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Alterations in autophagy are increasingly being recognized in the pathogenesis of proteinopathies like Alzheimer's disease (AD). This study was conducted to evaluate whether melatonin treatment could provide beneficial effects in an Alzheimer model related to tauopathy by improving the autophagic flux and, thereby, prevent cognitive decline. The injection of AAV-hTauP301L viral vectors and treatment/injection with okadaic acid were used to achieve mouse and human ex vivo, and in vivo tau-related models. Melatonin (10 µmol/L) impeded oxidative stress, tau hyperphosphorylation, and cell death by restoring autophagy flux in the ex vivo models. In the in vivo studies, intracerebroventricular injection of AAV-hTauP301L increased oxidative stress, neuroinflammation, and tau hyperphosphorylation in the hippocampus 7 days after the injection, without inducing cognitive impairment; however, when animals were maintained for 28 days, cognitive decline was apparent. Interestingly, late melatonin treatment (10 mg/kg), starting once the alterations mentioned above were established (from day 7 to day 28), reduced oxidative stress, neuroinflammation, tau hyperphosphorylation, and caspase-3 activation; these observations correlated with restoration of the autophagy flux and memory improvement. This study highlights the importance of autophagic dysregulation in tauopathy and how administration of pharmacological doses of melatonin, once tauopathy is initiated, can restore the autophagy flux, reduce proteinopathy, and prevent cognitive decline. We therefore propose exogenous melatonin supplementation or the development of melatonin derivatives to improve autophagy flux for the treatment of proteinopathies like AD.
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Enfermedad de Alzheimer/tratamiento farmacológico , Muerte Celular Autofágica/efectos de los fármacos , Disfunción Cognitiva/tratamiento farmacológico , Melatonina/farmacología , Estrés Oxidativo/efectos de los fármacos , Enfermedad de Alzheimer/inducido químicamente , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Animales , Disfunción Cognitiva/inducido químicamente , Disfunción Cognitiva/genética , Disfunción Cognitiva/metabolismo , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Ratones , Ratas , Ratas Sprague-DawleyRESUMEN
Aggregates, in particular high molecular weight species (HMWs), have been linked to increased immunogenicity. The current understanding on the impact of HMWs is mainly based on in vitro and nonclinical studies and there are only limited data available associating differences in HMWs in marketed monoclonal antibodies (mAbs) to clinical outcomes. Biosimilars offer a unique opportunity to study the potential association between quality parameters and clinical outcomes. We performed a retrospective evaluation to investigate the association between HMW content and reported antidrug-antibody (ADA) incidence in 30 full-length biosimilar mAbs approved in the European Union and the United States. Information for HMW content and ADA incidence were collected from publicly available sources. Differences in HMW content between biosimilars and their reference products (RPs) ranged from -0.75 to 1.65% with slightly higher differences observed in antineoplastic products as compared with immunosuppressant products. The difference in the ADA incidence between the RP and the biosimilar for the programs studied ranged from -11.0 to 18.5%. No association was observed between differences in HMW content and reported ADA incidence, in neither phase I nor phase III studies. Our results show that the limited differences in the content of HMWs between marketed biosimilars and reference mAbs were not associated with differences in reported immunogenicity, determined as incidence of ADAs and neutralizing ADAs in comparative clinical studies.
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Biosimilares Farmacéuticos , Humanos , Estados Unidos , Estudios Retrospectivos , Anticuerpos Monoclonales , Inmunosupresores , Unión EuropeaRESUMEN
Aggregates of the microtubule-associated protein tau are a common marker of neurodegenerative diseases collectively termed as tauopathies, such as Alzheimer's disease (AD) and frontotemporal dementia. Therapeutic strategies based on tau have failed in late stage clinical trials, suggesting that tauopathy may be the consequence of upstream causal mechanisms. As increasing levels of reactive oxygen species (ROS) may trigger protein aggregation or modulate protein degradation and, we had previously shown that the ROS producing enzyme NADPH oxidase 4 (NOX4) is a major contributor to cellular autotoxicity, this study was designed to evaluate if NOX4 is implicated in tauopathy. Our results show that NOX4 is upregulated in patients with frontotemporal lobar degeneration and AD patients and, in a humanized mouse model of tauopathy induced by AVV-TauP301L brain delivery. Both, global knockout and neuronal knockdown of the Nox4 gene in mice, diminished the accumulation of pathological tau and positively modified established tauopathy by a mechanism that implicates modulation of the autophagy-lysosomal pathway (ALP) and, consequently, improving the macroautophagy flux. Moreover, neuronal-targeted NOX4 knockdown was sufficient to reduce neurotoxicity and prevent cognitive decline, even after induction of tauopathy, suggesting a direct and causal role for neuronal NOX4 in tauopathy. Thus, NOX4 is a previously unrecognized causative, mechanism-based target in tauopathies and blood-brain barrier permeable specific NOX4 inhibitors could have therapeutic potential even in established disease.
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Enfermedad de Alzheimer , Demencia Frontotemporal , Tauopatías , Enfermedad de Alzheimer/genética , Animales , Encéfalo/metabolismo , Demencia Frontotemporal/metabolismo , Humanos , Ratones , NADPH Oxidasa 4/genética , NADPH Oxidasa 4/metabolismo , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Tauopatías/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismoRESUMEN
Tauopathies such as Alzheimer's disease are characterized by the accumulation of neurotoxic aggregates of tau protein. With aging and, especially, in Alzheimer's patients, the inducible enzyme heme oxygenase 1 (HO-1) progressively increases in microglia, causing iron accumulation, neuroinflammation, and neurodegeneration. The retina is an organ that can be readily accessed and can reflect changes that occur in the brain. In this context, we evaluated how the lack of microglial HO-1, using mice that do not express HO-1 in microglia (HMO-KO), impacts retinal macro and microgliosis of aged subjects (18 months old mice) subjected to tauopathy by intrahippocampal delivery of AAV-hTauP301L (TAU). Our results show that although tauopathy, measured as anti-TAUY9 and anti-AT8 positive immunostaining, was not observed in the retina of WT-TAU or HMO-KO+TAU mice, a morphometric study of retinal microglia and macroglia showed significant retinal changes in the TAU group compared to the WT group, such as: (i) increased number of activated microglia, (ii) retraction of microglial processes, (iii) increased number of CD68+ microglia, and (iv) increased retinal area occupied by GFAP (AROA) and C3 (AROC3). This retinal inflammatory profile was reduced in HMO-KO+TAU mice. Conclusion: Reduction of microglial HO-1 could be beneficial to prevent tauopathy-induced neuroinflammation.
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By replacing a phenolic ring of (E)-resveratrol with an 1,3,4-oxadiazol-2(3H)-one heterocycle, new resveratrol-based multitarget-directed ligands (MTDLs) were obtained. They were evaluated in several assays related to oxidative stress and inflammation (monoamine oxidases, nuclear erythroid 2-related factor, quinone reductase-2, and oxygen radical trapping) and then in experiments of increasing complexity (neurogenic properties and neuroprotection vs okadaic acid). 5-[(E)-2-(4-Methoxyphenyl)ethenyl]-3-(prop-2-yn-1-yl)-1,3,4-oxadiazol-2(3H)-one (4e) showed a well-balanced MTDL profile: cellular activation of the NRF2-ARE pathway (CD = 9.83 µM), selective inhibition of both hMAO-B and QR2 (IC50s = 8.05 and 0.57 µM), and the best ability to promote hippocampal neurogenesis. It showed a good drug-like profile (positive in vitro central nervous system permeability, good physiological solubility, no glutathione conjugation, and lack of PAINS or Lipinski alerts) and exerted neuroprotective and antioxidant actions in both acute and chronic Alzheimer models using hippocampal tissues. Thus, 4e is an interesting MTDL that could stimulate defensive and regenerative pathways and block early events in neurodegenerative cascades.
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Monoaminooxidasa , Fármacos Neuroprotectores , Antioxidantes/metabolismo , Antioxidantes/farmacología , Ligandos , Monoaminooxidasa/metabolismo , Inhibidores de la Monoaminooxidasa/farmacología , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Estrés Oxidativo , Resveratrol/farmacologíaRESUMEN
Heme oxygenase-1 (HO-1) is an inducible enzyme known for its anti-inflammatory, antioxidant and neuroprotective effects. However, increased expression of HO-1 during aging and age-related neurodegenerative diseases have been associated to neurotoxic ferric iron deposits. Being microglia responsible for the brain's innate immune response, the aim of this study was to understand the role of microglial HO-1 under inflammatory conditions in aged mice. For this purpose, aged wild type (WT) and LysMCreHmox1â³â³ (HMOX1M-KO) mice that lack HO-1 in microglial cells, were used. Aged WT mice showed higher basal expression levels of microglial HO-1 in the brain than adult mice. This increase was even higher when exposed to an inflammatory stimulus (LPS via i.p.) and was accompanied by alterations in different iron-related metabolism proteins, resulting in an increase of iron deposits, oxidative stress, ferroptosis and cognitive decline. Furthermore, microglia exhibited a primed phenotype and increased levels of inflammatory markers such as iNOS, p65, IL-1ß, TNF-α, Caspase-1 and NLRP3. Interestingly, all these alterations were prevented in aged HMOX1M-KO and WT mice treated with the HO-1 inhibitor ZnPPIX. In order to determine the effects of microglial HO-1-dependent iron overload, aged WT mice were treated with the iron chelator deferoxamine (DFX). DFX caused major improvements in iron, inflammatory and behavioral alterations found in aged mice exposed to LPS. In conclusion, this study highlights how microglial HO-1 overexpression contributes to neurotoxic iron accumulation providing deleterious effects in aged mice exposed to an inflammatory insult.
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Envejecimiento , Hemo-Oxigenasa 1 , Hierro/metabolismo , Microglía , Animales , Antiinflamatorios , Hemo-Oxigenasa 1/antagonistas & inhibidores , Hemo-Oxigenasa 1/genética , Lipopolisacáridos , Proteínas de la Membrana , RatonesRESUMEN
Neuroinflammation and oxidative stress are being recognized as characteristic hallmarks in many neurodegenerative diseases, especially those that portray proteinopathy, such as Alzheimer's disease (AD). Heme-oxygenase 1 (HO-1) is an inducible enzyme with antioxidant and anti-inflammatory properties, while microglia are the immune cells in the central nervous system. To elucidate the brain expression profile of microglial HO-1 in aging and AD-progression, we have used the 5xFAD (five familial AD mutations) mouse model of AD and their littermates at different ages (four, eight, 12, and 18 months). Total brain expression of HO-1 was increased with aging and such increase was even higher in 5xFAD animals. In co-localization studies, HO-1 expression was mainly found in microglia vs. other brain cells. The percentage of microglial cells expressing HO-1 and the amount of HO-1 expressed within microglia increased progressively with aging. Furthermore, this upregulation was increased by 2-3-fold in the elder 5xFAD mice. In addition, microglia overexpressing HO-1 was predominately found surrounding beta-amyloid plaques. These results were corroborated using postmortem brain samples from AD patients, where microglial HO-1 was found up-regulated in comparison to brain samples from aged matched non-demented patients. This study demonstrates that microglial HO-1 expression increases with aging and especially with AD progression, highlighting HO-1 as a potential biomarker or therapeutic target for AD.
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Alzheimer's disease is a chronic and irreversible pathological process that has become the most prevalent neurodegenerative disease. Currently, it is considered a multifactorial disease where oxidative stress and chronic neuroinflammation play a crucial role in its onset and development. Its characteristic neuronal loss has been related to the formation of neurofibrillary tangles mainly composed by hyperphosphorylated tau protein. Hyperphosphorylation of tau protein is related to the over-activity of GSK-3ß, a kinase that participates in several pathological mechanisms including neuroinflammation. Neuronal loss is also related to cytosolic Ca2+ homeostasis dysregulation that triggers apoptosis and free radicals production, contributing to oxidative damage and, finally, neuronal death. Under these premises, we have obtained a new family of 4,7-dihydro-2H-pyrazolo[3-b]pyridines as multitarget directed ligands showing potent antioxidant properties and able to scavenge both oxygen and nitrogen radical species, and also, with anti-inflammatory properties. Further characterization has demonstrated their capacity to inhibit GSK-3ß and to block L-type voltage dependent calcium channels. Novel derivatives have also demonstrated an interesting neuroprotective profile on in vitro models of neurodegeneration. Finally, compound 4g revokes cellular death induced by tau hyperphosphorylation in hippocampal slices by blocking reactive oxygen species (ROS) production. In conclusion, the multitarget profile exhibited by these compounds is a novel therapeutic strategy of potential interest in the search of novel treatments for Alzheimer's disease.
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Glycine-proline-glutamate (GPE) is a cleaved tripeptide of IGF-I that can be processed to cycloprolylglycine (cPG) in the brain. IGF-I protects the hippocampal somatostatinergic system from ß-amyloid (Aß) insult and although neither IGF-I-derived peptides bind to IGF-I receptors, they exert protective actions in several neurological disorders. As their effects on the hippocampal somatostatinergic system remain unknown, the objective of this study was to evaluate if cPG and/or GPE prevent the deleterious effects of Aß25-35 infusion on this system and whether changes in intracellular-related signaling and interleukin (IL) content are involved in their protective effect. We also determined the effect of cPG or GPE co-administration with Aß25-35 on IL secretion in glial cultures and the influence of these ILs on signaling activation and somatostatin synthesis in neuronal cultures. cPG or GPE co-administration reduced Aß-induced cell death and pro-inflammatory ILs, increased IL-4 and partially avoided the reduction of components of the somatostatinergic system affected by Aß25-35. GPE increased activation of Akt and CREB and reduced GSK3ß activation and astrogliosis, whereas cPG increased phosphorylation of extracellular signal-regulated kinases. Both peptides converged in the activation of mTOR and S6 kinase. Co-administration of these peptides with Aß25-35 to glial cultures increased IL-4 and reduced IL-1ß; this release of IL-4 could be responsible for activation of Akt and increased somatostatin in neuronal cultures. Our findings suggest that cPG and GPE exert protective effects against Aß on the somatostatinergic system by a reduction of the inflammatory environment that may activate different pro-survival pathways in these neurons.
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Péptidos beta-Amiloides/farmacología , Hipocampo/efectos de los fármacos , Inflamación/tratamiento farmacológico , Fármacos Neuroprotectores/uso terapéutico , Oligopéptidos/uso terapéutico , Fragmentos de Péptidos/farmacología , Péptidos Cíclicos/uso terapéutico , Somatostatina/metabolismo , Animales , Muerte Celular/efectos de los fármacos , Femenino , Hipocampo/metabolismo , Hipocampo/patología , Inflamación/metabolismo , Inflamación/patología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fármacos Neuroprotectores/farmacología , Oligopéptidos/farmacología , Péptidos Cíclicos/farmacología , Fosforilación/efectos de los fármacos , Ratas , Ratas Wistar , Receptores de Somatostatina/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
In this study we evaluate the chemical composition and neuroprotective effects of alkaloid fractions of the Amaryllidaceae species Rhodophiala pratensis, Rhodolirium speciosum, Phycella australis and Phaedranassa lehmannii. Gas chromatography-mass spectrometry (GC/MS) enable the identification of 41 known alkaloids. Rhodolirium speciosum and Rhodophiala pratensis were the most active extracts against acetylcholinesterase (AChE), with IC50 values of 35.22 and 38.13⯵g/mL, respectively. The protective effect of these extracts on human neuroblastoma cells (SH-SY5Y) subjected to mitochondrial oxidative stress with rotenone/oligomycin A (R/O) and toxicity promoted by okadaic acid (OA) was evaluated. Only Phycella australis and Rhodophiala pratensis at 0.75 and 1.5⯵g/mL, tend to reverse the cell death induced by R/O by around 12%. In OA assay, alkaloid fractions of Phycella Australis and Phaedranassa lehmannii displayed a concentration-dependent (0.375-3.0⯵g/mL) effect with a maximum neuroprotective response of 78% and 84%, respectively. Afterwards, neuroprotective effects of Phycella australis (3 and 6⯵g/mL) in mouse hippocampal slices stressed with oxygen glucose deprivation/reoxygenation (OGD/R), shown a protection greater than 14%. Finally, Phycella Australis (6⯵g/mL) reverted the cell viability from 65% to 90% in slices treated with OA, representing a protection of 25% attributable to the alkaloids of this species.
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Alcaloides de Amaryllidaceae/farmacología , Amaryllidaceae/química , Hipocampo/efectos de los fármacos , Isoquinolinas/farmacología , Neuroblastoma/metabolismo , Fármacos Neuroprotectores/farmacología , Estrés Oxidativo/efectos de los fármacos , Animales , Línea Celular Tumoral , Cromatografía de Gases y Espectrometría de Masas , Hipocampo/metabolismo , Humanos , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Neuroblastoma/patologíaRESUMEN
Acetylcholine α7 nicotinic receptors are widely expressed in the brain, where they are involved in the central processing of pain as well as in neuropsychiatric, neurodegenerative, and inflammatory processes. Positive allosteric modulators (PAMs) show the advantage of allowing the selective regulation of different subtypes of acetylcholine receptors without directly interacting with the agonist binding site. Here, we report the preparation and biological activity of a fluoro-containing compound, 1-(2',5'-dihydroxyphenyl)-3-(2-fluoro-4-hydroxyphenyl)-1-propanone (8, RGM079), that behaves as a potent PAM of the α7 receptors and has a balanced pharmacokinetic profile and antioxidant properties comparable or even higher than well-known natural polyphenols. In addition, compound RGM079 shows neuroprotective properties in Alzheimer's disease (AD)-toxicity related models. Thus, it causes a concentration-dependent neuroprotective effect against the toxicity induced by okadaic acid (OA) in the human neuroblastoma cell line SH-SY5Y. Similarly, in primary cultures of rat cortical neurons, RGM079 is able to restore the cellular viability after exposure to OA and amyloid peptide Aß1-42, with cell death almost completely prevented at 10 and 30 µM, respectively. Finally, compound RGM079 shows in vivo analgesic activity in the complete Freund's adjuvant (CFA)-induced paw inflammation model after intraperitoneal administration.
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Regulación Alostérica/efectos de los fármacos , Analgésicos/farmacología , Supervivencia Celular/efectos de los fármacos , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Dolor/tratamiento farmacológico , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo , Analgésicos/uso terapéutico , Animales , Línea Celular Tumoral , Humanos , Inflamación/metabolismo , Neuronas/metabolismo , Dolor/metabolismo , Dimensión del Dolor , RatasRESUMEN
Compound IG20 is a newly synthesised sulphated glycolipid that promotes neuritic outgrowth and myelinisation, at the time it causes the inhibition of glial proliferation and facilitates exocytosis in chromaffin cells. Here we have shown that IG20 at 0.3-10 µM afforded neuroprotection in rat hippocampal slices stressed with veratridine, glutamate or with oxygen plus glucose deprivation followed by reoxygenation (OGD/reox). Excess production of reactive oxygen species (ROS) elicited by glutamate or ODG/reox was prevented by IG20 that also restored the depressed tissue levels of GSH and ATP in hippocampal slices subjected to OGD/reox. Furthermore, the augmented iNOS expression produced upon OGD/reox exposure was also counteracted by IG20. Additionally, the IG20 elicited neuroprotection was prevented by the presence of inhibitors of the signalling pathways Jak2/STAT3, MEK/ERK1/2, and PI3K/Akt, consistent with the ability of the compound to increase the phosphorylation of Jak2, ERK1/2, and Akt. Thus, the activation of phase II response and the Nrf2/ARE pathway could explain the antioxidant and anti-inflammatory effects and the ensuing neuroprotective actions of IG20.