Aurel Popa Wagner

Seizure induced c-fos mRNA in the rat brain: comparison between young and aging animals

The molecular mechanisms associated with age-related alterations in the pharmacological and physiological properties of hippocampal and cortical neurons in response to chemically induced seizure are largely unknown. Administration of pentylenetetrazole (PTZ) (50 mg/kg body weight) to rats of various ages evoked tonic-colonic seizures. Using RNA gel blot analysis we found that 1 h after the onset of seizure, the mRNA for the protooncogene c-fos was increased in the hippocampus and cortex of 3-month-old rats. The levels of c-fos mRNA in the hippocampus and cortex of 3-month-old rats returned to control levels by 3 h after PTZ administration. The levels of c-fos mRNA in the hippocampus and cortex of 20-month-old and 30-month-old rats peaked at 3 h and returned to basal levels by 15 h following PTZ treatment. These results suggest that the induction of immediate-early gene expression, as exemplified by c-fos, is not impaired in the aged rat brain. However, the aged rat brain responded more slowly to chemically induced seizure and the levels of c-fos mRNA induction are decreased by about 49% in the cortex and by 27% in the hippocampus of 30-month-old rats, as compared to the levels expressed by 3-month-old rats.

Evidence that V+ fibronectin, GFAP, and S100ß mRNAs are increased in the hippocampus of aged rats

In the present study, using RNA gel-blot analysis, we characterized the developmental changes in the prevalence of mRNA coding for fibronectin (FN), glial fibrillary acidic protein (GFAP), neurotrophic protein S100 beta, and beta-actin mRNA in rat hippocampus and forebrain from 1 to 720 days of age. We found that the FN and mRNA containing the V segment (FN-V) was relatively abundant at early postnatal stages, but very few transcripts were detected in adult rats. However, the hybridization signal for the juvenile FN-V mRNA was up to approximately 8-fold increased in some but not all 24-versus 6-month-old rats. Also, GFAP and S100 beta transcripts were faintly expressed at an early developmental stage, then the level of expression steadily increased starting with day 21. The greatest increase averages approximately 1.8-fold for GFAP in 24-month-old rats, and approximately 1.5-fold for S100 beta in 15-month-old versus 6-month-old rats. As all these messages are localized primarily in astrocytes, we conclude that (a) astrocyte might play an active role in aging hippocampus and (b) an increase in S100 beta and GFAP mRNA expression may precede that for FN-V mRNA expression in a hypothetical pathway of molecular events underlying neurodegeneration in the hippocampus of old rats. We also note the considerable variability among the 24-month-old rats, suggesting that aging is an individual process.

Brain plasticity: to what extent do aged animals retain the capacity to coordinate gene activity in response to acute challenges.

The ability of the rodent brain to support plasticity-related phenomena declines with increasing age. A decreased coordination of genes implicated in brain plasticity may be one factor contributing to this decline. Synaptic rearrangement that occurs after seizure activity is regarded as a model of brain plasticity. In a rat model of seizure-related brain plasticity, we found that the induction of immediate-early genes, as exemplified by c-fos and tissue plasminogen activator (TPA) is not impaired in the aged rat brain. However, the aged rat brain responded more slowly to chemically induced seizure and the levels of c-fos and TPA mRNAs induction are decreased in the cortex and in the hippocampus of 30-month-old rats, as compared to the levels expressed by 3-month-old rats. In addition, at the peak induction the TPA transcripts were restricted to certain cortical layers of the older rats. Surprisingly, in applying the same experimental paradigm to late genes we found that there was a shift toward earlier times in the maximum expression of growth-related molecule, the microtubule-associated protein 1B (MAP1B) mRNA, which was very evident in 18-month-old rats. Aberrant immunolabeling of MAP1B occurred in cortical layer VI of the aged rats where, unlike in young rats, there was heavy staining of neuronal somata. These results suggest that (i) one consequence of aging, besides decreases in the levels of mRNA, is a progressive loss of coordination in gene activity following the administration of a stimulus; (ii) since c-fos, TPA and MAP1B have been implicated in neuronal plasticity, these findings could explain, in part, the limited plasticity of the aging brain.

Dynamics of gene expression for microtubule-associated protein MAP1B, embryonic α-tubulin and late neural β-tubulin mRNAs in the hippocampus of aged rats

Abstract In the present study we characterized the developmental changes in the prevalence of mRNA coding for microtubule-associated protein, MAP1B, embryonic α-tubulin and late neural β-tubulin in rat hippocampus and forebrain from 1 to 720 days of age using RNA gel-blot analysis. We find that (i) the microtubule-associated protein, MAP1B, signal was relatively abundant at early postnatal stages when compared with mature animals. The hybridization signal in the 24-month-old rats was was ∼1.7 times that observed in 6-month-old rats. (ii) Embryonic α-tubulin and late neural β-tubulin were differentially regulated during rat brain development. This regulation is characterized by a dramatic decrease in the amount of α-tubulin after day-1 and a coincident increase in the production of late neural β-tubulin. Both messages became stabilized at moderate levels during the subsequent developmental stages. However, the averaged signal for β-tubulin was then ∼1.8-fold increased in 24- vs. 6-month-old rats. These results are consistent with hypothesis of an age-associated increase in reactive synaptogenesis where the healthy neurons sprout new connections to compensate for neuronal loss occurring in neighboring neurons.

Evidence that the V+ fibronectin mRNA is increased in the hippocampus of aged rats

In this study we investigated the splicing pattern of fibronectin (FN) mRNA during development and aging by using Northern blot hybridization with probes that either recognize all forms of FN mRNA or that are specific for the different spliced forms (FN-EIIIA, FN-EIIIB, and FN-V). We find that (1) the FNmRNA in the hippocampus of some but not all old rats showed a pattern of splicing similar to that found in the forebrain of 21 day old rats and (2) the hybridization signal for the alternatively spliced FN mRNA containing the EIIIA, EIIIB, and V95 segments was relatively abundant at early postnatal stages but very few transcripts were detected in adult and old rats. However, the hybridization signal for the juvenile FN-V mRNA was markedly increased in some but not all two-year-old rats suggesting that aging is an individual process.

Dynamics of gene expression for immediate early- and late genes after seizure activity in aged rats

The ability of the rodent brain to support plasticity-related phenomena declines with increasing age. A decreased coordination of genes implicated in brain plasticity may be one factor contributing to this decline. Synaptic rearrangement that occurs after seizure activity is regarded as a model of brain plasticity. In a rat model of seizure-related brain plasticity, we found that the induction of immediate-early genes, as exemplified by c-fos and tissue plasminogen activator ( tPA), is not impaired in the aged rat brain. However, the aged rat brain responded more slowly to chemically induced seizure, and the levels of c-fos and tPA mRNAs induction are decreased in the cortex and in the hippocampus of 30 month old rats, as compared to the levels expressed by 3 month old rats. In addition, at the peak induction, the TPA transcripts were restricted to certain cortical layers of the older rats. Surprisingly, in applying the same experimental paradigm to late genes, we found that there was a shift toward earlier times in the maximum expression of growth-related molecules, the microtubule-associated protein 1B (MAP1B) mRNA, which was very evident in 18 month old rats. Aberrant immunolabeling of MAP1B occurred in cortical layer VI of the aged rats where, unlike in young rats, there was heavy staining of neuronal somata. These results suggest that (1) one consequence of aging, besides decreases in the levels of mRNA, is a progressive loss of coordination in gene activity following the administration of a stimulus; (2) since c-fos, TPA and MAP1B have been implicated in neuronal plasticity, these findings could explain, in part, the limited plasticity of the aging brain.

Age changes in the H1 group of histones from rat liver

Histones were obtained from young and old rat livers by extracting them in 0.25 N HCl. They were fractionated on 15% acid urea polyacrylamide gels containing 6.25 M urea and the changes in the ratio of the major histone fractions as a function of age were calculated. Data presented show a significant increase in the amount of H1 degree fraction in the liver of old rats as compared to young rats. This data is discussed and the possible involvement of H1 degree fraction in an increased resistance of old rat liver chromatin to micrococcal nuclease digestion of linker DNA is suggested. Finally, in connection with this increased resistance, some possible consequences in chromatin structure are discussed.

Altered expression of microtubule-associated protein 1B in cerebral cortical structures of pentylenetetrazole-treated rats

Using Northern blot, immunoblotting, immunocytochemistry, and in situ hybridization, we show that a single administration of the convulsant pentylenetetrazole leads to robust, long‐term changes in microtubule‐associated protein 1B and its mRNA, in the adult rat brain. The first increases in MAP1B mRNA were detected at 15 hr following pentylenetetrazole administration in the temporal (Te2) and perirhinal cortex followed by increases in microtubule‐associated protein 1B immunoreactivity at 72 hr postseizure. In contrast, the levels of microtubule‐associated protein 1B mRNA and protein in layers I–II of the retrosplenial and parietal cortex (Par2) declined visibly by 24 hr and 72 h, respectively, post‐seizure. The changes included loss of staining in layers I–II and development of structures resembling “strings‐of‐beads” along the fibers of projection neurons of layer V. The levels of microtubule‐associated protein 1B mRNA in the entorhinal cortex peaked at later times (72 h), especially in layers II–III, and returned to control levels by 10 days. Whereas the levels of microtubule‐associated protein 1B immunoreactivity in the retrosplenial and parietal cortex recovered by 5–10 days, it persisted at high levels through day 35 in layer V of the temporal cortex (Te2), layers II–III of the perirhinal cortex and layers I–II of the lateral entorhinal cortex. These results indicate that seizure activity leads to long‐term upregulation of genes coding for structural elements that are characteristic of the immature brain such as microtubule‐associated protein 1B. J. Neurosci. Res. 51:646–657, 1998.u2003© 1998 Wiley‐Liss, Inc.

Age changes of the isoelectric points of non-histone chromosomal proteins from rat liver in the pH range 5 to 8.

Non-histone chromosomal proteins were extracted from purified nuclei of young (2-3 months) and old (24-28 months) rat liver by a two-phase partition method (chloroform:-isoamyl alcohol, 24:1) after histone depletion with 0.25N HCl. The proteins were subjected to analytical isoelectric focusing in the pH range 5-8. On the densitograms derived from Coomasie brilliant blue R-250 stained gels, there were formally four areas separated having the following pH ranges: I) 5.80-6.10; II) 6.25-6.42; III) 6.80-7.00; IV) 7.20-7.35 for young rats, and I) absent; II) 6.35-6.75; III) 6.80-7.00; IV) 7.12-7.40 for old rats. Hence, the main result is the absence of a group of proteins from old rats having the pH range 5.80-6.10. The factors involved in protein separation by isoelectric focusing, like time-dependent post-translational charge modifications: phosphorylation, acetylation, ADP-ribosylation, deamidation, terminal peptide cleavage, glycosylation, and DNA-like contaminants are discussed.

A simple spectrophotometric method for the measurement of ribonuclease activity in biological fluids

We have developed a rapid and sensitive method for detecting ribonuclease (RNAase). The method makes use of a RNA-Pyronine Y complex which has a different absorption spectrum from that of Pyronine Y alone. When the RNA is hydrolyzed by RNAase, the spectrum of the complex changes to that of unbound Pyronine Y. The resultant decrease in absorbance at 572 nm is linear for final RNAase concentrations ranging from 2 to 45 ng/ml. Optimal assay conditions were 11.5 micrograms/ml Pyronine Y, 0.56 mg/ml RNA, 80 mumol/ml Tris-HCl buffer, pH 7.8, and 2-45 ng/ml RNAase. The effect of complex concentration, pH, molarity and temperature upon the rate of the reaction were determined. The assay is applicable to crude cell-free extracts.