Biology subjects

Mrestani, A.

Publications and source records attributed to Mrestani, A..

5 recordsLinked to original sources

bioRxiv2023

Rab3 mediates cyclic AMP-dependent presynaptic plasticity and olfactory learning

Presynaptic forms of plasticity occur throughout the nervous system and play an important role in learning and memory but the underlying molecular mechanisms are insufficiently understood. Here we show that the small GTPase Rab3 is a key mediator of cyclic AMP (cAMP)-induced presynaptic plasticity in Drosophila. Pharmacological and optogenetic cAMP production triggered concentration-dependent alterations of synaptic transmission, including potentiation and depression of evoked neurotransmitter release, as well as strongly facilitated spontaneous release. These changes correlated with a nanoscopic rearrangement of the active zone protein Unc13A and required Rab3. To link these results to animal behaviour, we turned to the established role of cAMP signalling in memory formation and demonstrate that Rab3 is necessary for olfactory learning. As Rab3 is dispensable for basal synaptic transmission, these findings highlight a molecular pathway specifically dedicated to tuning neuronal communication and adaptive behaviour.

neuroscience↗

bioRxiv2023

Drosophila Bchs overexpression recapitulates human WDFY3 neurodevelopmental phenotypes with implications for glial cell involvement in altered head circumference

The autophagy adaptor WDFY3 is linked to neurodevelopmental delay and altered brain size. Loss-of-function variants are associated with an increased brain size in both humans and mice. We thus, hypothesized that the microcephaly observed in some of the patients may be related to a gain-of-function of the WDFY3 gene product. While the role of WDFY3 loss-of-function has been studied extensively in neurons, little is known about the effects of WDFY3 overexpression in different neural cell types. We utilized a Drosophila melanogaster overexpression model to investigate the effect of the WDFY3 ortholog Bchs (blue cheese) on development, CNS size, and gene expression profiles. Glial and neuronal overexpression of Bchs impaired CNS development, locomotion and autophagy. Glial overexpression of Bchs also altered CNS size significantly. We identified 79 genes that were differentially expressed and overlapped in flies that overexpress Bchs in glial and neuronal cells, respectively. Additionally, upon neuronal Bchs overexpression differentially expressed genes clustered in gene ontology categories associated with autophagy and mitochondria. Our data indicate that WDFY3/Bchs overexpression in both neurons and glial cells results in impaired neural development, which corresponds to symptoms observed in WDFY3-related neurodevelopmental delay.

neuroscience↗

bioRxiv2022

Endogenous tagging of Unc-13 reveals nanocluster reorganization at active zones during presynaptic homeostatic potentiation

Neurotransmitter release at presynaptic active zones (AZs) requires concerted protein interactions within a dense 3D nano-hemisphere. Among the complex protein mesh-work the (M)unc-13 family member Unc-13 of Drosophila melanogaster is essential for docking of synaptic vesicles and transmitter release. We employ MiMIC-based gene editing using GFSTF (EGFP-FlAsH-StrepII-TEV-3xFlag) to endogenously tag all annotated Drosophila Unc-13 isoforms enabling visualization of endogenous Unc-13 expression within the central and peripheral nervous system. Electrophysiological characterization using two-electrode voltage clamp (TEVC) reveals that evoked and spontaneous synaptic transmission remain unaffected in unc-13GFSTF 3rd instar larvae and acute presynaptic homeostatic potentiation (PHP) can be induced at control levels. Furthermore, multi-color structured-illumination shows precise co-localization of Unc-13GFSTF, Bruchpilot and GluRIIA-receptor subunits within the synaptic mesoscale. Localization microscopy in combination with HDBSCAN algorithms detect Unc-13GFSTF nanoclusters that move towards the AZ center during PHP with unaltered Unc-13GFSTF protein levels.

neuroscience↗

bioRxiv2021

Channel opening duration in adult muscle nAChRs determined by activated external ACh binding site

We recorded currents through the cell membrane at single nAChR molecules, held at ACh or Epibatidine (Ebd) concentrations of 0.01, 0.1, 1, 10 or 100 {micro}M. The measured current amplitudes had an absolutely fixed value of 15 pA. This was valid for different agonists at all concentrations. Binding an agonist at one or both sites in the ring of subunits allowed to open the channel, the site that initiated the opening determined the duration of the final opening of the channel. In addition, the current flow was continuously interrupted by < 3 {micro}s shut times. The resolution of our records was optimized to reach 5 {micro}s, but was insufficient to resolve an unknown proportion of shorter shut times. Therefore, measured durations of openings are overestimated, and cited in brackets: {tau}o1 (3 {micro}s) elicited by agonist-binding at the {delta}-site, {tau}o2 and {tau}o3 (40 and 183 {micro}s) by binding at the {varepsilon}-site, and {tau}o4 (752 {micro}s) by binding at the {varepsilon}- and {delta}-site. Mono-liganded nAChRs trigger short bursts of 0.6 ms duration. Bi-liganded nAChRs generate long bursts that at low agonist concentrations last 12 ms. Above 10 {micro}M ACh, long bursts are shortened, with 100 {micro}M ACh, to 5 ms, and further at higher concentrations. While ACh was the main agonist, Ebd bound more effectively than ACh to the {varepsilon}-site. SIGNIFICANCETransmembrane currents were recorded from single nAChR molecules. Binding ACh at one of the external receptor sites opens the central current channel repeatedly for a period selected by the activated receptor site, and transmembrane current can flow with fixed 15 pA amplitude (same at various agonists and range of concentrations, unaffected by desensitization). Current flow is interrupted by fixed < 3 {micro}s blocks of the central channel that border each opening. Sets of current block and opening can be repeated for receptor-determined periods of time and form bursts of channel openings. The results may help to interpret the structural reactions in the center of the molecule that are addressed in the introduction.

neuroscience↗

bioRxiv2019

Active zone compaction for presynaptic strength

Brain function relies on neurotransmission which is stabilized by presynaptic homeostatic potentiation (PHP). PHP operates on time scales ranging from minute- to life-long adaptations and likely involves reorganization of presynaptic active zones (AZs). At Drosophila melanogaster neuromuscular junctions, earlier work ascribed AZ enlargement by incorporating more Bruchpilot (Brp) scaffold protein a central mechanistic role in PHP. We used localization microscopy (direct stochastic optical reconstruction microscopy, dSTORM) and hierarchical density-based spatial clustering of applications with noise (HDBSCAN) to study AZ plasticity during PHP. We found that both acute, philanthotoxin (PhTx)-induced and chronic, genetically-induced PHP lead to compaction of individual AZs without altering Brp copy numbers per AZ. This compaction even occurs within Brp subclusters of the AZ scaffold which also move towards AZ centers. Furthermore, lowering imaging resolution revealed how AZ compaction in PHP translates into apparent increases in AZ area and Brp protein content as implied earlier. Our results suggest AZ compaction in PHP as an effective mechanism to raise presynaptic protein density and transmitter release. SIGNIFICANCE STATEMENTHomeostatic plasticity stabilizes chemical synaptic transmission in multiple organisms ranging from insects to humans. Changes in active zones (AZs), membrane specializations of the presynapse where synaptic vesicles are discharged, are thought to be crucial in homeostatic adaptations. AZ growth by protein incorporation was proposed as a core mechanism in presynaptic homeostatic potentiation (PHP). Localization microscopy of an abundant AZ scaffold protein uncovered that instead of growing, AZs are compacted in acute and chronic PHP. At lower imaging resolution, however, AZs appear larger and brighter although protein numbers are not increased. In summary, our findings suggest AZ compaction as new and effective mechanism to raise presynaptic protein density and transmitter release in PHP.

neuroscience↗