Biology subjects

Brunner, J.

Publications and source records attributed to Brunner, J..

6 recordsLinked to original sources

bioRxiv2025

PAX3-FOXO1 drives targetable cell state-dependent metabolic vulnerabilities in rhabdomyosarcoma

PAX3-FOXO1, an oncogenic transcription factor, drives a particularly aggressive subtype of rhabdomyosarcoma (RMS) by enforcing gene expression programs that support malignant cell states. Here we show that PAX3-FOXO1+ RMS cells exhibit altered pyrimidine metabolism and increased dependence on enzymes involved in de novo pyrimidine synthesis, including dihydrofolate reductase (DHFR). Consequently, PAX3-FOXO1+ cells display increased sensitivity to inhibition of DHFR by the chemotherapeutic drug methotrexate, and this dependence is rescued by provision of pyrimidine nucleotides. Methotrexate treatment mimics the metabolic and transcriptional impact of PAX3-FOXO1 silencing, reducing expression of genes related to PAX3-FOXO1-driven malignant cell states. Accordingly, methotrexate treatment slows growth of multiple PAX3-FOXO1+ tumor xenograft models, but not fusion-negative counterparts. Taken together, these data demonstrate that PAX3-FOXO1 induces cell states characterized by altered pyrimidine dependence and nominate methotrexate as an addition to the current therapeutic arsenal for treatment of these malignant pediatric tumors.

cancer biology↗

bioRxiv2025

Optical recordings of unitary synaptic connections reveal high and random local connectivity between CA3 pyramidal cells

The hippocampal CA3 region is thought to play crucial roles in episodic memory functions because of the extensive recurrent connections between CA3 pyramidal cells (CA3PCs). However, different methods provided contradicting observations about the synaptic connectivity between CA3PCs. Therefore, we estimated the connectivity rate between individual CA3PCs using a new approach that is not affected by the confounds of conventional methods. Specifically, we used voltage imaging with the Voltron sensor in acute slices from rats of both sexes to test CA3PC connections by detecting spontaneous spiking and subthreshold responses in anatomically identified neurons. We detected 164 monosynaptic excitatory connections in 3078 tested CA3PC-CA3PC pairs. This 5.3% connectivity rate was much higher than that we observed with the theoretically more sensitive patch clamp method in similar experimental conditions, but it remained below the anatomically observed number of CA3PC-CA3PC contacts. We verified that the imaged excitatory connections were mediated by AMPA receptors. Our results also showed that the recurrent connections did not enrich into preferred connectivity motifs and followed a distribution that was consistent with random connectivity in general. Moreover, voltage imaging revealed CA3PCs with distinct firing properties and somatic locations corresponding to previously established heterogeneity and showed that specific connectivity rules create preferred information routes among these subpopulations. Finally, we showed that there is at least one condition, influencing patch clamp recordings but not voltage imaging, that affects the observable functional connections between CA3PCs. Altogether, our results obtained with a new voltage imaging approach argue for high local connectivity rates between CA3PCs. Significance StatementVoltage imaging offers new opportunities to measure synaptic connectivity between individual identified neurons. We employed the Voltron sensor to measure the monosynaptic connectivity between local CA3 pyramidal cells (CA3PCs) in acute slices from rats. Recurrent CA3PC connections are thought to play crucial roles in storing memory. However, the estimated connectivity rates between CA3PCs differ depending on the methods used. We showed that in spite of having a lower signal-to-noise ratio, voltage imaging results showed a much higher connectivity between CA3PCs than patch clamp recordings. Our results also suggest that the recurrent excitatory connections between CA3PCs are random, with preferred connectivity between and within subpopulations of CA3PCs.

neuroscience↗

bioRxiv2024

KIF5A regulates axonal repair and time-dependent axonal transport of SFPQ granules and mitochondria in human motor neurons

Mutations in the microtubule binding motor protein, kinesin family member 5A (KIF5A), cause the fatal motor neuron disease, Amyotrophic Lateral Sclerosis. While KIF5 family members transport a variety of cargos along axons, it is still unclear which cargos are affected by KIF5A mutations. We generated KIF5A null mutant human motor neurons to investigate the impact of KIF5A loss on the transport of various cargoes and its effect on motor neuron function at two different timepoints in vitro. The absence of KIF5A resulted in reduced neurite complexity in young motor neurons (DIV14) and significant defects in axonal regeneration capacity at all developmental stages. KIF5A loss did not affect neurofilament transport but resulted in decreased mitochondria motility and anterograde speed at DIV42. More prominently, KIF5A depletion strongly reduced anterograde transport of SFPQ-associated RNA granules in DIV42 motor neuron axons. We conclude that KIF5A most prominently functions in human motor neurons to promote axonal regrowth after injury as well as to anterogradely transport mitochondria and, to a larger extent, SFPQ-associated RNA granules in a time-dependent manner.

neuroscience↗

bioRxiv2024

A biosensor-based phage display selection method for automated, high-throughput Nanobody discovery

Biopanning methods to select target-specific Nanobodies(R) (Nbs) involve presenting the antigen, immobilized on plastic plates or magnetic beads, to Nb libraries displayed on phage. Most routines are operator-dependent, labor-intensive and often material- and time-consuming. Here we validate an improved panning strategy that uses biosensors to present the antigen to phage-displayed Nbs in a well. The use of automated Octet biolayer interferometry sensors (Sartorius) enables high throughput and precise control over each step. By playing with association and dissociation times and buffer composition, one can efficiently decrease the background of aspecific and low-affinity Nbs, reducing the rounds of panning needed for the enrichment of high-affinity binders. Octet panning also enables the use of unpurified target proteins and unpurified phage from a bacterial culture supernatant. Additionally, downscaling to a 384-well format significantly reduces the amount of protein required. Moreover, enrichment of binders can be quantified by monitoring phage binding to the target by interferometry, omitting additional phage titration steps. Routinely, up to three rounds of Octet panning can be performed in only five days to deliver target-specific binders, ready for screening and characterization using the same Octet instrument. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=144 SRC="FIGDIR/small/600780v2_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@72eda4org.highwire.dtl.DTLVardef@14fe6bcorg.highwire.dtl.DTLVardef@b57bc9org.highwire.dtl.DTLVardef@d1ca82_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Affective Symptoms in Pregnancy are Associated with the Vaginal Microbiome

Composition of the vaginal microbiome in pregnancy is associated with adverse maternal, obstetric, and child health outcomes. Identifying the sources of individual differences in the vaginal microbiome is therefore of considerable clinical and public health interest. The current study tested the hypothesis that vaginal microbiome composition during pregnancy is associated with an individuals experience of affective symptoms and stress exposure. Data were based on a prospective longitudinal study of a diverse and medically healthy community sample of 275 mother-infant pairs. Affective symptoms and stress exposure and select measures of associated biomarkers (diurnal salivary cortisol, serum measures of sex hormones) were collected at each trimester; self-report, clinical, and medical records were used to collect detailed data on socio-demographic factors and health behavior, including diet and sleep. Vaginal microbiome samples were collected in the third trimester (34-40 weeks) and characterized by 16S rRNA sequencing. Identified taxa were clustered into three community state types (CST1-3) based on dissimilarity of vaginal microbiota composition. Results indicate that depressive symptoms during pregnancy were reliably associated with individual taxa and CST3 in the third trimester. Prediction of functional potential from 16S taxonomy revealed a differential abundance of metabolic pathways in CST1-3 and individual taxa, including biosynthetic pathways for the neuroactive metabolites, serotonin and dopamine. With the exception of bioavailable testosterone, no significant associations were found between symptoms- and stress-related biomarkers and CSTs. Our results provide further evidence of how prenatal psychological distress during pregnancy alters the maternal-fetal microbiome ecosystem that may be important for understanding maternal and child health outcomes. ImportancePrenatal affective symptoms and stress are associated with maternal, obstetric, and child health outcomes, but the mechanisms underlying these links and their application to intervention remain unclear. The findings from this investigation extend prior microbiome-oriented research by demonstrating that the maternal vaginal microbiome composition has a biologically plausible mechanistic link with affective symptoms that also suggest additional clinical applications for assessment and intervention.

microbiology↗

bioRxiv2021

Small size of recorded neuronal structures confines the accuracy in direct axonal voltage measurements

Patch-clamp instruments including amplifier circuits and pipettes affect the recorded voltage signals. We hypothesized that realistic and complete in silico representation of recording instruments together with detailed morphology and biophysics of small recorded structures will precisely reveal signal distortions and provides a tool that predicts native signals from distorted voltage recordings. Therefore, we built a model that was verified by small axonal recordings. The model accurately recreated actual action potential measurements with typical recording artefacts and predicted the native electrical behavior. The simulations verified that recording instruments substantially filter voltage recordings. Moreover, we revealed that instrumentation directly interferes with local signal generation depending on the size of the recorded structures, which complicates the interpretation of recordings from smaller structures, such as axons. However, our model offers a straightforward approach that predicts the native waveforms of fast voltage signals and the underlying conductances even from the smallest neuronal structures.

neuroscience↗