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Selective postmortem degradation of inducible heat shock protein 70 (hsp70) mRNAs in rat brain
S Pardue1, A L Zimmerman, M Morrison-Bogorad
1Department of Neurology, University of Texas Southwestern Medical Center at Dallas 75235, USA.
Abstract:
1. Altered mRNA levels in postmortem brain tissue from persons with Alzheimer's disease (AD) or other neurological diseases are usually presumed to be characteristic of the disease state, even though both agonal state (the physiological state immediately premortem) and postmortem interval (PMI) (the time between death and harvesting the tissue) have the potential to affect levels of mRNAs measured in postmortem tissue. Although the possible effect of postmortem interval on mRNA levels has been more carefully evaluated than that of agonal state, many studies assume that all mRNAs have similar rates of degradation postmortem. 2. To determine the postmortem stability of inducible heat shock protein 70 (hsp70) mRNAs, themselves unstable in vivo at normal body temperature, rats were heat shocked in order to induce synthesis of the hsp70 mRNAs. hsp70 mRNA levels in cerebellum and cortex were then compared to those of their heat shock cognate 70 (hsc70) mRNAs, as well as to levels of 18S rRNAs, at 0 and at 24 hr postmortem. 3. Quantiation of northern blots after hybridization with an hsp70 mRNA-specific oligo probe indicated a massive loss of hsp70 mRNA signal in RNAs isolated from 24-hr postmortem brains; quantitation by slot-blot hybridization was 5- to 15-fold more efficient. Even using the latter technique, hsp70 mRNA levels were reduced by 59% in 24-hr-postmortem cerebellum and by 78% in cortex compared to mRNA levels in the same region of 0-hr-postmortem brain. There was little reduction postmortem in levels of the hsp70 mRNAs or of 18S rRNAs in either brain region. 4. In situ hybridization analysis indicated that hsp70 mRNAs were less abundant in all major classes of cerebellar cells after 24 hr postmortem and mRNAs had degraded severalfold more rapidly in neurons than in glia. There was no corresponding loss of intracellular 18S rRNA in any cell type. 5. We conclude from these results that the effect of postmortem interval on mRNA degradation must be carefully evaluated when analyzing levels of inducible hsp70 mRNAs, and perhaps other short-lived mRNAs, in human brain.
Insights
Postmortem interval significantly impacts inducible heat shock protein 70 (hsp70) mRNA levels in rat brains, with rapid degradation observed, especially in neurons. This highlights the need to consider mRNA stability when analyzing neurological disease states.
Area of Science:
- Neuroscience
- Molecular Biology
- Biochemistry
Background:
- Altered mRNA levels in postmortem brain tissue are often attributed to disease, but agonal state and postmortem interval (PMI) can influence these findings.
- Previous studies have not fully evaluated the impact of PMI on all mRNA types, assuming similar degradation rates.
- Inducible heat shock protein 70 (hsp70) mRNAs are known to be unstable in vivo.
Purpose of the Study:
- To determine the postmortem stability of inducible heat shock protein 70 (hsp70) mRNAs in rat brain.
- To compare the degradation rates of hsp70 mRNAs with heat shock cognate 70 (hsc70) mRNAs and 18S rRNAs.
- To investigate the differential degradation of hsp70 mRNAs in neurons versus glia.
Main Methods:
- Rats were heat shocked to induce hsp70 mRNA synthesis.
- mRNA levels (hsp70, hsc70, 18S rRNA) were quantified in cerebellum and cortex at 0 and 24 hours postmortem using northern and slot-blot hybridization.
- In situ hybridization was used to assess mRNA distribution in different cell types.
Main Results:
- A significant loss of hsp70 mRNA was observed within 24 hours postmortem, with levels reduced by 59% in cerebellum and 78% in cortex.
- hsc70 mRNAs and 18S rRNAs showed minimal reduction over the same postmortem interval.
- In situ hybridization revealed more rapid degradation of hsp70 mRNAs in neurons compared to glia.
Conclusions:
- The postmortem interval has a substantial impact on inducible hsp70 mRNA levels, necessitating careful evaluation.
- Inducible hsp70 mRNAs are unstable postmortem, degrading more rapidly than hsc70 mRNAs and rRNAs.
- Differential degradation rates in neurons versus glia should be considered when interpreting hsp70 mRNA data from postmortem human brain studies.