Efficient and Scalable Generation of Human Ventral Midbrain Astrocytes from Human-Induced Pluripotent Stem Cells

In Parkinson's disease, progressive dysfunction and degeneration of dopamine neurons in the ventral midbrain cause life-changing symptoms. Neuronal degeneration has diverse causes in Parkinson's, including non-cell autonomous mechanisms mediated by astrocytes. Throughout the CNS, astrocytes are essential for neuronal survival and function, as they maintain metabolic homeostasis in the neural environment. Astrocytes interact with the immune cells of the CNS, microglia, to modulate neuroinflammation, which is observed from the earliest stages of Parkinson's, and has a direct impact on the progression of its pathology. In diseases with a chronic neuroinflammatory element, including Parkinson's, astrocytes acquire a neurotoxic phenotype, and thus enhance neurodegeneration. Consequently, astrocytes are a potential therapeutic target to slow or halt disease, but this will require a deeper understanding of their properties and roles in Parkinson's. Accurate models of human ventral midbrain astrocytes for in vitro study are therefore urgently required. We have developed a protocol to generate high purity cultures of ventral midbrain-specific astrocytes (vmAstros) from hiPSCs that can be used for Parkinson's research. vmAstros can be routinely produced from multiple hiPSC lines, and express specific astrocytic and ventral midbrain markers. This protocol is scalable, and thus suitable for high-throughput applications, including for drug screening. Crucially, the hiPSC derived-vmAstros demonstrate immunomodulatory characteristics typical of their in vivo counterparts, enabling mechanistic studies of neuroinflammatory signaling in Parkinson's

ATG8-dependent LMX1B-autophagy crosstalk shapes human midbrain dopaminergic neuronal resilience

The LIM homeodomain transcription factors LMX1A and LMX1B are essential mediators of midbrain dopaminergic neuronal (mDAN) differentiation and survival. Here we show that LMX1A and LMX1B are autophagy transcription factors that provide cellular stress protection. Their suppression dampens the autophagy response, lowers mitochondrial respiration, and elevates mitochondrial ROS, and their inducible overexpression protects against rotenone toxicity in human iPSC-derived mDANs in vitro. Significantly, we show that LMX1A and LMX1B stability is in part regulated by autophagy, and that these transcription factors bind to multiple ATG8 proteins. Binding is dependent on subcellular localization and nutrient status, with LMX1B interacting with LC3B in the nucleus under basal conditions and associating with both cytosolic and nuclear LC3B during nutrient starvation. Crucially, ATG8 binding stimulates LMX1B-mediated transcription for efficient autophagy and cell stress protection, thereby establishing a novel LMX1B-autophagy regulatory axis that contributes to mDAN maintenance and survival in the adult brai

Nanoparticle-induced neuronal toxicity across placental barriers is mediated by autophagy and dependent on astrocytes

The potential for maternal nanoparticle (NP) exposures to cause developmental toxicity in the fetus without the direct passage of NPs has previously been shown, but the mechanism remained elusive. We now demonstrate that exposure of cobalt and chromium NPs to BeWo cell barriers, an in vitro model of the human placenta, triggers impairment of the autophagic flux and release of interleukin-6. This contributes to the altered differentiation of human neural progenitor cells and DNA damage in the derived neurons and astrocytes. Crucially, neuronal DNA damage is mediated by astrocytes. Inhibiting the autophagic degradation in the BeWo barrier by overexpression of the dominant-negative human ATG4BC74A significantly reduces the levels of DNA damage in astrocytes. In vivo, indirect NP toxicity in mice results in neurodevelopmental abnormalities with reactive astrogliosis and increased DNA damage in the fetal hippocampus. Our results demonstrate the potential importance of autophagy to elicit NP toxicity and the risk of indirect developmental neurotoxicity after maternal NP exposure

Isolation of a cationic polypeptide from human serum that stimulates proliferation of 3T3 cells.

A basic polypeptide that stimulates DNA synthesis and cell division in confluent populations of mouse Balb/c-3T3 cells has been isolated from whole human serum, and has been separated from the heterogenous group of molecules with insulin-like activity. This highly purified basic polypeptide has a molecular weight of 1.3 x 10(4) and an isoelectric point of 9.7. Approximately 10(7) polypeptide molecules in the growth medium allow the replication of one density-inhibited cell

Neutral endopeptidase-24.11 (NEP) activity in human fibroblasts during development and ageing

Neutral endopeptidase-24.11 (NEP, EC 3.4.24.11) is a cell surface Zn metallopeptidase that hydrolyzes bioactive regulatory peptides. Using a spectrofluorimetric procedure, we assessed NEP activity in plasma membranes of normal human skin and lung fibroblasts. We found a considerable increase in NEP activity during fetal-to-adult transition. Adult skin fibroblasts from an old donor exhibited significantly higher levels of NEP activity than cells from young donors. Interestingly, however, the NEP activity of fibroblasts from a centenarian donor was similar to that of cells from young donors. Increased levels of NEP activity were also found in in vitro aged lung fibroblasts. Finally, adrenocorticotropin hormone (ACTH (1-24)), a regulatory peptide that can be cleaved by NEP, provoked an increase in enzymic activity in fetal and young adult donor fibroblasts and a decrease in this activity in fibroblasts from adult and old donors. This finding Suggests that ageing may affect NEP activity. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved

Evaluation of three cultivation practices for early cotton establishment and improving crop profitability

Cultivation practices permitting earlier sowing of cotton (Gossypium hirsutum L.) in Greece are required to maximize yields and facilitate harvesting. An experiment was conducted for 2 years in Central Greece to evaluate two alternative systems. The experiment was carried out in a Vertic Cambisol and a Typic Regosol field. Cultivation practices tested were: (1) conventional tillage (CT) and sowing in a flat field, (2) ridge tillage (RT), using autumn ridging and (3) sowing in a flat field under clear plastic film (PF). Early and normal sowings were compared. The effects of the treatment on the crop establishment, growth and yield, as well as on the soil physical properties, were studied. Performance evaluation of the machinery was carried Out. The cost of cultivation practices was estimated. Results of soil physical properties were similar for both years. Soil water contents from sowing to plastic removal in 2000 were 14.2, 13.5 and 18.0 g/100 g and temperatures for the same period at 0.04 m depth were 17.7, 18.1 and 19.8 degrees C for CT, RT and PF, respectively. PF resulted in higher emergence and higher plants with smaller roots. Average yields of seed-cotton in early sowing were 4936, 4591 and 4033 kg/ha for PF, RT and CT, respectively. In late sowing yields in RT and in CT did not differ significantly. Ridge tillage machinery saved 13.6 kWh/ha (20.9%) compared to conventional tillage machinery. The higher yields under plastic film compensated for the higher cost of the practice at the present prices of seed-cotton. (c) 2005 Elsevier B.V. All rights reserved