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

Alzheimer's disease is characterized by the presence of extracellular deposits of amyloid, primarily composed of the amyloid ß-protein (Aß). A growing body of evidence indicates that oligomeric forms of Aß play a critical role in disease causation. Soybean isoflavones are flavonoids with an isoflavone backbone. Isoflavones have been reported to protect against Aß-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 Aß assembly and destabilize preformed Aß aggregates. Here, we show that isoflavones, especially glycitein and genistein, have anti-fibrillization, anti-oligomerization and fibril-destabilizing effects on Aß(1-40) and Aß(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 Aß monomers, oligomers and fibrils, indicating specific binding of glycitein to these Aß species. Glycitein also interacted with different Aß fragments (Aß(1-42), Aß(1-40), Aß(1-16) and Aß(25-35)), exhibiting the highest fluorescence enhancement with Aß(25-35). We speculated that glycitein's anti-amyloidogenic properties are specifically mediated by its binding to Aß monomers, oligomers and fibrils. Isoflavones may hold promise as a treatment option for preventative strategies targeting amyloid formation in Alzheimer's disease.

Severe Chronic Inflammatory Demyelinating Polyneuropathy Ameliorated following High-dose (3 g/kg) Intravenous Immunoglobulin Therapy.

We report the case of a 53-year-old woman with severe chronic inflammatory demyelinating polyneuropathy (CIDP) who developed progressive tetraplegia with respiratory failure despite receiving a standard dose of intravenous immunoglobulin therapy (IVIg), steroid pulse therapy, plasma exchange, and cyclosporine. We administered high-dose IVIg (3 g/kg; 0.6 g/kg/day for 5 consecutive days at monthly intervals). The patient's muscle weakness gradually improved after IVIg. She recovered completely 2 years after the onset of symptoms. The effects of IVIg treatment in individuals with CIDP may vary in each patient. In patients with refractory CIDP receiving standard-dose IVIg, repeated high-dose IVIg treatment can be considered.

Perineuritis Successfully Treated with Early Aggressive Immunotherapy.

Perineuritis is a rare type of peripheral neuropathy defined by swelling and cellular infiltration in the perineurium. We herein report a 52-year-old man who presented with subacute onset pain from the back to the lower limbs, muscle weakness and hypoesthesia. A sural nerve biopsy revealed perineuritis, consisting of inflammatory cell infiltration and swelling of the perineurium. Oral prednisolone, plasma exchange and intravenous immunoglobulin treatment were all effective, leading to significant improvement of the symptoms.

Cerebrospinal Fluid and Plasma Biomarkers in Neurodegenerative Diseases.

Cerebrospinal fluid (CSF) amyloid-ß (Aß)42 and tau are biomarkers for Alzheimer's disease (AD); however, the effects of other neurodegenerative processes on these biomarkers remain unclear. We measured Aß40, Aß42, total tau, phosphorylated-tau, and α-synuclein in CSF and plasma using matched samples from various neurodegenerative diseases to expand our basic knowledge on these biomarkers and their practical applications. A total of 213 CSF and 183 plasma samples were analyzed from cognitively unimpaired subjects, and patients with Alzheimer's disease dementia (ADD), mild cognitive impairment (MCI), non-AD dementias, and other neurological diseases. The CSF/plasma ratios of Aß40 and Aß42 were approximately 25:1. Aß40/42 ratios in CSF and plasma were both 10:1. The CSF total tau/P181tau ratio was 6:1. The CSF/plasma α-synuclein ratio was 1:65. Significantly decreased Aß42 levels and an increased Aß40/42 ratio in CSF in ADD/MCI suggested that these relationships were specifically altered in AD. Increased total tau levels in ADD/MCI, encephalopathy, and multiple system atrophy, and increased P181tau in ADD/MCI indicated that these biomarkers corresponded to neurodegeneration and tauopathy, respectively. Although CSF α-synuclein levels were increased in ADD/MCI, there was no merit in measuring α-synuclein in CSF or plasma as a biomarker. The combination of biomarkers by the Aß40/42 ratio×p181tau reflected specific changes due to the AD pathology in ADD/MCI. Thus, CSF Aß40, Aß42, p181tau, and tau were identified as biomarkers for aggregated Aß associated state (A), aggregated tau associated state (T), and neurodegeneration state (N) pathologies in AD based on the NIA-AA criteria. Overlaps in these biomarkers need to be considered in clinical practice for differential diagnoses of neurodegenerative diseases.

Oral Immunization with Soybean Storage Protein Containing Amyloid-ß 4-10 Prevents Spatial Learning Decline.

Amyloid-ß (Aß) plays a central role in the pathogenesis of Alzheimer's disease (AD). Because AD pathologies begin two decades before the onset of dementia, prevention of Aß amyloidosis has been proposed as a mean to block the pathological cascade. Here, we generate a transgenic plant-based vaccine, a soybean storage protein containing Aß4-10, named Aß+, for oral Aß immunization. One mg of Aß+ or control protein (Aß-) was administered to TgCRND8 mice once a week from 9 weeks up to 58 weeks. Aß+ immunization raised both anti-Aß antibodies and cellular immune responses. Spatial learning decline was prevented in the Aß+ immunized group in an extended reference memory version of Morris water maze test from 21 to 57 weeks. In Tris-buffered saline (TBS), sodium dodecyl sulfate (SDS), and formic acid (FA) serial extractions, all sets of Aß species from Aß monomer, low to high molecular weight Aß oligomers, and Aß smears had different solubility in TgCRND8 brains. Aß oligomers decreased in TBS fractions, corresponding to an increase in high molecular weight Aß oligomers in SDS extracts and Aß smears in FA fraction of the Aß+ treated group. There was significant inhibition of histological Aß burden, especially in diffuse plaques, and suppression of microglial inflammation. Processing of amyloid-ß protein precursor was not different between Aß+ and Aß- groups. No evidence of amyloid-related inflammatory angiopathy was observed. Thus, Aß+ oral immunization could be a promising, cheap, and long-term safe disease-modifying therapy to prevent the pathological process in AD.

Non-steroidal anti-inflammatory drugs have potent anti-fibrillogenic and fibril-destabilizing effects for alpha-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) [Parkinson's 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.

Aging and APOE-ε4 are determinative factors of plasma Aß42 levels.

OBJECTIVE: The aim of this study was to confirm determinative factors for plasma Aß and its association with cognitive function. METHODS: Fasting plasma Aß40 and Aß42 levels were measured by ELISA in 1019 participants in the Iwaki Health Promotion Project. The relationships between plasma Aß and health-related items, including physical characteristics, cognitive function tests, blood chemistry, and APOE-ε4 genotype were analyzed. RESULTS: The plasma levels of Aß40 and Aß42, and Aß40/42 ratio were found to significantly increase with aging. The age-dependent increase in Aß42 level was significantly suppressed by APOE-ε4. Renal function was an associated factor for the plasma Aß40 level. The plasma Aß42 level and Aß40/42 ratio correlated with cognitive function. INTERPRETATION: Age and APOE-ε4 are major determinative factors of plasma levels of Aß42 and the Aß40/42 ratio. These factors are critical adjustment factors for the usage of plasma Aß as a biomarker of central nervous system amyloidosis.

Non-steroidal anti-inflammatory drugs have anti-amyloidogenic effects for Alzheimer's beta-amyloid fibrils in vitro.

The pathogenesis of Alzheimer's disease (AD) is characterized by cerebral deposits of amyloid beta-peptides (A beta) and neurofibrillary tangles which are surrounded by inflammatory cells. Long-term use of non-steroidal anti-inflammatory drugs (NSAIDs) reduces the risk of developing AD and delays the onset of the disease. In the present study, we used fluorescence spectroscopy with thioflavin T and electron microscopy to examine the effects of NSAIDs such as ibuprofen, aspirin, meclofenamic acid sodium salt, diclofenac sodium salt, ketoprofen, flurbiprofen, naproxen, sulindac sulfide and indomethacin on the formation, extension, and destabilization of beta-amyloid fibrils (fA beta) at pH 7.5 at 37 degrees C in vitro. All examined NSAIDs dose-dependently inhibited formation of fA beta from fresh A beta(1-40) and A beta(1-42), as well as their extension. Moreover, these NSAIDs dose-dependently destabilized preformed fA betas. The overall activity of the molecules examined was in the following order: ibuprofen approximately sulindac sulfide >or= meclofenamic acid sodium salt>aspirin approximately ketoprofen >or= flurbiprofen approximately diclofenac sodium salt>naproxen approximately indomethacin. Although the mechanisms by which these NSAIDs inhibit fA beta formation from A beta, and destabilize preformed fA beta in vitro are still unclear, NSAIDs may be promising for the prevention and treatment of AD.

Reversible cortical lesions in primary Sjögren's syndrome presenting with meningoencephalitis as an initial manifestation.

We report a 50-year-old woman with primary Sjögren's syndrome (SjS) who initially showed forgetfulness, and later developed disturbance of consciousness. In addition to aseptic meningoencephalitis revealed by cerebrospinal fluid examination and magnetic resonance imaging (MRI), the presence of serum anti-SS-A and anti-SS-B antibodies and inflammatory findings in lip biopsy indicated primary SjS. Fluid attenuated inversion recovery (FLAIR) of MRI revealed well defined small, high signal intensity areas in the cortex involving the subcortical white matter. Corticosteroid therapy resulted in rapid and nearly complete resolution of the cortical lesions with marked improvement of the clinical manifestations. Memory disturbance is a rare initial manifestation in meningoencephalitis associated with SjS. Our patient with SjS showed inflammatory cortical lesions on MRI, which were reversed by corticosteroid therapy.

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

The folding of amyloid ß-protein (Aß) into oligomeric, protofibrillar, and fibrillar assemblies is hypothesized to be the key pathogenic event in Alzheimer's 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 Aß(1-40) and Aß(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 Aß 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.

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 Parkinson's 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.

Vitamin A has anti-oligomerization effects on amyloid-ß in vitro.

Inhibition of amyloid-ß (Aß) aggregation is an attractive therapeutic strategy for treatment of Alzheimer's disease (AD). We previously reported that vitamin A and ß-carotene inhibit fibrillation of Aß40 and Aß42 (Ono et al, 2004, Exp Neurol). In this study, we firstly examined the effects of vitamin A (retinoic acid, retinol, and retinal), ß-carotene, vitamin B2, vitamin B6, vitamin C, vitamin E, coenzyme Q10, and α-lipoic acid on oligomerization of Aß40 and Aß42 in vitro; vitamin A and ß-carotene dose-dependently inhibited oligomerization of Aß40 and Aß42. Furthermore, retinoic acid decreased cellular toxicity by inhibition of Aß42 oligomerization. Second, we analyzed how vitamin A inhibits Aß aggregation by using fluorescence spectroscopy and thioflavin T assay with two Aß fragments, Aß1-16 and Aß25-35. A fluorescence peak of retinoic acid was greatly restrained in the presence of Aß25-35, and retinoic acid inhibited aggregation of Aß25-35, but not of Aß1-16, which suggest the specific binding of retinoic acid to the C-terminal portion of Aß. Thus, vitamin A and ß-carotene might be key molecules for prevention of AD.

Anti-aggregation and fibril-destabilizing effects of sex hormones on alpha-synuclein fibrils in vitro.

The alpha-synuclein aggregation in the brain is the hallmark of Lewy body diseases, including Parkinson's disease and dementia with Lewy bodies, and multiple system atrophy. Some epidemiological studies have revealed that estrogen therapy reduces the risk of Parkinson's disease in females. We examined the effects of estriol, estradiol, estrone, androstenedione, and testosterone on the formation and destabilization of alpha-synuclein fibrils at pH 7.5 and 37 degrees C in vitro, using fluorescence spectroscopy with thioflavin S and electron microscopy. These sex hormones, especially estriol, significantly exert anti-aggregation and fibril-destabilizing effects; and hence, could be valuable preventive and therapeutic agents for alpha-synucleinopathies.

Alpha-synuclein assembly as a therapeutic target of Parkinson's disease and related disorders.

Lewy bodies (LBs) and Lewy neurites (LNs) in the brain constitute the main histopathological features of Parkinson's disease (PD) and dementia with Lewy bodies (DLB), and are comprised of amyloid-like fibrils composed of a small protein ( approximately 14 kDa) named alpha-synuclein (alphaS). As the aggregation of (alphaS in the brain has been implicated as a critical step in the development of the diseases, the current search for disease-modifying drugs is focused on modification of the process of (alpha S deposition in the brain. In this article, the recent developments on the molecules that inhibit the formation of alpha-synuclein fibrils (falphaS) as well as the oligomerization of alphaS are reviewed. Recently, various compounds such as curcumin, nicotine and wine-related polyphenols have been reported to inhibit the formation of falphaS, and to destabilize preformed falphaS at pH 7.5 at 37 degrees C in vitro. Although the mechanisms by which these compounds inhibit falphaS formation from falphaS, and destabilize preformed falphaS are still unclear, they could be key molecules for the development of preventives and therapeutics for PD and other alpha-synucleinopathies.

Non-steroidal anti-inflammatory drugs as anti-amyloidogenic compounds.

Amyloidosis is a clinical disorder caused by deposition of proteins that abnormally self-assemble into insoluble fibrils and impair organ function. More than 20 unrelated precursor proteins lose their native structure and misfold, leading to the formation of amyloid fibrils. The latter share cross-beta core structure in vivo and in vitro and gain abnormal functions. Local amyloid deposition occurs in the central nervous system in Alzheimer's disease (AD) and cerebral amyloid angiopathy. AD is the most common form of neurodegenerative disorder, with dementia in the elderly as well as dementia with Lewy bodies (DLB). Extracellular deposition of amyloid beta-peptide (Abeta) has been implicated as a critical step in the pathogenesis of AD. Involvement of neuroinflammation and microglial activation has been emphasized in the AD brain. Recent epidemiological studies have shown that long-term therapeutic use of non-steroidal anti-inflammatory drugs (NSAIDs) reduces the risk of developing AD and delayed the onset of AD. We review epidemiological studies of anti-AD effects of NSAIDs, experimental studies of anti-amyloidogenic as well as anti-inflammatory effects of NSAIDs, and recent clinical trials for AD with NSAIDs. We refer to the anti-fibrillogenic and fibril-destabilizing activities of NSAIDs for other proteins that can aggregate and form amyloid-like fibrils, including alpha-synuclein in DLB. The anti-amyloidogenic properties of some NSAIDs provide new insights for future therapeutic and preventative opportunities for AD and other amyloidoses, and protein-misfolding disorders.

Anti-fibrillogenic and fibril-destabilizing activity of nicotine in vitro: implications for the prevention and therapeutics of Lewy body diseases.

The aggregation of alpha-synuclein (alphaS) has been implicated as a critical step in the development of Lewy body diseases (LBD) and multiple system atrophy (MSA). Both retrospective and prospective epidemiological studies have consistently demonstrated an inverse association between cigarette smoking and Parkinson's disease (PD). We used fluorescence spectroscopy with thioflavin S, electron microscopy and atomic force microscopy to examine the effects of nicotine, pyridine, and N-methylpyrrolidine on the formation of alphaS fibrils (f alphaS) from wild-type alphaS (alphaS (WT)) and A53T mutant alphaS (A53T) and on preformed f alpha Ss. Nicotine dose-dependently inhibited the f alphaS formation from both alphaS (WT) and A53T. Moreover, nicotine dose-dependently destabilized preformed f alpha Ss. These effects of nicotine were similar to those of N-methylpyrrolidine. The anti-fibrillogenic activity of nicotine may be exerted not only by the inhibition of f alphaS formation but also by the destabilization of preformed f alphaS. Additionally, this effect may be attributed to N-methylpyrrolidine moieties of nicotine.

Anti-fibrillogenic and fibril-destabilizing activities of anti-Parkinsonian agents for alpha-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) and multiple system atrophy (MSA). Among the antioxidant strategies proposed, increasing evidence points to the possibility of achieving neuroprotection by dopamine agonists, as well as monoamine oxidase B inhibitors. We showed previously that the anti-Parkinsonian agents dose-dependently inhibited beta-amyloid fibrils (fAbeta)(1-40) and fAbeta(1-42) formation as well as destabilized preformed fAbetas. Using fluorescence spectroscopy with thioflavin S, electron microscopy, and atomic force microscopy, we examined the effects of anti-Parkinsonian agents, selegiline, dopamine, pergolide, bromocriptine, and trihexyphenidyl on the formation of alphaS fibrils (falphaS) and on preformed falphaS. All molecules except for trihexyphenidyl, dose-dependently inhibited the formation of falphaS. Moreover, these molecules dose-dependently destabilized preformed falphaS. The overall activity of the molecules examined was in the order of: selegiline = dopamine > pergolide > bromocriptine. These agents and other compounds related structurally could be key molecules for the development of therapeutics for LBD and MSA.

Estrogen has anti-amyloidogenic effects on Alzheimer's beta-amyloid fibrils in vitro.

Inhibition of the assembly of amyloid beta-peptide (Abeta) as well as the destabilization of preformed beta-amyloid fibrils (fAbeta) in the central nervous system could be valuable therapeutics of patients with Alzheimer's disease (AD). Epidemiological studies have indicated that estrogen therapy reduced 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 polymerization, extension and destabilization of fAbeta(1-42) and fAbeta(1-40) at pH 7.5 at 37 degrees C in vitro, using fluorescence spectroscopic analysis with thioflavin T and electron microscopic studies. E1, E2, and E3 dose-dependently inhibited the formation, as well as destabilization of fAbetas. The overall anti-amyloidogenic activity of these molecules was in the order of: E3>E2=E1>>AND=TES. Estrogen could be a potential therapeutic agent to prevent or delay AD progression.

The anti-amyloidogenic effect is exerted against Alzheimer's beta-amyloid fibrils in vitro by preferential and reversible binding of flavonoids to the amyloid fibril structure.

How various anti-amyloidogenic compounds inhibit the formation of Alzheimer's beta-amyloid fibrils (fAbeta) from amyloid beta-peptide (Abeta) and destabilize fAbeta remains poorly understood. Using spectrophotometry, spectrofluorometry, atomic force microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and surface plasmon resonance (SPR), we investigated the anti-amyloidogenic effects of five flavonoids on fAbeta in vitro. Oxidized flavonoids generally inhibited fAbeta(1-40) formation significantly more potently than fresh compounds. Characterization of the novel fluorescence of myricetin (Myr) emitted at 575 nm with an excitation maximum at 430 nm in the presence of fAbeta(1-40) revealed the specific binding of Myr to fAbeta(1-40). By SPR analysis, distinct association and dissociation reactions of Myr with fAbeta(1-40) were observed, in contrast to the very weak binding to the Abeta monomer. A significant decrease in the rate of fibril extension was observed when >0.5 microM Myr was injected into the SPR experimental system. These findings suggest that flavonoids, especially Myr, exert an anti-amyloidogenic effect in vitro by preferentially and reversibly binding to the amyloid fibril structure of fAbeta, rather than to Abeta monomers.