Biology subjects

Bonanno, S. L.

Publications and source records attributed to Bonanno, S. L..

3 recordsLinked to original sources

bioRxiv2025

Cytoplasmic and nuclear protein interaction networks of the synapto-nuclear messenger CRTC1 in neurons reveal cooperative chromatin binding between CREB1 and CRTC1, MEF2C and RFX3

Glutamatergic stimulation of excitatory neurons triggers the synapto-nuclear translocation of the cAMP response element (CRE) binding protein (CREB) regulated transcription coactivator 1 (CRTC1), resulting in the transcription of CREB1 target genes. Whether and how CRTC1 and CREB1 interact with other transcription factors to regulate activity-dependent transcription, and what the role of CRTC1 is in neurons beyond the activation of CREB1 regulated transcription, remains unknown. To address these questions in an unbiased manner, we used proximity labeling to identify CRTC1-proximal proteins in cytoplasmic and nuclear compartments of rodent forebrain neurons. The cytoplasmic CRTC1 proxisome included a variety of signaling pathways and downstream cellular processes involved in synaptic plasticity. In contrast, the nuclear CRTC1 proxisome included transcription factors that mediate activity-dependent transcription, chromatin factors, and splicing factors. Our data revealed that CRTC1 and CREB1 interact with MEF2C and RFX3 transcription factors in an activity-dependent manner. Thus, in chromatin immunoprecipitation-sequencing experiments, CREB1 was prebound to chromatin regions containing bZIP motifs in a manner that was unchanged by neuronal activity, while glutamatergic stimulation triggered the recruitment of CRTC1 and CREB1 to activity-dependent enhancers enriched in motifs for MEF2C and RFX3. Collectively, these results not only enhance our understanding of the role of cytoplasmic and nuclear CRTC1 in neurons, but also reveal a role for CRTC1 in promoting cooperativity of CREB1 with other transcription factors in response to synaptic activity.

neuroscience↗

bioRxiv2023

Constitutive and conditional epitope-tagging of endogenous G protein coupled receptors in Drosophila

To visualize the cellular and subcellular localization of neuromodulatory G-protein coupled receptors (GPCRs) in Drosophila, we implement a molecular strategy recently used to add epitope tags to ionotropic receptors at their endogenous loci. Leveraging evolutionary conservation to identify sites more likely to permit insertion of a tag, we generated constitutive and conditional tagged alleles for Drosophila 5-HT1A, 5-HT2A, 5-HT2B, Oct{beta}1R, Oct{beta}2R, two isoforms of OAMB, and mGluR. The conditional alleles allow for the restricted expression of tagged receptor in specific cell types, an option not available for any previous reagents to label these proteins. We show that 5-HT1A and 5-HT2B localize to the mushroom bodies and central complex respectively, as predicted by their roles in sleep. By contrast, the unexpected enrichment of Oct{beta}1R in the central complex and of 5-HT1A and 5-HT2A to nerve terminals in lobular columnar cells in the visual system suggest new hypotheses about their function at these sites. Using an additional tagged allele of the serotonin transporter, a marker of serotonergic tracts, we demonstrate diverse spatial relationships between postsynaptic 5-HT receptors and presynaptic 5-HT neurons, consistent with the importance of both synaptic and volume transmission. Finally, we use the conditional allele of 5-HT1A to show that it localizes to distinct sites within the mushroom bodies as both a postsynaptic receptor in Kenyon cells and a presynaptic autoreceptor. Significance StatementIn Drosophila, despite remarkable advances in both connectomic and genomic studies, antibodies to many aminergic GPCRs are not available. We have overcome this obstacle using evolutionary conservation to identify loci in GPCRs amenable to epitope-tagging, and CRISPR/Cas9 genome editing to generated eight novel lines. This method also may be applied to other GPCRs and allows cell-specific expression of the tagged locus. We have used the tagged alleles we generated to address several questions that remain poorly understood. These include the relationship between pre- and post-synaptic sites that express the same receptor, and the use of relatively distant targets by pre-synaptic release sites that may employ volume transmission as well as standard synaptic signaling.

neuroscience↗

bioRxiv2022

Mutation of the Drosophila serotonin transporter dSERT disrupts courtship and feeding and increases both daytime and nighttime sleep

The Serotonin Transporter (SERT) regulates extracellular serotonin levels and is the target of most current drugs used to treat depression. The mechanisms by which inhibition of SERT activity influences behavior are poorly understood. To address this question in the model organism Drosophila melanogaster, we developed new loss of function mutations in Drosophila SERT (dSERT). Previous studies in both flies and mammals have implicated serotonin as an important neuromodulator of sleep, and our newly generated dSERT mutants show an increase in total sleep and altered sleep architecture. Differences in daytime vs. nighttime sleep architecture as well as genetic rescue experiments unexpectedly suggest that distinct serotonergic circuits may modulate daytime versus nighttime sleep. dSERT mutants also show defects in copulation and food intake, akin to the clinical side effects of SSRIs. Starvation did not overcome the sleep drive in the mutants. Additionally in males, but not female dSERT mutants, the drive to mate also failed to overcome sleep drive. dSERT may be used to further explore the mechanisms by which serotonin regulates sleep and its interplay with other complex behaviors. Author SummaryMany medications used to treat depression and anxiety act by changing serotonin levels in the brain. Fruit flies also use serotonin and can be used as a model to study the brain. We have made a fly mutant for the serotonin transporter (SERT), which is the target of antidepressants in humans. The mutants sleep more, eat less, and have a decreased sex drive. These flies can be used to study the neuronal pathways by which serotonin regulates sleep, eating and sexual behaviors and may help us to understand the behavioral effects of antidepressants.

neuroscience↗