Amino acid analog toxicity in primary rat neuronal and astrocyte cultures: implications for protein misfolding and tdp-43 regulation

Amino acid analogs promote translational errors that result in aberrant protein synthesis, and have been used to understand the effects of protein misfolding in a variety of physiological and pathological settings. TDP-43 is a protein that is linked to protein aggregation and toxicity in a variety of neurodegenerative diseases. In this study we exposed primary rat neurons and astrocyte cultures to established amino acid analogs (Canavanine and Azetidine-2-carboxylic acid), and observed both cell types undergo a dose-dependent increase in toxicity, with neurons exhibiting a greater degree of toxicity as compared to astrocytes. Neurons and astrocytes exhibited similar increases in ubiquitinated and oxidized protein following analog treatment. Analog treatment increased Heat shock protein (Hsp) levels in both neurons and astrocytes. In neurons, and to a lesser extent astrocytes, the levels of TDP-43 increased in response to analog treatment. Taken together, these data indicate that neurons exhibit preferential toxicity and alterations in TDP-43, in response to increased protein misfolding, as compared to astrocytes

Protein Quality Control Systems and ER Stress as Key Players in SARS-CoV-2-Induced Neurodegeneration

The COVID-19 pandemic has brought to the forefront the intricate relationship between SARS-CoV-2 and its impact on neurological complications, including potential links to neurodegenerative processes, characterized by a dysfunction of the protein quality control systems and ER stress. This review article explores the role of protein quality control systems, such as the Unfolded Protein Response (UPR), the Endoplasmic Reticulum-Associated Degradation (ERAD), the Ubiquitin– Proteasome System (UPS), autophagy and the molecular chaperones, in SARS-CoV-2 infection. Our hypothesis suggests that SARS-CoV-2 produces ER stress and exploits the protein quality control systems, leading to a disruption in proteostasis that cannot be solved by the host cell. This disruption culminates in cell death and may represent a link between SARS-CoV-2 and neurodegeneration

Dysfunction of the unfolded protein response increases neurodegeneration in aged rat hippocampus following proteasome inhibition

Dysfunctions of the ubiquitin proteasome system (UPS) have been proposed to be involved in the aetiology and/or progression of several age-related neurodegenerative disorders. However, the mechanisms linking proteasome dysfunction to cell degeneration are poorly understood. We examined in young and aged rat hippocampus the activation of the unfolded protein response (UPR) under cellular stress induced by proteasome inhibition. Lactacystin injection blocked proteasome activity in young and aged animals in a similar extent and increased the amount of ubiquitinated proteins. Young animals activated the three UPR arms, IRE1α, ATF6α and PERK, whereas aged rats failed to induce the IRE1α and ATF6α pathways. In consequence, aged animals did not induce the expression of pro-survival factors (chaperones, Bcl-XL and Bcl-2), displayed a more sustained expression of proapoptotic markers (CHOP, Bax, Bak and JKN), an increased caspase-3 processing. At the cellular level, proteasome inhibition induced neuronal damage in young and aged animals as assayed using Fluorojade-B staining. However, degenerating neurons were evident as soon as 24 h postinjection in aged rats, but it was delayed up to 3 days in young animals. Our findings show evidence supporting age-related dysfunctions in the UPR activation as a potential mechanism linking protein accumulation to cell degeneration. An imbalance between pro-survival and pro-apoptotic proteins, because of noncanonical activation of the UPR in aged rats, would increase the susceptibility to cell degeneration. These findings add a new molecular vision that might be relevant in the aetiology of several age-related neurodegenerative disorders

Lipopolysacharide-induced neuroinflammation leads to the accumulation of ubiquitinated proteins and increases susceptibility to neurodegeneration induced by proteasome inhibition in rat hippocampus

BACKGROUND: Neuroinflammation and protein accumulation are characteristic hallmarks of both normal aging and age-related neurodegenerative diseases. However, the relationship between these factors in neurodegenerative processes is poorly understood. We have previously shown that proteasome inhibition produced higher neurodegeneration in aged than in young rats, suggesting that other additional age-related events could be involved in neurodegeneration. We evaluated the role of lipopolysaccharide (LPS)-induced neuroinflammation as a potential synergic risk factor for hippocampal neurodegeneration induced by proteasome inhibition. METHODS: Young male Wistar rats were injected with 1 μL of saline or LPS (5 mg/mL) into the hippocampus to evaluate the effect of LPS-induced neuroinflammation on protein homeostasis. The synergic effect of LPS and proteasome inhibition was analyzed in young rats that first received 1 μL of LPS and 24 h later 1 μL (5 mg/mL) of the proteasome inhibitor lactacystin. Animals were sacrificed at different times post-injection and hippocampi isolated and processed for gene expression analysis by real-time polymerase chain reaction; protein expression analysis by western blots; proteasome activity by fluorescence spectroscopy; immunofluorescence analysis by confocal microscopy; and degeneration assay by Fluoro-Jade B staining. RESULTS: LPS injection produced the accumulation of ubiquitinated proteins in hippocampal neurons, increased expression of the E2 ubiquitin-conjugating enzyme UB2L6, decreased proteasome activity and increased immunoproteasome content. However, LPS injection was not sufficient to produce neurodegeneration. The combination of neuroinflammation and proteasome inhibition leads to higher neuronal accumulation of ubiquitinated proteins, predominant expression of pro-apoptotic markers and increased neurodegeneration, when compared with LPS or lactacystin (LT) injection alone. CONCLUSIONS: Our results identify neuroinflammation as a risk factor that increases susceptibility to neurodegeneration induced by proteasome inhibition. These results highlight the modulation of neuroinflammation as a mechanism for neuronal protection that could be relevant in situations where both factors are present, such as aging and neurodegenerative diseases

Breast cancer cell line MCF7 escapes from G1/S arrest induced by proteasome inhibition through a GSK-3β dependent mechanism

Targeting the ubiquitin proteasome pathway has emerged as a rational approach in the treatment of human cancers. Autophagy has been described as a cytoprotective mechanism to increase tumor cell survival under stress conditions. Here, we have focused on the role of proteasome inhibition in cell cycle progression and the role of autophagy in the proliferation recovery. The study was performed in the breast cancer cell line MCF7 compared to the normal mammary cell line MCF10A. We found that the proteasome inhibitor MG132 induced G1/S arrest in MCF10A, but G2/M arrest in MCF7 cells. The effect of MG132 on MCF7 was reproduced on MCF10A cells in the presence of the glycogen synthase kinase 3β (GSK-3β) inhibitor VII. Similarly, MCF7 cells overexpressing constitutively active GSK-3β behaved like MCF10A cells. On the other hand, MCF10A cells remained arrested after MG132 removal while MCF7 recovered the proliferative capacity. Importantly, this recovery was abolished in the presence of the autophagy inhibitor 3-methyladenine (3-MA). Thus, our results support the relevance of GSK-3β and autophagy as two targets for controlling cell cycle progression and proliferative capacity in MCF7, highlighting the co-treatment of breast cancer cells with 3-MA to synergize the effect of the proteasome inhibition

Neuroprotective effect of rice bran enzymatic extract-supplemented diets in a murine model of Parkinsońs disease

Neurodegenerative disorders such as Parkinson's disease, present a global health concern with limited therapeutic options. In this context, dietary interventions have emerged as a potential strategy to counteract the oxidative stress and inflammation underlying these conditions. Rice bran enzymatic extract (RBEE), rich in bioactive compounds, has shown ability in modulating neuroinflammatory responses and improving mitochondrial function. This study investigates the neuroprotective potential of RBEE in a murine model of PD induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The results reveal that RBEE supplementation effectively preserves the dopaminergic population and reduces MPTP-induced gliosis in the Substantia Nigra (SN). Moreover, RBEE enhances mitochondrial Complex I activity in mouse brains. These findings underscore the potential of RBEE as a dietary supplement to mitigate neuronal loss, neuroinflammation, and mitochondrial dysfunction associated with PD. RBEE is shown as a promising novel candidate in neuroprotective strategies, offering hope in the quest for effective preventive and therapeutic measures in PD

“Farmaling”: Implementación y adaptación de una plataforma virtual de simulación para la docencia de Biología Molecular y Biotecnología

Tras una época de cambios sin precedentes en la educación mundial, tanto escuelas como universidades han adoptado métodos de enseñanza a distancia y virtual, haciendo que las herramientas que los educadores usan para sus cursos evolucionen. Las crisis globales presentes y futuras requieren soluciones científicas avanzadas, por lo que preparar a los estudiantes para carreras científicas y técnicas nunca ha sido más importante. Herramientas que ofrezcan soluciones sencillas y virtuales con pocos requerimientos (una dirección de correo electrónico y conexión a internet) son esenciales para que los profesores transmitan sus conocimientos y experiencia a sus estudiantes de forma efectiva. Este estudio implementa una plataforma virtual de Biología Molecular y Biotecnología (www.benchling.com) como una herramienta didáctica, centrando el aprendizaje del alumno en la resolución de problemas y la colaboración. Esta plataforma, basada en la nube, se utiliza para diseñar, registrar y analizar experimentos. Adaptada como una herramienta de aprendizaje virtual, puede ayudar a emular el proceso científico sin estar en el laboratorio, permitiendo al alumno familiarizarse con cada paso de un experimento, desde el diseño hasta el análisis de los datos y la documentación del proceso en libros de protocolos electrónicos. Proporciona amplias oportunidades para el aprendizaje práctico y exploratorio. Si bien las soluciones virtuales no sustituyen la experiencia práctica en el laboratorio, la familiaridad con el proceso experimental que aportan estas plataformas tiene beneficios clave que se extienden mucho más allá del aula. Su uso facilita el acceso homogéneo a los materiales e información, facilita el aprendizaje asíncrono, el trabajo en equipo, reduce el riesgo de exposición a sustancias peligrosas en el laboratorio a alumnos sin experiencia previa y proporciona un conjunto de habilidades transferibles para el próximo paso formativo o profesional de los estudiantes, ya sea académico o en el sector privado.After a period of unprecedented changes in worldwide education, both schools and universities have adopted distance and virtual teaching methods, forcing the tools that educators use for their courses to evolve. Present and future global crises require advanced scientific solutions, so preparing students for scientific and technical careers has never been more important. Tools that offer simple and virtual solutions with few requirements (an email address and internet connection) are essential for teachers to effectively communicate their knowledge and experience to their students. This study implements a virtual platform for Molecular Biology and Biotechnology (www.benchling.com) as a didactic tool, focusing student´s learning on problem solving and collaboration. This cloud-based platform is used to design, record, and analyze experiments. Adapted as a virtual learning tool, it can help emulate the scientific process without being in the laboratory, allowing the student to become familiar with each step of an experiment, from the initial design to data analysis and documentation of the process in electronic notebooks. While virtual solutions are not a substitute for hands-on experience in the lab, the familiarity with the experimental process that these platforms bring has key benefits that extend well beyond the classroom. Its use facilitates homogeneous access to materials and information, facilitates asynchronous learning, teamwork, reduces the risk of exposure to hazardous substances in the laboratory to students without previous experience, it offers abundant opportunities for exploratory learning, and provides a set of transferable skills for the student’s next professional or academic steps.IV Plan Propio de Docencia de la Universidad de Sevill