Indomethacin reverses the microglial response to amyloid beta-protein.

Alzheimer's disease (AD) brains display intense microglial immunoreactivity in the area of senile plaques, suggesting that amyloid beta-protein may stimulate microglial infiltration. The activated microglia may modulate an immune response in the brain. Non-steroidal anti-inflammatory drugs (NSAIDs) are candidate therapeutics for AD because their effects on immune system components may influence the course of the disease. The present study examined the effects of an NSAID (indomethacin) on amyloid beta-protein-induced microglial infiltration. Amyloid beta-protein was chronically infused into rat lateral ventricles for 2 weeks. Extracellular amyloid beta-protein deposited along the lining and diffused into the tissue surrounding the lateral ventricle. Immunocytochemical staining showed that animals receiving amyloid beta-protein exhibited dramatic microglial response when compared to vehicle-infused rats. Activated microglia surrounded immunopositive amyloid beta-protein deposits, but this response was significantly attenuated in animals receiving either concurrent i.c.v. or subcutaneous (s.c.) treatment with indomethacin. These results suggest that chronic amyloid beta-protein infusion induces the proliferation of activated microglia and that indomethacin may be an effective treatment for inhibiting microglial proliferation.

Preparation of a recombinant cDNA library from poly(A+) RNA of the Alzheimer brain. Identification and characterization of a cDNA copy encoding a glial-specific protein.

Studies were undertaken to assess the extent to which messenger RNA prepared from the postmortem Alzheimer's disease (AD) brain can be used for the successful preparation of a recombinant cDNA library. Initial experiments focused on the glial-specific marker glial fibrillary acidic protein (GFAP) since GFAP expression appeared to be a model for further studies on mRNAs that may continue to be expressed at high levels in the vicinity of lesioned sites in the AD brain. An AD cDNA library, prepared in the lambda gt11 expression vector system contained GFAP-specific recombinants. One of these was sequenced and the insert was shown to exhibit 88% homology with the similar sequence from mouse GFAP. As established by Northern blots, the size of the GFAP mRNA prepared from the routinely acquired postmortem AD cortex, approximately 2.7 kb, was the same as from a neurologically normal control brain. These results agree with earlier studies on GFAP mRNA from fresh mouse brain. The results demonstrate that in the postmortem AD brain, astroglial-specific mRNA remains sufficiently stable for molecular genetic analysis and may serve as a useful model for examining the genetic expression of mRNAs that may be related to the molecular pathogenesis and the etiology of AD.

Development of an anti-A beta monoclonal antibody for in vivo imaging of amyloid angiopathy in Alzheimer's disease.

We evaluated the efficacy of murine monoclonal antibodies (MAbs) targeted to the A beta amyloid of Alzheimer's disease for development of procedures for the in vivo identification of amyloid angiopathy (AA). MAbs to A beta were prepared and screened for effectiveness in visualizing AA and neuritic plaques in postmortem AD brain sections. They were assessed again after enzymatic cleavage to produce Fab fragments and after labeling with technetium-99m (99mTc) using a diamide dimercaptide ligand system. Modified and radiolabeled Fab fragments retained activity and specificity toward amyloid-laden blood vessels and neuritic plaques. A highly specific murine MAb, 10H3, was identified and characterized that fulfills criteria necessary for the development of an in vivo diagnostic imaging agent. Toxicity studies in rats showed the MAb to be safe. Biodistribution studies in mice demonstrated desirable properties for use as an imaging agent. Expansion and adaptation of these strategies may provide the methods and materials for the noninvasive analysis of AA in living patients, and permit assessment of the contribution of AA to the clinical and pathological features of AD.

Evidence for a diffusional model of Alzheimer amyloid A4 (beta-amyloid) deposition during neuritic plaque formation.

A recent study reported that Alzheimer senile plaques immunostained with monoclonal antibodies against the A4 (beta-amyloid) region of the amyloid precursor protein show gradients of density (Majocha R. E., Benes F. M., Reifel R. L., Rodenrys A. M. and Marotta C. A., Proc. natn. Acad. Sci. U.S.A. 85, 6182-6186, 1988). Although more than one explanation was suggested for this observation, the possible involvement of a diffusional process during plaque maturation was considered. In order to examine this hypothesis, specimens from prefrontal cortex, entorhinal area and hippocampal formation were immunoprocessed in a similar fashion and subjected to quantitative microdensitometric analyses of A4 amyloid reaction product. All plaques in the three brain areas examined showed a curvilinear relationship between the area of amyloid reaction product (expressed in pixel counts) and optical density (expressed as each of six grey scale levels). There was an increase in the area of amyloid at progressively lower density levels. When the area of amyloid reaction product at each density level was correlated with the overall size of individual plaques, it was found that there was a striking increase in the correlation coefficients at progressively lower grey scale levels, with r = 0.853 at the lowest level examined. When a second order derivation of these correlations was performed by expressing individual r-values with respect to an optical density index, an asymptotic relationship resulted with the lowest density levels showing an increasingly sharp rise toward unity. These data are consistent overall with a model for plaque maturation that involves diffusion of amyloid protein through the extracellular space from focal regions of high density where synthesis and/or release may occur.

Evidence for axonal loss in regions occupied by senile plaques in Alzheimer cortex.

The studies described have sought to determine what, if any, relationship exists between axons and the senile plaque, a hallmark histopathological feature of Alzheimer's disease. A double stain was performed on both early and late Alzheimer frontal cortex tissues in order to examine the interaction between axons stained with antibodies against the 200,000 mol. wt neurofilament subunit (NFP-200) of the axon cytoskeleton and Thioflavin-S, a fluorescent dye that stains plaques. Serial photomicrographs of plaques were taken and axon and plaque profiles were three-dimensionally reconstructed. Analysis of computer-processed images revealed that there were fewer axons within plaques than in regions lying one and two plaque distances away. When axons were observed passing through plaques, swelling and disruption of normal morphology was frequently present. Statistical analyses of axon counts within and around placques showed a gradient of axon density, with increased numbers occurring at progressive distances from the placque. Similar patterns were seen for early and late stages of the disease. The results of this study indicate that disruption of the axonal cytoskeleton may occur within the regions occupied by plaques.

Development of a monoclonal antibody specific for beta/A4 amyloid in Alzheimer's disease brain for application to in vivo imaging of amyloid angiopathy.

We evaluated the efficacy of murine monoclonal antibodies (Mabs) targeted to beta/A4 amyloid for development of procedures for the in vivo identification of amyloid angiopathy (AA) in Alzheimer's disease (AD). Mabs to beta/A4 amyloid were prepared and screened for effectiveness in visualizing AA and senile plaques in postmortem AD brain sections. They were assessed again after enzymatic cleavage to produce Fab fragments and after labeling with 99mTc using a diamide dimercaptide ligand system. Modified and radiolabeled Fab fragments retained activity and specificity towards amyloid-laden blood vessels and senile plaques. A highly specific murine Mab, 10H3, was identified and characterized that fulfills criteria necessary for the development of a diagnostic imaging agent. Expansion and adaptation of these strategies may provide the methods and materials for the noninvasive analysis of AA in living patients, and permit assessment of the contribution of AA to the clinical and pathological features of AD.

Molecular and cellular biology of Alzheimer amyloid.

Alzheimer's Disease (AD), a disorder of unknown etiology, is the most common form of adult-onset dementia and is characterized by severe intellectual deterioration. The definitive diagnosis of AD is made by postmortem examination of the brain, which reveals large quantities of neurofibrillary tangles (NFT) and senile plaques within the parenchyma. The NFT are composed of paired helical filaments associated with several cytoskeletal proteins. The primary protein component of senile plaques is beta/A4 amyloid, a 42-43 amino acid peptide derived from a much larger molecule, the amyloid precursor protein (APP). Vascular beta/A4 amyloidosis is also prevalent in the disease. The mechanism by which beta/A4 amyloid accumulates in the AD brain is unknown. Recent research has demonstrated that the precursor molecule, APP, is a transmembrane protein with a large extracytoplasmic domain, a membrane spanning region that includes the portion that gives rise to beta/A4 amyloid, and a short intracytoplasmic domain. The precursor has multiple forms among which are those that differ by a variable length insert within the extracytoplasmic domain. The insert has sequence homology to the family of Kunitz protease inhibitor proteins. Cellular and animal models have been developed to study the nature of APP processing and the biological and behavioral consequences of beta/A4 amyloidosis. The results of such studies indicate that the normal processing of APP involves enzymatic cleavage of the molecule within the beta/A4 amyloid region, thus preventing the accumulation of beta/A4 in the normal brain. The factors leading to abnormal processing of APP, and consequent beta/A4 amyloid accumulation within the AD brain, have yet to be identified. In cell culture, the biological effects associated with beta/A4 amyloid include neurotrophic and neurotoxic activities, while the peptide has also been shown to have dramatic behavioral effects in animal models.

Alzheimer cortical neurons containing abundant amyloid mRNA. Relationship to amyloid deposition and senile plaques.

Since the detailed molecular events leading to the formation of amyloid-containing senile plaques of the Alzheimer's disease (AD) brain are incompletely understood, the present studies were undertaken to address this issue using a combination of molecular and cytochemical approaches. Amyloid precursor protein riboprobes containing the A4 (beta-amyloid) domain were applied to cortex using the in situ hybridization method to examine the distribution of neuronal amyloid mRNA in relation to the laminar pattern of amyloid deposition and the localization of plaques. The derived data indicated that high levels of amyloid mRNA can be synthesized by AD cortical neurons that appeared to be morphologically intact. The distribution of these cells was not coincident with the cortical laminar pattern that is typical of amyloid deposits observed after immunostaining with anti-A4 monoclonal antibodies. Further, there was no obvious relationship between neurons containing abundant amyloid mRNA and the distribution of plaques identified by thioflavin S staining. While the neuronal synthesis of amyloid may be a significant factor at some point during plaque formation, it may not be the exclusive determinant. The possibility is raised that processes affecting secretion, diffusion, and/or transport of amyloid away from neuronal or non-neuronal cells of origin to sites of deposition may be meaningful aspects of the molecular pathology of Alzheimer's disease.

The postmortem Alzheimer brain is a source of structurally and functionally intact astrocytic messenger RNA.

Although the precise role of astrocytes in the pathogenesis of Alzheimer's disease (AD) is currently undefined, studies carried out at the molecular level may lead to new insights into the functioning of this class of brain cells in dementia. In order to facilitate such investigations, methods are described that establish that structurally and functionally intact messenger RNA (mRNA) for an astrocytic marker, glial fibrillary acidic protein (GFAP), is present in the postmortem Alzheimer's disease brain after long postmortem intervals. Rapid preparative procedures were used to obtain poly(A)+ RNA from postmortem control and AD cortices. In vitro protein synthesis was carried out in a reticulocyte system. Relative to controls, AD mRNA synthesized a two-fold higher level of a 50,000 mol.wt. protein that was immunologically identified as GFAP. High levels of GFAP synthesis by purified mRNA from AD cortices was independent of age at death and postmortem interval up to 24 h. Northern blot hybridization using a cloned human GFAP riboprobe was used to evaluate postmortem GFAP mRNA stability. No appreciable degradation products of GFAP mRNA were observed on Northern blots for at least 10 h postmortem in poly(A)+ RNA extracted from the AD brain. The described methodology demonstrates that the postmortem AD brain is an excellent source of functionally and structurally intact astrocyte-specific mRNA.

Cell surface extensions associated with overexpression of Alzheimer beta/A4 amyloid.

Deposition of beta/A4 amyloid in Alzheimer disease (AD) brain parenchyma and vasculature occurs by mechanisms that are currently undefined. Similarly the potential consequences of amyloid accumulation for disrupting cellular integrity have not been addressed in detail. To investigate the possible significance of amyloid deposits for cellular viability, PC12 cells were permanently transfected with DNA coding for the beta/A4-C terminal region of the amyloid precursor protein. The DNA represented 97 amino acids of the amyloid precursor protein of which 40 amino acids were derived from the beta/A4 region. Transfected clonal cell lines and controls were examined at both the light and electron microscopic levels for morphological abnormalities. beta/A4 amyloid accumulated in the cell membrane where the peptide was located at cellular processes resembling blebs and microvilli. These specialized structures at the cell surface were over-abundant in transfected cells that overexpressed the beta/A4 peptide but not in controls. Membranous processes may be involved in the delivery of the beta/A4 peptide to the external surface of the cell of origin and release into the extracellular space. Similar surface features of cells in the AD brain, should they occur, may indicate a role for membrane-associated processes in the pathophysiology of the disorder.

The noradrenergic system in cultured aggregates of fetal rat brain cells: morphology of the aggregates and pharmacological indices of noradrenergic neurons.

Spherical aggregates formed rapidly in culture by re-aggregation of trypsin-dissociated brain cells from the 17-day-old fetal rat. Over about 10 days in initially random distribution of cells evolved into a 3-layered arrangement; cells with characteristics of neurons were found largely in the intermediate layer. The survival of neuronal and glial cell types was evaluated histologically and verified by electron microscopy, which revealed synaptic and myelin structures that rapidly increased in number after 18 days in culture. Levels of norepinephrine (NE) and dopamine (DA) reached peaks of 9.5 and 4.4 ng/mg protein, respectively, at culture day 21. Uptake of [3H]NE paralleled these amine levels and was blocked by desipramine or pretreatment with either reserpine or 6-OH-DA. Autoradiographs of aggregates labeled with [3H]NE showed a high density of silver grains over cells, apparently neurons, with branching processes traced for 120 micrometer. Previously accumulated [3H]NE was released under depolarizing conditions (high [K+] or vertridine) only in the presence of Ca2+. Release was induced to a lesser extent by kainic greater than glutamic acid. Thus, such aggregates appear to contain catecholaminergic neurons capable of synthesis, uptake and release of NE. The time course of development of these functions supports suggestions that aggregate preparations might be useful in studying neurochemical or morphological aspects of brain development and function in vitro.

Membrane surface ruffling in cells that over-express Alzheimer amyloid beta/A4 C-terminal peptide.

Deposition of beta/A4 amyloid in brain is a defining characteristic of Alzheimer disease (AD); however, the extent to which amyloid deposits may interfere with normal cellular processes is incompletely understood. We examined this issue by means of PC12 cells. After transfection with DNA coding for 97 amino acids of the beta/A4 C-terminal region of the amyloid precursor protein, beta/A4 antigen was visible at the cell membrane. We report that normal unstimulated PC12 cells exhibit ruffling activity at the cell surface when plated on a plastic substrate. Relative to control cells, however, those that over-expressed the beta/A4 C-terminal peptide had significantly higher levels of ruffling activity, suggesting a structural and/or functional membrane modification. Similar cellular alterations, if present, in Alzheimer brain cells, may indicate disturbances in membrane-associated functions, including intercellular communication.

Conditioned media from the injured lower vertebrate CNS promote neurite outgrowth from mammalian brain neurons in vitro.

Unlike most pathways of the mature mammalian central nervous system (CNS), the CNS of lower vertebrates can regenerate after jury, a capacity that may be due to the secretion of neurite-promoting factors from the injured CNS. We report that conditioned media (CM) from the injured optic nerve of the mature goldfish promoted marked neurite outgrowth from dissociated embryonic rat cortical and hindbrain neurons in serum-free, neuron-enriched culture. This property was not shared by CM from intact goldfish optic nerve, or from intact or injured optic nerve of mature rats. Neurite-promoting activity was obtained at concentrations as low as 100 ng total protein/ml of CM from injured goldfish optic nerve, and was associated with a distinctive morphology of neurite outgrowth. Due its properties of non-dialyzability, heat lability, and trypsin sensitivity, the neurite-promoting factor(s) appeared to be one or more protein species of MW greater than 12,000. Factors secreted by the regenerating CNS of lower vertebrates can directly promote outgrowth of mammalian CNS neurons.

Effects of isoproterenol on the development of beta-adrenergic receptors in brain cell aggregates.

The development of binding sites for the beta-adrenergic receptor antagonist [125I]iodohydroxybenzylpindolol in reaggregate cultures of fetal rat brain has been investigated. beta-Adrenergic receptor density increased rapidly between 6 and 22 days in culture at which time maximal density was attained. When cultures were grown in the presence of the beta-adrenergic receptor agonist isoproterenol, the development of receptors was markedly inhibited. Cultures were also grown for 15 days in the presence of isoproterenol and then for 7-14 days in the absence of isoproterenol. Following the removal of the agonist, binding sites developed, but to only approximately 50% of the density seen in control cultures.

Soluble, phosphorylated forms of the high molecular weight neurofilament protein in perikarya of cultured neuronal cells.

The high molecular weight subunit of neurofilaments (NF-H) in mouse NB2a/d1 neuroblastoma cells is extensively phosphorylated and exhibits an apparent molecular weight of 200 kDa by SDS gel electrophoresis. In this study, we observed that extensively phosphorylated NF-H variants exist as both Triton-soluble and -insoluble forms, which display different cellular distributions. Perikarya and neurites of differentiated NB2a/d1 cells were immunostained by a polyclonal antiserum (anti-NF-H) that specifically recognizes the extensively phosphorylated NF-H forms and a monoclonal antibody (SMI-31) that recognizes phosphorylated epitopes of neurofilament proteins (NFPs). When cells were extracted with Triton X-100 to remove soluble proteins, however, only axonal neurites remained immunoreactive. Immunoblot analyses established the specificity of anti-NF-H and SMI-31 and demonstrated that both Triton-soluble and -insoluble NF-H subunits exhibit an apparent molecular weight of 200 kDa. Incorporation of radiolabeled phosphate into Triton-soluble NF-H following incubation of intact NB2a/d1 cells with 32P-orthophosphate confirmed that the Triton-soluble form of NF-H is a phosphoprotein. Most NF-H subunits in the Triton-soluble fraction sedimented after centrifugation at 100,000 g for 1 h, indicating that they may be present as oligomers. The implications of these data for the development of neurofibrillary pathology are discussed.

Molecular and genetic investigations on Alzheimer brain amyloid.

Amyloid-containing plaques are a characteristic feature of the Alzheimer's disease brain and have been the object of study for decades. Only recently, however, have molecular and genetic techniques been applied to examination of amyloid in order to understand the factors that contribute to the accumulation of plaques in dementia. Current investigations have focused on the structure and properties of the amyloid protein, its corresponding messenger RNA, its cellular site of production, and its chromosomal site of origin. These data are discussed in the present review.

A modular arrangement of neuronal processes in human cortex: disruption with aging and in Alzheimer's disease.

Studies were undertaken to assess whether or not neuron-specific immunostaining of the human brain can reveal unique cytoarchitectural features that may be affected by healthy aging and Alzheimer's disease (AD). Human prefrontal cortex (PFC) and anterior cingulate cortex (ACC) were stained with an antibody raised against the neurofilament protein 200,000 molecular weight subunit (NFP-200) using an avidin-biotin immunolocalization procedure. Immunostaining of cortex was neuron-specific and highlighted axons in particular. This staining has revealed an orderly arrangement of horizontal and vertical axon bundles which form latticelike compartments or modules throughout most of the matrix of the two cortical areas studied. The ACC shows this pattern to be intact in individuals through the ninth decade, while the PFC there was blurring of the modularity beyond the fifth decade. Irrespective of the age-related blurring of the latticelike arrangement of axons in PFC, neurologically normal elderly individuals nevertheless showed a highly organized appearance to the cortical matrix. By contrast, patients with AD showed a marked disruption of the modular arrangement of fibers in PFC, but not ACC. Differences between PFC fibers in controls and AD patients were also demonstrated with an axon-specific monoclonal antibody that reacted with phosphorylated epitopes of the NFP-200. The chaotic appearance of fiber staining in PFC seen in patients with AD was noted to be present in one subject who died in an early stage of the disease. The possible significance of this previously unknown aspect of cortical cytoarchitecture for normal cognitive functioning in humans is discussed.

Colocalization of amino terminal and A4 (beta-amyloid) antigens in Alzheimer plaques: evidence for coordinated processing of the amyloid precursor protein.

The mechanism by which the A4 (beta-amyloid) domain of the Alzheimer amyloid precursor protein (APP) is deposited in plaques is unknown, and limited information is available concerning the extent to which other APP sites are associated with plaques. To address these issues, we prepared antiserum to a peptide adjacent to the N-terminus of the APP (referred to as N1) and examined its distribution in brain relative to A4 by double-immunostaining techniques. Anti-N1 localized to both neurons and glia in control and Alzheimer patients. In the Alzheimer brain, anti-N1 detected plaques. Quantitation revealed that 85% of thioflavin-positive plaques, and 91% of A4-positive plaques were also N1 positive. Double-staining methods directly demonstrated colocalization of distant APP sites. The data suggest that suggest that proposed mechanisms for amyloid deposition during plaque formation must take into account the extracytoplasmic domain, in addition to the A4 region, rather than be confined exclusively to the A4 site.