Genistein attenuates amyloid-beta-induced cognitive impairment in rats by modulation of hippocampal synaptotoxicity and hyperphosphorylation of Tau

Alzheimer's disease is a progressive neurodegenerative disorder characterized by extracellular accumulation of amyloid-beta (Aβ) peptide, which induces synaptic dysfunction, alteration of intracellular signaling pathways, hyperphosphorylation of the Tau protein, and cognitive impairment. Genistein, one of the major isoflavones present in soy and soy products, has been shown to modulate some of the pathogenic events associated with the neurodegeneration process. However, its underlying mechanisms remain to be clarified. Therefore, the objectives of the present study were to evaluate the ability of genistein to protect against Aβ_1–42-induced cognitive impairment in rats and to elucidate some of the possible mechanisms involved in its neuroprotective effects in the hippocampus. Male Wistar rats received bilateral intracerebroventricular infusions of Aβ_1–42 (2 nmol) and genistein 10 mg/kg orally for 10 days. The Aβ-infused animals showed significant impairment of memory, which was accompanied by the following neurochemical alterations in the hippocampus: decreased levels of the synaptic proteins synaptophysin and postsynaptic density protein 95 (PSD-95), hyperphosphorylation of Tau with increased activation of glycogen synthase kinase-3β and c-Jun N-terminal kinase, and inactivation of ERK. Treatment with genistein improved Aβ-induced cognitive impairment by attenuation of synaptotoxicity, hyperphosphorylation of Tau, and inactivation of ERK. Furthermore, treatment with this soy isoflavone did not cause systemic toxicity. These findings provide further evidence of the neuroprotective effect of genistein in an in vivo model of Aβ toxicity and, importantly, extend the current knowledge concerning the mechanisms associated with the neuroprotective effects of this compound in the hippocampus.     

Truncation and Activation of Dual Specificity Tyrosine Phosphorylation-regulated Kinase 1A by Calpain I: A MOLECULAR MECHANISM LINKED TO TAU PATHOLOGY IN ALZHEIMER DISEASE*

Hyperphosphorylation and dysregulation of exon 10 splicing of Tau are pivotally involved in pathogenesis of Alzheimer disease (AD) and/or other tauopathies. Alternative splicing of Tau exon 10, which encodes the second microtubule-binding repeat, generates Tau isoforms containing three and four microtubule-binding repeats, termed 3R-Taus and 4R-Taus, respectively. Dual specificity tyrosine-phosphorylation-regulated kinase 1A (Dyrk1A) lies at the Down syndrome critical region of chromosome 21. Overexpression of this kinase may contribute to the early Tau pathology in Down syndrome via phosphorylation of Tau and dysregulation of Tau exon 10. Here, we report that Dyrk1A was truncated at the C terminus and was associated with overactivation of calpain I in AD brain. Calpain I proteolyzed Dyrk1A in vitro first at the C terminus and further at the N terminus and enhanced its kinase activity toward Tau via increased V_max but not K_m. C-terminal truncation of Dyrk1A resulted in stronger activity than its full-length protein in promotion of exon 10 exclusion and phosphorylation of Tau. Dyrk1A was truncated in kainic acid-induced excitotoxic mouse brains and coincided with an increase in 3R-Tau expression and phosphorylation of Tau via calpain activation. Moreover, truncation of Dyrk1A was correlated with an increase in the ratio of 3R-Tau/4R-Tau and Tau hyperphosphorylation in AD brain. Collectively, these findings suggest that truncation/activation of Dyrk1A by Ca²⁺/calpain I might contribute to Tau pathology via promotion of exon 10 exclusion and hyperphosphorylation of Tau in AD brain.     

Cellular Models of Aggregation-dependent Template-directed Proteolysis to Characterize Tau Aggregation Inhibitors for Treatment of Alzheimer Disease

Alzheimer disease (AD) is a degenerative tauopathy characterized by aggregation of Tau protein through the repeat domain to form intraneuronal paired helical filaments (PHFs). We report two cell models in which we control the inherent toxicity of the core Tau fragment. These models demonstrate the properties of prion-like recruitment of full-length Tau into an aggregation pathway in which template-directed, endogenous truncation propagates aggregation through the core Tau binding domain. We use these in combination with dissolution of native PHFs to quantify the activity of Tau aggregation inhibitors (TAIs). We report the synthesis of novel stable crystalline leucomethylthioninium salts (LMTX®), which overcome the pharmacokinetic limitations of methylthioninium chloride. LMTX®, as either a dihydromesylate or a dihydrobromide salt, retains TAI activity in vitro and disrupts PHFs isolated from AD brain tissues at 0.16 μm. The K_i value for intracellular TAI activity, which we have been able to determine for the first time, is 0.12 μm. These values are close to the steady state trough brain concentration of methylthioninium ion (0.18 μm) that is required to arrest progression of AD on clinical and imaging end points and the minimum brain concentration (0.13 μm) required to reverse behavioral deficits and pathology in Tau transgenic mice.     

Drosophila melanogaster as a model organism for Alzheimer’s disease

Drosophila melanogaster provides an important resource for in vivo modifier screens of neurodegenerative diseases. To study the underlying pathogenesis of Alzheimer’s disease, fly models that address Tau or amyloid toxicity have been developed. Overexpression of human wild-type or mutant Tau causes age-dependent neurodegeneration, axonal transport defects and early death. Large-scale screens utilizing a neurodegenerative phenotype induced by eye-specific overexpression of human Tau have identified several kinases and phosphatases, apoptotic regulators and cytoskeleton proteins as determinants of Tau toxicity in vivo. The APP ortholog of Drosophila (dAPPl) shares the characteristic domains with vertebrate APP family members, but does not contain the human Aβ42 domain. To circumvent this drawback, researches have developed strategies by either direct secretion of human Aβ42 or triple transgenic flies expressing human APP, β-secretase and Drosophila γ-secretase presenilin (dPsn). Here, we provide a brief overview of how fly models of AD have contributed to our knowledge of the pathomechanisms of disease.     

Role of Cyclin-Dependent Kinase 5 in the Neurodegenerative Process Triggered by Amyloid-Beta and Prion Peptides: Implications for Alzheimer’s Disease and Prion-Related Encephalopathies

Tau hyperphosphorylation, amyloid plaques, and neuronal death are major neuropathological features of Alzheimer’s disease (AD) and Prion-related encephalopathies (PRE). Cyclin-dependent kinase 5 (Cdk5) is a serine/threonine kinase, active in post-mitotic neurons, where it regulates survival and death pathways. Overactivation of Cdk5 is conferred by p25, a truncated fragment of the p35 activator formed upon calpain activation. Cdk5 deregulation causes abnormal phosphorylation of microtubule-associated protein tau, leading to neurodegeneration. In this work we investigated the involvement of Cdk5 in the neurodegeneration triggered by amyloid-beta (Aβ) and prion (PrP) peptides, the culprit agents of AD and PRE. As a work model, we used cultured rat cortical neurons treated with Aβ_1–40 and PrP_106–126 synthetic peptides. The obtained data show that apoptotic neuronal death caused by both the peptides was in part due to Cdk5 deregulation. After peptide treatment, p25 levels were significantly enhanced in a pattern consistent with the augment in calpain activity. Moreover, Aβ_1–40 and PrP_106–126 increased the levels of tau protein phosphorylated at Ser202/Thr205. Cdk5 (roscovitine) and calpain (MDL28170) inhibitors reverted tau hyperphosphorylation and prevented neuronal death caused by Aβ_1–40 and PrP_106–126. This study demonstrates, for the first time, that Cdk5 is involved in PrP-neurotoxicity. Altogether, our data suggests that Cdk5 plays an active role in the pathogenesis of AD and PRE.     

Inhibition of phospholipase A2 increases Tau phosphorylation at Ser214 in embryonic rat hippocampal neurons

BackgroundArachidonic acid is released from cellular membranes by the action of phospholipase A₂ (PLA₂) and is implicated in microtubule-associated protein Tau phosphorylation. Tau hyperphosphorylation affects its ability to stabilize microtubules.ObjectiveTo determine the effect of PLA₂ inhibition on the phosphorylation state of Tau phosphoepitopes in primary cultures of hippocampal neurons.Methods4 DIC neurons were incubated at different concentrations of methyl-arachidonylfluorophosphonate (MAFP), an irreversible inhibitor of cPLA₂ and iPLA₂. Changes on Tau phosphorylation were determined by Western blotting with a panel of anti-Tau antibodies (C-terminal, Ser199/202, Ser202/205, Ser396 and Ser214).ResultsThe Ser214 site was hyperphosphorylated upon MAFP treatment. Significant differences were observed with 10 μM (p=0.01), 50 μM (p=0.01) and 100 μM (p=0.05) of MAFP. Less-intense changes were found in other phosphoepitopes.ConclusionThe present findings indicate that the phosphorylation of Ser214 is regulated by c- and/or iPLA₂, whereas other phosphoepitopes primarily regulated by GKS3b were not affected.     

A combinatorial native MS and LC-MS/MS approach reveals high intrinsic phosphorylation of human Tau but minimal levels of other key modifications

Abnormal changes of neuronal Tau protein, such as phosphorylation and aggregation, are considered hallmarks of cognitive deficits in Alzheimer''s disease. Abnormal phosphorylation is thought to precede aggregation and therefore to promote aggregation, but the nature and extent of phosphorylation remain ill-defined. Tau contains ∼85 potential phosphorylation sites, which can be phosphorylated by various kinases because the unfolded structure of Tau makes them accessible. However, methodological limitations (e.g. in MS of phosphopeptides, or antibodies against phosphoepitopes) led to conflicting results regarding the extent of Tau phosphorylation in cells. Here we present results from a new approach based on native MS of intact Tau expressed in eukaryotic cells (Sf9). The extent of phosphorylation is heterogeneous, up to ∼20 phosphates per molecule distributed over 51 sites. The medium phosphorylated fraction P_m showed overall occupancies of ∼8 P_i (± 5) with a bell-shaped distribution; the highly phosphorylated fraction P_h had 14 P_i (± 6). The distribution of sites was highly asymmetric (with 71% of all P-sites in the C-terminal half of Tau). All sites were on Ser or Thr residues, but none were on Tyr. Other known posttranslational modifications were near or below our detection limit (e.g. acetylation, ubiquitination). These findings suggest that normal cellular Tau shows a remarkably high extent of phosphorylation, whereas other modifications are nearly absent. This implies that abnormal phosphorylations at certain sites may not affect the extent of phosphorylation significantly and do not represent hyperphosphorylation. By implication, the pathological aggregation of Tau is not likely a consequence of high phosphorylation.     

Drosophila Spaghetti and Doubletime Link the Circadian Clock and Light to Caspases,Apoptosis and Tauopathy

While circadian dysfunction and neurodegeneration are correlated, the mechanism for this is not understood. It is not known if age-dependent circadian dysfunction leads to neurodegeneration or vice-versa, and the proteins that mediate the effect remain unidentified. Here, we show that the knock-down of a regulator (spag) of the circadian kinase Dbt in circadian cells lowers Dbt levels abnormally, lengthens circadian rhythms and causes expression of activated initiator caspase (Dronc) in the optic lobes during the middle of the day or after light pulses at night. Likewise, reduced Dbt activity lengthens circadian period and causes expression of activated Dronc, and a loss-of-function mutation in Clk also leads to expression of activated Dronc in a light-dependent manner. Genetic epistasis experiments place Dbt downstream of Spag in the pathway, and Spag-dependent reductions of Dbt are shown to require the proteasome. Importantly, activated Dronc expression due to reduced Spag or Dbt activity occurs in cells that do not express the spag RNAi or dominant negative Dbt and requires PDF neuropeptide signaling from the same neurons that support behavioral rhythms. Furthermore, reduction of Dbt or Spag activity leads to Dronc-dependent Drosophila Tau cleavage and enhanced neurodegeneration produced by human Tau in a fly eye model for tauopathy. Aging flies with lowered Dbt or Spag function show markers of cell death as well as behavioral deficits and shortened lifespans, and even old wild type flies exhibit Dbt modification and activated caspase at particular times of day. These results suggest that Dbt suppresses expression of activated Dronc to prevent Tau cleavage, and that the circadian clock defects confer sensitivity to expression of activated Dronc in response to prolonged light. They establish a link between the circadian clock factors, light, cell death pathways and Tau toxicity, potentially via dysregulation of circadian neuronal remodeling in the optic lobes.     

Electrochemical detection of anti-tau antibodies binding to tau protein and inhibition of GSK-3β-catalyzed phosphorylation

Tau protein hyperphosphorylation triggers tau aggregation and its toxicity, leading to neuronal death and cell-to-cell toxicity. Hence, inhibition of protein kinases is a viable tool toward reduction of tau toxicity. By targeting various epitopes of Tau441 protein immobilized on Au surface, the protein kinase inhibition by anti-tau antibodies was measured by surface electrochemistry. The electrochemical impedance spectroscopy was used to measure the charge transfer resistance (R_ct) of nonphosphorylated tau–Au film (nTau–Au) and compared with the phosphorylated tau–Au film (pTau–Au). The pTau–Au films were characterized by X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (TOF–SIMS), which indicated high phosphorus content. The R_ct factor was used as the measure of inhibition efficacies by anti-tau antibodies (D8, A10, P262, and Tau46) in addition to antibody formulation intravenous immunoglobulin (IVIG). The R_ct factor for pTau–Au in the absence of antibodies was 0.25 ± 0.08, indicating a dramatic decrease in R_ct on phosphorylation. The R_ct factors for Tau46 and A10 were 0.57 ± 0.22 and 0.65 ± 0.26, respectively, indicating phosphorylation inhibition. All antibodies exhibited similar binding to nTau–Au. The proposed electrochemical assay may be used for detection of other posttranslational modifications.     

Absence of Tau triggers age‐dependent sciatic nerve morphofunctional deficits and motor impairment

Dementia is the cardinal feature of Alzheimer's disease (AD), yet the clinical symptoms of this disorder also include a marked loss of motor function. Tau abnormal hyperphosphorylation and malfunction are well‐established key events in AD neuropathology but the impact of the loss of normal Tau function in neuronal degeneration and subsequent behavioral deficits is still debated. While Tau reduction has been increasingly suggested as therapeutic strategy against neurodegeneration, particularly in AD, there is controversial evidence about whether loss of Tau progressively impacts on motor function arguing about damage of CNS motor components. Using a variety of motor‐related tests, we herein provide evidence of an age‐dependent motor impairment in Tau?/? animals that is accompanied by ultrastructural and functional impairments of the efferent fibers that convey motor‐related information. Specifically, we show that the sciatic nerve of old (17–22‐months) Tau?/? mice displays increased degenerating myelinated fibers and diminished conduction properties, as compared to age‐matched wild‐type (Tau+/+) littermates and younger (4–6 months) Tau?/? and Tau+/+ mice. In addition, the sciatic nerves of Tau?/? mice exhibit a progressive hypomyelination (assessed by g‐ratio) specifically affecting large‐diameter, motor‐related axons in old animals. These findings suggest that loss of Tau protein may progressively impact on peripheral motor system.     

Activation of Asparaginyl Endopeptidase Leads to Tau Hyperphosphorylation in Alzheimer Disease

Neurofibrillary pathology of abnormally hyperphosphorylated Tau is a key lesion of Alzheimer disease and other tauopathies, and its density in the brain directly correlates with dementia. The phosphorylation of Tau is regulated by protein phosphatase 2A, which in turn is regulated by inhibitor 2, I₂^PP2A. In acidic conditions such as generated by brain ischemia and hypoxia, especially in association with hyperglycemia as in diabetes, I₂^PP2A is cleaved by asparaginyl endopeptidase at Asn-175 into the N-terminal fragment (I_2NTF) and the C-terminal fragment (I_2CTF). Both I_2NTF and I_2CTF are known to bind to the catalytic subunit of protein phosphatase 2A and inhibit its activity. Here we show that the level of activated asparaginyl endopeptidase is significantly increased, and this enzyme and I₂^PP2A translocate, respectively, from neuronal lysosomes and nucleus to the cytoplasm where they interact and are associated with hyperphosphorylated Tau in Alzheimer disease brain. Asparaginyl endopeptidase from Alzheimer disease brain could cleave GST-I₂^PP2A, except when I₂^PP2A was mutated at the cleavage site Asn-175 to Gln. Finally, an induction of acidosis by treatment with kainic acid or pH 6.0 medium activated asparaginyl endopeptidase and consequently produced the cleavage of I₂^PP2A, inhibition of protein phosphatase 2A, and hyperphosphorylation of Tau, and the knockdown of asparaginyl endopeptidase with siRNA abolished this pathway in SH-SY5Y cells. These findings suggest the involvement of brain acidosis in the etiopathogenesis of Alzheimer disease, and asparaginyl endopeptidase-I₂^PP2A-protein phosphatase 2A-Tau hyperphosphorylation pathway as a therapeutic target.     

Alzheimer's Disease Related Markers,Cellular Toxicity and Behavioral Deficits Induced Six Weeks after Oligomeric Amyloid-β Peptide Injection in Rats

Alzheimer’s disease (AD) is a neurodegenerative pathology associated with aging characterized by the presence of senile plaques and neurofibrillary tangles that finally result in synaptic and neuronal loss. The major component of senile plaques is an amyloid-β protein (Aβ). Recently, we characterized the effects of a single intracerebroventricular (icv) injection of Aβ fragment (25–35) oligomers (oAβ_25–35) for up to 3 weeks in rats and established a clear parallel with numerous relevant signs of AD. To clarify the long-term effects of oAβ_25–35 and its potential role in the pathogenesis of AD, we determined its physiological, behavioral, biochemical and morphological impacts 6 weeks after injection in rats. oAβ_25–35 was still present in the brain after 6 weeks. oAβ_25–35 injection did not affect general activity and temperature rhythms after 6 weeks, but decreased body weight, induced short- and long-term memory impairments, increased corticosterone plasma levels, brain oxidative (lipid peroxidation), mitochondrial (caspase-9 levels) and reticulum stress (caspase-12 levels), astroglial and microglial activation. It provoked cholinergic neuron loss and decreased brain-derived neurotrophic factor levels. It induced cell loss in the hippocampic CA subdivisions and decreased hippocampic neurogenesis. Moreover, oAβ_25–35 injection resulted in increased APP expression, Aβ_1–42 generation, and increased Tau phosphorylation. In conclusion, this in vivo study evidenced that the soluble oligomeric forms of short fragments of Aβ, endogenously identified in AD patient brains, not only provoked long-lasting pathological alterations comparable to the human disease, but may also directly contribute to the progressive increase in amyloid load and Tau pathology, involved in the AD physiopathology.     

Protein Disulfide Isomerase Interacts with Tau Protein and Inhibits Its Fibrillization

Background

Tau protein is implicated in the pathogenesis of neurodegenerative disorders such as tauopathies including Alzheimer disease, and Tau fibrillization is thought to be related to neuronal toxicity. Physiological inhibitors of Tau fibrillization hold promise for developing new strategies for treatment of Alzheimer disease. Because protein disulfide isomerase (PDI) is both an enzyme and a chaperone, and implicated in neuroprotection against Alzheimer disease, we want to know whether PDI can prevent Tau fibrillization. In this study, we have investigated the interaction between PDI and Tau protein and the effect of PDI on Tau fibrillization.

Methodology/Principal Findings

As evidenced by co-immunoprecipitation and confocal laser scanning microscopy, human PDI interacts and co-locates with some endogenous human Tau on the endoplasmic reticulum of undifferentiated SH-SY5Y neuroblastoma cells. The results from isothermal titration calorimetry show that one full-length human PDI binds to one full-length human Tau (or human Tau fragment Tau_244–372) monomer with moderate, micromolar affinity at physiological pH and near physiological ionic strength. As revealed by thioflavin T binding assays, Sarkosyl-insoluble SDS-PAGE, and transmission electron microscopy, full-length human PDI remarkably inhibits both steps of nucleation and elongation of Tau_244–372 fibrillization in a concentration-dependent manner. Furthermore, we find that two molecules of the a-domain of human PDI interact with one Tau_244–372 molecule with sub-micromolar affinity, and inhibit both steps of nucleation and elongation of Tau_244–372 fibrillization more strongly than full-length human PDI.

Conclusions/Significance

We demonstrate for the first time that human PDI binds to Tau protein mainly through its thioredoxin-like catalytic domain a, forming a 1∶1 complex and preventing Tau misfolding. Our findings suggest that PDI could act as a physiological inhibitor of Tau fibrillization, and have applications for developing novel strategies for treatment and early diagnosis of Alzheimer disease.     

Molecular implication of PP2A and Pin1 in the Alzheimer's disease specific hyperphosphorylation of Tau

Background

Tau phosphorylation and dephosphorylation regulate in a poorly understood manner its physiological role of microtubule stabilization, and equally its integration in Alzheimer disease (AD) related fibrils. A specific phospho-pattern will result from the balance between kinases and phosphatases. The heterotrimeric Protein Phosphatase type 2A encompassing regulatory subunit PR55/Bα (PP2A_T55α) is a major Tau phosphatase in vivo, which contributes to its final phosphorylation state. We use NMR spectroscopy to determine the dephosphorylation rates of phospho-Tau by this major brain phosphatase, and present site-specific and kinetic data for the individual sites including the pS202/pT205 AT8 and pT231 AT180 phospho-epitopes.

Methodology/Principal Findings

We demonstrate the importance of the PR55/Bα regulatory subunit of PP2A within this enzymatic process, and show that, unexpectedly, phosphorylation at the pT231 AT180 site negatively interferes with the dephosphorylation of the pS202/pT205 AT8 site. This inhibitory effect can be released by the phosphorylation dependent prolyl cis/trans isomerase Pin1. Because the stimulatory effect is lost with the dimeric PP2A core enzyme (PP2A_D) or with a phospho-Tau T231A mutant, we propose that Pin1 regulates the interaction between the PR55/Bα subunit and the AT180 phospho-epitope on Tau.

Conclusions/Significance

Our results show that phosphorylation of T231 (AT180) can negatively influence the dephosphorylation of the pS202/pT205 AT8 epitope, even without an altered PP2A pool. Thus, a priming dephosphorylation of pT231 AT180 is required for efficient PP2A_T55α-mediated dephosphorylation of pS202/pT205 AT8. The sophisticated interplay between priming mechanisms reported for certain Tau kinases and the one described here for Tau phosphatase PP2A_T55α may contribute to the hyperphosphorylation of Tau observed in AD neurons.     

Phosphorylation of Tau at Thr212, Thr231, and Ser262 Combined Causes Neurodegeneration

Abnormal hyperphosphorylation of the microtubule-associated protein Tau is a hallmark of Alzheimer disease and related diseases called tauopathies. As yet, the exact mechanism by which this pathology causes neurodegeneration is not understood. The present study provides direct evidence that Tau abnormal hyperphosphorylation causes its aggregation, breakdown of the microtubule network, and cell death and identifies phosphorylation sites involved in neurotoxicity. We generated pseudophosphorylated Tau proteins by mutating Ser/Thr to Glu and, as controls, to Ala. These mutations involved one, two, or three pathological phosphorylation sites by site-directed mutagenesis using as backbones the wild type or FTDP-17 mutant R406W Tau. Pseudophosphorylated and corresponding control Tau proteins were expressed transiently in PC12 and CHO cells. We found that a single phosphorylation site alone had little influence on the biological activity of Tau, except Thr²¹², which, upon mutation to Glu in the R406W background, induced Tau aggregation in cells, suggesting phosphorylation at this site along with a modification on the C-terminal of the protein facilitates self-assembly of Tau. The expression of R406W Tau pseudophosphorylated at Thr²¹², Thr²³¹, and Ser²⁶² triggered caspase-3 activation in as much as 85% of the transfected cells, whereas the corresponding value for wild type pseudophosphorylated Tau was 30%. Cells transfected with pseudophosphorylated Tau became TUNEL-positive.     

Activation of mTOR Ameliorates Fragile X Premutation rCGG Repeat-Mediated Neurodegeneration

Fragile X associated tremor/ataxia syndrome (FXTAS) is a late onset neurodegenerative disorder caused by aberrant expansion of CGG repeats in 5′ UTR of FMR1 gene. The elevated mRNA confers a toxic gain-of-function thought to be the critical event of pathogenesis. Expressing rCGG₉₀ repeats of the human FMR1 5′UTR in Drosophila is sufficient to induce neurodegeneration. Rapamycin has been demonstrated to attenuate neurotoxicity by inducing autophagy in various animal models of neurodegenerative diseases. Surprisingly, we observed rapamycin exacerbated rCGG₉₀-induced neurodegenerative phenotypes through an autophagy-independent mechanism. CGG₉₀ expression levels of FXTAS flies exposed to rapamycin presented no significant differences. We further demonstrated that activation of the mammalian target of rapamycin (mTOR) signaling could suppress neurodegeneration of FXTAS. These findings indicate that rapamycin will exacerbate neurodegeneration, and that enhancing autophagy is insufficient to alleviate neurotoxicity in FXTAS. Moreover, these results suggest mTOR and its downstream molecules as new therapeutic targets for FXTAS by showing significant protection against neurodegeneration.     

Loss of tau results in defects in photoreceptor development and progressive neuronal degeneration in Drosophila

Accumulations of Tau, a microtubule‐associated protein (MAP), into neurofibrillary tangles is a hallmark of Alzheimer's disease and other tauopathies. However, the mechanisms leading to this pathology are still unclear: the aggregates themselves could be toxic or the sequestration of Tau into tangles might prevent Tau from fulfilling its normal functions, thereby inducing a loss of function defect. Surprisingly, the consequences of losing normal Tau expression in vivo are still not well understood, in part due to the fact that Tau knockout mice show only subtle phenotypes, presumably due to the fact that mammals express several MAPs with partially overlapping functions. In contrast, flies express fewer MAP, with Tau being the only member of the Tau/MAP2/MAP4 family. Therefore, we used Drosophila to address the physiological consequences caused by the loss of Tau. Reducing the levels of fly Tau (dTau) ubiquitously resulted in developmental lethality, whereas deleting Tau specifically in neurons or the eye caused progressive neurodegeneration. Similarly, chromosomal mutations affecting dTau also caused progressive degeneration in both the eye and brain. Although photoreceptor cells initially developed normally in dTau knockdown animals, they subsequently degenerated during late pupal stages whereas weaker dTau alleles caused an age‐dependent defect in rhabdomere structure. Expression of wild type human Tau partially rescued the neurodegenerative phenotype caused by the loss of endogenous dTau, suggesting that the functions of Tau proteins are functionally conserved from flies to humans. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 1210–1225, 2014     

Taurine in adipocytes prevents insulin-mediated H<Subscript>2</Subscript>o<Subscript>2</Subscript> generation and activates Pka and lipolysis

Among many actions assigned to taurine (Tau), the most abundant amino acid in numerous mammalian tissues, it prevents high-fat diet-induced obesity with increasing resting energy expenditure. To sustain this Tau action, the goal of the present study was to explore the acute effects of Tau on baseline and on adrenaline, insulin and their second messengers to modulate lipolysis in white adipose tissue (WAT) cells from rat epididymis. The Tau effects in this topic were compared with those recorded with Gly, Cys and Met. Tau on its own did not modify baseline lipolysis. Tau raised isoproterenol- and dibutyryl-cAMP (Bt₂cAMP)-activated glycerol release. Gly diminished Bt₂cAMP-activated glycerol release, and Cys and Met had no effect. Cyclic AMP-dependent activation of protein kinase A (PKA) in cell-free extracts decreased slightly by Gly and was unaltered by Cys, Met, and Tau. PKA catalytic activity in cell-free extracts was stimulated by Tau and unchanged by Cys, Gly and Met. Gly and Tau effects on PKA disappeared when these amino acids were withdrawn by gel filtration. Insulin-mediated NADPH-oxidase (NOX) raises H₂O₂ pool, which promotes PKA subunit oxidation, and precludes its cAMP activation; thus, lipolysis is diminished. Tau, but not Cys, Gly and Met, inhibited, by as much as 70%, insulin-mediated H₂O₂ pool increase. These data suggested that Tau raised lipolysis in adipocytes by two mechanisms: stimulating cAMP-dependent PKA catalytic activity and favoring PKA activation by cAMP as a consequence of lowering the H₂O₂ pool.     

Neurofibrillary tangles mediated human neuronal tauopathies: insights from fly models

Tauopathies represent a group of neurodegenerative disorder which are characterized by the presence of tau positive specialized argyrophilic and insoluble intraneuronal and glial fibrillar lesions known as neurofibrillary tangles (NFTs). Tau is a neuron specific microtubule binding protein which is required for the integrity and functioning of neuronal cells, and hyperphosphorylation of tau and its subsequent aggregation and paired helical filaments (PHFs) and NFTs has emerged as one of the major pathogenic mechanisms of tauopathies in human and mammalian model systems. Modeling of human tauopathies in Drosophila results in manifestation of associated phenotypes, and a recent study has demonstrated that similar to human and mammalian models, accumulation of insoluble tau aggregates in the form of typical neurotoxic NFTs triggers the pathogenesis of tauopathies in fly models. In view of the availability of remarkable genetic tools, Drosophila tau models could be extremely useful for in-depth analysis of the role of NFTs in neurodegeneration and tau aetiology, and also for the screening of novel gene(s) and molecule(s) which suppress the toxicity of tau aggregates.     

Novel function of Tau in regulating the effects of external stimuli on adult hippocampal neurogenesis

Tau is a microtubule‐associated neuronal protein found mainly in axons. However, its presence in dendrites and dendritic spines is particularly relevant due to its involvement in synaptic plasticity and neurodegeneration. Here, we show that Tau plays a novel in vivo role in the morphological and synaptic maturation of newborn hippocampal granule neurons under basal conditions. Furthermore, we reveal that Tau is involved in the selective cell death of immature granule neurons caused by acute stress. Also, Tau deficiency protects newborn neurons from the stress‐induced dendritic atrophy and loss of postsynaptic densities (PSDs). Strikingly, we also demonstrate that Tau regulates the increase in newborn neuron survival triggered by environmental enrichment (EE). Moreover, newborn granule neurons from Tau^?/? mice did not show any stimulatory effect of EE on dendritic development or on PSD generation. Thus, our data demonstrate that Tau^?/? mice show impairments in the maturation of newborn granule neurons under basal conditions and that they are insensitive to the modulation of adult hippocampal neurogenesis exerted by both stimulatory and detrimental stimuli.