Mihoko Usami

A novel brain-specific mRNA encoding nuclear protein (necdin) expressed in neurally differentiated embryonal carcinoma cells

A novel DNA sequence has been isolated from a subtraction cDNA library of P19 embryonal carcinoma cells treated with retinoic acid which induces neural differentiation of the stem cells. The cDNA insert (4B) hybridized with a single 1.7 kb mRNA, whose abundance was markedly increased in P19 cells after retinoic acid treatment. The 1.7 kb mRNA was also expressed in the brain, but not in other non-neuronal tissues. A 1.6 kb cDNA insert (4BFL), which was cloned by screening another cDNA library with the 4B probe, encodes a novel protein sequence of 325 amino acids (Mr 36,831). The protein expressed in 4BFL-transfected COS cells was translocated into the nuclei as detected with antibodies against subsequences of the predicted protein. The antibodies stained the nuclei of neurally differentiated P19 cells but not of the undifferentiated stem cells. This novel mRNA encoding the nuclear protein, termed necdin, may represent a useful marker for the differentiation and development of brain cells.

Tau in cerebrospinal fluids: establishment of the sandwich ELISA with antibody specific to the repeat sequence in tau

Clinical diagnosis for Alzheimers disease (AD) is provided by the criteria of DSMIV and clinical progress in addition to imaging analysis with MRI after negative screening. The final exclusive diagnosis is confirmed by the neuropathological findings of neurofibrillary tangles and senile plaques in autopsy brains. We developed a new ELISA system to measure the amount of tau in cerebrospinal fluids (CSF) using phosphorylation-independent and sequence-specific antibodies. The present ELISA was sensitive enough to detect tau in CSF of normal subjects. The amount of tau was significantly elevated in CSF of AD subjects compared with those of normal subjects and subjects with dementia of cerebrovascular disease, suggesting that tau in CSF reflects the massive and continuous neuronal cell death in the AD brain. In conclusion, we established an ELISA system which enabled us to detect tau in CSF and demonstrated that tau was significantly and specifically elevated in CSF of AD subjects. This assay system can provide us with a potent diagnostic tool for clinical AD.

Identification and characterization of presenilin I-467, I-463 and I-374.

We cloned a novel isoform of presenilin I (presenilin I‐374) besides previously published presenilin I‐467 and I‐463 in human lymphocytes. Presenilin I‐463 was identical to presenilin I‐467 except a 12 bp nucleotides deletion in its amino terminal region. Another isoform, presenilin I‐374 was produced by an alternative splicing with an additional exon consisting of 92 bp nucleotides (exon 11), which resulted in the frame shift with a stop codon to generate a truncated presenilin consisting of 374 amino acids. The transcripts for presenilin I‐4671463 was ubiquitously detected while that for presenilin I‐374 was selectively detected in liver, spleen, kidney. Abnormal behavior of presenilin I on gel electrophoresis was found with affinity‐purified antibodies against presenilin I.We cloned a novel isoform of presenilin I (presenilin I-374) besides previously published presenilin I-467 and I-463 in human lymphocytes. Presenilin I-463 was identical to presenilin I-467 except a 12 bp nucleotides deletion in its amino terminal region. Another isoform, presenilin I-374 was produced by an alternative splicing with an additional exon consisting of 92 bp nucleotides (exon 11), which resulted in the frame shift with a stop codon to generate a truncated presenilin consisting of 374 amino acids. The transcripts for presenilin I-4671463 was ubiquitously detected while that for presenilin I-374 was selectively detected in liver, spleen, kidney. Abnormal behavior of presenilin I on gel electrophoresis was found with affinity-purified antibodies against presenilin I.

Impaired cell cycle control of neuronal precursor cells in the neocortical primordium of presenilin-1-deficient mice

Recent studies have implicated presenilin‐1 (PS‐1) in the processing of the amyloid precursor protein and Notch‐1. We show that PS‐1 has biological effects on differentiation and cell cycle control of neuronal precursor cells in vivo using PS‐1‐deficient mice. The expression of Class III β‐tubulin was upregulated throughout the neocortical primordia of PS‐1‐deficient E14 embryos, especially on the ventricular surface. The increased speed of migration of the immature neurons from the ventricular zone outward in the PS‐1‐deficient neocortical primordia was indicated by an in vivo bromodeoxyuridine (BrdU)‐labeling assay and a DiI‐labeling assay in slice culture. Furthermore, we investigated the cell cycle of neuronal precursor cells in the neocortical ventricular zone using an in vivo cumulative BrdU‐labeling assay. The length of the cell cycle in the neocortical precursor cells of wild‐type mice was 11.4 hr whereas that of the PS‐1‐deficient mice was 15.4 hr. Among all phases of the cell cycle, S‐phase exhibited the most prominent change in length, increasing from 2.4 hr in the wild‐type mice to 7.4 hr in the mutant mice. The distribution of β–catenin was specifically affected in the ventricular zone of the PS‐1‐deficient mice. These findings suggest that PS‐1 is involved in the differentiation and the cell cycle control of neuronal precursor cells in the ventricular proliferating zone of the neocortical primordium.

Proteolytic processing of presenilin-1 (PS-1) is not associated with Alzheimer's disease with or without PS-1 mutations.

Cerebral presenilin‐1 protein (PS‐1) is normally composed of the amino‐terminal fragment (NTF) with M r 28 kDa and the carboxy‐terminal fragment (CTF) with 18 kDa. We analyzed human PS‐1 in brains with early‐onset familial Alzheimers disease (FAD) with and without PS‐1 mutations to study whether mutated PS‐1 was abnormally metabolized. Cerebral PS‐1 were found to be cleaved into two fragments of NTF and CTF independently of the occurrence of PS‐1 mutation in human brains. A small portion of PS‐1 was recently found to suffer another processing by caspase‐3, an apoptosis‐related cysteine protease. In contrast to the recent finding that the Volga‐German mutation on presenilin‐2 (PS‐2) affects the increasing caspase‐3 PS‐2 fragment, the PS‐1 mutation did not cause a significant change in PS‐1 fragmentation. We conclude that PS‐1 fragmentation and other (probably caspase‐3‐mediated) digestion following apoptosis occur independently of PS‐1 mutations.

The tau mutation (val337met) disrupts cytoskeletal networks of microtubules.

The missense point mutation found in the tau gene, which was segregated in a family with frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), has proved to be the causal molecule for widely spread dementia diseases. Here we examined the effects of the tau mutation using confocal analysis. When wild-type tau cDNA was introduced into cells, extensive cell processes and well-developed thick bundles of microtubules were induced. On the other hand, when altered tau cDNA with the mutation (valine337-methionine) was introduced, cell lost processes and microtubule networks resulted in more round cell shape but showed intact mitochondria or endoplasmic reticulum. We conclude that the tau mutation primarily affects the microtubules and resultantly causes the loss of cellular organization and function due to microtubule disruption.

A high incidence of apolipoprotein E ε4 allele in middle-aged non-demented subjects with cerebral amyloid β protein deposits

Abstract We examined the apolipoprotein E (ApoE) genotypes of 19 middle-aged non-demented subjects with cerebral amyloid β protein (Aβ) deposits, and compared the results with those of 16 patients with sporadic Alzheimer’s disease (AD) and those of 34 age-matched controls. The frequency of the ApoE ɛ4 allele was higher (P = 0.0256) in these 19 subjects (0.211) than in controls (0.059), and was close to that in AD patients (0.281). This result suggests that middle-aged non-demented subjects with cerebral Aβ deposits are at high risk of developing AD, and that the diffuse Aβ deposits in these cases represent an early stage of AD pathology. We speculate that in the majority of late-onset sporadic AD patients, cerebral Aβ deposition commences when these patients are in their forties or fifties, and that the pathological process progresses gradually, taking 20 to 30 years for clinical manifestation of dementia.

Mutant presenilin (A260V) affects Rab8 in PC12D cell

Most familial early-onset Alzheimers disease (FAD) is caused by mutations in the presenilin-1 (PS1) gene. Abeta is derived from amyloid precursor protein (APP) and an increased concentration of Abeta 42 is widely believed to be a pathological hallmark of abnormal PS function. Therefore, the interaction between PS1 and APP is a central theme in attempts to clarify the molecular mechanism of AD. To examine the effect of PS1 mutations on APP metabolism, we made PC12D cell lines that express human PS1 or mutant PS1 (A260V). In PC12D cells expressing the PS1A260V mutant, we found that Rab8, a GTPase involved in transport from the trans-Golgi network (TGN) to the plasma membrane (PM), was significantly reduced in PC12D cells expressing the A260V mutant and that APP C-terminal fragment (CTF), the direct precursor of Abeta, accumulated in the heavy membrane fraction including membrane vesicles involved in TGN-to-PM transport. Furthermore, the total intracellular Abeta production was reduced in these cells. Combined together, we have observed that PS1 mutation disturbs membrane vesicle transport, resulting in prolonged residence of APP CTF during TGN-to-PM transport pathway. Therefore, it is highly likely that reduction of Abeta is closely related to the retention of APP CTF during TGN-to-PM transport.

Mutation of Glu693 to Gln or Val717 to Ile has no effect on the processing of Alzheimer amyloid precursor protein expressed in COS-1 cells by cDNA transfection.

One of the features of Alzheimers disease (AD) is the formation of senile plaques, of which the main component is a 42 amino acid beta-protein (beta P). Molecular cloning of beta P revealed the presence of a 90-130 kDa precursor, amyloid precursor protein (APP). Since APP is expressed in normal brain without producing beta P, some abnormal processing is the cause of the formation of beta P in AD. Two kinds of mutations of APP, Glu693 to Gln and Val717 to Ile, were reported in AD-related diseases. Site-directed mutagenesis was applied, and the mutated APPs were expressed in COS-1 cells by cDNA transfection. They showed apparently the same processing as wild APP. This means that these mutations might not be a direct cause for the abnormal processing of APP or the formation of beta P in AD.

The Triplet of Lysine Residues (Lys724‐Lys725‐Lys726) of Alzheimer's Amyloid Precursor Protein Plays an Important Role in Membrane Anchorage and Processing

Abstract: One of the pathological changes of Alzheimers disease is the deposit of β/A4 protein, which is derived from Alzheimer amyloid precursor protein (APR). In the secretory pathway, APR is cleaved at an internal region of β/A4 protein by a hypothetical enzyme “secretase.” Our previous study showed that the site of cleavage of APR by secretase is determined by the length from the membrane‐spanning region. To investigate the role of the transmem‐ brane region in APR secretion, we constructed the mutations of triplet lysine residues (Lys724‐Lys725‐Lys726), which are located just in the carboxyl region after the proposed membrane domain. The mutations were as follows: VVK, Val724‐Val725‐Lys726; LLI, Leu724‐Leu725‐lle726; and EEE, Glu724‐Glu725‐Glu726. Wild‐type APR and mutant APPs were expressed transiently in COS‐1 cells by cDNA trans‐fection. The hydrophobic mutant VVK and LLI were processed and secreted in a way similar to that of the wild‐ type APR, although the rate of secretion was decreased. The acidic mutant EEE was not secreted into medium. Proteinase K treatment and cell surface biotinylation of the COS‐1 cells expressing APR revealed that APR was located in the plasma membrane with a short intracellular carboxyl region. However, EEE was completely digested by proteinase K treatment, which suggested that the whole residues of this mutant are located at the outer surface of the cell, including its proposed membrane domain and carboxyl region. This mutant was not cleaved at all by secretase. These findings suggested that the triplet lysine residues of APR after the predicted membrane spanning domain play an important role in the membrane anchorage. In addition, the membrane anchorage was also important for the normal processing by secretase.