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.

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 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.

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.

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.

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.

Neuronal and microvascular alterations induced by the cholinergic toxin AF64A in the rat retina.

The choline analogue ethylcholine mustard aziridinium ion (AF64A) produces both neuronal and non-neuronal alterations in the rat retina. The possible involvement of the retinal capillaries in the origin of the apparently non-specific lesions has been investigated. Two hours after a single intraocular injection of 5 nmol AF64A, ultrastructural alterations were observed in neurons of the inner nuclear layer and the ganglion cell layer, where cholinergic cells are located. One week later, the number of cholinergic neurons, identified by choline acetyltransferase immunohistochemistry, was decreased to 65% of control, the neurons located in the inner nuclear layer being more sensitive than those in the ganglion cell layer. The same dose of AF64A also induced ultrastructural changes in retinal capillaries, which showed a significant increase in the number of pinocytotic vesicles and microvilli in the endothelial cells, 2-5 h after the toxin administration. One day later, arterioles and capillaries presented contracted profiles and the lumen was occasionally lost. The sensitivity of endothelial cells to the toxic effects of AF64A may be explained by the presence in the cerebral endothelium of a choline transport mechanism with an affinity close to that of cerebral synaptosomes. In vitro, both neuronal and endothelial choline uptake systems were equally sensitive to the toxin inhibitory effect. The early and severe vascular alterations induced in the retinal microvessels by AF64A may produce changes in blood perfusion and capillary permeability that could account for the apparently non-specific histological damage.

Anterograde axoplasmic transport of peroxidase in the rat visual system after intraocular neurotoxin injection.

The effect of intraocular injection of the neurotoxin, kainic acid, on the retinofugal pathway has been examined by anterograde transport of horseradish peroxidase. Although less peroxidase was present in all the primary optic centers after the intraocular injection of kainic acid than in the controls, the area occupied by the retinal terminals remained fairly constant except for the pretectal region where there is a smaller area of peroxidase precipitate. These results suggest that certain ganglion cells are killed by kainic acid but some others survive and their terminals are present in the majority of the terminal fields.

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 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.

Biochemical and histological modifications of the rat retina induced by the cholinergic neurotoxin AF64A.

Intraocular injections of ethylcholine mustard aziridinium ion (AF64A) in the rat depressed retinal choline acetyltransferase (ChAT) activity in a dose-dependent manner without any significant change in the content of amino acid neurotransmitters GABA, glycine, aspartate and glutamate. ChAT reduction was already detected 24 h after the injection and persisted for at least one month. In vitro AF64A also inhibited retinal ChAT activity. No changes in muscarinic receptor sites were detected. The histological study showed light cells, characterized by cytoplasmic swelling in the innermost part of the inner nuclear layer and in the ganglion cell layer. We suggest that these light cells are the cholinergic retinal neurons affected by the toxin. In addition, dark cells in the inner nuclear layer, large empty spaces in the outer nuclear layer, inflammatory infiltrate and vascular alterations were also observed in treated retinas. Choline uptake systems in photoreceptors and in endothelial cells or cholinergic perivascular nerve endings may explain the lesions observed in the outer nuclear layer and the vascular alterations.

Kainic acid prevents peroxidase labeling of retinal ganglion cell bodies in the rat: a possible gate in retrograde axonal transport.

Kainic acid, a neurotoxic analogue of glutamate, injected into the vitreous body of the eye, interferes with the retrograde transport of the marker enzyme horseradish peroxidase. This interference is expressed by the absence of detectable peroxidase in the cell bodies of the retinal ganglion cells and by the presence of the marker in the intraretinal portions of the optic axons. These results suggest the hypothesis of the existence of some kind of gate at the proximal portion of the axons which would control the entry of retrogradely moving material into the cell body.

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.

Taste buds in vallate papillae grafted to the anterior chamber of the eye.

Vallate papillae from rats were grafted to the anterior chamber of the eye of the same animal. Longterm grafts showed small taste buds located in epitelial cavities which were probably the remnants of trench walls. These grafts have been reinnervated by local nerves. Excretory ducts and serous acini of Von Ebner's glands were also present. The latter showed secretory granules in their cells. The specificity of the nerve fibers and of the epithelium in relation to the induction of taste bud differentiation are discussed.

Visual evoked cortical potentials in the cat after retinal modifications induced by intraocular kainic acid.

Intraocular injections of kainic acid (KA) induce changes in visual-evoked cortical potentials in the cat at the level of the primary and secondary wave complexes. These changes are correlated with histological findings that show degeneration of cells in the inner nuclear layer and ganglion cell layer. These results indicate that despite the cell death produced by KA, certain retinal circuits continue to function. The fact that the first modifications induced by KA are observed at the level of the primary complex agrees well with the proposed retinal origin of this complex. The modifications of the secondary complex may imply a different transmission and/or processing of visual information at the central level.

The fine structural effect of sialectomy on the taste bud cells in the rat.

Taste buds in the rat and other mammals share a secretory activity with their transduction function as taste receptor. The present work shows the effect of bilateral removal of the main salivary glands on taste bud cells' components related to secretion in the vallate papilla of the rat. In the sialectomized rats remarkable changes were evidence in the dark and intermediate types of taste bud cells, which are known to be the secretory components. Such changes involve hypertrophy of either the protein synthetizing machinery, the smooth endoplasmic reticulum or the Golgi complex. Lucent and coated vesicles associated to Golgi cisternae increased in number but the amount of dense-core vesicles (secretory vesicles) at the apical cytoplasm of cells decreased. Images of exocytosis of secretory products were observed. The hypertrophy of Golgi complex components was clearly detected with the OsO4 impregnation method for light and electron microscopy. Alteration in the acid phosphatase activity of taste bud cells was not observed in the sialectomized rats. These findings suggest that sialectomy stimulates the entire secretory cycle of dark and intermediate taste bud cells. The light taste bud cells, which are not engaged in secretion, are hardly affected by the treatment. Although taste buds in mammals are neuro-dependent structures, present evidence indicates that they are also sensitive to non-neural influences.

[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.

[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 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.