Inhibition of heat shock proteins increases autophagosome formation, and reduces the expression of APP, Tau, SOD1 G93A and TDP-43.

Aberrant expression and denaturation of Tau, amyloid-beta and TDP-43 can lead to cell death and is a major component of pathologies such as Alzheimer's Disease (AD). AD neurons exhibit a reduced ability to form autophagosomes and degrade proteins via autophagy. Using genetically manipulated colon cancer cells we determined whether drugs that directly inhibit the chaperone ATPase activity or cause chaperone degradation and endoplasmic reticulum stress signaling leading to macroautophagy could reduce the levels of these proteins. The antiviral chaperone ATPase inhibitor AR12 reduced the ATPase activities and total expression of GRP78, HSP90, and HSP70, and of Tau, Tau 301L, APP, APP692, APP715, SOD1 G93A and TDP-43. In parallel, it increased the phosphorylation of ATG13 S318 and eIF2A S51 and caused eIF2A-dependent autophagosome formation and autophagic flux. Knock down of Beclin1 or ATG5 prevented chaperone, APP and Tau degradation. Neratinib, used to treat HER2+ breast cancer, reduced chaperone levels and expression of Tau and APP via macroautophagy, and neratinib interacted with AR12 to cause further reductions in protein levels. The autophagy-regulatory protein ATG16L1 is expressed as two isoforms, T300 or A300: Africans trend to express T300 and Europeans A300. We observed higher basal expression of Tau in T300 cells when compared to isogenic A300 cells. ATG16L1 isoform expression did not alter basal levels of HSP90, HSP70 or HSP27, however, basal levels of GRP78 were reduced in A300 cells. The abilities of both AR12 and neratinib to stimulate ATG13 S318 and eIF2A S51 phosphorylation and autophagic flux was also reduced in A300 cells. Our data support further evaluation of AR12 and neratinib in neuronal cells as repurposed treatments for AD.

Identification of cis-acting determinants mediating the unconventional secretion of tau.

The deposition of tau aggregates throughout the brain is a pathological characteristic within a group of neurodegenerative diseases collectively termed tauopathies, which includes Alzheimer's disease. While recent findings suggest the involvement of unconventional secretory pathways driving tau into the extracellular space and mediating the propagation of the disease-associated pathology, many of the mechanistic details governing this process remain elusive. In the current study, we provide an in-depth characterization of the unconventional secretory pathway of tau and identify novel molecular determinants that are required for this process. Here, using Drosophila models of tauopathy, we correlate the hyperphosphorylation and aggregation state of tau with the disease-related neurotoxicity. These newly established systems recapitulate all the previously identified hallmarks of tau secretion, including the contribution of tau hyperphosphorylation as well as the requirement for PI(4,5)P2 triggering the direct translocation of tau. Using a series of cellular assays, we demonstrate that both the sulfated proteoglycans on the cell surface and the correct orientation of the protein at the inner plasma membrane leaflet are critical determinants of this process. Finally, we identify two cysteine residues within the microtubule binding repeat domain as novel cis-elements that are important for both unconventional secretion and trans-cellular propagation of tau.

Cannabinoid receptor CB2 ablation protects against TAU induced neurodegeneration.

Tauopathies are a group of neurodegenerative diseases characterized by the alteration/aggregation of TAU protein, for which there is still no effective treatment. Therefore, new pharmacological targets are being sought, such as elements of the endocannabinoid system (ECS). We analysed the occurrence of changes in the ECS in tauopathies and their implication in the pathogenesis. By integrating gene expression analysis, immunofluorescence, genetic and adeno-associated virus expressing TAU mouse models, we found a TAU-dependent increase in CB2 receptor expression in hippocampal neurons, that occurs as an early event in the pathology and was maintained until late stages. These changes were accompanied by alterations in the endocannabinoid metabolism. Remarkably, CB2 ablation in mice protects from neurodegeneration induced by hTAUP301L overexpression, corroborated at the level of cognitive behaviour, synaptic plasticity, and aggregates of insoluble TAU. At the level of neuroinflammation, the absence of CB2 did not produce significant changes in concordance with a possible neuronal location rather than its classic glial expression in these models. These findings were corroborated in post-mortem samples of patients with Alzheimer's disease, the most common tauopathy. Our results show that neurons with accumulated TAU induce the expression of the CB2 receptor, which enhances neurodegeneration. These results are important for our understanding of disease mechanisms, providing a novel therapeutic strategy to be investigated in tauopathies.

Structure-Based Design of Potent Selective Nanomolar Type-II Inhibitors of Glycogen Synthase Kinase-3ß.

For the first time, the in silico design, screening, and in vitro validation of potent GSK-3ß type-II inhibitors are presented. In the absence of crystallographic evidence for a DFG-out GSK-3ß activation loop conformation, computational models were designed using an adapted DOLPHIN approach and a method consisting of Prime loop refinement, induced-fit docking, and molecular dynamics. Virtual screening of the Biogenics subset from the ZINC database led to an initial selection of 20 Phase I compounds revealing two low micromolar inhibitors in an isolated enzyme assay. Twenty more analogues (Phase II compounds) related to the hit [pyrimidin-2-yl]amino-furo[3,2-b]furyl-urea scaffold were selected for structure-activity relationship analysis. The Phase II studies led to five highly potent nanomolar inhibitors, with compound 23 (IC50 =0.087 µM) > 100 times more potent than the best Phase I inhibitor, and selectivity for GSK-3ß inhibition compared to homologous kinases was observed. Ex vivo experiments (SH-SY5Y cell lines) for tau hyperphosphorylation revealed promising neuroprotective effects at low micromolar concentrations. The type-II inhibitor design has been unraveled as a potential route toward more clinically effective GSK-3ß inhibitors.

Estrogens Inhibit Amyloid-ß-Mediated Paired Helical Filament-Like Conformation of Tau Through Antioxidant Activity and miRNA 218 Regulation in hTau Mice.

BACKGROUND: The risk of developing Alzheimer's disease as well as its progression and severity are known to be different in men and women, and cognitive decline is greater in women than in men at the same stage of disease and could be correlated at least in part on estradiol levels. OBJECTIVE: In our work we found that biological sex influences the effect of amyloid-ß42 (Aß42) monomers on pathological tau conformational change. METHODS: In this study we used transgenic mice expressing the wild-type human tau (hTau) which were subjected to intraventricular (ICV) injections of Aß peptides in nanomolar concentration. RESULTS: We found that Aß42 produces pathological conformational changes and hyperphosphorylation of tau protein in male or ovariectomized female mice but not in control females. The treatment of ovariectomized females with estradiol replacement protects against the pathological conformation of tau and seems to be mediated by antioxidant activity as well as the ability to modulate the expression of miRNA 218 linked to tau phosphorylation. CONCLUSION: Our study indicates that factors as age, reproductive stage, hormone levels, and the interplay with other risk factors should be considered in women, in order to identify the best appropriate therapeutic approach in prevention of cognitive impairment.

ACE2 activator diminazene aceturate ameliorates Alzheimer's disease-like neuropathology and rescues cognitive impairment in SAMP8 mice.

Previously, we revealed that brain Ang-(1-7) deficiency was involved in the pathogenesis of sporadic Alzheimer's disease (AD). We speculated that restoration of brain Ang-(1-7) levels might have a therapeutic effect against AD. However, the relatively short duration of biological effect limited the application of Ang-(1-7) in animal experiments. Since Ang-(1-7) is generated by its metabolic enzyme ACE2, we then tested the efficacy of an ACE2 activator diminazene aceturate (DIZE) on AD-like neuropathology and cognitive impairment in senescence-accelerated mouse prone substrain 8 (SAMP8) mice, an animal model of sporadic AD. Eight-month-old SAMP8 mice were injected intraperitoneally with vehicle or DIZE once a day for 30 consecutive days. DIZE markedly elevated brain Ang-(1-7) and MAS1 levels. Meanwhile, DIZE significantly reduced the levels of Aß1-42, hyperphosphorylated tau and pro-inflammatory cytokines in the brain. The synaptic and neuronal losses in the brain were ameliorated by DIZE. Importantly, DIZE improved spatial cognitive functions in the Morris water maze test. In conclusion, this study demonstrates that DIZE ameliorates AD-like neuropathology and rescues cognitive impairment in SAMP8 mice. These beneficial effects of DIZE may be achieved by activating brain ACE2/Ang-(1-7)/MAS1 axis. These findings highlight brain ACE2/Ang-(1-7)/MAS1 axis as a potential target for the treatment of sporadic AD.

Microtubule-associated protein tau (MAPT) is a promising independent prognostic marker and tumor suppressive protein in clear cell renal cell carcinoma.

INTRODUCTION: Microtubule-associated protein tau (MAPT) overexpression has been linked to poor prognosis in several cancers. MAPT-AS1 is a long noncoding RNA existing at the antisense strand of the MAPT promoter region. The clinical significance of MAPT and MAPT-AS-1 in clear cell renal cell carcinoma (ccRCC) is unknown. This study aimed to assess the expression and function of MAPT and MAPT-AS1 in ccRCC. METHODS: The expression of MAPT was determined using immunohistochemistry in ccRCC. The effects of MAPT knockdown on cell growth and invasion were evaluated and the interaction between MAPT and microtubule-associated protein tau antisense (MAPT-AS1) were analyzed. The expression of MAPT-AS1 was determined using quantitative reverse transcription polymerase chain reaction in ccRCC tissues. We investigated the effect of MAPT-AS1 knockdown on cell growth and invasion. We analyzed the regulation of MAPT and MAPT-AS1. RESULTS: Immunohistochemistry in 135 ccRCC cases showed that 61% of the cases were positive for MAPT. Kaplan-Meier analysis showed that the low expression of MAPT was associated with poor overall survival after nephrectomy. Knockdown of MAPT enhanced cell growth and invasion. quantitative reverse transcription polymerase chain reaction revealed a positive correlation between MAPT and MAPT-AS1. The expression of MAPT-AS1 was higher in ccRCC tissue than in nonneoplastic kidney tissue. Kaplan-Meier analysis showed that the low expression of MAPT-AS1 was associated with poor overall survival after nephrectomy by in silico analysis. MAPT-AS1 knockdown promoted cell growth and invasion activity. P53 knockout suppressed the expression of MAPT and MAPT-AS1. CONCLUSION: These results suggest that MAPT and MAPT-AS1 may be promising predictive biomarkers for survival and play a tumor-suppressive role in ccRCC.

Cre-inducible Adeno Associated Virus-mediated Expression of P301L Mutant Tau Causes Motor Deficits and Neuronal Degeneration in the Substantia Nigra.

Accumulation of microtubule associated protein tau in the substantia nigra is associated with several tauopathies including progressive supranuclear palsy (PSP). A number of studies have used mutant tau transgenic mouse model to mimic the neuropathology of tauopathies and disease phenotypes. However, tau expression in these transgenic mouse models is not specific to brain subregions, and may not recapitulate subcortical disease phenotypes of PSP. It is necessary to develop a new disease modeling system for cell and region-specific expression of pathogenic tau for modeling PSP in mouse brain. In this study, we developed a novel strategy to express P301L mutant tau to the dopaminergic neurons of substantia nigra by coupling tyrosine hydroxylase promoter Cre-driver mice with a Cre-inducible adeno-associated virus (iAAV). The results showed that P301L mutant tau was successfully transduced in the dopaminergic neurons of the substantia nigra at the presence of Cre recombinase and iAAV. Furthermore, the iAAV-tau-injected mice displayed severe motor deficits including impaired movement ability, motor balance, and motor coordination compared to the control groups over a short time-course. Immunochemical analysis revealed that tau gene transfer significantly resulted in loss of tyrosine hydroxylase-positive dopaminergic neurons and elevated phosphorylated tau in the substantia nigra. Our development of dopaminergic neuron-specific neurodegenerative mouse model with tauopathy will be helpful for studying the underlying mechanism of pathological protein propagation as well as development of new therapies.

Phospho-Tau Protein Expression in the Cell Cycle of SH-SY5Y Neuroblastoma Cells: A Morphological Study.

It has been reported that the main function of tau protein is to stabilize microtubules and promote the movement of organelles through the axon in neurons. In Alzheimer's disease, tau protein is the major constituent of the paired helical filament, and it undergoes post-translational modifications including hyperphosphorylation and truncation. Whether other functions of tau protein are involved in Alzheimer's disease is less clear. We used SH-SY5Y human neuroblastoma cells as an in vitro model to further study the functions of tau protein. We detected phosphorylated tau protein as small dense dots in the cell nucleus, which strongly colocalize with intranuclear speckle structures that were also labelled with an antibody to SC35, a protein involved in nuclear RNA splicing. We have shown further that tau protein, phosphorylated at the sites recognized by pT231, TG-3, and AD2 antibodies, is closely associated with cell division. Different functions may be characteristic of phosphorylation at specific sites. Our findings suggest that the presence of tau protein is involved in separation of sister chromatids in anaphase, and that tau protein also participates in maintaining the integrity of the DNA (pT231, prophase) and chromosomes during cell division (TG-3).

Novel model of secreted human tau protein reveals the impact of the abnormal N-glycosylation of tau on its aggregation propensity.

Alzheimer's disease (AD) is the most common neurodegenerative disorder and has no disease-modifying treatment yet. The hallmarks of AD are two amyloidogenic proteins: tau and amyloid ß (Aß). Tau undergoes several posttranslational modifications, including N-glycosylation. Tau was reported to be N-glycosylated in AD brains, but not in healthy counterparts, which may affect AD etiology. Here, we aimed to examine the effect of N-glycosylation on aggregation propensity of tau. To that end, a novel SH-SY5Y cell-based model was generated in which recombinant human tau (htau) is forced to be secreted from the cells. Secreted htau was found to localize in the secretory pathway compartments and to undergo N-glycosylation. Following N-glycan cleavage of the secreted htau, various biophysical results collectively indicated that the untreated N-glycosylated secreted htau is markedly less aggregative, contains thinner and shorter fibrils, as compared to treated de-glycosylated secreted htau. This finding shows that N-glycans attached to htau may affect its aggregation. This could help to better understand the effect of N-glycosylated htau on AD progression.

Overexpression of MAPT-AS1 is associated with better patient survival in breast cancer.

Breast cancer is the most frequent malignant disease in women worldwide. It is a heterogeneous and complex genetic disease with different molecular characteristics. MAPT-AS1, a long non-coding RNA (lncRNA) existing at the anti-sense strand of the MAPT (microtubule associated protein tau) promoter region, was believed to regulate MAPT, which was associated with disease state in Parkinson's disease. But the role of MAPT-AS1 in breast cancer has never been reported. In our study we found that MAPT-AS1 is overexpressed in breast cancer but not in triple negative breast cancer (TNBC), and that high expression of MAPT-AS1 was correlated with better patient survival. In addition, the level of MAPT-AS1 was correlated with the expression of MAPT, and MAPT was associated with survival time in breast cancer. Our study suggests that MAPT-AS1 may play a role and be a potential survival predictive biomarker in breast cancer.

SN56 neuronal cell death after 24 h and 14 days chlorpyrifos exposure through glutamate transmission dysfunction, increase of GSK-3ß enzyme, ß-amyloid and tau protein levels.

Chlorpyrifos (CPF) is an organophosphate insecticide described to induce cognitive disorders, both after acute and repeated administration. However, the mechanisms through which it induces these effects are unknown. CPF has been reported to produce basal forebrain cholinergic neuronal cell death, involved on learning and memory regulation, which could be the cause of such cognitive disorders. Neuronal cell death was partially mediated by oxidative stress generation, P75NTR and α7-nAChRs gene expression alteration triggered through acetylcholinesterase (AChE) variants disruption, suggesting other mechanisms are involved. In this regard, CPF induces Aß and tau proteins production and activation of GSK3ß enzyme and alters glutamatergic transmission, which have been related with basal forebrain cholinergic neuronal cell death and development of cognitive disorders. According to these data, we hypothesized that CPF induces basal forebrain cholinergic neuronal cell death through induction of Aß and tau proteins production, activation of GSK-3ß enzyme and disruption of glutamatergic transmission. We evaluated this hypothesis in septal SN56 basal forebrain cholinergic neurons, after 24 h and 14 days CPF exposure. This study shows that CPF increases glutamate levels, upregulates GSK-3ß gene expression, and increases the production of Aß and phosphorylated tau proteins and all these effects reduced cell viability. CPF increases glutaminase activity and upregulates the VGLUT1 gene expression, which could mediate the disruption of glutamatergic transmission. Our present results provide new understanding of the mechanisms contributing to the harmful effects of CPF, and its possible relevance in the pathogenesis of neurodegenerative diseases.

Estrogen-related receptor alpha is involved in Alzheimer's disease-like pathology.

Estrogen-related receptor alpha (ERRα) is a transcriptional factor associated with mitochondrial biogenesis and energy metabolism. However, little is known about the role of ERRα in Alzheimer's disease (AD). Here, we report that in APP/PS1 mice, an animal model of AD, ERRα protein and mRNA were decreased in a region- and age-dependent manner. In HEK293 cells that stably express human full-length ß-amyloid precursor protein (APP), overexpression of ERRα inhibited the amyloidogenic processing of APP and consequently reduced Aß1-40/1-42 level. ERRα overexpression also attenuated Tau phosphorylation at selective sites, with the concomitant reduction of glycogen synthase kinase 3ß (GSK3ß) activity. Interestingly, alterations of APP processing and Tau phosphorylation induced by hydrogen peroxide were reversed by ERRα overexpression in HEK/APP cells. These results indicated that ERRα plays a functional role in AD pathology. By attenuating both amyloidogenesis and Tau phosphorylation, ERRα may serve as a potential therapeutic target for AD.

Abnormal tau induces cognitive impairment through two different mechanisms: synaptic dysfunction and neuronal loss.

The hyperphosphorylated microtubule-associated protein tau is present in several neurodegenerative diseases, although the causal relationship remains elusive. Few mouse models used to study Alzheimer-like dementia target tau phosphorylation. We created an inducible pseudophosphorylated tau (Pathological Human Tau, PH-Tau) mouse model to study the effect of conformationally modified tau in vivo. Leaky expression resulted in two levels of PH-Tau: low basal level and higher upon induction (4% and 14% of the endogenous tau, respectively). Unexpectedly, low PH-Tau resulted in significant cognitive deficits, decrease in the number of synapses (seen by EM in the CA1 region), reduction of synaptic proteins, and localization to the nucleus. Induction of PH-Tau triggered neuronal death (60% in CA3), astrocytosis, and loss of the processes in CA1. These findings suggest, that phosphorylated tau is sufficient to induce neurodegeneration and that two different mechanisms can induce cognitive impairment depending on the levels of PH-Tau expression.

Inducible Expression of a Truncated Form of Tau in Oligodendrocytes Elicits Gait Abnormalities and a Decrease in Myelin: Implications for Selective CNS Degenerative Diseases.

The cytoskeleton protein Tau present in oligodendrocytes (OLGs) promotes cellular process outgrowth and myelination; whereas abnormally hyperphosphorylated Tau has been shown to be present in the most debilitating form of multiple sclerosis and in selective dementias. This research examined the functional consequences of expressing a truncated form of Tau in OLGs during the second postnatal life. In particular, this truncated form of Tau (∆Tau) retains the Fyn-binding domain but lacks the microtubule-binding domain. Similar to hyperphosphorylated Tau, ∆Tau cannot bind the cytoskeleton and is missorted. The Cre/loxP recombination system was used to generate transgenic (TG) founder lines, which contain a Floxed LacZ-STOP cassette to prevent expression of enhanced green fluorescence protein (EGFP)-∆Tau. The founder lines were then crossed with a Tamoxifen (TM)-inducible proteolipid protein (PLP)-dependent Cre driver line. Myelin PLP is the major myelin protein in the central nervous system (CNS). TM was given at postnatal day (p) 12 for 3 days, and CNS tissues were collected at p22. Only TG mice with both EGFP-∆Tau and Cre manifested an overt phenotype of loss of balance and stumbles starting around p18. CNS tissues obtained from TM-treated EGFP-∆Tau/Cre double transgenic mice had recombined PCR products, GFP, and diminished brain myelin. GFP was expressed in OLGs, but not in neurons or astrocytes. On the contrary, TM-treated TG mice with only one of the two transgenes, i.e., Cre or Tau, did not have recombinant PCR products, GFP, diminished myelin, or abnormal phenotype. Thus, this inducible model shows for the first time that a non-microtubule-associated Tau protein in OLGs elicits both myelin decrease and gait abnormalities, similar to the occurrence in selective demyelinating and neurodegenerative diseases.

Regulation of human MAPT gene expression.

The number of known pathologies involving deregulated Tau expression/metabolism is increasing. Indeed, in addition to tauopathies, which comprise approximately 30 diseases characterized by neuronal aggregation of hyperphosphorylated Tau in brain neurons, this protein has also been associated with various other pathologies such as cancer, inclusion body myositis, and microdeletion/microduplication syndromes, suggesting its possible function in peripheral tissues. In addition to Tau aggregation, Tau deregulation can occur at the expression and/or splicing levels, as has been clearly demonstrated in some of these pathologies. Here, we aim to review current knowledge regarding the regulation of human MAPT gene expression at the DNA and RNA levels to provide a better understanding of its possible deregulation. Several aspects, including repeated motifs, CpG island/methylation, and haplotypes at the DNA level, as well as the key regions involved in mRNA expression and stability and the splicing patterns of different mRNA isoforms at the RNA level, will be discussed.

Tau and PTEN status as predictive markers for response to trastuzumab and paclitaxel in patients with HER2-positive breast cancer.

Trastuzumab (H)-based chemotherapy has been an active treatment in patients with HER2-positive breast cancer; however, primary and secondary resistance has occurred in patients treated with H alone or in combination with chemotherapy. Biomarkers were searched using tissue microarrays (TMA) in HER2-positive advanced breast cancer patients treated with H and paclitaxel (P) combination chemotherapy between October 2004 and August 2010. Tumor blocks were analyzed for Tau-protein, beta-III tubulin, PTEN, p27, IGF-1R, c-Met, CD44, and MUC4 by immunohistochemical (IHC) analysis. The correlation between IHC status and clinical outcomes, including response rate (RR), progression free survival (PFS), and overall survival (OS), was investigated. With a median follow-up duration of 54.1 months (range, 42.3-72.7 months), 65 patients received H + P chemotherapy. The overall RR was 63 % (95 % CI, 51-75 %), and seven patients (11 %) with high Tau/low PTEN expression showed a significantly lower RR (14 % vs. 69 %; p = 0.008). The odds ratio for a poor response was 13.3 (95 % CI, 1.5-119.0; p = 0.020). In addition, patients with high Tau/low PTEN showed a trend of poor survival in terms of PFS (6.6 months vs. 9.6 months, p = 0.052). Subsequent multivariate analysis showed that high Tau/low PTEN (hazard ratio [HR] 2.40, 95 % CI, 1.06-5.47; p = 0.037) was the poor prognostic factor independently associated with PFS after adjusting for possible confounding factors such as recurrence/metastasis, age, performance status, visceral metastasis, and hormone receptor status. High Tau-protein and low PTEN expression showed a significant association with poor response to H + P chemotherapy in patients with HER2-positive advanced breast cancer.

Predictive value of microtubule-associated protein Tau in patients with recurrent and metastatic breast cancer treated with taxane-containing palliative chemotherapy.

Tau is a member of microtubule-associated proteins (MAPs) and expressed in normal breast epithelium and breast cancer cells. Tau expression levels in early breast cancer were correlated with the responsiveness of taxane-containing chemotherapy. However, it is unknown whether Tau contributes to breast cancer progression. Herein, Tau expression in recurrent and metastatic breast cancer (RMBC) and its predictive significance in taxane-containing palliative chemotherapy were investigated. Immunohistochemical (IHC) staining was conducted to detect Tau protein expression levels in biopsies from 285 patients with RMBC, and the correlation between Tau expression and sensitivity to taxane was evaluated. One hundred twenty-one (42.46 %, 121/285) patients were Tau positive in their tumor. One hundred ninety-four (68.07 %, 194/285) patients were effective clinical remission, which evaluated with response evaluation criteria in solid tumors (RECIST) criteria. In this group, 141 (85.98 %, 141/194) patients were Tau negative. We further analyzed the correlation between Tau expression and clinicopathological characteristics. Tau expression was positively correlated to estrogen receptor (ER) status. Multivariate logistic regression analysis showed that Tau expression significantly differentiated patients with effective response to treatment (95 % confidence interval (CI): 4.230-13.88, P < 0.01). Tau expression was identified as an independent factor to predict the sensitivity of tumors to taxane-containing palliative chemotherapy in RMBC, suggesting that Tau expression in RMBC may serve as a clinical predictor for taxane-containing palliative chemotherapy.

Uptake of silica nanoparticles: neurotoxicity and Alzheimer-like pathology in human SK-N-SH and mouse neuro2a neuroblastoma cells.

Growing concern has been raised over the potential adverse effects of engineered nanoparticles on human health due to their increasing use in commercial and medical applications. Silica nanoparticles (SiNPs) are one of the most widely used nanoparticles in industry and have been formulated for cellular and non-viral gene delivery in the central nerve system. However, the potential neurotoxicity of SiNPs remains largely unclear. In this study, we investigated the cellular uptake of SiNPs in human SK-N-SH and mouse neuro2a (N2a) neuroblastoma cells treated with 10.0 µg/ml of 15-nm SiNPs for 24 h by transmission electron microscopy. We found that SiNPs were mainly localized in the cytoplasm of the treated cells. The treatment of SiNPs at various concentrations impaired the morphology of SK-N-SH and N2a cells, characterized by increased number of round cells, diminishing of dendrite-like processes and decreased cell density. SiNPs significantly decreased the cell viability, induced cellular apoptosis, and elevated the levels of intracellular reactive oxygen species (ROS) in a dose-dependent manner in both cell lines. Additionally, increased deposit of intracellular ß-amyloid 1-42 (Aß(1-42)) and enhanced phosphorylation of tau at Ser262 and Ser396, two specific pathological hallmarks of Alzheimer's disease (AD), were observed in both cell lines with SiNPs treatment. Concomitantly, the expression of amyloid precursor protein (APP) was up-regulated, while amyloid-ß-degrading enzyme neprilysin was down-regulated in SiNP-treated cells. Finally, activity-dependent phosphorylation of glycogen syntheses kinase (GSK)-3ß at Ser9 (inactive form) was significantly decreased in SiNP-treated SK-N-SH cells. Taken together, these data demonstrated that exposure to SiNPs induced neurotoxicity and pathological signs of AD. The pre-Alzheimer-like pathology induced by SiNPs might result from the dys-regulated expression of APP/neprilysin and activation of GSK-3ß. This is the first study with direct evidence indicating that in addition to neurotoxicity induced by SiNPs, the application of SiNPs might increase the risk of developing AD.

Microtubule-associated protein tau facilitates the targeted killing of proliferating cancer cells in vitro and in a xenograft mouse tumour model in vivo.

BACKGROUND: Antibody drug conjugates (ADCs) and immunotoxins (ITs) are promising anticancer immunotherapeutics. Despite their encouraging performance in clinical trials, both ADCs and ITs often suffer from disadvantages such as stoichiometrically undefined chemical linkage of the cytotoxic payload (ADCs) and the potential immunogenicity of toxins derived from bacteria and plants (ITs). METHODS: Human microtubule-associated protein tau (MAP) was cloned in-frame with human EGF, expressed in E. coli and purified by standard chromatographic methods. The in vitro activity was confirmed by flow cytometry, cell viability assays and tubulin polymerisation assay. The in vivo efficacy was demonstrated using noninvasive far-red in vivo imaging. RESULTS: The EGF-MAP selectively induced apoptosis in EGFR-overexpressing proliferating cancer cells through stabilisation of microtubules. Nonproliferating cells were not affected, demonstrating superior selectivity of EGF-MAP for cancer cells. The EGF-MAP was well tolerated at high doses in mice compared with the ETA'-based control. The in vivo efficacy of EGF-MAP was demonstrated in a tumour xenograft mouse model. CONCLUSION: Our data indicate the general mechanism of action for a new class of human immunotherapeutic reagents suitable for the treatment of cancer. This approach combines the binding specificity of targeting ligands with the selective cytotoxicity of MAP towards proliferating cells.