Neurofilament distribution is altered in the Mnd (motor neuron degeneration) mouse.

Motor neuron degeneration (Mnd) is a genetic neurodegenerative disease of the mouse that is characterized by a progressive increase in motor dysfunction, moving from hind to fore limbs, leading to paralysis. An immunocytochemical analysis of the neurofilament distribution in spinal motor neurons in Mnd mice from all stages of the disease, including the presymptomatic, was performed using antibodies to different neurofilament subunits with different degrees of phosphorylation. Perikarya that stained with antibodies to phosphorylated neurofilaments were present in Mnd and control spinal cords, but the number of stained perikarya in Mnd was not significantly different from controls. There was a marked redistribution of neurofilaments within the cytoplasm of some motor neurons in Mnd cords. In Mnd but not controls, the immunoreaction product appeared marginated, leaving areas in the cytoplasm absent of immunostaining. These areas were observed in all stages of the disease, but less predictably in presymptomatics. Both the size of the areas and the number of motoneurons containing these areas appeared to increase with the severity of the disease. The number of anterior horn neurons in the hind limb region of lamina IX in spinal segment L4 of Mnd was lower than in controls, suggesting there is a loss of neurons in Mnd.

Quantitative decrease in synaptophysin message expression and increase in cathepsin D message expression in Alzheimer disease neurons containing neurofibrillary tangles.

Combining immunocytochemistry with in situ hybridization of Alzheimer disease (AD) hippocampus demonstrated a 50% reduction in grain density for synaptophysin message over CA1 pyramidal neurons containing neurofibrillary tangles (NFT) relative to near neighbor NFT-free neurons. This decrease was not global, but was selective since message grain density for the lysosomal protein, cathepsin D, increased 33% in these neurons (relative to NFT-free neurons). Poly A+ message grain density decreased by 25% in NFT neurons. Percent of the cell body containing NFT correlated -0.35 (p < 0.0001) with grain density for synaptophysin message. These data verify the concept of altered profiles of gene expression as a function of disease state within single cells and suggest that events associated with NFT formation may lead to altered expression of synaptic messages.

Cyclooxygenase-1 in human Alzheimer and control brain: quantitative analysis of expression by microglia and CA3 hippocampal neurons.

Epidemiological and clinical studies suggest that nonsteroidal anti-inflammatory drugs (NSAIDs) that inhibit cyclooxygenase (COX) slow the progression and delay the onset of Alzheimer disease (AD). Two isoforms of cyclooxygenase have been identified. Although much effort has recently been focused on the inducible COX-2 isoform, little is known about COX-1 expression in human brain. We report that COX-1 message and immunoreactivity are localized to human hippocampal CA3 and CA4 neurons, granular neurons in neocortical layer IV, and occasional cortical pyramidal neurons. Quantitative in situ hybridization showed no differences between COX-1 mRNA levels in control and AD CA3 hippocampal neurons. COX-1 immunoreactivity was also present in microglial cells in gray and white matter in all brain regions examined. COX-1 appeared to be expressed in microglial cells regardless of their activation state as determined by HLA-DR immunostaining. However, COX-1 immunopositive microglia were found in association with Abeta plaques, and the density of COX-1 immunopositive microglia in AD fusiform cortex was increased. This pattern suggests an overall increase of COX-1 expression in AD. Currently used NSAIDs inhibit both isoforms of cyclooxygenase. The present study shows that COX-1 is widely expressed in human brain, and raises the possibility that COX-1 may contribute to CNS pathology.

Neurons bearing neurofibrillary tangles are responsible for selected synaptic deficits in Alzheimer's disease.

The observation that neurons containing neurofibrillary tangles are usually adjacent to neurons free of any morphological indication of disease, suggests the hypothesis that it is NFT-bearing neurons that are primarily responsible for the loss of function in AD. Quantitative Golgi postmortem studies from our laboratories have indicated that there is in many regions of the brains of nondemented humans an age-related increase in dendritic extent of single neurons. In Alzheimer's disease, this normal, age-related increase in dendritic extent was not found, leading to the hypothesis that one of the neurobiological defects in AD is a failure of neuronal plasticity. Message levels of the growth-associated protein, GAP-43, in frontal association cortex (area 9/46) indicated that AD brains with the highest density of neurofibrillary tangle-bearing neurons, showed GAP-43 message levels decreased of the order of 6-fold relative to AD brains with the lowest density of NFT. Combined immunocytochemistry to differentiate tangle-bearing from tangle-free neurons with in situ hybridization to define relative GAP-43 message levels in single neurons revealed that grain density over tangle-bearing neurons containing nuclei was reduced 3-fold compared to that over adjacent tangle-free neurons. This reduction in expression of GAP-43 message in tangle-bearing neurons was selective, because using probes for other messages showed that grain density over tangle-bearing neurons was, on average, increased or similar to that over adjacent non-tangle-bearing neurons. Message levels for the synaptic vesicle-associated protein, synaptophysin, have also been found to be reduced in tangle-bearing neurons relative to adjacent tangle-free neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Preliminary evidence: decreased GAP-43 message in tangle-bearing neurons relative to adjacent tangle-free neurons in Alzheimer's disease parahippocampal gyrus.

Loss of synapses has been shown to correlate with the severity of dementia in Alzheimer's disease (AD). Intracellular neurofibrillary tangles (NFTs) have also been shown to correlate to the severity of AD dementia. We have been investigating the influence of NFTs on mRNAs related to neuronal plasticity and synaptic function. We recently reported a decrease in message for the plasticity marker, GAP-43, in AD cases with high tangle densities. The study did not permit us to determine if: a) the decrease in GAP-43 message was specific to the NFT-bearing neurons, b) a general decrease in GAP-43 message was occurring in all surviving neurons, or c) the decrease in GAP-43 message was due to a loss of neurons. It is unlikely a loss of neurons could explain the sixfold GAP-43 message loss we reported, because only a 19% excess decrease in density of hippocampal neurons occurs in AD cases with high tangle densities. Consequently, the study reported here was undertaken to determine if a general decrease in GAP-43 message was occurring in all surviving AD neurons or if the decrease in GAP-43 message was specific to NFT-bearing neurons. We combined immunocytochemistry for neurofibrillary tangles with in situ hybridization for GAP-43 message. We report here preliminary evidence indicating a decrease in GAP-43 message in NFT-bearing neurons compared to adjacent nontangle bearing neurons in parahippocampal cortex of AD patients.

Analysis of message expression in single neurons of Alzheimer's disease brain.

Because many cell types and disease states exist in the sample of cells in even a very small region of Alzheimer's disease (AD) brain tissue, optimal understanding of disease mechanisms requires study at the level of the single cell. Our Golgi studies of single neurons in the AD brain have revealed reduced dendritic extent in many, but not all, brain regions. This reduced dendritic extent is interpreted as reduced capacity of neurons in AD to proliferate new dendritic material. Studies of message expression in single neurons reveal that neurons containing neurofibrillary tangles (NFTs) show reduced expression of messages for proteins related to growth of neuronal processes and to synapses. Neighboring neurons free of NFTs express these messages at levels approximating the levels expressed by single neurons from control brain. This reduction of expression of messages related to growth of neuronal processes and to synapses is selective, because expression of message for the lysosomal enzyme, cathepsin D, is increased in neurons containing NFTs. Simultaneous analysis of the expression of multiple genes by single neurons using an aRNA technique offers powerful capacity to profile message expression as a function of disease state of single cells.

Changes in synaptic expression of clathrin assembly protein AP180 in Alzheimer's disease analysed by immunohistochemistry.

Clathrin assembly protein AP180 plays a regulatory role in clathrin-mediated synaptic vesicle recycling in synapses. Previously, using immunoblot analysis, we observed a significant reduction of AP180 protein in Alzheimer's disease neocortex. In this study, we examined immunohistochemically the expression of AP180 in post mortem brains with Alzheimer's disease (n = 5) in comparison with neurologically normal controls (n = 5). Overall, AP180 was revealed as immunoreactive punctate granules located in the neuropil, and around neuronal cell bodies and their processes, consistent with the typical expression of synaptic proteins. Reduced density of AP180 immunoreactive puncta was seen throughout all layers of the superior frontal gyrus in Alzheimer's disease, but the loss of AP180 immunoreactivity was not as prominent in the cerebellum. This regional difference is in agreement with our previous results from immunoblot analyses. In the hippocampus, cell body AP180 immunoreactivity normally seen in the hilus and the CA3 regions of control brains was completely lost in Alzheimer's disease. In addition, AP180 immunoreactivity in the molecular layer of the dentate gyrus showed several changes in Alzheimer's disease. These appeared to be expansion of the inner molecular layer and relative changes in immunoreactivity that resulted in clearer delineation of the inner and outer molecular layers. These results provide anatomical and spatial information on AP180 expression in Alzheimer's disease brains. The variations in altered AP180 immunoreactivity in different brain regions of Alzheimer's disease may underlie the dysfunction of the corresponding synapses.

Cytoplasmic inclusions in spinal neurons of the motor neuron degeneration (Mnd) mouse. I. Light microscopic analysis.

The motor neuron degeneration (Mnd) mutation in the mouse is a late onset, autosomal dominant, neurodegenerative disease in which ventral horn neurons have been shown to contain numerous, large cytoplasmic inclusions. Histochemical and immunocytochemical studies performed on spinal cord from Mnd/Mnd mice in late stages of the disease showed the inclusions to contain protein, lipid and carbohydrate moieties. Spinal neurons, especially those in spinal lamina IX, contained increased beta-glucuronidase activity in the form of large cytoplasmic inclusions. Such inclusions also contained increased acid phosphatase and trimetaphosphatase activity. When immunostained with antiubiquitin antibodies, intracellular ubiquitin deposits were present as accumulations of varying size; some were amorphous while others contained small granules. Extraneuronal ubiquitin deposits were detected in the neuropil. Immunostaining with monoclonal antibody ML30, used here to assay for the presence of a mitochondrial epitope in the inclusions, was widespread and punctate in white and grey matter from Mnd/Mnd and age-matched control spinal cords. The overall pattern of staining was similar for both tissue sources and did not correspond to any of the other probes which reacted with the inclusions in Mnd neurons. The presence of increased levels of lysosomal hydrolases and ubiquitinated molecules suggests that the two general systems for intracellular digestion are activated in Mnd/Mnd spinal neurons.

"I did so!" But can you prove it? Avoiding claims through documentation.

The author explains the need to document medical records to provide protection in litigation and to enhance medical care. Practical tips to make documentation easier and more efficient are given.

Cellular alterations in the alveolar wall in bleomycin-induced pulmonary fibrosis in rats. An ultrastructural morphometric study.

This study was designed to measure quantitatively changes in populations of major alveolar cell types, with a particular focus on nonmuscle, contractile interstitial cells, in lungs of rats 14 and 28 days after a single intratracheal instillation of bleomycin sulfate. Standard ultrastructural morphometric techniques, including stratified random sampling, were applied to airway-perfused lungs from bleomycin-instilled and control (saline-instilled) rats. Cell numbers and volumes were calculated for each major cell type populating the pulmonary parenchyma. At 14 days postinstillation, the number and volume of fibroblasts, contractile interstitial cells, Type II epithelial cells, and macrophages were increased significantly. At 28 days, the largest increase was in contractile interstitial cells, approximately tenfold in number and fourfold in volume over control cells. The number and volume of non-filament-containing fibroblasts did not differ between control and treated groups at this time. These data suggest proliferating fibroblasts undergo "differentiation" into cell types with increasing contractile capability during the development of fibrosis in this animal model. This study represents the first quantitative description of increases in contractile interstitial cells related to pulmonary fibrosis.

Expression profiles of multiple genes in single neurons of Alzheimer's disease.

Many changes have been described in the brains of Alzheimer's disease (AD) patients, including loss of neurons and formation of senile plaques and neurofibrillary tangles. The molecular mechanisms underlying these pathologies are unclear. Northern blot, dot-blot, and reverse transcription-coupled PCR analyses have demonstrated altered expression levels of multiple messages in AD brain. Because not all cells are equally affected by the disease, these methods obviously cannot study the changes in relation to disease states of individual cells. We address this problem by using antisense RNA profiling of single cells. We present expression profiles of single neurons at early and late stages of AD and describe statistical tools for data analysis. With multivariate canonical analysis, we were able to distinguish the disease state on the basis of altered expression of multiple messages. To validate this approach, we compared results obtained by this approach with results obtained by in situ hybridization analysis. When the neurofilament medium subunit was used as a marker, our results from an antisense RNA profiling revealed no change in neurofilament medium subunit expression between early- and late-stage AD, consistent with findings obtained with in situ hybridization. However, our results obtained by either analysis at the single-cell level differed from the reported decrease in AD neocortex obtained by Northern blot analysis [Kittur, S., Hoh, J., Endo, H., Tourtellotte, W., Weeks, B. S., Markesbery, W. & Adler, W. (1994) J. Geriatr. Psychiatry Neurol. 7, 153-158]. Thus, the strategy of using the single-cell antisense RNA approach to identify altered gene expression in postmortem AD brain, followed by detailed in situ hybridization studies for genes of interest, is valuable in the study of the molecular mechanisms underlying AD neuropathology.

DNA amplification fingerprinting analysis of bermudagrass (Cynodon): genetic relationships between species and interspecific crosses.

We have used DNA amplification fingerprinting (DAF) to study the genetic variation of bermudagrass (Cynodon) species and cultivars of interspecific crosses that exhibit leaf-blade textural characteristics ranging from coarse to fine. Arbitrary octamer primers produced complex and reproducible amplification profiles with high levels of polymorphic DNA. Phylogenetic analysis using parsimony (PAUP) and unweighted pair group cluster analysis using arithmetic means (UPGMA) grouped 13 bermudagrass cultivars into several clusters, including one containing the African-type bermudagrasses (C. transvaalensis) and another containing the common-type bermudagrasses (C. dactylon). The latter group included C. magennissii ('Sunturf') and a interspecific C. transvaalensisxC. dactylon cross ('Midiron'), 2 cultivars that exhibited leaf textural characteristics closer to the common-types. All other C. transvaalensisxC. dactylon crosses grouped between the African and common types. An extended screen of 81 octamer primers was needed to separate cultivar 'Tifway' from the irradiation-induced mutant 'Tifway II'. The use of either template endonuclease digestion prior to amplification or arbitrary mini-hairpin primers increased detection of polymorphic DNA and simplified the task of distinguishing these closely related cultivars. Alternatively, the use of capillary electrophoresis (CE) resolved fingerprints adequately and detected products with high sensitivity, thereby promising to increase throughput and the detection of polymorphic DNA. When used to fingerprint samples from commercial sources, DAF identified bermudagrass plant material on the basis of unique reference profiles generated with selected primers. DAF represents an excellent technique for bermudagrass cultivar verification, seed certification, varietal protection, and for the identification of mistakes in plantings, mislabeled plant materials, and contamination or substitutions of sod fields.