The number of neurotrophic factors found in the central nervous system is rapidly growing, but their functions in vivo are largely unknown. In the peripheral nervous system they promote the survival of developing and lesioned neurons and enhance nerve fibre growth and regeneration. Here we study the effects of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) on the largest tract system leading from the brain to the spinal cord, the corticospinal tract (CST). The developing CST grows down the spinal cord during the first postnatal days and innervates its targets after a waiting period by collateral sprouting. We find that NT-3 injected locally specifically enhances this sprouting, whereas BDNF has no effect. In adult rats, injection of NT-3 (but not BDNF) into the lesioned spinal cord increases the regenerative sprouting of the transected CST. The distance of growth of the sprouts is very restricted, but application of an antibody that neutralizes myelin-associated neurite growth inhibitory proteins results in long-distance regeneration of CST fibres.

Spinal cord trauma leads to loss of motor, sensory and autonomic functions below the lesion. Recovery is very restricted, due in part to neurite growth inhibitory myelin proteins, in particular Nogo-A. Two neutralizing antibodies against Nogo-A were used to study recovery and axonal regeneration after spinal cord lesions. Three months old Lewis rats were tested in sensory-motor tasks (open field locomotion, crossing of ladder rungs and narrow beams, the CatWalk(R) runway, reactions to heat and von Frey hairs). A T-shaped lesion was made at T8, and an intrathecal catheter delivered highly purified anti-Nogo-A monoclonal IgGs or unspecific IgGs for 2 weeks. A better outcome in motor behavior was obtained as early as two weeks after lesion in the animals receiving the Nogo-A antibodies. Withdrawal responses to heat and mechanical stimuli were not different between the groups. Histology showed enhanced regeneration of corticospinal axons in the anti-Nogo-A antibody groups. fMRI revealed significant cortical responses to stimulation of the hindpaw exclusively in anti-Nogo-A animals. These results demonstrate that neutralization of the neurite growth inhibitor Nogo-A by intrathecal antibodies leads to enhanced regeneration and reorganization of the injured CNS, resulting in improved recovery of compromised functions in the absence of dysfunctions.

Little is known about the functional role of axotomized cortical neurons that survive spinal cord injury. Large thoracic spinal cord injuries in adult rats result in impairments of hindlimb function. Using retrograde tracers, we found that axotomized corticospinal axons from the hindlimb sensorimotor cortex sprouted in the cervical spinal cord. Mapping of these neurons revealed the emergence of a new forelimb corticospinal projection from the rostral part of the former hindlimb cortex. Voltage-sensitive dye (VSD) imaging and blood-oxygen-level-dependent functional magnetic resonance imaging (BOLD fMRI) revealed a stable expansion of the forelimb sensory map, covering in particular the former hindlimb cortex containing the rewired neurons. Therefore, axotomized hindlimb corticospinal neurons can be incorporated into the sensorimotor circuits of the unaffected forelimb.

An assay based on the consumption of nitrilotriacetate (NTA) was developed to measure the activity of NTA monooxygenase (NTA-Mo) in cell extracts of "Chelatobacter" strain ATCC 29600 and to purify a functional, NTA-hydroxylating enzyme complex. The complex consisted of two components that easily dissociated during purification and upon dilution. Both components were purified to more than 95% homogeneity, and it was possible to reconstitute the functional, NTA-hydroxylating enzyme complex from pure component A (cA) and component B (cB). cB exhibited NTA-stimulated NADH oxidation but was unable to hydroxylate NTA. It had a native molecular mass of 88 kDa and contained flavin mononucleotide (FMN). cA had a native molecular mass of 99 kDa. No catalytic activity has yet been shown for cA alone. Under unfavorable conditions, NADH oxidation was partly or completely uncoupled from hydroxylation, resulting in the formation of H202.Optimum hydroxylating activity was found to be dependent on the molar ratio of the two components, the absolute concentration of the enzyme complex, and the presence of FMN. Uncoupling of the reaction was favored in the presence of high salt concentrations and in the presence of flavin adenine dinucleotide. The NTA-Mo complex was sensitive to sulfhydryl reagents, but inhibition was reversible by addition of excess dithiothreitol. The Km values for Mg2+-NTA, FMN, and NADH were determined as 0.5 mM, 1.3 ,uM, and 0.35 mM, respectively. Of 26 tested compounds, NTA was the only substrate for NTA-Mo.The complexing agent nitrilotriacetate (NTA) is used for a range of different purposes, and one of its most controversial applications is that as a substitute for sodium triphosphate in laundry detergents (28). Many representatives of both obligately aerobic and facultatively denitrifying microorganisms which can use NTA as a sole source of nitrogen, carbon and energy have been isolated. The majority of such isolates are gram-negative, obligately aerobic rods (1,5,10,14,29) which previously have been identified as Pseudomonas spp. Recently, it has been shown that these isolates belong to a new genus for which the name "Chelatobacter" has been proposed (6).The biochemical pathway for NTA degradation was first investigated in the two virtually identical "Chelatobacter" isolates T23 (1) and ATCC 29600 (9). In both strains, a monooxygenase was reported to be responsible for the oxidative conversion of NTA (24,25). In this paper, the characterization of the NTA-Mo in cell extracts, as well as the purification, reconstitution, and characterization of a functional twocomponent NTA-Mo, is reported. MATERIALS AND METHODSGrowth of the microorganism. "Chelatobacter" strain ATCC 29600 was obtained from the American Type Culture Collection, Rockville, Md., and was maintained on a synthetic medium containing 1 g of NTA liter-' as described previously (5). In order to avoid excretion of large amounts of ammonia, the strain was grown on a mixture of NTA and acetate (1 g of each liter-') for large-scale growth (100 lit...

Nogo-A, a membrane protein enriched in myelin of the adult CNS, inhibits neurite growth and regeneration; neutralizing antibodies or receptor blockers enhance regeneration and plasticity in the injured adult CNS and lead to improved functional outcome. Here we show that Nogo-A-specific knock-outs in backcrossed 129X1/SvJ and C57BL/6 mice display enhanced regeneration of the corticospinal tract after injury. Surprisingly, 129X1/SvJ Nogo-A knock-out mice had two to four times more regenerating fibers than C57BL/6 Nogo-A knock-out mice. Wild-type newborn 129X1/SvJ dorsal root ganglia in vitro grew a much higher number of processes in 3 d than C57BL/6 ganglia, confirming the stronger endogenous neurite growth potential of the 129X1/SvJ strain. cDNA microarrays of the intact and lesioned spinal cord of wild-type as well as Nogo-A knock-out animals showed a number of genes to be differentially expressed in the two mouse strains; many of them belong to functional categories associated with neurite growth, synapse formation, and inflammation/ immune responses. These results show that neurite regeneration in vivo, under the permissive condition of Nogo-A deletion, and neurite outgrowth in vitro differ significantly in two widely used mouse strains and that Nogo-A is an important endogenous inhibitor of axonal regeneration in the adult spinal cord.