Intravenous Injection of PHF-Tau Proteins From Alzheimer Brain Exacerbates Neuroinflammation, Amyloid Beta, and Tau Pathologies in 5XFAD Transgenic Mice.

Alzheimer's disease (AD) is characterized by the accumulation in the brain of intraneuronal aggregates of abnormally and hyperphosphorylated tau proteins and of extracellular deposits of amyloid-ß surrounded by dystrophic neurites. Numerous experimental models have shown that tau pathology develops in the brain after intracerebral injection of brain homogenates or pathological tau [paired helical filaments (PHF)-tau)] from AD brains. Further investigations are however necessary to identify or exclude potential extracerebral routes of tau pathology transmission, e.g., through the intravascular route. In this study, we have analyzed the effect of intravenous injection of PHF-tau proteins from AD brains on the formation of tau and amyloid pathologies in the brain of wild-type (WT) mice and of 5XFAD mice (an amyloid model). We observed that 5XFAD mice with a disrupted blood-brain barrier showed increased plaque-associated astrogliosis, microgliosis, and increased deposits of Aß40 and Aß42 after intravenous injection of PHF-tau proteins. In addition, an increased phosphotau immunoreactivity was observed in plaque-associated dystrophic neurites. These results suggest that blood products contaminated by PHF-tau proteins could potentially induce an exacerbation of neuroinflammation and AD pathologies.

Picalm reduction exacerbates tau pathology in a murine tauopathy model.

Genome-wide association studies (GWAS) have identified PICALM as one of the most significant susceptibility loci for late-onset Alzheimer's disease (AD) after APOE and BIN1. PICALM is a clathrin-adaptor protein and plays critical roles in clathrin-mediated endocytosis and in autophagy. PICALM modulates brain amyloid ß (Aß) pathology and tau accumulation. We have previously reported that soluble PICALM protein level is reduced in correlation with abnormalities of autophagy markers in the affected brain areas of neurodegenerative diseases including AD, sporadic tauopathies and familial cases of frontotemporal lobar degeneration with tau-immunoreactive inclusions (FTLD-tau) with mutations in the microtubule-associated protein tau (MAPT) gene. It remains unclarified whether in vivo PICALM reduction could either trigger or influence tau pathology progression in the brain. In this study, we confirmed a significant reduction of soluble PICALM protein and autophagy deficits in the post-mortem human brains of FTLD-tau-MAPT (P301L, S364S and L266V). We generated a novel transgenic mouse line named Tg30xPicalm+/- by crossing Tg30 tau transgenic mice with Picalm-haploinsufficient mice to test whether Picalm reduction may modulate tau pathology. While Picalm haploinsufficiency did not lead to any motor phenotype or detectable tau pathology in mouse brains, Tg30xPicalm+/- mice developed markedly more severe motor deficits than Tg30 by the age of 9 months. Tg30xPicalm+/- had significantly higher pathological tau levels in the brain, an increased density of neurofibrillary tangles compared to Tg30 mice and increased abnormalities of autophagy markers. Our results demonstrate that Picalm haploinsufficiency in transgenic Tg30 mice significantly aggravated tau pathologies and tau-mediated neurodegeneration, supporting a role for changes in Picalm expression as a risk/sensitizing factor for development of tau pathology and as a mechanism underlying the AD risk associated to PICALM.

Amyloid-ß pathology enhances pathological fibrillary tau seeding induced by Alzheimer PHF in vivo.

Neuropathological analysis in Alzheimer's disease (AD) and experimental evidence in transgenic models overexpressing frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17) mutant tau suggest that amyloid-ß pathology enhances the development of tau pathology. In this work, we analyzed this interaction independently of the overexpression of an FTDP-17 mutant tau, by analyzing tau pathology in wild-type (WT), 5xFAD, APP-/- and tau-/- mice after stereotaxic injection in the somatosensory cortex of short-length native human AD-PHF. Gallyas and phosphotau-positive tau inclusions developed in WT, 5xFAD, and APP-/- but not in tau-/- mice. Ultrastructural analysis demonstrated their intracellular localization and that they were composed of straight filaments. These seeded tau inclusions were composed only of endogenous murine tau exhibiting a tau antigenic profile similar to tau aggregates in AD. Insoluble tau level was higher and ipsilateral anteroposterior and contralateral cortical spreading of tau inclusions was more important in AD-PHF-injected 5xFAD mice than in WT mice. The formation of large plaque-associated dystrophic neurites positive for oligomeric and phosphotau was observed in 5xFAD mice injected with AD-PHF but never in control-injected or in non-injected 5xFAD mice. An increased level of the p25 activator of CDK5 kinase was found in AD-PHF-injected 5xFAD mice. These data demonstrate in vivo that the presence of Aß pathology enhances experimentally induced tau seeding of endogenous, wild-type tau expressed at physiological level, and demonstrate the fibrillar nature of heterotopically seeded endogenous tau. These observations further support the hypothesis that Aß enhances tau pathology development in AD through increased pathological tau spreading.

Amyloid precursor protein reduction enhances the formation of neurofibrillary tangles in a mutant tau transgenic mouse model.

Alzheimer's disease is characterized by the presence of 2 neuropathological lesions: neurofibrillary tangles, composed of tau proteins which are highly phosphorylated and phosphorylated on uncommon sites, and amyloid plaques, containing the Aß peptides generated from the amyloid precursor protein (APP). Reduction of some APP proteolytic derivatives in Alzheimer's disease such as sAPPα fragment has been reported and sAPPα has been shown to affect tau phosphorylation. To investigate in vivo the effect of absence of APP protein and its fragments on tau phosphorylation and the formation of neurofibrillary tangles, we have generated mice deleted for APP gene and overexpressing a human mutant tau protein and developing neurofibrillary tangles (APPKOTg30 mice). These APPKOTg30 mice showed more severe motor and cognitive deficits, increased tau phosphorylation, increased load of neurofibrillary tangles, and increased p25/35 ratio in the brain, compared with Tg30 mice. These data suggest that APP and/or its proteolytic derivatives interfere with the formation of neurofibrillary tangles in a transgenic mouse model that will be useful for investigating the relationship between APP and tau.

High-Molecular-Weight Paired Helical Filaments from Alzheimer Brain Induces Seeding of Wild-Type Mouse Tau into an Argyrophilic 4R Tau Pathology in Vivo.

In Alzheimer disease, the development of tau pathology follows neuroanatomically connected pathways, suggesting that abnormal tau species might recruit normal tau by passage from cell to cell. Herein, we analyzed the effect of stereotaxic brain injection of human Alzheimer high-molecular-weight paired helical filaments (PHFs) in the dentate gyrus of wild-type and mutant tau THY-Tau22 mice. After 3 months of incubation, wild-type and THY-Tau22 mice developed an atrophy of the dentate gyrus and a tau pathology characterized by Gallyas and tau-positive grain-like inclusions into granule cells that extended in the hippocampal hilus and eventually away into the alveus, and the fimbria. Gallyas-positive neuropil threads and oligodendroglial coiled bodies were also observed. These tau inclusions were composed only of mouse tau, and were immunoreactive with antibodies to 4R tau, phosphotau, misfolded tau, ubiquitin, and p62. Although local hyperphosphorylation of tau was increased in the dentate gyrus in THY-Tau22 mice, the development of neurofibrillary tangles made of mutant human tau was not accelerated in the hippocampus, indicating that wild-type human PHFs were inefficient in seeding tau aggregates made of G272V/P301S mutant human tau. Our results indicate thus that injection of human wild-type Alzheimer PHF seeded aggregation of wild-type murine tau into an argyrophilic 4R tau pathology, and constitutes an interesting model independent of expression of a mutant tau protein.

Increased misfolding and truncation of tau in APP/PS1/tau transgenic mice compared to mutant tau mice.

Neurofibrillary degeneration in transgenic models of tauopathies has been observed to be enhanced when these models are crossed with transgenic models developing an Aß pathology. The mechanisms leading to this enhanced tau pathology are not well understood. We have performed a detailed analysis of tau misprocessing in a new transgenic mouse model combining APP, PS1 and tau mutations (5xFAD×Tg30 mice) by comparison with littermates expressing only a FTD mutant tau (Tg30 mice). These 5xFAD×Tg30 mice showed a more severe deficient motor phenotype than Tg30 mice and developed with age a dramatically accelerated NFT load in the brain compared to Tg30 mice. Insoluble tau in 5xFAD×Tg30 mice compared to insoluble tau in Tg30 mice showed increased phosphorylation, enhanced misfolding and truncation changes mimicking more closely the post-translational changes characteristic of PHF-tau in Alzheimer's disease. Endogenous wild-type mouse tau was recruited at much higher levels in insoluble tau in 5xFAD×Tg30 than in Tg30 mice. Extracellular amyloid load, Aß40 and Aß42, ß-CTFs and ß-CTF phosphorylation levels were lower in 5xFAD×Tg30 mice than in 5xFAD mice. Despite this reduction of Aß, a significant hippocampal neuronal loss was observed in 5xFAD×Tg30 but not in 5xFAD mice indicating its closer association with increased tau pathology. This 5xFAD×Tg30 model thus mimics more faithfully tau pathology and neuronal loss observed in AD and suggests that additional post-translational changes in tau and self-recruitment of endogenous tau drive the enhanced tau pathology developing in the presence of Aß pathology.

Accelerated human mutant tau aggregation by knocking out murine tau in a transgenic mouse model.

Many models of human tauopathies have been generated in mice by expression of a human mutant tau with maintained expression of mouse endogenous tau. Because murine tau might interfere with the toxic effects of human mutant tau, we generated a model in which a pathogenic human tau protein is expressed in the absence of wild-type tau protein, with the aim of facilitating the study of the pathogenic role of the mutant tau and to reproduce more faithfully a human tauopathy. The Tg30 line is a tau transgenic mouse model overexpressing human 1N4R double-mutant tau (P301S and G272V) that develops Alzheimer's disease-like neurofibrillary tangles in an age-dependent manner. By crossing Tg30 mice with mice invalidated for their endogenous tau gene, we obtained Tg30xtau(-/-) mice that express only exogenous human double-mutant 1N4R tau. Although Tg30xtau(-/-) mice express less tau protein compared with Tg30, they exhibit signs of decreased survival, increased proportion of sarkosyl-insoluble tau in the brain and in the spinal cord, increased number of Gallyas-positive neurofibrillary tangles in the hippocampus, increased number of inclusions in the spinal cord, and a more severe motor phenotype. Deletion of murine tau accelerated tau aggregation during aging of this mutant tau transgenic model, suggesting that murine tau could interfere with the development of tau pathology in transgenic models of human tauopathies.

Deletion of murine tau gene increases tau aggregation in a human mutant tau transgenic mouse model.

We have reported previously a tau transgenic mouse model (Tg30tau) overexpressing human 4R1N double-mutant tau (P301S and G272V) and that develops AD (Alzheimer's disease)-like NFTs (neurofibrillary tangles) in an age-dependent manner. Since murine tau might interfere with the toxic effects of human mutant tau, we set out to analyse the phenotype of our Tg30tau model in the absence of endogenous murine tau with the aim to reproduce more faithfully a model of human tauopathy. By crossing the Tg30tau line with TauKO (tau-knockout) mice, we have obtained a new mouse line called Tg30xTauKO that expresses only exogenous human double-mutant 4R1N tau. Whereas Tg30xTauKO mice express fewer tau proteins compared with Tg30tau, they exhibit augmented sarkosyl-insoluble tau in the brain and an increased number of Gallyas-positive NFTs in the hippocampus. Taken together, exclusion of murine tau causes accelerated tau aggregation during aging of this mutant tau transgenic model.

Lithium treatment arrests the development of neurofibrillary tangles in mutant tau transgenic mice with advanced neurofibrillary pathology.

Neurofibrillary tangles (NFTs) made of phosphorylated tau proteins are a key lesion of Alzheimer's disease and other neurodegenerative diseases, and previous studies have indicated that lithium can decrease tau phosphorylation in tau transgenic models. In this study, we have reassessed the effectiveness of treatment per os with lithium on the prevention, the arrest, or the reversal of NFT development in a tau transgenic line (Tg30tau) developing severe neurofibrillary pathology in the brain and the spinal cord. Wild-type and Tgtau30 mice were treated per os with lithium carbonate or with natrium carbonate by chronic chow feeding for 8 months starting at the age of 3 months (to test for a preventive effect on NFT formation) or by oral gavage for 1 month starting at the age of 9 months (after development of NFTs). In mice treated by oral gavage, a decrease of tau phosphorylation and of Sarkosyl-insoluble aggregated tau was observed in the brain and in the spinal cord. The density of NFTs identified by Gallyas staining in the hippocampus and in the spinal cord was also significantly reduced and was similar to that observed at the beginning of the lithium treatment. In these animals, the level of brain beta-catenin was increased probably as a result of its stabilization by glycogen synthase kinase-3beta inhibition. Despite this inhibitory effect of lithium on NFT development, the motor and working memory deficits were not significantly rescued in these aged animals. Chronic chow feeding with lithium did not alter the development of NFT. Nevertheless, this study indicates that even a relatively short-term per os treatment leading to high blood concentration of lithium is effective in arresting the formation of NFTs in the hippocampus and the spinal cord of a tau transgenic model.

Impaired blood-brain and blood-spinal cord barriers in mutant SOD1-linked ALS rat.

Blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB) impairment is an additional accident occurring during the amyotrophic lateral sclerosis (ALS) progression. In this work, we aimed to decipher if BBB/BSCB leakage appeared before critical detrimental events and could serve as a marker preceding clinical symptoms. Three different BBB leakage markers: Evans blue, IgG and hemosiderin, were used to look at the SOD1-linked ALS rat model at presymptomatic and symptomatic stages. Although IgG and hemosiderin could be detected at presymptomatic stage, Evans blue extravasation which fits best with BBB/BSCB impairment could only be seen at symptomatic stages. BBB/BSCB impairment was further substantiate by showing at symptomatic stages decreased mRNA expression of ZO-1 and occludin as well as agrin, a basal membrane constituent. Electron microscopic data substantiate a toxic environment around endothelial cell and peri-vascular swollen astrocyte end-feet showing oedema-linked BBB opening.

Aquaporin-4 overexpression in rat ALS model.

Onset of motoneuron death characterizing amyotrophic lateral sclerosis (ALS) is closely linked to modified astrocytic and glial environments. Here, we show that in the spinal cord from transgenic rat overexpressing mutated human SOD1, aquaporin-4 mRNA and protein are specifically overexpressed in the gray matter at end stage of disease. Immunohistochemistry and double immunofluorescence allowed to detect, in the spinal cord gray matter of the ALS rat, increased aquaporin-4 surrounding both vessel and motoneuron perikarya. The use of pre-embedding immunohistochemistry at electron microscopic level confirmed such localization associated with swollen astrocytic processes surrounding the vessels. The AQP4 immunohistochemical labeling surrounding several motoneuron perikarya was only seen in ALS rats. Identification of this AQP4-positive cellular type remains unclear.

Gemals, a new drug candidate, extends lifespan and improves electromyographic parameters in a rat model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal disease involving selective and progressive degeneration and death of motor neurons. ALS is a multifactorial disease in which oxidative stress, glutamate excitotoxicity, intracellular aggregates, neurofilamentous disorganization, zinc excitotoxicity, mitochondrial damage, neuroinflammation, abnormalities in growth factors and apoptosis play a role. Any therapeutic approach to delay or stop the evolution of ALS should therefore ideally target these multiple pathways leading to motor neuron death. We have developed a combination therapy (Gemals) composed of functional polypeptides (fatty acids, free radical scavengers and amino acids linked to poly-L-lysine), chosen according to their known potentiality for regeneration or protection of neuronal components such as myelin, axon transport and mitochondria. We found that Gemals significantly extended lifespan and improved electromyographic parameters in a SOD1(G93A) rat model. The use of two drug concentrations indicated a possible dose dependence. These initial findings open the way to further investigation necessary to validate this new drug as a candidate for ALS treatment.

Early axonopathy preceding neurofibrillary tangles in mutant tau transgenic mice.

Neurodegenerative diseases characterized by brain and spinal cord involvement often show widespread accumulations of tau aggregates. We have generated a transgenic mouse line (Tg30tau) expressing in the forebrain and the spinal cord a human tau protein bearing two pathogenic mutations (P301S and G272V). These mice developed age-dependent brain and hippocampal atrophy, central and peripheral axonopathy, progressive motor impairment with neurogenic muscle atrophy, and neurofibrillary tangles and had decreased survival. Axonal spheroids and axonal atrophy developed early before neurofibrillary tangles. Neurofibrillary inclusions developed in neurons at 3 months and were of two types, suggestive of a selective vulnerability of neurons to form different types of fibrillary aggregates. A first type of tau-positive neurofibrillary tangles, more abundant in the forebrain, were composed of ribbon-like 19-nm-wide filaments and twisted paired helical filaments. A second type of tau and neurofilament-positive neurofibrillary tangles, more abundant in the spinal cord and the brainstem, were composed of 10-nm-wide neurofilaments and straight 19-nm filaments. Unbiased stereological analysis indicated that total number of pyramidal neurons and density of neurons in the lumbar spinal cord were not reduced up to 12 months in Tg30tau mice. This Tg30tau model thus provides evidence that axonopathy precedes tangle formation and that both lesions can be dissociated from overt neuronal loss in selected brain areas but not from neuronal dysfunction.

Osteosarcoma cell-calcium signaling through tissue factor-factor VIIa complex and factor Xa.

The cells responsible for bone formation express protease-activated receptors. Although serine protease thrombin has been shown to elicit functional responses in bone cells that impact on cell survival and alkaline phosphatase activity, nothing is known about tissue factor, factor VIIa, and factor Xa, the serine proteases that act upstream of thrombin in the coagulation cascade. This paper demonstrates that tissue factor is expressed in the osteoblast-like cell line SaOS-2 and, that tissue factor in a factor VIIa-bound complex induces a transient intracellular Ca(2+) increase through protease-activated receptor-2. In SaOS-2 cells, factor Xa induced a sustained intracellular Ca(2+) response, as does SLIGRL, a PAR2-activating peptide, and PAR-1-dependent cell viability.

In vitro pharmacological characterizations of the anti-angiogenic and anti-tumor cell migration properties mediated by microtubule-affecting drugs, with special emphasis on the organization of the actin cytoskeleton.

The aim of the present work is to investigate whether microtubule-affecting drugs including vincristine, vinblastine, vindesine and vinorelbine are able to produce an anti-angiogenic effect at non-cytotoxic doses in the same way of taxol. The cytotoxic effects were determined by means of the colorimetric MTT assay, and the anti-angiogenic effects on HUVEC cells growing on Matrigel and forming capillary networks. Sixteen additional drugs (camptothecin, SN38, topothecan, adriamycin, daunomycin, etoposide, bleomycin, melphalan, mitomycin C, TNP-470, cisplatin, carboplatin, 5-fluorouracil, methotrexate, suramin and batimastat) were used as control in order to test the specificity of the microtubule-affecting drug effects. We also investigated by means of videomicroscopy whether microtubule-affecting drugs could produce anti-migratory effects at non-cytotoxic doses on tumor cells. Finally, we used computer-assisted fluorescence microscopy to characterize the influence of microtubule-affecting drugs on the polymerization/depolymerization dynamics of the actin cytoskeleton in tumor cells. Our results show that taxol, vincristine and vindesine behave similarly in their ability to reduce the capillary network formation by HUVEC cells cultured on Matrigel. These anti-angiogenic effects appear at non-cytotoxic concentrations. In contrast, vinblastine and vinorelbine produce apparent anti-angiogenic effects by direct cytotoxicity. Microtubule-affecting agents are also able to significantly reduce the level of migration of tumor cells at non-cytotoxic concentrations, some of these effects may occur via modifications to the actin cytoskeleton organization. Several types of microtubule-affecting agents could be used as anti-angiogenic agents by administering them at non-cytotoxic concentrations, and some microtubule-affecting agents abandoned in pharmacological assays could turn out to be potent anti-migratory drugs acting on tumor cells, though without being too cytotoxic.