Laboratory of Molecular and Cellular Neurobiology: Jaworski Laboratory
Description of Current Research
The research of our team concentrates on the role of protein kinase mammalian Target of Rapamycin (mTOR) in the control of proper neuronal morphology in health and disease. Establishing proper neuronal morphology is required for proper brain function. Therefore, the mechanisms of axon targeting, dendritic arbor patterning, proper cell contact formation, and maintenance of plasticity of neuronal connectivity are at the center of interest of molecular neurobiology. Dendrites are the main site of information input onto neurons, and dendritic arbor shape is one of the crucial factors that determine how signals that originate from individual synapses are integrated. In fact, several neurodevelopmental pathologies are characterized by abnormalities in dendritic tree structure. Dendritic arbor development is a multistep process (Fig. 1) that depends, among other factors, on mTOR, a serine/threonine protein kinase known to merge extracellular instructions with information about cellular metabolic resources and control the rate of anabolic and catabolic processes accordingly. In neurons, mTOR has been also implicated in neuronal differentiation, axon elongation and directional movements, spinogenesis, long-term synaptic plasticity, and learning and memory. In neurons, mTOR is hypothesized to act primarily by controlling protein translation, including local protein synthesis in dendrites. Studies in different model systems (e.g., yeast, fruit flies, and cultured non-neuronal mammalian cells) strongly imply the involvement of mTOR in additional cellular processes, such as transcription, membrane trafficking, mitochondrial function, lipid metabolism, autophagy, and cytoskeleton dynamics (Fig. 2). Thus, considering the key role that mTOR plays in cell physiology, unsurprising is that mTOR signaling is disturbed under various neuropathological conditions.
Fig. 1. Dendritic arbor development is a multistage process. Dendritogenesis starts shortly after axon specification. Dendrites first elongate. Next they start to branch dynamically. The final shape of a dendritic arbor is achieved through repetitive rounds of branch additions and retractions that occur in response to a variety of extracellular signals. Once the final shape is reached the dendritic tree becomes stable, and structural plasticity occurs quite rarely under basal conditions. Drawing: Łukasz Świech
Fig. 2. Schematic representation of mTOR signaling network. Drawing: Małgorzata Urbańska
Altered mTOR activity has been reported in brain tumors, tuberous sclerosis, cortical dysplasia, and neurodegenerative disorders. However, in cases of either physiological processes or neuropathology, our knowledge of the molecular events downstream of mTOR, other than protein translation, is rather limited. We believe that expanding such knowledge is crucial for understanding the molecular biology of neurons and assessing the benefits and risks of the clinical use of mTOR inhibitors. Thus, our goal is to determine the mTOR-dependent proteins and cellular processes involved in neuronal development. For the past few years, our research has developed in three main areas:
1. Identifying mTOR partners and
regulated proteins involved in the processes of dendritic branching and synapse
formation and stabilization.
2. Establishing a link between local protein translation and physiological dendritic arbor development.
3. Characterizing both mTOR-regulated cellular processes and the role of local protein synthesis in pathologies of the central nervous system.
Identification of mTOR partners and mTOR regulated proteins involved in the process of dendritic branching.
In this area of interest our research goes in two directions. First, we focus on known, although controversial mTOR targets, not related to protein synthesis control, to provide the proof of principle that non-canonical mTOR functions contribute to neuronal development. Second, we perform medium-scale shRNA screens to identify novel mTOR targets involved in dendritogenesis. Using first approach we have recently shown that the mTOR-dependent regulation of dendritic arborization involves CLIP-170, a microtubule plus-end tracking protein (+TIP). Our studies describe a novel mechanism of control of dendritic arbor morphology in mammalian neurons likely involving an mTOR-dependent interaction between CLIP-170 and IQGAP1, an actin-regulating protein that coordinates cross-talk between dynamic microtubule plus-ends and the actin cytoskeleton (Fig 3). Our interest in +TIP proteins resulted also in discovery of important role of microtubules in dendritic spine stability (in collaboration with Casper Hoogenraad team, Utrecht University). Our major effort however, toward identifying mTOR regulated proteins involved in dendritic arborization has been to design a shRNA library against mRNA that encode those proteins and perform a screen in neurons cultured in vitro. We selected 150 proteins potentially regulated by the mTOR-Raptor complex based on a bioinformatic approach and designed a library of shRNAs against all selected candidates. We then performed a screen and identified 30 genes crucial for dendritic arbor development and stability using this library (Fig. 4). We also currently work on developing physiologically relevant in vivo models to study our positive hits. We focus on in vivo electroporation of new born mice brains to study dendritic arborization of neurons that migrated into olfactory bulbs after birth in subventricular zone.
Fig. 3. Forced actin stabilization prevents effects of CLIP-170 or IQGAP1 depletion in on dendritic arborization in developing hippocampal neurons. (A) Representative images of hippocampal neurons co-transfected at DIV7 with pSuperTRE vector or CLIP-170sh#3TRE treated with 1 μg/ml doxycycline and 10 nM jasplakinolide 24 h later and fixed 6 days after transfection. Cells were co-transfected with GFP encoding vector for morphology visualization. Scale bar = 20 μm. (B) Analysis of the total number of dendritic tips (TNDT) of transfected neurons. (C) TNDT of hippocampal neurons after IQGAP1 knockdown with sh#2 and 4 day treatment with 10 nM jasplakinolide. Error bars indicate SEM (***p < 0.001, **p < 0.01; Kruskal-Wallis test). (D) Pull-down of F-actin from vehicle or rapamycin-treated (100 nM) rat hippocampal homogenates. Silver stained gel confirming presence of actin bound with biotinylated phalloidin (left panel). Western blot analysis revealing rapamycinsensitive and IQGAP1 dependent interaction of CLIP-170 with F-actin resin (right panel). (E) A proposed model of a novel mTOR-dependent mechanism of dendritic arbor development regulation. Photo: Łukasz Świech
Fig. 4. Identification of mTOR regulated proteins involved in dendritic arborization of hippocampal neurons with use of shRNA library designed against mRNAs encoding proteins potentially regulated by this kinase. Exemplary images of neurons from the screen transfected with shRNAs Images in red frames highlight neurons with substantially changed dendritic morphology. Photo: Jacek Jaworski
Establishing a link between local protein translation and physiological dendritic arbor development.
Local protein synthesis is now a well established mechanism that underlies several important aspects of cell development and polarization. This is a phenomenon of regulated protein translation that occurs in cell subdomains, often distant from the major sites of protein production. For example, local translation that occurs in growth cones of navigating axons has been shown to be crucial for the proper connectivity of the neuronal network. Several lines of evidence suggest that mTOR kinase is one of the crucial regulators of local translation. The major difference between general and local translation is the spatial separation of these two processes, which requires the transportation of translationally dormant mRNA to the destination point where it gets derepressed and becomes translationally competent. Dendritically targeted mRNAs contain tagging sequences that are recognized and bound by protein complexes that provide both translational silencing and microtubule attachment. Zipcode binding protein 1 (ZBP1) is one of the proteins involved in this process and the formation of such ribonucleoprotein particles (RNPs) that transport mRNA in neurons. Our preliminary results suggest that ZBP1 can be phosphorylated by mTOR. Therefore, in the past 2 years, we studied ZBP1-dependent dendritic growth as a proof-of-concept that local mRNA targeting and translation are important for proper dendritic growth. Our work accomplished until now describes the essential contribution of ZBP1 to the development of proper dendritic morphology in hippocampal neurons (Fig 5). It also shows that β-actin, a well-known target of ZBP1 is a downstream effector during this process (Fig. 5).
Fig. 5. b-actin overexpression in hippocampal neurons with ZBP1 knockdown is sufficient for partial phenotype rescue. (A) Micrographs of hippocampal neurons transfected on DIV7 with pEGFP-C1 together with pSuper vector as a control, ZBP1sh#1, or ZBP1sh#1 together with EGFP-ZBP1*1, EGFP-β-actin, c-Myc, GW1-NR1, GFP-CamKIIα. Or EGFP-MAP2. Expression proceeded for 3 days. Neuronal morphology was visualized by staining for cotransfected β-gal. Scale bar = 50 μm (B, C) mean number of dendritic tips (B) ***p < 0.00033, **p < 0.0033 (Kruskal-Wallis test followed by Mann-Whitney post hoc test with Bonferroni adjustment; ns, not significant). (C) ***p < 0.00016, *p < 0.008 (Kruskal-Wallis test followed by Mann-Whitney post hoc test with Bonferroni adjustment; ns, not significant). Photo: Małgorzata Perycz
Characterizing both mTOR-regulated cellular processes and the role of local protein synthesis in pathologies of the central nervous system
In addition to the aforementioned activities, our important goal is to understand how physiological processes regulated by mTOR are disturbed in nervous system pathology. Towards this end launched research related to (i) the molecular role of mTOR in epileptogenesis (Polish-Norwegian Research Funds grant) (Fig. 6) and (ii) mTOR and GSK3 reciprocal communication in physiology and neurodegenerative disorders (FP7 “NeuroGSK3” project) and (iii) identification of mTOR targets in Tuberous Sclerosis. Currently, in this context, we are introducing to the lab the most recent clinically relevant model, i.e: reprogramming iPS cells to neurons (Era-Net Neuron grant).
Fig. 6. Seizures induced by kainic acid application in rats resulted in an increase in mTOR pathway activity (S6 protein phosphorylation), but with different kinetics depending on cell type. Neurons responded as early as 2 h while reactive astrocytes (highlighted by immunofluorescence against GFAP) turned on mTOR response later Photo: Matylda Macias
Swiech L., Blazejczyk M., Urbanska M., Pietruszka P., Dortland B., Malik A., Wulf P., Hoogenraad CC., Jaworski J. (2011) CLIP-170 and IQGAP1 cooperatively regulate dendrite morphology. J Neurosci., 31:4555-4568.
Perycz M., Urbanska AS., Krawczyk PS., Parobczak K., Jaworski J.(2011) Zipcode binding protein 1 regulates the development of dendritic arbors in hippocampal neurons. J Neurosci., 31:5271-5285.
Jaworski J., Kapitein LC., Montenegro Gouveia S., Dortland BR., Wulf PS., Grigoriev I., Camera P., Spangler SA., di Stefano P., Demmers J., Krugers H., Defilippi P., Akhmanova A. and Hoogenraad CC. (2009) Dynamic microtubules regulate dendritic spine morphology and synaptic plasticity. Neuron, 61:85-100.
Swiech L., Perycz M., Malik A., Jaworski J. (2008) Role of mTOR in physiology and pathology of the nervous system. Biochim. Biophys. Acta, 1784: 116-132.
Jaworski J., Spangler S., Seeburg DP., Hoogenraad CC., Sheng M. (2005) Control of dendritic arborization by the PI3-kinase – Akt - mTOR pathway, J. Neurosci. 25: 11300-11312.