Many organizations and individuals contributed in one way or another to lhi: r'ývision ol "The Effects of Nuclear Weapons," and their couperation is gratefully acknowledged. In particular, we wish io express our -Appreciation of tne help given us by L. J. Deal and W. W. S',hroebel of the Energy Research and De-elopment Administra!ion and by Cmdr. H. L. Hofpe of the Department Cf Defense.

Histone deacetylase 6 (HDAC6), a unique cytoplasmic deacetylase, likely plays a role in neurodegeneration by coordinating cell responses to abnormal protein aggregation. Here, we provide in vitro and in vivo evidence that HDAC6 interacts with tau, a microtubule-associated protein that forms neurofibrillary tangles in Alzheimer's disease. This interaction is mediated by the microtubule-binding domain on tau and the Ser/Glu tetradecapeptide domain on HDAC6. Treatment with tubacin, a selective inhibitor of tubulin deacetylation activity of HDAC6, did not disrupt HDAC6-tau interaction. Nonetheless tubacin treatment attenuated site-specific tau phosphorylation, as did shRNA-mediated knockdown of HDAC6. Proteasome inhibition potentiated HDAC6-tau interactions and facilitated the concentration and co-localization of HDAC6 and tau in a perinuclear aggresome-like compartment, independent of HDAC6 tubulin deacetylase activity. Furthermore, we observed that in Alzheimer's disease brains the protein level of HDAC6 was significantly increased. These findings establish HDAC6 as a tau-interacting protein and as a potential modulator of tau phosphorylation and accumulation.

In Alzheimer disease (AD) mitochondrial abnormalities occur early in the pathogenic process and likely play a significant role in disease progression. Tau is a microtubule-associated protein that is abnormally processed in AD, and a connection between tau pathology and mitochondrial impairment has been proposed. However, few studies have examined the relationship between pathological forms of tau and mitochondrial dysfunction. We recently demonstrated that inducible expression of tau truncated at Asp-421 to mimic caspase cleavage (T4C3) was toxic to immortalized cortical neurons compared with a fulllength tau isoform (T4). In this study we investigated the effects of T4C3 on mitochondrial function. Expression of T4C3 induced mitochondrial fragmentation and elevated oxidative stress levels in comparison with T4-expressing cells. Thapsigargin treatment of T4 or T4C3 cells, which causes an increase in intracellular calcium levels, resulted in a significant decrease in mitochondrial potential and loss of mitochondrial membrane integrity in T4C3 cells when compared with cells expressing T4. The mitochondrial fragmentation and mitochondrial membrane damage were ameliorated in T4C3 cells by pretreatment with cyclosporine A or FK506, implicating the calcium-dependent phosphatase calcineurin in these pathogenic events. Increased calcineurin activity has been reported in AD brain, and thus, inhibition of this phosphatase may provide a therapeutic target for the treatment of AD.Tau is a microtubule-associated protein which in a hyperphosphorylated state forms paired helical filaments; the major component of neurofibrillary tangles (NFTs) 3 (1, 2). These NFTs are one of the primary pathophysiological hallmarks of Alzheimer disease (AD) and were originally suggested to play a major role in facilitating neuronal degeneration (1). However, recent studies now suggest that mature tangles may not be the toxic species (3, 4). For example, in a repressible tau overexpression transgenic mouse model, turning off tau expression attenuated memory impairment and neuronal loss, whereas NFTs continued to accumulate (5). Furthermore, reduction of endogenous wild type tau attenuated behavioral abnormalities in an APP transgenic AD mouse model, in which substantial NFT pathology is absent (6). These and other findings suggest that a form or forms of tau that precede NFT formation may be the toxic species. There is increasing evidence that, in addition to aberrant phosphorylation, caspase cleavage of tau plays a role in the oligomerization and formation of a pathological tau species in AD (7,8). Tau is an in vitro substrate for caspase-3 and is readily cleaved at Asp-421, the caspase-3 cleavage site, located on the carboxyl-terminal end of the protein (7-10). This cleavage event results in a highly fibrillogenic tau isoform which in in vitro studies aggregates more readily and to a greater extent than full-length tau and facilitates aggregate formation of fulllength tau (7,8). Antibodies that specifically recognize Asp-421-truncated tau show ...

Glycogen synthase kinase 3 (GSK3) is a widely expressed Ser/Thr protein kinase that phosphorylates numerous substrates. This large number of substrates requires precise and specific regulation of GSK3 activity, which is achieved by a combination of phosphorylation, localization, and interactions with GSK3-binding proteins. Members of the Wnt canonical pathway have been shown to influence GSK3 activity. Through a yeast two-hybrid screen, we identified the Wnt canonical pathway co-receptor protein low density lipoprotein receptor-related protein 6 (LRP6) as a GSK3-binding protein. The interaction between the C terminus of LRP6 and GSK3 was also confirmed by in vitro GST pull-down assays and in situ coimmunoprecipitation assays. In vitro assays using immunoprecipitated proteins demonstrated that the C terminus of LRP6 significantly attenuated the activity of GSK3␤. In situ, LRP6 significantly decreased GSK3␤-mediated phosphorylation of tau at both primed and unprimed sites. Finally, it was also demonstrated that GSK3␤ phosphorylates the PPP(S/T)P motifs in the C terminus of LRP6. This is the first identification of a direct interaction between LRP6 and GSK3, which results in an attenuation of GSK3 activity. Glycogen synthase kinase 3 (GSK3)2 is a widely expressed protein kinase with high expression in the brain, and specifically within neurons (for a review, see Ref. 1). GSK3 is a unique Ser/Thr protein kinase that phosphorylates both primed (target Ser/Thr is 4 amino acids N-terminal to a prephosphorylated Ser/Thr) and unprimed (target Ser/Thr is flanked by a Pro) substrates (for a review, see Ref.2). A screen of a rat brain cDNA library revealed that GSK3 is encoded by two independent genes, GSK3␣ and GSK3␤, with molecular masses of 51 and 47 kDa, respectively (3). The two genes display 85% overall sequence identity, which is even higher in the catalytic domain (93%). In the brain, although GSK3␣ mRNA level is higher than GSK3␤, GSK3␤ protein level is higher (4). The poor relationship between transcription and translation in some tissues indicates that these two isoforms are subject to differential regulation, but little is known about the isoform-specific functions.More than 40 proteins have been reported to be phosphorylated by GSK3, including over a dozen transcription factors (reviewed in Ref.2). This large number of substrates illustrates the great potential of GSK3 to affect many cellular functions and suggests that the activity of GSK3 must be carefully regulated by individual mechanisms for each substrate. Although the mechanisms regulating GSK3 are not fully understood, precise control appears to be achieved by a combination of phosphorylation, localization, and interactions with GSK3-binding proteins (reviewed in Ref.2). Protein complexes that contain GSK3 are of major importance in regulating its actions. The best characterized of these complexes is involved in the Wnt canonical pathway, where GSK3-binding proteins control access to the GSK3 substrate, ␤-catenin, and generate a high degree of specifici...