Ameliorative Properties of Boronic Compounds in In Vitro and In Vivo Models of Alzheimer's Disease.

Alzheimer's disease (AD) is characterized by amyloid (Aß) aggregation, hyperphosphorylated tau, neuroinflammation, and severe memory deficits. Reports that certain boronic compounds can reduce amyloid accumulation and neuroinflammation prompted us to compare trans-2-phenyl-vinyl-boronic-acid-MIDA-ester (TPVA) and trans-beta-styryl-boronic-acid (TBSA) as treatments of deficits in in vitro and in vivo models of AD. We hypothesized that these compounds would reduce neuropathological deficits in cell-culture and animal models of AD. Using a dot-blot assay and cultured N2a cells, we observed that TBSA inhibited Aß42 aggregation and increased cell survival more effectively than did TPVA. These TBSA-induced benefits were extended to C. elegans expressing Aß42 and to the 5xFAD mouse model of AD. Oral administration of 0.5 mg/kg dose of TBSA or an equivalent amount of methylcellulose vehicle to groups of six- and 12-month-old 5xFAD or wild-type mice over a two-month period prevented recognition- and spatial-memory deficits in the novel-object recognition and Morris-water-maze memory tasks, respectively, and reduced the number of pyknotic and degenerated cells, Aß plaques, and GFAP and Iba-1 immunoreactivity in the hippocampus and cortex of these mice. These findings indicate that TBSA exerts neuroprotective properties by decreasing amyloid plaque burden and neuroinflammation, thereby preventing neuronal death and preserving memory function in the 5xFAD mice.

Acute toxicity testing of TiO2-based vs. oxybenzone-based sunscreens on clownfish (Amphiprion ocellaris).

Given the prevalence of skin cancer, sunscreens are recommended by dermatologists including the American Academy of Dermatology to protect skin from harmful ultraviolet rays. Unfortunately, this leads to an estimated 14,000 tons of sunscreen entering waterways each year. Many of the chemicals in sunscreens, such as oxybenzone and benzophenone-2, are indicated to have adverse effects on corals and other aquatic life. As an eco-conscious alternative, physical barrier sunscreens, such as non-nano-titanium dioxide (TiO2), have been suggested as a replacement. This study examines the impact of a non-nano-TiO2-based sunscreen over a nationally sold brand of sunscreen containing oxybenzone, on clownfish (Amphiprion ocellaris). Animals were evaluated for mortality, swimming behavior, and feeding behavior. Our data indicate that at an exposure level of 100 mg/L oxybenzone-containing sunscreen had a negative impact on mortality, leading to 25% death by the end of the 97-h testing period. Negative impacts on behavior were even more dramatic for the 100 mg/L oxybenzone-containing sunscreen, with 100% of the animals failing to feed over the first 49 h of testing and 100% of animals demonstrating abnormal swimming behavior over the entire testing period. By comparison, the non-nano-(TiO2) sunscreen at 100 mg/L had little (6.7%) negative impact on mortality and feeding. While swimming behavior was disrupted during the first 25 h of testing (26.7% abnormal movement), animals recovered well over the remainder of the testing period (out to 97 h).

Extensive and modular intrinsically disordered segments in C. elegans TTN-1 and implications in filament binding, elasticity and oblique striation.

TTN-1, a titin like protein in Caenorhabditis elegans, is encoded by a single gene and consists of multiple Ig and fibronectin 3 domains, a protein kinase domain and several regions containing tandem short repeat sequences. We have characterized TTN-1's sarcomere distribution, protein interaction with key myofibrillar proteins as well as the conformation malleability of representative motifs of five classes of short repeats. We report that two antibodies developed to portions of TTN-1 detect an approximately 2-MDa polypeptide on Western blots. In addition, by immunofluorescence staining, both of these antibodies localize to the I-band and may extend into the outer edge of the A-band in the obliquely striated muscle of the nematode. Six different 300-residue segments of TTN-1 were shown to variously interact with actin and/or myosin in vitro. Conformations of synthetic peptides of representative copies of each of the five classes of repeats--39-mer PEVT, 51-mer CEEEI, 42-mer AAPLE, 32-mer BLUE and 30-mer DispRep--were investigated by circular dichroism at different temperatures, ionic strengths and solvent polarities. The PEVT, CEEEI, DispRep and AAPLE peptides display a combination of a polyproline II helix and an unordered structure in aqueous solution and convert in trifluoroethanol to alpha-helix (PEVT, CEEEI, DispRep) and beta-turn (AAPLE) structures, respectively. The octads in BLUE motifs form unstable alpha-helix-like structures coils in aqueous solution and negligible heptad-based, alpha-helical coiled-coils. The alpha-helical structure, as modeled by threading and molecular dynamics simulations, tends to form helical bundles and crosses based on its 8-4-2-2 hydrophobic helical patterns and charge arrays on its surface. Our finding indicates that APPLE, PEVT, CEEEI and DispRep regions are all intrinsically disordered and highly reminiscent of the conformational malleability and elasticity of vertebrate titin PEVK segments. The proposed presence of long, modular and unstable alpha-helical oligomerization domains in the BLUE region of TTN-1 could bundle TTN-1 and stabilize oblique striation of the sarcomere.

Nuclear titin interacts with A- and B-type lamins in vitro and in vivo.

Lamins form structural filaments in the nucleus. Mutations in A-type lamins cause muscular dystrophy, cardiomyopathy and other diseases, including progeroid syndromes. To identify new binding partners for lamin A, we carried out a two-hybrid screen with a human skeletal-muscle cDNA library, using the Ig-fold domain of lamin A as bait. The C-terminal region of titin was recovered twice. Previous investigators showed that nuclear isoforms of titin are essential for chromosome condensation during mitosis. Our titin fragment, which includes two regions unique to titin (M-is6 and M-is7), bound directly to both A- and B-type lamins in vitro. Titin binding to disease-causing lamin A mutants R527P and R482Q was reduced 50%. Studies in living cells suggested lamin-titin interactions were physiologically relevant. In Caenorhabditis elegans embryos, two independent C. elegans (Ce)-titin antibodies colocalized with Ce-lamin at the nuclear envelope. In lamin-downregulated [lmn-1(RNAi)] embryos, Ce-titin was undetectable at the nuclear envelope suggesting its localization or stability requires Ce-lamin. In human cells (HeLa), antibodies against the titin-specific domain M-is6 gave both diffuse and punctate intranuclear staining by indirect immunofluorescence, and recognized at least three bands larger than 1 MDa in immunoblots of isolated HeLa nuclei. In HeLa cells that transiently overexpressed a lamin-binding fragment of titin, nuclei became grossly misshapen and herniated at sites lacking lamin B. We conclude that the C-terminus of nuclear titin binds lamins in vivo and might contribute to nuclear organization during interphase.

Three new isoforms of Caenorhabditis elegans UNC-89 containing MLCK-like protein kinase domains.

In Caenorhabditis elegans, the gene unc-89 is required for A-band organization of striated muscle. In mammals, a likely homolog of UNC-89, called obscurin, has been described and found to be localized at both the M-lines and Z-discs of striated muscle. Here, we show that the coding sequence for unc-89 is larger than originally thought, and that the gene encodes at least four major isoforms: UNC-89-A (original isoform, 732 kDa), UNC-89-B (potentially 900 kDa), and UNC-89-C and UNC-89-D (each 156 kDa). UNC-89-C and -D, except for unique N-terminal tails of eight and 11 residues, respectively, are co-linear with the C terminus of UNC-89-B. The unc-89 complex transcription unit contains at least three promoters: one directing UNC-89-A and -B primarily in body-wall and pharyngeal muscle, one internal promoter directing expression of UNC-89-C primarily in body-wall muscle, and one internal promoter directing expression of UNC-89-D primarily in a few muscle cells of the tail. Isoform-specific RNA interference resulted in a muscle structural phenotype similar to a typical unc-89 mutant, but with varying degrees of severity. Antibodies generated to the interkinase region shared by the UNC-89-B, -C and -D isoforms localize to the middle of A-bands, like previously-described UNC-89 antibodies, and detect proteins on immunoblots consistent with the proposed gene organization and additional isoforms. The three new UNC-89 isoforms contain two protein kinase domains, of the myosin light chain kinase (MLCK) family. UNC-89-B contains two complete protein kinase domains, designated PK1 and PK2. UNC-89-C and -D begin with partial kinase domains, PK1-C and PK1-D. Homology modeling suggests that PK2 is catalytically active, PK1 is inactive, and that PK1-C and PK1-D have similar structures at their N termini that may create sites for interaction with other proteins.

Caenorhabditis elegans UNC-98, a C2H2 Zn finger protein, is a novel partner of UNC-97/PINCH in muscle adhesion complexes.

To further understand the assembly and maintenance of the muscle contractile apparatus, we have identified a new protein, UNC-98, in the muscle of Caenorhabditis elegans. unc-98 mutants display reduced motility and a characteristic defect in muscle structure. We show that the major defect in the mutant muscle is in the M-lines and dense bodies (Z-line analogs). Both functionally and compositionally, nematode M-lines and dense bodies are analogous to focal adhesions of nonmuscle cells. UNC-98 is a novel 310-residue polypeptide consisting of four C2H2 Zn fingers and several possible nuclear localization signal and nuclear export signal sequences. By use of UNC-98 antibodies and green fluorescent protein fusions (to full-length UNC-98 and UNC-98 fragments), we have shown that UNC-98 resides at M-lines, muscle cell nuclei, and possibly at dense bodies. Furthermore, we demonstrated that 1) the N-terminal 106 amino acids are both necessary and sufficient for nuclear localization, and 2) the C-terminal (fourth) Zn finger is required for localization to M-lines and dense bodies. UNC-98 interacts with UNC-97, a C. elegans homolog of PINCH. We propose that UNC-98 is both a structural component of muscle focal adhesions and a nuclear protein that influences gene expression.

Transgenic zebrafish model of neurodegeneration.

In Alzheimer's disease (AD), the microtubule-associated protein, tau, is compromised in its normal association with microtubules and forms into paired helical filaments (PHF) that are the hallmark cytoskeletal pathology of the disease. Several posttranslational modifications of tau including phosphorylation have been implicated in AD pathogenesis. In addition, and importantly, mutations in the genes encoding human tau have recently been implicated in a variety of hereditary dementias, collectively termed frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17). This has rekindled interest in the importance of tau in neurodegenerative diseases (cf. Vogel [1998] Science 280:1524-1525; Goedert et al. [1998] Neuron 21:955-958; D'Souza et al. [1999] PNAS 96:5598-5603). Despite significant progress in the field of tau biology and neurodegenerative diseases, several important issues remain unresolved. The early functional consequences of tau alterations in living neurons is incompletely understood, and it is not clear how tau in neurodegenerative diseases becomes redistributed from its normal concentration in neuronal axons to pathological inclusions in neuronal soma known as neurofibrillary tangles (NFT). One of the reasons for these gaps in knowledge is the relative paucity of model systems to study these processes. We have developed a transgenic model system to study the functional consequences and trafficking patterns in zebrafish neurons of human tau either mutated on sites associated with hereditary dementias or altered at select posttranslational modification sites. The overall guiding hypothesis is that the model allows dissection of a hierarchy of events relevant to potential mechanisms of neurodegenerative diseases related to critical early stages in development of disease. We showed that a FTDP-17 mutant form of human tau expressed in zebrafish neurons produced a cytoskeletal disruption that closely resembled the NFT in human disease. This model system will prove useful in the study of other mutant taus in vertebrate neurons in vivo, and the approaches developed here will have broad usefulness in the study of functional consequences and potential genetic analyses of introducing into living vertebrate neurons other molecules involved in the pathogenesis of neurodegenerative diseases.

Titins in C.elegans with unusual features: coiled-coil domains, novel regulation of kinase activity and two new possible elastic regions.

We report that there are previously unrecognized proteins in Caenorhabditis elegans that are similar to the giant muscle proteins called titins, and these are encoded by a single approximately 90kb gene. The gene structure was predicted by GeneMark.hmm and then experimentally verified. The Ce titin gene encodes polypeptides of 2.2MDa, 1.2MDa and 301kDa. The 2.2MDa isoform resembles twitchin and UNC-89 in that it contains multiple Ig (56) and FnIII (11) domains, and a single protein kinase domain. In addition, however, the 2.2MDa isoform contains four classes of short, 14-51 residue, repeat motifs arranged mostly in many tandem copies. One of these tandem repeat regions is similar to the PEVK regions of vertebrate and fly titins. As the PEVK region is one of the main elastic elements of the titins and is also composed of short tandem repeats, this suggests that the repeat motifs in the Ce titins may have a similar elastic function. An interesting aspect of the two largest Ce titin isoforms, is that in contrast to other members of the twitchin/titin family, there are multiple regions which are likely to form coiled-coil structure. In transgenic animals, the first approximately 100 residues of the largest isoforms targets to dense bodies, the worm analogs of Z-discs. Anti-Ce titin antibodies show localization to muscle I-bands beginning at the L2-L3 larval stages and this pattern continues into adult muscle. Ce titins may not have a role in early myofibril assembly: (1) Ce titins are too short to span half a sarcomere, and the onset of their expression is well after the initial assembly of thick filaments. (2) Ce titins are not localized to I-bands in embryonic or L1 larval muscle. The Ce titin protein kinase domain is most similar to the kinase domains of the twitchins and projectin. The Ce titin kinase has protein kinase activity in vitro, and this activity is regulated by a novel mechanism.