Accumulation of a 50 kDa N-terminal fragment of beta-APP695 in Alzheimer's disease hippocampus and neocortex.

Proteolytic processing of beta-amyloid precursor protein (beta-APP) is a key event in the formation of beta-amyloid deposits in Alzheimer's disease (AD) brains and is likely to be accompanied by the accumulation of cleavage products other than the beta/A4 protein. Using a beta-APP695-specific monoclonal antibody in quantitative immunoblotting, a 50 kDa N-terminal fragment of beta-APP695 was detected in neocortex, hippocampus and cerebellum of AD patients and control individuals. The mean level of this fragment was higher in AD hippocampus and neocortex as compared to controls, suggesting that beta-APP695 fragments are generated in various brain regions but that the proteolytic processing is increased in pathologically affected brain areas.

Effects of aluminium chloride on cultured cells from rat brain hemispheres.

Neurofilamentous tangles have been induced by aluminium chloride in rat brain neurons cultivated on astroglial feeder layers. Monoclonal antibodies to neurofilaments were found to stain these aluminium-induced tangles. Immunostaining of these structures with anti-paired helical filament serum was always negative, though good staining of neuronal perikarya was achieved. This observation supports the hypothesis that aluminium-induced tangles are made up of neurofilament proteins. These tangles appear to be distinct immunochemically from Alzheimer-paired helical filaments.

Influence of basic fibroblast growth factor on carbonic anhydrase expression by rat glial cells in primary culture.

Modifications of the morphology, the proliferation and the synthesis of carbonic anhydrase of glial cells in primary cultures maintained in defined medium have been investigated under the action of basic fibroblast growth factor. Cultures contained essentially three cell types: astrocytes which expressed glial fibrillary acidic protein, oligodendrocytes which were characterized by the presence of carbonic anhydrase and precursor cells in which these two proteins were detected by immunocytochemistry. In the presence of basic fibroblast growth factor astrocytes and oligodendrocytes underwent morphological changes, characterized by a fibrous aspect; astroglial cells acquired essentially several long processes and oligodendroglial cells formed generally two long processes. The factor increased the proliferation of these two cell types. The quantity of carbonic anhydrase per oligodendrocyte was enhanced in treated cultures. The double-stained precursor cells were present between days 7 and 11 of culture in defined medium, while in the presence of fibroblast growth factor these cells were more numerous and were still present after 14 days. The basic fibroblast growth factor stimulated the proliferation of these young glial cells and modified their morphology. But the differentiation of precursor cells towards one glial cell type appeared to be delayed.

Synapse formation and development of neurotransmitter functions in neuronal cells from chick brain cultured in a serum-free, defined medium.

Cells dissociated from cerebral hemispheres of 8-day-old chick embryos were seeded on poly-L-lysine coated Petri dishes in serum-containing medium. After 24 hr the culture medium was switched to a serum-free, chemically defined medium. These cultures contain mainly neuronal cells until day 14, characterized by the presence of acetylcholinesterase activity and neurofilament proteins. After 2 weeks glial cells progressively contaminated the neuronal culture. Cultures were maintained for a period of 4 weeks. From day 6 on numerous synapses with clear vesicles were observed. The activity of choline acetyltransferase remained low throughout the culture period, while GABA levels increased in parallel with synaptogenesis. Our observations indicate that chick cerebral hemisphere neuronal cultures grown in serum-free, chemically defined medium contain GABAergic neurons that undergo maturation.

Immunocytochemical demonstration of both carbonic anhydrase isoenzyme II and glial fibrillary acidic protein in some immature rat glial cells in primary culture.

In glial cell cultures from newborn rat hemispheres we have demonstrated by immunocytochemistry the presence of carbonic anhydrase (CAII) and glial fibrillary acidic protein (GFAP) in the same cell. These double-stained cells are detected between day 5 and day 13 after seeding. These cells could be morphologically identified as oligodendroblasts. At 5 days in culture about 10% of the cells containing CAII also contained GFAP. At 11 days only 2% preserved this property. After 13 days in culture GFAP was only localized in typical astrocytes and CAII in oligodendroglial-like cells. The presumption that the immature glial cells, which contained specific markers of astrocytes and oligodendrocytes, became in our culture conditions essentially oligodendroglial cells is discussed.

Rapidly progressing dementia with filopodia-like cytoskeletal neuritic anomalies, but without Alzheimer changes.

An unusual case of dementia is reported. The patient was a woman who died at the age of 69 years and 6 months after a two years history of organic dementia. Possibly the disease was familial. Examination of the brain at autopsy revealed no atrophy. In routine histology the brain seemed to be normal. However, when the sections were stained with highly sensitive techniques such as an antibody to phosphorylated Tau (PHF-1), widespread neuritic pathology was discovered. Probably both, axons and dendrites were involved. Only few perikarya were reactive with the antibody. In some of them, morphologic alterations were reminiscent of Pick's disease.

[The neuron theory: one of the main scientific achievements of the 19th century]. / La théorie du neurone: une des principales conquêtes scientifiques du XIXe siècle.

The cell theory, enunciated by Theodor Schwann in 1839, stated that all tissues in the body are composed of individual cells. The theory gained immediate acceptance for all organs except for the nervous system where some basic problems were encountered, among them the difficulties in establishing the relation between nerve cells, nerve fibers and terminal branches. Deiters observations (1865) had provided evidence that the basic structure of a nerve cell was made up of a cell body, protoplasmic prolongations (dendrites), and a single long axon. This helped to define the nerve cell as the basic unit of the nervous tissue but it remained then to understand how the nerve cells are connected since they do not make direct contact through their cell bodies and are separated by the 'neuroglia'. Two fundamentally different views of the organization of neurons arose, one holding that neurons are individual and contiguous units, connected in chains to form specific pathways (His, Forel, Nansen, Ramón y Cajal), the other that thin nerve cell branches from continuous diffuse networks through which the neuronal activity propagates (Gerlach, Golgi). The key technological advance that led to the resolution of most of these uncertainties came in 1873 with the introduction by Camillo Golgi of a new method of staining individual nerve cells. In 1887, Santiago Ramón y Cajal stumbled on the Golgi stain and began an intense study of neuronal morphology throughout the nervous system. As far back as 1887 it was shown that the nervous system is not a mass of fused cells showing a common cytoplasm, but a highly intricate network of discrete cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Alzheimer's changes in non-demented and demented patients: a statistical approach to their relationships.

The anterior hippocampus and the entorhinal cortex of 167 non-demented individuals (controls) and 22 patients with Alzheimer's dementia were investigated. Senile plaques and neurofibrillary tangles were counted on paraffin sections stained with methenamin silver and Gallyas silver iodide, respectively. The results showed that both senile plaques and neurofibrillary tangles areal densities (lesions per mm2) correlated significantly with age in the control collective. No significant correlation between Alzheimer's disease lesions and age was found in patients with Alzheimer's dementia. Positive and significant correlation between senile plaques and neurofibrillary tangles was found in the control group but not in the Alzheimer group. Control individuals (n = 43, matched for age with the Alzheimer group) displayed both plaques and tangles. In this subgroup neither plaques nor tangles correlation with age; however, there was a significant correlation between senile plaques and neurofibrillary tangles. We hypothesize that the continuous activity of underlying processes associated with senile plaques and neurofibrillary tangles formation or resolution might lead to the unbalanced production of these lesions in Alzheimer's disease.

A molecular mechanism for the induction of neurofilament bundling by aluminum ions.

A1 induces neurofibrillary tangles in the perikaryon of neurons in vivo and in culture. The effect of A1 ions complexed with maltol, a plant-derived ligand of A1, on purified neurofilament preparations was studied in vitro. The binding of A1 to the arm-like projections of the high (H)- and medium (M)-molecular-weight neurofilament subunits causes a conformational change of the molecule (intrafilamentous reaction), characterized by an altered migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In addition, A1 compounds strongly stimulate the interaction between neurofilaments (interfilamentous reaction). The possibility that phosphate groups of the H and M sidearms are involved in binding A1 ions is discussed with regard to the migration on SDS-PAGE of dephosphorylated neurofilaments incubated with A1 compounds and the alteration by A1 ions of neurofilament phosphorylation in vitro by the associated kinase. Immunoblotting analysis of neurofilaments in cultivated neurons intoxicated with A1 compounds revealed a similar A1-dependent alteration of the neurofilament subunit conformation. This result suggest that the mechanism of A1-induced bundling of neurofilaments derived from in vitro studies might be involved in the formation of tangles in situ.

Early expression of carbonic anhydrase II (CAII) in transitory glial cells of the developing murine nervous system.

Using immunocytochemical methods carbonic anhydrase II (CAII) has been detected in the embryonic mouse central nervous system as early as in stage E16. In the spinal cord and the brain stem, the enzyme first appeared in transitory cells probably derived from the radial glia. In the cerebrum such transitory cells were never stained with anti-CAII sera. The bodies of CAII-positive cells were never visualized in the ventricular layer. The controversial cellular specificity of CAII made it impossible to specify the glial lineage to which these transitory CAII-positive cells are committed.

Aluminium-induced tangles in cultured rat neurones. Enhanced effect of aluminium by addition of maltol.

Neurofilamentous tangles have been induced in cultured neurones from rat brain hemispheres by application of both aluminium and maltol. Quantitative evaluation revealed a significantly higher percentage of tangle containing neurones when using the aluminium-maltol mixture than after application of aluminium alone. Tangles were found to be consistently stained with monoclonal antibodies to neurofilament proteins but failed to react with polyclonal antibodies against microtubule-associated proteins 1, 2 and tau.

Deposition of beta/A4 protein along neuronal plasma membranes in diffuse senile plaques.

The origin of the extracellular beta-amyloid protein (beta/A4) found in senile plaques and the cellular mechanisms responsible for its deposition in cerebral tissues are still an unresolved issue in Alzheimer's disease. In this study we analyzed in detail the distribution of various epitopes of beta/A4 in relation to local cellular elements in diffuse plaques of the hippocampal region. We also correlated our findings with the presence and distribution of non-beta/A4 epitopes of the amyloid precursor protein (APP) and with synaptophysin immunoreactivity in the cortical neuropil. Discontinuous beta/A4-immunoreactive deposits were found along dendrites, and around the soma of neurons included in the plaques. Furthermore, increased synaptophysin reactivity with slightly dilated synaptophysin-immunolabeled presynaptic terminals were found in diffuse plaques. APP epitopes could not be found in diffuse plaques. However, some of the APP antibodies, mainly those to the C-terminal portion of APP, and antibodies to beta/A4 recognized clusters of flat vesicular profiles (0.6-1.4 micron in width and 2-3 microns in length) in the neuropil of cortical areas where plaques had developed. Our findings are compatible with a neuronal origin of beta/A4 in diffuse plaques and with a primary release of beta/A4 at synaptic sites along the immunostained neurites. They also suggest that diffuse plaques might be preceded by minute lesions of the neuropil where beta/A4 is not yet released from the precursor molecule.

Pick's disease: hyperphosphorylated tau protein segregates to the somatoaxonal compartment.

Pick bodies and ballooned cells of Pick's disease and the neurofibrillary lesions of Alzheimer's disease are characterized by the presence of hyperphosphorylated microtubule-associated protein tau. Little is known about the mechanisms underlying tau hyperphosphorylation in Pick's disease and the distribution of abnormal tau in affected neurons. We have used a panel of phosphorylation-dependent (AT270, AT8, AT180, 12E8, PHF-1, AT10 and Tau-1) and phosphorylation-independent anti-tau antibodies (N-tau 5 and 134) to stain brain tissue sections from subjects with Pick's disease and Alzheimer's disease. These antibodies labeled Pick bodies and neurofibrillary lesions in a similar way, with the exception of antibody 12E8, which stained a subset of neurofibrillary tangles, but no Pick bodies. Moreover, abundant AT8- and PHF-1-positive neuritic profiles were observed in cortical areas rich in Pick bodies, even in the complete absence of neurofibrillary lesions. Unlike the Gallyas-positive neuropil threads of Alzheimer's disease, which were of variable diameter and covered by spiny appendages, neuritic profiles of Pick's disease showed a regular diameter, appeared smooth and were Gallyas-negative. In contrast to Alzheimer's disease, dendritic branches of neurons containing Pick bodies were not labeled by anti-tau antibodies. In the hippocampus, numerous tau-positive axon terminals were found along dendrites of the polymorphic layer of the dentate gyrus. Our results indicate that tau proteins in Pick's disease and Alzheimer's disease share similar phosphorylated residues, with the exception of serine 262, which is phosphorylated in Alzheimer tangles but not in Pick bodies or neuritic profiles. Furthermore, we show that hyperphosphorylated tau segregates to different neuronal compartments in the two diseases, with a somatoaxonal distribution in Pick's disease and a somatodendritic distribution in Alzheimer's disease.

Argyrophilic grain disease: widespread hyperphosphorylation of tau protein in limbic neurons.

Argyrophilic grains (ArG) and coiled bodies of argyrophilic grain disease (AgD) and the neurofibrillary lesions of Alzheimer's disease (AD) share similar antigenic determinants, among them hyperphosphorylated microtubule-associated protein tau. Nothing is known about the mechanisms underlying tau hyperphosphorylation in AgD, the hyperphosphorylated sites or the intracellular distribution of abnormally phosphorylated tau. We have analysed brain tissue sections from 41 subjects with AgD with a panel of phosphorylation-dependent (AT270, AT8, Tau-1, AT180, 12E8, PHF-1 and AT100) and phosphorylation-independent anti-tau antibodies (N-tau 5, 304, 189 and 134). All antibodies labelled ArG, coiled bodies and neurofibrillary lesions, with the exception of antibody 12E8, which stained a subset of neurofibrillary tangles, but no ArG or coiled bodies. Most pyramidal neurons in areas rich in ArG showed diffuse granular tau labelling in cell bodies and dendrites. Only very few tau-positive cells also contained neurofibrillary tangles. Phosphorylation-dependent anti-tau antibodies also stained a felt-like network of Gallyas-negative filiform neurites in layer CA1 of the hippocampus and in layer pre-B of the transentorhinal cortex. These results demonstrate a widespread hyperphosphorylation of tau protein in the somatodendritic domain of neurons in AgD, in addition to silver grains in the neuropil. Unlike in AD, tau hyperphosphorylation in the somatodendritic domain in AgD does not appear to be followed by neurofibrillary tangle formation, even in the presence of widespread ArG in the neuropil. Furthermore, our data suggest that no strict correlation exists between the presence or density of ArG in the limbic area and the occurrence of dementia.

Progressive supranuclear palsy: extensive neuropil threads in addition to neurofibrillary tangles. Very similar antigenicity of subcortical neuronal pathology in progressive supranuclear palsy and Alzheimer's disease.

Light microscopic immunohistochemical investigations were performed on neurofibrillary tangles (NFT) in four histologically confirmed cases of Alzheimer's disease (AD) and in five patients with a progressive supranuclear palsy (PSP). The antibody panel included antisera to the neuronal microtubule-associated protein, tau, and to isolated paired helical filaments (PHF), as well as mouse monoclonal antibodies (MAbs) to phosphorylated epitopes on high and medium molecular weight neurofilament subunits (RT97 and BF10, respectively). Paraffin sections were also impregnated with the Gallyas silver method, which specifically stains tangles and cortical neuropil threads in AD, but does not stain normal neurofilaments. All tangles in PSP and AD showed consistent immunostaining with antibodies to tau protein and isolated PHF, regardless of their localization. MAbs RT97 and BF10, however, did not stain or only weakly stained, subcortical tangles in PSP and AD, whereas most cortical NFT in AD were intensely immunostained. All tangles in PSP were as heavily impregnated with Gallyas as they were in AD. Furthermore there were extensive networks of Gallyas-positive, tau- and PHF-immunoreactive neurites in subcortical gray areas containing NFT, and bundles of positive axons in white matter tracts interconnecting subcortical nuclei of PSP. Our studies indicate a much more extensive disruption of fibrillar proteins in PSP subcortical neurons than previously reported. They furthermore indicate a very similar antigenic profile of NFT in PSP and AD, as far as subcortical neurons are concerned.

Argyrophilic grain disease: distribution of grains in patients with and without dementia.

In a previous study we reported on a late onset dementia which occurred in only half of the patients with argyrophilic grain disease (AgD) investigated. To find a correlation between the distribution of argyrophilic grains (ArG) and the occurrence of a late onset dementia, we examined the limbic area in 35 subjects who had ArG as the main neuropathological finding. A retrospective clinical analysis was performed by collecting information from hospital charts supplemented by standardized interviews based on DSM IV criteria for dementia. Sections from the rostral and caudal hippocampal regions, including the entorhinal/transentorhinal and parahippocampal cortex on both sides, were strained by the Gallays method. Nineteen subjects were diagnosed as demented according to these criteria; 16 were considered to have been cognitively normal. High numbers of ArG were observed in the anterior part of the CA1 subfield in all cases. However, the posterior half of CA1 was involved significantly more often and more severely in demented than in non-demented individuals (P < 0.01). Moreover, the distribution of ArG in the entorhinal/transentorhinal and parahippocampal cortex was more widespread in the group of demented patients (P < 0.05). These results show that the intellectual status of patients with AgD was related to the extension of ArG in the limbic area. We suggest that AgD is a progressive neurodegenerative disorder with early subclincial lesions in the anterior part of the hippocampal formation. To provide a more accurate clinicopathological correlation, the rostrocaudal extension of ArG in the limbic area should be evaluated in AgD cases.

Cytoskeletal immunohistochemistry of Alzheimer's dementia and related diseases. A study with monoclonal antibodies.

Mabs directed against phosphorylated epitopes on the heavy and medium neurofilament protein were used to immunostain histological sections from brains of patients without neurological disease and patients suffering from SDAT, Pick's disease, Parkinson's disease, progressive supranuclear palsy and encephalomalacias of the white matter inducing chromatolysis in the overlying cortex. In normal brains only axons but never perikarya were stained. In the pathological brains, however, swollen neurons with chromatolysis and swollen cells in Pick's disease, NFT in SDAT, Pick bodies in Pick's disease, the centers of Lewy bodies in Parkinson's disease and some tangles in progressive supranuclear palsy were stained. These changes are perikaryal alterations. The results are discussed in relation to the formation of NFT in SDAT, i.e. the PHF as seen by electron microscopy. It is concluded that in spite of the reliable staining of NFT with some of our mabs, with sera directed against PHF, MAPs and other cytoskeletal proteins there is no absolutely specific immunoreaction for PHF. The most similar pattern to that observed in NFTs of SDAT is seen in the Pick bodies of Pick's disease, although these do not consist of PHF when looked at with the electron microscope, and although they behave differently from NFT in some 'conventional' histological stains. From this nonspecificity of the immunoreaction and from the presence of multiple cytoskeletal epitopes in NFT it is concluded that NFT (i.e. PHF) are probably not derived from one particular cytoskeletal element but are reassembled from proteolytic breakdown fragments of several of these elements. In this regard the similarities and dissimilarities with the alterations of Pick's disease might be specially relevant and deserve further studies, especially as the clinical features of SDAT and Pick's disease can be very similar.

Accumulation of flotillin-1 in tangle-bearing neurones of Alzheimer's disease.

The protein flotillin-1 is associated with the 'lipid rafts', that is, membrane microdomains that are enriched in cholesterol and sphingolipids. We compared flotillin-1 immunoreactivity in the hippocampus, amygdala and isocortex (Brodmann area 22) of six controls and 13 Alzheimer's disease (AD) cases (10 sporadic and three familial). A diffuse labelling of the neuropil was observed in most of the samples. The intensity of this labelling was not correlated with the density of neurofibrillary tangles (NFT) or of senile plaques. Some neuronal cell bodies were diffusely labelled in patients as in controls. Immunostained granular bodies were found in the cell body of a few neurones. The density of neuronal profiles containing large granular bodies (diameter > or =2 microm) was significantly higher in AD cases and was correlated with the density of NFTs in the three regions that were studied. Sections stained by double immunofluorescence methods and examined with confocal microscopy suggested that flotillin-1 accumulated most often in tangle-bearing neurones (76% of flotillin-1-positive neurones contained a NFT). Flotillin-1 immunoreactivity, even when found in a tangle-bearing neurone, was not colocalized with tau protein indicating that the two proteins were not in close contact and probably in different subcellular compartments. Flotillin-1-positive granular bodies were also found in neurones containing Pin1-positive vesicles but were not colocalized with them. Flotillin-1 immunoreactivity was colocalized with cathepsin D, a lysosomal marker. These data indicate that flotillin-1, a marker of rafts, accumulates in lysosomes of tangle-bearing neurones in the course of AD.