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"Nutraceuticals" are well-tolerated natural dietary compounds with drug-like properties that make them attractive as Alzheimer's disease (AD) therapeutics. Combination therapy for AD has garnered attention following a recent National Institute on Aging mandate, but this approach has not yet been fully validated. In this report, we combined the two most promising nutraceuticals with complementary anti-amyloidogenic properties: the plant-derived phenolics (-)-epigallocatechin-3-gallate (EGCG, an α-secretase activator) and ferulic acid (FA, a β-secretase modulator). We used transgenic mice expressing mutant human amyloid β-protein precursor and presenilin 1 (APP/PS1) to model cerebral amyloidosis. At 12 months of age, we orally administered EGCG and/or FA (30 mg/kg each) or vehicle once daily for 3 months. At 15 months, combined EGCG-FA treatment reversed cognitive impairment in most tests of learning and memory, including novel object recognition and maze tasks. Moreover, EGCG- and FA-treated APP/PS1 mice exhibited amelioration of brain parenchymal and cerebral vascular β-amyloid deposits and decreased abundance of amyloid β-proteins compared with either EGCG or FA single treatment. Combined treatment elevated nonamyloidogenic soluble APP-α and α-secretase candidate and down-regulated amyloidogenic soluble APP-β, β-C-terminal APP fragment, and β-secretase protein expression, providing evidence for a shift toward nonamyloidogenic APP processing. Additional beneficial co-treatment effects included amelioration of neuroinflammation, oxidative stress, and synaptotoxicity. Our findings offer preclinical evidence that combined treatment with EGCG and FA is a promising AD therapeutic approach.

To date, there is no effective Alzheimer's disease (AD)-modifying therapy. Nonetheless, combination therapy holds promise, and nutraceuticals (natural dietary compounds with therapeutic properties) and their synthetic derivatives are well-tolerated candidates. We tested whether combination therapy with octyl gallate (OG) and ferulic acid (FA) improves cognition and mitigates AD-like pathology in the presenilin-amyloid β-protein precursor (PSAPP) transgenic mouse model of cerebral amyloidosis. One-year-old mice with established β-amyloid plaques received daily doses of OG and FA alone or in combination for 3 months. PSAPP mice receiving combination therapy had statistically significant improved cognitive function versus OG or FA single treatment on some (but not all) measures. We also observed additional statistically significant reductions in brain parenchymal and cerebral vascular β-amyloid deposits as well as brain amyloid β-protein abundance in OG- plus FA-treated versus singly-treated PSAPP mice. These effects coincided with enhanced nonamyloidogenic amyloid β-protein precursor (APP) cleavage, increased α-secretase activity, and β-secretase inhibition. We detected elevated expression of nonamyloidogenic soluble APP-α and the α-secretase candidate, a disintegrin and metalloproteinase domain-containing protein 10. Correspondingly, amyloidogenic β-carboxyl-terminal APP fragment and β-site APP-cleaving enzyme 1 expression levels were reduced. In parallel, the ratio of β- to α-carboxyl-terminal APP fragment was decreased. OG and FA combination therapy strikingly attenuated neuroinflammation, oxidative stress, and synaptotoxicity. Co-treatment afforded additional statistically significant benefits on some, but not all, of these outcome measures. Taken together, these data provide preclinical proof-of-concept for AD combination therapy.

Amyloid precursor protein (APP) proteolysis is required for production of amyloid-β (Aβ) peptides that comprise β-amyloid plaques in the brains of patients with Alzheimer disease (AD). Here, we tested whether the experimental agent methylene blue (MB), used for treatment of methemoglobinemia, might improve AD-like pathology and behavioral deficits. We orally administered MB to the aged transgenic PSAPP mouse model of cerebral amyloidosis and evaluated cognitive function and cerebral amyloid pathology. Beginning at 15 months of age, animals were gavaged with MB (3 mg/kg) or vehicle once daily for 3 months. MB treatment significantly prevented transgene-associated behavioral impairment, including hyperactivity, decreased object recognition, and defective spatial working and reference memory, but it did not alter nontransgenic mouse behavior. Moreover, brain parenchymal and cerebral vascular β-amyloid deposits as well as levels of various Aβ species, including oligomers, were mitigated in MB-treated PSAPP mice. These effects occurred with inhibition of amyloidogenic APP proteolysis. Specifically, β-carboxyl-terminal APP fragment and β-site APP cleaving enzyme 1 protein expression and activity were attenuated. Additionally, treatment of Chinese hamster ovary cells overexpressing human wild-type APP with MB significantly decreased Aβ production and amyloidogenic APP proteolysis. These results underscore the potential for oral MB treatment against AD-related cerebral amyloidosis by modulating the amyloidogenic pathway.

DNase X is the first mammalian DNase to be isolated that is homologous to DNase I. In this study, we have examined its function using a novel monoclonal antibody and showed it to be expressed on the cell surface as a glycosylphosphatidylinositolanchored membrane protein. High level expression was observed in human muscular tissues and in myotubes obtained in vitro from RD rhabdomyosarcoma cells. We observed that RD myotubes incorporated a foreign gene, lacZ, by endocytosis but that expression of the encoded coding product, beta-galactosidase, was strongly inhibited. Overexpression of DNase X inhibited endocytosis-mediated gene transfer, whereas knockdown of DNase X with small interfering RNA had the opposite effect. These results reveal that DNase X provides a cell surface barrier to endocytosis-mediated gene transfer.

This research was originally published in the Journal of Biological Chemistry. Young-Ho Lee, Kosuke Tamura, Masahiro Maeda, Masaru Hoshino, Kazumasa Sakurai, Satoshi Takahashi, Takahisa Ikegami, Toshiharu Hase, and Yuji Goto. Cores and pH-dependent Dynamics of Ferredoxin-NADP+ Reductase Revealed by Hydrogen/Deuterium Exchange. J. Biol. Chem. 2007; 282, 5959-5967. © the American Society for Biochemistry and Molecular Biology, NMR-detected hydrogen/deuterium (H/D) exchange of amide protons is a powerful way for investigating the residue-based conformational stability and dynamics of proteins in solution. Maize ferredoxin-NADP+ reductase (FNR) is a relatively large protein with 314 amino acid residues, consisting of flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADP+)-binding domains. To address the structural stability and dynamics of FNR, H/D exchange of amide protons was performed using heteronuclear NMR at pDr values 8.0 and 6.0, physiologically relevant conditions mimicking inside of chloroplasts. At both pDr values, the exchange rate varied widely depending on the residues. The profiles of protected residues revealed that the highly protected regions matched well with the hydrophobic cores suggested from the crystal structure, and that the NADP+-binding domain can be divided into two subdomains. The global stability of FNR obtained by H/D exchange with NMR was higher than that by chemical denaturation, indicating that H/D exchange is especially useful for analyzing the residue-based conformational stability of large proteins, for which global unfolding is mostly irreversible. Interestingly, more dynamic conformation of the C-terminal subdomain of the NADP+-binding domain at pDr 8.0, the daytime pH in chloroplasts, than at pDr 6.0 is likely to be involved in the increased binding of NADP+ for elevating the activity of FNR. In light of photosynthesis, the present study provides the first structure-based relationship of dynamics with function for the FNR-type family in solution.

The mechanisms of energy coupling and catalytic co-operativity are not yet understood for H(+)-ATPase (ATP synthase). An Escherichia coli gamma subunit frameshift mutant (downstream of Thr-gamma 277) could not grow by oxidative phosphorylation because both mechanisms were defective (Iwamoto, A., Miki, J., Maeda, M., and Futai, M. (1990) J. Biol. Chem. 265, 5043-5048). The defect(s) of the gamma frameshift was obvious, because the mutant subunit had a carboxyl terminus comprising 16 residues different from those in the wild type. However, in this study, we surprisingly found that an Arg-beta 52-->Cys or Gly-beta 150-->Asp replacement could suppress the deleterious effects of the gamma frameshift. The membranes of the two mutants (gamma frameshift/Cys-beta 52 with or without a third mutation, Val-beta 77-->Ala) exhibited increased oxidative phosphorylation, together with 70-100% of the wild type ATPase activity. Similarly, the gamma frameshift/Asp-beta 150 mutant could grow by oxidative phosphorylation, although this mutant had low membrane ATPase activity. These results suggest that the beta subunit mutation suppressed the defects of catalytic cooperativity and/or energy coupling in the gamma mutant, consistent with the notion that conformational transmission between the two subunits is pertinent for this enzyme.

The gamma subunit mutations, gamma Met-23-->Lys or Arg, in the Escherichia coli ATP synthase were previously reported to cause dramatically inefficient energy coupling between ATPase catalysis and H+ translocation (Shin, K., Nakamoto, R.K., Maeda, M., and Futai, M. (1992) J. Biol. Chem. 267, 20835-20839). In this paper, we report that second-site mutations in the gamma subunit can suppress the effects of gamma Met-23-->Lys. By screening randomly mutagenized uncG (gamma Met-23-->Lys), eight mutations in the carboxyl-terminal region were identified; strains carrying gamma Arg-242-->Cys, gamma Gln-269-->Arg, gamma Ala-270-->Val, gamma Ile-272-->Thr, gamma Thr-273-->Ser, gamma Glu-278-->Gly, gamma Ile-279-->Thr, or gamma Val-280-->Ala in combination with gamma Met-23-->Lys were able to grow by oxidative phosphorylation. H+ pumping assayed in membranes prepared from double mutation strains demonstrated that efficient ATP-dependent H+ transport was restored. Interestingly, the single mutations, gamma Gln-269-->Arg or gamma Thr-273-->Ser, caused reduced growth by oxidative phosphorylation; however, when these mutations were in combination with gamma Met-23-->Lys, growth was substantially increased. Furthermore, strains carrying gamma Met-23-->Lys, gamma Gln-269-->Arg, or gamma Thr-273-->Ser as single mutations were temperature sensitive, whereas, strains with the double mutations, gamma Met-23-->Lys/gamma Gln-269-->Arg or gamma Met-23-->Lys/gamma Thr-273-->Ser, were thermally stable. Taken together, these results strongly suggest that gamma Met-23, gamma Arg-242, and the region between gamma Gln-269 to gamma Val-280 are close to each other and interact to mediate efficient energy coupling.

Arabidopsis thaliana has two genes (atpC1, atpC2) coding for gamma subunits of chloroplast ATP synthase. The atpC1 and atpC2 were cloned and sequenced. They had no introns within the reading frames and coded for proteins of 373 and 386 amino acid residues, respectively, including putative transit sequences (50 and 60 amino acid residues, respectively). In contrast, the spinach gamma subunit gene had two introns within the reading frame. The mature sequences coded by the two genes of A. thaliana (atpC1, 323 residues; atpC2, 326 residues) were homologous with that of spinach (J. Miki, M. Maeda, Y. Mukohata, and M. Futai (1988) FEBS Lett. 232, 221-226): the homologies of gamma subunits coded by atpC1 and atpC2 were 72%, those of the subunits coded by atpC1 and spinach cDNA were 84%, and those of the proteins coded by atpC2 and spinach cDNA were 71%. Like the spinach subunit, the gamma subunits coded by the two genes had unique regulatory domains not found in mitochondrial or bacterial subunits. Poly(A)+ mRNAs corresponding to atpC1 (1.5 kilobases) and atpC2 (2.5 kilobases) were detected in illuminated plants, the amount of the former being at least 140 times that of the latter. The atpC1 mRNA was not found in dark-adapted plants. Nuclear protein(s) specifically bound to the upstream region of atpC1 was detected by gel shift assay and its binding was shown to be inhibited by the GT-1 element of the gene encoding the ribulose-1,5-bisphosphate carboxylase small subunit, which is expressed under illumination (P. J. Green, S. A. Kay, and N. H. Chau (1987) EMBO J. 6, 2543-2549). Consistent with these findings, an increased amount of the gamma subunit was detected immunochemically in illuminated plants.

Mutants of the uncC gene for the epsilon subunit (138 amino acid residues) of Escherichia coli H+-ATPase were isolated: strain KF53 (Gln-72----end) and KF148(SD-) (two base substitutions in the Shine-Dalgarno sequence, GGAGG----AAAGG). These strains did not have F1 bound to membranes and were unable to grow by oxidative phosphorylation. A series of plasmids carrying truncated uncC genes were constructed and introduced into strain KF148(SD-). Analyses of KF148(SD-) cells with different plasmids indicated that the amino-terminal fragment of the epsilon subunit of 78-80 amino acid residues was capable of forming active membrane-bound F1-ATPase, whereas that of 73 residues was not, indicating that the carboxyl-terminal half of the epsilon subunit is not necessary for the active enzyme. Furthermore, results indicated that residues between 73 and 78-80 may have a critical role(s) in binding F1 to F0. Truncated epsilon subunits of 80 and 93 residues were identified in purified F1 from cells carrying the respective uncC genes, and only the latter subunit had intrinsic activity to inhibit ATPase of F1, suggesting that residues between 80 and 93 are essential for the inhibitory activity.

The uncD gene for the beta subunit of Escherichia coli H+-ATPase was cloned downstream of the lac promoter and mutagenized (Glu-185----Gln or Lys) by an oligonucleotide-directed procedure. The recombinant plasmid was introduced into a strain in which the unc operon for subunits of H+-ATPase was deleted. The wild-type or mutant beta subunit synthesized amounted to about 10% total cell protein and was mainly found in the cytoplasmic fraction. These subunits could be purified to almost homogeneity by conventional procedures. The wild-type and two mutant beta subunits had essentially the same Kd values for 8-anilinonaphthalene-1-sulfonate, aurovertin, and ATP, although the fluorescence intensities of 8-anilinonaphthalene-1-sulfonate and aurovertin were significantly less when bound to the two mutant beta subunits than when bound to the wild-type subunit. The three beta subunits showed essentially the same circular dichroism spectra, indicating alpha-helical contents of about 16-18%. Thus, the mutations did not cause marked change of the secondary structure of the subunit. However, measurements of theta 208 during linear increase in temperature suggested that replacement of Glu-185 by Gln or Lys slightly changed the stability of the secondary structure. Only trace amounts of alpha beta gamma complexes could be reconstituted using the two mutant beta subunits. These results suggest that Glu-185 or the region in its vicinity may be essential for subunit assembly. The methods developed in this study should be useful for further studies on the beta subunit.

Mutant alleles for the alpha subunit of H+-translocating ATPase (FoF1) were cloned from Escherichia coli strains isolated in this laboratory. Determination of their DNA sequence revealed four nonsense mutations (KF3 and KF9, Gln-20----end; KF24, Trp-111----end; KF2, Trp-231----end; KF70, Gln-252----end) and one missense mutation (KF45, Pro-143----Ser). The membranes of all the mutants except strain KF9 (KF3) had 50-70% of ATPase activities of the wild-type. Unlike the F1-ATPase of the wild-type, those of the mutants were insensitive to dicyclohexylcarbodiimide and were easier to solubilize from membranes. As membranes of strain KF24 had F1-ATPase activity, these results suggest that at least a part of the F1-binding sites could be formed without a region between residues 111 and the carboxyl terminus of the alpha subunit. However, normal interactions between Fo and F1 require regions between residues 252 and 271 (carboxyl terminus) and in the vicinity of Pro-143. Membranes of strain KF45 were capable of forming a low ATP-driven H+ gradient, whereas other membranes were not. The possibility that the region between residues 252 and 271 is involved in H+ translocation is discussed.

A sequence of 10 amino acids (I-C-S-D-K-T-G-T-L-T) of ion motive ATPases such as Na+/K+-ATPase is similar to the sequence of the beta subunit of H+-ATPases, including that of Escherichia coli (I-T-S-T-K-T-G-S-I-T) (residues 282-291). The Asp (D) residue phosphorylated in ion motive ATPase corresponds to Thr (T) of the beta subunit. This substitution may be reasonable because there is no phosphoenzyme intermediate in the catalytic cycle of F1-ATPase. We replaced Thr-285 of the beta subunit by an Asp residue by in vitro mutagenesis and reconstituted the alpha beta gamma complex from the mutant (or wild-type) beta and wild-type alpha and gamma subunits. The uni- and multisite ATPase activities of the alpha beta gamma complex with mutant beta subunits were about 20 and 30% of those with the wild-type subunit. The rate of ATP binding (k1) of the mutant complex under uni-site conditions was about 10-fold less than that of the wild-type complex. These results suggest that Thr-285, or the region in its vicinity, is essential for normal catalysis of the H+-ATPase. The mutant complex could not form a phosphoenzyme under the conditions where the H+/K+-ATPase is phosphorylated, suggesting that another residue(s) may also be involved in formation of the intermediate in ion motive ATPase. The wild-type alpha beta gamma complex had slightly different kinetic properties from the wild-type F1, possibly because it did not contain the epsilon subunit.

The atpA and atpB genes coding for the alpha and beta subunits, respectively, of membrane ATPase were cloned from a methanogen Methanosarcina barkeri, and the amino acid sequences of the two subunits were deduced from the nucleotide sequences. The methanogenic alpha (578 amino acid residues) and beta (459 amino acid residues) subunits were highly homologous to the large and small subunits, respectively, of vacuolar H+-ATPases; 52% of the residues of the methanogenic alpha subunit were identical with those of the large subunit of vacuolar enzyme of carrot or Neurospora crassa, respectively, and 59, 60, and 59% of the residues of the methanogenic beta subunit were identical with those of the small subunits of N. crassa, Arabidopsis thaliana, and Sacharomyces cerevisiae, respectively. The methanogenic subunits were also highly homologous to the corresponding subunits of Sulfolobus acidocaldarius ATPase. The methanogenic alpha and beta subunits showed 22 and 24% identities with the beta and the alpha subunits of Escherichia coli F1, respectively. Furthermore, important amino acid residues identified genetically in the E. coli enzyme were conserved in the methanogenic enzyme. This sequence conservation suggests that vacuolar, F1, methanogenic, and S. acidocaldarius ATPases were derived from a common ancestral enzyme.