Oligomer-Targeting Prevention of Neurodegenerative Dementia by Intranasal Rifampicin and Resveratrol Combination - A Preclinical Study in Model Mice.

Amyloidogenic protein oligomers are thought to play an important role in the pathogenesis of neurodegenerative dementia, including Alzheimer's disease, frontotemporal dementia, and dementia with Lewy bodies. Previously we demonstrated that oral or intranasal rifampicin improved the cognition of APP-, tau-, and α-synuclein-transgenic mice by reducing the amount of Aß, tau, and α-synuclein oligomers in the brain. In the present study, to explore more effective and safer medications for dementia, we tested the drug combination of rifampicin and resveratrol, which is a multifunctional natural polyphenol with the potential to antagonize the adverse effects of rifampicin. The mixture was intranasally administered to APP-, tau-, and α-synuclein-transgenic mice, and their memory and oligomer-related pathologies were evaluated. Compared with rifampicin and resveratrol alone, the combinatorial medicine significantly improved mouse cognition, reduced amyloid oligomer accumulation, and recovered synaptophysin levels in the hippocampus. The plasma levels of liver enzymes, which reflect hepatic injury and normally increase by rifampicin treatment, remained normal by the combination treatment. Notably, resveratrol alone and the combinatorial medicine, but not rifampicin alone, enhanced the levels of brain-derived neurotrophic factor (BDNF) and its precursor, pro-BDNF, in the hippocampus. Furthermore, the combination showed a synergistic effect in ameliorating mouse cognition. These results show the advantages of this combinatorial medicine with regards to safety and effectiveness over single-drug rifampicin. Our findings may provide a feasible means for the prevention of neurodegenerative dementia that targets toxic oligomers.

Proteomic analysis of the hippocampus in Alzheimer's disease model mice by using two-dimensional fluorescence difference in gel electrophoresis.

We previously identified the E693Δ mutation in amyloid precursor protein (APP) in patients with Alzheimer's disease (AD) and then generated APP-transgenic mice expressing this mutation. As these mice possessed abundant Aß oligomers from 8 months of age but no amyloid plaques even at 24 months of age, they are a good model to study pathological effects of amyloid ß (Aß) oligomers. The two-dimensional fluorescence difference in gel electrophoresis (2D-DIGE) technology, using a mixed-sample internal standard, is now recognized as an accurate method to determine and quantify proteins. In this study, we examined the proteins for which levels were altered in the hippocampus of 12-month-old APP(E693Δ)-transgenic mice using 2D-DIGE and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Fourteen proteins were significantly changed in the hippocampus of APP(E693Δ)-transgenic mice. Actin cytoplasmic 1 (ß-actin), heat shock cognate 71kDa, γ-enolase, ATP synthase subunit ß, tubulin ß-2A chain, clathrin light chain B (clathrin) and dynamin-1 were increased. Heat shock-related 70kDa protein 2, neurofilament light polypeptide (NFL), stress-induced-phosphoprotein 2, 60kDa heat shock protein (HSP60), α-internexin, protein kinase C and casein kinase substrate in neurons protein 1 (Pacsin 1), α-enolase and ß-actin were decreased. Western blotting also validated the changed levels of HSP60, NFL, clathrin and Pacsin 1 in APP(E693Δ)-transgenic mice. The identified proteins could be classified as cytoskeleton, chaperons, neurotransmission, energy supply and signal transduction. Thus, proteomics by 2D-DIGE and LC-MS/MS has provided knowledge of the levels of proteins in the early stages of AD brain.

Proteomic analysis of the brain tissues from a transgenic mouse model of amyloid ß oligomers.

Amyloid ß (Aß) oligomers are presumed to be one of the causes of Alzheimer's disease (AD). Previously, we identified the E693Δ mutation in amyloid precursor protein (APP) in patients with AD who displayed almost no signals of amyloid plaques in amyloid imaging. We generated APP-transgenic mice expressing the E693Δ mutation and found that they possessed abundant Aß oligomers from 8months of age but no amyloid plaques even at 24months of age, indicating that these mice are a good model to study pathological effects of Aß oligomers. To elucidate whether Aß oligomers affect proteome levels in the brain, we examined the proteins and phosphoproteins for which levels were altered in 12-month-old APP(E693Δ)-transgenic mice compared with age-matched non-transgenic littermates. By two-dimensional gel electrophoresis (2DE) followed by staining with SYPRO Ruby and Pro-Q Diamond and subsequent mass spectrometry techniques, we identified 17 proteins and 3 phosphoproteins to be significantly changed in the hippocampus and cerebral cortex of APP(E693Δ)-transgenic mice. Coactosin like-protein, SH3 domain-bind glutamic acid-rich-like protein 3 and astrocytic phosphoprotein PEA-15 isoform 2 were decreased to levels less than 0.6 times those of non-transgenic littermates, whereas dynamin, profilin-2, vacuolar adenosine triphosphatase and creatine kinase B were increased to levels more than 1.5 times those of non-transgenic littermates. Furthermore, 2DE Western Blotting validated the changed levels of dynamin, dihydropyrimidinase-related protein 2 (Dpysl2), and coactosin in APP(E693Δ)-transgenic mice. Glyoxalase and isocitrate dehydrogenase were increased to levels more than 1.5 times those of non-transgenic littermates. The identified proteins could be classified into several groups that are involved in regulation of different cellular functions, such as cytoskeletal and their interacting proteins, energy metabolism, synaptic component, and vesicle transport and recycling. These findings indicate that Aß oligomers altered the levels of some proteins and phosphoproteins in the hippocampus and cerebral cortex, which could illuminate novel therapeutic avenues for the treatment of AD.

In vivo positron emission tomographic imaging of glial responses to amyloid-beta and tau pathologies in mouse models of Alzheimer's disease and related disorders.

Core pathologies of Alzheimer's disease (AD) are aggregated amyloid-ß peptides (Aß) and tau, and the latter is also characteristic of diverse neurodegenerative tauopathies. These amyloid lesions provoke microglial activation, and recent neuroimaging technologies have enabled visualization of this response in living brains using radioligands for the peripheral benzodiazepine receptor also known as the 18 kDa translocator protein (TSPO). Here, we elucidated contributions of Aß and tau deposits to in vivo TSPO signals in pursuit of mechanistic and diagnostic significance of TSPO imaging in AD and other tauopathies. A new antibody to human TSPO revealed induction of TSPO-positive microgliosis by tau fibrils in tauopathy brains. Emergence of TSPO signals before occurrence of brain atrophy and thioflavin-S-positive tau amyloidosis was also demonstrated in living mice transgenic for mutant tau by positron emission tomography (PET) with two classes of TSPO radioligands, [(11)C]AC-5216 and [(18)F]fluoroethoxy-DAA1106. Meanwhile, only modest TSPO elevation was observed in aged mice modeling Aß plaque deposition, despite the notably enhanced in vivo binding of amyloid radiotracer, [(11)C]Pittsburgh Compound-B, to plaques. In these animals, [(11)C]AC-5216 yielded better TSPO contrasts than [(18)F]fluoroethoxy-DAA1106, supporting the possibility of capturing early neurotoxicity with high-performance TSPO probes. Furthermore, an additional line of mice modeling intraneuronal Aß accumulation displayed elevated TSPO signals following noticeable neuronal loss, unlike TSPO upregulation heralding massive neuronal death in tauopathy model mice. Our data corroborate the utility of TSPO-PET imaging as a biomarker for tau-triggered toxicity, and as a complement to amyloid scans for diagnostic assessment of tauopathies with and without Aß pathologies.

Regulation of cholesterol efflux by amyloid beta secretion.

Amyloid beta (Abeta) is a key molecule in the pathogenesis of Alzheimer's disease, but its physiological function remains unclear. Abeta is produced from amyloid precursor protein (APP) by beta- and gamma-secretases, which is enhanced by high levels of cellular cholesterol, so cholesterol is a risk factor for Alzheimer's disease. This linkage led us to hypothesize that Abeta is produced to regulate cellular cholesterol levels in response to high-cholesterol stimulation. Here we show that Abeta production caused a reduction of cellular cholesterol levels in transfected HEK293 cells and neuronal IMR-32 and Neuro2a cells, which was accompanied by an increase in efflux of cholesterol from cells. Fractionation of the culture media by ultracentrifugation and subsequent immunoelectron microscopic observation revealed that Abeta assembled high-density lipoprotein-like particles with cellular cholesterol during its secretion. This assembly was mediated by the ATP-binding cassette transporter A1. APP transgenic and knockout mice exhibited lower and higher levels of cellular cholesterol in their brains, suggesting that Abeta-mediated regulation of cellular cholesterol is physiological. Furthermore, we found that, when injected into mouse cerebral ventricle, reconstituted lipoproteins with Abeta were excreted into the peripheral tissues more efficiently than those without Abeta. This result suggests that Abeta mediates cholesterol transport from the brain to the circulation. We propose, based on these findings, a novel, apolipoprotein-like function for Abeta that is involved in maintenance of cellular and cerebral cholesterol homeostasis.

Amyloid-beta E22Delta variant induces synaptic alteration in mouse hippocampal slices.

We recently identified a novel amyloid precursor protein mutation (E693Delta) in familial Alzheimer's-type dementia. This mutation produces amyloid-beta (Abeta) variant lacking glutamate-22 (E22Delta), which showed enhanced oligomerization but no fibrillization. Here, we examined in-vitro toxicity of Abeta E22Delta peptide. Wild-type Abeta1-42 showed a dose-dependent (1 nM to 1 microM) cytotoxicity to cultured neuronal cells in the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide assay, whereas Abeta1-42 E22Delta was toxic only weakly at 1 microM. In mouse hippocampal slices, however, Abeta1-42 E22Delta caused a dose-dependent (0.1-10 microM) decrease of synaptophysin, whereas wild-type Abeta1-42 was trophic at 0.1-1 microM and toxic at 10 microM. These results suggest that extracellular Abeta E22Delta causes more potent synaptic alteration, but lower neurodegeneration, than wild-type Abeta probably because of its unique aggregation property.

Cerebral vascular accumulation of Dutch-type Abeta42, but not wild-type Abeta42, in hereditary cerebral hemorrhage with amyloidosis, Dutch type.

Hereditary cerebral hemorrhage with amyloidosis, Dutch type (HCHWA-D), is an autosomal dominant disorder caused by the Dutch mutation (E693Q) in the beta-amyloid precursor protein. This mutation produces an aberrant amyloid beta (Abeta) species (AbetaE22Q) and causes severe meningocortical vascular Abeta deposition. We analyzed the Abeta composition of the vascular amyloid in the brains of HCHWA-D patients. Immunohistochemistry demonstrated that the vascular amyloid contained both Abeta40 and Abeta42, with a high Abeta40/Abeta42 ratio. In Western blotting of cerebral microvessel fractions isolated from the brains, both wild-type and Dutch-type Abeta40 were observed as major species. Reverse-phase HPLC-mass spectrometric analysis of the fractions revealed both wild-type and Dutch-type Abeta38 as the other main components of the vascular amyloid. Moreover, we detected peaks corresponding to Dutch-type Abeta42 but not to wild-type Abeta42. These results suggest a pathogenic role for the mutant Abeta42 in addition to the mutant Abeta40 in the cerebral amyloid angiopathy of HCHWA-D.

Syntaxin 5 interacts specifically with presenilin holoproteins and affects processing of betaAPP in neuronal cells.

The specific roles of syntaxin 5 (Syx 5) in the interaction with presenilin (PS) and the accumulation of beta-amyloid precursor protein (betaAPP), as well as the secretion of beta-amyloid peptide (Abeta peptide) were examined in NG108-15 cells. Syx 5, which localizes from the endoplasmic reticulum (ER) to the Golgi, bound to PS holoproteins, while the other Syxs studied did not. Among familial Alzheimer's disease (FAD)-linked PS mutants, PS1deltaE9, which lacks the endoproteolytic cleavage site, showed markedly decreased binding to Syx 5. The interaction domains in Syx 5 were mapped to the transmembrane region and to the cytoplasmic region containing the alpha-helical domains, which are distinct from the H3 (SNARE motif). Among all of the Syxs examined, only overexpression of Syx 5 resulted in the accumulation of betaAPP in the ER to cis-Golgi compartment, an attenuation of the amount of the C-terminal fragment (APP-CTF) of betaAPP, and a reduction in the secretion of Abeta peptides. Furthermore, co-expression of Syx 5 with C99 resulted in an increase in APP-CTF and suppressed Abeta secretion. Taken together, these results indicate that Syx 5 may play a specific role in the modulation of processing and/or trafficking of FAD-related proteins in neuronal cells by interaction with PS holoproteins in the early secretory compartment of neuronal cells.

Syntaxin 5 interacts with presenilin holoproteins, but not with their N- or C-terminal fragments, and affects beta-amyloid peptide production.

Mutations in presenilins 1 and 2 (PS1 and PS2) account for the majority of cases of early-onset familial Alzheimer's disease. However, the trafficking and interaction of PSs with other proteins in the early secretory pathways are poorly understood. Using co-immunoprecipitation, we found that PS bound to Syx5 (syntaxin 5), which is a target-soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor involved in endoplasmic reticulum (ER)-Golgi vesicular transport in vivo. Syx5 interacted only with the full-length PS holoproteins and not with the naturally occurring N- or C-terminal fragments. The PS holoproteins co-immunoprecipitated with the mutant Syx5, which localized to the ER and Golgi compartments, despite the substitution of the transmembrane region with that of syntaxin 1A. In contrast, the transmembrane deletion mutant that localized to the cytosol, but not to the ER or Golgi compartments, did not co-immunoprecipitate the PS holoproteins. The PS1 variant linked to familial Alzheimer's disease (PS1DeltaE9), lacking the region that contains the endoproteolytic cleavage site in the cytoplasmic loop, showed markedly decreased binding to Syx5. Immunofluorescence and sucrose-density-gradient fractionation analyses showed that the full-length PS holoproteins co-localized with Syx5 to the ER and cis-Golgi compartments. Furthermore, Syx5 overexpression resulted in the accumulation of PS holoproteins and the beta-amyloid precursor protein, and reduced the secretion of the Abeta (amyloid beta) peptide in COS-7 cells. In summary, these results indicate that Syx5 binds to full-length PSs and affects the processing and trafficking of beta-amyloid precursor protein in the early secretory compartments.

The distributions of tau short and long isoforms fused with EGFP in cultured cells.

Frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) are caused by mutations of the TAU gene. Many such mutations are located near the splicing site of exon 10 and affect the splicing ratio of 3-repeat/4-repeat tau isoforms (referred to as 3R-tau and 4R-tau) which contain 3 and 4 microtubule-binding domains, respectively. Little is known, however, concerning cellular localization of 3R-tau and 4R-tau. We examined the subcellular localization of tau isoforms in IMR-32 cells under differentiated conditions using the fusion proteins of tau isoforms probed with fluorescent protein (EGFP). 3R-tau was observed in spotty and rarely linear distributions while 4R-tau was observed in linear and sometimes spotty distributions. Together with findings of phase-contrast microscopy of cultured cells, these results indicated that 3R- and 4R-tau were predominantly localized at growth tips/branching points and along neurite processes, respectively. Due to their different localizations, balanced expression of 3R- and 4R-tau may coordinate plastic morphogenesis and stabilization of neurite processes.

Human neprilysin is capable of degrading amyloid beta peptide not only in the monomeric form but also the pathological oligomeric form.

Amyloid beta-peptide (Abeta) is widely believed to play a central role in Alzheimer's disease (AD). Coordinate regulation of cerebral Abeta level is important in the pathogenesis of AD since either increased production of Abeta from amyloid precursor protein or decreased degradation causes elevated levels of Abeta, leading to accumulation of cerebral plaque formation or amyloid angiopathy. Here we studied neprilysin, a putative proteolytic enzyme for Abeta, and found that it degraded not only monomeric but also oligomeric forms of Abeta1-40. Moreover, neprilysin was found to be capable of degradation of the oligomeric form of Abeta1-42, a significant Abeta species in early pathogenesis. Neprilysin to decrease cerebral Abeta is suggested to be inevitable factor as a vital therapeutic target.

Mutation (D472Y) in the type 3 repeat domain of cartilage oligomeric matrix protein affects its early vesicle trafficking in endoplasmic reticulum and induces apoptosis.

Cartilage oligomeric matrix protein (COMP) is a large pentameric extracellular glycoprotein found in cartilage, tendon, and synovium, and plays structural roles in cartilage as the fifth member of the thrombospondin family. Familial mutations in type 3 repeats of COMP are known to cause pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (EDM1). Although such mutations induce enlarged rough endoplasmic reticulum (rER) as a morphological change, the metabolic trafficking of mutated COMP remains unclear. In transfected COS7 cells, wild-type COMP was rapidly secreted into culture medium, while the great majority of COMP with the type 3 repeats mutation (D472Y) remained in the cells and a small portion of mutated COMP was secreted. This finding was followed up with a confocal study with an antibody specific to COMP, which demonstrated mutated COMP tightly associated with abnormally enlarged rER. Phosphorylated eIF2alpha, an ER stress protein, was expressed as a pathological reaction in virtually all COS7 cells expressing mutated but not wild-type COMP. Moreover, COS7 cells expressing mutated COMP exhibited significantly more apoptotic reaction than those expressing wild-type COMP. Pathological accumulation of COMP in rER and apoptosis in COS7 cells that were induced by the mutation (D472Y) in COMP imply that COMP mutations play a role in the pathogenesis of PSACH.