Biology subjects

Stahlberg, H.

Publications and source records attributed to Stahlberg, H..

At least 19 recordsLinked to original sources

bioRxiv2026

Cryo-ET and Sub-Volume Analysis of Fibrillar Pathology in the Neurodegenerative Human Brain

Structural studies of amyloid fibrils extracted from brain tissue have identified disease-specific fibril polymorphs. However, the mechanisms driving distinct polymorphs remain unclear because no method currently links the cellular context, composition and ultrastructure of individual aggregates to their constituent fibril polymorphs. Here, we present a workflow for in situ cryo-electron tomography (cryo-ET) to study amyloids within individual aggregates in the human neurodegenerative postmortem brain. Using chemically fixed tissue, we studied in situ tau fibrils in the hippocampus of an Alzheimer's disease (AD) donor and -synuclein fibrils within a glial nuclear inclusion in the cingulate gyrus of a multiple systems atrophy (MSA) donor. Subtomogram averaging and helical reconstruction of fibril subvolumes yielded low-resolution (~30-35A) density maps, which were compared to existing ex vivo structures. Preserved ultrastructure enabled detailed observations of pathology, including an alpha-synuclein fibril penetrating the nuclear envelope, providing mechanistic insight into intranuclear aggregate formation. Combined with existing high-resolution fibril structures lacking spatial context, cryo-ET offers a powerful approach to understanding amyloid polymorphism in neurodegenerative diseases, advancing anti-amyloid therapies and diagnostic tools.

neuroscience↗

bioRxiv2026

Ultrastructural remodelling of tau fibrils during ghost tangle formation in Alzheimer's disease brain

Tau aggregation into intracellular neurofibrillary tangles (NFTs) is one of the major hallmarks of Alzheimers disease (AD). Based on neuropathological studies, NFTs have been classified into pre-tangles, mature tangles, and ghost tangles, however the ultrastructural transitions between these stages remain poorly understood. Here, we used correlative light and electron microscopy (CLEM) to structurally characterize tau tangle maturity states in post-mortem human AD brain tissue. Pre-tangles showed no consistent fibrillar ultrastructure. Mature tangles contained densely packed, highly aligned paired helical filaments (PHF) and straight filaments (SF), often organized in spatially distinct bundles within the neuronal soma. Ghost tangles lacked cellular organelles and were composed predominantly of thin fibrils compartmentalized by membranous structures, with fibril morphology differing between compartmentalized and non-compartmentalized regions. Electron tomography and fibril segmentation demonstrated that these fibrils were significantly thinner than PHFs and SFs while immunogold labeling using the 2E9 tau marker confirmed the presence of tau within both mature and ghost tangle fibrils. GFAP-positive astrocytic processes infiltrated fibril-rich compartments within ghost tangles, linking astrocytic engagement with the emergence of this distinct ultrastructural organization. Together, our findings show that ghost-tangles contain a structurally distinct population of tau fibrils, suggesting that tau aggregates undergo astrocytic-mediated structural remodeling at late stages of pathology.

pathology↗

bioRxiv2026

Deconvolving the structural heterogeneity of alpha-Synuclein in vitro and in situ

The structural states of proteins in cells and tissues provide important insight into their functional states, but studying protein structures in situ remains challenging. Furthermore, a single protein can adopt multiple conformations in cells, which typically cannot be assessed by most structural approaches. Here we developed a novel approach, based on structural proteomics fingerprints, for the quantitative analysis of the distribution of structural states of a protein in vitro and in situ. We applied it to the Parkinsons disease hallmark protein alpha-synuclein (aSyn), for which various structural states (disordered, helical, oligomeric and amyloid fibrillar, among others) have been characterized in vitro, but for which the in vivo structural states remain hotly debated. We measured structure-specific proteolytic fingerprints from well-characterized aSyn in vitro conformations and used them to quantitatively determine the aSyn conformational composition in samples of interest. We first benchmarked our approach using ground truth datasets of known composition and showed that, during in vitro amyloid fibril formation, we could simultaneously detect a time-dependent decrease in disordered monomeric aSyn, an increase in {beta}-sheet-rich oligomers, and a delayed rise in amyloid fibrils. We then applied the method to complex, biologically relevant samples. In a S. cerevisiae aSyn overex-pression model, aSyn was predominantly helical, with an increased helical fraction accompanying its relocalization from the plasma membrane to cytosolic lipid droplets. This shift was linked to proteome-wide changes in lipid droplet homeostasis and fatty acid and ergosterol metabolism, underscoring the role of lipid metabolism and droplet formation in aSyn biology. Importantly, we also detected helical aSyn in human iPSC-derived cortical neurons, supporting the physiological relevance of this conformation. Finally, neurons differentiated from PD patient-derived iPSCs showed elevated levels of {beta}-sheet-rich aSyn compared to wild-type cells. Our approach allowed the in situ identification and quantification of different structural states of aSyn directly in cell lysates. Since several proteins can adopt multiple, functionally-relevant conformations in cells, our approach should be broadly applicable to in situ, quantitative structural and functional studies of proteins.

neuroscience↗

bioRxiv2026

Temperature-dependent ligand relocation reveals plasticity of TRPM4 inhibition

Transient receptor potential melastatin 4 (TRPM4) is a Ca{superscript 2}-activated cation channel whose pharmacology is shaped by its molecular environment. It remains poorly understood how temperature and membrane context influence inhibitor recognition. Here we combine cryo-electron microscopy of membrane-derived vesicles and detergent-solubilized TRPM4 to investigate lipid-associated architecture and binding of the potent anthranilic anilide inhibitor PBA. We find that membrane vesicles preserve a native-like paralipid environment and reveal lipid binding patterns highly similar to those observed in GDN, supporting detergent-solubilized TRPM4 as a structurally relevant system for ligand analysis. Strikingly, PBA occupies distinct binding pockets at 8 {degrees}C and 37 {degrees}C. At low temperature, PBA binds in a previously described inhibitor pocket formed by S3, S4, the S4-S5 linker and the TRP helix, whereas at physiological temperature it relocates to a distinct site within the S1-S4 domain proximal to the Ca{superscript 2} regulatory region. These findings reveal temperature-dependent plasticity in TRPM4 ligand recognition.

biophysics↗

bioRxiv2025

Cryo-EM structure of alpha-Synuclein Fibrils Harboring the Dementia with Lewy Bodies - Associated E83Q Mutation

Aggregation of -synuclein (-Syn) into amyloid fibrils underlies the pathology of synucleinopathies. Rare familial mutations can modulate -Syn aggregation and may give rise to distinct fibril conformations linked to disease heterogeneity. The E83Q mutation, identified in a Dementia with Lewy Body disease (DLB) patient with atypical Lewy body distribution, has been shown to accelerate -Syn aggregation and enhance neuronal seeding, yet its structural basis remains unclear. Here, we determined the cryo-electron microscopy structure at 3.4 [A] resolution of recombinant full-length human -Syn fibrils harboring the E83Q mutation. The fibrils adopt a double-protofilament architecture with a conserved Greek-key-like core spanning residues 36-99, stabilized by intra- and inter-filament salt bridges. Despite this conserved fold, the E83Q mutation induces a distinct local rearrangement within the hydrophobic region: substitution of Glu83 with Gln reorients residue 83 inward, abolishing solvent exposure and altering interactions with the N-terminal region. This structural shift brings the hydrophobic region closer to the N-terminus, shielding residues implicated in post-translational modification and ligand binding. Our findings reveal how a single charge-neutralizing mutation reshapes -Syn fibril architecture, providing a structural framework to understand the enhanced aggregation and pathogenic properties associated with the E83Q variant.

neuroscience↗

bioRxiv2025

Correlative Ultrastructural Mapping of Lewy Pathology Reveals Regional Diversity in Parkinsons and Dementia with Lewy bodies

Lewy body diseases, including Parkinsons disease (PD) and dementia with Lewy bodies (DLB), are defined by neuronal accumulation of misfolded -synuclein (-Syn), yet the ultrastructural diversity of these inclusions across brain regions and disease contexts remains unclear. Here, we applied large-scale correlative light and electron microscopy (CLEM) to map -Syn pathology across cortical regions (entorhinal cortex, ENT; anterior cingulate cortex, AC; hippocampal CA2 region) and substantia nigra (SN) in clinically and pathologically confirmed PD and DLB donors. We identified pronounced regional heterogeneity in Lewy pathology, with cortical inclusions showing diverse maturation stages at the ultrastructural level, ranging from low-density fibrils interspersed with organelles to highly compact fibrillar inclusions. In the SN of DLB donors, we observed the full range of classical nigral LB morphologies previously described in PD. We additionally characterized diverse neuritic -Syn pathologies in DLB and identified a distinct population of electron-dense, degenerating, -Syn-positive cortical neurons not previously reported. Importantly, we found no significant difference in LB ultrastructure between PD and DLB in either cortical or nigral pathology. In contrast, quantitative analysis of >10,000 mitochondria revealed disease- and region-specific signatures of altered mitochondrial homeostasis. PD showed increased mitochondrial density and enlargement in the SN, whereas DLB showed increased mitochondrial density only in the ENT. Mitochondrial enlargement was exclusive to PD. These findings indicate that LB ultrastructure alone does not distinguish PD from DLB; instead, region-specific mitochondrial phenotypes may better reflect disease identity and regional susceptibility. Overall, we provide a high-resolution framework for human Lewy pathology in PD and DLB, revealing that ultrastructural responses to -Syn pathology are driven primarily by neuronal identity and regional vulnerability. Our results highlight the need for disease- and region-specific models that capture human phenotypes to advance mechanistic understanding and therapeutic targeting of synucleinopathies.

pathology↗

bioRxiv2025

CryoWriter: A Robotic Solution for Improved Cryo-EM Grid Preparation

Cryo-electron microscopy (cryo-EM) structure determination relies on preparing thin, vitreous films of sample solution on EM grids. Cryo-EM is a mature technology, but preparing the grids remains a major bottleneck. Here, we evaluate the cryoWriter, a blotting-free, microfluidic grid-preparation robot that writes nanoliter volumes onto EM grids in a controlled environment. Using capillary-writing in spiral or line patterns, we prepared high-quality grids from minimal sample volumes and obtained near-atomic reconstructions for test specimens, including TMV, apoferritin, and the membrane protein TRPM4. We further demonstrate programmable deposition modes, such as writing the sample twice to boost particle density, or two-line writing for on-grid mixing to visualize time-resolved protein-ligand binding. In a challenging case (NrS-1 DNA polymerase), the cryoWriter grids exhibited reduced orientation bias relative to conventional blotting, enabling a more isotropic reconstruction. These results show that the cryoWriter provides a versatile platform for reproducible low volume cryo-EM grid preparation and for on-grid biochemical workflows.

biophysics↗

bioRxiv2025

Structural basis for a filamentous morpheein model of human cystathionine beta-synthase

Human cystathionine beta-synthase (CBS) is a vital enzyme that regulates sulfur amino acid metabolism, hydrogen sulfide production, and cellular redox balance. Using a multidisciplinary approach, we demonstrate that CBS functions as a filamentous morpheein, with its stability, turnover, and activity governed by dynamic quaternary structural transitions. Three distinct filamentous assemblies were resolved by cryo-EM and are mediated by the oligomerization loop (residues 516-525): (i) ligand-free trans-dimers that form trans-basal filaments with basal stability and activity, (ii) adenosylornithine-bound cis-dimers that assemble into stabilized cis-basal filaments and (iii) S-adenosylmethionine-bound allo-dimers, which, together with cis-dimers, form highly stable, allo-activated stacked filaments. These reversible filamentous assemblies redefine CBS biology by integrating oligomerization and allosteric regulation within a morpheein framework. These findings provide a transformative perspective on CBS function and open new avenues for pharmacological targeting of dysregulated CBS in various diseases including homocystinuria, cancer, and Down syndrome.

biochemistry↗

bioRxiv2025

Pulsed-electron illumination does not reduce beam damage for imaging biological macromolecules

Radiation damage remains a fundamental limitation in cryo-electron microscopy (cryo-EM), constraining the total electron dose that can be used and thus limiting high-resolution imaging of biological specimens. Recent studies have proposed that temporally structured or pulsed electron beams could reduce radiation damage by allowing time for energy dissipation between individual electron interactions. To evaluate this hypothesis, we conducted a systematic investigation using a radiofrequency (RF) driven 300 kV Titan Krios microscope equipped with cold field emission gun (c-FEG) to generate highly regular pulsed electron beams for specimens under cryogenic conditions. We compared radiation damage in three representative samples: paraffin 2D crystals, bacteriorhodopsin (purple membrane) 2D crystals, and plunge-frozen tobacco mosaic virus (TMV) in vitreous ice, under both pulsed and conventional (= random) illumination, while keeping all other imaging conditions constant. Radiation damage was quantified by tracking the decay of computed diffraction intensities to determine the critical dose (Ne). We observed no statistically significant difference in critical dose between pulsed and random illumination across all 3 samples. Our findings provide a critical reference point for future development and evaluation of temporally modulated electron sources in cryo-EM instrumentation.

molecular biology↗

bioRxiv2025

Multiscale analysis of myelin alterations in skin biopsies from synucleinopathies.

Loss of myelin and demyelination play a role in the pathophysiology of Parkinsons disease (PD) and related neurodegenerative diseases, but little is known about the ultrastructure of the myelin-axon unit in the peripheral nervous system of subjects diseased with synucleinopathies. We here present an analysis of the myelin ultrastructure and the myelin protein abundance that characterize myelinated axons of nerve fiber bundles in cervical skin biopsies of 45 pathologically confirmed PD, DLB, MSA and non-neurological control donors. We calculated a myelin damage score and classified over 1100 myelin sheaths by looking at myelin fragmentation and swellings with correlative light and electron microscopy. We found a higher load of myelin damage in the PD compared to MSA and control groups. Quantification with ELISA did not reveal any differences in myelin protein zero (MPZ) concentrations in skin tissue homogenates between synucleinopathies. The observed structural abnormalities in the myelin sheaths may help to discriminate among subjects with synucleinopathies and control subjects and to understand the involvement of the peripheral innervation in the diseases. Our multiscale analysis of peripheral nerves highlights their potential as future biomarkers for the detection and differentiation of synuclein diseases.

neuroscience↗

bioRxiv2024

Structural basis of SIRT7 nucleosome engagement and substrate specificity

Chromatin-modifying enzymes selectively target distinct residues within histones to finetune gene expression profiles. SIRT7 is an NAD+-dependent histone deacylase often deregulated in cancer, which deacetylates either H3 lysine 36 (H3K36) or H3K18 with high specificity within nucleosomes. Here, we report structures of nucleosome-bound SIRT7, and uncover the structural basis of its specificity towards H3K36 and K18 deacylation, combining a mechanism-based cross-linking strategy, cryo-EM, mutagenesis and enzymatic assays. We show that the SIRT7 N-terminus represents a unique, extended nucleosome-binding domain, reaching across the nucleosomal surface to the acidic patch. The catalytic domain binds at the H3-tail exit site, engaging both DNA gyres of the nucleosome. Contacting H3K36 versus H3K18 requires a change in enzyme binding pose, and results in structural changes in both SIRT7 and the nucleosome. These structures reveal interactions critical for target lysine specificity, allowing us to engineer enzyme activity towards H3K18 or 36, and provides a basis for small molecule modulator development.

biophysics↗

bioRxiv2024

Ultrastructural diversity of alpha-Synuclein pathology in the post-mortem brain of Parkinson patients: implications for Lewy Body formation

Lewy bodies, the defining pathological feature of Parkinsons disease, are intraneuronal inclusions enriched in aggregated alpha-synuclein (Syn). We used correlative light and electron microscopy to selectively investigate phosphorylated Syn (SynpS129)-positive inclusions in the substantia nigra of end-stage postmortem Parkinsons disease brain. Here we show that somatic SynpS129 inclusions in nigral dopaminergic neurons are consistently fibrillar, whereas the membranous-type inclusions are restricted to neuritic processes. These neuritic inclusions displayed marked ultrastructural heterogeneity, ranging from predominantly membranous to mixed membranous-fibrillar forms. The selective targeting of defined inclusions enabled detailed structural characterization of Lewy pathology, rather than quantitative or disease-stage comparisons. Our findings highlight clear ultrastructural differences between somatic and neuritic SynpS129 pathology and demonstrate the structural complexity and heterogeneity of Lewy pathology in human Parkinsons disease brain.

neuroscience↗

bioRxiv2024

Biased agonism of carvedilol in the beta1-adrenergic receptor is governed by conformational exclusion

Only two non-visual arrestins recognize many hundreds of different, intracellularly phosphorylated G protein-coupled receptors (GPCRs). Due to the highly dynamic nature of GPCR*arrestin complexes, the critical determinants of GPCR-arrestin recognition have remained largely unclear. We show here that arrestin2 recruitment to the {beta}1-adrenergic receptor ({beta}1AR) can be induced by an arrestin-activating phosphopeptide that is not covalently linked to the receptor and that the recruitment is independent of the presence and type of the orthosteric receptor ligand. Apparently, the arrestin-receptor interaction is driven by the conformational switch within arrestin induced by the phosphopeptide, whereas the electrostatic attraction towards the receptor phosphosites may only play an auxiliary role. Extensive NMR observations show that in contrast to previous static GPCR*arrestin complex structures, the {beta}1AR complex with the beta-blocker carvedilol and arrestin2 is in a G protein-inactive conformation. The insensitivity to the specific receptor conformation provides a rationale for arrestins promiscuous recognition of GPCRs and explains the arrestin-biased agonism of carvedilol, which largely blocks G protein binding, while still enabling arrestin engagement. Significance statementG protein-coupled receptors regulate cellular signaling through G proteins and arrestins. While G protein interactions are well understood, the molecular basis of arrestin recognition remains unclear due to the dynamic nature of GPCR*arrestin complexes. We show that arrestin recognition of the {beta}1-adrenergic receptor occurs independently of the receptors conformational state or ligand binding. Using NMR, cryo-EM, and biochemical assays, we find that arrestin engagement is driven by a conformational change within arrestin itself, triggered by a non-receptor-attached phosphopeptide, whereas electrostatic attraction towards receptor phosphosites may only play an auxiliary role. These findings provide new insights into arrestin activation, explain its ability to recognize diverse GPCRs, with significant implications for understanding biased signaling mechanisms and designing of selective therapeutic strategies.

biochemistry↗

bioRxiv2024

1.94 Angstrom structure of synthetic alpha-synuclein fibrils seeding MSA neuropathology

Summary ParagraphMultiple system atrophy (MSA) is a rapidly progressive neurodegenerative disease of unknown cause, typically affecting individuals aged 50-60 and leading to death within a decade1-3. It is characterized by glial cytoplasmic inclusions (GCIs) composed of fibrillar alpha-synuclein (aSyn)4-8, whose formation shows parallels with prion propagation9,10. While fibrils extracted from MSA brains have been structurally characterized11, their ability to replicate in a "protein-only" manner has been questioned12, and their capacity to induce GCIs in vivo remains unexplored. By contrast, the synthetic fibril strain 1B13,14, assembled from recombinant human aSyn, self-replicates in vitro and induces GCIs in mice15 - suggesting direct relevance to MSA - but awaited scrutiny at an atomic scale. Here, we report high-resolution structural analyses of 1B fibrils and of fibrils extracted from diseased mice injected with 1B that developed GCIs (1BP). We show in vivo that conformational templating enables fibril strain replication, resulting in MSA-like inclusion pathology. Remarkably, the structures of 1B and 1BP are highly similar and mimic the fold of aSyn observed in one protofilament of fibrils isolated from MSA patients11. Moreover, reinjection of crude mouse brain homogenates containing 1BP into new mice reproduces the same MSA-like pathology induced by the parent synthetic seed 1B. Our findings identify 1B as a synthetic pathogen capable of self-replication in vivo and reveal structural features of 1B/1BP that may underlie MSA pathology, offering insights for therapeutic strategies.

neuroscience↗

bioRxiv2024

A versatile correlative light and electron microscopy protocol for human brain and other biological models

Correlative light and electron microscopy (CLEM) combines light microscopy, for identifying a target via genetic labels, dyes, antibodies, and morphological features, with electron microscopy, for analyzing high-resolution subcellular ultrastructures. Here, we describe the step-by-step instructions to perform a CLEM experiment, optimized for the investigation of ultrastructural features in human brain tissue. The procedure is carried out at room-temperature and can be also adapted to other human and animal tissue samples. The procedure requires 8-days to complete and includes the stages of sample fixation for optimal ultrastructural preservation, immunofluorescence staining, image acquisition, multi-modal image correlation, and is executable within standard EM laboratories. Serving as a critical tool for characterizing human tissue and disease models, room-temperature CLEM facilitates the identification and quantification of subcellular morphological features across brain regions. Key pointsO_LIThe protocol for correlative light and electron microscopy (CLEM) is optimized for analyzing chemically fixed human brain tissues. It focuses on maintaining the integrity of ultrastructural features, thereby minimizing artifacts and structural alterations. C_LIO_LIExamining brain tissues at the ultrastructural level can provide an unprecedented amount of detail which may help advance our understanding of the mechanisms underlying neurodegenerative disorders. C_LI

neuroscience↗

bioRxiv2024

Mouse α-synuclein fibrils are structurally and functionally distinct from human fibrils associated with Lewy body diseases

Short AbstractThe intricate process of -synuclein aggregation and fibrillization hold pivotal roles in Parkinsons disease (PD) and multiple system atrophy (MSA). While mouse -synuclein can fibrillize in vitro, whether these fibrils commonly used in research to induce this process or form can reproduce structures in the human brain remains unknown. Here we report the first atomic structure of mouse -synuclein fibrils, which was solved in parallel by two independent teams. The structure shows striking similarity to MSA-amplified and PD-associated E46K fibrils. However, mouse -synuclein fibrils display altered packing arrangements, reduced hydrophobicity, heightened fragmentation sensitivity, and evoke only weak immunological responses. Furthermore, mouse -synuclein fibrils exhibit exacerbated pathological spread in neurons and humanized -synuclein mice. These findings provide new insights into the structural underpinnings of -synuclein pathogenicity and emphasize a need to reassess the role of mouse -synuclein fibrils in the development of related diagnostic probes and therapeutic interventions.

neuroscience↗

bioRxiv2024

Low-dose cryo-electron ptychography of proteins at sub-nanometer resolution

Cryo-transmission electron microscopy (cryo-EM) of frozen hydrated specimens is an efficient method for the structural analysis of purified biological molecules. However, cryo-EM and cryo-electron tomography are limited by the low signal-to-noise ratio (SNR) of recorded images, making detection of smaller particles challenging. For dose-resilient samples often studied in the physical sciences, electron ptychography - a coherent diffractive imaging technique using 4D scanning transmission electron microscopy (4D-STEM) - has recently demonstrated excellent SNR and resolution down to tens of picometers for thin specimens imaged at room temperature. Here we applied 4D-STEM and ptychographic data analysis to frozen hydrated proteins, reaching sub-nanometer resolution 3D reconstructions. We employed low-dose cryo-EM with an aberration-corrected, convergent electron beam to collect 4D-STEM data for our reconstructions. The high frame rate of the electron detector allowed us to record large datasets of electron diffraction patterns with substantial overlaps between the interaction volumes of adjacent scan positions, from which the scattering potentials of the samples were iteratively reconstructed. The reconstructed micrographs show strong SNR enabling the reconstruction of the structure of apoferritin protein at up to 5.8 [A] resolution. We also show structural analysis of the Phi92 capsid and sheath, tobacco mosaic virus, and bacteriorhodopsin at slightly lower resolutions.

biophysics↗

bioRxiv2024

Identification of a Binding Site for Small Molecule Inhibitors Targeting Human TRPM4

Transient receptor potential (TRP) melastatin 4 (TRPM4) protein is a calcium-activated monovalent cation channel associated with various genetic and cardiovascular disorders. The anthranilic acid derivative NBA is a potent and specific TRPM4 inhibitor, but its binding site in TRPM4 has been unknown, although this information is crucial for drug development targeting TRPM4. We determined three cryo-EM structures of full-length human TRPM4 embedded in native lipid nanodiscs without inhibitor, bound to NBA, and a new anthranilic acid derivative, IBA. We found that the small molecules NBA and IBA were bound in a pocket formed between the S3, S4, and TRP helices and the S4-S5 linker of TRPM4. Our structural data and results from patch clamp experiments enable validation of a binding site for small molecule inhibitors, paving the way for further drug development targeting TRPM4.

biochemistry↗