Novel drugs affecting tau behavior in the treatment of Alzheimer's disease and tauopathies.

The anomalous aggregation of proteins into pathological filaments is a common feature of a many human diseases, often related to aging. In this context, neurodegenerative pathologies such as Alzheimer's disease (AD) account for a major part of these protein misfolding diseases. AD is characterized by pathological aggregation of two proteins, tau and Aß-amyloid. The intracellular neurofibrillary tangles (NFTs) and neuropil threads consists of filaments of the modified microtubule-associated protein tau, while extracellular amyloid plaques consists of filaments of Aß-peptide. It is noteworthy that tau oligomers with a prefilamentous structure appear to play a role at early stages of AD and tauopathies, but also in asymptomatic patients with Braak-stage I neuropathology, where clinical symptoms of AD and NFTs in frontal cortex are absent. This suggests that an increase in tau oligomers levels occurs before individuals manifest clinical symptoms of AD. NFTs are one of the hallmarks of Alzheimer disease and other tauphaties. These aggregates are thought to be toxic to neurons, either by causing some neurotoxic signalling defects or by obstructing the cell function. Factors contributing to accumulation of tau aggregates include the increased rate of protein misfolding, generation of amyloidogenic oligomers, underactivity of repair systems such as chaperones and ubiquitin-proteasome system, or a failure of energy supply and antioxidant defense mechanisms. There is not clear evidence if the aggregated tau or oligomers cause cellular damage, but on the basis of the emergent need to have an early and effective treatment, lowering the production or removal of these aggregates appears as a pathway toward alleviating the disease. In the context of some of most relevant reports, we analyze why tau protein seems to be an interesting target for AD treatment, and the importance to understand the pathways of tau. aggregation. This knowledge will allow us to identify and optimize potential inhibitors that interact with aggregated forms of tau and hyperphosphorylated tau before the formation of the NFTs, offering a possible therapeutic route for AD treatment.

Molecular targets in the rational design of AD specific PET tracers: tau or amyloid aggregates?

A major limitation in finding therapeutic solutions for Alzheimer's disease (AD) has been the lack of a reliable method for early diagnosis of this devastating disease. Besides the development of biomarkers in biological fluids of patients, the search for a pathology-specific neuroimaging tools is critical at the present stage in which almost 30 million people suffer this disease worldwide. Several interesting approaches have been developed, however their clinical impact has been low. One of the difficulties has been to find the proper molecular tracers to specifically tag pathognomonic lesions in AD brain, including not only amyloid aggregates but also filaments of the modified microtubule-associated protein tau. In this review, we analyze the evidence towards developing pathology-specific diagnostic tools for AD. We analyze the current evidence and clinical implications of new imaging technologies for AD, and how tau hypothesis and the amyloid cascade hypothesis will impact on these scientific efforts in the near future.

Mechanisms of tau self-aggregation and neurotoxicity.

Pathological tau protein aggregates can be found in brain of patients with some of the neurodegenerative diseases collectively known as tauopathies, which include Alzheimer's disease (AD). Since tau post-translational modifications including phosphorylations, glycosylations, truncation and the subsequent aggregation in oligomers, paired helical filaments (PHFs) and neurofibrillary tangles (NFTs), correlate with cognitive impairment and neurodegeneration in AD, a pathogenic role for tau and its modifications has been raised. Here we summarize the current status of knowledge about tau modifications under pathologic conditions and the evidence supporting neurotoxic - or neuroprotective - roles of the diverse forms of modified and aggregated tau. Finally, we analyze the structural and functional tau alterations found in different tauopathies and how these modifications are related to the pathophysiologic mechanisms of neurodegeneration.

IL-3 controls tau modifications and protects cortical neurons from neurodegeneration.

Interleukin-3 (IL-3) regulates the proliferation, survival and differentiation of haematopoietic cells via interaction with specific cell-surface receptors. IL-3 is expressed in several non-hematopoietic cell types. Studies have demonstrated the presence of IL-3 in the central nervous system, however, its physiological role in these cells is poorly understood. Previously we have been demonstrated that IL-3 prevents neuronal death induced by fibrillary ß amyloid in these cells, by PI 3-kinase and Jak/STAT pathway activation. In this study, we demonstrated that IL-3 significantly reduced Aß-promoted neurite degeneration and toxicity. Thus, this cytokine provides cellular protection against Aß neurotoxicity in primary cortical neuronal cells, by modulating microtubular dynamics and prevention of tau cleavage and hyperphosphorylation. We also demonstrates that IL-3 is expressed in the "in vivo" mouse model of AD, Tg2576, which also expresses human AßPP with the Swedish mutation. In summary, these results suggest that IL-3 could play a neuroprotective role in AD.

In vivo effects of 3-iodocytisine: pharmacological and genetic analysis of hypothermia and evaluation of chronic treatment on nicotinic binding sites.

Several cytisine derivatives have been developed in the search for more selective drugs at nicotinic acetylcholine receptors (nAChR). Binding experiments in transfected cell lines showed that the iodination of cytisine in the position 3 of the pyridone ring increased potency at alpha7-nAChR and to a lesser extent at the alpha4beta2 subtypes, both of which are widely expressed in the brain. However, no in vivo studies have been published on this compound. Inhibition curves presented here using wild type, beta2, and beta4-null mutant mice confirm that 3-IC binds to alpha4beta2 *, alpha7 * and alpha3beta4 * receptors with higher affinity than cytisine (asterisk indicates the receptor may contain additional subunits, Lukas et al., 1999). Intraperitoneal injection of 3-iodocytisine (3-IC) induced considerable dose-dependent hypothermia in DBA/2J and C57BL/6J mice. This response was blocked by mecamylamine and partially inhibited by hexamethonium. beta4-null mice displayed significantly less 3-IC-induced hypothermia than wild-type mice, beta2-null mice were somewhat less affected than wild types, while responses of alpha7 *-null mice were similar to wild types. Mice treated chronically with 3-IC display a marked increase in alpha7 * and alpha4beta2 * binding sites determined by radioligand binding in membrane preparations from cerebral cortex and hippocampus. Quantitative autoradiographic analysis of 28 brain regions of mice treated with 3-IC was consistent with the membrane binding, detecting an increase of cytisine-sensitive [(125)I]epibatidine binding sites, while cytisine-resistant [(125)I]epibatidine sites were unchanged. [(125)I]alpha-Bungarotoxin binding sites also exhibited up-regulation. These results give a first evaluation of in vivo consequences of 3-IC as a potent agonist with marked effects on mice.

Role of the JAKs/STATs pathway in the intracellular calcium changes induced by interleukin-6 in hippocampal neurons.

Recent studies show that inflammation has an active role in the onset of neurodegenerative diseases. It is known that in response to extracellular insults microglia and/or astrocytes produce inflammatory agents. These contribute to the neuropathological events in the aging process and neuronal degeneration. Interleukin-6 (IL-6) has been involved in the pathogenesis of neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases. Here, we show that IL-6 treatment of rat hippocampal neurons increases the calcium influx via NMDA-receptor, an effect that is prevented by the specific NMDA receptor antagonist MK-801 (dizocilpine). We also show that this calcium influx is mediated by the JAKs/STATs pathway, since the inhibitor of JAKs/STATs pathway, JAK 3 inhibitor, blocks calcium influx even in the presence of IL-6. This increase in calcium signal was dependent on external sources, since this signal was not observed in the presence of EGTA. Additional studies indicate that the increase in cytosolic calcium induces tau protein hyperphosphorylation, as revealed by using specific antibodies against Alzheimer phosphoepitopes. This anomalous tau hyperphosphorylation was dependent on both the JAKs/STATs pathway and NMDA receptor. These results suggest that IL-6 would induce a cascade of molecular events that produce a calcium influx through NMDA receptors, mediated by the JAKs/STATs pathway, which subsequently modifies the tau hyperphosphorylation patterns.

The molecular bases of Alzheimer's disease and other neurodegenerative disorders.

Alzheimer's disease, the cause of one of the most common types of dementia, is a brain disorder affecting the elderly and is characterized by the formation of two main protein aggregates: senile plaques and neurofibrillary tangles, which are involved in the process leading to progressive neuronal degeneration and death. Neurodegeneration in Alzheimer's disease is a pathologic condition of cells rather than an accelerated way of aging. The senile plaques are generated by a deposition in the human brain of fibrils of the beta-amyloid peptide (Abeta), a fragment derived from the proteolytic processing of the amyloid precursor protein (APP). Tau protein is the major component of paired helical filaments (PHFs), which form a compact filamentous network described as neurofibrillary tangles (NFTs). Experiments with hippocampal cells in culture have indicated a relationship between fibrillary amyloid and the cascade of molecular signals that trigger tau hyperphosphorylations. Two main protein kinases have been shown to be involved in anomalous tau phosphorylations: the cyclin-dependent kinase Cdk5 and glycogen synthase kinase GSK3beta. Cdk5 plays a critical role in brain development and is associated with neurogenesis as revealed by studies in brain cells in culture and neuroblastoma cells. Deregulation of this protein kinase as induced by extracellular amyloid loading results in tau hyperphosphorylations, thus triggering a sequence of molecular events that lead to neuronal degeneration. Inhibitors of Cdk5 and GSK3beta and antisense oligonucleotides exert protection against neuronal death. On the other hand, there is cumulative evidence from studies in cultured brain cells and on brains that oxidative stress constitutes a main factor in the modification of normal signaling pathways in neuronal cells, leading to biochemical and structural abnormalities and neurodegeneration as related to the pathogenesis of Alzheimer's disease. This review is focused on the main protein aggregates responsible for neuronal death in both sporadic and familial forms of Alzheimer's disease, as well as on the alterations in the normal signaling pathways of functional neurons directly involved in neurodegeneration. The analysis is extended to the action of neuroprotective factors including selective inhibitors of tau phosphorylating protein kinases, estrogens, and antioxidants among other molecules that apparently prevent neuronal degeneration.

Expression pattern of DMAP-85 during Drosophila embryonic development.

Microtubule-associated proteins (MAPs) play major regulatory roles on the organization and integrity of the cytoskeletal network. Previously, we identified DMAP-85, a Drosophila MAP that promotes tubulin polymerization in vitro. In this work, we examine the distribution of DMAP-85 and its association pattern with microtubules at embryonic stages. Immunoblots revealed that DMAP-85 was present throughout embryogenesis, but it was most abundant in stages 6-9. Immunofluorescence studies showed that DMAP-85 was associated with sub-populations of stable microtubules during embryo cellularization, and after gastrulation with interphase microtubule arrays. At late embryonic stages, it was preferentially found in the ventral nerve cord, co-localizing with axonal microtubules. These observations are in agreement with previous reports on DMAP-85 functions, suggesting that DMAP-85 might be required for the stabilization and organization of cytoplasmic microtubules during embryonic development.

A Cdk5-p35 stable complex is involved in the beta-amyloid-induced deregulation of Cdk5 activity in hippocampal neurons.

The cdk5 and its activator p35 constitute one of the main tau-phosphorylating systems in neuronal cells. Under normal conditions for neurons, its activity is required for modulating tau involvement in neuronal polarity and in development of the mammalian central nervous system. Recently, we reported that the treatment of rat hippocampal cells in culture with fibrillary beta-amyloid (Abeta) results in deregulation of the protein kinase cdk5. The neurotoxic effects of Abeta fibrils were prevented by inhibition of cdk5 activity by butyrolactone I or by using antisense oligonucleotides that control the expression of this kinase. Here, we show that the Abeta-promoted increase of cdk5 activity is associated with changes in tau phosphorylation patterns and in the intraneuronal distribution of tau. In addition to hippocampal cells, deregulation of cdk5 was observed in other cell types. However, butyrolactone I prevented Abeta-induced cell death only in neuronal cells in which cdk5 activation was sensitive to Abeta fibrils. This lost of cdk5 regulation in hippocampal cells exposed to Abeta fibrils appears to be associated with an increase in the cdk5-p35 complex stability. Complex stabilization was sensitive to phosphorylation of cdk5. However, no changes in cdk5 and p35 mRNAs were observed, suggesting that the main effects on cdk5 occur at the posttranslational level. These studies indicate that cdk5 phosphorylation and the formation of an abnormally active cdk5-p35 complex are directly involved in the molecular paths leading to the neurodegenerative process of rat hippocampal neurons triggered by Abeta fibrils.

The protein kinase Cdk5. Structural aspects, roles in neurogenesis and involvement in Alzheimer's pathology.

A set of different protein kinases have been involved in tau phosphorylations, including glycogen synthase kinase 3beta (GSK3 beta), MARK kinase, MAP kinase, the cyclin-dependent kinase 5 (Cdk5) system and others. The latter system include the catalytic component Cdk5 and the regulatory proteins p35, p25 and p39. Cdk5 and its neuron-specific activator p35 are essential molecules for neuronal migration and for the laminar configuration of the cerebral cortex. Recent evidence that the Cdk5/p35 complex concentrates at the leading edge of axonal growth cones, together with the involvement of this system in the phosphorylation of neuronal microtubule-asociated proteins (MAPs), provide further support to the role of this protein kinase in regulating axonal extension in developing brain neurons. Although the aminoacid sequence of p35 has little similarity with those of normal cyclins, studies have shown that its activation domain may adopt a conformation of the cyclin-folded structure. The computed structure for Cdk5 is compatible with experimental data obtained from studies on the Cdk5/p35 complex, and has allowed predictions on the protein interacting domains. This enzyme exhibits a wide cell distribution, even though a regulated Cdk5 activity has been shown only in neuronal cells. Cdk5 has been characterized as a proline-directed Ser/Thr protein kinase, that contributes to phosphorylation of human tau on Ser202, Thr205, Ser235 and Ser404. Cdk5 is active in postmitiotic neurons, and it has been implicated in cytoskeleton assembly and its organization during axonal growth. In addition to tau and other MAPs, Cdk5 phosphorylates the high molecular weight neurofilament proteins at their C-terminal domain. Moreover, nestin, a protein that regulates cytoskeleton organization of neuronal and muscular cells during development of early embryos, and several other regulatory proteins appear to be substrates of Cdk5 and are phosphorylated by this kinase. Studies also suggest, that in addition to Cdk5 involvement in neuronal differentiation, its activity is induced during myogenesis, however, the mechanisms of how this activity is regulated during muscular differentiation has not yet been elucidated. Recent studies have shown that the beta-amyloid peptide (A beta) induces a deregulation of Cdk5 in cultured brain cells, and raises the question on the possible roles of this tau-phosphorylating protein kinase in the sequence of molecular events leading to neuronal death triggered by A beta. In this context, there are evidence that Cdk5 is involved in tau hyperphosphorylation promoted by A beta in its fibrillary form. Cdk5 inhibitors protect hippocampal neurons against both tau anomalous phosphorylations and neuronal death. The links between the studies on the Cdk5/p35 system in normal neurogenesis and its claimed participation in neurodegeneration, provide the framework to understand the regulatory relevance of this kinase system, and changes in its regulation that may be implicated in disturbances such as those occurring in Alzheimer disease.

Increase in the expression of the neuronal cyclin-dependent protein kinase cdk-5 during differentiation of N2A neuroblastoma cells.

Cytoskeleton organization is sensitive to regulatory signals at both spatial and temporal levels. In differentiating neurons, regulation of cell architecture is specially relevant, and tau plays a major role in the outgrowth of neurites and axonal development. Tau activity in determining neuronal polarity is modulated by protein kinases including cdk5. A significant increase in the expression of cdk5 was observed in N2A neuroblastoma cells induced to differentiate in the presence of dibutyryl cAMP. This induction of cdk5 was concomitant with changes in the distribution of tau, and with an increase in the microtubule assembling activity of neuronal extracts of cells undergoing differentiation. The course of cdk5 expression with time followed a linear relationship within a 48 h period. These findings were corroborated by RT-PCR in which higher levels of the transcripts for cdk5 were detected in N2A cells with differentiated morphology, as compared with undifferentiated cells. Studies suggest that the role of tau in the sequence of molecular events leading to extension of neurites in neuroblastoma cells is mediated by selective phosphorylations by cdk5.

Nuclear and cytoplasmic tau proteins from human nonneuronal cells share common structural and functional features with brain tau.

The heterogeneous family of tau proteins interacts with microtubules, actin filaments, and intermediate filaments. The tau isoforms have been shown to play a major role in neuronal polarity. However, tau-like proteins have been found in several other types of cells. Previous studies have also indicated the presence of a nuclear tau. The relationships between nuclear and cytoplasmic tau as well as the functional aspects of the nuclear tau are unknown. In this study, we demonstrate by reverse transcriptase polymerase chain reaction using specific primers that a transcript with features of neuronal tau is present in human fibroblast and Huh-7 hepatoma cell lines. Additionally, we present the first isolation and characterization of cytosolic and nuclear tau-like proteins from nonneuronal cells. Nonneuronal cytosolic tau components were isolated using the perchloric acid precipitation approach, while nuclear tau was isolated after selective extractions using high-ionic strength buffers. The cytoplasmic tau of nonneuronal cells is composed of at least three isoforms, whereas two main isoforms were detected in nuclear tau. Interestingly, the cytoplasmic and nuclear tau components exhibited the capacity to promote tubulin polymerization in vitro. Immunofluorescence studies using monoclonal anti-tau antibodies indicated a discrete distribution of tau protein in both the interphase and mitotic nucleus. In the latter, tau colocalized with the chromosomal scaffold. These studies, together with previous evidence on tau roles in modulating microtubule growth from centrosomes, and its role in the interaction patterns that stabilize the integrity of the cytoskeletal network, strongly support the idea that tau is a multifunctional protein involved in fundamental cellular processes.

Differential association of tau with subsets of microtubules containing posttranslationally-modified tubulin variants in neuroblastoma cells.

Neuronal cells display different subsets of dynamic microtubules. In axons and extending neurites, this intrinsic dynamics is modulated by the microtubule-associated protein tau. Moreover, posttranslational modifications of tubulin, namely acetylation, tyrosination or glutamylation are directly involved in determining the stability of neuronal microtubules. Studies were carried out to analyze the interaction patterns of tau with subsets of microtubules in N2A neuroblastoma cells, which can differentiate in the presence of dibutyryl cAMP. Double labeling studies showed a differential pattern of tau association with microtubules containing acetylated and tyrosinated tubulin. Furthermore, studies using depolymerizing drugs revealed a selectivity in the association of tau with microtubular polymers and microfilaments, within the organization of the neuronal cytoskeleton. In order to study the association of specific tau isoforms with microtubules containing modified tubulin variants, immunoprecipitation studies were carried out. The coimmunoprecipitation data indicated a selective binding of specific tau isoforms to either modified tubulin variant. To assess the hypothesis on the roles of tau isoforms in the stabilization of microtubules containing modified tubulins, the association of those variants with tau isoforms was analyzed in overlay experiments. A preferential binding of acetylated tubulin from undifferentiated N2A cell extracts, to at least one slow-migrating tau isoform was revealed. However, acetylated tubulin from N2A cells containing long neurites displayed a preferential association with two isoforms of tau. On the other hand, tyrosinated tubulin from N2A extracts bound to the entire set of neuronal tau isoforms. These studies, along with the tau association with microtubules with different stability, indicate that tau segregates into subsets of microtubules in the axonal processes. The studies also suggest that these interactions may respond to a functional versatility of these polymers in differentiating neurons.

Inhibition of tau phosphorylating protein kinase cdk5 prevents beta-amyloid-induced neuronal death.

The key target of this study was the tau protein kinase II system (TPK II) involving the catalytic subunit cdk5 and the regulatory component p35. TPK II is one of the tau phosphorylating systems in neuronal cells, thus regulating its functions in the cytoskeletal dynamics and the extension of neuronal processes. This research led to demonstration that the treatment of rat hippocampal cells in culture with fibrillary beta-amyloid (Abeta) results in a significant increase of the cdk5 enzymatic activity. Interestingly, the data also showed that the neurotoxic effect of 1-20 microM Abeta on primary cultures markedly diminished with co-incubation of hippocampal cells with the amyloid fibers plus the cdk5 inhibitor butyrolactone I. This inhibitor protected brain cells against Abeta-induced cell death in a concentration dependent fashion. Moreover, death was also prevented by a cdk5 antisense probe, but not by an oligonucleotide with a random sequence. The cdk5 antisense also reduced neuronal expression of cdk5 compared with the random oligonucleotide. The studies indicate that cdk5 plays a major role in the molecular path leading to the neurodegenerative process triggered by the amyloid fibers in primary cultures of rat hippocampal neurons. These findings are of interest in the context of the pathogenesis of Alzheimer's disease.

The beta-isoform of heat shock protein hsp-90 is structurally related with human microtubule-interacting protein Mip-90.

Through major research advances in the study of cytoskeletal organization, an integrated view of the complexity of this system has emerged. Recent findings on the microtubule-interacting protein Mip-90, which associates with microtubules and actin filaments in different cell domains, have shed light on its roles in cytoskeletal regulation. In order to study structural features of Mip-90, we sequenced several peptide fragments. A comparative sequence analysis revealed a high degree of similarity between the primary structure of this protein and the human heat shock protein of 90 kDa (hsp-90). Taken together, the present studies indicate the identity between Mip-90 and the the beta-isoform of hsp-90 (hsp-90beta). Western blot assays with an anti-hsp-90 monoclonal antibody showed cross-reactivity of hsp-90 and Mip-90 affinity purified from HeLa cells. Furthermore, the observed structural identity of Mip-90 with the hsp-90beta was sustained by immunoblot assays using monoclonal antibodies that specifically recognize the alpha- and beta-forms of hsp-90. Comparative fingerprinting analysis, along with the evidence of a remarkably similar biochemical behavior of both hsp-90 and Mip-90 in different affinity chromatographic systems, supported these observations. These studies, along with previous investigations, provide new data to elucidate the functional significance of these interesting cellular components and its relationships with other proteins linked to the cell architecture.

Regulatory roles of microtubule-associated proteins in neuronal morphogenesis. Involvement of the extracellular matrix.

As a result of recent investigations, the cytoskeleton can be viewed as a cytoplasmic system of interconnected filaments with three major integrative levels: self-assembling macromolecules, filamentous polymers, e.g., microtubules, intermediate filaments and actin filaments, and supramolecular structures formed by bundles of these filaments or networks resulting from cross-bridges between these major cytoskeletal polymers. The organization of this biological structure appears to be sensitive to fine spatially and temporally dependent regulatory signals. In differentiating neurons, regulation of cytoskeleton organization is particularly relevant, and the microtubule-associated protein (MAP) tau appears to play roles in the extension of large neuritic processes and axons as well as in the stabilization of microtubular polymers along these processes. Within this context, tau is directly involved in defining neuronal polarity as well as in the generation of neuronal growth cones. There is increasing evidence that elements of the extracellular matrix contribute to the control of cytoskeleton organization in differentiating neurons, and that these regulations could be mediated by changes in MAP activity. In this brief review, we discuss the possible roles of tau in mediating the effects of extracellular matrix components on the internal cytoskeletal arrays and its organization in growing neurons.

Regulatory roles of microtubule-associated proteins in neuronal morphogenesis. Involvement of the extracellular matrix

As a result of recent investigations, the cytoskeleton can be viewed as a cytoplasmic system of interconnected filaments with three major integrative levels: self-assembling macromolecules, filamentous polymers, e.g., microtubules, intermediate filaments and actin filaments, and supramolecular structures formed by bundles of these filaments or networks resulting from cross-bridges between these major cytoskeletal polymers. The organization of this biological structure appears to be sensitive to fine spatially and temporally dependent regulatory signals. In differentiating neurons, regulation of cytoskeleton organization is particularly relevant, and the microtubule-associated protein (MAP) tau appears to play roles in the extension of large neuritic processes and axons as well as in the stabilization of microtubular polymers along these processes. Within this context, tau is directly involved in defining neuronal polarity as well as in the generation of neuronal growth cones. There is increasing evidence that elements of the extracellular matrix contribute to the control of cytoskeleton organization in differentiating neurons, and that these regulations could be mediated by changes in MAP activity. In this brief review, we discuss the possible roles of tau in mediating the effects of extracellular matrix components on the internal cytoskeletal arrays and its organization in growing neurons

Modification of tau to an Alzheimer's type protein interferes with its interaction with microtubules.

The microtubule associated protein tau is the main structural component of paired helical filaments (PHFs), aberrant polymers found intracellularly in neurons of brains with the Alzheimer's disease. Glycation is one of the posttranslational modifications that has been found in tau from PHFs, but not in normal brain tau. Studies were carried out with purified tau protein subjected to chemical modifications, in order to further investigate the mechanisms of tau self-association into PHFs. Tau was subjected to modifications affecting reactive lysyl residues, e.g., carbamoylation with potassium cyanate and glycation reaction with glucose. The effects of these modifications to produce functional alterations in tau capacity to bind brain tubulin and to induce microtubule assembly were investigated. Chemically-modified tau and tau of Alzheimer's type exhibited a similar microtubule interaction behavior as analysed by overlay assays, but those were different than normal tau controls. On the other hand, studies of the microtubule assembly kinetics indicated that the reported tau modifications resulted in a loss of its capacity to promote microtubule assembly from purified tubulin preparations. The data on the differences in the electrophoretic profiles, Western blots and the overlay patterns, along with those on the microtubule polymerisation of normal brain tau as compared with both modified and Alzheimer's tau, suggest changes in the functional behavior of this protein as a result of its structural modifications. These studies were complemented with an immunogold analysis at the electron microscope level, which indicated that the modified tau did not incorporate into assembled microtubules. These findings, combined with the results on tau chemical modifications suggest that the reactive lysine residues within functional domains on tau, e.g., those of the repetitive binding motifs, were affected by these modifications. Furthermore, these observations provide new clues to understand the anomalous interactions of tau in Alzheimer's disease.

The compound 14-keto-stypodiol diacetate from the algae Stypopodium flabelliforme inhibits microtubules and cell proliferation in DU-145 human prostatic cells.

We investigated the effects of the drug 14-keto-stypodiol diacetate (SDA) extracted from the seaweed product Stypopodium flabelliforme, in inhibiting the cell growth and tumor invasive behavior of DU-145 human prostate cells. In addition, the molecular action of the drug on microtubule assembly was analyzed. The effects of this diterpenoid drug in cell proliferation of DU-145 tumor cells in culture revealed that SDA at concentrations of 5 microM decreased cell growth by 14%, while at 45 microM a 61% decrease was found, as compared with control cells incubated with the solvent but in the absence of the drug. To study their effects on the cell cycle, DU-145 cells were incubated with increasing concentrations of SDA and the distribution of cell-cycle stages was analyzed by flow cytometry. Interestingly, the data showed that 14-keto-stypodiol diacetate dramatically increased the proportion of cells in the G2/M phases, and decreased the number of cells at the S phase of mitosis, as compared with appropriate controls. Studies on their action on the in vitro assembly of microtubules using purified brain tubulin, showed that SDA delayed the lag period associated to nucleation events during assembly, and decreased significantly the extent of polymerization. The studies suggest that this novel derivative from a marine natural product induces mitotic arrest of tumor cells, an effect that could be associated to alterations in the normal microtubule assembly process. On the other hand, a salient feature of this compound is that it affected protease secretion and the in vitro invasive capacity, both properties of cells from metastases. The secretion of plasminogen activator (u-PA) and the capacity of DU-145 cells to migrate through a Matrigel-coated membrane were significantly inhibited in the presence of micromolar concentrations of SDA. These results provide new keys to analyze the functional relationships between protease secretion, invasive behavior of tumor cells and the microtubule network.

The association of tau-like proteins with vimentin filaments in cultured cells.

There is increasing evidence that the different polymers that constitute the cytoskeleton are interconnected to form a three-dimensional network. The macromolecular interaction patterns that stabilize this network and its intrinsic dynamics are the basis for numerous cellular processes. Within this context, in vitro studies have pointed to the existence of specific associations between microtubules, microfilaments, and intermediate filaments. It has also been postulated that microtubule-associated proteins (MAPs) are directly involved in mediating these interactions. The interactions of tau with vimentin filaments, and its relationships with other filaments of the cytoskeletal network, were analyzed in SW-13 adenocarcinoma cells, through an integrated approach that included biochemical and immunological studies. This cell line has the advantage of presenting a wild-type clone (vim+) and a mutant clone (vim-) which is deficient in vimentin expression. We analyzed the cellular roles of tau, focusing on its interactions with vimentin filaments, within the context of its functional aspects in the organization of the cytoskeletal network. Cosedimentation experiments of microtubular protein with vimentin in cell extracts enriched in intermediate filaments, combined with studies on the direct interaction of tau with nitrocellulose-bound vimentin and analysis of tau binding to vimentin immobilized in single-strand DNA affinity columns, indicate that tau interacts with the vimentin network. These studies were confirmed by a quantitative analysis of the immunofluorescence patterns of cytoskeleton-associated tubulin, tau, and vimentin using flow cytometry. In this regard, a decrease in the levels of tau associated to the cytoskeletal network in the vim- cell mutant compared with the wild-type clones was observed. However, immunofluorescence data on SW-13 cells suggest that the absence of a structured network of vimentin in the mutant vim- cells does not affect the cytoplasmic organization formed by microtubules and actin filaments, when compared with the wild-type vim+ cells. These studies suggest that tau associates with vimentin filaments and that these interactions may play a structural role in cells containing these filaments.