Neurotoxic Peptide Research Articles

Phikud navakot extract acts as an ER stress inhibitor to ameliorate ER stress and neuroinflammation

The prevalence of neurological disorders (NDs) such as Alzheimer's disease (AD) is increasing globally, and the lack of effective pharmacological interventions presents a significant health risk. Multiple mechanisms including the activation of oxidative stress, amyloid pathway, ER stress, and neuroinflammation have been implicated in AD; therefore, multi-targeted agents against these mechanisms may be preferable to single-target agents. Phikud Navakot (PN), a Thai traditional medicine combining nine herbs, has been shown to reduce oxidative stress and neuroinflammation of neuronal and microglia cells and the coculture between them, indicating the promising role of PN extract as anti-AD. This study evaluated the neuroprotective effects of PN extract against oxidative stress, amyloid pathway, endoplasmic reticulum stress (ER stress), and neuroinflammation using neuronal and microglia cells, as well as in a Drosophila model of AD. Results showed that PN extract reduced oxidative stress, lipid peroxidation, pro-inflammatory cytokines, amyloid pathway, and ER stress induced by aluminum chloride (AlCl3, AD-induced agent) or thapsigargin (TG, an ER stress activator) in both neurons and microglia cells. PN extract also reduced oxidative stress, ER-stress-related genes, and neurotoxic peptides (amyloid beta) in a Drosophila model of AD. Data indicated that PN extract may function as an anti-AD agent by targeting multiple mechanisms as described. This research also revealed for the first time that PN extract acted as an ER stress inhibitor.

SIRT1 Regulates Mitochondrial Damage in N2a Cells Treated with the Prion Protein Fragment 106-126 via PGC-1α-TFAM-Mediated Mitochondrial Biogenesis.

Mitochondrial damage is an early and key marker of neuronal damage in prion diseases. As a process involved in mitochondrial quality control, mitochondrial biogenesis regulates mitochondrial homeostasis in neurons and promotes neuron health by increasing the number of effective mitochondria in the cytoplasm. Sirtuin 1 (SIRT1) is a NAD+-dependent deacetylase that regulates neuronal mitochondrial biogenesis and quality control in neurodegenerative diseases via deacetylation of a variety of substrates. In a cellular model of prion diseases, we found that both SIRT1 protein levels and deacetylase activity decreased, and SIRT1 overexpression and activation significantly ameliorated mitochondrial morphological damage and dysfunction caused by the neurotoxic peptide PrP106-126. Moreover, we found that mitochondrial biogenesis was impaired, and SIRT1 overexpression and activation alleviated PrP106-126-induced impairment of mitochondrial biogenesis in N2a cells. Further studies in PrP106-126-treated N2a cells revealed that SIRT1 regulates mitochondrial biogenesis through the PGC-1α-TFAM pathway. Finally, we showed that resveratrol resolved PrP106-126-induced mitochondrial dysfunction and cell apoptosis by promoting mitochondrial biogenesis through activation of the SIRT1-dependent PGC-1α/TFAM signaling pathway in N2a cells. Taken together, our findings further describe SIRT1 regulation of mitochondrial biogenesis and improve our understanding of mitochondria-related pathogenesis in prion diseases. Our findings support further investigation of SIRT1 as a potential target for therapeutic intervention of prion diseases.

Cell Penetrating Peptide Enhances the Aphidicidal Activity of Spider Venom-Derived Neurotoxin.

HxTx-Hv1h, a neurotoxic peptide derived from spider venom, has been developed for use in commercial biopesticide formulations. Cell Penetrating Peptides (CPPs) are short peptides that facilitate the translocation of various biomolecules across cellular membranes. Here, we evaluated the aphidicidal efficacy of a conjugated peptide, HxTx-Hv1h/CPP-1838, created by fusing HxTx-Hv1h with CPP-1838. Additionally, we aimed to establish a robust recombinant expression system for HxTx-Hv1h/CPP-1838. We successfully achieved the secretory production of HxTx-Hv1h, its fusion with Galanthus nivalis agglutinin (GNA) forming HxTx-Hv1h/GNA and HxTx-Hv1h/CPP-1838 in yeast. Purified HxTx-Hv1h exhibited contact toxicity against Megoura crassicauda, with a 48 h median lethal concentration (LC50) of 860.5 μg/mL. Fusion with GNA or CPP-1838 significantly enhanced its aphidicidal potency, reducing the LC50 to 683.5 μg/mL and 465.2 μg/mL, respectively. The aphidicidal efficacy was further improved with the addition of surfactant, decreasing the LC50 of HxTx-Hv1h/CPP-1838 to 66.7 μg/mL-over four times lower compared to HxTx-Hv1h alone. Furthermore, we engineered HxTx-Hv1h/CPP-1838 multi-copy expression vectors utilizing the BglBrick assembly method and achieved high-level recombinant production in laboratory-scale fermentation. This study is the first to document a CPP fusion strategy that enhances the transdermal aphidicidal activity of a natural toxin like HxTx-Hv1h and opens up the possibility of exploring the recombinant production of HxTx-Hv1h/CPP-1838 for potential applications.

Pathological Defects in a Drosophila Model of Alzheimer's Disease and Beneficial Effects of the Natural Product Lisosan G.

Alzheimer's disease (AD) brains are histologically marked by the presence of intracellular and extracellular amyloid deposits, which characterize the onset of the disease pathogenesis. Increasing evidence suggests that certain nutrients exert a direct or indirect effect on amyloid β (Aβ)-peptide production and accumulation and, consequently, on AD pathogenesis. We exploited the fruit fly Drosophila melanogaster model of AD to evaluate in vivo the beneficial properties of Lisosan G, a fermented powder obtained from organic whole grains, on the intracellular Aβ-42 peptide accumulation and related pathological phenotypes of AD. Our data showed that the Lisosan G-enriched diet attenuates the production of neurotoxic Aβ peptides in fly brains and reduces neuronal apoptosis. Notably, Lisosan G exerted anti-oxidant effects, lowering brain levels of reactive oxygen species and enhancing mitochondrial activity. These aspects paralleled the increase in autophagy turnover and the inhibition of nucleolar stress. Our results give support to the use of the Drosophila model not only to investigate the molecular genetic bases of neurodegenerative disease but also to rapidly and reliably test the efficiency of potential therapeutic agents and diet regimens.

Temporal-Focusing Multiphoton Excitation Single-Molecule Localization Microscopy Using Spontaneously Blinking Fluorophores.

Single-molecule localization microscopy (SMLM) based on temporal-focusing multiphoton excitation (TFMPE) and single-wavelength excitation is used to visualize the three-dimensional (3D) distribution of spontaneously blinking fluorophore-labeled subcellular structures in a thick specimen with a nanoscale-level spatial resolution. To eliminate the photobleaching effect of unlocalized molecules in out-of-focus regions for improving the utilization rate of the photon budget in 3D SMLM imaging, SMLM with single-wavelength TFMPE achieves wide-field and axially confined two-photon excitation (TPE) of spontaneously blinking fluorophores. TPE spectral measurement of blinking fluorophores is then conducted through TFMPE imaging at a tunable excitation wavelength, yielding the optimal TPE wavelength for increasing the number of detected photons from a single blinking event during SMLM. Subsequently, the TPE fluorescence of blinking fluorophores is recorded to obtain a two-dimensional TFMPE-SMLM image of the microtubules in cancer cells with a localization precision of 18±6 nm and an overall imaging resolution of approximately 51 nm, which is estimated based on the contribution of Nyquist resolution and localization precision. Combined with astigmatic imaging, the system is capable of 3D TFMPE-SMLM imaging of brain tissue section of a 5XFAD transgenic mouse with the pathological features of Alzheimer's disease, revealing the distribution of neurotoxic amyloid-beta peptide deposits.

PEDF-derived peptide protects against Amyloid-β toxicity in vitro and prevents retinal dysfunction in rats

Amyloid-beta (Aβ), a family of aggregation-prone and neurotoxic peptides, has been implicated in the pathophysiology of age-related macular degeneration (AMD). We have previously shown that oligomeric and fibrillar species of Aβ42 exerted retinal toxicity in rats, but while the consequences of exposure to amyloid were related to intracellular effects, the mechanism of Aβ42 internalization in the retina is not well characterized. In the brain, the 67 kDa laminin receptor (67LR) participates in Aβ-related neuronal cell death. A short peptide derived from pigment epithelium-derived factor (PEDF), formerly designated PEDF-335, was found to mitigate experimental models of ischemic retinopathy via targeting of 67LR. In the present study, we hypothesized that 67LR mediates the uptake of pathogenic Aβ42 assemblies in the retina, and that targeting of this receptor by PEDF-335 may limit the internalization of Aβ, thereby ameliorating its retinotoxicity. To test this assumption ARPE-19 cells in culture were incubated with PEDF-335 before treatment with fibrillar or oligomeric structures of Aβ42. Immunostaining confirmed that PEDF-335 treatment substantially prevented amyloid internalization into ARPE-19 cells and maintained their viability in the presence of toxic oligomeric and fibrillar Aβ42 entities in vitro. FRET competition assay was performed and confirmed the binding of PEDF-335 to 67LR in RPE-like cells. Wild-type rats were treated with intravitreal PEDF-335 in the experimental eye 2 days prior to administration of retinotoxic Aβ42 oligomers or fibrils to both eyes. Retinal function was assessed by electroretinography through 6 weeks post injection. The ERG responses in rats treated with oligomeric or fibrillar Aβ42 assemblies were near-normal in eyes previously treated with intravitreal PEDF-335, whereas those measured in the control eyes treated with injection of the Aβ42 assemblies alone showed pathologic attenuation of the retinal function through 6 weeks. The retinal presence of 67LR was determined ex vivo by immunostaining and western blotting. Retinal staining demonstrated the constitutional expression of 67LR mainly in the retinal nuclear layers. In the presence of Aβ42, the levels of 67LR were increased, although its retinal distribution remained largely unaltered. In contrast, no apparent differences in the retinal expression level of 67LR were noted following exposure to PEDF-335 alone, and its pattern of localization in the retina remained similarly concentrated primarily in the inner and outer nuclear layers. In summary, we found that PEDF-335 confers protection against Aβ42-mediated retinal toxicity, with significant effects noted in cells as well as in vivo in rats. The effects of PEDF-335 in the retina are potentially mediated via binding to 67LR and by at least partial inhibition of Aβ42 internalization. These results suggest that PEDF-335 may merit further consideration in the development of targeted inhibition of amyloid-related toxicity in the retina. More broadly, our observations provide evidence on the importance of extracellular versus intracellular Aβ42 in the retina and suggest concepts on the molecular mechanism of Aβ retinal pathogenicity.

Determining Structural Changes for Ligand Recognition between Human and Rat Phosphorylated BACE1 in Silico and Its Phosphorylation by GSK3β at Thr252 by in Vitro Studies.

Alzheimer's disease (AD) is a neurodegenerative disease affecting older adults. AD pathogenesis involves the production of the highly neurotoxic amyloid-β peptide 1-42 (Aβ1-42) from β-site amyloid precursor protein cleaving enzyme 1 (BACE1). The phosphorylation of BACE1 at Thr252 increases its enzymatic activity. This study examined the phosphorylation of BACE1 from human and rat BACE1 in silico through phosphorylation predictors. Besides, we explored how phosphorylation at various sites affected the BACE1 structure and its affinity with amyloid precursor protein (APP) and six BACE1 inhibitors. Additionally, we evaluated the phosphorylation of Thr252-BACE1 by glycogen synthase kinase 3 β (GSK3β) in vitro. The phosphorylation predictors showed that Thr252, Ser59, Tyr76, Ser71, and Ser83 could be phosphorylated. Also, Ser127 in rat BACE1 can be phosphorylated, but human BACE1 has a Gly at this position. Molecular dynamics simulations showed that Ser127 plays an important role in the open and closed BACE1 conformational structures. Docking studies and the molecular mechanics generalized Born surface area (MMGBSA) approach showed that human BACE1 phosphorylated at Thr252 and rat BACE1 phosphorylated at Ser71 have the best binding and free energy with APP, forming hydrogen bonds with Asp672. Importantly, inhibitors have a higher affinity for the phosphorylated rat BACE1 than for its human counterpart, which could explain their failure during clinical trials. Finally, in vitro experiments showed that GSK3β could phosphorylate BACE1. In conclusion, BACE1 phosphorylation influences the BACE1 conformation and its recognition of ligands and substrates. Thus, these features should be carefully considered in the design of BACE1 inhibitors.

Exploring the Potential of Fenamate NSAID Analogs for the Treatment of Alzheimer’s Disease

AbstractBackgroundAlzheimer’s disease (AD) is the most common form of dementia, and its prevalence continues to increase with absence of available interventions. Drug repurposing accelerates the drug discovery process as therapeutics with known safety and toxicology profiles are considered. Although the blood‐brain barrier (BBB) prevents uptake of most pharmaceuticals, our laboratory previously showed that tolfenamic acid (TA), a fenamate non‐steroidal anti‐inflammatory drug (NSAID) with known anti‐cancer effects, can cross the BBB and slow the progression of AD (Zawia et al, 2018).MethodIn the present study, we test 2‐(2‐Cyanophenylthio) benzoic acid (CBA) and Niflumic acid (NA), small molecule analogs of TA that showed better safety and efficacy at targeting neurodegenerative pathways in vitro, and a similar mechanism of action to TA. We aimed to examine the effects of the analogs on in mice using proteomic and transcriptomic analyses.Single dose study of TA, CBA, and NA was carried out in C57BL/6 mice following oral administration at 100 mg/kg. Differential expression of genes and proteomic biomarkers in mice cortices were analyzed between each treatment group and a corresponding control group.ResultsAnalysis of the number of perturbed genes revealed that the drugs demonstrate limited off‐target effects. Bioinformatic analyses revealed that the therapeutics targeted cancer and neurodegenerative pathways including autophagy, inflammation, and neuronal trafficking. CBA and NA shared common downstream targets and pathways suggesting that they may have a similar mode of action.ConclusionWe found a set of differentially expressed genes that have been linked to processes involved in AD pathogenesis such as the production and clearance of neurotoxic Aβ peptides, tau phosphorylation, regulation of oxidative stress, and inflammatory processes. These results suggest that the drugs may influence pathways related to the pathogenesis of AD and support the potential of drug repurposing of fenamate NSAIDs such as TA, and its analogs CBA and NA, as a strategy for cancer and neurodegenerative disease therapy. Further research is needed to confirm the effect of the analogs on AD biomarkers and their efficacy as therapeutics for AD.

PHB2 Alleviates Neurotoxicity of Prion Peptide PrP106-126 via PINK1/Parkin-Dependent Mitophagy.

Prion diseases are a group of neurodegenerative diseases characterized by mitochondrial dysfunction and neuronal death. Mitophagy is a selective form of macroautophagy that clears injured mitochondria. Prohibitin 2 (PHB2) has been identified as a novel inner membrane mitophagy receptor that mediates mitophagy. However, the role of PHB2 in prion diseases remains unclear. In this study, we isolated primary cortical neurons from rats and used the neurotoxic prion peptide PrP106-126 as a cell model for prion diseases. We examined the role of PHB2 in PrP106-126-induced mitophagy using Western blotting and immunofluorescence microscopy and assessed the function of PHB2 in PrP106-126-induced neuronal death using the cell viability assay and the TUNEL assay. The results showed that PrP106-126 induced mitochondrial morphological abnormalities and mitophagy in primary cortical neurons. PHB2 was found to be indispensable for PrP106-126-induced mitophagy and was involved in the accumulation of PINK1 and recruitment of Parkin to mitochondria in primary neurons. Additionally, PHB2 depletion exacerbated neuronal cell death induced by PrP106-126, whereas the overexpression of PHB2 alleviated PrP106-126 neuronal toxicity. Taken together, this study demonstrated that PHB2 is indispensable for PINK1/Parkin-mediated mitophagy in PrP106-126-treated neurons and protects neurons against the neurotoxicity of the prion peptide.

Hexa-Histidine, a Peptide with Versatile Applications in the Study of Amyloid-β(1-42) Molecular Mechanisms of Action.

Amyloid β (Aβ) oligomers are the most neurotoxic forms of Aβ, and Aβ(1-42) is the prevalent Aβ peptide found in the amyloid plaques of Alzheimer's disease patients. Aβ(25-35) is the shortest peptide that retains the toxicity of Aβ(1-42). Aβ oligomers bind to calmodulin (CaM) and calbindin-D28k with dissociation constants in the nanomolar Aβ(1-42) concentration range. Aβ and histidine-rich proteins have a high affinity for transition metal ions Cu2+, Fe3+ and Zn2+. In this work, we show that the fluorescence of Aβ(1-42) HiLyteTM-Fluor555 can be used to monitor hexa-histidine peptide (His6) interaction with Aβ(1-42). The formation of His6/Aβ(1-42) complexes is also supported by docking results yielded by the MDockPeP Server. Also, we found that micromolar concentrations of His6 block the increase in the fluorescence of Aβ(1-42) HiLyteTM-Fluor555 produced by its interaction with the proteins CaM and calbindin-D28k. In addition, we found that the His6-tag provides a high-affinity site for the binding of Aβ(1-42) and Aβ(25-35) peptides to the human recombinant cytochrome b5 reductase, and sensitizes this enzyme to inhibition by these peptides. In conclusion, our results suggest that a His6-tag could provide a valuable new tool to experimentally direct the action of neurotoxic Aβ peptides toward selected cellular targets.

De Novo Assembly of Venom Gland Transcriptome of Tropidolaemus wagleri (Temple Pit Viper, Malaysia) and Insights into the Origin of Its Major Toxin, Waglerin.

The venom proteome of Temple Pit Viper (Tropidolaemus wagleri) is unique among pit vipers, characterized by a high abundance of a neurotoxic peptide, waglerin. To further explore the genetic diversity of its toxins, the present study de novo assembled the venom gland transcriptome of T. wagleri from west Malaysia. Among the 15 toxin gene families discovered, gene annotation and expression analysis reveal the dominating trend of bradykinin-potentiating peptide/angiotensin-converting enzyme inhibitor-C-type natriuretic peptide (BPP/ACEI-CNP, 76.19% of all-toxin transcription) in the transcriptome, followed by P-III snake venom metalloproteases (13.91%) and other toxins. The transcript TwBNP01 of BPP/ACEI-CNP represents a large precursor gene (209 amino acid residues) containing the coding region for waglerin (24 residues). TwBNP01 shows substantial sequence variations from the corresponding genes of its sister species, Tropidolaemus subannulatus of northern Philippines, and other viperid species which diversely code for proline-rich small peptides such as bradykinin-potentiating peptides (BPPs). The waglerin/waglerin-like peptides, BPPs and azemiopsin are proline-rich, evolving de novo from multiple highly diverged propeptide regions within the orthologous BPP/ACEI-CNP genes. Neofunctionalization of the peptides results in phylogenetic constraints consistent with a phenotypic dichotomy, where Tropidolaemus spp. and Azemiops feae convergently evolve a neurotoxic trait while vasoactive BPPs evolve only in other species.

Transcriptomic Analysis in the Hippocampus and Retina of Tg2576 AD Mice Reveals Defective Mitochondrial Oxidative Phosphorylation and Recovery by Tau 12A12mAb Treatment.

Increasing evidence implicates decreased energy metabolism and mitochondrial dysfunctions among the earliest pathogenic events of Alzheimer's disease (AD). However, the molecular mechanisms underlying bioenergetic dysfunctions in AD remain, to date, largely unknown. In this work, we analyzed transcriptomic changes occurring in the hippocampus and retina of a Tg2576 AD mouse model and wild-type controls, evaluating their functional implications by gene set enrichment analysis. The results revealed that oxidative phosphorylation and mitochondrial-related pathways are significantly down-regulated in both tissues of Tg2576 mice, supporting the role of these processes in the pathogenesis of AD. In addition, we also analyzed transcriptomic changes occurring in Tg2576 mice treated with the 12A12 monoclonal antibody that neutralizes an AD-relevant tau-derived neurotoxic peptide in vivo. Our analysis showed that the mitochondrial alterations observed in AD mice were significantly reverted by treatment with 12A12mAb, supporting bioenergetic pathways as key mediators of its in vivo neuroprotective and anti-amyloidogenic effects. This study provides, for the first time, a comprehensive characterization of molecular events underlying the disrupted mitochondrial bioenergetics in AD pathology, laying the foundation for the future development of diagnostic and therapeutic tools.

Drosophila X virus-like particles as delivery carriers for improved oral insecticidal efficacy of scorpion Androctonus australis peptide against the invasive fruit fly, Drosophila suzukii.

Insect-specific neurotoxic peptides derived from the venoms of scorpions and spiders can cause acute paralysis and death when injected into insects, offering a promising insecticidal component for insect pest control. However, effective delivery systems are required to help neurotoxic peptides pass through the gut barrier into the hemolymph, where they can act. Here, we investigated the potential of a novel nanocarrier, Drosophila X virus-like particle (DXV-VLP), for delivering a neurotoxin from the scorpion Androctonus australis Hector (AaIT) against the invasive pest fruit fly, Drosophila suzukii. Our results show that the fusion proteins of DXV polyproteins with AaIT peptide at their C-termini could be sufficiently produced in Lepidoptera Hi5 cells in a soluble form using the recombinant baculovirus expression system, and could self-assemble into VLPs with similar particle morphology and size to authentic DXV virions. In addition, the AaIT peptides displayed on DXV-VLPs retained their toxicity, as demonstrated in injection bioassays that resulted in severe mortality (72%) in adults after 72 h. When fed to adults, mild mortality was observed in the group treated with DXV-AaIT (38%), while no mortality occurred in the group treated with AaIT peptide, thus indicating the significant role of DXV-VLPs in delivering AaIT peptides. Overall, this proof-of-concept study demonstrates for the first time that VLPs can be exploited to enhance oral delivery of insect-specific neurotoxic peptides in the context of pest control. Moreover, it provides insights for further improvements and potentially the development of neurotoxin-based bioinsecticides and/or transgenic crops for insect pest control.

A Metalloproteinase Cocktail from the Venom of Protobothrops flavoviridis Cleaves Amyloid Beta Peptides at the α-Cleavage Site.

A disintegrin and metalloproteinase (ADAM) family proteins are a major class of membrane-anchored multidomain proteinases that are responsible for the shedding of cell surface protein ectodomains, including amyloid precursor protein (APP). Human ADAM 9, 10, and 17 proteolyze APPs and produce non-amyloid-genic p3 peptides, instead of neurotoxic amyloid-β peptides (Aβs; Aβ40 and Aβ42), which form fibrils and accumulate in the brain of patients with Alzheimer's disease (AD). The ADAM family is closely related to snake venom metalloproteinases (SVMPs), which are derived from ancestral ADAMs but act as soluble proteinases. To test the therapeutic potential of SVMPs, we purified SVMPs from Protobothrops flavoviridis venom using metal ion affinity and pooled into a cocktail. Thus, 9 out of 11 SVMPs in the P. flavoviridis genome were identified in the cocktail. SVMPs inhibited Aβ secretion when added to human cell culture medium without affecting APP proteolysis. SVMPs degraded synthetic Aβ40 and Aβ42 peptides at the same cleavage site (α-site of APP) as ADAM9, 10, and 17. SVMPs did not degrade Aβ fibrils but interfered with their formation, assessed using thioflavin-T. Thus, SVMPs have therapeutic potential for AD as an Aβ-degrading protease, and the finding adds to the discovery of bioactive peptides from venoms as novel therapeutics.

Mammalian neurotoxins, Blarina paralytic peptides, cause hyperpolarization of human T-type Ca channel hCav3.2 activation

Among the rare venomous mammals, the short-tailed shrew Blarina brevicauda has been suggested to produce potent neurotoxins in its saliva to effectively capture prey. Several kallikrein-like lethal proteases have been identified, but the active substances of B.brevicauda remained unclear. Here, we report Blarina paralytic peptides (BPPs) 1 and 2 isolated from its submaxillary glands. Synthetic BPP2 showed mealworm paralysis and a hyperpolarization shift (-11mV) of a human T-type Ca2+ channel (hCav3.2) activation. The amino acid sequences of BPPs were similar to those of synenkephalins, which are precursors of brain opioid peptide hormones that are highly conserved among mammals. However, BPPs rather resembled centipede neurotoxic peptides SLPTXs in terms of disulfide bond connectivity and stereostructure. Our results suggested that the neurotoxin BPPs were the result of convergent evolution as homologs of nontoxic endogenous peptides that are widely conserved in mammals. This finding is of great interest from the viewpoint of the chemical evolution of vertebrate venoms.

Protective Effects of Cirsium japonicum var. maackii Flower on Amyloid Beta25-35-Treated C6 Glial Cells.

Amyloid beta (Aβ) is a neurotoxic peptide and a key factor causing Alzheimer's disease. Cirsium japonicum var. maackii (CJM) has neuroprotective effects, but the protective effects of the flower from CJM (FCJM) on the neural system remain unclear. This study aimed to identify the fraction of FCJM with the highest neuroprotective potential and investigate its protective mechanisms against Aβ25-35-induced inflammation in C6 glial cells. The cell viability and generation of reactive oxygen species (ROS) were measured to investigate the positive effect of FCJM on oxidative stress. Treatment with the FCJM extract or fractions increased the cell viability to 60-70% compared with 52% in the Aβ25-35-treated control group and decreased ROS production to 84% compared with 100% in the control group. The ethyl acetate fraction of FCJM (EFCJM) was the most effective among all the extracts and fractions. We analyzed the protective mechanisms of EFCJM on Aβ25-35-induced inflammation in C6 glial cells using Western blot. EFCJM downregulated amyloidogenic pathway-related proteins, such as Aβ precursor protein, β-secretase, presenilin 1, and presenilin 2. Moreover, EFCJM attenuated the Bax/Bcl-2 ratio, an index of apoptosis, and upregulated the oxidative stress-related protein, heme oxygenase-1. Therefore, this study demonstrated that FCJM improves cell viability and inhibits ROS in Aβ25-35-treated C6 glial cells. Furthermore, EFCJM exhibits neuroprotective effects in Aβ25-35-induced inflammation in C6 glial cells by modulating oxidative stress and amyloidogenic and apoptosis signaling pathways. FCJM, especially EFCJM, can be a promising agent for neurodegenerative disease prevention.

Role of oxidative stress in Alzheimer's disease

Alzheimer's disease is the leading cause of disability in people over 65. Amyloid (Aβ) peptide deposition, neurofibrillary tangles of hyperphosphorylated protein, and dementia are hallmarks of AD [1]. The neurotoxic oligomer Aβ peptide and tau protein [2] mediate neurodegeneration, one of the main causes of the disease. Oxidative stress [3], an imbalance between antioxidants and oxidants, causes and exacerbates these symptoms. Free radicals, which have one or more unpaired electrons in their outer shell, can augment or reduce antioxidant defence, causing this imbalance. The reduction of molecular oxygen in water produces superoxide, which produces hydrogen peroxide by adding an electron. Reactive oxygen species (ROS) [4]—hydroxyl radicals produced by hydrogen peroxide reduction—can react with lipids, proteins, nucleic acids, and other molecules and change their structures and activities. Due to its makeup, ROS affects tissues and organs, particularly the sensitive brain. High oxygen usage and highly oxidizable lipids make up the brain.

α7- and α9-Containing Nicotinic Acetylcholine Receptors in the Functioning of Immune System and in Pain.

Nicotinic acetylcholine receptors (nAChRs) present as many different subtypes in the nervous and immune systems, muscles and on the cells of other organs. In the immune system, inflammation is regulated via the vagus nerve through the activation of the non-neuronal α7 nAChR subtype, affecting the production of cytokines. The analgesic properties of α7 nAChR-selective compounds are mostly based on the activation of the cholinergic anti-inflammatory pathway. The molecular mechanism of neuropathic pain relief mediated by the inhibition of α9-containing nAChRs is not fully understood yet, but the role of immune factors in this process is becoming evident. To obtain appropriate drugs, a search of selective agonists, antagonists and modulators of α7- and α9-containing nAChRs is underway. The naturally occurring three-finger snake α-neurotoxins and mammalian Ly6/uPAR proteins, as well as neurotoxic peptides α-conotoxins, are not only sophisticated tools in research on nAChRs but are also considered as potential medicines. In particular, the inhibition of the α9-containing nAChRs by α-conotoxins may be a pathway to alleviate neuropathic pain. nAChRs are involved in the inflammation processes during AIDS and other viral infections; thus they can also be means used in drug design. In this review, we discuss the role of α7- and α9-containing nAChRs in the immune processes and in pain.

A proteomic overview of the major venom components from Tityus championi from Panama

In recent years, morbidity caused by scorpion sting of the species Tityus championi has increased in Panama. Therefore, the LD50 was determined by intravenous injection in 2.9 mg/kg and the venom of T. championi was separated using a HPLC system and their fractions were tested for biological activities in mice to identify the most toxic fractions to mammals. In addition, the venom fractions were also tested against invertebrates to look for insect-specific toxin peptides. The most toxic fractions were analyzed by MS/MS spectrometry. The primary structures of T. championi venom peptides with the most relevant activity were obtained, and the primary structure of one of most neurotoxic peptides was found at least in other four species of Tityus from Panama. This neurotoxin is quite important to be used as a protein target to be neutralized if developing antivenoms against the sting of this Panamanian scorpion or other relevant species of genera Tityus in the country.

Lipid Dys-Homeostasis Contributes to APOE4-Associated AD Pathology.

The association of the APOE4 (vs. APOE3) isoform with an increased risk of Alzheimer's disease (AD) is unequivocal, but the underlying mechanisms remain incompletely elucidated. A prevailing hypothesis incriminates the impaired ability of APOE4 to clear neurotoxic amyloid-β peptides (Aβ) from the brain as the main mechanism linking the apolipoprotein isoform to disease etiology. The APOE protein mediates lipid transport both within the brain and from the brain to the periphery, suggesting that lipids may be potential co-factors in APOE4-associated physiopathology. The present study reveals several changes in the pathways of lipid homeostasis in the brains of mice expressing the human APOE4 vs. APOE3 isoform. Carriers of APOE4 had altered cholesterol turnover, an imbalance in the ratio of specific classes of phospholipids, lower levels of phosphatidylethanolamines bearing polyunsaturated fatty acids and an overall elevation in levels of monounsaturated fatty acids. These modifications in lipid homeostasis were related to increased production of Aβ peptides as well as augmented levels of tau and phosphorylated tau in primary neuronal cultures. This suite of APOE4-associated anomalies in lipid homeostasis and neurotoxic protein levels may be related to the accrued risk for AD in APOE4 carriers and provides novel insights into potential strategies for therapeutic intervention.