Athena Andreadis

Phosphorylation of Tau by Fyn: Implications for Alzheimer's Disease

The abnormal phosphorylation of tau protein on serines and threonines is a hallmark characteristic of the neurofibrillary tangles of Alzheimers disease (AD). The discovery that tau could be phosphorylated on tyrosine and evidence that Aβ signal transduction involved tyrosine phosphorylation led us to question whether tyrosine phosphorylation of tau occurred during the neurodegenerative process. In this study we determined that human tau tyr18 was phosphorylated by the src family tyrosine kinase fyn. By developing both polyclonal and monoclonal probes specific for phospho-tyr18, we found that the phosphorylation of tau at tyr18 occurred at early developmental stages in mouse but was absent in the adult. Our phosphospecific probes also revealed that paired helical filament preparations exhibited phospho-tyr18 reactivity that was sensitive to phosphotyrosine-specific protein phosphatase treatment. Moreover, immunocytochemical studies indicated that tyrosine phosphorylated tau was present in the neurofibrillary tangles in AD brain. However, the staining pattern excluded neuropil threads and dystrophic neurites indicating that tyrosine phosphorylated tau was distributed in AD brain in a manner dissimilar from other abnormally phosphorylated tau. We also found evidence suggesting that differentially phosphorylated tau existed within degenerating neurons. Our data add new support for a role for fyn in the neurodegenerative process.

A retinoic acid synthesizing enzyme in ventral retina and telencephalon of the embryonic mouse

Most retinoic acid (RA) in the embryonic mouse is generated by three retinaldehyde dehydrogenases (RALDHs). RALDH1 (also called E1, AHD2 or ALDH1) is expressed in the dorsal retina, and RALDH2 (V2, ALDH11) generates most RA in the embryonic trunk. The third one, RALDH3 (V1), synthesizes the bulk of RA in the head of the early embryo. We show here that RALDH3 is a mouse homologue to ALDH6, an aldehyde dehydrogenase cloned from adult human salivary gland (Hsu, L.C., Chang, W.-C., Hiraoka, L., Hsien, C.-L., 1994. Molecular cloning, genomic organization, and chromosomal localization of an additional human aldehyde dehydrogenase gene, ALDH6. Genomics 24, 333-341), which was recently reported to act as a RALDH (Yoshida, A., Rzhetsky, A., Hsu, L.C., Chang, C., 1998. Human aldehyde dehydrogenase gene family. Eur. J. Biochem. 251, 549-557). RALDH3 expression begins in the surface ectoderm over the optic recess. In rapidly changing expression patterns it labels the appearance of several ectodermal structures: it marks the formation of the lens and the olfactory organ from ectodermal placodes, and it delineates the beginning eyelid field. Within the optic vesicle, RALDH3 is expressed in the ventral retina and the dorsal pigment epithelium. In the telencephalon, RALDH3 is expressed at high levels in the lateral part of the ganglionic eminence. From here it extends via the piriform cortex into the lower part of the septum. Of the three RALDHs, RALDH3 shows the strongest predilection for epithelia.

Pathogenic Forms of Tau Inhibit Kinesin-Dependent Axonal Transport through a Mechanism Involving Activation of Axonal Phosphotransferases

Aggregated filamentous forms of hyperphosphorylated tau (a microtubule-associated protein) represent pathological hallmarks of Alzheimers disease (AD) and other tauopathies. While axonal transport dysfunction is thought to represent a primary pathogenic factor in AD and other neurodegenerative diseases, the direct molecular link between pathogenic forms of tau and deficits in axonal transport remain unclear. Recently, we demonstrated that filamentous, but not soluble, forms of wild-type tau inhibit anterograde, kinesin-based fast axonal transport (FAT) by activating axonal protein phosphatase 1 (PP1) and glycogen synthase kinase 3 (GSK3), independent of microtubule binding. Here, we demonstrate that amino acids 2–18 of tau, comprising a phosphatase-activating domain (PAD), are necessary and sufficient for activation of this pathway in axoplasms isolated from squid giant axons. Various pathogenic forms of tau displaying increased exposure of PAD inhibited anterograde FAT in squid axoplasm. Importantly, immunohistochemical studies using a novel PAD-specific monoclonal antibody in human postmortem tissue indicated that increased PAD exposure represents an early pathogenic event in AD that closely associates in time with AT8 immunoreactivity, an early marker of pathological tau. We propose a model of pathogenesis in which disease-associated changes in tau conformation lead to increased exposure of PAD, activation of PP1-GSK3, and inhibition of FAT. Results from these studies reveal a novel role for tau in modulating axonal phosphotransferases and provide a molecular basis for a toxic gain-of-function associated with pathogenic forms of tau.

Tau Protects Microtubules in the Axon from Severing by Katanin

Microtubules in the axon are more resistant to severing by katanin than microtubules elsewhere in the neuron. We have hypothesized that this is because of the presence of tau on axonal microtubules. When katanin is overexpressed in fibroblasts, the microtubules are severed into short pieces, but this phenomenon is suppressed by the coexpression of tau. Protection against severing is also afforded by microtubule-associated protein 2 (MAP2), which has a tau-like microtubule-binding domain, but not by MAP1b, which has a different microtubule-binding domain. The microtubule-binding domain of tau is required for the protection, but within itself, provides less protection than the entire molecule. When tau (but not MAP2 or MAP1b) is experimentally depleted from neurons, the microtubules in the axon lose their characteristic resistance to katanin. These results, which validate our hypothesis, also suggest a potential explanation for why axonal microtubules deteriorate in neuropathies involving the dissociation of tau from the microtubules.

The alternative splicing of tau exon 10 and its regulatory proteins CLK2 and TRA2-BETA1 changes in sporadic Alzheimer's disease

Pathological inclusions containing fibrillar aggregates of hyperphosphorylated tau protein are a characteristic feature in tauopathies, which include Alzheimers disease (AD). Tau is a microtubule‐associated protein whose transcript undergoes alternative splicing in the brain. Exon 10 encodes one of four microtubule‐binding repeats. Exon 10 inclusion gives rise to tau protein isoforms containing four microtubule‐binding repeats (4R) whereas exclusion leads to isoforms containing only three repeats (3R). The ratio between 3R and 4R isoforms is tightly controlled via alternative splicing in the human adult nervous system and distortion of this balance results in neurodegeneration. Previous studies showed that several splicing regulators, among them hTRA2‐beta1 and CLK2, regulate exon 10 alternative splicing. Like most splicing factors, htra2‐beta and clk2 pre‐mRNAs are regulated by alternative splicing. Here, we investigated whether human postmortem brain tissue of AD patients reveal differences in alternative splicing patterns of the tau, htra2‐beta, presenilin 2 and clk2 genes when compared with age‐matched controls. We found that the splicing patterns of all four genes are altered in affected brain areas of sporadic AD patients. In these affected areas, the amount of mRNAs of tau isoforms including exon 10, the htra2‐beta1 isoform and an inactive form of clk2 are significantly increased. These findings suggest that a misregulation of alternative splicing seems to contribute to sporadic AD.

Tau exon 10, whose missplicing causes frontotemporal dementia, is regulated by an intricate interplay of cis elements and trans factors

Tau is a microtubule‐associated protein whose transcript undergoes complex regulated splicing in the mammalian nervous system. In humans, exon 10 of the gene is an alternatively spliced cassette which is adult‐specific and which codes for a microtubule binding domain. Mutations that affect splicing of exon 10 have been shown to cause inherited frontotemporal dementia (FTDP). In this study, we reconstituted naturally occurring exon 10 FTDP mutants and classified their effects on its splicing. We also carried out a comprehensive survey of the influence of splicing regulators on exon 10 inclusion and tentatively identified the site of action for several of these factors. Lastly, we identified the domains of regulators SWAP and hnRNPG, which are required for regulation of exon 10 splicing.

Mapping of a disease locus for familial rapidly progressive frontotemporal dementia to chromosome 17q12-21

Familial frontotemporal dementia (FTD) is a complex disorder with lack of distinctive histopathological markers found in other types of dementia. Most of the linkage reports from FTD families map the disease loci to chromosome 17q21-22. However, FTD is genetically heterogeneous, as linkage also has been reported to chromosome 3. In the present study, we investigated the genetics of a Swedish family with an early-onset type of rapidly progressive FTD, associated with muscular rigidity and akinetic movements. Neuropathological features such as severe frontal lobe degeneration, spongy changes, and gliosis were present in affected family members. We here report probable linkage to chromosome 17q12-21 with a maximum two-point lod score of 2.76 at theta = 0 for marker D17S806, and a peak multipoint lod score of 2.86 for the same marker. Linkage to chromosome 3 was excluded, as two-point lod scores of -2.79, and -2.27 at theta = 0.01 for markers D3S1603 and D3S1552, respectively, were obtained. Sequencing of the translated exons of a strong candidate gene in the linked region of chromosome 17, the tau gene, failed to identify any mutations segregating with the disease.

Regulation of Alternative Splicing of Human Tau Exon 10 by Phosphorylation of Splicing Factors

Tau is a microtubule-associated protein whose transcript undergoes regulated splicing in the mammalian nervous system. Exon 10 of the gene is an alternatively spliced cassette that is adult-specific and encodes a microtubule-binding domain. Mutations increasing the inclusion of exon 10 result in the production of tau protein which predominantly contains four microtubule-binding repeats and were shown to cause frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). Here we show that exon 10 usage is regulated by CDC2-like kinases CLK1, 2, 3, and 4 that phosphorylate serine-arginine-rich proteins, which in turn regulate pre-mRNA splicing. Cotransfection experiments suggest that CLKs achieve this effect by releasing specific proteins from nuclear storage sites. Our results show that changing pre-mRNA-processing pathways through phosphorylation could be a new therapeutic concept for tauopathies.

Tau gene polymorphisms and apolipoprotein E ε4 may interact to increase risk for Alzheimer’s disease

Abstract In an effort to analyze the genetic role of tau in Alzheimers disease (AD), 17 polymorphisms were identified. Eleven of these polymorphisms were in complete linkage disequilibrium and segregated as two haplotypes, A and B. The A and B haplotypes were investigated in 269 AD cases and 238 controls from two different sources, a clinic-based group (mean age of onset 65±9 years), and a population-based group (mean age of onset 80±5 years). A synergistic effect between the common tau genotype AA and apolipoprotein E (APOE e4) was found in the clinic-based AD group. Our study suggests that the common tau genotype AA may interact with APOE e4 in increasing the risk of AD in a subgroup of the AD population.

Tau exons 2 and 10, which are misregulated in neurodegenerative diseases, are partly regulated by silencers which bind a SRp30c.SRp55 complex that either recruits or antagonizes htra2beta1.

Tau is a microtubule-associated protein whose transcript undergoes complex regulated splicing in the mammalian nervous system. Exon 2 modulates the tau N-terminal domain, which interacts with the axonal membrane. Exon 10 codes for a microtubule binding domain, increasing the affinity of tau for microtubules. Both exons are excluded from fetal brain, but their default behavior is inclusion, suggesting that silencers are involved in their regulation. Exon 2 is significantly reduced in myotonic dystrophy type 1, whose symptoms include dementia. Mutations that affect exon 10 splicing cause frontotemporal dementia (FTDP). In this study, we investigated three regulators of exon 2 and 10 splicing: serine/arginine-rich (SR) proteins SRp55, SRp30c, and htra2β1. The first two inhibit both exons; htra2β1 inhibits exon 2 but activates exon 10. By deletion analysis, we identified splicing silencers located at the 5′ end of each exon. Furthermore, we demonstrated that SRp30c and SRp55 bind to both silencers and to each other. In exon 2, htra2β1 binds to the inhibitory heterodimer through its RS1 domain but not to exon 2, whereas in exon 10 the heterodimer may sterically interfere with htra2β1 binding to a purine-rich enhancer (defined by FTDP mutation E10-Δ5 = Δ280K) directly downstream of the silencer. Increased exon 10 inclusion in FTDP mutant ENH (N279K) may arise from abolishing SRp30c binding. Also, htra2β3, a naturally occurring variant of htra2β1, no longer inhibits exon 2 splicing but can partially rescue splicing of exon 10 in FTDP mutation E10-Δ5. This work provides interesting insights into the splicing regulation of the tau gene.