RESUMEN
Alzheimer's disease (AD), the leading cause of dementia in older adults, is a double proteinopathy characterized by amyloid-ß (Aß) and tau pathology. Despite enormous efforts that have been spent in the last decades to find effective therapies, late pharmacological interventions along the course of the disease, inaccurate clinical methodologies in the enrollment of patients, and inadequate biomarkers for evaluating drug efficacy have not allowed the development of an effective therapeutic strategy. The approaches followed so far for developing drugs or antibodies focused solely on targeting Aß or tau protein. This paper explores the potential therapeutic capacity of an all-D-isomer synthetic peptide limited to the first six amino acids of the N-terminal sequence of the A2V-mutated Aß, Aß1-6A2V(D), that was developed following the observation of a clinical case that provided the background for its development. We first performed an in-depth biochemical characterization documenting the capacity of Aß1-6A2V(D) to interfere with the aggregation and stability of tau protein. To tackle Aß1-6A2V(D) in vivo effects against a neurological decline in genetically predisposed or acquired high AD risk mice, we tested its effects in triple transgenic animals harboring human PS1(M146 V), APP(SW), and MAPT(P301L) transgenes and aged wild-type mice exposed to experimental traumatic brain injury (TBI), a recognized risk factor for AD. We found that Aß1-6A2V(D) treatment in TBI mice improved neurological outcomes and reduced blood markers of axonal damage. Exploiting the C. elegans model as a biosensor of amyloidogenic proteins' toxicity, we observed a rescue of locomotor defects in nematodes exposed to the brain homogenates from TBI mice treated with Aß1-6A2V(D) compared to TBI controls. By this integrated approach, we demonstrate that Aß1-6A2V(D) not only impedes tau aggregation but also favors its degradation by tissue proteases, confirming that this peptide interferes with both Aß and tau aggregation propensity and proteotoxicity.
Asunto(s)
Enfermedad de Alzheimer , Lesiones Traumáticas del Encéfalo , Humanos , Animales , Ratones , Anciano , Proteínas tau/metabolismo , Caenorhabditis elegans/metabolismo , Fragmentos de Péptidos/metabolismo , Péptidos beta-Amiloides/metabolismo , Enfermedad de Alzheimer/metabolismo , Encéfalo/metabolismo , Ratones Transgénicos , Modelos Animales de Enfermedad , Precursor de Proteína beta-Amiloide/metabolismoRESUMEN
We developed and validated a technology platform for designing and testing peptides inhibiting the infectivity of SARS-CoV-2 spike protein-based pseudoviruses. This platform integrates target evaluation, in silico inhibitor design, peptide synthesis, and efficacy screening. We generated a cyclic peptide library derived from the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and the angiotensin-converting enzyme 2 (ACE2) receptor. The cell-free validation process by ELISA competition assays and Surface Plasmon Resonance (SPR) studies revealed that the cyclic peptide c9_05, but not its linear form, binds well to ACE2. Moreover, it effectively inhibited the transduction in HEK293, stably expressing the human ACE2 receptor of pseudovirus particles displaying the SARS-CoV-2 spike in the Wuhan or UK variants. However, the inhibitory efficacy of c9_05 was negligible against the Omicron variant, and it failed to impede the entry of pseudoviruses carrying the B.1.351 (South African) spike. These variants contain three or more mutations known to increase affinity to ACE2. This suggests further refinement is needed for potential SARS-CoV-2 inhibition. Our study hints at a promising approach to develop inhibitors targeting viral infectivity receptors, including SARS-CoV-2's. This platform also promises swift identification and evaluation of inhibitors for other emergent viruses.
Asunto(s)
COVID-19 , Virus ARN , Humanos , SARS-CoV-2 , Enzima Convertidora de Angiotensina 2 , Células HEK293 , Péptidos/farmacología , Péptidos Cíclicos , Biblioteca de Péptidos , Tecnología , Unión ProteicaRESUMEN
The formation of neurofibrillary tangles and amyloid plaques accompanies the progression of Alzheimer's disease. Tangles are made of fibrillar aggregates formed by the microtubule-associated protein tau, whereas plaques comprise fibrillar forms of amyloid-beta (Aß). Both form toxic oligomers during aggregation and are thought to interact synergistically to each promote the accumulation of the other. Recent in vitro studies have suggested that the monomeric nonphosphorylated full-length tau protein hinders the aggregation of Aß1-40 peptide, but whether the same is true for the more aggregation-prone Aß1-42 was not determined. We used in vitro and in vivo techniques to explore this question. We have monitored the aggregation kinetics of Aß1-42 by thioflavine T fluorescence in the presence or the absence of different concentrations of nonphosphorylated tau. We observed that elongation of Aß1-42 fibrils was inhibited by tau in a dose-dependent manner. Interestingly, the fibrils were structurally different in the presence of tau but did not incorporate tau. Surface plasmon resonance indicated that tau monomers bound to Aß1-42 oligomers (but not monomers) and hindered their interaction with the anti-Aß antibody 4G8, suggesting that tau binds to the hydrophobic central core of Aß recognized by 4G8. Tau monomers also antagonized the toxic effects of Aß oligomers in Caenorhabditis elegans. This suggests that nonphosphorylated tau might have a neuroprotective effect by binding Aß1-42 oligomers formed during the aggregation and shielding their hydrophobic patches.
Asunto(s)
Péptidos beta-Amiloides/antagonistas & inhibidores , Amiloide/antagonistas & inhibidores , Caenorhabditis elegans/crecimiento & desarrollo , Larva/crecimiento & desarrollo , Fármacos Neuroprotectores/farmacología , Fragmentos de Péptidos/antagonistas & inhibidores , Proteínas tau/farmacología , Péptidos beta-Amiloides/toxicidad , Animales , Caenorhabditis elegans/efectos de los fármacos , Humanos , Cinética , Larva/efectos de los fármacos , Fragmentos de Péptidos/toxicidadRESUMEN
A clear relationship between the tau assemblies and toxicity has still to be established. To correlate the tau conformation with its proteotoxic effect in vivo, we developed an innovative cell-worm-based approach. HEK293 cells expressing tau P301L under a tetracycline-inducible system (HEK T-Rex) were employed to produce different tau assemblies whose proteotoxic potential was evaluated using C. elegans. Lysates from cells induced for five days significantly reduced the worm's locomotor activity. This toxic effect was not related to the total amount of tau produced by cells or to its phosphorylation state but was related to the formation of multimeric tau assemblies, particularly tetrameric ones. We investigated the applicability of this approach for testing compounds acting against oligomeric tau toxicity, using doxycycline (Doxy) as a prototype drug. Doxy affected tau solubility and promoted the disassembly of already formed toxic aggregates in lysates of cells induced for five days. These effects translated into a dose-dependent protective action in C. elegans. These findings confirm the validity of the combined HEK T-Rex cells and the C. elegans-based approach as a platform for pharmacological screening.
Asunto(s)
Tauopatías , Animales , Caenorhabditis elegans , Doxiciclina/farmacología , Células HEK293 , Humanos , Proteínas tauRESUMEN
Gelsolin amyloidosis (AGel) is characterized by multiple systemic and ophthalmic features resulting from pathological tissue deposition of the gelsolin (GSN) protein. To date, no cure is available for the treatment of any form of AGel. More than ten single-point substitutions in the GSN gene are responsible for the occurrence of the disease and, among them, D187N/Y is the most widespread variant. These substitutions undergo an aberrant proteolytic cascade, producing aggregation-prone peptides of 5 and 8 kDa, containing the Gelsolin Amyloidogenic Core, spanning residues 182-192 (GAC182-192). Following a structure-based approach, we designed and synthesized three novel sequence-specific peptidomimetics (LB-5, LB-6, and LB-7) built on a piperidine-pyrrolidine unnatural amino acid. LB-5 and LB-6, but not LB-7, efficiently inhibit the aggregation of the GAC182-192 amyloidogenic peptides at sub-stoichiometric concentrations. These peptidomimetics resulted also effective in vivo, in a C. elegans-based assay, in counteracting the proteotoxicity of aggregated GAC182-192. These data pave the way to a novel pharmacological strategy against AGel and also validate a toolbox exploitable in other amyloidogenic diseases.
Asunto(s)
Amiloidosis Familiar , Amiloidosis , Peptidomiméticos , Animales , Gelsolina/metabolismo , Peptidomiméticos/farmacología , Caenorhabditis elegans/metabolismo , Amiloidosis Familiar/genética , Amiloide/metabolismo , Proteínas Amiloidogénicas/metabolismo , Amiloidosis/metabolismo , Péptidos/farmacología , Péptidos/metabolismoRESUMEN
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.
Asunto(s)
Cobre/metabolismo , Cadenas Ligeras de Inmunoglobulina/química , Cadenas Ligeras de Inmunoglobulina/metabolismo , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas/metabolismo , Sustitución de Aminoácidos , Animales , Caenorhabditis elegans , Calorimetría , Modelos Animales de Enfermedad , Histidina/metabolismo , Humanos , Cadenas Ligeras de Inmunoglobulina/toxicidad , Modelos Moleculares , Conformación Proteica , Especies Reactivas de Oxígeno/metabolismoRESUMEN
A significant portion of the world's plastic is not properly disposed of and, through various processes, is degraded into microscopic particles termed micro- and nanoplastics. Marine and terrestrial faunae, including humans, inevitably get in contact and may inhale and ingest these microscopic plastics which can deposit throughout the body, potentially altering cellular and molecular functions in the nervous and other systems. For instance, at the cellular level, studies in animal models have shown that plastic particles can cross the blood-brain barrier and interact with neurons, and thus affect cognition. At the molecular level, plastics may specifically influence the folding of proteins, induce the formation of aberrant amyloid proteins, and therefore potentially trigger the development of systemic and local amyloidosis. In this review, we discuss the general issue of plastic micro- and nanoparticle generation, with a focus on their effects on protein folding, misfolding, and their possible clinical implications.
Asunto(s)
Amiloidosis , Contaminantes Químicos del Agua , Proteínas Amiloidogénicas , Amiloidosis/etiología , Animales , Humanos , Microplásticos , Plásticos , Pliegue de Proteína , Contaminantes Químicos del Agua/análisisRESUMEN
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.
Asunto(s)
Lesiones Traumáticas del Encéfalo/metabolismo , Caenorhabditis elegans , Actividad Motora/fisiología , Enfermedades Neurodegenerativas/metabolismo , Unión Neuromuscular/metabolismo , Proteínas tau/metabolismo , Animales , Lesiones Traumáticas del Encéfalo/fisiopatología , Ratones , Enfermedades Neurodegenerativas/fisiopatología , Unión Neuromuscular/fisiopatología , Tauopatías/metabolismo , Tauopatías/fisiopatologíaRESUMEN
AGel amyloidosis is a genetic degenerative disease characterized by the deposition of insoluble gelsolin protein aggregates in different tissues. Until recently, this disease was associated with two mutations of a single residue (Asp187 to Asn/Tyr) in the second domain of the protein. The general opinion is that pathogenic variants are not per se amyloidogenic but rather that the mutations trigger an aberrant proteolytic cascade, which results in the production of aggregation prone fragments. Here, we report the crystal structure of the second domain of gelsolin carrying the recently identified Gly167Arg mutation. This mutant dimerizes through a three-dimensional domain swapping mechanism, forming a tight but flexible assembly, which retains the structural topology of the monomer. To date, such dramatic conformational changes of this type have not been observed. Structural and biophysical characterizations reveal that the Gly167Arg mutation alone is responsible for the monomer to dimer transition and that, even in the context of the full-length protein, the pathogenic variant is prone to form dimers. These data suggest that, in addition to the well-known proteolytic-dependent mechanism, an alternative oligomerization pathway may participate in gelsolin misfolding and aggregation. We propose to integrate this alternative pathway into the current model of the disease that may also be relevant for other types of AGel amyloidosis, and other related diseases with similar underlying pathological mechanisms.
Asunto(s)
Amiloidosis/genética , Gelsolina/química , Gelsolina/genética , Mutación , Amiloide/genética , Amiloide/metabolismo , Amiloidosis/metabolismo , Cristalografía por Rayos X/métodos , Dimerización , Gelsolina/metabolismo , Humanos , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Dominios ProteicosRESUMEN
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.
Asunto(s)
Proteínas de Caenorhabditis elegans/biosíntesis , Degeneración Nerviosa/metabolismo , Agregado de Proteínas/fisiología , Tauopatías/metabolismo , Proteínas tau/biosíntesis , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Degeneración Lobar Frontotemporal/genética , Degeneración Lobar Frontotemporal/metabolismo , Degeneración Lobar Frontotemporal/patología , Humanos , Degeneración Nerviosa/genética , Degeneración Nerviosa/patología , Tauopatías/genética , Tauopatías/patología , Proteínas tau/genéticaRESUMEN
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.
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Compuestos de Bifenilo/efectos adversos , Compuestos de Bifenilo/farmacocinética , Compuestos de Bifenilo/toxicidad , Lignanos/efectos adversos , Lignanos/farmacocinética , Lignanos/toxicidad , Animales , Compuestos de Bifenilo/análisis , Interacciones Farmacológicas , Humanos , Lignanos/análisis , Magnolia/química , Pruebas de Mutagenicidad , Extractos Vegetales/química , Distribución TisularRESUMEN
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.
Asunto(s)
Péptidos beta-Amiloides/antagonistas & inhibidores , Caenorhabditis elegans/efectos de los fármacos , Clusterina/farmacología , Fármacos Neuroprotectores/farmacología , Fragmentos de Péptidos/antagonistas & inhibidores , Faringe/efectos de los fármacos , Agregación Patológica de Proteínas/prevención & control , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/síntesis química , Péptidos beta-Amiloides/toxicidad , Animales , Bioensayo , Caenorhabditis elegans/fisiología , Clusterina/aislamiento & purificación , Humanos , Cinética , Larva/efectos de los fármacos , Larva/fisiología , Fármacos Neuroprotectores/aislamiento & purificación , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/toxicidad , Faringe/fisiología , Agregado de Proteínas/efectos de los fármacos , Agregación Patológica de Proteínas/patología , Unión ProteicaRESUMEN
RATIONALE: Butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) are widely used to prevent oxidation and rancidity in foodstuffs, pharmaceutical preparations and cosmetic formulations. Although their safety has been thoroughly investigated, possible endocrine side-effects have been suggested. A useful method for the determination of BHA and BHT in foods is needed to estimate their daily intake through the diet. METHODS: We selected commercial chewing gums as a model of a complex food matrix and developed a new method based on gas chromatography/mass spectrometry. This allows the determination of 130 pg/gum of BHA and 9 pg/gum of BHT. RESULTS: Analysis of different chewing gums from the European market indicated that the two antioxidants were never used together and that the content of BHA was in the range of 220-348 µg/gum and BHT ranged from 278 up to 479 µg/gum. These amounts correspond to 86-157 mg/kg gum for BHA and 170-185 mg/kg gum for BHT, and are both within the maximum levels established by the European Food Safety Authority. Chewing a piece of gum for 15 min resulted in the release of up to 28% of BHA, but no release of BHT was detectable. CONCLUSIONS: A new, simple and rapid method for the determination of BHA and BHT in chewing gums was described. This analytical method, based on headspace sampling, did not require the extraction of antioxidants from chewing gum samples, assuring the absence of any gum material contaminants that might affect the instrumentation. It is also automatable, employing a sequential automatic sampler. This method could be of interest to academic researchers and to food industrialists looking for a new methodological approach for BHA and BHT determination in foodstuffs with complex matrices. Copyright © 2017 John Wiley & Sons, Ltd.
RESUMEN
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.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Mutación/genética , Síndromes de Neurotoxicidad , Fragmentos de Péptidos/metabolismo , Secuencia de Aminoácidos , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/toxicidad , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Productos del Gen tat/genética , Productos del Gen tat/metabolismo , Humanos , Técnicas In Vitro , Trastornos del Movimiento/etiología , Unión Neuromuscular/fisiopatología , Síndromes de Neurotoxicidad/complicaciones , Síndromes de Neurotoxicidad/etiología , Síndromes de Neurotoxicidad/genética , Parálisis/etiología , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/toxicidad , Unión Proteica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Resonancia por Plasmón de SuperficieRESUMEN
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.
Asunto(s)
Amiloidosis/diagnóstico , Caenorhabditis elegans , Cardiopatías/diagnóstico , Cadenas Ligeras de Inmunoglobulina/inmunología , Adulto , Anciano , Amiloidosis/inmunología , Animales , Bioensayo , Cardiotoxinas/aislamiento & purificación , Cardiotoxinas/farmacología , Supervivencia Celular/efectos de los fármacos , Femenino , Cardiopatías/inmunología , Humanos , Masculino , Persona de Mediana Edad , Mieloma Múltiple/inmunología , Faringe/citología , Faringe/efectos de los fármacos , Faringe/fisiologíaRESUMEN
We describe the multigram synthesis and in vivo efficacy studies of a donepezilâhuprine 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.
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Enfermedad de Alzheimer/tratamiento farmacológico , Aminoquinolinas/administración & dosificación , Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/metabolismo , Compuestos Heterocíclicos de 4 o más Anillos/administración & dosificación , Indanos/administración & dosificación , Piperidinas/administración & dosificación , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Aminoquinolinas/química , Aminoquinolinas/uso terapéutico , Precursor de Proteína beta-Amiloide/genética , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans , Modelos Animales de Enfermedad , Donepezilo , Células Hep G2 , Compuestos Heterocíclicos de 4 o más Anillos/química , Compuestos Heterocíclicos de 4 o más Anillos/uso terapéutico , Humanos , Indanos/química , Indanos/uso terapéutico , Ratones , Estructura Molecular , Piperidinas/química , Piperidinas/uso terapéuticoRESUMEN
A hallmark of Alzheimer disease (AD) is the accumulation of the amyloid-ß (Aß) peptide in the brain. Considerable evidence suggests that soluble Aß oligomers are responsible for the synaptic dysfunction and cognitive deficit observed in AD. However, the mechanism by which these oligomers exert their neurotoxic effect remains unknown. Recently, it was reported that Aß oligomers bind to the cellular prion protein with high affinity. Here, we show that N1, the main physiological cleavage fragment of the cellular prion protein, is necessary and sufficient for binding early oligomeric intermediates during Aß polymerization into amyloid fibrils. The ability of N1 to bind Aß oligomers is influenced by positively charged residues in two sites (positions 23-31 and 95-105) and is dependent on the length of the sequence between them. Importantly, we also show that N1 strongly suppresses Aß oligomer toxicity in cultured murine hippocampal neurons, in a Caenorhabditis elegans-based assay, and in vivo in a mouse model of Aß-induced memory dysfunction. These data suggest that N1, or small peptides derived from it, could be potent inhibitors of Aß oligomer toxicity and represent an entirely new class of therapeutic agents for AD.
Asunto(s)
Péptidos beta-Amiloides/química , Priones/química , Enfermedad de Alzheimer/metabolismo , Proteínas Amiloidogénicas/química , Animales , Benzotiazoles , Caenorhabditis elegans/metabolismo , Hipocampo/metabolismo , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Enfermedades Neurodegenerativas/metabolismo , Neuronas/metabolismo , Péptidos/química , Unión Proteica , Estructura Terciaria de Proteína , Resonancia por Plasmón de Superficie , Sinapsis/metabolismo , Tiazoles/químicaRESUMEN
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-673âvaline-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.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/toxicidad , Neuronas/metabolismo , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/toxicidad , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/genética , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans , Humanos , Locomoción/efectos de los fármacos , Mutación , Neuronas/efectos de los fármacos , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genéticaRESUMEN
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.
Asunto(s)
Compuestos Orgánicos/toxicidad , Contaminantes del Suelo/toxicidad , Pruebas de Toxicidad/métodos , Animales , Células 3T3 BALB , Caenorhabditis elegans , Línea Celular Tumoral , Cucumis sativus , Conducta Alimentaria/efectos de los fármacos , Células Hep G2 , Humanos , Italia , Lepidium sativum , Neoplasias Hepáticas/inducido químicamente , Ratones , Compuestos Orgánicos/normas , Contaminantes del Suelo/normas , Sorghum , Pruebas de Toxicidad/normasRESUMEN
To increase the therapeutic efficacy of nanoparticle (NP)-assisted photothermal therapy (PTT) and allow for a transition toward the clinical setting, it is pivotal to characterize the thermal effect induced in cancer cells and correlate it with the cell biological response, namely cell viability and cell death pathways. This study quantitatively evaluated the effects of gold nanorod (GNR)-assisted near-infrared (NIR) PTT on two different cancer cell lines, the 4T1 triple-negative breast cancer cells and the Pan02 pancreatic cancer cells. The interaction between nanomaterials and biological matrices was investigated in terms of GNR internalization and effect on cell viability at different GNR concentrations. GNR-mediated PTT was executed on both cell lines, at the same treatment settings to allow a straightforward comparison, and real-time monitored through thermographic imaging. A thermal analysis based on various parameters (i.e., maximum absolute temperature, maximum temperature change, temperature variation profile, area under the time-temperature change curve, effective thermal enhancement (ETE), and time constants) was performed to evaluate the treatment thermal outcome. While GNR treatment and NIR laser irradiation alone did not cause cell toxicity in the selected settings, their combination induced a significant reduction of cell viability in both cell lines. At the optimal experimental condition (i.e., 6 µg/mL of GNRs and 4.5 W/cm2 laser power density), GNR-assisted PTT reduced the cell viability of 4T1 and Pan02 cells by 94% and 87% and it was associated with maximum temperature changes of 25 °C and 29 °C (i.e., â¼1.8-fold increase compared to the laser-only condition), maximum absolute temperatures of 55 °C and 54 °C, and ETE values of 78% and 81%, for 4T1 and Pan02 cells, correspondingly. Also, the increase in the GNR concentration led to a decrease in the time constants, denoting faster heating kinetics upon irradiation. Furthermore, the thermal analysis parameters were correlated with the extent of cell death. Twelve hours after NIR exposure, GNR-assisted PTT was found to mainly trigger secondary apoptosis in both cell lines. The proposed study provides relevant insights into the relationship between temperature history and biological responses in the context of PTT. The findings contribute to the development of a universal methodology for evaluating thermal sensitivity upon NP-assisted PTT on different cell types and lay the groundwork for future translational studies.