Biology subjects

Metzger, J. M.

Publications and source records attributed to Metzger, J. M..

6 recordsLinked to original sources

bioRxiv2026

Replicable generation of rhesus macaque iPSCs for in vitro modeling of genetic frontotemporal dementia

At the Wisconsin National Primate Research Center, we have identified a family of rhesus carrying the microtubule-associated protein tau (MAPT) R406W mutation linked to frontotemporal dementia (FTD). Rhesus induced pluripotent stem cells (RhiPSCs) derived from these monkeys present a unique opportunity for in vitro modeling and comparison with cells derived from MAPT R406W human carriers. Here, we report the development of a reproducible method to generate RhiPSCs compliant with the standards of the International Society for Stem Cell Research (ISSCR) to support in vitro modeling of FTD-MAPT R406W. Our stepwise approach identified efficient methods for fibroblast derivation, fibroblast reprogramming to RhiPSC, and RhiPSC maintenance over continued culture. To derive fibroblasts from MAPT wild type (WT) and R406W monkeys, a combination of manual processing and overnight enzymatic digestion was required to maximize the number of low passage fibroblasts available for reprogramming. Fibroblast reprogramming to RhiPSC using Sendai viral vectors versus oriP/EBNA1 episomal plasmids revealed the latter as most efficient. Electroporation conditions for oriP/EBNA1 reprogramming were optimized to maximize plasmid uptake and cell survival. Ultimately, eight RhiPSC lines were derived from 4 donor rhesus monkeys (n=2 WT, n=2 R406W; two clonal lines per donor) and fully characterized according to ISSCR standards. RhiPSC stemness and genetic stability was best maintained on mouse embryonic fibroblast feeders in Universal Primate Pluripotency Stem Cell medium, as opposed to Essential 12 medium supplemented with IWR1, which produced cytogenetic abnormalities. Rhesus neural progenitor cells were generated using a monolayer protocol and expressed PAX6 and NESTIN after 21 days of differentiation. Our reliable method will be useful to labs seeking to derive RhiPSCs for preclinical studies. Overall, the RhiPSCs generated from MAPT R406W carriers will be a critical resource for evaluating the molecular underpinnings of tau-related neurodegeneration across primate species.

cell biology↗

bioRxiv2025

Elucidating cancer cachexia-mediated aberrant cardiac wasting signaling in human iPSC-derived cardiac muscle

Cancer cachexia is a highly debilitating clinical syndrome of involuntary body mass loss featuring profound muscle wasting leading to high mortality. Notably, cardiac wasting is prominent in cancer patients and cancer survivors. Cachexia studies present significant challenges due to the absence of human models and mainly short-term animal studies. To address this translational gap, we have developed a robust human-based cachexia experimental approach characterized by marked cardiac muscle wasting and contractile dysfunction, with increased expression of protein degradation markers. Using human iPSC-derived cardiac muscle, we investigated morphological, functional, and metabolic alterations in the key stages of cachexia and in the post-cachexia phase. C26 and HCT116 tumor cell lines were used to induce cachexia by two methods, pulse addition of cancer cell conditioned media or in transwell-adapted co-culture. Cachectic cardiac myocytes exhibited reduced contraction amplitude, prolonged relaxation time, and increased oxygen consumption rate (OCR), as assessed by video-based and Seahorse analyses. Mechanistic investigations centered on the Atrogin-1/Calcineurin A/NFAT axis revealed this signaling pathway as a central driver of cachexia-induced cardiac atrophy. Cachectic cardiac myocytes exhibited significant upregulation of Atrogin-1, leading to a marked decrease in Calcineurin A protein levels. This, in turn, impaired nuclear translocation of NFAT, thereby suppressing its transcriptional activity and downstream cell growth signaling. These molecular changes were accompanied by increased autophagic flux, as indicated by elevated LC3BII/LC3BI ratios. Furthermore, withdrawal of cachexia-inducing stimuli followed by regular media changes for one week led to normalization of Atrogin-1 and autophagy markers; however, functional impairments and metabolic dysregulation persisted, highlighting delayed recovery. Our new findings establish the Atrogin-1/Calcineurin A/NFAT axis as a key regulatory mechanism in cardiac muscle wasting and suggest this aberrant signaling axis may serve as a targetable mechanism for treatment of cachexia-induced cardiac dysfunction. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=154 HEIGHT=200 SRC="FIGDIR/small/670675v2_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@12e9borg.highwire.dtl.DTLVardef@19ad494org.highwire.dtl.DTLVardef@1fa664corg.highwire.dtl.DTLVardef@5232a6_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

Gene transfer of human cardiomyopathy beta-MyHC mutant R403Qdirectly alters intact cardiac myocyte calcium homeostasis and causes hyper-contractility

The R403Q mutation of human cardiac {beta}-myosin heavy chain was the first missense mutation of a sarcomeric protein identified as being causal for hypertrophic cardiomyopathy (HCM), in humans. The direct effect of the R403Q mutant myosin on intracellular calcium homeostasis and contractility is not fully known. Here we have used in vitro gene transfer of the R403Q mutant human {beta}-myosin to study its direct effects on single intact adult cardiac myocyte contractility and calcium homeostasis. In the first experiments, adult cardiac myocytes transduced with the R403Q mutant myosin recombinant viral vectors were compared to myocytes transduced with wild-type human {beta}-myosin (wtMYH7). Efficiency of gene transfer was high in both groups (>98%) and the degree of stoichiometric myofilament incorporation of either the mutant or normal myosin was comparable at [~]40% in quiescent myocytes in primary culture. Sarcomere structure and cellular morphology were unaffected by R403Q myosin expression and myofilament incorporation. Functionally, in electrically paced cardiac myocytes, the R403Q mutant myosin caused a significant increase in intracellular calcium concentration and myocyte hyper-contractility. At the sub-cellular myofilament level, the mutant myosin increased the calcium sensitivity of steady state isometric tension development and increased isometric cross-bridge cycling kinetics. R403Q myocytes became arrhythmic after {beta}-adrenergic stimulation with spontaneous calcium transients and contractions in between electrical stimuli. These results indicate that human R403Q mutant myosin directly alters myofilament function and intracellular calcium cycling. Elevated calcium levels may provide a trigger for the ensuing hypertrophy and susceptibility to arrhythmia that are characteristic of HCM.

physiology↗

bioRxiv2023

Striatonigral distribution of a fluorescent reporter following intracerebral delivery of genome editors

IntroductionTargeted gene editing is proposed as a therapeutic approach for numerous disorders, including neurological diseases. As the brain is organized into neural networks, it is critical to understand how anatomically connected structures are affected by genome editing. For example, neurons in the substantia nigra pars compacta (SNpc) project to the striatum, and the striatum contains neurons that project to the substantia nigra pars reticulata (SNpr). MethodsHere, we report the effect of injecting genome editors into the striatum of Ai14 reporter mice, which have a LoxP-flanked stop cassette that prevents expression of the red fluorescent protein tdTomato. Two weeks following intracerebral delivery of either synthetic nanocapsules (NCs) containing CRISPR ribonucleoprotein targeting the tdTomato stop cassette or adeno-associated virus (AAV) vectors expressing Cre recombinase, the brains were collected, and the presence of tdTomato was assessed in both the striatum and SN. ResultsTdTomato expression was observed at the injection site in both the NC- and AAV-treated groups and typically colocalized with the neuronal marker NeuN. In the SN, tdTomato-positive fibers were present in the pars reticulata, and SNpr area expressing tdTomato correlated with the size of the striatal genome edited area. ConclusionThese results demonstrate in vivo anterograde axonal transport of reporter gene protein products to the SNpr following neuronal genome editing in the striatum.

bioengineering↗

bioRxiv2022

Efficient in vivo neuronal genome editing in the mouse brain using nanocapsules containing CRISPR-Cas9 ribonucleoproteins

Genome editing of somatic cells via clustered regularly interspaced short palindromic repeats (CRISPR) offers promise for new therapeutics to treat a variety of genetic disorders, including neurological diseases. However, the dense and complex parenchyma of the brain and the post-mitotic state of neurons make efficient genome editing challenging. In vivo delivery systems for CRISPR-Cas proteins and single guide RNA (sgRNA) include both viral vectors and non-viral strategies, each presenting different advantages and disadvantages for clinical application. We developed non-viral and biodegradable PEGylated nanocapsules (NCs) that deliver preassembled Cas9-sgRNA ribonucleoproteins (RNPs). Here, we show that the RNP NCs led to robust genome editing in neurons following intracerebral injection into the mouse striatum. Genome editing was predominantly observed in medium spiny neurons (>80%), with occasional editing in cholinergic, calretinin, and parvalbumin interneurons. Glial activation was minimal and was localized along the needle tract. Our results demonstrate that the RNP NCs are capable of safe and efficient neuronal genome editing in vivo. SIGNIFICANCE STATEMENTModifying the DNA of cells in the brain could present opportunities for new treatments of neurological diseases. In this report, we describe a nanocapsule system designed to deliver the elements needed to modify the DNA of brain cells, also known as genome editing. These nanocapsules are created by chemically encapsulating the genome editing components, such that the nanocapsules are stable when prepared and biodegradable to release their payload upon entering cells. When injected into the mouse brain, our research shows that the nanocapsules lead to safe and efficient editing of DNA in neurons.

neuroscience↗

bioRxiv2020

Oxidative stress pathogenically remodels the cardiac myocyte cytoskeleton via structural alterations to the microtubule lattice

In the failing heart, the cardiac myocyte microtubule network is remodeled, which increases cellular stiffness and disrupts contractility, contributing to heart failure and death. However, the origins of this deleterious cytoskeletal reorganization are unknown. We now find that oxidative stress, a condition characteristic of failing heart cells, leads to cysteine oxidation of microtubules. Further, our electron and fluorescence microscopy experiments revealed regions of structural damage within the oxidized microtubule lattice. These damaged regions led to the lengthening, realignment, and acetylation of dynamic microtubules within cardiac myocytes. Thus, we found that oxidative stress acts inside of cardiac myocytes to facilitate a dramatic, pathogenic shift from a dynamic, multifaceted microtubule network into a highly acetylated, longitudinally aligned, and static microtubule network. Our results demonstrate how a disease condition characterized by oxidative stress can trigger a molecular oxidation event, which propagates a toxic cellular-scale transformation of the cardiac myocyte microtubule network.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cell biology↗