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D E Weinstein

Publications and source records attributed to D E Weinstein.

At least 19 recordsLinked to original sources
PubMed2004

A role for schwann cells in the neuroregenerative effects of a non-immunosuppressive fk506 derivative, jnj460.

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.

Animals↗
PubMed2001

CD81 regulates neuron-induced astrocyte cell-cycle exit.

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.

Animals↗
PubMed2001

The POU gene Brn-5 is induced by neuregulin and is restricted to myelinating Schwann cells.

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.

Animals↗
PubMed2001

Autologous Schwann cells drive regeneration through a 6-cm autogenous venous nerve conduit.

Peripheral nerve regeneration is a complex series of events, involving bi-directional interactions between regenerating axons and Schwann cells. These authors have demonstrated in their laboratory that nerves will regenerate through a 3-cm autogenous venous nerve conduit (AVNC) in an animal model and, with Dr. David Chiu, a similar distance in the human. They have shown that the upper limit of nerve regeneration in an empty AVNC was 3 cm, with no evidence of nerve growth at the 6-cm mark (no-growth model). Most recently, they have demonstrated rapid growth at 1 month in a 3-cm AVNC filled with Schwann cells, compared to poor-to-no-regrowth at 1 month in controls. While, in theory, Schwann-cell-derived growth factor should be sufficient to supplant the requirement for Schwann cells, in practice, therapies with growth factors have failed in clinical trials, with some resulting in severe morbidity and mortality for the subjects. The present study showed excellent nerve regeneration through a 6-cm AVNC with the addition of autologous Schwann cells, breaking the barrier in the previous no-growth model. In the first stage, autologous Schwann cells were harvested from the contralateral peroneal nerve of the rabbit and expanded in culture. The Schwann cells were purified to >99 percent homogeneity using differential adhesion and antibody-compliment-mediated cytolysis. In the second stage, 6 cm of gluteal vein were harvested and used as a conduit that was filled with either Matrigel or a slurry of Matrigel and 10(6)/ml autologous Schwann cells (n=6 control and 6 experimental animals). The non-donor side peroneal was exposed and transected, leaving a gap of 6 cm. The filled gluteal vein graft (AVNC) was then anastomosed to the proximal and distal peroneal nerve stumps, and the rabbits were allowed to recover. Four months postoperatively, the animals were subjected to transcardiac perfusion with EM grade fixative. The grafts were analyzed at the light and electronmicroscopic levels, and showed excellent growth of nerve at 6 cm, the distal end of the AVNC.

Animals↗
PubMed2000

Caveolin-1 expression inhibits Wnt/beta-catenin/Lef-1 signaling by recruiting beta-catenin to caveolae membrane domains.

Caveolin-1 is a principal component of caveolae membranes. In NIH 3T3 cells, caveolin-1 expression is dramatically up-regulated in confluent cells and localizes at areas of cell-cell contact. However, it remains unknown whether caveolin-1 is involved in cell-cell signaling. Here, we examine the potential role of caveolin-1 in regulating beta-catenin signaling. beta-Catenin plays a dual role in the cell, linking E-cadherin to the actin cytoskeleton and in Wnt signaling by forming a complex with members of the lymphoid enhancing factor (Lef-1) family of transcription factors. We show that E-cadherin, beta-catenin, and gamma-catenin (plakoglobin) are all concentrated in caveolae membranes. Moreover, we demonstrate that activation of beta-catenin/Lef-1 signaling by Wnt-1 or by overexpression of beta-catenin itself is inhibited by caveolin-1 expression. We also show that recombinant expression of caveolin-1 in caveolin-1 negative cells is sufficient to recruit beta-catenin to caveolae membranes, thereby blocking beta-catenin-mediated transactivation. These results suggest that caveolin-1 expression can modulate Wnt/beta-catenin/Lef-1 signaling by regulating the intracellular localization of beta-catenin.

3T3 Cells↗
PubMed1999

Targeted expression of an oncogenic adaptor protein v-Crk potentiates axonal growth in dorsal root ganglia and motor neurons in vivo.

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.

Animals↗
PubMed1998

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.

Animals↗
PubMed1997

Abnormal nerve conduction studies in mice expressing a mutant form of the POU transcription factor SCIP.

We have previously described transgenic mice that harbor a dominant-negative antagonist of the POU protein SCIP (termed deltaSCIP). Native SCIP is expressed in promyelinating Schwann cells, where it represses expression of the myelin structural genes. The deltaSCIP 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 deltaSCIP transgene had similar maximum conduction velocities in caudal, sural, and tibial nerves compared to wild-type controls. Therefore, conduction in deltaSCIP animals was faster than predicted by axon diameter alone. Compound amplitude responses were 38% higher in the deltaSCIP caudal nerve. DeltaSCIP 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 deltaSCIP phenotype. In addition, these studies address the physiologic sequelae of altering the g-ratio in the absence of demyelination or axonal degeneration.

Animals↗
PubMed1997

Prognostic significance of the delayed plantar reflex following spinal cord injury.

The delayed plantar reflex (DPR) is a pathologic flexor variant which requires unusually strong stimulation of the sole of the foot and is characterized by a prolonged interval between the stimulus and the response, slow and protracted plantarflexion of the great toe and/or other toes and slow return to the neutral position. The purpose of this study was to evaluate prospectively the functional outcome of spinal cord injury (SCI) patients based on the presence or absence of a DPR immediately following injury. Thirty-six subjects were evaluated within one week of injury. A complete neurological evaluation following American Spinal Injury Association (ASIA) standards was performed and the presence or absence of a DPR was noted on admission. Ambulation status and ASIA Impairment Scales were recorded at discharge from the rehabilitation unit and the data were analyzed by the Chi-square method with Yates' correction for continuity. The data demonstrated a high correlation of the DPR with motor complete injuries (p < 0.01) and a poor prognosis for recovery of ambulation (p < 0.01). Clinicians should recognize this abnormal reflex, which may be used in conjunction with a complete neurological examination, to help prognosticate future function in the acute SCI patient.

Adolescent↗
PubMed1996

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.

Animals↗
PubMed1995

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.

Animals↗
PubMed1991

Suppression by antisense mRNA demonstrates a requirement for the glial fibrillary acidic protein in the formation of stable astrocytic processes in response to neurons.

The glial fibrillary acidic protein (GFAP) is a glial-specific intermediate filament protein, which is expressed in astrocytes in the central nervous system, as well as in astrocytoma cell lines. To investigate the function of GFAP, we have studied the human astrocytoma cell line, U251, which constitutively expresses GFAP and vimentin in the same 10-nm filaments. These cells respond to neurons in vitro in the same way as primary astrocytes: they withdraw from the cell cycle, support neuronal cell survival and neurite outgrowth, and they extend complex, GFAP-positive processes. To determine the role of GFAP in these responses, we have specifically suppressed its expression by stably transfecting the U251 cells with an antisense GFAP construct. Two stable antisense cell lines from separate transfections were isolated and were shown to be GFAP negative by Northern and Western blot analyses, and by immunofluorescence studies. The antisense cell lines were inhibited in their ability to extend significant glial processes in response to neurons. In culture with primary neurons, the average increase in process length of the U251 cells was nearly 400%, as compared to only 14% for the antisense transfectants. The other neuron induced responses of astrocytes, i.e., proliferative arrest and neuronal support, were not affected in these cell lines. These data support the conclusion that the glial-specific intermediate filament protein, GFAP, is required for the formation of stable astrocytic processes in response to neurons.

Animals↗
PubMed1990

C17, a retrovirally immortalized neuronal cell line, inhibits the proliferation of astrocytes and astrocytoma cells by a contact-mediated mechanism.

We have investigated the ability of various cell lines to effect contact-mediated inhibition of astrocytic cells. Of the lines tested, only C17, a mouse cell line from postnatal day 0 cerebellum immortalized by infection with a retroviral construct containing the avian myc gene, and U251, a human astrocytoma line, were able to inhibit the proliferation of astrocytic cells. When co-cultured with either primary astrocytes from rat cerebellum or the U251 line, the C17 cells induced a rapid cessation of glial cell division as well as complex astrocytic process extension. The effects on glial mitosis were cell-dose-dependent, with ten C17 cells/glial cell being the optimal ratio. At this ratio [3H]thymidine incorporation into the U251 cells was reduced by greater than 80% and there was a virtual stasis in glial cell number at 48 hours. Fixed C17 cells as well as partially purified C17 membranes were also potent inhibitors of astrocytic proliferation, suggesting that the gliastatic effect of the C17 cell line is membrane associated. However, neither of these preparations induced astrocytic process formation. We also confirmed earlier reports that U251 cells inhibited their own proliferation in a density-dependent manner but at a lower efficiency than the C17 cells.

Animals↗
PubMed1988

Clustering with dendritic cells precedes and is essential for T-cell proliferation in a mitogenesis model.

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.

Animals↗