Habituation is a major mechanism to decrease responsiveness to repetitive or prolonged non-reinforced stimuli. Filtering such events of low significance is likely the foundation of selective attention, necessary for learning and memory. Defective habituation has been associated with schizophrenia, learning disabilities such as Attention Deficit Hyperactivity Disorder (ADHD) and migraines among other conditions. We established a system to study habituation to two qualitatively different stimuli and identified and characterized at least one mutant in the process. We are conducting genetic screens aiming to elucidate the molecular basis of habituation for which very little is known and to map neurons within the brain that mediate habituation and protection from premature habituation. In addition, we are screening drugs likely to reverse the ADHD-like premature habituation of the mutants.

Molecular mechanisms of learning and memory

Identification of the molecular components of neuronal signalling cascades and determination of their role in neuroplasticity is essential to understanding learning and memory. To that end, we are employing a multidisciplinary approach based on the knowledge that 5000 specific neurons in the Drosophila brain organized in a characteristic structure called mushroom bodies (MBs) are required for olfactory learning and memory. We have shown that particular regulators of members of the RAS/RAF/MAPK signalling cascade expressed in MB neurons when mutated precipitate learning and memory deficits. This lead us to hypothesize that unlike their explored activities in differentiation, proliferation and oncogenicity members of the cascade have unique roles and regulation in postdevelopmental neurons mediated via novel interactors or alternative signalling routes. We are pursuing this hypothesis in order to elucidate the role of this cascade in learning and memory especially in combination with cAMP signalling which has been established as essential for these processes in multiple experimental systems. Additionally we are investigating Notch signalling, whose essential developmental role is unquestionable and more recently calcium and calmodulin activated kinase- CamK-II.

Molecular and Behavioural Neurobiology of Cognitive and Denenerative disorders

We use transgenic models that express human genes associated with common disorders such as Alzheimer's, Parkinsons, and other dementias to investigate the molecular mechanisms responsible for the earliest possible hallmarks of each condition, learning and memory deficits and attempt their pharmacological amelioration.
A. TAU-dependent disorders. Recently we established that mere accumulation of the neuronal microtubule associated protein TAU, akin to that observed in neurons of Frontotemporal Dementia with Parkinsonism and Alzheimer's patients prior to visible degeneration and neurofibrillary tangle (NFT) formation, precipitates large deficits in learning and memory. We are using this model to attempt pharmacological amelioration of the disease using the behavioural phenotypes as indicators in an effort to halt, or inhibit progression to NFT formation and degeneration. In addition, in an effort to elucidate the mechanisms of disease progression and the cause(s) for the observed behavioural deficits, we are using genetic tools to identify interacting proteins and signalling pathways.
B. Iron and the nervous system. A number of human behavioural and cognitive disorders such as Friedeich's ataxia, Hallevorden-Spatz syndrome and neuroferritinopathies are associated with perturbations of the level of free and protein bound iron. In addition, iron accumulates in the hippocampus of Alzheimer's patients and normal aged individuals and in Lewy bodies and degenerating neurons of Parkinson's patients. To elucidate the contribution of iron in neurodegenerative diseases and the mechanisms of cognitive impairment in individuals with abnormal levels of iron, in collaboration with Dr. T. Roualt and F. Missirlis (NIH, USA) we are investigating the effects of changes in the levels of free and stored iron in particular neurons and glia on longevity, behavior, learning and memory and neurodegeneration and its interactions with models of TAU-dependent cognitive and neurodegenerative conditions described above and Parkinson's.

Molecular Biology of 14-3-3 proteins

The 14-3-3 proteins comprise a highly conserved family of small acidic molecules present in all eukaryotes. 14-3-3s share a common structure composed of nine anti-parallel α-helices forming a palisade around a central negatively charged groove of largely invariant amino acids. All 14-3-3s form homo- and heterodimers. A phosphoprotein binding surface formed by conserved amino acids in the groove interacts with target proteins that contain the motifs RSxpS/TxP, or RxxxpS/TxP (where x=any amino acid, pS/T=phosphoserine or phosphothreonine). 14-3-3 binding on a target protein may protect it from dephosphorylation or proteolysis, modulate its activity, alter its ability to interact with other partners, or modify its cytoplasmic/nuclear partition. Therefore, it is not unusual that 14-3-3 proteins have been implicated in a diverse number of processes and biochemical pathways.

Unlike vertebrates which contain seven genes Drosophila melanogaster contains two 14-3-3 genes that generate two proteins for leonardo (LEO I and LEO II) with 88% identity to mammalians. The D14-3-3ε gene encodes a single protein 82% identical to the mammalian isoform. As in vertebrates, the LEO proteins are found enriched in adult brain and in low levels throughout the body, as well as embryos, larvae and pupae. Similarly, D14-3-3ε is present in all developmental stages and in all tissues examined with only slight enrichment in the adult brain. Therefore, Drosophila provides a simple genetically tractable model system to study 14-3-3 isotype specific functions and interactions in vivo.

We have generated and characterized multiple mutant alleles and transgenic constructs of the fly proteins and used them to understand their functions using mutant analyses. We use transgenic Drosophila expressing each of the seven vertebrate isoforms and our understanding of the phenotypes of 14-3-3 mutations in the fly, as a "test tube" to investigate in vivo and in tissues where activity of these proteins is essential, a problem typically addressed by in vitro or in culture experiments. Namely, whether the high degree of identity among the vertebrate isoforms results in a high degree of redundancy in function, or whether each has unique functions in vivo. We are asking whether and which of the vertebrate proteins are capable to functionally reverse the phenotypes associated with loss of the Drosophila proteins in a tissue or process-specific manner.

Furthermore, we have identified 14-3-3 interacting proteins and we are in the process of characterizing them and investigate their roles in neurons. Since mutants are not available thus far, we are generating phenocopies utilizing transgenes capable of eliminating these proteins in a tissue specific manner using RNA-interference techniques.

Collaborative research

The expertise available in our laboratory has allowed the initiation of diverse collaborative projects with other research groups as well as biotechnology and pharmaceutical companies, both in Greece and abroad. These include: Transgenic models of the vertebrate gene BM88 to investigate its potential neurogenic activity. Neuropharmacological projects with a biotechnology company in the USA. Behavioral analyses of novel mutations in genes expressed in the adult nervous system. Finally, development of a model for Fetal Alcohol Syndrome in Drosophila.