Neuronal Depolarization Drives Increased Dopamine Synaptic Vesicle Loading via VGLUT.

The ability of presynaptic dopamine terminals to tune neurotransmitter release to meet the demands of neuronal activity is critical to neurotransmission. Although vesicle content has been assumed to be static, in vitro data increasingly suggest that cell activity modulates vesicle content. Here, we use a coordinated genetic, pharmacological, and imaging approach in Drosophila to study the presynaptic machinery responsible for these vesicular processes in vivo. We show that cell depolarization increases synaptic vesicle dopamine content prior to release via vesicular hyperacidification. This depolarization-induced hyperacidification is mediated by the vesicular glutamate transporter (VGLUT). Remarkably, both depolarization-induced dopamine vesicle hyperacidification and its dependence on VGLUT2 are seen in ventral midbrain dopamine neurons in the mouse. Together, these data suggest that in response to depolarization, dopamine vesicles utilize a cascade of vesicular transporters to dynamically increase the vesicular pH gradient, thereby increasing dopamine vesicle content.

Dysregulation of microRNA-219 promotes neurodegeneration through post-transcriptional regulation of tau.

Tau is a highly abundant and multifunctional brain protein that accumulates in neurofibrillary tangles (NFTs), most commonly in Alzheimer's disease (AD) and primary age-related tauopathy. Recently, microRNAs (miRNAs) have been linked to neurodegeneration; however, it is not clear whether miRNA dysregulation contributes to tau neurotoxicity. Here, we determined that the highly conserved brain miRNA miR-219 is downregulated in brain tissue taken at autopsy from patients with AD and from those with severe primary age-related tauopathy. In a Drosophila model that produces human tau, reduction of miR-219 exacerbated tau toxicity, while overexpression of miR-219 partially abrogated toxic effects. Moreover, we observed a bidirectional modulation of tau levels in the Drosophila model that was dependent on miR-219 expression or neutralization, demonstrating that miR-219 regulates tau in vivo. In mammalian cellular models, we found that miR-219 binds directly to the 3'-UTR of the tau mRNA and represses tau synthesis at the post-transcriptional level. Together, our data indicate that silencing of tau by miR-219 is an ancient regulatory mechanism that may become perturbed during neurofibrillary degeneration and suggest that this regulatory pathway may be useful for developing therapeutics for tauopathies.

Miniature neurotransmission regulates Drosophila synaptic structural maturation.

Miniature neurotransmission is the transsynaptic process where single synaptic vesicles spontaneously released from presynaptic neurons induce miniature postsynaptic potentials. Since their discovery over 60 years ago, miniature events have been found at every chemical synapse studied. However, the in vivo necessity for these small-amplitude events has remained enigmatic. Here, we show that miniature neurotransmission is required for the normal structural maturation of Drosophila glutamatergic synapses in a developmental role that is not shared by evoked neurotransmission. Conversely, we find that increasing miniature events is sufficient to induce synaptic terminal growth. We show that miniature neurotransmission acts locally at terminals to regulate synapse maturation via a Trio guanine nucleotide exchange factor (GEF) and Rac1 GTPase molecular signaling pathway. Our results establish that miniature neurotransmission, a universal but often-overlooked feature of synapses, has unique and essential functions in vivo.

Tramtrack regulates different morphogenetic events during Drosophila tracheal development.

Tramtrack (Ttk) is a widely expressed transcription factor, the function of which has been analysed in different adult and embryonic tissues in Drosophila. So far, the described roles of Ttk have been mainly related to cell fate specification, cell proliferation and cell cycle regulation. Using the tracheal system of Drosophila as a morphogenetic model, we have undertaken a detailed analysis of Ttk function. Ttk is autonomously and non-autonomously required during embryonic tracheal formation. Remarkably, besides a role in the specification of different tracheal cell identities, we have found that Ttk is directly involved and required for different cellular responses and morphogenetic events. In particular, Ttk appears to be a new positive regulator of tracheal cell intercalation. Analysis of this process in ttk mutants has unveiled cell shape changes as a key requirement for intercalation and has identified Ttk as a novel regulator of its progression. Moreover, we define Ttk as the first identified regulator of intracellular lumen formation and show that it is autonomously involved in the control of tracheal tube size by regulating septate junction activity and cuticle formation. In summary, the involvement of Ttk in different steps of tube morphogenesis identifies it as a key player in tracheal development.

Egfr is essential for maintaining epithelial integrity during tracheal remodelling in Drosophila.

A fundamental requirement during organogenesis is to preserve tissue integrity to render a mature and functional structure. Many epithelial organs, such as the branched tubular structures, undergo a tremendous process of tissue remodelling to attain their final pattern. The cohesive properties of these tissues need to be finely regulated to promote adhesion yet allow flexibility during extensive tissue remodelling. Here, we report a new role for the Egfr pathway in maintaining epithelial integrity during tracheal development in Drosophila. We show that the integrity-promoting Egfr function is transduced by the ERK-type MAPK pathway, but does not require the downstream transcription factor Pointed. Compromising Egfr signalling, by downregulating different elements of the pathway or by overexpressing the Mkp3 negative regulator, leads to loss of tube integrity, whereas upregulation of the pathway results in increased tissue stiffness. We find that regulation of MAPK pathway activity by Breathless signalling does not impinge on tissue integrity. Egfr effects on tissue integrity correlate with differences in the accumulation of markers for cadherin-based cell-cell adhesion. Accordingly, downregulation of cadherin-based cell-cell adhesion gives rise to tracheal integrity defects. Our results suggest that the Egfr pathway regulates maintenance of tissue integrity, at least in part, through the modulation of cell adhesion. This finding establishes a link between a developmental pathway governing tracheal formation and cell adhesiveness.