Protein and DNA oxidation in spinal injury: neurofilaments--an oxidation target.

This study measured the time courses of protein and DNA oxidation following spinal cord injury (SCI) in rats and characterized oxidative degradation of proteins. Protein carbonyl content-a marker of protein oxidation-significantly increased at 3-9 h postinjury and the ratio 8-hydroxy-2-deoxyguanosine/deoxyguanosine-an indicator of DNA oxidation-was significantly higher at 3-6 h postinjury in the injured cords than in the sham controls. This suggests that oxidative modification of proteins and DNA contributes to secondary damage in SCI. Densities of selected bands on coomassie-stained gels indicated that most proteins were degraded. Neurofilament protein (NFP) was particularly evaluated immunohistochemically; its light chain (NFP-68) was gradually degraded in nerve fibers, neuron bodies, and large dendrites following SCI. A mixture of Mn (III) tetrakis (4-benzoic acid) porphyrin (10 mg/kg)-a novel SOD mimetic-and nitro-L-arginine (1 mg/kg)-an inhibitor of nitric oxide synthase-injected intraperitoneally, increased NFP-68 immunoreactivity and the numbers of NFP-positive nerve fibers post-SCI, correlating NFP degradation in SCI to free radical-triggered oxidative damage for the first time. Therefore, blockage of protein and DNA oxidation in the secondary injury stage may improve long-term recovery-important information for development of the SCI therapies.

Insulin-like growth factor I prevents the development of sensitivity to kainate neurotoxicity in cerebellar granule cells.

This study reports that insulin-like growth factor I (IGF-I) prevents cerebellar granule cells from developing sensitivity to kainate neurotoxicity. Sensitivity to kainate neurotoxicity normally develops 5-6 days after switching cultures to a serum-free medium containing 25 mM K(+). Addition of either IGF-I or insulin to the serum-free medium at the time of the switch prevented the development of sensitivity to kainate, whereas brain-derived neurotrophic factor, neurotrophin-3, neurotrophin-4, and nerve growth factor did not. The dose-response curves indicated IGF-I was more potent than insulin, favoring the assignment of the former as the physiological protective agent. The phosphatidylinositol 3-kinase (PI 3-K) inhibitors wortmannin (10-100 nM) and LY 294002 (0.3-1 microM) abolished the protection afforded by IGF-I. The p70 S6 kinase (p70(S6k)) inhibitor rapamycin (5-50 nM:) also abolished the protection afforded by IGF-I. The activities of both enzymes decreased in cultures switched to serum-free medium but increased when IGF-I was included; wortmannin (100 nM) lowered the activity of PI 3-K from 2 to 5 days after medium switch, whereas rapamycin (50 nM) prevented the increase observed for p70(S6k) activity over the same interval. The mitogen-activated protein kinase kinase inhibitor U 0126 and the mitogen-activated protein kinase inhibitor SB 203580 did not abolish IGF-I protection. Kainate neurotoxicity was not prevented by Joro spider toxin; therefore, the development of kainate neurotoxicity could not be explained by the formation of calcium-permeable alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors. These results indicate that IGF-I functions through a signal transduction pathway involving PI 3-K and p70(S6k) to prevent the development of sensitivity to kainate neurotoxicity in cerebellar granule cells.

L-type voltage-gated calcium channels modulate kainic acid neurotoxicity in cerebellar granule cells.

This study reports on the regulation of kainate neurotoxicity in cerebellar granule cells by calcium entry through voltage-gated calcium channels and by calcium release from internal cellular stores. Kainate neurotoxicity was prevented by the AMPA selective antagonist LY 303070 (10 microM). Kainate neurotoxicity was potentiated by cadmium, a general voltage-gated calcium channel blocker, and the L-type voltage-gated calcium channel blocker nifedipine. The antagonists of intracellular Ca2+ ([Ca2+]i) release, thapsigargin and ryanodine, were also able to potentiate kainate neurotoxicity. Kainate treatment elevated [Ca2+]i concentration with a rapid initial increase that peaked at 1543 nM and then declined to plateau at approximately 400 nM. Nifedipine lowered the peak response to 764 nM and the plateau response to approximately 90 nM. Thapsigargin also lowered the kainate-induced increase in [Ca2+]i (640 nM peak, 125 nM plateau). The ryanodine receptor agonist caffeine eliminated the kainate-induced increase in [Ca2+]i, and reduced kainate neurotoxicity. Kainate neurotoxicity potentiated by nifedipine was not prevented by RNA or protein synthesis inhibitors, nor by the caspase inhibitors YVAD-CHO and DEVD-CHO. Neither DNA laddering nor the number of apoptotic nuclei were increased following treatment with kainate and nifedipine. Increased nuclear staining with the membrane impermeable dye propidium iodide was observed immediately following kainate treatment, indicating a loss of plasma membrane integrity. Thus, kainate neurotoxicity is prevented by calcium entry through L-type calcium channels.

Protein synthesis within dendrites: ionic and neurotransmitter modulation of synthesis of particular polypeptides characterized by gel electrophoresis.

This study evaluates whether physiological variables differentially affect the local synthesis of protein constituents of synapses in subcellular fractions containing pinched-off dendrites (synaptodendrosomes). Synaptodendrosomes were pulse-labeled in a medium containing 35S-methionine with 3 or 25 mM KCl and in the presence or absence of 0.5 mM EGTA or 10 microM glutamate. Synaptodendrosomes were then subfractionated to prepare synaptic plasma membranes and synaptic junctional complexes. The protein constituents of the synaptic plasma membrane and synaptic junctional complex fractions that were locally synthesized were identified using SDS-PAGE and two-dimensional gel electrophoresis and the extent of labeling of individual bands was analyzed using a Phosphorimager. Analysis of incorporation into individual bands resolved by SDS-PAGE revealed that depolarizing conditions (25 mM KCl) increased the extent of labeling of different bands to a different extent (ranging from 10-70% increases in labeling). Addition of 0.5 mM EGTA decreased the extent of labeling of the same group of bands in both 3 mM KCl and 25 mM KCl conditions. Addition of 10 microM glutamate reduced incorporation especially in the synaptodendrosomes incubated in 25 mM KCl. Two-dimensional gel electrophoresis analyses revealed that the labeled spots that showed differential labeling under the different conditions did not correspond to the most prominent Coomassie-stained spots. These results indicate that the proteins that are synthesized in synaptodendrosomes and regulated by physiological variables are not amongst the more abundant protein constituents of the fractions. Taken together, these results are consistent with the idea that protein synthesis within dendrites may be regulated by synaptic activity.

Isolation of cDNA clones encoding RICH: a protein induced during goldfish optic nerve regeneration with homology to mammalian 2',3'-cyclic-nucleotide 3'-phosphodiesterases.

Using data derived from peptide sequencing of p68/70, a protein doublet induced during optic nerve regeneration in goldfish, we have isolated cDNAs that encode RICH (regeneration-induced CNPase homolog) from a goldfish regenerating retina cDNA library. The predicted RICH protein comprises 411 amino acids, possesses a pI of 4.48, and shows significant homology to the mammalian myelin marker enzyme 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase; EC 3.1.4.37). The mRNA encoding RICH was demonstrated, by both Northern blot analysis and RNase protection assays, to be induced as much as 8-fold in regenerating goldfish retinas at 20 days after nerve crush. Analysis of total RNA samples from various tissues showed a broad distribution of RICH mRNA, with the highest levels observed in gravid ovary. The data obtained strongly suggest that RICH is identical or very similar to p68/70. The molecular cloning of RICH provides the means for a more detailed analysis of its function in nerve regeneration. Additionally, the homology of RICH and CNPase suggests that further investigation may provide additional insight into the role of these proteins in the nervous system.

Purification and characterization of p68/70, regeneration-associated proteins from goldfish brain.

Two acidic proteins (p68/70) previously shown to be associated with regeneration of the goldfish optic nerve were purified 887-fold from brain homogenates of Carassius auratus. Purification to homogeneity was achieved by sequential chromatography of a 100,000 g brain supernatant fraction on DEAE-Sephacel, Cu(2+)-charged iminodiacetic acid agarose, and gel filtration. The Stokes radius of the doublet was determined to be 5.8 nm, and the sedimentation coefficient calculated to be 5.2. From these values a molecular mass of 128 kDa and a frictional coefficient ratio of 1.6 were calculated. Chromatofocusing on a high-resolution DEAE column resolved the protein doublet into three dimeric species of p68, p68/70, and p70. These results indicate that the proteins are highly elongated and associate as homodimers or as a heterodimer. Subcellular localization and membrane extraction experiments indicated p68/70 to be a component of the plasma membrane associated primarily through hydrophobic interactions. p68/70 demonstrated biphasic behavior in phase partition experiments using Triton X-114. Analysis of hydrolytic products indicated p68/70 to be a glycoprotein, containing 11% carbohydrate.