Biology subjects

Fink, K.

Publications and source records attributed to Fink, K..

9 recordsLinked to original sources

bioRxiv2026

Advancing Neurological Liquid Biopsy via Six-Base Sequencing of Cerebrospinal Fluid Cell-Free DNA

Real-time molecular insights into the human brain are crucial for understanding neurological conditions and development of soluble biomarkers but challenged by the limited access to target tissue. Here we utilize 6-base sequencing to profile DNA modifications of cell-free DNA released from neural cells into cerebrospinal fluid. This strategy provides molecular, quantitative and qualitative insights into the ongoing neurodegenerative biology, which paves the way for liquid biopsy approaches to study neurological conditions.

neuroscience↗

bioRxiv2026

Cortical Organoid Model of PPP2R5D Genetic Intellectual Disability Models Disease Severity Phenotype

Jordans Syndrome (JS) is a rare, neurodevelopmental disorder caused by de novo missense mutations in protein phosphatase 2 regulatory subunit Bdelta (PPP2R5D). JS is characterized by severe neurological impairments starting in early life. PPP2R5D encodes for B56{delta}, one of the regulatory subunits of protein phosphatase 2A (PP2A). PP2A is a heterotrimeric protein serine/threonine phosphatase that is highly expressed in the brain and the liver. Past studies have focused on PP2As role in liver and little is known about the holoenzymes behavior in neuronal cells. Although B56{delta} is known to play an important role in the substrate specificity of PP2A, the identification of validated downstream substrates in JS remains unclear. To better understand how the mutations affect neuronal cells, we developed cerebral cortical-like organoids from an engineered allele series of the most common JS mutations to characterize the physiological changes throughout different stages of neurodevelopment. Organoids were assessed for transcriptomic, protein, and electrophysiological changes utilizing bulk RNA sequencing, immunocytochemistry, Western Blot, and high-density MicroElectrode Array. The results identify differentially expressed genes and translated proteins, potential neuronal substrates, and significant electrophysiological signatures that suggest mutations in B56{delta} lead to variant-specific dysfunction of PP2A. Overexpression of PPP2R5D through AAV transduction of organoids rescued several phenotypes in the variants, suggesting different pathogenetic etiology underneath. Our findings successfully characterized cerebral cortical-like organoids in JS cell lines and demonstrated its potential as a model for studying neurodevelopmental disorder and for screening therapeutic approaches.

cell biology↗

bioRxiv2026

Reliable quantification of multiplexed genetically encoded biosensors responsiveness in plant tissues

Genetically encoded biosensors are one of the essential tools in biological research. They enable visualization of molecules of interest from the subcellular level to entire organism level in vivo and can be used to monitor presence of small molecules, gene expression, protein activity, and protein degradation. However, multiplexing fluorescent biosensors in plants is notoriously difficult due to signal bleed-through and strong autofluorescence from chlorophyll. In this study, we investigated the potential of multiplexing biosensors based on the selection of reporter fluorescent proteins. We characterized the emission spectra, fluorescence lifetimes, and relative brightness of diverse fluorescent proteins in plant leaves. We show that selected proteins exhibit comparable brightness, supporting their use in co-expression experiments and reliable quantification of individual signals. To separate three overlapping signals, we applied two different linear unmixing approaches and compared them to results obtained without unmixing. We identified channel separation unmixing approach as the most suitable for biosensors. Additionally, we show how unmixing with the selected approach can be applied to separate autofluorescence and five fluorescent proteins. We further validated this approach in virus-infected cells by following organelle dynamics in vivo. Finally, we demonstrate the feasibility of high-throughput segmentation and quantification with a custom MATLAB workflow for nuclei, chloroplasts, and cytoplasm signal analysis. Overall, our work demonstrates that biosensors can be multiplexed, even when their emission spectra overlap. Significance statementMultiplexing genetically encoded biosensors in plants has been limited by overlapping fluorescent signals and strong autofluorescence. This study presents an optimized framework for linear unmixing and provides a MATLAB-based organelle segmentation tool, allowing precise quantification of multiple fluorescent reporters in vivo and advancing real-time visualization of complex cellular processes in plants.

plant biology↗

bioRxiv2026

ZC4H2 loss of function is associated with temporal dysregulation of neural stem proliferation and neuron development

ZC4H2 is an X-linked zinc finger transcription factor essential for early neurodevelopment. Pathogenic variants in ZC4H2 are associated with both central and peripheral nervous system pathologies. The molecular and cellular mechanisms driving these phenotypes remain poorly understood, particularly in human female models that have undergone X chromosome inactivation. Neuronal models were differentiated from a human female cell line with a de novo Xq11.2 deletion causing ZC4H2 loss of function, associated with arthrogryposis multiplex congenita and cognitive impairment. Using iPSC-derived neural stem cells and cortical organoids, we identified premature neuronal differentiation, reduced BMP-SMAD signaling, and decreased SMAD1/5 phosphorylation. In cortical organoids, ZC4H2 deficiency altered neurogenesis timing, retaining proliferative progenitors while prematurely activating neuronal programs, leading to enlarged organoids with persistent dysregulation of gene programs required for complete neuronal maturation. We identified ZC4H2 target genes likely to mediate these phenotypes and tested a codon-optimized transgene showing both the restoration of SMAD1/5 phosphorylation, BMPR2 gene expression, and improved neuronal complexity. These results demonstrate effective ZC4H2 restoration in complex human models and highlight therapeutic potential for ZC4H2-linked neurodevelopmental disorders.

neuroscience↗

bioRxiv2026

RNA Polymerase III subunit Polr3a is required for craniofacial cartilage and bone development

Transcription by RNA Polymerase III (Pol III) is essential for ribosome biogenesis and translation in all cells, but pathogenic variants in genes encoding subunits of Pol III lead to tissue-specific phenotypes including craniofacial differences. To understand the function of Pol III in craniofacial development, we examined polr3a mutant zebrafish. These mutants display hypoplasia of the neural crest cell-derived craniofacial cartilage and bone but, surprisingly, no significant changes were observed in neural crest cell proliferation or survival during embryogenesis. At larval stages, increased cell death was observed throughout the head, including in the craniofacial cartilage. These changes coincide with reduced transcription of transfer RNAs and reduced ribosome biogenesis in polr3a mutant zebrafish. To determine tissue-specific transcriptional changes, we performed single-cell RNA-sequencing. Analysis revealed both global and cartilage-specific changes, including upregulation of tp53. However, Tp53 inhibition alone was not sufficient to rescue craniofacial cartilage and bone, indicating that additional factors are important to support cartilage and bone growth in polr3a mutants. Altogether, our study provides new mechanistic insights into the functions of Pol III in craniofacial development. Author SummaryCraniofacial anomalies account for around 33% of all congenital birth defects and many of these are associated with defects in neural crest cell development. Disruptions in RNA Polymerase III, which plays important roles in ribosome biogenesis and translation, can result in tissue-specific phenotypes including craniofacial anomalies. However, the mechanisms underlying these craniofacial anomalies are not well understood. Here, we establish a zebrafish model to understand how a mutation in Pol III subunit polr3a affects craniofacial development. We observe hypoplasia of the craniofacial cartilage and bone in polr3a mutant zebrafish along with diminished transcription of transfer RNAs and reduced ribosome biogenesis. This leads to reduced proliferation and increased cell death throughout the head, but we do not detect any differences in early neural crest cell development. Using single-cell RNA-sequencing, we examine the transcriptional changes both broadly throughout the head and specifically within the craniofacial cartilage and identify upregulation of the Tp53 pathway. Inhibition of tp53 only partially rescues cartilage and bone development, suggesting that Tp53-independent mechanisms contribute to cranioskeletal development in polr3a mutant zebrafish. Altogether, these studies highlight the critical function of Pol III during development and specifically in the differentiation and growth of craniofacial cartilage and bone.

developmental biology↗

bioRxiv2025

Molecular profiling of glioblastoma-derived extracellular vesicles identifies small nucleolar RNAs as candidate liquid biomarkers for radiation-induced senescence

Radiation-induced senescence (RIS) in glioblastoma (GBM) is an undesirable cell fate that reduces tumor cell death and supports resistance and outgrowth. While senescence-targeting drugs are promising adjuvants, their clinical application will require proper patient selection based on post-treatment RIS burden. Current methods to evaluate senescence, however, are tissue-based, and given GBMs difficult anatomical location, post-treatment biopsies are impractical. Innovative and less invasive biomarkers for RIS are urgently needed. To this end, we aimed to identify candidate extracellular vesicle (EV) liquid biomarkers for RIS by profiling senescence-associated cargo changes within GBM EVs. Using a panel of GBM patient-derived cell cultures, we show that RIS is the primary functional state following radiation exposure and is associated with significant alterations in the cargo of senescent-derived EVs (senEVs). In particular, senEV transcriptomes have an increased abundance of senescence-associated RNA genes and gene sets. Most striking, however, was that senEVs are most differentiated by the significant enrichment of a panel of snoRNAs. This signature was conserved in 4/5 GBM models of RIS and was validated by qRT-PCR. We further confirmed snoRNA enrichment in the senEVs of a breast cancer cell line, as well as the lack of snoRNA enrichment following senescence-independent drug exposure. Analysis by mass spectrometry revealed that snoRNAs are likely co-packaged with their associating proteins, as senEVs had concurrent increases in these binding partners. We examined whether packaging is associated with nucleolar stress during RIS, but found that upregulation in senEVs is likely due to a tightly controlled cellular abundance rather than nucleoli fragmentation and release of nucleolar components to the cytoplasm. Finally, using a preliminary cohort of longitudinal plasma samples from four GBM patients, we determined the feasibility of detecting senescence-associated and snoRNA species in the extracellular vesicles of patient biofluids. Of interest, post-treatment EVs had increased senescence-associated RNAs like CDKN2B and GLB1 and the snoRNA SNORA49. Altogether, this data suggests that senEV RNA species, and particularly snoRNAs, are a promising analyte for RIS-biomarker development. With further study, this work may open avenues for a companion diagnostic for senotherapeutics.

cancer biology↗

bioRxiv2025

Anoctamin-2-specific T Cells Link Epstein-Barr Virus to Multiple Sclerosis

Multiple sclerosis (MS) occurs when the central nervous system (CNS) is damaged by misguided adaptive immune responses, likely caused by a combination of environmental factors in genetically susceptible individuals. A known prerequisite for disease is Epstein-Barr virus (EBV) infection, and previous studies have demonstrated elevated Epstein-Barr virus nuclear antigen 1 (EBNA1) antibodies which cross-react with the calcium-activated chloride channel anoctamin-2 (ANO2) in persons with MS (pwMS). ANO2-reactive antibodies have been associated with greater neuroaxonal damage in MS, although their exact effector function is still uncertain. Here, we demonstrate that ANO2 is also the target of IFN{gamma}-producing CD4+ T cells, which are more frequent in untreated and natalizumab-treated pwMS compared to control individuals. Immunisation of SJL/J mice with either ANO2 or EBNA1 elicited cross-reactive CD4+ T cell and antibody responses in vivo. Pre-immunisation of young mice with ANO2 worsened proteolipid protein (PLP)-induced experimental autoimmune encephalomyelitis (EAE), which in older mice included atypical clinical phenotype, immune infiltration into the brain and reduced survival. EAE exacerbation was recapitulated with the adoptive co-transfer of ANO2 and myelin antigen-specific CD4+ T cells, and ANO2-specific T cells alone could induce the cell death of ANO2-expressing glial cells in vitro. T cell clones with cross-reactivity to both EBNA1 and ANO2 antigens could be isolated from natalizumab-treated pwMS. Single cell sequencing of EBNA1 and ANO2-specific T cell receptors (TCR) from four pwMS revealed a significant overlap between their antigen-specific expanded TCR repertoires within donors and transcriptomic analysis showed cross-reactive T cells to have predominantly activated and cytotoxic phenotypes. In summary, we report the first mechanistic evidence that EBNA1 CD4+ T cells can target the MS-associated autoantigen ANO2, thereby establishing a link between EBV infection and development of autoimmune neuroinflammatory disease.

immunology↗

bioRxiv2025

Large-scale discovery of neural enhancers for cis-regulation therapies

CRISPR-based gene activation (CRISPRa) has emerged as a promising therapeutic approach for neurodevelopmental disorders (NDD) caused by haploinsufficiency. However, scaling this cis-regulatory therapy (CRT) paradigm requires pinpointing which candidate cis-regulatory elements (cCREs) are active in human neurons, and which can be targeted with CRISPRa to yield specific and therapeutic levels of target gene upregulation. Here, we combine Massively Parallel Reporter Assays (MPRAs) and a multiplex single cell CRISPRa screen to discover functional human neural enhancers whose CRISPRa targeting yields specific upregulation of NDD risk genes. First, we tested 5,425 candidate neuronal enhancers with MPRA, identifying 2,422 that are active in human neurons. Selected cCREs also displayed specific, autonomous in vivo activity in the developing mouse central nervous system. Next, we applied multiplex single-cell CRISPRa screening with 15,643 gRNAs to test all MPRA-prioritized cCREs and 761 promoters of NDD genes in their endogenous genomic contexts. We identified hundreds of promoter- and enhancer-targeting CRISPRa gRNAs that upregulated 200 of the 337 NDD genes in human neurons, including 91 novel enhancer-gene pairs. Finally, we confirmed that several of the CRISPRa gRNAs identified here demonstrated selective and therapeutically relevant upregulation of SCN2A, CHD8, CTNND2 and TCF4 when delivered virally to patient cell lines, human cerebral organoids, and a humanized mouse model of hTcf4. Our results provide a comprehensive resource of active, target-linked human neural enhancers for NDD genes and corresponding gRNA reagents for CRT development. More broadly, this work advances understanding of neural gene regulation and establishes a generalizable strategy for discovering CRT gRNA candidates across cell types and haploinsufficient disorders.

genomics↗

bioRxiv2020

Structural and functional analysis of a potent sarbecovirus neutralizing antibody

SARS-CoV-2 is a newly emerged coronavirus responsible for the current COVID-19 pandemic that has resulted in more than one million infections and 73,000 deaths1,2. Vaccine and therapeutic discovery efforts are paramount to curb the pandemic spread of this zoonotic virus. The SARS-CoV-2 spike (S) glycoprotein promotes entry into host cells and is the main target of neutralizing antibodies. Here we describe multiple monoclonal antibodies targeting SARS-CoV-2 S identified from memory B cells of a SARS survivor infected in 2003. One antibody, named S309, potently neutralizes SARS-CoV-2 and SARS-CoV pseudoviruses as well as authentic SARS-CoV-2 by engaging the S receptor-binding domain. Using cryo-electron microscopy and binding assays, we show that S309 recognizes a glycan-containing epitope that is conserved within the sarbecovirus subgenus, without competing with receptor attachment. Antibody cocktails including S309 along with other antibodies identified here further enhanced SARS-CoV-2 neutralization and may limit the emergence of neutralization-escape mutants. These results pave the way for using S309 and S309-containing antibody cocktails for prophylaxis in individuals at high risk of exposure or as a post-exposure therapy to limit or treat severe disease.

immunology↗