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

A novel type of binding specificity to phospholipids for rat mannose-binding proteins isolated from serum and liver

AbstractMannose-binding protein (MBP) belongs to the collectin subgroup of C-type lectins with specificity for mannose and N-acetylglucosamine sugars. We investigated whether rat MBPs isolated from serum (S-MBP) and liver (L-MBP) interact with phospholipids using antibody against each MBP. Both S- and L-MBPs bound to phosphatidylinositol coated onto microtiter wells in a concentration- and a Ca2+-dependent manner. L-MBP also bound to phosphatidylglycerol and weakly to phosphatidylserine. MBPs interacted with liposomes composed of these lipids. S- and L-MBPs bound to phosphatidylinositol 4-monophosphate. L-MBP also bound to cardiolipin. These results provide evidence for a novel type of ligand binding specificity for MBPs, and raise the possibility that phospholipids are ligands for collectins

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

Effects of atelocollagen on neural stem cell function and its migrating capacity into brain in psychiatric disease model

Abstract Stem cell therapy is well proposed as a potential method for the improvement of neurodegenerative damage in the brain. Among several different procedures to reach the cells into the injured lesion, the intravenous (IV) injection has benefit as a minimally invasive approach. However, for the brain disease, prompt development of the effective treatment way of cellular biodistribution of stem cells into the brain after IV injection is needed. Atelocollagen has been used as an adjunctive material in a gene, drug and cell delivery system because of its extremely low antigenicity and bioabsorbability to protect these transplants from intrabody environment. However, there is little work about the direct effect of atelocollagen on stem cells, we examined the functional change of survival, proliferation, migration and differentiation of cultured neural stem cells (NSCs) induced by atelocollagen in vitro. By 72-h treatment 0.01-0.05 % atelocollagen showed no significant effects on survival, proliferation and migration of NSCs, while 0.03-0.05 % atelocollagen induced significant reduction of neuronal differentiation and increase of astrocytic differentiation. Furthermore, IV treated NSCs complexed with atelocollagen (0.02 %) could effectively migrate into the brain rather than NSC treated alone using chronic alcohol binge model rat. These experiments suggested that high dose of atelocollagen exerts direct influence on NSC function but under 0.03 % of atelocollagen induces beneficial effect on regenerative approach of IV administration of NSCs for CNS disease

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

Calcium-dependent regulation of tumour necrosis factor-alpha receptor signalling by copine.

The role of copines in regulating signalling from the TNF-alpha (tumour necrosis factor-alpha) receptor was probed by the expression of a copine dominant-negative construct in HEK293 (human embryonic kidney 293) cells. The construct was found to reduce activation of the transcription factor NF-kappaB (nuclear factor-kappaB) by TNF-alpha. The introduction of calcium into HEK293 cells either through the activation of muscarinic cholinergic receptors or through the application of the ionophore A23187 was found to enhance TNF-alpha-dependent activation of NF-kappaB. This effect of calcium was completely blocked by the copine dominant-negative construct. TNF-alpha was found to greatly enhance the expression of endogenous copine I, and the responsiveness of the TNF-alpha signalling pathway to muscarinic stimulation increased in parallel with the increased copine I expression. The copine dominant-negative construct also inhibited the TNF-alpha-dependent degradation of IkappaB, a regulator of NF-kappaB. All of the effects of the dominant-negative construct could be reversed by overexpression of full-length copine I, suggesting that the construct acts specifically through competitive inhibition of copine. One of the identified targets of copine I is the NEDD8-conjugating enzyme UBC12 (ubiquitin C12), that promotes the degradation of IkappaB through the ubiquitin ligase enzyme complex SCF(betaTrCP). Therefore the copine dominant-negative construct might inhibit TNF-alpha signalling by dysregulation or mislocalization of UBC12. Based on these results, a hypothesis is presented for possible roles of copines in regulating other signalling pathways in animals, plants and protozoa

Elucidation of Melanogenesis Cascade for Identifying Pathophysiology and Therapeutic Approach of Pigmentary Disorders and Melanoma

Melanogenesis is the biological and biochemical process of melanin and melanosome biosynthesis. Melanin is formed by enzymic reactions of tyrosinase family proteins that convert tyrosine to form brown-black eumelanin and yellow-red pheomelanin within melanosomal compartments in melanocytes, following the cascades of events interacting with a series of autocrine and paracrine signals. Fully melanized melanosomes are delivered to keratinocytes of the skin and hair. The symbiotic relation of a melanocyte and an associated pool of keratinocytes is called epidermal melanin unit (EMU). Microphthalmia-associated transcription factor (MITF) plays a vital role in melanocyte development and differentiation. MITF regulates expression of numerous pigmentation genes for promoting melanocyte differentiation, as well as fundamental genes for maintaining cell homeostasis. Diseases involving alterations of EMU show various forms of pigmentation phenotypes. This review introduces four major topics of melanogenesis cascade that include (1) melanocyte development and differentiation, (2) melanogenesis and intracellular trafficking for melanosome biosynthesis, (3) melanin pigmentation and pigment-type switching, and (4) development of a novel therapeutic approach for malignant melanoma by elucidation of melanogenesis cascade