Shin-ichi Tatsumi

The 28‐amino acid form of an APLP1‐derived Aβ‐like peptide is a surrogate marker for Aβ42 production in the central nervous system

Surrogate markers for the Alzheimer disease (AD)‐associated 42‐amino acid form of amyloid‐β (Aβ42) have been sought because they may aid in the diagnosis of AD and for clarification of disease pathogenesis. Here, we demonstrate that human cerebrospinal fluid (CSF) contains three APLP1‐derived Aβ‐like peptides (APL1β) that are generated by β‐ and γ‐cleavages at a concentration of ∼4.5 nM. These novel peptides, APL1β25, APL1β27 and APL1β28, were not deposited in AD brains. Interestingly, most γ‐secretase modulators (GSMs) and familial AD‐associated presenilin1 mutants that up‐regulate the relative production of Aβ42 cause a parallel increase in the production of APL1β28 in cultured cells. Moreover, in CSF from patients with pathological mutations in presenilin1 gene, the relative APL1β28 levels are higher than in non‐AD controls, while the relative Aβ42 levels are unchanged or lower. Most strikingly, the relative APL1β28 levels are higher in CSF from sporadic AD patients (regardless of whether they are at mild cognitive impairment or AD stage), than those of non‐AD controls. Based on these results, we propose the relative level of APL1β28 in the CSF as a candidate surrogate marker for the relative level of Aβ42 production in the brain.

The production ratios of AICDε51 and Aβ42 by intramembrane proteolysis of βAPP do not always change in parallel

Background:  During intramembrane proteolysis of β‐amyloid protein precursor (βAPP) by presenilin (PS)/γ‐secretase, ε‐cleavages at the membrane‐cytoplasmic border precede γ‐cleavages at the middle of the transmembrane domain. Generation ratios of Aβ42, a critical molecule for Alzheimers disease (AD) pathogenesis, and the major Aβ40 species might be associated with ε48 and ε49 cleavages, respectively. Medicines to downregulate Aβ42 production have been investigated by many pharmaceutical companies. Therefore, the ε‐cleavages, rather than the γ‐cleavage, might be more effective upstream targets for decreasing the relative generation of Aβ42. Thus, one might evaluate compounds by analyzing the generation ratio of the βAPP intracellular domain (AICD) species (ε‐cleavage‐derived), instead of that of Aβ42.

Macrophage colony stimulating factor is associated with excretion of amyloid‐β peptides from cerebrospinal fluid to peripheral blood

Background:  The process of aggregation of brain amyloid‐β peptides (Aβ) is thought to be associated with the pathogenesis of Alzheimers disease (AD). Amyloid‐β peptides are produced by sequential endoproteolysis by β‐site amyloid‐β protein precursor‐cleaving enzyme (BACE) followed by presenilin (PS)/γ‐secretase. There are several species of Aβ due to cleavage diversity of PS/γ‐secretase. The predominant species in human cerebrospinal fluid (CSF) or plasma is Aβ40, whereas Aβ42 is much more aggregatable and accumulated in senile plaques. The level of Aβ in the brain is determined by the balance between the generation and clearance of Aβ, including transport across the brain–blood barrier (BBB). Although the processes of Aβ generation and degradation have been studied in some detail, knowledge of the Aβ transport process across the BBB is limited. So far, low‐density lipoprotein receptor‐related protein (LRP1), P‐glycoprotein (P‐gp), and insulin‐like growth factor‐1 (IGF‐1) have been identified to modify the excretion of brain Aβ to the blood.

Determination of amino acid sequences of several thousand peptides in human cerebrospinal fluid (CSF)

balance ability was measured by a stabilometer, G6100 (Anima Ltd.) The training prolonged the balance holding time while standing on one foot with both eyes closed or eyes opend, and reduced the locus length per unit area both while standing on one foot with and without eyes closed, shortened the performance time of Kazufusen, but did not show a significant effect on Kana Pick Up Test. The leg muscle strength and body weight ratio were not changed before and after the training. These results indicate the existence of interaction between the cognitive and vestibular function.

Trial for detection of Aβ-like peptides in peripheral blood

protein microarray technology, showed a significant difference in mild AD participants or subjects after cognitive decline. Of the ratios observed to be significantly altered, 93% (14 of 15) involved either heme oxygenase 1, biliverdin reductase A or biliverdin reductase B. Using mass spectrometry we have identified several other functional protein groups that may be potential biomarkers for AD, such as metal/transition metal binding proteins, proteins with associated receptor activity/binding and proteins with lipid transporter activity. Conclusions: Several proteins have been identified in serum that could potentially serve as biomarkers for the early diagnosis of Alzheimer’s disease. Proteins involved in heme degradation and oxidative stress, such as heme oxygenase 1 and biliverdin reductase A and B, were shown to be promising targets for future biomarker validation using a large and blinded study set. We conclude, that although the brain is the center of pathology for AD, peripheral blood is a promising source for AD biomarker development.

Loose membrane-bound endogenous factors might modulates the intramembrane cleavages of βAPP

Mitochondria were isolated according to Rajapakse et al. (2001). M-aconitase activity was measured by monitoring formation of cis-aconitate at 240 nM. Results: In the more damaged FC of SFAD and AD brain, the enzyme activity was decreased compared with Co region whereas in the less damaged OPC of SFAD and AD brain the parameter was not changed. Oxidants Ab(25-35), H2O2 and homocysteine (Hcy) dose-dependently inhibited maconitase. In Co FC, maximal inhibitory effect increased in sequence: 0.5 mM Hcy (15%) < 1mM H2O2 (34%) < 0.001 mM Ab(25-35) (40%). In AD FC, Ab(25-35) and H2O2 induced weaker enzyme inhibition (26%) than in Co FC whereas in SFAD FC inhibition was lowest (18%). Co and AD brain OPC showed lower inhibition of m-aconitase by oxidants than FC. SFAD brain revealed no regional difference of inhibition. In contrast to oxidants, the antioxidants melatonin (Mel), glutathione (GSH) and 17b-estradiol (17bE) stimulated m-aconitase in Co, AD and SFAD brain. In Co FC, maximal stimulatory effect increased in sequence: 0.01 mM 17bE (21%) < 1.0 mM GSH (46 %) < 0.01 mM Mel (67%). In SFAD FC, the effects were weaker than in Co FC. In OPC, antioxidant-induced stimulation of m-aconitase did not differ significantly from that in FC. Conclusions: Thus, m-aconitase of human brain reveals opposite response to endogenous proand antioxidants. The mitochondrial oxidant-antioxidant relationship may play a principal role in the regulation of m-aconitase activity and, thereby, Krebs cycle rate in aged, AD and SFAD brain regions.

Proteolytic processing of membrane anchored Neuregulin1 in cell culture

Background: BACE1/2 play an essential role for Neuregulin1 (NRG1)-related peripheral myelination. Here, we studied sequential proteolytic processing of NRG1. Methods: NRG1b1/1b2 were stably expressed and analyzed their degradation in cultured cell. Soluble ectodomain NRG1, generated by proteolysis at the juxtamembrane region of the full-length form, and secreted Abeta-like peptides were searched. Results: We revealed that Abeta-like peptides derived from NRG1b1 but not NRG1b2 were produced mainly by a combination of the alphaand gamma-secretases. The mechanism is reminiscent of production of Nbeta/p3 peptides. We still have not reached any conclusion whether BACE1/2 play roles in the production of soluble ectodomain NRG1b1/1b2. Conclusions: Further studies are necessary how BACE1/2 involve in NRG1-related peripheral myelination.

P2-366: Inhibition of Aβ production without perturbing Notch and Neuregulin signaling

biological activity. Methods: Affinity selection of random peptide displaying phage library was performed using the anti-fibril scFv, B6 as a mold at 3-rounds biopanning. The isolated phage clones were analyzed their motif DNA sequences by the ABI Genetic Analyzer PRISM 3100. The motif sequences were chemically synthesized and tested their effect on A 1-42 fibril formation by thioflavin T and precipitation assay (Yoshihara, et al., J. Biochem., 2, 2008). Results: We isolated phage clones from peptide phage display libraries and identified several different peptide motifs (B6L1, B6-L10 and B7-C15). The deduced amino acid sequences of these peptide motifs have little homology to A 1-42 peptide. The synthetic peptides based on these phage motifs (B6-L1, B7-C15) had inhibitory effect on A 1-42 fibrillation by binding to prefibrillar soluble oligomers. Conclusions: Taken these results together with the sequence homology analysis, it is suggested that B6-L1 and B7-C15 might be mimics of A 1-42 fibril. To investigate this possibility, we characterize the specificity of immune response elicited with these peptides in mice.

P3-339: Characteristics of membrane-bound factors affecting presenilin/γ-secretase cleavages

Neuron-specific enolase levels were measured by direct ELISA and used to adjust ACE measurements in the cortex for neuronal loss. ACE protein and activity were determined in post-mortem CSF samples from a subset of these cases. Results: Adjusted ACE protein and activity in frontal cortex were highly correlated (p 0.0001, r 0.82) and were significantly increased in AD (p 0.05). The levels rose with increasing Braak stage and correlated with parenchymal A load (p 0.0069, r 0.27). In CSF samples from AD cases, ACE enzyme activity was increased (p 0.058) but ACE protein levels were decreased (p 0.05) and correlated inversely with Braak stage. ACE protein levels in CSF were lower in cases with ACE-1 II (carrying increased risk for AD) than those with DD (protective against AD) alleles. No effect of ACE-1 genotype on ACE protein or activity was evident in frontal cortex. Conclusions: ACE protein and activity are increased in frontal cortex in AD. The levels correlate directly with Braak stage and A load, raising the possibility that ACE expression is upregulated by A . The association between ACE-1 II and reduced ACE protein in the CSF mirrors previous findings in plasma. The disparity between ACE protein and activity in the CSF suggests that post-translational processes are involved in determining ACE activity.