Biomedical subjects
Paul D Coleman
Publications and source records attributed to Paul D Coleman.
Single-channel quantitative multiplex reverse transcriptase-polymerase chain reaction for large numbers of gene products differentiates nondemented from neuropathological Alzheimer's disease.
Effective approaches using array technologies are critical to understand the molecular bases of human diseases. The results obtained using such procedures require analysis and validation procedures that are still under development. In the context of Alzheimer's disease, in which the identification of molecular mechanisms of underlying pathologies is vital, we describe a robust assay that is the first real-time reverse transcriptase-polymerase chain reaction-based high-throughput approach that can simultaneously quantitate the expression of a large number of genes at the copy number level from a minute amount of starting material. Using this approach within the human brain, we were able to quantitate as many as 19 genes at a time with only one type of fluorescent probe. The number of genes included can be considerably increased. Examples of consistent changes in Alzheimer's disease within these 19 candidate genes included reductions in targets related to the dendritic and synaptic apparatus. These changes were specific to Alzheimer's disease when compared with Parkinson's disease cases. We also present comparison data with microarray analysis from the same brain region and the same patients. The high sensitivity and reproducibility of this technology coupled with appropriate multivariate analysis is proposed here to form a biotechnology platform that can be widely used for diagnostic purposes as well as basic research.
Synaptic slaughter in Alzheimer's disease.
Synaptic loss is currently established as the best neurobiological correlate of the cognitive deficits of Alzheimer's disease (AD) [Ann. Neurol. 27 (1990) 457; Ann. Neurol. 30 (1991) 572]. We provide evidence that still living neurons lose synapses in AD, in addition to the synapse loss due to death of neurons. We also provide evidence indicating that in addition to loss of synapses, synaptic function is also affected in AD by decrements in transcript species related to synaptic vesicle trafficking.
Defects in expression of genes related to synaptic vesicle trafficking in frontal cortex of Alzheimer's disease.
Loss of synapses correlates with cognitive decline in Alzheimer's disease (AD). However, molecular mechanisms underlying the synaptic dysfunction and loss are not well understood. In this study, microarray analysis of brain tissues from five AD cases revealed a reduced expression of a group of related genes, all of which are involved in synaptic vesicle (SV) trafficking. By contrast, several synaptic genes with functions other than vesicle trafficking remained unchanged. Quantitative RT-PCR confirmed and expanded the microarray findings. Furthermore, immunoblotting showed that the protein level of at least one of these gene products, dynamin I, correlated with its reduced transcript. Immunhistochemical analysis exhibited an altered distribution of dynamin I immunolabeling in AD neurons. Microarray analysis of transgenic mice with mutated amyloid precursor protein showed that although the transcript levels for some of the SV trafficking-related genes are also decreased, the change in dynamin did not replicate the AD pattern. The results suggest a link among SV vesicle-trafficking pathways, synaptic malfunction, and AD pathogenesis.
Generation of single-frequency coherent transition radiation by a prebunched electron beam traversing a vacuum beam tunnel in a periodic medium.
A classical Maxwell equation longitudinal boundary value problem analysis of a prebunched ac electron beam traversing a vacuum tunnel in a periodic layered dielectric medium is used to calculate the single-frequency coherent transition radiation power generated per unit length. For low voltage electron beams in the kilovolt range, only transition radiation is produced, the Cerenkov effect being below threshold. A numerical example indicates that power levels of the order of milliwatts per centimeter can be produced in the 35 GHz range with 3-10 keV beams. An interesting aspect of transition versus Cerenkov radiation is that the transition "cone" of radiation is in the backward direction of the charge beam.
High-resolution localization of clathrin assembly protein AP180 in the presynaptic terminals of mammalian neurons.
Synaptic vesicles (SVs) assemble at the presynaptic compartment through a clathrin-dependent mechanism that involves one or more assembly proteins (APs). The assembly protein AP180 is especially efficient at facilitating clathrin cage formation, but its precise ultrastructural localization in neurons is unknown. Using immunoelectron microscopy, we demonstrate the presynaptic localization of AP180 in axon terminals of rat cerebellar neurons. In contrast, the assembly protein AP2 was associated with both the presynaptic plasma membrane and the cytosolic side of the membrane at postsynaptic and extrasynaptic sites. Furthermore, ultrastructural analysis of primate retina showed that AP180 immunoreactivity was preferentially and highly enriched at ribbon synapses, where glutamate is released tonically at high levels and rapid vesicle turnover is essential. To maintain functional synaptic transmission, neurotransmitter-filled SVs must be readily available, and this requires proper reassembly of new vesicles. The expression of AP180, in addition to AP-2, in the clathrin-mediated endocytic pathway might add another level of control to SV reformation for efficient assembly of clathrin, effectively controlling the size of assembled vesicles and faithfully recovering SV-specific components.
Single-cell antisense RNA amplification and microarray analysis as a tool for studying neurological degeneration and restoration.
Neurodegenerative diseases typically affect subpopulations of neurons. Characterizing these vulnerable cells and identifying the factors that make them susceptible to damage while neighboring cells remain resistant are essential to the understanding of molecular pathogenesis that underlies neurodegenerative diseases. Classically, molecular analysis of the central nervous system involves the identification and isolation of an anatomic region of interest; next, the relevant tissue is pulverized, and the resulting homogenate is analyzed. Although this method provides useful data, its effectiveness diminishes when used in areas of high cellular diversity or in instances in which one cell type is lost as a consequence of selective cell death or quiescence. A technique that affords the ability to assess molecular events in a very precise anatomical site would provide a powerful tool for this research discipline. In this review, we discuss the amplification of messenger RNA from single neural cells and the subsequent use of the RNA to probe DNA microarrays in an effort to create cell-specific molecular profiles. Specifically, recent work in single-cell expression profiling in Alzheimer's and Huntington's diseases is discussed. We also review some new work with neural stem cells and their application to restorative neurobiology. Finally, we discuss the use of cell-specific molecular profiles to better understand the basics of neuronal cell biology.
Progressive reduction of synaptophysin message in single neurons in Alzheimer disease.
The data presented here examine 2 hypotheses: 1) that viable but vulnerable single neurons remaining in the Alzheimer brain lose synaptic markers, and 2) that the extent of this loss is related to the disease state of these single neurons when disease state is defined by immunoreactivity. We used double immunohistochemistry (IHC) to define neurofibrillary tangle (NFT) and phosphorylation status of tau at selected defined epitopes. This double IHC was combined with quantitative in situ hybridization for message for the synaptic marker, synaptophysin, in 1,127 single hippocampal CA1 pyramidal neurons from 15 Alzheimer disease (AD) and 4 control cases. We found that there is a graded, progressive, decrease of synaptophysin message expressed by single neurons related to immunohistochemical markers of tau status, and that neurons in similar immunohistochemically defined classes show similar losses of synaptophysin message regardless of whether they were sampled from clinical control brains or advanced AD. The resulting conclusions are consistent with a suggestion that differences among clinically defined AD and control status are defined by the numbers of neurons in various disease states.
Immunohistochemical characterization of clathrin assembly protein AP180 and synaptophysin in human brain.
Neurons rely on clathrin-mediated endocytosis for retrieving synaptic vesicles (SVs) at the presynaptic compartment after the release of neurotransmitters. The clathrin assembly protein AP180 is shown to be a regulator for this clathrin-dependent SV recycling pathway. AP180 is efficient in facilitating the formation of clathrin-coated vesicles and regulating their size, but its exact location in synapse is not clear. In this study, we compared the expression of AP180 with synaptophysin in the aged human brain using confocal immunofluorescence microscopy. Synaptophysin is well characterized for its association with SVs and therefore a commonly used presynaptic marker. We achieved satisfactory immunofluorescent labeling by using an autofluorescence blocker Sudan Black B and more photostable Alexa Fluor dyes. Although we found that AP180 had an overall expression similar to synaptophysin, the immunoreactivity for the two proteins did not always co-localize.
Facial nerve axotomy in aged and young adult rats: analysis of the glial response.
With increasing age, there is a trend towards greater morbidity and injury extent with brain injury. Because several reports have suggested that microglia and astrocytes have an exacerbated response to brain injury in the aged, we set out to explore glial responses to facial nerve axotomy. This model was chosen because the glial responses are well-characterized in young rats and there is no perturbation of the blood-brain barrier (BBB). Immunohistochemistry was performed for glial fibrillary acidic protein (GFAP), leukocyte common antigen, type 3 complement receptor, and major histocompatability complex classes I and II. Quantitative analysis showed that age does not affect the glial response to axotomy in the lesioned facial nucleus; however, an aging-related contralateral effect with enhanced GFAP-labeling was observed. Interestingly, despite a lack of infiltrating neutrophils, a T cell influx was observed in both young and aged rats. Overall, these results suggest that neutrophil extravasion and BBB breakdown are underappreciated with regards to aging and injury exacerbation.