staff

Rafael Koch

Research assistant in Neuroscience & neurodegeneration

  • T: +41 22 379 34 91
  • office 3014 (Sciences III)

  • The utility and caveat of split-GAL4s in the study of neurodegeneration. Fly (Austin) 2023 Dec;17(1):2192847. PMC10038051. 10.1080/19336934.2023.2192847.

    abstract

    Parkinson's disease (PD) is the second most common neurodegenerative disorder, afflicting over 1% of the population of age 60 y and above. The loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) is the primary cause of its characteristic motor symptoms. Studies using and other model systems have provided much insight into the pathogenesis of PD. However, little is known why certain cell types are selectively susceptible to degeneration in PD. Here, we describe an approach to identify vulnerable subpopulations of neurons in the genetic background linked to PD in , using the split-GAL4 drivers that enable genetic manipulation of a small number of defined cell populations. We identify split-GAL4 lines that target neurons selectively vulnerable in a model of ()-linked familial PD, demonstrating the utility of this approach. We also show an unexpected caveat of the split-GAL4 system in ageing-related research: an age-dependent increase in the number of GAL4-labelled cells.

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  • Uncovering the Roles of Clocks and Neural Transmission in the Resilience of Circadian Network. Front Physiol 2021 ;12():663339. 10.3389/fphys.2021.663339. PMC8188733.

    abstract

    Studies of circadian locomotor rhythms in gave evidence to the preceding theoretical predictions on circadian rhythms. The molecular oscillator in flies, as in virtually all organisms, operates using transcriptional-translational feedback loops together with intricate post-transcriptional processes. Approximately150 pacemaker neurons, each equipped with a molecular oscillator, form a circuit that functions as the central pacemaker for locomotor rhythms. Input and output pathways to and from the pacemaker circuit are dissected to the level of individual neurons. Pacemaker neurons consist of functionally diverse subclasses, including those designated as the Morning/Master (M)-oscillator essential for driving free-running locomotor rhythms in constant darkness and the Evening (E)-oscillator that drives evening activity. However, accumulating evidence challenges this dual-oscillator model for the circadian circuit organization and propose the view that multiple oscillators are coordinated through network interactions. Here we attempt to provide further evidence to the revised model of the circadian network. We demonstrate that the disruption of molecular clocks or neural output of the M-oscillator during adulthood dampens free-running behavior surprisingly slowly, whereas the disruption of both functions results in an immediate arrhythmia. Therefore, clocks and neural communication of the M-oscillator act additively to sustain rhythmic locomotor output. This phenomenon also suggests that M-oscillator can be a pacemaker or a downstream path that passively receives rhythmic inputs from another pacemaker and convey output signals. Our results support the distributed network model and highlight the remarkable resilience of the circadian pacemaker circuit, which can alter its topology to maintain locomotor rhythms.

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  • Nitric Oxide Mediates Neuro-Glial Interaction that Shapes Drosophila Circadian Behavior. PLoS Genet. 2020 Jun;16(6):e1008312. 10.1371/journal.pgen.1008312. PGENETICS-D-19-01150.

    abstract

    Drosophila circadian behavior relies on the network of heterogeneous groups of clock neurons. Short- and long-range signaling within the pacemaker circuit coordinates molecular and neural rhythms of clock neurons to generate coherent behavioral output. The neurochemistry of circadian behavior is complex and remains incompletely understood. Here we demonstrate that the gaseous messenger nitric oxide (NO) is a signaling molecule linking circadian pacemaker to rhythmic locomotor activity. We show that mutants lacking nitric oxide synthase (NOS) have behavioral arrhythmia in constant darkness, although molecular clocks in the main pacemaker neurons are unaffected. Behavioral phenotypes of mutants are due in part to the malformation of neurites of the main pacemaker neurons, s-LNvs. Using cell-type selective and stage-specific gain- and loss-of-function of NOS, we also demonstrate that NO secreted from diverse cellular clusters affect behavioral rhythms. Furthermore, we identify the perineurial glia, one of the two glial subtypes that form the blood-brain barrier, as the major source of NO that regulates circadian locomotor output. These results reveal for the first time the critical role of NO signaling in the Drosophila circadian system and highlight the importance of neuro-glial interaction in the neural circuit output.

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  • Parallel roles of transcription factors dFOXO and FER2 in the development and maintenance of dopaminergic neurons. PLoS Genet. 2018 Mar;14(3):e1007271. 10.1371/journal.pgen.1007271. PGENETICS-D-17-00703.

    abstract

    Forkhead box (FOXO) proteins are evolutionarily conserved, stress-responsive transcription factors (TFs) that can promote or counteract cell death. Mutations in FOXO genes are implicated in numerous pathologies, including age-dependent neurodegenerative disorders, such as Parkinson's disease (PD). However, the complex regulation and downstream mechanisms of FOXOs present a challenge in understanding their roles in the pathogenesis of PD. Here, we investigate the involvement of FOXO in the death of dopaminergic (DA) neurons, the key pathological feature of PD, in Drosophila. We show that dFOXO null mutants exhibit a selective loss of DA neurons in the subgroup crucial for locomotion, the protocerebral anterior medial (PAM) cluster, during development as well as in adulthood. PAM neuron-targeted adult-restricted knockdown demonstrates that dFOXO in adult PAM neurons tissue-autonomously promotes neuronal survival during aging. We further show that dFOXO and the bHLH-TF 48-related-2 (FER2) act in parallel to protect PAM neurons from different forms of cellular stress. Remarkably, however, dFOXO and FER2 share common downstream processes leading to the regulation of autophagy and mitochondrial morphology. Thus, overexpression of one can rescue the loss of function of the other. These results indicate a role of dFOXO in neuroprotection and highlight the notion that multiple genetic and environmental factors interact to increase the risk of DA neuron degeneration and the development of PD.

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  • Transforming Growth Factor β/Activin signaling in neurons increases susceptibility to starvation. PLoS ONE 2017 ;12(10):e0187054. 10.1371/journal.pone.0187054. PONE-D-17-08341.

    abstract

    Animals rely on complex signaling network to mobilize its energy stores during starvation. We have previously shown that the sugar-responsive TGFβ/Activin pathway, activated through the TGFβ ligand Dawdle, plays a central role in shaping the post-prandial digestive competence in the Drosophila midgut. Nevertheless, little is known about the TGFβ/Activin signaling in sugar metabolism beyond the midgut. Here, we address the importance of Dawdle (Daw) after carbohydrate ingestion. We found that Daw expression is coupled to dietary glucose through the evolutionarily conserved Mio-Mlx transcriptional complex. In addition, Daw activates the TGFβ/Activin signaling in neuronal populations to regulate triglyceride and glycogen catabolism and energy homeostasis. Loss of those neurons depleted metabolic reserves and rendered flies susceptible to starvation.

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  • A screening of UNF targets identifies Rnb, a novel regulator of Drosophila circadian rhythms. J. Neurosci. 2017 Jun;():. JNEUROSCI.3286-16.2017. 10.1523/JNEUROSCI.3286-16.2017.

    abstract

    Behavioral circadian rhythms are controlled by multi-oscillator networks comprising functionally different subgroups of clock neurons. Studies have demonstrated that molecular clocks in the fruit fly Drosophila melanogaster are regulated differently in clock neuron subclasses to support their specific functions (Lee et al., 2016; Top et al., 2016). The nuclear receptor unfulfilled (unf) represents a regulatory node that provides the small ventral Lateral Neurons (s-LNvs) unique characteristics as the master pacemaker (Beuchle et al., 2012). We previously showed that UNF interacts with the s-LNv molecular clocks by regulating transcription of the core clock gene period (per) (Jaumouille et al., 2015). To gain more insight into the mechanisms by which UNF contributes to the functioning of the circadian master pacemaker, we identified UNF target genes using chromatin immunoprecipitation. Our data demonstrate that a previously uncharacterized gene CG7837, which we termed R and B (Rnb), acts downstream of UNF to regulate the function of s-LNvs as the master circadian pacemaker. Mutations and LNv-targeted adult-restricted knockdown of Rnb impair locomotor rhythms. RNB localizes to the nucleus and its loss-of-function blunts the molecular rhythms and output rhythms of the s-LNvs, particularly the circadian rhythms in PDF accumulation and axonal arbor remodeling. These results establish a second pathway by which UNF interacts with the molecular clocks in the s-LNvs and highlight the mechanistic differences in the molecular clockwork within the pacemaker circuit.SIGNIFICANCE STATEMENTCircadian behavior is generated by a pacemaker circuit comprising diverse classes of pacemaker neurons, each of which contains a molecular clock. In addition to the anatomical and functional diversity, recent studies have shown the mechanistic differences in the molecular clockwork among the pacemaker neurons in Drosophila Here, we identified the molecular characteristics distinguishing the s-LNvs, the master pacemaker of the locomotor rhythms, from other clock neuron subtypes. We demonstrated that a newly identified gene Rnb is a s-LNv-specific regulator of the molecular clock and essential for the generation of circadian locomotor behavior. Our results provide additional evidence to the emerging view that the differential regulation of the molecular clocks underlies the functional differences among the pacemaker neuron subgroups.

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  • USP2-45 Is a Circadian Clock Output Effector Regulating Calcium Absorption at the Post-Translational Level. PLoS ONE 2016 ;11(1):e0145155. 10.1371/journal.pone.0145155. PONE-D-15-15381. PMC4710524.

    abstract

    The mammalian circadian clock influences most aspects of physiology and behavior through the transcriptional control of a wide variety of genes, mostly in a tissue-specific manner. About 20 clock-controlled genes (CCGs) oscillate in virtually all mammalian tissues and are generally considered as core clock components. One of them is Ubiquitin-Specific Protease 2 (Usp2), whose status remains controversial, as it may be a cogwheel regulating the stability or activity of core cogwheels or an output effector. We report here that Usp2 is a clock output effector related to bodily Ca2+ homeostasis, a feature that is conserved across evolution. Drosophila with a whole-body knockdown of the orthologue of Usp2, CG14619 (dUsp2-kd), predominantly die during pupation but are rescued by dietary Ca2+ supplementation. Usp2-KO mice show hyperabsorption of dietary Ca2+ in small intestine, likely due to strong overexpression of the membrane scaffold protein NHERF4, a regulator of the Ca2+ channel TRPV6 mediating dietary Ca2+ uptake. In this tissue, USP2-45 is found in membrane fractions and negatively regulates NHERF4 protein abundance in a rhythmic manner at the protein level. In clock mutant animals (Cry1/Cry2-dKO), rhythmic USP2-45 expression is lost, as well as the one of NHERF4, confirming the inverse relationship between USP2-45 and NHERF4 protein levels. Finally, USP2-45 interacts in vitro with NHERF4 and endogenous Clathrin Heavy Chain. Taken together these data prompt us to define USP2-45 as the first clock output effector acting at the post-translational level at cell membranes and possibly regulating membrane permeability of Ca2+.

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  • Transcriptional regulation via nuclear receptor crosstalk required for the Drosophila circadian clock. Curr. Biol. 2015 Jun;25(11):1502-8. S0960-9822(15)00430-3. 10.1016/j.cub.2015.04.017. PMC4454776.

    abstract

    Circadian clocks in large part rely on transcriptional feedback loops. At the core of the clock machinery, the transcriptional activators CLOCK/BMAL1 (in mammals) and CLOCK/CYCLE (CLK/CYC) (in Drosophila) drive the expression of the period (per) family genes. The PER-containing complexes inhibit the activity of CLOCK/BMAL1 or CLK/CYC, thereby forming a negative feedback loop [1]. In mammals, the ROR and REV-ERB family nuclear receptors add positive and negative transcriptional regulation to this core negative feedback loop to ensure the generation of robust circadian molecular oscillation [2]. Despite the overall similarities between mammalian and Drosophila clocks, whether comparable mechanisms via nuclear receptors are required for the Drosophila clock remains unknown. We show here that the nuclear receptor E75, the fly homolog of REV-ERB α and REV-ERB β, and the NR2E3 subfamily nuclear receptor UNF are components of the molecular clocks in the Drosophila pacemaker neurons. In vivo assays in conjunction with the in vitro experiments demonstrate that E75 and UNF bind to per regulatory sequences and act together to enhance the CLK/CYC-mediated transcription of the per gene, thereby completing the core transcriptional feedback loop necessary for the free-running clockwork. Our results identify a missing link in the Drosophila clock and highlight the significance of the transcriptional regulation via nuclear receptors in metazoan circadian clocks.

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