mammals.

physiology↗

bioRxiv2023

Metabolic benefits of 17α-estradiol in liver are partially mediated by ERβ in male mice

Metabolic dysfunction underlies several chronic diseases. Dietary interventions can reverse metabolic declines and slow aging but remaining compliant is difficult. 17-estradiol (17-E2) treatment improves metabolic parameters and slows aging in male mice without inducing significant feminization. We recently reported that estrogen receptor is required for the majority of 17-E2-mediated benefits in male mice, but that 17-E2 also attenuates fibrogenesis in liver, which is regulated by estrogen receptor {beta} (ER{beta})-expressing hepatic stellate cells (HSC). The current studies sought to determine if 17-E2-mediated benefits on systemic and hepatic metabolism are ER{beta}-dependent. We found that 17-E2 treatment reversed obesity and related systemic metabolic sequela in both male and female mice, but this was partially blocked in female, but not male, ER{beta}KO mice. ER{beta} ablation in male mice attenuated 17-E2-mediated benefits on hepatic stearoyl-coenyzme A desaturase 1 (SCD1) and transforming growth factor {beta}1 (TGF-{beta}1) production, which play critical roles in HSC activation and liver fibrosis. We also found that 17-E2 treatment suppresses SCD1 production in cultured hepatocytes and hepatic stellate cells, indicating that 17-E2 directly signals in both cell-types to suppress drivers of steatosis and fibrosis. We conclude that ER{beta} partially controls 17-E2-mediated benefits on systemic metabolic regulation in female, but not male, mice, and that 17-E2 likely signals through ER{beta} in HSCs to attenuate pro-fibrotic mechanisms.

physiology↗

bioRxiv2022

Skeletal muscle nuclei in mice are not post-mitotic

The skeletal muscle research field generally accepts that nuclei in skeletal muscle fibers (i.e., myonuclei) are post-mitotic and unable to proliferate. Because our deuterium oxide (D2O) labeling studies showed DNA synthesis in skeletal muscle tissue, we hypothesized that resident myonuclei can replicate in vivo. To test this hypothesis, we used a mouse model that temporally labeled myonuclei with GFP followed by D2O labeling during normal cage activity, functional overload, and with satellite cell ablation. During normal cage activity, we observed deuterium enrichment into myonuclear DNA in 7 out of 7 plantaris (PLA), 6 out of 6 tibialis anterior (TA), 5 out of 7 gastrocnemius (GAST) and 7 out of 7 quadriceps (QUAD). The average fractional synthesis rates (FSR) of DNA in myonuclei were: 0.0202 {+/-} 0.0093 in PLA, 0.0239 {+/-} 0.0040 in TA, 0.0076 {+/-} 0. 0058 in GAST, and 0.0138 {+/-} 0.0039 in QUAD, while there was no replication in myonuclei from EDL. These FSR values were largely reproduced in the overload and satellite cell ablation conditions although there were higher synthesis rates in the overloaded PLA muscle. We further provided evidence that myonuclear replication is through endoreplication that results in polyploidy. These novel findings contradict the dogma that skeletal muscle nuclei are post-mitotic and open potential avenues to harness the intrinsic replicative ability of myonuclei for muscle maintenance and growth. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/513426v2_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1e4d210org.highwire.dtl.DTLVardef@19b52daorg.highwire.dtl.DTLVardef@5c9a53org.highwire.dtl.DTLVardef@197dc0d_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2022

Blocking necroptosis reduces inflammation and tumor incidence in a mouse model of diet-induced hepatocellular carcinoma

Background & AimsNonalcoholic fatty liver disease (NAFLD) is one of the etiologies that contribute to hepatocellular carcinoma (HCC), and chronic inflammation is one of the proposed mediators of HCC. As necroptosis is a cell death pathway that induces inflammation, we tested whether necroptosis- induced inflammation contributes to the progression of NAFLD to HCC in a mouse model of diet- induced HCC. MethodsMale and female wild-type (WT) mice or mouse models where necroptosis is blocked (Ripk3-/- or Mlkl-/- mice) were fed a control diet or choline-deficient low fat diet (CD-LFD) or CD-high fat diet (CD-HFD) for 6 months. Changes in inflammation, immune cell infiltration, activation of oncogenic pathways, and tumor incidence were assessed by gene expression analysis, western blotting, and flow cytometry. RNA sequencing (RNA-seq) was performed to assess the changes in liver transcriptome. ResultsBlocking necroptosis by deleting either Ripk3 or Mlkl reduced markers of inflammation [proinflammatory cytokines (TNF, IL-6, and IL-1{beta}), F4/80+ve macrophages, CCR2+ve infiltrating monocytes], inflammation associated oncogenic pathways (JNK, PD-L1/PD-1, {beta}-catenin), and HCC in male mice. In female mice, blocking necroptosis reduced HCC independent of inflammation. Blocking necroptosis reduced cell senescence markers in males and females, suggesting a novel cross-talk between necroptosis and cell senescence. ConclusionsOur data show that hepatic necroptosis promotes recruitment and activation of liver macrophages leading to chronic inflammation, which in turn trigger oncogenic pathways leading to the progression of NAFLD to HCC in male mice. In female mice necroptosis contributes to HCC independent of inflammation. Thus, our study suggests that necroptosis is a valid target for NAFLD-mediated HCC. SynopsisNecroptosis is a cell death pathway that mediate inflammation. Blocking necroptosis attenuated chronic inflammation by reducing recruitment and activation of liver macrophages, which in turn reduced activation of oncogenic pathways and progression of NAFLD to HCC in mice. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/502666v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@1bf82d7org.highwire.dtl.DTLVardef@26fc68org.highwire.dtl.DTLVardef@18538f4org.highwire.dtl.DTLVardef@15e338b_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2022

Senolytic Treatment Reduces Cell Senescence and Necroptosis in Sod1 Knockout Mice that is Associated with Reduced Inflammation and Hepatocellular Carcinoma

The goal of this study was to test the role cellular senescence plays in the increase in inflammation, chronic liver disease, and hepatocellular carcinoma, which are seen in mice null for Cu/Zn-Superoxide dismutase (Sod1KO). To inhibit senescence, six-month-old wildtype (WT) and Sod1KO mice were given the senolytics, dasatinib and quercetin (D+Q) for seven months. D+Q treatment reduced the expression of p16 in the livers of Sod1KO mice to WT levels as well as the expression of several SASP (senescence associated secretory phenotype) factors (IL-6, IL-1{beta}, CXCL-1, and GDF-15). D+Q treatment also reduced markers of inflammation in livers of the Sod1KO mice, e.g., cytokines, chemokines, macropthage levels, and Kupffer cell clusters. D+Q treatment had no effect on various markers of liver fibrosis in the Sod1KO mice but reduced the expression of genes involved in liver cancer (Myc, Tgfbr2, Socs3, and Cdkn2a) as well as dramatically reducing the incidence of hepatocellular carcinoma. Surprisingly, D+Q also reduced markers of necroptosis (phosphorylated and oligomerized MLKL) in the Sod1KO mice to WT levels. We also found that inhibiting necroptosis in the Sod1KO mice with necrostatin-1s reduced the markers of cellular senescence (p16, p21, and p53). The data from our study suggest that an interaction occurs between cellular senescence and necroptosis in the liver of Sod1KO mice. We propose that these two cell fates interact through a positive feedback loop resulting in a cycle amplifying both cellular senescence and necroptosis leading to inflammaging and age-associated pathology in the Sod1KO mice.

biochemistry↗

bioRxiv2021

Necroptosis contributes to chronic inflammation and fibrosis in aging liver

Inflammaging, characterized by an increase in low-grade chronic inflammation with age, is a hallmark of aging and is strongly associated with various age-related diseases, including chronic liver disease (CLD) and hepatocellular carcinoma (HCC). Because necroptosis is a cell death pathway that induces inflammation through the release of DAMPs, we tested the hypothesis that age-associated increase in necroptosis contributes to chronic inflammation in aging liver. Phosphorylation of MLKL and MLKL-oligomers, markers of necroptosis, as well as phosphorylation of RIPK3 and RIPK1 were significantly upregulated in the livers of old mice relative to young mice and this increase occurred in the later half of life (i.e., after 18 months of age). Markers of M1 macrophages, expression of proinflammatory cytokines (TNF, IL6 and IL-1{beta}), and markers of fibrosis were significantly upregulated in the liver with age and the change in necroptosis paralleled the changes in inflammation and fibrosis. Hepatocytes and liver macrophages isolated from old mice showed elevated levels of necroptosis markers as well as increased expression of proinflammatory cytokines relative to young mice. Short term treatment with the necroptosis inhibitor, necrostatin-1s (Nec-1s), reduced necroptosis, markers of M1 macrophages, expression of proinflammatory cytokines, and markers of fibrosis in the livers of old mice. Thus, our data show for the first time that liver aging is associated with increased necroptosis and necroptosis contributes to chronic inflammation in the liver, which in turn appears to contribute to liver fibrosis and possibly CLD. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=158 SRC="FIGDIR/small/460953v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@15ab761org.highwire.dtl.DTLVardef@bd7336org.highwire.dtl.DTLVardef@1d6e3bcorg.highwire.dtl.DTLVardef@10159eb_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

Reference-free cell-type deconvolution of pixel-resolution spatially resolved transcriptomics data

Recent technological advancements have enabled spatially resolved transcriptomic profiling but at multi-cellular pixel resolution, thereby hindering the identification of cell-type-specific spatial patterns and gene expression variation. To address this challenge, we developed STdeconvolve as a reference-free approach to deconvolve underlying cell-types comprising such multi-cellular pixel resolution spatial transcriptomics (ST) datasets. Using simulated as well as real ST datasets from diverse spatial transcriptomics technologies comprising a variety of spatial resolutions such as Spatial Transcriptomics, 10X Visium, DBiT-seq, and Slide-seq, we show that STdeconvolve can effectively recover cell-type transcriptional profiles and their proportional representation within pixels without reliance on external single-cell transcriptomics references. STdeconvolve provides comparable performance to existing reference-based methods when suitable single-cell references are available, as well as potentially superior performance when suitable single-cell references are not available. STdeconvolve is available as an open-source R software package with the source code available at https://github.com/JEFworks-Lab/STdeconvolve.

bioinformatics↗

bioRxiv2021

Phytochemical Nrf2 activator attenuates skeletal muscle mitochondrial dysfunction and impaired proteostasis in a preclinical model of musculoskeletal aging

Musculoskeletal dysfunction is an age-related syndrome associated with impaired mitochondrial function and proteostasis. However, few interventions have tested targeting two drivers of musculoskeletal decline. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that stimulates transcription of cytoprotective genes and improves mitochondrial function. We hypothesized daily treatment with a Nrf2 activator in Hartley guinea pigs, a model of age-related musculoskeletal dysfunction, attenuates the progression of skeletal muscle mitochondrial dysfunction and impaired proteostasis, preserving musculoskeletal function. We treated 2-month- and 5-month-old male and female Hartley guinea pigs for 3 and 10 months, respectively, with the phytochemical Nrf2 activator PB125 (Nrf2a). Longitudinal assessments of voluntary mobility were measured using Any-Maze open-field enclosure monitoring. Cumulative skeletal muscle protein synthesis rates were measured using deuterium oxide over the final 30 days of treatment. Mitochondrial oxygen consumption in permeabilized soleus muscles was measured using ex vivo high resolution respirometry. In both sexes, Nrf2a 1) increased electron transfer system capacity; 2) attenuated the disease/age-related decline in coupled and uncoupled mitochondrial respiration; and 3) attenuated declines in protein synthesis in the myofibrillar, mitochondrial, and cytosolic subfractions of the soleus. These improvements were not associated with statistically significant prolonged maintenance of voluntary mobility in guinea pigs. Collectively, these results demonstrate that treatment with an oral Nrf2 activator contributes to maintenance of skeletal muscle mitochondrial function and proteostasis in a pre-clinical model of musculoskeletal decline. Further investigation is necessary to determine if these improvements are also accompanied by slowed progression of other aspects of musculoskeletal decline.

physiology↗

bioRxiv2019

Inducible cell-specific mouse models for paired epigenetic and transcriptomic studies of microglia and astroglia

Epigenetic regulation of gene expression occurs in a cell type-specific manner. Current cell-type specific neuroepigenetic studies rely on cell sorting methods that can alter cell phenotype and introduce potential confounds. Here we demonstrate and validate a Nuclear Tagging and Translating Ribosome Affinity Purification (NuTRAP) approach for temporally controlled labeling and isolation of ribosomes and nuclei, and thus RNA and DNA, from specific CNS cell types. Paired analysis of the transcriptome and DNA modifications in astrocytes and microglia demonstrates differential usage of DNA methylation and hydroxymethylation in CG and non-CG contexts that corresponds to cell type-specific gene expression. Application of this approach in LPS treated mice uncovers microglia-specific transcriptome and epigenome changes in inflammatory pathways that cannot be detected with tissue-level analysis. The NuTRAP model and the validation approaches presented can be applied to any CNS cell type for which a cell type-specific cre is available.

neuroscience↗