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Banos-Mateos S.,CSIC - Institute of Physical Chemistry "Rocasolano" | Chaves-Sanjuan A.,CSIC - Institute of Physical Chemistry "Rocasolano" | Mansilla A.,Institute Cajal | Ferrus A.,Institute Cajal | Sanchez-Barrena M.J.,CSIC - Institute of Physical Chemistry "Rocasolano"
Acta Crystallographica Section F:Structural Biology Communications | Year: 2014

Drosophila melanogaster contains two calcium-binding proteins, Frq1 and Frq2, in the nervous system that control the number of synapses and the probability of release. To understand the differential function of the two proteins, whose sequence is only 5% dissimilar, the crystal structures of Frq1 and Frq2 are needed. Here, the cloning, expression, purification, crystallization and preliminary crystallographic analysis of Frq2 are presented. The full-length protein was purified using a two-step chromatographic procedure. Two different diffracting crystal forms were obtained using a progressive streak-seeding method and detergents. © 2014 International Union of Crystallography All rights reserved.

Romero-Pozuelo J.,Institute Cajal | Romero-Pozuelo J.,German Cancer Research Center | Dason J.S.,University of Toronto | Mansilla A.,Institute Cajal | And 12 more authors.
Journal of Cell Science | Year: 2014

The conserved Ca2+-binding protein Frequenin (homolog of the mammalian NCS-1, neural calcium sensor) is involved in pathologies that result from abnormal synapse number and probability of neurotransmitter release per synapse. Both synaptic features are likely to be co-regulated but the intervening mechanisms remain poorly understood. We show here that Drosophila Ric8a (a homolog of mammalian synembryn, which is also known as Ric8a), a receptor-independent activator of G protein complexes, binds to Frq2 but not to the virtually identical homolog Frq1. Based on crystallographic data on Frq2 and site-directed mutagenesis on Frq1, the differential amino acids R94 and T138 account for this specificity. Human NCS-1 and Ric8a reproduce the binding and maintain the structural requirements at these key positions. Drosophila Ric8a and Gas regulate synapse number and neurotransmitter release, and both are functionally linked to Frq2. Frq2 negatively regulates Ric8a to control synapse number. However, the regulation of neurotransmitter release by Ric8a is independent of Frq2 binding. Thus, the antagonistic regulation of these two synaptic properties shares a common pathway, Frq2- Ric8a-Gαs, which diverges downstream. These mechanisms expose the Frq2-Ric8a interacting surface as a potential pharmacological target for NCS-1-related diseases and provide key data towards the corresponding drug design. © 2014. Published by The Company of Biologists Ltd.

LLorens-Martin M.,Institute Cajal | Torres-Aleman I.,Institute Cajal | Trejo J.L.,Institute Cajal
Molecular and Cellular Neuroscience | Year: 2010

While physical exercise clearly has beneficial effects on the brain, fomenting neuroprotection as well as promoting neural plasticity and behavioural modifications, the cellular and molecular mechanisms mediating these effects are not yet fully understood. We have analyzed sedentary and exercised animals to examine the effects of activity on behaviour (spatial memory and anxiety - as measured by a fear/exploration conflict test), as well as on adult hippocampal neurogenesis (a well-known form of neural plasticity). We have found that the difference in activity between sedentary and exercised animals induced a decrease in the fear/exploration conflict scores (a measure usually accepted as an anxiolytic effect), while no changes are evident in terms of spatial memory learning. The short-term anxiolytic-like effect of exercise was IGF1-dependent and indeed, the recall of hippocampus-dependent spatial memory is impaired by blocking serum IGF1 (as observed by measuring serum IGF levels in the same animals used to analyze the behaviour), irrespective of the activity undertaken by the animals. On the other hand, activity affected neurogenesis as reflected by counting the numbers of several cell populations, while the dependence of this effect on IGF1 varied according to the differentiation state of the new neurons. Hence, while proliferating precursors and postmitotic immature neurons (measured by means of doublecortin and calretinin) are influenced by serum IGF1 levels in both sedentary and exercised animals, premitotic immature neurons (an intermediate stage) respond to exercise independently of serum IGF1. Therefore, we conclude that physical exercise has both serum IGF1-independent and -dependent effects on neural plasticity. Furthermore, several effects mediated by serum IGF1 are induced by physical activity while others are not (both in terms of behaviour and neural plasticity). These findings help to delimit the role of serum IGF1 as a mediator of the effects of exercise, as well as to extend the role of serum IGF1 in the brain in basal conditions. Moreover, these data reveal the complexity of the interaction between neurogenesis, behaviour, and IGF1 under different levels of physical activity. © 2010 Elsevier Inc.

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