Malika Hamdane

Biochemistry of Tau in Alzheimer's disease and related neurological disorders

Microtubule-associated Tau proteins belong to a family of factors that polymerize tubulin dimers and stabilize microtubules. Tau is strongly expressed in neurons, localized in the axon and is essential for neuronal plasticity and network. From the very beginning of Tau discovery, proteomics methods have been essential to the knowledge of Tau biochemistry and biology. In this review, we have summarized the main contributions of several proteomic methods in the understanding of Tau, including expression, post-translational modifications and structure, in both physiological and pathophysiological aspects. Finally, recent advances in proteomics technology are essential to develop further therapeutic targets and early predictive and discriminative diagnostic assays for Alzheimer’s disease and related disorders.

Increased expression of BIN1 mediates Alzheimer genetic risk by modulating tau pathology

Genome-wide association studies (GWAS) have identified a region upstream the BIN1 gene as the most important genetic susceptibility locus in Alzheimer’s disease (AD) after APOE. We report that BIN1 transcript levels were increased in AD brains and identified a novel 3 bp insertion allele ∼28 kb upstream of BIN1, which increased (i) transcriptional activity in vitro, (ii) BIN1 expression levels in human brain and (iii) AD risk in three independent case-control cohorts (Meta-analysed Odds ratio of 1.20 (1.14–1.26) (P=3.8 × 10−11)). Interestingly, decreased expression of the Drosophila BIN1 ortholog Amph suppressed Tau-mediated neurotoxicity in three different assays. Accordingly, Tau and BIN1 colocalized and interacted in human neuroblastoma cells and in mouse brain. Finally, the 3 bp insertion was associated with Tau but not Amyloid loads in AD brains. We propose that BIN1 mediates AD risk by modulating Tau pathology.

PUFA induce antidepressant-like effects in parallel to structural and molecular changes in the hippocampus.

Epidemiological data suggest that omega-3 polyunsaturated fatty acids (PUFA) consumption may be inversely correlated to the prevalence and severity of depression but little is known about the underlying mechanisms. In this study, we experimentally investigated whether a chronic supplementation with PUFA may induce antidepressant-like effects in mice in parallel to brain structural and molecular changes. Six weeks feeding with a PUFA-enriched diet induced behavioral changes in the Forced Swim Test (FST), the Tail Suspension Test and the Novelty-Suppressed Feeding Test. Moreover, more than 5 weeks supplementation with a PUFA blend containing 70% alpha-linolenic acid induced antidepressant-like effects in the FST with an increase in both swimming and climbing behaviors. The combination of a shorter duration of PUFA supplementation with a low dose of imipramine also induced an additive effect in the FST. Finally, PUFA supplementation was associated with an increase in the hippocampal volume, an over-expression of both synaptophysin and BDNF, and a raise in the number of newborn cells. Besides the possible modulation of brain plasticity, present results highlight the effectiveness of PUFA given alone or in combination with antidepressant drug as potential treatment of depressive disorders.

Alkalizing Drugs Induce Accumulation of Amyloid Precursor Protein By-products in Luminal Vesicles of Multivesicular Bodies

Amyloid precursor protein (APP) metabolism is central to the pathogenesis of Alzheimer disease. We showed recently that the amyloid intracellular domain (AICD), which is released by γ-secretase cleavage of APP C-terminal fragments (CTFs), is strongly increased in cells treated with alkalizing drugs (Vingtdeux, V., Hamdane, M., Bégard, S., Loyens, A., Delacourte, A., Beauvillain, J.-C., Buée, L., Marambaud, P., and Sergeant, N. (2007) Neurobiol. Dis. 25, 686–696). Herein, we aimed to determine the cell compartment in which AICD accumulates. We show that APP-CTFs and AICD are present in multivesicular structures. Multivesicular bodies contain intraluminal vesicles (known as exosomes) when released in the extracellular space. We demonstrate that APP, APP-CTFs, and AICD are integrated and secreted within exosomes in differentiated neuroblastoma and primary neuronal culture cells. Together with recent data showing that amyloid-β is also found in exosomes, our data show that multivesicular bodies are essential organelles for APP metabolism and that all APP metabolites can be secreted in the extracellular space.

Mitotic-like tau phosphorylation by p25-Cdk5 kinase complex

Among tau phosphorylation sites, some phosphoepitopes referred to as abnormal ones are exclusively found on tau aggregated into filaments in Alzheimers disease. Recent data suggested that molecular mechanisms similar to those encountered during mitosis may play a role in abnormal tau phosphorylation. In particular, TG-3 phosphoepitope is associated with early stages of neurofibrillary tangles (NFTs). In this study, we reported a suitable cell model consisting of SH-SY5Y cells stably transfected with an inducible p25 expression vector. It allows investigation of tau phosphorylation by p25-Cdk5 kinase complex in a neuronal context and avoiding p25-induced cytotoxicity. Immunoblotting analyses showed that p25-Cdk5 strongly phosphorylates tau protein not only at the AT8 epitope but also at the AT180 epitope and at the Alzheimers mitotic epitope TG-3. Further biochemical analyses showed that abnormal phosphorylated tau accumulated in cytosol as a microtubule-free form, suggesting its impact on tau biological activity. Since tau abnormal phosphorylation occurred in dividing cells, TG-3 immunoreactivity was also investigated in differentiated neuronal ones, and both TG-3-immunoreactive tau and nucleolin, another early marker for NFT, were also generated. These data suggest that p25-Cdk5 is responsible for the mitotic-like phosphoepitopes present in NFT and argue for a critical role of Cdk5 in neurodegenerative mechanisms.

p25/Cdk5-mediated retinoblastoma phosphorylation is an early event in neuronal cell death

In large models of neuronal cell death, there is a tight correlation between Cdk5 deregulation and cell-cycle dysfunction. However, pathways that link Cdk5 to the cell cycle during neuronal death are still unclear. We have investigated the molecular events that precede p25/Cdk5-triggered neuronal death using a neuronal cell line that allows inducible p25 expression. In this system, no sign of apoptosis was seen before 24 hours of p25 induction. Thus, at that time, cell-cycle-regulatory proteins were analysed by immunoblotting and some of them showed a significant deregulation. Interestingly, after time-course experiments, the earliest feature correlated with p25 expression was the phosphorylation of the retinoblastoma protein (Rb). Indeed, this phosphorylation was observed 6 hours after p25 induction and was abolished in the presence of a Cdk5 inhibitor, roscovitine, which does not inhibit the usual Rb cyclin-D kinases Cdk4 and Cdk6. Furthermore, analyses of levels and subcellular localization of Cdk-related cyclins did not reveal any change following Cdk5 activation, arguing for a direct effect of Cdk5 activity on Rb protein. This latter result was clearly demonstrated by in vitro kinase assays showing that the p25-Cdk5 complex in our cell system phosphorylates Rb directly without the need for any intermediary kinase activity. Hence, Rb might be an appropriate candidate that connects Cdk5 to cell-cycle deregulation during neuronal cell death.

Activation of p65 NF-κB protein by p210BCR-ABL in a myeloid cell line (P210BCR-ABL activates p65 NF-κB)

The chimeric tyrosine kinase p210BCR – ABL is involved in the pathogenesis of chronic myelogenous leukemia. It transforms immature hematopoietic cells in vitro and abrogates IL-3-dependent growth. The mechanisms by which p210BCR – ABL mediates its oncogenicity are not well elucidated. Identifying transcription factors targeted by the chimeric protein may help to clarify these mechanisms. We have analysed the effect of p210BCR – ABL expression on NF-κB activity in DA1 cells (an IL-3-dependent murine myeloid progenitor cell line). A specific stimulation of NF-κB activity by kinase-active wild-type p210BCR – ABL has been evidenced by transcriptional activation assays. Electrophoretic mobility supershift assays revealed the presence of p65 protein (RelA) DNA binding activity in p210BCR – ABL transformed DA1 cells but not in parental DA1 cells. Activation of RelA in transformed DA1 cells may occur by protein stabilization. Experiments using oligonucleotides antisense to RelA showed that p210BCR – ABL transfected cells failed to survive after IL-3 removal. Moreover, inhibition of cellular growth was shown following treatment of p210BCR – ABL transformed DA1 cells by p65 antisense oligonucleotides. This study suggests that p65 NF-κB may be an effector for p210BCR – ABL and probably contributes to its induced transformation process.

Beneficial effects of exercise in a transgenic mouse model of Alzheimer's disease-like Tau pathology.

Tau pathology is encountered in many neurodegenerative disorders known as tauopathies, including Alzheimers disease. Physical activity is a lifestyle factor affecting processes crucial for memory and synaptic plasticity. Whether long-term voluntary exercise has an impact on Tau pathology and its pathophysiological consequences is currently unknown. To address this question, we investigated the effects of long-term voluntary exercise in the THY-Tau22 transgenic model of Alzheimers disease-like Tau pathology, characterized by the progressive development of Tau pathology, cholinergic alterations and subsequent memory impairments. Three-month-old THY-Tau22 mice and wild-type littermates were assigned to standard housing or housing supplemented with a running wheel. After 9 months of exercise, mice were evaluated for memory performance and examined for hippocampal Tau pathology, cholinergic defects, inflammation and genes related to cholesterol metabolism. Exercise prevented memory alterations in THY-Tau22 mice. This was accompanied by a decrease in hippocampal Tau pathology and a prevention of the loss of expression of choline acetyltransferase within the medial septum. Whereas the expression of most cholesterol-related genes remained unchanged in the hippocampus of running THY-Tau22 mice, we observed a significant upregulation in mRNA levels of NPC1 and NPC2, genes involved in cholesterol trafficking from the lysosomes. Our data support the view that long-term voluntary physical exercise is an effective strategy capable of mitigating Tau pathology and its pathophysiological consequences.

The Peptidylprolyl cis/trans-Isomerase Pin1 Modulates Stress-induced Dephosphorylation of Tau in Neurons IMPLICATION IN A PATHOLOGICAL MECHANISM RELATED TO ALZHEIMER DISEASE

Deregulation of Tau phosphorylation is a key question in Alzheimer disease pathogenesis. Recently, Pin1, a peptidylprolyl cis/trans-isomerase, was proposed to be a new modulator in Tau phosphorylation in Alzheimer disease. In vitro, Pin1 was reported to present a high affinity for both Thr(P)-231, a crucial site for microtubule binding, and Thr(P)-212. In fact, Pin1 may facilitate Thr(P)-231 dephosphorylation by protein phosphatase 2A through trans isomerization of the Thr(P)-Pro peptide bound. However, whether Pin1 binding to Tau leads to isomerization of a single site or of multiple Ser/Thr(P)-Pro sites in vivo is still unknown. In the present study, Pin1 involvement was investigated in stress-induced Tau dephosphorylation with protein phosphatase 2A activation. Both oxidative (H2O2) and heat stresses induced hypophosphorylation of a large set of phospho-Tau epitopes in primary cortical cultures. In both cases, juglone, a Pin1 pharmacological inhibitor, partially prevented dephosphorylation of Tau at Thr-231 among a set of phosphoepitopes tested. Moreover, Pin1 is physiologically found in neurons and partially co-localized with Tau. Furthermore, in Pin1-deficient neuronal primary cultures, H2O2 stress-induced Tau dephosphorylation at Thr(P)-231 was significantly lower than in wild type neurons. Finally, Pin1 transfection in Pin1-deficient neuronal cell cultures allowed for rescuing the effect of H2O2 stress-induced Tau dephosphorylation, whereas a Pin1 catalytic mutant did not. This is the first demonstration of an in situ Pin1 involvement in a differential Tau dephosphorylation on the full-length multiphosphorylated substrate.

Beneficial effects of caffeine in a transgenic model of Alzheimer's disease-like tau pathology

Tau pathology found in Alzheimers disease (AD) is crucial in cognitive decline. Epidemiologic evidences support that habitual caffeine intake prevents memory decline during aging and reduces the risk to develop Alzheimers disease. So far, experimental studies addressed the impact of caffeine in models mimicking the amyloid pathology of AD. However, in vivo effects of caffeine in a model of AD-like tauopathy remain unknown. Here, we evaluated effects of chronic caffeine intake (0.3 g/L through drinking water), given at an early pathologic stage, in the THY-Tau22 transgenic mouse model of progressive AD-like tau pathology. We found that chronic caffeine intake prevents from the development of spatial memory deficits in tau mice. Improved memory was associated with reduced hippocampal tau phosphorylation and proteolytic fragments. Moreover, caffeine treatment mitigated several proinflammatory and oxidative stress markers found upregulated in the hippocampus of THY-Tau22 animals. Together, our data support that moderate caffeine intake is beneficial in a model of AD-like tau pathology, paving the way for future clinical evaluation in AD patients.