Yujiro Kashiwagi

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.

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.

Major phosphorylation site (Ser55) of neurofilament L by cyclic AMP-dependent protein kinase in rat primary neuronal culture.

Abstract: Ser55 of neurofilament L (NF‐L) is reported to be partly phosphorylated in neurons and to be phosphorylated by cyclic AMP‐dependent protein kinase (PKA). Bovine NF‐L was phosphorylated by PKA in a low concentration of MgCl2 (0.3 mM) and digested by trypsin. Trypsin‐digested fragments were assigned by MALDI/TOF (matrix‐assisted laser desorption and ionization/time‐of‐flight) mass spectrometry. Phosphorylation sites were found at Ser41, Ser55, and Ser62 in the head region, with Ser55 considered the preferred site. A site‐specific phosphorylation‐dependent antibody against Ser55 rendered NF‐L phosphorylated at Ser55 detectable in primary cultured rat neurons. One‐hour treatment with 20 nM okadaic acid increased the phosphorylation level of Ser55, and co‐treatment with 10 μM forskolin enhanced it. However, forskolin alone did not elevate the phosphorylation level. As a consequence, NF‐L may be phosphorylated at Ser55 by PKA or by a PKA‐like kinase in vivo; however, the phosphorylation level of Ser55 may be modulated by certain phosphatases sensitive to okadaic acid.

Localized phosphorylation of vimentin by Rho-kinase in neuroblastoma N2a cells

Vimentin, which is one of the intermediate filaments, is the major cytoskeletal component in developing neurones or neuroblastoma cells. Rho‐associated kinase (Rho‐kinase), is rich in neurones and is found downstream of Rho. It is involved in the agonist‐induced neurite retraction of neuronal cells, and phosphorylates vimentin at Ser‐38 and Ser‐71 resulting in in vitro disassembly of the filaments.

Casein kinase II is responsible for phosphorylation of NF‐L at Ser‐473

Ser‐473 is solely phosphorylated in vivo in the tail region of neurofilament L (NF‐L). With peptides including the native phosphorylation site, it was not possible to locate responsible kinases. We therefore adopted full‐length dephosphorylated NF‐L as the substrate, and employed MALDI/TOF (matrix‐assisted laser desorption and ionization/time of flight) mass spectrometry and a site‐specific phosphorylation‐dependent antibody recognizing Ser‐473 phosphorylation. The antibody showed that casein kinase I (CK I) as well as casein kinase II (CK II) phosphorylated Ser‐473 in vitro, while neither GSK‐3β nor calcium/calmodulin‐dependent protein kinase II did so. However, the mass spectra of the tail fragments of the phosphorylated NF‐L indicated that CK II was the kinase mediating Ser‐473 phosphorylation in vitro as opposed to CK I, because CK I phosphorylated another site as well as Ser‐473 in vitro. The antibody also demonstrated that NF‐L phosphorylated at Ser‐473 was abundant in the neuronal perikarya of the rat cortex, indicating that phosphorylation of Ser‐473 may take place there. This result may support the suggestion that CK II is the kinase responsible for Ser‐473 phosphorylation. Despite many reports showing that CK I mediates phosphorylation of neurofilaments, CK II may phosphorylate NF‐L in vivo.

Abnormal distribution of neurofilament L in neurons with Alzheimer's disease

Abnormality of cytoskeletal proteins is closely related to the pathology of Alzheimers disease. As neurofilament proteins are major cytoskeletal components of neurons, abnormality of neurofilaments is proposed in brain with Alzheimers disease. Free-floating sections of the hippocampus with Alzheimers disease were studied immunohistochemically, using a polyclonal antibody specifically bound to the tail region of neurofilament L (NF-L). In brains with early onset type of Alzheimers disease, many neurons and dystrophic neurites were labeled by the antibody, while these observations were not seen in either brains with late onset type or control brains. Double immunohistochemical staining of NF-L and tau protein demonstrated that abnormal deposition of NF-L was not always accompanied with that of tau protein, indicating that the abnormal deposition of NF-L might not occur in parallel with that of tau protein. These observations suggest the involvement of neurofilament proteins on the pathology of Alzheimers disease in a different way than tau protein.

Phosphorylation of neurofilament-L during LTD

We recently reported that CaMKII-dependent phosphorylation of the neurofilament-L (NF-L) head domain was induced in the apical dendrites during long-term potentiation. Long-term depression (LTD) is another cellular model for neuronal plasticity. In the present study, we examined the phosphorylation of NF-L during hippocampal LTD using a series of phospho-specific antibodies against the NF-L head domain. During LTD, these antibodies visualized NF-L phosphorylation at Ser57 in the apical dendrites of the hippocampal pyramidal neurons. The assembly and disassembly of NF-L filaments are regulated by phosphorylation of its head domain. Thus, our results indicated that NF-L phosphorylation might be associated with alterations of the neuronal structure during LTD.

Pulse exposure of cultured rat neurons to aluminum-maltol affected the axonal transport system

Although chronic aluminum neurotoxicity has been well established, the mechanism of the toxicity has not been elucidated yet. In order to simplify the study of the aluminum neurotoxicity, we employed the pulse exposure of cultured rat cortical neurons to 250 microM aluminum-maltol for 1 h at the early stage (6 h after plating), which resulted in abnormal distribution of neurofilament L (NFL) and fast axonal transported proteins, whereas the axonal transport of tubulin, actin, and clathrin were not impaired. Otherwise, the pulse exposure of neurons at the late stage (4 days after plating) to the same concentration of aluminum-maltol did not affect the cell morphology and the distribution of NFL. The pulse exposure of cultured neurons to aluminum-maltol at the early stage might affect the axonal transport system of NFL and fast axonal transported proteins.

Altered adhesion efficiency and fibronectin content in fibroblasts from schizophrenic patients

Cultured fibroblasts from the cutaneous tissue of 16 schizophrenic patients were compared with 16 control cultured fibroblasts from the healthy subjects. The fibroblasts from the schizophrenic patients showed a decreased adhesion efficiency within 30 min after plating compared to that of the control subjects. However, after 90 min, there was no significant difference between the groups, where more than 90% of the cells from both groups had adhesed to the plate. By immunohistochemistry and western blotting using the antibodies against integrin (VLA5), talin, vinculin, fodrin, vimentin, ankyrin, plectin, fibronectin, and focal adhesion kinase (FAK), there was no significant difference in localization and amount between the groups. The amount of fibronectin released into the medium in which the fibroblast had already kept confluency showed no significant difference between the groups. However, the fibronectin content in cell lysate within 48 h after plating was significantly lower in the schizophrenic group.

Increased tau protein level in postmortem cerebrospinal fluid

Abstract Microtubule‐associated protein tau has been reported to be significantly increased in cerebrospinal fluid (CSF) of the patients with Alzheimers disease (AD), which suggests that it is possibly a biological marker for the diagnosis of AD. The underlying mechanism of the increased tau level in CSF, however, is not known. In this study, the tau levels were compared between antemortem and postmortem CSF. The postmortem tau levels in CSF were significantly increased in all groups including AD, neurological control, and nondemented control. A striking elevation of CSF tau was observed during the postmortem change with the nondemented subjects. These findings may offer some insight into the understanding of the mechanism of the increased tau level in CSF with AD and other related disorders.