Biology subjects

van Woerden, G. M.

Publications and source records attributed to van Woerden, G. M..

4 recordsLinked to original sources

bioRxiv2024

Simultaneous loss of CAMK2A and CAMK2B reveals endogenous in vivo substrates

Ca2+/calmodulin-dependent protein kinase 2 (CAMK2) plays a critical role in calcium signaling. Recent gene knockout studies show that CAMK2A and CAMK2B can have distinct roles yet also partially compensate for each other in yet unknown brain functions. In order to provide insight into potential novel CAMK2 functions, we performed parallel phosphoproteomic analyses on non-stimulated cortex tissue from inducible Camk2a and Camk2b double knockout (Camk2af/f;Camk2bf/f;CAG-CreESR) mice and from wild type mice. A total of 5622 phosphorylated peptides derived from 2080 proteins were identified. Phosphorylation at serine/threonine residues in 130 proteins were downregulated in the double knockout mice, including residues in 113 proteins that have not previously been identified as potential CAMK2 substrates. Comparison of amino acid sequences surrounding the downregulated phosphorylation residues provided new insights into the CAMK2-substrate consensus sequences in vivo. This dataset provides an important resource for future studies examining novel roles for CAMK2 in the brain.

neuroscience↗

bioRxiv2024

The non-canonical thioreductase TMX2 is essential for neuronal survival during embryonic brain development

Biallelic variants in thioredoxin-related transmembrane 2 protein (TMX2) can cause a brain malformation of cortical development (MCD), characterized by primary microcephaly, polymicrogyria and pachygyria by an unknown mechanism. To better understand and visualize how TMX2 loss disrupts brain development in vivo we investigated the function of TMX2, using the zebrafish embryo as a model system. We generated zebrafish deficient for TMX2 ortholog tmx2b, which during the first 2 days post fertilization (dpf) showed normal behavioral activity and brain developmental hallmarks. From 3 dpf onwards however, tmx2b mutants failed to exhibit locomotor activity, which was accompanied by cell death primarily in the brain, but not in other organs or in the spinal cord. Strikingly, cell death in tmx2b mutants occurs specifically in newborn neurons within a [~]1.5-hour timeframe, whereas neuronal progenitor and other glial cells are preserved, and could be suppressed by inhibiting neuronal activity. In vivo GCaMP6s calcium imaging showed a persistent [~]2-fold increase in calcium in neurons after the onset of cell death. This suggests that calcium homeostasis underlies the tmx2b mutant brain phenotype. Altogether, our results indicate that TMX2 is an evolutionary conserved, protective regulator essential specifically for newborn neurons to survive after their differentiation in the vertebrate embryonic brain.

developmental biology↗

bioRxiv2024

autoMEA: Machine learning-based burst detection for multi-electrode array datasets

Neuronal activity in the highly organized networks of the central nervous system is the vital basis for various functional processes, such as perception, motor control, and cognition. Understanding interneuronal connectivity and how activity is regulated in the neuronal circuits is crucial for interpreting how the brain works. Multi-electrode arrays (MEAs) are particularly useful for studying the dynamics of neuronal network activity and their development as they allow for real-time, high-throughput measurements of neural activity. At present, the key challenge in the utilization of MEA data is the sheer complexity of the measured datasets. Available software offers semi-automated analysis for a fixed set of parameters that allow for the definition of spikes and bursts. However, this analysis remains time-consuming, user-biased, and limited by pre-defined parameters. Here, we present autoMEA, software for machine learning-based automated burst detection in MEA datasets. We exemplify autoMEA efficacy on neuronal network activity of primary hippocampal neurons from wild-type mice monitored using 24-well multiwell MEA plates. To validate and benchmark the software, we showcase its application using wild-type neuronal networks and two different neuronal networks modeling neurodevelopmental disorders to assess network phenotype detection. Detection of network characteristics typically reported in literature, such as synchronicity and rhythmicity, could be accurately detected compared to manual analysis using the autoMEA software. Additionally, autoMEA could detect reverberations, a more complex burst dynamic present in hippocampal cultures. Furthermore, autoMEA burst detection was sufficiently sensitive to detect changes in the synchronicity and rhythmicity of networks modeling neurodevelopmental disorders as well as detecting changes in their burst dynamics. Thus, we show that autoMEA reliably analyses neural networks measured with the multi-well MEA setup with the precision and accuracy compared to that of a human expert.

neuroscience↗

bioRxiv2020

GHB confers neuroprotection by stabilizing the CaMKIIα hub domain

Ca2+/calmodulin-dependent protein kinase II alpha (CaMKII) is an abundant neuronal signaling protein involved in synaptic plasticity and memory formation1,2. The central hub domain regulates the activity of CaMKII by organizing the holoenzyme complex into functional oligomers3-6. Recent findings have suggested that the hub is also an allosteric determinant of kinase activity7, and is thus an emerging target for therapies to correct CaMKII dysregulation8,9. However, pharmacological modulation of the hub domain has never been demonstrated. Here we show that stabilization of the CaMKII hub domain confers neuroprotection. By combining photoaffinity labeling and chemical proteomics using small molecule analogs of the natural metabolite {gamma}-hydroxybutyrate (GHB)10 we reveal that CaMKII is the selective target for GHB. We further find that these GHB analogs bind to the hub interior by solving a 2.2 [A] crystal structure of CaMKII with bound ligand. Using differential scanning fluorimetry, we show that binding of ligands to the hub interior increases the thermal stability of hub oligomers in a concentration-dependent manner. Moreover, we demonstrate the functional significance of this hub stabilization by showing substantial neuroprotective effects in cellular excitotoxicity assays and in a mouse model of cerebral ischemia. Together, our results reveal that CaMKII hub stabilization is the mechanism by which GHB provides endogenous neuroprotection and that small-molecule CaMKII-selective ligands have therapeutic potential.

biochemistry↗