Ichiro Yoshida

Depletion of intracellular Ca2+ store itself may be a major factor in thapsigargin-induced ER stress and apoptosis in PC12 cells.

The mechanisms of intracellular calcium store depletion and store-related Ca(2+) dysregulation in relation to apoptotic cell death in PC12 cells were investigated at physiological temperatures with a leak-resistant fluorescent indicator dye Fura-PE3/AM by a cooled CCD imaging analysis system. Electron microscopic observations have shown thapsigargin (TG; 100 nM)-induced apoptosis in PC12 cells. Thorough starvation of stored Ca(2+) by BAPTA/AM (50 microM), or La(3+) (100 microM) enhanced while dantrolene (100 microM) attenuated the TG-induced apoptosis by preventing a calcium release from internal stores. An immunoblotting analysis revealed an enhanced expression of GRP78, the hallmark of endoplasmic reticulum (ER) stress when cells were treated by TG along with BAPTA/AM. These results indicate that the depletion of the intracellular Ca(2+) stores itself induces the ER stress and apoptosis in PC12 cells without any involvement of the capacitative calcium entry (CCE) or a sustained elevation of intracellular Ca(2+) concentrations ([Ca(2+)](i)).

The relationship between the aggregational state of the amyloid-β peptides and free radical generation by the peptides

In the present study, we investigated whether or not the amyloid‐β protein (Aβ) peptide itself spontaneously generates free radicals using electron spin resonance (ESR) spectroscopy while also monitoring the aggregational state of Aβ and Aβ‐induced cytotoxicity. The present results demonstrated a four‐line spectrum in the presence of both Aβ40 and Aβ42 with Ntert‐butyl‐α‐phenylnitrone (PBN), but not in the presence of PBN alone in phosphate‐buffered saline (PBS). The fact that the four‐line spectrum obtained for the Aβ/PBN in PBS was completely abolished in the presence of the iron‐chelating agent Desferal demonstrated the observed four‐line spectrum to be iron‐dependent. The present study also revealed that either Aβ40 or Aβ42 with PBN in phosphate buffer (PB) did not produce any definite four‐line spectrum. Both a thioflavine‐T (Th‐T) fluorometric assay and circular dichroism (CD) spectroscopy showed the amyloid fibril formation of Aβ in PBS to be much higher than that of Aβ in PB. Moreover, Aβ‐induced cytotoxicity assays showed Aβ incubated in PBS to be more cytotoxic than that incubated in PB. These results thus suggest that Aβ‐associated free radical generation is strongly influenced by the aggregational state of the peptides.

Amyloid-β-protein (Aβ) (25–35)-associated free radical generation is strongly influenced by the aggregational state of the peptides

We investigated whether or not the Amyloid-β-protein (Aβ) itself spontaneously generates free radicals using electron spin resonance (ESR) spectroscopy while also monitoring the aggregational state of Aβ and Aβ-induced cytotoxicity. The present results demonstrated a four-line spectrum in the presence of Aβ25–35 with N-tert-butyl-α-phenylnitrone (PBN) but not in the presence of PBN alone in phosphate-buffered saline (PBS). The fact that the four-line spectrum obtained for the Aβ25–35/PBN in PBS was completely abolished in the presence of the iron-chelating agent Desferal demonstrated the observed four-line spectrum to be iron-dependent. On the other hand, Aβ25–35 with PBN in phosphate buffer (PB) did not produce any definite four-line spectrum. the present results showed the amyloid fibril formation of Aβ25–35 in PBS to be much higher than that of Aβ25–35 in PB. Moreover, Aβ-induced cytotoxicity assays showed Aβ incubated in PBS to be more cytotoxic than that incubated in PB. These results thus demonstrate that Aβ(25–35) - associated free radical generation is strongly influenced by the aggregational state of the peptides.

Inhibition of Aβ fibril formation and Aβ-induced cytotoxicity by senile plaque-associated proteins

Aβ neurotoxicity is generally believed to require Aβ fibril formation. The prevention of Aβ fibril formation thus seems to be a promising strategy for the treatment of AD. Recent studies have shown senile plaque-associated proteins such as laminin to have an inhibitory effect on both Aβ40 and Aβ42 fibril formation in vitro. In the present study, we thus investigated whether or not midkine (MK) and α2-macroglobulin (α2M), both of which are also senile plaque-associated proteins like laminin, affect Aβ fibril formation and Aβ-induced cytotoxicity. The present study demonstrated that both MK and α2M inhibit both Aβ fibril formation and Aβ-induced cytotoxicity in PC12 cells. The confirmation of the present results based on in vivo experiments is called for in future studies to clarify whether or not senile plaque-associated proteins such as MK and α2M can be a model for therapeutic agents in the treatment of AD.

Laminin inhibits both Aβ40 and Aβ42 fibril formation but does not affect Aβ40 or Aβ42-induced cytotoxicity in PC12 cells

Laminin has recently been reported to inhibit both Aβ40 and Aβ42 fibril formation in vitro. Laminin was thus suggested to be an effective therapeutic agent for Alzheimers disease. However, some recent reports have shown that Aβ fibril formation may not necessarily be linked to the development of Aβ neutotoxicity. In the present study, we thus investigated whether or not laminin affects Aβ40 and Aβ42-induced neurotoxicity. The findings of the present study by using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) dye reduction test showed laminin not to have an inhibitory effect on Aβ40 or Aβ42-induced cytotoxicity in PC12 cells while Aβ fibril formation was inhibited under the conditions used in the present study. The findings of the present study therefore do not support the hypothesis that Aβ fibril formation is absolutely required for the development of Aβ cytotoxicity.

Laminin inhibits Aβ42 fibril formation in vitro

Abstract In the present study, we investigated whether or not laminin inhibits A β 42 fibril formation in the same manner as A β 40. Both a thioflavine-T fluorometric assay and electron microscopy by negative staining demonstrated laminin to have a concentration-dependent inhibitory effect on A β 42 fibril formation. The amyloid fibril formation was inhibited approximately by 70% due to the presence of 1.0 mg/ml laminin co-incubated with 1.0 mg/ml A β 42 peptide (molar ratio; A β 42 peptide:laminin=200:1). These results thus suggested that laminin or its derivatives may be effective as therapeutic agents to either prevent or slow down the progression of amyloidogenesis in Alzheimers disease.

Identification of a heparin binding site and the biological activities of the laminin α1 chain carboxy-terminal globular domain

The carboxy‐terminal globular domain (G‐domain) of the laminin α1 chain has been shown to promote heparin binding, cell adhesion, and neurite outgrowth. In this study, we defined the potential sequences originating from the G‐domain of laminin α1 chain which possess these functional activities. A series of peptides were synthesized from the G‐domain, termed LG peptides (LG‐1 to LG‐6) and were tested for their various biological activities. In the direct [3H]heparin binding assays, LG‐6 (residues 2,335–2,348: KDFLSIELVRGRVK) mediated high levels of [3H]heparin binding, and this peptide also directly promoted cell adhesion and spreading, including B16F10, M2, HT1080, and PC12 cells. The peptide LG‐6 also promoted the neurite outgrowth of PC12 cells, mouse granule cells, and chick telencephalic cells. An anti‐peptide LG‐6 antibody inhibited laminin‐1 and peptide LG‐6–mediated cell adhesion and neurite outgrowth. Furthermore, an anti‐integrin α2 antibody also inhibited the cell adhesion activity. These results suggest that peptide LG‐6 plays a functional role as a heparin binding site in the G‐domain of the laminin α1 chain, and this sequence was thus concluded to play a crucial role in regulating cell adhesion and spreading and neurite outgrowth which is related to integrin α2. J. Cell. Physiol. 179:18–28, 1999.

Laminin inhibits Aβ40 fibril formation promoted by apolipoprotein E4 in vitro

Abstract The aggregation of soluble Aβ into insoluble amyloid fibrils is believed to be an important step in the pathogenesis of Alzheimers disease (AD) and the prevention of this process therefore seems to be a promising strategy for the treatment of AD. Both apolipoprotein E(apoE) and laminin are known to play important roles in the regeneration of the central nervous system and both are known to accumulate in the senile plaques of the AD brains. In the present study, we therefore investigated whether or not laminin has any effect on Aβ40 fibril formation promoted by apoE4 in vitro. A thioflavine-T fluorometric assay and electron microscopic observations using negative staining together demonstrated that laminin inhibits Aβ40 fibril formation in vitro while it also inhibits Aβ40 fibril formation promoted by apoE4. These results suggested that either laminin or its derivatives may thus be effective as therapeutic agents for AD.

The inhibitory effect of laminin 1 and synthetic peptides deduced from the sequence in the laminin α1 chain on Aβ40 fibril formation in vitro

We investigated whether or not laminin 1 and the two different synthetic peptides deduced from the sequence in the laminin α1 chain, both of which mediate cell attachment and neurite outgrowth in PC12 cells, have an effect on Aβ40 fibril formation in vitro. A thioflavine-T fluorometric assay showed a synthetic peptide containing the YFQRYLI sequence from the laminin α1 chain to inhibit Aβ40 fibril formation while the inhibitory effect of this peptide was found to be somewhat less than that of intact laminin 1. These results were confirmed by electron microscopic observations using negative staining. The findings of the present study suggested that the synthetic peptide derived from the laminin α1 chain may thus be an effective therapeutic agent for either preventing or slowing down the progression of amyloidogenesis in Alzheimers disease.

A patient with Cotard syndrome who showed an improvement in single photon emission computed tomography findings after successful treatment with antidepressants.

We report the case of a presenile woman with Cotard syndrome, in the context of major depression, who showed an improvement in bilateral frontal hypoperfusion in a SPECT study using 99mTc-HMPAO after undergoing successful treatment with antidepressant therapy. We also retrospectively evaluated her clinical course based on the clinical stages. The symptoms of Cotard syndrome have been reported to change dramatically according to the stages. This peculiarity made it difficult for us to rapidly diagnose Cotard syndrome in the context of major depression, and not dementia, and thereby adequately treat the patient in our case. Differences in the reduced blood flow regions and a time lag from psychiatric remission were observed before the improvement in the SPECT findings when comparing our case with a previously reported case of Cotard syndrome. These differences suggest that the mechanism of Cotard syndrome is still not well understood at the present time.