Oligodendrocyte precursor cells (OPCs) persist near the demyelinated axons arising in MS but inefficiently differentiate into oligodendrocytes and remyelinate these axons. The pathogenesis of differentiation failure remains elusive. We initially hypothesized that injured axons fail to present Contactin, a positive ligand for the oligodendroglial Notch1 receptor to induce myelination, and thus tracked axoglial Contactin/Notch1 signaling in situ, using immunohistochemistry in brain tissue from MS patients containing chronic demyelinated lesions. Instead, we found that Contactin was saturated on demyelinated axons, Notch1-positive OPCs accumulated in Contactin-positive lesions, and the receptor was engaged, as demonstrated by cleavage to Notch1-intracellular domain (NICD). However, nuclear translocalization of NICD, required for myelinogenesis, was virtually absent in these cells. NICD and related proteins carrying nuclear localization signals were associated with the nuclear transporter Importin but were trapped in the cytoplasm. Abnormal expression of TIP30, a direct inhibitor of Importin, was observed in these OPCs. Overexpression of TIP30 in a rat OPC cell line resulted in cytoplasmic entrapment of NICD and arrest of differentiation upon stimulation with ContactinFc. Our results suggest that extracellular inhibitory factors as well as an intrinsic nucleocytoplasmic transport blockade within OPCs may be involved in the pathogenesis of remyelination failure in MS.

The 24-residue peptide Humanin (HN), containing two Ser residues at positions 7 and 14, protects neuronal cells from insults of various Alzheimer's disease (AD) genes and Ab. It was not known why the rescue function of (S14G)HN is more potent than HN by two to three orders of magnitude. Investigating the possibility that the post-translational modification of Ser14 might play a role, we found that HN with D-Ser at position 14 exerts neuroprotection more potently than HN by two to three orders of magnitude, whereas D-Ser7 substitution does not affect the rescue function of HN. On the other hand, S7A substitution nullified the HN function. Multiple series of experiments indicated that Ser7 is necessary for self-dimerization of HN, which is essential for neuroprotection by this factor. These findings indicate that the rescue function of HN is quantitatively modulated by D-isomerization of Ser14 and Ser7-relevant dimerization, allowing for the construction of a very potent HN derivative that was fully neuroprotective at 10 pM against 25 lM Ab1-43. This study provides important clues to the understanding of the neuroprotective mechanism of HN, as well as to the development of novel AD therapeutics. Keywords: Alzheimer's disease, dimerization, Humanin, neuronal death, neuroprotection, D-serine isomerization. Alzheimer's disease (AD) is the most prevalent neurodegenerative disease associated with progressive dementia. No fundamental therapy for this disease has so far been established. As brain atrophy is the central abnormality in AD, pathological mechanisms leading to neuronal loss must be understood to develop future curative therapy. Three forms of known mutant genes cause familial AD (FAD): amyloid precursor protein (APP) mutants, presenilin (PS)1 mutants, and PS2 mutants (Shastry and Giblin 1999). Multiple groups have found that all examined FAD mutants cause or enhance cell death when they are expressed in neuronal cells (Niikura et al. 2002 for review). In an attempt to find the molecules that suppress neuronal cell death by these AD-related insults, we used 'death-trap' screening, developed by D'Adamio et al. (1997) -an unbiased functional screening of molecules that allow dying cells to survive.We applied this method to V642I-APP-inducible neuronal cells with our unique modification using an expression cDNA library constructed from an occipital lobe of the brain of an autopsy-diagnosed AD patient. As a result of this screening, we identified an ORF cDNA, encoding a novel short peptide MAP-RGFSCLLLLTSEIDLPVKRRA, that suppresses neuronal cell death by various FAD genes [APP mutants (V642I, K595M/N596L, A617G, and L648P), PS1 mutants (M146L, H163R, A246E, L286V, and C410Y), and a PS2 mutant Received February 20, 2003; accepted March 7, 2003. Address correspondence and reprint requests to Ikuo Nishimoto, Departments of Pharmacology and Anatomy, KEIO University School of Medicine, Medical Research Center, 6th Floor, 35 Shinanomachi, Tokyo 160-8582, Japan. E-mail: nisimoto@sc.itc.keio.ac.jp 1 These two ...

Humanin (HN) is a newly identified neuroprotective peptide that specifically suppresses Alzheimer's disease (AD)-related neurotoxicity. HN peptide has been detected in the human AD brain as well as in mouse testis and colon by immunoblot and immunohistochemical analyses. By means of yeast two-hybrid screening, we identified TRIM11 as a novel HN-interacting protein. TRIM11, which is a member of protein family containing a tripartite motif (TRIM), is composed of a RING finger domain, which is a putative E3 ubiquitin ligase, a B-box domain, a coiled-coil domain and a B30.2 domain. Deletion of the B30.2 domain in TRIM11 abolished the interaction with HN, whereas the B30.2 domain alone did not interact with HN. For their interaction, at least the coiled-coil domain was indispensable together with the B30.2 domain. The intracellular level of glutathione S-transferase-fused or EGFP-fused HN peptides or plain HN was drastically reduced by the coexpression of TRIM11. Disruption of the RING finger domain by deleting the first consensus cysteine or proteasome inhibitor treatment significantly diminished the effect of TRIM11 on the intracellular level of HN. These results suggest that TRIM11 plays a role in the regulation of intracellular HN level through ubiquitin-mediated protein degradation pathways.

Humanin (HN) is a 24-amino acid peptide that protects neuronal cells from death caused by Alzheimer's disease (AD)-related genes and amyloid-beta (Abeta). Multiple studies have revealed its biochemical and neuroprotective characteristics in vitro; however, little has been known regarding whether HN is effective in vivo in AD model systems. We examined the effect of S14G-HN, a 1,000-fold more potent derivative of HN in vitro, on amnesia induced by Abeta25-35 in mice. The Y-maze test revealed that at least 50 pmol of S14G-HN by intracerebroventricular injection prevented Abeta-induced impairment of short-term/spatial working memory; however, 5 nmol of S14A-HN, a neuroprotection-defective mutant in vitro, did not prevent Abeta-induced amnesia. These results are in agreement with the structure-function correlation shown previously in vitro. In the water-finding task, S14G-HN prevented prolongation of finding latency (the time to find water) observed in Abeta-amnesic mice, indicating that S14G-HN also blocked Abeta-induced impairment of latent learning. In accordance with these observations, immunohistochemical analysis showed that S14G-HN sustained the number of cholinergic neurons in the basal forebrain and the striata nearly to the normal level. Furthermore, genistein, a specific inhibitor of tyrosine kinases, blocked recovery from scopolamine-induced amnesia by S14G-HN, suggesting that certain tyrosine kinase(s) are involved in the inhibitory function of S14G-HN in vivo. Taking these findings together, we conclude that S14G-HN has rescue activity against memory impairment caused by AD-related insults in vivo by activating the same intracellular neuroprotective machinery as elucidated previously in vitro.

The biological function of full-length amyloid-␤ protein precursor (A␤PP), the precursor of A␤, is not fully understood. Multiple laboratories have reported that antibody binding to cell surface A␤PP causes neuronal cell death. Here we examined whether induced dimerization of the cytoplasmic domain of A␤PP (A␤PP CD ) triggers neuronal cell death. In neurohybrid cells expressing fusion constructs of the epidermal growth factor (EGF) receptor with A␤PP CD (EGFR/A␤PP hybrids), EGF drastically enhanced neuronal cell death in a manner sensitive to acetyl-Laspartyl-L-glutamyl-L-valyl-L-aspartyl-aldehyde (Ac-DEVD-CHO; DEVD), GSH-ethyl ester (GEE), and pertussis toxin (PTX). Dominant-negative apoptosis signal-regulating kinase 1 (ASK1) blocked this neuronal cell death, but not ␣-synuclein-induced cell death. Constitutively active ASK1 (caASK1) caused DEVD/GEEsensitive cell death in a manner resistant to PTX and sensitive to Humanin, which also suppressed neuronal cell death by EGFR/ A␤PP hybrid. ASK1 formed a complex with A␤PP CD via JIP-1b, the c-Jun N-terminal kinase (JNK)-interacting protein. EGFR/ A␤PP hybrid-induced and caASK1-induced neuronal cell deaths were specifically blocked by SP600125 (anthra[1,9-cd]pyrazol-6(2H)-one), a specific JNK inhibitor. Combined with our earlier study, these data indicate that dimerization of A␤PP CD triggers ASK1/JNK-mediated neuronal cell death. We also noticed a potential role of ASK1/JNK in sustaining the activity of this mechanism after initial activation by A␤PP, which allows for the achievement of cell death by short-term anti-A␤PP antibody treatment. Understanding the function of A␤PP CD and its downstream pathway should lead to effective anti-Alzheimer's disease therapeutics.