Alzheimer disease: curly fibers and tangles in organs other than brain.

The filamentous brain lesions that define Alzheimer disease (AD) consist of senile plaques and neurofibrillary tangles. Undulated pathological filaments--curly fibers or neuropil threads--also occur in the neuropil. Beta-amyloid precursor proteins are synthesized by many cells outside the central nervous system and recently, deposition of beta-amyloid-protein was reported to occur in non-neuronal tissues. In addition, increasing data claim the importance of chronic inflammation in the pathogenesis of AD. These observations suggest that AD may be a widespread systemic disorder. Here we report that pathological argyrophilic filaments with histochemical properties of amyloid showing striking morphological similarity to curly fibers and/or tangles accumulate not only in ependymal layer and in epithelial cells of choroid plexus, but also in several other organs (e.g. liver, pancreas, ovary, testis, thyroid) in AD. The ependyma, choroid plexus, and various organs of 39 autopsy cases were analyzed. In search of curly fiber and tangle-like changes in organs other than brain, 395 blocks from 21 different tissues of 24 AD cases, 5 cases with discrete or moderate AD-type changes, and 10 control cases were investigated. We found in non-neuronal cells "curly fibers" or "tangles" immunoreactive with antibodies to P component, Tau-protein, ubiquitin, fibronectin, and Apolipoprotein-E, but lacking immunoreactivity with antibodies to neurofilament proteins. Ultrastructurally they consist of densely packed straight and paired helical filaments and closely resemble neurofibrillary tangles and neuropil threads. These observations indicate that the formation of "curly fibers" and "tangles" is not unique to the central nervous system. The results suggest that AD might be a systemic disorder or that similar fibrillary changes to tangles and curly fibers may also be associated with other amyloidosis than beta-amyloidosis. Further investigations are necessary to understand the pathogenetic interest of these fibrillary changes outside the CNS.

Differential phosphoproteomics of fragmenting yeast vacuoles

Many organelles exist in an equilibrium of fragmentation into smaller units and fusion into larger structures, which is coordinated with cell division, the increase in cell mass, and envi¬ronmental conditions. In yeast cells, organelle homeostasis can be studied using the yeast vacuole (lysosome) as a model system. Yeast vacuoles are the main compartment for degrada¬tion of cellular proteins and storage of nutrients, ions and metabolites. Fission and fusion of vacuoles can be induced by hyper- and hypotonic shock in vivo, respectively, and have also been reconstituted in vitro using isolated vacuoles. The conserved serine/threonine kinase TOR (target of rapamycin) is a central nutrient sensor and regulates cell growth and metabolism. In yeast, there are two TOR proteins, Torlp and Tor2p, which are part of larger protein complexes, TORCI and TORC2. Only TORCI is rapamycin-sensitive. Disregulation of TOR signaling is linked to a multitude of diseases in humans, e.g. cancer, neurodegenerative diseases and metabolic syndrome. It has been shown that TORCI localizes to the vacuole membrane, and recent findings of our laboratory demonstrated that TORCI positively regulates vacuole fragmentation. This suggests that the fragmentation machinery should contain target proteins phosphorylated by TORCI. I explored the rapamycin-and fission-dependent vacuolar phosphoproteome during frag¬mentation, using a label-free mass-spectrometry approach. I identified many vacuolar factors whose phosphorylation was downregulated in a TORCI- and fission-dependent manner. Among them were known protein complexes that are functionally linked to fission or fusion, like the HOPS, VTC and FAB1 complexes. Hence, TORCI-dependent phosphorylations might positively regulate vacuole fission. Several candidates were chosen for detailed microscopic analysis of in vivo vacuole frag-mentation, using deletion mutants. I was able to identify novel factors not previously linked to fission phenotypes, e.g. the SEA complex, Pib2, and several vacuolar amino acid transporters. Transport of neutral and basic amino acids across the membrane seems to control vacuole fission, possibly via TORCI. I analyzed vacuolar fluxes of amino acids in wildtype yeast cells and found evidence for a selective vacuolar export of basic amino acids upon hyperosmotic stress. This leads me to propose a model where vacuolar export of amino acids is necessary to reshape the organelle under salt stress. - Le nombre et la taille de certaines organelles peut être déterminé par un équilibre entre la fragmentation qui produit des unités plus petites et la fusion qui génère des structures plus larges. Cet équilibre est coordonné avec la division cellulaire, l'augmentation de la masse cellulaire, et les conditions environnementales. Dans des cellules de levure, l'homéostasie des organelles peut être étudié à l'aide d'un système modèle, la vacuole de levure (lysosome). Les vacuoles constituent le principal compartiment de la dégradation des protéines et de stockage des nutriments, des ions et des métabolites. La fragmentation et la fusion des vacuoles peuvent être respectivement induites par un traitement hyper- ou hypo-tonique dans les cellules vivantes. Ces processus ont également été reconstitués in vitro en utilisant des vacuoles isolées. La sérine/thréonine kinase conservée TOR (target of rapamycin/cible de la rapamycine) est un senseur de nutriments majeur qui régule la croissance cellulaire et le métabolisme. Chez la levure, il existe deux protéines TOR, Torlp et Tor2p, qui sont les constituants de plus grands complexes de protéines, TORCI et TORC2. TORCI est spécifiquement inhibé par la rapamycine. Une dysrégulation de la signalisation de TOR est liée à une multitude de maladies chez l'homme comme le cancer, les maladies neurodégénératives et le syndrome métabolique. Il a été montré que TORCI se localise à la membrane vacuolaire et les découvertes récentes de notre laboratoire ont montré que TORCI régule positivement la fragmentation de la vacuole. Ceci suggère que le mécanisme de fragmentation doit être contrôlé par la phosphorylation de certaines protéines cibles de TORCI. J'ai exploré le phosphoprotéome vacuolaire lors de la fragmentation, en présence ou absence de rapamycine et dans des conditions provoquant la fragmentation des organelles. La méthode choisie pour réaliser la première partie de ce projet a été la spectrométrie de masse différentielle sans marquage. J'ai ainsi identifié plusieurs facteurs vacuolaires dont la phosphorylation est régulée d'une manière dépendante de TORCI et de la fragmentation. Parmi ces facteurs, des complexes protéiques connus qui sont fonctionnellement liées à fragmentation ou la fusion, comme les complexes HOPS, VTC et FAB1 ont été mis en évidence. Par conséquent, la phosphorylation dépendante de TORCI peut réguler positivement la fragmentation des vacuoles. Plusieurs candidats ont été choisis pour une analyse microscopique détaillée de la fragmentation vacuolaire in vivo en utilisant des mutants de délétion. J'ai été en mesure d'identifier de nouveaux facteurs qui n'avaient pas été encore associés à des phénotypes de fragmentation tels que les complexes SEA, Pib2p, ainsi que plusieurs transporteurs vacuolaires d'acides aminés. Le transport des acides aminés à travers la membrane semble contrôler la fragmentation de la vacuole. Puisque ces transporteurs sont phosphorylés par TORCI, ces résultats semblent confirmer la

SET translocation is associated with increase in caspase cleaved amyloid precursor protein in CA1 of Alzheimer and Down syndrome patients.

Caspase cleaved amyloid precursor protein (APPcc) and SET are increased and mislocalized in the neuronal cytoplasm in Alzheimer Disease (AD) brains. Translocated SET to the cytoplasm can induce tau hyperphosphorylation. To elucidate the putative relationships between mislocalized APPcc and SET, we studied their level and distribution in the hippocampus of 5 controls, 3 Down syndrome and 10 Alzheimer patients. In Down syndrome and Alzheimer patients, APPcc and SET levels were increased in CA1 and the frequency of both localizations in the neuronal cytoplasm was high in CA1, and low in CA4. As the increase of APPcc is already present at early stages of AD, we overexpressed APPcc in CA1 and the dentate gyrus neurons of adult mice with a lentiviral construct. APPcc overexpression in CA1 and not in the dentate gyrus induced endogenous SET translocation and tau hyperphosphorylation. These data suggest that increase in APPcc in CA1 neurons could be an early event leading to the translocation of SET and the progression of AD through tau hyperphosphorylation.

Involvement of 4E-BP1 in the protection induced by HDLs on pancreatic beta cells.

High-density lipoproteins (HDLs) protect pancreatic beta cells against apoptosis. This property might relate to the increased risk to develop diabetes in patients with low HDL blood levels. The mechanisms by which HDLs protect beta cells are poorly characterized however. Here we used a transcriptomic approach to identify genes differentially modulated by HDLs in beta cells subjected to apoptotic stimuli. The transcript encoding 4E-BP1 was up-regulated by serum starvation and HDLs blocked this increase. 4E-BP1 inhibits cap-dependent translation in its non- or hypo-phosphorylated state but it looses this ability when hyper-phosphorylated. At the protein level, 4E-BP1 was also up-regulated in response to starvation and IL1beta and this was blunted by HDLs. While an ectopic increase of 4E-BP1 expression induced beta cell death, silencing 4E-BP1 increase with shRNAs inhibited the apoptotic-inducing capacities of starvation. HDLs can therefore protect beta cells by blocking 4E-BP1 protein expression but this is not the sole protective mechanism activated by HDLs. Indeed, HDLs blocked apoptosis induced by ER stress with no associated decrease in total 4E-BP1 induction. Although, HDLs favored the phosphorylation, and hence the inactivation of 4E-BP1 in these conditions, this appeared not to be required for HDL protection. Our results indicate that HDLs can protect beta cells through modulation of 4E-BP1 depending on the type of stress stimuli.

Sustained antipsychotic effect of metacognitive training in psychosis: a randomized-controlled study.

Persistent psychotic symptoms represent a major challenge for psychiatric care. Basic research has shown that psychotic symptoms are associated with cognitive biases. Metacognitive training (MCT) aims at helping patients to become aware of these biases and to improve problem-solving. Fifty-two participants fulfilling diagnostic criteria of schizophrenia or schizoaffective disorders and persistent delusions and stabilized antipsychotic medication were enrolled in this study. Following baseline assessment patients were randomized either to treatment as usual (TAU) conditions or TAU+MCT. The intervention consisted of eight weekly 1-hour sessions (maximum: 8 hours). Participants were assessed at 8 weeks and 6-months later by blind assessors. Participants were assessed with the Psychotic Symptoms Rating Scales (PSYRATS) and the positive subscale of the PANSS. Between-group differences in post- and pre-test values were significant at a medium effect size in favor of the MCT for the PSYRATS delusion scale and the positive scale of the PANSS both at post and follow-up. The results of this study indicate that MCT training has a surplus antipsychotic effect for patients suffering from schizophrenia-related disorders who demonstrate only a partial response to antipsychotic treatment and that the effect of the intervention persists for at least 6 months after the intervention.

Homeodomain binding motifs modulate the probability of odorant receptor gene choice in transgenic mice.

Odorant receptor (OR) genes constitute with 1200 members the largest gene family in the mouse genome. A mature olfactory sensory neuron (OSN) is thought to express just one OR gene, and from one allele. The cell bodies of OSNs that express a given OR gene display a mosaic pattern within a particular region of the main olfactory epithelium. The mechanisms and cis-acting DNA elements that regulate the expression of one OR gene per OSN - OR gene choice - remain poorly understood. Here, we describe a reporter assay to identify minimal promoters for OR genes in transgenic mice, which are produced by the conventional method of pronuclear injection of DNA. The promoter transgenes are devoid of an OR coding sequence, and instead drive expression of the axonal marker tau-β-galactosidase. For four mouse OR genes (M71, M72, MOR23, and P3) and one human OR gene (hM72), a mosaic, OSN-specific pattern of reporter expression can be obtained in transgenic mice with contiguous DNA segments of only ~300 bp that are centered around the transcription start site (TSS). The ~150bp region upstream of the TSS contains three conserved sequence motifs, including homeodomain (HD) binding sites. Such HD binding sites are also present in the H and P elements, DNA sequences that are known to strongly influence OR gene expression. When a 19mer encompassing a HD binding site from the P element is multimerized nine times and added upstream of a MOR23 minigene that contains the MOR23 coding region, we observe a dramatic increase in the number of transgene-expressing founders and lines and in the number of labeled OSNs. By contrast, a nine times multimerized 19mer with a mutant HD binding site does not have these effects. We hypothesize that HD binding sites in the H and P elements and in OR promoters modulate the probability of OR gene choice.

Role of the JNK-interacting protein 1/islet brain 1 in cell degeneration in Alzheimer disease and diabetes.

Numerous epidemiological studies and some pharmacological clinical trials show the close connection between Alzheimer disease (AD) and type 2 diabetes (T2D) and thereby, shed more light into the existence of possible similar pathogenic mechanisms between these two diseases. Diabetes increases the risk of developing AD and sensitizers of insulin currently used as diabetes drugs can efficiently slow cognitive decline of the neurological disorder. Deposits of amyloid aggregate and hyperphosphorylation of tau, which are hallmarks of AD, have been also found in degenerating pancreatic islets beta-cells of patients with T2D. These events may have a causal role in the pathogenesis of the two diseases. Increased c-Jun NH(2)-terminal kinase (JNK) activity is found in neurofibrillary tangles (NFT) of AD and promotes programmed cell death of beta-cells exposed to a diabetic environment. The JNK-interacting protein 1 (JIP-1), also called islet brain 1 (IB1) because it is mostly expressed in the brain and islets, is a key regulator of the JNK pathway in neuronal and beta-cells. JNK, hyperphosphorylated tau and IB1/JIP-1 all co-localize with amyloids deposits in NFT and islets of AD and patients with T2D. This review discusses the role of the IB1/JIP-1 and the JNK pathway in the molecular pathogenesis of AD and T2D.

Bcl10 controls TCR- and FcgammaR-induced actin polymerization.

Bcl10 plays an essential role in the adaptive immune response, because Bcl10-deficient lymphocytes show impaired Ag receptor-induced NF-kappaB activation and cytokine production. Bcl10 is a phosphoprotein, but the physiological relevance of this posttranslational modification remains poorly defined. In this study, we report that Bcl10 is rapidly phosphorylated upon activation of human T cells by PMA/ionomycin- or anti-CD3 treatment, and identify Ser(138) as a key residue necessary for Bcl10 phosphorylation. We also show that a phosphorylation-deficient Ser(138)/Ala mutant specifically inhibits TCR-induced actin polymerization yet does not affect NF-kappaB activation. Moreover, silencing of Bcl10, but not of caspase recruitment domain-containing MAGUK protein-1 (Carma1) induces a clear defect in TCR-induced F-actin formation, cell spreading, and conjugate formation. Remarkably, Bcl10 silencing also impairs FcgammaR-induced actin polymerization and phagocytosis in human monocytes. These results point to a key role of Bcl10 in F-actin-dependent immune responses of T cells and monocytes/macrophages.

Oxidation and ubiquitination in neurodegeneration.

It is widely accepted that protein oxidation is involved in a variety of diseases, including neurodegenerative diseases. Especially during aging, a reduction in anti-oxidant defence mechanisms leads to an increased formation of free radical oxygen species and consequently results in a damage of proteins, including mitochondrial and synaptic ones. Even those proteins involved in repair and protein clearance via the ubiquitin proteasome and lysosomal system are subject to damage and show a reduced function. Here, we will discuss a variety of mechanisms and provide examples where cognition is affected and where repair mechanisms are no longer sufficient to compensate for a dysfunction of damaged proteins or even may become toxic. Next to physiological deficits, an accumulation of deficient proteins in aggresomes may occur and result in a formation of pathological hallmark structures typical for aging and disease. A major challenge is how to prevent aberrant oxidation, given that oxidation plays an essential role in aging and neurodegenerative diseases. Particularly interesting are the possibilities to reduce the formation of radical oxygen species leading to a dysfunction of protein repair and protein clearance, or to a formation of toxic byproducts accelerating neurodegeneration.

Control of the proliferation of activated CD4+ T cells by connexins.

As expression of Cxs in cells of the immune system increases upon cellular activation, we investigated whether Cxs and especially CxHcs play a major role during T cell-mediated responses. In particular, we studied the expression of Cx43Hc following CD4(+) T cell stimulation using flow cytometry, real-time PCR, and Western blot analysis. We showed that expression of Cx43 and its phosphorylated isoforms increased in response to the engagement of CD3 and CD28. Cx43Hcs were found to be involved in sustaining proliferation of T cells, as assessed by cell cycle staining, thymidine incorporation assays, and CFSE analysis of cells exposed to mimetic peptide inhibitors of the plasma membrane Cx channels and antibodies generated to an extracellular region of Cx. The reduction of T cell proliferation mediated by Cx channel inhibitors suppressed cysteine uptake but not cytokine production. We conclude that upon antigen recognition, T cells require CxHc to sustain their clonal expansion.

Ubiquitination and cysteine nitrosylation during aging and Alzheimer's disease.

Protein oxidation and ubiquitination of brain proteins are part of mechanisms that modulate protein function or that inactivate proteins and target misfolded proteins to degradation. In this study, we focused on brain aging and on mechanism involved in neurodegeneration such as events occurring in Alzheimer's disease (AD). The goal was to identify differences in nitrosylated proteins - at cysteine residues, and in the composition of ubiquinated proteins between aging and Alzheimer's samples by using a proteomic approach. A polyclonal anti-S-nitrosyl-cysteine, a mono- and a polyclonal anti-ubiquitin antibody were used for the detection of modified or ubiquitinated proteins in middle-aged and aged human entorhinal autopsy brains tissues of 14 subjects without neurological signs and 8 Alzheimer's patients. Proteins were separated by one- and two-dimensional gel electrophoresis and analyzed by Coomassie blue and immuno-blot staining. We identified that the glial fibrillary acidic and tau proteins are more ubiquitinated in brain tissues of Alzheimer's patients. Furthermore, glial fibrillary proteins were also found in nitrosylated state and further characterized by 2D Western blots and identified. Since reactive astrocytes localized prominently around senile plaques one can speculate that elements of plaques such as beta-amyloid proteins may activate surrounding glial elements and proteins.

Motivational intervention to reduce cannabis use in young people with psychosis: a randomized controlled trial.

Background: Cannabis use has a negative impact on psychosis. Studies are needed to explore the efficacy of psychological interventions to reduce cannabis use in psychosis. Our aim is to study the efficacy of a specific motivational intervention on young cannabis users suffering from psychosis. Methods: Participants (aged less than 35 years) were randomly assigned to treatment as usual (TAU) alone, or treatment as usual plus motivational intervention (MI + TAU). TAU was comprehensive and included case management, early intervention and mobile team when needed. Assessments were completed at baseline and at 3, 6 and12 months follow-up. Results: Sixty-two participants (32 TAU and 30 MI + TAU) were included in the study. Cannabis use decreased in both groups at follow-up. Participants who received MI in addition to TAU displayed both a greater reduction in number of joints smoked per week and greater confidence to change cannabis use at 3 and 6 months follow-up, but differences between groups were nonsignificant at 12 months. Conclusions: MI is well accepted by patients suffering from psychosis and has a short-term impact on cannabis use when added to standard care. However, the differential effect was not maintained at 1-year follow-up. MI appears to be a useful active component to reduce cannabis use which should be integrated in routine clinical practice.

Thrombin-induced ischemic tolerance is prevented by inhibiting c-jun N-terminal kinase.

We have studied ischemic tolerance induced by the serine protease thrombin in two different models of experimental ischemia. In organotypic hippocampal slice cultures, we demonstrate that incubation with low doses of thrombin protects neurons against a subsequent severe oxygen and glucose deprivation. L-JNKI1, a highly specific c-jun N-terminal kinase (JNK) inhibitor, and a second specific JNK inhibitor, SP600125, prevented thrombin preconditioning (TPC). We also show that the exposure to thrombin increases the level of phosphorylated c-jun, the major substrate of JNK. TPC, in vivo, leads to significantly smaller lesion sizes after a 30-min middle cerebral artery occlusion (MCAo), and the preconditioned mice were better off in the three tests used to evaluate functional recovery. In accordance with in vitro results, TPC in vivo was prevented by administration of L-JNKI1, supporting a role for JNK in TPC. These results, from two different TPC models and with two distinct JNK inhibitors, show that JNK is likely to be involved in TPC.

Activity-dependent phosphorylation of SNAP-25 in hippocampal organotypic cultures.

Synaptosomal-associated protein of 25 kDa (SNAP-25) is thought to play a key role in vesicle exocytosis and in the control of transmitter release. However, the precise mechanisms of action as well as the regulation of SNAP-25 remain unclear. Here we show by immunoprecipitation that activation of protein kinase C (PKC) by phorbol esters results in an increase in SNAP-25 phosphorylation. In addition, immunochemical analysis of two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels shows that SNAP-25 focuses as three or four distinct spots in the expected range of molecular weight and isoelectric point. Changing the phosphorylation level of the protein by incubating the slices in the presence of either a PKC agonist (phorbol 12,13-dibutyrate) or antagonist (chelerythrine) modified the distribution of SNAP-25 among these spots. Phorbol 12,13-dibutyrate increased the intensity of the spots with higher molecular weight and lower isoelectric point, whereas chelerythrine produced the opposite effect. This effect was specific for regulators of PKC, as agonists of other kinases did not produce similar changes. Induction of long-term potentiation, a property involved in learning mechanisms, and production of seizures with a GABA(A) receptor antagonist also increased the intensity of the spots with higher molecular weight and lower isoelectric point. This effect was prevented by the PKC inhibitor chelerythrine. We conclude that SNAP-25 can be phosphorylated in situ by PKC in an activity-dependent manner.

Association atypique d'une démence sémantique, d'un syndrome cortico-basal et d'une tauopathie 4R = [Atypical association of semantic dementia, corticobasal syndrome and 4R tauopathy]

Enjeu et contexte de la recherche La dégénérescence lobaire fronto-temporale (DLFT) est une pathologie neurodégénérative aussi fréquente que la maladie d'Alzheimer parmi les adultes de moins de 65 ans. Elle recouvre une constellation de syndromes neuropsychiatriques et moteurs dont les caractéristiques cliniques et anatomo-pathologiques se recoupent partiellement. La plupart des cas de démence sémantique ne présentent pas de troubles moteurs et révèlent à l'autopsie des lésions ubiquitine-positives. Son association à un syndrome cortico-basal et à une tauopathie 4R est donc très inhabituelle. Le cas que nous présentons est le premier à disposer d'une description clinique complète, tant sur le plan cognitif que moteur, et d'une analyse génétique et histopathologique. Résumé de l'article Il s'agit d'un homme de 57 ans, sans antécédents familiaux, présentant une démence sémantique accompagnée de symptômes inhabituels dans ce contexte, tels qu'une dysfonction exécutive et en mémoire épisodique, une désorientation spatiale et une dyscalculie. Le déclin physique et cognitif fut rapidement progressif. Une année et demie plus tard, il développait en effet des symptômes moteurs compatibles initialement avec un syndrome de Richardson, puis avec un syndrome cortico-basal. Son décès survint à l'âge de 60 ans des suites d'une pneumonie sur broncho-aspiration. L'autopsie cérébrale mit en évidence une perte neuronale et de nombreuses lésions tau-4R-positives dans les lobes frontaux, pariétaux et temporaux, les ganglions de la base et le tronc cérébral. Aucune mutation pathologique n'a été décelée dans le gène MAPT (microtubule-associated protein tau). L'ensemble de ces éléments sont discutés dans le cadre des connaissances actuelles sur la DLFT. Conclusions et perspectives Ce cas illustre le recoupement important des différents syndromes de la DLFT, parfois appelée le « complexe de Pick ». De plus, la démence sémantique pourrait s'avérer cliniquement moins homogène que prévu. Les définitions actuelles de la démence sémantique omettent la description des symptômes cognitifs extra-sémantiques malgré l'accumulation de preuves de leur existence. La faible prévalence de la démence sémantique, ainsi que des différences dans les examens neuropsychologiques, peuvent expliquer en partie la raison de cette omission. La variabilité histopathologique de chaque phénotype de DLFT peut également induire des différences dans leur expression clinique. Dans un domaine aussi mouvant que la DLFT, la co- occurrence ou la succession de plusieurs syndromes cliniques est en outre probablement la règle plutôt que l'exception.