Cellular factors modulating the mechanism of tau protein aggregation

Pathological accumulation of the microtubule-associated protein tau, in the form of neurofibrillary tangles, is a major hallmark of Alzheimer’s disease, the most prevalent neurodegenerative condition worldwide. In addition to Alzheimer’s disease, a number of neurodegenerative diseases, called tauopathies, are characterized by the accumulation of aggregated tau in a variety of brain regions. While tau normally plays an important role in stabilizing the microtubule network of the cytoskeleton, its dissociation from microtubules and eventual aggregation into pathological deposits is an area of intense focus for therapeutic development. Here we discuss the known cellular factors that affect tau aggregation, from post-translational modifications to molecular chaperones.     

Transport and diffusion of Tau protein in neurons

In highly polarized and elongated cells such as neurons, Tau protein must enter and move down the axon to fulfill its biological task of stabilizing axonal microtubules. Therefore, cellular systems for distributing Tau molecules are needed. This review discusses different mechanisms that have been proposed to contribute to the dispersion of Tau molecules in neurons. They include (1) directed transport along microtubules as cargo of tubulin complexes and/or motor proteins, (2) diffusion, either through the cytosolic space or along microtubules, and (3) mRNA-based mechanisms such as transport of Tau mRNA into axons and local translation. Diffusion along the microtubule lattice or through the cytosol appear to be the major mechanisms for axonal distribution of Tau protein in the short-to-intermediate range over distances of up to a millimetre. The high diffusion coefficients ensure that Tau can distribute evenly throughout the axonal volume as well as along microtubules. Motor protein-dependent transport of Tau dominates over longer distances and time scales. At low near-physiological levels, Tau is co-transported along with short microtubules from cell bodies into axons by cytoplasmic dynein and kinesin family members at rates of slow axonal transport.     

Developing pharmacological therapies for Alzheimer disease

Alzheimer disease (AD), while chronic and progressive with an average progression of 7 – 10 years, is both multifactorial and heterogeneous. Thus, AD offers a large window of opportunity and a large number of therapeutic targets to inhibit it. The selection of a therapeutic target, however, is one of the biggest challenges in developing a pharmacological treatment of this multifactorial disease. Inhibition of a pivotal downstream event is likely to benefit more patients than inhibition of an upstream event in AD pathogenesis. Neurofibrillary degeneration of abnormally hyperphosphorylated tau offers such a pivotal therapeutic target. Abnormal hyperphosphorylation of tau and not its aggregation into filaments appears to be the most deleterious step in neurofibrillary degeneration. Tau can be abnormally hyperphosphorylated by downregulation of protein phosphatase-2A activity or by upregulation of more than one tau kinase. Restoration of the phosphatase activity which is downregulated in AD brain or inhibition of GSK-3β and cdk5, which are required for AD-type abnormal hyperphosphorylation of tau, are among the most promising therapeutic strategies.     

RNA and protein-dependent mechanisms in tauopathies: consequences for therapeutic strategies

Tauopathies are a group of neurodegenerative diseases characterised by intracellular deposits of the microtubule-associated protein tau. The most typical example of a tauopathy is Alzheimer’s disease. The importance of tau in neuronal dysfunction and degeneration has been demonstrated by the discovery of dominant mutations in the MAPT gene, encoding tau, in some rare dementias. Recent developments have shed light on the significance of tau phosphorylation and aggregation in pathogenesis. Furthermore, emerging evidence reveals the central role played by tau pre-mRNA processing in tauopathies. The present review focuses on the current understanding of tau-dependent pathogenic mechanisms and how realistic therapies for tauopathies can be developed. Received 3 December 2006; received after revision 23 February 2007; accepted 20 March 2007     

Microtubule associated protein tau binds to double-stranded but not single-stranded DNA

Tau, a major microtubule-associated protein of the neuron, which is known to promote the assembly of and to stabilize microtubules, has also been seen associated with chromatin in neuronal cell lines, but its role in this subcellular compartment is still unknown. In this study, the binding of tau to DNA was investigated using the electrophoretic mobility shift assay. Using polynucleotide as probe, we found that tau bound to double-stranded but not to single-stranded DNA. Formation of tau-polynucleotide complex was disrupted by alkaline pH and a high concentration of NaCl, but was not affected by dithiothreitol. Electron microscopy revealed that the protein associated with the nucleic acid in a necklacelike manner. DNA-cellulose chromatography and radioimmunodot-blot analyses showed that calf thymus histones VI-S, VII-S and VIII-S could replace both recombinant human brain tau₃₅₂ (tau-23) and tau₄₄₁ (tau-40) from DNA. Thus, tau appears to bind to DNA reversibly in the presence of histones. Received 24 November 2002; received after revision 28 December 2002; accepted 30 December 2002 RID="*" ID="*"Corresponding author.     

The p38/MAPK pathway regulates microtubule polymerization through phosphorylation of MAP4 and Op18 in hypoxic cells

In both cardiomyocytes and HeLa cells, hypoxia (1% O₂) quickly leads to microtubule disruption, but little is known about how microtubule dynamics change during the early stages of hypoxia. We demonstrate that microtubule associated protein 4 (MAP4) phosphorylation increases while oncoprotein 18/stathmin (Op18) phosphorylation decreases after hypoxia, but their protein levels do not change. p38/MAPK activity increases quickly after hypoxia concomitant with MAP4 phosphorylation, and the activated p38/MAPK signaling leads to MAP4 phosphorylation and to Op18 dephosphorylation, both of which induce microtubule disruption. We confirmed the interaction between phospho-p38 and MAP4 using immunoprecipitation and found that SB203580, a p38/MAPK inhibitor, increases and MKK6(Glu) overexpression decreases hypoxic cell viability. Our results demonstrate that hypoxia induces microtubule depolymerization and decreased cell viability via the activation of the p38/MAPK signaling pathway and changes the phosphorylation levels of its downstream effectors, MAP4 and Op18.     

Back to the tubule: microtubule dynamics in Parkinson’s disease

Cytoskeletal homeostasis is essential for the development, survival and maintenance of an efficient nervous system. Microtubules are highly dynamic polymers important for neuronal growth, morphology, migration and polarity. In cooperation with several classes of binding proteins, microtubules regulate long-distance intracellular cargo trafficking along axons and dendrites. The importance of a delicate interplay between cytoskeletal components is reflected in several human neurodegenerative disorders linked to abnormal microtubule dynamics, including Parkinson’s disease (PD). Mounting evidence now suggests PD pathogenesis might be underlined by early cytoskeletal dysfunction. Advances in genetics have identified PD-associated mutations and variants in genes encoding various proteins affecting microtubule function including the microtubule-associated protein tau. In this review, we highlight the role of microtubules, their major posttranslational modifications and microtubule associated proteins in neuronal function. We then present key evidence on the contribution of microtubule dysfunction to PD. Finally, we discuss how regulation of microtubule dynamics with microtubule-targeting agents and deacetylase inhibitors represents a promising strategy for innovative therapeutic development.     

Phosphorylation-dependent prolyl isomerization: a novel signaling regulatory mechanism

Protein phosphorylation on serine or threonine residues preceding proline (Ser/Thr-Pro) plays an essential role for regulating various cellular processes, including cell cycle progression. Although phosphorylation has been proposed to regulate the function of a protein by inducing conformational changes, much less is known about what phosphate additions actually do and how the functions of phosphoproteins are coordinated. Proline is important for determining protein structure because it exists in cis or trans conformation and can put kinks into a polypeptide chain. We have shown that phosphorylation on Ser/Thr-Pro motifs reduces the cis/trans isomerization rate of Ser/Thr-Pro bonds. At the same time, proteins containing phosphorylated Ser/Thr-Pro motifs are substrates for the prolyl isomerase Pin1. The WW domain of Pin1 acts as a phosphoserine/threonine-binding module binding a defined subset of mitosis-specific phosphoproteins, such as Cdc25 and tau. These interactions target the enzymatic activity of Pin1 close to its substrates. In contrast to other prolyl isomerases (peptidyl-prolyl isomerases, PPlases), Pin1 has an extremely high degree of substrate specificity, specifically isomerizing phosphorylated Ser/Thr-Pro bonds. Therefore, Pin1 binds and regulates the function of a defined subset of phosphoproteins. Furthermore, inhibiting Pin1 function is lethal for dividing cells. Interestingly, Pin1, which can restore the biological function of phosphorylated tau, is sequestered in the neurofibrillary tangles in Alzheimer's brains. Thus, we have proposed a novel signaling regulatory mechanism, where protein phosphorylation creates binding sites for Pin1, which can then latch on to and isomerize the phosphorylated Ser/Thr-Pro peptide bond. In turn, this may change the shape of the protein, regulating its activity, dephosphorylation, degradation or location in the cell. This new post-phosphorylation regulatory mechanism appears to play an important role in normal cell function, such as mitotic progression, and in the pathogenesis of some human pathologies, such as Alzheimer's disease.     

Synthetic alpha-synuclein fibrils cause mitochondrial impairment and selective dopamine neurodegeneration in part via iNOS-mediated nitric oxide production

Intracellular accumulation of α-synuclein (α-syn) are hallmarks of synucleinopathies, including Parkinson’s disease (PD). Exogenous addition of preformed α-syn fibrils (PFFs) into primary hippocampal neurons induced α-syn aggregation and accumulation. Likewise, intrastriatal inoculation of PFFs into mice and non-human primates generates Lewy bodies and Lewy neurites associated with PD-like neurodegeneration. Herein, we investigate the putative effects of synthetic human PFFs on cultured rat ventral midbrain dopamine (DA) neurons. A time- and dose-dependent accumulation of α-syn was observed following PFFs exposure that also underwent phosphorylation at serine 129. PFFs treatment decreased the expression levels of synaptic proteins, caused alterations in axonal transport-related proteins, and increased H2AX Ser139 phosphorylation. Mitochondrial impairment (including modulation of mitochondrial dynamics-associated protein content), enhanced oxidative stress, and an inflammatory response were also detected in our experimental paradigm. In attempt to unravel a potential molecular mechanism of PFFs neurotoxicity, the expression of inducible nitric oxide synthase was blocked; a significant decline in protein nitration levels and protection against PFFs-induced DA neuron death were observed. Combined exposure to PFFs and rotenone resulted in an additive toxicity. Strikingly, many of the harmful effects found were more prominent in DA rather than non-DA neurons, suggestive of higher susceptibility to degenerate. These findings provide new insights into the role of α-syn in the pathogenesis of PD and could represent a novel and valuable model to study DA-related neurodegeneration.     

Regulation of end-binding protein EB1 in the control of microtubule dynamics

The regulation of microtubule dynamics is critical to ensure essential cell functions, such as proper segregation of chromosomes during mitosis or cell polarity and migration. End-binding protein 1 (EB1) is a plus-end-tracking protein (+TIP) that accumulates at growing microtubule ends and plays a pivotal role in the regulation of microtubule dynamics. EB1 autonomously binds an extended tubulin-GTP/GDP-Pi structure at growing microtubule ends and acts as a molecular scaffold that recruits a large number of regulatory +TIPs through interaction with CAP-Gly or SxIP motifs. While extensive studies have focused on the structure of EB1-interacting site at microtubule ends and its role as a molecular platform, the mechanisms involved in the negative regulation of EB1 have only started to emerge and remain poorly understood. In this review, we summarize recent studies showing that EB1 association with MT ends is regulated by post-translational modifications and affected by microtubule-targeting agents. We also present recent findings that structural MAPs, that have no tip-tracking activity, physically interact with EB1 to prevent its accumulation at microtubule plus ends. These observations point out a novel concept of “endogenous EB1 antagonists” and emphasize the importance of finely regulating EB1 function at growing microtubule ends.     

d-Ribosylated Tau forms globular aggregates with high cytotoxicity

Although the glycation of Tau that is involved in paired helical filament formation in Alzheimer’s disease has been widely studied, little attention has been paid to the role of d-ribose in the glycation of Tau. Here, we show that Tau is rapidly glycated in the presence of d-ribose, resulting in oligomerization and polymerization. Glycated derivatives appeared after 24 h incubation. Western blotting indicated the formation of advanced glycation end-products (AGEs) during initial stages of glycation. Thioflavin T-positive (ThT-positive) aggregations that appeared from day 4 indicated the globular-like features. Atomic force microscopy revealed that the surface morphology of ribosylated Tau40 was globular-like. Kinetic studies suggested that d-ribosylated Tau is slowly oligomerized and rapidly polymerized with ThT-positive features. Moreover, d-ribosylated Tau aggregates were highly toxic to SHSY5Y cells and resulted in both apoptosis and necrosis. This work has demonstrated that d-ribose reacted with Tau protein rapidly, producing ThT-positive aggregations which had high cytotoxicity. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Contrasting models for kinetochore microtubule attachment in mammalian cells

Kinetochore function is mediated through its interaction with microtubule plus ends embedded in the kinetochore outer plate. Here, we compare and evaluate current models for kinetochore microtubule attachment, beginning with a brief review of the molecular, biochemical, cellular, and structural studies upon which these models are based. The majority of these studies strongly support a model in which the kinetochore outer plate is a network of fibers that form multiple weak attachments to each microtubule, chiefly through the Ndc80 complex. Multiple weak attachments enable kinetochores to remain attached to microtubule plus ends that are continually growing and shrinking. It is unlikely that rings or “kinetochore fibrils” have a significant role in kinetochore microtubule attachment, but such entities could have a role in stabilizing attachment, modifying microtubule dynamics, and harnessing the energy released from microtubule disassembly. It is currently unclear whether kinetochores control and coordinate the dynamics of individual kinetochore microtubules.     

Genetic analysis of the kinome and phosphatome in cancer

Protein phosphorylation is a well-characterized biochemical process for reversible regulation of protein activity. Protein kinases and protein phosphatases are the key complementary players in this process, and through their coordinated activity cell homeostasis is tightly controlled. If these enzymes display aberrant activity, cells may undergo unrestrained growth, thus giving rise to complex diseases such as cancer. The technological platform gathered during the Human Genome Project recently allowed the systematic identifi cation of the genetic alterations present in the kinase (the kinome) and the phosphatase (the phosphatome) gene families. These studies suggest that most if not all human tumors carry genetic alterations in at least one phosphatase or kinase gene. Here we integrate the biochemical knowledge on the properties of these molecules with the information collected through their systematic genetic analysis in cancer. We also analyze why the molecular profi ling of the kinome and phosphatome in individual cancers is revolutionizing basic and clinical oncology.Received 13 May 2005; received after revision 30 May 2005; accepted 22 June 2005     

Analysis of <Emphasis Type="Italic">Dictyostelium</Emphasis> TACC reveals differential interactions with CP224 and unusual dynamics of <Emphasis Type="Italic">Dictyostelium</Emphasis> microtubules

We have localized TACC to the microtubule-nucleating centrosomal corona and to microtubule plus ends. Using RNAi we proved that Dictyostelium TACC promotes microtubule growth during interphase and mitosis. For the first time we show in vivo that both TACC and XMAP215 family proteins can be differentially localized to microtubule plus ends during interphase and mitosis and that TACC is mainly required for recruitment of an XMAP215-family protein to interphase microtubule plus ends but not for recruitment to centrosomes and kinetochores. Moreover, we have now a marker to study dynamics and behavior of microtubule plus ends in living Dictyostelium cells. In a combination of live cell imaging of microtubule plus ends and fluorescence recovery after photobleaching (FRAP) experiments of GFP-α-tubulin cells we show that Dictyostelium microtubules are dynamic only in the cell periphery, while they remain stable at the centrosome, which also appears to harbor a dynamic pool of tubulin dimers.     

Inhibition of protein misfolding and aggregation by natural phenolic compounds

Protein misfolding and aggregation into fibrillar deposits is a common feature of a large group of degenerative diseases affecting the central nervous system or peripheral organs, termed protein misfolding disorders (PMDs). Despite their established toxic nature, clinical trials aiming to reduce misfolded aggregates have been unsuccessful in treating or curing PMDs. An interesting possibility for disease intervention is the regular intake of natural food or herbal extracts, which contain active molecules that inhibit aggregation or induce the disassembly of misfolded aggregates. Among natural compounds, phenolic molecules are of particular interest, since most have dual activity as amyloid aggregation inhibitors and antioxidants. In this article, we review many phenolic natural compounds which have been reported in diverse model systems to have the potential to delay or prevent the development of various PMDs, including Alzheimer’s and Parkinson’s diseases, prion diseases, amyotrophic lateral sclerosis, systemic amyloidosis, and type 2 diabetes. The lower toxicity of natural compounds compared to synthetic chemical molecules suggest that they could serve as a good starting point to discover protein misfolding inhibitors that might be useful for the treatment of various incurable diseases.     

Transformation of amyloid β(1–40) oligomers into fibrils is characterized by a major change in secondary structure

Alzheimer’s disease (AD) is a neurodegenerative disorder occurring in the elderly. It is widely accepted that the amyloid beta peptide (Aβ) aggregation and especially the oligomeric states rather than fibrils are involved in AD onset. We used infrared spectroscopy to provide structural information on the entire aggregation pathway of Aβ(1–40), starting from monomeric Aβ to the end of the process, fibrils. Our structural study suggests that conversion of oligomers into fibrils results from a transition from antiparallel to parallel β-sheet. These structural changes are described in terms of H-bonding rupture/formation, β-strands reorientation and β-sheet elongation. As antiparallel β-sheet structure is also observed for other amyloidogenic proteins forming oligomers, reorganization of the β-sheet implicating a reorientation of β-strands could be a generic mechanism determining the kinetics of protein misfolding. Elucidation of the process driving aggregation, including structural transitions, could be essential in a search for therapies inhibiting aggregation or disrupting aggregates.