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.

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.

Anoctamin 1 (Ano1) is required for glucose-induced membrane potential oscillations and insulin secretion by murine ß-cells.

Anions such as Cl(-) and HCO3 (-) are well known to play an important role in glucose-stimulated insulin secretion (GSIS). In this study, we demonstrate that glucose-induced Cl(-) efflux from ß-cells is mediated by the Ca(2+)-activated Cl(-) channel anoctamin 1 (Ano1). Ano1 expression in rat ß-cells is demonstrated by reverse transcriptase-polymerase chain reaction, western blotting, and immunohistochemistry. Typical Ano1 currents are observed in whole-cell and inside-out patches in the presence of intracellular Ca(++): at 1 µM, the Cl(-) current is outwardly rectifying, and at 2 µM, it becomes almost linear. The relative permeabilities of monovalent anions are NO3 (-) (1.83 ± 0.10) > Br(-) (1.42 ± 0.07) > Cl(-) (1.0). A linear single-channel current-voltage relationship shows a conductance of 8.37 pS. These currents are nearly abolished by blocking Ano1 antibodies or by the inhibitors 2-(5-ethyl-4-hydroxy-6-methylpyrimidin-2-ylthio)-N-(4-(4-methoxyphenyl)thiazol-2-yl)acetamide (T-AO1) and tannic acid (TA). These inhibitors induce a strong decrease of 16.7-mM glucose-stimulated action potential rate (at least 87 % on dispersed cells) and a partial membrane repolarization with T-AO1. They abolish or strongly inhibit the GSIS increment at 8.3 mM and at 16.7 mM glucose. Blocking Ano1 antibodies also abolish the 16.7-mM GSIS increment. Combined treatment with bumetanide and acetazolamide in low Cl(-) and HCO3 (-) media provokes a 65 % reduction in action potential (AP) amplitude and a 15-mV AP peak repolarization. Although the mechanism triggering Ano1 opening remains to be established, the present data demonstrate that Ano1 is required to sustain glucose-stimulated membrane potential oscillations and insulin secretion.

Astrocytes are an early target in osmotic demyelination syndrome.

Abrupt osmotic changes during rapid correction of chronic hyponatremia result in demyelinative brain lesions, but the sequence of events linking rapid osmotic changes to myelin loss is not yet understood. Here, in a rat model of osmotic demyelination syndrome, we found that massive astrocyte death occurred after rapid correction of hyponatremia, delineating the regions of future myelin loss. Astrocyte death caused a disruption of the astrocyte-oligodendrocyte network, rapidly upregulated inflammatory cytokines genes, and increased serum S100B, which predicted clinical manifestations and outcome of osmotic demyelination. These results support a model for the pathophysiology of osmotic brain injury in which rapid correction of hyponatremia triggers apoptosis in astrocytes followed by a loss of trophic communication between astrocytes and oligodendrocytes, secondary inflammation, microglial activation, and finally demyelination.

Functional model of rat thyroid follicles cultured in Matrigel.

BACKGROUND: Long-term maintenance of functional activity of thyroid cells is an essential requirement for basic in vitro studies on the physiology and pathology of the thyroid. An important prerequisite of thyrocytes' functional activity in vivo and in vitro is their follicle organization. AIM: This study aimed at developing a method of cultivation of functionally active rat thyroid follicles in Matrigel under three-dimensional conditions. METHODS: Undamaged rat thyroid follicles were isolated by enzymatic digestion with collagenase/dispase, then embedded into Matrigel, and cultivated for 2 weeks. Thyroglobulin, thyroxine and zonula occludens-1 (ZO-1) localization were revealed by immunofluorescence analysis. Iodide organification was tested by protein-bound 125I (PBI) measurement. RESULTS: Integrity of the follicles was preserved during the whole period of cultivation and was confirmed by 3D reconstruction of ZO-1 localization. Thyroglobulin was detected in the thyrocyte cytoplasm, as well as in the intrafollicular lumen. Thyroxine was observed predominantly at the apical side of thyrocytes. Also, generated cultures were characterized by a high level of iodide organification: PB125I represented 39% of the total radioactivity in the Matrigel drop embedding the follicles; at the same time, methimazole almost totally inhibited this process (0.2% of total radioactivity). CONCLUSION: The method of rat thyrocyte cultivation in Matrigel, as described here allows to maintain the structural integrity and the functional activity of thyroid follicles in vitro and could be used for wide ranges of basic and applied researches in thyroidology.

Bimodal modulation of tau protein phosphorylation and conformation by extracellular Zn2+ in human-tau transfected cells.

Abnormal homeostasis of heavy metals is a well-documented physiopathological mechanism in Alzheimer's disease. An exacerbation of these abnormalities is best illustrated in the amyloid plaques in Alzheimer's disease brain tissue, in which zinc reaches the enormous concentration of 1000 microM. Zinc in the plaques is thought to originate from impaired glutamatergic neurons distributed in the associative cortex and limbic structures of normal brain. Although the characteristics of zinc binding to Abeta and its role in promotion of Abeta aggregation have been intensively studied, the contribution of zinc to the development of tau pathology remains elusive. To further document the effect of zinc we have investigated the modifications of tau phosphorylation, conformation and association to microtubules induced by zinc in clonal cell lines expressing a human tau isoform. A bimodal dose dependent effect of zinc was observed. At 100 microM zinc induced a tau dephosphorylation on the PHF-1 epitope, and at higher zinc concentrations induced the appearance of the abnormal tau conformational epitope MC1 and reduced the electrophoretic mobility of tau, known to be associated to increased tau phosphorylation. High zinc concentrations also increased glycogen synthase kinase-3beta (GSK-3beta) phosphorylation on tyrosine 216, a phosphorylation associated with increased activity of this tau kinase. Live imaging of tau-EGFP expressing cells demonstrated that high zinc concentrations induced a release of tau from microtubules. These results suggest that zinc plays a significant role in the development of tau pathology associated to Alzheimer's disease.

DUOX1-mediated hydrogen peroxide release regulates sodium transport in H441 bronchiolar epithelial cells.

AIM: Dexamethasone has been shown to induce the formation of epithelial domes by bronchiolar H441 cells. It stimulates the expression of both amiloride inhibitable epithelial sodium channels (ENaC) and dual oxidase-1 (DUOX1). We therefore ask the question whether DUOX1 expression and production of submillimolar amounts of H2 O2 is instrumental for the sodium channel upregulation observed in H441 cells. METHODS: In vitro cell culture, nystatin-perforated whole-cell patch-clamp technique, immunocytochemistry and RT-PCR methods have been used. RESULTS: Cells forming epithelial domes induced by dexamethasone (0.1 µmol L-1 , 24 hours) and by 5-aza-2'-deoxytidine (1 µmol L-1 , 48 hours) expressed more DUOX1 protein compared with other cells in the monolayer. Dome formation could be inhibited by exogenous catalase in a concentration-dependent manner and by the NADPH oxidase inhibitor diphenyliodonium, which suggested the involvement of H2 O2 . While single application of 0.2 mmol L-1 H2 O2 induced transient dome formation, lower doses were ineffective and higher doses disrupted the cell monolayer. Hydrogen peroxide (0.1 mmol L-1 ) activated acutely amiloride-sensitive whole-cell currents from 3.91 ± 0.79 pA pF-1 to 4.76 ± 0.98 pA pF-1 in dome-forming cells and had no effect in cells outside of domes. ENaC but not DUOX1 transcription was potentiated by catalase in the presence of dexamethasone, which suggested negative feedback of H2 O2 on ENaC gene expression. CONCLUSION: Our observations suggest that tonic production of H2 O2 by DUOX1 participates in maintaining the level of vectorial sodium transport by lung epithelial cells. Moreover, the system appears to be well tuned as it would allow H2 O2 -dependent innate immunity without inducing airway/alveolar sodium and fluid hyperabsorption.

Expression, Localization, and Regulation of the Sodium Bicarbonate Cotransporter NBCe1 in the Thyroid.

BACKGROUND: The intrafollicular space of thyroid follicles is the storage compartment for thyroid hormones. Its pH has been established at around 7.6 at least after thyrotropin (TSH) stimulation. This alkaline intrafollicular pH is thought to be critical for iodide coupling to thyroglobulin and internalization of iodinated thyroglobulin. At least in mice, this alkalinization requires the expression of pendrin (Slc26a4) within the apical membrane, and a lack of pendrin results in acidic follicular lumen pH. Yet, the mechanism importing HCO3- into the cytoplasm is unknown. This study investigated whether the rather ubiquitous sodium bicarbonate cotransporter NBCe1 (SLC4A4) might play this role. It also examined which variant was expressed and where it was localized in both rat and human thyroid tissue. Lastly, the dependence of its expression on TSH was studied. METHODS: Reverse transcription polymerase chain reaction, immunofluorescence, and Western blotting were used to test whether TSH stimulated NBCe1 protein expression in vivo. Subcellular localization of NBCe1 was performed using immunofluorescence in both rat and human thyroid. Cultured thyroid cells were also used to attempt to define how TSH affects NBCe1 expression. RESULTS: Only transcripts of the NBCe1-B variant were detected in both rat and human thyroid. Of interest, NBCe1-C was not detected in human tissues, not even in the brain. On immunofluorescence microscopy, the immunostaining of NBCe1 mainly appeared in the basolateral membrane upon stimulation with TSH. This TSH induction of basolateral membrane expression of NBCe1 protein was confirmed in vivo in rat thyroid and in vitro on human thyroid slices. CONCLUSIONS: This study demonstrates the expression of the sodium bicarbonate cotransporter NBCe1-B in rat and human thyroid. Additionally, the data suggest that TSH blocks the degradation of NBCe1 protein by trafficking it to the basolateral membrane. Hence, TSH increases NBCe1 half-life without increasing its synthesis.

A neuronal aging pattern unique to humans and common chimpanzees.

Lipofuscin pigment accumulation is among the most prominent markers of cellular aging in postmitotic cells. The formation of lipofuscin is related to oxidative enzymatic activity and free radical-induced lipid peroxidation. In various mammals such as rat, dog, macaque as well as in cheirogaleid primates, most of the large neurons, such as cerebellar Purkinje cells and neocortical pyramidal cells, show heavy lipofuscin accumulation in adulthood. In contrast, a well-known yet poorly studied feature of the aging human brain is that although lipofuscin accumulation is most marked in large neurons of the cerebral cortex, the large neurons of the cerebellar cortex-the Purkinje cells-appear to remain free of lipofuscin accumulation. It is however, not known whether this characteristic of human Purkinje cells is shared with other primates or other mammals. This study reports results from histological observation of Purkinje cells in humans, non-human primates, and other mammals. Procedures include histochemistry, immunocytochemistry, and fluorescence microscopy. Abundant lipofuscin deposition was observed in Purkinje cells of all the species we examined except Homo sapiens (including Alzheimer's disease cases) and Pan troglodytes. In contrast, lipofuscin deposition was observed in neurons of the dentate nucleus. Our findings suggest that when compared with other primates, Purkinje cells in chimpanzees and humans might share a common aging pattern that involves mechanisms for neuroprotection. This observation is important when considering animal models of aging.

Astrocytic calcium/zinc binding protein S100A6 over expression in Alzheimer's disease and in PS1/APP transgenic mice models.

Astrocytes recruitment and activation are a hallmark of many neurodegenerative diseases including Alzheimer's disease (AD). We have previously observed an overexpression for S100A6 protein, a Ca(2+)/Zn(2+) binding protein presenting more affinity for zinc than for calcium, in amyotrophic lateral sclerosis (ALS). Here we demonstrated in AD patients but also in two different AD mouse models, that astrocytic S100A6 protein was homogeneously up-regulated within the white matter. However, within the grey matter, almost all S100A6 immunoreactivity was concentrated in astrocytes surrounding the Abeta amyloid deposits of senile plaques. These S100A6 neocortex labelled astrocytes were also positive for the glial fibrillary acidic protein (GFAP) and S100B protein. Contrasting with S100A6, the distribution for S100B and GFA astrocytic labelled cells was not restricted to the Abeta amyloid deposit in grey matter, but widely distributed throughout the neocortex. Coupling the knowledge that biometals such as zinc are highly concentrated in the amyloid deposits in AD and S100A6 having a high affinity for Zn(2+) may suggest that S100A6 plays a role in AD neuropathology.

S100A6 overexpression within astrocytes associated with impaired axons from both ALS mouse model and human patients.

Astrogliosis is one of the earliest pathological changes observed in neurodegenerative diseases in general and in amyotrophic lateral sclerosis (ALS) in particular. ALS is characterized by selective degeneration of motoneurons. There are 2 forms of the disease: sporadic ALS (SALS), comprising 90%-95% of cases, and familial ALS (FALS), comprising 5%-10% of cases. FALS is an age-dependent autosomal dominant disorder in which mutations in the homodimeric enzyme Cu/ Zn superoxide dismutase 1 (SOD1) is linked to the disease. The animal model for this disease is a transgenic mouse expressing the mutated human SOD1(G93A) gene. Here we show by immunohistochemistry and double immunofluorescence that astrocytes located near impaired axons of motoneurons that were selectively programmed to die overexpressed S100A6, a Ca2+/Zn2+ binding protein able to translocate into the nucleus. Transgenic mice overexpressing the mutated human SOD1 gene and patients suffering from SALS showed this selective astrocytic S100A6 expression. For instance, the pyramidal tract could be macroscopically detected on S100A6-labeled spinal cord and brainstem sections from SALS patients. Transgenic mice overexpressing the non-mutated SOD1 gene did not overexpress S100A6, although glial fibrillary associated protein astrogliosis was seen. Although these results do not give any clue about the beneficial or detrimental role played by S100A6, its induction may be assumed to appropriately serve some function(s).

Subcellular structural plasticity caused by the absence of the fast Ca(2+) buffer calbindin D-28k in recurrent collaterals of cerebellar Purkinje neurons.

Purkinje cells (PC) control spike timing of neighboring PC by their recurrent axon collaterals. These synapses underlie fast cerebellar oscillations and are characterized by a strong facilitation within a time window of <20 ms during paired-pulse protocols. PC express high levels of the fast Ca(2+) buffer protein calbindin D-28k (CB). As expected from the absence of a fast Ca(2+) buffer, presynaptic action potential-evoked [Ca(2+)]i transients were previously shown to be bigger in PC boutons of young (second postnatal week) CB-/- mice, yet IPSC mean amplitudes remained unaltered in connected CB-/- PC. Since PC spine morphology is altered in adult CB-/- mice (longer necks, larger spine head volume), we summoned that morphological compensation/adaptation mechanisms might also be induced in CB-/- PC axon collaterals including boutons. In these mice, biocytin-filled PC reconstructions revealed that the number of axonal varicosities per PC axon collateral was augmented, mostly confined to the granule cell layer. Additionally, the volume of individual boutons was increased, evidenced from z-stacks of confocal images. EM analysis of PC-PC synapses revealed an enhancement in active zone (AZ) length by approximately 23%, paralleled by a higher number of docked vesicles per AZ in CB-/- boutons. Moreover, synaptic cleft width was larger in CB-/- (23.8 ± 0.43 nm) compared to wild type (21.17 ± 0.39 nm) synapses. We propose that the morphological changes, i.e., the larger bouton volume, the enhanced AZ length and the higher number of docked vesicles, in combination with the increase in synaptic cleft width likely modifies the GABA release properties at this synapse in CB-/- mice. We view these changes as adaptation/homeostatic mechanisms to likely maintain characteristics of synaptic transmission in the absence of the fast Ca(2+) buffer CB. Our study provides further evidence on the functioning of the Ca(2+) homeostasome.

Inhibition of insulin-stimulated hydrogen peroxide production prevents stimulation of sodium transport in A6 cell monolayers.

Insulin-stimulated sodium transport across A6 cell (derived from amphibian distal nephron) monolayers involves the activation of a phosphatidylinositol (PI) 3-kinase. We previously demonstrated that exogenous addition of H2O2 to the incubation medium of A6 cell monolayers provokes an increase in PI 3-kinase activity and a subsequent rise in sodium transport (Markadieu N, Crutzen R, Blero D, Erneux C, Beauwens R. Am J Physiol Renal Physiol 288: F1201-F1212, 2005). We therefore questioned whether insulin would produce an intracellular burst of H2O2 leading to PI 3-kinase activation and subsequent increase in sodium transport. An acute production of reactive oxygen species (ROS) in A6 cells incubated with the oxidation-sensitive fluorescent probe 5,6-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate was already detected after 2 min of insulin stimulation. This fluorescent signal and the increase in sodium transport were completely inhibited in monolayers incubated with peggylated catalase, indicating that H2O2 is the main intracellular ROS produced upon insulin stimulation. Similarly, preincubation of monolayers with different chelators of either superoxide (O2(*-); nitro blue tetrazolium, 100 microM) or H2O2 (50 microM ebselen), or blockers of NADPH oxidase (Nox) enzymes (diphenyleneiodonium, 5 microM; phenylarsine oxide, 1 microM and plumbagin, 30 microM) prevented both insulin-stimulated H2O2 production and insulin-stimulated sodium transport. Furthermore, diphenyleneiodonium pretreatment inhibited the recruitment of the p85 PI 3-kinase regulatory subunit in an anti-phosphotyrosine immunoprecipitate in insulin-stimulated cells. In contrast, PI-103, an inhibitor of class IA PI 3-kinase, inhibited insulin-stimulated sodium transport but did not significantly reduce insulin-stimulated H2O2 production. Taken together, our data suggest that insulin induces an acute burst of H2O2production which participates in an increase in phosphatidylinositol 3,4,5-trisphosphate production and subsequently stimulation of sodium transport.

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.

Expression and localization of cystic fibrosis transmembrane conductance regulator in the rat endocrine pancreas.

Impaired glucose tolerance and overt diabetes mellitus are becoming increasingly common complications of cystic fibrosis (CF), most probably merely as a result of increased life expectancy. In order to understand the pathophysiology of cystic fibrosis-related diabetes (CFRD), knowledge on the possible expression and cell distribution of the cystic fibrosis transmembrane conductance regulator (CFTR) protein within the endocrine pancreas is required. In this report, we establish the first evidence for expression of CFTR protein in rat pancreatic islets by using independent techniques. First reverse transcriptase-polymerase chain reaction (RT-PCR) amplification showed that CFTR mRNA is present in isolated islets of Langerhans. Furthermore, the analysis of flow cytometry-separated islet cells indicated that the level of CFTR transcripts is significantly higher in the non-beta than in beta-cell populations. The expression of CFTR protein in rat islet cells was also demonstrated by Western blotting and the level of expression was also found significantly higher in the non-beta than in beta-cell populations. Last, in situ immunocytochemistry studies with two monoclonal antibodies recognizing different CFTR epitopes indicated that CFTR expression occurs mainly in glucagon-secreting alpha-cells.

Phosphatidylinositol 3,4,5-trisphosphate: an early mediator of insulin-stimulated sodium transport in A6 cells.

Insulin stimulates sodium transport across A6 epithelial cell monolayers. Activation of phosphatidylinositol 3-kinase (PI 3-kinase) was suggested as an early step in the insulin-stimulated sodium reabsorption (Ref. 35). To establish that the stimulation of the PI 3-kinase signaling cascade is causing stimulation of apical epithelial Na channel, we added permeant forms of phosphatidylinositol (PI) phosphate (P) derivatives complexed with a histone carrier to A6 epithelium. Only PIP(3) and PI(3,4)P(2) but not PI(4,5)P(2) stimulated sodium transport, although each of them penetrated into A6 cell monolayers as assessed using fluorescent permeant phosphoinositides derivatives. By Western blot analysis of A6 cell extracts, the inositol 3-phosphatase PTEN and the protein kinase B PKB were both detected. To further establish that the stimulation of sodium transport induced by insulin is related to PIP(3) levels, we transfected A6 cells with human PTEN cDNA and observed a 30% decrease in the natriferic effect of insulin. Similarly, the increase in sodium transport observed by addition of permeant PIP(3) was also reduced by 30% in PTEN-overexpressing cells. PKB, a main downstream effector of PI 3-kinase, was phosphorylated at both Thr 308 and Ser 473 residues upon insulin stimulation of the A6 cell monolayer. PKB phosphorylation in response to insulin stimulation was reduced in PTEN-overexpressing cells. Permeant PIP(3) also increased PKB phosphorylation. Taken together, the present results establish that the d-3-phosphorylated phosphoinositides PIP(3) and PI(3,4)P(2) mediate the effect of insulin on sodium transport across A6 cell monolayers.