Expresión y purificación de una proteína que funciona como un canal iónico de potasio (KcsA)

La importancia de los canales iónicos radica, no sólo en su implicación en procesos biológicos clave, sino en el hecho de que son dianas terapéuticas reales y potenciales. Entre estos canales iónicos encontramos los canales de potasio, involucrados en numerosos procesos celulares de vital importancia. Debido a la complejidad de estas proteínas, durante los últimos años se ha prestado especial atención a la expresión de sus homólogos procariotas, en este sentido, el canal KcsA constituye un modelo sencillo para el estudio de canales de potasio eucariotas más complejos. Durante este proyecto, se ha llevado a cabo la expresión y caracterización funcional de un canal de potasio de origen procariota: KcsA. Para ello se han utilizado dos cepas de E.coli: BL21 y M15, debido a su capacidad de expresión de la proteína de interés. Con este trabajo, se pretende determin ar el rendimiento de expresión de la proteína en ambas cepas y estudiar sus características funcionales. Tras finalizar los experimentos de expresión de la proteína y purificación, se obtuvo que la cepa BL21 presentó mayor rendimiento que la cepa M15 a la hora de expresar el canal KcsA. Por otra parte, mediante medidas electrofisiológicas se obtuvieron registros de rampas y continuos a -150 y +150mV similares en KcsA-WT expresado en ambas cepas. Por todo lo expuesto se concluye que se puede expresar indistintamente KcsA-WT en ambas cepas con resultados satisfactorios, siendo la cepa BL21 la que presenta un mayor rendimiento

Oligomeric and Fibrillar Species of Aβ42 Diversely Affect Human Neural Stem Cells.

Amyloid-β 42 peptide (Aβ1-42 (Aβ42)) is well-known for its involvement in the development of Alzheimer's disease (AD). Aβ42 accumulates and aggregates in fibers that precipitate in the form of plaques in the brain causing toxicity; however, like other forms of Aβ peptide, the role of these peptides remains unclear. Here we analyze and compare the effects of oligomeric and fibrillary Aβ42 peptide on the biology (cell death, proliferative rate, and cell fate specification) of differentiating human neural stem cells (hNS1 cell line). By using the hNS1 cells we found that, at high concentrations, oligomeric and fibrillary Aβ42 peptides provoke apoptotic cellular death and damage of DNA in these cells, but Aβ42 fibrils have the strongest effect. The data also show that both oligomeric and fibrillar Aβ42 peptides decrease cellular proliferation but Aβ42 oligomers have the greatest effect. Finally, both, oligomers and fibrils favor gliogenesis and neurogenesis in hNS1 cells, although, in this case, the effect is more prominent in oligomers. All together the findings of this study may contribute to a better understanding of the molecular mechanisms involved in the pathology of AD and to the development of human neural stem cell-based therapies for AD treatment.This work was supported by grants from the Spanish Ministry of Science and Innovation (RTI2018-101663-B-100), MICINN-ISCIII (PI-10/00291 and MPY1412/09), MINECO (SAF2015-71140-R), and Comunidad de Madrid (NEUROSTEMCM consortium; S2010/BMD-2336).S

Advances in Alzheimer’s Disease Research: Human Cerebral Organoids

Alzheimer’s disease (AD) is the main neurodegenerative disorder in old age, causing memory impairment and dependency. The histopathology of AD is characterized by the presence of amyloid plaques and neurofibrillary tangles formed by Aβ peptide and hyperphosphorylated Tau, respectively. There is still no cure or effective treatment for AD. This could be due, in part, to the lack of suitable research models since animal models do not recapitulate the full physiological complexity of the human brain. With the development of induced pluripotent stem cells (iPSCs), these limitations could be overcome. Even so, the bi-dimensional (2D) culture models still do not allow to recapitulate all types of brain cells and do not show a three-dimensional (3D) arrangement. Since obtaining 3D cultures called organoids, a new opportunity arises to overcome the limitations of previous models. Human Cerebral Organoids (hCOs) represent a pioneering model, in which part of the complexity of the human brain is present. For this reason, they are fast becoming a very remarkable model for the study of the evolution of the molecular and cellular pathology of AD. This review provides a brief overview of AD research, focusing on the most recent advances achieved through the development of stem cell and cerebral organoid technologyThe authors would like to thank to financing entities: i. State R+D+i Program Oriented to the Challenges of Society. Ministry of Science and Innovation (PID2021-126715OB-I00). ii. Strategic Action in Intramural Health (PI22/00055). iii. Ministry of Science and Innovation and Universities, within the program “R&D Projects «Retos Investigación» (RTI2018-101663-B-100).S

Oligomeric and Fibrillar Species of Aβ42 Diversely Affect Human Neural Stem Cells

Amyloid-β 42 peptide (Aβ1-42 (Aβ42)) is well-known for its involvement in the development of Alzheimer’s disease (AD). Aβ42 accumulates and aggregates in fibers that precipitate in the form of plaques in the brain causing toxicity; however, like other forms of Aβ peptide, the role of these peptides remains unclear. Here we analyze and compare the effects of oligomeric and fibrillary Aβ42 peptide on the biology (cell death, proliferative rate, and cell fate specification) of differentiating human neural stem cells (hNS1 cell line). By using the hNS1 cells we found that, at high concentrations, oligomeric and fibrillary Aβ42 peptides provoke apoptotic cellular death and damage of DNA in these cells, but Aβ42 fibrils have the strongest effect. The data also show that both oligomeric and fibrillar Aβ42 peptides decrease cellular proliferation but Aβ42 oligomers have the greatest effect. Finally, both, oligomers and fibrils favor gliogenesis and neurogenesis in hNS1 cells, although, in this case, the effect is more prominent in oligomers. All together the findings of this study may contribute to a better understanding of the molecular mechanisms involved in the pathology of AD and to the development of human neural stem cell-based therapies for AD treatment

Efectos de los péptidos Ab40 y Ab42 en la biología de las células madre neurales humanas y la generación de organoides cerebrales humanos

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 03-07-2020Esta tesis tiene embargado el acceso al texto completo hasta el 03-01-2022Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by memory loss and cognitive decline that represents the most common cause of dementia in the elderly population. One of the main histopathologic features of AD is the formation and accumulation of amyloid plaques in the brain. They are formed by extracellular fibrillary aggregates of amyloid-β peptides (Aβ) generated by the enzymatic processing of amyloid precursor protein (APP). Aβ40 and Aβ42 peptides are the main isoforms found in these amyloid plaques. Although considered neurotoxic, Aβ peptides also play an important biological role in the adult brain and during embryonic brain development. However, the physiological function of these Aβ peptides remains poorly understood and results are controversial. Aβ peptides can be detected in non-aggregated (monomeric) and aggregated forms (oligomeric and fibrillary) and each form has different cytotoxic and physiological properties. In order to better understand the physiological/pathological role of these Aβ peptides, in this thesis, we studied the effects of both Aβ peptides (Aβ40 and Aβ42) at different concentrations and different aggregation states (monomeric, oligomeric and fibrillary), on cell death, cell proliferation and cell fate specification of human neural stem cells (human NSCs) by immunocytochemistry and qRT-PCR. Our results showed that higher concentrations of both Aβ peptides are cytotoxic for these cells in all cases, but the fibrillary form is the most toxic. Regarding cell proliferation, we observed a significant increase after treatment with both Aβ peptides in monomeric form; however, we observed a decrease or no change when we studied the other forms. Finally, concerning cell fate specification, our data showed that treatment with Aβ peptides, in all states tested, significantly affects differentiation of human NSCs. Together, our results suggest that Aβ peptides affect human NSCs biology and provide us with information about their physiological/pathological role. NSCs have the capability of self-renewal and differentiate into functional glial and neuronal cells, making these cells a good tool for the study of neurodegenerative diseases and for future therapeutic applications in diseases such as AD. However, although highly valuable, they are devoid of the tridimensional component necessary for normal organ development. It has recently been described that pluripotent stem cells in a suitable environment are capable of generating three-dimensional (3D) structures called "cerebral organoids”. These 3D structures have revealed important similarities and differences between their development and that of the brain in vivo, and therefore represent a promising in vitro model to understand how the brain develops, to study the physiological pathways during development and the molecular pathology associated with neurodegenerative disorders, such as A

Physiological and pathological effects of amyloid-β species in neural stem cell biology

Although amyloid-β peptide is considered neurotoxic, it may mediate several physiological processes during embryonic development and in the adult brain. The pathological function of amyloid-β peptide has been extensively studied due to its implication in Alzheimer's disease, but its physiological function remains poorly understood. Amyloid-β peptide can be detected in non-aggregated (monomeric) and aggregated (oligomeric and fibrillary) forms. Each form has different cytotoxic and/or physiological properties, so amyloid-β peptide and its role in Alzheimer's disease need to be studied further. Neural stem cells and neural precursor cells are good tools for the study on neurodegenerative diseases and can provide future therapeutic applications in diseases such as Alzheimer's disease. In this review, we provide an outline of the effects of amyloid-β peptide, in monomeric and aggregated forms, on the biology of neural stem cells/neural precursor cells, and discuss the controversies. We also describe the possible molecular targets that could be implicated in these effects, especially GSK3β. A better understanding of amyloid-β peptide (both physiological and pathological), and the signaling pathways involved are essential to advance the field of Alzheimer's disease.Funding: This work was supported by grants from the MICINN-ISCIII (PI-10/00291 and MPY1412/09), MINECO (SAF2015-71140-R) and Comunidad de Madrid (NEUROSTEMCM consortium; S2010/BMD-2336)S

Physiological effects of amyloid precursor protein and its derivatives on neural stem cell biology and signaling pathways involved

The pathological implication of amyloid precursor protein (APP) in Alzheimer's disease has been widely documented due to its involvement in the generation of amyloid-β peptide. However, the physiological functions of APP are still poorly understood. APP is considered a multimodal protein due to its role in a wide variety of processes, both in the embryo and in the adult brain. Specifically, APP seems to play a key role in the proliferation, differentiation and maturation of neural stem cells. In addition, APP can be processed through two canonical processing pathways, generating different functionally active fragments: soluble APP-α, soluble APP-β, amyloid-β peptide and the APP intracellular C-terminal domain. These fragments also appear to modulate various functions in neural stem cells, including the processes of proliferation, neurogenesis, gliogenesis or cell death. However, the molecular mechanisms involved in these effects are still unclear. In this review, we summarize the physiological functions of APP and its main proteolytic derivatives in neural stem cells, as well as the possible signaling pathways that could be implicated in these effects. The knowledge of these functions and signaling pathways involved in the onset or during the development of Alzheimer's disease is essential to advance the understanding of the pathogenesis of Alzheimer's disease, and in the search for potential therapeutic targets.Financial support: This work was supported by grants from the Ministerio de Ciencia e Innovación-Instituto de Salud Carlos III (PI-10/00291 and MPY1412/09), Ministerio de Economía y Competitividad (SAF2015-71140-R) and Comunidad de Madrid (Neurostem-Comunidad de Madrid consortium; S2010/BMD-2336). RC was supported by grants from Plan de Empleo Juvenil-Ministerio de Economía y Competitividad.S

Evaluation and Optimization of Poly-d-Lysine as a Non-Natural Cationic Polypeptide for Gene Transfer in Neuroblastoma Cells

Cationic polypeptides and cationic polymers have cell-penetrating capacities and have been used in gene transfer studies. In this study, we investigate the capability of a polymer of d-lysine (PDL), a chiral form of α–Poly-lysine, as a possible nonviral vector for releasing genetic materials to neuroblastoma cells and evaluate its stability against proteases. We tested and compared its transfection effectiveness in vitro as a vehicle for the EGFP plasmid DNA (pDNA) reporter in the SH-SY5Y human neuroblastoma, HeLa, and 3T3 cell lines. Using fluorescent microscopy and flow cytometry, we demonstrated high transfection efficiencies based on EGFP fluorescence in SH-SY5Y cells, compared with HeLa and 3T3. Our results reveal PDL as an efficient vector for gene delivery specifically in the SH-SY5Y cell line and suggest that PDL can be used as a synthetic cell-penetrating polypeptide for gene therapy in neuroblastoma cells

Efficient generation of human cerebral organoids directly from adherent cultures of pluripotent stem cells

Human cerebral organoids (hCOs) offer the possibility of deepening the knowledge of human brain development, as well as the pathologies that affect it. The method developed here describes the efficient generation of hCOs by going directly from two-dimensional (2D) pluripotent stem cell (PSC) cultures to three-dimensional (3D) neuroepithelial tissue, avoiding dissociation and aggregation steps. This has been achieved by subjecting 2D cultures, from the beginning of the neural induction step, to dual-SMAD inhibition in combination with CHIR99021. This is a simple and reproducible protocol in which the hCOs generated develop properly presenting proliferative ventricular zones (VZs) formed by neural precursor and radial glia (RG) that differentiate to give rise to mature neurons and glial cells. The hCOs present additional cell types such as oligodendrocyte precursors, astrocytes, microglia-like cells, and endothelial-like cells. This new approach could help to overcome some of the existing limitations in the field of organoid biotechnology, facilitating its execution in any laboratory setting.The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Grant PID2021-126715OB-I00 funded by MCIN/AEI/10.13039/501100011033 and “ERDF A way of making Europe,” by the Grant of Instituto de Salud Carlos III (ISCIII) PI22CIII/00055, grant RTI2018-101663-B-100 funded by MCIN/AEI/ and the UFIECPY 398/19, PEJ2018-004965 grant to RGS funded by AEI.S

Neurogenesis Is Increased in Human Neural Stem Cells by Aβ40 Peptide

Amyloid-β 40 peptides [Aβ1-40 (Aβ40)] are present within amyloid plaques in the brains of patients with Alzheimer's disease (AD). Even though Aβ peptides are considered neurotoxic, they can mediate many biological processes, both in adult brains and throughout brain development. However, the physiological function of these Aβ peptides remains poorly understood, and the existing data are sometimes controversial. Here, we analyze and compare the effects of monomeric Aβ40 on the biology of differentiating human neural stem cells (human NSCs). For that purpose, we used a model of human NSCs called hNS1. Our data demonstrated that Aβ40 at high concentrations provokes apoptotic cellular death and the damage of DNA in human NSCs while also increasing the proliferation and favors neurogenesis by raising the percentage of proliferating neuronal precursors. These effects can be mediated, at least in part, by β-catenin. These results provide evidence of how Aβ modulate/regulate human NSC proliferation and differentiation, suggesting Aβ40 may be a pro-neurogenic factor. Our data could contribute to a better understanding of the molecular mechanisms involved in AD pathology and to the development of human NSC-based therapies for AD treatment, since these results could then be used in diagnosing the disease at early stages and be applied to the development of new treatment options.This work was supported by grants from the Spanish Ministry of Science and Innovation (RTI2018-101663-B-100), MICINN-ISCIII (PI-10/00291 and MPY1412/09); MINECO (SAF2015-71140-R) and Comunidad de Madrid (NEUROSTEMCM consortium; S2010/BMD-2336)S