[Show abstract][Hide abstract] ABSTRACT: Rapamycin is an immunosuppressive immunophilin ligand reported as having neurotrophic activity. We show that modification of rapamycin at the mammalian target of rapamycin (mTOR) binding region yields immunophilin ligands, WYE-592 and ILS-920, with potent neurotrophic activities in cortical neuronal cultures, efficacy in a rodent model for ischemic stroke, and significantly reduced immunosuppressive activity. Surprisingly, both compounds showed higher binding selectivity for FKBP52 versus FKBP12, in contrast to previously reported immunophilin ligands. Affinity purification revealed two key binding proteins, the immunophilin FKBP52 and the beta1-subunit of L-type voltage-dependent Ca(2+) channels (CACNB1). Electrophysiological analysis indicated that both compounds can inhibit L-type Ca(2+) channels in rat hippocampal neurons and F-11 dorsal root ganglia (DRG)/neuroblastoma cells. We propose that these immunophilin ligands can protect neurons from Ca(2+)-induced cell death by modulating Ca(2+) channels and promote neurite outgrowth via FKBP52 binding.
Proceedings of the National Academy of Sciences 02/2008; 105(1):33-8. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Immunophilins are protein receptors for the immunosuppressant drugs FK506, cyclosporin A (CsA), and rapamycin. Two categories of immunophilins are the FK506-binding proteins (FKBPs), which bind to FK506, rapamycin, and CCI-779 and the cyclophilins, which bind to CsA. Reports have shown that immunophilins are expressed in the brain and spinal cord, are 10-100-fold higher in CNS tissue than immune tissue, and their expression is increased following nerve injury, suggesting that their chemical ligands may have therapeutic utility in the treatment of neurodegenerative diseases. In this study, we report the development and utility of a rapid neurofilament (NF) enzyme-linked immunosorbent assay (ELISA) to quantify neuronal survival and the Cellomics ArrayScan platform to quantify neurite outgrowth following treatment with immunophilin ligands. Cultured neurons or F-11 cells were treated with various immunophilin ligands for 72 or 96h and their promotion of neuronal survival and neurite outgrowth were determined. The results showed that all immunophilin ligands, in a concentration-dependent manner, significantly increased neuronal survival and neurite outgrowth, when compared to control cultures. Taken together, these results demonstrate the potential utility of the neurofilament ELISA and Cellomics ArrayScan platform to efficiently quantify neurotrophic effects of immunophilin ligands on cultured neurons and cell lines.
Journal of Neuroscience Methods 08/2007; 163(2):310-20. · 1.96 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 3-Normeridamycin (1), isolated from fermentation extracts of the soil actinomycete Streptomyces sp. LL-C31037, demonstrated potent neuroprotective activity. When challenged with the neurotoxin 1-methyl-4-phenylpyridinium (MPP+), known to induce parkinsonism, 1 restored functional dopamine uptake in a concentration-dependent manner, with an EC50 of 110 nM in dopaminergic neurons. The structure of 1 was determined via spectroscopic methods, and the immunosuppressive and immunophilin binding properties of the compound were also measured.
The Journal of Antibiotics 04/2006; 59(3):184-9. · 2.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: PACAP is a peptide with neuroprotective activity, which induces adenylate cyclase and protein kinase A (PKA) activity. PACAP has also been shown to induce neurite outgrowth in PC12 cells and dorsal root ganglion (DRG) neurons. Here, we report that exogenous PACAP38 promotes neurite outgrowth in the F11 neuroblastoma/dorsal DRG hybrid cell line. Using an automated microscopy system, we show that PACAP38 induces a 170-fold increase in neurite length, with an EC50 of 3.1 nM, compared to 3.7 microM for forskolin and 143.4 microM for dibutyril cyclic AMP (dbcAMP). PACAP38 induced a 4-fold increase in the level of phosphorylation of cAMP-responsive element binding protein (CREB) in F11 cells with an EC50 of 130 pM. In contrast a peptide related to PACAP, vasoactive intestinal peptide (VIP) failed to induce CREB phosphorylation or neurite outgrowth in F11 cells. Addition of the nonselective phosphodiesterase inhibitor, isobutyl methylxanthine (IBMX) increased the potency of PACAP at inducing neurite outgrowth by ten-fold. The PKA inhibitor, H89, was a potent inhibitor of PACAP38-induced neurite outgrowth. The delta-opioid receptor agonist, SNC 80, did not inhibit PACAP-induced neurogenesis even though it did reduce CREB phosphorylation. In contrast to previous studies in PC12 cells, PACAP38 failed to show MEK1 activation in F11 cells. PACAP is upregulated in DRG neurons as a result of injury, and F11 cells provide an easily accessible in vitro model for understanding mechanisms underlying PACAP differentiation and neurogenesis.
Brain Research 04/2006; 1077(1):16-23. · 2.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A brief ischemic injury to the gerbil forebrain that caused selective damage in the CA1 region of the hippocampus also enhanced the production of new cells in the hippocampal neurogenic area. When evaluated 1 week after bromodeoxyuridine (BrdU) injection, approximately ten times more labeled cells were detected in the hippocampal dentate gyrus in ischemic animals than controls, indicating a stimulation of mitotic activity. To assess the temporal course of the survival and fate of these newborn cells, we monitored BrdU labeling and cell marker expression up to 60 days after ischemia (DAI). Loss of BrdU-positive cells was observed from both control and ischemic animals, but at 30 DAI and afterward, the ischemic group maintained more than 3 times as many BrdU-positive cells as the control group. In addition, ischemic injury also fostered the neuronal differentiation of these cells beyond the capacity observed in control animals and facilitated the migration of developing neurons to a neuronal cellular layer. The establishment of a temporal correlation between differentiation and migration provides evidence of the functional maturation of these cells. Surprisingly, we found that ischemic injury induced activation of caspase-3, not only in the CA1 region as expected, but also in the dentate subgranular zone (SGZ). Active caspase-3 immunoreactivity in the subgranular layer was co-localized with an early neuronal marker, suggesting that caspase-mediated apoptosis could mediate the loss of neurogenic cells in the SGZ. Inhibiting caspase-3 in the context of ischemia-induced neurogenesis might provide an opportunity for functional repair and a therapeutic outcome in the wake of ischemic injury.
Brain research 11/2005; 1058(1-2):167-77. · 2.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Oxygen-glucose deprivation (OGD) induced neuron-specific cell death in organotypic hippocampal slice cultures. Neuronal death was first evident in the CA1 region 24 h after the injury as assessed by propidium iodide (PI) labeling, and continued to extend to the CA3/4 region up to 72 h. At 6 days post-OGD, PI labeling was weak and diffuse with no clear demarcation of pyknotic nuclei. To characterize biochemical changes produced by OGD, cellular efflux of three key amino acid neurotransmitters was evaluated. OGD elicited large increases in the release of GABA and aspartate (55- and 4.5-fold increase over basal, respectively), while there were no detectable changes in extracellular glutamate levels. In order to ascertain the existence of the synaptic pool of glutamate, sister cultures were treated with sodium azide. This evoked a strong increase in glutamate release, suggesting the intactness of the glutamate system. Further studies revealed a time-dependent activation of caspase 3 following OGD, shown by immunoblot analysis as well as by confocal laser scanning microscopy. While we did not observe the activation of caspases 1, 2, or 8 in our model, the activation of caspase 9 was evident, peaking at 12 h post-OGD. Despite no apparent increase in glutamate release by ischemic slices, treatment with a N-methyl-D-aspartate (NMDA) antagonist or an alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) antagonist significantly reduced neuronal death. Furthermore, a pan-caspase inhibitor (zVAD-fmk), but not the caspase 3 inhibitor (DEVD-fmk), provided partial neuroprotection. Inhibition of a Ca(2+)-dependent cysteine protease, calpain, by MDL28170 also elicited partial neuroprotective effects.
Neurochemistry International 08/2004; 45(1):117-27. · 2.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To determine whether hippocampal pyramidal neurons retain authentic functional properties in mature organotypic culture, hippocampal slice cultures were established from young adult rats (P20-21). Cultures maintained 7 days in vitro retained tight organization of neuronal layers, as opposed to the widening restructure of pyramidal neurons often observed in perinatal slices. CA3 and CA1 pyramidal neurons fired action potentials in response to current injection and exhibited spontaneous and evoked synaptic currents, indicating intact neuronal function and normal hippocampal neural circuitry. We also tested neuronal sensitivity of slice cultures to ischemic injury. Acute ischemic paradigm resulted in selective death of pyramidal neurons in the CA1 region, which was prevented by treatment with an NMDA-antagonist, MK-801. Robust efflux of excitatory and inhibitory amino acid neurotransmitters was detected during ischemia, consistent with changes shown in acute slices. In summary, hippocampal organotypic cultures prepared from young adult rats maintained neuronal architecture and synaptic activity in vitro and can be used in parallel with an acute slice system to model mature brain tissue to examine ischemic pathophysiology and neuroprotective treatment.
Brain Research 04/2004; 1001(1-2):125-32. · 2.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Rodent models of focal and global ischemia were used to examine caspase activation. Several readouts were employed on identical tissue to provide correlative measurement of caspase induction, activation and enzymatic activity. In a rat focal ischemia model, caspase-3 enzymatic activity, as recorded by DEVD-AMC cleavage, peaked in penumbral cortex at 6-12 h following ischemia, correlating with increases in caspase 3-cleaved substrates of PARP and alpha-spectrin and subsequent disappearance of caspase-3 zymogen. Although induction of caspases 8 and 2 proteins was detectable as early as 6 h following ischemia, examination of the same tissues for caspase 8 or 2 enzymatic activities did not show significant modulation up to 12 h after ischemic insult. Caspase 9 induction was evident only after 24 h postischemia and did not correlate with elevated LDHD-AMC cleavage. Following global ischemia in gerbils, levels of caspase-3 enzyme activity peaked at 12 h in hippocampal tissue extracts. Cleaved caspase-3 signal was prominent in NeuN-positive layers in the CA1 region 6-12 h following ischemia. Interestingly, strong caspase-3 immunoreactivity was also detected in the subgranular zone of the dentate gyrus, a known region of ischemia-induced neurogenesis. Caspase-3 activation may be responsible for the loss of these cells, thereby hindering the endogenous recovery process.
Brain Research 09/2003; 982(2):146-55. · 2.83 Impact Factor