Biology subjects

Pielage, J.

Publications and source records attributed to Pielage, J..

8 recordsLinked to original sources

bioRxiv2026

Mitochondrial protein import stress causes progressive neurodegeneration opposed by PERK - eIF2α signalling

Mitochondrial dysfunction and impairments of the mitochondrial protein import system are often linked to neurodegenerative disease, but whether import stress per se causes neurodegeneration has not been tested. Here, we adapted the yeast clogger system to Drosophila motoneurons to block TOM-TIM23-mediated import with temporal control. Sustained import stress converts somatic mitochondria into donut-shaped structures, depletes functional mitochondria from synaptic terminals, and causes progressive neurodegeneration with impaired neurotransmitter release and locomotor dysfunction. This neurodegeneration is mechanistically distinct from mitochondrial absence, as miro mutant neurons that completely lack presynaptic mitochondria do not degenerate. Import-stressed motoneurons activate multiple protective programmes, including chaperone remodelling, metabolic repression, and translational control through the eIF2 kinase PERK. Both pharmacological PERK inhibition and reversal of translational attenuation via ISRIB accelerate neurodegeneration, whereas PERK overexpression alone is sufficient to cause it, defining a protective range of eIF2-dependent translational control. The observation that PERK inhibition is protective in protein misfolding models but detrimental during import stress shows that the nature of mitochondrial dysfunction determines the molecular consequence of translational control in neurodegeneration.

neuroscience↗

bioRxiv2026

Loss of NudE-mediated dynein activation at synaptic terminals causes progressive axon length-dependent neurodegeneration

Progressive, axon length-dependent degeneration of nerve terminals is a defining feature of dying-back neuropathies; yet, whether defects in axonal transport initiate or are a consequence of this process remains unresolved. Here, we show that NudE, a scaffold for dynein motor activation, is required for the initiation of retrograde axonal transport at Drosophila motoneuron synapses in vivo. Loss of NudE impairs dynein activation at the synaptic terminal, severely reducing the proportion of cargo entering retrograde transport and decreasing retrograde motor velocity. Live imaging and temporal analysis establish that this transport initiation defect is the earliest event in a degenerative cascade, followed by progressive microtubule destabilization, impaired synaptic transmission, and structural degeneration in a distal-to-proximal gradient that recapitulates dying-back neuropathies. Structure-function analysis validates the biochemically defined NudE-dynein binding domains in vivo, with graded disruption producing correspondingly graded phenotypes. Genetic two-hit experiments uncover a reciprocal dependence between transport initiation and microtubule maintenance: perturbations in either process that are individually tolerated synergistically trigger degeneration when combined, while increasing microtubule levels alone cannot compensate for failed dynein activation. These findings reveal retrograde transport initiation as a critical vulnerability point in motoneurons and provide a mechanistic basis for how defects in dynein activation produce progressive neurodegeneration.

neuroscience↗

bioRxiv2025

Protein phosphatase 4 is required for centrosome asymmetry in fly neural stem cells

Asymmetric cell division is used by stem cells to create diverse cell types while self-renewing the stem cell population. Biased segregation of molecularly distinct centrosomes could provide a mechanism to maintain stem cell fate, induce cell differentiation or both. However, the molecular mechanisms generating molecular and functional asymmetric centrosomes remain incompletely understood. Here, we show that in asymmetrically dividing fly neural stem cells, Protein phosphatase 4 (Pp4) is necessary for correct centrosome asymmetry establishment during mitosis, and microtubule organizing center (MTOC) maintenance in interphase. Using in-vivo live cell imaging we show that while wild type neural stem cells always maintain one active MTOC, Pp4 mutant neuroblasts contain two inactive centrioles in interphase. Furthermore, centrosomes of Pp4 mutant neural stem cells mature in mitosis but fail to correctly transfer the centriolar protein Centrobin (Cnb) from the mother to the daughter centriole. Using superresolution imaging, we find that phosphomimetic Centrobin fails to accurately relocalize in mitosis. We propose that Pp4 regulates the timely relocalization of Cnb in mitosis to establish two molecularly distinct centrosomes. In addition, Pp4 is also necessary to maintain MTOC activity in interphase, ensuring biased centrosome segregation. Mechanistically, Pp4 could regulate centrosome asymmetry by dephosphorylating both Cnb and gamma-Tubulin. SIGNIFICANCE STATEMENTO_LIAsymmetric centrosome segregation occurs in stem cells and has been linked with cell fate decisions. C_LIO_LIProtein phosphatase 4 (Pp4), a conserved Serine/Threonine phosphatase, regulates centrosome asymmetry in Drosophila neural stem cells by acting upon gamma tubulin and Centrobin. C_LIO_LIPp4 regulates centrosome asymmetry establishment in mitosis and interphase, necessary for biased centrosome segregation. C_LI

cell biology↗

bioRxiv2023

Darkness inhibits autokinase activity of bacterial bathy phytochromes

Bathy phytochromes are a subclass of bacterial biliprotein photoreceptors that carry a biliverdin IX chromophore. In contrast to prototypical phytochromes that adopt a Pr ground state, the Pfr-form is the thermally stable ground state of bathy phytochromes. Although the photobiology of bacterial phytochromes has been extensively studied since their discovery in the late 1990s, our understanding of the signal transduction process to the connected transmitter domains, which are often histidine kinases, remains insufficient. Initiated by the analysis of the bathy phytochrome PaBphP from Pseudomonas aeruginosa, we performed a systematic analysis of five different bathy phytochromes with the aim to derive a general statement on the correlation of photostate and autokinase output. While all proteins adopt different Pr/Pfr-fractions in response to red, blue, and far-red light, only darkness leads to a pure or highly-enriched Pfr-form, directly correlated with the lowest level of autokinase activity. Using this information, we developed a method to quantitatively correlate the autokinase activity of phytochrome samples with well-defined stationary Pr/Pfr-fractions. We demonstrate that the off-state of the phytochromes is the Pfr-form and that different Pr/Pfr-fractions enable the organisms to fine-tune their kinase output in response to a certain light environment. Furthermore, the output response is regulated by the rate of dark reversion, which differs significantly from 5 seconds to 50 minutes half-life. Overall, our study indicates that bathy phytochromes function as sensors of light and darkness, rather than red and far-red light, as originally postulated.

microbiology↗

bioRxiv2023

Optimized design and in vivo application of optogenetically functionalized Drosophila dopamine receptors

Neuromodulatory signaling via G protein-coupled receptor (GPCRs) plays a pivotal role in regulating neural network function and animal behavior. Recent efforts have led to the development of optogenetic tools to induce G protein-mediated signaling, with the promise of acute and cell type-specific manipulation of neuromodulatory signals. However, designing and deploying optogenetically functionalized GPCRs (optoXRs) with accurate specificity and activity to mimic endogenous signaling in vivo remains challenging. Here we optimized the design of optoXRs by considering evolutionary conserved GPCR-G protein interactions and demonstrate the feasibility of this approach using two Drosophila Dopamine receptors (optoDopRs). We validated these optoDopRs showing that they exhibit high signaling specificity and light sensitivity in vitro. In vivo we detected receptor and cell type-specific effects of dopaminergic signaling in various behaviors including the ability of optoDopRs to rescue loss of the endogenous receptors. This work demonstrates that OptoXRs can enable optical control of neuromodulatory receptor specific signaling in functional and behavioral studies.

neuroscience↗

bioRxiv2022

MitoStores: Chaperone-controlled protein granules store mitochondrial precursors in the cytosol

Hundreds of mitochondrial precursor proteins are synthesized in the cytosol and imported into mitochondria in a post-translational reaction. The early processes associated with mitochondrial protein targeting remain poorly understood. Here we show that in bakers yeast, the cytosol has the capacity to transiently store matrix-destined precursors in dedicated deposits which we named MitoStores. MitoStores are strongly enhanced when protein import into mitochondria is competitively inhibited by a clogging of mitochondrial import sites, but also formed under physiological conditions when cells grow on non-fermentable carbon sources. MitoStores are enriched for a specific subset of nuclear encoded mitochondrial proteins, in particular those containing N-terminal mitochondrial targeting sequences. MitoStore formation is controlled by the heat shock proteins Hsp42 and Hsp104, potentially to suppress the toxic potential of accumulating mitochondrial precursor proteins. Thus, the cytosolic protein quality control system plays an active role during early stages in mitochondrial protein targeting by the coordinated and localized sequestration of mitochondrial precursor proteins. SummaryThe yeast cytosol can deposit precursors of mitochondrial proteins in specific granules called MitoStores. MitoStores are controlled by the cytosolic chaperone system, in particular by Hsp42 and Hsp104. MitoStore formation suppresses the toxicity arising from non-imported mitochondrial precursor proteins.

biochemistry↗

bioRxiv2020

Dendritic signal integration in Drosophila Mushroom Body Output Neuron (MBON) essential for learning and memory

The ability to associate neutral stimuli with valence information and to store these associations as memories forms the basis for decision making. To determine the underlying computational principles, we build a realistic computational model of a central decision module within the Drosophila mushroom body (MB), the flys center for learning and memory. Our model combines the electron microscopy-based architecture of one MB output neuron (MBON-3), the synaptic connectivity of its 948 presynaptic Kenyon cells (KCs), and its membrane properties obtained from patch-clamp recordings. We show that this neuron is electrotonically compact and that synaptic input corresponding to simulated odor input robustly drives its spiking behavior. Therefore, sparse innervation by KCs can efficiently control and modulate MBON activity in response to learning with minimal requirements on the specificity of synaptic localization. This architecture allows efficient storage of large numbers of memories using the flexible stochastic connectivity of the circuit.

neuroscience↗

bioRxiv2019

Integration of Odor-Induced Activity of Kenyon Cells in an Electrotonically Compact Drosophila Mushroom Body Output Neuron (MBON)

The formation of an ecologically useful lasting memory requires that the brain has an accurate internal representation of the surrounding environment. In addition, it must have the ability to integrate a variety of different sensory stimuli and associate them with rewarding and aversive behavioral outcomes. Over the previous years, a number of studies have dissected the anatomy and elucidated some of the working principles of the Drosophila mushroom body (MB), the flys center for learning and memory. As a consequence, we now have a functional understanding of where and how in the MB sensory stimuli converge and are associated. However, the molecular and cellular dynamics at the critical synaptic intersection for this process, the Kenyon cell-mushroom body output neuron (KC-MBON) synapse, are largely unknown. Here, we introduce a first approach to understand this integration process and the physiological changes occurring at the KC-MBON synapse during Kenyon cell (KC) activation. We use the published connectome of the Drosophila MB to construct a functional computational model of the MBON-3 dendritic structure. We simulate synaptic input by individual KC-MBON synapses by current injections into precisely (m) identified local dendritic sections, and the input from a model population of KCs representing an odor by a spatially distributed cluster of current injections. By recording the effect of the simulated current injections on the membrane potential of the neuron, we show that the MBON-3 is electrotonically compact. This suggests that odor-induced MBON activity is likely governed by input strength while the positions of KC input synapses are largely irrelevant.

neuroscience↗