Nogo-66 receptor antagonist peptide (NEP1-40) administration promotes functional recovery and axonal growth after lateral funiculus injury in the adult rat.

OBJECTIVE: The myelin protein Nogo inhibits axon regeneration by binding to its receptor (NgR) on axons. Intrathecal delivery of an NgR antagonist (NEP1-40) promotes growth of injured corticospinal axons and recovery of motor function following a dorsal hemisection. The authors used a similar design to examine recovery and repair after a lesion that interrupts the rubrospinal tract (RST). METHODS: Rats received a lateral funiculotomy at C4 and NEP1-40 or vehicle was delivered to the cervical spinal cord for 4 weeks. Outcome measures included motor and sensory tests and immunohistochemistry. RESULTS: Gait analysis showed recovery in the NEP1-40-treated group compared to operated controls, and a test of forelimb usage also showed a beneficial effect. The density of labeled RST axons increased ipsilaterally in the NEP1-40 group in the lateral funiculus rostral to the lesion and contralaterally in both gray and white matter. Thus, rubrospinal axons exhibited diminished dieback and/or growth up to the lesion site. This was accompanied by greater density of 5HT and calcitonin gene-related peptide axons adjacent to and into the lesion/matrix site in the NEP1-40 group. CONCLUSIONS: NgR blockade after RST injury is associated with axonal growth and/or diminished dieback of severed RST axons up to but not into or beyond the lesion/matrix site, and growth of serotonergic and dorsal root axons adjacent to and into the lesion/matrix site. NgR blockade also supported partial recovery of function. The authors' results indicate that severed rubrospinal axons respond to NEP1-40 treatment but less robustly than corticospinal, raphe-spinal, or dorsal root axons.

Death in the balance: alternative participation of the caspase-2 and -9 pathways in neuronal death induced by nerve growth factor deprivation.

The data presented here demonstrate that sympathetic neurons have the potential to activate two alternative caspase-dependent pathways either of which is capable of mediating death induced by NGF deprivation and that these neurons have the potential to switch from one pathway to the other. The presence of these two alternative pathways to trophic factor deprivation-induced death may have implications for ensuring the correct development of the nervous system. In wild-type neurons, a caspase-2-dependent pathway is required for death, and a caspase-9-dependent pathway appears to be suppressed by endogenous inhibitors of apoptosis proteins (IAPs). In contrast, for caspase-2-null neurons, death is dependent on the caspase-9 pathway. The mechanism underlying the shift is the result of a threefold compensatory elevation of caspase-9 expression and a doubling of levels of direct IAP binding protein with low pI/(DIABLO)/second mitochondria-derived activator of caspase (Smac), an IAP inhibitor, both at the mRNA and protein levels [corrected]. These findings resolve seemingly discrepant findings regarding the roles of various caspases after NGF deprivation and raise a cautionary note regarding the interpretation of findings with caspase-null animals. The choice of the death-mediating caspase pathway in the sympathetic neurons is thus dependent on the regulated relative expression of components of the pathways including those of caspases, IAPs, and IAP inhibitors.

beta-Amyloid-induced neuronal apoptosis requires c-Jun N-terminal kinase activation.

beta-Amyloid (A beta) has been strongly implicated in the pathophysiology of Alzheimer's disease (AD), but the means by which the aggregated form of this molecule induces neuronal death have not been fully defined. Here, we examine the role of the c-Jun N-terminal kinases (JNKs) and of their substrate, c-Jun, in the death of cultured neuronal PC12 cells and sympathetic neurons evoked by exposure to aggregated A beta. The activities of JNK family members increased in neuronal PC12 cells within 2 h of A beta treatment and reached 3--4-fold elevation by 6 h. To test the role of these changes in death caused by A beta, we examined the effects of CEP-1347 (KT7515), an indolocarbazole that selectively blocks JNK activation. Inclusion of CEP-1347 (100--300 nM) in the culture medium effectively blocked the increases in cellular JNK activity caused by A beta and, at similar concentrations, protected both PC12 cells and sympathetic neurons from A beta-evoked-death. Effective protection required addition of CEP-1347 within 2 h of A beta treatment, indicating that the JNK pathway acts relatively proximally and as a trigger in the death mechanism. A dominant-negative c-Jun construct also conferred protection from A beta-evoked death, supporting a model in which JNK activation contributes to death via activation of c-Jun. Finally, CEP-1347 blocked A beta-stimulated activation of caspase-2 and -3, placing these downstream of JNK activation. These observations implicate the JNK pathway as a required element in death evoked by A beta and hence identify it as a potential therapeutic target in AD.

Caspase-2 mediates neuronal cell death induced by beta-amyloid.

beta-amyloid (Abeta) has been proposed to play a role in the pathogenesis of Alzheimer's disease (AD). Deposits of insoluble Abeta are found in the brains of patients with AD and are one of the pathological hallmarks of the disease. It has been proposed that Abeta induces death by oxidative stress, possibly through the generation of peroxynitrite from superoxide and nitric oxide. In our current study, treatment with nitric oxide generators protected against Abeta-induced death, whereas inhibition of nitric oxide synthase afforded no protection, suggesting that formation of peroxynitrite is not critical for Abeta-mediated death. Previous studies have shown that aggregated Abeta can induce caspase-dependent apoptosis in cultured neurons. In all of the neuronal populations studied here (hippocampal neurons, sympathetic neurons, and PC12 cells), cell death was blocked by the broad spectrum caspase inhibitor N-benzyloxycarbonyl-val-ala-asp-fluoromethyl ketone and more specifically by the downregulation of caspase-2 with antisense oligonucleotides. In contrast, downregulation of caspase-1 or caspase-3 did not block Abeta(1-42)-induced death. Neurons from caspase-2 null mice were totally resistant to Abeta(1-42) toxicity, confirming the importance of this caspase in Abeta-induced death. The results indicate that caspase-2 is necessary for Abeta(1-42)-induced apoptosis in vitro.

BDNF and NT4/5 promote survival and neurite outgrowth of pontocerebellar mossy fiber neurons.

The neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3), and NT4/5 are all found in the developing cerebellum. Granule cells, the major target neurons of mossy fibers, express BDNF during mossy fiber synaptogenesis. To determine whether neurotrophins contribute to the development of cerebellar afferent axons, we characterized the effects of neurotrophins on the growth of mossy fiber neurons from mice and rats in vitro. For a mossy fiber source, we used the basilar pontine nuclei (BPN), the major source of cerebellar mossy fibers in mammals. BDNF and NT4/5 increased BPN neuron survival, neurite outgrowth, growth cone size, and elongation rate, while neither NT3 nor NGF increased survival or outgrowth. In addition, BDNF and NT4/5 reduced the size of neurite bundles. Consistent with these effects, in situ hybridization on cultured basilar pontine neurons revealed the presence of mRNA encoding the TrkB receptor which binds both BDNF and NT4/5 with high affinity. We detected little or no message encoding the TrkC receptor which preferentially binds NT3. BDNF and NT4/5 also increased TrkB mRNA levels in BPN neurons. In addition to previously established functions as an autocrine/paracrine trophic factor for granule cells, the present results indicate that cerebellar BDNF may also act as a target-derived trophic factor for basilar pontine mossy fibers.

Collapsin-1/semaphorin-III/D is regulated developmentally in Purkinje cells and collapses pontocerebellar mossy fiber neuronal growth cones.

Most axons in the CNS innervate specific subregions or layers of their target regions and form contacts with specific types of target neurons, but the molecular basis of this process is not well understood. To determine whether collapsin-1/semaphorin-III/D, a molecule known to repel specific axons, might guide afferent axons within their cerebellar targets, we characterized its expression by in situ hybridization and observed its effects on mossy and climbing fiber extension and growth cone size in vitro. In newborn mice sema-D is expressed by cerebellar Purkinje cells in parasagittal bands located medially and in some cells of the cerebellar nuclei. Later, sema-D expression in Purkinje cells broadens such that banded expression is no longer prominent, and expression is detected in progressively more lateral regions. By postnatal day 16, expression is observed throughout the cerebellar mediolateral axis. Collapsin-1 protein, the chick ortholog of sema-D, did not inhibit the extension of neurites from explants of inferior olivary nuclei, the source of climbing fibers that innervate Purkinje cells. In contrast, when it was applied to axons extending from basilar pontine explants, a source of mossy fiber afferents of granule cells, collapsin-1 caused most pontine growth cones to collapse, as evidenced by a reduction in growth cone size of up to 59%. Moreover, 63% of pontine growth cones arrested their extension or retracted. Its effects on mossy fiber extension and its distribution suggest that sema-D prevents mossy fibers from innervating inappropriate cerebellar target regions and cell types.

Protection of retinal ganglion cells from natural and axotomy-induced cell death in neonatal transgenic mice overexpressing bcl-2.

Approximately half of the retinal ganglion cells (RGCs) present in the rodent retina at birth normally die during early development. Overexpression of the photo-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 study in vivo the 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.

A developmentally regulated nerve growth factor-induced gene, VGF, is expressed in geniculocortical afferents during synaptogenesis.

The expression of the nerve growth factor-inducible gene VGF has been examined by in situ hybridization. Western blot and immunohistochemical studies in the developing and adult rat central nervous system, with particular emphasis on the visual system. Both the messenger RNA and the protein are particularly abundant in the developing dorsal lateral geniculate nucleus, appearing, respectively, at embryonal day 16 and 18. After its onset at E16, VGF messenger RNA expression increases progressively in the dorsal lateral geniculate nucleus and remains high during the first two post-natal weeks; afterwards, it gradually decreases and, at the offset of the plasticity period, it reaches very low levels maintained in adulthood. A similar time course has been observed for VGF protein in the dorsal lateral geniculate nucleus area, by semi-quantitative Western blots. In addition to the presence of the protein in the geniculate neurons, a strong, transient immunoreactivity has been found at the embryonic cortical subplate at E18, reflecting the presence of the antigen in axonal terminals originating from thalamic neurons. Interestingly, we found that the blockade of afferent electrical activity by intraocular injection of tetrodotoxin strongly reduces the level of VGF messenger RNA in the dorsal lateral geniculate nucleus. Although the function of the VGF protein is not known, it had been previously proposed that VGF could be a precursor for neuropeptide/s. The spatiotemporal expression of VGF, together with the observation of a regulation by electrical activity, suggest that this protein may be relevant in the process of synaptogenesis and/or synaptic stabilization in the developing geniculocortical connections.

Nerve growth factor reduces apoptosis of axotomized retinal ganglion cells in the neonatal rat.

It has recently been reported that the degeneration of retinal ganglion cells induced by transection of the optic nerve in the neonatal rat is due to an active process of apoptosis, as opposed to passive necrosis. Here we tested whether the administration of the trophic factor nerve growth factor could prevent the apoptotic death of the axotomized cells. We administered nerve growth factor by two intraocular injections, one immediately after the lesion and the second 12 h later. The retinas were taken at 24 h post-lesion and stained as whole mounts with Cresyl Violet. Pyknotic as well as surviving cells were counted in the retinal ganglion cell layer. In this layer at least 95% of the total cell population is composed by ganglion cells, as revealed by retrogradely labelling these cells with horseradish peroxidase injected in the superior colliculi. We found that intraocular administration of nerve growth factor diminishes the degeneration induced by optic nerve transection in the neonatal rat. After nerve growth factor injection, in fact, the number of pyknotic cells is reduced by 39% compared with controls (lesioned, injected with saline); in addition, nerve growth factor also increases the survival of retinal ganglion cells by 30% at 24 h post-lesion.

Apoptotic cell death induced by optic nerve lesion in the neonatal rat.

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.

Role of the target in synapse elimination: studies in cerebellum of developing lurcher mutants and adult chimeric mice.

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)

A positional marker for the dorsal embryonic retina is homologous to the high-affinity laminin receptor.

In a search for determinants of positional information in the embryonic eye, we isolated two monoclonal antibodies that label strongly the dorsal part of the undifferentiated embryonic retina in mammals, bird and cold-blooded vertebrates. In the chick, the optic tectum is labeled in a corresponding fashion, the ventral tectum more heavily than the dorsal tectum. Through biochemical and molecular analysis both antibodies were found to recognize a protein that has been cloned repeatedly, first in a screen with antibodies to the '68K-laminin receptor' (Wewer et al. (1986) Cancer Res. 47, 5691-5698), a name that may not exhaustively describe its function. Western blots show the protein to be present in most or all tissues, and Western and Southern blots reveal a high degree of conservation in the detected signals up to invertebrates and bacteria. Despite the very strong and selective labeling of the dorsal retina in conventional immunohistochemical preparations, the protein and its mRNA are present in even amounts throughout the embryonic retina, as demonstrated by Western and Northern blots of bisected retinas, and immunohistochemically in retinas fixed with ethylene glycole bissuccinimide (EGS), an NH2-group crosslinker with very long spacer arm. This indicates that the dorsoventral asymmetry in the embryonic retina is not in the amount but in the configuration of this protein; whether this difference relates to laminin binding is not known.