Silymarin's Inhibition and Treatment Effects for Alzheimer's Disease.

As a longstanding problem, Alzheimer's disease (AD) has stymied researchers in the medical field with its increasing incidence and enormous treatment difficulty. Silymarin has always been valued by researchers for its good efficacy and safety in treating liver disease. Recent studies have shown that silymarin also has good pharmacological activity in the nervous system, especially for the treatment of AD. Silymarin can control the production of Aß by inhibiting the precursor substance of Aß (ß-amyloid precursor protein), and it can inhibit the polymerization of Aß. Silymarin can also increase the acetylcholine content in the nervous system by inhibiting cholinesterase activity. At the same time, it also has the effect of resisting oxidative stress and the inflammatory response of the nervous system. These pharmacological activities contribute to the inhibition of the onset of AD. The good efficacy of silymarin on AD and its high safety and availability give it huge potential for the treatment of AD.

Paradigm shift from diagnosing patients based on common symptoms to categorizing patients into subtypes with different pathogenic mechanisms to guide treatment for Alzheimer's disease.

Alzheimer's disease (AD) is a major cause of dementia in the elderly, and the number of AD patients is rapidly growing as life expectancy increases. However, disease-modifying drugs are not yet available. According to the amyloid hypothesis, disease onset is triggered by aggregation and accumulation of amyloid-ß peptide, followed by the formation of neurofibrillary tangles composed of hyperphosphorylated tau, and synaptic loss/neuronal cell death leading to dementia. Based on this hypothesis, various clinical trials for treatment of AD have been conducted, but most were discontinued due to failure to achieve cognitive improvement or appearance of adverse effects. Here we discuss the reasons for the failure of these trials. We suggest that biomarkers of specific, distinct molecular mechanisms of amyloidogenesis should be developed concomitantly with disease-modifying drugs (the so-called companion diagnosis) to aid the proper design of clinical trials, as well as to enable personalized treatment of individual AD patients.

Multiple mechanisms of age-dependent accumulation of amyloid beta protein in rat brain: Prevention by dietary supplementation with N-acetylcysteine, α-lipoic acid and α-tocopherol.

The aged brain may be used as a tool to investigate altered metabolism of amyloid beta protein (Aß42) that may have implications in the pathogenesis of Alzheimer's disease (AD). In the present study, we have observed a striking increase in the amyloid precursor protein (APP) level in the brain cortex of aged rats (22-24 months) along with a mild but statistically significant increase in the level of APP mRNA. Moreover, the activity of ß secretase is elevated (nearly 55%) and that of neprilysin diminished (48%) in brain cortex of aged rats compared to that in young rats (4-6 months). All these changes lead to a markedly increased accumulation of Aß42 in brain cortical tissue of aged rats. Long-term dietary supplementation of rats with a combination of N-acetylcysteine, α-lipoic and α-tocopherol from 18 months onwards daily till the sacrifice of the animals by 22-24 months, attenuates the age-related alterations in amyloid beta metabolism. In separate experiments, a significant impairment of spatial learning and memory has been observed in aged rats, and the phenomenon is remarkably prevented by the dietary supplementation of the aged animals by the same combination of N-acetylcysteine, α-lipoic acid and α-tocopherol. The results call for further explorations of this combination in suitable animal models in ameliorating AD related brain deficits.

Anti-Aß single-chain variable fragment antibodies exert synergistic neuroprotective activities in Drosophila models of Alzheimer's disease.

Both active and passive immunotherapy protocols decrease insoluble amyloid-ß42 (Aß42) peptide in animal models, suggesting potential therapeutic applications against the main pathological trigger in Alzheimer's disease (AD). However, recent clinical trials have reported no significant benefits from humanized anti-Aß42 antibodies. Engineered single-chain variable fragment antibodies (scFv) are much smaller and can easily penetrate the brain, but identifying the most effective scFvs in murine AD models is slow and costly. We show here that scFvs against the N- and C-terminus of Aß42 (scFv9 and scFV42.2, respectively) that decrease insoluble Aß42 in CRND mice are neuroprotective in Drosophila models of Aß42 and amyloid precursor protein neurotoxicity. Both scFv9 and scFv42.2 suppress eye toxicity, reduce cell death in brain neurons, protect the structural integrity of dendritic terminals in brain neurons and delay locomotor dysfunction. Additionally, we show for the first time that co-expression of both anti-Aß scFvs display synergistic neuroprotective activities, suggesting that combined therapies targeting distinct Aß42 epitopes can be more effective than targeting a single epitope. Overall, we demonstrate the feasibility of using Drosophila as a first step for characterizing neuroprotective anti-Aß scFvs in vivo and identifying scFv combinations with synergistic neuroprotective activities.

Docosahexaenoic acid reduces ER stress and abnormal protein accumulation and improves neuronal function following traumatic brain injury.

In this study, we investigated the development of endoplasmic reticulum (ER) stress after traumatic brain injury (TBI) and the efficacy of post-TBI administration of docosahexaenoic acid (DHA) in reducing ER stress. TBI was induced by cortical contusion injury in Sprague-Dawley rats. Either DHA (16 mg/kg in DMSO) or vehicle DMSO (1 ml/kg) was administered intraperitoneally at 5 min after TBI, followed by a daily dose for 3-21 d. TBI triggered sustained expression of the ER stress marker proteins including phosphorylated eukaryotic initiation factor-2α, activating transcription factor 4, inositol requiring kinase 1, and C/EBP homologous protein in the ipsilateral cortex at 3-21 d after TBI. The prolonged ER stress was accompanied with an accumulation of abnormal ubiquitin aggregates and increased expression of amyloid precursor protein (APP) and phosphorylated tau (p-Tau) in the frontal cortex after TBI. The ER stress marker proteins were colocalized with APP accumulation in the soma. Interestingly, administration of DHA attenuated all ER stress marker proteins and reduced the accumulation of both ubiquitinated proteins and APP/p-Tau proteins. In addition, the DHA-treated animals exhibited early recovery of their sensorimotor function after TBI. In summary, our study demonstrated that TBI induces a prolonged ER stress, which is positively correlated with abnormal APP accumulation. The sustained ER stress may play a role in chronic neuronal damage after TBI. Our findings illustrate that post-TBI administration of DHA has therapeutic potentials in reducing ER stress, abnormal protein accumulation, and neurological deficits.

In vitro BACE1 inhibitory activity of geraniin and corilagin from Geranium thunbergii.

Generation of amyloid ß peptide through the proteolytic process of amyloid precursor protein by ß-secretase and γ-secretase is a main casual factor of Alzheimer's disease, since amyloid ß peptide is a major and crucial component of senile plaques in Alzheimer's disease brains. In the process of searching for ß-secretase inhibitors from natural resources, the EtOAc soluble fraction of Geranium thunbergii exhibited significant ß-secretase inhibitory activity. Two compounds, geraniin and corilagin, isolated from the most active EtOAc fraction of G. thunbergii, exhibited predominant inhibition against ß-secretase with IC50 values of 4.0 × 10⁻6 M and 3.4 × 10⁻5 M, respectively. Dixon plot of geraniin and corilagin demonstrated that the ß-secretase inhibition was noncompetitive with the substrate, thus clearly suggesting that these compounds might bind either to the ß-secretase subsites or to another regulatory domain with Ki values of 2.8 × 10⁻6 M and 7.9 × 10⁻5 M, respectively. Both compounds exhibited no significant inhibition against α-secretase and other serine proteases including trypsin and chymotrypsin, showing that they were relatively specific and selective inhibitors of ß-secretase. These novel findings suggest that geraniin and corilagin from G. thunbergii may be effective therapeutic agents for further drug development in Alzheimer's disease.

Supplementation of Convolvulus pluricaulis attenuates scopolamine-induced increased tau and amyloid precursor protein (AßPP) expression in rat brain.

AIM: Scopolamine is known to produce amnesia due to blockade of the cholinergic neurotransmission. The present study investigated the potential of Convolvulus pluricaulis (CP) to attenuate scopolamine (2 mg/kg, i.p) induced increased protein and mRNA levels of tau, amyloid precursor protein (AßPP), amyloid ß (Aß) levels and histopathological changes in rat cerebral cortex. MATERIALS AND METHODS: The study was conducted on male Wistar rats (250 ± 20 g) divided into four groups of eight animals each. Groups 1 and 2 served as controls receiving normal saline and scopolamine for 4 weeks, respectively. Group 3 received rivastigmine (standard) and group 4 received aqueous extract of CP simultaneously with scopolamine. Western blot and RT-PCR analysis were used to evaluate the levels of protein and mRNA of amyloid precursor protein (AßPP) and tau in rat cortex and ELISA was used to measure the amyloid ß (Aß) levels. Histopathology was also performed on cortical section of all groups. RESULT: Oral administration of CP extract (150 mg/kg) to scopolamine treated rats reduced the increased protein and mRNA levels of tau and AßPP levels followed by reduction in Aß levels compared with scopolamine treated group. The potential of extract to prevent scopolamine neurotoxicity was reflected at the microscopic level as well, indicative of its neuroprotective effects. CONCLUSION: CP treatment alleviated neurotoxic effect of scopolamine reflects its potential as potent neuroprotective agent.

Huperzine A alleviates synaptic deficits and modulates amyloidogenic and nonamyloidogenic pathways in APPswe/PS1dE9 transgenic mice.

Huperzine A (HupA) is a potent acetylcholinesterase inhibitor (AChEI) used in the treatment of Alzheimer's disease (AD). Recently, HupA was shown to be active in modulating the nonamyloidogenic metabolism of ß-amyloid precursor protein (APP) in APP-transfected human embryonic kidney cell line (HEK293swe). However, in vivo research concerning the mechanism of HupA in APP transgenic mice has not yet been fully elucidated. The present study indicates that the loss of dendritic spine density and synaptotagmin levels in the brain of APPswe/presenilin-1 (PS1) transgenic mice was significantly ameliorated by chronic HupA treatment and provides evidence that this neuroprotection was associated with reduced amyloid plaque burden and oligomeric ß-amyloid (Aß) levels in the cortex and hippocampus of APPswe/PS1dE9 transgenic mice. Our findings further demonstrate that the amelioration effect of HupA on Aß deposits may be mediated, at least in part, by regulation of the compromised expression of a disintegrin and metalloprotease 10 (ADAM10) and excessive membrane trafficking of ß-site APP cleavage enzyme 1 (BACE1) in these transgenic mice. In addition, extracellular signal-regulated kinases 1/2 (Erk1/2) phosphorylation may also be partially involved in the effect of HupA on APP processing. In conclusion, our work for the first time demonstrates the neuroprotective effect of HupA on synaptic deficits in APPswe/PS1dE9 transgenic mice and further clarifies the potential pharmacological targets for this protective effect, in which modulation of nonamyloidogenic and amyloidogenic APP processing pathways may be both involved. These findings may provide adequate evidence for the clinical and experimental benefits gained from HupA treatment.

Ginsenoside Rg1 attenuates ß-amyloid generation via suppressing PPARγ-regulated BACE1 activity in N2a-APP695 cells.

The level of ß-site APP-cleaving enzyme 1 (BACE1) has been documented to increase in the brains of patients with Alzheimer's disease, which has resulted in elevation of ß-amyloid (Aß) peptides. As a transcription factor binding site of the BACE1 promoter, peroxisome proliferator-activated receptor-γ (PPARγ) response element regulates the activity of the BACE1 promoter activity, indicating that PPARγ may become a potential target for Alzheimer's disease treatment. Recent studies have demonstrated that ginsenoside Rg1 which is an effective component of extracts of ginseng can prevent memory loss and improve cognitive function in a variety of animal models. However, the underlying mechanism remains unclear. In the present study, we found that Rg1 decreased the levels of Aß1₋40 and Aß1₋42 secreted in N2a-APP695 cells. The expression levels of both BACE1 mRNA and protein as well as ß-CTFs, a cleavaged C-terminal fragment of APP by BACE1, were reduced in cells treated with Rg1. Moreover, Rg1 treatment led to a translocation of PPARγ from cytoplasm to nuclear. Intriguingly, Rg1, like pioglitazone (a PPARγ agonist), suppressed BACE1 activity in N2a-APP695 cells, while its effect on BACE1 activity was attenuated by GW9662 (a PPARγ antagonist). These results indicate that Rg1 may be a PPARγ agonist to enhance the binding of nuclear PPARγ to the BACE1 promoter, which may in turn inhibit the transcription and translation of BACE1, suppress the activity of BACE1, and ultimately attenuate Aß generation. Therefore, ginsenoside Rg1 may serve as a promising agent in modulating Aß-related pathology in Alzheimer's disease.

Design, synthesis and structure-activity relationship of novel [3.3.1] bicyclic sulfonamide-pyrazoles as potent γ-secretase inhibitors.

The structure-activity relationship (SAR) of a novel, potent and metabolically stable series of sulfonamide-pyrazoles that attenuate ß-amyloid peptide synthesis via γ-secretase inhibition is detailed herein. Sulfonamide-pyrazoles that are efficacious in reducing the cortical Aßx-40 levels in FVB mice via a single PO dose, as well as sulfonamide-pyrazoles that exhibit selectivity for inhibition of APP versus Notch processing by γ-secretase, are highlighted.

Berberine: a potential multipotent natural product to combat Alzheimer's disease.

With the accelerated aging of human society Alzheimer's disease (AD) has become one of the most threatening diseases in the elderly. However, there is no efficient therapeutic agent to combat AD. Berberine is a natural isoquinoline alkaloid that possesses a wide range of pharmacological effects. In the present paper, we review the multiple activities of berberine, including antioxidant, acetylcholinesterase and butyrylcholinesterase inhibitory, monoamine oxidase inhibitory, amyloid-b peptide level-reducing and cholesterol-lowering activities, which suggest that berberine may act as a promising multipotent agent to combat AD.

Rapid and efficient screening of Alzheimer's disease ß-amyloid inhibitors using label-free gold nanoparticles.

Herein we report that a visual, label-free gold nanoparticle-based assay for rapid and efficient screening of Alzheimer's disease ß-amyloid inhibitors.

Docosahexaenoic acid reduces amyloid-ß(1-42) secretion in human AßPP-transfected CHO-cells by mechanisms other than inflammation related to PGE2.

The effect of supplementation with the omega 3 polyunsaturated fatty acid (n3 PUFA) docosahexaenoic acid (DHA) on membrane composition and amyloid-ß1₋42 (Aß42) secretion was studied in human amyloid-ß protein precursor-transfected Chinese Hamster Ovary (CHO) cells. Twenty-four hour incubation with a range of DHA concentrations resulted in a dose-dependent increase in membrane DHA and eicosapentaenoic acid content and a decrease in arachidonic acid content. In addition, DHA supplementation caused a dose-dependent reduction in the secreted Aß42 levels and resulted in a 4-8 fold decrease in extracellular prostaglandin E2 (PGE2) levels. Tocopherol, which was added to DHA to prevent oxidation, may have contributed to the effect of DHA, since it slightly decreased extracellular Aß42 and PGE2 levels when given alone. The addition of selective COX2 inhibitors Celebrex and curcumin to the culture medium resulted in a significant and comparable inhibition of PGE2 release, but did not inhibit Aß42 secretion, and even significantly increased Aß42 production in this cell system. Together, the present data show that, whereas both DHA and COX2 inhibitors may reduce PGE2 production, only DHA in the presence of tocopherol significantly reduced Aß42 production and concurrently changed membrane lipid composition in CHO cells. It is concluded that in this in vitro setting DHA reduced Aß42 secretion through membrane-related, but not PGE2-related mechanisms.

Antioxidants have a rapid and long-lasting effect on neuritic abnormalities in APP:PS1 mice.

Senile plaques are a major pathological hallmark of Alzheimer's disease (AD). Compelling evidence suggests that senile plaques lead to structural alterations of neuronal processes and that local toxicity may be mediated by increased oxidative stress. Anti-oxidant therapy can alleviate the neuronal abnormalities in APP mice, but the time-course of this beneficial effect is unknown. We used multiphoton microscopy to assess in vivo the characteristics of antioxidant treatment on senile plaques and neurites in AD model mice (APPswe/PS1dE9). We observed that α-phenyl-N-tert-butyl nitrone (PBN), Ginkgo biloba extract (EGb 761) and Trolox had no effect on the size of existing senile plaques. However, all anti-oxidants had a straightening effect on curved neurites. This effect was detected as soon as 4 days after commencing the treatment, and was maintained after 1 month of daily treatment, with no further increase in the effect. The straightening of neurites persisted 15 days after stopping the treatment. These data indicate that neuronal plasticity is fast and still active in adult animals, and suggest that amelioration of the neuritic distortions associated with senile plaques with antioxidants is both rapid and long lasting.

Identification of novel small molecule inhibitors of amyloid precursor protein synthesis as a route to lower Alzheimer's disease amyloid-beta peptide.

A wealth of independent research with transgenic mice, antibodies, and vaccines has pointed to a causative role of the amyloid-beta peptide (A beta) in Alzheimer's disease (AD). Based on these and earlier associative studies, A beta represents a promising target for development of therapeutics focused on AD disease progression. Interestingly, a cholinesterase inhibitor currently in clinical trials, phenserine, has been shown to inhibit production of both amyloid precursor protein (APP) and A beta. We have shown that this inhibition occurs at the post-transcriptional level with a specific blocking of the synthesis of APP relative to total protein synthesis (Shaw et al., 2001). However, the dose of phenserine necessary to block APP production is far higher than that needed to elicit its anticholinesterase activity, and it is these latter actions that are dose limiting in vivo. The focus of this study was to screen 144 analogs of phenserine to identify additional small molecules that inhibit APP protein synthesis, and thereby A beta production, without possessing potent acetylcholinesterase (AChE) inhibitory activity. An enzyme-linked immunosorbent assay was used to identify analogs capable of suppressing APP production following treatment of human neuroblastoma cells with 20 muM of compound. Eight analogs were capable of dose dependently reducing APP and A beta production without causing cell toxicity in further studies. Several of these analogs had little to no AChE activities. Translation of APP and A beta actions to mice was demonstrated with one agent. They thus represent interesting lead molecules for assessment in animal models, to define their tolerance and utility as potential AD therapeutics.

FDA-preapproved drugs targeted to the translational regulation and processing of the amyloid precursor protein.

The 5' untranslated region (5'UTR) of the transcript encoding the Alzheimer's amyloid precursor protein (APP) is a key regulatory sequence that determines the amount of intracellular APP holoprotein present in brain derived cells. Using neuroblastoma cells (SY5Y) we developed a transfection based screen of a library of FDA drugs to identify compounds that limited APP luciferase reporter expression translated from the APP 5'UTR. Paroxetine (Paxil trade mark ), dimercaptopropanol, phenserine, desferrioxamine, tetrathiolmobdylate, and azithromycin were six leads that were subsequently found to also suppress APP holoprotein levels or to alter APP cleavage (azithromycin). Since APP holoprotein levels are proportionate to Abeta peptide output in many systems we tested the efficacy of paroxetine and dimercaptopropanol to limit Abeta secretion as measured by ELISA assays. Paroxetine and dimercaptopropanol limited Abeta peptide secretion from lens epithelial cells (B3 cells). Interestingly, paroxetine changed the steady-state levels of transferrin receptor mRNAs. These data suggested that this serotonin reuptake inhibitor (SSRI) provided extra pharmacological action to chelate interacellular iron or change the intracellular iron distribution. An altered iron distribution would be predicted to indirectly limit APP holoprotein expression and Abeta peptide secretion.