12 Dec 1998

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). 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, University of Pennsylvania, 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 282:1914. 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 three repeats (3R) in FTD. Interestingly, many of these FTD cases contained tau proteins that migrated on SDS-PAGE gels as two bands (68 and 64 kDa) whereas in Alzheimer's disease (AD) an additional band of 60 kDα 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 of 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 α-helical content of tau protein, the FTD mutations are predicted to cause an extension of adjoining α-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, University of Massachusetts, 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 of Northwestern University 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 β-sheets that appeared to be stacked in a manner such that they were both tilted and with a slight rotation.

Eva-Maria Mandelkow of 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 four 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 of the University 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.—June Kinoshita

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Further Reading

Papers

1. Goedert M, Crowther RA, Spillantini MG. Tau mutations cause frontotemporal dementias. Neuron. 1998 Nov;21(5):955-8. PubMed.