Biology subjects

Fagan, R. R.

Publications and source records attributed to Fagan, R. R..

6 recordsLinked to original sources

bioRxiv2026

Primary cilia facilitate GPCR neurotransmission through the canonical cAMP cascade

The primary cilium is increasingly recognized to be a discrete site for initiating neurotransmission through G protein-coupled receptors (GPCRs), yet how such neurotransmission is propagated downstream remains unclear. A canonical pathway of downstream GPCR neurotransmission is the cyclic AMP (cAMP) / cAMP-dependent protein kinase (PKA) cascade. While cAMP can be locally generated in cilia and produce important effects that are functionally compartmentalized there, neurotransmission through GPCRs must propagate to effectors outside of cilia. Thus, a prevailing current view is that ciliary neurotransmission is restricted to non-canonical, cAMP-dependent pathways. Revisiting this view, we show here that the primary cilium of striatal medium spiny neurons is a major site from which endogenous D1 dopamine receptors initiate cAMP and PKA activity elevations that propagate throughout the extraciliary cytoplasm. We also show that the primary cilium spatially aligns essentially this entire long-range signaling axis from intercalating with numerous dopaminergic axon varicosities that initiate neurotransmission to enforcing proximity with somatic PKA stores that are activated by cAMP downstream. Together, these results extend the concept of ciliary neurotransmission to dopaminergic signaling in striatal neurons and reveal that, in this system, the primary cilium facilitates rather than restricts neurotransmission through the canonical cAMP / PKA cascade.

neuroscience↗

bioRxiv2026

Diet-dependent sleep modulation by the Drosophila amino acid transporter ANIDRA

Sleep is a conserved animal behavior necessary for survival. It is under tight circadian and homeostatic control, and modulated by diet. Here, we identify the amino acid transporter ANIDRA (ANID) as an important sleep regulator in Drosophila. Flies lacking ANID show decreased and poorly consolidated daytime and nighttime sleep. Contrary to wild-type controls, anid mutant flies are unable to adjust their sleep to their diet, behaving as if they were constantly on a complete diet rich in amino acids. ANID is expressed in ensheathing and cortex glia, where it inhibits mTOR activity in a diet-dependent manner. Moreover, pharmacological inhibition of mTOR attenuates the anid mutant sleep phenotypes. Interestingly, DH44-expressing brain neurons, which promote arousal and sense amino acids, are constantly active in ANIDs absence. We therefore propose that ANID mediates detection of dietary amino acids by ensheathing and cortex glia to regulate the activity of arousal-promoting neurons.

neuroscience↗

bioRxiv2023

Rit2 loss in dopaminergic neurons drives a progressive Parkinsonian phenotype

Parkinsons disease (PD) is the second most prevalent neurodegenerative disease and arises from dopamine (DA) neuron death selectively in the substantia nigra pars compacta (SNc). Rit2 is a reported PD risk allele, and recent single cell transcriptomic studies identified a major RIT2 cluster in PD DA neurons, potentially linking Rit2 expression loss to a PD patient cohort. However, it is still unknown whether Rit2 loss itself is causative for PD or PD-like symptoms. Here we report that conditional Rit2 silencing in mouse DA neurons drove motor dysfunction that occurred earlier in males than females and was rescued at early stages by either inhibiting the DA transporter (DAT) or with L-DOPA treatment. Motor dysfunction was accompanied by decreased DA release, striatal DA content, phenotypic DAergic markers, DA neurons, and DAergic terminals, with increased pSer129-alpha synuclein and pSer935-LRRK2 expression. These results provide the first evidence that Rit2 loss is causal for SNc cell death and a PD-like phenotype, and reveal key sex-specific differences in the response to Rit2 loss.

neuroscience↗

bioRxiv2021

Dopaminergic Ric GTPase activity impacts amphetamine sensitivity and sleep quality in a dopamine transporter-dependent manner in Drosophila melanogaster

Dopamine (DA) is required for movement, sleep, and reward, and DA signaling is tightly controlled by the presynaptic DA transporter (DAT). Therapeutic and addictive psychostimulants, including methylphenidate (Ritalin; MPH), cocaine, and amphetamine (AMPH), markedly elevate extracellular DA via their actions as competitive DAT inhibitors (MPH, cocaine) and substrates (AMPH). DAT silencing in mice and invertebrates results in hyperactivity, reduced sleep, and blunted psychostimulant responses, highlighting DATs essential role in DA-dependent behaviors. DAT surface expression is not static; rather it is dynamically regulated by endocytic trafficking. PKC-stimulated DAT endocytosis requires the neuronal GTPase, Rit2, and Rit2 silencing in mouse DA neurons impacts psychostimulant sensitivity. However, it is unknown whether or not Rit2-mediated changes in psychostimulant sensitivity are DAT-dependent. Here, we leveraged Drosophila melanogaster to test whether the Drosophila Rit2 ortholog, Ric, impacts dDAT function, trafficking, and DA-dependent behaviors. Orthologous to hDAT and Rit2, dDAT and Ric directly interact, and the constitutively active Ric mutant Q117L increased dDAT surface levels and function in cell lines and ex vivo Drosophila brains. Moreover, DAergic RicQ117L expression caused sleep fragmentation in a DAT-dependent manner but had no effect on total sleep and daily locomotor activity. Importantly, we found that Rit2 is required for AMPH-stimulated DAT internalization in mouse striatum, and that DAergic RicQ117L expression significantly increased Drosophila AMPH sensitivity in a DAT-dependent manner, suggesting a conserved impact of Ric-dependent DAT trafficking on AMPH sensitivity. These studies support that the DAT/Rit2 interaction impacts both baseline behaviors and AMPH sensitivity, potentially by regulating DAT trafficking.

neuroscience↗

bioRxiv2019

Hedgehog signaling regulates neurogenesis in the larval and adult zebrafish hypothalamus

While neurogenesis in the adult hypothalamus is now known to be essential for proper function, the cell-cell signaling events that regulate neurogenesis in this evolutionarily conserved brain region remain poorly understood. Here we show that Hedgehog (Hh)/Gli signaling positively regulates hypothalamic neurogenesis in both larval and adult zebrafish and is necessary and sufficient for normal hypothalamic proliferation rates. Hedgehog-responsive cells are relatively rapidly proliferating pluripotent neural precursors that give rise to dopaminergic, serotonergic, and GABAergic neurons. in situ and transgenic reporter analyses revealed substantial heterogeneity in cell-cell signaling within the hypothalamic niche, with slow cycling Nestin-expressing cells residing among distinct and overlapping populations of Sonic Hh (Shh)-expressing, Hh-responsive, Notch-responsive, and Wnt-responsive radial glia. This work shows for the first time that Hh/Gli-signaling is a key component of the complex cell-cell signaling environment that regulates hypothalamic neurogenesis throughout life.

neuroscience↗

bioRxiv2019

Dopamine transporter trafficking and Rit2 GTPase:In vivo impact and mechanism of action

Following its evoked release, DA signaling is rapidly terminated by presynaptic reuptake, mediated by the cocaine-sensitive DAT. DAT surface availability is dynamically regulated by endocytic trafficking, and direct PKC activation acutely diminishes DAT surface expression by accelerating DAT internalization. Previous cell line studies demonstrated that PKC-stimulated DAT endocytosis requires both Ack1 inactivation, which releases a DAT-specific endocytic brake, and the neuronal GTPase, Rit2, which binds DAT. However, it is unknown whether Rit2 is required for PKC-stimulated DAT endocytosis in DAergic terminals, or whether there are region- and/or sex-dependent differences in PKC-stimulated DAT trafficking. Moreover, the mechanisms by which Rit2 controls PKC-stimulated DAT endocytosis are unknown. Here, we directly examined these important questions. Ex vivo studies revealed that PKC activation acutely decreased DAT surface expression selectively in ventral, but not dorsal, striatum. AAV-mediated, conditional Rit2 knockdown in DAergic neurons impacted baseline DAT surface:intracellular distribution in DAergic terminals from female ventral, but not dorsal, striatum. Further, Rit2 was required for PKC-stimulated DAT internalization in both male and female ventral striatum. FRET and surface pulldown studies in cell lines revealed that PKC activation drives DAT-Rit2 surface dissociation, and that the DAT N-terminus is required for both PKC-mediated DAT-Rit2 dissociation and DAT internalization. Finally, we found that Rit2 and Ack1 independently converge on DAT to facilitate PKC-stimulated DAT endocytosis. Together, our data provide greater insight into mechanisms that mediate PKC-regulated DAT internalization, and reveal unexpected region-specific differences in PKC-stimulated DAT trafficking in bona fide DAergic terminals.

neuroscience↗