Karina F. Meiri

Tacrolimus (FK506) increases neuronal expression of GAP-43 and improves functional recovery after spinal cord injury in rats.

Tacrolimus (FK506), a widely used immunosuppressant drug, has neurite-promoting activity in cultured PC12 cells and peripheral neurons. The present study investigated whether tacrolimus affects the expression of the neuronal growth-associated protein, GAP-43, as well as functional recovery after photothrombotic spinal cord injury in the rat. In injured animals receiving tacrolimus, the number of neurons expressing GAP-43 mRNA and protein approximately doubled compared to that in injured animals receiving vehicle alone. This increase in GAP-43-positive cells was paralleled by a significant improvement in neurological function evaluated by open-field and inclined plane tests. Another FKBP-12 ligand (V-10,367) had similar effects on GAP-43 expression and functional outcome, indicating that the observed effects of tacrolimus do not involve inhibition of the phosphatase calcineurin. Thus, tacrolimus, a drug which is already approved for use in humans, as well as other FKBP-12 ligands which do not inhibit calcineurin, could potentially enhance functional outcome after CNS injury in humans.

The Earliest Patterns of Neuronal Differentiation and Migration in the Mammalian Central Nervous System

With the use of four independent cell markers and Brd-U birthdating we have charted the earliest stages of neuronal differentiation and migration in the developing rat central nervous system, including the cortex, spinal cord, and retina. One of the markers, the monoclonal antibody 2G12, labeled a large subpopulation of differentiating cells that uniformly lined the ventricles throughout these CNS regions at unexpectedly early ages. Immunocytochemistry demonstrated that, in cortex, the 2G12 antigen could appear in cells during mitosis. More mature looking 2G12-positive cell types initially had a primitive radial morphology and were axonless. However, in strict spatio-temporal sequences, the most mature looking 2G12-positive cells had the ability to sprout GAP-43-positive axons before or after the cell body left the ventricular surface and before or after detachment of their pial or ventricular endfoot processes. Double label experiments with 2G12 and Brd-U showed that none of these three 2G12-positive cell types incorporated Brd-U after a short pulse. The primitive neuroepithelial shape of the immature neurons was verified with a polyclonal GAP-43 antibody, a type III beta tubulin antibody, and DiI labeling from a distal portion of the axon. In the cortex and retina, the 2G12 marker persisted in cells that had reached prospective neuronal layers. However, in all CNS regions observed, 2G12 immunoreactivity disappeared from the cell body as the axon extended from the young neuron. Based on the smooth progression of changing 2G12-positive cell shapes, but also because of the transient nature of this label, we can only speculate that the 2G12 epitope may be marking a continuum of neuronal cell states throughout the earliest period of differentiation and migration. Thus, our hypothesis suggests that many of the youngest CNS neurons may have a widespread distribution and may begin their differentiation, and even remain axonless for a time, while retaining a neuroepithelial morphology. Once differentiation resumes, a major mode of transformation into mature neurons during the earliest stages of development could occur via translocation of the cell soma into the pial process. Importantly, these markers have verified at later stages, and especially in cortex, that multiple mechanisms exist for neuronal migration in the CNS depending on the region and stage of development.

Demonstration of presynaptic protein kinase C activation following long-term potentiation in rat hippocampal slices.

Pharmacological and biochemical evidence implicate the Ca2+ and phospholipid-dependent protein kinase C in long-term potentiation. The in vitro hippocampal slice preparation was used to demonstrate redistribution of protein kinase C from cytosol to membrane and protein kinase C-dependent phosphorylation of the presynaptic growth-associated protein-43 substrate following long-term potentiation induction in area CA1. Protein kinase C translocation was assessed using both quantitative immunoblotting with a monoclonal antibody recognizing a common epitope in the alpha and beta isoforms of protein kinase C and Ca2+ and phospholipid-dependent phosphorylation of exogenous histone substrate. Slices examined 5 min after tetanus-induced spike potentiation showed no change in protein kinase C redistribution, whereas slices examined at 15-, 30- and 60-min intervals all showed a similar degree of protein kinase C translocation to membrane, although only at 15 min was the effect statistically significant. Additionally, an increase in protein kinase C-dependent growth-associated protein 43 phosphorylation was observed 10 min after high-frequency stimulation. The translocation of protein kinase C and phosphorylation of growth-associated protein 43 were dependent upon high-frequency (repetitive 400 Hz) afferent stimulation, as no effects were observed in slices receiving low-frequency (1 Hz) or no stimulation. The N-methyl-D-aspartate receptor antagonist, DL-2-amino-5-phosphonovaleric acid (50 microM), inhibited induction of long-term potentiation, redistribution of protein kinase C and phosphorylation of growth-associated protein 43. A significant redistribution of the predominantly presynaptic protein kinase C isoform, protein kinase C-alpha, was also detected 15 min after induction of long-term potentiation using an alpha-isoform-specific monoclonal antibody. These observations support a presynaptic role for protein kinase C and growth-associated protein 43 in the early maintenance phase of LTP, and further suggest that a retrograde messenger produced postsynaptically following N-methyl-D-aspartate receptor activation mediates these effects.

Nerve growth factor stimulation of GAP-43 phosphorylation in intact isolated growth cones

Phosphorylation of the nervous system-specific growth cone protein GAP- 43 by kinase C in vivo occurs exclusively in growth cones and distal axons, and the onset of this phosphorylation is delayed relative to the onset of axonogenesis, with the delay predicted on the time needed for axons to reach the vicinity of their targets (Meiri et al., 1991). We have used a subcellular fraction of intact growth cones (IGCs) to investigate whether this induction of GAP-43 phosphorylation can be influenced by target-derived substances, and show here that increased phosphorylation of GAP-43 can be both stimulated and maintained by NGF at concentrations of 2 x 10(-10) M. This low concentration of NGF and the subsequent phosphorylation of GAP-43 are both consistent with the interpretation that phosphorylation is due to the binding of NGF to a biologically active high-affinity receptor. Second, we used the monoclonal antibody 2G12 to show that the NGF-stimulated phosphorylation of GAP-43 occurs on serine, the kinase C phosphorylation site, consistent with the results seen in vivo. Levels of phosphorylated GAP-43 in the intact IGCs are also modulated by calcium-stimulated dephosphorylation that could be inhibited by EGTA but not okadaic acid and that therefore resembled the calcineurin- stimulated dephosphorylation reported in vitro. The results suggest that the spatial and temporal regulation of GAP-43 phosphorylation that occurs during axonogenesis in vivo can be regulated by target-derived neurotropic molecules, specifically NGF.

Isolation and characterization of detergent-resistant microdomains responsive to NCAM-mediated signaling from growth cones.

It is still largely unclear how cell adhesion molecule (CAM)-mediated signaling evokes responses from the growth cone cytoskeleton. Here we used TX-114 extraction of growth cones followed by equilibrium gradient centrifugation to isolate subfractions of detergent-resistant microdomains (DRMs) that could be structurally and functionally distinguished on the basis of localization and activation of components of CAM-mediated signaling pathways. DRMs enriched in cholesterol, caveolin, NCAM140, GPI-linked NCAM120, fyn, and GAP-43, all conventional markers of microdomains or rafts, were located in areas 2 and 3 of the gradient. Coimmunoprecipitation of specific components of CAM signaling pathways by GAP-43 then identified distinct subpopulations of DRMs. GAP-43 from area 2 DRMs coprecipitated GPI-linked NCAM120 and was inactive, i.e., PKC phosphorylation had not been stimulated. In contrast the GAP-43 from area 3 DRMs coprecipitated both transmembrane NCAM140 and caveolin and was active, i.e., highly phosphorylated by PKC. A different subset of DRMs from both area 2 and area 3 contained fyn that could not be coprecipitated with GAP-43 antibodies. In this case area 2 DRMs contained activated fyn that was phosphorylated on Y415. In contrast area 3 DRMs contained inactive fyn. Hence fyn and GAP-43, both targets of NCAM signaling, are located in distinct populations of DRMs, and their activated forms are reciprocally distributed on the gradient. A detergent-resistant membrane fraction recovered from area 4 was enriched in NCAM140, phosphorylated GAP-43, and actin, but not cholesterol, caveolin, or fyn. Immunoelectron microscopy revealed that phosphorylated GAP-43 was localized where the membranes and F-actin interacted. Our results provide evidence for NCAM-mediated signaling in DRMs and suggest that the DRMs responsible for fyn and PKC/GAP-43-mediated NCAM signaling are structurally distinct and differentially distributed in growth cones.

Effects of nerve compression on fast axonal transport in streptozotocin-induced diabetes mellitus. An experimental study in the sciatic nerve of rats.

SummaryThe hypothesis that nerves in diabetes mellitus exhibit an increased susceptibility to compression was experimentally tested. Inhibition of fast axonal transport was induced by local compression in sciatic nerves of rats with streptozotocin-induced diabetes mellitus. Fast anterograde axonal transport was measured after application of3H-leucine to the motor neurone cell bodies in the spinal cord. The sciatic nerve as subjected to local, graded compression in vivo by a small compression chamber. The amount of accumulation of proteins was quantified by calculation of a transport block ratio. Compression at 30 mm Hg for 3 h induced a significantly greater (p<0.05) accumulation of axonally transported proteins at the site of compression in nerves of diabetic animals (transport block ratio: 1.01±0.35; n=7) than in nerves of controls (0.67±0.16;n=7). Accumulation was significantly higher in ligature experiments of both control (1.34±0.44;n=8;p< 0.01) and diabetic animals (1.45±0.30;n=8 ;p< 0.05), indicating that the block of transport in compressed nerves was incomplete. Neither sham compressed diabetic (0.50±0.09;n=6) nor control (0.49±0.11;n=6) nerves showed any block of axonal transport. The possible causes of the increased inhibition of fast axonal transport in diabetic rats are discussed. The results indicate that diabetes may lead to an increased susceptibility of peripheral nerves to compression.

Failure to Express GAP-43 during Neurogenesis Affects Cell Cycle Regulation and Differentiation of Neural Precursors and Stimulates Apoptosis of Neurons

GAP-43 is first expressed in proliferating neuroblasts and is required for maturation of neurons. When GAP-43 is not expressed in differentiating embryonal carcinoma P19 cells, reduced numbers of neurons were generated. Here we show that neuronal differentiation is initially disrupted at the onset of cell-cycle arrest in aggregated, proliferating neuronal precursors. The ratio of nestin:beta-tubulin-labeled progeny generated at this stage suggests that the differentiation is asymmetric. Apoptosis of immature neurons subsequently produced was also significantly induced. In vivo, too, proliferation of neuroblasts was significantly reduced in cortex of GAP-43(-/-) mice at E14.5. These data demonstrate that when GAP-43 is not expressed in proliferating neuroblasts, neural differentiation is not initiated appropriately, inducing apoptosis. Moreover, the concurrent inhibition of Ca2+-dependent adhesion between differentiating P19 cells in aggregates implicates GAP-43 in CAM-mediated signaling during neurogenesis, as has been previously shown in growth cones.

Alterations in hippocampal GAP-43 phosphorylation and protein level following contextual fear conditioning.

C57BL/6 (B6) mice display better contextual learning than the DBA/2 (D2) mice. The possibility that GAP-43, is differentially affected as a function of strain and learning was investigated in the present study. No basal difference between C57BL/6J (B6) and DBA/2J (D2) mice in the amount of hippocampal GAP-43 was observed, but naive D2 mice have slightly lower basal levels of GAP-43 phosphorylation than do B6 mice. Interestingly, alterations in hippocampal GAP-43 protein levels and phosphorylation state in response to training for contextual learning were observed only in B6 mice. Immediate-shocked mice, serving as nonlearning controls, showed no GAP-43 alterations, nor did D2 mice subjected to either training condition. These results suggest that modulation of hippocampal GAP-43 may be important for contextual learning and that strain-specific alterations in GAP-43 may be part of a disrupted pathway in D2 mice that is essential for learning.

Lipid rafts and regulation of the cytoskeleton during T cell activation.

The ability of polarized cells to initiate and sustain directional responses to extracellular signals is critically dependent on direct communication between spatially organized signalling modules in the membrane and the underlying cytoskeleton. Pioneering work in T cells has shown that the assembly of signalling modules critically depends on the functional compartmentalization of membrane lipids into ordered microdomains or lipid rafts. The significance of rafts in T cell activation lies not only in their ability to recruit the signalling partners that eventually assemble into a mature immunological synapse but also in their ability to regulate actin dynamics and recruit cytoskeletal associated proteins, thereby achieving the structural polarization underlying stability of the synapse—a critical prerequisite for activation to be sustained. Lipid rafts vary quite considerably in size and visualizing the smallest of them in vivo has been challenging. Nonetheless it is now been shown quite convincingly that a surprisingly large proportion—in the order of 50%—of external membrane lipids (chiefly cholesterol and glycosphingolipids) can be dynamically localized in these liquid ordered rafts. Complementary inner leaflet rafts are less well characterized, but contain phosphoinositides as an important functional component that is crucial for regulating the behaviour of the actin cytoskeleton. This paper provides an overview of the interdependency between signalling and cytoskeletal polarization, and in particular considers how regulation of the cytoskeleton plays a crucial role in the consolidation of rafts and their stabilization into the immunological synapse.

GAP-43 is essential for the neurotrophic effects of BDNF and positive AMPA receptor modulator S18986

Positive α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor modulators include benzamide compounds that allosterically modulate AMPA glutamate receptors. These small molecules that cross the blood–brain barrier have been shown to act as a neuroprotectant by increasing the levels of endogenous brain-derived neurotrophic factor (BDNF). Positive AMPA receptor modulators have also been shown to increase the levels of growth-associated protein-43 (GAP-43). GAP-43 plays a major role in many aspects of neuronal function in vertebrates. The goal of this study was to determine whether GAP-43 was important in mediating the actions of positive AMPA receptor modulator (S18986) and BDNF. Using cortical cultures from GAP-43 knockout and control mice, we show that (1) GAP-43 is upregulated in response to S18986 and BDNF in control cultures; (2) this upregulation of GAP-43 is essential for mediating the neuroprotective effects of S18986 and BDNF; (3) administration of S18986 and BDNF leads to an increase in the expression of the glutamate transporters GLT-1 and GLAST that are key to limiting excitotoxic cell death and this increase in GLT-1 and GLAST expression is completely blocked in the absence of GAP-43. Taken together this study concludes that GAP-43 is an important mediator of the neurotrophic effects of S18986 and BDNF on neuronal survival and plasticity, and is essential for the success of positive AMPA receptor modulator-BDNF-based neurotrophin therapy.