Biology subjects

Tilgner, H. U.

Publications and source records attributed to Tilgner, H. U..

15 recordsLinked to original sources

bioRxiv2026

Humanized tauopathy chimeras uncover microglial and lncRNA strategies for neuroprotection

Human genetics implicates innate immunity as a key modifier of tau toxicity, yet human-specific neuroimmune mechanisms remain difficult to test in vivo. Here, we developed HuMiNAX, the first humanized iPSC-based neuroimmune xenograft model of tau-associated neurodegeneration, enabling human microglia to interact with human neurons and astrocytes in the adult mouse brain. In HuMiNAX, tau seeding induced aggregation only in mutation-carrying human neural grafts, causing neuron loss and inflammatory activation of human microglia. Progranulin-overexpressing human microglia dampened tau-associated inflammation, preserved neurons, and restored neuronal gene-expression and RNA-splicing programs, supporting microglial control of neuronal resilience. CRISPRi knockdown of the human-specific lncRNA HNRNPK-AS1 also protected neurons in HuMiNAX. These findings establish HuMiNAX as a human neuroimmune model of tauopathy and identify microglial and RNA-mediated strategies of neuronal resilience.

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

Spatial isoform sequencing at sub-micrometer single-cell resolution reveals novel patterns of spatial isoform variability in brain cell types

Spatial long-read technologies are becoming increasingly common but lack nanometer and therefore often single-cell resolution. This leaves the question unanswered of whether spatially variable isoforms represent spatial variability within one cell type or differences in cell-type composition between different regions. Here, we developed Spl-ISO-Seq2 (220nm spot size and 500nm resolution), and the accompanying software packages Spl-IsoQuant-2 and Spl-IsoFind, enabling long-read sequencing using up to 500 million barcodes compared to 80,000 previously. Applying this to the adult mouse brain, we compared spatial variability by examining (a) differential isoform abundance between known brain regions and (b) spatial isoform patterns that do not align with predefined regions. While the former revealed more spatial isoform differences, both approaches identified overlapping hits, e.g., Rps24 in oligodendrocytes. For Snap25, previously known to exhibit spatial isoform variation, we now show that this variability occurs in excitatory neurons. The second approach also uncovered patterns not captured by predefined-region comparisons, e.g., Tnnc1 in excitatory neurons. Furthermore, we show that a surprising number of spatial isoform signals is not driven by cell-type composition alone. Finally, we applied our software to newly generated Visium HD 3 long-read data to demonstrate its applicability and strong reproducibility across protocols and biological replicates. Taken together, our experimental and analytical methods enrich spatial transcriptomics with a so-far elusive isoform view of spatial variation for individual cell types.

neuroscience↗

bioRxiv2025

A spatial long-read approach at near-single-cell resolution reveals developmental regulation of splicing and polyadenylation sites in distinct cortical layers and cell types.

Genome-wide single-cell and spatial long-read approaches have gained traction, but mostly lack single-cell resolution - and yield limited read lengths. Here, we introduce spatial ISOform sequencing (Spl-ISO-Seq), which reveals exons and polyadenylation sites from long reads with near-single-cell resolution. Spl-ISO-Seq selects long cDNAs and doubles to triples read lengths compared to standard preparations. Adding a highly specific software tool (Spl-ISOquant) and comparing human post-mortem pre-puberty samples of the visual cortex (8-11 years) to post-puberty samples (16-19 years), we find that cortical layers harbor stronger splicing and poly(A)-site regulation than the adjacent white matter, with enrichment of multiple protein-domain types. For oligodendrocytes however, developmental splicing changes are stronger in white matter. Among cortical layers, layer 4 has the most developmental changes in alternative-exon inclusion in excitatory neurons and in poly(A) sites. We also find many repeat elements, especially ERV1 long terminal repeats downstream of developmentally-regulated layer 4 exons. Overall, alternative splicing changes are linked to synapses - specifically at the post-synapse. Age-linked splicing changes in layers 1-3 and 4 are associated with autism spectrum disorder but not with schizophrenia, amyotrophic lateral sclerosis and Alzheimers disease. These results root developmental splicing changes during puberty and the resulting protein changes in specific layers and cell types. More generally, our new technologies enable new observations for any complex tissue.

genomics↗

bioRxiv2025

Neuronal cell adhesion molecule (NRCAM) variant defined by microexon skipping is an essential, antigenically distinct, and targetable surface proteoform in high-grade glioma

To overcome the paucity of known tumor-specific surface antigens in pediatric high-grade glioma (pHGG), we contrasted splicing patterns in pHGGs and normal brain samples. Among alternative splicing events affecting extracellular protein domains, the most pervasive alteration was the skipping of [≤]30 nucleotide-long exons. Several of these skipped microexons mapped to L1-IgCAM family members, such as NRCAM. Bulk and single-nuclei short- and long-read RNA-seq revealed uniform skipping of NRCAM microexons 5 and 19 in virtually every pHGG sample. Importantly, the {Delta}ex5{Delta}ex19 (but not the full-length) NRCAM proteoform was essential for pHGG cell migration and invasion in vitro and tumor growth in vivo. We developed a monoclonal antibody selective for {Delta}ex5{Delta}ex19 NRCAM and demonstrated that "painting" of pHGG cells with this antibody enables killing by T cells armed with an FcRI-based universal immune receptor. Thus, pHGG-specific NRCAM and possibly other L1-IgCAM proteoforms are promising and highly selective targets for adoptive immunotherapies.

cancer biology↗

bioRxiv2024

ScISOr-ATAC reveals convergent and divergent splicing and chromatin specificities between matched cell types across cortical regions, evolution, and in Alzheimer's disease.

Multimodal measurements have become widespread in genomics, however measuring open chromatin accessibility and splicing simultaneously in frozen brain tissues remains unconquered. Hence, we devised Single-Cell-ISOform-RNA sequencing coupled with the Assay-for-Transposase-Accessible-Chromatin (ScISOr-ATAC). We utilized ScISOr-ATAC to assess whether chromatin and splicing alterations in the brain convergently affect the same cell types or divergently different ones. We applied ScISOr-ATAC to three major conditions: comparing (i) the Rhesus macaque (Macaca mulatta) prefrontal cortex (PFC) and visual cortex (VIS), (ii) cross species divergence of Rhesus macaque versus human PFC, as well as (iii) dysregulation in Alzheimers disease in human PFC. We found that among cortical-layer biased excitatory neuron subtypes, splicing is highly brain-region specific for L3-5/L6 IT_RORB neurons, moderately specific in L2-3 IT_CUX2.RORB neurons and unspecific in L2-3 IT_CUX2 neurons. In contrast, at the chromatin level, L2-3 IT_CUX2.RORB neurons show the highest brain-region specificity compared to other subtypes. Likewise, when comparing human and macaque PFC, strong evolutionary divergence on one molecular modality does not necessarily imply strong such divergence on another molecular level in the same cell type. Finally, in Alzheimers disease, oligodendrocytes show convergently high dysregulation in both chromatin and splicing. However, chromatin and splicing dysregulation most strongly affect distinct oligodendrocyte subtypes. Overall, these results indicate that chromatin and splicing can show convergent or divergent results depending on the performed comparison, justifying the need for their concurrent measurement to investigate complex systems. Taken together, ScISOr-ATAC allows for the characterization of single-cell splicing and chromatin patterns and the comparison of sample groups in frozen brain samples.

genomics↗

bioRxiv2023

Pronounced early differentiation underlies zebra finch gonadal germ cell development.

The diversity of germ cell developmental strategies has been well documented across many vertebrate clades. However, much of our understanding of avian primordial germ cell (PGC) specification and differentiation has derived from only one species, the chicken (Gallus gallus). Of the three major classes of birds, chickens belong to Galloanserae, representing less than 4% of species, while nearly 95% of extant bird species belong to Neoaves. This represents a significant gap in our knowledge of germ cell development across avian species, hampering efforts to adapt genome editing and reproductive technologies developed in chicken to other birds. We therefore applied single-cell RNA sequencing to investigate inter-species differences in germ cell development between chicken and zebra finch (Taeniopygia castanotis), a Neoaves songbird species and a common model of vocal learning. Analysis of early embryonic male and female gonads revealed the presence of two distinct early germ cell types in zebra finch and only one in chicken. Both germ cell types expressed zebra finch Germline Restricted Chromosome (GRC) genes, present only in songbirds among birds. One of the zebra finch germ cell types expressed the canonical PGC markers, as did chicken, but with expression differences in several signaling pathways and biological processes. The second zebra finch germ cell cluster was marked by proliferation and fate determination markers, indicating beginning of differentiation. Notably, these two zebra finch germ cell populations were present in both male and female zebra finch gonads as early as HH25. Using additional chicken developmental stages, similar germ cell heterogeneity was identified in the more developed gonads of females, but not males. Overall, our study demonstrates a substantial heterochrony in zebra finch germ cell development compared to chicken, indicating a richer diversity of avian germ cell developmental strategies than previously known.

cell biology↗

bioRxiv2023

Single-cell long-read mRNA isoform regulation is pervasive across mammalian brain regions, cell types, and development

RNA isoforms influence cell identity and function. Until recently, technological limitations prevented a genome-wide appraisal of isoform influence on cell identity in various parts of the brain. Using enhanced long-read single-cell isoform sequencing, we comprehensively analyze RNA isoforms in multiple mouse brain regions, cell subtypes, and developmental timepoints from postnatal day 14 (P14) to adult (P56). For 75% of genes, full-length isoform expression varies along one or more axes of phenotypic origin, underscoring the pervasiveness of isoform regulation across multiple scales. As expected, splicing varies strongly between cell types. However, certain gene classes including neurotransmitter release and reuptake as well as synapse turnover, harbor significant variability in the same cell type across anatomical regions, suggesting differences in network activity may influence cell-type identity. Glial brain-region specificity in isoform expression includes strong poly(A)-site regulation, whereas neurons have stronger TSS regulation. Furthermore, developmental patterns of cell-type specific splicing are especially pronounced in the murine adolescent transition from P21 to P28. The same cell type traced across development shows more isoform variability than across adult anatomical regions, indicating a coordinated modulation of functional programs dictating neural development. As most cell-type specific exons in P56 mouse hippocampus behave similarly in newly generated data from human hippocampi, these principles may be extrapolated to human brain. However, human brains have evolved additional cell-type specificity in splicing, suggesting gain-of-function isoforms. Taken together, we present a detailed single-cell atlas of full-length brain isoform regulation across development and anatomical regions, providing a previously unappreciated degree of isoform variability across multiple scales of the brain.

genetics↗

bioRxiv2022

Comparing 10x Genomics single-cell 3' and 5' assay in short-and long-read sequencing

Barcoding strategies are fundamental to droplet-based single-cell sequencing, and understanding the biases and caveats between approaches is essential. Here, we comprehensively evaluated both short and long reads of the cDNA obtained through the two marketed approaches from 10x Genomics, the "3 assay" and the "5 assay", which attach barcodes at different ends of the mRNA molecule. Although the barcode detection, cell-type identification, and gene expression profile are similar in both assays, the 5 assay captured more exonic molecules and fewer intronic molecules compared to the 3 assay. We found that 13.7% of genes sequenced have longer average read lengths and are more complete (spanning both polyA-site and TSS) in the long reads from the 5 assay compared to the 3 assay. These genes are characterized by long average transcript length, high intron number, and low expression overall. Despite these differences, cell-type-specific isoform profiles observed from the two assays remain highly correlated. This study provides a benchmark for choosing the single-cell assay for the intended research question, and insights regarding platform-specific biases to be mindful of when analyzing data, particularly across samples and technologies.

bioinformatics↗

bioRxiv2022

Sequencing of individual barcoded cDNAs on Pacific Biosciences and Oxford Nanopore reveals platform-specific error patterns

Long-read transcriptomics requires understanding error sources inherent to technologies. Current approaches cannot compare methods for an individual RNA molecule. Here, we present a novel platform comparison method that combined barcoding strategies and long-read sequencing to sequence cDNA copies representing an individual RNA molecule on both Pacific Biosciences and Oxford Nanopore. We compared these long reads pairs in terms of sequence content and splicing structure. Although individual read pairs show high similarity, we found differences in (i) aligned length, (ii) TSS and (iii) polyA-site assignment, and (iv) exon-intron structures. Overall 25% of read pairs disagreed on either TSS, polyA-site, or a splice site. Intron-chain disagreement typically arises from alignment errors of microexons and complicated splice sites. Our single-molecule technology comparison revealed that inconsistencies are often caused by sequencing-error induced inaccurate ONT alignments, especially to downstream GTNNGT donor motifs. However, annotation-disagreeing upstream shifts in NAGNAG acceptors in ONT are often confirmed by PacBio and thus likely real. In both barcoded and non-barcoded ONT reads, we found that intron number and proximity of other GT/AGs better predict inconsistency with the annotation than read quality alone. We summarized these findings in an annotation-based algorithm for spliced alignment correction that improves subsequent transcript construction with ONT reads.

bioinformatics↗

bioRxiv2021

Single-nuclei isoform RNA sequencing reveals combination patterns of transcript elements across human brain cell types

Single-nuclei RNA-Seq is being widely employed to investigate cell types, especially of human brain and other frozen samples. In contrast to single-cell approaches, however, the majority of single-nuclei RNA counts originate from partially processed RNA leading to intronic cDNAs, thus hindering the investigation of complete isoforms. Here, using microfluidics, PCR-based artifact removal, target enrichment, and long-read sequencing, we developed single-nuclei isoform RNA-sequencing ( SnISOr-Seq), and applied it to the analysis of human adult frontal cortex samples. We found that exons associated with autism exhibit coordinated and more cell-type specific inclusion than exons associated with schizophrenia or ALS. We discovered two distinct modes of combination patterns: first, those distinguishing cell types in the human brain. These are enriched in combinations of TSS-exon, exon-polyA site, and distant (non-adjacent) exon pairs. Second, those with all isoform combinations found within one neural cell type, which are enriched in adjacent exon pairs. Furthermore, adjacent exon pairs are predominantly mutually associated, while distant pairs are frequently mutually exclusive. Finally, we observed that human-specific exons are as tightly coordinated as conserved exons, pointing to an efficient evolutionary mechanism underpinning coordination. SnISOr-Seq opens the door to single-nuclei long-read isoform analysis in the human brain, and in any frozen, archived or hard-to-dissociate sample.

genomics↗

bioRxiv2021

Induction of an immortalized songbird cell line allows for gene characterization and knockout by CRISPR-Cas9

The zebra finch is a powerful model for several biological fields, particularly neuroscience and vocal communication. However, this species lacks a robust cell line for molecular biology research and reagent optimization. Here we describe a cell line, CFS414, generated from zebra finch embryonic fibroblasts using the SV40 large and small T antigens. This cell line demonstrates an improvement over previous songbird cell lines through continuous and density-independent growth, allowing for indefinite culture and monoclonal line derivation. Cytogenetic, genomic, and transcriptomic profiling established the provenance of this cell line and identified the expression of genes relevant to ongoing songbird research. Using this cell line, we demonstrated a stress-dependent localization response of the zebra finch song nuclei specialized gene, SAP30L, and disrupted endogenous gene sequences using S.aureus Cas9. The utility of this cell line enhances the molecular potential of the zebra finch and validates cell immortalization strategies in a songbird species.

cell biology↗

bioRxiv2020

Cell-type, single-cell, and spatial signatures of brain-region specific splicing in postnatal development

Alternative RNA splicing varies across brain regions, but the single-cell resolution of such regional variation is unknown. Here we present the first single-cell investigation of differential isoform expression (DIE) between brain regions, by performing single cell long-read transcriptome sequencing in the mouse hippocampus and prefrontal cortex in 45 cell types at postnatal day 7 (www.isoformAtlas.com). Using isoform tests for brain-region specific DIE, which outperform exon-based tests, we detect hundreds of brain-region specific DIE events traceable to specific cell-types. Many DIE events correspond to functionally distinct protein isoforms, some with just a 6-nucleotide exon variant. In most instances, one cell type is responsible for brain-region specific DIE. Cell types indigenous to only one anatomic structure display distinctive DIE, where for example, the choroid plexus epithelium manifest unique transcription start sites. However, for some genes, multiple cell-types are responsible for DIE in bulk data, indicating that regional identity can, although less frequently, override cell-type specificity. We validated our findings with spatial transcriptomics and long-read sequencing, yielding the first spatially resolved splicing map in the postnatal mouse brain (www.isoformAtlas.com). Our methods are highly generalizable. They provide a robust means of quantifying isoform expression with cell-type and spatial resolution, and reveal how the brain integrates molecular and cellular complexity to serve function.

neuroscience↗

bioRxiv2019

Targeted, High-Resolution RNA Sequencing Of Non-Coding Genomic Regions Associated With Neuropsychiatric Functions

The human brain is one of the last frontiers of biomedical research. Genome-wide association studies (GWAS) have succeeded in identifying thousands of haplotype blocks associated with a range of neuropsychiatric traits, including disorders such as schizophrenia, Alzheimers and Parkinsons disease. However, the majority of single nucleotide polymorphisms (SNPs) that mark these haplotype blocks fall within non-coding regions of the genome, hindering their functional validation. While some of these GWAS loci may contain cis-acting regulatory DNA elements such as enhancers, we hypothesized that many are also transcribed into non-coding RNAs that are missing from publicly available transcriptome annotations. Here, we use targeted RNA capture ( RNA CaptureSeq) in combination with nanopore long-read cDNA sequencing to transcriptionally profile 1,023 haplotype blocks across the genome containing non-coding GWAS SNPs associated with neuropsychiatric traits, using post-mortem human brain tissue from three neurologically healthy donors. We find that the majority (62%) of targeted haplotype blocks, including 13% of intergenic blocks, are transcribed into novel, multi-exonic RNAs, most of which are not yet recorded in GENCODE annotations. We validated our findings with short-read RNA-seq, providing orthogonal confirmation of novel splice junctions and enabling a quantitative assessment of the long-read assemblies. Many novel transcripts are supported by independent evidence of transcription including cap analysis of gene expression (CAGE) data and epigenetic marks, and some show signs of potential functional roles. We present these transcriptomes as a preliminary atlas of non-coding transcription in human brain that can be used to connect neurological phenotypes with gene expression.

genomics↗

bioRxiv2018

Single-cell isoform RNA sequencing (ScISOr-Seq) across thousands of cells reveals isoforms of cerebellar cell types.

Full-length isoform sequencing has advanced our knowledge of isoform biology1-11. However, apart from applying full-length isoform sequencing to very few single cells12,13, isoform sequencing has been limited to bulk tissue, cell lines, or sorted cells. Single splicing events have been described for <=200 single cells with great statistical success14,15, but these methods do not describe full-length mRNAs. Single cell short-read 3 sequencing has allowed identification of many cell sub-types16-23, but full-length isoforms for these cell types have not been profiled. Using our new method of single-cell-isoform-RNA-sequencing (ScISOr-Seq) we determine isoform-expression in thousands of individual cells from a heterogeneous bulk tissue (cerebellum), without specific antibody-fluorescence activated cell sorting. We elucidate isoform usage in high-level cell types such as neurons, astrocytes and microglia and finer sub-types, such as Purkinje cells and Granule cells, including the combination patterns of distant splice sites6-9,24,25, which for individual molecules requires long reads. We produce an enhanced genome annotation revealing cell-type specific expression of known and 16,872 novel (with respect to mouse Gencode version 10) isoforms (see isoformatlas.com).\n\nScISOr-Seq describes isoforms from >1,000 single cells from bulk tissue without cell sorting by leveraging two technologies in three steps: In step one, we employ microfluidics to produce amplified full-length cDNAs barcoded for their cell of origin. This cDNA is split into two pools: one pool for 3 sequencing to measure gene expression (step 2) and another pool for long-read sequencing and isoform expression (step 3). In step two, short-read 3-sequencing provides molecular counts for each gene and cell, which allows clustering cells and assigning a cell type using cell-type specific markers. In step three, an aliquot of the same cDNAs (each barcoded for the individual cell of origin) is sequenced using Pacific Biosciences (\"PacBio\")1,2,4,5,26 or Oxford Nanopore3. Since these long reads carry the single-cell barcodes identified in step two, one can determine the individual cell from which each long read originates. Since most single cells are assigned to a named cluster, we can also assign the cells cluster name (e.g. \"Purkinje cell\" or \"astrocyte\") to the long read in question (Fig 1A) - without losing the cell of origin of each long read.\n\nO_FIG O_LINKSMALLFIG WIDTH=180 HEIGHT=200 SRC=\"FIGDIR/small/364950_fig1.gif\" ALT=\"Figure 1\">\nView larger version (66K):\norg.highwire.dtl.DTLVardef@4df0cdorg.highwire.dtl.DTLVardef@fc4beborg.highwire.dtl.DTLVardef@1dc485forg.highwire.dtl.DTLVardef@1138a3e_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1:C_FLOATNO (A) Outline of our ScISOr-Seq approach. (B) TSNE-plot depicting cell clusters, marker genes and names given to clusters, including: Bergman glia (BG), External granule cell layer neurons (EGL), Internal granule cell layer and other neurons in the interior of the cerebellum (IGL), two clusters of Purkinje cell layer neurons (PCL), oligodendrocyte progenitor cells (OPCs), Atoh1+ neuronal progenitors, Ptf1a+ neuronal progenitors and other neuronal progenitors (NPCs) (C) In-situ hybridization images from the Allen Brain Atlas depicting expression of marker genes in specific layers. (D) Expression patterns of selected marker genes across cell types.\n\nC_FIG

molecular biology↗