Tsuneo Yamazaki

Consensus Report of the Working Group on : Molecular and Biochemical Markers of Alzheimer's Disease

The ideal biomarker for Alzheimers disease (AD) should detect a fundamental feature of neuropathology and be validated in neuropathologically-confirmed cases; it should have a sensitivity >80% for detecting AD and a specificity of >80% for distinguishing other dementias; it should be reliable, reproducible, non-invasive, simple to perform, and inexpensive. Recommended steps to establish a biomarker include confirmation by at least two independent studies conducted by qualified investigators with the results published in peer-reviewed journals. Our review of current candidate markers indicates that for suspected early-onset familial AD, it is appropriate to search for mutations in the presenilin 1, presenilin 2, and amyloid precursor protein genes. Individuals with these mutations typically have increased levels of the amyloid Abeta42 peptide in plasma and decreased levels of APPs in cerebrospinal fluid. In late-onset and sporadic AD, these measures are not useful, but detecting an apolipoprotein E e4 allele can add confidence to the clinical diagnosis. Among the other proposed molecular and biochemical markers for sporadic AD, cerebrospinal fluid assays showing low levels of Abeta42 and high levels of tau come closest to fulfilling criteria for a useful biomarker.The ideal biomarker for Alzheimers disease (AD) should detect a fundamental feature of neuropathology and be validated in neuropathologically-confirmed cases: it should have a sensitivity >80% for detecting AD and a specificity of >80% for distinguishing other dementias: it should be reliable, reproducible non-invasive, simple to perform, and inexpensive. Recommended steps to establish a biomarker include confirmation by at least two independent studies conducted by qualified investigators with the results published in peer-reviewed journals. Our review of current candidate markers indicates that for suspected early-onset familial AD. it is appropriate to search for mutations in the presenilin 1, presenilin 2, and amyloid precursor protein genes. Individuals with these mutations typically have increased levels of the amyloid Aβ 42 peptide in plasma and decreased levels of APPs in cerebrospinal fluid. In late-onset and sporadic AD. these measures are not useful. but detecting an apolipoprotein E e4 allele can add confidence to the clinical diagnosis. Among the other proposed molecular and biochemical markers for sporadic AD. cerebrospinal fluid assays showing low levels of Aβ 42 and high levels of tau come closest to fulfilling criteria for a useful biomarker.

Intracellular generation and accumulation of amyloid beta-peptide terminating at amino acid 42.

Amyloid β-peptide (Aβ) is known to accumulate in senile plaques of Alzheimer’s disease (AD) patients and is now widely believed to play a major role in the disease. Two populations of peptides occur terminating either at amino acid 40 or at amino acid 42 (Aβ1–40 and Aβ1–42). Alternative N-terminal cleavages produce additional heterogeneity (Aβx-40 and Aβx-42). Peptides terminating at amino acid 42 are believed to be the major player in sporadic AD as well as familial AD (FAD). Whereas the cellular mechanism for the generation of Aβ terminating at amino acid 40 is well understood, very little is known about the cleavage of Aβ after amino acid 42. By using two independent methods we demonstrate intracellular Aβ1–42 as well as Aβx-42 but less Aβx-40 and Aβ1–40 in kidney 293 cells stably transfected with wild type β-amyloid precursor protein (βAPP) or the FAD-associated Val/Gly mutation. Moreover, retention of βAPP within the endoplasmic reticulum (ER) by treatment with brefeldin A does not block the cleavage at amino acid 42 but results in an increased production of all species of Aβ terminating at amino acid 42. This indicates that the cleavage after amino acid 42 can occur within the ER. Treatment of cells with monensin, which blocks transport of (βAPP) within the Golgi causes a marked accumulation of intracellular Aβx-42 and Aβx-40. Therefore these experiments indicate that the γ-secretase cleavage of Aβ after amino acid 42 can occur within the ER and later within the secretory pathway within the Golgi. Moreover inhibition of reinternalization by cytoplasmic deletions of βAPP as well as inhibition of intracellular acidification by NH4Cl does not block intracellular Aβ1–42 or Aβx-42 production.

Amyloid β-protein (Aβ) Accumulation in the Leptomeninges during Aging and in Alzheimer Disease

The results of well-characterized two-site enzyme immunoassays showed that the crude leptomeninges (consisting of the pin matter, arachnoid matter, and leptomeningeal vessels [LV]) from aged control brains and brains affected by Alzheimer disease (AD) contain very high levels of amyloid β-protein (Aβ). To learn about the source of Aβ, we carefully dissected out both leptomeninges (LM) and LV under a dissecting microscope and determined the levels of soluble Aβ in each. The purity of these dissected tissues was confirmed by the absence or presence of α-smooth muscle actin representing LV by Western blotting. Surprisingly, the amounts of Aβ in each dissected sample were nearly equivalent on a weight basis. In each compartment from aged controls the level of Aβ1–42 was comparable to that of Aβ1–140, while in AD brain Aβ1–40 was a predominant species in both LM and LV. In some cases careful immunocytochemical examination revealed the presence of Aβ deposits that were immunolabeled by several Aβ monoclonal antibodies in leptomeningeal layers (most often in the arachnoid matter). The extent of Aβ deposition in LM appeared to be much less than that explained by the soluble Aβ levels, suggesting that immunocytochemically undetectable Aβ can accumulate in LM. These observations indicate that leptomeninges are a large reservoir of Aβ in normal aged individuals and in AD patients.

Truncated product of the bifunctional DLST gene involved in biogenesis of the respiratory chain.

Dihydrolipoamide succinyltransferase (DLST) is a subunit enzyme of the α‐ketoglutarate dehydrogenase complex of the Krebs cycle. While studying how the DLST genotype contributes to the pathogenesis of Alzheimers disease (AD), we found a novel mRNA that is transcribed starting from intron 7 in the DLST gene. The novel mRNA level in the brain of AD patients was significantly lower than that of controls. The truncated gene product (designated MIRTD) localized to the intermembrane space of mitochondria. To investigate the function of MIRTD, we established human neuroblastoma SH‐SY5Y cells expressing a maxizyme, a kind of ribozyme, that specifically digests the MIRTD mRNA. The expression of the maxizyme specifically eliminated the MIRTD protein and the resultant MIRTD‐deficient cells exhibited a marked decrease in the amounts of subunits of complexes I and IV of the mitochondrial respiratory chain, resulting in a decline of activity. A pulse‐label experiment revealed that the loss of the subunits is a post‐translational event. Thus, the DLST gene is bifunctional and MIRTD transcribed from the gene contributes to the biogenesis of the mitochondrial respiratory complexes.

Amyloid β-protein (Aβ) Accumulation in the Putamen and Mammillary Body during Aging and in Alzheimer Disease

Immunocytochemical studies clearly showed that amyloid β-protein (Aβ) deposits are widely distributed in the subcortical regions as well as the cortices of normal aged and Alzheimer disease (AD) brains. To investigate the temporal profile of Aβ accumulation in the subcortical region, we quantitated Aβ40 and Aβ42 levels, using sensitive enzyme immunoassays, in the putamen and mammillary bodies of normal individuals aged 24 to 87 years and of AD patients. In these two regions, Aβ42 was the predominant species; in particular, Aβ42 was the only Aβ species detected in the putamen. In several cases the mammillary body contained only Aβ40, but not Aβ42. Although the extent of Aβ accumulation in the 2 subcortical regions was much less than that in the cortex of the same subject, Aβ42 appears to accumulate in both subcortical regions at the same time as in the cortex and leptomeninges. In addition, the Aβ42 levels in the putamen or in the mammillary body correlated with those in the occipitotemporal cortex. This strongly suggests that the extent of Aβ42 accumulation in the brain is determined not only by the duration of Aβ accumulation but also by other unknown regional factors. Western blotting showed that the initial Aβ species to accumulate in the putamen or mammillary body varied among individuals. In some cases, an Aβ42 stable dimer was the most predominant species, while in other cases a 3 or 4 kD Aβ42 monomer was more abundant, suggesting that the clearance rates of the Aβ42 stable dimer and monomer are different in vivo.

Proteolytic processing of Alzheimer's disease associated proteins.

Amyloid beta-peptide (A beta), the major component of senile plaques, is generated by proteolytic processing from the beta-amyloid precursor protein (beta APP). Mutations within the beta APP gene cause early onset familial AD (FAD) by affecting A beta generation. Interestingly, the much more abundant mutations within the presenilin (PS) genes also result in the abnormal generation of a 42 residue A beta (A beta 42), thus clearly supporting a pivotal role of A beta for the pathology of AD. PS proteins are proteolytically processed into stable 30 kDa N-terminal fragments (NTF) and 20 kDa C-terminal fragments (CTF). Beside the conventional proteolytic pathway. PS proteins can also be cleaved further C-terminal by proteases of the caspase superfamily. PS proteins were localized within the endoplasmic reticulum (ER) and early Golgi, compartments which we have demonstrated to be involved in A beta 42 generation and intracellular accumulation. Using Caenorhabditis elegans as a simple animal model, we demonstrate that PS proteins are involved in NOTCH signaling FAD causing mutations interfere with the biological function of PS proteins in NOTCH signaling.

Effects of Specific Protease Inhibitors on Amyloid β-Protein 42 Secretion

Amyloid beta-protein (Abeta) is classified into two subspecies defined by its C-terminal length, designated Abeta40 and Abeta42. Although Abeta42 accounts for only approximately 10% of secreted Abeta, this particular species is the most dominant species in Abeta deposits in Alzheimers disease (AD) and normal aged brains and appears to be the initially deposited species. Secretion levels of Abeta42 have been shown to increase in patients affected by any form of early-onset familial AD. Thus, the suppression of Abeta42 production or secretion could be a therapeutic strategy for AD. In this study, we examined whether protease inhibition affects the Abeta42 secretion ratio (Abeta42: total Abeta). Using specific inhibitors, we determined that the inhibition of calpain but not proteasome induces an increased Abeta42 secretion in cultured cells. These data suggest that calpain differentially affects the gamma-secretases generating Abeta40 and Abeta42 and indicate the possibility of developing compounds that reduce Abeta42 production and secretion though this pathway.

The Cellular Biology of Presenilin Proteins and a Novel Mechanism of Amyloid β-Peptide Generation

Presenilin (PS) proteins are involved in numerous cases of familial Alzheimer’s disease (reviewed by Tanzi et al. 1996) and are therefore key players in the pathogenesis of the disease. To understand the biology of the PS proteins we searched for a potential biological function and analyzed the biochemistry of the two homologous PS proteins. We also determined the subcellular localization of presenilis and took advantage of this knowledge to search for a cellular mechanism that is involved in Aβ42 generation. These three main topics will be reviewed in detail.

Molecular pathology of Alzheimer's disease

Recent molecular and cellular approaches to the nature of Alzheimers disease (AD) have revealed that the disease has a heterogenous genetic background. Thus, AD is not a disease but a syndrome. In the present article we review the significance of the causative and susceptibility genes of AD identified to date and discuss future perspectives of AD research.