Department of Neuropathology
Professor Yasuo Ihara, M.D.
Lecturer Maho Morishima, Ph.D.
Associate Satoru Funamoto, Ph.D.
Tomohiro Miyasaka, Ph.D.

The long-term goal of the research in our department is to determine the molecular events that lead to the development of Alzheimer's disease (AD). For this purpose, we are analyzing two proteins, amyloid s-protein (As) and tau, and their related molecules that have been implicated in the AD pathogenesis:.

Current goals include:
1 Characterization of the enzymatic properties of ?-secretase
[Background] We and other groups found that APP is cleaved by ?-secretase, not only in the middle of the transmembrane domain (?-cleavage), but also near the membrane-cytoplasm boundary (referred to as ?-cleavage). This ?-cleavage site is located a few residues inside the membrane from the boundary, and is very close to site 3 for cleavage of Notch. The major product of ?-cleavage is an APP intracellular domain (AICD) that begins at Val-50, while the minor one is AICD49-99.
Most importantly, there is a link between AICD50-99 and As40 production, and a link between AICD49-99 and As42 production. A potential link between As40/42 and AICD50-99/49-99 raises further questions. Which cleavage, ?- or ?-cleavage, comes first, and how is one related to the other? Because we failed to detect a particular AICD, longer than AICD49-99, one possibility is that sCTF (s-stub of APP, an immediate substrate for ?-secretase) is first cleaved at the ?-sites, and the products generated (As1-48 and 1-49) undergo ?-cleavage, generating As40/42.
A number of substrates for ?-secretase thus far identified are type 1 membrane proteins, and appear to be cleaved at a very similar site to that of ?-cleavage. This suggests that the ?-site is the primary cleavage position for a fraction of type 1 membrane proteins. The water molecules absolutely required for proteolysis may be facilitated to gain entry into this peripheral region of the membrane. We hypothesized that ?-cleavage comes first, and ?-cleavage follows, generating As40 and 42. To test this hypothesis, we seek to address the following issues.
a) Do those longer Ass exist in the cell lysate?
Here the longer Ass indicate As43-As49. If this is the case, it has important implications for understanding the mechanism of intramembranous cleavage for APP, and probably for the degradation of many type 1 membrane proteins (see above).
b) Catalytic properties of the ?-secretase
According to our recent observations, DAPT (a potent, dipeptidic ?-secretase inhibitor) leads to suppression of As40 production and an accumulation of As43. Its increasing doses cause suppression of As43 production, which is associated with an accumulation of As46. Although the mode of action of DAPT remains unknown, this observation strongly suggests that As46 is a precursor for As43, As43 is a precursor for As40, and that the cleavages progress in a unidirectional and successive manner from the carboxyl side of the transmembrane domain of sCTF to the middle of the membrane.
The transmembrane domain of sCTF is postulated to adopt an ?-helix that needs 3.6 residues for one complete turn. According to this model, the cleavage sites for As49, As46, As43, and As40 are aligned on the ?-helical surface of the sCTF molecule, while those for As48, As45, and As42 are aligned on the other ?-helical surface. Thus, we suggest that As40 may be produced from As49 by cleaving at every three residues, while As42 is produced similarly from As48. As40-producing secretase and As42-producing secretase may be topographically distinct from each other.
2 Alternate mechanisms of As accumulation in the brain
[Background] At present, the pathogenesis of Familial AD can be understood by altered enzyme-substrate relationship: Mutations in enzyme (presenilin) or substrate (APP) cause an (small) increase in the As42 production, which eventually leads to AD. Patients with presenilin or APP mutants usually show higher levels of As42 in the plasma. This altered relationship can be detected by increased levels of As42 in the plasma. The majority of AD patients do not appear to have increased levels of As42 in the plasma, thus suggesting another pathway(s) to the formation of senile plaques and the development of AD. In particular, the ?4 allele that brings earlier deposition of As42 in the brain is probably mediated through other pathways. One possibility is altered environments induced by aging: Altered lipid composition and/or generation of a certain factor may promote As aggregation. In this regard, it may be important to investigate the significance of As accumulation in the raft.
3 The mechanisms of neuronal cell death in AD
[Background] FTDP-17 (Frontotemporal dementia and parkinsonism linked to chromosome 17) is caused by a number of mutations in the tau gene, and characterized neuropathologically by tauopathy and neuronal loss. Currently, the most important question about tau is why and how tau kills the neuron, rather than why and how it aggregates into neurofibrillary tangles (NFT).
We have constructed C. elegans model of tauopathy. When wild-type tau is expressed in the mechanosensory neuron of the worm, its touch response decreases in an age-dependent manner. Furthermore, the expression of P301L and R406W (two representative FTDP-17 tau mutations) aggravated the symptoms. Morphological investigations showed that tau accumulates age-dependently, and importantly, degeneration of the neuron appear to start without accumulation of tau. This observation is consistent with our view that the initial step to neuronal death may be shared by the pathway to NFT formation, and but that neuronal death occurs without tau accumulation.
a) Microarray analysis of mutant worms
Using those transgenic worms, we would like to proceed to microarray study. Now we have a couple of lines which panneuronally express wild-type, P301L or R406W tau, and show unc phenotypes. By comparing the these lines of transgenic worms, we would pick up abnormally up- or down-regulated genes, which may be involved in tau-mediated neuronal degeneration. Particular genes that are involved in the neuronal degeneration should be shared by all three kinds of transgenic worms, and most aberrantly expressed in the worms developing the phenotype earlier.

References
  1. Lee S, Kim JH, Lee CS, Kim JH, Kim Y, Heo K, Ihara Y, Goshima Y, Suh PG, Ryu SH: Collapsin response mediator protein-2 inhibits neuronal phospholipase D2 activity by direct interaction. J Biol Chem 277: 6542-6549, 2002
  2. Qi Y, Morishima-Kawashima M, Sato T, Mitsumori R, lhara Y: Distinct mechanisms by mutant presenilin 1 and 2 leading to increased intracellular levels of amyloid s-protein 42 in Chinese hamster ovary cells. Biochemistry 42: 1042-1052, 2003
  3. Sato T, Dohmae N, Qi Y, Kakuda N, Misonou H, Mitsumori R, Maruyama H, Koo EH, Haass C, Takio K, Morishima-Kawashima M, Ishiura S, Ihara Y: Potential link between amyloid s-protein 42 and C-terminal fragment ?49-99 of s-amyloid precursor protein. J Biol Chem 278: 24294-24301, 2003
  4. Kanamori T, Nishimaki K, Asoh S, Ishibashi Y, Takata I, Kuwabara T, Taira K, Yamaguchi H, Sugihara S, Yamazaki T, Ihara Y, Nakano K, Matuda S, Ohta S. Truncated product of the bifunctional DLST gene involved in biogenesis of the respiratory chain. EMBO J 22: 2913-2923, 2003
  5. Wada S, Morishima-Kawashima M, Qi Y, Misono H, Shimada Y, Ohno-Iwashita Y, Ihara Y: ?-Secretase activity is present in rafts, but not cholesterol-dependent. Biochemistry 42: 13977-13986, 2003
  6. Watanabe N, Araki W, Chui DH, Makifuchi T, Ihara Y, Tabira T: Glypican-1 as an As binding HSPG in the human brain: Its localization in DIG domains and possible roles in the pathogenesis of Alzheimer's disease. FASEB J in press
  7. Oyama F, Kotliarova S, Harada A, Ito M, Miyazaki H, Ueyama Y, Hirokawa N, Nukina N, Ihara Y: Gem GTPase and tau: morphological changes induced by Gem GTPase in CHO cells are antagonized by tau. J Biol Chem in press
Annual Report of the Graduate School of Medicine and The Faculty of Medicine The University of Tokyo Reports for the Period April 2002 - March 2004