Biology subjects

Cramer, A.

Publications and source records attributed to Cramer, A..

6 recordsLinked to original sources

bioRxiv2026

Integrated morphological, multi-omics, and functional profiling reveals microglial plasticity driven by AD risk genes

Microglia, the resident macrophages of the central nervous system, are highly dynamic cells essential for brain homeostasis. While genome-wide association studies (GWAS) strongly implicate microglial dysfunction in Alzheimer's disease (AD), the mechanistic links coordinating their diverse transcriptional, morphological, and functional states remain poorly understood. Using human induced pluripotent stem cell-derived microglia (iMicroglia) and single-cell RNA sequencing, we identified six distinct transcriptional profiles and mapped them to specific morphological phenotypes via targeted immunofluorescence, establishing a link between microglial morphology and molecular identity. Transcriptomic and morphological profiling further demonstrated profound microglial plasticity, revealing distinct, stimulus-specific responses to AD-relevant pathologies, including Tau PFF, amyloid-beta, and apoptotic neurons. To assess how AD risk variants perturb these states, we performed high-efficiency CRISPR-Cas9 ribonucleoprotein (RNP) knockouts of specific AD GWAS genes. Bulk RNA-seq profiling revealed extensive transcriptional remodeling following genetic perturbation. Crucially, we show that depletion of these AD risk genes disrupts baseline morpho-transcriptomic coupling and fundamentally alters microglial phagocytic capacity. Together, this study reveals how AD GWAS genes may drive microglia into dysfunctional states characterized by altered morphology, distinct multi-omic signatures, and impaired functions.

neuroscience↗

bioRxiv2025

Spatial and single-cell transcriptomics capture two distinct cell states in plant immunity

Unlike animals, plants are sessile organisms that must adapt to localized and fluctuating environmental stimuli, including abiotic and biotic stresses. While animals use mobile immune cells to eliminate pathogens, plants rely on localized cells in contact with the pathogen to detect and mount immune responses. Although bulk RNA sequencing (RNA-seq) has enabled the assessment of plant responses to pathogen infection at the whole transcriptome level, the spatial coordination of plant immune responses remains elusive. In this study, we performed both spatial and single-nuclei transcriptomic experiments to capture the spatial pattern of soybean plant responses to Asian soybean rust infection caused by the pathogen Phakopsora pachyrhizi. Through the analysis of both spatial and single-nuclei transcriptomics data, we identified two distinct host cell states with specific spatial localization in response to pathogen infection: the infected regions with the presence of the pathogen and the surrounding regions bordering the infected regions. Importantly, the surrounding regions exhibited higher expression of defense response-related genes than the infected regions, despite having minimal presence of the pathogen, indicating a cell non-autonomous defense response in the surrounding regions. Additionally, gene co-expression network analysis with single-cell resolution identified a key immune response-related gene module activated in the stressed cells captured in our single-nuclei RNA-seq data. This study reveals the intricate spatial coordination of plant defense responses against pathogen infection and enhances our understanding of the importance of localized cell non-autonomous defense responses in plant-pathogen interactions.

plant biology↗

bioRxiv2024

SARM1 orthosteric base exchange inhibitors cause subinhibitory SARM1 activation

SARM1, an octameric NADase, is a key regulator of axon degeneration and an emerging target in small molecule drug discovery to treat a wide range of neurodegenerative diseases. Recently, a structurally diverse series of adduct-forming, orthosteric SARM1 inhibitors have been discovered. Here, we show that subinhibitory concentrations of these orthosteric inhibitors, under mildly SARM1 activating conditions, cause sustained SARM1 activation. This synergistic adverse effect leads to increased nicotinamide adenine dinucleotide (NAD) consumption, neurodegeneration and release of the biomarker neurofilament-light (NfL) in cultured cortical neurons. In two distinct animal models, we found that low-dose treatment with these orthosteric SARM1 inhibitors results in increased plasma NfL and adverse events when combined with cellular stress or injury conditions. This may present a critical liability for orthosteric SARM1 inhibitors in certain patient populations.

neuroscience↗

bioRxiv2023

Enveloped viruses pseudotyped with mammalian myogenic cell fusogens target skeletal muscle for gene delivery

Entry of enveloped viruses into cells is mediated by fusogenic proteins that form a complex between membranes to drive rearrangements needed for fusion. Skeletal muscle development also requires membrane fusion events between progenitor cells to form multinucleated myofibers. Myomaker and Myomerger are muscle-specific cell fusogens, but do not structurally or functionally resemble classical viral fusogens. We asked if the muscle fusogens could functionally substitute for viral fusogens, despite their structural distinctiveness, and fuse viruses to cells. We report that engineering of Myomaker and Myomerger on the membrane of enveloped viruses leads to specific transduction of skeletal muscle. We also demonstrate that locally and systemically injected virions pseudotyped with the muscle fusogens can deliver micro-Dystrophin (Dys) to skeletal muscle of a mouse model of Duchenne muscular dystrophy. Through harnessing the intrinsic properties of myogenic membranes, we establish a platform for delivery of therapeutic material to skeletal muscle.

cell biology↗

bioRxiv2023

A scalable, GMP-compatible, autologous organotypic cell therapy for Dystrophic Epidermolysis Bullosa

BackgroundGene editing in induced pluripotent stem (iPS) cells has been hailed to enable new cell therapies for various monogenetic diseases including dystrophic epidermolysis bullosa (DEB). However, manufacturing, efficacy and safety roadblocks have limited the development of genetically corrected, autologous iPS cell-based therapies. MethodsWe developed Dystrophic Epidermolysis Bullosa Cell Therapy (DEBCT), a new generation GMP-compatible (cGMP), reproducible, and scalable platform to produce autologous clinical-grade iPS cell-derived organotypic induced skin composite (iSC) grafts to treat incurable wounds of patients lacking type VII collagen (C7). DEBCT uses a combined high-efficiency reprogramming and CRISPR-based genetic correction single step to generate genome scar- free, COL7A1 corrected clonal iPS cells from primary patient fibroblasts. Validated iPS cells are converted into epidermal, dermal and melanocyte progenitors with a novel 2D organoid differentiation protocol, followed by CD49f enrichment and expansion to minimize maturation heterogeneity. iSC product characterization by single cell transcriptomics was followed by mouse xenografting for disease correcting activity at 1 month and toxicology analysis at 1-6 months. Culture-acquired mutations, potential CRISPR-off targets, and cancer-driver variants were evaluated by targeted and whole genome sequencing. FindingsiPS cell-derived iSC grafts were reproducibly generated from four recessive DEB patients with different pathogenic mutations. Organotypic iSC grafts onto immune-compromised mice developed into stable stratified skin with functional C7 restoration. Single cell transcriptomic characterization of iSCs revealed prominent holoclone stem cell signatures in keratinocytes and the recently described Gibbin-dependent signature in dermal fibroblasts. The latter correlated with enhanced graftability. Multiple orthogonal sequencing and subsequent computational approaches identified random and non-oncogenic mutations introduced by the manufacturing process. Toxicology revealed no detectable tumors after 3-6 months in DEBCT- treated mice. InterpretationDEBCT successfully overcomes previous roadblocks and represents a robust, scalable, and safe cGMP manufacturing platform for production of a CRISPR-corrected autologous organotypic skin graft to heal DEB patient wounds.

bioengineering↗

bioRxiv2021

A suitable murine model for studying respiratory coronavirus infection and therapeutic countermeasures in BSL-2 laboratories

Several animal models are being used to explore important features of COVID-19, nevertheless none of them recapitulates all aspects of the disease in humans. The continuous refinement and development of other options of in vivo models are opportune, especially ones that are carried out at BSL-2 (Biosafety Level 2) laboratories. In this study, we investigated the suitability of the intranasal infection with the murine betacoronavirus MHV-3 to recapitulate multiple aspects of the pathogenesis of COVID-19 in C57BL/6J mice. We demonstrate that MHV-3 replicated in lungs 1 day after inoculation and triggered respiratory inflammation and dysfunction. This MHV-model of infection was further applied to highlight the critical role of TNF in cytokine-mediated coronavirus pathogenesis. Blocking TNF signaling by pharmacological and genetic strategies greatly increased the survival time and reduces lung injury of MHV-3-infected mice. In vitro studies showed that TNF blockage decreased SARS-CoV-2 replication in human epithelial lung cells and resulted in the lower release of IL-6 and IL-8 cytokines beyond TNF itself. Taken together, our results demonstrate that this model of MHV infection in mice is a useful BSL-2 screening platform for evaluating pathogenesis for human coronaviruses infections, such as COVID-19.

microbiology↗