Monji A, Zhang Y, Kumar NGV, Guillermier C, Kim S, Olenchock B, Steinhauser ML. A Cycle of Inflammatory Adipocyte Death and Regeneration in Murine Adipose Tissue. Diabetes (New York, N.Y.). 2022;71 (3) :412–423.Abstract
Adipose tissue (AT) expands by a combination of two fundamental cellular mechanisms: hypertrophic growth of existing adipocytes or through generation of new adipocytes, also known as hyperplastic growth. Multiple lines of evidence suggest a limited capacity for hyperplastic growth of AT in adulthood and that adipocyte number is relatively stable, even with fluctuations in AT mass. If the adipocyte number is stable in adulthood, despite well-documented birth and death of adipocytes, then this would suggest that birth may be coupled to death in a regenerative cycle. To test this hypothesis, we examined the dynamics of birth of new fat cells in relationship to adipocyte death by using high-fidelity stable isotope tracer methods in C57Bl6 mice. We discovered birth of new adipocytes at higher frequency in histological proximity to dead adipocytes. In diet-induced obesity, adipogenesis surged after an adipocyte death peak beyond 8 weeks of high-fat feeding. Through transcriptional analyses of AT and fractionated adipocytes, we found that the dominant cell death signals were inflammasome related. Proinflammatory signals were particularly evident in hypertrophied adipocytes or with deletion of a constitutive oxygen sensor and inhibitor of hypoxia-inducible factor, Egln1. We leveraged the potential role for the inflammasome in adipocyte death to test the adipocyte death-birth hypothesis, finding that caspase 1 loss of function attenuated adipocyte death and birth in murine visceral AT. These data collectively point to a regenerative cycle of adipocyte death and birth as a driver of adipogenesis in adult murine AT.
Turck CW, Webhofer C, Reckow S, Moy J, Wang M, Guillermier C, Poczatek JC, Filiou MD. Antidepressant treatment effects on hippocampal protein turnover: Molecular and spatial insights from mass spectrometry. PROTEOMICS. 2022;n/a (n/a) :2100244. Publisher's VersionAbstract
Abstract A major challenge in managing depression is that antidepressant drugs take a long time to exert their therapeutic effects. For the development of faster acting therapies, it is crucial to get an improved understanding of the molecular mechanisms underlying antidepressant mode of action. Here, we used a novel mass spectrometry-based workflow to investigate how antidepressant treatment affects brain protein turnover at single cell and subcellular resolution. We combined stable isotope metabolic labeling, quantitative Tandem Mass Spectrometry (qTMS) and Multi-isotope Imaging Mass Spectrometry (MIMS) to simultaneously quantify and image protein synthesis and turnover in hippocampi of mice treated with the antidepressant paroxetine. We identified changes in turnover of individual hippocampal proteins that reveal altered metabolism-mitochondrial processes and report on subregion-specific turnover patterns upon paroxetine treatment. This workflow can be used to investigate brain protein turnover changes upon pharmacological interventions at a resolution and precision that has not been possible with other methods to date. Our results reveal acute paroxetine effects on brain protein turnover and shed light on antidepressant mode of action. This article is protected by copyright. All rights reserved
McMahon G, Lechene CP. High-Resolution Multi-Isotope Imaging Mass Spectrometry (MIMS) Imaging Applications in Stem Cell Biology. Current Protocols. 2021;1 (11) :e290. Publisher's VersionAbstract
Abstract Multi-isotope imaging mass spectrometry (MIMS) allows the measurement of turnover of molecules within intracellular compartments with a spatial resolution down to 30 nm. We use molecules enriched in stable isotopes administered to animals by diet or injection, or to cells through the culture medium. The stable isotopes used are, in general, 15N, 13C, 18O, and 2H. For stem cell studies, we essentially use 15N-thymidine, 13C-thymidine, and 81Br from BrdU. This protocol describes the practical use of MIMS with specific reference to applications in stem cell research. This includes choice and administration of stable isotope label(s), sample preparation, best practice for high-resolution imaging, secondary ion mass spectrometry using the Cameca NanoSIMS 50L, and methods for robust statistical analysis of label incorporation in regions of interest (ROI). © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Stable isotope labeling of DNA in cultured cells Basic Protocol 2: Stable isotope labeling of DNA in animals Basic Protocol 3: Preparation of Si chips, the general sample support for NanoSIMS analysis Basic Protocol 4: Stable isotope analysis of DNA replication in single nuclei in a population of cells with NanoSIMS Basic Protocol 5: Data reduction and processing
Hark TJ, Rao NR, Castillon C, Basta T, Smukowski S, Bao H, Upadhyay A, Bomba-Warczak E, Nomura T, O’Toole ET, et al. Pulse-Chase Proteomics of the App Knockin Mouse Models of Alzheimer’s Disease Reveals that Synaptic Dysfunction Originates in Presynaptic Terminals. Cell systems. 2021;12 (2) :141–158.e9.Abstract
Compromised protein homeostasis underlies accumulation of plaques and tangles in Alzheimer’s disease (AD). To observe protein turnover at early stages of amyloid beta (Aβ) proteotoxicity, we performed pulse-chase proteomics on mouse brains in three genetic models of AD that knock in alleles of amyloid precursor protein (APP) prior to the accumulation of plaques and during disease progression. At initial stages of Aβ accumulation, the turnover of proteins associated with presynaptic terminals is selectively impaired. Presynaptic proteins with impaired turnover, particularly synaptic vesicle (SV)-associated proteins, have elevated levels, misfold in both a plaque-dependent and -independent manner, and interact with APP and Aβ. Concurrent with elevated levels of SV-associated proteins, we found an enlargement of the SV pool as well as enhancement of presynaptic potentiation. Together, our findings reveal that the presynaptic terminal is particularly vulnerable and represents a critical site for manifestation of initial AD etiology. A record of this paper’s transparent peer review process is included in the Supplemental Information. [Display omitted] •Axon terminals are selective sites of impaired protein degradation in App KI brains•Presynaptic proteins have elevated levels at early stages of Aβ accumulation•Synaptic-vesicle-associated proteins accumulate with Aβ peptides and plaques•Aβ disrupts vesicle fusion and leads to a larger readily releasable vesicle pool Hark et al. use dynamic metabolic 15N labeling and mass-spectrometry-based proteomics to investigate changes to protein turnover in App knockin mice. In this preclinical Alzheimer’s disease mouse model, they find that proteostasis in the presynaptic terminal is specifically altered. Synaptic vesicle (SV)-associated proteins functioning in exo- and endocytosis have impaired degradation and elevated levels in the cortex and hippocampus. Finally, the readily releasable SV pool and presynaptic potentiation is enhanced at the earliest stages of amyloid beta accumulation.
El Khoudary SR, Fabio A, Yester JW, Steinhauser ML, Christopher AB, Gyngard F, Adams PS, Morell VO, Viegas M, Da Silva JP, et al. Design and rationale of a clinical trial to increase cardiomyocyte division in infants with tetralogy of Fallot. International Journal of Cardiology. 2021;339 :36–42. PDF
Gyngard F, Trakimas L, Steinhauser ML. High-Fidelity Quantification of Cell Cycle Activity with Multi-Isotope Imaging Mass Spectrometry. In: Cardiac Regeneration. Humana, New York, NY ; 2021. pp. 257–268. PDF
Yester JW, Liu H, Gyngard F, Ammanamanchi N, Little KC, Thomas D, Sullivan MLG, Lal S, Steinhauser ML, Kühn B. Use of stable isotope-tagged thymidine and multi-isotope imaging mass spectrometry (MIMS) for quantification of human cardiomyocyte division. Nature protocols. 2021;16 (4) :1995–2022. PDF
Narendra DP, Steinhauser ML. Metabolic Analysis at the Nanoscale with Multi-Isotope Imaging Mass Spectrometry (MIMS). Current Protocols in Cell Biology. 2020;88 (1) :e111. Publisher's VersionAbstract
Abstract Incorporation of a stable-isotope metabolic tracer into cells or tissue can be followed at submicron resolution by multi-isotope imaging mass spectrometry (MIMS), a form of imaging secondary ion microscopy optimized for accurate isotope ratio measurement from microvolumes of sample (as small as ∼30 nm across). In a metabolic MIMS experiment, a cell or animal is metabolically labeled with a tracer containing a stable isotope. Relative accumulation of the heavy isotope in the fixed sample is then measured as an increase over its natural abundance by MIMS. MIMS has been used to measure protein turnover in single organelles, track cellular division in vivo, visualize sphingolipid rafts on the plasma membrane, and measure dopamine incorporation into dense-core vesicles, among other biological applications. In this article, we introduce metabolic analysis using NanoSIMS by focusing on two specific applications: quantifying protein turnover in single organelles of cultured cells and tracking cell replication in mouse tissues in vivo. These examples illustrate the versatility of metabolic analysis with MIMS. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Metabolic labeling for MIMS Basic Protocol 2: Embedding of samples for correlative transmission electron microscopy and MIMS with a genetically encoded reporter Alternate Protocol: Embedding of samples for correlative light microscopy and MIMS Support Protocol: Preparation of silicon wafers as sample supports for MIMS Basic Protocol 3: Analysis of MIMS data
Zhang Y, Guillermier C, De Raedt T, Cox AG, Maertens O, Yimlamai D, Lun M, Whitney A, Maas RL, Goessling W, et al. Imaging Mass Spectrometry Reveals Tumor Metabolic Heterogeneity. iScience. 2020;23 (8) :101355–101355.Abstract
Malignant tumors exhibit high degrees of genomic heterogeneity at the cellular level, leading to the view that subpopulations of tumor cells drive growth and treatment resistance. To examine the degree to which tumors also exhibit metabolic heterogeneity at the level of individual cells, we employed multi-isotope imaging mass spectrometry (MIMS) to quantify utilization of stable isotopes of glucose and glutamine along with a label for cell division. Mouse models of melanoma and malignant peripheral nerve sheath tumors (MPNSTs) exhibited striking heterogeneity of substrate utilization, evident in both proliferating and non-proliferating cells. We identified a correlation between metabolic heterogeneity, proliferation, and therapeutic resistance. Heterogeneity in metabolic substrate usage as revealed by incorporation of glucose and glutamine tracers is thus a marker for tumor proliferation. Collectively, our data demonstrate that MIMS provides a powerful tool with which to dissect metabolic functions of individual cells within the native tumor environment. [Display omitted] •A method to measure substrate utilization at single cancer cell resolution•Heterogeneity of glucose and glutamine utilization in murine tumors•Metabolic heterogeneity in proliferating and non-proliferating tumor cells•Metabolic heterogeneity correlates with proliferative growth and treatment resistance Biological Sciences; Cancer Systems Biology
Narendra DP, Guillermier C, Gyngard F, Huang X, Ward ME, Steinhauser ML. Coupling APEX labeling to imaging mass spectrometry of single organelles reveals heterogeneity in lysosomal protein turnover. Journal of Cell Biology. 2020;219 (1). PDF
Chinchore Y, Begaj T, Guillermeir C, Steinhauser ML, Punzo C, Cepko CL. Transduction of gluconeogenic enzymes prolongs cone photoreceptor survival and function in models of retinitis pigmentosa. bioRxiv. 2019. Publisher's VersionAbstract
The hereditary nature of many retinal degenerative disorders makes them potentially amenable to corrective gene therapies. Numerous clinical trials are ongoing with the goal to rectify the genetic defect in the afflicted cell types. However, the personalized nature of these approaches excludes many patients for whom the underlying mutation is not mapped, or the number of affected individuals is too few to develop a commercially viable therapy (vide infra). Thus, a therapy that can delay visual impairment irrespective of the underlying genetic etiology can satisfy this unmet medical need. Here, we demonstrate the utility of such an approach in retinitis pigmentosa (RP) by promoting survival of cone photoreceptors by targeting metabolic stress. These cells are not primarily affected by the inherited mutation, but their non-autonomous demise leads to a decline in daylight vision, greatly reducing the quality of life. We designed adeno-associated virus (AAV) vectors that promote gluconeogenesis- a pathway found in the liver which produces glucose in response to hypoglycemia. Retinal transduction with these vectors resulted in improved cone survival and delayed a decline in visual acuity in three different RP mouse models. Because this approach extended visual function independent of the primary mutation, therapies emanating from this approach could be used as a treatment option for a genetically heterogenous cohort of patients.
Guillermier C, Doherty SP, Whitney AG, Babaev VR, Linton MRF, Steinhauser ML, Brown JD. Imaging mass spectrometry reveals heterogeneity of proliferation and metabolism in atherosclerosis. JCI insight. 2019;4 (11).Abstract
Atherosclerotic plaques feature local proliferation of leukocytes and vascular smooth muscle cells (VSMCs) and changes in cellular metabolism. Yet the relationship between glucose utilization and proliferation has been technically impossible to study directly in cells of atherosclerotic plaques in vivo. We used multi-isotope imaging mass spectrometry (MIMS), a quantitative imaging platform, to measure coincident cell division and glucose utilization at suborganelle resolution in atherosclerotic plaques. In established plaques, 65% of intimal foam cells and only 4% of medial VSMCs were labeled with N-15-thymidine after 1 week of isotope treatment. Dividing cells demonstrated heightened glucose labeling. MIMS detected H-2-glucose label in multiple subcellular compartments within foam cells, including lipid droplets, the cytosol, and chromatin. Unexpectedly, we identified an intensely focal region of H-2-label in VSMCs underlying plaques. This signal diminished in regions of aorta without atherosclerosis. In advanced plaques. N-15-thymidine and H-2-glucose labeling in foam cells and VSMCs significantly decreased. These data demonstrate marked heterogeneity in VSMC glucose metabolism that was dependent on both proliferative status and proximity of VSMCs to plaques. Furthermore, these results reveal how quantitative mass spectrometry coupled with isotope imaging can complement other methods used to study cell biology directly in the growing atherosclerotic plaque in vivo.
Gyngard F, Steinhauser ML. Biological explorations with nanoscale secondary ion mass spectrometry. Journal of analytical atomic spectrometry. 2019;34 (8) :1534–1545. PDF
Lu J, Baccei A, Lummertz da Rocha E, Guillermier C, McManus S, Finney LA, Zhang C, Steinhauser ML, Li H, Lerou PH. Single-cell RNA sequencing reveals metallothionein heterogeneity during hESC differentiation to definitive endoderm. Stem cell research. 2018;28 (C) :48–55.Abstract
Differentiation of human pluripotent stem cells towards definitive endoderm (DE) is the critical first step for generating cells comprising organs such as the gut, liver, pancreas and lung. This in-vitro differentiation process generates a heterogeneous population with a proportion of cells failing to differentiate properly and maintaining expression of pluripotency factors such as Oct4. RNA sequencing of single cells collected at four time points during a 4-day DE differentiation identified high expression of metallothionein genes in the residual Oct4-positive cells that failed to differentiate to DE. Using X-ray fluorescence microscopy and multi-isotope mass spectrometry, we discovered that high intracellular zinc level corresponds with persistent Oct4 expression and failure to differentiate. This study improves our understanding of the cellular heterogeneity during in-vitro directed differentiation and provides a valuable resource to improve DE differentiation efficiency. •Definitive endoderm cells differentiated from hESCs are mixed with an Oct4-positive subpopulation.•Single-cell RNA sequencing identified high expression of metallothionein genes in the residual Oct4-positive cells.•High intracellular zinc level corresponds with persistent Oct4 expression.
Wildburger NC, Gyngard F, Guillermier C, Patterson BW, Elbert D, Mawuenyega KG, Schneider T, Green K, Roth R, Schmidt RE, et al. Amyloid-β plaques in clinical Alzheimer’s disease brain incorporate stable isotope tracer in vivo and exhibit nanoscale heterogeneity. Frontiers in neurology. 2018;9 :169. PDF
Vujic A, Lerchenmüller C, Mittag S, Wang A, Rabolli C, Gyngard F, Guillermier C, Steinhauser ML, Rosenzweig A, Lee RT. Exercise-Induced New Cardiomyocyte Formation in the Aged Mammalian Heart. Nature Communications. 2018;140 (Suppl\_1) :A14692–A14692. PDF
Filiou M, Guillermier C, Poczatek C, Wang M, Chen A, Turck C, Lechene C. Paroxetine treatment alters hippocampal protein turnover, in Pharmacopsychiatry. Vol 50. Stuttgart · New York: Georg Thieme Verlag KG ; 2017.
Guillermier C, Poczatek JC, Taylor WR, Steinhauser ML. Quantitative imaging of deuterated metabolic tracers in biological tissues with nanoscale secondary ion mass spectrometry. International Journal of Mass Spectrometry. 2017.Abstract
In the field of secondary ion mass spectrometry at nanometer scale (NanoSIMS), configuration of parallel detectors to routinely measure isotope ratios in sub-100 nm domains brings classical stable isotope tracer studies from the whole tissue level down to the suborganelle level. Over the past decade, the marriage of stable isotope tracers with NanoSIMS has been applied to a range of fundamental biological questions that were largely inaccessible by other means. Although multiplexed measurement of different stable isotope tracers is feasible, in practice there remains a gap in the current analytical capacity to efficiently measure stable isotopes commonly utilized in tracer studies. One such example is the measurement of deuterated tracers. The most obvious approach to measuring deuterium/hydrogen isotope ratios is at mass 2/1. However, the radius of the magnetic sector limits concomitant measurement of other masses critical to multiplexed exploration of biological samples. Here we determine the experimental parameters to measure deuterated tracers in biological samples using the C2H polyatomic ion species (C2D/C2H) while operating the NanoSIMS at a reduced Mass Resolving Power of 14,000. Through control of the sputtering parameters, we demonstrate that there is an analytical window during which the C2D/C2H isotope ratio can be measured with sufficient precision for biological studies where the degree of D-labeling is typically well above natural abundance. We provide validation of this method by comparing the C2D measurement of D-water labeling in the murine small intestine relative to measurements of native D/H conducted in the same analytical fields. Additional proof-of-concept demonstrations include measurement of D-water, D-glucose, and D-thymidine in biological specimens. Therefore, this study provides a practical template for deuterium-based tracer studies in biological systems.
Guillermier C, Fazeli PK, Kim S, Lun M, Zuflacht JP, Milian J, Steinhauser ML. Imaging mass spectrometry demonstrates age-related decline in human adipose plasticity. JCI Insight. 2017;2 (5).Abstract
Quantification of stable isotope tracers has revealed the dynamic state of living tissues. A new form of imaging mass spectrometry quantifies isotope ratios in domains much smaller than a cubic micron, enabling measurement of cell turnover and metabolism with stable isotope tracers at the single-cell level with a methodology we refer to as multi-isotope imaging mass spectrometry. In a first-in-human study, we utilize stable isotope tracers of DNA synthesis and de novo lipogenesis to prospectively measure cell birth and adipocyte lipid turnover. In a study of healthy adults, we elucidate an age-dependent decline in new adipocyte generation and adipocyte lipid turnover. A linear regression model suggests that the aging effect could be mediated by a decline in insulin-like growth factor-1 (IGF-1). This study therefore establishes a method for measurement of cell turnover and metabolism in humans with subcellular resolution while implicating the growth hormone/IGF-1 axis in adipose tissue aging.
Kumar S, Rajagopalan S, Sarkar P, Dorward DW, Peterson ME, Liao H-S, Guillermier C, Steinhauser ML, Vogel SS, Long EO. Zinc-Induced Polymerization of Killer-Cell Ig-like Receptor into Filaments Promotes Its Inhibitory Function at Cytotoxic Immunological Synapses. Molecular Cell. 2016;62 (1) :21-33. Publisher's VersionAbstract

The inhibitory function of killer cell immunoglobulin-like receptors (KIR) that bind HLA-C and block activation of human natural killer (NK) cells is dependent on zinc. We report that zinc induced the assembly of soluble KIR into filamentous polymers, as detected by electron microscopy, which depolymerized after zinc chelation. Similar KIR filaments were isolated from lysates of cells treated with zinc, and membrane protrusions enriched in zinc were detected on whole cells by scanning electron microscopy and imaging mass spectrometry. Two independent mutations in the extracellular domain of KIR, away from the HLA-C binding site, impaired zinc-driven polymerization and inhibitory function. KIR filaments formed
spontaneously, without the addition of zinc, at functional inhibitory immunological synapses of NK cells with HLA-C + cells. Adding to the recent paradigm of signal transduction through higher order molecular assemblies, zinc-induced polymerization of inhibitory KIR represents an unusual mode of signaling
by a receptor at the cell surface.