Biology subjects

Wuttke, T. V.

Publications and source records attributed to Wuttke, T. V..

8 recordsLinked to original sources

bioRxiv2026

Local translation drives glioblastoma heterogeneity and tumor invasion

O_FIG O_LINKSMALLFIG WIDTH=174 HEIGHT=200 SRC="FIGDIR/small/722387v1_ufig1.gif" ALT="Figure 1"> View larger version (96K): org.highwire.dtl.DTLVardef@1da020corg.highwire.dtl.DTLVardef@1bc9b32org.highwire.dtl.DTLVardef@171198aorg.highwire.dtl.DTLVardef@43ce6f_HPS_FORMAT_FIGEXP M_FIG C_FIG Glioblastoma is characterized by diffuse brain invasion, yet the subcellular mechanisms enabling this aggressive behavior remain poorly understood. A subpopulation of glioblastoma cells forms invasive tumor microtubes (TMs), neurite-like extensions that drive whole-brain colonization. Here, we establish local protein translation as a fundamental driver of TM dynamics and invasive cell states. Developing a subcellular transcriptomics approach - integrating subcellular organelle organization with spatially resolved transcriptomics and functional readouts - we reveal that TM gene expression drives cell state identity. Invasive cells further exhibit significantly elevated local translation in protruding TMs, directly linking subcellular protein synthesis to functional invasive states associated with neurodevelopmental programs of axonal growth cones. Targeted disruption of TM-localized translation via photoswitchable puromycin, and specific knockdowns of the TM-enriched proteins GPM6A and GAP43, impaired TM dynamics, suppressed invasion, and reduced tumor growth. Together, these findings define local translation as a key determinant of tumor heterogeneity and glioblastoma invasion.

cancer biology↗

bioRxiv2024

Comprehensive Analysis of Human Dendritic Spine Morphology and Density: Exploring Diversity of Human Dendritic Spines

Dendritic spines, small protrusions on neuronal dendrites, play a crucial role in brain function by changing shape and size in response to neural activity. So far, in depth analysis of dendritic spines in human brain tissue is lacking. This study presents a comprehensive analysis of human dendritic spine morphology and density using a unique dataset from human brain tissue from 27 patients (8 females, 19 males, aged 18-71) undergoing tumor or epilepsy surgery at three neurosurgery sites. We used acute slices and organotypic brain slice cultures to examine dendritic spines, classifying them into the three main morphological subtypes: Mushroom, Thin, and Stubby, via 3D reconstruction using ZEISS arivis Pro software. A deep learning model, trained on 39 diverse datasets, automated spine segmentation and 3D reconstruction, achieving a 74% F1-score and reducing processing time by over 50%. We show significant differences in spine density by sex, dendrite type, and tissue condition. Females had higher spine densities than males, and apical dendrites were denser in spines than basal ones. Acute tissue showed higher spine densities compared to cultured human brain tissue. With time in culture, Mushroom spines decreased, while Stubby and Thin spine percentages increased, particularly from 7-9 to 14 days in vitro, reflecting potential synaptic plasticity changes. Our study underscores the importance of using human brain tissue to understand unique synaptic properties and shows that integrating deep learning with traditional methods enables efficient large-scale analysis, revealing key insights into sex- and tissue-specific dendritic spine dynamics relevant to neurological diseases. New and NoteworthyThis study presents a dataset of nearly 4,000 morphologically reconstructed human dendritic spines across different ages, gender, and tissue conditions. The dataset was further used to evaluate a deep learning algorithm for three-dimensional spine reconstruction, offering a scalable method for semi-automated spine analysis across various tissues and microscopy setups. The findings enhance understanding of human neurology, indicating potential connections between spine morphology, brain function, and the mechanisms of neurological and psychiatric diseases.

neuroscience↗

bioRxiv2024

Morphotype-specific calcium signaling in human microglia

Key functions of Ca2+ signaling in rodent microglia include monitoring the brain state or the surrounding neuronal activity and sensing the danger or damage in their vicinity. Microglial Ca2+ dyshomeostasis is a disease hallmark in many mouse models of neurological disorders but the Ca2+ signal properties of human microglia remain unknown. Using a newly developed toolbox, we analyzed in situ Ca2+ signaling of decades-old human cortical microglia. The data revealed marked compartmentalization of Ca2+ signals, with signal properties differing across the compartments and resident morphotypes. The basal Ca2+ levels were low in ramified and high in ameboid microglia. The fraction of cells with ongoing Ca2+ signaling, the fraction and the amplitude of process Ca2+ signals and the duration of somatic Ca2+ signals decreased when moving from ramified via hypertrophic to ameboid microglia. In contrast, the size of active compartments, the fraction and amplitude of somatic Ca2+ signals and the duration of process Ca2+ signals increased along this pathway.

immunology↗

bioRxiv2024

Electrophysiological signatures of a developmental delay in a stem cell model of KCNQ2 developmental and epileptic encephalopathy

BackgroundKCNQ2, encoding KV7.2 ion channels, has emerged as one of the prominent genes causing early onset seizures with developmental delay (KCNQ2 developmental and epileptic encephalopathy; KCNQ2-DEE). KCNQ2 de novo loss-of-function (LOF) and associated neuronal hyperexcitability have been accepted as mechanisms contributing to seizures. To investigate the developmental impact of KCNQ2 LOF, we generated patient iPSC-derived models for two previously reported de novo variants, p.(Arg325Gly) and p.(Gly315Arg), linked to severe congenital DEE. MethodsFunctional investigation of the two variants was initially performed in Xenopus laevis oocyte system. Patient-derived iPSC lines were differentiated using NGN2- and embryoid body-based protocols yielding neurons roughly corresponding to mid- and mid-late gestational stages, respectively. KV7- mediated M-current, passive neuronal properties, action potential generation and spontaneous oscillatory network activities were analysed with whole-cell patch clamping. FindingsStudied KCNQ2 variants showed LOF with a dominant-negative effect in the heterologous system. Reduced M-currents in patient iPSC-derived neurons corroborated a LOF as the main pathomechanism. Interestingly, this led to the reduced neuronal firing of the early neurons and to a disruption of complex oscillatory activity, with significantly reduced duration and amplitude of these events in patient iPSC-derived neurons. InterpretationWe provide experimental evidence for changing roles of the M-current throughout development and place disease variant-mediated M-current reduction in the context of the neuronal maturation in the prenatal brain. Based on the reduced neuronal firing and disrupted oscillatory activity seen in patient iPSC-derived neurons, we propose that a delayed/impaired maturation of neuronal and network properties underlies KCNQ-DEE caused by LOF variants.

neuroscience↗

bioRxiv2023

Long-term human organotypic brain slice cultures: a detailed protocol to provide a comprehensive framework for single-neuron and neuronal network investigations

BackgroundThe investigation of the human brain at cellular and microcircuit level remains challenging due to the fragile viability of neuronal tissue, inter- and intra-variability of the samples and limited availability of human brain material. New methodHere, we present an optimized work-up to use resected tissue from brain surgeries for live cell experiments in vitro. Comparison with existing methodsWe provide a reworked, detailed protocol of the production, culturing and viral transduction of human organotypic brain slice cultures for research purposes. ResultsWe highlight the critical pitfalls of the culturing process of the human brain tissue and present results on viral expression, single-cell Patch-Clamp recordings, as well as multi-electrode array recordings over a prolonged period of time. Additionally, our statistics show that brain tissue from patients of any age and morbidity can be used for organotypic brain slice cultures if carefully selected. ConclusionsOrganotypic brain slice cultures are of great value for basic neuroscience and disease modeling over a time course of three weeks. HighlightsO_LILong-term human organotypic brain slice cultures are viable for 2-3 weeks and provide a framework for basic neuroscience and disease modeling C_LIO_LIWe provide a reworked, detailed protocol for organotypic human brain slice culture production and maintenance C_LIO_LIWe show results of long-term culturing of human organotypic brain slice cultures in terms of viral transduction, whole-cell Patch-Clamp recordings and multi-electrode array recordings C_LIO_LIStatistics of 16 surgeries show the correlation between the overall success rates of efficient culturing and viral transduction with the patient age and morbidity C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/561508v1_fig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@17c985aorg.highwire.dtl.DTLVardef@f651fdorg.highwire.dtl.DTLVardef@180d32eorg.highwire.dtl.DTLVardef@a896b5_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1, Graphical abstract.C_FLOATNO Schematic overview of preparation, maintenance and experimental workup of human organotypic brain slice cultures. After surgical resection, human brain tissue is prepared, sliced and cultured at air-liquid-interface between human cerebrospinal fluid and defined incubator atmosphere, allowing for week-long viability. This enables extensive experimental workup such as viral transduction, single cell and multi-electrode array electrophysiological recordings. C_FIG

neuroscience↗

bioRxiv2023

Axonopathy and altered synaptic development in early hippocampal epileptogenesis of Dravet syndrome

Dravet syndrome caused by SCN1A variants is a severe developmental epileptic encephalopathy (DEE) characterized by pharmaco-resistant epileptic seizures and progressive neurodevelopmental decline with cognitive impairment and autism-spectrum-traits. Numerous preceding studies indicate that the initial pathophysiology due to impaired NaV1.1 function mainly derives from reduced interneuron firing leading to a network hyperexcitability (Bender et al. 2012). However, little is known how epileptogenesis and generally disease pathogenesis progress from the inborn molecular defect to infantile seizure onset. We address this question in a Dravet mouse model by comprehensive single-cell RNA sequencing and selected downstream analysis via single-cell electrophysiology, histology, live cell imaging and electron microscopy. Our data reveal a continuum of early primary (preseizure) and secondary (post-seizure onset) transcriptomic changes in various cell populations in the hippocampus. Focusing on cornu ammonis, we find a number of transcriptional pathways that are dysregulated including synaptic transmembrane adhesion molecules of the neurexin superfamily and voltage-gated ion channels. Further investigations support an ultrastructural and functional axonopathy and synaptopathy of parvalbumin interneurons. These processes precede somatic firing impairment and seizures suggesting they underlie fundamental early-phase disease mechanisms. Taken together we provide a cellularly resolved transcriptomic resource of early disease phases of Dravet syndrome and demonstrate epileptogenesis beyond NaV1.1 loss-of-function during an early developmental time window of CNS maturation. Altogether these data establish proof-of principle that the concept of epileptogenesis, originally devised for acquired forms of epilepsy, similarly applies to genetic epilepsies and DEEs.

neuroscience↗

bioRxiv2021

In vitro effects of S-Licarbazepine as a potential precision therapy on SCN8A variants causing neuropsychiatric disorders

Background and PurposeAmong genetic epilepsies, variants in sodium channel coding genes constitute a major subgroup. Variants in SCN8A, the coding gene for NaV1.6 channels, are characterized by a variety of symptoms including intractable epileptic seizures, psychomotor delay, progressive cognitive decline, and others such as autistic features, ataxia or dystonia. Standard anticonvulsant treatment has only limited impact on the course of disease. Experimental ApproachPersonalized therapeutic regimens tailored to disease-causing pathophysiological mechanisms may offer the specificity required to overcome intractability. Toward this aim, we investigated in vitro in neuroblastoma cells the effects of S-Licarbazepine, a third-generation dibenzazepine and enhancer of slow inactivation of voltage gated sodium channels, on three gain-of-function NaV1.6 variants linked to representative phenotypes of mild epilepsy (G1475R), developmental and epileptic encephalopathy (M1760I) and intellectual disability without epilepsy (A1622D). Key ResultsS-Licarbazepine strongly enhances the slow and - less pronounced - the fast inactivation of NaV1.6 wildtype channels. It acts similarly on all tested variants and irrespective of their particular biophysical dysfunction mechanism. Beyond that S-Licarbazepine has variant-specific effects including a partial reversal of pathologically slowed fast inactivation dynamics (A1622D, M1760I) and a trend to reduce the enhanced persistent Na+ current by A1622D variant channels. Conclusion and ImplicationsThese data bring out that S-Licarbazepine not only owns substance-specific effects, but also holds variant-specific effects, which can variably contribute to functional compensation of distinct channel-specific biophysical properties and thereby highlighting the role of personalized approaches, which likely will be key to improved and successful treatment not only of SCN8A-related disease. Bullet pointsO_LIWhat is already known? S-Lic strongly modulates slow and - to a less extend - fast inactivation of wild-type NaV1.6 channels. C_LIO_LIWhat this study adds? Differential modulatory effects of S-Lic extend to NaV1.6 A1622D, M1760I and G1475R variant channels irrespective of their leading biophysical mode of gain-of-function and variably contribute to variant-specific functional compensation of their altered biophysical properties. C_LIO_LIClinical significance: These data suggest therapeutic potential of S-Lic for SCN8A neuropsychiatric disorders and highlight the role of personalized approaches aimed at increasingly precise correction of underlying pathophysiological mechanisms. C_LI

pharmacology and toxicology↗

bioRxiv2021

Dravet variant SCN1AA1783V impairs interneuron firing predominantly by altered channel activation

Dravet syndrome (DS) is a developmental epileptic encephalopathy mainly caused by functional NaV1.1 haploinsufficiency in interneurons (IN). Recently, a new conditional mouse model expressing the recurrent human p.A1783V missense variant has become available. Here we provide an electrophysiological characterization of this variant in tsA201 cells, revealing both altered voltage-dependence of activation and slow inactivation without reduced sodium peak current density. Simulating IN excitability in a Hodgkin-Huxley one-compartment model suggested surprisingly similar firing deficits for Scn1aA1783V and full haploinsufficiency as caused by heterozygous truncation variants. Impaired NaVA1783V channel activation was predicted to have a significantly larger impact on channel function than altered slow inactivation and is therefore proposed as the main mechanism underlying IN dysfunction. The computational model was validated in cortical organotypic slice cultures derived from conditional Scn1aA1783V mice. Pan-neuronal activation of the p.A1783V variant in vitro confirmed the predicted IN firing deficit while demonstrating normal excitability of pyramidal neurons. Taken together these data demonstrate that despite maintained physiological peak currents density LOF gating properties may match effects of full haploinsufficiency on neuronal level, thereby causing DS. HighlightsNaV1.1A1783V alters voltage-dependence of activation and slow inactivation while not affecting fast inactivation. Depolarizing and hyperpolarizing shifts of activation and slow inactivation curves result in combined channel loss of function (LOF). Simulations of NaV1.1A1783V interneuronal properties indicate reduced action potential firing rates comparable to full SCN1A haploinsufficiency, which is often found in Dravet syndrome. In silico modelling identifies impaired channel activation as the predominant mechanism of channel LOF. Panneuronal induction of Scn1a+/A1783V in a cortical slice culture model confirms restriction of loss of function and its restriction to interneurons.

neuroscience↗