Faculty Bibliography

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.

Low extracellular glutamate content is maintained primarily by high-affinity sodium-dependent glutamate transport. Three glutamate transporter proteins have been cloned: GLT-1 and GLAST are astroglial, whereas EAAC1 is neuronal. The effects of axotomy on glutamate transporter expression was evaluated in adult rats following unilateral fimbria-fornix and corticostriatal lesions. The hippocampus and striatum were collected at 3, 7, 14, and 30 days postlesion. Homogenates were immunoblotted using antibodies directed against GLT-1, GLAST, EAAC1, and glial fibrillary acidic protein and assayed for glutamate transport by D-[3H]aspartate binding. GLT-1 immunoreactivity was decreased within the ipsilateral hippocampus and striatum at 14 days postlesion. GLAST immunoreactivity was decreased within the ipsilateral hippocampus and striatum at 7 and 14 days postlesion. No alterations in EAAC1 immunoreactivity were observed. D-[3H]Aspartate binding was decreased at 14 days postlesion within the ipsilateral hippocampus and at 7 and 14 days postlesion within the ipsilateral striatum. By 30 days postlesion, glutamate transporters and D-[3H]aspartate binding returned to control levels. This study demonstrates the down-regulation of primarily glial, and not neuronal, glutamate transporters following regional disconnection.

Deposition of beta-amyloid (A beta) in senile plaques is a major pathological characteristic of Alzheimer's disease. A beta is generated by proteolytic processing of amyloid precursor proteins (APP). APP is a member of a family of related polypeptides that includes amyloid precursor-like proteins APLP1 and APLP2. To examine the distribution of APLP2 in the nervous system, we generated antibodies specific for APLP2 and used these reagents in immunocytochemical and biochemical studies of the rodent nervous system. In this report, we document that in cortex and hippocampus, APLP2 is enriched in postsynaptic compartments. In the olfactory system, however, APLP2 is abundant in olfactory sensory axons, and axon terminals in glomeruli. Confocal microscopy revealed that APLP2 is present in both pre- and postsynaptic compartments in the olfactory bulb. Notably, mRNA encoding chondroitin sulfate glycosaminoglycan (CS GAG)-modified forms of APLP2 are enriched in the olfactory epithelium, relative to alternatively-spliced mRNA, encoding CS GAG-free forms of APLP2. In addition, we demonstrate that CS-modified APLP2 forms accumulate in the olfactory bulb. CS proteoglycans are known to play an important role in regulating cell migration and neuronal outgrowth. Since sensory neurons in the olfactory epithelium are in a state of continual turnover, axons of newly generated cells must establish synaptic connections with neurons in the olfactory bulb in adult life. The presence of APLP2 in olfactory sensory axons and glomeruli is consistent with the view that this protein may play an important role in axonal pathfinding and/or synaptogenesis.