Tau protein in Neurodegenerative Diseases

A special interest subgroup meeting on 'Tau protein in Neurodegenerative Diseases' was organized by Gloria Lee, University of Iowa. This was a timely and stimulating meeting, that was energized in large part by the recent discovery that dominant mutations in tau genes are linked to neurodegenerative disorders that are characterized principally by pronounced atrophy of the frontal and temporal lobes of the cortex, called frontotemporal dementias (FTD).

Multiple tau mutations leading to FTD have been discovered by several different groups (reviewed by Goedert et al., 1998. Neuron 21, p955). The study of tau protein has been of considerable interest to cell biologists. Tau was discovered in the laboratory of one of the godfathers of modern cell biology, Marc Kirschner, who in 1975 described a protein that copurified with brain microtubules (first thought to be a contaminant!) that turned out to promote microtubule (MT) assembly. Named tau, this protein was subsequently found in humans to be comprised of six isoforms produced by differential splicing of a gene with 14 exons that spans about 150 kbp. The isoforms differ by containing either none, one, or two repeats of 29 amino acids (aa) of unknown function in the N-terminal region, and 3 or 4 repeats of 31 or 32 aa that are responsible for MT binding in the C-terminal half of the protein. Implication that tau protein is involved in neurodegenerative diseases has been long-standing, ever since its discovery as a major component of neurofibrillary tangles of AD (see Milestone papers).

Today's session started with Kirk Wilhelmsen (UCSF) describing how genetic-linkage analysis was used to identify mutations in tau genes in families with FTD. He also described the remarkable pathology of the affected brains, showing there was substantial loss of neurons in the frontal cortex with rare ballooned neurons that contain abnormal filamentous aggregates. The FTD affected brains are remarkable in that they contain no evidence for senile plaques, indicating that in these diseases amyloid pathology in not likely to be involved. Evidence for neuronal loss in the entorhinal cortex and substantia nigra was especially evident in some of the FTD affected brains, yet other regions of the brain appeared remarkably unaffected. Wilhelmsen alluded that they had evidence for new tau mutations, including some in introns. He described several other familial FTD cases, especially a large family in the Lunde region of Europe, in which tau mutations were not obvious, but could not rule out that mutations exist in unsequenced introns or flanking sequences.

Virginia Lee (U Penn) went on to describe the biochemical properties of tau proteins isolated from these and other FTD cases. A synopsis of these elegant findings were described last week in Science 282, p1914. The major findings were that there is a noticeable and quantitative increase in the ratio of tau isotypes containing four MT binding repeats (4R) compared to isoforms with 3 repeats (3R) in FTD. Interestingly, many of these FTD cases contained tau proteins that migrated on SDS-PAGE gels as 2 bands (68 and 64 kDa) whereas in Alzheimer's disease (AD) an additional band of 60 kDa is usually found. These tau proteins were unusually hyperphosphorylated leading to reduced MT binding. Furthermore, reduced MT binding was observbed with recombinant bacterially expressed tau containing many of mutations associated with FTD, although all of the mutants were able to promote an equivalent mass of MT polymer over time. Lee speculated that the deficiency of tau binding to MT may have catastrophic consequences to neurons, since stabilization of the MT cytoskeleton network may be critical for maintenance of axonal structure and transport.

Peter Davies (Albert Einstein College of Medicine) described evidence that in most of the FTD cases they had examined, there was extensive neuronal loss in the frontal cortex with hardly any neurons spared. Very rare examples of surviving neurons that were immunoreactive with an anti-PHF-1 antibody could be found in brains of the FTD cases they had examined. Davies suggested that it may not be surprising that biochemical analysis of tau from these brains would be different due to such gross loss of neurons. Davies also demonstrated that purified recombinant tau protein containing FTD mutations (such as G272V, P301L, V337M, and R406W, numbered according to the longest tau isoform) had abnormal CD spectra compared to wild type tau protein. Davies suggested that by using computer programs to predict the alpha-helical content of tau protein, the FTD mutations are predicted to cause an extension of adjoining alpha-helical domains leading to altered tau conformation. This conformational abnormality, he suggested, is similar to tau in AD where altered conformation, evident by immunoreactivity with conformational specific PHF antibodies such as Alz-50, is well documented. He suggested that tau with altered conformation could potentially interact with proteins or factors producing a gain of function.

Garth Hall (Univ. of Mass.) described production of filamentous-like inclusions upon overexpression of tau protein in neurons of a primitive organism, the lamprey central neurons. The exact composition of these filaments are not known but appeared to be specifically induced due to expression of wild type human tau.

Lester Binder (Northwestern Univ.) described in vitro methods his group has devised for assembling tau into filaments. These in vitro assembled filaments share many similarities with filaments composed of tau proteins isolated from AD and other neurodegenerative disorders. Binder described how tau in physiological concentrations can be efficiently polymerized into filaments in the presence of free fatty acid (arachodonic acid) under reducing conditions and physiological salt concentrations. During this treatment tau appears to undergo a time-dependent conformational change. Filaments assembled by this procedure appear to have polarity due to preferential growth at one end. Also, tau proteins with 4R assembled better than those with 3R. Binder speculated that enhanced polymerization of tau by fatty acids may be relevant to AD since tau filaments have been shown by other investigators to be associated with membranes. This hypothesis is also attractive since perturbations in membrane function could potentially be elicited by the mutations in amlyoid precursor protein and presenilin proteins that are associated with early onset development of AD, leading to tau polymerization into PHF structures in these diseases.

Hanna Kziezak-Reding (Albert Einstein College of Medicine) used scanning transmission electron microscopy (STEM) to measure the mass per unit length (also mass/density) of tau filaments assembled under different conditions. She showed that tau filaments assembled by the so called 'hanging-drop' method differed from tau filaments isolated from AD-affected human brains. Interestingly, tau filaments assembled by the procedure described by Binder were more similar to those isolated from AD-affected human brains in terms of mass/density. It was unclear however, if the fatty acid used for propagating tau assembly contributed towards the mass/density measurements.

Lori Kohlstaedt (UCSB) showed x-ray diffraction data of tau filaments suggesting that tau forms beta sheets that appeared to be stacked in a manner such that they were both tilted and with a slight rotation.

Eva-Maria Mandelkow (Max Planck Institute) described how phosphorylation of Ser 214 of tau by PKA is sufficient to cause its detachment from MT. She also described the cloning of 4 isoforms of a tau kinase which they termed MARK. MARK is ubiquitously expressed and phosphorylates tau on residue Ser 262. Phosphorylation of tau by MARK leads to destabilization of MT. Finally, by elegant transfection studies Mandelkow showed that the overexpression of normal tau can cause aggregation of tau at mitotic organizing centers (centrosomes) and this gross overexpression can lead to sequestration of mitochondria and the ER, due to loss of MT-plus-end directed transport. The relationship of this defect to AD is unclear as tau in these experiments could bind MT, whereas tau in AD does not appear to bind to MT and should theoretically not interfere with MT-based transport.

Gloria Lee (Univ. of Iowa), concluded the session by describing results from her laboratory showing that tau interacts with Fyn kinase by several criteria, including coimmunoprecipitation assays. Fyn kinase is myristoylated, and this fatty acid modification is thought to be important for targeting of Fyn to the innerside of the plasma membrane. Lee showed that co-expression of tau and Fyn leads to tau and MT recruitment to the plasma membrane with the resulting phosphorylation of the protein on tyrosine. The interaction of tau with Fyn suggests that the apart from binding to MT, tau may bind to this and potentially other proteins in the cell. This notion may be especially important in the light of the remarkable and selective loss of neurons in the frontal cortex of FTD-affected brains. Mutation in tau by itself cannot account for this selective loss. There was speculation that perhaps tau-interacting proteins expressed in selective regions of brain may either be protective or deleterious, thereby accounting for the regional loss of neurons in FTD.

There was also discussion at the meeting that it is possible that tau in FTD, and AD, adopts abnormal conformation or conformations that could potentially lead to disease due to some detrimental gain-of-function. This issue was emphasized by Davies, and Binder, suggesting that the time has come to think about whether this is the case, as this could potentially lead to therapeutic interventions to prevent these neurodegenerative disorders. In this regard it is interesting to note that only a few years ago N. Hirokawa had shown at a previous ASCB meeting that mice disrupted of the tau gene survive and are quite normal. Therefore tau may not serve any useful function, but instead could potentially be bad for you! It should be interesting to determine if there are any humans who fail to express tau and what the consequence of the loss of this protein has on development and aging. The conclusion from the meeting was that it is still unclear exactly how tau mutations cause neurodegeneration. The power of genetics has provided us with new insights into tau neuropathologies but the cell biology and molecular mechanisms by which these mutations lead to disease is just beginning.

Novel Protein binds to APP and Microtubules Zheng et al. (Abstract 516) described the characterization of PAT1, a novel protein that binds to an 11-residue segment of the amyloid precursor protein (APP) in yeast 2-hybrid assays. PAT1 also bind to microtubules (MT). When coexpressed in HeLa and MDCK cells, PAT1 and APP partially co-localize by double immunofluorescence microscopy. The two proteins also cofractionate in sucrose gradients. The authors suggest that PAT1 is involved in sorting or trafficking of APP since heterologous expression of a HAT reporter fused to a segment of APP is differentially modulated when coexpressed with PAT1. Thus HAT activity increased when PAT1 was coexpressed in its sense orientation, but decreased when PAT was expressed in its anti-sense orientation.

Chaperone Clings to A-beta Holtzman et al. (Abstract 612) reported that 95% of immunoreactive beta-amyloid in human cerebral spinal fluid is bound to the ER chaperone Erp57. Anti-Erp57 and anti-A-beta antibodies were found to react with an approximately 62 kDa band even after SDS-PAGE suggesting that these two proteins form a tight complex. Immunoreactivity of protein extracts from Alzheimer's Disease (AD) CSF was not studied.

A Lethal Double Knockout Harada et al. (Abstract 902) have now disrupted the MAP2 gene in mouse. Like tau and MAP1B knock-out mice, MAP-2 knock out mice are fertile and have apparently normal brain cytoarchitecture. Interestingly, double tau and MAP1B knock-out mice (abstract 2284) die before they are 4 weeks old and show abnormalities of brain structures, particularly the corpus callosum, indicating that redundant functions between MAPS may not be compensated when two or more MAP genes are disrupted.

Glutamate Receptor Breakdown Chan et al. (Abstract 1421) used immunoblot analysis to quantitatively demonstrate that the polypeptides corresponding to AMPA (GluR2 and 3) and NMDA subfamilies of the glutamate receptors are decreased in AD brain lysates compared to brain lysates from most non-demented controls. Furthermore, they showed that treatment of 8 day old cortical cultures with A-beta induced similar breakdown of the Glu receptors. This breakdown could be protected by addition of zVAD-FMK, a broad spectrum caspase inhibitor. Their data suggest that cleavage of the Glu receptors by caspases slows calcium responses, making them more susceptible to death by apoptosis rather than necrosis.

Presenilin-binding Proteins Abound Mervyn Monteiro (the author of this report) and colleagues described the identification and characterization of two different proteins that interact with presenilins in the yeast-2 hybrid interaction trap (Y2H). Stabler et al. (abstract 1056) described the interaction of a calcium-binding-myristoylated protein with homology to calcineurin, which we have termed calmyrin, with the loop region of presenilin-2. Interestingly, calmyrin bound preferentially and with 10-fold greater affinity to the PS2-loop compared to the corresponding PS1-loop in Y2H assays. When coexpressed in HeLa cells, calmyrin and PS2 co-localized, and caused additive cell death.

Mah et al. (Abstract 1058) described the cloning of a novel ubitiquously expressed 66 kDa protein that interacted strongly with the C-terminal region of the presenilins in Y2H assays. A monospecific antibody raised against the novel protein reveals punctate vesicle-like staining throughout the cell. When the novel protein and PS2 are coexpressed the two proteins co-localize by double immunofluorescence microscopy. The novel protein has homology to genes of unknown function in yeast and Caenorhabditis elegans uncovered in the genome sequencing effort. The novel protein contains a ubiquitin-like motif. Many basic functions of this protein are still unknown, including its regulation and expression in AD.

Smine et al. (Abstract 2099) presented elegant biochemical and immunological data that the C-terminal region (residues 379-467) of PS1 binds the Go form of G-proteins selectively. PS1 and Go proteins coimmunoprecipitated, and the two proteins partially co-localized by double immunofluorescence microscopy. PS1 interaction with Go may be important in G-protein activation which they demonstrated, as expected, was Mg-dependent.

Presenilins Arrest Cell Cycle Monteiro and Janicki et al. (Abstract 1057) showed by BrdU labeling of HeLa cells that overexpression of both PS1 and PS2 arrest cells in the G1 phase of the cell cycle. This arrest presumably precedes apoptosis of the cells which this group has reported in an earlier publication. The mechanisms by which presenilin induces cell cycle arrest was not revealed, as simple changes in protein levels of cell cycle regulators such as, p53, p21, p27, or myc were not seen.

More About Tau Maas et al. (Abstract 2285) studied how phosphorylation of two different regions in tau proteins affects binding of tau protein to the plasma-membrane (PM). The two major phosphorylation sites studied were those located just upstream of the MT-binding repeats (whose phosphorylation can be monitored using the phosphorylation-dependent Tau-1 antibody) and those downstream of the MT repeats (phosphorylation of which can be detected using the PHF-1 antibody). Their results indicate that the tau that binds to the PM is mainly Tau-1 immunoreactive, and not PHF-1 immunoreactive. They then simulated phosphorylation in the two regions by substituting charged residues (glutamate in place of serine), and remarkably these two sets of mutants displayed selectivity of PM binding, with substitution of Glu residues in the C-terminal region inhibiting PM binding.

Ko et al. (Abstract 2286) investigated the role of tau glycation by raising an antibody specific for carboxymethyl-lysine (CML), the latter being a major glycoxidation product of advance glycation. The anti-CML antibody reacted with 3 major polypeptides of soluble PHF-tau preparations, but did not react with similar bands of PHF insoluble tau. Interestingly, PHF aggregates stained positively by immunogold labelling with the anti-CML antibody leading the investigators to conclude that tau-glycation probably occurs late in PHF formation.

Liao et al. (Abstract 2293) reported that protein phosphatase 1 (PP1) binds to the N-terminal 50-173 amino acid region of tau protein. They showed that PP1 co-localizes with both tau and MT in transfected cells. They also demonstrated that PP1 could dephosphorylate MAP2. They suggest that tau acts to target PP1 to microtubules where it may function in regulation of protein dephosphorylation.


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