Plasma Biomarkers in Alzheimer’s Disease

Biomarker study on dementia has developed widely. In applying biomarkers, there seems to be several utilizations such as presymptomatic- and early-stage detection, differential diagnosis, and evaluation of treatment effect. Currently, most reliable fluid markers are amyloid peptide (Aβ) with microtubule-associated protein tau (TAU) and phosphorylated TAU (P-TAU) detected in cerebrospinal fluid (CSF). Aβ42 correlates with plaque pathology, TAU reflects the intensity of neuroaxonal degeneration, and P-TAU may correlate with neurofibrillary tangle (NFT) pathology. An attenuation of the level of Aβ42 and elevation in the ratio of Aβ42 relative to the shorter major species of Aβ42 peptide with 40 amino acid residues (Aβ40) has been identified as significant events in the early stage of Alzheimer’s disease (AD) pathology. In addition, there is great interest in blood-based markers of AD since blood extraction is much less invasive. Moreover, plasma biomarkers can be measured at relatively low expense once a standard system of measurement is established. Although there is not yet an established or validated diagnostic test for plasma biomarkers, there is great interest in blood-based markers. We will summarize reported biomarkers, describe our novel potential plasma biomarker for AD (annexin A5), offering a strategy for selecting candidates, and show our results and evaluation

Milk fat globule-EGF-factor 8 is induced from neuronal cells upon stimulation of Aβ oligomer and specifi cally localizes in amyloid plaques in the brain of mouse model for Alzheimer’s disease.

Amyloid β (Aβ), a proteolytic product of amyloid precursor protein, plays an important role for apoptosis of neuronal cells and closely links to Alzheimer’s disease, though a detailed molecular mechanism remains studied. This is partly due to a lack of molecular information on affected neurons by Aβ peptides. To get more insights of this characteristic condition, we took an advantage of proteomic approach studying culture supernatant of neuronal cells treated with Aβ peptide oligomer and screened proteins preferentially bound for phosphatidylserine (PS), an acidic phospholipid expressed on outer leafl ets of apoptotic cell membranes, by employing a modifi ed liposome. Upon stimulation by Aβ, a number of proteins were detected in culture supernatant including milk fat globule-EGF-factor 8 (MFG-E8). MFG-E8 is found to be increased in the supernatant and preferentially localized in cytoplasm of neurons but not in glial cells with Aβ treatment in cultures. Quite interestingly, localization of MFG-E8 is apparently detected in cored plaques of mouse brain of Tg2576, a model for Alzheimer’s disease carrying the Swedish mutation of APP (APPK670N, M671L). The distribution of MFG-E8 is found to be limited inside of plaques and surrounded by Aβ in all plaques examined. This expression is barely detectable in control mice brain. Signifi cant increase of MFG-E8 is further observed in plasma of Alzheimer’s disease patients. Collectively, MFG-E8 is a new component of amyloid plaques and seemed to be closely linked with Aβ primed neuronal pathology. The specifi c expression of MFG-E8 in amyloid plaques may suggest an important role of apoptotic process controlled by this particular molecule. (255 words)departmental bulletin pape

Metallothionein contributes to neuropathic pain in partial sciatic nerve ligated rats

Neuropathic pain is a chronic pain state caused by nerve injury or diseases. The symptoms involve spontaneous pain, hyperalgesia and allodynia. Neuropathic pain develops by the mechanisms both central nervous system and peripheral nervous system. Moreover,both neuronal cells and glia cells are involved in the development of neuropathic pain. However, the pathogenic mechanism of neuropathic pain is not clearly understood. We previously reported that metallothionein lacked in peripheral nerve from patients of complex regional pain syndrome by proteomic approach. In this report, we examined whether the level of metallothionein (MT) is changed in partial sciatic nerve ligation (PSL) rats as the model animal of neuropathic pain and the administration of metallothionein affects behavior against physical and thermal stimulus to PSL rats. MT-I/II expression was gradually decreased in the distal region of the injury site. At day 28, MT-I/II expression was markedly decreased in both proximal and distal region at the same level. The administration of MT signifi cantly improved allodynia and thermal hyperalgesia comparing to the administration of PBS. Moreover,GAP43, a marker protein of nerve regeneration, increased in the distal region and g lial fibrillar acidic protein, a marker protein of infl ammation, decreased in the proximal region of the injury site. These results suggest that metallothionein is deeply related to occurrence of neuropathic pain and regeneration of the injured nerve in PSL rats.departmental bulletin pape

Early expression of serum CCL8 closely correlates to non-relapse mortality after allogeneic hematopoietic stem cell transplantation

To explore the role of Chemokine (C-C motif) ligand 8 (CCL8) as a potential biomarker for acute graft-versus-host disease (aGVHD), we retrospectively analyzed the sera and clinical course of 31 patients with grade II?IV aGVHD. No deaths occurred in the ten patients with serum CCL8 concentrations less than 213 pg/mL, whereas 11 of the 21 patients with more than 213 pg/mL died within 180 days post-transplantation. This landmark analysis revealed a significantly lower urvival rate of patients with a CCL8 serum concentration greater than 213 pg/mL. Thus, elevated serum CCL8 concentration before day 100 post-transplantation may predict aGVHD prognosi

Isolation of cDNA Encoding 7H6-Reactive Polypeptide Defines a New Class of Protein with alpha-Helical Coiled-Coil Structure and DA-Box Similar to Yeast Chromosomal Segregation Proteins

7H6 monoclonal antibody was recently developed in our laboratory by im-munizing mice with a bile canaliculus-rich fraction of the rat liver. The anti-body reacted with a novel 155 Kd polypeptide designated 7H6 antigen that specifically localizes at tight junctions of various epithelia. Correlations of the paracellular barrier function of the tight junction with expression of the 7H6 antigen at the cell border have suggested important roles of this polypeptide for the maintenance of tight junctional functions. As the first step for the analysis of the antigen at the molecular level, we isolated a series of cDNA clones encod-ing 7H6-reactive polypeptides. Five clones were isolated by immunoscreening. Among them a clone designated RL5.3 which carries the largest 5.3Kb insert was characterized in this study. Both plaque screening and immunoblotting of the fusion protein produced by the RL5.3 clone with lysogen confirmed that the pro-tein specifically reacts with the 7H6 monoclonal antibody. Using DNA fragmentsof the RL5.3 clone, 21 clones were further identified. Studies with restriction enzymes and probe hybridization revealed that all the cDNA clones were derived from a single class of transcripts. A partial sequence identified one open reading frame with an α-helical coiled-coil structure and highly conserved aspartate (D)- alanine (A) residues with a helix-loop-helix structure corresponding to DA- box. Since this domain has been specifically found in yeast chromosomal segregation proteins (SMC1, CUT3 and CUT14), the polypeptide encoded by the RL5.3 clone provides the first rodent counterpart of these protein family. Yeast is known to be lethal when SMC and CUT proteins are deleted, suggesting essential roles of these proteins for cell cycle progression as a regulator for chromosomal segregation. Identification of a mammalian counterpart of this pro-tein family may give us some clues for a better understanding of fundamental regulatory mechanisms in the function of tigh junctions

Apolipoprotein E4 Frequencies in a Japanese Population with Alzheimer's Disease and Dementia with Lewy Bodies

BACKGROUND: The apolipoprotein E (APOE) ε4 allele has been reported to be a risk factor for Alzheimer's disease (AD) and dementia with Lewy bodies (DLB). Previous neuropathological studies have demonstrated similar frequencies of the APOE ε4 allele in AD and DLB. However, the few ante-mortem studies on APOE allele frequencies in DLB have shown lower frequencies than post-mortem studies. One reason for this may be inaccuracy of diagnosis. We examined APOE genotypes in subjects with AD, DLB, and a control group using the latest diagnostic criteria and MRI, SPECT, and MIBG myocardial scintigraphy. METHODS: The subjects of this study consisted of 145 patients with probable AD, 50 subjects with probable DLB, and a control group. AD subjects were divided into two groups based on age of onset: early onset AD (EOAD) and late onset AD (LOAD). All subjects had characteristic features on MRI, SPECT, and/or myocardial scintigraphy. RESULTS: The rate of APOE4 carrier status was 18.3% and the frequency of the ε4 allele was 9.7% in controls. The rate of APOE4 carrier status and the frequency of the ε4 allele were 47% and 27% for LOAD, 50% and 31% for EOAD, and 42% and 31% for DLB, respectively. CONCLUSION: The APOE4 genotypes in this study are consistent with previous neuropathological studies suggesting accurate diagnosis of AD and DLB. APOE4 genotypes were similar in AD and DLB, giving further evidence that the ε4 allele is a risk factor for both disorders

Effect of Collagen Oligopeptide Injection on Rabbit Tenositis

Effects of the collagen oligopeptide (COP) were examined by its repeat injection into the inflammatory rabbit Achilles tendon (Tendo calcaneus communis), in which tenositis was induced by injection of bacterial collagenase. COP was evaluated 5 times over a period of 3 weeks to 1 month after injection of collagenase. At 1 month after treatment, the therapeutic effect of COP was evaluated by examining the structure of collagen fibrils, amount and components of glycosaminoglycans (GAGs) and matrix metalloproteinases (MMPs), and compared with the saline injection, control, and normal groups. The Achilles tendon of rabbit in the control group (no COP injection) and saline injection group showed a notable increase in the number of fine collagen fibrils, a change in GAG composition and increase in the amount of pro-MMP-2, indicating the weakening of the tendon. In contrast, the size distribution of collagen fibrils, GAG composition and the amount of pro-MMP-2 was similar to those in the normal group. These results suggest that COP injection promotes healing processes of the tendon injury