Akiyoshi Morinaga

Epidemiology of familial amyloid polyneuropathy in Japan: Identification of a novel endemic focus.

BACKGROUND Familial amyloid polyneuropathy (FAP) is distributed worldwide with several endemic foci including two major foci in Japan. OBJECTIVE To elucidate a nationwide epidemiology of FAP in Japan. DESIGN, SETTING, AND PATIENTS (i) We analyzed the data of FAP patients registered by the Ministry of Health, Labour, and Welfare, Japan, during 2003-2005. (ii) As Ishikawa prefecture was found to be a novel endemic focus, we examined 27 FAP patients in Ishikawa to characterize their clinical and genetic features in comparison with other endemic foci. RESULTS (i) The prevalence of familial amyloidosis in Japan was estimated to be 0.87-1.1 per 1,000,000 persons. Nagano prefecture had the highest prevalence (11-15.5), followed by Kumamoto (10.1-10.3), and then Ishikawa (3.5-4.2). (ii) All the FAP patients in Ishikawa had transthyretin (TTR) type FAP; all the families had a TTR Val30Met mutation except one family with a Leu58Arg mutation. FAP with Val30Met mutation in Ishikawa was characterized by late onset, high penetrance, and moderate autonomic dysfunction. CONCLUSIONS Ishikawa prefecture is the third endemic focus of FAP in Japan. FAP with TTR Val30Met mutation in Japan can be classified to (i) early-onset and endemic (Nagano and Kumamoto), (ii) late-onset and endemic (Ishikawa), and (iii) late-onset and non-endemic types.

Critical role of interfaces and agitation on the nucleation of Aβ amyloid fibrils at low concentrations of Aβ monomers

Amyloid deposits are pathological hallmarks of various neurodegenerative diseases including Alzheimers disease (AD), where amyloid beta-peptide (Abeta) polymerizes into amyloid fibrils by a nucleation-dependent polymerization mechanism. The biological membranes or other interfaces as well as the convection of the extracellular fluids in the brain may influence Abeta amyloid fibril formation in vivo. Here, we examined the polymerization kinetics of 2.5, 5, 10 and 20 microM Abeta in the presence or absence of air-water interface (AWI) using fluorescence spectroscopy and fluorescence microscopy with the amyloid specific dye, thioflavin T. When the solutions were incubated with AWI and in quiescence, amyloid fibril formation was observed at all Abeta concentrations examined. In contrast, when incubated without AWI, amyloid fibril formation was observed only at higher Abeta concentrations (10 and 20 microM). Importantly, when the 5 microM Abeta solution was incubated with AWI, a ThT-reactive film was first observed at AWI without any other ThT-reactive aggregates in the bulk. When 5 microM Abeta solutions were voltexed or rotated with AWI, amyloid fibril formation was considerably accelerated, where a ThT-reactive film was first observed at AWI before ThT-reactive aggregates were observed throughout the mixture. When 5 microM Abeta solutions containing a polypropylene disc were rotated without AWI, amyloid fibril formation was also considerably accelerated, where fine ThT-reactive aggregates were first found attached at the edge of the disc. These results indicate the critical roles of interfaces and agitation for amyloid fibril formation. Furthermore, elimination of AWI may be essential for proper evaluation of the roles of various biological molecules in the amyloid formation studies in vitro.

Non-steroidal anti-inflammatory drugs have potent anti-fibrillogenic and fibril-destabilizing effects for α-synuclein fibrils in vitro

The aggregation of alpha-synuclein (alphaS) in the brain has been implicated as a critical step in the development of Lewy body diseases (LBD) [Parkinsons disease (PD)/dementia with Lewy bodies (DLB)] and multiple system atrophy (MSA). The involvement of neuroinflammation and microglial activation has been emphasized in the pathogenesis of PD. Recent epidemiological studies have revealed that therapeutic use of non-steroidal anti-inflammatory drugs (NSAIDs) reduces the risk of developing PD. Here, we examined the effects of NSAIDs, such as ibuprofen, aspirin, acetaminophen, meclofenamic acid sodium salt, sulindac sulfide, ketoprofen, flurbiprofen, diclofenac sodium salt, naproxen, and indomethacin, on the formation and destabilization of alphaS fibrils (falphaS) at pH 7.5 and 37 degrees C in vitro, using fluorescence spectroscopy with thioflavin S and electron microscopy. All examined NSAIDs, except for naproxen and indomethacin, inhibited the formation of falphaS in a dose-dependent manner. Moreover, these molecules dose-dependently destabilized preformed falphaS. The overall activity was in the order: ibuprofen approximately aspirin approximately acetaminophen approximately meclofenamic acid sodium salt approximately sulindac sulfide>ketoprofen approximately flurbiprofen approximately diclofenac sodium salt>naproxen approximately indomethacin. These findings indicate that NSAIDs could be key molecules for the development of therapeutic or preventive agents for LBD and MSA.

A Comparison of the Diagnostic Sensitivity of MRI, CBF-SPECT, FDG-PET and Cerebrospinal Fluid Biomarkers for Detecting Alzheimer's Disease in a Memory Clinic

Background/Aim: Magnetic resonance imaging (MRI), cerebral blood flow single photon emission computed tomography (CBF-SPECT), fluorodeoxyglucose-positron emission tomography (FDG-PET) and cerebrospinal fluid (CSF) biomarkers are used for the diagnosis of Alzheimer’s disease (AD). We aimed to reveal the relative sensitivity of these tools in a memory clinic setting. Methods: In 207 patients with probable AD in our memory clinic, medial temporal lobe atrophy on MRI, hypoperfusion/hypometabolism of the parietotemporal lobe and posterior cingulate gyrus in ethylcysteinate dimer-CBF-SPECT/FDG-PET, and abnormalities of CSF amyloid β-protein 1–42, total tau and phosphorylated tau were evaluated as findings characteristic of AD. Results: The AD findings were observed in 77.4% of all AD patients with MRI, 81.6% with CBF-SPECT, 93.1% with FDG-PET and 94.0% with CSF biomarkers. At the stage of Clinical Dementia Rating (CDR) 0.5, CSF biomarkers were the most sensitive (90.0%); at the stage of CDR 1, FDG-PET (96.7%) and CSF biomarkers (95.5%) were highly sensitive. At the stage of CDR 2, all tools showed high positive percentages. Conclusion: The diagnosis of AD was most often supported by CSF biomarkers and FDG-PET at the early stage of dementia (CDR 1) and by CSF biomarkers at the earlier stage (CDR 0.5).

Effects of sex hormones on Alzheimer's disease-associated β-amyloid oligomer formation in vitro.

The folding of amyloid β-protein (Aβ) into oligomeric, protofibrillar, and fibrillar assemblies is hypothesized to be the key pathogenic event in Alzheimers disease (AD), with oligomeric assemblies thought to be the most neurotoxic. Inhibitors of oligomer formation, therefore, could be valuable therapeutics for patients with AD. Epidemiological studies have indicated that estrogen therapy reduces the risk of developing AD in women. Here, we examined the effects of estrogen (estrone (E1), estradiol (E2), and estriol (E3)) and related sexual steroids (androstenedione (AND) and testosterone (TES)) on the in vitro oligomer formation of Aβ(1-40) and Aβ(1-42) using a method of photo-induced cross-linking of unmodified proteins (PICUP) and electron microscopic studies. Estrogens (E1, E2, and E3) inhibited low-order Aβ oligomer formation, and among them, E3 had the strongest in vitro activity. Estrogen could be a potential therapeutic agent to prevent or delay AD progression, and further understanding of the fact that these very similar molecules have different anti-oligomeric effects would contribute to the development of new agents.

Vitamin A has Anti-Oligomerization Effects on Amyloid-β In Vitro

Inhibition of amyloid-β (Aβ) aggregation is an attractive therapeutic strategy for treatment of Alzheimers disease (AD). We previously reported that vitamin A and β-carotene inhibit fibrillation of Aβ40 and Aβ42 (Ono et al, 2004, Exp Neurol). In this study, we firstly examined the effects of vitamin A (retinoic acid, retinol, and retinal), β-carotene, vitamin B2, vitamin B6, vitamin C, vitamin E, coenzyme Q10, and α-lipoic acid on oligomerization of Aβ40 and Aβ42 in vitro; vitamin A and β-carotene dose-dependently inhibited oligomerization of Aβ40 and Aβ42. Furthermore, retinoic acid decreased cellular toxicity by inhibition of Aβ42 oligomerization. Second, we analyzed how vitamin A inhibits Aβ aggregation by using fluorescence spectroscopy and thioflavin T assay with two Aβ fragments, Aβ1-16 and Aβ25-35. A fluorescence peak of retinoic acid was greatly restrained in the presence of Aβ25-35, and retinoic acid inhibited aggregation of Aβ25-35, but not of Aβ1-16, which suggest the specific binding of retinoic acid to the C-terminal portion of Aβ. Thus, vitamin A and β-carotene might be key molecules for prevention of AD.

Anti-amyloidogenic effects of soybean isoflavones in vitro: Fluorescence spectroscopy demonstrating direct binding to Aβ monomers, oligomers and fibrils

Alzheimers disease is characterized by the presence of extracellular deposits of amyloid, primarily composed of the amyloid β-protein (Aβ). A growing body of evidence indicates that oligomeric forms of Aβ play a critical role in disease causation. Soybean isoflavones are flavonoids with an isoflavone backbone. Isoflavones have been reported to protect against Aβ-induced neurotoxicity in cultured cell systems, the molecular mechanisms remain unclear. Our previous studies demonstrated that red wine-related flavonoids with a flavone backbone are able to inhibit Aβ assembly and destabilize preformed Aβ aggregates. Here, we show that isoflavones, especially glycitein and genistein, have anti-fibrillization, anti-oligomerization and fibril-destabilizing effects on Aβ(1-40) and Aβ(1-42)in vitro at physiological pH and temperature, by using nucleation-dependent polymerization monitored by thioflavin T fluorescence, atomic force microscopy, electron microscopy, and photo-induced cross-linking of unmodified proteins followed by SDS-PAGE. Our three-dimensional fluorescence spectroscopic analyses demonstrated that glycitein interacted with Aβ monomers, oligomers and fibrils, indicating specific binding of glycitein to these Aβ species. Glycitein also interacted with different Aβ fragments (Aβ(1-42), Aβ(1-40), Aβ(1-16) and Aβ(25-35)), exhibiting the highest fluorescence enhancement with Aβ(25-35). We speculated that glyciteins anti-amyloidogenic properties are specifically mediated by its binding to Aβ monomers, oligomers and fibrils. Isoflavones may hold promise as a treatment option for preventative strategies targeting amyloid formation in Alzheimers disease.

Cerebrospinal fluid from patients with multiple system atrophy promotes in vitro α-synuclein fibril formation

The aggregation of α-synuclein (αS) in the central nervous system (CNS) is the hallmark of multiple system atrophy (MSA) and Lewy body diseases including Parkinsons disease (PD) and dementia with Lewy bodies (DLB) (α-synucleinopathies). To test the hypothesis that patients with α-synucleinopathies have a CNS environment favorable for αS aggregation, we examined the influence of cerebrospinal fluid (CSF) from patients with MSA (n=20), DLB (n=8), and PD (n=10) on in vitro αS fibril (fαS) formation at pH 7.5 and 37°C using fluorescence spectroscopy with thioflavin S, compared with those with hereditary spinocerebellar ataxia (hSCA) (n=16), and tension-type headache (n=7). CSF from MSA patients (MSA-CSF) promoted fαS formation more strongly than PD-, hSCA-, or headache-CSF. By electron microscopic analyses, the width of fαS formed in MSA-CSF was significantly greater than others. MSA may have a CSF environment particularly favorable for fαS formation.

Microscopic polyangitis presenting with temporal arteritis and multiple cranial neuropathies

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis affects vessels of various diameters in various tissues or organs, sometimes associated with neurological complications. A 77-year-old man developed dysphagia, hoarseness, dysgeusia, gait unsteadiness, and right temporalgia; neurological examination revealed multiple cranial neuropathies. Laboratory studies demonstrated severe inflammatory responses, elevation of perinuclear ANCA, and mild proteinuria. Magnetic resonance imaging of the brain showed dural enhancement in the cerebellar tentorium. Biopsy revealed necrotizing glomerulonephritis in the kidney, and temporal arteritis without giant cells in the temporal artery. The patient was diagnosed with microscopic polyangitis presenting with temporal arteritis and multiple cranial nerve involvement, and was treated with predonisolone, after which the symptoms and laboratory data showed improvement. This is the first case of ANCA-associated vasculitis with pathologically verified lesions in the temporal artery as well as in the kidney. Thus, ANCA-associated vasculitis may simultaneously affect large vessels such as temporal artery, as well as microvessels in the kidney, nerves and other organs.

A novel presenilin 1 mutation (L282F) in familial Alzheimer’s disease

Sirs, Mutations in the genes of b-amyloid precursor protein (APP), presenilin 1 (PSEN1) and presenilin 2 (PSEN2) have been identified as causative in familial Alzheimer’s disease (FAD) [9]. Mutations in PSEN1 are most common in FAD, and 168 pathogenic PSEN1 mutations have been reported to date [3]. We report a novel mutation of PSEN1 (L282F) in a patient with FAD. A 57-year-old woman consulted our hospital because of progressive memory loss. She had noticed forgetfulness since age 53 and had been treated with donepezil at a local clinic since age 55. She presented with impairment of recent memory and orientation for date and scored 24/30 on the mini-mental state examination without any other neurological deficits. Findings of routine blood tests were normal. Serum apolipoprotein E phenotype was E3/E4. Mild medial temporal atrophy was demonstrated on brain magnetic resonance imaging. Fluoro-2-deoxy-D-glucose positron emission tomography (FDG-PET) revealed glucose hypometabolism in the bilateral parietal cortices and posterior cingulate gyri. Her family history suggested FAD, but we could not determine the hereditary pattern of FAD (Fig. 1). After written consent was obtained from the patient and her family, all exons of APP, PSEN1 and PSEN2 were analyzed by direct cycle sequencing as described previously [5, 10]. The analysis of PSEN1 demonstrated a C ? T transversion at the first position of codon 282 in exon 8 (genomic accession number: g. 50029C[T), which predicted a L282F substitution (Fig. 2). There were no other mutations detected in the analyzed genes. We also searched for this mutation in 192 Japanese patients with Alzheimer’s disease (AD) or other forms of dementia, and none demonstrated this PSEN1 mutation. We diagnosed this patient as having probable AD according to the NINCDS-ADRDA criteria [6]. The pattern of brain hypometabolism on FDG-PET supported the diagnosis [2, 7]. Although this L282F mutation has not been described previously, two other mutations at the same