Nancy R. Nichols

Evidence for apoptotic cell death in Alzheimer's disease

We provide evidence for apoptosis in Alzheimers disease using the in situ labeling technique TUNEL (terminal transferase-mediated dUTP-biotin nick end labeling). The technique specifically detects apoptotic cells by utilizing terminal transferase to incorporate biotinylated nucleotides into the fragmented DNA of apoptotic cells. The labeled cells are visualized by reaction with avidin peroxidase and a suitable substrate. Sections from the hippocampus of Alzheimer-diseased (AD) brains and non-AD brains were examined for apoptosis. While considerable variation in the quantity of apoptotic cells was observed among individual samples, the incidence of apoptosis in AD brains was elevated in comparison to age-matched, non-AD brains in specific regions of the hippocampal formation. Immunostaining indicated that both neurons and astrocytes were undergoing apoptosis, although the majority of the TUNEL-positive cells appeared to be glial, based on the location of the stained cells. These data suggest that apoptosis may be involved in both the primary neuronal cell loss and in the glial response that is a component of AD.

GFAP mRNA increases with age in rat and human brain

Glial fibrillary acidic protein (GFAP) mRNA was examined by RNA blot hybridization in three age groups of two cohorts of male F-344 rats and in 47 human postmortem brain samples. GFAP mRNA increased in the hippocampus and striatum of 24 versus 6- to 7-month-old rats. Another astrocytic molecular marker, glutamine synthetase mRNA, did not change with age in rat brain. Rat GFAP mRNA prevalence was inversely correlated with serum testosterone but not correlated with serum corticosterone. In human hippocampus, frontal and temporal cortex, GFAP mRNA also increased in older (60-79 years) compared with middle-aged (25-59 years) individuals. In contrast, mitochondrial cytochrome oxidase subunit 1 mRNA did not change between age groups in any region. By combining the three regions for further analysis, GFAP mRNA increased with age irregardless of gender, alcoholism in the middle-aged group, or whether brains were classified as normal or neuropathologic (excluding Alzheimers disease pathology). These data indicate that increased GFAP protein or GFAP-immunoreactive astrocytes in rats and humans may result from transcriptional or post-transcriptional regulation and extend the number to three species (including mouse) showing an increase in GFAP mRNA with age. Factors that are known to regulate GFAP mRNA expression in young brains are considered as possible causes of age-related increases.

Glial fibrillary acidic protein: regulation by hormones, cytokines, and growth factors.

Levels of glial fibrillary acidic protein (GFAP), an astrocyte‐specific intermediate filament protein, are altered during development and aging, GFAP also responds dynamically to neurodegenerative lesions. Changes in GFAP expression can occur at both transcriptional and translational levels. Modulators of GFAP expression include steroids, cytokines, and growth factors. GFAP expression also shows brain region‐specific responses to sex steroids and of astrocyte‐neuronal interactions. The 5′‐upstream sequences of rat, mouse, and human are compared for the presence of response elements that are candidates for transcriptional regulation of GFAP. We propose that the regulation of the GFAP gene has evolved a system of controls that allow integrated responses to neuroendocrine and inflammatory modulators.

TGF-β1 mRNA Increases in Macrophage/Microglial Cells of the Hippocampus in Response to Deafferentation and Kainic Acid-Induced Neurodegeneration

This study examined TGF-beta 1 mRNA levels and cellular localization in the F344 rat hippocampus following deafferentation or kainic acid (KA)-induced neurodegeneration. By RNA solution hybridization, TGF-beta 1 transcripts were at low prevalence in intact adult rat hippocampus (0.02 pg/microgram total RNA). Four days after unilateral entorhinal cortex lesioning (ECL), TGF-beta 1 mRNA increased threefold in the ipsilateral hippocampus. This increase was localized to the outer molecular layer of the dentate gyrus, where gliosis, synapse loss, and synaptic reorganization occur. TGF-beta 1 mRNA also increased in the hippocampus after KA-induced limbic seizures, particularly in the areas of the hippocampus undergoing neurodegeneration. Microglia [OX-42 immunoreactive (IR) cells] responded to these two lesions with distinct morphological changes. Combined immunocytochemistry-in situ hybridization showed that TGF-beta 1 mRNA was localized to reactive microglia (OX-42-IR, with blunt processes), but not to resting ramified microglia (OX-42-IR, with numerous fine processes) or to astrocytes (GFAP-IR). After ECL, round macrophage-like cells (OX-42-IR with TGF-beta 1 mRNA) were seen at the wound site. Thus, brain macrophage/microglial cells produce TGF-beta 1 mRNA in the hippocampus in response to deafferentation and neurodegeneration.

Messenger RNA for glial fibrillary acidic protein is decreased in rat brain following acute and chronic corticosterone treatment

RNA coding for a 50 kDa polypeptide decreased by 50% in 5 brain regions after corticosterone (CORT) treatment (40 mg/kg for 3 days). By hybrid selection and in vitro translation, the 50 kDa polypeptide is identified as glial fibrillary acidic protein (GFAP). Hippocampal GFAP mRNA (2.9 kb) decreases in a dose-dependent manner in response to CORT by RNA blot hybridization using a mouse GFAP cRNA probe; a similar decrease in response to the glucocorticoid agonist, RU 28362, is consistent with a type II glucocorticoid receptor-mediated effect. GFAP mRNA is decreased in both hippocampus and cortex following acute (1-3 days) and chronic (3 days to 3 months) CORT treatment. GFAP gene expression is disinhibited in the rat hippocampus by 7 days post adrenalectomy but not by 3 days. Finally, two clones (CR46 and CR59) that were isolated from a rat hippocampal cDNA library by differential hybridization, show decreased RNA abundance in CORT-treated rats compared to controls. A partial DNA sequence derived from the two clones exhibits 94% nucleotide identity and 96% derived amino acid identity with mouse GFAP mRNA. These results indicate that GFAP mRNA is under negative regulation by glucocorticoids and suggests that glucocorticoids may be used to inhibit GFAP gene expression in vivo in order to assess the role of GFAP in temporal aspects of central nervous system damage.

Transforming growth factor-β1 induces neuronal and astrocyte genes: Tubulin α1, glial fibrillary acidic protein and clusterin

Transforming growth factor-beta 1 was studied as a possible regulator of messenger RNAs in astrocytes and neurons that increase after hippocampal deafferentation by perforant path transection: tubulin alpha 1, clusterin and glial fibrillary acidic protein messenger RNA. Because transforming growth factor-beta 1 messenger RNA is increased after this lesion, we examined which messenger RNA lesion responses could be induced by transforming growth factor-beta 1 alone. Porcine transforming growth factor-beta 1 infused into the lateral ventricle elevated the messenger RNAs for tubulin alpha 1, clusterin and glial fibrillary acidic protein 24 h after infusion in the ipsilateral hippocampus. As assayed by nuclear run-on, the transcription of glial fibrillary acidic protein RNA was increased in the ipsilateral hippocampus after perforant path transection and in primary rat astrocyte cultures by transforming growth factor-beta 1. In contrast, transforming growth factor-beta 1 did not change apolipoprotein-E messenger RNA or transcription, or growth associated protein-43 messenger RNA levels. We conclude that transforming growth factor-beta 1 increases subsets of neuronal and astrocyte messenger RNAs coding for cytoskeletal proteins that are also elevated in response to experimental lesions and Alzheimers disease. This suggests that transforming growth factor-beta 1 might be a local organizing factor of neuronal and astrocyte responses to brain injury.

Transforming growth factor β1 and fibronectin messenger RNA in rat brain: Responses to injury and cell-type localization

Transforming growth factor-beta 1 rapidly increases in adult rat brain in response to experimental lesions. This study characterized the schedule of changes, regional distribution, and cellular localization of striatal transforming growth factor-beta 1 messenger RNA and fibronectin messenger RNA following partial striatal deafferentation by frontal cortex ablation. Frontal cortex ablation induced striatal transforming growth factor-beta 1 messenger RNA elevations that coincided temporally and overlapped anatomically with the course of degeneration of cortico-striatal afferent fibers. Within three days post-lesioning, transforming growth factor-beta 1 messenger RNA was localized at the cortical wound. By 10 days, the anatomical site of transforming growth factor-beta 1 messenger RNA expression shifted to the dorsal half of the deafferented striatum and co-localized with OX-42+ immunostained microglia-macrophage at the site of degenerating afferent terminals. Similarly, fibronectin messenger RNA also shifted from the cortical wound to the deafferented striatum by 10 days post-lesioning. Fibronectin messenger RNA was localized to glial fibrillary acidic protein+ immunostained astrocytes surrounding degenerating corticostriatal afferents. Infusion of transforming growth factor-beta 1 peptide elevated striatal and cortical fibronectin messenger RNA. These findings suggest that microglia-macrophage associated with degenerating afferent fibres can upregulate transforming growth factor-beta 1 messenger RNA and may influence fibronectin messenger RNA synthesis in reactive astrocytes. This study suggests that transforming growth factor-beta 1 has a role in controlling extracellular matrix synthesis following brain injury, which is analogous to that in peripheral wound healing.

Tumor Suppressor p53 Induction and DNA Damage in Hippocampal Granule Cells after Adrenalectomy

Tumor suppressor p53 encodes a protein that is an important regulator of the cell cycle. However, under certain conditions increased p53 expression results in programmed cell death or apoptosis. We used in situ hybridization histochemistry to investigate the role of p53 in the adrenalectomy-induced degeneration of hippocampal granule cells. Three days after adrenalectomy, a subpopulation of granule cells exhibiting morphological features of apoptosis expressed increased amounts of p53 mRNA. Both adrenalectomy-induced p53 expression and granule cell degeneration were prevented by daily administration of corticosterone. In situ end-labeling of nuclei containing fragmented DNA revealed a distribution similar to that of cells with increased p53 expression. These results demonstrate an association between p53 induction and apoptosis in the central nervous system and support the idea that cell cycle-related genes play a role in neuronal death pathways.

Corticosterone differentially regulates the bilateral response of astrocyte mRNAs in the hippocampus to entorhinal cortex lesions in male rats

This study examined the effect of adrenalectomy (ADX) and corticosterone (CORT) replacement on the levels of two astrocyte mRNAs during responses to unilateral entorhinal cortex lesions (ECL) to identify molecular mechanisms involved in glucocorticoid modulation of astrocyte activation following deafferentation. Both glial fibrillary acidic protein (GFAP) and sulfated glycoprotein-2 (SGP-2) mRNA were increased in the ipsilateral hippocampus 4 days following unilateral ECL. In unlesioned ADX rats CORT replacement decreased both messages in the hippocampus. CORT replacement suppressed the ECL-induced increase of GFAP mRNA in the contralateral, but not ipsilateral hippocampus of ADX rats. In contrast, CORT decreased SGP-2 mRNA both ipsi- and contralaterally. It is clear that several regulatory mechanisms are responsible for maintaining a physiological balance of astrocyte activity in the adult brain, and that changes in circuit integrity and the endocrine milieu can alter this balance.

Food Restriction Delays the Age-Related Increase in GFAP mRNA in Rat Hypothalamus

Astrogliosis with advancing age is correlated with increased expression of glial fibrillary acidic protein (GFAP). Hypothalamic GFAP mRNA prevalence was determined in male F344 rats of different ages that were fed ad lib (AL) and compared with that of rats that were food-restricted (FR) to 60% of AL levels. Hypothalamic GFAP mRNA increased 3-fold at 24 to 25 months in AL rats compared with 3 and 6 month groups. There were no differences in GFAP mRNA levels between AL and FR rats from 3 to 18 months. However, GFAP mRNA was significantly lower in FR than in AL rats at 24 to 25 months; FR rats reached the level of GFAP mRNA in 24 to 25 months AL rats by 33 months. Hypothalamic glutamine synthetase mRNA also increased with age in both dietary groups but did not differ between dietary groups at any age. The observation that FR delays the increased expression of GFAP in the hypothalamus during aging lends support to the hypothesis that upregulation of GFAP mRNA is a biomarker of brain aging.