Neurotransmitter release

Mechanisms regulating neurotransmitter release

We are currently involved in a research project (Progetto di Eccellenza Fondazione Cariparo; leader Prof C. Montecucco) aimed at analysing the crucial steps of vesicle exo- and endo- cytosis during neurotransmitter release at neuronal chemical synapses. In particular the project aims to deepen the understanding of the molecular and cellular pathogenesis of botulism and of snake envenomation and the process of regeneration of the toxin-blocked neuromuscular junction. This project entails the use of different experimental models (whole animal and cellular cultures; vertebrates and invertebrates) in which the UPFNL will employ the powerful genetic tools available for Drosophila melanogaster in combination with sophisticated psychophysiological, neurophysiological, as well as functional and morphological imaging techniques.The Drosophila CASK (caki) null mutant is characterized by a remarkable increase in spontaneous neurotrasmitter release (miniature end-plate potentials or minis) and an early onset of synaptic fatigue during evoked neurotrasmitter release engendered by continuous high frequency stimulation. Such functional alterations are not accompanied by neuronal degeneration, and are possibly responsible for impaired neurophysiological responses at the level of the optomotor circuits as well at the level of flight motor circuits. In our lab, we have studied the Drosophila null mutant of the mammalian homologue CASK, a protein which is part of a multiprotein membrane complex which also includesMunc18-1.The SNAREs and the SM protein Munc18-1 are probably the most central components of the exocytotic apparatus. The SNAREs have been proposed to account for the specificity of membrane fusion and to directly execute fusion by forming a tight complex (the SNARE or core complex). The SNARE complex brings the synaptic vesicle and plasma membranes together, while Munc18-1 has been suggested to assist in the formation of this complex.Many proteins are involved in neurotransmitter release and they are evolutionarily conserved. In fact there is evidence that the mechanism of neurotransmitter release itself has been conserved during evolution in very different organisms. The proteins involve in this process include Sec18/N-ethylmaleimide-sensitive fusion protein (NSF) homologues, Sec17/soluble NSF attachment proteins (SNAPs), SNAP receptors (SNAREs), Sec1/Munc18 homologues (SM proteins) and small GTPases of the Rab family. In addition, proteins such as synaptotagmin 1, Munc13-1 and the complexins are crucial for Ca2+-triggered exocytosis, (but not for other forms of membrane-traffic), and are specialized for the tight spatial and temporal regulation of neurotransmitter release.Neurotransmitter release is localized in specialized regions of the pre-synaptic plasma membrane known as active zones. Synaptic vesicles dock at active zones and then undergo a priming reaction that prepares them for exocytosis when Ca2+ channels open in response to an action potential. Neurotrasmitter release is very fast (less than 0.5 ms after Ca2+ influx). The speed of synaptic transmission is also facilitated by the close apposition of the active zones to specialized sites on the post-synaptic plasma membrane which are enriched in neurotransmitter receptors.During the last several years, the application of classical (physical or chemical) as well as transposon-based mutagenesis, or methods for gene silencing based on double-stranded RNAi, has led to the generation of several fly lines carrying null or hypomorphic alleles of genes expressing proteins involved in neurotrasmitter release. These have constituted the ideal object for experimental approaches based on psychophysiological, electrophysiological and both functional as well as morphological imaging techniques. Neurons communicate at the level of specialized junctions: synapses. Through synapses, information is transmitted from one neuron to the next leading to the complex functional organization of the nervous system into circuits. Information processing in the nervous system depends upon the integration of information received by each single neuron through synapses (both excitatory and inhibitory). Thus, it is important to understand the general (basic) mechanisms regulating synaptic activity. From an evolutionary standpoint, these mechanisms are well conserved throughout the animal kingdom. Thus it is possible to study them in the simplest nervous systems where specific circuits or even synapses are easily accessible. The best animal models should be amenable to genetic manipulation which allows the genetics and molecular dissection of the components involved in synaptic function. The model which actually appears to conjugate these two basic requirements is Drosophila melanogaster.

Neurological Diseases

In a recent work, made in collaboration with K Nowikowsky (Max Perutz, Wien), we analysed the nervous system behavioural and electrophysiological phenotype in Drosophila LETM1 homolog silenced lines. LETM1 is one of the genes linked to the Wolf-Hirschhorn Syndrome, a congenital syndrome characterised by developmental alterations, mental retardation and epilepsy. Another gene which homolog was analysed in collaboration with R.Costa (Dep. of Biology, University of Padova) and M.Zeviani (Istituto Neurologico Besta, Milano) is surf1 which is linked in humans with the Leigh Syndrome, the most commun mitochondrial encephalomiopathy. Using the RNAi technique, D.melanogaster surf1 homolog was silenced by creating transgenic lines bearing the interferring dsRNA against Drosophila surf1 under the control of an UAS promoter. After crossing the transgenic line with a Gal4 line, the Gal4 production in F1 individuals induces the UAS activation in cells expressing Gal4. In this way, using the Gal4/UAS binary system it was possible to silence surf1 ubiquitously or in specific tissues (nervous system or muscle system). On this line are the studies, in collaboration with R.Costa (Dep of Biology, University of Padova) on zasp/cypher, a gene expressing a protein of the Z-line in striated muscle which alteration is responsible of a group of mild dystrophies in humans.Our aim is to specifically disrupt identified homologs in D.melanogaster using the powerful genetics available for this animal model (P-elements or RNAi) and analyse the nervous system phenotype, physiology and morphology in mutant lines.Between 60 to 70 % homologs of genes associated to inherited human genetic diseases are present in Drosophila melanogaster genome. Among these are genes linked to inherited nervous system diseases and also to other neurologic disturbances of uncertain origin like Alzheimer disease, Parkinsons disease, Schizophrenia or epilepsy.