Loss of KEAP1 causes an accumulation of nondegradative organelles

KEAP1 is a cytoplasmic protein that functions as an adaptor for the Cullin-3-based ubiquitin E3 ligase system, which regulates the degradation of many proteins, including NFE2L2/NRF2 and p62/SQSTM1. Loss of KEAP1 leads to an accumulation of protein ubiquitin aggregates and defective autophagy. To better understand the role of KEAP1 in the degradation machinery, we investigated whether Keap1 deficiency affects the endosome-lysosomal pathway. We used KEAP1-deficient mouse embryonic fibroblasts (MEFs) and combined Western blot analysis and fluorescence microscopy with fluorometric and pulse chase assays to analyze the levels of lysosomal-endosomal proteins, lysosomal function, and autophagy activity. We found that the loss of keap1 downregulated the protein levels and activity of the cathepsin D enzyme. Moreover, KEAP1 deficiency caused lysosomal alterations accompanied by an accumulation of autophagosomes. Our study demonstrates that KEAP1 deficiency increases nondegradative lysosomes and identifies a new role for KEAP1 in lysosomal function that may have therapeutic implications

El factor de transcripción Nrf2 como nueva diana terapéutica para la Enfermedad de Parkinson

Casi doscientos años después de ser descrita por el doctor inglés James Parkinson, la enfermedad de Parkinson (EP) continúa estando rodeada por un halo de misterio, que la hace hasta la fecha una patología incurable. En su enorme complejidad y etiología, radica la falta de éxito para su control, ya que en la EP se ven inmersos múltiples factores internos entre los que se encuentran genes (genes PARK) y sus productos las proteínas, metabolitos, procesos electrofisiológicos… así como agentes externos, influidos por el propio estilo de vida, entre los que destacan la exposición a herbicidas (paraquat, rotenona..) y otros factores medioambientales. A pesar de que el estudio de estos factores parece resultar insuficiente para el conocimiento completo de la enfermedad, todo apunta que es en la interacción entre todos ellos dónde existe la clave para su total conocimiento. Por ejemplo, la interactuación de los factores mencionados anteriormente afecta a la maquinaria antioxidante y a los sistemas de degradación y desintoxicación celular, entre los que se encuentran la autofagia, el sistema ubiquitina-proteasoma y la vía antioxidante Nrf2/Keap1, y éstos a su vez están íntimamente asociados a la EP. De manera que el efecto acumulativo, sinérgico y subyacente del fallo de estos procesos bioquímicos, que abarcan múltiples niveles dentro de la jerarquía biológica, son los que promueven la pérdida neuronal a lo largo del tiempo. El objeto de la presente tesis doctoral es el estudio del papel neuroprotector que ejerce la vía antioxidante Nrf2/Keap1 así como de su íntima relación con el proceso autofágico, demostrándose una mayor sensibilidad en las células expuestas a agentes medioambientales que presentan una alteración en dicha vía. La tesis se ha estructurado en seis bloques: 1) introducción a la EP; 2) enumeración de los objetivos planteados en este trabajo; 3) descripción de la metodología seguida; 4) exposición de los resultados obtenidos; 5) discusión de los datos dentro del entorno científico; 6) conclusiones extraídas.Almost two hundred years past after it was fisrt described by the English doctor James Parkinson, Parkinson’s disease (PD) still remains to be mysterious and incurable. Our lack of real success in controlling the disease is in large part due to its enormous complexity. PD involves uncounted internal factors, such as genes (PARK genes) and their products the proteins, metabolites, electrophysiological features and external factors related to lifestyle choices and, presumably, exposures to pesticides (paraquat, rotenone) and other enviromental factors. But it is not enough by a long shot to list the many contributors to the disease. Instead, it is the system of functional interactions among these contributors that makes the disease so complicated. For instance, the factors listed above could affect the machinery of the antioxidant defense system and degradation, detoxication systems as autophagy, ubiqutin-proteasome or the Nrf2/Keap1 pathway, which are critically associated with PD. So the acumulative, synergistic and failure underlying of these biochemical processes, that span multiple levels in the biological hierarchy, promote neuronal loss over time. The purpose of this thesis is to study the neuroprotective role exerted by Nrf2/Keap1 antioxidant pathway and its intimate relationship with the autophagic process, demonstrating greater sensitivity in cells exposed to environmental agents that have an alteration in this pathway . The thesis is structured in six sections: 1) introduction to PD, 2) a list of the goals outlined in this paper, 3) description of the methodology followed, 4) presentation of the results obtained, 5) discussion of data within the scientific environment, 6) conclusions.Esta tesis doctoral ha sido subvencionada por los siguientes proyectos: • GR10054 del Gobierno de Extremadura; • PI11/00040 del Ministerio de Economia y Competitividad; • PRIS11014, RIS11019 de FUNDESALUD; • CB01/11, CB06/05/0041 de CIBERNED--‐Instituto de Salud Carlos III

El factor de transcripción Nrf2 como nueva diana terapéutica para la Enfermedad de Parkinson

Casi doscientos años después de ser descrita por el doctor inglés James Parkinson, la enfermedad de Parkinson (EP) continúa estando rodeada por un halo de misterio, que la hace hasta la fecha una patología incurable. En su enorme complejidad y etiología, radica la falta de éxito para su control, ya que en la EP se ven inmersos múltiples factores internos entre los que se encuentran genes (genes PARK) y sus productos las proteínas, metabolitos, procesos electrofisiológicos… así como agentes externos, influidos por el propio estilo de vida, entre los que destacan la exposición a herbicidas (paraquat, rotenona..) y otros factores medioambientales. A pesar de que el estudio de estos factores parece resultar insuficiente para el conocimiento completo de la enfermedad, todo apunta que es en la interacción entre todos ellos dónde existe la clave para su total conocimiento. Por ejemplo, la interactuación de los factores mencionados anteriormente afecta a la maquinaria antioxidante y a los sistemas de degradación y desintoxicación celular, entre los que se encuentran la autofagia, el sistema ubiquitina-proteasoma y la vía antioxidante Nrf2/Keap1, y éstos a su vez están íntimamente asociados a la EP. De manera que el efecto acumulativo, sinérgico y subyacente del fallo de estos procesos bioquímicos, que abarcan múltiples niveles dentro de la jerarquía biológica, son los que promueven la pérdida neuronal a lo largo del tiempo. El objeto de la presente tesis doctoral es el estudio del papel neuroprotector que ejerce la vía antioxidante Nrf2/Keap1 así como de su íntima relación con el proceso autofágico, demostrándose una mayor sensibilidad en las células expuestas a agentes medioambientales que presentan una alteración en dicha vía. La tesis se ha estructurado en seis bloques: 1) introducción a la EP; 2) enumeración de los objetivos planteados en este trabajo; 3) descripción de la metodología seguida; 4) exposición de los resultados obtenidos; 5) discusión de los datos dentro del entorno científico; 6) conclusiones extraídas.Almost two hundred years past after it was fisrt described by the English doctor James Parkinson, Parkinson’s disease (PD) still remains to be mysterious and incurable. Our lack of real success in controlling the disease is in large part due to its enormous complexity. PD involves uncounted internal factors, such as genes (PARK genes) and their products the proteins, metabolites, electrophysiological features and external factors related to lifestyle choices and, presumably, exposures to pesticides (paraquat, rotenone) and other enviromental factors. But it is not enough by a long shot to list the many contributors to the disease. Instead, it is the system of functional interactions among these contributors that makes the disease so complicated. For instance, the factors listed above could affect the machinery of the antioxidant defense system and degradation, detoxication systems as autophagy, ubiqutin-proteasome or the Nrf2/Keap1 pathway, which are critically associated with PD. So the acumulative, synergistic and failure underlying of these biochemical processes, that span multiple levels in the biological hierarchy, promote neuronal loss over time. The purpose of this thesis is to study the neuroprotective role exerted by Nrf2/Keap1 antioxidant pathway and its intimate relationship with the autophagic process, demonstrating greater sensitivity in cells exposed to environmental agents that have an alteration in this pathway . The thesis is structured in six sections: 1) introduction to PD, 2) a list of the goals outlined in this paper, 3) description of the methodology followed, 4) presentation of the results obtained, 5) discussion of data within the scientific environment, 6) conclusions.Esta tesis doctoral ha sido subvencionada por los siguientes proyectos: • GR10054 del Gobierno de Extremadura; • PI11/00040 del Ministerio de Economia y Competitividad; • PRIS11014, RIS11019 de FUNDESALUD; • CB01/11, CB06/05/0041 de CIBERNED--‐Instituto de Salud Carlos III

Parkinson's Disease: Leucine-Rich Repeat Kinase 2 and Autophagy, Intimate Enemies

Parkinson's disease is the second common neurodegenerative disorder, after Alzheimer's disease. It is a clinical syndrome characterized by loss of dopamine-generating cells in the substancia nigra, a region of the midbrain. The etiology of Parkinson's disease has long been through to involve both genetic and environmental factors. Mutations in the leucine-rich repeat kinase 2 gene cause late-onset Parkinson's disease with a clinical appearance indistinguishable from Parkinson's disease idiopathic. Autophagy is an intracellular catabolic mechanism whereby a cell recycles or degrades damage proteins and cytoplasmic organelles. This degradative process has been associated with cellular dysfunction in neurodegenerative processes including Parkinson's disease. We discuss the role of leucine-rich repeat kinase 2 in autophagy, and how the deregulations of this degradative mechanism in cells can be implicated in the Parkinson's disease etiology

Is the Modulation of Autophagy the Future in the Treatment of Neurodegenerative Diseases?

The pathogenesis of neurodegenerative diseases involves altered activity of proteolytic systems and accumulation of protein aggregates. Autophagy is an intracellular process in which damaged organelles and long-lived proteins are degraded and recycled for maintaining normal cellular homeostasis. Disruption of autophagic activity in neurons leads to modify the cellular homeostasis, causing deficient elimination of abnormal and toxic protein aggregates that promotes cellular stress and death. Therefore, induction of autophagy has been proposed as a reasonable strategy to help neurons to clear abnormal protein aggregates and survive. This review aims to give an overview of some of the main modulators of autophagy that are currently being studied as possible alternatives in the search of therapies that slow the progression of neurodegenerative diseases, which are incurable to date

G2019S Mutation of LRRK2 Increases Autophagy via MEK/ERK Pathway

Abstract Parkinson’s disease (PD) is a neurodegenerative disorder characterized by mitochondrial dysfunction, oxidative stress, and later neuronal death. Several genetics and environmental factors have been implicated in the {PD} pathogenesis. Mutations in leucine-rich repeat kinase 2 (LRRK2) are a major cause of familial parkinsonism, and the {G2019S} mutation of {LRRK2} is one of the most prevalent. The deregulation of autophagic process in nerve cells is thought to be a possible cause of PD. {G2019S} mutant fibroblasts exhibited higher autophagic activity that can trigger cell death. In this sense, {G2019S} mutant cells displayed increased apoptosis hallmarks and enough susceptibility to MPP+ (1-methyl-4-phenylpyridinium). {G2019S} {LRRK2} heightens the phosphorylation of MAPK/ERK kinases (MEK). The use of 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene (U0126) reduced the enhanced autophagy suggesting that the {G2019S} mutation induces autophagy via MEK/ERK pathway. Further, the inhibition of this exacerbated autophagy reduces the sensitivity remarked in {G2019S} mutant cells

mRNA and protein dataset of autophagy markers (LC3 and p62) in several cell lines

We characterized the dynamics of autophagy in vitro using four different cell systems and analyzing markers widely used in this field, i.e. LC3 (microtubule-associated protein 1 light chain 3; protein recruited from the cytosol (LC3-I) to the autophagosomal membrane where it is lipidated (LC3-II)) and p62/SQSTM1 (adaptor protein that serves as a link between LC3 and ubiquitinated substrates), (Klionsky et al., 2016) [1]. Data provided include analyses of protein levels of LC3 and p62 by Western-blotting and endogenous immunofluorescence experiments, but also p62 mRNA levels obtained by quantitative PCR (qPCR). To monitor the turnover of these autophagy markers and, thus, measure the flux of this pathway, cells were under starvation conditions and/or treated with bafilomycin A1 (Baf. A1) to block fusion of autophagosomes with lysosomes. Keywords: Autophagy, LC3, p62, Western-blo

PINK1 deficiency enhances autophagy and mitophagy induction

Parkinson's disease (PD) is a neurodegenerative disorder with poorly understood etiology. Increasing evidence suggests that age-dependent compromise of the maintenance of mitochondrial function is a key risk factor. Several proteins encoded by PD-related genes are associated with mitochondria including PTEN-induced putative kinase 1 (PINK1), which was first identified as a gene that is upregulated by PTEN. Loss-of-function PINK1 mutations induce mitochondrial dysfunction and, ultimately, neuronal cell death. To mitigate the negative effects of altered cellular functions cells possess a degradation mechanism called autophagy for recycling damaged components; selective elimination of dysfunctional mitochondria by autophagy is termed mitophagy. Our study indicates that autophagy and mitophagy are upregulated in PINK1-deficient cells, and is the first report to demonstrate efficient fluxes by one-step analysis. We propose that autophagy is induced to maintain cellular homeostasis under conditions of non-regulated mitochondrial quality control