IFN-γ stimulates Paneth cell secretion through necroptosis mTORC1 dependent.

Immune mediators affect multiple biological functions of intestinal epithelial cells (IECs) and, like Paneth and Paneth-like cells, play an important role in intestinal epithelial homeostasis. IFN-γ a prototypical proinflammatory cytokine disrupts intestinal epithelial homeostasis. However, the mechanism underlying the process remains unknown. In this study, using in vivo and in vitro models we demonstrate that IFN-γ is spontaneously secreted in the small intestine. Furthermore, we observed that this cytokine stimulates mitochondrial activity, ROS production, and Paneth and Paneth-like cell secretion. Paneth and Paneth-like secretion downstream of IFN-γ, as identified here, is mTORC1 and necroptosis-dependent. Thus, our findings revealed that the pleiotropic function of IFN-γ also includes the regulation of Paneth cell function in the homeostatic gut.

Association of α-1-Antichymotrypsin Expression with the Development of Conformational Changes of Tau Protein in Alzheimer's Disease Brain.

In Alzheimer's disease (AD), two mutually exclusive amino-terminal-dependent conformations have been reported to occur during the aggregation of Tau protein into neurofibrillary tangles (NFTs). An early conformation of full-length Tau, involving the bending of the amino terminus over the third repeated domain, is recognized by the Alz-50 antibody, followed by a second conformation recognized by Tau-66 antibody that depends on the folding of the proline-rich region over the third repeated domain in a molecule partially truncated at the amino- and carboxyl-termini. α-1-antichymotrypsin (ACT) is an acute phase serum glycoprotein that accumulates abnormally in the brain of AD patients, and since it is considered to promote the in vitro and in vivo aggregation of amyloid-ß, we here seek further evidence that ACT may also contribute to the abnormal aggregation of Tau in AD. By analyzing brain samples from a population of AD cases under immunofluorescence and high-resolution confocal microscopy, we demonstrate here the abundant expression of ACT in hippocampal neurons, visualized as a granular diffuse accumulation, frequently reaching the nuclear compartment. In a significant number of these neurons, intracellular NFTs composed of abnormally phosphorylated and truncated Tau at Asp421 were also observed to coexist in separated regions of the cytoplasm. However, we found strong colocalization between ACT and diffuse aggregates of Tau-66-positive granules, which was not observed with Alz-50 antibody. These results suggest that ACT may play a role during the development of Tau conformational changes facilitating its aggregation during the formation of the neurofibrillary pathology in AD.

Current Evidence on the Protective Effects of Recombinant Human Erythropoietin and Its Molecular Variants against Pathological Hallmarks of Alzheimer's Disease.

Substantial evidence in the literature demonstrates the pleiotropic effects of the administration of recombinant human erythropoietin (rhEPO) and its molecular variants in different tissues and organs, including the brain. Some of these reports suggest that the chemical properties of this molecule by itself or in combination with other agents (e.g., growth factors) could provide the necessary pharmacological characteristics to be considered a potential protective agent in neurological disorders such as Alzheimer's disease (AD). AD is a degenerative disorder of the brain, characterized by an aberrant accumulation of amyloid ß (Aß) and hyperphosphorylated tau (tau-p) proteins in the extracellular and intracellular space, respectively, leading to inflammation, oxidative stress, excitotoxicity, and other neuronal alterations that compromise cell viability, causing neurodegeneration in the hippocampus and the cerebral cortex. Unfortunately, to date, it lacks an effective therapeutic strategy for its treatment. Therefore, in this review, we analyze the evidence regarding the effects of exogenous EPOs (rhEPO and its molecular variants) in several in vivo and in vitro Aß and tau-p models of AD-type neurodegeneration, to be considered as an alternative protective treatment to this condition. Particularly, we focus on analyzing the differential effect of molecular variants of rhEPO when changes in doses, route of administration, duration of treatment or application times, are evaluated for the improved cellular alterations generated in this disease. This narrative review shows the evidence of the effectiveness of the exogenous EPOs as potential therapeutic molecules, focused on the mechanisms that establish cellular damage and clinical manifestation in the AD.

Early but not late conformational changes of tau in association with ubiquitination of neurofibrillary pathology in Alzheimer's disease brains.

In Alzheimer's disease, tau protein undergoes post-translational modifications including hyperphosphorylation and truncation, which promotes two major conformational changes associated with progressive N-terminal folding. Along with the development of the disease, tau ubiquitination was previously shown to emerge in the early and intermediate stages of the disease, which is closely associated with early tau truncation at aspartic acid 421, but not with a subsequently truncated tau molecule at glutamic acid 391. In the same group of cases, using multiple immunolabeling and confocal microscopy, a possible relationship between the ubiquitin-targeting of tau and the progression of conformational changes adopted by the N-terminus of this molecule was further studied. A comparable number of neurofibrillary tangles was found displaying ubiquitin, an early conformation recognized by the Alz-50 antibody, and a phosphorylation. However, a more reduced number of neurofibrillary tangles were immunoreactive to Tau-66 antibody, a late tau conformational change marker. When double-labeling profiles of neurofibrillary tangles were assessed, ubiquitination was clearly demonstrated in tau molecules undergoing early N-terminal folding, but was barely observed in late conformational changes of the N-terminus adopted by tau. The same pattern of colocalization was visualized in neuritic pathology. Overall, these results indicate that a more intact conformation of the N-terminus of tau may facilitate tau ubiquitination, but this modification may not occur in a late truncated and more compressed folding of the N-terminus of the tau molecule.

Fragmentation of the Golgi Apparatus in Neuroblastoma Cells Is Associated with Tau-Induced Ring-Shaped Microtubule Bundles.

Abnormal fibrillary aggregation of tau protein is a pathological condition observed in Alzheimer's disease and other tauopathies; however, the presence and pathological significance of early non-fibrillary aggregates of tau remain under investigation. In cell and animal models expressing normal or modified tau, toxic effects altering the structure and function of several membranous organelles have also been reported in the absence of fibrillary structures; however, how these abnormalities are produced is an issue yet to be addressed. In order to obtain more insights into the mechanisms by which tau may disturb intracellular membranous elements, we transiently overexpressed human full-length tau and several truncated tau variants in cultured neuroblastoma cells. After 48 h of transfection, either full-length or truncated tau forms produced significant fragmentation of the Golgi apparatus (GA) with no changes in cell viability. Noteworthy is that in the majority of cells exhibiting dispersion of the GA, a ring-shaped array of cortical or perinuclear microtubule (Mt) bundles was also generated under the expression of either variant of tau. In contrast, Taxol treatment of non-transfected cells increased the amount of Mt bundles but not sufficiently to produce fragmentation of the GA. Tau-induced ring-shaped Mt bundles appeared to be well-organized and stable structures because they were resistant to Nocodazole post-treatment and displayed a high level of tubulin acetylation. These results further indicate that a mechanical force generated by tau-induced Mt-bundling may be responsible for Golgi fragmentation and that the repeated domain region of tau may be the main promoter of this effect.

Plasmonic/Magnetic Multifunctional nanoplatform for Cancer Theranostics.

A multifunctional magneto-plasmonic CoFe2O4@Au core-shell nanoparticle was developed by iterative-seeding based method. This nanocargo consists of a cobalt ferrite kernel as a core (Nk) and multiple layers of gold as a functionalizable active stratum, (named as Nk@A after fifth iteration). Nk@A helps in augmenting the physiological stability and enhancing surface plasmon resonance (SPR) property. The targeted delivery of Doxorubicin using Nk@A as a nanopayload is demonstrated in this report. The drug release profile followed first order rate kinetics optimally at pH 5.4, which is considered as an endosomal pH of cells. The cellular MR imaging showed that Nk@A is an efficient T2 contrast agent for both L6 (r2-118.08 mM-1s-1) and Hep2 (r2-217.24 mM-1s-1) cells. Microwave based magnetic hyperthermia studies exhibited an augmentation in the temperature due to the transformation of radiation energy into heat at 2.45 GHz. There was an enhancement in cancer cell cytotoxicity when hyperthermia combined with chemotherapy. Hence, this single nanoplatform can deliver 3-pronged theranostic applications viz., targeted drug-delivery, T2 MR imaging and hyperthermia.

Expression of Tau Produces Aberrant Plasma Membrane Blebbing in Glial Cells Through RhoA-ROCK-Dependent F-Actin Remodeling.

Abnormal aggregation of Tau in glial cells has been reported in Alzheimer's disease (AD) and other tauopathies; however, the pathological significance of these aggregates remains unsolved to date. In this study, we evaluated whether full-length Tau (Tau441) and its aspartic acid421-truncated Tau variant (Tau421) produce alterations in the normal organization of the cytoskeleton and plasma membrane (PM) when transiently expressed in cultured C6-glial cells. Forty-eight hours post-transfection, abnormal microtubule bundling was observed in the majority of the cells, which expressed either Tau441 or Tau421. Moreover, both variants of Tau produced extensive PM blebbing associated with cortical redistribution of filamentous actin (F-Actin). These effects were reverted when Tau-expressing cells were incubated with drugs that depolymerize F-Actin. In addition, when glial cells showing Tau-induced PM blebbing were incubated with inhibitors of the Rho-associated protein kinase (ROCK) signaling pathway, both formation of abnormal PM blebs and F-Actin remodeling were avoided. All of these effects were initiated upstream by abnormal Tau-induced microtubule bundling, which may release the microtubule-bound guanine nucleotide exchange factor-H1 (GEF-H1) into the cytoplasm in order to activate its major effector RhoA-GTPase. These results may represent a new mechanism of Tau toxicity in which Tau-induced microtubule bundling produces activation of the Rho-GTPase-ROCK pathway that in turn mediates the remodeling of cortical Actin and PM blebbing. In AD and other tauopathies, these Tau-induced abnormalities may occur and contribute to the impairment of glial activity.

Alterations in the nuclear architecture produced by the overexpression of tau protein in neuroblastoma cells.

Abnormal intracellular aggregation of tau protein is a pathological condition leading to neuronal death in Alzheimer's disease. Fibrillar and nonfibrillar aggregates of tau protein alter the normal functioning of neurons by disturbing important cellular processes and distinct membranous organelles. However, tau-caused alterations in the nuclear compartment are not totally established so far. In our study we evaluated whether tau protein and its Asp421-truncated variant produce alterations in the normal architecture of the nucleus when expressed in cultured neuroblastoma cells. After 48 hours of transfection, significant deformity of the nuclear compartment with extensive lobulations along the nuclear envelope was observed in SH-SY5Y cells expressing either full-length tau or Asp421-truncated tau. This aberrant formation did not involve either nuclear fragmentation or cell death. The lobulated nuclei were devoid of tau protein, which mostly remained in the cytoplasm in a nonfibrillar state. Degradation of nuclear Lamins was not observed in tau-expressing SH-SY5Y cells, and a cell-cycle analysis did not show aberrant chromosome accumulation. Thus multiple division defects leading to multinucleation were discarded. The lobulated nuclei in tau-expressing SH-SY5Y cells seem to more resemble the multilobular phenotype of the nuclear envelope seen in Lamin-mutated cells from those pathological conditions leading to premature aging. Nevertheless, in our tau-expressing cells, the abnormal formation of cortical and perinuclear rings of tubulin generated by tau binding may be a more feasible mechanism of a nuclear-cytoskeleton generating force that causes the nuclear deformation.

Myotonic dystrophy type 1-associated CTG repeats disturb the expression and subcellular distribution of microtubule-associated proteins MAP1A, MAP2, and MAP6/STOP in PC12 cells.

To study the effect of DM1-associated CTG repeats on neuronal function, we developed a PC12 cell-based model that constitutively expresses the DMPK gene 3'-untranslated region with 90 CTG repeats (CTG90 cells). As CTG90 cells exhibit impaired neurite outgrowth and as microtubule-associated proteins (MAPs) are crucial for microtubule stability, we analyzed whether MAPs are a target of CTG repeats. NGF induces mRNA expression of Map2, Map1a and Map6 in control cells (PC12 cells transfected with the empty vector), but this induction is abolished for Map2 and Map1a in CTG90 cells. MAP2 and MAP6/STOP proteins decrease in NGF-treated CTG90 cells, whereas MAP1A increases. Data suggest that CTG repeats might alter somehow the expression of MAPs, which appears to be related with CTG90 cell-deficient neurite outgrowth. Decreased MAP2 levels found in the hippocampus of a DM1 mouse model indicates that targeting of MAPs expression by CTG repeats might be relevant to DM1.

Ubiquitin is associated with early truncation of tau protein at aspartic acid(421) during the maturation of neurofibrillary tangles in Alzheimer's disease.

Pathological processing of tau protein during the formation and maturation of neurofibrillary tangles (NFTs) includes abnormal phosphorylation, conformational changes and truncation of the C-terminus at aspartic-acid(421) (apoptotic product) and glutamic-acid(391) residues. Abnormal phosphorylation and misfolding may serve as recognition signals for ubiquitin-targeting and proteosomal processing. For this reason, we sought to determine whether ubiquitin-targeting of tau is associated with particular tau modifications that herald specific stages of NFTs maturation in the hippocampus of Alzheimer's disease cases. Using multiple tau antibodies, we found that 30% of the total load of NFTs is ubiquitin-associated. As reported previously ubiquitin immunoreactivity was associated with markers of phosphorylated tau in certain NFTs; however, a strong association was also found between ubiquitin and the earliest known truncation event at aspartic-acid(421) . These findings indicate that tau protein in the NFTs may be dually subjected to both apoptotic and proteosomal processing. By contrast ubiquitin immunoreactivity was poorly associated with truncation of tau at glutamic-acid(391) , suggesting that this proteolytic event may be independent of proteosomal activity. It would appear, therefore, that ubiquitin targeting of tau protein occurs at NFTs in the early and intermediate stages of the maturation.

Neuronal differentiation modulates the dystrophin Dp71d binding to the nuclear matrix.

The function of dystrophin Dp71 in neuronal cells remains unknown. To approach this issue, we have selected the PC12 neuronal cell line. These cells express both a Dp71f cytoplasmic variant and a Dp71d nuclear isoform. In this study, we demonstrated by electron and confocal microscopy analyses of in situ nuclear matrices and Western blotting evaluation of cell extracts that Dp71d associates with the nuclear matrix. Interestingly, this binding is modulated during NGF-induced neuronal differentiation of PC12 cells with a twofold increment in the differentiated cells, compared to control cells. Also, distribution of Dp71d along the periphery of the nuclear matrix observed in the undifferentiated cells is replaced by intense fluorescent foci localized in the center of the nucleoskeletal structure. In summary, we revealed that Dp71d is a dynamic component of nuclear matrix that might participate in the nuclear modeling occurring during neuronal differentiation.

Truncation of tau protein and its pathological significance in Alzheimer's disease.

Abnormal posttranslational modifications of tau protein lead it to aggregate into paired helical filaments in Alzheimer's disease (AD). The mechanisms involved in the early pathological processing of tau and the induction of a polymeric state seem to progress through a sequential pattern of changes mainly involving abnormal phosphorylation, conformational changes and truncation. While proteolytic cleavage of tau protein during the progression of AD has not been comprehensively analyzed, tau is a substrate for several intracellular proteases. Furthermore, abnormal regulation of proteolytic events, including those associated with apoptosis, may generate truncated tau subproducts which in turn may be toxic to neurons per se and capable of polymerization at a faster rate. Accumulation of tau fibrils has long been controversial, with much debate concerning the true toxicity of polymerized tau. The development of different transgenic mice overexpressing tau protein, the generation of cell models expressing tau, and the in vitro polymerization paradigms have significantly enhanced our understanding of the biophysics and pathological properties of tau polymers in AD, as well as in other tau pathologies. This review will discuss the pathological role of truncated tau protein in the context of toxicity and neurofibrillary tangle formation and maturation and its significance in clinical dementia.

Accumulation of aspartic acid421- and glutamic acid391-cleaved tau in neurofibrillary tangles correlates with progression in Alzheimer disease.

Truncations of tau protein at aspartic acid421 (D421) and glutamic acid391 (E391) residues are associated with neurofibrillary tangles (NFTs) in the brains of Alzheimer disease (AD) patients. Using immunohistochemistry with antibodies to D421- and E391-truncated tau (Tau-C3 and MN423, respectively), we correlated the presence of NFTs composed of these truncated tau proteins with clinical and neuropathologic parameters in 17 AD and 23 non-AD control brains. The densities of NFTs composed of D421- or E391-truncated tau correlated with clinical dementia index and Braak staging in AD. Glutamic acid391 tau truncation was prominent in the entorhinal cortex, whereas D421 truncation was prominent in the subiculum, suggesting that NFTs composed of either D421- or E391-truncated tau may be formed mutually exclusively in these areas. Both truncations were associated with the prevalence of the apolipoprotein E epsilon4 allele. By double labeling, intact tau in NFTs was commonly associated with D421-cleaved tau but not with E391-truncated tau; D421-cleaved tau was never associated with E391-truncated tau. These results indicate that tau is not randomly proteolyzed at different domains, and that proteolysis occurs sequentially from the C-terminus to inner regions of tau in AD progression. Identification of NFTs composed of tau at different stages of truncation may facilitate assessment of neurofibrillary pathology in AD.