Protein kinase MARK/PAR-1 is required for neurite outgrowth and establishment of neuronal polarity

Protein kinases of the microtubule affinity-regulating kinase (MARK) family were originally discovered because of their ability to phosphorylate certain sites in tau protein (KXGS motifs in the repeat domain). This type of phosphorylation is enhanced in abnormal tau from Alzheimer brain tissue and causes the detachment of tau from microtubules. MARK-related kinases (PAR-1 and KIN1) occur in various organisms and are involved in establishing and maintaining cell polarity. Herein, we report the ability of MARK2 to affect the differentiation and outgrowth of cell processes from neuroblastoma and other cell models. MARK2 phosphorylates tau protein at the KXGS motifs; this results in the detachment of tau from microtubules and their destabilization. The formation of neurites in N2a cells is blocked if MARK2 is inactivated, either by transfecting a dominant negative mutant, or by MARK2 inhibitors such as hymenialdisine. Alternatively, neurites are blocked if the target KXGS motifs on tau are rendered nonphosphorylatable by point mutations. The results suggest that MARK2 contributes to the plasticity of microtubules needed for neuronal polarity and the growth of neurites.     

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.     

Tau splicing and the intricacies of dementia

Tau is a microtubule-associated protein that fulfills several functions critical for neuronal formation and health. Tau discharges its functions by producing multiple isoforms via regulated alternative splicing. These isoforms modulate tau function in normal brain by altering the domains of the protein, thereby influencing its localization, conformation, and post-translational modifications and hence its availability and affinity for microtubules and other ligands. Disturbances in tau expression result in disruption of the neuronal cytoskeleton and formation of tau structures (neurofibrillary tangles) found in brains of dementia sufferers. More specifically, aberrations in tau splicing regulation directly cause several neurodegenerative diseases, which lead to dementia. In this review, I present our cumulative knowledge of tau splicing regulation in connection with neurodegeneration and also briefly go over the still-extensive list of questions that are connected to tau (dys)function.     

The disordered neuronal cytoskeleton in Alzheimer's disease.

Evidence continues to accrue in support of the notion that normal adult human tau is converted into the protein subunits of Alzheimer's disease paired helical filaments as a result of the abnormal phosphorylation of tau at aberrant sites. Although the biological consequences of the generation of these abnormal tau derivatives in neurons remain uncertain, it is plausible that this process could destabilize microtubules and have a deleterious effect on the function and survival of neurons. Recent studies that probe the mechanisms whereby normal tau, a component of the neuronal cytoskeleton, undergoes profound alterations to become paired helical filaments in the Alzheimer's diseased brain are discussed.     

Cell cycle-dependent phosphorylation and microtubule binding of tau protein stably transfected into Chinese hamster ovary cells.

Tau protein, a neuronal microtubule-associated protein, is phosphorylated in situ and hyperphosphorylated when aggregated into the paired helical filaments of Alzheimer's disease. To study the phosphorylation of tau protein in vivo, we have stably transfected htau40, the largest human tau isoform, into Chinese hamster ovary cells. The distribution and phosphorylation of tau was monitored by gel shift, autoradiography, immunofluorescence, and immunoblotting, using the antibodies Tau-1, AT8, AT180, and PHF-1, which are sensitive to the phosphorylation of Ser202, Thr205, Thr231, Ser235, Ser396, and Ser404 and are used in the diagnosis of Alzheimer tau. In interphase cells, tau becomes phosphorylated to some extent, partly at these sites; most of the tau is associated with microtubules. In mitosis, the above Ser/Thr-Pro sites become almost completely phosphorylated, causing a pronounced shift in M(r) and an antibody reactivity similar to that of Alzheimer tau. Moreover, a substantial fraction of tau is found in the cytoplasm detached from microtubules. Autoradiographs of metabolically labeled Chinese hamster ovary cells in interphase and mitosis confirmed that tau protein is more highly phosphorylated during mitosis. The understanding of tau phosphorylation under physiological conditions might help elucidate possible mechanisms for the hyperphosphorylation in Alzheimer's disease.     

Distinct FTDP-17 missense mutations in tau produce tau aggregates and other pathological phenotypes in transfected CHO cells

Multiple tau gene mutations are pathogenic for hereditary frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), with filamentous tau aggregates as the major lesions in the CNS of these patients. Recent studies have shown that bacterially expressed recombinant tau proteins with FTDP-17 missense mutations cause functional impairments, i.e., a reduced ability of mutant tau to bind to or promote the assembly of microtubules. To investigate the biological consequences of FTDP-17 tau mutants and assess their ability to form filamentous aggregates, we engineered Chinese hamster ovary cell lines to stably express tau harboring one or several different FTDP-17 mutations and showed that different tau mutants produced distinct pathological phenotypes. For example, delta K, but not several other single tau mutants (e.g., V337 M, P301L, R406W), developed insoluble amorphous and fibrillar aggregates, whereas a triple tau mutant (VPR) containing V337M, P301L, and R406W substitutions also formed similar aggregates. Furthermore, the aggregates increased in size over time in culture. Significantly, the formation of aggregated delta K and VPR tau protein correlated with reduced affinity of these mutants to bind microtubules. Reduced phosphorylation and altered proteolysis was also observed in R406W and delta K tau mutants. Thus, distinct pathological phenotypes, including the formation of insoluble filamentous tau aggregates, result from the expression of different FTDP-17 tau mutants in transfected Chinese hamster ovary cells and implies that these missense mutations cause diverse neurodegenerative FTDP-17 syndromes by multiple mechanisms.     

The development of cell processes induced by tau protein requires phosphorylation of serine 262 and 356 in the repeat domain and is inhibited by phosphorylation in the proline-rich domains

The differentiation of neurons and the outgrowth of neurites depends on microtubule-associated proteins such as tau protein. To study this process, we have used the model of Sf9 cells, which allows efficient transfection with microtubule-associated proteins (via baculovirus vectors) and observation of the resulting neurite-like extensions. We compared the phosphorylation of tau23 (the embryonic form of human tau) with mutants in which critical phosphorylation sites were deleted by mutating Ser or Thr residues into Ala. One can broadly distinguish two types of sites, the KXGS motifs in the repeats (which regulate the affinity of tau to microtubules) and the SP or TP motifs in the domains flanking the repeats (which contain epitopes for antibodies diagnostic of Alzheimer's disease). Here we report that both types of sites can be phosphorylated by endogenous kinases of Sf9 cells, and that the phosphorylation pattern of the transfected tau is very similar to that of neurons, showing that Sf9 cells can be regarded as an approximate model for the neuronal balance between kinases and phosphatases. We show that mutations in the repeat domain and in the flanking domains have opposite effects. Mutations of KXGS motifs in the repeats (Ser262, 324, and 356) strongly inhibit the outgrowth of cell extensions induced by tau, even though this type of phosphorylation accounts for only a minor fraction of the total phosphate. This argues that the temporary detachment of tau from microtubules (by phosphorylation at KXGS motifs) is a necessary condition for establishing cell polarity at a critical point in space or time. Conversely, the phosphorylation at SP or TP motifs represents the majority of phosphate (>80%); mutations in these motifs cause an increase in cell extensions, indicating that this type of phosphorylation retards the differentiation of the cells.     

Mutant (R406W) human tau is hyperphosphorylated and does not efficiently bind microtubules in a neuronal cortical cell model

Frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17) is an autosomal dominant neurodegenerative disorder caused by mutations in the gene that encodes for tau, a microtubule-binding protein. Neuropathologically the disease is characterized by extensive neuronal loss in the frontal and temporal lobes and the filamentous accumulation of hyperphosphorylated tau. The R406W missense mutation was originally described in an American and a Dutch family. Although R406W tau is hyperphosphorylated in FTDP-17 cases, R406W tau expressed in cell model systems has not shown increased phosphorylation. The purpose of this study was to establish a neuronal model system in which the phosphorylation of R406W tau is increased and thus more representative of the in vivo situation. To accomplish this goal immortalized mouse cortical cells that express low levels of endogenous tau were stably transfected with human wild type or R406W tau. In this neuronal model R406W tau was more highly phosphorylated at numerous epitopes and showed decreased microtubule binding compared with wild type tau, an effect that could be reversed by dephosphorylation. In addition the expression of R406W tau in the cortical cells resulted in increased cell death as compared with wild type tau-expressing cells when the cells were exposed to an apoptotic stressor. These results indicate that in an appropriate cellular context R406W tau is hyperphosphorylated, which leads to decreased microtubule binding. Furthermore, expression of R406W tau sensitized cells to apoptotic stress, which may contribute to the neuronal cell loss that occurs in this FTDP-17 tauopathy.     

Tau mutations in frontotemporal dementia FTDP-17 and their relevance for Alzheimer's disease

Alzheimer's disease is characterised by the degeneration of selected populations of nerve cells that develop filamentous inclusions prior to degeneration. The neuronal inclusions of Alzheimer's disease are made of the microtubule-associated protein tau, in a hyperphosphorylated state. Abundant filamentous tau inclusions are not limited to Alzheimer's disease. They are the defining neuropathological characteristic of frontotemporal dementias, such as Pick's disease, and of progressive supranuclear palsy and corticobasal degeneration. The discovery of mutations in the tau gene in familial frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) has provided a direct link between tau dysfunction and dementing disease. Known mutations produce either a reduced ability of tau to interact with microtubules, or an overproduction of tau isoforms with four microtubule-binding repeats. This leads in turn to the assembly of tau into filaments similar or identical to those found in Alzheimer's disease brain. Several missense mutations also have a stimulatory effect on heparin-induced tau filament formation. Assembly of tau into filaments may be the gain of toxic function that is believed to underlie the demise of affected brain cells.     

Tau story: from frontotemporal dementia to other tauopathies

Tau proteins belong to the family of microtubule-associated proteins. They are mainly expressed in neurons where they play an important role in the assembly of tubulin monomers into microtubules to constitute the neuronal microtubules network. Tau proteins are translated from a single gene located on chromosome 17. Their expression is developmentally regulated by an alternative splicing mechanism and six different isoforms exist in the human adult brain. Tau proteins are the major constituents of fibrillar lesions described in Alzheimer's disease and numerous neurodegenerative disorders referred to as 'tauopathies'. Molecular analysis has revealed that an abnormal phosphorylation might be one of the important events in the process leading to their aggregation. Moreover, a specific set of pathological tau proteins exhibiting a typical biochemical pattern, and a different regional and laminar distribution could characterize each of these disorders. Finally, the recent discovery of tau gene mutations in fronto-temporal dementia with parkinsonism linked to chromosome 17 has reinforced the direct role attributed to tau proteins in the pathogenesis of neurodegenerative disorders, and underlined the fact that distinct sets of tau isoforms expressed in different neuronal populations could lead to different pathologies. Conversely, recent data in myotonic dystrophy has demonstrated that indirect effect (CTG repeat expansion) leading to variations in tau alternative splicing also produce neurofibrillary degeneration.     

Monoclonal antibody 2G13, a new axonal growth cone marker

Growth cones are specialized sensorimotor structures at the tips of neurites implicated in pathfinding decisions and axonal outgrowth during neuronal development. We generated a mouse monoclonal antibody (mAb 2G13) against chick tectum and found that the antibody exclusively labelled axonal growth cones, particularly their filopodia and lamellipodia, in developing rat CNS and in embryonic neurons in culture. The high fidelity of the staining of growth cones by mAb 2G13 means that the antibody will be a useful marker for identifying growth cones. In growth cones of cultured neurons, mAb 2G13 labelling is intracellular and mainly associated with the filamentous actin cytoskeleton. Experiments with cytochalasins, which depolymerise filamentous actin, showed that 2G13p (the protein recognised by mAb 2G13) is physically associated with filamentous actin in growth cones. These properties of 2G13p suggest a role in growth cone motility.     

Tau phosphorylation at serine 396 and serine 404 by human recombinant tau protein kinase II inhibits tau's ability to promote microtubule assembly

In Alzheimer's disease, hyperphosphorylated tau is an integral part of the neurofibrillary tangles that form within neuronal cell bodies and fails to promote microtubule assembly. Dysregulation of the brain-specific tau protein kinase II is reported to play an important role in the pathogenesis of Alzheimer's disease (Patrick, G. N., Zukerberg, L., Nikolic, M., De La Monte, S., Dikkes, P., and Tsai, L.-H. (1999) Nature 402, 615-622). We report here that in vitro phosphorylation of human tau by human recombinant tau protein kinase II severely inhibits the ability of tau to promote microtubule assembly as monitored by tubulin polymerization. The ultrastructure of tau-mediated polymerized tubulin was visualized by electron microscopy and compared with phosphorylated tau. Consistent with the observed slower kinetics of tubulin polymerization, phosphorylated tau is compromised in its ability to generate microtubules. Moreover, we show that phosphorylation of microtubule-associated tau results in tau's dissociation from the microtubules and tubulin depolymerization. Mutational studies with human tau indicate that phosphorylation by tau protein kinase II at serine 396 and serine 404 is primarily responsible for the functional loss of tau-mediated tubulin polymerization. These in vitro results suggest a possible role for tau protein kinase II-mediated tau phosphorylation in initiating the destabilization of microtubules.     

Tau phosphorylation by cyclin-dependent kinase 5/p39 during brain development reduces its affinity for microtubules

The microtubule-associated protein tau is a developmentally regulated neuronal phosphoprotein. The phosphorylation of tau reduces its ability to bind and stabilize axonal microtubules during axonal growth. Although tau is phosphorylated by cyclin-dependent kinase 5 (Cdk5) in vitro, its in vivo roles remain unclear. Here, we show that tau is phosphorylated by Cdk5/p39 during brain development, resulting in a reduction of its affinity for microtubules. The activity of Cdk5 is tightly regulated by association with its neuronal activators, p35 or p39. The p35 and p39 expression levels were investigated in the developing mouse brain; the p39 expression level was higher in embryonic hind brain and spinal cord and in postnatal cerebral cortex, whereas that of p35 was most prominent in cerebral cortex at earlier stages of development. The ability of Cdk5 to phosphorylate tau was higher when in association with p39 than in association with p35. Tau phosphorylation at Ser-202 and Thr-205 was decreased in Cdk5-/- mouse brain but not in p35-/- mouse brain, suggesting that Cdk5/p39 is responsible for the in vivo phosphorylation of tau at these sites. Our data suggest that tau phosphorylation by Cdk5 may provide the neuronal microtubules with dynamic properties in a region-specific and developmentally regulated manner.     

Phosphorylation of type III β-tubulin in PC 12 cell neurites during NGF-induced process outgrowth

Nerve growth factor (NGF) produces both rapid and delayed cellular responses that are involved in neuronal differentiation. Neurite formation, a conspicuous delayed response, is accompanied by phosphorylation of β-tubulin in PC12 cells. The present work provides further characterization of the phospho form of β-tubulin in this neuronal model system with regard to isotype, cellular localization, and the circumstances that favor its formation. The results indicate that neuron-specific type III β-tubulin (βIII-tubulin) is selectively affected during neurite formation. This phosphorylation occurs relatively late in the NGF signal transduction cascade and increases progressively with increasing duration of NGF treatment concomitant with more extensive neurite growth. The subcellular distribution of βIII-tubulin is not markedly different from that of total tubulin, but the phosphorylated protein is uniquely associated with microtubules that are calcium and cold labile. Although NGF is capable of inducing phosphorylation of βIII-tubulin, it is not necessarily sufficient. Based on experiments that employ either nonpermissive substrate conditions or microtubule-depolymerizing drugs, this phosphorylation requires neurite outgrowth. Direct measurements of the phospho form in neurites versus cell bodies by means of a microculture system indicate that phosphorylated βIII-tubulin is enriched in neurites. The enrichment of phospho-βIII-tubulin in calcium- and cold-labile polymer within neurites and its near absence in nonneurite bearing, NGF-treated cells suggests a role for this posttranslationally modified protein in the regulation of dynamic microtubules involved in neurite formation. © 1996 John Wiley & Sons, Inc.     

Ubiquitination and abnormal phosphorylation of paired helical filaments in Alzheimer's disease

The most characteristic cellular change in Alzheimer's disease is the accumulation of aberrant filaments, the paired helical filaments (PHF), in the affected neurons. There is growing evidence from a number of laboratories that dementia correlates better with the accumulation of PHF than of the extracellular amyloid, the second major lesion of Alzheimer's disease. PHF are both morphologically and biochemically unlike any of the normal neurofibrils. The major polypeptides in isolated PHF are microtubule-associated protein tau. Tau in PHF is phosphorylated differently from tau in microtubules. This abnormal phosphorylation of tau in PHF occurs at several sites. The accumulation of abnormally phosphorylated tau in the affected neurons in Alzheimer's disease brain precedes both the formation and the ubiquitination of the neurofibrillary tangles. In Alzheimer's disease brain, tubulin is assembly competent, but the in vitro assembly of microtubules is not observed. In vitro, the phosphate groups in PHF are less accessible than those of tau to alkaline phosphatase. The in vitro dephosphorylated PHF polypeptides stimulate microtubule assembly from bovine tubulin. It is hypothesized that a defect in the protein phosphorylation/dephosphorylation system is one of the earliest events in the cytoskeletal pathology in Alzheimer's disease. Production of nonfunctional tau by its phosphorylation and its polymerization into PHF most probably contributes to a microtubule assembly defect, and consequently, to a compromise in both axoplasmic flow and neuronal function. Index Entries: Alzheimer's disease; mechanisms of neuronal degeneration; neurofibrillary changes; paired helical filaments: biochemistry; microtubule-associated protein tau; abnormal phosphorylation; ubiquitination; microtubule assembly; axoplasmic flow; protein phosphorylation/dephosphorylation.     

Computational predictions of volatile anesthetic interactions with the microtubule cytoskeleton: implications for side effects of general anesthesia

The cytoskeleton is essential to cell morphology, cargo trafficking, and cell division. As the neuronal cytoskeleton is extremely complex, it is no wonder that a startling number of neurodegenerative disorders (including but not limited to Alzheimer's disease, Parkinson's disease and Huntington's disease) share the common feature of a dysfunctional neuronal cytoskeleton. Recently, concern has been raised about a possible link between anesthesia, post-operative cognitive dysfunction, and the exacerbation of neurodegenerative disorders. Experimental investigations suggest that anesthetics bind to and affect cytoskeletal microtubules, and that anesthesia-related cognitive dysfunction involves microtubule instability, hyper-phosphorylation of the microtubule-associated protein tau, and tau separation from microtubules. However, exact mechanisms are yet to be identified. In this paper the interaction of anesthetics with the microtubule subunit protein tubulin is investigated using computer-modeling methods. Homology modeling, molecular dynamics simulations and surface geometry techniques were used to determine putative binding sites for volatile anesthetics on tubulin. This was followed by free energy based docking calculations for halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) on the tubulin body, and C-terminal regions for specific tubulin isotypes. Locations of the putative binding sites, halothane binding energies and the relation to cytoskeleton function are reported in this paper.     

The natural osmolyte trimethylamine N-oxide (TMAO) restores the ability of mutant tau to promote microtubule assembly

Coding region and intronic mutations in the gene for microtubule-associated protein tau cause frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17). Most coding region mutations effect a reduced ability of tau protein to interact with microtubules and lead to the formation of a filamentous pathology made of hyperphosphorylated tau. Here we show that trimethylamine N-oxide (TMAO) restores the ability of tau with FTDP-17 mutations to promote microtubule assembly. To mimic phosphorylation, serine and threonine residues in tau were singly or multiply mutated to glutamic acid, resulting in a reduced ability of tau to promote microtubule assembly. With the exception of the most heavily substituted protein (27 glutamic acid residues), TMAO increased the ability of mutant tau to promote microtubule assembly. However, it had no significant effect on heparin-induced assembly of tau into filaments.     

MARKK, a Ste20-like kinase, activates the polarity-inducing kinase MARK/PAR-1

MARK, a kinase family related to PAR-1 involved in establishing cell polarity, phosphorylates microtubule-associated proteins (tau/MAP2/MAP4) at KXGS motifs, causes detachment from microtubules, and their disassembly. The sites are prominent in tau from Alzheimer's disease brains. We studied the activation of MARK and identified the upstream kinase, MARKK, a member of the Ste20 kinase family. It phosphorylates MARK within the activation loop (T208 in MARK2). A fraction of MARK in brain tissue is doubly phosphorylated (at T208/S212), reminiscent of the activation of MAP kinase; however, the phosphorylation of the second site in MARK (S212) is inhibitory. In cells the activity of MARKK enhances microtubule dynamics through the activation of MARK and leads to phosphorylation and detachment of tau or equivalent MAPs from microtubules. Overexpression of MARK eventually leads to microtubule breakdown and cell death, but in neuronal cells the primary effect is to allow the development of neurites during differentiation.     

Phosphorylation of microtubule-associated protein STOP by calmodulin kinase II

STOP proteins are microtubule-associated, calmodulin-regulated proteins responsible for the high degree of stabilization displayed by neuronal microtubules. STOP suppression in mice induces synaptic defects affecting both short and long term synaptic plasticity in hippocampal neurons. Interestingly, STOP has been identified as a component of synaptic structures in neurons, despite the absence of microtubules in nerve terminals, indicating the existence of mechanisms able to induce a translocation of STOP from microtubules to synaptic compartments. Here we have tested STOP phosphorylation as a candidate mechanism for STOP relocalization. We show that, both in vitro and in vivo, STOP is phosphorylated by the multifunctional enzyme calcium/calmodulin-dependent protein kinase II (CaMKII), which is a key enzyme for synaptic plasticity. This phosphorylation occurs on at least two independent sites. Phosphorylated forms of STOP do not bind microtubules in vitro and do not co-localize with microtubules in cultured differentiating neurons. Instead, phosphorylated STOP co-localizes with actin assemblies along neurites or at branching points. Correlatively, we find that STOP binds to actin in vitro. Finally, in differentiated neurons, phosphorylated STOP co-localizes with clusters of synaptic proteins, whereas unphosphorylated STOP does not. Thus, STOP phosphorylation by CaMKII may promote STOP translocation from microtubules to synaptic compartments where it may interact with actin, which could be important for STOP function in synaptic plasticity.