Introduction: The aim of this prospective study was to see whether LD-1227, a quality-controlled marine nutraceuticals shown to protect experimental stress-induced hyppocampal degeneration, could beneficially modulate BDNF, as measured in the serum, in otherwise healthy but work-stressed individuals.Materials and Methods: Forty-eight men and women between the ages of 38 and 62 reporting high-demanding work activity but with an overall positive attitude towards their personal life were recruited. Subjects were divided in two group (24 patients each) and blindly supplemented for 2 month with: a) LD-1227 400mg or b) placebo. A third group of healthy non-stressed subjects was used as well. Blood samples were taken before and after the supplementation period. Unstimulated saliva was collected and tested for amylase while serum levels were used to measure BDNF. State Trait Anxiety Inventory (STAI), Pittsburgh Sleep Quality Index (PSQI) and psychological wellbeing assessment (PSWB) were measured too. Patients with Val66Met functional polymorphism of BDNF excluded those given their reported association with an impaired release of BDNF.Results: Results showed that, as compared to healthy, non-stressed individuals, stressed ones has a trend decrease of BDNF and this was significantly increased by LD 12-1227 supplementation and the same inverse phenomenon occurred to salivary amylase (p<0.05). No change was noted in the PSQI score but, either STAI or PSWB tests scored better in LD-1227 supplemented subjects.Conclusion: The present data suggest that LD-1227 is beneficially affecting neuromodulation and related symptoms during common stressful life conditions and may have the potential as tools in a neuroprotective clinical strategy.

The aim of this study is to test the activity of a marine bioactive compound containing high-purity caviar-derived DNA, collagen elastin and protein extracts from sturgeon (LD-1227, Caviarlieri, Laboratoires Dom, Switzerland) to exert neuroprotective properties in an experimental setting while also being potential triggers of neurogenesis in a separate in vitro study. Supplementation with high-DHA mixture of LD-1227 was applied for 30 days to stress model rats. Both supplementations significantly mitigated the histological brain damage when analyzing hippocampal subregions and corticosterone level. However, LD-1227 was most significantly efficient in preventing SOD, Catalase and ascorbic acid decrease in brain tissue. Both supplementations stimulated neurogenesis in vitro and neuron markers in particular but og olygodendrocyte markers and glia increased only in LD-1227-enriched medium. Taken together, these data suggest that LD-1227 is able to significantly protect the brain structure redox system to higher degree than DHA. Moreover, from in vitro study it appears that marine bioactive compound, through it wide array of small unsaturated fatty acids, phospholipids and neurotransmitter precursors, is likely to influence neuronal and glial lineage to act differently from a DHA-rich mixture.

The main object of this study is to examine the effect of Klamin®, a nutraceutical containing phenylethylamine, phycocyanins, mycosporine-like aminoacids and aphanizomenon flos aquae-phytochrome on the learning and memory ability, the oxidative status and cerebral erythropoietin and its receptor EPO/EPOR system in prematurely senescent (PS) mice. A total of 28 PS mice, selected according to a prior T-maze test, and 26 non-prematurely senescent mice (NPS) mice were chosen. PS animals were divided into 3 groups and followed for 4 weeks: A) normal chow diet; B) added with Klamin® at 20 mg/kg/day (low dose); C) added with Klamin® at 100mg/kg/day (high dose). A further group of NPS mice given either normal food (group D) or high dose Klamin® (group E) was also considered. The behavioral procedures of spatial learning ability (Morris test) showed that PS mice had significantly longer learning time as compared to their NPS counterpart (p<0.01), but this effect was prevented especially in mice supplemented with high-dose Klamin® (p<0.05) which improved performances in NPS mice (p<0.05). High-dose Klamin® supplementation restored the depleted total thiol concentration in the brain observed in PS mice while normalizing their increased malonildialdehyde level (p<0.05). Moreover, the high-dosage only caused a significant upregulation of EPO/EPOR system both in PS and in NPS animals (p<0.05). Taken together, these data suggest that this specific alga Klamath extract has considerable antioxidant and adaptogenic properties, also through a stimulatory effect of cerebral EPO/EPO system.

The aim of this study is to gain further insights into the possible nutraceutical effect on redox balance via thioredoxin (Trx) modulation and on the intrinsic susceptibility of monocytes to generate an inflammatory response. The study group consisted of thirty-two patients with compensated Child A-C, HCV-related cirrhosis. The patients were supplemented for 6 months with 6g/day of a certified fermented papaya preparation (FPP). Fifteen unsupplemented, age/gender-matched healthy subjects served as controls. The patients filled in a detailed diet-life style questionnaire, and blood samples were collected to test routine biochemistry, Trx, redox status (GSH, GSSG, GSH/GSSG ratio, 4-HNE and alpha-tocopherol). Moreover, isolated monocytes were tested for ex-vivo LPS-stimulated TNF-alpha production and TNF-alpha mRNA. As compared to control, patients with liver cirrhosis showed a significantly higher serum level of Trx. A significant correlation occurred with GSH/GSSG ratio in Child B and C patients. FPP supplementation brought about a significant reduction of Trx with levels comparable to the ones of healthy controls. Ten patients Child C (31.2 percent) showed borderline low levels of alpha-tocopherol while all cirrhotic patients, as a whole, showed a significantly abnormal redox balance. Supplementation with FPP did not modify alpha-tocopherol depletion but significantly improved redox balance parameters. Patients with liver cirrhosis showed a significantly upregulated TNF-alpha production in a time-dependent manner and this effect was more pronounced in more advanced stages of the disease and showed a significant correlation with alpha-tocopherol level. Supplementation with FPP significantly, although partially, downregulated TNF-alpha production from monocytes. Taken altogether, it would appear that the typical oxidative-inflammatory biochemical milieu of these patients is mirrored by a significant TNF-alpha upregulation at a monocyte level while a targeted nutraceutical might be a potentially amenable intervention to be part of validated scheduled treatments.

This study aims to determine the effects of different alkaline supplementations on high protein diet-induced abnormalities affecting bone metabolism in rats which were also undergoing physical exercise of moderate intensity. Sixty elderly Sprague-Dawley rats were randomly divided into four groups of 10 rats each and treated for 16 weeks as follows: baseline control group fed normal food (C); acidic high-protein diet supplemented group (chronic acidosis, CA group), bicarbonate-based alkaline formula (Basenpulver, Named, Italy) supplemented chronic acidosis (BB-CA) and citrate-based alkaline supplement (CB-CA). Throughout the supplementation period, rats were put on a treadmill training mimicking a moderate level of exercise. In the CA group, 24-hour urinary calcium (Ca) and phosphorus (P) excretion were increased over 30 percent (p<0.05 vs normal diet controls). However serum Ca was not significantly changed. Femural and tibial BMD and BMC was significantly decreased in the CA group (p<0.05) but both alkaline supplementations prevented such phenomenon (p<0.05 vs CA), without significant difference between the two formulations although the BB-CA group showed significantly more preserved trabecular bone volume (p<0.05 vs CB-CA group). An increased level of over 50 percent of urinary Dpd observed in the CA group (p<0.001) was reverted to normal by both supplementations (p<0.001 vs CA group). The same applied to urinary net acid excretion (p<001) with BB-supplementation performing better than CB-supplementation (p<0.05). Moreover, while the latter did not modify Nterminal telopeptide value, BB-supplementation significantly normalized this parameter (p<0.05 vs CA group) which exercise and acidic protein diet had modified (p<0.01 vs control diet). Overall, the present study shows that a bicarbonate-based alkaline formula, when administered to a dose amenable to clinical use, may significantly protect bone structure in exercising aged animals to a greater extent than a quali/quantitavely similar citrate-based formula.

There is increasing evidence that psychosocial stress can be viewed as a system-wide derangement of cellular homeostasis, with heightened oxidative stress and triggered proinflammatory mechanisms. The aim of this study is twofold: a) to replicate findings that psychological stress increases oxidative damage and b) to determine whether a fermented papaya preparation known to exert significant protective antioxidant properties could buffer such increases in nuclear DNA damage while also inducing epigenetic protective mechanisms. Twenty-eight sedentary men and women (age range: 28-52), who reported living a stressful lifestyle but with an overall positive attitude, were recruited for this study. Chronic diseases as well as severe burnout and use of drugs for anxiety constituted exclusion criteria. Subjects were supplemented for 1 month with 9 g/day (4.5 g twice a day) of a certified fermented papaya preparation. All subjects were given a stress and sleep quality questionnaire together with a diet and life style assessment. Blood was collected at 2 and 4 week, erythrocyte and leukocyte were separated to assess redox balance and heme oxygenase-1 (HO-1) gene expression while bilirubin oxidized metabolites (BOMs) were tested in the urine. Stressed individuals showed a significant abnormality of redox status with increased MDA of erythrocyte and increased level of 8-0HdG in leukocyte and BOMs excretion (p<0.05). Nutraceutical supplementation brought about a normalization of such values already at the 2 week observation (p<0.05) together with a significant upregulation of HO-1 (p<0.01). Taken together, the results of this study confirm that stressful occupational life per se, without any overt psychiatric illness, may be associated to increased oxidative stress. Supplementation with functional food affecting redox regulation may be part of the therapeutic armamentarium to be considered in this clinical setting.

Diabetic nephropathy (DN) is a severe and life-threatening complication of long-standing diabetes. As one of the main causes of end-stage renal disease, the prevention and treatment of DN in early stage, and the slowing down of DN progression are of utmost importance and are topics of several ongoing research studies. Nutraceuticals endowed with antioxidant-anti-inflammatory properties may offer an opportunity of integrative treatment for this condition. Male Wistar rats were randomly assigned to two groups. One group of rats (diabetic group) received a single tail-vein injection of STZ compound (50 mg/kg) under light anaesthesia. A protective dose of 0.5 ml of 5 percent dextrose was given intraperitoneally 30 min before the administration of STZ. One diabetic group was fed a normal pellet diet (group A) while group B was fed the diet added with DTS (panax pseudoginseng, eucommia ulmoides), (Kyotsu Jigyo, Tokyo, Japan) in the proportion of 50/25 (percent weight/weight), at the dose of 50 mg/kg/day throughout the experimental period. At the end of 8 weeks, 24-hour urine was collected for the measurement of the albumin concentration: blood samples were collected for serum biochemistry and the rats were sacrificed for kidney measurement of oxidative stress and histomorphological features. Nephrin and Macrophage Chemoattractant Protein-1 (MCP-1) gene expression were also assessed by fluorescence real-time quantitative PCR after RNA extraction and cDNA synthesis. STZ-treated animals showed significantly increased in lipid peroxidation in the kidney and in proteinuria. DTS supplementation did not affect plasma glucose but significantly decreased malonyldialdehyde (MDA) plasma level and the overall redox parameters together with a partial mitigation of proteinuria. Histological analysis showed also that DTS significantly reduced the glomerular volume together with glomerulosclerosis and interstitial fibrosis score (p less than 0.05), the latter two being correlated to proteinuria (p less than 0.05). DTS supplementation also enabled a reduction of diabetes-induced decrease of nephrin mRNA expression and a 67 percent reduction of MCP-1 mRNA up-regulation (p less than 0.01). Taken altogether, these data show that, besides the mandatory control of glycemia, intervention with a nutraceutical with antioxidant and anti-inflammatory properties may have beneficial effects when integrated in the mainstream of the therapeutic regimen.

This study aims to determine the effects of a high protein diet and alkaline supplementation on bone metabolic turnover in rats. Eight-week-old male Sprague-Dawley rats were investigated by bone status, including bone mineral density (BMD) and biomechanical markers from blood and urine. Thirty rats were randomly divided into three groups and treated for 8 weeks as follows: baseline control group (n. 10, C), high-protein supplemented diet group (n. 10, chronic acidosis, CA group) and supplemented chronic acidosis (n.10, SCA). Diet-treated rats were fed an acidic high-protein diet and the supplementation consisted in a modified alkaline formula (Basenpulver, NaMed, Italy). At the end of the experimental period, the rats were sacrificed, blood samples were drawn and femur and tibia were removed for analysis of bone mineral density (BMD) by dual energy X-ray absorptiometry (DEXA). In the CA group, 24-hour urinary calcium (Ca) and phosphorus (P) excretion were increased 2.1-fold (p<0.05 vs normal diet controls) as well as kidney weight. However, serum Ca and P concentration, as well as urinary Dpd excretion were not significantly changed. Femural and tibial BMD was significantly decreased in the CA group (p<0.05), but alkaline supplementation prevented such phenomenon (p<0.05 vs CA). These results suggest that blood Ca and P concentrations in chronic acidosis condition during the 12-week supplementation might be maintained by hypercalciuria and hyperphosphaturia at the expenses of bone structure. However, modified alkaline supplementation is able to prevent such derangements.

Ovariectomized Wistar rats received orally 15 mg/kg of a phytoestrogen compound (genistein, daidzein, glycitein, black cohosh, angelica sin., licorice, vitex agnus) for 2 weeks to test its ability to modulate inflammatory microglia response. Microglial proliferation was tested by trypan blue and by absorbance. Serial supernatant sampling was performed for 24 h to check TNF-alpha, IL-beta, IL-6, and TGF-beta. LPS caused a time course increase of all cytokines, with IL-beta and TNF-alpha peaking at the 12th hour, whereas IL-6 and TGF-beta peaked at the 24 h observation. Rats fed with the phytoestrogen displayed a significantly lower level of proinflammatory cytokines and a higher level of TGF-beta, as shown also by Western blot analysis. This finding may offer promise in the field of nutraceutical intervention.

Mutations in the presenilin 1 ( PS1) gene are responsible for the early onset of familial Alzheimer disease ( FAD). Accumulating evidence shows that PS1 is involved in gamma-secretase activity and that FAD-associated mutations of PS1 commonly accelerate A beta(1-42) production, which causes Alzheimer disease ( AD). Recent studies suggest, however, that PS1 is involved not only in A beta production but also in other processes that lead to neurodegeneration. To better understand the causes of neurodegeneration linked to the PS1 mutation, we analyzed the development of tau pathology, another key feature of AD, in PS1 knock-in mice. Hippocampal samples taken from FAD mutant ( I213T) PS1 knock-in mice contained hyperphosphorylated tau that reacted with various phosphodependent tau antibodies and with Alz50, which recognizes the conformational change of PHF tau. Some neurons exhibited Congo red birefringence and Thioflavin T reactivity, both of which are histological criteria for neurofibrillary tangles ( NFTs). Biochemical analysis of the samples revealed SDS-insoluble tau, which under electron microscopy examination, resembled tau fibrils. These results indicate that our mutant PS1 knock-in mice exhibited NFT-like tau pathology in the absence of A beta deposition, suggesting that PS1 mutations contribute to the onset of AD not only by enhancing A beta(1-42) production but by also accelerating the formation and accumulation of filamentous tau.

The amyloid beta-protein (Abeta) ending at 42 plays a pivotal role in Alzheimer's disease (AD). We have reported previously that intracellular Abeta42 is associated with neuronal apoptosis in vitro and in vivo. Here, we show that intracellular Abeta42 directly activated the p53 promoter, resulting in p53-dependent apoptosis, and that intracellular Abeta40 had a similar but lesser effect. Moreover, oxidative DNA damage induced nuclear localization of Abeta42 with p53 mRNA elevation in guinea-pig primary neurons. Also, p53 expression was elevated in brain of sporadic AD and transgenic mice carrying mutant familial AD genes. Remarkably, accumulation of both Abeta42 and p53 was found in some degenerating-shape neurons in both transgenic mice and human AD cases. Thus, the intracellular Abeta42/p53 pathway may be directly relevant to neuronal loss in AD. Although neurotoxicity of extracellular Abeta is well known and synaptic/mitochondrial dysfunction by intracellular Abeta42 has recently been suggested, intracellular Abeta42 may cause p53-dependent neuronal apoptosis through activation of the p53 promoter; thus demonstrating an alternative pathogenesis in AD.

Previous studies have suggested that heparan sulfate proteoglycans (HSPGs) play a role in deposition of beta-amyloid protein (Abeta) in the Alzheimer's disease (AD) brain. In the present study, we demonstrated that glypican-1 can bind fibrillar Abeta, and the binding is mainly mediated by heparan sulfate (HS) chains. Further analysis revealed that glypican-1 is the major HSPG localized in detergent-insoluble glycosphingolipid-enriched (DIG) domains where all machineries for Abeta production exist and Abeta is accumulated as monomeric and oligomeric forms. Immunohistochemical studies demonstrated that glypican-1 is localized in primitive plaques as well as classic plaques. Moreover, overexpression of glypican-1 and amyloid precursor protein in SH-SY5Y cells resulted in reduced cell viability and made cells more susceptible to thapsigargin-induced stress and Abeta toxicity. The results raise the possibility that glypican-1 interacts with oligomerized or polymerized Abeta in such a specific compartment as DIG, resulting not only in amyloid deposition in senile plaques of AD brain, but also in accelerating neuronal cell death in response to stress and Abeta.

Neurofibrillary tangles (NFTs), which are composed of hyperphosphorylated and ubiquitylated tau, are exhibited at regions where neuronal loss occurs in neurodegenerative diseases; however, the mechanisms of NFT formation remain unknown. Molecular studies of frontotemporal dementia with parkinsonism-17 demonstrated that increasing the ratio of tau with exon 10 insertion induced fibrillar tau accumulation. Here, we show that carboxyl terminus of Hsc70-interacting protein (CHIP), a U-box protein, recognizes the microtubule-binding repeat region of tau and preferentially ubiquitylates four-repeat tau compared with three-repeat tau. Overexpression of CHIP induced the prompt degradation of tau, reduced the formation of detergent-insoluble tau and inhibited proteasome inhibitor-induced cell death. NFT bearing neurons in progressive supranuclear palsy, in which four-repeat tau is a component, showed the accumulation of CHIP. Thus, CHIP is a ubiquitin ligase for four-repeat tau and maintains neuronal survival by regulating the quality control of tau in neurons.

Alzheimer's disease (AD) may be caused by toxic aggregates formed from amyloid-beta (Abeta) peptides. By using Thioflavin T, a dye that specifically binds to beta-sheet structures, we found that highly toxic forms of Abeta-aggregates were formed at the initial stage of fibrillogenesis, which is consistent with recent reports on Abeta oligomers. Formation of such aggregates depends on factors that affect both nucleation and elongation. As reported previously, addition of Abeta42 systematically accelerated the nucleation of Abeta40, most likely because of the extra hydrophobic residues at the C terminus of Abeta42. At Abeta42-increased specific ratio (Abeta40: Abeta42 = 10: 1), on the other hand, not only accelerated nucleation but also induced elongation were observed, suggesting pathogenesis of early-onset AD. Because a larger proportion of Abeta40 than Abeta42 was still required for this phenomenon, we assumed that elongation does not depend only on hydrophobic interactions. Without any change in the C-terminal hydrophobic nature, elongation was effectively induced by mixing wild type Abeta40 with Italian variant Abeta40 (E22K) or Dutch variant (E22Q). We suggest that Abeta peptides in specific compositions that balance hydrophilic and hydrophobic interactions promote the formation of toxic beta-aggregates. These results may introduce a new therapeutic approach through the disruption of this balance.

We have examined Notch3 mutations in patients with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) whose samples were submitted to us in Japan. The subjects were composed of 21 Japanese, 1 Iranian, 1 Korean and 1 Canadian families. Mutations in the Notch3 gene were found in 7 of 24 families examined. These were R133C in two unrelated Japanese families, and R213K, C174F and R169C in each Japanese family. In addition, we have found R90C in an Iranian family and C174R in a Korean family. Thus, contribution of Notch3 gene mutations is less than one fourth of Japanese CADASIL families, suggesting the existence of other causative genes in CADASIL. It is also of interest to know that Notch3 mutant CADASIL exists in other Asian countries. We next examined the localization of Notch3 protein in the tissue by immunohistochemistry. It was restricted to the wall of arterioles in the brain and other organs. In the brain, there was no difference in the staining pattern among arterioles in the cortex, white matter and meninges. The staining was negative in the venule and capillaries as well as in neurons and glial cells. From the staining pattern, it was recognized to be expressed in the vascular smooth muscle cells in the adult tissue. In an autopsy case with R213K mutation, we could see numerous cerebral infarcts and arteriole wall degeneration with deposits of granular osmiophilic material (GOM). However, it is interesting to note that occlusion of arterioles was rarely observed and the GOM was negative for Notch3 staining. These findings suggest that hemodynamic abnormalities due to smooth muscle cell degeneration may be important in the pathogenesis of CADASIL.

We reviewed Alzheimer's cases with spastic paresis and cotton wool type plaques in five Japanese and nine Caucasian cases. Most were early onset familial Alzheimer's disease with presenilin 1 mutations. The cotton wool type plaques were related to extremely high production of A beta 42, due mainly to presenilin 1 mutations and low immune responses. Cotton wool plaques were numerous in the entire central nervous system, including basal ganglia, brainstem and even in spinal cord. Cotton wool type plaques were composed of slightly electron dense synaptic structures, but amyloid fibrils were rarely found. Such a high accumulation of A beta 42 may cause degeneration of the pyramidal tract and basal ganglia from an early stage of Alzheimer's disease.