Lynn T. Landmesser

Polysialic acid in the vertebrate nervous system: a promoter of plasticity in cell-cell interactions

Polysialic acid (PSA), a homopolymer attached to the neural cell adhesion molecule (NCAM), serves as a modulator of cell interactions. Polysialic acid exhibits a highly regulated expression pattern. During embryonic development its abundant expression is closely correlated with axon pathfinding and targeting, and with certain aspects of muscle formation. Its level also can be altered by synaptic activity. During neonatal development and in the adult brain, PSA expression is more restricted, being primarily associated with regions capable of morphological or physiological plasticity. The ability to perturb PSA in vivo by a specific glycosidase and by the creation of NCAM-deficient mice has led to extensive analysis of its biological function. These studies suggest that the primary role of PSA is to promote changes in cell interactions and thereby facilitate plasticity in the structure and function of the nervous system.

Normal Patterns of Spontaneous Activity Are Required for Correct Motor Axon Guidance and the Expression of Specific Guidance Molecules

Rhythmic spontaneous electrical activity occurs in many parts of the developing nervous system, where it plays essential roles in the refinement of neural connections. By blocking or slowing this bursting activity, via in ovo drug applications at precise developmental periods, we show that such activity is also required at much earlier stages for spinal motoneurons to accurately execute their first major dorsal-ventral pathfinding decision. Blockade or slowing of rhythmic bursting activity also prevents the normal expression patterns of EphA4 and polysialic acid on NCAM, which may contribute to the pathfinding errors observed. More prolonged (E2-5) blockade resulted in a downregulation of LIM homeodomain transcription factors, but since this occurred only after the pathfinding errors and alterations in guidance molecules, it cannot have contributed to them.

Axonin-1, Nr-CAM, and Ng-CAM play different roles in the in vivo guidance of chick commissural neurons.

Immunoglobulin/fibronectin type III-like cell adhesion molecules have been implicated in axon pathfinding based on their expression pattern in the developing nervous system and on their complex interactions described in vitro. The present in vivo study demonstrates that interactions by two of these molecules, axonin-1 on commissural growth cones and Nr-CAM on floor plate cells, are required for accurate pathfinding at the midline. When axonin-1 or Nr-CAM interactions were perturbed, many commissural axons failed to cross the midline and turned instead along the ipsilateral floor plate border. In contrast, though perturbation of Ng-CAM produced a defasciculation of the commissural neurites, it did not affect their guidance across the floor plate.

Polysialic acid regulates growth cone behavior during sorting of motor axons in the plexus region

Removal of polysialic acid (PSA) from N-CAM during the time when chick motoneuron axons are segregating into target-specific fascicles at the base of the limb was previously shown to result in motoneuron projection errors. Here, it is established that these errors are associated with altered growth cone behavior in the plexus. In contrast to control embryos, in which individual axons were observed to exhibit dramatic changes in direction and extensive divergence, axonal trajectories following the removal of PSA were relatively straight. To determine whether enhanced axon-axon fasciculation following PSA removal had prevented growth cones from responding appropriately to guidance cues at the base of the limb, we also examined the role of L1, a major mediator of axon-axon fasciculation in this system. Anti-L1 reversed the effects of PSA removal on both growth cone trajectories and projection errors. These results indicate that PSA plays a permissive role, attenuating axon-axon interactions in the plexus and thereby allowing the axonal reorganization that is essential for the formation of specific motoneuron projections.

Interference with Axonin-1 and NrCAM Interactions Unmasks a Floor-Plate Activity Inhibitory for Commissural Axons

Axonin-1 and NrCAM were previously shown to be involved in the in vivo guidance of commissural growth cones across the floor plate of the embryonic chicken spinal cord. To further characterize their role in axon pathfinding, we developed a two-dimensional coculture system of commissural and floor-plate explants in which it was possible to study the behavior of growth cones upon floor-plate contact. Although commissural axons readily entered the floor plate under control conditions, perturbations of either axonin-1 or NrCAM interactions prevented the growth cones from entering the floor-plate explants. The presence of antiaxonin-1 resulted in the collapse of commissural growth cones upon contact with the floor plate. The perturbation of NrCAM interactions also resulted in an avoidance of the floor plate, but without inducing growth-cone collapse. Therefore, axonin-1 and NrCAM are crucial for the contact-mediated interaction between commissural growth cones and the floor plate, which in turn is required for the proper guidance of the axons across the ventral midline and their subsequent rostral turn into the longitudinal axis.

Alterations in transmission, vesicle dynamics, and transmitter release machinery at NCAM-deficient neuromuscular junctions

Although functional neuromuscular junctions (NMJs) form in NCAM-deficient mice, they exhibit multiple alterations in presynaptic organization and function. Profound depression and unusual periodic total transmission failures with repetitive stimulation point to a defect in vesicle mobilization/cycling, and these defects were mimicked in (+/+) NMJs by inhibitors of myosin light chain kinase, known to affect vesicle mobilization. Two separate release mechanisms, utilizing different endocytic machinery and Ca(2+) channels, were shown to coexist in (-/-) terminals, with the mature process targeted to presynaptic membrane opposed to muscle, and an abnormally retained immature process targeted to the remainder of the presynaptic terminal and axon. Thus, NCAM plays a critical and heretofore unsuspected role in the molecular organization of the presynaptic NMJ.

Axon guidance at choice points

The common theme in many recent axonal pathfinding studies, both in vertebrates and invertebrates, is the demonstration of the importance of a balance between positive and negative cues. The integration of multiple and often opposing molecular interactions at each site along the axons trajectory, especially at choice points, helps to fine tune the directional response of its growth cone, which continuously samples its environment for guidance cues. The dynamic regulation of the receptors for such cues, in response to extrinsic signals, also enhances the behavioral repertoire of growth cones at different points along their trajectory. Some of the molecules identified as being important for axon guidance at choice points are conserved between invertebrates and vertebrates (e.g. Robo and netrin), whereas other molecules have been identified, so far, only in invertebrates (e.g. Comm) or vertebrates (e.g. axonin-1 and NrCAM).

Cholinergic Input Is Required during Embryonic Development to Mediate Proper Assembly of Spinal Locomotor Circuits

Rhythmic limb movements are controlled by pattern-generating neurons within the ventral spinal cord, but little is known about how these locomotor circuits are assembled during development. At early stages of embryogenesis, motor neurons are spontaneously active, releasing acetylcholine that triggers the depolarization of adjacent cells in the spinal cord. To investigate whether acetylcholine-driven activity is required for assembly of the central pattern-generating (CPG) circuit, we studied mice lacking the choline acetyltransferase (ChAT) enzyme. Our studies show that a rhythmically active spinal circuit forms in ChAT mutants, but the duration of each cycle period is elongated, and right-left and flexor-extensor coordination are abnormal. In contrast, blocking acetylcholine receptors after the locomotor network is wired does not affect right-left or flexor-extensor coordination. These findings suggest that the cholinergic neurotransmitter pathway is involved in configuring the CPG during a transient period of development.

The acquisition of motoneuron subtype identity and motor circuit formation.

Experiments in chick embryos using classical transplantation techniques introduced by Viktor Hamburger are reviewed; these demonstrated that chick‐limb innervating motoneurons become specified by extrinsic signals prior to axon outgrowth and that they selectively grow to appropriate muscles by actively responding to guidance cues within the limb. More recent experiments reveal that fast/slow and flexor/extensor subclasses of motoneurons are distinct by E4–5 and that they exhibit patterned spontaneous activity while still growing to their targets. These observations are then related to the combinatorial code of LIM transcription factor expression, which has been hypothesized to specify motoneuron subtypes.

Reduced Survival of Motor Neuron (SMN) Protein in Motor Neuronal Progenitors Functions Cell Autonomously to Cause Spinal Muscular Atrophy in Model Mice Expressing the Human Centromeric (SMN2) Gene

Spinal muscular atrophy (SMA) is a common (∼1:6400) autosomal recessive neuromuscular disorder caused by a paucity of the survival of motor neuron (SMN) protein. Although widely recognized to cause selective spinal motor neuron loss when deficient, the precise cellular site of action of the SMN protein in SMA remains unclear. In this study we sought to determine the consequences of selectively depleting SMN in the motor neurons of model mice. Depleting but not abolishing the protein in motor neuronal progenitors causes an SMA-like phenotype. Neuromuscular weakness in the model mice is accompanied by peripheral as well as central synaptic defects, electrophysiological abnormalities of the neuromuscular junctions, muscle atrophy, and motor neuron degeneration. However, the disease phenotype is more modest than that observed in mice expressing ubiquitously low levels of the SMN protein, and both symptoms as well as early electrophysiological abnormalities that are readily apparent in neonates were attenuated in an age-dependent manner. We conclude that selective knock-down of SMN in motor neurons is sufficient but may not be necessary to cause a disease phenotype and that targeting these cells will be a requirement of any effective therapeutic strategy. This realization is tempered by the relatively mild SMA phenotype in our model mice, one explanation for which is the presence of normal SMN levels in non-neuronal tissue that serves to modulate disease severity.