Biology subjects

Engelhardt, M.

Publications and source records attributed to Engelhardt, M..

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

MEG-informed navigated TMS for individualized speech cortical mapping

Speech cortical mapping by means of navigated repetitive transcranial magnetic stimulation (SCM nrTMS) provides neurosurgeons with noninvasive prior information about individuals cortical speech network. Individualized mapping is required, since the exact locations and activation patterns of speech production show high variability between individuals. We hypothesized that magnetoencephalography (MEG) data of an individuals speech production could guide the SCM TMS process temporally and spatially, leading to higher error rates at MEG-defined locations with TMS pulse timings coinciding with MEG activity. 13 healthy subjects participated in MEG and TMS measurements, where the timing of the TMS pulse (PTI; picture-to-TMS interval) was adjusted based on the individuals MEG activation in a picture naming task. At the group level, significant correlations were observed between the latency of the peak MEG activation and the PTI that produced the highest speech error rate. The MEG peak preceded the best PTI by 132 ms (R=0.713, p=0.006) across the entire stimulation area in the lateral left hemisphere, and by 103 ms (R=0.673, p=0.012) in the left frontal regions. We found 17 combinations of PTI and stimulation area in which the subjects speech error rate increased significantly compared to their average error rate. Our findings suggest that optimal PTIs are highly individual, and that individualizing the PTI according to MEG activation provides a straightforward method for accounting individual variability in speech function and may increase the sensitivity and utility of SCM TMS.

neuroscience↗

bioRxiv2025

HIF1α-dependent induction of the T-Type calcium channel CaV3.2 mediates hypoxia-induced neuronal hyperexcitability

Post-stroke epilepsy (PSE) is a leading cause of acquired epilepsy in adults, yet the molecular mechanisms linking post-ischemic hypoxia to neuronal hyperexcitability remain poorly understood. The transcription factor hypoxia-inducible factor 1 (HIF1) is a central mediator of the cellular response to hypoxia and may contribute to epileptogenesis by regulating ion channel expression. Here, we identify the T-type calcium channel CaV3.2 (encoded by Cacna1h) as a direct transcriptional target of HIF1 and demonstrate its role in hypoxia-induced network hyperexcitability. Oxygen-glucose deprivation followed by reoxygenation (OGD/R) induced a persistent increase in neuronal firing rate. In murine and human organotypic brain slices, hypoxia led to a marked increase in HIF1 and Cacna1h expression at both transcript and protein levels. Using neuronal cell lines and primary cortical neurons we show that HIF1 activation through HIF1 overexpression, consistently increases Cacna1h expression. In NS20Y cells, overexpression of a normoxia-stable HIF1 variant increased Cacna1h promoter activity in both fluorescent and dual-luciferase reporter assays. The same effect was observed in primary cortical neurons, where HIF1 overexpression also elevated network activity measured by multielectrode array recordings, replicating the effect of the OGD/R model. Together, these results establish HIF1 as a key transcriptional regulator of CaV3.2 in neurons and reveal a conserved hypoxia-HIF1-CaV3.2 pathway that enhances neuronal excitability. This mechanism may underlie hypoxia-induced network hyperactivity and contribute to the pathogenesis of post-ischemic epileptogenesis, offering a potential molecular target for intervention.

neuroscience↗

bioRxiv2025

Unreliable homeostatic action potential broadening in cultured dissociated neurons

Homeostatic plasticity preserves neuronal activity against perturbations. Recently, somatic action potential broadening was proposed as a key homeostatic adaptation to chronic inactivity in neocortical neurons. Since action potential shape critically controls calcium entry and neuronal function, broadening provides an attractive homeostatic feedback mechanism to regulate activity. Here, we report that chronic inactivity induced by sodium channel block does not broaden action potentials in neocortical neurons under a wide range of conditions. In contrast, action potentials were broadened in CA3 neurons of organotypic hippocampal cultures by chronic sodium channel block and in hippocampal dissociated cultures by chronic synaptic block. Mechanistically, BK-type potassium channels were proposed to underly inactivity-induced action potential broadening. However, BK channels did not affect action potential duration in our recordings. Our results indicate that action potential broadening can occur in specific neurons and conditions but is not a general mechanism of homeostatic plasticity in cultured neurons.

neuroscience↗

bioRxiv2025

Low extent of sex-based venom variation in Earth's most widespread viper, the common adder (Vipera berus)

Snake venom is an ecologically critical functional trait, primarily applied for foraging and accordingly shaped by selective pressures. Recent insights underpinned the high variability of snake venoms down to the intraspecific level, with regional, ontogenetic, and seasonal variation being mostly investigated. In contrast, sex-based venom variation has received considerably less attention so far, and its influence on venom compositions in vipers is virtually unknown. The common adder (Vipera berus) is a promising model species to explore this subject because of a described sexual dimorphism and its wide distribution, which promises a noteworthy degree of adaptability and venom plasticity. Here, we tested for sex-based venom variation in Central European V. berus by comparing venom profiles and bioactivity. Proteomics, paired with SDS-PAGE and RP-HPLC, revealed highly similar venom profiles. Likewise, phospholipases A2 and proteases bioactivity profiling, and bioassays targeting the coagulation cascade and mammalian cell lines revealed similar activity spectra. Hence, our analysis does not show a noteworthy extent of sex-based intraspecific venom variation in V. berus. We further discuss our data in light of the species venom profile at larger geographic scales, its clinical relevance, and the need for more standardized approaches when investigating venom variability. Our work provides novel insights into the venom biology of Earths most widespread viper, and serves as a foundation upon which future works can build.

biochemistry↗

bioRxiv2024

Variability of axon initial segment geometry and its impact on hippocampal pyramidal cell function

Action potentials, the primary information units of the nervous system, are usually generated at the axon initial segment (AIS). Changes in the length and position of the AIS are associated with alterations in neuronal excitability but there is only limited information about the baseline structural variability of the AIS. This work provides a comprehensive atlas of the diversity of proximal cell geometries across all anatomical axes of the murine hippocampus, encompassing dorsal-ventral, superficial-deep, and proximal-distal regions. We analyzed the morphology of 3,936 hippocampal pyramidal neurons in 12 animals of both sexes, focusing on AIS length, position, and their association with proximal cellular features such as the soma and dendritic geometries. Notably, neurons with axon-carrying dendrites were significantly more common in ventral compared to dorsal hippocampal areas, suggesting a functional adaptation to regional demands. Validation of this finding in human samples confirms the translational relevance of our murine model. We employed NEURON simulations to assess the functional implications of this variability. Here, variation in proximal geometry only minimally contributed to neuronal homeostasis, but instead increased heterogeneity of response patterns across neurons.

neuroscience↗

bioRxiv2023

Axon onset remodeling in response to network activity in mouse cortical neurons

In certain subtypes of pyramidal neurons, axons originate from basal dendrites, resulting in an axon-carrying dendrite branch with unique functional characteristics (AcD cells). The number of AcD cells increases during development, however, it is so far unclear whether neurons remodel their axon emergence throughout their lifetime in response to changes in network activity. To test our hypothesis of such large-scale morphological plasticity, we utilized in vitro and in vivo strategies in mouse primary somatosensory cortex to test whether network activity impacts axon onset. Based on data obtained by immunofluorescence, confocal microscopy and/or live-cell imaging, we show that neurons are indeed capable of changing the onset of their axon origin from somatic to dendritic and vice versa within a few days in vitro and that peripheral whisker manipulation and thus changes in sensory input drives large-scale morphological plasticity in vivo.

neuroscience↗

bioRxiv2023

Live imaging of excitable axonal microdomains in ankyrin-G-GFP mice

The axon initial segment (AIS) constitutes not only the site of action potential initiation, but also a hub for activity-dependent modulation of output generation. Recent studies shedding light on AIS function used predominantly post-hoc approaches since no robust murine in vivo live reporters exist. Here, we introduce a reporter line in which the AIS is intrinsically labeled by an ankyrin-G-GFP fusion protein activated by Cre recombinase, tagging the native Ank3 gene. Using confocal, superresolution, and two-photon microscopy as well as whole-cell patch-clamp recordings in vitro, ex vivo, and in vivo, we confirm that the subcellular scaffold of the AIS and electrophysiological parameters of labeled cells remain unchanged. We further uncover rapid AIS remodeling following increased network activity in this model system, as well as highly reproducible in vivo labeling of AIS over weeks. This novel reporter line allows longitudinal studies of AIS modulation and plasticity in vivo in real-time and thus provides a unique approach to study subcellular plasticity in a broad range of applications.

neuroscience↗

bioRxiv2023

Assessment of lisavanbulin (BAL101553) as an anti-lymphoma agent

Microtubules are major components of the cellular cytoskeleton, ubiquitously founded in all eukaryotic cells. They are involved in mitosis, cell motility, intracellular protein and organelle transport, and maintenance of cytoskeletal shape. Avanbulin (BAL27862) is a microtubule-targeted agent (MTA) that promotes tumor cell death by destabilization of microtubules. Due to its unique binding to the colchicine site of tubulin, differently from other MTAs, avanbulin has previously shown activity in solid tumor cell lines. Its prodrug, lisavanbulin (BAL101553), has shown early signs of clinical activity, especially in tumors with high EB1 expression. Here, we assessed the preclinical anti-tumor activity of avanbulin in diffuse large B cell lymphoma (DLBCL) and the pattern of expression of EB1 in DLBCL cell lines and clinical specimens. Avanbulin showed a potent in vitro anti-lymphoma activity, which was mainly cytotoxic with potent and rapid apoptosis induction. Median IC50 was around 10nM in both activated B cell like (ABC) and germinal center B cell like (GCB) DLBCL. Half of the cell lines tested showed an induction of apoptosis already in the first 24h of treatment, the other half in the first 48h. EB1 showed expression in DLBCL clinical specimens, opening the possibility for a cohort of patients that could potentially benefit from treatment with lisavanbulin. These data show the basis for further preclinical and clinical evaluation of lisavanbulin in the lymphoma field.

cancer biology↗

bioRxiv2021

Neocortical pyramidal neurons with axons emerging from dendrites are frequent in non-primates, but rare in monkey and human

The canonical view of neuronal function is that inputs are received by dendrites and somata, become integrated in the somatodendritic compartment and upon reaching a sufficient threshold, generate axonal output with axons emerging from the cell body. The latter is not necessarily the case. Instead, axons may originate from dendrites. The terms "axon carrying dendrite" (AcD) and "AcD neurons" have been coined to describe this feature. Here, we report on the diversity of axon origins in neocortical pyramidal cells. We found that in non-primates (rodent, cat, ferret, pig), 10-21% of pyramidal cells of layers II-VI had an AcD. In marked contrast, in macaque and human, this proportion was lower, and it was particularly low for supragranular neurons. Unexpectedly, pyramidal cells in the white matter of postnatal cat and aged human cortex exhibit AcDs to much higher percentages. In rodent hippocampus, AcD cells are functionally privileged, since inputs here can circumvent somatic integration and lead to immediate action potential initiation in the axon. Our findings expand the current knowledge regarding the distribution and proportion of AcD cells in neocortial regions of non-primate taxa, which strikingly differs from primates where these cells are mainly found in deeper layers and white matter.

neuroscience↗

bioRxiv2020

Single-dose ethanol intoxication causes acute and lasting neuronal changes in the brain

Alcohol intoxication at early ages is a risk factor for development of addictive behavior. To uncover neuronal molecular correlates of acute ethanol intoxication, we used stable-isotope labeled mice combined with quantitative mass spectrometry to screen over 2000 hippocampal proteins of which 72 changed synaptic abundance up to two-fold after ethanol exposure. Among those were mitochondrial proteins and proteins important for neuronal morphology, including MAP6 and Ankyrin-G. Based on these candidate proteins, we found acute and lasting molecular, cellular, and behavioral changes following a single intoxication in alcohol-naive mice. Immunofluorescence analysis revealed a shortening of axon initial segments. Longitudinal two-photon in vivo imaging showed increased synaptic dynamics and mitochondrial trafficking in axons. Knockdown of mitochondrial trafficking in dopaminergic neurons abolished conditioned alcohol preference in Drosophila. This introduces mitochondrial trafficking as a process implicated in reward learning, and highlights the potential of high-resolution proteomics to identify cellular mechanisms relevant for addictive behavior.

neuroscience↗

bioRxiv2020

Sensory input drives rapid homeostatic scaling of the axon initial segment in mouse barrel cortex

The axon initial segment (AIS) is an important axonal microdomain for action potential initiation and implicated in the regulation of neuronal excitability during activity-dependent cortical plasticity. While structural AIS plasticity has been suggested to fine-tune neuronal activity when network states change, whether it acts as a homeostatic regulatory mechanism in behaviorally relevant contexts remains poorly understood. Using an in vivo model of the mouse whisker-to-barrel pathway in combination with immunofluorescence, confocal analysis and patch-clamp electrophysiological recordings, we observed bidirectional AIS plasticity. Furthermore, we find that structural and functional AIS remodeling occurs in distinct temporal domains: long-term sensory deprivation elicits an AIS length increase, accompanied with an increase in neuronal excitability, while sensory enrichment results in a rapid AIS shortening, accompanied by a decrease in action potential generation. Our findings highlight a central role of the AIS in the homeostatic regulation of neuronal input-output relations.

neuroscience↗