Yu Nakamura

Abnormal distribution of cathepsins in the brain of patients with Alzheimer's disease

Formalin-fixed paraffin-embedded hippocampal sections of brains with early-onset and late-onset Alzheimers disease were studied immunohistochemically with antisera against cathepsin D and cathepsin B. In addition to the staining of neuronal perikarya, some of the senile plaques visualized by Bielshowsky silver staining and some of reactive astrocytes were positively stained with the antisera against cathepsin D and cathepsin B in brains with Alzheimers disease. Abnormal localization of cathepsin D and cathepsin B immunoreactivity in neuronal perikarya was observed in brains with early-onset Alzheimers disease. These findings demonstrate that the distribution of lysosomal proteases was altered in brains with Alzheimers disease, suggesting the primary and/or secondary involvement of the lysosomal proteases in the pathological process of Alzheimers disease.

Amyloid β-protein precursor deposition in rat hippocampus lesioned by ibotenic acid injection

Ibotenic acid was injected into 3 parts of the lateral rat hippocampus. The animals were sacrificed 100 days after treatment, and studied immunohistochemically. The lesioned side of the hippocampus was highly atrophic with extensive neuronal loss and gliosis. Although silver staining revealed no particular structures such as neurofibrillary tangles or senile plaques, globular and granular depositions of amyloid beta-protein precursor (APP) immunoreactivity was observed by immunostaining in the lesion with the monoclonal antibody (clone 22C11). Increased immunoreactivities of glial fibrillary acidic protein (GFAP) and ubiquitin were found in the lesioned area, while the immunoreactivities of microtubule-associated protein 2 (MAP2) and 200 kDa neurofilament subunit protein (NF-H) were diminished. The results indicate that APP deposition is formed in the lesioned area where neuronal degeneration is produced by ibotenic acid in rat hippocampus.

Effect of phosphorylation on 68 KDa neurofilament subunit protein assembly by the cyclic AMP dependent protein kinase in vitro

The effect of phosphorylation by cyclic AMP dependent protein kinase on the assembly of the core-forming 68 KDa neurofilament subunit protein (NF-L) was studied in vitro by fluorescence energy transfer and electron microscopy. Phosphorylation of unassembled NF-L in a low ionic strength buffer by cyclic AMP dependent protein kinase led to the incorporation of 1-2 phosphate groups/mole protein. Assembly of this phosphorylated NF-L was inhibited significantly; compared to non-phosphorylated NF-L, the critical concentration of phosphorylated NF-L was raised by greater than 30-fold. Assembled NF-L filaments could also be phosphorylated by cyclic AMP dependent protein kinase indicating that the sites were accessible. Phosphorylation of NF-L in the filamentous state induced their disassembly. The results suggest that phosphorylation by cyclic AMP dependent protein kinase is a possible means to modulate the assembly state of NF-L.

Lysosome instability in aged rat brain

The study of the age-dependent change in lysosomal enzyme activities of the cerebral tissue showed the significant increase of cathepsin D in the aged rat brain, while those of beta-glucuronidase and acid phosphatase remained unchanged. The subcellular distribution study of cathepsin D and beta-glucuronidase revealed the increased activity of these enzymes in the cytosolic fraction from the aged brain. In vitro incubation of the lysosome fraction from the aged rat brain resulted in more leakage of these two enzymes, indicating the instability of the lysosome in the aged brain, which resembled the effect of L-Leu-methyl ester to the lysosome.

Are cerebrovascular factors involved in Alzheimer's disease?

Recent epidemiological studies have shown that vascular risk factors may be involved in Alzheimers disease (AD) as well as dementia in general. To investigate the relation between a vascular disorder and AD pathology, current criteria are defective because most depend on exclusion of a cerebrovascular disorder. Epidemiological studies have indicated the possibilities that arteriosclerosis, abnormal blood pressure, diabetes mellitus and smoking may be related to the pathogenesis of AD. As for the mechanism that vascular disorders influence AD, it is presumed that amyloid deposition may be caused by a vascular disorder. Alternatively, a vascular event may cause progression of subclinical AD to a clinical stage. Insulin resistance and apolipoprotein E may also be involved in these mechanisms. Our studies show that ischemia-induced the Alzheimer-associated gene presenilin 1 (PS1) and endoplasmic reticulum-stress, generated from a vascular disorder, may unmask clinical AD symptoms caused by presenilin mutation, suggesting that a vascular factor might be involved in the onset of familial AD.

Cerebrospinal fluid tau in dementia disorders: a large scale multicenter study by a Japanese study group

A large scale multicenter study of cerebrospinal fluid (CSF) tau levels was conducted to determine the cut-off value, sensitivity and specificity for clinical usage as a biomarker of Alzheimers disease (AD). Its use for early and differential diagnosis and the factors that increase CSF tau levels were also examined. CSF samples from a total of 1,031 subjects including 366 patients with AD, 168 patients with non-Alzheimer type dementia (NA), 316 patients with non-dementia neurological diseases (ND) and 181 normal controls (NC) were measured using ELISA for tau. The cut-off value of tau, 375 pg/ml, showed 59.1% sensitivity and 89.5% specificity for diagnosis of AD compared with the other groups. The tau levels were increased from the early to late stages of AD. Elevation of CSF tau in the non-tauopathy and tauopathy dementia groups, chronic and acute damage to the cerebrum, and meningeal disturbance were other factors that required attention for clinical practice. Measurement of CSF tau was useful as a biomarker for early and differential diagnosis of AD.

Site-specific phosphorylation of neurofilament-L is mediated by calcium/calmodulin-dependent protein kinase II in the apical dendrites during long-term potentiation.

Abstract : Neurofilament‐L (NF‐L), one subunit of the neuronal intermediate filaments, is a major element of neuronal cytoskeletons. The dynamics of NF‐L are regulated by phosphorylation of its head domain. The phosphorylation sites of the NF‐L head domain by protein kinase A, protein kinase C, and Rho‐associated kinase have been previously identified, and those by calcium/calmodulin‐dependent protein kinase II (CaMKII) were identified in this study. A series of site‐ and phosphorylation state‐specific antibodies against NF‐L was prepared to investigate NF‐L phosphorylation in neuronal systems. Long‐term potentiation (LTP) is a cellular model of neuronal plasticity that is thought to involve the phosphorylation of various proteins. NF‐L is considered a possible substrate for phosphorylation. During LTP stimulation of mouse hippocampal slices, the series of antibodies demonstrated the increase in the phosphorylation level of Ser57 in NF‐L and the visualization of the localized distribution of Ser57 phosphorylation in a subpopulation of apical dendrites of the pyramidal neurons. Furthermore, Ser57 phosphorylation during LTP is suggested to be mediated by CaMKII. Here we show that NF‐L is phosphorylated by CaMKII in a subpopulation of apical dendrites during LTP, indicating that Ser57 is a novel phosphorylation site of NF‐L in vivo related to the neuronal signal transduction.

An immunohistochemical study of MAP2 and clathrin in gerbil hippocampus after cerebral ischemia.

Changes in MAP2 and clathrin immunoreactivity were studied in gerbil hippocampus after transient cerebral ischemia. MAP2 immunoreactivity decreased significantly by 1 h in the subiculum-CA1 and CA2 areas which correspond to reactive change, while no decrease was observed in CA1 until day 4. Before the initiation of delayed neuronal death, MAP2 immunoreactivity was not changed in CA1. On the other hand clathrin immunoreactivity increased in the pyramidal cell layer of CA1 by 3 h after ischemia and remained high for 2 days. Clathrin immunoreactivity in the pyramidal cell layer of CA1 diminished after delayed neuronal death. The transient change of clathrin was noted especially in CA1 in the period prior to delayed neuronal death. These results imply an abnormal change in clathrin turnover after ischemia, which may participate in the pathogenesis of delayed neuronal death.

Tau protein is a potential biological marker for normal pressure hydrocephalus

A biological marker for normal pressure hydrocephalus (NPH) is beneficial for evaluation of its severity and of indications for shunt operation. Tau protein was initially considered as a biological marker in cerebrospinal fluid (CSF) from Alzheimer’s patients. Recently, it has been demonstrated that degeneration in the brain causes elevation of tau in CSF. Therefore, the tau level in CSF from NPH patients was evaluated. Tau levels in CSF from NPH patients were significantly higher than that in controls. The tau levels were correlated with the severity of dementia, urinary incontinence, and gait disturbance in NPH. These results suggest that CSF tau may be useful as a biological marker for NPH to determine the level of neuronal degeneration.

Functional and possible physical association of scavenger receptor with cytoplasmic tyrosine kinase Lyn in monocytic THP-1-derived macrophages

Acetyl LDL (modified low‐density lipoprotein), which is thought to be taken up through scavenger receptor A (SR‐A), rapidly induced the appearance of phosphotyrosine proteins in monocytic THP‐1‐derived macrophages in vitro. The two alternative forms of Lyn (p53 and p56) were found to be tyrosine‐phosphorylated within 30 s after the stimulation with acetyl LDL. The catalytic activity of Lyn measured by an in vitro kinase assay had also increased in acetyl LDL‐stimulated THP‐1‐derived macrophages. Furthermore, Lyn could be co‐immunoprecipitated with SR‐A from the cell lysate. These observations suggest a functional and possible physical association of SR‐A with Lyn in THP‐1‐derived macrophages, and also imply a possible involvement of Lyn in SR‐A signal transduction.