Biology subjects

Kirkeby, A.

Publications and source records attributed to Kirkeby, A..

11 recordsLinked to original sources

bioRxiv2025

Activation of transposable elements is linked to a region- and cell-type-specific interferon response in Parkinson's disease

Parkinsons disease (PD) is a neurodegenerative disorder involving a neuroinflammatory response, the cause of which remains unclear. Transposable elements (TE) have been linked to inflammation, but their potential role in PD remains unexplored. Using bulk- and single-nuclei RNAseq of postmortem brain tissue from four brain regions, we studied TE transcription and its correlation with PD neuroinflammation. Over a thousand TEs, including LINE-1s and ERVs, were expressed in a cell-type and region-specific manner in the human brain. Increased TE expression was found in microglia and neurons in the substantia nigra and putamen of PD brains, but not amygdala or prefrontal cortex, compared to controls. This TE activation correlated with an innate immune response in the same brain regions. The link between an interferon response and TE activation was mechanistically confirmed using human pluripotent stem cell-derived microglia and neurons. Our findings provide insights into TE transcription in the PD brain and suggest TEs may contribute to neuroinflammation and pathological progression in PD. Teaser: Transposable elements are linked to a cell type specific inflammatory state in Parkinsons Disease.

neuroscience↗

bioRxiv2025

Mapping early patterning events in human neural development usingan in-vitro microfluidic stem cell model

Stem cell models can provide insights into human brain development at embryonic stages which are normally inaccessible. We previously developed the Microfluidic Stem Cell Regionalisation (MiSTR) model, which recapitulates the rostro-caudal patterning of human neural tube through a WNT activation (WNTa) gradient. Through temporal single cell transcriptomics of rostro-caudal and dorso-ventral gradient-patterned MiSTR, we found that rostro-caudal subtypes were regionally specified and fate-determined already during the late epiblast stage, several days before onset of neuralisation at day 3-4. Rostral cells were characterised by expression of HESX1 and SHISA2 during pre-neuralisation and PAX6 during early neuralisation, whereas caudal cells expressed FST and HOXA1 during pre-neuralisation and SOX1 as the dominant neuralising factor. In contrast to the early rostro-caudal specification, response to ventralisation in telencephalic progenitors was developmentally delayed and occurred around day 9. We further uncovered temporal events in human midbrain-hindbrain boundary formation and ventral forebrain patterning, contributing new knowledge on early human neural region-specification.

neuroscience↗

bioRxiv2025

Gene regulatory networks linked to GABA signalling emerge as relevant for glioblastoma pathogenesis

Glioblastoma (GB) is the most common and aggressive adult brain tumor. Recent evidence shows that GB cells form glutamatergic synapses with neurons, promoting tumor growth and invasion, yet the gene regulatory networks (GRNs) underlying this interaction remain underexplored. GRNs coordinate gene expression through interactions among transcription factors (TFs), enhancers, and promoters, and are often disrupted in cancer due to epigenetic and chromatin architecture changes. To identify GRNs involved in GB pathogenesis, we applied the machine learning-based MOBILE (Multi-Omics Binary Integration via Lasso Ensembles) pipeline to integrate multi-omics data. GABA-signalling emerged as a previously unrecognized contributor to GB, involving relevant TFs as ARX, GSX2 and DLX family, key regulators of GABAergic interneuron development. Co-culture assays further demonstrated that GABAergic input promotes GB proliferation through non-synaptic mechanisms likely involving metabolic or paracrine interactions. Our findings reveal novel GRNs in GB, positioning GABA signalling as a potential therapeutic target to disrupt neuron-glioma interactions. SignificanceThis study uncovers novel gene regulatory networks driving glioblastoma progression through integration of multi-omics data with machine-learning tools. By identifying GABAergic signalling as a previously unrecognized contributor to tumour growth, it reveals new transcriptional regulators, providing valuable insights into neuron-glioma interactions. These findings open new therapeutic avenues for brain-cancer treatment. GRAPHICAL ABSTRAC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=61 SRC="FIGDIR/small/651564v2_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@ed95e1org.highwire.dtl.DTLVardef@b1d9forg.highwire.dtl.DTLVardef@1c5bc00org.highwire.dtl.DTLVardef@b9d833_HPS_FORMAT_FIGEXP M_FIG C_FIG Integration of multi-omics data from glioblastoma (GB) patients identified networks related to GABA signalling as relevant for GB pathogenesis. In co-culture assays, GABAergic interneurons increase the proliferation of GB cells.

genomics↗

bioRxiv2025

LINE-1 retrotransposons regulate the exit of human pluripotency and early brain development

Long interspersed nuclear element 1 (L1) retrotransposons represent a vast source of divergent genetic information. However, mechanistic analysis of whether and how L1s contribute to human developmental programs is lacking, in part due to the challenges associated with specific profiling and manipulation of human L1 expression. Here we show that thousands of hominoid-specific L1 integrants are expressed in human induced pluripotent stem cells and cerebral organoids. The activity of individual L1 promoters is surprisingly divergent and correlates with an active epigenetic state. Efficient on-target CRISPRi silencing of L1s revealed nearly a hundred co-opted L1-derived chimeric transcripts and L1 silencing resulted in changes in neural differentiation programs and reduced cerebral organoid size. Together, these data implicate L1s and L1-derived transcripts in hominoid-specific CNS developmental processes.

developmental biology↗

bioRxiv2024

Interspecies Organoids Reveal Human-Specific Molecular Features of Dopaminergic Neuron Development and Vulnerability

The disproportionate expansion of telencephalic structures during human evolution involved tradeoffs that imposed greater connectivity and metabolic demands on midbrain dopaminergic neurons. Despite the central role of dopaminergic neurons in human-enriched disorders, molecular specializations associated with human-specific features and vulnerabilities of the dopaminergic system remain unexplored. Here, we establish a phylogeny-in-a-dish approach to examine gene regulatory evolution by differentiating pools of human, chimpanzee, orangutan, and macaque pluripotent stem cells into ventral midbrain organoids capable of forming long-range projections, spontaneous activity, and dopamine release. We identify human-specific gene expression changes related to axonal transport of mitochondria and reactive oxygen species buffering and candidate cis- and trans-regulatory mechanisms underlying gene expression divergence. Our findings are consistent with a model of evolved neuroprotection in response to tradeoffs related to brain expansion and could contribute to the discovery of therapeutic targets and strategies for treating disorders involving the dopaminergic system.

evolutionary biology↗

bioRxiv2024

Generation of human appetite-regulating neurons and tanycytes from stem cells

The balance between energy intake and expenditure is controlled by the hypothalamus, a small brain region characterised by high neuronal diversity. Specifically, the arcuate nucleus (ARC) and ventromedial hypothalamus (VMH) are key hypothalamic nuclei controlling appetite through behavioural response to circulating humoral signals. Yet, despite their physiological importance, the cellular and functional characteristics of this highly specialised neural region has been studied mainly in animals due to a lack of human models. Here, we fine-tuned the differentiation of human pluripotent stem cells toward the ARC and VMH hypothalamic nuclei and identified key subtype-specific progenitor markers of these subregions. We demonstrate that the timing for initiation and termination of bone morphogenetic protein (BMP) signalling is essential for controlling subregional specification of tuberal hypothalamic progenitors along the anterior-posterior axis, balancing VMH versus ARC fates. A particular population of SHH-/NKX2.1+/FGF10high/RAXhigh/TBX3high posterior tuberal progenitors was identified as the source for generation of ARC-associated agouti-related peptide (AGRP) neurons and tanycytes whilst anterior tuberal SHH+/NKX2.1+/FGF10low/RAXlow/TBX3low progenitors generated VMH phenotypes including NR5A1 neurons. Upon maturation in vitro and in xenografts, ARC-patterned progenitors gave rise to key appetite-regulating cell types including those producing AGRP, prepronociceptin (PNOC), growth hormone-releasing hormone (GHRH), thyrotropin-releasing hormone (TRH) and pro-opiomelanocortin (POMC), as well as tanycyte glial cells. Differentiated ARC cultures showed high transcriptomic similarity to the human ARC and displayed evidence of functionality by AGRP secretion and responsiveness to leptin and fibroblast growth factor 1 (FGF1). In summary, our work provides insights into the developmental lineages underlying hypothalamic subregional specification and enables access to highly characterised human ARC and VMH cultures, which will provide novel opportunities for investigating the cellular and molecular pathways triggered by obesity-associated genetic variants and weight-regulating stimuli.

neuroscience↗

bioRxiv2024

Patterning effects of FGF17 and cAMP on generation of dopaminergic progenitors for cell replacement therapy in Parkinson's disease

Cell replacement therapies using human pluripotent stem cell-derived ventral midbrain (VM) dopaminergic (DA) progenitors are currently in clinical trials for treatment of Parkinsons disease (PD). Recapitulating developmental patterning cues, such as fibroblast growth factor 8 (FGF8), secreted at the midbrain-hindbrain boundary (MHB), is critical for the in vitro production of authentic VM DA progenitors. Here, we explore the application of alternative MHB-secreted FGF-family members, FGF17 and FGF18, for VM DA pro-genitor patterning. We show that while FGF17 and FGF18 both recapitulate VM DA progenitor patterning events, FGF17 induced expression of key VM DA progenitor markers at higher levels than FGF8 and transplanted FGF17-patterned progenitors fully reversed motor deficits in a rat PD model. Early activation of the cAMP pathway mimicked FGF17-induced patterning, although strong cAMP activation came at the expense of EN1 expression. In summary, we identified FGF17 as a promising candidate for more robust VM DA progenitor patterning, with the potential to improve cell products for treatment of PD. Summary statementIn this article we find that FGF17 induces high expression of key dopamine progenitor markers and that these cells can provide functional rescue in a rat model of Parkinsons disease.

developmental biology↗

bioRxiv2024

Decoding morphogen patterning of human neural organoids with a multiplexed single-cell transcriptomic screen

Morphogens, secreted signalling molecules that direct cell fate and tissue development, are used to direct neuroepithelial progenitors towards discrete regional identities across the central nervous system. Neural tissues derived from pluripotent stem cells in vitro (neural organoids) provide new models for studying neural regionalization, however, we lack a comprehensive survey of how the developing human neuroepithelium responds to morphogen cues. Here, we produce a detailed map of morphogen-induced effects on the axial and regional specification of human neural organoids using a multiplexed single-cell transcriptomics screen. We find that the timing, concentration, and combination of morphogens strongly influence organoid cell type and regional composition, and that cell line and neural induction method strongly impact the response to a given morphogen condition. We apply concentration gradients in microfluidic chips or a range of static concentrations in multi-well plates to explore how human neuroepithelium interprets morphogen concentrations and observe similar dose-dependent induction of patterned domains in both scenarios. Altogether, we provide a detailed resource that supports the development of new regionalized neural organoid protocols and enhances our understanding of human central nervous system patterning.

developmental biology↗

bioRxiv2022

Ribosomal RNA 2'-O-methylation dynamics impact cell fate decisions

Translational regulation impacts both pluripotency maintenance and cell differentiation. To what degree the ribosome itself exerts control over this process remains unanswered. Accumulating evidence has demonstrated heterogeneity in ribosome composition in various organisms. 2-O-methylation of rRNA represents an important source of heterogeneity, where site-specific alteration of methylation levels can modulate translation. Here we explore changes in rRNA 2-O-methylation during mouse brain development and during tri-lineage differentiation of human embryonic stem cells. We find distinct alterations between brain regions, as well as clear dynamics during cortex development and germ layer differentiation. We identify a methylation site which impacts neuronal differentiation. Modulation of its methylation levels affects ribosome association of the Fragile X Mental Retardation Protein and translation of WNT pathway-related mRNAs. Together, the data reveals ribosome heterogeneity through rRNA 2-O-methylation during early development and differentiation and suggests a direct role for ribosomes in regulating translation during cell fate acquisition.

developmental biology↗

bioRxiv2020

Neural tube patterning: from a minimal model for rostrocaudal patterning towards an integrated 3D model

The rostrocaudal patterning of the neural tube is a key event in early brain development. This process is mainly driven by a gradient of WNT, which defines the fate of the present neural progenitor cells in a dose dependent matter and leads to a subdivision of the tube into forebrain, midbrain and hindbrain. Although this process is extensively studied experimentally both in vivo and in vitro, an integrated view of the responsible genetic circuitry is currently lacking. In this work, we present a minimal gene regulatory model for rostrocaudal neural tube patterning. The model's nodes and architecture are determined in a data driven way, leading to a tristable configuration of mutually repressing brain regions. Analysis of the parameter sensitivity and simulations of knockdown and overexpression cases show that repression of hindbrain fate is a promising strategy for the improvement of current protocols for the generation of dopaminergic neurons in vitro. Furthermore, we combine the model with an existing model for dorsoventral neural tube patterning, to test its capabilities in an in vivo setting, by predicting the steady state pattern of a realistic three-dimensional neural tube. This reveals that the rostrocaudal pattern stacks dorsoventrally in the caudal half of the neural tube. Finally, we simulate morphogen secretion overexpression, which highlights the sensitivity of neural tube patterning to the morphogen levels.

systems biology↗

bioRxiv2019

Single Cell Gene Expression Analysis Reveals Human Stem Cell-Derived Graft Composition in a Cell Therapy Model of Parkinson’s Disease

Since the pioneering studies using fetal cell transplants in Parkinsons disease (PD), brain repair by cell replacement has remained a long-standing and realistic goal for the treatment of neurodegenerative disorders including PD. Authentic and functional midbrain dopamine (DA) neurons can now be generated from human pluripotent stem cells (hPSCs) via a floor plate intermediate1,2, and these cell preparations are both safe and functional when transplanted to animal models of PD3. However, although resulting grafts from fetal brain tissue and hPSCs contain large numbers of desired DA neurons, these therapeutic cells are a minor component of the grafts. Moreover, the cellular composition of the graft has remained difficult to assess due to limitations in histological methods that rely on pre-conceived notions concerning cell types. Here, we used single cell RNA sequencing (scRNA-seq) combined with comprehensive histological analyses to characterize intracerebral grafts from ventral midbrain (VM)-patterned human embryonic stem cells (hESCs) and VM fetal tissue after long-term survival and functional maturation in a pre-clinical rat model of PD. The analyses revealed that while both cell preparations gave rise to neurons and astrocytes, oligodendrocytes were only detected in grafts of fetal tissue. On the other hand, a cell type closely resembling a class of newly identified perivascular-like cells was identified as a unique component of hESC-derived grafts. The presence of these cells was confirmed in transplants from three different hESC lines, as well as from iPSCs. Thus, these experiments have addressed one of the major outstanding questions in the field of cell replacement in neurological disease by revealing graft composition and differences between hESC- and fetal cell-derived grafts, which can have important implications for clinical trials.

neuroscience↗