Biology subjects

Bruestle, O.

Publications and source records attributed to Bruestle, O..

6 recordsLinked to original sources

bioRxiv2026

Presynaptic mechanism of epileptiform activities in forward-programmed human excitatory neuronal networks

Epilepsy is one of the most common neurological disorders, yet the mechanisms controlling seizure termination remain poorly understood. In particular, why rhythmic spike-wave discharges decelerate before stopping is unexplained. Here, using human iPSC-derived excitatory neurons differentiated via targeted forward-programming, we report a similar deceleration phenomenon in cultured neuronal networks. These networks exhibit glutamate-dependent, epileptiform 'super-bursts' with a slowing rhythm from ~4 Hz to ~2 Hz. Combining in silico simulations and in vitro experiments, we correlate this activity pattern with the hierarchical organization of presynaptic vesicle pools. Nested bursts link to the recycling pool (RP), and sub-bursts associate with the readily releasable pool (RRP). Decelerating RP-to-RRP vesicle translocation shortened the super-bursts, indicating that epileptiform dynamics depend heavily on this translocation process. These findings depict human neuronal networks derived from forward-programmed cells as a model for epileptology, revealing a presynaptic framework for rhythmic discharges in excitatory networks.

neuroscience↗

bioRxiv2025

Protracted fate acquisition and epigenetic de-aging during induced neural stem cell conversion of human blood cells

Transcription factor-based direct conversion of somatic cells represents an interesting avenue for generating induced neural stem cells (iNSCs) from peripheral blood without transit through a pluripotent stage. While this paradigm has been shown to be associated with epigenetic de-aging, the dynamics of this process have remained unclear. Here, we used overexpression of the two reprogramming factors SOX2 and cMYC to generate iNSC from erythroid progenitors of donors ranging from neonatal to 101 years of age. Using an epigenetic clock algorithm, we corroborated our previous finding that iNSCs generated from aged donors show pronounced epigenetic de-aging, preserving around 13 % and 5 % of the original donor age at low and high passages, respectively. Studying the dynamic of epigenetic de-aging during iNSC conversion across time, we found that this process is largely protracted, continuing for several weeks and even beyond forced neuronal differentiation of iNSCs. Transcriptomic differences between young and old donor-derived iNSCs dissipate with extended time in conversion, too. Concordant with this observation, established iNSC lines lack age-associated cellular hallmarks, similar to induced pluripotent stem cells and their derivatives. Interestingly, time course analysis of DNA methylation and RNA sequencing data revealed that acquisition of a bona fide NSC signature extends greatly beyond the time point when proliferative PAX6-positive iNSCs emerge. The unexpected slow dynamics of these processes makes iNSC conversion an attractive model for dissecting the mechanisms underlying somatic transdifferentiation and de-aging. Statement of Ethical ApprovalsThe collection of human somatic material (i.e., blood) from healthy donors for iPSC reprogramming and iNSC conversion was conducted in accordance with German law and approved by the Ethics Committee of the University of Bonn Medical Center (approval number: 275/08). All subjects gave written informed consent.

neuroscience↗

bioRxiv2024

Modelling inflammation-induced peripheral sensitization in a dish - more complex than expected?

Peripheral sensitization of nociceptors is believed to be a key driver of chronic pain states. Here, we sought to study the effects of a modified version of inflammatory soup on the excitability of human stem-cell derived sensory neurons. For this, we used a pre-existing and a novel stem cell line, modified to stably express the calcium sensor GCamP6f. Upon treatment with inflammatory soup, we observed no changes in neuronal transcription or functional responses upon calcium imaging, and only a very minor increase in resting membrane potential via whole cell patch clamping. Similarly small changes were observed when treating mouse primary sensory neurons with inflammatory soup. A semi-systematic re-examination of past literature further indicated that observed effects of inflammatory mediators on dissociated sensory neuron cultures are generally very small. We conclude that modelling inflammation-induced peripheral sensitization in vitro is non-trivial and will require careful selection of mediators and/or more complex, longitudinal multi-cellular setups. Especially in the latter, our novel GCamP6f induced-pluripotent stem cell line may be of value.

neuroscience↗

bioRxiv2021

Imaging Three-Dimensional Brain Organoid Architecture from Meso- to Nanoscale across Development

Organoids are human stem cell-derived three-dimensional cultures offering a new avenue to model human development and disease. Brain organoids allow studying various aspects of human brain development in the finest details in vitro in a tissue-like context. However, spatial relationships of subcellular structures such as synaptic contacts between distant neurons are hardly accessible by conventional light microscopy. This limitation can be overcome by systems that quickly image the entire organoid in three dimensions and in super-resolution. To that end we have developed a setup combining tissue expansion and light sheet fluorescence microscopy for imaging and quantifying diverse spatial parameters during organoid development. This technique enables zooming from a mesoscopic perspective into super-resolution within a single imaging session, thus revealing cellular and subcellular structural details in three spatial dimensions, including unequivocal delineation of mitotic cleavage planes as well as the alignment of pre- and postsynaptic proteins. We expect light sheet fluorescence expansion microscopy (LSFEM) to facilitate qualitative and quantitative assessment of organoids in developmental and disease-related studies. Summary statementThe combination of light sheet fluorescence and expansion microscopy enables imaging of mature human brain organoids in toto and down to synaptic resolution

neuroscience↗

bioRxiv2021

GDAP1 loss of function inhibits the mitochondrial pyruvate dehydrogenase complex by altering the actin cytoskeleton

Charcot-Marie-Tooth (CMT) disease 4A is an autosomal-recessive polyneuropathy caused by mutations of ganglioside-induced differentiation-associated protein 1 (GDAP1), a putative glutathione transferase, which affects mitochondrial shape and alters cellular Ca2+ homeostasis. Here, we identify the underlying mechanism. We found that patient-derived motoneurons and GDAP1 knockdown SH-SY5Y cells display two phenotypes: more tubular mitochondria and a metabolism characterized by glutamine dependence and fewer cytosolic lipid droplets. GDAP1 interacts with the actin-depolymerizing protein Cofilin-1 in a redoxdependent manner, suggesting a role for actin signaling. Consistently, GDAP1 loss causes less F-actin close to mitochondria, which restricts mitochondrial localization of the fission factor dynamin-related protein 1, instigating tubularity. Changes in the actin cytoskeleton also disrupt mitochondria-ER contact sites. This results in lower mitochondrial Ca2+ levels and inhibition of the pyruvate dehydrogenase complex, explaining the metabolic changes upon GDAP1 loss of function. Together, these findings reconcile GDAP1-associated phenotypes and implicate disrupted actin signaling in CMT4A pathophysiology.

neuroscience↗

bioRxiv2020

Nutrient signaling pathways regulate amyloid clearance and synaptic loss in Alzheimer's disease

Extra-cellular accumulation of Amyloid-{beta} (A{beta}) plaques is causatively associated with Alzheimers disease (AD). However, mechanisms that mediate the pre-pathological state of amyloid plaque formation remain elusive. Here, using paired RNAi and kinase inhibitor screens, we discovered that AKT-mediated insulin/nutrient signaling suppresses lysosomal clearance of A{beta} and promotes amyloid formation. This mechanism is cell-autonomous and functions in multiple systems, including iPSC-derived human neurons and in vivo. Nutrient signaling regulates amyloid formation via distinct lysosomal functional mechanisms, while enhanced amino acid signaling promotes amyloid formation by transcriptionally suppressing lysosome biogenesis, and high intracellular cholesterol levels suppress lysosomal clearance of amyloid by increasing the number of non-functional lysosomes. The nutrient signaling pathway, present in both neurons and microglia, regulates lysosomal clearance of amyloid and microglia mediated synapse loss, both in vitro and in vivo. Clinically, older hyperlipidemic patients showed less synapse loss through microglia and performed better in cognitive tests. Thus, our results reveal a bi-partite cellular quality control system regulated by the insulinnutrient signaling that in neurons regulates A{beta} peptide clearance and in microglia regulates synaptic loss, both processes causally associated with AD. Our results also caution against reducing amyloid through such processes as this might also result in synapse loss.

neuroscience↗