Ultrastructural localization of fructose-1,6-bisphosphatase in mouse brain.

Fructose-1,6-bisphosphatase has been studied in adult mouse brain of different ages using an antibody directed against the liver isoform. The presence of liver fructose-1,6-bisphosphatase in cerebellum, cerebral cortex, and hippocampus was assayed using Western blot and different immunocytochemical techniques. Immunocytochemistry with peroxidase reaction product was used to locate fructose-1,6-bisphosphatase in both neurons and astrocytes in the same areas, as well as in the rest of the brain, at light and electron microscope levels. Double immunofluorescence with neuronal or astrocytic markers confirmed the neuronal and astrocytic location of fructose-1,6-bisphosphatase in confocal microscope images. At the subcellular level, fructose-1,6-bisphosphatase was located in the nuclear and cytoplasmic compartments of both neurons and astrocytes, at all ages studied. Ultrastructurally, immunostaining appeared as small patches in the nucleus and the cytosol. In addition, immunostaining was present over the outer mitochondrial membrane, the plasma membrane, and the membranes of the rough endoplasmic reticulum and nuclear envelope, but not over Golgi membranes. In the neuropil fructose-1,6-bisphosphatase was located in dendritic spines, as well as in abundant astrocytic processes that, in some cases, surrounded immunopositive synapses. The possible role of fructose-1,6-bisphosphatase in neurons and astrocytes is discussed.

Neuroimaging studies in Alzheimer's disease. A review.

Alzheimer's Disease (AD) is a progressive neurodegenerative disease associated with memory loss and gradual behavioral, functional and cognitive impairment. Conventional imaging studies, such as magnetic resonance or computed tomography have played a secondary role in AD diagnosis: While other causes of memory loss and cognitive deficit can be evaluated by these imaging methods, AD structural changes are not detected until very late in the course of the disease. Recent and more precise techniques have been developed to detect subtle changes not visualized with those imaging methods. This article presents a review of the neuroimaging techniques used as a diagnostic aid for AD.

Hippocampal neuronal subpopulations are differentially affected in double transgenic mice overexpressing frontotemporal dementia and parkinsonism linked to chromosome 17 tau and glycogen synthase kinase-3beta.

Glycogen synthase kinase-3beta (GSK-3beta) has been proposed as the main kinase able to phosphorylate tau aberrantly in Alzheimer's disease and in related tauopathies. We have previously generated a double transgenic mouse line overexpressing the enzyme GSK-3beta and tau protein carrying a triple frontotemporal dementia and parkinsonism linked to chromosome 17 mutation whose expression patterns overlap in CA1 (pyramidal neurons) and dentate gyrus (granular neurons). Here, we have used this transgenic model to analyze how axonal and somatodendritic neuronal compartments are affected in the hippocampus. Our data demonstrate that neuronal subpopulations respond differentially to increased GSK-3 activity. Thus, dentate gyrus granular neurons undergo apoptotic death with subsequent degeneration of the mossy fibers, while CA1 pyramidal neurons accumulate hyperphosphorylated tau both in the axonal and in the somatodendritic compartments. These studies also allow us to propose a model of spreading of pathology through the hippocampus as a consequence of GSK-3 and tau dysregulation.

FTDP-17 mutations in tau transgenic mice provoke lysosomal abnormalities and Tau filaments in forebrain.

The tauopathies, which include Alzheimer's disease (AD) and frontotemporal dementias, are a group of neurodegenerative disorders characterized by filamentous Tau aggregates. That Tau dysfunction can cause neurodegeneration is indicated by pathogenic tau mutations in frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). To investigate how Tau alterations provoke neurodegeneration we generated transgenic mice expressing human Tau with four tubulin-binding repeats (increased by FTDP-17 splice donor mutations) and three FTDP-17 missense mutations: G272V, P301L, and R406W. Ultrastructural analysis of mutant Tau-positive neurons revealed a pretangle appearance, with filaments of Tau and increased numbers of lysosomes displaying aberrant morphology similar to those found in AD. Lysosomal alterations were confirmed by activity analysis of the marker acid phosphatase, which was increased in both transgenic mice and transfected neuroblastoma cells. Our results show that Tau modifications can provoke lysosomal aberrations and suggest that this may be a cause of neurodegeneration in tauopathies.

Ubiquitinated granular structures and initial neurofibrillary changes in the human brain.

Ubiquitin-positive dots and granular structures from insular, temporopolar, hippocampal and parahippocampal cortices of nondemented and Alzheimer's disease patients have been studied with both light and electron microscopes. The relationship of both types of ubiquitin-positive elements with pretangle neurons and neurofibrillary tangles has been analyzed by comparing adjacent or nearly adjacent sections immunostained for either ubiquitin or an antibody that recognizes hyperphosphorylated tau protein (AT-8). Moreover, a double protocol with both antibodies was used in order to obtain double-stained sections. The presence of ubiquitin-positive dots and granular structures precedes the appearance of pretangle neurons in the youngest cases. In aged and Alzheimer disease cases, both types of ubiquitin-positive elements decrease in number as pretangle neurons are replaced by mature and ghost tangles. Ultrastructurally, dots and granular structures appear to be degenerating neuronal processes and/or terminals. Our results suggest that the degeneration of these processes and/or terminals might be related with the initiation of neurofibrillary degeneration.

Apolipoprotein E immunoreactivity in neurons and neurofibrillary degeneration of aged non-demented and Alzheimer's disease patients.

Apolipoprotein E (ApoE) genotype is a risk factor for Alzheimer's disease (AD) but its relationship with neurofibrillary degeneration remains obscure. To further analyze this relationship, hippocampal, entorhinal, temporopolar, and insular cortices of 10 non-demented and 7 Alzheimer disease brains were studied with both light and electron microscopy. Focus was directed on pretangles and neurons starting to accumulate tangles. ApoE immunolabeling in neurons and tangles was independent of ApoE individual genotype. The majority of the neurons in all of the brains were ApoE-negative, but virtually every brain also contained groups of ApoE-immunoreactive neurons, with diffuse cytoplasmic labeling. Most of the ApoE-positive tangles were extracellular, but a few tangles were shown to be intraneuronal when studied ultrastructurally. No ApoE immunoreactivity was found in neuropil threads, as well as in neurites associated with senile plaques. Double protocols with both AT-8 and anti-ApoE antibodies, performed to determine whether ApoE-positive neurons were pretangle neurons, did not detect cytoplasmic AT-8 in ApoE-positive neurons. Though careful electron microscopy studies found ApoE reaction product in an occasional ApoE-positive pretangle-like neuron and a few intracellular tangles, these findings do not support that ApoE is necessary for the accumulation of hyperphosphorylated tau protein. The more consistent colocalization of anti-ApoE and AT-8 in extracellular tangles reveals that ApoE mainly binds to tangles once they are in the extracellular space, in a manner similar to that described for amyloid fibrils.

Decreased nuclear beta-catenin, tau hyperphosphorylation and neurodegeneration in GSK-3beta conditional transgenic mice.

Glycogen synthase kinase-3beta (GSK-3beta) has been postulated to mediate Alzheimer's disease tau hyperphosphorylation, beta-amyloid-induced neurotoxicity and presenilin-1 mutation pathogenic effects. By using the tet-regulated system we have produced conditional transgenic mice overexpressing GSK-3beta in the brain during adulthood while avoiding perinatal lethality due to embryonic transgene expression. These mice show decreased levels of nuclear beta-catenin and hyperphosphorylation of tau in hippocampal neurons, the latter resulting in pretangle-like somatodendritic localization of tau. Neurons displaying somatodendritic localization of tau often show abnormal morphologies and detachment from the surrounding neuropil. Reactive astrocytosis and microgliosis were also indicative of neuronal stress and death. This was further confirmed by TUNEL and cleaved caspase-3 immunostaining of dentate gyrus granule cells. Our results demonstrate that in vivo overexpression of GSK-3beta results in neurodegeneration and suggest that these mice can be used as an animal model to study the relevance of GSK-3beta deregulation to the pathogenesis of Alzheimer's disease.

Ultrastructural aspects of neurofibrillary tangle formation in aging and Alzheimer's disease.

Neurofibrillary tangles, one of the neuropathological signs of Alzheimer's disease, are frequently present in brains of aged nondemented people. Ultrastructurally, neurofibrillary tangles appear as paired helical and straight filaments. Both types of filaments, made of hyperphosphorylated tau protein, are present in neurons with neurofibrillary tangles. Neurons with neurofibrillary tangles have been described to undergo an evolution, starting with the accumulation of hyperphosphorylated tau, followed by the progressive appearance of both types of filaments, and ending in the death of the neuron. We ultrastructurally studied this evolution, using immunocytochemistry with an antibody against phosphorylated tau protein, in both nondemented aged and Alzheimer's disease brains. No differences were found between nondemented and demented brains, thus indicating the occurrence of the same process in both cases. Our results also suggest that hyperphosphorylated tau protein first appears as granular material, which becomes organized into short and disordered paired helical filaments. These filaments elongate and gradually become arranged into bundles whose core regions are occupied by straight filaments.

Association of cholinesterases with amyloid in extracellular neurofibrillary tangles.

Double-labeling techniques conclusively demonstrated that extracellular neurofibrillary tangles immunoreactive for beta protein were also positive for cholinesterases. Ultrastructurally, cholinesterases decorated bundles of amyloid-like filaments, which appeared intermingled with straight filaments and cellular processes. A lighter labeling of esterases was also seen over most straight filaments. These observations extend the consistent association of cholinesterases with fibrillary beta-amyloid protein to a new location in neurofibrillary tangles.

Ultrastructural localization of butyrylcholinesterase in senile plaques in the brains of aged and Alzheimer disease patients.

Histochemical localization of butyrylcholinesterase has been carried out in primitive, perivascular, and classic plaques in the brains of both nondemented and Alzheimer disease (AD) patients. Butyrylcholinesterase histochemistry has been compared to amyloid beta-protein (A beta P) immunocytochemistry in adjacent sections. In small primitive plaques, most of the butyrylcholinesterase reaction product appears ultrastructurally located over plasma membranes of healthy-looking cell processes. In more extensive primitive plaques, butyrylcholinesterase reaction product also decorates amyloid filaments, which become identifiable as delicate wisps. In classic plaques, large aggregates of butyrylcholinesterase reaction product colocalize with bundles of amyloid filaments, as well as with the compact amyloid core. Thus, deposition of butyrylcholinesterase in senile plaques follows a close parellelism with the progressive aggregation of amyloid beta-protein, supporting the possibility that cholinesterases may play some role in the maturation of these structures.

Alzheimer's disease-type neurofibrillary degeneration in verrucose dysplasias of the cerebral cortex.

Verrucose dysplasias, found at autopsy in the cerebral cortex of three elderly individuals (two without neurological disorders and one with motor neuron disease), are shown to present neurofibrillary degeneration of Alzheimer's disease type. This neurofibrillary degeneration immunoreacted with antibodies against abnormally phosphorylated tau (5E2 and AT8), disclosed acetyl- and butyrylcholinesterase activity, and was consistently stained with thioflavin-S. Cortical dysplasias, found either as isolated verrucose nodules or comprising multiple nodules, contained cell-sparse areas around which a peak of neurofibrillary changes was seen. Cell-sparse areas were sometimes bridged by stripes of neurons and fibers arranged in a radial fashion, and many of these neurons showed neurofibrillary degeneration. Cytoskeletal abnormalities were conspicuous in layers II and III at the external borders of the dysplasias, as well as in neurons located in layers V and VI, and in the white matter beneath layer VI in central zones of each lesion. The morphology of cells undergoing neurofibrillary changes (from early non-fibrillar stages to late extracellular ones) suggests that neurons disturbed in their migration toward the site to which they had been committed may become vulnerable to cytoskeletal changes. Micro-environmental disturbances related to hypoxia-ischemia in the affected cortex are proposed as likely contributing factors for the long-term production of this neurofibrillary degeneration.

Microtubule-associated protein MAP1B showing a fetal phosphorylation pattern is present in sites of neurofibrillary degeneration in brains of Alzheimer's disease patients.

Alzheimer's disease results in the appearance of cytoskeletal disorders yielding pathological structures such a neurofibrillary tangles or dystrophic neurites. It has been previously described that the microtubule-associated protein, tau, modified by phosphorylation in serines adjacent to prolines, is a major component of these structures. Here, we show that another microtubule associated protein, MAP1B, aberrantly phosphorylated by a proline-dependent protein kinase, is a component of these previously mentioned structures. Thus, a possible common phosphorylation of axonal MAPs such as tau or MAP1B may correlate with their association with those aberrant cytoskeletal structures present in AD.

Ultrastructural localization of butyrylcholinesterase on neurofibrillary degeneration sites in the brains of aged and Alzheimer's disease patients.

Butyrylcholinesterase histochemical techniques were applied to vibratome sections of several cortical areas from the brains of non-demented aged and of Alzheimer's disease patients. At the light microscope level, all the senile plaque types and all the structures with neurofibrillary degeneration showed butyrylcholinesterase reaction product, whereas nearby neuronal perikarya and axons on the same slides remained unstained. Areas containing stained elements were selected, re-sectioned, and finally observed under the electron microscope. Focusing on the sites of neurofibrillary degeneration, butyrylcholinesterase reaction product was found in both intra- and extracellular neurofibrillary tangles, in neurites associated with plaques, and in neuropil threads that were either axons or dendrites. This reaction product was exclusively located over filament bundles, and sometimes covered them so completely that they could not be identified. When the filaments were only partially covered, it was possible to identify them as either paired helical filaments or straight filaments. Occasionally, neurofibrillary tangles, neuropil threads and plaque-associated neurites, all of them containing either paired helical filaments or straight filaments, were found to be completely free of butyrylcholinesterase reaction product. The origin and possible role of butyrylcholinesterase, which is ultrastructurally localized over elements presenting neurofibrillary degeneration, is discussed.

Cholinesterases colocalize with sites of neurofibrillary degeneration in aged and Alzheimer's brains.

Acetylcholinesterase and butyrylcholinesterase have been associated with structures undergoing neurofibrillary degeneration, as well as with all types of senile plaques, in non-demented aged and Alzheimer's brains. At the electron microscope level, the reaction product of both enzymes, appeared to decorate paired helical filaments, straight filaments and beta A4 amyloid fibrils. Recent studies showed that cholinesterases were associated with amyloid at early stages, e.g., in diffuse plaques. In the present study, the interrelationship of cholinesterases to structures undergoing neurofibrillary degeneration was analyzed further. Tau immunoreactivity was compared to the staining pattern observed with the two esterases. Double protocols consecutively performed on the same sections, and counterstaining with thioflavin-S, confirmed the presence of cholinesterases in all structures with neurofibrillary degeneration. The conclusion that cholinesterases consistently colocalize with both neurofibrillary bundles and beta A4 amyloid fibrils at all stages of their accumulation, allows us to speculate on the possible role that these enzymes may play in either the formation or the consolidation of fibrillary aggregates.

[Importance of neurofibrillary degeneration in the diagnosis of Alzheimer's disease]. / Importancia de la degeneración neurofibrilar en el diagnóstico de la enfermedad de Alzheimer.

For the pathological diagnosis of Alzheimer's disease ADRA-NIH recommend the quantification of plaques, without distinguishing their types, and neurofibrillary tangles. However, diffuse plaques may not be specifically associated to such diagnosis, as suggested by several authors and a previous paper. The neurofibrillary degeneration, present not only in neurofibrillary tangles but also in neuropil threads, either free in the neuropil or associated in plaques, could be more closely correlated with the decrease of cognitive functions than the plaques by themselves. In the present work, the efficiency of cholinesterase histochemical techniques and thioflavin-S is compared to the immunocytochemistry for abnormal tau, in two aspects: 1) the quantification of neurofibrillary degeneration, 2) the distribution of this degeneration, when present, within the plaques. It is concluded that immunocytochemistry for tau is the technique that better fulfills both goals, showing a better agreement with the cognitive state. For that reason tau immunocytochemistry is highly recommended in order to improve the anatomopathological diagnosis of Alzheimer's disease.

Colocalization of cholinesterases with beta amyloid protein in aged and Alzheimer's brains.

The colocalization of beta amyloid protein with the enzymes acetyl- and butyrylcholinesterase was assessed using immunocytochemistry for beta amyloid protein and a sensitive histochemical technique for cholinesterases. In non-demented aged and Alzheimer's disease brains, double-stained sections for cholinesterases and thioflavin-S showed that all thioflavin-S-positive plaques were also positive for cholinesterases, indicating the presence of these enzymes in all plaques with beta-pleated amyloid protein. When amyloid angiopathy was present, cholinesterases were also observed in amyloid-laden vessels walls. Comparison of series of adjacent sections alternatively stained for acetylcholinesterase, beta amyloid protein and butyrylcholinesterase, as well as by double histo-immunocytochemical staining, showed either cholinesterase in a proportion of the preamyloid diffuse plaques. These data indicate that cholinesterases are associated with the amyloid protein from very early stages, when the beta-pleated structure is being formed. Novel functions attributed to acetyl- and butyrylcholinesterase, such us their proteolytic activity either by themselves or in association with heparan sulfate proteoglycans, may play a role in the aggregation or the consolidation processes taking place at the early stages of diffuse plaque formation.

Cholinesterase histochemistry in the human brain: effect of various fixation and storage conditions.

Human brains fixed in 3 conventional solutions were used to compare acetylcholinesterase and butyrylcholinesterase labeling after being processed by the same histochemical procedure. The best fixative was found to be 4% paraformaldehyde in a 0.1 M phosphate buffer for 24-42 h. The addition of 2% glutaraldehyde to 2% paraformaldehyde for the same fixation time reduced the staining intensity of both enzymes. Formalin after 2 days resulted in a lighter staining intensity of fibers, senile plaques and neurofibrillary tangles, whereas after 10 days it caused the near disappearance of acetylcholinesterase-positive fibers. Finally, after more than 10 days in formalin, a certain number of senile plaques and a few neurofibrillary tangles could be observed, and those only in the case in which the substrate concentration and incubation time were increased. The fixed tissue, in either blocks or sections, can be stored for more than 1 year in a glycol solution at -17 degrees C, without the inconvenience of freezing, with good histology and histochemical preservation of both enzymes.

[Diagnosis of Alzheimer's disease. Evaluation of senile plaques of the diffuse type]. / Diagnóstico de la enfermedad de Alzheimer. Valoración de las placas seniles de tipo difuso.

The different types of plaques throughout the thickness of the pole of the temporal lobule of brains from autopsies of a patient with Alzheimer's disease and two elderly patients with normal cognitive function were quantified. Thioflavin-S techniques and cholinesterase marking indicating amyloid aggregates were employed. If the Khachaturian criteria had been followed one of the controls would have also been diagnosed with involvement of Alzheimer's disease. The aforementioned control and patients with Alzheimer's disease had numerous senile plaques, most of which were diffuse, however, only the patient with senile dementia also had neuritic plaques. This fact indicates the convenience of reviewing the pathologic diagnosis of Alzheimer's disease in two ways: firstly, specific techniques for neurofibrillary degeneration should be included and secondly, the plaques with neurofibrillary degeneration should be quantified since diffuse plaques without degeneration appear to have a questionable significance with respect to this diagnosis.

Ultrastructural localization of acetylcholinesterase in neurofibrillary tangles, neuropil threads and senile plaques in aged and Alzheimer's brain.

Acetylcholinesterase (AChE) histochemistry was used to evaluate the accumulation of this enzyme in senile plaques, neurofibrillary tangles and neuropil threads using light and electron microscopy in Alzheimer's disease as well as non-demented aged brains. Under the electron microscope, a crystalline-like AChE precipitate was localized over paired helical filaments and straight filaments in both neurofibrillary tangles and neuropil threads. AChE reaction product also decorated the amyloid fibrils in diffuse plaques as well as the halo and the heavy accumulation of amyloid which forms the core of classical plaques. In both diffuse plaques and the halo of classical plaques, we found AChE-positive structures resembling cell processes, which in some cases appeared to contain amyloid fibrils. The possible origin and significance of AChE localized over paired helical filaments, straight filaments and amyloid is discussed.

Ultrastructural effects of colchicine on perikarya and initial segment of rat retinal ganglion cells.

Our ultrastructural study was focused on the perikaryal region and initial segment of the axon of rat retinal ganglion cells in controls and after intraocular injections of colchicine. In control rats that region contained, among other organelles, elements of the Golgi complex and, close to them, short isolated microtubules oriented preferentially toward the axon where they funnel and aggregate in bundles. One day after sufficient doses of colchicine to inhibit axoplasmic transport (2-20 micrograms) these cytoplasmic microtubules were absent, whereas some axonal microtubules were still present but reduced in number. In addition, colchicine induced an altered distribution of organelles, leaving empty spaces in the periphery and most organelles concentrated in the perinuclear region, especially around Golgi elements where numerous vesicles and tubules accumulate at the trans face of Golgi elements. These results suggest that the vesicles that leave the Golgi and have been directed towards axoplasmic transport may need the cytoplasmic microtubules located between Golgi elements and the axonal initial segments to reach the axon.