Biology subjects

Villmann, C.

Publications and source records attributed to Villmann, C..

6 recordsLinked to original sources

bioRxiv2024

Neuropathic pain and distinct CASPR2 autoantibody IgG subclasses drive neuronal hyperexcitability

Patients with autoantibodies (aAbs) against the contactin-associated protein-like 2 (CASPR2) suffer from a variety of clinical syndromes including neuropathic pain, in some patients even as the only symptom. CASPR2 is an adhesion protein of the neurexin IV family and part of the voltage-gated potassium channel complex (VGKC) in neurons of dorsal root ganglia (DRG). The subsequent pathological mechanisms following the binding of CASPR2 aAbs and their association with pain are only partially understood. CASPR2 aAbs are mainly of the IgG4 subclass. Previous studies have neglected subclass-dependent effects. Here we investigated 49 subclassified patient serum samples positive for CASPR2 aAbs. To unravel underlying molecular mechanisms, we used a combination of super-resolution lattice structural illumination microscopy (SIM2) and functional readouts by calcium imaging and electrophysiological recordings. CASPR2-positive patient sera subclassified in IgG4 together with at least one other IgG subclass (IgGX) and patients with only IgG4 were further subdivided into the pain and no pain group. Patient subclassification shed further light on the pathological mechanisms of CASPR2 aAbs. A decrease of CASPR2 expression after long-term exposure to CASPR2 aAbs was only observed for the patient group without pain. Upon withdrawal of the CASPR2 aAbs, CASPR2 expression returned to normal level. Structural alterations were obtained by increased distances between CASPR2 and associated potassium channels along DRG axons using high-resolution lattice SIM2 microscopy but only following binding of CASPR2 aAbs from patients with pain. Similarly, CASPR2 aAbs of patients with pain significantly increased overall neuronal excitability of cultured DRG neurons as measured by calcium imaging. Patch-clamp recordings revealed significantly decreased current amplitudes of voltage-gated potassium (Kv) channels after incubation with all four CASPR2 aAbs subclassifications with the most prominent effect of serum samples harboring IgG4 aAbs. Notably, a patient serum sample lacking IgG4 did not alter Kv channel function. Withdrawal of aAbs rescued Kv channel function to normal levels suggesting that the affected potassium channel function is rather due to a functional block of the VGKC rather than altered structural integrity of the VGKC. Taken together, we found IgG4 aAbs to be a major modifier of potassium channel function. The increase in DRG excitability is primarily due to impaired Kv channel conductance as a consequence of CASPR2 aAbs binding but additional and so far unidentified signal pathways contribute to this process in patients with neuropathic pain.

neuroscience↗

bioRxiv2023

7,8-Dihydroxyflavone is a direct inhibitor of pyridoxal phosphatase

Vitamin B6 deficiency has been linked to cognitive impairment in human brain disorders for decades. Still, the molecular mechanisms linking vitamin B6 to these pathologies remain poorly understood, and whether vitamin B6 supplementation improves cognition is unclear as well. Pyridoxal phosphatase (PDXP), an enzyme that controls levels of pyridoxal 5-phosphate (PLP), the co-enzymatically active form of vitamin B6, may represent an alternative therapeutic entry point into vitamin B6-associated pathologies. However, pharmacological PDXP inhibitors to test this concept are lacking. We now identify a PDXP and age-dependent decline of PLP levels in the murine hippocampus that provides a rationale for the development of PDXP inhibitors. Using a combination of small molecule screening, protein crystallography and biolayer interferometry, we discover, visualize and analyze 7,8-dihydroxyflavone (7,8-DHF) as a direct and potent PDXP inhibitor. 7,8-DHF binds and reversibly inhibits PDXP with low micromolar affinity and sub-micromolar potency. In mouse hippocampal neurons, 7,8-DHF increases PLP in a PDXP-dependent manner. These findings validate PDXP as a druggable target. Of note, 7,8-DHF is a well-studied molecule in brain disorder models, although its mechanism of action is actively debated. Our discovery of 7,8-DHF as a PDXP inhibitor offers novel mechanistic insights into the controversy surrounding 7,8-DHF-mediated effects in the brain.

biochemistry↗

bioRxiv2021

Identification of a stereotypic molecular arrangement of glycine receptors at native spinal cord synapses

Precise quantitative information about the molecular architecture of synapses is essential to understanding the functional specificity and downstream signaling processes at specific populations of synapses. Glycine receptors (GlyRs) are the primary fast inhibitory neurotransmitter receptors in the spinal cord and brainstem. These inhibitory glycinergic networks crucially regulate motor and sensory processes. Thus far the nanoscale organization of GlyRs underlying the different network specificities has not been defined. Here, we have quantitatively characterized the molecular arrangement and ultra-structure of glycinergic synapses in spinal cord tissue using quantitative super-resolution correlative light and electron microscopy (SR-CLEM). We show that endogenous GlyRs exhibit equal receptor-scaffold occupancy and constant packing densities of about 2000 GlyRs m-2 at synapses across the spinal cord and throughout adulthood, even though ventral horn synapses have twice the total copy numbers, larger postsynaptic domains and more convoluted morphologies than dorsal horn synapses. We demonstrate that this stereotypic molecular arrangement is maintained at glycinergic synapses in the oscillator mouse model of the neuromotor disease hyperekplexia despite a decrease in synapse size, indicating that the molecular organization of GlyRs is preserved in this hypomorph. We thus conclude that the morphology and size of inhibitory postsynaptic specializations rather than differences in GlyR packing determine the postsynaptic strength of glycinergic neurotransmission in motor and sensory spinal cord networks.

molecular biology↗

bioRxiv2021

Sylites: Multipurpose markers for the visualization of inhibitory synapses

We introduce Sylites - small and versatile fluorogenic affinity probes for high-contrast visualization of inhibitory synapses. Having stoichiometric labeling and exceptional selectivity for neuronal gephyrin, a hallmark protein of the inhibitory post-synapse, Sylites enable superior synapse staining compared with antibodies. Combined with super-resolution microscopy, Sylites allow precise nanoscopic measurements of the synapse. In brain tissue, Sylites reveal the three-dimensional distribution of inhibitory synapses within just an hour.

neuroscience↗

bioRxiv2020

Constitutively active TrkB kinase signalling reduces actin filopodia dynamics and cell migration

Trk receptors and gene fusions of NTRK are targets in precision oncology. Classical Trk signalling concepts fail to explain ligand-independent signalling of intracellular TrkB or NTRK fusion proteins. Here, we show that abundance of the intracellular domain of TrkB is sufficient for ligand-independent autophosphorylation. This constitutive TrkB signalling reduced actin filopodia dynamics, could phosphorylate FAK, and changed cell morphology. Mutating Y705 in the kinase domain of TrkB alone specifically blocked these pathways. Engineered intracellular kinase domain proteins and a cancer-related intracellular NTRK2-fusion protein (SQSTM1-NTRK2) also underwent constitutive activation. In migrating glioblastoma-like U87MG cells, self-active TrkB kinase reduced cell migration. Moreover, we found evidences for constitutively active, intracellular TrkB in tissue of human grade IV glioblastoma. Structural modelling of the kinase domain let us postulate that release from cis-autoinhibition by abundance is sufficient for TrkB/FAK/Actin signalling via Y705. These constitutive signalling pathways could be fully blocked within minutes by clinically approved, anti-tumorigenic Trk inhibitors. In conclusion, our data provide an explanation and biological function for TrkB kinase domain signalling in the absence of a ligand.

neuroscience↗

bioRxiv2020

Pyridoxal Kinase Inhibition by Artemisinins Downregulates Inhibitory Neurotransmission

The anti-malarial artemisinins have also been implicated in the regulation of various other cellular pathways. Despite their widespread application, the cellular specificities and molecular mechanisms of target recognition by artemisinins remain poorly characterized. We recently demonstrated how these drugs modulate inhibitory postsynaptic signaling by direct binding to the scaffolding protein gephyrin. Here, we report the crystal structure of the central metabolic enzyme pyridoxal kinase (PDXK), which catalyzes the production of the active form of vitamin-B6 (also known as pyridoxal 5-phosphate, PLP), in complex with artesunate at 2.4-[A] resolution. Partially overlapping binding of artemisinins with the substrate pyridoxal inhibits PLP biosynthesis as demonstrated by kinetic measurements. Electrophysiological recordings from hippocampal slices and activity measurements of glutamic acid decarboxylase (GAD), a PLP-dependent enzyme synthesizing the neurotransmitter {gamma}-aminobutyric acid (GABA), define how artemisinins interfere presynaptically with GABAergic signaling. Our data provide a comprehensive picture of artemisinin-induced effects on inhibitory signaling in the brain.

neuroscience↗