Diplazium esculentum (Retz.) Sw. reduces BACE-1 activities and amyloid peptides accumulation in Drosophila models of Alzheimer's disease.

Alzheimer's disease (AD), one type of dementia, is a complex disease affecting people globally with limited drug treatment. Thus, natural products are currently of interest as promising candidates because of their cost-effectiveness and multi-target abilities. Diplazium esculentum (Retz.) Sw., an edible fern, inhibited acetylcholinesterase in vitro, inferring that it might be a promising candidate for AD treatment by supporting cholinergic neurons. However, evidence demonstrating anti-AD properties of this edible plant via inhibiting of neurotoxic peptides production, amyloid beta (Aß), both in vitro and in vivo is lacking. Thus, the anti-AD properties of D. esculentum extract both in vitro and in Drosophila models of Aß-mediated toxicity were elucidated. Findings showed that an ethanolic extract exhibited high phenolics and flavonoids, contributing to antioxidant and inhibitory activities against AD-related enzymes. Notably, the extract acted as a BACE-1 blocker and reduced amyloid beta 42 (Aß42) peptides in Drosophila models, resulting in improved locomotor behaviors. Information gained from this study suggested that D. esculentum showed potential for AD amelioration and prevention. Further investigations in vertebrates or humans are required to determine the effective doses of D. esculentum against AD, particularly via amyloidogenic pathway.

Label-free photothermal disruption of cytotoxic aggregates rescues pathology in a C. elegans model of Huntington's disease.

Aggregation of proteins is a prominent hallmark of virtually all neurodegenerative disorders including Alzheimer's, Parkinson's and Huntington's diseases. Little progress has been made in their treatment to slow or prevent the formation of aggregates by post-translational modification and regulation of cellular responses to misfolded proteins. Here, we introduce a label-free, laser-based photothermal treatment of polyglutamine (polyQ) aggregates in a C. elegans nematode model of huntingtin-like polyQ aggregation. As a proof of principle, we demonstrated that nanosecond laser pulse-induced local photothermal heating can directly disrupt the aggregates so as to delay their accumulation, maintain motility, and extend the lifespan of treated nematodes. These beneficial effects were validated by confocal photothermal, fluorescence, and video imaging. The results obtained demonstrate that our theranostics platform, integrating photothermal therapy without drugs or other chemicals, combined with advanced imaging to monitor photothermal ablation of aggregates, initiates systemic recovery and thus validates the concept of aggregate-disruption treatments for neurodegenerative diseases in humans.

Identification of Crocin as a New hIAPP Amyloid Inhibitor via a Simple Yet Highly Biospecific Screening System.

Amylin (hIAPP) amyloid formation plays an important role in the pathogenesis of type 2 diabetes (T2D), which makes it a promising therapeutic target for T2D. In this study, we established a screening tool for identifying chemicals affecting hIAPP amyloid formation based on a reported genetic tool, which constantly tracks protein aggregates in Saccharomyces cerevisiae. In order to obtain the hIAPP with better aggregation ability, the gene of hIAPP was tandemly ligated to create 1×, 2×, 4× or 6×-hIAPP expressing strains. By measuring the cell density and fluorescence intensity of green fluorescent protein (GFP) regulated by the aggregation status of hIAPP, it was found that four intramolecular ligated hIAPP (4×hIAPP) could form obvious amyloids with mild toxicity. The validity and reliability of the screening tool were verified by testing six reported hIAPP inhibitors, including curcumin, epigallocatechin gallate and so on. Combined with surface plasmon resonance (SPR) and the screening tool, which could be a screening system for hIAPP inhibitors, we found that crocin specifically binds to hIAPP and acts inhibit amyloid formation of hIAPP. The effect of crocin was further confirmed by Thioflavin T (ThT) fluorescence and transmission electron microscopy (TEM) analysis. Thus, a screening system for hIAPP amyloid inhibitors and a new mechanism of crocin on anti-T2D were obtained as a result of this study.

The translocator protein ligands as mitochondrial functional modulators for the potential anti-Alzheimer agents.

Small molecule modulators of mitochondrial function have been attracted much attention in recent years due to their potential therapeutic applications for neurodegenerative diseases. The mitochondrial translocator protein (TSPO) is a promising target for such compounds, given its involvement in the formation of the mitochondrial permeability transition pore in response to mitochondrial stress. In this study, we performed a ligand-based pharmacophore design and virtual screening, and identified a potent hit compound, 7 (VH34) as a TSPO ligand. After validating its biological activity against amyloid-ß (Aß) induced mitochondrial dysfunction and in acute and transgenic Alzheimer's disease (AD) model mice, we developed a library of analogs, and we found two most active compounds, 31 and 44, which restored the mitochondrial membrane potential, ATP production, and cell viability under Aß-induced mitochondrial toxicity. These compounds recovered learning and memory function in acute AD model mice with improved pharmacokinetic properties.

2,3,5,6-Tetramethylpyrazine protects retinal photoreceptors against endoplasmic reticulum stress by modulating ATF4-mediated inhibition of PRP aggregation.

Endoplasmic reticulum (ER) stress is a common threat to photoreceptors during the pathogenesis of chronic retinopathies and often results in irreversible visual impairment. 2,3,5,6-Tetramethylpyrazine (TMP), which possesses many beneficial pharmacological activities, is a potential drug that could be used to protect photoreceptors. In the present study, we found that the cellular growth rate of 661 W cells cultured under low glucose conditions was lower than that of control cells, while the G2/M phase of the cell cycle was longer. We further found that the mitochondrial membrane potential (ΔΨm) was lower and that ER stress factor expression was increased in 661 W cells cultured under low glucose conditions. TMP reversed these trends. Visual function and cell counts in the outer nuclear layer (ONL) were low and the TUNEL-positive rate in the ONL was high in a C3H mouse model of spontaneous retinal degeneration. Similarly, visual function was decreased, and the TUNEL-positive rate in the ONL was increased in fasted C57/BL6j mice compared with control mice. On the other hand, ER stress factor expression was found to be increased in the retinas of both mouse models, as shown by reverse transcription real-time PCR (RT-qPCR) and western blotting. TMP reversed the physiological and molecular biological variations observed in both mouse models, and ATF4 expression was enhanced again. Further investigation by using western blotting illustrated that the proportion of insoluble prion protein (PRP) versus soluble PRP was reduced both in vitro and in vivo. Taken together, these results suggest that TMP increased the functions of photoreceptors by alleviating ER stress in vitro and in vivo, and the intrinsic mechanism was the ATF4-mediated inhibition of PRP aggregation. TMP may potentially be used clinically as a therapeutic agent to attenuate the functional loss of photoreceptors during the pathogenesis of chronic retinopathies. KEY MESSAGES: • Already known: TMP is a beneficial drug mainly used in clinic to enhance organ functions, and the intrinsic mechanism is still worthy of exploring. • New in the study: We discovered that TMP ameliorated retinal photoreceptors function via ER stress alleviation, which was promoted by ATF4-mediated inhibition of PRP aggregation. • Application prospect: In prospective clinical practices, TMP may potentially be used in the clinic as a therapeutic agent to attenuate the photoreceptors functional reduction in chronic retinopathies.

Inhibition of Aß aggregates in Alzheimer's disease by epigallocatechin and epicatechin-3-gallate from green tea.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive accumulation of senile plaques, which are primarily composed of misfolded amyloid ß-peptide (Aß). Aß aggregates are believed to be a key factor in the pathogenesis of AD, affecting the nervous system in human body. The therapeutic potential of tea-derived polyphenolic compounds, (-)-epigallocatechin (EGC) and (-)-epicatechin-3-gallate (ECG), for AD was investigated by assessing their effects on the Cu2+/Zn2+-induced or self-assembled Aß40 aggregation using thioflavine T fluorescent spectrometry, inductively coupled plasma mass spectrometry, UV-Vis spectroscopy, transmission electron microscope, silver staining, immunohistochemistry, and immunofluorescence assays. EGC and ECG mildly bind to Cu2+ and Zn2+, and diminish the Cu2+- or Zn2+-induced or self-assembled Aß aggregates; they also modulate the Cu2+/Zn2+-Aß40 induced neurotoxicity on mouse neuroblastoma Neuro-2a cells by reducing the production of ROS. Metal chelating, hydrogen bonding or Van Der Waals force may drive the interaction between the polyphenolic compounds and Aß. The results demonstrate that green tea catechins EGC and ECG are able to alleviate the toxicity of Aß oligomers and fibrils. Particularly, ECG can cross the blood-brain barrier to reduce the Aß plaques in the brain of APP/PS1 mice, thereby protecting neurons from injuries. The results manifest the potential of green tea for preventing or ameliorating the symptoms of AD.

POSCAbilities: The Application of the Prion Organotypic Slice Culture Assay to Neurodegenerative Disease Research.

Prion diseases are fatal, transmissible neurodegenerative disorders whose pathogenesis is driven by the misfolding, self-templating and cell-to-cell spread of the prion protein. Other neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and Huntington's disease, share some of these prion-like features, with different aggregation-prone proteins. Consequently, researchers have begun to apply prion-specific techniques, like the prion organotypic slice culture assay (POSCA), to these disorders. In this review we explore the ways in which the prion phenomenon has been used in organotypic cultures to study neurodegenerative diseases from the perspective of protein aggregation and spreading, strain propagation, the role of glia in pathogenesis, and efficacy of drug treatments. We also present an overview of the advantages and disadvantages of this culture system compared to in vivo and in vitro models and provide suggestions for new directions.

Ability of selenium species to inhibit metal-induced Aß aggregation involved in the development of Alzheimer's disease.

Extracellular accumulation of amyloid beta peptide (Aß) is believed to be one of the main factors responsible for neurodegeneration in Alzheimer's disease (AD). Metals could induce Aß aggregation, by their redox activity or binding properties to amyloid ß fibrils, leading to their accumulation and deposition outside neurons. For this reason, metal chelation may have an acknowledged part to play in AD prevention and treatment. In the current work, the role of different selenium species, including selenium nanoparticles, in Aß aggregation, was studied by evaluating their metal-chelating properties and their ability both to inhibit metal-induced Aß1-42 aggregation fibrils and to disaggregate them once formed. Transition biometals such as Fe(II), Cu(II), and Zn(II) at 50 µM were selected to establish the in vitro models. The DPPH assay was used to determine the antioxidant capacity of the evaluated selenium species. Selenium nanoparticles stabilized with chitosan (Ch-SeNPs) and with both chitosan and chlorogenic acid polyphenol (CGA@ChSeNPs) showed the highest antioxidant properties with EC50 of 0.9 and 0.07 mM, respectively. UV-Vis and d1(UV-Vis) spectra also revealed that selenium species, in particular selenomethionine (SeMet), were able to interact with metals. Regarding Aß1-42 incubation experiments, Fe(II), Cu(II), and Zn(II) induced Aß aggregation, in a similar way to most of the evaluated selenium species. However, Ch-SeNPs produced a high inhibition of metal-induced Aß aggregation, as well as a high disaggregation capacity of Aß fibrils in both the presence and absence of biometals, in addition to reducing the length and width (20% of reduction in the presence of Zn(II)) of the generated Aß fibrils. Graphical abstract.

Carbonyl-protein content increases in brain and blood of female rats after chronic oxycodone treatment.

BACKGROUND: Opioids are the most effective drugs commonly prescribed to treat pain. Due to their addictive nature, opioid pain relievers are now second to marijuana, ahead of cocaine with respect to dependence. Ours and other studies suggest potential toxic effects of chronic opioid administration leading to neuronal degeneration. It has been suggested that protein carbonylation may represent a sensitive biomarker of cellular degeneration. To evaluate whether prolonged oxycodone administration is associated with accumulation of protein aggregates that may contribute to neuronal degeneration we measured protein carbonylation levels in brain and also in blood plasma of rats after 30-days of 15 mg/kg daily oxycodone administration. RESULTS: We observed a significant increase in the level of carbonylated proteins in rat brain cortex after 30-days of oxycodone treatment compare to that in water treated animals. Also, oxycodone treated rats demonstrated accumulation of insoluble carbonyl-protein aggregates in blood plasma. CONCLUSIONS: Our data suggests that tests detecting insoluble carbonyl-protein aggregates in blood may serve as an inexpensive and minimally invasive method to monitor neuronal degeneration in patients with a history of chronic opioid use. Such methods could be used to detect toxic side effects of other medications and monitor progression of aging and neurodegenerative diseases.

The Traditional Formula Kai-Xin-San Alleviates Polyglutamine-Mediated Neurotoxicity by Modulating Proteostasis Network in Caenorhabditis elegans.

The inherited polyglutamine (polyQ) expansion diseases are characterized by progressive accumulation of aggregation-prone polyQ proteins, which may provoke proteostasis imbalance and result in significant neurotoxicity. Using polyQ transgenic Caenorhabditis elegans models, we find that Kai-Xin-San (KXS), a well-known herbal formula traditionally used to treat mental disorders in China, can alleviate polyQ-mediated neuronal death and associated chemosensory deficiency. Intriguingly, KXS does not reduce polyQ aggregation in vitro as demonstrated by Thioflavin-T test, but does inhibit polyQ aggregation in C. elegans models, indicating an indirect aggregation-inhibitory mechanism. Further investigation reveals that KXS can modulate two key arms of the protein quality control system, that is, heat shock response and autophagy, to clear polyQ aggregates, but has little effect on proteasome activity. In addition, KXS is able to reduce oxidative stress, which is involved in proteostasis and neurodegeneration, but has no effect on life span or dietary restriction response. To examine potential interaction of the four component herbs of KXS, a dissection strategy was used to study the effects of differential herbal combinations in C. elegans polyQ models. While the four herbs do contribute additively to KXS function, Panax ginseng is found to be the most effective constituent. Taken together, these findings not only demonstrate the neuroprotective ability of KXS but also suggest its potential as a proteostasis regulator in protein aggregation disorders and provide an insight into the mechanism studies of traditionally used complex prescriptions and their rationality.

Effect of catechins and high-temperature-processed green tea extract on scavenging reactive oxygen species and preventing Aß1-42 fibrils' formation in brain microvascular endothelium.

The present study investigated the effect of high-temperature-processed green tea extract (HTP_GTE) and its bioactive components on the reduction of reactive oxygen species (ROS) and amyloid-beta (Aß) protein in human microvascular endothelial cells. Compared to Aß1-42-only treatment, pretreatment of HTP_GTE was revealed to effectively inhibit ROS generation (P<0.05). HTP_GTE and catechins not only inhibit Aß1-42 fibril formation but also destabilize preformed Aß1-42 fibrils. The presence of HTP_GTE, Aß1-42 fibril formation was significantly inhibited in a dose-dependent manner at 12.5-100 µg/ml of HTP_GTE, showing 86-56%, respectively. Treatment of various concentrations of HTP_GTE and catechins steadily destabilized the preformed Aß1-42 fibrils for 24 h in a dose-dependent manner. It was observed that the gallated groups such as epigallocatechin gallate, epicatechin gallate, gallocatechin gallate, and catechin gallate more effectively disturbed Aß1-42 fibril formation and destabilized the preformed Aß1-42 fibrils than the non-gallated group. Taken together, these findings supported that sterilized green tea could be promising natural anti-amyloidogenic agents associated with therapeutic approaches in Alzheimer's disease by scavenging ROS generation and Aß fibril in the brain tissue.

Biological Evaluation of 8-Hydroxyquinolines as Multi-Target Directed Ligands for Treating Alzheimer's Disease.

BACKGROUND: Accumulating evidence suggests that multi-target directed ligands have great potential for the treatment of complex diseases such as Alzheimer's Disease (AD). OBJECTIVE: To evaluate novel chimeric 8-hydroxyquinoline ligands with merged pharmacophores as potential multifunctional ligands for AD. METHODS: Nitroxoline, PBT2 and compounds 2-4 were evaluated in-vitro for their inhibitory potencies on cathepsin B, cholinesterases, and monoamine oxidases. Furthermore, chelation, antioxidative properties and the permeability of Blood-Brain Barrier (BBB) were evaluated by spectroscopy-based assays and the inhibition of Amyloid ß (Aß) aggregation was determined in immunoassay. Cell-based assays were performed to determine cytotoxicity, neuroprotection against toxic Aß species, and the effects of compound 2 on apoptotic cascade. RESULTS: Compounds 2-4 competitively inhibited cathepsin B ß-secretase activity, chelated metal ions and were weak antioxidants. All of the compounds inhibited Aß aggregation, whereas only compound 2 had a good BBB permeability according to the parallel artificial membrane permeability assay. Tested ligands 2 and 3 were not cytotoxic to SH-SY5Y and HepG2 cells at 10 µM. Compound 2 exerted neuroprotective effects towards Aß toxicity, reduced the activation of caspase-3/7 and diminished the apoptosis of cells treated with Aß1-42. CONCLUSION: Taken together, our data suggest that compound 2 holds a promise to be used as a multifunctional ligand for AD.

Amyloid-ß Aggregation Inhibitory and Neuroprotective Effects of Xanthohumol and its Derivatives for Alzheimer's Diseases.

BACKGROUND: Xanthohumol has been reported to have cytoprotection through activation of Nrf2-ARE signaling pathway and; it has capability of scavenging free radicals, suggesting its potential for the prevention of neurodegeneration. However, the bio-incompatibility and blood-brain barrier impermeability of xanthohumol hindered its in vivo efficacy potential for treating Alzheimer's disease (AD). OBJECTIVE: We designed and prepared a series of xanthohumol derivatives to enhance the desirable physical, biological and pharmacological properties in particular the blood-brain barrier permeability for intervention of AD. METHODS: We designed and synthesized a novel series of 9 xanthohumol derivatives. Their inhibitory effect on amyloid-ß (1-42), Aß1-42, oligomerization and fibrillation as well as neuroprotection against amyloid-ß induced toxicities, were explored. RESULTS: Among the 9 xanthohumol derivatives, some of them exhibited a moderate to high inhibitory effect on Aß1-42 oligomerization and fibrillation. They were biocompatible and neuroprotective to the SH-SY5Y cells by reducing the ROS generation and calcium uploading that were induced by the amyloid- ß. Importantly, two of the derivatives were found to be blood-brain barrier permeable showing promising potential for AD treatment. CONCLUSION: Two derivatives have been identified to be biocompatible, non-toxic, neuroprotective against Aß-induced toxicities and blood-brain barrier permeable highlighting their promising potential as AD drug candidates for future clinical use.

Cellular models of alpha-synuclein toxicity and aggregation.

Misfolding and aggregation of alpha-synuclein (α-synuclein) with concomitant cytotoxicity is a hallmark of Lewy body related disorders such as Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. Although it plays a pivotal role in pathogenesis and disease progression, the function of α-synuclein and the molecular mechanisms underlying α-synuclein-induced neurotoxicity in these diseases are still elusive. Many in vitro and in vivo experimental models mimicking α-synuclein pathology such as oligomerization, toxicity and more recently neuronal propagation have been generated over the years. In particular, cellular models have been crucial for our comprehension of the pathogenic process of the disease and are beneficial for screening of molecules capable of modulating α-synuclein toxicity. Here, we review α-synuclein based cell culture models that reproduce some features of the neuronal populations affected in patients, from basic unicellular organisms to mammalian cell lines and primary neurons, to the cutting edge models of patient-specific cell lines. These reprogrammed cells known as induced pluripotent stem cells (iPSCs) have garnered attention because they closely reproduce the characteristics of neurons found in patients and provide a valuable tool for mechanistic studies. We also discuss how different cell models may constitute powerful tools for high-throughput screening of molecules capable of modulating α-synuclein toxicity and prevention of its propagation. This article is part of the Special Issue "Synuclein".

Near-Infrared Activated Black Phosphorus as a Nontoxic Photo-Oxidant for Alzheimer's Amyloid-ß Peptide.

The inhibition of amyloid-ß (Aß) aggregation by photo-oxygenation has become an effective way of treating Alzheimer's disease (AD). New near-infrared (NIR) activated treatment agents, which not only possess high photo-oxygenation efficiency, but also show low biotoxicity, are urgently needed. Herein, for the first time, it is demonstrated that NIR activated black phosphorus (BP) could serve as an effective nontoxic photo-oxidant for amyloid-ß peptide in vitro and in vivo. The nanoplatform BP@BTA (BTA: one of thioflavin-T derivatives) possesses high affinity to the Aß peptide due to specific amyloid selectivity of BTA. Importantly, under NIR light, BP@BTA can significantly generate a high quantum yield of singlet oxygen (1 O2 ) to oxygenate Aß, thereby resulting in inhibiting the aggregation and attenuating Aß-induced cytotoxicity. In addition, BP could finally degrade into nontoxic phosphate, which guarantees the biosafety. Using transgenic Caenorhabditis elegans CL2006 as AD model, the results demonstrate that the 1 O2 -generation system could dramatically promote life-span extension of CL2006 strain by decreasing the neurotoxicity of Aß.

Transthyretin Amyloid Fibril Disrupting Activities of Extracts and Fractions from Juglans mandshurica Maxim. var. cordiformis (Makino) Kitam.

Transthyretin-related amyloidosis is a slowly progressive disorder caused by deposition of insoluble amyloid plaques formed by fibrillization of mutant or defective transthyretin (TTR) monomers that leads to neurodegeneration and organ failure. Thus, any compound exhibiting TTR amyloid formation inhibitory activity or TTR amyloid fibril disrupting activity might be a potential candidate for the development of therapies for these disorders. Our aim in this study was the evaluation of the TTR amyloid fibril disrupting potential of extracts of leaves and immature fruits of two Juglans plants, i.e., Juglans mandshurica var. sachalinensis and Juglans mandshurica var. cordiformis. The TTR amyloid fibril disrupting activity was measured by Thioflavin-T (ThT) assay and PROTEOSTAT® Protein aggregation assay methods. A fifty percent acetone extract of the fruits of Juglans mandshurica var. cordiformis showed strong amyloid fibril disrupting activity, and was further fractionated using different solvents. Ethyl acetate and n-butanol fractions showed significant activity in both assays. Syringic acid was isolated and identified as main compound in both of these fractions; however, it did not show any activity. Furthermore, some of the previously reported compounds from Juglans plants including naphthoquinone derivatives and phenolic compounds were evaluated to identify the potential bioactive compounds. Among them, juglone, a naphthoquinone derivative showed promising activity. However, juglone also showed strong cytotoxicity in HEK293 cells. Thus, future studies should focus on the isolation and identification of naphthoquinone derivatives or other compounds from Juglans plan ts with potent bioactivity and low cytotoxicity.

Proinflammatory and amyloidogenic S100A9 induced by traumatic brain injury in mouse model.

Traumatic brain injury (TBI) represents a significant risk factor for development of neurodegenerative diseases such as Alzheimer's and Parkinson's. The S100A9-driven amyloid-neuroinflammatory cascade occurring during primary and secondary TBI events can serve as a mechanistic link between TBI and Alzheimer's as demonstrated recently in the human brain tissues. Here by using immunohistochemistry in the controlled cortical impact TBI mouse model we have found pro-inflammatory S100A9 in the brain tissues of all mice on the first and third post-TBI days, while 70% of mice did not show any S100A9 presence on seventh post-TBI day similar to controls. This indicates that defensive mechanisms effectively cleared S100A9 in these mouse brain tissues during post-TBI recovery. By using sequential immunohistochemistry we have shown that S100A9 was produced by both neuronal and microglial cells. However, Aß peptide deposits characteristic for Alzheimer's disease were not detected in any post-TBI animals. On the first and third post-TBI days S100A9 was found to aggregate intracellularly into amyloid oligomers, similar to what was previously observed in human TBI tissues. Complementary, by using Rayleigh scatting, intrinsic fluorescence and atomic force microscopy we demonstrated that in vitro S100A9 self-assembles into amyloid oligomers within minutes. Its amyloid aggregation is highly dependent on changes of environmental conditions such as variation of calcium levels, pH, temperature and reduction/oxidation, which might be relevant to perturbation of cellular and tissues homeostasis under TBI. Present results demonstrate that S100A9 induction mechanisms in TBI are similar in mice and humans, emphasizing that S100A9 is an important marker of brain injury and therefore can be a potential therapeutic target.

Targeting Ubiquitin Proteasome Pathway with Traditional Chinese Medicine for Treatment of Spinocerebellar Ataxia Type 3.

Nine autosomal dominant spinocerebellar ataxias (SCAs) are caused by an abnormal expansion of CAG trinucleotide repeats that encodes a polyglutamine (polyQ) tract within different genes. Accumulation of aggregated mutant proteins is a common feature of polyQ diseases, leading to progressive neuronal dysfunction and degeneration. SCA type 3 (SCA3), the most common form of SCA worldwide, is characterized by a CAG triplet expansion in chromosome 14q32.1 ATXN3 gene. As accumulation of the mutated polyQ protein is a possible initial event in the pathogenic cascade, clearance of aggregated protein by ubiquitin proteasome system (UPS) has been proposed to inhibit downstream detrimental events and suppress neuronal cell death. In this study, Chinese herbal medicine (CHM) extracts were studied for their proteasome-activating, polyQ aggregation-inhibitory and neuroprotective effects in GFPu and ATXN3/Q 75 -GFP 293/SH-SY5Y cells. Among the 14 tested extracts, 8 displayed increased proteasome activity, which was confirmed by 20S proteasome activity assay and analysis of ubiquitinated and fused GFP proteins in GFPu cells. All the eight extracts displayed good aggregation-inhibitory potential when tested in ATXN3/Q 75 -GFP 293 cells. Among them, neuroprotective effects of five selected extracts were shown by analyses of polyQ aggregation, neurite outgrowth, caspase 3 and proteasome activities, and ATXN3-GFP, ubiquitin, BCL2 and BAX protein levels in neuronal differentiated ATXN3/Q 75 -GFP SH-SY5Y cells. Finally, enhanced proteasome function, anti-oxidative activity and neuroprotection of catalpol, puerarin and daidzein (active constituents of Rehmannia glutinosa and Pueraria lobata) were demonstrated in GFPu and/or ATXN3/Q 75 -GFP 293/SH-SY5Y cells. This study may have therapeutic implication in polyQ-mediated disorders.

Screening of a neuronal cell model of tau pathology for therapeutic compounds.

We have developed a cell-based phenotypic automated high-content screening approach for N2a cells expressing the pro-aggregant repeat domain of tau protein (tauRDΔK), which allows analysis of a chemogenomic library of 1649 compounds for their effect on the inhibition or stimulation of intracellular tau aggregation. We identified several inhibitors and stimulators of aggregation and achieved a screening reproducibility >85% for all data. We identified 18 potential inhibitors (= 1.1% of the library) and 10 stimulators (= 0.6% of the library) of tau aggregation in this cell model of tau pathology. The results provide insights into the regulation of cellular tau aggregation and the pathways involved in this process (e.g., involving signaling via p38 mitogen-activated protein kinase, histone deacetylases, vascular endothelial growth factor, rho/ROCK). For example, inhibitors of protein kinases (e.g., p38) can reduce tau aggregation, whereas inhibitors of deacetylases (histone deacetylases) can enhance aggregation. These observations are compatible with reports that phosphorylated or acetylated tau promotes pathology.

Biaryl scaffold-focused virtual screening for anti-aggregatory and neuroprotective effects in Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is a primary cause of dementia in ageing population affecting more than 35 million people around the globe. It is a chronic neurodegenerative disease caused by defected folding and aggregation of amyloid beta (Aß) protein. Aß is formed by the cleavage of membrane embedded amyloid precursor protein (APP) by using enzyme 'transmembrane aspartyl protease, ß-secretase'. Inhibition of ß-secretase is a viable strategy to prevent neurotoxicity in AD. Another strategy in the treatment of AD is inhibition of acetylcholinesterase. This inhibition reduces the degradation of acetylcholine and temporarily restores the cholinergic function of neurons and improves cognitive function. Monoamine oxidase and higher glutamate levels are also found to be linked with Aß peptide related oxidative stress. Oxidative stress leads to reduced activity of glutamate synthase resulting in significantly higher level of glutamate in brain. The aim of this study is to perform in silico screening of a virtual library of biaryl scaffold containing compounds potentially used for the treatment of AD. Screening was done against the primary targets of AD therapeutics, acetylcholinesterase, ß-secretase (BACE1), Monoamine oxidases (MAO) and N-Methyl-D-aspartate (NMDA) receptor. Compounds were screened for their inhibitory potential by employing molecular docking approach using AutoDock vina. Binding energy scores were embodied in the heatmap to display varies strengths of interactions of the ligands targeting AD. RESULTS: Several ligands showed notable interaction with at least two targets, but the strong interaction with all the targets is shown by very few ligands. The pharmacokinetics of the interacting ligands was also predicted. The interacting ligands have good drug-likeness and brain availability essential for drugs with intracranial targets. CONCLUSION: These results suggest that biaryl scaffold may be pliable to drug development for neuroprotection in AD and that the synthesis of further analogues to optimize these properties should be considered.