Keisuke Kouyama

A rescue factor abolishing neuronal cell death by a wide spectrum of familial Alzheimer's disease genes and Aβ

Through functional expression screening, we identified a gene, designated Humanin (HN) cDNA, which encodes a short polypeptide and abolishes death of neuronal cells caused by multiple different types of familial Alzheimers disease genes and by Aβ amyloid, without effect on death by Q79 or superoxide dismutase-1 mutants. Transfected HN cDNA was transcribed to the corresponding polypeptide and then was secreted into the cultured medium. The rescue action clearly depended on the primary structure of HN. This polypeptide would serve as a molecular clue for the development of new therapeutics for Alzheimers disease targeting neuroprotection.

Involvement of c-Jun N-terminal kinase in amyloid precursor protein-mediated neuronal cell death

Amyloid precursor protein (APP), the precursor of Aβ, has been shown to function as a cell surface receptor that mediates neuronal cell death by anti‐APP antibody. The c‐Jun N‐terminal kinase (JNK) can mediate various neurotoxic signals, including Aβ neurotoxicity. However, the relationship of APP‐mediated neurotoxicity to JNK is not clear, partly because APP cytotoxicity is Aβ independent. Here we examined whether JNK is involved in APP‐mediated neuronal cell death and found that: (i) neuronal cell death by antibody‐bound APP was inhibited by dominant‐negative JNK, JIP‐1b and SP600125, the specific inhibitor of JNK, but not by SB203580 or PD98059; (ii) constitutively active (ca) JNK caused neuronal cell death and (iii) the pharmacological profile of caJNK‐mediated cell death closely coincided with that of APP‐mediated cell death. Pertussis toxin (PTX) suppressed APP‐mediated cell death but not caJNK‐induced cell death, which was suppressed by Humanin, a newly identified neuroprotective factor which inhibits APP‐mediated cytotoxicity. In the presence of PTX, the PTX‐resistant mutant of Gαo, but not that of Gαi, recovered the cytotoxic action of APP. These findings demonstrate that JNK is involved in APP‐mediated neuronal cell death as a downstream signal transducer of Go.

A humanin derivative, S14G-HN, prevents amyloid-β-induced memory impairment in mice

Humanin (HN) is a 24‐amino acid peptide that protects neuronal cells from death caused by Alzheimers disease (AD)‐related genes and amyloid‐β (Aβ). Multiple studies have revealed its biochemical and neuroprotective characteristics in vitro; however, little has been known regarding whether HN is effective in vivo in AD model systems. We examined the effect of S14G‐HN, a 1,000‐fold more potent derivative of HN in vitro, on amnesia induced by Aβ25–35 in mice. The Y‐maze test revealed that at least 50 pmol of S14G‐HN by intracerebroventricular injection prevented Aβ‐induced impairment of short‐term/spatial working memory; however, 5 nmol of S14A‐HN, a neuroprotection‐defective mutant in vitro, did not prevent Aβ‐induced amnesia. These results are in agreement with the structure–function correlation shown previously in vitro. In the water‐finding task, S14G‐HN prevented prolongation of finding latency (the time to find water) observed in Aβ‐amnesic mice, indicating that S14G‐HN also blocked Aβ‐induced impairment of latent learning. In accordance with these observations, immunohistochemical analysis showed that S14G‐HN sustained the number of cholinergic neurons in the basal forebrain and the striata nearly to the normal level. Furthermore, genistein, a specific inhibitor of tyrosine kinases, blocked recovery from scopolamine‐induced amnesia by S14G‐HN, suggesting that certain tyrosine kinase(s) are involved in the inhibitory function of S14G‐HN in vivo. Taking these findings together, we conclude that S14G‐HN has rescue activity against memory impairment caused by AD‐related insults in vivo by activating the same intracellular neuroprotective machinery as elucidated previously in vitro.

Molecular characterization of neurohybrid cell death induced by Alzheimer's amyloid‐β peptides via p75NTR/PLAIDD

One of the most important pathological features of Alzheimers disease (AD) is extracellular senile plaques, whose major component is amyloid‐β peptides (Aβ). Aβ binds to the extracellular domain of p75NTR (p75 neurotrophin receptor) and induces neuronal cell death. We investigated the molecular mechanism of Aβ‐induced neurotoxicity in detail from the standpoint of interaction between p75NTR and its recently identified relative, PLAIDD (p75‐like apoptosis‐inducing death domain). Using F11 neuronal hybrid cells, we demonstrate that there are two distinct pathways for Aβ‐induced toxicity mediated by p75NTR. One pathway that has been previously elucidated, is mediated by p75NTR, Go, JNK, NADPH oxidase and caspase3‐related caspases. We found that PLAIDD and Gi proteins, heterotrimeric G proteins, are involved in the alternative Aβ‐induced neurotoxicity mediated by p75NTR. The alternative pathway triggered by Aβ is thus mediated by p75NTR, PLAIDD, Gi, JNK, NADPH oxidase and caspase3‐related caspases. In addition, we found that HN, ADNF, IGF‐I, or bFGF inhibits both pathways of Aβ‐induced neurotoxicity mediated by p75NTR.

Targeted introduction of V642I mutation in amyloid precursor protein gene causes functional abnormality resembling early stage of Alzheimer's disease in aged mice

While the exact aetiology of Alzheimers disease (AD) is unknown, distinct genetic mutations have been identified for the rare cases of familial AD (FAD). V642I mutation in amyloid precursor protein (APP) co‐segregates with FAD with perfect penetration, and the clinicopathological characteristics of patients with this mutation resemble that of sporadic AD. To examine the pathogenic process of this FAD‐linked trait in vivo, we produced a mouse with the corresponding point mutation in the APP gene using homologous recombination and Cre‐loxP site‐specific recombination (‘knock‐in’ technique). Mice with the heterozygous V642I‐APP allele most precisely reflected the genotype of humans bearing this mutation. For the observation period of 2.5 years the mutants stayed apparently indistinguishable from the wild‐type littermates. However, behavioural analysis revealed significantly deteriorated long‐term memory in mutants when examined for the retention of spatial attention. Interestingly, acquisition of spatial memory was slightly affected but short‐term working memory was not deteriorated at all. Histological examination was negative for formation of neuritic plaques or neurofibrillary tangles, whereas the relative amount of longer form of β‐amyloid species Aβ42(43) was significantly increased against that of the shorter form (Aβ40) in the mutant brain homogenates. We conclude that a V642I‐APP mutant allele in aged mice confers functional components, but not organic components, of the AD‐related phenotype that are observed in the early stage of AD. This V642I‐APP knock‐in mutant line may serve as a model to study the early pathogenic processes of AD in vivo and to develop therapeutics for this stage.

The Association between Primary Open-Angle Glaucoma and Motor Vehicle Collisions

PURPOSE To investigate and compare the prevalence of motor vehicle collisions (MVCs) in individuals with or without primary open-angle glaucoma (POAG). METHODS A total of 265 subjects were consecutively enrolled: 121 (79 men, 42 women; age, 62.1 ± 8.0 years) with POAG; and 144 (95 men, 49 women; age, 61.2 ± 7.9 years) who were free of ocular disease. Participants answered a questionnaire on MVC experience during the previous 10 years, past driving experience, and daily driving habits. The POAG group was subdivided into three groups according to disease severity (mild, moderate, or severe), to assess the relationship between POAG severity and MVC. RESULTS A statistically significant association between POAG severity and MVC frequency was observed; 3.5% of the controls, 0.0% of the mild POAG group, 3.9% of the moderate POAG group, and 25.0% of the severe POAG group had experienced MVCs (P = 0.007, Cochran-Armitage trend test). The severe POAG group had experienced a much higher frequency of MVCs during the surveyed period than had the control group (P < 0.010; Fishers exact test). Logistic regression analyses to account for confounding factors (age, presence of diabetes mellitus, driving history, time spent driving per day, and best corrected visual acuity in the better or worse eye) produced consistent results. CONCLUSIONS Advanced POAG with marked visual field defects may be a risk factor for MVCs.

p53-independent apoptosis is induced by the p19ARF tumor suppressor.

p19(ARF) is a potent tumor suppressor. By inactivating Mdm2, p19(ARF) upregulates p53 activities to induce cell cycle arrest and sensitize cells to apoptosis in the presence of collateral signals. It has also been demonstrated that cell cycle arrest is induced by overexpressed p19(ARF) in p53-deficient mouse embryonic fibroblasts, only in the absence of the Mdm2 gene. Here, we show that apoptosis can be induced without additional apoptosis signals by expression of p19(ARF) using an adenovirus-mediated expression system in p53-intact cell lines as well as p53-deficient cell lines. Also, in primary mouse embryonic fibroblasts (MEFs) lacking p53/ARF, p53-independent apoptosis is induced irrespective of Mdm2 status by expression of p19(ARF). In agreement, p19(ARF)-mediated apoptosis in U2OS cells, but not in Saos2 cells, was attenuated by coexpression of Mdm2. We thus conclude that there is a p53-independent pathway for p19(ARF)-induced apoptosis that is insensitive to inhibition by Mdm2.

The association between visual field defect severity and fear of falling in primary open-angle glaucoma.

PURPOSE To determine if glaucomatous visual field defect severity is associated with fear of falling. METHODS This is a cross-sectional study. A total of 387 consecutive subjects with POAG were enrolled in this study along with 293-ocular disease-free control subjects, who were screened at the same institutions. We defined mild POAG as MD of -6 dB or better, moderate POAG as MD of -6 to -12 dB, and severe POAG as MD of -12 dB or worse in the better eye. All participants were requested to answer a questionnaire on fear of falling. Associations between POAG severity and the prevalence of fear of falling were evaluated with the Cochran-Armitage trend test. Multivariable factors including age-adjusted odds ratios (ORs) for the prevalence of fear of falling and 95% confidence intervals (CIs) were evaluated with logistic regression models. RESULTS The prevalence of fear of falling was 35/293 (11.9%) in the control group, 38/313 (12.1%) in the mild POAG group, 12/48 (25.0%) in the moderate POAG group, and 6/26 (23.1%) in the severe POAG group, and the trend was statistically significant (P = 0.028 Cochran-Armitage trend test). The adjusted ORs for prevalence in the mild, moderate, and severe POAG groups compared with that in the control group were 1.44 (95% CI: 0.83-2.51), 2.33 (95% CI: 1.00-5.44), and 4.06 (95% CI: 1.39-11.90), respectively. CONCLUSIONS Among patients with POAG, the severity of visual field defects is associated with fear of falling. (http://www.umin.ac.jp/ctr/index.htm number, UMIN000005574.).

Preoperative and intraoperative assessment of myometrial invasion in endometrial cancer: comparison of magnetic resonance imaging and frozen sections.

To compare the diagnostic characteristics of the evaluation of myometrial invasion (MI) retrospectively between preoperative magnetic resonance imaging (MRI) and intraoperative frozen sections.

Flexible hysteroscopy with narrow band imaging (NBI) for endoscopic diagnosis of malignant endometrial lesions

Narrow band imaging (NBI) for detection of blood vessels and microstructures on the mucosal surface is used in gastrointestinal endoscopy since it can improve qualitative diagnosis and detection of lesion. However, there are no studies on flexible hysteroscopy using NBI. We performed flexible hysteroscopy with NBI for outpatients to investigate the sensitivity and specificity of endoscopic diagnosis of malignant endometrial lesions. Of patients who attended our hospital for suspected lesions in the uterine cavity between April 2009 and May 2010, 104 subjects underwent hysteroscopy with NBI, in addition to white light. Using the pathological diagnosis as the gold-standard, we evaluated the sensitivity and specificity of NBI hysteroscopy for detecting atypical endometrial hyperplasia (AEH) or carcinoma. The results were also compared with historical data (n=209) for conventional hysteroscopy using white light only in 2008. The sensitivities were 97.2% [95% confidence interval (95% CI): 90.3-99.7%] and 82.6% (95% CI: 74.4-89.0%) for NBI hysteroscopy and conventional hysteroscopy, respectively. The 95% CIs for the two methods did not overlap and the sensitivity of lesion detection was higher with NBI hysteroscopy. Specificities were comparable, 90.6% (95% CI: 75.0-98.0%) and 85.1% (95% CI: 76.3-91.6%) between the methods. NBI hysteroscopy has increased sensitivity for detection of atypical endometrial hyperplasia (AEH) or carcinoma. A comparison with historical data suggested that NBI may be useful for diagnosis of malignant endometrial lesions. As far as we are aware, this is the first evaluation of flexible hysteroscopy with NBI for diagnosis of malignant endometrial lesions.