Autophagy and protein aggregation as a mechanism of dopaminergic degeneration in a primary human dopaminergic neuronal model.

The pathophysiology underlying the loss of dopaminergic neurons in Parkinson's disease (PD) is unclear. A gap of knowledge in the molecular and cellular events leading to degeneration of the nigrostriatal DA system is a major barrier to the development of effective therapies for PD. 1-methyl-4-phenylpyridinium (MPP+) is used as a reliable in vitro model of PD in dopaminergic neurons; however, the molecular mechanisms that lead to cell death with this model are not fully understood. Additionally, there is a lack of translational in vitro models to fully understand progressive dopaminergic neurotoxicity. Here, we propose cultures of primary human dopaminergic neuronal precursor cells (HDNPCs) as a model to study progressive dopaminergic toxicity and neuronal damage in PD. We evaluated the concentration-response of MPP+ (0-10 mM) at 24 h, using cell viability and mitochondrial activity assays (LDH, XTT, Live/Dead staining, and MitoTracker). Based on concentration-response data, we chose two concentrations (1.0 and 2.5 mM) of MPP+ to evaluate markers of autophagy and dopaminergic status [tyrosine hydroxylase (TH)] after a 24-h exposure. Exposure to MPP+ induced cytotoxicity, reduced cell viability, and decreased mitochondrial activity. MPP+ at 1.0 and 2.5 mM also induced expression of lysosome-associated membrane protein 1 (LAMP-1) and increased the ratio of light chain 3 (LC3), LC3BII/LC3BI. The expression of TH also decreased. Furthermore, α-synuclein (α-SYN) and parkin were evaluated by immunofluorescence (IF) at 1.0 and 2.5 mM MPP+ after 24 h. A qualitative analysis revealed decreased parkin expression while α-SYN aggregation was observed in the cytoplasm and the nucleus. These data suggest that in HDNPCs MPP+ can cause cytotoxicity and neuronal damage. This damage may be mediated by autophagy, dopamine synthesis, and protein aggregation. The combination of HDNPCs and MPP+ may serve as valuable in vitro model of progressive dopaminergic neurotoxicity for research into potential treatments for PD.

The dynamics of epidermal stratification during post-larval development in zebrafish.

BACKGROUND: The epidermis, as a defensive barrier, is a consistent trait throughout animal evolution. During post-larval development, the zebrafish epidermis thickens by stratification or addition of new cell layers. Epidermal basal stem cells, expressing the transcription factor p63, are known to be involved in this process. Zebrafish post-larval epidermal stratification is a tractable system to study how stem cells participate in organ growth. METHODS: We used immunohistochemistry, in combination with EdU cell proliferation detection, to study zebrafish epidermal stratification. For this procedure, we selected a window of post-larval stages (5-8 mm of standard length or SL, which normalizes age by size). Simultaneously, we used markers for asymmetric cell division and the Notch signaling pathway. RESULTS: We found that epidermal stratification is the consequence of several events, including changes in cell shape, active cell proliferation and asymmetrical cell divisions. We identified a subset of highly proliferative epidermal cells with reduced levels of p63, which differed from the basal stem cells with high levels of p63. Additionally, we described different mechanisms that participate in the stratification process, including the phosphorylation of p63, asymmetric cell division regulated by the Par3 and LGN proteins, and expression of Notch genes.

Amyloid Beta 25-35 induces blood-brain barrier disruption in vitro.

The amyloid ß-peptide (Aß) is transported across the blood-brain barrier (BBB) by binding with the receptor for advanced glycation end products (RAGE). Previously, we demonstrated that the Aß fraction 25-35 (Aß25-35) increases RAGE expression in the rat hippocampus, likely contributing to its neurotoxic effects. However, it is still debated if the interaction of Aß with RAGE compromises the BBB function in Alzheimer' disease (AD). Here, we evaluated the effects of Aß25-35 in an established in vitro model of the BBB. Rat brain microvascular endothelial cells (rBMVECs) were treated with 20 µM active Aß25-35 or the inactive Aß35-25 (control), for 24 h. Exposure to Aß25-35 significantly decreased cell viability, increased cellular necrosis, and increased the production of reactive oxygen species (ROS), which triggered a decrease in the enzyme glutathione peroxidase when compared to the control condition. Aß25-35 also increased BBB permeability by altering the expression of tight junction proteins (decreasing zonula occludens-1 and increasing occludin). Aß25-35 induced monolayer disruption and cellular disarrangement of the BBB, with RAGE being highly expressed in the zones of disarrangement. Together, these data suggest that Aß25-35-induces toxicity by compromising the functionality and integrity of the BBB in vitro. Graphical abstract Aß25-35 induces BBB dysfunction in vitro, wich is likely mediated by OS and ultimately leads to disruption of BBB integrity and cell death.

A stem cell proliferation burst forms new layers of P63 expressing suprabasal cells during zebrafish postembryonic epidermal development.

Organ growth during development is a highly regulated process with both temporal and spatial constraints. Epidermal stratification is essential for skin growth and development. Although the zebrafish has been well studied, it is not known when and how epidermal stratification occurs. This is because beyond the first five days of development our knowledge is currently limited. We found that epidermal stratification in zebrafish begins when the larvae reach a standard length (SL) of 6 mm at approximately 25 days of age. Over the next four days (from a SL of 6 to 9 mm), epidermis thickness increases almost four-fold. This represents a sudden increase in organ size, since for the previous 20 days of development, the epidermis has been only two layers thick. This pattern is different from that observed in mammals that undergo continuous stratification from E14.5-E18.5. To study how stem cell proliferation gives rise to the new epidermal layers, we used a combination of markers: one for cell proliferation (proliferating cell nuclear-antigen PCNA) and one for epidermal stem cells (P63 transcription factor). We identified, throughout the stratification process, two different waves of cell division. Initially, the most basal epidermal cells divided and generated a subset of suprabasal cells (possibly transient-amplifying cells); within the next several days, the basal cells stopped dividing, and the suprabasal cells began proliferation, giving rise to most of the cell types in the new layers. This part of the process is similar to what has been recently found during epidermal stratification in mammals.

The zebrafish mutants for the V-ATPase subunits d, ac45, E, H and c and their variable pigment dilution phenotype.

BACKGROUND: The V-ATPase is a proton pump that creates an acidic medium, necessary for lysosome function and vesicular traffic. It is also essential for several developmental processes. Many enzymes, like the V-ATPase, are assemblies of multiple subunits, in which each one performs a specific function required to achieve full activity. In the zebrafish V-ATPase 15 different subunits form this multimeric complex and mutations in any of these subunits induce hypopigmentation or pigment dilution phenotype. We have previously found variability in the pigment dilution phenotype among five of the V-ATPase zebrafish mutants. This work presents additional information about such differences and is an update from a previous report. FINDINGS: We describe the variable phenotype severity observed among zebrafish V-ATPase pigment dilution mutants studying mRNA expression levels from their corresponding genes. At the same time we carried out phylogenetic analysis for this genes. CONCLUSIONS: Based in the similarities between different pigment dilution mutants we suggest that there is an essential role for V-ATPases in melanosome biogenesis and melanocyte survival. Neither variable expression levels for the different V-ATPase subunits studied here or the presence of duplicated genes seems to account for the variable phenotype severity from this group of mutants. We believe there are some similarities between the pigment dilution phenotype from zebrafish V-ATPase insertional mutants and pigment mutants obtained in a chemical screening ("Tubingen pigmentation mutants"). As for some of these "Tubingen mutants" the mutated gene has not been found we suggest that mutations in V-ATPase genes may be inducing their defects.

[Physiopathology of prion diseases]. / Fisiopatología de las enfermedades por priones.

Prion diseases are a group of degenerative disorders characterized by being progressive, fast growing, and fatal, they affect humans and animals. Due to their physiopathogeny, these disorders can be sporadic, genetic, or infectious. Prions are cellular proteins that lack nucleic acids; they are not viruses or microorganisms. Prions induce neuronal death, brain spongiosis, which are a hallmark of these diseases, as well as amyloid prion protein plaque aggregates. Although the causes that favor pathogenic prion proteins remain uncertain, it is possible that conformational changes of the prion protein allow them to create copies of themselves to form aggregates and induce neuronal death. Other theories suggest that quantitative and qualitative changes in the glycosylation pattern induce the pathological prion form. The latter allows to explain some of their interactions and to understand better the conformational changes and the physico-chemical properties of the prion protein. We review some of the first biological functions (as a transporter of Cu2+ ions) that have been described to this molecule. The present review focuses on different aspects of prion diseases aimed at understanding better their physiopathogenic characteristics.

Fisiopatología de las enfermedades por priones / Physiopathology of prion diseases

Las enfermedades por priones, son trastornos neurodegenerativos progresivos rápidos e invariablemente fatales que afectan tanto a seres humanos como a animales. Tienen formas de presentación esporádica, genética e infecciosa. Los priones son proteínas celulares. No contienen ácidos nucleicos y no son virus o microorganismos. En todos los casos, provocan muerte neuronal, espongiosis común del cerebro, que caracteriza a estas enfermedades, así como agregación de la proteína amiloide prión en forma de placa. La teoría más importante hasta el momento, es la que trata de explicar el cambio de conformación de la proteína prión para producir copias de sí misma y para su agregación y la muerte de las neuronas. Sin embargo, nuevas formas de explicación toman auge actualmente. Una de las más importantes se basa en entender el contenido y cambio de la glicosilación de la proteína prión patológica. Esto permite explicar algunas de sus interacciones, para entender el cambio de conformación y las propiedades físico-químicas de la proteína. Así como algunas de las primeras funciones biológicas (como transportador de iones Cu++2) descritas para esta molécula. En esta revisión abordamos todos los tópicos importantes acerca de estas patologías por demás fascinantes.

Prion diseases are a group of degenerative disorders characterized by being progressive, fast growing, and fatal, they affect humans and animals. Due to their physiopathogeny, these disorders can be sporadic, genetic, or infectious. Prions are cellular proteins that lack nucleic acids; they are not viruses or microorganisms. Prions induce neuronal death, brain spongiosis, which are a hallmark of these diseases, as well as amyloid prion protein plaque aggregates. Although the causes that favor pathogenic prion proteins remain uncertain, it is possible that conformational changes of the prion protein allow them to create copies of themselves to form aggregates and induce neuronal death. Other theories suggest that quantitative and qualitative changes in the glycosylation pattern induce the pathological prion form. The latter allows to explain some of their interactions and to understand better the conformational changes and the physico-chemical properties of the prion protein. We review some of the first biological functions (as a transporter of Cu2+ ions) that have been described to this molecule. The present review focuses on different aspects of prion diseases aimed at understanding better their physiopathogenic characteristics.

Characterization of an O-glycosylated plaque-associated protein from Alzheimer disease brain.

In this work we characterized a 90-kDa glycoprotein from Alzheimer disease (9OAzgp) brain extracts that is recognized by the GalNAc-specific lectin from Amaranthus leucocarpus (ALL), as determined through Western blot. The 90Azgp was purified by electro-elution, and its amino acid sequence determined from peptides obtained after trypsin digestion through MALDI-TOF (Matrix-assisted laser desorption ionization-time of flight), and compared with the relative values obtained from the NCBInr (Swiss-Prot 10/01/2001) database. The 90Azgp showed 32% and 42% homology with the KIAA0310 protein from human brain and the human gastric mucin, respectively. Presence of O-glycosidically linked glycans in the proteins recognized by ALL was confirmed by inhibition of the lectin-glycoprotein interaction through hapten-inhibition assays and also by elimination of the O-glycosidically linked glycans after treatment with O-glycanase from Diplococcus pneumoniae. Electron transmission microscopy confirmed that the receptor recognized by the lectin is processed in the Golgi apparatus of AD neurons. Although the specific role of this glycoprotein has not been identified, considering that the presence of this lectin receptor co-localized with neuritic plaques and in AD sprouting neurons, it could suggest that the O-glycosyl-protein identified by the A. leucocarpus lectin participates in the pathogenesis of neurodegenerative diseases.