Biology subjects

Siegert, S.

Publications and source records attributed to Siegert, S..

12 recordsLinked to original sources

bioRxiv2026

Nanoscale remodeling of synaptic glutamate receptors and calcium channels in temporal lobe epilepsy

Temporal lobe epilepsy (TLE) is the most common form of focal epilepsy and remains drug-resistant in approximately one-third of patients, for whom surgical resection of the epileptogenic zone is currently the most effective treatment option. One strategy to improve therapeutic outcomes involves identifying the molecular mechanisms and key regulators underlying synaptic function. Although altered glutamatergic neurotransmission is considered a major contributor to epileptic hyperexcitability, the nanoscale organization of key synaptic proteins in the human epileptic cortex is still not well characterized. In particular, it remains unclear whether pre- and postsynaptic proteins undergo synapse-type-specific remodeling in TLE. Here, we used high-resolution freeze-fracture replica immunolabeling to map the nanoscale organization of postsynaptic AMPA receptors (AMPARs) and presynaptic P/Q-type voltage-gated calcium channels (CaV2.1) in glutamatergic synapses of cortical layers II/III of surgically resected human tissue. We examined perilesional, cytologically unremarkable, non-epileptogenic cortical tissue obtained from patients undergoing tumor resection (52-63 years; two female and three male patients) and compared these data with cortical samples from patients with refractory TLE (22-53 years; two female and four male patients). To assess whether pathological remodeling differs across synapse populations, we analyzed both excitatory dendritic spine and shaft synapses. We found a robust, highly consistent increase in AMPAR density at both spine and shaft synapses in TLE patients indicating enhanced postsynaptic excitatory signaling. However, AMPAR spatial reorganization differed between synapse types. Monte Carlo simulations demonstrated that spine synapses largely preserved a random receptor distribution without a significant clustering pattern, whereas shaft synapses exhibited a redistribution from clustering toward random receptor arrangements together with reduced center-preference within the postsynaptic density. In contrast, presynaptic CaV2.1 remodeling was more variable and predominantly associated with excitatory spine synapses. TLE spine synapses exhibited an overall increase in CaV2.1 density together with significantly larger and more numerous CaV2.1 nanoclusters within the active zone. This difference was particularly pronounced in male patients. In comparison, shaft synapses displayed only minor presynaptic alterations. CaV2.1 channels remained highly clustered under both control and epileptic conditions, indicating preservation of core active-zone organizational features despite disease-associated nanoscale remodeling. Together, these findings provide direct ultrastructural support that pathological plasticity in human TLE involves an overall increase in excitatory postsynaptic responsiveness and a target cell-specific increase of excitatory release probability at cortical synapses.

neuroscience↗

bioRxiv2026

Chromatic topological mapping reveals organelle-specific spatial organization within microglia

In branched cells, including neurons and glia, intracellular organelles are distributed across complex cellular arbors where their spatial arrangement supports transport, signaling, and compartmentalized function. Although intracellular organelle organization is increasingly recognized as an important feature of cellular state and function, existing approaches assess organelle abundance or spatial position without accounting for the branching architecture that shapes cellular function. Here, we introduce the chromatic topological morphology descriptor (chromatic TMD), a framework that quantitatively resolves intracellular organization in relation to branching morphology. Applied to reconstructed microglia with annotated lysosomal and mitochondrial compartments across retinal layers and after optic nerve crush injury, chromatic TMD identifies distinct organelle-specific spatial programs: CD68+-endosomal-lysosomes undergo layer-dependent branch-specific redistribution, revealing selective intracellular reorganization after injury, whereas mitochondrial organization remains closely coupled to branching morphology. These findings establish intracellular organization as an additional layer of cellular architecture that can be systematically analyzed across branched neural cells.

neuroscience↗

bioRxiv2026

Self-organized tiling generates tissue-scale hyperuniformity during development

Biological tissues require branched cellular architectures to maximize spatial coverage while minimizing redundancy. Yet, how cells decode local spatial information to collectively tile territories without a global blueprint remains a key open question. Here, we develop a biophysical theory of interacting branched cells, and show that coupling their growth to short-range repulsion drives efficient tiling with minimal territorial overlap. Our model predicts that the same local mechanism simultaneously suppresses long-range density fluctuations, driving the cellular collective toward hyperuniformity. We confirm these theoretical predictions with experiments on microglial patterning in the developing retina, and show that perturbations resulting in limited cell growth disrupt both tiling and fluctuation suppression. Our results reveal that two seemingly distinct optimization principles of biological patterning, large-scale regularity and efficient tiling, are intimately linked and can arise from a single growth-repulsion feedback, suggesting a general principle for self-organized tissue coverage.

biophysics↗

bioRxiv2025

Corticosterone-linked microglial activity underpins sexually dimorphic neuroplasticity after ketamine anesthesia.

Anesthesia recovery is critical for resuming normal physiological and neuronal functions; however, the mechanisms involved remain elusive. Here, we identify a female-selective corticosterone-mediated microglia-neuron interaction in vivo during ketamine anesthesia recovery, absent in males. This microglia-neuron interaction induces plastic and functional neuronal changes, as evidenced by increased spine density and mEPSC frequency, which is occluded upon microglia depletion. We show that this process is driven through upregulation of the stress-responsive co-chaperone Fkbp5 mRNA and its protein, FKBP51, in female microglia. Fkbp5/FKBP51 is a key intermediary in a corticosteroid-induced stress response, and its involvement points towards a critical interface between endocrine signaling and microglia. Thus, to counteract the observed KXA-mediated corticosterone increase in the blood, we remove the primary source of corticosterone through adrenalectomy. Close microglia-neuron interaction was absent, but was reinstated after corticosterone injection. Our findings offer a new mechanism of microglia-mediated neuronal plasticity during anesthesia recovery, which is mediated through corticosterone, enhancing our understanding of sex differences in brain function.

neuroscience↗

bioRxiv2024

Microglia determine an immune-challenged environment and facilitate ibuprofen action in human retinal organoids.

Prenatal immune challenges pose significant risks to human embryonic brain and eye development. However, we still lack knowledge about the safe usage of anti-inflammatory drugs during pregnancy. While human induced pluripotent stem cells (hIPSC)-derived brain organoid models have started to explore functional consequences upon viral stimulation, these models commonly lack microglia. Yet, microglia are susceptible to and promote inflammation and will influence the effects. Here, we generate hIPSC-derived microglia precursor cells and assemble them into retinal organoids to dissect their interplay with the developing nervous system and expose them to the immunostimulant poly(I:C). We first identified successful hIPSC-derived microglia (iMG) integration into the retinal organoid at the time when the outer plexiform layer forms. To improve ganglion cell surveillance, we adapted the retinal organoid model and found that the ganglion cell number significantly decreased only with iMG presence. While poly(I:C) exposure alters the iMG phenotype, it does not hinder their interaction with ganglion cells. Furthermore, iMG significantly enhances the supernatants inflammatory secretome and increases retinal cell proliferation. Simultaneous exposure with the non-steroidal anti-inflammatory drug (NSAID) ibuprofen dampens the poly(I:C)-mediated changes of the iMG phenotype and ameliorates cell proliferation. Remarkably, while poly(I:C) disrupts neuronal calcium dynamics independent of iMG, ibuprofen rescues this effect only if iMG are present. This effect depends on cyclooxygenase 1 in microglia and cyclooxygenase 2, common NSAID targets. These findings underscore the importance of microglia in the context of prenatal immune challenges and provide insight into the mechanisms by which ibuprofen exerts its protective effects during embryonic development.

neuroscience↗

bioRxiv2024

Optic nerve crush does not induce retinal ganglion cell loss in the contralateral eye.

PurposeOptic nerve crush (ONC) is a model for studying optic nerve trauma. Unilateral ONC induces massive retinal ganglion cell (RGC) degeneration in the affected eye, leading to vision loss within a month. A common assumption has been that the non-injured contralateral eye is unaffected due to the minimal anatomical decussation of the RGC projections at the chiasm. Yet, recently, microglia, the brain-resident macrophages, have shown a responsive phenotype in the contralateral eye after ONC. Whether RGC loss accompanies this phenotype is still controversial. MethodsUsing the available RGCode algorithm and developing our own RGC-Quant deep-learning-based tool, we quantify RGCs total number and density across the entire retina after ONC. ResultsWe confirm a short-term microglia response in the contralateral eye after ONC, but this did not affect microglia number. Furthermore, we cannot confirm the previously reported RGC loss between naive and contralateral retinas five weeks after ONC induction across the commonly used Cx3cr1creERT2 and C57BL6/J mouse models. Neither sex nor the direct comparison of the RGC markers Brn3a and RBPMS, with Brn3a co-labeling, on average, 89% of the RBPMS+-cells, explained this discrepancy, suggesting that the early microglia-responsive phenotype does not have immediate consequences on the RGC number. ConclusionsOur results corroborate that unilateral optic nerve injury elicits a microglial response in the uninjured contralateral eye but without RGC loss. Therefore, the contralateral eye should be treated separately and not as an ONC control.

neuroscience↗

bioRxiv2022

Sex, stress and UCP2: mitochondria illuminate microglial identity beyond the dichotomy

Balanced and dynamic mitochondrial networks are essential for cell survival and function. Mitochondrial networks remodel their connectivity, content and subcellular localization to support optimized energy production under conditions of increased stress. In vivo, stressors can arise from the environment, such as in neuronal injury, or from mutation-induced cellular dysfunction. Cells programmed to identify and respond to these stress signals, like microglia, rely on optimized mitochondrial function, however we know very little about mitochondrial networks of microglia in vivo or their adaptation to environmental or cellular stressors. Here, we define the mitochondrial networks of retinal microglia in physiological conditions in vivo and evaluate network alterations by taking advantage of a microglia-selective mitochondria-labeled mouse model. First, we demonstrate significant differences in the mitochondrial networks of microglia in vivo and in vitro. Then, we induced neuronal injury in the in vivo environment using optic nerve crush, where responsive microglia exhibit more fragmented mitochondrial networks with increased content and perinuclear localization, supporting a state of increased cellular stress. Surprisingly, when we selectively increase cellular stress by knocking out the mitochondria-associated gene uncoupling protein 2 (UCP2), only male UCP2KO microglia establish a hyperfused mitochondrial network after injury, indicating sex differences in microglial stress mitigation. Ovariectomy in UCP2KO females elicits a shift toward the male hyperfused mitochondrial phenotype suggesting that circulating estrogens are a contributing factor to the differences in microglial stress mitigation.

neuroscience↗

bioRxiv2022

Uncovering brain tissue architecture across scales with super-resolution light microscopy

Mapping the complex and dense arrangement of cells and their connectivity in brain tissue demands nanoscale spatial resolution imaging. Super-resolution optical microscopy excels at visualizing specific molecules and individual cells but fails to provide tissue context. Here we developed Comprehensive Analysis of Tissues across Scales (CATS), a technology to densely map brain tissue architecture from millimeter regional to nanoscopic synaptic scales in diverse chemically fixed brain preparations, including rodent and human. CATS leverages fixation-compatible extracellular labeling and advanced optical readout, in particular stimulated-emission depletion and expansion microscopy, to comprehensively delineate cellular structures. It enables 3D-reconstructing single synapses and mapping synaptic connectivity by identification and tailored analysis of putative synaptic cleft regions. Applying CATS to the hippocampal mossy fiber circuitry, we demonstrate its power to reveal the systems molecularly informed ultrastructure across spatial scales and assess local connectivity by reconstructing and quantifying the synaptic input and output structure of identified neurons.

neuroscience↗

bioRxiv2022

A systematic characterization of intrinsically formed microglia-like cells during retinal organoid differentiation.

Brain organoids differentiated from human induced pluripotent stem cells provide a unique opportunity to investigate the development, organization and connectivity of neurons in a complex cellular environment. However, organoids usually lack microglia, brain-resident immune cells which are both present in the early human embryonic brain and participate in neuronal circuit development. Here, we find that microglia innately develop in unguided retinal organoid differentiation between week 3 and 4 in 2.5D culture and appear later in floating, non-pigmented, 3D-cystic compartments. We enriched for cystic structures using a low-dosed BMP4 application and performed mass spectrometry, thus defining the protein composition of microglia-containing compartments. We found that cystic compartments expressed both mesenchymal and epithelial markers with microglia enriched in the mesenchymal region. Interestingly, microglia-like cells started to express the border-associated macrophage marker CD163. The preferential localization of human microglia to a mesenchymal compartment provides insight into the behavior and migration of microglia. The model will ultimately allow detailed study of these enigmatic cells and how they enter and distribute within the human brain.

neuroscience↗

bioRxiv2021

Microglial MorphOMICs unravel region- and sex-dependent morphological phenotypes from postnatal development to degeneration

Microglia contribute to tissue homeostasis in physiological conditions with environmental cues influencing their ever-changing morphology. Strategies to identify these changes usually involve user-selected morphometric features, which, however, have proved ineffective in establishing a spectrum of context-dependent morphological phenotypes. Here, we have developed MorphOMICs, a topological data analysis approach to overcome feature-selection-based biases and biological variability. We extracted a spatially heterogeneous and sexually-dimorphic morphological phenotype for seven adult brain regions, with ovariectomized females forming their own distinct cluster. This sex-specific phenotype declines with maturation but increases over the disease trajectories in two neurodegeneration models, 5xFAD and CK-p25. Females show an earlier morphological shift in the immediately-affected brain regions. Finally, we demonstrate that both the primary- and the short terminal processes provide distinct insights to morphological phenotypes. MorphOMICs maps microglial morphology into a spectrum of cue-dependent phenotypes in a minimally-biased and semi-automatic way.

neuroscience↗

bioRxiv2021

Chimeric GPCRs mimic distinct signaling pathways and modulate microglia responses

G protein-coupled receptors (GPCRs) regulate multiple processes ranging from cell growth and immune responses to neuronal signal transmission. However, ligands for many GPCRs remain unknown, suffer from off-target effects or have poor bioavailability. Additional challenges exist to dissect cell type-specific responses when the same GPCR is expressed on different cells within the body. Here, we overcome these limitations by engineering DREADD-based GPCR chimeras that selectively bind their agonist clozapine-N-oxide (CNO) and mimic a GPCR-of-interest. We show that the chimeric DREADD-{beta}2-adrenergic receptor ({beta}2AR/ADRB2) triggers comparable responses to levalbuterol on second messenger and kinase activity, post-translational modifications, and protein-protein interactions. Moreover, we successfully recapitulate {beta}2AR-mediated filopodia formation in microglia, a {beta}2AR-expressing immune cell that can drive inflammation in the nervous system. To further dissect microglial inflammation, we compared DREADD-{beta}2AR with two additionally designed DREADD-based chimeras mimicking GPR65 and GPR109A/HCAR2, both enriched in microglia. DREADD-{beta}2AR and DREADD-GPR65 modulate the inflammatory response with a similar profile as endogenously expressed {beta}2AR, while DREADD-GPR109A had no impact. Our DREADD-based approach allows investigation of cell type-dependent signaling pathways and function without known endogenous ligands.

synthetic biology↗

bioRxiv2021

Optimizing AAV2/6 microglial targeting identified enhanced efficiency in the photoreceptor degenerative environment

Adeno-associated viruses (AAVs) are widely used to deliver genetic material in vivo to distinct cell types such as neurons or glial cells allowing for targeted manipulation. Transduction of microglia is mostly excluded from this strategy likely due to the cells heterogeneous state upon environmental changes, which makes AAV design challenging. Here, we established the retina as a model system for microglial AAV validation and optimization. First, we show that AAV2/6 transduced microglia in both synaptic layers, where layer preference corresponds to the intravitreal or subretinal delivery method. Surprisingly, we observed significantly enhanced microglial transduction during photoreceptor degeneration. Thus, we modified the AAV6 capsid to reduce heparin binding resulting in increased microglial transduction in the outer plexiform layer. Finally, to improve microglial-specific transduction, we validated a Cre-dependent transgene delivery cassette.Together, our results provide a foundation for future studies optimizing AAV-mediated microglia transduction and highlight that environmental conditions influence microglial transduction efficiency.

neuroscience↗