The postmortem Alzheimer brain is a source of structurally and functionally intact astrocytic messenger RNA

Although the precise role of astrocytes in the pathogenesis of Alzheimer's disease (AD) is currently undefined, studies carried out at the molecular level may lead to new insights into the functioning of this class of brain cells in dementia. In order to facilitate such investigations, methods are described that establish that structurally and functionally intact messenger RNA (mRNA) for an astrocytic marker, glial fibrillary acidic protein (GFAP), is present in the postmortem Alzheimer's disease brain after long postmortem intervals. Rapid preparative procedures were used to obtain poly(A)+ RNA from postmortem control and AD cortices. In vitro protein synthesis was carried out in a reticulocyte system. Relative to controls, AD mRNA synthesized a two-fold higher level of a 50,000 mol.wt. protein that was immunologically identified as GFAP. High levels of GFAP synthesis by purified mRNA from AD cortices was independent of age at death and postmortem interval up to 24 h. Northern blot hybridization using a cloned human GFAP riboprobe was used to evaluate postmortem GFAP mRNA stability. No appreciable degradation products of GFAP mRNA were observed on Northern blots for at least 10 h postmortem in poly(A)+ RNA extracted from the AD brain. The described methodology demonstrates that the postmortem AD brain is an excellent source of functionally and structurally intact astrocyte-specific mRNA.     

Developmental profile of polyadenylated and non-polyadenylated GABAA receptor subunit mRNAs.

The ratio of mRNA not selected for polyadenylation (non-poly(A)+ selected) to mRNA selected for polyadenylation (poly(A)+) for the beta 1, alpha 1 and gamma 2 subunits of the GABAA receptor complex was examined in rats as a function of age. RNA was extracted from whole brain of rats that were either 0, 1, 3, 5 or over 60 days of postnatal age. Poly(A)+ mRNA was purified by oligo(dT)-cellulose chromatography. Non-poly(A)+ selected mRNA and poly(A)+ mRNA for the GABAA receptor beta 1, alpha 1 and gamma 2 subunits were examined by Northern blot analysis using cDNA probes specific for these subunits. Levels of GABAA receptor beta 1 subunit mRNA were also examined by solution hybridization analysis with a beta 1 riboprobe. Analysis of Northern blots revealed that levels of poly(A)+ beta 1 subunit mRNA were highest at 0 days of age, but decreased and reached adult levels by 5 days of postnatal age. However, levels of the beta 1 subunit message extracted from non-poly(A)+ selected mRNA were not significantly different at any of the ages examined, suggesting the existence of a population of beta 1 subunit mRNA that is not polyadenylated. The age-related discrepancy between beta 1 subunit levels measured in non-poly(A)+ selected mRNA and poly(A)+ mRNA was also observed using solution hybridization analysis. In contrast, levels of both non-poly(A)+ selected mRNA and poly(A)+ mRNA for the alpha 1 subunit of the GABAA complex increased from 0 days of age to adulthood. Similarly, levels of both non-poly(A)+ selected mRNA and poly(A)+ mRNA for the GABAA receptor gamma 2 subunit increased with age.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning of non-polyadenylated RNAs from rat brain.

Rodent brain has been reported to contain a fraction of non-polyadenylated (poly(A)-) mRNA that includes about 100,000 different sequences, most of which are not found in the poly(A)+ fraction. We have prepared a cDNA library of low-abundance poly(A)- RNAs from rat brain polysomes, and have characterized three clones in detail. Two of the clones hybridize on Northern blots to poly(A)+ RNAs from brain. Dot blot hybridization and RNase protection assays demonstrate that although the bulk of the RNA complementary to these clones is present in the poly(A)- fraction, a small portion (7-21%) is present in the poly(A)+ fraction. Our results suggest that the poly(A)-mRNA fraction from rat brain may not contain sequences that are different from those in the poly(A)+ fraction.     

Presence of beta-hexosaminidase A alpha-chain mRNA in two different variants of GM2-gangliosidosis

Tay-Sachs disease displays a variety of forms on the clinical and biochemical level. On the molecular level it has been shown, that poly (A)+ RNA preparations from fibroblasts of patients with classical Tay-Sachs disease lack detectable alpha-chain message when analyzed by Northern blotting with complementary DNA encoding the alpha-chain of human beta-hexosaminidase A. In this report the p beta H alpha-5 clone was used to investigate whether patients with two different variants of Tay-Sachs disease also lack the alpha-chain message. On the basis of RNA hybridization analyses, we could show that our patients which synthesize an altered alpha-chain, as judged by testing enzyme activity and substrate specificity, have the 2.1 kb mRNA which is also seen in healthy control patients.