A. Defossez

Alzheimer's disease: Tau proteins, the promoting factors of microtubule assembly, are major components of paired helical filaments

A rabbit antiserum was raised against paired helical filaments (PHF). This antiserum stains specifically neurofibrillary tangles (NFT) at the light-microscopic level and PHF at the electron-microscopic level in sections of Alzheimer neocortex and hippocampus. We studied the nature of the antigens recognized by this antiserum by immunocytochemistry, immunoblots and immunoadsorption. These approaches showed that the anti-PHF specifically labels a set of low molecular weight 65-50 kDa microtubule-associated proteins, named Tau proteins, which are promoting factors of microtubule assembly. Furthermore, antisera against Tau proteins stained NFT. It is concluded that neurofibrillary tangles are very likely composed of aggregated Tau proteins. This process might be due to an abnormal Tau protein synthesis or to an unknown lesion of certain pyramidal neurons leading to an aggregation of Tau proteins.

Neuronal-specific expression of human copper-zinc superoxide dismutase gene in transgenic mice: animal model of gene dosage effects in Down's syndrome

It has been suggested that copper-zinc superoxide dismutase (CuZn SOD) increment, by accelerating hydrogen peroxide formation, might promote oxidative damage within trisomy 21 cells and might be involved in the various neurobiological abnormalities found in Downs syndrome such as premature aging and Alzheimer-type neurological lesions. In order to test this hypothesis, we have developed strains of transgenic mice carrying the human CuZn SOD gene. The human transgene expression resulted in increased CuZn SOD activity predominantly in the brain (1.93 fold). Immunohistochemical and in situ hybridization analysis of brain sections revealed that human CuZn SOD protein and mRNA was preferentially expressed in neurons, particularly in pyramidal cells of Ammons horn and granule cells of gyrus dentate. The amount of thiobarbituric acid (TBA)-reactive material was significantly higher in transgenic brains compared to controls, strongly suggesting an increased level of peroxidation in vivo. These results support the notion that CuZn SOD gene dosage effect could play a role in the pathogenesis of rapid aging features in the brain of Downs syndrome patients.

Characterization of two pathological Tau protein variants in Alzheimer brain cortices

Tau proteins were detected in brain tissue homogenates from 10 patients with Alzheimers disease versus 10 age-matched controls using the immunoblot technique and 2 polyclonal antibodies: anti-paired helical filaments (PHF) and anti-human native Tau proteins. In control brains, both antisera detected identically the normal set of Tau proteins, with molecular weight (MW) ranging from 45 to 62 kDa. Moreover, in association areas of neocortex from Alzheimer brains, the antisera detected 2 additional Tau variants of 64 and 69 kDa. Tau 64 and 69 were not found in regions of Alzheimer brains where the Alzheimer pathology was absent (caudate nucleus or cerebellum for example). The heavy MW of Tau 64 and 69 is due to their phosphorylation state as shown by the decrease of their MW after alkaline phosphatase treatment. Therefore, Tau 64 and Tau 69 are specific markers of the Alzheimers disease neuronal degenerating process and their characterization demonstrates that an abnormal phosphorylation of Tau really occurs during the disease. Tau 64 and 69 were isolated with normal Tau proteins while the PHF were insoluble. Therefore, Tau proteins are likely to be abnormally phosphorylated prior to their incorporation in the PHF structure. Consequently, they might appear before the lesions and might be instrumental for the search of biochemical deregulations that precede the neurofibrillary degeneration.

Pathological proteins Tau 64 and 69 are specifically expressed in the somatodendritic domain of the degenerating cortical neurons during Alzheimer's disease

SummaryBundles of paired helical filaments (PHF) accumulate in the pyramidal neurons that degenerate during Alzheimers disease. This neurofibrillary degeneration is highly correlated with clinical signs of dementia. During the degenerating process, Tau proteins, which are the major antigenic components of PHF, are abnormally phosphorylated and two pathological isoforms named Tau 64 and 69 are expressed. We have studied their immunoblot distribution in the cortical gray and white matter from different regions of normal and Alzheimer brains, to determine if the degenerating process preferentially affects the somatodendritic or the axonal domain. Two categories of antibodies were used. The first category consisted of anti-human native Tau, anti-Tau proteins from different vertebrates, anti-PHF, monoclonal antibody Alz-50 and an anti-C terminal repeated region of Tau. In control brains, these antibodies strongly detected normal Tau proteins in the gray matter while Tau immunodetection was weak in the white matter. In Alzheimer brain cortices, each antibody detected Tau 64 and 69 in gray matter extracts but not at all in white matter extracts. The second category of anti-Tau consisted of the anti-PHF saturated with normal brain protein extracts. This antiserum only probed the abnormally phosphorylated Tau proteins. It detected Tau 64 and 69 exclusively in the cortical gray matter of Alzheimer brains. Moreover, a 55-kDa Tau protein was also immunolabelled, which might be an intermediary form between normal Tau and Tau 64 and 69. Our results demonstrate that Tau proteins are normal and major components of the somatodendritic domain and that Tau pathology, reflected by the presence of Tau 64 and 69, affects preferentially this domain during Alzheimers disease.

Immunolocalization of Inhibin and Activin α and βB Subunits and Expression of Corresponding Messenger RNAs in the Human Adult Testis

Abstract Inhibin B is a testicular peptide hormone that regulates FSH secretion in a negative feedback loop. Inhibin B is a dimer of an α and a βB subunit. In adult testes, the cellular site of production is still controversial, and it was hypothesized that germ cells contribute to inhibin B production. To determine which cell types in the testes may produce inhibin B, the immunohistochemical localization of the two subunits of inhibin B were examined in adult testicular biopsies with normal spermatogenesis, spermatogenic arrest, or Sertoli cell only (SCO) tubules. Moreover, using in situ hybridization with mRNA probes, the mRNA expression patterns of inhibin α and inhibin/activin βB subunits have been investigated. In all testes, Sertoli cells and Leydig cells showed positive immunostaining for inhibin α subunit and expressed inhibin α subunit mRNA. Using inhibin βB subunit immunoserum on testes with normal spermatogenesis and with spermatogenic arrest, intense labeling was located in germ cells from pachytene spermatocytes to round spermatids but not in Sertoli cells. Inhibin βB subunit mRNA expression was intense in germ cells from spermatogonia to round spermatids and in Sertoli cells in these testes. In testes with SCO, high inhibin βB subunit mRNA labeling density was observed in both Sertoli cells and Leydig cells, whereas βB subunit immunostaining was negative for Sertoli cells and faintly positive for Leydig cells. These results agree with the recent opinion that inhibin B in adult men is possibly a joint product of Sertoli cells and germ cells.

Alzheimer's disease: A new evidence for common epitopes between microtubule associated protein Tau and paired helical filaments (PHF): Demonstration at the electron microscope level by a double immunogold labelling

Paired helical filaments (PHF) are neuronal landmarks of Alzheimers disease. These pathological filaments are antigenically related to proteins present in the normal cytoskeleton, particularly to microtubule associated protein Tau. The evidence for these common epitopes was studied on sections of cortex from Alzheimer brains after Araldite embedding. Two rabbit immunsera were used: one was raised against PHF isolated from Alzheimer cortex; the other against Tau proteins extracted from bovine cortex. The comparison of adjacent semi-thin sections alternatively treated with anti-PHF and anti-Tau immunesera reveals that both stained degenerating neurofibrils in pyramidal perikaria and in neurites surrounding senile plaques. On ultra-thin sections, double immunogold labelling of PHF was obtained. These results are in accordance with the hypothesis that Tau proteins are major antigenic components of PHF.

Observation of morphological relationships between angiopathic blood vessels and degenerative neurites in Alzheimer's disease

Two main techniques are used to stain the three types of brain lesions characteristic of Alzheimers disease: Neurofibrillary tangles (NFT), senile plaques (SP) and congophilic angiopathy. Thioflavine-S is an histochemical marker of the amyloid substance located essentially in the central core of senile plaques and in the walls of the pathological blood vessels. Specific antibodies against Paired Helical Filaments (PHF), the ultra-structural elements of NFT, reveal neuron cell bodies with NFT and numerous dystrophic neurites, mostly around neuritic plaques. Using simultaneous histochemical and immunohistochemical labellings on the same tissue sections of Alzheimer cortex (association cortex and hippocampus), the different lesions were stained with great sensivity and specificity. Moreover, an unusual morphological relationship between two types of lesions was detected in two Alzheimer brains with prominent congophilic angiopathy: we observed a well marked concentration of dystrophic neurites, immunolabelled with anti-PHF, around blood vessels with Thioflavine-S stained amyloid angiopathy. These lesions were distributed like a sleeve around 1/10 of dyshoric or congophilic blood vessels. The significance of such lesions is unknown but they probably represent a step of the pathogenesis of Alzheimer brain lesions and may explain the general mechanism of lesion formation in Alzheimers disease.

Dystrophic peptidergic neurites in senile plaques of Alzheimer's disease hippocampus precede formation of paired helical filaments

The relationship between peptidergic dystrophic neurites and paired helical filament (PHF)-positive neurites in Alzheimers disease (AD) senile plaques (SPs) was studied using combined fluorescence and bright-field optics. Cryostat sections of AD hippocampi were first stained with thioflavine-S and immunolabelled with antisera raised against different neuropeptides: somatostatin-28(1-12), somatostatin-14, neuropeptide Y, cholecystokinin (CCK) and substance P. Secondly, using the elution-restaining procedure, sections were immunolabelled with anti-tau/PHF. In immature SPs, clusters of abnormal, swollen neurites were found. The dystrophic, strongly peptidic-positive neurites contained fewer PHFs than the poorly positive ones. Cell bodies, exhibiting a peptidic content, could be found within SPs without any alteration. These results suggest the following sequence of events: an extracellular poisoning mechanism, perhaps the amyloid substance, first changes the structure of presynaptic endings and causes the formation of ballooning dystrophic neurites filled with their normal peptidic content. Subsequently, intracellular degradation occurs with formation of the PHFs. Then the other structures such as dendrites and perikarya are damaged by the same mechanism. Therefore, this phenomenon seems to precede any formation of PHFs in SPs.

Cortical angiopathy in Alzheimer's disease: The formation of dystrophic perivascular neurites is related to the exudation of amyloid fibrils from the pathological vessels

We studied the organization of dystrophic neurites around pathological vessels in Alzheimer cortex. Two techniques were used simultaneously on serial sections: thioflavine staining of amyloid substance and immunohistochemistry with immune sera against Paired Helical Filaments (anti-PHF) and native Tau proteins (anti-Tau). We observed different distributions of dystrophic neurites (immunolabelled with anti-PHF or anti-Tau) around thioflavine-stained angiopathic arterioles. The wall of the vessels with large diameter (>100 µm) presented a congophilic angiopathy without neuropil reaction. In vessels with lesser diameter (<100 µm), dystrophic neurites constituted a discontinuous sleeve around vessels, always in close contact with amyloid substance outside the wall (dyshoric angiopathy). We observed structures similar to senile plaques around capillaries (diameter: 10–15 µm). The sleeve of dystrophic neurites with aggregated Tau proteins were always observed in the close vicinity of the amyloid substance which exuded from the pathological blood vessels. Thus, the exudation of these amyloid fibrils seems to induce the formation of dystrophic neurites (neuritic reaction).

Anti-PHF antibodies: an immunohistochemical marker of the lesions of the Alzheimer's disease

An immune serum raised against paired helical filaments (PHF) was able to stain senils plaques (SP) and neurofibrillary tangles (NFT) specifically, the two characteristic lesions of the dementia of Alzheimer-type. This polyclonal antibody against PHF was characterized by immunochemistry and also compared with the classical Bodian silver staining. NFT and SP were observed where they were expected: in the fronto-temporal neo-cortex and hippocampus of Alzheimer-type patients, and also in hippocampus of non-demented elderley subjects. The pattern of SP visualized by the two methods was identical whereas NFT were not detected specifically by silver salts, specially in the nervous tissue where NFT were in discrete quantities. Since the preparation of the antigen is very easy and the resulting antibodies are specific, we conclude that this technique will be of considerable interest for routine neuropathological diagnosis. Finally, the properties of our anti-PHF antibody are compared with those reported in the literature. This antibody will probably be a good tool for the identification of the chemical nature of PHF components.