Improving therapeutic potential of GDNF family ligands

The last decade has been a frustrating time for investigators who had envisioned major advances in the treatment of Parkinson's disease using neurotrophic factors. The first trials of glial cell line-derived neurotrophic factor for treating Parkinson's disease were very promising. Later blinded control trials were disappointing, not reaching the predetermined outcomes for improvement in motor function. Consideration of the problems in the studies as well as the biology of the neurotrophins used can potentially lead to more effective therapies. Parkinson's disease presents a multitude of opportunities for the cell biologist wanting to understand its pathology and to find possible new avenues for treatment.Peer reviewe

Developing therapeutically more efficient Neurturin variants for treatment of Parkinson's disease

In Parkinson's disease midbrain dopaminergic neurons degenerate and die. Oral medications and deep brain stimulation can relieve the initial symptoms, but the disease continues to progress. Growth factors that might support the survival, enhance the activity, or even regenerate degenerating dopamine neurons have been tried with mixed results in patients. As growth factors do not pass the blood-brain barrier, they have to be delivered intracranially. Therefore their efficient diffusion in brain tissue is of crucial importance. To improve the diffusion of the growth factor neurturin (NRTN), we modified its capacity to attach to heparan sulfates in the extracellular matrix. We present four new, biologically fully active variants with reduced heparin binding. Two of these variants are more stable than WT NRTN in vitro and diffuse better in rat brains. We also show that one of the NRTN variants diffuses better than its close homolog GDNF in monkey brains. The variant with the highest stability and widest diffusion regenerates dopamine fibers and improves the conditions of rats in a 6-hydroxydopamine model of Parkinson's disease more potently than GDNF, which previously showed modest efficacy in clinical trials. The new NRTN variants may help solve the major problem of inadequate distribution of NRTN in human brain tissue. (C) 2016 Elsevier Inc. All rights reserved.Peer reviewe

Overexpression of the aspartic protease ASPG1 gene confers drought avoidance in Arabidopsis

Drought is one of the most severe environmental stresses affecting plant growth and limiting crop production. Although many genes involved in adaptation to drought stress have been disclosed, the relevant molecular mechanisms are far from understood. This study describes an Arabidopsis gene, ASPG1 (ASPARTIC PROTEASE IN GUARD CELL 1), that may function in drought avoidance through abscisic acid (ABA) signalling in guard cells. Overexpression of the ASPG1 gene enhanced ABA sensitivity in guard cells and reduced water loss in ectopically overexpressing ASPG1 (ASPG1-OE) transgenic plants. In ASPG1-OE plants, some downstream targets in ABA and/or drought-signalling pathways were altered at various levels, suggesting the involvement of ASPG1 in ABA-dependent drought avoidance in Arabidopsis. By analysing the activities of several antioxidases including superoxide dismutase and catalase in ASPG1-OE plants, the existence was demonstrated of an effective detoxification system for drought avoidance in these plants. Analysis of ProASPG1-GUS lines showed a predominant guard cell expression pattern in various aerial tissues. Moreover, the protease activity of ASPG1 was characterized in vitro, and two aspartic acid sites, D180 and D379, were found to be key residues for ASPG1 aspartic protease activity in response to ABA. In summary, these findings suggest that functional ASPG1 may be involved in ABA-dependent responsiveness and that overexpression of the ASPG1 gene can confer drought avoidance in Arabidopsis

RET PLCγ Phosphotyrosine Binding Domain Regulates Ca2+ Signaling and Neocortical Neuronal Migration

The receptor tyrosine kinase RET plays an essential role during embryogenesis in regulating cell proliferation, differentiation, and migration. Upon glial cell line-derived neurotrophic factor (GDNF) stimulation, RET can trigger multiple intracellular signaling pathways that in concert activate various downstream effectors. Here we report that the RET receptor induces calcium (Ca2+) signaling and regulates neocortical neuronal progenitor migration through the Phospholipase-C gamma (PLCγ) binding domain Tyr1015. This signaling cascade releases Ca2+ from the endoplasmic reticulum through the inositol 1,4,5-trisphosphate receptor and stimulates phosphorylation of ERK1/2 and CaMKII. A point mutation at Tyr1015 on RET or small interfering RNA gene silencing of PLCγ block the GDNF-induced signaling cascade. Delivery of the RET mutation to neuronal progenitors in the embryonic ventricular zone using in utero electroporation reveal that Tyr1015 is necessary for GDNF-stimulated migration of neurons to the cortical plate. These findings demonstrate a novel RET mediated signaling pathway that elevates cytosolic Ca2+ and modulates neuronal migration in the developing neocortex through the PLCγ binding domain Tyr1015

Positive Selection for New Disease Mutations in the Human Germline: Evidence from the Heritable Cancer Syndrome Multiple Endocrine Neoplasia Type 2B

Multiple endocrine neoplasia type 2B (MEN2B) is a highly aggressive thyroid cancer syndrome. Since almost all sporadic cases are caused by the same nucleotide substitution in the RET proto-oncogene, the calculated disease incidence is 100–200 times greater than would be expected based on the genome average mutation frequency. In order to determine whether this increased incidence is due to an elevated mutation rate at this position (true mutation hot spot) or a selective advantage conferred on mutated spermatogonial stem cells, we studied the spatial distribution of the mutation in 14 human testes. In donors aged 36–68, mutations were clustered with small regions of each testis having mutation frequencies several orders of magnitude greater than the rest of the testis. In donors aged 19–23 mutations were almost non-existent, demonstrating that clusters in middle-aged donors grew during adulthood. Computational analysis showed that germline selection is the only plausible explanation. Testes of men aged 75–80 were heterogeneous with some like middle-aged and others like younger testes. Incorporating data on age-dependent death of spermatogonial stem cells explains the results from all age groups. Germline selection also explains MEN2B's male mutation bias and paternal age effect. Our discovery focuses attention on MEN2B as a model for understanding the genetic and biochemical basis of germline selection. Since RET function in mouse spermatogonial stem cells has been extensively studied, we are able to suggest that the MEN2B mutation provides a selective advantage by altering the PI3K/AKT and SFK signaling pathways. Mutations that are preferred in the germline but reduce the fitness of offspring increase the population's mutational load. Our approach is useful for studying other disease mutations with similar characteristics and could uncover additional germline selection pathways or identify true mutation hot spots

Sorting of proteins to vacuoles in plant cells

for correspondence