Components in the tuberous sclerosis complex - target of rapamycin (TSC-TOR) pathway have been implicated in a number of human behavioral and neurological disorders including autism, epilepsy, and Huntington disease. While the TSC-TOR pathway is known to regulate cell growth, actin cytoskeletal organization, and autophagy, its role in axon guidance is largely unstudied. Here we demonstrate using genetic mosaic strategies that mutations in multiple components of this pathway disrupt retinal axon pathfinding in Drosophila. Photoreceptors mutant for the small GTPase Rheb, a positive regulator of TOR, fail to make proper connections with their target neurons in the optic lobe, stopping short of their target. Conversely, photoreceptors mutant for the negative regulators TSC1 or PTEN project past their targets in the medulla and fail to defasciculate. These phenotypes are not the result of delayed developmental timing, as photoreceptors were competent to reach their targets when mutant for Minute genes, a phenot...

During embryonic development, motor and sensory axons innervate the chick hindlimb precisely. Axons from different segmental levels converge in a plexus region at the base of the limb, pause for 24 hr, and then enter the limb in distinct dorsal and ventral pathways around St. 24.5 (E4.5). We previously showed that axons wait in the plexus region for maturational changes in the limb, but the molecular mechanisms responsible are unknown. Recently, we found that Raldh2, an enzyme required for synthesis of retinoic acid (RA), is first expressed in both dorsal and ventral regions of the hindlimb around St. 24, just before axons leave the plexus region and grow into the limb. The timing and pattern of Raldh2 expression suggest that RA could potentially regulate the waiting period. To investigate this possibility, we prematurely increased RA in the limb by implanting beads soaked in all-trans-RA just distal to the plexus region in St. 22-23 (E4) embryos. Both motor and sensory axons exited the plexus region and g...

Visual cortex sends out long range, transient axonal projections to spinal cord in early postnatal development and selectively eliminates these axons by the second postnatal week in the rat. The cellular mechanism for elimination of the transient visual-spinal axons is unknown and no detailed study has been carried out in the mouse. The aim of this study is to define the time course of selective axon pruning of the visual-spinal projection and to reveal how these transient axons and their terminals are eliminated. We injected the tracer DiI or biotinylated dextran amine (BDA) into the mouse visual cortex at postnatal day 3 (P3) and examined the visual-spinal axon projection between P7 and P15. From P7 to P9, a substantial number of labeled fibers from the visual cortex passed the caudal pons, entered the pyramid and a small number of fibers decussated and descended in the cervical segment of the spinal cord. Some axon collaterals derived from the dorsal tract innervated the gray matter. After P15, no visua...

Schwann cell phenotype is usually described as myelinating or non-myelinating. However, we have recently shown that Schwann cells of cutaneous nerve differ substantially from those of ventral root in their growth factor expression (NSci Abst 373.2, 2004). Cutaneous nerve, denervated or reinnervated by cutaneous axons, dramatically upregulates NGF and BDNF but expresses little GDNF or Pleitrophin. Under similar conditions, ventral root upregulates GDNF and Pleitrophin 20-40 fold while expressing virtually no NGF or BDNF. Cutaneous and motor Schwann cells thus exhibit distinct phenotypes. The current experiments examine the effect of cutaneous and motor Schwann cell phenotype on cutaneous axon regeneration. Experiments were performed on the femoral nerve of Lewis rats. Two-cm-long grafts of femoral cutaneous nerve (1,600 myelinated axons) or L5 ventral root (2200 myelinated axons) were sewn to the transected femoral cutaneous nerve to be reinnervated by femoral cutaneous axons. Regeneration was assayed after...

Cdc2 kinase is a prototypical cyclin-dependent kinase critical for G2 to M phase cell cycle transition. Yet, its function in the nervous system is largely unknown. Here, we investigated possible role of Cdc2 in axonal regeneration using sciatic nerve system in rat. Cdc2 protein levels and activity were increased in the injured sciatic nerves 3 and 7 days after crush injury and then decreased to basal level 14 days later. Administration of Cdc2 kinase inhibitor roscovitine in vivo at the time of crush injury significantly inhibited axonal regeneration when regrowing axons were analyzed using anterograde and retrograde tracers. Immunohistochemical analysis showed that induced Cdc2 protein was localized in the Schwann cells, and Cdc2 protein levels in cultured Schwann cells which were prepared from sciatic nerves 7 days after crush injury were much higher compared with those from uninjured sciatic nerves, suggesting that Cdc2 protein expression was primarily induced in the Schwann cells. To further investigat...

Denervation is associated with neurodegenerative diseases and acute injuries in skeletal muscle. To promote functional recovery from denervation type injuries or disease processes, it is necessary for injured axons to regenerate and navigate back to their target to reestablish synapses. To better understand these processes at the neuromuscular junction, we crushed peripheral nerves in transgenic mice with cytoplasmic expression of yellow fluorescent protein (YFP) in their motoneurons and allowed them to regenerate to their former synaptic sites. We then used correlated confocal and serial electron microscopy to examine ultrastructural features of the regenerating axons prior to endplate arrival and during initial reoccupation of the denervated endplate. Using this approach, we found that the tips of regenerating axons contain no active zones and are guided to former endplates within tubes filled with hypertrophied Schwann cell processes. These axons do not appear to associate with basal lamina material. Th...

Within neocortical layer 5, pyramidal neurons can be differentiated into at least two major subclasses on the basis of their projections to the brainstem and thalamus (type A), or to the contralateral cortex and basal ganglia (type B). These two types c...

Previous work has shown that cultured hippocampal neurons extend axons in the direction of an increasing substrate-bound laminin gradient. However, these gradients are either difficult to manufacture or lack sufficient spatial resolution to guide individual axons for experiments requiring patterned cultures. To circumvent these problems, we have developed a tapered line pattern that has the growth properties of a laminin gradient with high spatial resolution and used it to direct the growth of single axons in culture. Poly(dimethylsiloxane) microstamps with line patterns 5µm wide at one end tapering to a width of 2µm over a distance of 2mm were coated 100µg/mL laminin. The tapered line laminin pattern was then transferred to glass cover slips by microcontact printing and background regions were coated with 100µg/mL polylysine to promote cell attachment. Finally, rat E18 hippocampal neurons were cultured on the laminin patterns and axons of individual cells were scored for alignment to the laminin pattern a...

Following spinal cord injury, CNS neurons require a permissive environment, rich in growth-promoting cues, for axonal growth, guidance, regeneration and recovery. Neuronal cell adhesion molecules (CAMs) mediate axon growth, branching, fasciculation, guidance and synapse formation. They are essential for proper development of the nervous system, including the corticospinal tract. As different CAMs have distinct structures and functions, the intracellular signaling mechanisms they employ must be distinct as well, yet these differences are poorly understood. Our objective is to elucidate the molecular mechanisms that underlie CAM regulated axon growth and branching; in particular, to characterize the mechanisms stimulated by the L1 immunoglobulin superfamily CAM and compare them to those employed by the calcium-dependent CAM, N-cadherin, and the extracellular matrix protein laminin. We are conducting a “high-content” screen to determine how approximately 500 chemical compounds, which regulate diverse intracel...

Spinal Muscular Atrophy (SMA) is an autosomal recessive disease that affects 1 in every 6,000-10,000 individuals at birth, making it the leading genetic cause of infant mortality. There are effective treatments for SMA, but not all patients respond to t...