Endocytosis and Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder and is the most common cause of dementia. The pathogenesis of AD still remains unclear, including two main hypotheses: amyloid cascade and tau hyperphosphorylation. The hallmark neuropathological changes of AD are extracellular deposits of amyloid-ß (Aß) plaques and intracellular neurofibrillary tangles (NFTs). Endocytosis plays an important role in a number of cellular processes including communication with the extracellular environment, nutrient uptake, and signaling by the cell surface receptors. Based on the results of genetic and biochemical studies, there is a link between neuronal endosomal function and AD pathology. Taking this into account, we can state that in the results of previous research, endolysosomal abnormality is an important cause of neuronal lesions in the brain. Endocytosis is a central pathway involved in the regulation of the degradation of amyloidogenic components. The results of the studies suggest that a correlation between alteration in the endocytosis process and associated protein expression progresses AD. In this article, we discuss the current knowledge about endosomal abnormalities in AD.

Macrophages in the reticuloendothelial system inhibit early induction stages of mouse apolipoprotein A-II amyloidosis.

Amyloidosis refers to a group of degenerative diseases that are characterized by the deposition of misfolded protein fibrils in various organs. Deposited amyloid may be removed by a phagocyte-dependent innate immune system; however, the precise mechanisms during disease progression remain unclear. We herein investigated the properties of macrophages that contribute to amyloid degradation and disease progression using inducible apolipoprotein A-II amyloidosis model mice. Intravenously injected AApoAII amyloid was efficiently engulfed by reticuloendothelial macrophages in the liver and spleen and disappeared by 24 h. While cultured murine macrophages degraded AApoAII via the endosomal-lysosomal pathway, AApoAII fibrils reduced cell viability and phagocytic capacity. Furthermore, the depletion of reticuloendothelial macrophages before the induction of AApoAII markedly increased hepatic and splenic AApoAII deposition. These results highlight the physiological role of reticuloendothelial macrophages in the early stages of pathogenesis and suggest the maintenance of phagocytic integrity as a therapeutic strategy to inhibit disease progression.

Volatile Anesthetic Sevoflurane Precursor 1,1,1,3,3,3-Hexafluoro-2-Propanol (HFIP) Exerts an Anti-Prion Activity in Prion-Infected Culture Cells.

Prion disease is a neurodegenerative disorder with progressive neurologic symptoms and accelerated cognitive decline. The causative protein of prion disease is the prion protein (PrP), and structural transition of PrP from the normal helix rich form (PrPC) to the abnormal ß-sheet rich form (PrPSc) occurs in prion disease. While so far numerous therapeutic agents for prion diseases have been developed, none of them are still useful. A fluorinated alcohol, hexafluoro isopropanol (HFIP), is a precursor to the inhalational anesthetic sevoflurane and its metabolites. HFIP is also known as a robust α-helix inducer and is widely used as a solvent for highly aggregated peptides. Here we show that the α-helix-inducing activity of HFIP caused the conformational transformation of the fibrous structure of PrP into amorphous aggregates in vitro. HFIP added to the ScN2a cell medium, which continuously expresses PrPSc, reduced PrPSc protease resistance after 24-h incubation. It was also clarified that ScN2a cells are more susceptible to HFIP than any of the cells being compared. Based on these findings, HFIP is expected to develop as a therapeutic agent for prion disease.

Isomerization of Asp is essential for assembly of amyloid-like fibrils of αA-crystallin-derived peptide.

Post-translational modifications are often detected in age-related diseases associated with protein misfolding such as cataracts from aged lenses. One of the major post-translational modifications is the isomerization of aspartate residues (L-isoAsp), which could be non-enzymatically and spontaneously occurring in proteins, resulting in various effects on the structure and function of proteins including short peptides. We have reported that the structure and function of an αA66-80 peptide, corresponding to the 66-80 (66SDRDKFVIFLDVKHF80) fragment of human lens αA-crystallin, was dramatically altered by the isomerization of aspartate residue (Asp) at position 76. In the current study, we observed amyloid-like fibrils of L-isoAsp containing αA66-80 using electron microscopy. The contribution of each amino acid for the peptide structure was further evaluated by circular dichroism (CD), bis-ANS, and thioflavin T fluorescence using 14 alanine substituents of αA66-80, including L-isoAsp at position 76. CD of 14 alanine substituents demonstrated random coiled structures except for the substituents of positively charged residues. Bis-ANS fluorescence of peptide with substitution of hydrophobic residue with alanine revealed decreased hydrophobicity of the peptide. Thioflavin T fluorescence also showed that the hydrophobicity around Asp76 of the peptide is important for the formation of amyloid-like fibrils. One of the substitutes, H79A (SDRDKFVIFL(L-isoD)VKAF) demonstrated an exact ß-sheet structure in CD and highly increased Thioflavin T fluorescence. This phenomenon was inhibited by the addition of protein-L-isoaspartate O-methyltransferase (PIMT), which is an enzyme that changes L-isoAsp into Asp. These interactions were observed even after the formation of amyloid-like fibrils. Thus, isomerization of Asp in peptide is key to form fibrils of αA-crystallin-derived peptide, and L-isoAsp on fibrils can be a candidate for disassembling amyloid-like fibrils of αA-crystallin-derived peptides.

Nuclear Envelope and Nuclear Pore Complexes in Neurodegenerative Diseases-New Perspectives for Therapeutic Interventions.

Transport of proteins, transcription factors, and other signaling molecules between the nucleus and cytoplasm is necessary for signal transduction. The study of these transport phenomena is particularly challenging in neurons because of their highly polarized structure. The bidirectional exchange of molecular cargoes across the nuclear envelope (NE) occurs through nuclear pore complexes (NPCs), which are aqueous channels embedded in the nuclear envelope. The NE and NPCs regulate nuclear transport but are also emerging as relevant regulators of chromatin organization and gene expression. The alterations in nuclear transport are regularly identified in affected neurons associated with human neurodegenerative diseases. This review presents insights into the roles played by nuclear transport defects in neurodegenerative disease, focusing primarily on NE proteins and NPCs. The subcellular mislocalization of proteins might be a very desirable means of therapeutic intervention in neurodegenerative disorders.

PrP (122-139) is a covert mitochondrial targeting signal of prion protein and it specifically triggers the perinuclear clustering of mitochondria in neuronal culture cells.

In many neurodegenerative diseases, mitochondria are actively involved in the onset and/or progression of diseases because the energy depletion of the neuronal cells directly leads to the dysfunction and degeneration of cells. In the case of prion diseases, mitochondrial involvement has been reported recently and evidence that prion protein (PrP) is localized in mitochondria is increasing. Despite these findings, the precise molecular mechanism by which PrP targets mitochondria remains unclear. PrP is a secretory protein and does not have a pre-sequence that targets the mitochondria, therefore, we thought that there was a covert signal in the amino acid sequence of PrP. To find the sequence, we constructed various GFP-fused PrP-truncations and colocalization with mitochondria was verified by live-cell imaging. Consequently, we found that 18 amino acids, PrP (122-139), are indispensable for the mitochondrial targeting of PrP. In addition, fluorescent microscopy observation revealed that PrP-localized mitochondria were accumulated at the perinuclear region in neuronal cells such as mouse neuroblastoma Neuro2a (N2a) and prion persistent infection N2a strain (ScN2a), anterograde movement of the mitochondria toward the cell membrane was completely inhibited because of the stacking of PrP on the outer membrane. The cristae formation of perinuclear accumulated mitochondria was disappeared indicating the reduced mitochondrial activity. Surprisingly, PrP-dependent mitochondrial perinuclear accumulation was specifically occurred on neuronal cells, whereas in epithelial HeLa cells and fibroblast COS-7 cells, no perinuclear accumulation observed even after the mitochondrial targeting of PrP.

Needle-shaped amyloid deposition in rat mammary gland: evidence of a novel amyloid fibril protein.

Amyloidosis is an extremely rare event in rats. In this study, we report that lipopolysaccharide binding protein (LBP) is the most likely amyloidogenic protein in rat mammary amyloidosis. Histologically, corpora amylacea (CA) and stromal amyloid (SA) were observed in rat mammary glands, and needle-shaped amyloid (NA) was also observed on the surface or gap of CA and SA. Following surveillance in aged rats, NA was observed in 62% of mammary tumours, 25% of male mammary glands and 83% of female mammary glands. Proteomic analysis showed that lactadherin was a major constitutive protein of CA and SA, and both were positive following immunohistochemistry with anti-lactadherin antibodies. In the same analysis, LBP was detected as a prime candidate protein in NA, and NA was positive following immunohistochemistry and immunoelectron microscopy with anti-LBP antibody. Furthermore, synthetic peptides derived from rat LBP formed amyloid fibrils in vitro. Overall, these results provide evidence that LBP is an amyloid precursor protein of NA in rat mammary glands.

WITHDRAWN: Association Between Alzheimer's Disease and Cancer - A Short Overview

The following article has been withdrawn at the request of the authors and editor of the journal Current Medicinal Chemistry: Title: Association between Alzheimer's Disease and Cancer - A Short Overview. Authors: Katarzyna Szczechowiak, Anna Brzecka, Naomi Hachiya, Joanna Wyka and Jerzy Leszek* Bentham Science apologizes to the readers of the journal for any inconvenience this may cause. Bentham Science Disclaimer: It is a condition of publication that manuscripts submitted to this journal have not been published and will not be simultaneously submitted or published elsewhere. Furthermore, any data, illustration, structure or table that has been published elsewhere must be reported, and copyright permission for reproduction must be obtained. Plagiarism is strictly forbidden, and by submitting the article for publication the authors agree that the publishers have the legal right to take appropriate action against the authors, if plagiarism or fabricated information is discovered. By submitting a manuscript the authors agree that the copyright of their article is transferred to the publishers if and when the article is accepted for publication.

Comprehensive proteomic profiles of mouse AApoAII amyloid fibrils provide insights into the involvement of lipoproteins in the pathology of amyloidosis.

Amyloidosis is a disorder characterized by extracellular fibrillar deposits of misfolded proteins. The amyloid deposits commonly contain several non-fibrillar proteins as amyloid-associated proteins, but their roles in amyloidosis pathology are still unknown. In mouse senile amyloidosis, apolipoprotein A-II (ApoA-II) forms extracellular amyloid fibril (AApoAII) deposits with other proteins (AApoAII-associated proteins) in many organs. We previously reported that R1.P1-Apoa2c mice provide a reproducible model of AApoAII amyloidosis. In order to investigate the sequential alterations of AApoAII-associated protein, we performed a proteomic analysis of amyloid fibrils extracted from mouse liver tissues that contained different levels of AApoAII deposition. We identified 6 AApoAII-associated proteins that constituted 20 of the top-ranked proteins in mice with severe AApoAII deposition. Although the amount of AApoAII-associated proteins increased with the progression of amyloidosis, the relative abundance of AApoAII-associated proteins changed little throughout the progression of amyloidosis. On the other hand, plasma levels of these proteins showed dramatic changes during the progression of amyloidosis. In addition, we confirmed that AApoAII-associated proteins were significantly associated with lipid metabolism based on functional enrichment analysis, and lipids were co-deposited with AApoAII fibrils from early stages of development of amyloidosis. Thus, these results demonstrate that lipoproteins are involved in AApoAII amyloidosis pathology. SIGNIFICANCE: This study presented proteomic profiles of AApoAII amyloidosis during disease progression and it revealed co-deposition of lipids with AApoAII deposits based on functional analyses. The relative abundance of AApoAII-associated proteins in the amyloid fibril fractions did not change over the course of development of AApoAII amyloidosis pathology. However, their concentrations in plasma changed dramatically with progression of the disease. Interestingly, several AApoAII-associated proteins have been found as constituents of lipid-rich lesions of other degenerative diseases, such as atherosclerosis and age-related macular degeneration. The common protein components among these diseases with lipid-rich deposits could be accounted for by a lipoprotein retention model.

TRIM32-Cytoplasmic-Body Formation Is an ATP-Consuming Process Stimulated by HSP70 in Cells.

The spontaneous and energy-releasing reaction of protein aggregation is typically prevented by cellular quality control machinery (QC). TRIM32 is a member of the TRIM (tripartite motif-containing) ubiquitin E3 ligases, and when overexpressed in cultured cells, readily forms spherical inclusions designated as cytoplasmic bodies (CBs) even without proteasome inhibition. Here, we show that HSP70, a central QC component, is a primary binding factor of overexpressed TRIM32. Contrary to expectation, however, we find that this molecular chaperone facilitates and stabilizes CB assembly depending on intrinsic ATPase activity, rather than preventing CB formation. We also show that the HSP70-TRIM32 complex is biochemically distinct from the previously characterized 14-3-3-TRIM32 phospho-complex. Moreover, the two complexes have opposing roles, with HSP70 stimulating CB formation and 14-3-3 retaining TRIM32 in a diffuse form throughout the cytosol. Our results suggest that CB inclusion formation is actively controlled by cellular QC and requires ATP, similar to protein folding and degradation reactions.

The pathological and biochemical identification of possible seed-lesions of transmitted transthyretin amyloidosis after domino liver transplantation.

The most serious issue in domino liver transplantation (DLT) using liver grafts from patients with transthyretin (TTR)-related familial amyloid polyneuropathy (FAP) is the development of iatrogenic transmitted amyloidosis (de novo amyloidosis) in DLT-recipients. However, little is known regarding the mechanisms of the initial stage of amyloid formation in these recipients. We detected initial lesions (possible seed-lesions) of this iatrogenic amyloidosis in two recipients following liver grafting from FAP patients. Patient 1 underwent DLT at age 65 from an FAP patient with a Val30Met TTR variant and patient 2 received DLT from an FAP patient with a Val30Leu TTR variant at age 32. Patient 2 was started on diflunisal administration from 4 years after DLT. While neither patient had symptoms of FAP, small amyloid deposits were detected on the gastroduodenal mucosae 14 months and 12 years after DLT in patient 1 and patient 2, respectively. The amyloid was analyzed using a laser microdissection system and tandem mass spectrometry. Biochemical analysis indicated that the amyloid was composed mostly of variant TTR produced from the transplanted liver in both patients. In patient 1, wild-type TTR amyloid was detectable in the duodenal mucosa obtained 2 years after DLT. This is the first study to successfully capture the pathological and biochemical features of initial-stage amyloid lesions in DLT recipients. The findings clearly indicate that amyloid deposition can start by deposition of variant TTR followed by deposition of wild-type TTR, and blocking of amyloid seed formation from variant TTR may be a key to prevent or delay the development of DLT-associated amyloidosis.

14-3-3 proteins sequester a pool of soluble TRIM32 ubiquitin ligase to repress autoubiquitylation and cytoplasmic body formation.

Deregulated expression of tripartite motif-containing protein 32 (TRIM32, an E3 ubiquitin-protein ligase) contributes to various diseases. Here we report, using quantitative proteomics and biochemistry, that 14-3-3 proteins bind to phosphorylated TRIM32 and prevent TRIM32 autoubiquitylation and the formation of TRIM32-containing cytoplasmic bodies, which are potential autoregulatory mechanisms that can reduce the concentration of soluble free TRIM32. The 14-3-3-TRIM32 interaction is dependent on protein-kinase-A-catalyzed phosphorylation of TRIM32 at Ser651. We found that the inhibitory effect of 14-3-3 is, in part, a consequence of disrupting the propensity of TRIM32 to undergo higher-order self-association without affecting its dimerization. Consequently, dimerized TRIM32 bound to 14-3-3 was sequestered in a distinct cytoplasmic pool away from the microtubule network, whereas a TRIM32 mutant that cannot bind 14-3-3 underwent multimerization and was unavailable to facilitate cell growth. Our results reveal a novel connection between ubiquitylation and phosphorylation pathways, which could modulate a variety of cell events by stimulating the formation of the 14-3-3-TRIM32 signaling complex.

The involvement of P2X1 receptor in pyramidal cell degeneration in the rat hippocampus after trimethyltin administration.

The P2 family of receptors for adenosine 5'-triphosphate (ATP) is involved in several neuronal and glial cell functions in the central nervous system (CNS), and impaired function of these receptors is associated with both neuronal and glial dysfunction. Using quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and immunohistochemical analysis, we examined the expression profiles of P2 subtype receptors in the rat hippocampus following treatment with the neurotoxicant trimethyltin (TMT). Among the subtypes, P2X1 exhibited a unique profile, with an increase in expression prior to the onset of cell death after TMT administration, and a gradual decrease thereafter in neuronal cells in the rat hippocampus. This expression pattern was similar to that of cyclooxygenase-2 (COX-2) following TMT administration. The P2X1 antagonist NF499 strongly prevented neuronal cell death induced by TMT in the CA1 region, and successfully suppressed locomotor hyperreactivity. Furthermore, NF449 administration also inhibited COX-2 expression in the CA1 region on day 3 following TMT treatment, whereas no change was observed in the CA3. These findings suggest that P2X1 plays a primary role in TMT-induced neuronal cell death in the CA1 region.

Possible involvement of calpain-like activity in normal processing of cellular prion protein.

Time-lapse imaging analysis was previously used to show that spontaneous proteolysis of PrP(C), which is fluorescence-labeled at both NH(2)- and COOH-termini, occurred in mouse neuroblastoma neuro2a (N2a) cells susceptible to PrP(Sc). We demonstrated that, unlike other protease inhibitors, a calpain inhibitor, calpastatin, drastically inhibited endoproteolysis of PrP(C), as observed with time-lapse imaging in living cells, suggesting calpain-like activity. Calpastatin also inhibited cleavage of endogenous PrP(C), and unprocessed molecules and the double-labeled PrP(C) accumulated around the perinuclear region. The molecular weight of PrP(C) fragments generated by spontaneous proteolysis was identical to those produced when PrP(C) synthesized in vitro was exposed to exogenous calpain. These results suggest that a calpain-like activity mediates normal processing of PrP(C) in N2a cells.

Detection system based on the conformational change in an aptamer and its application to simple bound/free separation.

Aptamers are good molecular recognition elements for biosensors. Especially, their conformational change, which is induced by the binding to the target molecule, enables the development of several types of useful detection systems. We applied this property to bound/free separation, which is a crucial process for highly sensitive detection. We designed aptamers which change their conformation upon binding to the target molecule and thereby expose a single-strand bearing the complementary sequence to the capture probe immobilized onto the support. We named the designed aptamers "capturable aptamers" and the capture probe "capture DNA". Three capturable aptamers were designed based on the PrP aptamer, which binds to prion protein. One of these capturable aptamers was demonstrated to recognize prion protein and change its conformation upon binding to it. A detection system using this designed capturable aptamer for prion protein was developed. Capturable aptamers and capture DNA allow us to perform simple bound/free separation with only one target ligand.

14-3-3zeta is indispensable for aggregate formation of polyglutamine-expanded huntingtin protein.

Huntington's disease (HD) is an autosomal dominant progressive neurodegenerative disorder caused by polyglutamine (polyQ) expansions in the huntingtin (Htt) protein. A hallmark of HD is the presence of aggregates-predominantly composed of NH(2)-terminal fragments of polyQ-expanded Htt-in the nucleus and cytoplasm of affected neurons. We previously proposed that 14-3-3zeta might act as a sweeper of misfolded proteins by facilitating the formation of aggregates possibly for neuroprotection; these aggregates are referred to as inclusion bodies. However, evidence available in this regard is indirect and circumstantial. In this study, analysis of the aggregation-prone protein Htt encoded by HD gene exon 1 containing polyglutamine expansions (Htt86Q) revealed that 17 residues in the NH(2)-terminal of this protein are indispensable for its aggregate formation. Immunoprecipitation assays revealed that 14-3-3beta, gamma, eta, and zeta interact with Htt86Q transfected in N2a cells. Interestingly, the small interfering ribonucleic acid (siRNA) suppression of 14-3-3zeta exclusively abolished Htt86Q aggregate formation, whereas 14-3-3beta or eta siRNA suppression did not. This indicates that 14-3-3zeta participates in aggregate formation under nonnative conditions. Our data support a novel role for 14-3-3zeta in the aggregate formation of nonnative, aggregation-prone proteins.