Cu(II) Binding Increases the Soluble Toxicity of Amyloidogenic Light Chains.

Light chain amyloidosis (AL) is caused by the aberrant overproduction of immunoglobulin light chains (LCs). The resulting abnormally high LC concentrations in blood lead to deposit formation in the heart and other target organs. Organ damage is caused not only by the accumulation of bulky amyloid deposits, but extensive clinical data indicate that circulating soluble LCs also exert cardiotoxic effects. The nematode C. elegans has been validated to recapitulate LC soluble toxicity in vivo, and in such a model a role for copper ions in increasing LC soluble toxicity has been reported. Here, we applied microscale thermophoresis, isothermal calorimetry and thermal melting to demonstrate the specific binding of Cu2+ to the variable domain of amyloidogenic H7 with a sub-micromolar affinity. Histidine residues present in the LC sequence are not involved in the binding, and yet their mutation to Ala reduces the soluble toxicity of H7. Copper ions bind to and destabilize the variable domains and induce a limited stabilization in this domain. In summary, the data reported here, elucidate the biochemical bases of the Cu2+-induced toxicity; moreover, they also show that copper binding is just one of the several biochemical traits contributing to LC soluble in vivo toxicity.

C. elegans detects toxicity of traumatic brain injury generated tau.

Traumatic brain injury (TBI) is associated with widespread tau pathology in about 30% of patients surviving late after injury. We previously found that TBI in mice induces the formation of an abnormal form of tau (tauTBI) which progressively spreads from the site of injury to remote brain regions. Intracerebral inoculation of TBI brain homogenates into naïve mice induced progressive tau pathology, synaptic loss and late cognitive decline, suggesting a pivotal role of tauTBI in post-TBI neurodegeneration. However, the possibility that tauTBI was a marker of TBI-associated neurodegeneration rather than a toxic driver of functional decline could not be excluded. Here we employed the nematode C. elegans as a biosensor to test the pathogenic role of TBI generated tau. The motility of this nematode depends on efficient neuromuscular transmission and is exceptionally sensitive to the toxicity of amyloidogenic proteins, providing a tractable model for our tests. We found that worms exposed to brain homogenates from chronic but not acute TBI mice, or from mice in which tauTBI had been transmitted by intracerebral inoculation, had impaired motility and neuromuscular synaptic transmission. Results were similar when worms were given brain homogenates from transgenic mice overexpressing tau P301L, a tauopathy mouse model, suggesting that TBI-induced and mutant tau have similar toxic properties. P301L brain homogenate toxicity was similar in wild-type and ptl-1 knock-out worms, indicating that the nematode tau homolog protein PTL-1 was not required to mediate the toxic effect. Harsh protease digestion to eliminate the protein component of the homogenates, pre-incubation with anti-tau antibodies or tau depletion by immunoprecipitation, abolished the toxicity. Homogenates of chronic TBI brains from tau knock-out mice were not toxic to C. elegans, whereas oligomeric recombinant tau was sufficient to impair their motility. This study indicates that tauTBI impairs motor activity and synaptic transmission in C. elegans and supports a pathogenic role of tauTBI in the long-term consequences of TBI. It also sets the groundwork for the development of a C. elegans-based platform for screening anti-tau compounds.

V363I and V363A mutated tau affect aggregation and neuronal dysfunction differently in C. elegans.

Mutations in the microtubule-associated protein tau (MAPT) gene have been linked to a heterogeneous group of progressive neurodegenerative disorders commonly called tauopathies. From patients with frontotemporal lobar degeneration with distinct atypical clinical phenotypes, we recently identified two new mutations on the same codon, in position 363 of the MAPT gene, which resulted in the production of Val-to-Ala (tauV363A) or Val-to-Ile (tauV363I) mutated tau. These substitutions specifically affected microtubule polymerization and propensity of tau to aggregate in vitro suggesting that single amino acid modification may dictate the fate of the neuropathology. To clarify whether tauV363A and tauV363I affect protein misfolding differently in vivo driving certain phenotypes, we generated new transgenic C. elegans strains. Human 2N4R tau carrying the mutation was expressed in all the neurons of worms. The behavioral defects, misfolding and proteotoxicity caused by the tauV363A and tauV363I mutated proteins were compared to that induced by the expression of wild-type tau (tauwt). Pan-neuronal expression of human 2N4R tauWT in worms resulted in a neuromuscular defect with characteristics of a neurodegenerative phenotype. This defect was worsened by the expression of mutated proteins which drive distinct neuronal dysfunctions and synaptic impairments involving, in transgenic worms expressing tauV363A (V363A) also a pharyngeal defect never linked before to other mutations. The two mutations differently affected the tau phosphorylation and misfolding propensities: tauV363I was highly phosphorylated on epitopes corresponding to Thr231 and Ser202/Thr205, and accumulated as insoluble tau assemblies whereas tauV363A showed a greater propensity to form soluble oligomeric assemblies. These findings uphold the role of a single amino acid substitution in specifically affecting the ability of tau to form soluble and insoluble assemblies, opening up new perspectives in the pathogenic mechanism underlying tauopathies.

Safety and Toxicology of Magnolol and Honokiol.

Magnolia officinalis and Magnolia obovata bark extracts have been used for thousands of years in Chinese and Japanese traditional medicines and are still widely employed as herbal preparations for their sedative, antioxidant, anti-inflammatory, antibiotic, and antispastic effects. Neolignans, particularly magnolol and honokiol, are the main substances responsible for the beneficial properties of the magnolia bark extract (MBE). The content of magnolol and honokiol in MBE depends on different factors, including the Magnolia plant species, the area of origin, the part of the plant employed, and the method used to prepare the extract. The biological and pharmacological activities of magnolol and honokiol have been extensively investigated. Here we review the safety and toxicological properties of magnolol and honokiol as pure substances or as components of concentrated MBE, including the potential side-effects in humans after oral intake. In vitro and in vivo genotoxicity studies indicated that concentrated MBE has no mutagenic and genotoxic potential, while a subchronic study performed according to OECD (Organisation for Economic Co-operation and Development) guidelines established a no adverse effect level for concentrated MBE > 240 mg/kg b.w/d. Similar to other dietary polyphenols, magnolol and honokiol are subject to glucuronidation, and despite a relatively quick clearance, an interaction with pharmaceutical active principles or other herbal constituents cannot be excluded. However, intervention trials employing concentrated MBE for up to 1 y did not report adverse effects. In conclusion, over the recent years different food safety authorities evaluated magnolol and honokiol and considered them safe.

Clusterin Binds to Aß1-42 Oligomers with High Affinity and Interferes with Peptide Aggregation by Inhibiting Primary and Secondary Nucleation.

The aggregation of amyloid ß protein (Aß) is a fundamental pathogenic mechanism leading to the neuronal damage present in Alzheimer disease, and soluble Aß oligomers are thought to be a major toxic culprit. Thus, better knowledge and specific targeting of the pathways that lead to these noxious species may result in valuable therapeutic strategies. We characterized some effects of the molecular chaperone clusterin, providing new and more detailed evidence of its potential neuroprotective effects. Using a classical thioflavin T assay, we observed a dose-dependent inhibition of the aggregation process. The global analysis of time courses under different conditions demonstrated that clusterin has no effect on the elongation rate but mainly interferes with the nucleation processes (both primary and secondary), reducing the number of nuclei available for further fibril growth. Then, using a recently developed immunoassay based on surface plasmon resonance, we obtained direct evidence of a high-affinity (KD= 1 nm) interaction of clusterin with biologically relevant Aß1-42oligomers, selectively captured on the sensor chip. Moreover, with the same technology, we observed that substoichiometric concentrations of clusterin prevent oligomer interaction with the antibody 4G8, suggesting that the chaperone shields hydrophobic residues exposed on the oligomeric assemblies. Finally, we found that preincubation with clusterin antagonizes the toxic effects of Aß1-42oligomers, as evaluated in a recently developedin vivomodel inCaenorhabditis elegans.These data substantiate the interaction of clusterin with biologically active regions exposed on nuclei/oligomers of Aß1-42, providing a molecular basis for the neuroprotective effects of the chaperone.

The new ß amyloid-derived peptide Aß1-6A2V-TAT(D) prevents Aß oligomer formation and protects transgenic C. elegans from Aß toxicity.

One attractive pharmacological strategy for Alzheimer's disease (AD) is to design small peptides to interact with amyloid-ß (Aß) protein reducing its aggregation and toxicity. Starting from clinical observations indicating that patients coding a mutated Aß variant (AßA2V) in the heterozygous state do not develop AD, we developed AßA2V synthetic peptides, as well as a small peptide homologous to residues 1-6. These hindered the amyloidogenesis of Aß and its neurotoxicity in vitro, suggesting a basis for the design of a new small peptide in D-isomeric form, linked to the arginine-rich TAT sequence [Aß1-6A2V-TAT(D)], to allow translocation across biological membranes and the blood-brain barrier. Aß1-6A2V-TAT(D) was resistant to protease degradation, stable in serum and specifically able to interfere with Aß aggregation in vitro, reducing the appearance of toxic soluble species and protecting transgenic C. elegans from toxicity related to the muscular expression of human Aß. These observations offer a proof of concept for future pharmacological studies in mouse models of AD, providing a foundation for the design of AßA2V-based peptidomimetic molecules for therapeutic purposes.

A Caenorhabditis elegans-based assay recognizes immunoglobulin light chains causing heart amyloidosis.

Poor prognosis and limited therapeutic options characterize immunoglobulin light-chain (AL) amyloidosis with major heart involvement. Reliable experimental models are needed to study light-chain (LC)/heart interactions and to explore strategies for prevention of cardiac damage. We have exploited the nematode Caenorhabditis elegans as a novel tool, because its pharynx is evolutionarily related to the vertebrate heart. Our data demonstrate that the pharyngeal pumping of C elegans is significantly and selectively reduced by LCs from AL patients suffering from cardiomyopathy, but not by amyloid LCs with different organ tropism or nonamyloidogenic LCs from multiple myeloma. This functional alteration is dependent on the LC concentration and results in persistent pharyngeal dysfunction and in a significant reduction of the worms' lifespan. These manifestations are paralleled by an increase of mitochondrial reactive oxygen species and can be prevented by treatment with antioxidant agents. In conclusion, these data indicate that this nematode-based assay is a promising surrogate model for investigating the heart-specific toxicity of amyloidogenic LCs and for a rapid screening of new therapeutic strategies.

Multigram synthesis and in vivo efficacy studies of a novel multitarget anti-Alzheimer's compound.

We describe the multigram synthesis and in vivo efficacy studies of a donepezil‒huprine hybrid that has been found to display a promising in vitro multitarget profile of interest for the treatment of Alzheimer's disease (AD). Its synthesis features as the key step a novel multigram preparative chromatographic resolution of intermediate racemic huprine Y by chiral HPLC. Administration of this compound to transgenic CL4176 and CL2006 Caenorhabditis elegans strains expressing human Aß42, here used as simplified animal models of AD, led to a significant protection from the toxicity induced by Aß42. However, this protective effect was not accompanied, in CL2006 worms, by a reduction of amyloid deposits. Oral administration for 3 months to transgenic APPSL mice, a well-established animal model of AD, improved short-term memory, but did not alter brain levels of Aß peptides nor cortical and hippocampal amyloid plaque load. Despite the clear protective and cognitive effects of AVCRI104P4, the lack of Aß lowering effect in vivo might be related to its lower in vitro potency toward Aß aggregation and formation as compared with its higher anticholinesterase activities. Further lead optimization in this series should thus focus on improving the anti-amyloid/anticholinesterase activity ratio.

Expression of A2V-mutated Aß in Caenorhabditis elegans results in oligomer formation and toxicity.

Although Alzheimer's disease (AD) is usually sporadic, in a small proportion of cases it is familial and can be linked to mutations in ß-amyloid precursor protein (APP). Unlike the other genetic defects, the mutation [alanine-673→valine-673] (A673V) causes the disease only in the homozygous condition with enhanced amyloid ß (Aß) production and aggregation; heterozygous carriers remain unaffected. It is not clear how misfolding and aggregation of Aß is affected in vivo by this mutation and whether this correlates with its toxic effects. No animal models over-expressing the A673V-APP gene or alanine-2-valine (A2V) mutated human Aß protein are currently available. Using the invertebrate Caenorhabditis elegans, we generated the first transgenic animal model to express the human Aß1-40 wild-type (WT) in neurons or possess the A2V mutation (Aß1-40A2V). Insertion of an Aß-mutated gene into this nematode reproduced the homozygous state of the human pathology. Functional and biochemical characteristics found in the A2V strain were compared to those of transgenic C. elegans expressing Aß1-40WT. The expression of both WT and A2V Aß1-40 specifically reduced the nematode's lifespan, causing behavioral defects and neurotransmission impairment which were worse in A2V worms. Mutant animals were more resistant than WT to paralysis induced by the cholinergic agonist levamisole, indicating that the locomotor defect was specifically linked to postsynaptic dysfunctions. The toxicity caused by the mutated protein was associated with a high propensity to form oligomeric assemblies which accumulate in the neurons, suggesting this to be the central event involved in the postsynaptic damage and early onset of the disease in homozygous human A673V carriers.

Soil quality in the Lomellina area using in vitro models and ecotoxicological assays.

Soil quality is traditionally evaluated by chemical characterization to determine levels of pollutants. Biological tools are now employed for soil monitoring since they can take account of the global biological effects induced by all xenobiotics. A combined monitoring of soils based on chemical analyses, human-related in vitro models and ecotoxicological assay was applied in the Lomellina, a semirural area of northern Italy. Chemical characterization indicated overall good quality of the soils, with low levels of toxic and carcinogenic pollutants such as heavy metals, PAHs, PCDD/Fs and PCBs. HepG2 cells were used as a model for the human liver and BALB/c 3T3 cells to evaluate carcinogenic potential. Cells were treated with soil extractable organic matter (EOM) and the MTS assay, DNA release and morphological transformation were selected as endpoints for toxicity and carcinogenicity. Soil EOMs induced dose-dependent inhibition of cell growth at low doses and cytotoxicity only at doses of 500 and 1000 mg soil equivalents/ml. Potential issues for human health can be hypothesized after ingestion of soil samples from some sites. No statistically significant inductions of foci were recorded after exposure to EOMs, indicating that the levels of the soil-extracted organic pollutants were too low to induce carcinogenesis in our experimental conditions. An acute phytotoxicity test and studies on Caenorhabditis elegans were used as ecotoxicological assays for plants and small invertebrates. No significant alerts for ecotoxicity were found. In this proposed case study, HepG2 cells detected differences in the toxicity of soil EOMs, indicating that this cell line could be appropriate to assess the potential harm caused by the ingestion of contaminated soil. Additional information on the carcinogenic potential of mixtures was provided by the cell transformation assay, strengthening the combined approach.

Nanobodies counteract the toxicity of an amyloidogenic light chain by stabilizing a partially open dimeric conformation.

Light chain amyloidosis (AL) is a systemic disease where fibrillar deposition of misfolded immunoglobulin light chains (LCs) severely affects organ function and results in poor prognosis for patients, especially when heart involvement is severe. Particularly relevant in this context is the cardiotoxicity exerted by still uncharacterized soluble LC species. Here, with the final goal of identifying alternative therapeutic strategies to tackle AL amyloidosis, we produced five llama-derived nanobodies (Nbs) specific against H3, a well-characterized amyloidogenic and cardiotoxic LC from an AL patient with severe cardiac involvement. We found that Nbs are specific and potent agents capable of abolishing H3 soluble toxicity in C. elegans in vivo model. Structural characterization of H3-Nb complexes revealed that the protective effect of Nbs is related to their ability to bind to the H3 VL domain and stabilise an unexpected partially open LC dimer in which the two VL domains no longer interact with each other. Thus, while identifying potent inhibitors of LC soluble toxicity, we also describe the first non-native structure of an amyloidogenic LC that may represent a crucial step in toxicity and aggregation mechanisms.

Antitumour drugs targeting tau R3 VQIVYK and Cys322 prevent seeding of endogenous tau aggregates by exogenous seeds.

Emerging experimental evidence suggests tau pathology spreads between neuroanatomically connected brain regions in a prion-like manner in Alzheimer's disease (AD). Tau seeding, the ability of prion-like tau to recruit and misfold naïve tau to generate new seeds, is detected early in human AD brains before the development of major tau pathology. Many antitumour drugs have been reported to confer protection against neurodegeneration, supporting the repurposing of approved and experimental or investigational oncology drugs for AD therapy. In this study, we evaluated whether antitumour drugs that abrogate the generation of seed-competent aggregates of tau Repeat 3 (R3) domain peptides can prevent tau seeding and toxicity in Tau-RD P301S FRET Biosensor cells and Caenorhabditis elegans. We demonstrate that drugs that interact with the N-terminal VQIVYK or the C-terminal region housing the Cys322 prevent R3 dimerisation, abolishing the generation of prion-like R3 seeds. Preformed R3 seeds (fibrils) capped with, or R3 seeds formed in the presence of VQIVYK- or Cys322-targeting drugs have a reduced potency to cause aggregation of naïve tau in biosensor cells and protect worms from aggregate toxicity. These findings indicate that VQIVYK- or Cys322-targeting drugs may act as prophylactic agents against tau seeding.

Aryl Hydrocarbon Receptor-Dependent and -Independent Pathways Mediate Curcumin Anti-Aging Effects.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor whose activity can be modulated by polyphenols, such as curcumin. AhR and curcumin have evolutionarily conserved effects on aging. Here, we investigated whether and how the AhR mediates the anti-aging effects of curcumin across species. Using a combination of in vivo, in vitro, and in silico analyses, we demonstrated that curcumin has AhR-dependent or -independent effects in a context-specific manner. We found that in Caenorhabditis elegans, AhR mediates curcumin-induced lifespan extension, most likely through a ligand-independent inhibitory mechanism related to its antioxidant activity. Curcumin also showed AhR-independent anti-aging activities, such as protection against aggregation-prone proteins and oxidative stress in C. elegans and promotion of the migratory capacity of human primary endothelial cells. These AhR-independent effects are largely mediated by the Nrf2/SKN-1 pathway.

Antioxidant and Anti-Inflammaging Ability of Prune (Prunus Spinosa L.) Extract Result in Improved Wound Healing Efficacy.

Prunus spinosa L. fruit (PSF) ethanol extract, showing a peculiar content of biologically active molecules (polyphenols), was investigated for its wound healing capacity, a typical feature that declines during aging and is negatively affected by the persistence of inflammation and oxidative stress. To this aim, first, PSF anti-inflammatory properties were tested on young and senescent LPS-treated human umbilical vein endothelial cells (HUVECs). As a result, PSF treatment increased miR-146a and decreased IRAK-1 and IL-6 expression levels. In addition, the PSF antioxidant effect was validated in vitro with DPPH assay and confirmed by in vivo treatments in C. elegans. Our findings showed beneficial effects on worms' lifespan and healthspan with positive outcomes on longevity markers (i.e., miR-124 upregulation and miR-39 downregulation) as well. The PSF effect on wound healing was tested using the same cells and experimental conditions employed to investigate PSF antioxidant and anti-inflammaging ability. PSF treatment resulted in a significant improvement of wound healing closure (ca. 70%), through cell migration, both in young and older cells, associated to a downregulation of inflammation markers. In conclusion, PSF extract antioxidant and anti-inflammaging abilities result in improved wound healing capacity, thus suggesting that PSF might be helpful to improve the quality of life for its beneficial health effects.

Machine learning analyses of antibody somatic mutations predict immunoglobulin light chain toxicity.

In systemic light chain amyloidosis (AL), pathogenic monoclonal immunoglobulin light chains (LC) form toxic aggregates and amyloid fibrils in target organs. Prompt diagnosis is crucial to avoid permanent organ damage, but delayed diagnosis is common because symptoms usually appear only after strong organ involvement. Here we present LICTOR, a machine learning approach predicting LC toxicity in AL, based on the distribution of somatic mutations acquired during clonal selection. LICTOR achieves a specificity and a sensitivity of 0.82 and 0.76, respectively, with an area under the receiver operating characteristic curve (AUC) of 0.87. Tested on an independent set of 12 LCs sequences with known clinical phenotypes, LICTOR achieves a prediction accuracy of 83%. Furthermore, we are able to abolish the toxic phenotype of an LC by in silico reverting two germline-specific somatic mutations identified by LICTOR, and by experimentally assessing the loss of in vivo toxicity in a Caenorhabditis elegans model. Therefore, LICTOR represents a promising strategy for AL diagnosis and reducing high mortality rates in AL.

An across-species comparison of the sensitivity of different organisms to Pb-based perovskites used in solar cells.

Organic-inorganic perovskite solar cells (PSCs) are promising candidates as photovoltaic cells. Recently, they have attracted significant attention due to certified power conversion efficiencies exceeding 23%, low-cost engineering, and superior electrical/optical characteristics. These PSCs extensively utilize a perovskite-structured composite with a hybrid of Pb-based nanomaterials. Operation of them may cause the release of Pb-based nanoparticles. However, limited information is available regarding the potential toxicity of Pb-based PSCs on various organisms. This study conducted a battery of in vitro and in vivo toxicity bioassays for three quintessential Pb-based PSCs (CH3NH3PbI3, NHCHNH3PbBr3, and CH3NH3PbBr3) using progressively more complex forms of life. For all species tested, the three different perovskites had comparable toxicities. The viability of Caco-2/TC7 cells was lower than that of A549 cells in response to Pb-based PSC exposure. Concentration-dependent toxicity was observed for the bioluminescent bacterium Vibrio fischeri, for soil bacterial communities, and for the nematode Caenorhabditis elegans. Neither of the tested Pb-based PSCs particles had apparent toxicity to Pseudomonas putida. Among all tested organisms, V. fischeri showed the highest sensitivity with EC50 values (30 min of exposure) ranging from 1.45 to 2.91 mg L-1. Therefore, this study recommends that V. fischeri should be preferably utilized to assess. PSC toxicity due to its increased sensitivity, low costs, and relatively high throughput in a 96-well format, compared with the other tested organisms. These results highlight that the developed assay can easily predict the toxic potency of PSCs. Consequently, this approach has the potential to promote the implementation of the 3Rs (Replacement, Reduction, and Refinement) principle in toxicology and decrease the dependence on animal testing when determining the safety of novel PSCs.

Inherent Biophysical Properties Modulate the Toxicity of Soluble Amyloidogenic Light Chains.

In light chain amyloidosis (AL), fibrillar deposition of monoclonal immunoglobulin light chains (LCs) in vital organs, such as heart, is associated with their severe dysfunction. In addition to the cellular damage caused by fibril deposition, direct toxicity of soluble prefibrillar amyloidogenic proteins has been reported, in particular, for cardiotoxicity. However, the molecular bases of proteotoxicity by soluble LCs have not been clarified. Here, to address this issue, we rationally engineered the amino acid sequence of the highly cardiotoxic LC H6 by introducing three residue mutations, designed to reduce the dynamics of its native state. The resulting mutant (mH6) is less toxic than its parent H6 to human cardiac fibroblasts and C. elegans. The high sequence and structural similarity, together with the different toxicity, make H6 and its non-toxic designed variant mH6 a test case to shed light on the molecular properties underlying soluble toxicity. Our comparative structural and biochemical study of H6 and mH6 shows closely matching crystal structures, whereas spectroscopic data and limited proteolysis indicate that H6 displays poorly cooperative fold, higher flexibility, and kinetic instability, and a higher dynamic state in its native fold. Taken together, the results of this study show a strong correlation between the overall conformational properties of the native fold and the proteotoxicity of cardiotropic LCs.

Methacycline displays a strong efficacy in reducing toxicity in a SCA3 Caenorhabditis elegans model.

BACKGROUND: We have previously demonstrated the neuroprotective activity of tetracycline on a Spinocerebellar Ataxia 3 nematode model. Here, we present the screening of a small library of tetracycline congeners in order to identify the most effective compound in preventing ataxin-3 aggregation. METHODS: We performed the assays on the Josephin Domain as it is directly involved in the onset of fibrillation. We used thioflavin T and solubility assays to spot out the most effective tetracycline congeners; Fourier transform infrared and NMR spectroscopies to characterize their mode of action. We employed an ataxic Caenorhabditis elegans model to evaluate the pharmacological efficacy of tetracycline congeners. RESULTS: Methacycline was identified as the most effective compound. Like tetracycline, methacycline neither significantly affected the aggregation kinetics nor did it change the secondary structures of the final aggregates but increased the solubility of the aggregated species. Saturation transfer NMR experiments demonstrated methacycline capability to only bind the oligomeric species of Josephin Domain. Competition assays also showed that methacycline binds to the Josephin Domain more tightly than tetracycline. The treatment with methacycline induced a significant improvement in motility and locomotion of the transgenic C. elegans without changing its lifespan. The efficacy was distinctly stronger than that of tetracycline. Noteworthy, unlike tetracycline, methacycline was able to retard aging-related decline in motility of even the healthy worms used. CONCLUSIONS: The apparent absence of toxic effects displayed by methacycline, along with its stronger efficacy in contrasting expanded ataxin-3 toxicity, makes it a possible candidate for a chronic treatment of the disease.

Humanin Specifically Interacts with Amyloid-ß Oligomers and Counteracts Their in vivo Toxicity.

The 24-residue peptide humanin (HN) has been proposed as a peptide-based inhibitor able to interact directly with amyloid-ß (Aß) oligomers and interfere with the formation and/or biological properties of toxic Aß species. When administered exogenously, HN, or its synthetic S14G-derivative (HNG), exerted multiple cytoprotective effects, counteracting the Aß-induced toxicity. Whether these peptides interact directly with Aß, particularly with the soluble oligomeric assemblies, remains largely unknown. We here investigated the ability of HN and HNG to interact directly with highly aggregating Aß42, and interfere with the formation and toxicity of its oligomers. Experiments were run in cell-free conditions and in vivo in a transgenic C. elegans strain in which the Aß toxicity was specifically due to oligomeric species. Thioflavin-T assay indicated that both HN and HNG delay the formation and reduce the final amount of Aß42 fibrils. In vitro surface plasmon resonance studies indicated that they interact with Aß42 oligomers favoring the formation of amorphous larger assemblies, observed with turbidity and electron microscopy. In vivo studies indicated that both HN and HNG decrease the relative abundance of A11-positive prefibrillar oligomers as well as OC-positive fibrillar oligomers and had similar protective effects. However, while HN possibly decreased the oligomers by promoting their assembly into larger aggregates, the reduction of oligomers caused by HNG can be ascribed to a marked decrease of the total Aß levels, likely the consequence of the HNG-induced overexpression of the Aß-degrading enzyme neprilysin. These findings provide information on the mechanisms underlying the anti-oligomeric effects of HN and HNG and illustrate the role of S14G substitution in regulating the in vivo mechanism of action.

Inhibition of Aß Amyloid Growth and Toxicity by Silybins: The Crucial Role of Stereochemistry.

The self-assembling of the amyloid ß (Aß) peptide into neurotoxic aggregates is considered a central event in the pathogenesis of Alzheimer's disease (AD). Based on the "amyloid hypothesis", many efforts have been devoted to designing molecules able to halt disease progression by inhibiting Aß self-assembly. Here, we combine biophysical (ThT assays, TEM and AFM imaging), biochemical (WB and ESI-MS), and computational (all-atom molecular dynamics) techniques to investigate the capacity of four optically pure components of the natural product silymarin (silybin A, silybin B, 2,3-dehydrosilybin A, 2,3-dehydrosilybin B) to inhibit Aß aggregation. Despite TEM analysis demonstrated that all the four investigated flavonoids prevent the formation of mature fibrils, ThT assays, WB and AFM investigations showed that only silybin B was able to halt the growth of small-sized protofibrils thus promoting the formation of large, amorphous aggregates. Molecular dynamics (MD) simulations indicated that silybin B interacts mainly with the C-terminal hydrophobic segment 35MVGGVV40 of Aß40. Consequently to silybin B binding, the peptide conformation remains predominantly unstructured along all the simulations. By contrast, silybin A interacts preferentially with the segments 17LVFF20 and 27NKGAII32 of Aß40 which shows a high tendency to form bend, turn, and ß-sheet conformation in and around these two domains. Both 2,3-dehydrosilybin enantiomers bind preferentially the segment 17LVFF20 but lead to the formation of different small-sized, ThT-positive Aß aggregates. Finally, in vivo studies in a transgenic Caenorhabditis elegans strain expressing human Aß indicated that silybin B is the most effective of the four compounds in counteracting Aß proteotoxicity. This study underscores the pivotal role of stereochemistry in determining the neuroprotective potential of silybins and points to silybin B as a promising lead compound for further development in anti-AD therapeutics.