H. M. Wisniewski

Cell death in Alzheimer's disease evaluated by DNA fragmentation in situ.

Loss of nerve cells is a hallmark of the pathology of Alzheimers 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.

Abnormal fibrils from scrapie-infected brain

SummaryAbnormal fibrillary structures, designated “scrapie-associated fibrils” (SAF), have been observed using negative stain techniques in subfractions of brains from scrapie-affected animals. SAF have been observed in all combinations of strain of scrapie agent and strain or species of host examined, regardless of their histopathology, in particular the presence or absence of amyloid plaques. SAF consist either of two or four filaments. They are morphologically dissimilar to the normal brain fibrils — microtubules, neurofilaments, glial filaments, and F actin. However, SAF do bear a resemblance to amyloid.

Adult fragile X syndrome

SummaryFragile X syndrome [fra (X)] is currently accepted as the second most frequent chromosomal disorder associated with developmental disability. Although next to Down syndrome in frequency, no postmortem studies of confirmed adult cases had been reported.The autopsy examination of a 62-year-old, moderately retarded man with the fra (X) syndrome confirmed the preferential involvement of cerebral and testicular structures in this disorder.Dendritic spine abnormalities of the type observed in trisomic chromosomal disorders were associated with synaptic immaturity. Severe testicular hypogonadism accompanied bilateralmacro-orchidism, normal penis, and unilateral hydrocele. Valvular, articular, and testicular interstitial compartments showed normal histochemical staining characteristics for glycoproteins and lipids.

Brain aluminum in aging and Alzheimer disease.

Aluminum was assayed by atomic absorption spectroscopy in 274 brain samples, and assayed in neurons isolated in bulk from the frontal cortex of patients with Alzheimer dementia and from age-matched patients with no neurologic disease. Brain aluminum concentration increased with age, from late middle age to old age. There were no statistically significant differences in brain aluminum concentration between the 10 patients with Alzheimer disease (mean, 2.7 μg per gram dry weight of tissue; mean age, 81 years), and the 9 nonneurological controls (mean, 2.5 μg per gram; mean age, 73 years). In both groups, the hippocampus had the highest concentration of aluminum (5.6 μg per gram), and the corpus callosum the lowest (1.5 μg per gram).

Ultrastructure of the microglia that phagocytose amyloid and the microglia that produce β-amyloid fibrils

SummaryThe function of microglia associated with β-amyloid deposits still remains a controversial issue. On the basis of recent ultrastructural data, microglia were postulated to be cells that form amyloid fibrils, not phagocytes that remove amyloid deposits. In this electron microscopic study, we examined the ability of microglia to ingest and digest exogenous amyloid fibrils in vitro. We demonstrate that amyloid fibrils are ingested by cultured microglial cells and collected and stored in phagosomes. The ingested, nondegraded amyloid remains within phagosomes for up to 20 days, suggesting a very limited effectiveness of microglia in degrading β-amyloid fibrils. On the other hand, we showed that in microglial cells of classical plaques in brain cortex of patients with Alzheimers disease, amyloid fibrils appear first in altered endoplasmic reticulum and deep infoldings of cell membranes. These differences in intracellular distribution of amyloid fibrils in microglial cells support our observations that microglial cells associated with amyloid plaques are engaged in production of amyloid, but not in phagocytosis.

Ultrastructural Studies of the Cells Forming Amyloid Fibers in Classical Plaques

Three-dimensional reconstruction and ultrastructural studies of classical plaques from the cortex of patients with Alzheimers disease showed that microglial cells of the plaques are the amyloid-forming cells. The amyloid star of the single plaque represents the product of five or six microglial cells covering about 80% of the amyloid star surface. The amyloid fibers appear to be formed within altered cisterns of the endoplasmic reticulum. Distended cisterns form channels filled with amyloid fibers. Numerous vesicles derived from the Golgi apparatus appear to be attached to or fused with the amyloid-filled channels. Reconstruction of the amyloid star and the microglia cell pole that forms the amyloid star reveals three different zones of distribution of cytoplasmic organelles and amyloid deposits. The peripheral zone comprises channels filled with loosely packed amyloid fibers arranged in a parallel manner. The transient zone consists of a mixture of fusing amyloid channels and products of disintegration of cytoplasmic pockets, dense bodies and fragments of cellular membranes. The core of the amyloid star is composed of condensed, densely packed amyloid fibers that are free of cellular debris. Formation of the three zones supports the idea that the microglia/macrophages are not phagocytes but instead are the cells manufacturing the amyloid fibers.

Alzheimer paired helical filaments: Bulk isolation, solubility, and protein composition

SummaryA method has been developed for the bulk isolation of Alzheimer neurofibrillary tangles (ANT) of paired helical filaments (PHF) from histopathologically confirmed cases of Alzheimer disease/senile dementia of the Alzheimer type (AD/SDAT). The fresh or frozen autopsied cerebral cortex affected with Alzheimer neurofibrillary changes is dissociated by homogenization and sieving through nylon bolting cloth and the ANT are separated by a combination of sucrose discontinuous density gradient centrifugation, glass bead column chromatography, and sodium dodecyl sulfate (SDS) treatment. The isolated ANT produce red-green birefringence when viewed through polarized light after staining with Congo red. Ultrastructurally, the isolated PHF are well preserved and have the dimensions of the PHF seen in situ. Two major Populations of ANT which exist in different proportions in AD/SDAT brains are identified on the basis of their solubility in SDS. The ANT I and the ANT II are soluble and insoluble respectively on treatment with 2% SDS at room temperature for 5 min. Solubilization of the ANT II requires several repeated extractions with a solution containing 10% each of SDS and β-mercaptoethanol (BME) at 100°C for 10 min. Sonication of the ANT II greatly facilitates their solubilization. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated ANT reveals the presence of two major polypeptides with molecular weights (MW) of 62,000 and 57,000, several minor polypeptides with MW below 57,000, and a significant amount of material not entering the stacking and the resolving gels. Re-electrophoresis of polypeptides extracted from various areas of the resolving gel or of the material which does not enter the gel generates the same polypeptide profile as on the first gel, suggesting that the PHF material which does not enter the gel may result from the reaggregation of the polypeptides that enter the resolving gel. None of the polpeptides observed in the isolated PHF comigrate in the SDS-PAGE with any of the neurofilament polypeptides, tubulin, actin, or myosin.

Ultrastructural studies of the cells forming amyloid in the cortical vessel wall in Alzheimer's disease

SummaryUltrastructural studies of serial sections of the vessels with amyloid deposits in the brain cortex of patients with Alzheimers disease showed that cells in the position of pericytes — perivascular cells - and perivascular microglial cells are producers of amyloid fibrils in the vascular wall. Three types of changes from normal are distinguishable in the vessel wall: (1) semicircular or circular thickening of vascular wall containing a large amount of amorphous material and various number of amyloid fibrils, (2) tuberous amyloid deposits containing both amorphous material and amyloid fibrils, some of the fibrils being arranged in strata and others arranged radially, and (3) amyloid star composed of a predominantly radial arrangement of bundles of amyloid fibrils and a less prominent amorphous component. A mixture of amorphous material and amyloid fibrils is present in cell membrane envaginations of perivascular cells, and occasionally perivascular microglial cells. Bundles of amyloid fibrils are found in altered cisternae of the endoplasmic reticulum and in the channels confluent with the infoldings of the plasma membrane of perivascular microglial cells. The amyloid deposition in the wall of the vessel causes degeneration of endothelial cells and the reduction of, and in some vessels obliteration of, the vessel lumen. In areas affected by amyloid angiopathy, extensive degeneration both of the neuropil and of neurons was observed. These changes were accompanied by astrogliosis. This study demonstrates similarities in amyloid formation in amyloid angiopathy and in β-amyloid plaques in the neuropil and suggests that cells of the mononuclear phagocyte system of the brain (perivascular cells and perivascular microglia) are engaged in amyloid fibril formation.

Spectrum of morphological appearance of amyloid deposits in Alzheimer's disease

SummaryImmunocytochemical staining with monoclonal antibodies to the β-protein on tissue sections which have been pretreated with formic acid is not only a very specific but also a highly sensitive method for the detection of amyloid deposits in the brains of Alzheimers disease victims. We report here a spectrum of morphological appearance of the brain amyloid deposits which are one of the main histopathological correlates of this disorder. Deposits of the β-protein are not only found in the well-known lesions [congophilic angiopathy and senile (neuritic) plaques] but are also seen under various morphological forms for which the word “plaques” does not appear an appropriate term: amyloid fibrils are found as large areas of diffuse infiltration of the neuropil, as ribbon-like infiltration in the subpial layer of the cerebral cortex, as granular deposits in the white matter, as diffuse deposits in the molecular layer of the cerebellum and the basal ganglia and as star-shaped deposits in the cerebellar Purkinje cell layer. The morphology of these deposits seems to depend on the cyto-and fibroarchitectonics of the brain region in which they are found, on the amount of amyloid deposited, and also on the type of staining technique used. It is only under specific circumstances that the deposition of amyloid in the neuropil is accompanied by the formation of paired helical filaments in nerve cell processes and their parent perikarya. In conclusion, our studies suggest that the extent of brain amyloidosis in Alzheimers disease is much wider than so far appreciated.

Relationships between regional neuronal loss and neurofibrillary changes in the hippocampal formation and duration and severity of Alzheimer disease

The total numbers of neurons with and without neurofibrillary changes in the hippocampal subdivisions were estimated in 16 subjects with Alzheimer disease (AD) and in 5 normal elderly controls. On the basis of clinical symptoms, AD patients were subdivided into relatively less (AD-1, Functional Assessment Staging [FAST] stages 7a to 7c) and more severely affected (AD-2, FAST stages 7e to 7f) patient groups. In the AD-1 group relative to controls, the total number of neurons was reduced only in CA1 and in the subiculum. In the AD-2 group, neuronal losses were found in all sectors of the cornu Ammonis and in the subiculum and ranged from 53% in CA3 to 86% in CA1. The dentate gyrus was the only hippocampal subdivision without significant neuronal loss. Within the combined AD patient groups, significant correlations were noted between both clinical stage and duration of AD and both the total number of neurons and the percentage of neurons with neurofibrillary changes in CA1, CA4, and the subiculum. Regression analyses predicted neuronal losses over the maximal observed duration of 22 years of 87% in CA1, 63% in CA4, and 77% in the subiculum. Our data suggest that over the course of AD, continuous neurofibrillary tangle formation and continuous neuronal loss occur in the hippocampal subdivisions. The rate of neuronal loss appears to be similar for CA1, CA4, and the subiculum.