Two-dimensional gel analysis of secreted proteins induced by interleukin-1 beta in rat astrocytes.

Interleukin-1 beta (IL-1 beta) is a pro-inflammatory cytokine produced in the brain by endogenous microglial cells responding to injury. Levels of IL-1 beta are elevated in several neurodegenerative disorders, including Alzheimer's disease. IL-1 beta, which can act as a mitogen for astrocytes, also elicits the expression and secretion of multiple factors and paracrine 'second messengers' such as other cytokines, nerve growth factor, prostaglandins and nitric oxide that may in turn modulate neuronal and glial responses to injury. Utilizing giant, high-resolution two-dimensional gel electrophoresis, we have sought to more fully define the potential range of protein mediators that are secreted by astrocytes treated with IL-1 beta. In cultured rat astrocytes, we observe dramatic increases in the secretion of eight different protein species after 24 h of treatment with human recombinant IL-1 beta (1 U/ml). Seven of the proteins are also induced by tumor necrosis factor-alpha or basic fibroblast growth factor. Based on immunoprecipitation with specific antisera, we have identified three of these proteins as plasminogen activator inhibitor type-1, ceruloplasmin, and complement component C3. The identities of the other proteins, including the IL-1 beta-specific induction, are currently unknown. Characterization of these downstream modulators of IL-1 beta action complements gene-based approaches and will provide a better understanding of astrocyte responses to injury as well as markers for astrocyte activation in neurodegenerative diseases.

Clathrin assembly protein AP-2 is detected in both neurons and glia, and its reduction is prominent in layer II of frontal cortex in Alzheimer's disease.

There are several adaptor proteins associated with clathrin coated vesicles. Among them are AP180 and AP-2. We and others have previously described synaptic localization of AP180. AP180 immunoreactivity is altered in both the superior frontal gyrus and hippocampus in Alzheimer's disease (AD). We here investigate the location and alteration of another adaptor protein, AP-2. In contrast to AP180, we have found that AP-2 is expressed by both neurons and glia. Furthermore, the only noticeable change of AP-2 in AD is a loss of its immunoreactivity in layer II of the superior frontal gyrus.

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.

Non-HPLC separation of water-soluble choline metabolites by two-dimensional high voltage electrophoresis and thin layer chromatography.

In cholinergic neurons choline is directed to three main pathways; (1) conversion to phosphorylcholine (PCh) and cytidine diphosphate choline (CDP-choline) for the synthesis of phosphatidylcholine, (2) acylation to the neurotransmitter acetylcholine and (3) oxidation to betaine for the formation of methionine. Thus, the distribution of choline among the different metabolites is important for a better understanding of the regulation of these pathways in neurons. A non-HPLC method for the simultaneous separation of five choline metabolites found in neurons is described. High voltage electrophoresis (HVE) was combined with thin layer chromatography (TLC) to separate choline, PCh, CDP-choline, acetylcholine and betaine. This method is useful in studying the distribution of choline among its different metabolites in radiotracer experiments. Aqueous metabolites from leukemia inhibitory factor treated LA-N-2 cells labeled with [methyl-3H]choline were separated by HVE followed by TLC in the same dimension. Although the separation appeared to be complete, some 'tailing' by PCh significantly elevated the radioactivity measured in CDP-choline. This tailing of PCh was confirmed by subjecting radiolabeled PCh alone to this multiple separation method. Contamination of CDP-choline by PCh was eliminated by subjecting the samples to HVE followed by TLC in the second dimension. This two-dimensional approach was consistently reproducible and achieved excellent resolution of all five metabolites. In addition, this technique also resolved a sixth choline-containing metabolite, glycerophosphorylcholine (GPC), a breakdown product of phosphatidylcholine.

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.

Neurons may live for decades with neurofibrillary tangles.

Neurons containing neurofibrillary tangles (NFT) are one of the pathological hallmarks of Alzheimer disease (AD). It is known that this population of neurons express gene products and thus function to some degree, but it is unknown how long these neurons may survive with NFT. It is also thought that the formation of NFT results in the death of neurons. Using quantitative data on neuron loss and NFT formation as a function of disease duration, we have generated a computer program that models both the degeneration of CA1 hippocampal neurons and the formation of NFT in these neurons in AD. Modeling various neuron survival times with NFT and altering selected assumptions upon which the models are based, we arrive at the conclusions that 1) CA1 hippocampal neurons survive with NFT for about 20 years, and 2) NFT may not be obligatory for death of CA1 hippocampal neurons in AD.

Reduced O-glycosylated clathrin assembly protein AP180: implication for synaptic vesicle recycling dysfunction in Alzheimer's disease.

Synapse loss is one of the neuropathologies in Alzheimer's disease (AD) that may play a crucial role in the mechanism of its distinct cognitive impairment and dementia. In a previous study [18], a significant reduction of O-glycosylated clathrin assembly protein AP180 was observed in neocortex of AD. The reduction correlated with the density of neurofibrillary tangles. In this study we further determine that the O-GlcNAc/AP180 ratio is not changed, but the level of AP180 protein decreases in AD. Furthermore, whereas the level of neurofilament (NF-M) remains relatively unchanged, another clathrin assembly protein, AP-2, is also reduced in AD along with a small loss of synaptophysin. Our findings suggest that synaptic vesicle recycling dysfunction may be involved in the pathology of synapse loss in AD.

Identification of a neuronal calmodulin-binding peptide, CAP-19, containing an IQ motif.

Neurons produce polypeptides which can bind the calcium-poor or pre-activated form of calmodulin. It is expected that this class of peptide will serve an important role in maintaining cellular homeostasis since it would modulate calcium-dependent target regulation and redirect intracellular signaling. The lack of conserved sequence has made the identification of these peptides difficult, consequently leading us to exploit their property of binding calcium-poor calmodulin as a means of finding new species. A new peptide termed Calmodulin-Associated Peptide-19 (CAP-19) was purified and characterized. The protein-sequence information was employed in order to recover a cDNA clone from rat which included the entire reading frame for the peptide. Like its counterparts, neuromodulin (GAP-43), neurogranin (RC3) and PEP-19, it contains an IQ motif although the remainder of the peptide is quite different. Northern blot analysis of ribonucleic acid (RNA) from animals of differing ages indicated that the message appears at birth and then persists into adulthood. Antibodies to synthetic peptide were employed for localizing CAP-19. The results indicated that the peptide was localized to neurons in several brain regions. CAP-19 is similar to other calmodulin-binding proteins in that the domain spanning the IQ motif was demonstrated to participate in binding to calmodulin. Database searching showed CAP-19 to be homologous to the silkworm protein, multiprotein bridging factor 1 (MBF1). This homology suggests a potential new role for calmodulin-associated proteins in cellular homeostasis.

PEP-19 immunohistochemistry defines the basal ganglia and associated structures in the adult human brain, and is dramatically reduced in Huntington's disease.

We have investigated the distribution of PEP-19, a neuron-specific protein, in the adult human brain. Immunohistochemistry for PEP-19 appears to define the basal ganglia and related structures. The strongest immunoreactivity is seen in the caudate nucleus and putamen, each of which showed both cell body and neuropil PEP-19 immunoreactivity. The substantia nigra and both segments of the globus pallidus showed PEP-19 immunoreactivity only in the neuropil. Cell bodies and dendrites of the thalamic nuclei ventralis lateralis and ventralis anterioralis were less strongly immunoreactive. Cerebellar Purkinje cells and their dendrites were immunoreactive, as were the presubiculum/subiculum regions and dentate gyrus granule cells of the hippocampus. The CA zones of the hippocampus were not immunoreactive. Preliminary data from immunoblotting experiments indicate that PEP-19 immunoreactivity is significantly reduced in cerebellum in Alzheimer's disease. While there were no apparent alterations of immunoreactivity in Down's syndrome or in Parkinson's disease, immunohistochemical analysis showed a massive loss of PEP-19 immunoreactivity in the caudate nucleus, putamen, globus pallidus and substantia nigra in Huntington's disease. These results show that PEP-19, a neuron-specific, calmodulin-binding protein, is distributed in specific areas of the adult human brain. The reduction in PEP-19 immunoreactivity in Alzheimer's disease and Huntington's disease suggests that PEP-19 may play a role in the pathophysiology of these diseases through a mechanism of calcium/calmodulin disregulation. This may be especially apparent in Huntington's disease where the distribution of the product of the abnormal gene, huntingtin, alone is not sufficient to explain the pattern of pathology. Abnormal huntingtin associates more strongly with calmodulin than does normal huntingtin [Bao et al. (1996) Proc. natn. Acad. Sci. U.S.A., 93, 5037-5042] suggesting a disruption of calmodulin-mediated intracellular mechanism(s), very likely involving PEP-19.

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.

Developmental regulation and PKC dependence of Alzheimer's-type tau phosphorylations in cultured fetal rat hippocampal neurons.

Attempts to describe a mechanism of neurofibrillary tangle formation often focus on site specific phosphorylations of tau protein. These have typically been described in both Alzheimer's disease and developing brains. Therefore, study of the developmental regulation of Alzheimer epitope tau phosphorylations may help explain their persistence or recurrence during Alzheimer's disease. Using fetal rat hippocampal cultures, we report a spatial and temporal expression of tau phosphorylation during neuronal differentiation. We have examined phosphorylation at the epitopes recognized by monoclonal antibodies, PHF-1 and Tau 1. Tau was highly phosphorylated at the PHF-1 epitope at all culture ages examined using both immunohistochemical staining and Western blots. Tau was heavily phosphorylated at the Tau 1 epitope only in older cultures. The populations of tau recognized by the two antibodies also exhibited different solubilities, suggesting different microtubule binding behaviors: tau phosphorylated at PHF-1 was retained in axons following solubilization whereas Tau 1 immunoreactive tau was not retained in any cell compartment. Finally, in this culture system, maintenance of phosphorylation at the PHF-1 epitope, but not the Tau 1 epitope, required protein kinase C activity. These results indicate unique regulatory mechanisms and roles for each of these phosphorylated tau epitopes.

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.

Reduction of O-linked N-acetylglucosamine-modified assembly protein-3 in Alzheimer's disease.

Abnormal protein processing and modification is associated with Alzheimer's disease (AD) pathology. The role of phosphorylation in AD has been studied extensively because the presumed abnormal phosphorylation of tau protein is believed to play a role in the formation of paired helical filaments. Glycosylation with O-linked N-acetylglucosamine (O-GlcNAc) to serine and threonine residues is a dynamic protein modification of intracellular proteins, and it shares similar features with protein phosphorylation. In this study, O-GlcNAc glycosylation of proteins from autopsied human brains with confirmed AD and non-AD age-matched controls was examined. O-GlcNAcylation was demonstrated by labeling protein extracts with [3H]galactose in the presence of galactosyltransferase and subsequent analyses of saccharide-protein linkage and saccharide structure. The number of O-GlcNAc-containing proteins and the overall O-GlcNAc level do not appear to be different between AD and control brain tissues. The only significant change observed is a marked reduction of O-GlcNAcylated clathrin assembly protein-3 (AP-3) in AD. The reduction is more evident in brain neocortical regions, and there appears to be a negative correlation between O-glycosylated AP-3 and the density of neurofibrillary tangles. These data suggest a possible association between the O-glycosylated AP-3 and AD pathology.

Interleukin-1 beta stimulates mitogen-activated protein kinase in U373 astrocytoma cells without the production of lipid second messengers.

IL-1 beta is one of the cytokines known to affect astroglial cells in normal brain development, brain injury and neurodegenerative diseases. IL-1 beta causes astrocytes to become more reactive, alter the expression and release of molecules and in some cases to proliferate. We have investigated the mitogenic effect and signal transduction pathway induced by IL-1 beta in U373 cells, a human astrocytoma cell-line. Recombinant human IL-1 beta induced mitogenesis of U373 cells in a dose-dependent fashion as assessed by tritiated thymidine incorporation. The following signal transduction mechanisms, reported to be induced in other systems by IL-1 beta, were investigated in U373 cells: (1) activation of phosphatidylcholine-specific phospholipase C as assayed by incorporation of tritiated choline into cellular phospholipids, (2) production of diacylglycerol, a lipid second messenger, (3) activation of sphingomyelinase, and (4) activation of mitogen-activated protein kinase (MAPK). Of these, IL-1 beta activated only MAPK. In cultured rat astrocytes, IL-1 beta caused activation of MAPK without inducing proliferation.

Low initial tau phosphorylation in human brain biopsy samples.

A rapid reversible tau phosphorylation at Ser 396/404 was observed in adult human cortical biopsy tissue and rat primary cortical cell cultures. Tau phosphorylation increased usually during the first 20-30 min in phosphate-buffered saline, followed by a decrease. The time course of tau phosphorylation and dephosphorylation in biopsy tissue could be lengthened by culturing in defined, oxygenated medium, instead of in phosphate-buffered saline. Phosphorylation of total protein in biopsy tissue occurred in two phases, with peaks at 30 and 90 min. The first peak of total protein phosphorylation coincided with the peak of tau phosphorylation, although both the first and second peaks of total protein phosphorylation coincided with the first and second peaks of neurofilament-H phosphorylation.

Isolation of single immunohistochemically identified whole neuronal cell bodies from post-mortem human brain for simultaneous analysis of multiple gene expression.

In Alzheimer's disease (AD), one cell in the brain may clearly be affected, while an adjacent cell appears healthy or unaffected. Previous technology has allowed us to examine one message at a time, at the level of a single cell (in situ hybridization, ISH), or multiple messages in a heterogeneous population of cells (Northern analysis). We have developed a methodology to build up a profile of multiple mRNA expression in single, whole, post-mortem cells that have been immunohistochemically (IHC) characterized. Fresh post-mortem tissue is spread into a layer one cell thick and fixed. Neurons are identified using an antibody to neurofilament and isolated using a micropipette. The mRNA is reverse transcribed and PCR carried out to confirm that material is present. A radioactively labeled antisense aRNA probe, which is representative of the messages contained in the cell is then amplified. This aRNA is used as a probe for a reverse Northern blot, allowing us to profile many genes from one cell at the same time. This technology has the potential to be applied to a wide variety of diseases encompassing many different cell types.

Research uses of neuropathological data.

The study of relationships between neuropathological characteristics and behavioral, structural, chemical, and molecular variables offers immense promise for understanding the basic pathophysiology of Alzheimer's disease. This position paper examines the need for standardized procedures and quantitation if neuropathological data are to be optimally useful among laboratories investigating the biology of Alzheimer's disease. These requirements include standardized fixation, embedding, sectioning, staining, brain regions and sampling methods. In addition, the definition of the structures to be quantified, such as plaque type(s), needs to be rigorously specified. Unbiased stereological methods for quantification should be used. These needs for optimal research utility exceed the needs and practicality for diagnostic purposes, suggesting a two-tiered approach to the neuropathology of Alzheimer's disease: diagnostic and research.

Prostaglandin G/H synthase-2 (cyclooxygenase-2) mRNA expression is decreased in Alzheimer's disease.

Recent evidence suggests that the use of nonsteroidal anti-inflammatory drugs (NSAIDS) is beneficial for therapy or prevention of Alzheimer's disease (AD). The major anti-inflammatory action of NSAIDS is to inhibit prostaglandin G/H synthase-2 (PGHS-2), the first committed enzymatic step for prostaglandin biosynthesis. We have previously shown that PGHS-2 message is induced by Interleukin-1 beta and other inflammatory mediators in primary cultures of rodent astrocytes. To determine whether similar elevations of PGHS-2 occur as part of the gliosis in AD, we quantified PGHS-2 mRNA levels in control and AD brain by Northern hybridization analysis. To our surprise we found that PGHS-2 mRNA levels were reduced threefold in AD neocortex relative to control brain tissue. In contrast, levels were not reduced in putamen, an area that is relatively spared in AD. To localize PGHS-2 mRNA production in control and AD brain, sections of neocortex and hippocampus were hybridized with a 35S-labeled riboprobe for human PGHS-2 followed by immunocytochemistry with antibodies against neuron specific enolase (NSE) or glial fibrillary acidic protein (GFAP). Our findings indicate that PGHS-2 message is primarily localized to cells that stain for NSE rather than GFAP. Furthermore, in the three cases we examined, PGHS-2 hybridization per neuron appeared to be reduced in AD. Thus, the decrease we observe in overall PGSH-2 mRNA levels is likely to reflect both the known decline in numbers of neurons in AD as well as a lowered capacity for neuronal synthesis of PGHS-2, perhaps due to dysfunction or a loss of synaptic input.