Faculty Bibliography

Cytoplasmic RNA species have been identified recently within neurofibrillary tangles and senile plaques of Alzheimer's disease brain. To determine whether RNA sequestration is a common feature of other lesions found in progressive neurodegenerative disorders, acridine orange histofluorescence was employed, alone or in combination with immunohistochemistry and thioflavine-S staining to identify RNA species in paraffin-embedded brain tissue sections. Postmortem samples came from 39 subjects with the following diagnoses: Alzheimer's disease, amyotrophic lateral sclerosis/parkinsonism-dementia complex of Guam, corticobasal degeneration, diffuse Lewy body disease, normal controls, multiple system atrophy, Parkinson's disease, Pick's disease, progressive supranuclear palsy, and Shy-Drager syndrome. RNAs were detected in neurofibrillary tangles and neuritic senile plaques as well as in Pick bodies. However, Lewy bodies, Hirano bodies, and cytoplasmic glial inclusions did not contain abundant cytoplasmic RNA species. These observations demonstrate the selective localization of RNA species to distinct pathological lesions of neurodegenerative disease brains

The fimbria-fornix transection paradigm has been used as a model of retrograde neurodegeneration within the medial septal nucleus and anterograde degeneration of axon terminals within the lateral septal nucleus. Because the maintenance and survival of neurons may depend on the integrity of both efferents and afferents, the ultrastructure of neurons in the medial septal nucleus and dorsolateral septal nucleus was analysed at three, seven, 14, 30 days, and six months following unilateral transection of the fimbria-fornix in adult rats. Degeneration of axonal and somatodendritic compartments occurred in both nuclei on the side ipsilateral to fimbria-fornix transection. Degeneration of axons and terminals was present by three days and dissipated thereafter, although degenerating axodendritic and axosomatic terminals were still detected at 14-30 days postlesion. Dendrosomal alterations in both septal nuclei manifested as redistribution of organelles, dispersion and loss of rough endoplasmic reticulum, formation of membrane-bound vacuolar cisternae and membranous inclusions, loss of cytoplasmic matrix, and dispersion of chromatin throughout the nucleoplasmic matrix. These changes occurred in the absence of apparent ultrastructural damage to mitochondria and condensation of the nucleus. Dendritic pathology in both the medial and dorsolateral septal nuclei was most prominent at 14-30 days postlesion, but the neuropil recovered to control appearance by six months postlesion. In contrast, the cytoplasmic rarefaction and vacuolation of neuronal cell bodies were persistent in both the medial septal nucleus and the dorsolateral septal nucleus. We conclude that, following disconnection from the hippocampus, ultrastructural abnormalities occur within neurons in both the medial and lateral septal nuclei. The characteristics and time-course for these changes are similar in both nuclei. The neuropilar degeneration was transient, in contrast to the neuronal cell body injury which was persistent and was morphologically consistent with long-term neuronal atrophy.

In screening amplified poly(A) mRNA from hippocampal dendrites and growth cones in culture to determine candidates for local translation, we found that select transcription factor mRNAs were present. We hypothesized that synthesis of transcription factor proteins within dendrites would provide a direct signaling pathway between the distal dendrite and the nucleus resulting in modulation of gene expression important for neuronal differentiation. To evaluate this possibility, radiolabeled amplified antisense RNA was used to probe slot blots of transcription factor cDNAs as well as arrayed blots of zinc finger transcription factors. The mRNAs encoding the cAMP response element binding protein (CREB), zif 268, and one putative transcription factor were detected. We expanded upon these results showing that CREB protein is present in dendrites, that translation of CREB mRNA in isolated dendrites is feasible and that CREB protein found in dendrites can interact with the cis-acting cyclic AMP reponse element DNA sequence by using an in situ Southwestern assay. Further, CREB protein in dendrites is not transported to this site from the cell body because fluorescently tagged CREB microperfused into the soma did not diffuse into the dendrites. In addition, CREB protein microperfused into dendrites was rapidly transported to the nucleus, its likely site of bioactivity. Lastly, by using the isolated dendrite system we show that phosphorylation of Ser-133 on CREB protein can occur in isolated dendrites independent of the nucleus. These data provide a regulatory pathway in which transcription factors synthesized and posttranslationally modified in dendrites directly alter gene expression bypassing the integration of signal transduction pathways that converge on the nucleus.

The polypeptide composition of neurofibrillary tangles (NFTs) and senile plaques (SPs) has been characterized extensively within the Alzheimer's disease (AD) brain. Because few data exist on the nonproteinaceous components of these lesions, we sought to determine if NFTs, neuropil threads (NTs), and SPs contain RNA species. To accomplish this, acridine orange (AO) histofluorescence was employed, alone or in combination with thioflavine S (TS) staining and immunohistochemistry to identify RNAs in paraffin-embedded tissue sections of hippocampus and entorhinal cortex. Postmortem brain samples came from 32 subjects including AD and elderly Down's syndrome (DS) patients, age-matched normal controls, and non-AD diseased controls. AO stained the cytoplasm of normal hippocampal and entorhinal neurons in all of the cases, while NFTs, NTs, and SPs were AO-positive in the same regions of AD and DS brains. Cytoplasmic AO histofluorescence was abolished with RNase, but not DNase or proteinase K, indicating the relative specificity of AO for RNA species. Quantitative analysis of double-labeled sections demonstrated that approximately 80% of TS-positive NFTs also were AO-positive, whereas approximately 55% of TS-stained SPs contained AO labeling. These novel observations demonstrate the presence of RNAs in NFTs, NTs, and SPs.

The effects of CNS axotomy on glutamate transporter and glutamate receptor expression were evaluated in adult rats following unilateral fimbria-fornix transections. The septum and hippocampus were collected at 3, 7, 14, and 30 days postlesion. Homogenates were immunoblotted by using antibodies directed against glutamate transporters (GLT-1, GLAST, and EAAC1) and glutamate receptors (GluR1, GluR2/3, GluR6/7, and NMDAR1), and they were assayed for glutamate transport by D-[3H]aspartate binding. GLT-1 was decreased at 7 and 14 days postlesion within the ipsilateral septum and at 7 days postlesion in the hippocampus. GLAST was decreased within the ipsilateral septum and hippocampus at 7 and 14 days postlesion. No postlesion alterations in EAAC1 immunoreactivity were observed. D-[3H]Aspartate binding was decreased at 7, 14, and 30 days postlesion within the ipsilateral septum and 14 days postlesion in the hippocampus. GluR2/3 expression was down-regulated at 30 days postlesion within the ipsilateral septum, whereas GluR1, GluR6/7, and NMDAR1 immunoreactivity was unchanged. In addition, no alterations in glutamate receptor expression were detected within hippocampal homogenates. This study demonstrates a selective down-regulation of primarily glial, and not neuronal, glutamate transporters and a delayed, subtype-specific down-regulation of septal GluR2/3 receptor expression after regional deafferentation within the CNS.