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The growth of injured axons in the adult mammalian CNS is limited after injury. Three myelin proteins, Nogo, MAG (myelin-associated glycoprotein), and OMgp (oligodendrocyte myelin glycoprotein), bind to the Nogo-66 receptor (NgR) and inhibit axonal growthin vitro. Transgenic or viral blockade of NgR function allows axonal sproutingin vivo. Here, we administered the soluble function-blocking NgR ectodomain [aa 27-310; NgR(310)ecto] to spinal-injured rats. Purified NgR(310)ecto-Fc protein was delivered intrathecally after midthoracic dorsal over-hemisection. Axonal sprouting of corticospinal and raphespinal fibers in NgR(310)ecto-Fc-treated animals correlates with improved spinal cord electrical conduction and improved locomotion. The ability of soluble NgR(310)ecto to promote axon growth and locomotor recovery demonstrates a therapeutic potential for NgR antagonism in traumatic spinal cord injury.

The Nogo66 receptor (NgR1) is a neuronal, leucinerich repeat (LRR) protein that binds three central nervous system (CNS) myelin proteins, Nogo, myelin-associated glycoprotein, and oligodendrocyte myelin glycoprotein, and mediates their inhibitory effects on neurite growth. Although the LRR domains on NgR1 are necessary for binding to the myelin proteins, the exact epitope(s) involved in ligand binding is unclear. Here we report the generation and detailed characterization of an anti-NgR1 monoclonal antibody, 7E11. The 7E11 monoclonal antibody blocks Nogo, myelin-associated glycoprotein, and oligodendrocyte myelin glycoprotein binding to NgR1 with IC50 values of 120, 14, and 4.5 nm, respectively, and effectively promotes neurite outgrowth of P3 rat dorsal root ganglia neurons cultured on a CNS myelin substrate. Further, we have defined the molecular epitope of 7E11 to be DNAQLR located in the third LRR domain of rat NgR1. Our data demonstrate that anti-NgR1 antibodies recognizing this epitope, such as 7E11, can neutralize CNS myelin-dependent inhibition of neurite outgrowth. Thus, specific anti-NgR1 antibodies may represent a useful therapeutic approach for promoting CNS repair after injury.

In many instances, the establishment of highly specific neuronal connections during development results from the rearrangement of axonal projections through the trimming of exuberant collaterals or the elimination of functional synapses or both. Although the involvement of the N-methyl D-aspartate (NMDA) subtype of the glutamate receptor has been demonstrated in the shaping of axonal arbors, its participation in the process of selective stabilization of synapses remains an open issue. In this study, the effects of chronic in vivo application of D,L-2-amino-5-phosphonovaleric acid (D,L-APV), a selective antagonist of the NMDA receptor, on the synapse elimination process that takes place in the developing cerebellum of the rat have been analyzed. D,L-APV treatment prevented the regression of supernumerary climbing fiber synapses in 49 percent of the recorded Purkinje cells, while the inactive isomer L-APV was ineffective. Thus, activation of the NMDA receptor is a critical step in the regression of functional synapses during development.

Approximately half of the retinal ganglion cells (RGCs) present in the rodent retina at birth normally die during early development. Overexpression of the proto-oncogene bcl-2 recently has been shown to rescue some neuronal populations from natural cell death and from degeneration induced by axotomy of nerves within the peripheral nervous system. Here we studyin vivothe role of the overexpression of bcl-2 in the natural cell death of RGCs and in the degenerative process induced in these cells by transection of the optic nerve. We find that in newborn bcl-2 transgenic mice, the number of RGCs undergoing natural cell death is considerably lower than in wild-type pups. Consistently, a vast majority (90%) of the ganglion cells found in the retina of neonatal transgenics are maintained in adulthood, whereas only 40% survive in wild-type mice.After transection of the optic nerve, the number of degenerating ganglion cells, determined by counting pyknotic nuclei or nuclei with fragmented DNA, is substantially reduced in transgenic mice. In wild-type animals, almost 50% of ganglion cells degenerate in the 24 hr after the lesion, whereas almost the entire ganglion cell population survives axotomy in transgenic mice. Therefore, overexpression of bcl-2 is effective in preventing degeneration of this neuronal population, raising the possibility that ganglion cells are dependent on the endogenous expression of bcl-2 for survival. The remarkable rescue capacity of bcl-2 overexpression in these neurons makes it an interesting model for studying natural cell death and responses to injury in the CNS.

Cell death can be ascribed to one of two distinct modes of degeneration: apoptosis (programmed or active cell death) or necrosis (passive degeneration). While apoptosis is generally assumed to occur in physiological conditions such as normal development or tissue turnover, necrotic cell degeneration is induced in pathological situations. Here we report that also in a pathological situation, such as after axotomy in the CNS, apoptotic type of cell death comes into play: following intracranial transection of the optic nerve in the neonatal rat in vivo, retinal ganglion cells undergo an active, apoptotic cell death. In fact, the administration of protein synthesis inhibitors (actinomycin D and cycloheximide) prevents the appearance of pyknotic nuclei as well as of fragmented DNA of ganglion cells at 24 hr postlesion. Correspondingly, the number of surviving cells after actinomycin D and cycloheximide treatment is comparable to normal, unlesioned retinas. In addition, cycloheximide decreases the number of pyknotic ganglion cells during spontaneous cell death.

As the mature nervous system is sculpted out of its embryonic anlage, regressive events are a surprisingly common feature. As one example, the establishment of adult innervation in the CNS and PNS often involves a massive withdrawal of previously formed functional synapses. In the cerebellum, the one-to-one relationship of inferior olivary climbing fibers to Purkinje cells is preceded by a transient stage in which each Purkinje cell is multiply innervated. The regulation of this regressive event is still not fully understood; previous studies suggested that some stimulus from the maturing granule cells is necessary. We have used the lurcher (Lc) mutation as a model system in which to study this phenomenon. In lurcher mice, Purkinje cells degenerate during the first few postnatal weeks, after receiving synaptic contacts from both inferior olivary neurons and granule cells. We have recorded the climbing fiber responses both in lurcher mutants at postnatal days 14–20 (P14-P20) and in adult lurcher chimeras. In the latter, experimental genetics are used to create a situation in which untreated wild-type Purkinje cells are present in an environment that ranges from 100% wild-type to nearly mutant. We found that in P14-P16 lurcher mutants, most of the cells recorded (75%) remained polyinnervated, whereas in wild-type control mice, only 10% of the Purkinje cells retained their multiple innervation. By P18-20, it was difficult to find Purkinje cells in the lurcher mutants that would withstand an intracellular electrode. Nonetheless, in those cells that were successfully impaled, most remained multiply innervated. By this age in wild-type mice, 100% of the Purkinje cells are monoinnervated.(ABSTRACT TRUNCATED AT 250 WORDS)