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Alzheimer's disease is an age-dependent neuro-degenerative disorder that is characterized by a progressive decline in cognitive function. [gamma]-secretase dysfunction is evident in many cases of early onset familial Alzheimer's disease. However, the mechanism by which [gamma]-secretase dysfunction results in memory loss and neurodegeneration is not fully understood. Here, we demonstrate that [gamma]-secretase is localized at synapses and regulates spine formation. We identify EphA4, one of the Ephrin receptor family members, asa substrate of [gamma]-secretase, and find that EphA4 processing is enhanced by synaptic activity. Moreover, overexpression of EphA4 intracellular domain increases the number of dendritic spines by activating the Rac signaling pathway. These findings reveal a function for EphA4-mediated intracellular signaling in the morphogenesis of dendritic spines and suggest that the processing of EphA4 by [gamma]-secretase affects the pathogenesis of Alzheimer's disease.

Protein kinase C [Delta] (PKC [Delta]) is normally activated by diacylglycerol produced from receptor-mediated hydrolysis of inositol phospholipids. On stimulation of cells with [H.sub.2][O.sub.2], the enzyme is tyrosine phosphorylated, with a concomitant increase in enzymatic activity. This activation does not appear to accompany its translocation to membranes. In the present study, the tyrosine phosphorylation sites of PKC [Delta] in the [H.sub.2][O.sub.2]-treated cells were identified as Tyr-311, Tyr-332, and Tyr-512 by mass spectrometric analysis with the use of the precursor-scan method and by immunoblot analysis with the use of phosphorylation site-specific antibodies. Tyr-311 was the predominant modification site among them. In an in vitro study, phosphorylation at this site by Lck, a non-receptor-type tyrosine kinase, enhanced the basal enzymatic activity and elevated its maximal velocity in the presence of diacylglycerol. The mutation of Tyr-311 to phenylalanine prevented the increase in this maximal activity, but replacement of the other two tyrosine residues did not block such an effect. The results indicate that phosphorylation at Tyr-311 between the regulatory and catalytic domains is a critical step for generation of the active PKC [Delta] in response to [H.sub.2][O.sub.2].

Background: Soluble amyloid‐β (Aβ) aggregates including protofibrils (PFs) have been reported to be more toxic than insoluble fibrils. Recently, pharmaceutical antibodies targeting these soluble Aβ aggregates have been developed. The structural dynamics and heterogeneity of soluble Aβ aggregates have made it difficult to analyze the structural characteristics and tertiary structure. To investigate conformational recognition of lecanemab to Aβ aggregates, the structural characteristics of Aβ PFs and their change upon binding to lecanemab, an antibody with preferential binding to Aβ protofibrils, were studied using hydrogen‐deuterium exchange mass spectrometry (HDX‐MS). Method: Aβ (1‐42) PFs were prepared in vitro. The hydrogen/deuterium (H/D) exchange status for Aβ (1‐42) PFs and monomeric Aβ (1‐40) were studied by HDX‐MS in phosphate buffered saline (PBS) to explore characteristics of their regional structural dynamics. The effects of the anti–Aβ PF antibody lecanemab on H/D status of Aβ (1‐42) PFs and monomeric Aβ (1‐40) were studied to explore the structural basis for the characteristics of antibody‐ Aβ interaction and specificity against Aβ PFs. Result: Aβ (1‐42) PFs were more protected from H/D exchange throughout the molecule than monomeric Aβ (1‐40), though the N‐terminal and the mid region of Aβ still showed relatively higher H/D exchange in Aβ (1‐42) PFs. Upon interaction with lecanemab, the mid region of Aβ (1‐42) PFs was also protected further in addition to the N‐terminal region, and its protection level of Aβ (1‐42) PFs was higher than monomeric Aβ (1‐40). The higher protection from H/D exchange both on N‐terminal and mid regions of Aβ PFs is considered to be characteristics of interaction between Aβ (1‐42) PFs and lecanemab. Conclusion: The results indicate that the mid region of Aβ has relatively high flexibility in PFs in addition to N‐terminal region. The mid region is suggested to be involved in the interaction with lecanemab as well as its N‐terminal region, and the conformational epitope formation was expected, though there are still possibility of structural change in the mid region upon lecanemab binding. The results indicated that the combinational action on the N‐terminal and mid regions of Aβ are involved in the modulation of Aβ PFs by lecanemab. [ABSTRACT FROM AUTHOR]

The calmodulin-binding domain of myristoylated alanine-rich C kinase substrate (MARCKS), which interacts with various targets including calmodulin, actin and membrane lipids, has been suggested to function as a crosstalk point among several signal transduction pathways. We present here the crystal structure at 2 A resolution of a peptide consisting of the MARCKS calmodulin (CaM)-binding domain in complex with Ca2+-CaM. The domain assumes a flexible conformation, and the hydrophobic pocket of the calmodulin N-lobe, which is a common CaM-binding site observed in previously resolved Ca2+-CaM-target peptide complexes, is not involved in the interaction. The present structure presents a novel target-recognition mode of calmodulin and provides insight into the structural basis of the flexible interaction module of MARCKS. [ABSTRACT FROM AUTHOR]

The paired helical filaments (PHF) found in Alzheimer’s disease (AD) brain are composed mainly of the hyperphosphorylated form of microtubule-associated protein tau (PHF-tau). It is well known that tau is a good in vitro substrate for Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). To establish the phosphorylation sites, the longest human tau (hTau40) was bacterially expressed and phosphorylated by CaM kinase II, followed by digestion with lysyl endoprotease. The digests were subjected to liquid chromatography/mass spectrometry. We found that 5 of 22 identified peptides were phosphorylated. From the tandem mass spectrometry, two phosphorylation sites (serines 262 and 356) were identified in the tubulin binding sites. When tau was phosphorylated by CaM kinase II, the binding of tau to taxol-stabilized microtubules was remarkably impaired. As both serines 262 and 356 are reportedly phosphorylated in PHF-tau, CaM kinase II may be involved in hyperphosphorylation of tau in AD brain. [Copyright &y& Elsevier]

Examines the phosphorylation sites of protein kinase C delta (PKC delta) in H[sub 2]O[sub 2]-treated cells. Activation PKC delta by tyrosine kinase; Increase in enzymatic activity due to stimulation of cells with H[sub 2]O[sub 2].