Increase in Tau Pathology in P290S Mapt Knock-In Mice Crossed with App NL-G-F Mice.

Alzheimer's Disease (AD) is characterized by the pathologic assembly of amyloid ß (Aß) peptide, which deposits into extracellular plaques, and tau, which accumulates in intraneuronal inclusions. To investigate the link between Aß and tau pathologies, experimental models featuring both pathologies are needed. We developed a mouse model featuring both tau and Aß pathologies by knocking the P290S mutation into murine Mapt and crossing these Mapt P290S knock-in (KI) mice with the App NL-G-F KI line. Mapt P290S KI mice developed a small number of tau inclusions, which increased with age. The amount of tau pathology was significantly larger in App NL-G-F xMapt P290S KI mice from 18 months of age onward. Tau pathology was higher in limbic areas, including hippocampus, amygdala, and piriform/entorhinal cortex. We also observed AT100-positive and Gallyas-Braak-silver-positive dystrophic neurites containing assembled filamentous tau, as visualized by in situ electron microscopy. Using a cell-based tau seeding assay, we showed that Sarkosyl-insoluble brain extracts from both 18-month-old Mapt P290S KI and App NL-G-F xMapt P290S KI mice were seed competent, with brain extracts from double-KI mice seeding significantly more than those from the Mapt P290S KI mice. Finally, we showed that App NL-G-F xMapt P290S KI mice had neurodegeneration in the piriform cortex from 18 months of age. We suggest that App NL-G-F xMapt P290S KI mice provide a good model for studying the interactions of aggregation-prone tau, Aß, neuritic plaques, neurodegeneration, and aging.

Structural mapping of post-translational modifications in human interleukin-24: role of N-linked glycosylation and disulfide bonds in secretion and activity.

Human interleukin-24 (IL-24) is unique among the IL-10 superfamily as there is considerable evidence that it possesses multiple anti-cancer properties, including direct tumor cell cytotoxicity, helper T cell (TH1) immune stimulation, and anti-angiogenic activities. The primary sequence of human IL-24 differs from homologous cytokines, because it possesses three consensus N-linked glycosylation sites and the potential for a single disulfide bond. To address the significance of these modifications in human IL-24, we analyzed the relationship between post-translational modifications and the cytokine activity of the human IL-24 protein. In contrast to related interleukins, we identified a relationship between net glycosylation, protein solubility, and cytokine activity. In addition, abrogation of the two cysteine residues by mutagenesis dramatically altered the ability of IL-24 to secrete from host cells and resulted in the concomitant loss of IL-24 activity. We conclude that, unlike other IL-10 family members, human IL-24 must be glycosylated to maintain solubility and bioavailability. Further, a single, unique disulfide bond is required for secretion and activity. These structure-function relationships show that, although IL-24 is a member of the IL-19 subfamily of IL-10-like cytokines by sequence similarity, its surface properties and its distinctive disulfide arrangement make it unique. These observations could explain the novel biological activities measured of this cytokine. Understanding the structural basis of IL-24 activity will be important in the interpretation of the function of this cytokine and in the development of scale-up strategies for biophysical and clinical applications.

A manual collection of Syt, Esyt, Rph3a, Rph3al, Doc2, and Dblc2 genes from 46 metazoan genomes--an open access resource for neuroscience and evolutionary biology.

BACKGROUND: Synaptotagmin proteins were first identified in nervous tissue, residing in synaptic vesicles. Synaptotagmins were subsequently found to form a large family, some members of which play important roles in calcium triggered exocytic events. These members have been investigated intensively, but other family members are not well understood, making it difficult to grasp the meaning of family membership in functional terms. Further difficulty arises as families are defined quite legitimately in different ways: by common descent or by common possession of distinguishing features. One definition does not necessarily imply the other. The evolutionary range of genome sequences now available, can shed more light on synaptotagmin gene phylogeny and clarify family relationships. The aim of compiling this open access collection of synaptotagmin and synaptotagmin-like sequences, is that its use may lead to greater understanding of the biological function of these proteins in an evolutionary context. RESULTS: 46 metazoan genomes were examined and their complement of Syt, Esyt, Rph3a, Rph3al, Doc2 and Dblc2 genes identified. All of the sequences were compared, named, then examined in detail. Esyt genes were formerly named Fam62. The species in this collection are Trichoplax, Nematostella, Capitella, Helobdella, Lottia, Ciona, Strongylocentrotus, Branchiostoma, Ixodes, Daphnia, Acyrthosiphon, Tribolium, Nasonia, Apis, Anopheles, Drosophila, Caenorhabditis, Takifugu, Tetraodon, Gasterosteus, Oryzias, Danio, Xenopus, Anolis, Gallus, Taeniopygia,Ornithorhynchus, Monodelphis, Mus and Homo. All of the data described in this paper is available as additional files. CONCLUSIONS: Only a subset of synaptotagmin proteins appear able to function as calcium triggers. Syt1, Syt7 and Syt9 are ancient conserved synaptotagmins of this type. Some animals carry extensive repertoires of synaptotagmin genes. Other animals of no less complexity, carry only a small repertoire. Current understanding does not explain why this is so. The biological roles of many synaptotagmins remain to be understood. This collection of genes offers prospects for fruitful speculation about the functional roles of the synaptotagmin repertoires of different animals and includes a great range of biological complexity. With reference to this gene collection, functional relationships among Syt, Esyt, Rph3a, Rph3al, Doc2 and Dblc2 genes, which encode similar proteins, can better be assessed in future.

Evolutionary genomics of plant genes encoding N-terminal-TM-C2 domain proteins and the similar FAM62 genes and synaptotagmin genes of metazoans.

BACKGROUND: Synaptotagmin genes are found in animal genomes and are known to function in the nervous system. Genes with a similar domain architecture as well as sequence similarity to synaptotagmin C2 domains have also been found in plant genomes. The plant genes share an additional region of sequence similarity with a group of animal genes named FAM62. FAM62 genes also have a similar domain architecture. Little is known about the functions of the plant genes and animal FAM62 genes. Indeed, many members of the large and diverse Syt gene family await functional characterization. Understanding the evolutionary relationships among these genes will help to realize the full implications of functional studies and lead to improved genome annotation. RESULTS: I collected and compared plant Syt-like sequences from the primary nucleotide sequence databases at NCBI. The collection comprises six groups of plant genes conserved in embryophytes: NTMC2Type1 to NTMC2Type6. I collected and compared metazoan FAM62 sequences and identified some similar sequences from other eukaryotic lineages. I found evidence of RNA editing and alternative splicing. I compared the intron patterns of Syt genes. I also compared Rabphilin and Doc2 genes. CONCLUSION: Genes encoding proteins with N-terminal-transmembrane-C2 domain architectures resembling synaptotagmins, are widespread in eukaryotes. A collection of these genes is presented here. The collection provides a resource for studies of intron evolution. I have classified the collection into homologous gene families according to distinctive patterns of sequence conservation and intron position. The evolutionary histories of these gene families are traceable through the appearance of family members in different eukaryotic lineages. Assuming an intron-rich eukaryotic ancestor, the conserved intron patterns distinctive of individual gene families, indicate independent origins of Syt, FAM62 and NTMC2 genes. Resemblances among these large, multi-domain proteins are due not only to shared ancestry (homology) but also to convergent evolution (analogy). During the evolution of these gene families, duplications and other gene rearrangements affecting domain composition, have occurred along with sequence divergence, leading to complex family relationships with accordingly complex functional implications. The functional homologies and analogies among these genes remain to be established empirically.

Abundant tau filaments and nonapoptotic neurodegeneration in transgenic mice expressing human P301S tau protein.

The identification of mutations in the Tau gene in frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) has made it possible to express human tau protein with pathogenic mutations in transgenic animals. Here we report on the production and characterization of a line of mice transgenic for the 383 aa isoform of human tau with the P301S mutation. At 5-6 months of age, homozygous animals from this line developed a neurological phenotype dominated by a severe paraparesis. According to light microscopy, many nerve cells in brain and spinal cord were strongly immunoreactive for hyperphosphorylated tau. According to electron microscopy, abundant filaments made of hyperphosphorylated tau protein were present. The majority of filaments resembled the half-twisted ribbons described previously in cases of FTDP-17, with a minority of filaments resembling the paired helical filaments of Alzheimer's disease. Sarkosyl-insoluble tau from brains and spinal cords of transgenic mice ran as a hyperphosphorylated 64 kDa band, the same apparent molecular mass as that of the 383 aa tau isoform in the human tauopathies. Perchloric acid-soluble tau was also phosphorylated at many sites, with the notable exception of serine 214. In the spinal cord, neurodegeneration was present, as indicated by a 49% reduction in the number of motor neurons. No evidence for apoptosis was obtained, despite the extensive colocalization of hyperphosphorylated tau protein with activated MAP kinase family members. The latter may be involved in the hyperphosphorylation of tau.

Comparative analysis of tandem C2 domains from the mammalian synaptotagmin family.

Intracellular membrane traffic is governed by a conserved set of proteins, including Syts (synaptotagmins). The mammalian Syt family includes 15 isoforms. Syts are membrane proteins that possess tandem C2 domains (C2AB) implicated in calcium-dependent phospholipid binding. We performed a pair-wise amino acid sequence comparison, together with functional studies of rat Syt C2ABs, to examine common and divergent properties within the mammalian family. Sequence analysis indicates three different C2AB classes, the members of which share a high degree of sequence similarity. All the other C2ABs are highly divergent in sequence. Nearly half of the Syt family does not exhibit calcium/phospholipid binding in comparison to Syt I, the major brain isoform. Syts do, however, possess a more conserved function, namely calcium-independent binding to target SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) heterodimers. All tested isoforms, except Syt XII and Syt XIII, bound the target SNARE heterodimer comprising syntaxin 1 and SNAP-25 (25 kDa synaptosome-associated protein). Our present study suggests that many Syt isoforms can function in membrane trafficking to interact with the target SNARE heterodimer on the pathway to calcium-triggered membrane fusion.

Synaptotagmin gene content of the sequenced genomes.

BACKGROUND: Synaptotagmins exist as a large gene family in mammals. There is much interest in the function of certain family members which act crucially in the regulated synaptic vesicle exocytosis required for efficient neurotransmission. Knowledge of the functions of other family members is relatively poor and the presence of Synaptotagmin genes in plants indicates a role for the family as a whole which is wider than neurotransmission. Identification of the Synaptotagmin genes within completely sequenced genomes can provide the entire Synaptotagmin gene complement of each sequenced organism. Defining the detailed structures of all the Synaptotagmin genes and their encoded products can provide a useful resource for functional studies and a deeper understanding of the evolution of the gene family. The current rapid increase in the number of sequenced genomes from different branches of the tree of life, together with the public deposition of evolutionarily diverse transcript sequences make such studies worthwhile. RESULTS: I have compiled a detailed list of the Synaptotagmin genes of Caenorhabditis, Anopheles, Drosophila, Ciona, Danio, Fugu, Mus, Homo, Arabidopsis and Oryza by examining genomic and transcript sequences from public sequence databases together with some transcript sequences obtained by cDNA library screening and RT-PCR. I have compared all of the genes and investigated the relationship between plant Synaptotagmins and their non-Synaptotagmin counterparts. CONCLUSIONS: I have identified and compared 98 Synaptotagmin genes from 10 sequenced genomes. Detailed comparison of transcript sequences reveals abundant and complex variation in Synaptotagmin gene expression and indicates the presence of Synaptotagmin genes in all animals and land plants. Amino acid sequence comparisons indicate patterns of conservation and diversity in function. Phylogenetic analysis shows the origin of Synaptotagmins in multicellular eukaryotes and their great diversification in animals. Synaptotagmins occur in land plants and animals in combinations of 4-16 in different species. The detailed delineation of the Synaptotagmin genes presented here, will allow easier identification of Synaptotagmins in future. Since the functional roles of many of these genes are unknown, this gene collection provides a useful resource for future studies.

Tau gene (MAPT) sequence variation among primates.

Filamentous tau deposits are a defining feature of a number of human neurodegenerative diseases. Apes and monkeys have been reported to be differentially susceptible to developing tau pathology. Despite this, only little is known about the organisation and sequence of Tau from nonhuman primates. Here we have sequenced Tau exons 1-13, including flanking intronic regions, and the region in intron 9 that contains Saitohin in chimpanzees, gorillas, and gibbons. Partial sequences were obtained for cynomolgus macaque and green monkey. Chimpanzee brain tau was 100% identical to human tau. Identities were 99.5% for gorilla tau and 99.0% for gibbon tau. Chimpanzee DNA was polymorphic for a repeat in intron 9, which was present in human and gorilla tau, and for the nucleotide at position +29 of the intron that follows exon 10. As was the case of the other nonhuman primates examined, chimpanzee DNA was homozygous for nucleotides used to define the H2 haplotype in human Tau. These differences between human and chimpanzee Tau may contribute to the apparent resistance of chimpanzee brain to developing tau pathology. Sequencing of Saitohin revealed an intact open reading frame in chimpanzee and gorilla, but not in gibbon or macaque.

Synuclein proteins of the pufferfish Fugu rubripes: sequences and functional characterization.

In humans, three genes encode the related alpha-, beta-, and gamma-synucleins, which function as lipid-binding proteins in vitro. They are being widely studied, mainly because of the central involvement of alpha-synuclein in a number of neurodegenerative diseases, including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. In these diseases, the normally soluble alpha-synuclein assembles into abnormal filaments. Here, we have identified and characterized the synuclein gene family from the pufferfish Fugu rubripes. It consists of four genes, which encode alpha-, beta-, gamma1-, and gamma2-synucleins. They range from 113 to 127 amino acids in length and share many of the characteristics of human synucleins, including the presence of imperfect amino-terminal repeats of 11 amino acids, a hydrophobic middle region, and a negatively charged carboxy-terminus. All four synucleins are expressed in the Fugu brain. Recombinant Fugu synucleins exhibited differential liposome binding, which was strongest for alpha-synuclein, followed by beta-, gamma2-, and gamma1-synucleins. In assembly experiments, Fugu alpha-, gamma1-, and gamma2-synucleins formed filaments more readily than human alpha-synuclein. Fugu beta-synuclein, by contrast, failed to assemble in bulk. Filament assembly of synucleins was directly proportional to their degree of hydrophobicity and their tendency to form beta-sheet structure, and correlated inversely with their net charge.

Synaptotagmin interaction with the syntaxin/SNAP-25 dimer is mediated by an evolutionarily conserved motif and is sensitive to inositol hexakisphosphate.

Synaptotagmins are membrane proteins that possess tandem C2 domains and play an important role in regulated membrane fusion in metazoan organisms. Here we show that both synaptotagmins I and II, the two major neuronal isoforms, can interact with the syntaxin/synaptosomal-associated protein of 25 kDa (SNAP-25) dimer, the immediate precursor of the soluble NSF attachment protein receptor (SNARE) fusion complex. A stretch of basic amino acids highly conserved throughout the animal kingdom is responsible for this calcium-independent interaction. Inositol hexakisphosphate modulates synaptotagmin coupling to the syntaxin/SNAP-25 dimer, which is mirrored by changes in chromaffin cell exocytosis. Our results shed new light on the functional importance of the conserved polybasic synaptotagmin motif, suggesting that synaptotagmin interacts with the t-SNARE dimer to up-regulate the probability of SNARE-mediated membrane fusion.