Topographic mapping from the retina to the midbrain is controlled by relative but not absolute levels of EphA receptor signaling.

Topographic maps are a fundamental feature of sensory representations in nervous systems. The formation of one such map, defined by the connection of ganglion cells in the retina to their targets in the superior colliculus of the midbrain, is thought to depend upon an interaction between complementary gradients of retinal EphA receptors and collicular ephrin-A ligands. We have tested this hypothesis by using gene targeting to elevate EphA receptor expression in a subset of mouse ganglion cells, thereby producing two intermingled ganglion cell populations that express distinct EphA receptor gradients. We find that these two populations form separate maps in the colliculus, which can be predicted as a function of the net EphA receptor level that a given ganglion cell expresses relative to its neighbors.

Induction of postnatal schwann cell death by the low-affinity neurotrophin receptor in vitro and after axotomy.

Schwann cells express the low-affinity neurotrophin receptor (p75), but no role for either the neurotrophins or their cognate receptors in Schwann cell development has been established. We have found that Schwann cells isolated from postnatal day 1 (P1) or P2 mice that were p75-deficient exhibited potentiated survival compared to wild-type cells after growth factor and serum withdrawal. There was, however, no disparity in the survival of p75-deficient and wild-type Schwann cells isolated at embryonic day 15, suggesting that the death-inducing effects of p75 are developmentally regulated. A comparable degree of cell death was also observed in the sciatic nerves of both wild-type and p75-deficient mice at P1. However, 24 hr after axotomy, there was a 13-fold increase in the percentage of apoptotic nuclei in the distal nerve stumps of the transected sciatic nerves of neonatal wild-type but not p75-deficient mice. The expression of both the p75 and nerve growth factor (NGF) genes was upregulated after axotomy in neonatal wild-type nerves. Collectively, these results suggest that NGF-mediated activation of p75 is likely to be an important mediator of Schwann cell apoptosis in the context of peripheral nerve injury.

Evectins: vesicular proteins that carry a pleckstrin homology domain and localize to post-Golgi membranes.

We have identified two vesicular proteins, designated evectin (evt)-1 and -2. These proteins are approximately 25 kDa in molecular mass, lack a cleaved N-terminal signal sequence, and appear to be inserted into membranes through a C-terminal hydrophobic anchor. They also carry a pleckstrin homology domain at their N termini, which potentially couples them to signal transduction pathways that result in the production of lipid second messengers. evt-1 is specific to the nervous system, where it is expressed in photoreceptors and myelinating glia, polarized cell types in which plasma membrane biosynthesis is prodigious and regulated; in contrast, evt-2 is widely expressed in both neural and nonneural tissues. In photoreceptors, evt-1 localizes to rhodopsin-bearing membranes of the post-Golgi, an important transport compartment for which specific molecular markers have heretofore been lacking. The structure and subcellular distribution of evt-1 strongly implicate this protein as a mediator of post-Golgi trafficking in cells that produce large membrane-rich organelles. Its restricted cellular distribution and genetic locus make it a candidate gene for the inherited human retinopathy autosomal dominant familial exudative vitreoretinopathy and suggest that it also may be a susceptibility gene for multiple sclerosis.

Accelerated nerve regeneration mediated by Schwann cells expressing a mutant form of the POU protein SCIP.

After injury, the peripheral nervous system (PNS) is capable of full regeneration and recovery of function. Many molecular events that are the hallmarks of the regenerating PNS are recapitulations of developmental processes. The expression of one such molecule, the POU transcription factor suppressed cAMP-inducible POU protein (SCIP), is required for the establishment of normal nerves and is reexpressed during regeneration. Here we describe markedly accelerated regeneration and hypertrophy of both myelin and axons in transgenic mice that express an amino-terminal deletion of the SCIP molecule. This mutant SCIP molecule retains the POU-specific and POU homeodomain moieties, which allow for both DNA binding and some protein-protein interaction. We demonstrate that the transgene indirectly effects dramatic axonal changes. This is the first demonstration of a genetically controlled acceleration of neural regeneration.

A transgenic mouse model for human hereditary neuropathy with liability to pressure palsies.

Mutations in the gene encoding peripheral myelin protein 22 (PMP22) account for several inherited peripheral neuropathies in humans. We now show that transgenic mice expressing antisense PMP22 RNA exhibit modestly reduced levels of PMP22 together with a phenotype that is reminiscent of hereditary neuropathy with liability to pressure palsies (HNPP), a human disease caused by a 1.5-Mb deletion of a chromosome 17 region that contains the PMP22 gene. Transgenic antisense homozygotes display a striking movement disorder and a slowing of nerve conduction that worsens with age. Morphological analysis of peripheral nerves demonstrates that a subset of axons have thickened myelin sheaths and tomacula in young adults, with significant myelin degeneration detected in older animals. Together with other recent work, these data suggest that dosage of the PMP22 gene alone underlies the pathophysiology observed in HNPP and related disorders.

Increased proliferation of precursor cells in the adult rat brain after targeted lesioning.

There have been a number of reports on the proliferation of a subset of precursor cells in the subventricular zone of the lateral ventricles in the adult mammalian brain. Here we report on studies that sought to ascertain whether these cells could respond to a targeted lesion of the adult brain by increasing their proliferative rate. We have lesioned the fimbria fornix, a major pathway of septal cholinergic fibers. Previous reports demonstrated that such a lesion results in the loss of neurons in both the basal forebrain and in the CA1 field of hippocampus, without direct injury to either tissue. Ten days after making such a lesion in adult rats, the animals were given serial injections of [3H]thymidine and sacrificed after a final injection. The brains were processed for both immunocytochemistry and autoradiography. Our data demonstrate a two-fold increase over the basal proliferative rate in animals that had received such a lesion. We used a panel of antibodies to ascertain the identity of the proliferating cells. The only clearly identifiable cells that were [3H]thymidine-positive were astrocytes, based on GFAP staining. The remainder of the cells were of a null phenotype.

Cell death in the Schwann cell lineage and its regulation by neuregulin.

The development of Schwann cells, the myelin-forming glial cells of the vertebrate peripheral nervous system, involves a neonatal phase of proliferation in which cells migrate along and segregate newly formed axons. Withdrawal from the cell cycle, around postnatal days 2-4 in rodents, initiates terminal differentiation to the myelinating state. During this time, Schwann cell number is subject to stringent regulation such that within the first postnatal week, axons and myelinating Schwann cells attain the one-to-one relationship characteristic of the mature nerve. The mechanisms that underly this developmental control remain largely undefined. In this report, we examine the role of apoptosis in the determination of postnatal Schwann cell number. We find that Schwann cells isolated from postnatal day 3 rat sciatic nerve undergo apoptosis in vitro upon serum withdrawal and that Schwann cell death can be prevented by beta forms of neuregulin (NRG-beta) but not by fibroblast growth factor 2 or platelet-derived growth factors AA and BB. This NRG-beta-mediated Schwann cell survival is apparently transduced through an ErbB2/ErbB3 receptor heterodimer. We also provide evidence that postnatal Schwann cells undergo developmentally regulated apoptosis in vivo. Together with other recent findings, these results suggest that Schwann cell apoptosis may play an important role in peripheral nerve development and that Schwann cell survival may be regulated by access to axonally derived NRG.

Premature Schwann cell differentiation and hypermyelination in mice expressing a targeted antagonist of the POU transcription factor SCIP.

The transcription factor SCIP is expressed by immature neurons and Schwann cells of the developing central and peripheral nervous systems, but this expression is largely extinguished when these cells fully differentiate. In immature Schwann cells in vitro, SCIP acts as a repressor of the myelin-specific genes that mark full differentiation. We have generated transgenic mice that express a dominant-negative antagonist of SCIP, specifically targeted to developing Schwann cells. This antagonist--designated delta SCIP--is transcriptionally inactive, but retains full DNA-binding activity. Mice that express delta SCIP exhibit a debilitating peripheral neuropathy that results from developmentally advanced Schwann cell differentiation, over-expression of myelin-specific gene products, and hypermyelination. These results suggest that SCIP functions as a transcriptional sensor of differentiation cues and thereby regulates the time and place at which Schwann cells differentiate.

Neocortical pathology in chronic murine retroviral encephalitis.

Retroviral infection of the central nervous system (CNS) causes chronic functional and morphological damage in a wide variety of mammals. Neuropathological studies have focused on subcortical pathology, however, the neocortex is also affected. Because studies of human CNS pathology have been limited to the use of material from terminal stages of disease, we used two neuropathogenic murine leukemia virus (MuLV) models to study the development of neocortical damage. MuLV infection caused spongiform change in the spinal cord, brainstem and cerebellum but not in the cerebrum. However, over the course of disease, we observed a reduction of neocortical thickness, accompanied by diminished neuronal and dendritic spine density. Electron microscopic studies showed minimal to no ultrastructural alterations of dendritic spines. Since there was no evidence of extensive direct viral infection of the neocortical neurons or glia at the ultrastructural level, we hypothesize that neocortical damage may be an indirect effect of subcortical retroviral infection.

In vivo model of HIV infection of the human brain.

Approximately one quarter of the AIDS patients have severe HIV encephalitis with diffuse neuronal damage that may be mediated by immune factors secreted by CNS macrophages. Based on an in vitro brain microsphere model, we developed an in vivo system in which human embryonic brain tissue survives for several months in the interscapular fat pad of SCID mice. Coculture of human brain tissue with macrophages prior to transplantation resulted in infiltration of the microspheres by activated macrophages. When the macrophages were infected in vitro with a neurotropic HIV strain, viral particles were detected in vivo up to 3 months after transplantation. HIV-infected transplants contained multinucleated giant cells similar to those seen in HIV encephalitis. However, the neuroglial component degenerated in the fat pad of SCID mice. The absence of synaptogenesis in the human transplants suggests that the murine fat pad lacks adequate stimuli or support for human neuronal differentiation. To study neurologic damage associated with HIV infection, sites of implantation that stimulate synaptogenesis (e.g. murine CNS) will need to be explored. Based on these findings we conclude that transplantation of brain microspheres with HIV-infected macrophages into SCID mice may be an achievable model of HIV encephalitis.

Development of spongiform encephalopathy in retroviral infected mice.

The Cas-Br-E strain of murine leukemia virus is a neurovirulent retrovirus that induces progressive noninflammatory degeneration of the central nervous system (CNS). The molecular clone pNE-8 retains pathogenic properties of Cas-Br-E. The neurotropic determinants are known; however, the mechanism of neuropathogenesis is unknown. We examined the temporal development of disease after infection of SWR/J mice with pNE-8 virus. Development of CNS lesions, cellular targets of viral replication, accumulation of ubiquitinated proteins and integrity of blood-brain barrier were determined in mice infected with pNE-8 virus; and compared with uninfected, sham-infected, and nonneuropathogenic virus-infected mice. During 24 weeks of pNE-8 infection, noninflammatory spongiform lesions developed initially in the lumbar spinal cord and progressed to involve the brainstem and deep cerebellar nuclei. Virions and viral antigens accumulated for 18 weeks postinfection and then declined. Major sites of viral infection outside the CNS were splenic megakaryocytes, and skeletal muscle. Cellular targets of viral replication in the CNS included neurons, oligodendrocytes, and capillary endothelium. No astrocytic infection was observed; however, a reactive gliosis marked the development of clinical symptoms and histopathology. Spongiform lesions began as swelling of perivascular astrocytic processes. Intramyelinic vacuoles with splitting of myelin at major dense lines were prominent around dystrophic axons at later time points. Dendritic processes showed vacuolization and local degeneration. Viral particles were most commonly observed in extracellular spaces and within rough endoplasmic reticulum of neurons, oligodendrocytes, and splenic megakaryocytes. Infected megakaryocytes and regions of spleen containing viral aggregates showed accumulation of ubiquitinated proteins. Areas of histopathology in the CNS showed accumulation of ubiquitinated proteins but unlike spleen, viral proteins were not highly ubiquitinated. Disruption of the blood brain barrier was only evident at late stages of infection. In conclusion, the neuropathogenic damage associated with pNE-8 infection appears to be tightly associated with direct viral infection of oligodendroglia and neurons.

Immunoelectron microscopy of Alzheimer and Pick brain tissue labelled with the monoclonal antibody Alz-50.

Previous studies have shown that Alzheimer and Pick brains contain abnormally elevated amounts of a 68 Kd protein detected by the monoclonal antibody Alz-50. We have used immunoperoxidase and immunological techniques to localize Alz-50-reactive epitopes in sections from Alzheimer and Pick brains at the ultrastructural level. Detectable immunoreactivity was restricted to the paired helical filaments of Alzheimer neurofibrillary tangles and to the paired helical and straight filaments of Pick bodies. In Alzheimer tissue, the antibody also labelled scattered neuronal paired helical filaments that were not aggregated into neurofibrillary tangles. Amorphous components of Pick bodies and other constituents in the Alzheimer and Pick brain tissue were not immunostained.

Immuno-gold localization of prion filaments in scrapie-infected hamster brains.

The brains of scrapie-infected hamsters have been examined for the presence of structures antigenically related to the prion protein (PrP 27-30). Glutaraldehyde-perfused hamster brains, 72 days postinfection, were immunostained using rabbit monospecific antisera raised against synthetic peptides corresponding to the N-terminal 13 or 15 amino acids of PrP 27-30, and using rabbit antisera raised against infectious prions or PrP 27-30 purified from scrapie-infected hamster brains. Antisera to the synthetic peptides stained extracellular filaments in agreement with previous immunoperoxidase studies which used affinity-purified PrP 27-30 antibodies; in addition to subependymal and subpial localization, we show ventricular and perivascular staining. Using a colloidal gold-secondary antibody technique, we have demonstrated that the antibodies labeled filaments measuring 7 to 17 nm in diameter. Whereas most of the periventricular and perivascular filaments appeared extracellular, some appeared to be within processes intimately associated with ependymal cells, degenerating membranes of astrocytes, and neurites.