Biology subjects

Olah, M.

Publications and source records attributed to Olah, M..

12 recordsLinked to original sources

bioRxiv2026

Dense CRISPR Mapping of the WAVE Complex Identifies Pharmacological Modulators of AD-Linked Myeloid Migration

Although genetic risk for Alzheimers disease (AD) strongly converges on microglial pathways, the druggable functional protein regions that control disease-relevant microglial behaviors remain largely unknown. Here, we applied dense CRISPR-Cas9 mutagenesis and CRISPRtile-based functional mapping to the WAVE regulatory complex (WRC), a central regulator of actin remodeling and cell migration. In a pooled CCL2-directed migration assay in human THP-1 myeloid cells, perturbation of NCKAP1L, CYFIP1, and BRK1 impaired migration and revealed divergent effects among WRC paralogs. Residue-level analysis mapped discrete migration-associated functional regions within CYFIP1 and NCKAP1L, including a CYFIP1 regulatory hotspot and a prioritized NCKAP1L region nominated for pharmacological targeting. Human single-nucleus datasets identified NCKAP1L as a microglia-enriched WRC component expressed across diverse microglial states. Machine-learning-guided compound prediction nominated Montelukast sodium and Piperacetazine, which we experimentally validated as negative and positive modulators of chemokine-directed migration, respectively. These findings establish WRC-dependent migration as a pharmacologically tunable myeloid process relevant to AD.

neuroscience↗

bioRxiv2026

cGAS inhibition delays TDP-43-driven ALS Pathogenesis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder marked by motor neuron loss and cytoplasmic mislocalization of TAR DNA-binding protein 43 (TDP-43), a key regulator of RNA splicing. However, the upstream modulators of this process remain poorly defined. Here we identify cyclic GMP-AMP synthase (cGAS) as a central mediator of TDP-43 pathology and associated mis-splicing. cGAS expression was elevated in ALS patient brains and enriched across activated microglia. In human iPSC-derived microglia-motor neuron co-cultures, neuronal TDP-43 pathology triggered microglial cGAS activation, whereas pharmacological inhibition with a potent human cGAS inhibitor reduced phosphorylated TDP-43, restored lysosomal and phagocytic programs, normalized microglial reactivity, and reversed TDP-43-associated RNA splicing defects. In vivo, cGAS inhibition in TDP-43 Q331K mice reversed widespread RNA splicing abnormalities across neurons and oligodendrocyte lineage cells, attenuated neurodegenerative pathology, and preserved motor function. Together, these findings identify cGAS as a druggable upstream regulator linking innate immune signaling to TDP-43-dependent RNA mis-splicing and neurodegeneration, and establish cGAS inhibition as a promising therapeutic strategy for ALS.

neuroscience↗

bioRxiv2025

A proteogenomic tool uncovers protein markers for human microglial states

Human microglial heterogeneity has been largely described using transcriptomic data. Here, we introduce a microglial proteomic data resource and a Cellular Indexing of Transcriptomes and Epitopes by Sequencing panel enhanced with antibodies targeting 17 microglial cell surface proteins (mCITE-Seq). We evaluated mCITE-Seq on HMC3 microglia-like cells, induced-pluripotent stem cell-derived microglia (iMG), and freshly isolated primary human microglia. We identified novel protein microglial markers such as CD51 and relate expression of 101 cell surface proteins to transcriptional programs. This results in the identification and validation of three protein marker combinations with which to purify microglia enriched with each of 23 transcriptional programs; for example, CD49D, HLA-DR and CD32 enrich for GPNMBhigh ("disease associated") microglia. Further, we identify and validate proteins - SIRPA, PDPN and CD162 - that differentiate microglia from infiltrating macrophages. The mCITE-Seq panel enables the transition from RNA-based classification and facilitates the functional characterization and harmonization of model systems.

neuroscience↗

bioRxiv2025

A factor-based analysis of individual human microglia uncovers regulators of an Alzheimer-related transcriptional signature

Human microglial heterogeneity is only beginning to be appreciated at the molecular level. Here, we present a large, single-cell atlas of expression signatures from 441,088 live microglia broadly sampled across a diverse set of brain regions and neurodegenerative and neuroinflammatory diseases obtained from 161 donors sampled at autopsy or during a neurosurgical procedure. Using single-cell hierarchical Poisson factorization (scHPF), we derived a 23-factor model for continuous gene expression signatures across microglia which capture specific biological processes (e.g., metabolism, phagocytosis, antigen presentation, inflammatory signaling, disease-associated states). Using external datasets, we evaluated the aspects of microglial phenotypes that are encapsulated in various in vitro and in vivo microglia models and identified and replicated the role of two factors in human postmortem tissue of Alzheimers disease (AD). Further, we derived a complex network of transcriptional regulators for all factors, including regulators of an AD-related factor enriched for the mouse disease-associated microglia 2 (DAM2) signature: ARID5B, CEBPA, MITF, and PPARG. We replicated the role of these four regulators in the AD-related factor and then designed a multiplexed MERFISH panel to assess our microglial factors using spatial transcriptomics. We find that, unlike cells with high expression of the interferon-response factor, cells with high expression of the AD DAM2-like factor are widely distributed in neocortical tissue. We thus propose a novel analytic framework that provides a taxonomic approach for microglia that is more biologically interpretable and use it to uncover new therapeutic targets for AD.

neuroscience↗

bioRxiv2024

Single-cell transcriptomic landscape of the neuroimmune compartment in amyotrophic lateral sclerosis brain and spinal cord

Development of therapeutic approaches that target specific microglia responses in amyotrophic lateral sclerosis (ALS) is crucial due to the involvement of microglia in ALS progression. Our study identifies the predominant microglia subset in human ALS primary motor cortex and spinal cord as an undifferentiated phenotype with dysregulated respiratory electron transport. Moreover, we find that the interferon response microglia subset is enriched in donors with aggressive disease progression, while a previously described potentially protective microglia phenotype is depleted in ALS. Additionally, we observe an enrichment of non-microglial immune cell, mainly NK/T cells, in ALS central nervous system, primarily in the spinal cord. These findings pave the way for the development of microglia subset-specific therapeutic interventions to slow or even stop ALS progression.

neuroscience↗

bioRxiv2022

FLASH and Conventional Radiation Induce Differential Immune Responses in Diffuse Intrinsic Pontine Glioma, Highlighting Potential for Combination Immunotherapy

PurposeDiffuse Midline Glioma (DMG) is a fatal tumor traditionally treated with radiotherapy (RT) and previously characterized as having a non-inflammatory tumor immune microenvironment (TIME). FLASH is a novel RT technique using ultra-high dose rate, which is associated with decreased toxicity and effective tumor control. However, the effect of FLASH and conventional (CONV) RT on the DMG TIME have not yet been explored. MethodsHere, we perform single-cell RNA sequencing and flow cytometry on immune cells isolated from an orthotopic syngeneic murine model of brainstem DMG following the use of FLASH (90Gy/sec) or CONV (2Gy/min) dose-rate RT, and compare to unirradiated tumor (SHAM). ResultsAt day 4 post-RT, FLASH exerts similar effects as CONV in the predominant microglial (MG) population, including the presence of two activated subtypes. However, at day 10 post-RT, we observe a significant increase in type 1 interferon alpha receptor (IFNAR+) in MG in CONV and SHAM compared to FLASH. In the non-resident myeloid clusters of macrophages (MACs) and dendritic cells (DCs), we find increased type 1 interferon (IFN1) pathway enrichment for CONV compared to FLASH and SHAM by scRNA-seq. We observe this trend by flow cytometry at day 4 post-RT in IFNAR+ MACs and DCs, which equalizes by day 10 post-RT. DMG control and murine survival are equivalent between RT dose rates. ConclusionOur work is the first to map CONV and FLASH immune alterations of the DMG TIME with single-cell resolution. While DMG tumor control and survival are similar between CONV and FLASH, we find that changes in immune compartments differ over time. Importantly, while both RT modalities increase IFN1, we find that the timing of this response is cell-type and dose-rate dependent. These temporal differences, particularly in the context of tumor control, warrant further study.

immunology↗

bioRxiv2022

A cross-disease human microglial framework identifies disease-enriched subsets and tool compounds for microglial polarization.

Human microglia play a pivotal role in neurological diseases, but few targeted therapies that directly modulate microglial state or function exist due to an incomplete understanding of microglial heterogeneity. We use single-cell RNA sequencing to profile live human microglia from autopsies or surgical resections across diverse neurological diseases and central nervous system regions. We observe a central divide between oxidative and heterocyclic metabolism and identify subsets associated with antigen presentation, motility, and proliferation. Specific subsets are enriched in susceptibility genes for neurodegenerative diseases or the disease-associated microglial signature. We validate subtypes in situ with an RNAscope-immunofluorescence pipeline and leverage our dataset as a classification resource, finding that iPSC model systems recapitulate substantial in vivo heterogeneity. Finally, we identify and validate candidates for chemically inducing subtype-specific states in vitro, showing that Camptothecin downregulates the transcriptional signature of disease-enriched subsets and upregulates a signature previously shown to be depleted in Alzheimers.

neuroscience↗

bioRxiv2020

Epigenomic features related to microglia are associated with attenuated effect of APOE ε4 on Alzheimer's disease risk in humans

INTRODUCTIONNot all APOE {varepsilon}4 carriers who survive to advanced age develop Alzheimers disease (AD); factors attenuating the risk of {varepsilon}4 on AD may exist. METHODSGuided by the top {varepsilon}4-attenuating signals from methylome-wide association analyses (N=572, {varepsilon}4+ and {varepsilon}4-) of neurofibrillary tangles and neuritic plaques, we conducted a meta-analysis for pathological AD within the {varepsilon}4+ subgroups (N=235) across four independent collections of brains. Cortical RNA-seq and microglial morphology measurements were used in functional analyses. RESULTSThree out of the four significant CpG dinucleotides were captured by one principle component (PC1), which interacts with {varepsilon}4 on AD, and is associated with expression of innate immune genes and activated microglia. In {varepsilon}4 carriers, reduction in each unit of PC1 attenuated the odds of AD by 58% (OR=2.39, 95%CI=[1.64,3.46], P=7.08x10-6). DISCUSSIONAn epigenomic factor associated with a reduced proportion of activated microglia appears to attenuate the risk of {varepsilon}4 on AD.

neuroscience↗

bioRxiv2019

BIN1 protein isoforms are differentially expressed in astrocytes, neurons, and microglia: neuronal and astrocyte BIN1 implicated in Tau pathology

Identified as an Alzheimers disease (AD) susceptibility gene by genome wide-association studies, BIN1 has 10 isoforms that are expressed in the Central Nervous System (CNS). The distribution of these isoforms in different cell types, as well as their role in AD pathology still remains unclear. Utilizing antibodies targeting specific BIN1 epitopes in human postmortem tissue and analyzing RNA expression data from purified microglia, we identified three isoforms expressed specifically in neurons (isoforms 1, 2 and 3) and four isoforms expressed in microglia (isoforms 6, 9, 10 and 12). The abundance of selected peptides, which correspond to groups of BIN1 protein isoforms, was measured in dorsolateral prefrontal cortex, and their relation to neuropathological features of AD was assessed. Peptides contained in exon 7 of BIN1s N-BAR domain were found to be significantly associated with AD-related traits and, particularly, tau pathology. Since only isoforms 1, 2 and 3 contain exon 7, it appears that decreased protein expression of the N-BAR domain of BIN1 is associated with greater accumulation of tau pathology and subsequent cognitive decline, with astrocytic rather than neuronal BIN1 being the more likely culprit. These effects are independent of the BIN1 AD risk variant, suggesting that targeting specific BIN1 isoforms might be a novel therapeutic approach to prevent the accumulation of tau pathology.

cell biology↗

bioRxiv2018

A single cell-based atlas of human microglial states reveals associations with neurological disorders and histopathological features of the aging brain

Recent studies of bulk microglia have provided insights into the role of this immune cell type in central nervous system development, homeostasis and dysfunction. Nonetheless, our understanding of the diversity of human microglial cell states remains limited; microglia are highly plastic and have multiple different roles, making the extent of phenotypic heterogeneity a central question, especially in light of the development of therapies targeting this cell type. Here, we investigated the population structure of human microglia by single-cell RNA-sequencing. Using surgical- and autopsy-derived cortical brain samples, we identified 14 human microglial subpopulations and noted substantial intra- and inter-individual heterogeneity. These putative subpopulations display divergent associations with Alzheimers disease, multiple sclerosis, and other diseases. Several states show enrichment for genes found in disease-associated mouse microglial states, suggesting additional diversity among human microglia. Overall, human microglia appear to exist in different functional states with varying levels of involvement in different brain pathologies.

neuroscience↗

bioRxiv2017

A cortical immune network map identifies a subset of human microglia involved in Tau pathology

Microglial dysfunction has been proposed as one of the many cellular mechanisms that can contribute to the development of Alzheimer's disease (AD). Here, using a transcriptional network map of the human frontal cortex, we identify five gene modules of co-expressed genes related to microglia and assess their role in the neuropathologic features of AD in 541 subjects from two cohort studies of brain aging. Two of these transcriptional programs - modules 113 and 114 - relate to the accumulation of {beta}-amyloid, while module 5 relates to tau pathology. These modules are also detectable in the human brain's epigenome, where we replicate these associations. In terms of tau, we propose that module 5, a marker of activated microglia, may lead to tau accumulation and subsequent cognitive decline. We validate our model further by showing that VASP, a representative module 5 gene, encodes a protein that is upregulated in activated microglia in AD.

neuroscience↗

bioRxiv2017

Differential Mast Cell Outcomes Are Sensitive to FcϵRI-Syk Binding Kinetics

Crosslinking of IgE-bound Fc{varepsilon}RI triggers multiple cellular responses, including degranulation and cytokine production. Signaling is dependent on recruitment of Syk via docking of its dual SH2 domains to phosphorylated tyrosines within the Fc{varepsilon}RI immunoreceptor tyrosine-based activation motifs. Using single molecule imaging in live cells, we directly visualized and quantified the binding of individual mNeonGreen-tagged Syk molecules as they associated with the plasma membrane after Fc{varepsilon}RI activation. We found that Syk colocalizes transiently to Fc{varepsilon}RI and that Syk-Fc{varepsilon}RI binding dynamics are independent of receptor aggregate size. Substitution of glutamic acid for tyrosine between the Syk SH2 domains (SykY130E) led to an increased Syk-Fc{varepsilon}RI off-rate, loss of site-specific Syk autophosphorylation, and impaired downstream signaling. CRISPR-Cas9 engineered cells expressing only SykY130E were deficient in antigen-stimulated calcium release, degranulation and production of some cytokines (TNF-a, IL-3) but not others (MCP-1, IL-4). We propose that kinetic discrimination along the Fc{varepsilon}RI signaling pathway occurs at the level of Syk-Fc{varepsilon}RI interactions, with key outcomes dependent upon sufficiently long-lived Syk binding events.\n\nSummarySchwartz et al. use single molecule imaging to quantify the transient nature of Fc{varepsilon}RI-Syk interactions in live mast cells. A functional mutation that increases Syk off-rate leads to loss of site-specific Syk phosphorylation and impaired signaling, highlighting the importance of finely tuned protein interactions in directing cellular outcomes.

cell biology↗