Discovery of BAY-390, a Selective CNS Penetrant Chemical Probe as Transient Receptor Potential Ankyrin 1 (TRPA1) Antagonist.

Transient receptor potential ankyrin 1 (TRPA1) is a voltage-dependent, ligand-gated ion channel, and activation thereof is linked to a variety of painful conditions. Preclinical studies have demonstrated the role of TRPA1 receptors in a broad range of animal models of acute, inflammatory, and neuropathic pain. In addition, a clinical study using the TRPA1 antagonist GRC-17536 (Glenmark Pharmaceuticals) demonstrated efficacy in a subgroup of patients with painful diabetic neuropathy. Consequently, there is an increasing interest in TRPA1 inhibitors as potential analgesics. Herein, we report the identification of a fragment-like hit from a high-throughput screening (HTS) campaign and subsequent optimization to provide a novel and brain-penetrant TRPA1 inhibitor (compound 18, BAY-390), which is now being made available to the research community as an open-source in vivo probe.

Actin filament turnover drives leading edge growth during myelin sheath formation in the central nervous system.

During CNS development, oligodendrocytes wrap their plasma membrane around axons to generate multilamellar myelin sheaths. To drive growth at the leading edge of myelin at the interface with the axon, mechanical forces are necessary, but the underlying mechanisms are not known. Using an interdisciplinary approach that combines morphological, genetic, and biophysical analyses, we identified a key role for actin filament network turnover in myelin growth. At the onset of myelin biogenesis, F-actin is redistributed to the leading edge, where its polymerization-based forces push out non-adhesive and motile protrusions. F-actin disassembly converts protrusions into sheets by reducing surface tension and in turn inducing membrane spreading and adhesion. We identified the actin depolymerizing factor ADF/cofilin1, which mediates high F-actin turnover rates, as an essential factor in this process. We propose that F-actin turnover is the driving force in myelin wrapping by regulating repetitive cycles of leading edge protrusion and spreading.

A high-speed vertical optical trap for the mechanical testing of living cells at piconewton forces.

Although atomic force microscopy is often the method of choice to probe the mechanical response of (sub)micrometer sized biomaterials, the lowest force that can be reliably controlled is limited to ≈0.1 nN. For soft biological samples, like cells, such forces can already lead to a strain large enough to enter the non-elastic deformation regime. To be able to investigate the response of single cells at lower forces we developed a vertical optical trap. The force can be controlled down to single piconewtons and most of the advantages of atomic force microscopy are maintained, such as the symmetrical application of forces at a wide range of loading rates. Typical consequences of moving the focus in the vertical direction, like the interferometric effect between the bead and the coverslip and a shift of focus, were quantified and found to have negligible effects on our measurements. With a fast responding force feedback loop we can achieve deformation rates as high as 50 µm/s, which allow the investigation of the elastic and viscous components of very soft samples. The potential of the vertical optical trap is demonstrated by measuring the linearity of the response of single cells at very low forces and a high bandwidth of deformation rates.

Molecular evolution of myelin basic protein, an abundant structural myelin component.

Rapid nerve conduction in jawed vertebrates is facilitated by the myelination of axons, which evolved in ancient cartilaginous fish. We aim to understand the coevolution of myelin and the major myelin proteins. We found that myelin basic protein (MBP) derived from living cartilaginous fish (sharks and rays) associated with the plasma membrane of glial cells similar to the phosphatidylinositol (4,5)-bisphosphate (PIP2)-binding marker PH-PLCδ1, and that ionomycin-induced PIP2-hydrolysis led to its cellular redistribution. We identified two paralogous mbp genes in multiple teleost species, consistent with a genome duplication at the root of the teleost clade. Zebrafish mbpb is organized in a complex transcription unit together with the unrelated gene-of-the-oligodendrocyte-lineage (golli) while mbpa does not encode GOLLI. Moreover, the embryonic expression of mbpa and mbpb differed, indicating functional specialization after duplication. However, both mbpa and mbpb-mRNAs were detected in mature oligodendrocytes and Schwann cells, MBPa and MBPb were mass spectrometrically identified in zebrafish myelin, both associated with the plasma membrane via PIP2, and the ratio of nonsynonymous to synonymous nucleotide-substitution rates (Ka/Ks) was low. Together, this indicates selective pressure to conserve many aspects of the cellular expression and function of MBP across vertebrate species. We propose that the PIP2-binding function of MBP is evolutionarily old and that its emergence in ancient gnathostomata provided glial cells with the competence to myelinate.

Cell visco-elasticity measured with AFM and optical trapping at sub-micrometer deformations.

The measurement of the elastic properties of cells is widely used as an indicator for cellular changes during differentiation, upon drug treatment, or resulting from the interaction with the supporting matrix. Elasticity is routinely quantified by indenting the cell with a probe of an AFM while applying nano-Newton forces. Because the resulting deformations are in the micrometer range, the measurements will be affected by the finite thickness of the cell, viscous effects and even cell damage induced by the experiment itself. Here, we have analyzed the response of single 3T3 fibroblasts that were indented with a micrometer-sized bead attached to an AFM cantilever at forces from 30-600 pN, resulting in indentations ranging from 0.2 to 1.2 micrometer. To investigate the cellular response at lower forces up to 10 pN, we developed an optical trap to indent the cell in vertical direction, normal to the plane of the coverslip. Deformations of up to two hundred nanometers achieved at forces of up to 30 pN showed a reversible, thus truly elastic response that was independent on the rate of deformation. We found that at such small deformations, the elastic modulus of 100 Pa is largely determined by the presence of the actin cortex. At higher indentations, viscous effects led to an increase of the apparent elastic modulus. This viscous contribution that followed a weak power law, increased at larger cell indentations. Both AFM and optical trapping indentation experiments give consistent results for the cell elasticity. Optical trapping has the benefit of a lower force noise, which allows a more accurate determination of the absolute indentation. The combination of both techniques allows the investigation of single cells at small and large indentations and enables the separation of their viscous and elastic components.

Phosphatidylinositol 4,5-bisphosphate-dependent interaction of myelin basic protein with the plasma membrane in oligodendroglial cells and its rapid perturbation by elevated calcium.

Myelin basic protein (MBP) is an essential structural component of CNS myelin. The electrostatic association of this positively charged protein with myelin-forming membranes is a crucial step in myelination, but the mechanism that regulates myelin membrane targeting is not known. Here, we demonstrate that phosphatidylinositol 4,5-bisphosphate (PIP2) is important for the stable association of MBP with cellular membranes. In oligodendrocytes, overexpression of synaptojanin 1-derived phosphoinositide 5-phosphatase, which selectively hydrolyzes membrane PIP2, causes the detachment of MBP from the plasma membrane. In addition, constitutively active Arf6/Q67L induces the formation of PIP2-enriched endosomal vacuoles, leading to the redistribution of MBP to intracellular vesicles. Fluorescence resonance energy transfer imaging revealed an interaction of the PIP2 sensing probe PH-PLCdelta1 with wild-type MBP, but not with a mutant MBP isoform that fails to associate with the plasma membrane. Moreover, increasing intracellular Ca(2+), followed by phospholipase C-mediated PIP2 hydrolysis, as well as reduction of the membrane charge by ATP depletion, resulted in the dissociation of MBP from the glial plasma membrane. When the corpus callosum of mice was analyzed in acute brain slices by electron microscopy, the reduction of membrane surface charge led to the loss of myelin compaction and rapid vesiculation. Together, these results establish that PIP2 is an essential determinant for stable membrane binding of MBP and provide a novel link between glial phosphoinositol metabolism and MBP function in development and disease.

Neuregulin-1/ErbB signaling serves distinct functions in myelination of the peripheral and central nervous system.

Understanding the control of myelin formation by oligodendrocytes is essential for treating demyelinating diseases. Neuregulin-1 (NRG1) type III, an EGF-like growth factor, is essential for myelination in the PNS. It is thus thought that NRG1/ErbB signaling also regulates CNS myelination, a view suggested by in vitro studies and the overexpression of dominant-negative ErbB receptors. To directly test this hypothesis, we generated a series of conditional null mutants that completely lack NRG1 beginning at different stages of neural development. Unexpectedly, these mice assemble normal amounts of myelin. In addition, double mutants lacking oligodendroglial ErbB3 and ErbB4 become myelinated in the absence of any stimulation by neuregulins. In contrast, a significant hypermyelination is achieved by transgenic overexpression of NRG1 type I or NRG1 type III. Thus, NRG1/ErbB signaling is markedly different between Schwann cells and oligodendrocytes that have evolved an NRG/ErbB-independent mechanism of myelination control.