Alberto Ferrús

Shaker encodes a family of putative potassium channel proteins in the nervous system of Drosophila.

The Shaker locus of Drosophila contains a very large transcription unit. It is expressed predominantly in the nervous system by multiple, differential as well as alternative, splicing mechanisms into different, but functionally related proteins. The structure of the Shaker transcription unit and the properties of the encoded Shaker protein family provide a molecular basis for A channel diversity in excitable cells.

Frequenin—A novel calcium-binding protein that modulates synaptic efficacy in the drosophila nervous system

The T(X;Y)V7 rearrangement in Drosophila has originally been recognized as a Shaker-like mutant because of its behavioral and electrophysiological phenotype. The gene whose expression is altered by the V7 rearrangement has been characterized. It encodes a novel Ca(2+)-binding protein named frequenin, which is related to recoverin and visinin. In vitro, the frequenin protein functions like recoverin as a Ca(2+)-sensitive guanylyl cyclase activator. Anti-frequenin antibodies stain the central and peripheral nervous system in Drosophila embryos and in larval and adult tissue sections. Frequenin appears to be particularly enriched in synapses, such as the motor nerve endings at neuromuscular junctions. Neuromuscular junctions of transgenic flies, which overexpress frequenin upon heat shock, exhibit an extraordinarily enhanced, frequency-dependent facilitation of neurotransmitter release, with properties identical to those observed in V7 junctions. We propose that frequenin represents a new element for the Ca(2+)-dependent modulation of synaptic efficacy.

Tachykinin-related peptides modulate odor perception and locomotor activity in Drosophila

The invertebrate tachykinin-related peptides (TKRPs) constitute a conserved family, structurally related to the mammalian tachykinins, including members such as substance P and neurokinins A and B. Although their expression has been documented in the brains of insects and mammals, their neural functions remain largely unknown, particularly in behavior. Here, we have studied the role of TKRPs in Drosophila. We have analyzed the olfactory perception and the locomotor activity of individuals in which TKRPs are eliminated in the nervous system specifically, by using RNAi constructs to silence gene expression. The perception of specific odorants and concentrations is modified towards a loss of sensitivity, thus resulting in a significant change of the behavioral response towards indifference. In locomotion assays, the TKRP-deficient flies show hyperactivity. We conclude that these peptides are modulators of olfactory perception and locomotion activity in agreement with their abundant expression in the olfactory lobes and central complex. In these brain centers, TKRPs seem to enhance the regulatory inhibition of the neurons in which they are expressed.

Phosphoinositide-3-Kinase Activation Controls Synaptogenesis and Spinogenesis in Hippocampal Neurons

The possibility of changing the number of synapses may be an important asset in the treatment of neurological diseases. In this context, the synaptogenic role of the phosphoinositide-3-kinase (PI3K) signaling cascade has been previously demonstrated in Drosophila. This study shows that treatment with a PI3K-activating transduction peptide is able to promote synaptogenesis and spinogenesis in primary cultures of rat hippocampal neurons, as well as in CA1 hippocampal neurons in vivo. In culture, the peptide increases synapse density independently of cell density, culture age, dendritic complexity, or synapse type. The induced synapses also increase neurotransmitter release from cultured neurons. The synaptogenic signaling pathway includes PI3K-Akt. Furthermore, the treatment is effective on adult neurons, where it induces spinogenesis and enhances the cognitive behavior of treated animals in a fear-conditioning assay. These findings demonstrate that functional synaptogenesis can be induced in mature mammalian brains through PI3K activation.

Cellular and molecular features of axon collaterals and dendrites

Neural geometry is the major factor that determines connectivity and, possibly, functional output from a nervous system. Recently some of the proteins and pathways involved in specific modes of branch formation or maintenance, or both, have been described. To a variable extent, dendrites and axon collaterals can be viewed as dynamic structures subject to fine modulation that can result either in further growth or retraction. Each form of branching results from specific molecular mechanisms. Cell-internal, substrate-derived factors and functional activity, however, can often differ in their effect according to cell type and physiological context at the site of branch formation. Neural branching is not a linear process but an integrative one that takes place in a microenvironment where we have only a limited experimental access. To attain a coherent mechanism for this phenomenon, quantitative in situ data on the proteins involved and their interactions will be required.

Age-Independent Synaptogenesis by Phosphoinositide 3 Kinase

Synapses are specialized communication points between neurons, and their number is a major determinant of cognitive abilities. These dynamic structures undergo developmental- and activity-dependent changes. During brain aging and certain diseases, synapses are gradually lost, causing mental decline. It is, thus, critical to identify the molecular mechanisms controlling synapse number. We show here that the levels of phosphoinositide 3 kinase (PI3K) regulate synapse number in both Drosophila larval motor neurons and adult brain projection neurons. The supernumerary synapses induced by PI3K overexpression are functional and elicit changes in behavior. Remarkably, PI3K activation induces synaptogenesis in aged adult neurons as well. We demonstrate that persistent PI3K activity is necessary for synapse maintenance. We also report that PI3K controls the expression and localization of synaptic markers in human neuroblastoma cells, suggesting that PI3K synaptogenic activity is conserved in humans. Thus, we propose that PI3K stimulation can be applied to prevent or delay synapse loss in normal aging and in neurological disorders.

Molecular basis of altered excitability in Shaker mutants of Drosophila melanogaster.

Mutations in the Shaker (Sh) locus of Drosophila melanogaster have differing effects on action potential duration and repolarization in neurons as well as on A‐type K+ channels (IA) in muscle. The molecular basis of three exemplary Sh alleles (ShKS133, ShE62 and Sh5) has been identified. They are point mutations in the Sh transcription unit expressing aberrant voltage‐gated A‐type K+ channels. Replicas of each mutation have been introduced by in vitro mutagenesis into Sh cDNA. The expression of in vitro transcribed mutant Sh cRNA in Xenopus laevis oocytes reproduced the specific phenotypic traits of each Sh allele. The lack of IA in ShKS133 is due to a missense mutation within a sequence motif occurring in all hitherto characterized voltage‐gated K+ channel forming proteins. The reduction of IA in ShE62 is due to a mutation in an AG acceptor site. The intervening sequence between exons 19 and 20 is not spliced in ShE62 RNA. As a consequence, ShE62 flies do not contain the full complement of Sh K+ forming proteins. Finally, the Sh5 mutation leads to an altered voltage dependence of K+ channel activation and inactivation as well as to an accelerated rate of recovery from inactivation. This is due to a missense mutation altering the amino acid sequence of the proposed transmembrane segment S5 of the Sh K+ channels. Segment S5 is located adjacently to the presumed voltage sensor of voltage‐gated ion channels. The results explain the altered properties of excitable cells in Sh mutants and provide a general model for the possible role of A‐type K+ channels in modulating action potential profiles.

Action Potentials in Normal and Shaker Mutant Drosophila

Intracellular microelectrode recordings from the cervical giant fiber of normal Drosophila show a characteristic action potential waveform for this identified neuron. The action potential has a rapid initial spike followed by a prominent depolarizing afterpotential. Pharmacological experiments suggest that the giant fiber action potential depends on inward currents carried by Na+ and outward currents carried by K+. Abnormal action potentials are seen in Shaker (Sh) mutant Drosophila. This study compares the effects of six Sh alleles. In each case, abnormalities are limited to action potential repolarization. There are, however, allelic differences. Five alleles cause delayed repolarization and increased action potential durations. Going from most to least extreme, these alleles are: Sh102 greater than ShKS133 greater than ShM greater than ShE62 greater than ShrKO120. Compared to normal action potentials, durations in the extreme mutants are longer by an order of magnitude or more. One mutant allele, Sh5 appears to cause an incompletely repolarized action potential, rather than a repolarization delay.

Nerve Terminal Excitability and Neuromuscular Transmission in T(X;Y)V7 and Shaker Mutants of Drosophila Melanogaster

We investigated the neuromuscular transmission in relation with genetic neuronal excitability changes in mutants T(X;Y)V7 and ShK,S133 of Drosophila. These mutations affect two different genes belonging to the Shaker gene complex which encode different yet functionally related proteins. Experiments were performed on neuromuscular junctions from Drosophila larvae by recording pre- and postsynaptic membrane currents using external electrodes. It was found that the neuromuscular electrophysiological phenotype of T(X;Y)V7 is caused by presynaptic membrane hyperexcitability probably in relation with a Ca2(+)-dependent down regulation of voltage dependent K channels. By contrast, the ShKS133 phenotype can be explained solely by action potential widening due to the absence of type-A K channels.

Genetic analysis of muscle development in Drosophila melanogaster

The different thoracic muscles of Drosophila are affected specifically in the mutants: stripe (sr), erect wing (ewg), vertical wings (vtw), and nonjumper (nj). We have tested the extent of this specificity by means of a genetic analysis of these loci, multiple mutant combinations, and gene dosage experiments. A quantitative, rather than a qualitative, specificity is found in the mutant phenotypes. All muscles are altered by mutations in any given gene, but the severity of these alterations is muscle specific. The locus stripe seems to have a polar organization where different allelic combinations show quantitative specificity in the muscle affected. In addition to the muscle phenotypes, neural alterations are detected in these mutants. The synergism found between ewg, vtw and ewg, sr as well as the dosage effect of the distal end of the X chromosome upon the expression of ewg and sr suggests the existence of functional relationships among the loci analyzed.