Bernhard K. Mueller

The Rho/ROCK pathway mediates neurite growth-inhibitory activity associated with the chondroitin sulfate proteoglycans of the CNS glial scar

Axons fail to regenerate in the central nervous system after injury. Chondroitin sulfate proteoglycans (CSPG) expressed in the scar significantly contribute to the nonpermissive properties of the central nervous system environment. To examine the inhibitory activity of a CSPG mixture on retina ganglion cell (RGC) axon growth, we employed both a stripe assay and a nerve fiber outgrowth assay. We show that the inhibition exerted by CSPGs in vitro can be blocked by application of either C3 transferase, a specific inhibitor of the Rho GTPase, or Y27632, a specific inhibitor of the Rho kinase. These results demonstrate that CSPG-associated inhibition of neurite outgrowth is mediated by the Rho/ROCK signaling pathway. Consistent with these results, we found that retina ganglion cell axon growth on glial scar tissue was enhanced in the presence of C3 transferase and Y27632, respectively. In addition, we show that the recently identified inhibitory CSPG Te38 is upregulated in the lesioned spinal cord.

RGM is a repulsive guidance molecule for retinal axons

Axons rely on guidance cues to reach remote targets during nervous system development. A well-studied model system for axon guidance is the retinotectal projection. The retina can be divided into halves; the nasal half, next to the nose, and the temporal half. A subset of retinal axons, those from the temporal half, is guided by repulsive cues expressed in a graded fashion in the optic tectum, part of the midbrain. Here we report the cloning and functional characterization of a membrane-associated glycoprotein, which we call RGM (repulsive guidance molecule). This molecule shares no sequence homology with known guidance cues, and its messenger RNA is distributed in a gradient with increasing concentration from the anterior to posterior pole of the embryonic tectum. Recombinant RGM at low nanomolar concentration induces collapse of temporal but not of nasal growth cones and guides temporal retinal axons in vitro, demonstrating its repulsive and axon-specific guiding activity.

RGMa inhibition promotes axonal growth and recovery after spinal cord injury

Repulsive guidance molecule (RGM) is a protein implicated in both axonal guidance and neural tube closure. We report RGMa as a potent inhibitor of axon regeneration in the adult central nervous system (CNS). RGMa inhibits mammalian CNS neurite outgrowth by a mechanism dependent on the activation of the RhoA–Rho kinase pathway. RGMa expression is observed in oligodendrocytes, myelinated fibers, and neurons of the adult rat spinal cord and is induced around the injury site after spinal cord injury. We developed an antibody to RGMa that efficiently blocks the effect of RGMa in vitro. Intrathecal administration of the antibody to rats with thoracic spinal cord hemisection results in significant axonal growth of the corticospinal tract and improves functional recovery. Thus, RGMa plays an important role in limiting axonal regeneration after CNS injury and the RGMa antibody offers a possible therapeutic agent in clinical conditions characterized by a failure of CNS regeneration.

Electron-capture supernovae as the origin of elements beyond iron

We examine electron-capture supernovae (ECSNe) as sources of elements heavier than iron in the solar system and in Galactic halo stars. Nucleosynthesis calculations are performed on the basis of thermodynamic histories of mass elements from a fully self-consistent, two-dimensional hydrodynamic explosion model of an ECSN. We find that neutron-rich convective lumps with an electron fraction down to Y e,min = 0.40, which are absent in the one-dimensional counterpart, allow for interesting production of elements between the iron group and N = 50 nuclei (from Zn to Zr, with little Ga) in nuclear (quasi-)equilibrium. Our models yield very good agreement with the Ge, Sr, Y, and Zr abundances of r-process-deficient Galactic halo stars and constrain the occurrence of ECSNe to ~4% of all stellar core-collapse events. If tiny amounts of additional material with slightly lower Y e,min down to ~0.30-0.35 were also ejected?which presently cannot be excluded because of the limitations of resolution and two dimensionality of the model?a weak r-process can yield elements beyond N = 50 up to Pd, Ag, and Cd as observed in the r-process-deficient stars.

The Repulsive Guidance Molecule RGMa Is Involved in the Formation of Afferent Connections in the Dentate Gyrus

In the developing dentate gyrus, afferent fiber projections terminate in distinct laminas. This relies on an accurately regulated spatiotemporal network of guidance molecules. Here, we have analyzed the functional role of the glycosylphosphatidylinositol (GPI)-anchored repulsive guidance molecule RGMa. In situ hybridization in embryonic and postnatal brain showed expression of RGMa in the cornu ammonis and hilus of the hippocampus. In the dentate gyrus, RGM immunostaining was confined to the inner molecular layer, whereas the outer molecular layers targeted by entorhinal fibers remained free. To test the repulsive capacity of RGMa, different setups were used: the stripe and explant outgrowth assays with recombinant RGMa, and entorhino–hippocampal cocultures incubated either with a neutralizing RGMa antibody (Ab) or with the GPI anchor-digesting drug phosphatidylinositol-specific phospholipase C. Entorhinal axons were clearly repelled by RGMa in the stripe and outgrowth assays. After disrupting the RGMa function, the specific laminar termination pattern in entorhino–hippocampal cocultures was lost, and entorhinal axons entered inappropriate hippocampal areas. Our data indicate an important role of RGMa for the layer-specific termination of the perforant pathway as a repulsive signal that compels entorhinal fibers to stay in their correct target zone.

Neogenin and repulsive guidance molecule signaling in the central nervous system.

The repulsive guidance molecule (RGM) is a membrane-bound protein that was originally identified as an axon guidance molecule in the visual system. Functional studies have revealed that it has roles in axon guidance and laminar patterning in Xenopus and chick embryos, and in controlling cephalic neural tube closure in mouse embryos. The recent identification of neogenin as a receptor for RGM has provided evidence of the diverse functions of this ligand-receptor pair. Re-expression of RGM is observed after injury in the adult human and rat central nervous systems. Inhibition of RGM enhances growth of injured axons and promotes functional recovery after spinal cord injury in rats. Thus, re-expression of embryonic repulsive cues in adult tissues contributes to failure of axon regeneration in the central nervous system.

Expression of receptor tyrosine phosphatases during development of the retinotectal projection of the chick

Receptor tyrosine kinases and receptor protein tyrosine phosphatases (RPTPs) appear to coordinate many aspects of neural development, including axon growth and guidance. Here, we focus on the possible roles of RPTPs in the developing avian retinotectal system. Using both in situ hybridization analysis and immunohistochemistry, we show for the first time that five RPTP genes--CRYPalpha, CRYP-2, PTPmu, PTPgamma, and PTPalpha--have different but overlapping expression patterns throughout the retina and the tectum. PTPalpha is restricted to Muller glia cells and radial glia of the tectum, indicating a possible function in controlling neuronal migration. PTPgamma expression is restricted to amacrine neurons. CRYPalpha and CRYP-2 mRNAs in contrast are expressed throughout the retinal ganglion cell layer from where axons grow out to their tectal targets. PTPmu is expressed in a subset of these ganglion cells. CRYPalpha, CRYP-2, and PTPmu proteins are also localized in growth cones of retinal ganglion cell axons and are present in defined laminae of the tectum. Thus, the spatial and temporal expression of three distinct RPTP subtypes--CRYPalpha, CRYP-2, and PTPmu--are consistent with the possibility of their involvement in axon growth and guidance of the retinotectal projection.

The role of repulsive guidance molecules in the embryonic and adult vertebrate central nervous system.

During the development of the nervous system, outgrowing axons often have to travel long distances to reach their target neurons. In this process, outgrowing neurites tipped with motile growth cones rely on guidance cues present in their local environment. These cues are detected by specific receptors expressed on growth cones and neurites and influence the trajectory of the growing fibres. Neurite growth, guidance, target innervation and synapse formation and maturation are the processes that occur predominantly but not exclusively during embryonic or early post-natal development in vertebrates. As a result, a functional neural network is established, which is usually remarkably stable. However, the stability of the neural network in higher vertebrates comes at an expensive price, i.e. the loss of any significant ability to regenerate injured or damaged neuronal connections in their central nervous system (CNS). Most importantly, neurite growth inhibitors prevent any regenerative growth of injured nerve fibres. Some of these inhibitors are associated with CNS myelin, others are found at the lesion site and in the scar tissue. Traumatic injuries in brain and spinal cord of mammals induce upregulation of embryonic inhibitory or repulsive guidance cues and their receptors on the neurites. An example for embryonic repulsive directional cues re-expressed at lesion sites in both the rat and human CNS is provided with repulsive guidance molecules, a new family of directional guidance cues.

Myosin IIA is required for neurite outgrowth inhibition produced by repulsive guidance molecule

Although myelin‐associated neurite outgrowth inhibitors express their effects through RhoA/Rho‐kinase, the downstream targets of Rho‐kinase remain unknown. We examined the involvement of myosin II, which is one of the downstream targets of Rho‐kinase, by using blebbistatin – a specific myosin II inhibitor – and small interfering RNA targeting two myosin II isoforms, namely, MIIA and MIIB. We found that neurite outgrowth inhibition by repulsive guidance molecule (RGMa) was mediated via myosin II, particularly MIIA, in cerebellar granule neurons. RGMa induced myosin light chain (MLC) phosphorylation by a Rho‐kinase‐dependent mechanism. After spinal cord injury in rats, phosphorylated MLC in axons around the lesion site was up‐regulated, and this effect depends on Rho‐kinase activity. Further, RGMa‐induced F‐actin reduction in growth cones and growth cone collapse were mediated by MIIA. We conclude that Rho‐kinase‐dependent activation of MIIA via MLC phosphorylation induces F‐actin reduction and growth cone collapse and the subsequent neurite retraction/outgrowth inhibition triggered by RGMa.

LIM-only protein 4 interacts directly with the repulsive guidance molecule A receptor Neogenin

Repulsive guidance molecule A (RGM A) was recently described as a potent inhibitor of neuroregeneration in a rat spinal cord injury model. The receptor mediating RGM A’s repulsive activity was shown to be Neogenin, a member of the Deleted in Colorectal Cancer (DCC) family of netrin receptors. Binding of RGM A to Neogenin induces activation of the small GTPase RhoA and of its effector Rho‐kinase by an unknown mechanism. Here we show, that the cytoplasmic tail of Neogenin interacts directly with the transcriptional coactivator LIM domain only 4 (LMO4) in human SH‐SY5Y cells, human Ntera neurons, and in embryonic rat cortical neurons. RGM A binding to Neogenin but not binding of Netrin‐1, induces release of LMO4 from Neogenin. Down‐regulation of LMO4 neutralizes the repulsive activity of RGM A in neuronal cell lines and embryonic rat cortical neurons and prevents RhoA activation. These results show for the first time that an interaction of Neogenin with LMO4 is involved in the RGM A – Neogenin signal transduction pathway for RhoA activation.