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SUMMARY Experimental hydrocephalus was induced by the infusion of a non-inflammatory silicone oil into the cisterna magna of adult rabbits and infant dogs. In an histological and electron-microscopical study, the brain tissue damage in these animals was compared with that in cerebral biopsies from hydrocephalic human infants. The main features of ependymal disruption, with nerve fibre destruction and clear-fluid (probably csf) extracellular oedema of the periventricular white matter, were present to varying degrees in all the severe hydrocephalic brains. Very much less disruption occurs in the unmyelinated infant brain than in the partially or fully myelinated animal brains. It appears that the extent of the tissue damage bears some relationship to the degree of white matter myelination. RESUME Une hydrocephalie experimentale a ete provoqutee par l'injection d'huile de silicone non inflammatoire dans la grande citerne de lapins adultes et de chiots. Une etude histo-logique et electromicroscopique a permis de comparer les lesions cerebrales chez ces animaux avec les biopsies cerebrales provenant de jeunes hydrocephales humains. Les principales caracteristiques de l'epanchement ependymaire, avec destruction des fibres nerveuses et oedeme de liquide clair (probablement LCR) de la substance blanche periventriculaire, ont ete observees a des degres varies dans tous les cas d'hydrocephalies severes. Un epanchement beaucoup moindre se produit dans le cas du cerveau non myelinise du jeune enfant que dans les cerveaux animaux, partiellement ou totalement myilinises. II apparait donc une relation entre l'etendue des lesions tissulaires cerebrales et le degre de myelinisation de la substance blanche. ZUSAMMENFASSUNG Ein experimenteller Hydrocephalus wurde durch die Infusion von nicht entzundendem Siliconol in die Cysterna magna von erwachsenen Kaninchen und jungen Hunden hervorge-rufen. In einer histologischen und elektronenmikroskopischen Untersuchung wurde die Schadigung des Gehirngewebes verglichen mit der in cerebralen Biopsien von hydro-cephalischen Kindern beobachteten Schadigung. Die hauptsachlichen Merkmale der ependymalen Zerreiβung mit Zerstorung von Nervenfasern und einem klarussigen (wahrscheinlich CSF) extrazellularen Oedem der periventrikularen weiβen Substanz fanden sich in unterschiedlichem Ausmaβ in allen schwer hydrocephalischen Gehirnen. In den nicht myelinisierten kindlichen Gehirnen kommt sehr viel weniger Zerreiβung vor als in den teilweise oder ganz myelinisierten Gehirnen der Tiere. Es scheint so, daβ das Ausmaβ der Gewebeschadigung in einem gewissen Verhaltnis zur Myelinisierung der weiβen Substanz steht. RESUMEN Se produjo una hidrocefalia experimental por la infusion de un aceite siliconico ininflam-able en la cisterna magna de conejos adultos y de perrillos lactantes. En un estudio histologico y electromicroscopico, se comparo el dano a los tejidos cerebrales en estos animales con el dano revelado por biopsias cerebrales en lactantes humanos con hidrocefalia. Las caracteristicas mas importantes de disrupcion ependimaria, con destruccion de las fibras nerviosas y edema extracelular de liquido claro (probablemente liquido cefalorra-quideo) de la substancia blanca periventricular se hallaban a un grado variable en todos los cerebros hidrocefalos mas graves. Mucho menos disrupcion ocurre en el cerebro no mieli-nico del lactante que en los cerebros animales parcial o completamente mielinicos. Parece que la extension del dano a los tejidos tiene relacion con el grado de mielinizacion de la substancia blanca.

Calbindin D 28K (CB) expression was analyzed in the rat hippocampus following 10-min-cardiac arrest-induced ischemia within a year after reperfusion. In rats examined 3 days after ischemia, CB immunoreactivity disappeared completely from CA1 pyramidal neurons and from most CA2 pyramids. In the stratum granulosum of the dentate gyrus, mossy fibers, and hippocampal interneurons, CB immunoreactivity was preserved, although staining was somewhat paler than that in control rats. A similar pattern of CB immunoreactivity was found in rats sacrificed 14 days and 1 month after cardiac arrest. From the 14 th postischemic day, neuronal loss in the stratum pyramidale of CA1 but not in that of CA2 became apparent. The reappearance of CB immunoreactivity in CA1 and CA2 pyramidal neurons was noticed 6 months after ischemia, and the pattern was identical to that observed in animals sacrificed 12 months after the ictus. The prolonged loss and delayed reappearance of CB immunoreactivity in the hippocampus demonstrate that ischemia may induce long-term disturbances of protein expression, which may in turn result in impairment of hippocampal functioning.

The claustrum is a subcortical structure lying under the insular and piriform cortices, whose function is still not clear. Although data exist on connections of the claustrum and the limbic cortex, the topography of the limbic zone in the rabbit and rat claustrum has not been studied extensively. The study was performed on 17 adult Wistar rats and 12 New Zealand rabbits. Two percent water solutions of fluorescent retrograde tracers fast blue and nuclear yellow were injected into the various regions of the limbic cortex. The limbic zone is localized throughout the whole rostrocaudal extent of the claustrum, mainly in its ventromedial portion lying close to the external capsule. Although this zone of the claustrum is localized similarly in both rat and rabbit, some differences between these two species exist. In the rat, neurons projecting to all limbic areas are localized mainly in the anterior and central parts of the claustrum, whereas in the rabbit, the majority of the neurons projecting to the cingulate cortex are present in the anterior and central parts of this structure, while neurons sending axons to the retrosplenial cortex are localized in the central and posterior parts. In both species, double-labeling study showed that neurons projecting to various limbic regions are intermingled and that neurons sending axons into two different limbic regions are seen only occasionally. Our findings give support to the role of the claustrum in integrating information between different areas of the cerebral cortex and the limbic system.

Previous epidemiological evidence suggested that in some instances a vector and/or reservoir is involved in the occurrence and spread of transmissible spongiform encephalopathies (TSEs). In a preliminary study, hay mite preparations from five Icelandic farms with a history of scrapie were injected into mice, and some of these mice became sick after long incubation periods. To confirm that the disease was scrapie, subsequent passages in mice were performed. In addition, the characteristics of the disease process in these passages were assessed and the results compared to those findings with standard scrapie strains. As expected for scrapie, subsequent passages in the same host led to shortened incubation periods compared to those in primary isolate mice, and all mice had spongiform changes in brain. Results were similar for three of four isolates with regard to clinical manifestations, the incubation periods in mice of the three scrapie incubation-period genotypes (s7s7, s7p7, p7p7), and the PrPSc Western blot (WB) pattern. The characteristics of the fourth isolate were markedly different from the other three isolates with regard to these parameters. Comparison of the characteristics of standard mouse-adapted scrapie strains and the four isolates revealed differences; these differences were particularly pronounced for the fourth isolate.

The function of the neuronal high molecular weight microtubule-associated proteins (MAPs) MAP1b and MAP2 is regulated by the degree of their phosphorylation, which in turn is controlled by the activities of protein kinases and protein phosphatases (PP). To investigate the role of PP in the regulation of the phosphorylation of MAP1b and MAP2, we used okadaic acid and cyclosporin A to selectively inhibit PP2A and PP2B activities, respectively, in metabolically competent rat brain slices. The alteration of the phosphorylation levels of MAP1b and MAP2 was examined by Western blots using several phosphorylation-dependent antibodies to these proteins. The inhibition of PP2A, and to a lesser extent of PP2B, was found to induce an increased phosphorylation of MAP1b and inhibit its microtubule binding activity. Immunocytochemically, a marked increase in neuronal staining in inhibitor-treated tissue was observed with antibodies to the phosphorylated MAP1b. The inhibition of PP2A but not of PP2B also induced phosphorylation of MAP2 at multiple sites and impaired its microtubule binding activity. These results suggest that PP2A might be the major PP that participates in regulation of the phosphorylation of MAP1b and MAP2 and their biological activities., This study was supported by the New York State Office of Mental Retardation and Developmental Disabilities, and NIH Grants AG05892, AG08076, AG14875 and NS18105.

The pattern of neuronal loss in the rat hippocampus following 10-min-long cardiac arrest-induced global ischemia was analyzed using the unbiased, dissector morphometric technique and hierarchical sampling. On the third day after ischemia, the pyramidal layer of sector CA1 demonstrated significant (27%) neuronal loss (P

Two (P117L; M146L) familial Alzheimer's disease (FAD)-causing presenilin-1 (PS1) mutations have been tested fortheir effect in stably transfected mouse neuroblastoma (N2a) cell lines. The P117L mutation is associated with the earliest onset of AD reported so far (24 years), while the M146L is less pathogenic with the onset at about 43 years. Overexpression of wild-type (wt) PS1 gene was associated with the marked increase in the number and the length of neuritic outgrowths accompanied by accumulation of PS1 immunoreactivity in neurites. The highly pathogenic P117L mutation completely suppressed this effect and the pattern of PS1 immunolabeling resembled a cup structure with all immunoreactivity gathered at one pole of the cell. The effect of less pathogenic M146L mutation was similar, but not as pronounced. These findings suggest that one of the normal functions of PS1 may be the control of neurite outgrowth, and the inhibitory effect of two FAD-linked mutations stresses its importance in the cellular mechanism that leads to the development of Alzheimer's disease (AD).

Canine smooth muscle cells (SMCs), cultured from amyloid-affected brain blood vessels accumulate Alzheimer amyloid-beta peptide (Abeta) intracellularly, either spontaneously or after treatment with apolipoprotein E (apoE). ApoE is codeposited with Abeta, which suggests that apoE participates in Abeta accumulation. We tested the hypothesis that apoE-induced accumulation of Abeta in SMCs is caused by an increased production of amyloid-beta precursor protein (AbetaPP) and/or its altered metabolism. We found that 24 hours of treatment with apoE3 or apoE4 induced intracellular accumulation of Abeta-immunoreactive deposits in SMCs but did not influence AbetaPP production and processing. The treatment with apoE3 or E4 for 3 days resulted in the following: increased Abeta-accumulation; reduced levels of secreted Abeta; increased production and cellular retention of mature AbetaPP770; and reduced culture growth, cell proliferation, and viability. ApoE4, but not apoE3, increased cellular levels of mRNA AbetaPP 770 (the main form produced in SMCs) about ninefold. ApoE3 stimulated production and cellular retention of endogenous apoE. We hypothesize that Abeta accumulation is triggered by apoE, which may bind and immobilize soluble Abeta produced in SMCs. The newly formed Abeta deposits may further accelerate Abeta accumulation by altering metabolism of AbetaPP.

In Alzheimer's disease (AD), neurofibrillary degeneration of neurons starts in the transentorhinal cortex and spreads in a time-dependent manner to the entorhinal cortex, which provides a major input to the hippocampus--a key structure of the memory system. People with Down's syndrome (DS) develop neurofibrillary changes more than 30 years earlier than those with sporadic AD. To characterize AD-related pathology in the entorhinal cortex in DS, we examined seven subjects with DS of 60-74 years of age who died in the end stage of AD, and four age-matched control subjects. The volume of the entorhinal cortex in brains of subjects with DS was 42% less than that in control cases; however, the total number of neurons free of neurofibrillary changes was reduced in DS by 90%: from 9,619,000 +/- 914,000 (mean +/- standard deviation) to 932,000 +/- 504,000. The presence of 2,488,000 +/- 544,000 neurofibrillary tangles in the entorhinal cortex of people with DS, the prevalence of end-stage tangles, and the significant negative correlation between the total number of intact neurons and the percentage of neurons with neurofibrillary changes indicate that neurofibrillary degeneration is a major cause of neuronal loss in the entorhinal cortex of people with DS. The relatively low amyloid load (7 +/- 1%) and lack of correlation between the amyloid load and the volumetric or neuronal loss suggest that the contribution of beta-amyloid to neuronal loss in the entorhinal cortex is unsubstantial.

Morphometry of the cerebellum of 11 subjects who died in the severe, final stage of Alzheimer's disease (AD) and of five age-matched subjects without dementia revealed significant atrophy in the AD group, with a decrease in the volume of the molecular layer by 24% and of the granular layer by 22% in comparison with controls. The 32% decrease in the total number of Purkinje cells that was observed correlates with the atrophy of the molecular layer, whereas the 30% reduction in the total number of granule cells correlates with the atrophy of the molecular and granular layers. A unique pattern of Alzheimer-type pathology was observed in the cerebellum: (1) there were no neurofibrillary changes in the cerebellum of either the control or the AD subjects, (2) there was almost the same extent of leptomeningeal and cortical amyloid angiopathy in the normal aged subjects and in the AD patients, and (3) the presence of plaques was noted in the AD group, but not in the control group. This pattern of pathology suggests that two factors might be considered in the etiopathogenesis of cerebellar atrophy: (1) transneuronal degeneration and neuronal loss resulting from primary pathologic changes in cerebral structures and (2) parenchymal cerebellar ss-amyloidosis. The correlation between the temporal duration of AD and both the decrease of the total number of granule cells (r=0.86, p

Two-laser and two-color approaches were used to observe the colocalization of the calcium-binding proteins, calbindin D28k and parvalbumin, and the retrograde tracer, Fluoro-Gold (FG) in the basolateral amygdala of the rat. The study was performed on five adult rats into which FG was injected to the frontal association cortex. Then, the localization of the retrogradely labeled neurons in the basolateral amygdala was compared with the localization of the neurons labeled by calcium-binding proteins. The present study showed that most of the retrogradely labeled neurons in the posterior part of the basolateral amygdala are also calbindin-positive. Even though a lot of parvalbumin-positive endings were present at the surface of the retrogradely labeled cells, we did not observe the colocalization of the parvalbumin and projective neurons.

An important gap in our understanding of the pathogenesis of the amyloidoses is the identification of the cellular events that lead from synthesis of an amyloid precursor protein to its conversion to the amyloid fiber subunit. We address this question by characterizing the effects of an amyloidogenic mutation on the intracellular processing of its protein product. The protein, a mutant of the cysteine protease inhibitor cystatin C, is the amyloid precursor protein in Hereditary Cerebral Hemorrhage with Amyloidosis--Icelandic type (HCHWA-I). The amyloid fibers are composed of mutant cystatin C (L68Q) that lacks the first 10 amino acids. We have previously shown that processing of wild-type cystatin C entails formation of a transient intracellular dimer that dissociates prior to secretion, such that extracellular cystatin C is monomeric. We report here that the cystatin C mutation engenders several alterations in its intracellular trafficking. It forms a stable intracellular dimer that is partially retained in the endoplasmic reticulum and degraded. The bulk of mutant cystatin C that is secreted does not dissociate and is secreted as an inactive dimer. Thus, formation of the stable mutant cystatin C dimer is an early event in the pathogenesis of this disease.

Alzheimer's disease (AD), a progressive neurodegenerative disorder, is diagnosed definitively by increased numbers of beta-amyloid plaques and neurofibrillary tangles in brain biopsy or autopsy specimens. There are no simple straightforward laboratory tests currently available for clinical diagnosis. We have found consistent reduction in mitotic index levels in skin fibroblast cultures from AD individuals compared with age- and sex-matched controls. These differences were enhanced by overnight exposure to colcemid (p = 0.04). Results suggest that mitotic index in skin fibroblasts cultures should be further investigated as a potential diagnostic indicator for AD.

A characteristic feature of the parvopyramidal layer of the presubiculum of 6 individuals with Alzheimer disease (AD) was the presence of large, evenly distributed amyloid-beta (A beta) deposits, which in the end stage of the disease occupy 80.9 +/- 12.2% of the parvopyramidal layer. The strong reaction of A beta deposits with antibodies 4G8 (17-24 amino acids, aa), 6E10 (1-17 aa), and R165 (32-42 aa), and their weak reaction with antibody R162 (32-40 aa) indicate that potentially highly fibrillogenic A beta1-42 is a major constituent of presubicular amyloid. However, A beta deposits in the presubiculum are thioflavin-S- and Congo red-negative--and thus, nonfibrillar--even after 11 to 19 years of AD. The unique properties of presubicular amyloid appear to be related to their origin; amyloid-associated proteins such as apolipoproteins E, and AI, alpha1-antichymotrypsin, and heparan sulfate proteoglycan, which are promoters of fibrillization or stabilizers of A beta in neuritic plaques, are absent; activated astrocytes, which are the source of these proteins, are also absent. The unchanged number and distribution and the resting appearance of microglial cells revealed with RCA-I histochemistry suggest that they do not respond to diffuse A beta deposits. The source of nonfibrillar presubicular A beta is probably local neurons or neuronal projections to the parvocellular layer of the presubiculum. Neuronal, lake-like A beta deposition appears to be characteristic of AD pathology. The presubiculum is most likely the model brain structure for the study of amyloid of exclusively neuronal origin. The parvopyramidal layer of the presubiculum reveals only a small population of the neurons (2.5 +/- 2%) affected by neurofibrillary pathology.

Scrapie is a degenerative disease of the central nervous system of sheep and goats. The causative agent has been passaged to a number of laboratory species, including mice and hamster. Amyloid plaque formation and vacuolation, the signs of senile dementia, are found in the brains of mice infected with 87V scrapie agent. Dopamine (DA) and norepinephrine (NE) concentrations in the brains of scrapie-infected mice were measured with high-performance liquid chromatography-electrochemical detector (HPLC-ECD). A significant decrease in NE level was exhibited in all regions tested, whereas the level of DA decreased significantly only in cerebral cortex. Immunohistochemistry was used to examine immunoreactive catecholamine neurons in substantia nigra and locus ceruleus using antisera against tyrosine hydroxylase (TH). The population of TH-immunoreactive neurons in the substantia nigra and locus ceruleus were significantly decreased in scrapie-infected mice compared to controls. These data suggest that both the noradrenergic and dopaminergic system are sensitive to the action of scrapie agent 87V and that changes in the catecholamine levels in the brains of scrapie-infected mice may contribute to some of the clinical symptoms of the diseases, such as ataxia and apraxia.

Amyloid beta protein 1-40 (A beta40) and A beta42 levels were quantitated in plasma from 43 persons with Down syndrome (DS; 26-68 years of age), 43 age-matched normal controls, and 19 non-DS mentally retarded (MR) persons (26-91 years of age) by using a sandwich enzyme linked immunosorbent assay. A beta40 levels were higher in DS and MR than controls, but were similar between DS and MR groups. A beta42 levels were higher in DS than controls or MR persons. The ratios of A beta42/A beta40 were higher in DS than controls or MR persons. The findings are consistent with those seen in DS brains.

This study investigated the time of expression of two endothelial cell adhesion molecules, ICAM-1/CD54 and PECAM-1/CD31 in the developing postpartum mouse blood-brain barrier (BBB). Immunoultrastructural studies demonstrated that both adhesion molecules are initially expressed primarily on the luminal endothelial cell surfaces at birth or shortly thereafter, they increase in an intensity at approximately 1 week postpartum and then decrease to a weak labeling of the luminal endothelial cell surfaces at 2 weeks after birth. In the adult animals, little or no adhesion molecule labeling was expressed on blood vessels. Our results present immunocytochemical evidence that upregulation of ICAM-1 and PECAM-1 occurs prior to structural maturity of the BBB. Moreover, these data support a notion that these adhesion molecules play an important role for angiogenesis in the developing murine BBB.

To define the cellular processing of human cystatin C as well as to lay the groundwork for investigating its contribution to lcelandic Hereditary Cerebral Hemorrhage with Amyloidosis (HCHWA-I), we have characterized the trafficking, secretion, and extracellular fate of human cystatin C in transfected Chinese hamster ovary (CHO) cells. It is constitutively secreted with an intracellular half-life of 72 min. Gel filtration of cell lysates revealed the presence of three cystatin C immunoreactive species; an 11 kDa species corresponding to monomeric cystatin C, a 33 kDa complex that is most likely dimeric cystatin C and immunoreactive material, > or = 70 kDa, whose composition is unknown. Intracellular monomeric cystatin C is functionally active as a cysteine protease inhibitor, while the dimer is not. Medium from the transfected CHO cells contained only active monomeric cystatin C indicating that the cystatin C dimer, formed during intracellular trafficking, is converted to monomer at or before secretion. Cells in which exit from the endoplasmic reticulum (ER) was blocked with brefeldin A contained the 33 kDa species, indicating that cystatin C dimerization occurs in the ER. After removal of brefeldin A, there was a large increase in intracellular monomer suggesting that dimer dissociation occurs later in the secretion pathway, after exiting the ER but prior to release from the cell. Extracellular monomeric cystatin C was found to be internalized into lysosomes where it again dimerized, presumably as a consequence of the low pH of late endosome/lysosomes. As a dimer, cystatin C would be prevented from inhibiting the lysosomal cysteine proteases. These results reveal a novel mechanism, transient dimerization, by which cystatin C is inactivated during the early part of its trafficking through the secretory pathway and then reactivated prior to secretion. Similarly, its uptake by the cell also leads to its redimerization in the lysosomal pathway.

Smooth muscle cells cultured from amyloid-beta-affected arteries accumulate amyloid-beta peptide A beta. We now show that accumulation of "A beta" deposits in this model can be significantly reduced by culture in conditioned media from microglia and monocytes. Reduced A beta accumulation was associated with (i) lower secretion of A beta, (ii) increased secretion, but not cellular levels of amyloid-beta-precursor protein (A beta PP), and (iii) increased cell proliferation and metabolic activity. We suggest that improper regulation of A beta PP metabolism by monokines may facilitate vascular amyloidogenesis.

Monoclonal antibodies (mAbs) D3G6 and C8A5, specific for amino acid residues 160-168 of S182 protein, immunolabeled neurons, ependymal and choroid plexus cells, and myocytes in brain sections from normal subjects and people with Alzheimer disease or Down syndrome and in rats and mice. Oligodendroglia, microglia, and the majority of astrocytes were negative. S182 protein or a fragment of the protein detected with these mAbs is not a constituent of amyloid-beta deposits or tangles.

In a search for Alzheimer beta-amyloid peptide precursor ligands, Potempska et al. (Arch. Biochem. Biophys. (1993) 304, 448) found that histones bind with high affinity and specificity to the secreted precursor. Because exogenous histones can be cytotoxic, we compared the effects of histones on the viability of cells which produce little beta-amyloid peptide precursor (U-937) to those on cells that produce twenty times as much precursor (COS-7). Addition of purified histones caused necrosis of U-937 cells (histone H4, LD50 = 1.5 microM). Extracellular A beta precursor in the submicromolar range prevented histone-induced U-937 cell necrosis. Cell-surface precursor also reduced histone toxicity: COS-7 cells were less sensitive to the toxic effects of histone H4 (LD50 = 5.4 microM). COS-7 cells in which the expression of an APP mRNA-directed ribozyme reduced the synthesis of the protein by up to 80% were more sensitive to histone H4 (LD50 = 3.2 microM) than cells that expressed the vector alone. Histone H4 binds to cell-associated A beta precursor. Cells expressing the A beta precursor-directed ribozyme bound less 125I-labeled histone H4 than those expressing the vector alone. In the limited extracellular space of tissues in vivo, both secreted and cell-surface A beta precursor protein may play significant roles in trapping chromatin or histones and removing them from the extracellular milieu.

Smooth muscle cells cultured from leptomeningeal vessels from old dogs with amyloid-angiopathy accumulate intracellular deposits that are immunoreactive for amyloid-beta peptide (A beta). We used this cellular model in the present study to examine the influence of sera and cerebrospinal fluid on intracellular accumulation of A beta-immunoreactive deposits and on secretion of soluble A beta into culture media. We found that sera from old dogs significantly increased the percentage of A beta-positive smooth muscle cells in culture. The enhanced accumulation of A beta was associated with (a) lower secretion of A beta into media, (b) altered maturation of amyloid-beta-precursor protein (A betaPP) into A betaPP751-770 with faster electrophoretic mobility, (c) increased accumulation of C-terminal fragments of A betaPP (12-15 kD, 10kD and less), and (d) increased secretion of A betaPP into culture media. These findings suggest that age- or disease-related serum factors increase accumulation of A beta by affecting production and processing of A betaPP In contrast, cerebrospinal fluids reduced accumulation of A beta. Involvement of A beta-carrier proteins-apolipoprotein E and transthyretin-in accumulation of A beta is demonstrated. Accumulation of A beta in cultured smooth muscle cells-a model of beta-amyloidosis-may be regulated by factors that alter production and processing of A betaPP as well as the fate of soluble A beta in extracellular space.

Proinflammatory cytokines (interleukin [IL]-1beta, tumor necrosis factor [TNF]-alpha, and IL-6) and soluble intercellular adhesion molecule-1 (sICAM-1) were measured in paired cerebrospinal fluid (CSF) and serum samples from patients with subacute sclerosing panencephalitis (SSPE), multiple sclerosis (MS), or other neurologic diseases (OND) by ELISA. IL-1beta was significantly increased in CSF of the SSPE group compared with levels in the MS or OND group. IL-1beta CSF/serum ratios were higher in the SSPE than in the MS or OND group. TNF-alpha and IL-6 levels were similar in the 3 groups. CSF sICAM-1 was higher in the SSPE group than in the MS or OND group. sICAM-1 CSF/serum ratios were higher in the SSPE than the OND group. The increased CSF/serum ratios of IL-1beta and sICAM-1 in SSPE indicate synthesis of IL-1beta and sICAM-1 in the central nervous system and may be important in the pathogenesis of disease.

We developed a method for the preparation of ultrathin longitudinal sections of chromosomes enabling TEM studies of whole chromosomes. By using a novel "repeat chill" method of exposing the glass slide and plastic block interface to liquid nitrogen, it was possible to separate consistently hardened epoxy resin-embedded chromosome spreads from glass slides for ultrathin longitudinal sectioning of entire spreads and of individual chromosomes. The method was applied to analyze the fragile X chromosome. The ultrastructure of centromeres, telomeres, fragile sites and other chromosomal areas can now be studied in detail.

Amyloid beta-protein (Aβ) is the major constituent of amyloid fibrils composing β-amyloid plaques and cerebrovascular amyloid in Alzheimer's disease (AD). We studied the effect of metal cations on preformed fibrils of synthetic Aβ by Thioflavin T (ThT) fluorescence spectroscopy and electronmicroscopy (EM) in negative staining. The amount of cross beta-pleated sheet structure of Aβ 1–40 fibrils was found to decrease by metal cations in a concentration-dependent manner as measured by ThT fluorescence spectroscopy. The order of defibrillization of Aβ 1–40 fibrils by metal cations was: Ca2+ and Zn2+ (IC50 = 100 μM) > Mg2+ (IC50 = 300 μM) > Al3+ (IC50 =1.1 mM). EM analysis in negative staining showed that Aβ 1–40 fibrils in the absence of cations were organized in a fine network with a little or no amorphous material. The addition of Ca2+, Mg2+, and Zn2+ to preformed Aβ 1–40 fibrils defibrillized the fibrils or converted them into short rods or to amorphous material. Al3+ was less effective, and reduced the fibril network by about 80 % of that in the absence of any metal cation. Studies with Aβ 1–42 showed that this peptide forms more dense network of fibrils as compared to Aβ 1–40. Both ThT fluorescence spectroscopy and EM showed that similar to Aβ 1–40, Aβ 1–42 fibrils are also defibrillized in the presence of millimolar concentrations of Ca2+. These studies suggest that metal cations can defibrillize the fibrils of synthetic Aβ.

In vitro aggregation and fibrillization of synthetic amyloid beta-protein Aβ 1–40 was assessed in the conditioned media from rhabdomyosarcoma (CRL 1598, HTB 82, HTB 153, CCL 136), adenocarcinoma (CCL 218), neuroblastoma (SY5Y), and COS cells cultured in the absence and presence of 10% heat-inactivated fetal bovine serum (FBS). The aggregation and formation of cross β-pleated sheet structures in Aβ was quantitated by Thioflavin T (ThT) fluorescence spectroscopy, while the morphology of Aβ fibrils was examined in negative staining in the electronmicroscope (EM). In cultures supplemented with 10% FBS, the conditioned media from CRL 1598, HTB 82, CCL 218, and SY5Y cell cultures stimulated Aβ aggregation in a time-dependent manner as compared to that of control (serum-containing medium that had not been exposed to cells). The order of stimulation was SY5Y > CRL 1598 ≥ HTB 82 > CCL 218, and the stimulation was higher in 2 week cultures than in 1 week cultures. Similar studies using media from HTB 153, CCL 136 and COS cell cultures showed no effect on Aβ 1–40 aggregation. In serum-free cell cultures, only media from SY5Y and CRL 1598 could promote significant aggregation of Aβ 1–40. Negative staining in EM revealed Aβ fibril formation only with conditioned media from SY5Y and CRL 1598 cultured under serum free conditions; no Aβ fibrils were noticed in media from cell cultures supplemented with 10% FBS. We propose that both the SY5Y neuroblastoma cell line and the CRL 1598 rhabdomyosarcoma cell line may serve as experimental models for in vitro studies of extracellular aggregation and fibrillization of Aβ-protein in cell cultures, while rhabdomyosarcoma HTB 82 and adenocarcinoma CCL 218 may be models for study of Aβ aggregation only.

We examined the effect of cerebrospinal fluid (CSF) from 23 Alzheimer's disease (AD) patients and 22 age-matched non-demented controls with apolipoprotein E4/4, 3/3, or 3/2 phenotypes on in vitro aggregation of amyloid beta-protein (A beta) 1-40 by Thioflavin T fluorescence spectroscopy. CSF from both AD and control groups inhibited A beta aggregation, as compared to that of phosphate buffered saline, in agreement with an earlier report (Wisniewski et al., 1993). However, there was significantly less aggregation of A beta in presence of CSF from AD than that from non-demented controls. The presence of CSF from controls with apoE3/3 phenotype resulted in higher A beta aggregation as compared to other phenotypes. There was a positive correlation between CSF apoE concentrations and A beta aggregation; whereas age, CSF soluble A beta levels or severity of dementia did not correlate with A beta aggregation. These results suggest that mechanism of sequestration of A beta in CSF may not be defective in AD. Amyloid formation in AD may be impact of altered balance of other factors such as amyloid-associated proteins/extracellular matrix components that can immobilize A beta in the brain, and promote its fibrillogenesis in AD.

Dulbecco's modified Eagle's medium promotes aggregation and fibrillization of the synthetic amyloid beta 1-40 and beta 1-42 peptides more than RPMI and OPTI media. Fibrillization in all of these media is faster than in phosphate-buffered saline and Tris buffer. Normal and heat-inactivated fetal bovine and human serum abolish amyloid fibril formation in buffers and cell culture media. Fibrillar amyloid formed during 2-day-long incubation in cell culture media and buffers is defibrillized by 1-day-long treatment with human and bovine serum. This study indicates that amyloid beta fibrillogenesis in cell culture should be studied in serum-free media or in media with a low concentration of serum.

In this article, we present a clinical description of Alzheimer's disease, review extant literature regarding the prevalence of dementia of the Alzheimer's type in people with Down syndrome, and discuss the relationship between Alzheimer's-type neuropathology and Alzheimer's type dementia in people with Down syndrome. After reaching the age of 30 years, virtually all adults with Down syndrome develop beta-amyloid plaques and tangles, the neuropathological changes of Alzheimer disease. However, substantially fewer than 100% of these adults develop dementia of the Alzheimer's type. When adults with Down syndrome do develop dementia of the Alzheimer's type, it typically does not occur until they are over 50 years of age, a full 15 to 20 years after the inferred development of the neuropathology. This discrepancy may be explained by subtleties in the characteristics of the neuropathology (such as the types and distributions of beta-amyloid plaques and the densities and distributions of neurofibrillary tangles). One variable that may affect the overall neuropathological picture is the type of apo-E allele on chromosome 19. Finally, given the considerable range of individual differences in both neuropathological and clinical expression of Alzheimer's disease, it is possible that as-yet-unidentified aging genes are located on chromosome 21 and influence the rate of precocious aging. These genes may be of greater importance in the clinical expression of Alzheimer's disease than is the triplication of the beta-amyloid gene, which also is located on chromosome 21. © 1996 Wiley-Liss, Inc.

Garlic is known as a potent spice and a medicine with broad therapeutic properties ranging from antibacterial to anticancer, antidiabetic, and anticoagulant. Two major proteins of 40 KD and 14 KD constituting approximately 96% of total garlic proteins have been recently purified at our Institute. This immunocytochemical and ultrastructural study revealed that the 40 KD protein was localized in the parenchyma sheath cells (PSC) of garlic bulbs, whereas the 14 KD protein was present in the cortical cells (CC). Immunogold electron microscopy study indicated that the 40 KD protein was specifically localized in the globular granules of the cytoplasmic area of PSC. Each globular granule was amorphous and homogenous with membrane limiting its outermost layer. The yellowish color of PSC in freshly cut slices of garlic bulb suggested that PSC may have sulfur-containing compounds such as allicin, the primary contributor of the pungency and medicinal properties of garlic. Ellman's reagent test quantitatively revealed that there were 17.8 n moles sulfhydryl (SH)/ml of 40 KD garlic protein. Microtubule tubulin in mitotic figures from PHA-stimulated human short-term whole blood cultures reacted strongly with antitubulin antibody but reacted negatively with anti-40 KD garlic protein antibodies and therefore was not related to the 40 KD garlic protein immunocytochemically.

That the topography, severity, and progression of beta-amyloid deposition in the brain of Alzheimer's disease (AD) and Down's syndrome (DS) cases is not uniform is well documented. We have addressed at present, the issue of whether the structural composition of beta-peptide (A beta) within the early amyloid deposits might contribute to this phenomenon. The cerebral cortex, the caudate/putamen, and the cerebellum from 10 AD and 8 DS cases were immunostained with antibodies that recognize the 1-17; 17-24 amino acid residues of A beta, and the COOH-terminus of A beta 42 variant, thus to the epitopes of A beta located amino- and carboxy-terminally to the site of the putative alpha-secretase cleavage. We demonstrate that numerous diffuse, early plaques in AD and especially in DS cases show predominance of the carboxy-terminally located epitopes of A beta; the most prominent in the cerebellum, less pronounced in the cerebral cortex, and only marginal, or absent in the striatum, except for some DS cases. These data suggest that the deposition of the carboxy-terminal fragment of A beta truncated at the position of alpha-secretase cleavage or close to it in diffuse plaques may be brain-region-specific, reflecting either dissimilar processing of amyloid precursor protein or the resolution of early A beta deposits, and may substantially contribute to different progression of beta-amyloidosis in various brain regions.

Loss of nerve cells is a hallmark of the pathology of Alzheimer's disease (AD), yet the patterns of cell death are unknown. By analyzing DNA fragmentation in situ we found evidence for cell death not only of nerve cells but also of oligodendrocytes and microglia in AD brains. In average, 30 times more brain cells showed DNA fragmentation in AD as compared to age-matched controls. Nuclear alterations suggestive of apoptosis were rare in degenerating cells. Even though the majority of degenerating cells were not located within amyloid deposits and did not contain neurofibrillary tangles, neurons situated within areas of amyloid deposits or affected by neurofibrillary degeneration revealed a higher risk of DNA fragmentation and death than cells not exposed to these AD changes.

A total of 66 skin biopsies from persons with Alzheimer's disease (AD) or Down's syndrome (DS) and from persons without AD were used in this study. The age range was from 7 to 89 years. Positive immunoreactivity of skin biopsies to monoclonal antibody 4G8, which is reactive to amino acid residue 17-24 of synthetic amyloid beta protein (A beta), and 4G8-Fab (the antigen-binding fragment of 4G8 IgG, reactive only to amyloid plaque) was observed in the epidermis-dermis junction or the basement membrane of the epidermis and in some blood vessels of the biopsy skins of 13/18 (72%) AD, 9/10 (90%) DS, and 14/38 (37%) non-AD control cases. The Fisher exact probability test revealed a significant difference (P = 0.0415 one-tailed) in immunoreactivity between AD and age-matched controls. There was also a significant difference (P = 0.0152 one-tailed; P = 0.0200 two-tailed) between DS and age-matched control in the same test. Immuno-gold electron microscopy examination of these cases with positive immunoreactivity revealed that the gold particles were deposited along the basement membrane of the epidermis. Amyloid fibrils were not observed in the regions with gold particles. Results of this study suggest that A beta is associated with the basement membrane of skin and is present in amorphous, non-fibrillar form as soluble A beta.

In dogs under 17 years of age no amyloid deposits were observed in the claustrum. After that age amyloid deposits were found in all the brains studied. The number of amyloid deposits increases rapidly between 17 and 19 years of age. In none of the animals were neurofibrillary tangles found. However, in almost all claustra with amyloid deposits the number of neurones was smaller than in those without amyloid pathology. The loss of neurones resulted in a decrease in the volume of claustrum, which is about 22% smaller in dogs over 17 years of age than in younger animals. No considerable age-related differences were found in the average size of neurones and their nuclei.

Aluminum, the third most common element in the earth's crust (second to oxygen and silicon) and recently suspected by some investigators to be implicated in Alzheimer disease etiology, has been studied in relation to its effect on mitogenesis, mitosis, and cell cycle. We have observed that 2–4 mM concentrations of AlCl3 have decreased the number of cells that undergo mitogenesis (PHA-induced blast transformation) and mitosis in human short term whole blood cultures. We have also shown that the rate of the cell cycle was slowed down, i.e., cell cycle time was increased in the presence of AlCl3. Also, we have demonstrated a reversible effect on aluminum-induced reduced mitotic index in long-term EBV-transformed lymphoblastoid cultures. Although safeguards such as limiting aluminum serum concentrations have been recommended to protect individuals undergoing dialysis, it should be realized that concentration accumulations of aluminum may increase over chronic exposures. Accordingly, if the number of cells stimulated by PHA is reduced in the presence of AlCl3, there may be a reduction of immune competence, since the degree of PHA stimulation has been used as an indicator of immune response. Similar reductions in mitotic index could affect every tissue involved with cell division. Although it may not be the same for higher concentrations, from our results, we have also shown that decreased mitotic rates were reversible in long-term EBV-transformed lymphoblastoid cultures. Increased numbers of mitoses were observed in human short-term whole blood cultures that were exposed to 2 μM concentrations of aluminum chloride. The concentration is close to those found in normal human serum and within the “safeguard” range recommended for dialysis patients. A similar trend for aluminum sulfate was also observed, while preliminary results for three other aluminum species, lactate, citrate, and maltol, were also reported. Although previous reports have indicated a positive effect of aluminum on mitosis in vitro or in vivo, this is the first such report involving human material. It is clear that higher concentrations of aluminum chloride at 2.0–4.0 mM reversibly inhibit mitosis while more dilute concentrations of 1–2 μM, closer to those found in normal serum, enhance mitosis. The present results, as well as those in the literature, suggest that aluminum may be an essential element in cellular processes for optimal growth, development, and health maintenance. Future research will further test this hypothesis.

The clinical histories of 102 schizophrenics who died at 70 years of age or older were reviewed. The incidence of neurofibrillary tangles (NFTs) was two times higher in the patients who received (74%) than in those who did not receive (36%) treatment with neuroleptics. The development of NFTs started earlier in the treated group. Further studies comparing brains of nine schizophrenics (average age, 86 years) who did not receive treatment with neuroleptics and seven age-matched cases who received neuroleptics, both with neurofibrillary pathology and neuritic plaques, showed characteristic differences. The numerical density of NFTs was slightly greater in the cornu Ammonis (CA1 and CA2) and subiculum of treated patients. Significantly lower numerical density and lower percentage of pretangles (stage 0) and early and mature tangles (stages 1 and 2) and increased number of end-stage tangles (stage 3) were found in the CA, subicular complex, and cerebral cortex of the treated group. These changes suggest accelerated neurofibrillary degeneration in neurons. A significant increase in the numerical density of tau-1-positive plaques was observed in sector CA1 of the CA (from 0.15/mm2 to 17.36/mm2), subiculum (from 0/mm2 to 16.62/mm2), temporal cortex (from 0.14/mm2 to 9.46/mm2), and occipital cortex (from 0.08/mm2 to 0.39/mm2). The higher numerical density of tau-1-positive plaques, but not of 4G8-positive plaques, indicates acceleration of neurofibrillary changes in the plaques of patients treated with neuroleptics. The significant decrease (20-25%) in the numerical density of neurons in the pyramidal layer of sectors 2-4 in the CA appears to be associated with accelerated neurofibrillary changes in neurons and plaques in the treated group. This study demonstrates that chronic treatment with neuroleptics--not schizophrenia itself--significantly increases the risk of more frequent, earlier, and accelerated development of neurofibrillary pathology in the brains of elderly schizophrenics.

Meningeal and cerebral blood vessels were studied in Alzheimer's disease (AD), Down's syndrome (DS), and control brain specimens. Serial sections were double-and triple-immunostained for β-protein, smooth muscle cells, and leukocytes or macrophages/monocytes. At early stages of amyloidogenesis, β-protein immunoreactive material is present in vascular tunica media in the cytoplasm of myocytes or extracellularly between smooth muscle cells. The sites of β-protein deposition exhibit increased immunoreactivity for C-terminal βPP, but not for N-terminal βPP. The cells around small β-protein deposits express actin, myosin, and vimentin but most lack another smooth muscle-specific protein—desmin. These muscle cells often have swollen nuclei and express the proliferating cell nuclear antigen. At more advanced stages of amyloidosis, the tunica media is replaced by amyloid deposits with only scanty smooth muscle cells. Macrophages and leukocytes are not present at the sites of amyloid formation. The results indicat...

We measured beta 2-microglobulin (beta 2-M), soluble interleukin-2 receptor (sIL-2R), and soluble CD8 (sCD8) antigen levels in paired cerebrospinal fluid (CSF) and sera from patients with subacute sclerosing panencephalitis (SSPE), multiple sclerosis (MS), and other neurological diseases (OND) using enzyme-linked immunosorbent assay. beta 2-M was significantly increased in CSF of the SSPE group compared to the MS or the OND group. Similarly, beta 2-M in the MS versus OND group was significantly increased in CSF. Although serum levels of beta 2-M were similar in the three groups, the CSF/serum ratios were higher in SSPE versus the MS group and in the MS versus the OND group. Levels of sIL-2R and sCD8 were higher in SSPE CSF than OND CSF; however, there were no differences between levels in SSPE and MS CSF. The levels of sIL-2R were increased in SSPE sera compared to those of MS or the OND group, whereas levels of sCD8 in serum from the three groups were similar. The findings of increased CSF/serum ratio of beta 2-M and higher levels of serum sIL-2R and CSF sCD8 in SSPE patients are consistent with those seen in patients with acute and chronic viral infections. When the levels between the initial and follow-up CSF and serum samples from SSPE patients were compared, the data showed that CSF levels of sCD8 elevated during periods of clinical worsening and decreased during clinical improvement. In contrast, serum beta 2-M decreased during periods of worsening and increased during improvement. The measurement of serum beta 2-M and CSF sCD8 may be useful in SSPE patients as markers to monitor disease activity.

Alzheimer neurofibrillary tangles (NFT) and senile plaque core amyloid (SPCA) isolated from the brain of patients with Alzheimer's disease were freeze-dried and replicated with a new platinum-carbon (Pt-C) vertical deposition method for high-resolution transmission electron microscopy (TEM). The resolution of this vertical Pt-C replication method is superior to either of the more conventional 20 degrees rotary replication or 45 degrees unidirectional replication methods and is dependent on the Pt-C film thickness coating the specimen. The paired helical filaments (PHF) observed within the tangles were right-handed helices with a fairly regular twist period averaging 79.3 +/- 5.9 nm and a fairly regular maximum width averaging 14.9 +/- 1.0 nm. The PHF regions of minimum width were not regular and fell into three size categories: 2.4 +/- 0.3 nm, 4.9 +/- 0.6 nm and 9.6 +/- 1.4 nm. In addition to the PHF found in the tangles, a new filament was found within all the tangles. These 2.1 +/- 0.2 nm diameter filaments were triple-stranded left helices with 1.0 +/- 0.2 nm diameter strands with a structure identical to bovine tau. Like bovine tau polymer a number of filaments (130 nm to 238 nm) were longer than a fully stretched tau monomer of 96 nm. Images of neuritic senile plaque core amyloid (SPCA) showed that amyloid had a more solid appearance than the NFT and its branched filament structures were unlike the approximately 2.1 nm diameter filaments or the PHF found in NFT.

To explore the utility of cultured skin fibroblasts in investigating diseases of the nervous system in which constituents characteristic of neurons are involved, sensitive immunochemical methods were used to test for the presence in skin fibroblasts of low amounts of proteins normally used as neuronal markers. The presence of each of the neurofilament triplet proteins and of neuron-specific enolase was demonstrated by immunoblotting and by immunocytochemistry, and of an 86-kDa synapsin-like material by immunoblotting. These observations agree with previous suggestions that readily available cultured fibroblasts may be useful in investigations of disorders in which molecules are involved which are typically associated with neurons in vivo, such as Alzheimer's disease.

Summary Point mutations in codon 331 of mitochondrial NADH dehydrogenase subunit 2 (ND2) were detected in 10 of 19 Alzheimer's brains but not in 11 normal brains. The same mutations were also detected in 2 of 6 patients with amyotrophic lateral sclerosis (ALS). However, neurofibrillary tangles and neuritic plaques characteristic of Alzheimer's disease were found histologically in the brain of one ALS patient who was positive of the mutation. The finding suggests that a point mutation in ND2 is a potential risk factor for Alzheimer's disease.

One characteristic of Alzheimer's disease (A beta disease) is the accumulation of amyloid deposits within the extracellular space of the brain and meninges. A 40 amino acid peptide called beta-peptide or A4 protein is the subunit of the amyloid fibrils found in these deposits. The sequence of beta-peptide is contained within those of a family of larger proteins called the Alzheimer beta-amyloid peptide precursor (APP). These APPs contain, in addition to a signal sequence, a hydrophobic sequence that is believed to span cell membranes. Although biochemical studies indicate that some APPs have properties of integral membrane proteins, morphological confirmation of this has not been reported. We recently described an expression system in which human APP751 cDNA was placed under the transcriptional regulation of the polyhedrin gene promoter in the baculovirus Autographica californica infecting a Spodoptera frugiperda cell line (Ramakrishna et al., Biochem Biophys Res Commun 174:983-989, 1991). As part of a larger biochemical and molecular biological study of APP, we have carried out an immunocytochemical study using antibodies directed against several epitopes within APP to reveal, at both the light and the electron microscopic levels, the cellular localization of APP in the baculovirus expression system. These studies demonstrate that APP751 is abundantly synthesized and inserted into certain of the membrane compartments of the cell. As early as 24 hr postinfection, APP751 is found associated with all membrane compartments excepting mitochondrial membranes. The patterns of immunolabeling are consistent with our biochemical findings that the protein is processed in these cells so as to release the extracellular domain and to retain a transmembrane and intracellular segment. These data provide the first morphological demonstration of the membrane location of APP751, its posttranslational processing to a secreted fragment, and its exclusion from the mitochondrial membranes. This system is especially valuable for identifying conditions under which antibodies raised against APP or appropriate synthetic peptides will react with native APP.

Brain and spinal cord blood vessels from mice subjected to chronic relapsing experimental allergic encephalomyelitis were examined by scanning (SEM) and high-voltage electron microscopy (HVEM). SEM analysis of veins and venules from affected tissue regions demonstrated inflammatory cells (ICs), primarily lymphocytes or monocytes, attached to the luminal endothelial cell (EC) surface adjacent to the junctional complexes. In transverse section these cells were shown by HVEM to extend and to insert filopodia (lymphocytes) or flap-like lamellapodia (monocytes) into the luminal EC surfaces. Affected ECs often expressed increased microvillar projection as well as parajunctional crater-like structures on their luminal surfaces. Based on scanning and high-voltage electron microscopy, we present morphological evidence that some populations of sensitized ICs do not penetrate the EC junctions initially during EC attachment but instead insert pseudopodial projections into specialized openings in the ECs that are formed in response to chronic inflammation.

Amyloid beta-protein (A beta), the major protein of cerebrovascular and plaque amyloid in Alzheimer disease, is considered a primary factor in the pathology of this disease. The effect of synthetic A beta (1-40) on the activity of protein kinase C (PKC) was studied with histones for a substrate in a mixed micellar assay, and with calmodulin-depleted soluble brain proteins in a liposomal system. We report here that A beta affects PKC activity in a biphasic manner. An initial stimulation of PKC was noted at low concentrations of A beta (less than 2.5 microM); while PKC-inhibition was observed in a concentration-dependent manner at higher concentrations of A beta. The in vitro phosphorylation of 20, 47, and 87 kDa brain proteins (known PKC substrates) was significantly reduced by 60 microM A beta. The role of 20 kDa in memory storage, of 87 kDa in neurotransmission and neurosecretory processes, and of 47 kDa in long-term potentiation or memory is well recognized, and A beta is known to have both neurotrophic and neurotoxic effects. Since PKC plays an important role in neuronal function, it is suggested that dual modulation of PKC by A beta may be linked to its neurotrophic and neurotoxic effects. We propose that at low concentrations A beta, by stimulating PKC, may contribute to neurites generation; and at higher concentrations A beta, by inhibiting PKC activity, might lead first to memory impairment, and then to neuronal loss.

Neurofibrillary changes are characteristic of the brains of individuals with Alzheimer''s disease (AD) and of those with Down syndrome. Neurofibrillary tangles (NFT) are also seen in small numbers in the brains of normal old people. They also occur in subacute sclerosing panencephalitis (SSPE), a fatal inflammatory disease induced by measles virus. The aim of this study of 44 autopsy cases of SSPE was to estimate the frequency of NFT in this subpopulation, their topography and distribution, as well as the stage of NFT maturation. Nine specimens from the cortex, hippocampus, mesencephalon, and pons of 38 brains and 6 specimens from the cortex of six other brains were used. We used silver impregnation (Bodian-PAS and Bielschowsky techniques), thioflavine S, and immunoreactivity with monoclonal antibodies (mAbs) to tau-1, paired helical filaments (PHF)/ubiquitin 3–39, GFAP, and beta-amyloid protein 4G8. NFT were found in 8 cases (18.2%). Their number was extremely variable individually. In 5 cases, morphometric studies were performed. The average number of pretangle- and NFT -positive neurons in the brain structures examined ranged from 1.1/mm2 in the case with the shortest duration of disease (27 months) to 43.8/mm2 in the case with the longest duration (14 years). In spite of the young age (9–31 years) of the patients with SSPE, the number of NFT in some cases was comparable to the number observed in AD. In 5 cases, NFT were present in the hippocampus as well as in the entorhinal, temporal, and insular cortices. In 6 cases, the frontal, and in 4 cases, the parietal and occipital cortices were involved. About 25% (SE= 4.42) of the abnormal neurons in the cortex constituted pretangle neurons with granular, argentophilic material that is tau-1 positive, but PHF/ubiquitin 3–39 negative. Another 27.4% were early tangles (stage 1) (SE = 0.82). Mature tangles (stage 2) were more numerous and constituted 47.55% (SE = 3.93) of all tangles. Ghost tangles (stage 3) were not found.