Marcelle Morrison-Bogorad

Expression of heat shock protein 70 and heat shock cognate 70 messenger RNAs in rat cortex and cerebellum after heat shock or amphetamine treatment.

Abstract: The expression of strictly inducible hsp70 mRNAs and constitutively expressed hsc70 mRNAs was compared in cerebellum and cerebral cortex of control rats, heat‐shocked rats, and rats made hyperthermic with amphetamine. An hsc70‐specific oligonucleotide probe identified a 2.55‐kb mRNA in cerebellum and cerebral cortex of all rats. An hsp70‐specific oligonucleotide probe identified a 3.05‐kb mRNA and a 3.53‐kb mRNA in cerebellum and cerebral cortex of heat‐shocked and amphetamine‐treated rats, but not in control rats. Quantitation demonstrated that both hsp70 and hsc70 mRNA levels, relative to 18S rRNA levels, were increased following each treatment. The relative levels of both mRNAs were higher in cerebellum than in cerebral cortex. In amphetamine‐treated rats, hsc70 mRNA relative levels increased at body temperatures greater than 39°C, whereas hsp70 mRNA synthesis was induced at temperatures greater than 40°C. Total thermal response values and relative levels of both mRNAs were compared. The results suggested that both the transcription and turnover of hsp70 mRNAs differed between cerebellum and cerebral cortex. At equivalent total thermal response values, amphetamine‐treated rats had higher relative levels of hsp70 mRNAs than heat‐shocked rats, suggesting that amphetamine enhanced the induction of hsp70 mRNAs.

Developmental expression of mRNAs encoding thymosins β4 and β10 in rat brain and other tissues

In the course of screening a fetal rat cerebellum cDNA library for developmentally regulated sequences, we have identified a cDNA clone identical in sequence to that encoding a protein originally isolated from thymus, thymosin β10. Based on northern hybridization analyses with gene-specific oligonucleotide probes derived from the 3′-untranslated regions of thymosin β10 mRNA and the closely related β4 mRNA, we showed that both thymosin mRNAs were present at highest levels in fetal cortex and cerebellum but also were present at varying levels in all other fetal tissues examined (thymus, spleen, lung, kidney, adrenal, heart, and liver). Steady-state levels of thymosin β10 mRNA in cerebellum declined to negligible levels after day 14 of postnatal development. Its levels exhibited a similar pattern in developing cortex, although the adult cortex had slightly higher thymosin β10 mRNA levels. These results suggest that thymosin β10 mRNA is subject to strong developmental regulation in the rat central nervous system. Reduction of thymosin β10 mRNA levels also was seen during development of kidney, heart, and liver. Levels of both thymosin β10 and β4 mRNAs remained relatively constant during development of thymus, spleen, and lung. Thymosin β4 mRNA levels dropped much less sharply during brain development than did levels of the β10 mRNA. Testis and ovary contained the highest relative levels of thymosin β10 mRNA among adult tissues, but little thymosin β4 mRNA. A novel thymosin β10 mRNA species unique to adult testis was detected. These results indicate that both thymosins must function in the development of brain and many other organs, as well as in different subsets of organs in the adult.

Heat-Shock 70 Messenger RNA Levels in Human Brain: Correlation with Agonal Fever

Abstract: Systematic review of antemortem clinical information on randomly selected Alzheimer disease (AD) patients revealed that ∼40% of the patients had a recorded fever of ≥39.2°C at or near death. Using isolation and quantitation techniques appropriate for analysis of human brain mRNAs, we found that low levels of inducible heat‐shock protein 70 (hsp70) mRNAs were present in cerebellum of afebrile AD patients and that mRNA levels were usually lower in two brain regions affected in AD, i.e., hippocampus and temporal cortex. Levels of hsp70 mRNAs were increased three‐ to 33‐fold in cerebellum of febrile patients compared with levels in patients whose recorded temperatures were ≤37.5°C. Levels of hsp70 mRNAs were also increased in hippocampus and cortex of these febrile patients, but to a lesser extent than cerebellum. Heat‐shock cognate 70 (hsc70) mRNAs were present at highest levels in afebrile cerebellum and were also present in the other brain regions. In cerebellum of patients with the highest temperatures, hsc70 mRNAs were induced severalfold over basal levels. Although there was a low and variable induction of hsc70 mRNAs in temporal cortex of these patients, there was no evidence for any induction in hippocampus. Increased heat‐shock 70 mRNA levels did not correlate with hypoxia, coma, hypertension, hypoglycemia, seizures, or medication. These results indicate that a specific agonal stress, namely fever, can increase the levels of heat shock 70 mRNAs in AD brain; however, there is no evidence to suggest that affected regions of AD brain have higher overall levels of these mRNAs. Failure to obtain adequate agonal state information could result in inaccurately identifying short‐term stress‐related changes in postmortem brain as neuropathology characteristic of a chronic disease state.

Alterations in Actin-Binding β-Thymosin Expression Accompany Neuronal Differentiation and Migration in Rat Cerebellum

The β4‐and β10‐thymosins, recently identified as actin monomer‐sequestering proteins, are developmentally regulated in brain. Using specific mRNA and protein probes, we have used in situ hybridization and immunohis‐tochemical techniques to investigate the distribution of the β‐thymosin mRNAs and their proteins in developing rat cerebellum. Early in postnatal development, both β‐thymosin mRNAs were expressed at highest levels in the postmitotic, premigratory granule cells of the external granular layer; expression diminished as granule cells migrated to and differentiated within the developing internal granular layer. In addition, both β‐thymosin proteins were present in bundles of cerebellar afferent fibers in the white matter at this time. Throughout the maturation period, both proteins were present in elongating parallel fibers in the upper portion of the molecular layer. Later in cerebellar development, thymosin β4, but not thymosin β10, was expressed in Golgi epithelial cells and Bergmann processes. Thymosin β4 was expressed in a small population of cells with microglial morphology scattered throughout the gray and white matter. Thymosin β10 was detected in an even smaller population of glia. Expression of thymosin β4 and thymosin β10 in premigratory granule cells and in growing neuronal processes is consistent with the possibility that both β‐thymosins are involved in the dynamics of actin polymerization during migration and process extension of neurons.

Selective postmortem degradation of inducible heat shock protein 70 (hsp70) mRNAs in rat Brain

Summary1. Altered mRNA levels in postmortem brain tissue from persons with Alzheimers 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 unstablein 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.

Developmental Characterization of Thymosin β4 and β10 Expression in Enriched Neuronal Cultures from Rat Cerebella

Abstract: The β4 and β10 thymosins are G‐actin binding proteins that exhibit complex patterns of expression during rat cerebellar development. Their expression in vivo is initially high in immature granule cells and diminishes as they migrate and differentiate, ceasing altogether by postnatal day 21. Thymosin β4 is present in a subset of glia throughout postnatal development, and its synthesis is also induced in maturing Bergmann glia. In contrast, thymosin β10 is only present at very low levels in a very small subpopulation of glia in the adult cerebellum. To study the factors differentially regulating expression of the β‐thymosins, we characterized their patterns of expression in primary cultures of rat cerebellum. Both β‐thymosins were initially expressed in granule cells, although expression, especially of thymosin β4, was truncated compared with the in vivo time course. As in vivo, thymosin β4 was synthesized at much higher levels in astrocytes and microglia in cultures from postnatal cerebellum than was thymosin β10. Unlike in vivo, the latter was expressed in glia cultured from fetal cerebellum. The similarities between the in vivo and in vitro expression of the β‐thymosins show that modulation of tissue culture conditions could be used to identify factors regulating β‐thymosin expression in vivo. The differences would identify regulatory mechanisms that are not evident from the in vivo studies alone.

Differential expression of heat shock 70 proteins in primary cultures from rat cerebellum

While a number of studies have described the heat shock response in established cell lines and in primary cultures of cells derived from the nervous system, there has been no systematic analysis comparing expression and localization of the inducible heat shock 70 (hsp70) proteins and the constitutively synthesized members of the family (hsc70) in neurons and glia. In the present communication, we utilized specific probes to compare the expression of hsp70 and hsc70 mRNAs and proteins in two types of primary cultures, astroglial and neuro-astroglial, from postnatal rat cerebellum. Conditions were adjusted to maintain physiological numbers of microglia in both types of culture, and cultures were analyzed at a number of different time points following a precisely defined heat shock. The northern, in situ hybridization and immunohistochemical analyses resulted in a number of novel observations concerning the nature of the heat shock response in these neuronal and glial cells. In postnatal day 4-5 cultures, hsp70 mRNA levels were elevated for at least 10 h in both types of culture, but in situ hybridization analysis showed no evidence for hsp70 mRNAs in neurons. Microglia were the only cell type in which hsp70 was detected in non-stressed cultures and this cell type contained the highest concentrations of hsp70 proteins in stressed cultures. Hsc70 mRNA levels were also increased after heat shock, but the increase was more transient. Hsc70 mRNAs and proteins were present in all cell types, again with the highest concentrations being present in microglia. Hsc70 mRNAs and proteins were localized in the cytoplasm at all time points examined, with hsc70 protein also being localized in nucleoli. Hsp70 mRNAs and proteins were diffusely localized over nuclei of astrocytes, as well as of most microglia. Hsp70, but not hsc70, was localized on chromosomes in glia once they had resumed cell division after heat shock, suggesting a role for hsp70 either in targeting damaged chromosomal proteins or in cell division. Some cytoplasmic hsp70 was observed in astrocytes of the mixed neuro-astroglial cultures and a delayed hsp70 immunoreactivity was observed in granule neurons in these cultures, suggesting either that translation of low levels of hsp70 mRNAs was more efficient in neurons, or that glial-neuronal translocation of hsp70 proteins had taken place. These results suggest that metabolism and functions of different heat shock protein family members may not always be identical and that care must be taken in extrapolation of results from one cell type to another.

Polyadenylated messenger RNA in paired helical filament-immunoreactive neurons in Alzheimer disease.

SummaryAn antibody raised against isolated paired helical filaments (PHF) was used to identify tangle-bearing (PHF+) neurons in autopsy brain tissue from six Alzheimer disease (AD) patients and six age-matched controls (AMC). A comparison of the levels of polyadenylated messenger RNA [poly(A)+ mRNA] in PHF+ and PHF∼ neurons of similar cross-sectional area in temporal and parietal lobe and cerebellum from four AD and four AMC brains was made by analysis of in situ hybridization of [3H] polyuridylate [poly(U)] to intracellular poly(A)+ mRNA. In PHF+ neurons, the level of poly(A)+ mRNA was approximately two-thirds that in similar-sized PHF-neurons in either AD or AMC. The level of poly(A)+ mRNA in PHF- neurons in regions of the brain that have more of the histopathologically defined effects in AD was similar to that in regions with less effects.

Ribonuclease Activities and Distribution in Alzheimer's and Control Brains

Abstract Levels of free and total alkaline ribonuclease, and levels of acidic ribonuclease, were measured postmortem in control brains and in the brains of patients with Alzheimers disease. In each brain region assayed, whether control or Alzheimers, there was a statistically significant difference between the levels of free and total alkaline ribonuclease. Between 59 and 90% of the enzyme activity was associated with alkaline ribonuclease inhibitor in an inactive complex. Levels of free and total alkaline ribonuclease varied widely among different brains and brain regions, and were always lower in cerebellum than in temporal cortex and occipital pole. There was no significant difference in the levels of total alkaline ribonuclease, free alkaline ribonuclease, or acidic ribonu‐cleases between corresponding regions of Alzheimers and control brains. There was also no qualitative difference in the subcellular distribution of the alkaline and acidic ribonucleases between Alzheimers and control brain. No significant relationships were found between ribonuclease levels and age, neuritic plaque density, postmortem interval, or storage time.

Quantitative in situ hybridization analysis of glutamic acid decarboxylase messenger RNA in developing rat cerebellum.

The appearance and relative amounts of GAD mRNA in rat cerebellar neurons during postnatal development was studied by in situ hybridization. GAD mRNA content within all GABAergic neurons increased during the first month of postnatal development, but the degree and time course of the increase varied among different neuronal types. In newborn rats, GAD mRNA was present only in the prenatally-formed Purkinje and Golgi cells. GAD mRNA in Golgi cells had reached adult levels by postnatal day 14, while GAD mRNA levels in Purkinje cells reached adult levels one week later. Most basket cells expressed GAD mRNA by postnatal day 14, and final levels were attained one week later. Stellate cells in the bottom two-thirds of the molecular layer attained their final GAD mRNA content by postnatal day 21 whereas stellate cells in close proximity to the pial surface were not yet mature at this age. No GAD mRNA was detected within the external granular layer at any time during development. In adult rat, approximately 40% of cerebellar GAD mRNA was contained within the Purkinje cell population, 38% within the stellate cells, 17% within the basket cells, and only 5% within the Golgi cells. Increases in GAD mRNA within GABAergic neurons during cerebellar development correlated with the timing of neuronal maturation and synaptogenesis in these cell populations, suggesting that synaptic activity affects GAD gene expression in developing cerebellum.