UNLABELLED:FK506 and its non-immunosuppressive derivatives represent a class of pharmacological agents referred to as immunophilin ligands that have been reported to promote neuroregeneration and survival in several experimental models; however their cellular and molecular mechanisms of action have not been well established. Here we characterize a new immunophilin ligand that interacts with both FK506 binding protein 12 (FKBP12) and FKBP52, and demonstrate that JNJ460 induces neurite outgrowth from freshly explanted dorsal root ganglia (DRG) in a Schwann cell-dependent manner. Purified cultures of neurons fail to respond to these drugs, but cultures containing Schwann cells and neurons respond with neurite outgrowth, as do neurons grown in conditioned medium from JNJ460-treated Schwann cells. Using microarray analysis and a transcription reporter assay, we show that JNJ460 induces a series of transcriptional changes that occur in a temporal cascade. Among the Schwann cell-expressed genes upregulated following JNJ460 treatment is the POU transcription factor SCIP, which has been shown to regulate Schwann cell gene transcription and differentiation. JNJ460 potentiated transforming growth factor beta (TGF-beta)-induced transcriptional activation and SCIP induction in Schwann cells, by altering the interaction between FKBP12 and the TGF-beta type I receptor, TbetaR1. Finally, to test whether JNJ460 enhances neurite regeneration in vivo, we treated animals with JNJ460 for 30 days following mechanical transection of the sciatic nerve and demonstrated myelin and axonal hypertrophy at the ultrastructural level. Collectively, these data suggest that Schwann cells play an important role in the biological effects of immunophilin ligands by affecting neuron-glial signaling during regeneration. SUMMARY:The cellular and molecular mechanisms responsible for the regenerative effects of immunophilin ligands are not well understood. Here we show that the neuritogenic effects of JNJ460 in a DRG model depend on interactions between neurons and Schwann cells. Treatment of purified Schwann cells with JNJ460 alters Schwann cell gene expression, and promotes the generation of factors that act on neurons. These data indicate that Schwann cells play an important role in the actions of immunophilin ligands.

Astrocytes are a major cell type in the mammalian central nervous system (CNS). The ability to obtain virtually pure populations of these cells makes it possible to study their function as isolated cells or in mixed populations where they support the growth and survival of surrounding neurons. Unlike other mature CNS cells, mature astrocytes maintain the lifelong ability to reenter the cell cycle. The first isolation procedure described in this unit takes advantage of the proliferative ability of these cells, as does the second, except that no antibody or complement treatment is required. A procedure for detecting glial fibrillary acidic protein (GFAP), which is present in most astrocytes in vivo and virtually all astrocytes in vitro and is a useful marker for assessing the purity of cultures, is also presented.

Astrocytes respond to contact with neurons by cell-cycle arrest and complex process formation. In our effort to discover the molecular mechanisms that underlie this phenomenon we have identified a known tetraspanin, CD81, as a critical component of astrocyte responses to neuronal differentiation signals. Here we show that CD81 is expressed on the surface of the astrocyte and that its expression level can be modulated by contact with neurons. Further, using three separate antibodies, 2F7, Eat1, and Eat2, which recognize unique epitopes in the extracellular domains of the CD81 protein, we show that there is a unique domain, recognized by Eat1, that is required for astrocyte cell-cycle withdrawal in response to neurons. This is likely due to conformational changes in the CD81 molecule, as inclusion of 2F7 actually augments neuron-induced astrocyte growth arrest. The critical nature of CD81 in normal astrocyte-neuron biology was confirmed by using mice in which CD81 had been deleted by homologous recombination. Astrocytes null at the CD81 locus were blind to the proliferative arrest encoded on the neuronal cell surface. Taken together, these data strongly suggest that CD81 is a critical regulator of neuron-induced astrocytic differentiation.

The POU family of transcription factors plays a vital role in controlling cell-fate determination and the timing of cellular events in a number of tissues, including the nervous system. One such POU protein, SCIP, is expressed by Schwann cells in a tightly delimited developmental window termed promyelination. In the PNS, promyelination is functionally defined as the period following Schwann cell exit from the cell-cycle, but prior to the onset of myelination. Previous transgenic and gene ablation studies have shown that SCIP is a myelin-competence factor in the Schwann cell, where it is required for entry into, and the subsequent maintenance of promyelination. To further understand the molecular biology of the promyelination-to-myelination transition in the Schwann cell, we have undertaken a series of DDRTPCR studies to identify genes that are expressed during this phenotypic flux. Through these studies we have identified another POU gene, Brn-5, the expression of which has not previously been appreciated in the Schwann cell. Here we show that the developmental expression patterns of Brn-5 and SCIP are inverse, with Brn-5 stably expressed in the adult myelinating Schwann cell, but virtually absent during promyelination. Further, we show that the induction of the two genes is independent, with SCIP induction requiring activation of adenyl cyclase, whereas Brn-5 induction requires only GGF2. In addition, the induction of Brn-5 is exquisitely sensitive to neuregulin concentration, with higher levels inhibiting its expression. Following nerve injury, when GGF2 levels are elevated in the distal nerve, Brn-5 expression disappears, and SCIP is reexpressed.

4 showed that M. leprae binds specifically to the G-domain, which is located at the C-terminal end of the a2 chain of laminin-2.

Leprosy, caused by infection with Mycobacterium leprae, has widely varied clinical manifestations. Despite the systemic clinical heterogeneity, there is universal involvement of the peripheral nerves in all leprosy patients 1 Pfaltzgraff R.E. Bryceson A. Hastings R.C. Leprosy. Churchill Livingston, 1985: 134-176 Google Scholar . Even in patients with minimal or no clinical manifestations of disease, histopathological examination of skin frequently reveals infection of one or more peripheral nerves. Consequently, this infection is the leading cause of all non-traumatic peripheral neuropathy worldwide 2 Nations S.P. et al. Semin. Neurol. 1998; 18: 113-124 Crossref PubMed Google Scholar ; infection can result in chronic demyelination/remyelination, often leading to calcification and permanent loss of neural function 3 Sabin T.D. Swift T.R. Jacobson R.R. Dyck P.K.T.P.J. Peripheral Neuropathy. W.B. Saunders, 1984: 1354-1379 Google Scholar . M. leprae appears to have a specific tropism for peripheral nerves, where it resides primarily in the myelinating Schwann cells 4 Stoner G.L. Lancet. 1979; ii: 994-996 Abstract Scopus (62) Google Scholar and in macrophages that have migrated through the blood–nerve barrier into the endoneural space (Fig. 1). Although it is clear that the Schwann cells are targeted, the molecular mechanisms that facilitate M. leprae entry into the nerve and the subsequent infection of Schwann cells have been elusive.

Schwann cells are the glial cells of the peripheral nervous system (PNS) which both myelinate and provide trophic support for their associated axons. Their functions are critical for proper development, homeostasis, and regeneration of the PNS. Schwann cells can be isolated and expanded in culture. Such a culture can be used as a source of myelinating glia in culture or as a source of Schwann cells for various cell and molecular biological experiments. The proliferative ability of these cells is exploited in the isolation procedures presented here.

The ability of neurons to survive and to target axonal growth requires a coordinated series of cell extrinsic and intrinsic events. Previously, in a cellular model for neuronal differentiation, we showed that pheochromocytoma (PC12) cells expressing v-Crk, an oncogenic form of the SH2/SH3-containing c-Crk adaptor protein, potentiates axonal growth and prolongs nerve growth factor (NGF)-independent survival. In the present study, we have generated transgenic mice that express v-Crk in sensory, motor, and enteric neurons by placing v-crk under the control of the neuron-specific peripherin promoter. In contrast to wild-type (wt) mice, dorsal root ganglia (DRG) neurons explanted from post-natal day 1 transgenic mice demonstrated a reduced dependence on trophic factors for both survival and axonogenesis. v-Crk also caused an increase in the number of surviving spinal motor neurons (SMN), and interestingly, upon staining of sternomastoid muscle fibers with rhodamine conjugated alpha-bungarotoxin, many muscle fibers displayed an apparent increase in volume of motor end plates, and an increase in complexity of neuromuscular junctions (NMJ). Our data suggest that v-Crk may be involved in transducing extracellular signals to regulate cytoskeletal organization, and may act on an intrinsic determinant for axonal growth in a variety of neural types including sensory and motor neurons during development.

The difference in regenerative capacity between the PNS and the CNS is not due to an intrinsic inability of central neurons to extend fibers. Rather, it is probably related to the environment in the CNS that is either repulsive to axonal outgrowth and/or nonsupportive of axonal elongation. In contrast, the PNS both supports and allows for axonal elongation after injury. The Schwann cell, which is the glial cell of the PNS, is strictly required for peripheral regeneration. Here we discuss recent work describing the biology of Schwann cell- dependent regeneration, discuss what is known of the molecular basis of this phenomenon, and how it might apply to the damaged CNS. NEUROSCIENTIST 5:208-216, 1999

The formation of the myelin sheath requires the coordinated regulation of a number of gene products, both glial and axonal. These include the myelin structural genes, enzymes involved in lipid biosynthetic pathways, transcriptional regulators, trophic and recognition factors. Alterations in gene dosage or gene expression timing can have profoundly deleterious effects on either the establishment or maintenance of the myelin sheath. In this review, the major structural genes are described, as are the consequences of changes in gene dosage which occur as a result of either natural mutations or experimentally mediated events. While it is clear that our knowledge has expanded greatly with regard to these events, there continue to be large gaps, especially within the genetics of gene regulation and the mechanisms of glial axon interactions. MRDD Research Reviews 1998; 4:179–186 © 1998 Wiley-Liss, Inc.

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.

We have previously described transgenic mice that harbor a dominant-negative antagonist of the POU protein SCIP (termed ΔSCIP). Native SCIP is expressed in promyelinating Schwann cells, where it represses expression of the myelin structural genes. The ΔSCIP mice display morphologic and behavioral abnormalities, including decreased axonal diameter, increased myelin thickness, developmentally early myelination, and clinical features of neuropathy. To assess the neurophysiologic correlates of these abnormalities, a series of electrophysiologic tests was performed. Despite having smaller diameter axons, mice expressing the ΔSCIP transgene had similar maximum conduction velocities in caudal, sural, and tibial nerves compared to wild-type controls. Therefore, conduction in ΔSCIP animals was faster than predicted by axon diameter alone. Compound amplitude responses were 38% higher in the ΔSCIP caudal nerve. ΔSCIP tibial F-wave responses showed less difference between minimum and maximum latencies than controls, suggesting less variance between fastest and slowest conducting fibers. These data further characterize the functional components of the ΔSCIP phenotype. In addition, these studies address the physiologic sequelae of altering the g-ratio in the absence of demyelination or axonal degeneration. J. Neurosci. Res. 50:821–828, 1997. © 1997 Wiley-Liss, Inc.

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.

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.

Several antigen-specific immune responses are known to occur in discrete aggregates of dendritic cells (DC) and lymphocytes. We have used a polyclonal model, the mitogenesis of T cells that have been modified with sodium periodate, to evaluate the significance of cell-cell clustering. Firstly, we found that clustering precedes the onset of DNA synthesis by a day. Within 2 hr, virtually all of the added dendritic cells and most of the T cells that will respond have formed clusters. The T cells then progressively release and become responsive to interleukin-2 over 18 hr and DNA synthesis begins at 24 hr. Secondly, clustering with dendritic cells appears to be essential for mitogenesis. If dendritic cells are eliminated, the clusters disassemble and subsequent proliferation is reduced. Clustering and proliferation can be restored with dendritic cells that are syngeneic or allogeneic with the initial inoculum. DC are inactive if they are treated with ultraviolet light, formaldehyde or heat. Thirdly, the non-clustered cells do not synthesize DNA even when mixed with the clusters. However, non-clusters will respond when supplemented with additional DC. We conclude that clustering with DC precedes and seems essential for T-cell mitogenesis in the periodate model.

The extent to which M. leprae and its products induced suppression of T lymphocyte proliferation in vitro was evaluated. M. leprae antigens suppressed T cell proliferation in response to mitogens and antigens in both lepromatous and tuberculoid patients, as well as controls never exposed to M. leprae or M. leprae endemic areas. Both soluble and particulate fractions of M. leprae were found to suppress proliferation in a dose-dependent manner. The extent of suppression was inversely related to the proliferative response of the donors mononuclear cells to M. leprae. Evidence indicates that M. leprae contains both stimulatory and suppressive molecules for T cells. One such suppressive antigen, Lipoarabinomannan (LAM)-B of M. leprae, also suppressed the proliferative response of tuberculoid patients. Suppression was also observed with the LAM-B of M. tuberculosis. The suppressive effects observed were not due to the toxicity of the antigen. Some of the suppressive activity was mediated by T8+ suppressor cells and was expressed in both lepromatous and tuberculoid patients. We suggest that previous sensitization to M. leprae and other cross-reactive mycobacterial antigens determines the sensitivity of T cells to the suppressive effects of M. leprae antigens.