Publications by authors named "Rüdiger Klein"

115 Publications

Fluc-EGFP reporter mice reveal differential alterations of neuronal proteostasis in aging and disease.

EMBO J 2021 Oct 19;40(19):e107260. Epub 2021 Aug 19.

Department of Molecules - Signaling - Development, Max Planck Institute of Neurobiology, Martinsried, Germany.

The cellular protein quality control machinery is important for preventing protein misfolding and aggregation. Declining protein homeostasis (proteostasis) is believed to play a crucial role in age-related neurodegenerative disorders. However, how neuronal proteostasis capacity changes in different diseases is not yet sufficiently understood, and progress in this area has been hampered by the lack of tools to monitor proteostasis in mammalian models. Here, we have developed reporter mice for in vivo analysis of neuronal proteostasis. The mice express EGFP-fused firefly luciferase (Fluc-EGFP), a conformationally unstable protein that requires chaperones for proper folding, and that reacts to proteotoxic stress by formation of intracellular Fluc-EGFP foci and by reduced luciferase activity. Using these mice, we provide evidence for proteostasis decline in the aging brain. Moreover, we find a marked reaction of the Fluc-EGFP sensor in a mouse model of tauopathy, but not in mouse models of Huntington's disease. Mechanistic investigations in primary neuronal cultures demonstrate that different types of protein aggregates have distinct effects on the cellular protein quality control. Thus, Fluc-EGFP reporter mice enable new insights into proteostasis alterations in different diseases.
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http://dx.doi.org/10.15252/embj.2020107260DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8488555PMC
October 2021

FLRT2 and FLRT3 Cooperate in Maintaining the Tangential Migratory Streams of Cortical Interneurons during Development.

J Neurosci 2021 Sep 23;41(35):7350-7362. Epub 2021 Jul 23.

Lleida Biomedical Research Institute, University of Lleida, Lleida 25198, Spain

Neuron migration is a hallmark of nervous system development that allows gathering of neurons from different origins for assembling of functional neuronal circuits. Cortical inhibitory interneurons arise in the ventral telencephalon and migrate tangentially forming three transient migratory streams in the cortex before reaching the final laminar destination. Although migration defects lead to the disruption of inhibitory circuits and are linked to aspects of psychiatric disorders such as autism and schizophrenia, the molecular mechanisms controlling cortical interneuron development and final layer positioning are incompletely understood. Here, we show that mouse embryos with a double deletion of and genes encoding cell adhesion molecules exhibit an abnormal distribution of interneurons within the streams during development, which in turn, affect the layering of somatostatin+ interneurons postnatally. Mechanistically, FLRT2 and FLRT3 proteins act in a noncell-autonomous manner, possibly through a repulsive mechanism. In support of such a conclusion, double knockouts deficient in the repulsive receptors for FLRTs, Unc5B and Unc5D, also display interneuron defects during development, similar to the / mutants. Moreover, FLRT proteins are chemorepellent ligands for developing interneurons , an effect that is in part dependent on FLRT-Unc5 interaction. Together, we propose that FLRTs act through Unc5 receptors to control cortical interneuron distribution in a mechanism that involves cell repulsion. Disruption of inhibitory cortical circuits is responsible for some aspects of psychiatric disorders such as schizophrenia or autism. These defects include interneuron migration during development. A crucial step during this process is the formation of three transient migratory streams within the developing cortex that determine the timing of interneuron final positioning and the formation of functional cortical circuits in the adult. We report that FLRT proteins are required for the proper distribution of interneurons within the cortical migratory streams and for the final laminar allocation in the postnatal cortex. These results expand the multifunctional role of FLRTs during nervous system development in addition to the role of FLRTs in axon guidance and the migration of excitatory cortical neurons.
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http://dx.doi.org/10.1523/JNEUROSCI.0380-20.2021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8412983PMC
September 2021

Natural loss of function of ephrin-B3 shapes spinal flight circuitry in birds.

Sci Adv 2021 Jun 11;7(24). Epub 2021 Jun 11.

Department of Medical Neurobiology, IMRIC, Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel.

Flight in birds evolved through patterning of the wings from forelimbs and transition from alternating gait to synchronous flapping. In mammals, the spinal midline guidance molecule ephrin-B3 instructs the wiring that enables limb alternation, and its deletion leads to synchronous hopping gait. Here, we show that the ephrin-B3 protein in birds lacks several motifs present in other vertebrates, diminishing its affinity for the EphA4 receptor. The avian gene lacks an enhancer that drives midline expression and is missing in galliforms. The morphology and wiring at brachial levels of the chicken embryonic spinal cord resemble those of null mice. Dorsal midline decussation, evident in the mutant mouse, is apparent at the chick brachial level and is prevented by expression of exogenous at the roof plate. Our findings support a role for loss of ephrin-B3 function in shaping the avian brachial spinal cord circuitry and facilitating synchronous wing flapping.
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http://dx.doi.org/10.1126/sciadv.abg5968DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8195482PMC
June 2021

Recent advances in inter-cellular interactions during neural circuit assembly.

Curr Opin Neurobiol 2021 08 28;69:25-32. Epub 2020 Dec 28.

Department of Translational Neuroscience, University Medical Center (UMC) Utrecht Brain Center, Utrecht University, Universiteitsweg 100, 3584 CG, Utrecht, The Netherlands. Electronic address:

Neural circuit assembly is regulated by inter-cellular interactions involving secreted and surface-tethered guidance molecules. Here, we review recent progress in our understanding of their mechanisms-of-action and biological effects. We discuss mechanisms through which the secreted cue Netrin-1 regulates neuron migration and highlight novel roles for axon-derived secreted guidance cues. We cover recent structural work at atomic resolution that provides new insights into the activation mechanisms of axon guidance receptors and into protein complexes containing cell adhesion molecules, such as Teneurin (Ten), Latrophilin (Lphn) and FLRT. Ten-Ten homophilic, Ten-Lphn heterophilic, and Ten-Lphn-FLRT tripartite complexes seem to elicit distinct context-dependent cellular responses. Seemingly opposite responses can also be triggered by the Eph/ephrin signaling system. Here, recent work provides a simple mechanism for the decision of forming a new synapse versus rejection of the pre-synaptic partner. These studies identify novel regulatory mechanisms and biological functions that will apply generally in developing neural systems.
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http://dx.doi.org/10.1016/j.conb.2020.12.004DOI Listing
August 2021

Spinal Inhibitory Ptf1a-Derived Neurons Prevent Self-Generated Itch.

Cell Rep 2020 11;33(8):108422

Department Molecules-Signaling-Development, Max Planck Institute of Neurobiology, Am Klopferspitz 18, 82152 Martinsried, Germany. Electronic address:

Chronic itch represents an incapacitating burden on patients suffering from a spectrum of diseases. Despite recent advances in our understanding of the cells and circuits implicated in the processing of itch information, chronic itch often presents itself without an apparent cause. Here, we identify a spinal subpopulation of inhibitory neurons defined by the expression of Ptf1a, involved in gating mechanosensory information self-generated during movement. These neurons receive tactile and motor input and establish presynaptic inhibitory contacts on mechanosensory afferents. Loss of Ptf1a neurons leads to increased hairy skin sensitivity and chronic itch, partially mediated by the classic itch pathway involving gastrin-releasing peptide receptor (GRPR) spinal neurons. Conversely, chemogenetic activation of GRPR neurons elicits itch, which is suppressed by concomitant activation of Ptf1a neurons. These findings shed light on the circuit mechanisms implicated in chronic itch and open novel targets for therapy developments.
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http://dx.doi.org/10.1016/j.celrep.2020.108422DOI Listing
November 2020

The Insula Cortex Contacts Distinct Output Streams of the Central Amygdala.

J Neurosci 2020 11 13;40(46):8870-8882. Epub 2020 Oct 13.

Department of Molecules, Signaling, Development, Max-Planck Institute of Neurobiology, Martinsried 82152, Germany

The emergence of genetic tools has provided new means of mapping functionality in central amygdala (CeA) neuron populations based on their molecular profiles, response properties, and importantly, connectivity patterns. While abundant evidence indicates that neuronal signals arrive in the CeA eliciting both aversive and appetitive behaviors, our understanding of the anatomy of the underlying long-range CeA network remains fragmentary. In this study, we combine viral tracings, electrophysiological, and optogenetic approaches to establish in male mice, a wiring chart between the insula cortex (IC), a major sensory input region of the lateral and capsular part of the CeA (CeL/C), and four principal output streams of this nucleus. We found that retrogradely labeled output neurons occupy discrete and likely strategic locations in the CeL/C, and that they are disproportionally controlled by the IC. We identified a direct line of connection between the IC and the lateral hypothalamus (LH), which engages numerous LH-projecting CeL/C cells whose activity can be strongly upregulated on firing of IC neurons. In comparison, CeL/C neurons projecting to the bed nucleus of the stria terminalis (BNST) are also frequently contacted by incoming IC axons, but the strength of this connection is weak. Our results provide a link between long-range inputs and outputs of the CeA and pave the way to a better understanding of how internal, external, and experience dependent information may impinge on action selection by the CeA. Our current knowledge of the circuit organization within the central amygdala (CeA), a critical regulator of emotional states, includes independent information about its long-range efferents and afferents. We do not know how incoming sensory information is appraised and routed through the CeA to the different output channels. We address this issue by using three different techniques to investigate how a sensory region, the insula cortex (IC), connects with the motor, physiological and autonomic output centers of the CeA. We uncover a strong connection between the IC and the lateral hypothalamus (LH) with a monosynaptic relay in the CeA and shed new light on the previously described functions of IC and CeA through direct projections to the LH.
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http://dx.doi.org/10.1523/JNEUROSCI.0567-20.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7659460PMC
November 2020

Structural Basis of Teneurin-Latrophilin Interaction in Repulsive Guidance of Migrating Neurons.

Cell 2020 01 9;180(2):323-339.e19. Epub 2020 Jan 9.

Department of Biochemistry, Oxford University, Oxford OX1 3QU, UK. Electronic address:

Teneurins are ancient metazoan cell adhesion receptors that control brain development and neuronal wiring in higher animals. The extracellular C terminus binds the adhesion GPCR Latrophilin, forming a trans-cellular complex with synaptogenic functions. However, Teneurins, Latrophilins, and FLRT proteins are also expressed during murine cortical cell migration at earlier developmental stages. Here, we present crystal structures of Teneurin-Latrophilin complexes that reveal how the lectin and olfactomedin domains of Latrophilin bind across a spiraling beta-barrel domain of Teneurin, the YD shell. We couple structure-based protein engineering to biophysical analysis, cell migration assays, and in utero electroporation experiments to probe the importance of the interaction in cortical neuron migration. We show that binding of Latrophilins to Teneurins and FLRTs directs the migration of neurons using a contact repulsion-dependent mechanism. The effect is observed with cell bodies and small neurites rather than their processes. The results exemplify how a structure-encoded synaptogenic protein complex is also used for repulsive cell guidance.
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http://dx.doi.org/10.1016/j.cell.2019.12.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6978801PMC
January 2020

Identification of Spinal Neurons Contributing to the Dorsal Column Projection Mediating Fine Touch and Corrective Motor Movements.

Neuron 2019 11 2;104(4):749-764.e6. Epub 2019 Oct 2.

Department of Molecules - Signaling - Development, Max Planck Institute of Neurobiology, 82152 Martinsried, Germany. Electronic address:

Tactile stimuli are integrated and processed by neuronal circuits in the deep dorsal horn of the spinal cord. Several spinal interneuron populations have been implicated in tactile information processing. However, dorsal horn projection neurons that contribute to the postsynaptic dorsal column (PSDC) pathway transmitting tactile information to the brain are poorly characterized. Here, we show that spinal neurons marked by the expression of Zic2cre mediate light touch sensitivity and textural discrimination. A subset of Zic2cre neurons are PSDC neurons that project to brainstem dorsal column nuclei, and chemogenetic activation of Zic2 PSDC neurons increases sensitivity to light touch stimuli. Zic2 neurons receive direct input from the cortex and brainstem motor nuclei and are required for corrective motor movements. These results suggest that Zic2 neurons integrate sensory input from cutaneous afferents with descending signals from the brain to promote corrective movements and transmit processed touch information back to the brain. VIDEO ABSTRACT.
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http://dx.doi.org/10.1016/j.neuron.2019.08.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7814917PMC
November 2019

Gulp1 controls Eph/ephrin trogocytosis and is important for cell rearrangements during development.

J Cell Biol 2019 10 13;218(10):3455-3471. Epub 2019 Aug 13.

Max Planck Institute of Neurobiology, Department of Molecules-Signaling-Development, Munich-Martinsried, Germany

Trogocytosis, in which cells nibble away parts of neighboring cells, is an intercellular cannibalism process conserved from protozoa to mammals. Its underlying molecular mechanisms are not well understood and are likely distinct from phagocytosis, a process that clears entire cells. Bi-directional contact repulsion induced by Eph/ephrin signaling involves transfer of membrane patches and full-length Eph/ephrin protein complexes between opposing cells, resembling trogocytosis. Here, we show that the phagocytic adaptor protein Gulp1 regulates EphB/ephrinB trogocytosis to achieve efficient cell rearrangements of cultured cells and during embryonic development. Gulp1 mediates trogocytosis bi-directionally by dynamic engagement with EphB/ephrinB protein clusters in cooperation with the Rac-specific guanine nucleotide exchange factor Tiam2. Ultimately, Gulp1's presence at the Eph/ephrin cluster is a prerequisite for recruiting the endocytic GTPase dynamin. These results suggest that EphB/ephrinB trogocytosis, unlike other trogocytosis events, uses a phagocytosis-like mechanism to achieve efficient membrane scission and engulfment.
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http://dx.doi.org/10.1083/jcb.201901032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6781437PMC
October 2019

Cortical circuit alterations precede motor impairments in Huntington's disease mice.

Sci Rep 2019 04 29;9(1):6634. Epub 2019 Apr 29.

Department of Molecules - Signaling - Development, Max Planck Institute of Neurobiology, 82152, Martinsried, Germany.

Huntington's disease (HD) is a devastating hereditary movement disorder, characterized by degeneration of neurons in the striatum and cortex. Studies in human patients and mouse HD models suggest that disturbances of neuronal function in the neocortex play an important role in disease onset and progression. However, the precise nature and time course of cortical alterations in HD have remained elusive. Here, we use chronic in vivo two-photon calcium imaging to longitudinally monitor the activity of identified single neurons in layer 2/3 of the primary motor cortex in awake, behaving R6/2 transgenic HD mice and wildtype littermates. R6/2 mice show age-dependent changes in cortical network function, with an increase in activity that affects a large fraction of cells and occurs rather abruptly within one week, preceeding the onset of motor defects. Furthermore, quantitative proteomics demonstrate a pronounced downregulation of synaptic proteins in the cortex, and histological analyses in R6/2 mice and human HD autopsy cases reveal a reduction in perisomatic inhibitory synaptic contacts on layer 2/3 pyramidal cells. Taken together, our study provides a time-resolved description of cortical network dysfunction in behaving HD mice and points to disturbed excitation/inhibition balance as an important pathomechanism in HD.
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http://dx.doi.org/10.1038/s41598-019-43024-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6488584PMC
April 2019

Spatiotemporal Proteomic Profiling of Huntington's Disease Inclusions Reveals Widespread Loss of Protein Function.

Cell Rep 2017 Nov;21(8):2291-2303

Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany. Electronic address:

Aggregation of polyglutamine-expanded huntingtin exon 1 (HttEx1) in Huntington's disease (HD) proceeds from soluble oligomers to late-stage inclusions. The nature of the aggregates and how they lead to neuronal dysfunction is not well understood. We employed mass spectrometry (MS)-based quantitative proteomics to dissect spatiotemporal mechanisms of neurodegeneration using the R6/2 mouse model of HD. Extensive remodeling of the soluble brain proteome correlated with insoluble aggregate formation during disease progression. In-depth and quantitative characterization of the aggregates uncovered an unprecedented complexity of several hundred proteins. Sequestration to aggregates depended on protein expression levels and sequence features such as low-complexity regions or coiled-coil domains. In a cell-based HD model, overexpression of a subset of the sequestered proteins in most cases rescued viability and reduced aggregate size. Our spatiotemporally resolved proteome resource of HD progression indicates that widespread loss of cellular protein function contributes to aggregate-mediated toxicity.
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http://dx.doi.org/10.1016/j.celrep.2017.10.097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5714591PMC
November 2017

In Situ Architecture and Cellular Interactions of PolyQ Inclusions.

Cell 2017 Sep 7;171(1):179-187.e10. Epub 2017 Sep 7.

Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany.

Expression of many disease-related aggregation-prone proteins results in cytotoxicity and the formation of large intracellular inclusion bodies. To gain insight into the role of inclusions in pathology and the in situ structure of protein aggregates inside cells, we employ advanced cryo-electron tomography methods to analyze the structure of inclusions formed by polyglutamine (polyQ)-expanded huntingtin exon 1 within their intact cellular context. In primary mouse neurons and immortalized human cells, polyQ inclusions consist of amyloid-like fibrils that interact with cellular endomembranes, particularly of the endoplasmic reticulum (ER). Interactions with these fibrils lead to membrane deformation, the local impairment of ER organization, and profound alterations in ER membrane dynamics at the inclusion periphery. These results suggest that aberrant interactions between fibrils and endomembranes contribute to the deleterious cellular effects of protein aggregation. VIDEO ABSTRACT.
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http://dx.doi.org/10.1016/j.cell.2017.08.009DOI Listing
September 2017

Central amygdala circuits modulate food consumption through a positive-valence mechanism.

Nat Neurosci 2017 Oct 21;20(10):1384-1394. Epub 2017 Aug 21.

Molecules-Signaling-Development, Max Planck Institute of Neurobiology, Martinsried, Germany.

The complex behaviors underlying reward seeking and consumption are integral to organism survival. The hypothalamus and mesolimbic dopamine system are key mediators of these behaviors, yet regulation of appetitive and consummatory behaviors outside of these regions is poorly understood. The central nucleus of the amygdala (CeA) has been implicated in feeding and reward, but the neurons and circuit mechanisms that positively regulate these behaviors remain unclear. Here, we defined the neuronal mechanisms by which CeA neurons promote food consumption. Using in vivo activity manipulations and Ca imaging in mice, we found that GABAergic serotonin receptor 2a (Htr2a)-expressing CeA neurons modulate food consumption, promote positive reinforcement and are active in vivo during eating. We demonstrated electrophysiologically, anatomically and behaviorally that intra-CeA and long-range circuit mechanisms underlie these behaviors. Finally, we showed that CeA neurons receive inputs from feeding-relevant brain regions. Our results illustrate how defined CeA neural circuits positively regulate food consumption.
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http://dx.doi.org/10.1038/nn.4623DOI Listing
October 2017

Placental labyrinth formation in mice requires endothelial FLRT2/UNC5B signaling.

Development 2017 07 2;144(13):2392-2401. Epub 2017 Jun 2.

Department of Vascular Biology, The Sakaguchi Laboratory, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan

The placental labyrinth is the interface for gas and nutrient exchange between the embryo and the mother; hence its proper development is essential for embryogenesis. However, the molecular mechanism underlying development of the placental labyrinth, particularly in terms of its endothelial organization, is not well understood. Here, we determined that fibronectin leucine-rich transmembrane protein 2 (FLRT2), a repulsive ligand of the UNC5 receptor family for neurons, is unexpectedly expressed in endothelial cells specifically in the placental labyrinth. Mice lacking FLRT2 in endothelial cells exhibited embryonic lethality at mid-gestation, with systemic congestion and hypoxia. Although they lacked apparent deformities in the embryonic vasculature and heart, the placental labyrinths of these embryos exhibited aberrant alignment of endothelial cells, which disturbed the feto-maternal circulation. Interestingly, this vascular deformity was related to endothelial repulsion through binding to the UNC5B receptor. Our results suggest that the proper organization of the placental labyrinth depends on coordinated inter-endothelial repulsion, which prevents uncontrolled layering of the endothelium.
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http://dx.doi.org/10.1242/dev.149757DOI Listing
July 2017

Regulation of Cerebral Cortex Folding by Controlling Neuronal Migration via FLRT Adhesion Molecules.

Cell 2017 05;169(4):621-635.e16

Max Planck Institute of Neurobiology, Am Klopferspitz 18, 82152 Martinsried, Germany; Munich Cluster for Systems Neurology (SyNergy), 80336 Munich, Germany. Electronic address:

The folding of the mammalian cerebral cortex into sulci and gyri is thought to be favored by the amplification of basal progenitor cells and their tangential migration. Here, we provide a molecular mechanism for the role of migration in this process by showing that changes in intercellular adhesion of migrating cortical neurons result in cortical folding. Mice with deletions of FLRT1 and FLRT3 adhesion molecules develop macroscopic sulci with preserved layered organization and radial glial morphology. Cortex folding in these mutants does not require progenitor cell amplification but is dependent on changes in neuron migration. Analyses and simulations suggest that sulcus formation in the absence of FLRT1/3 results from reduced intercellular adhesion, increased neuron migration, and clustering in the cortical plate. Notably, FLRT1/3 expression is low in the human cortex and in future sulcus areas of ferrets, suggesting that intercellular adhesion is a key regulator of cortical folding across species.
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http://dx.doi.org/10.1016/j.cell.2017.04.012DOI Listing
May 2017

Intrinsic Circuits in the Lateral Central Amygdala.

eNeuro 2017 Jan-Feb;4(1). Epub 2017 Mar 24.

Queensland Brain Institute, The University of Queensland , Brisbane 4072, Queensland, Australia.

Network activity in the lateral central amygdala (CeL) plays a crucial role in fear learning and emotional processing. However, the local circuits of the CeL are not fully understood and have only recently begun to be explored in detail. Here, we characterized the intrinsic circuits in the CeL using paired whole-call patch-clamp recordings, immunohistochemistry, and optogenetics in C57BL/6J wild-type and somatostatin-cre (SOM-Cre) mice. Our results revealed that throughout the rostrocaudal extent of the CeL, neurons form inhibitory connections at a rate of ∼29% with an average amplitude of 20 ± 3 pA (at -40 mV). Inhibitory input from a single neuron is sufficient to halt firing in the postsynaptic neuron. immunostaining for protein kinase Cδ (PKCδ) in wild-type mice and paired recordings in SOM-Cre mice demonstrated that the most common local connections were PKCδ(-) → PKCδ(-) and SOM(+) → SOM(+). Finally, by optogenetically activating either SOM(+) or SOM(-) neurons, we found that almost all neurons in the CeL were innervated by these neuronal populations and that connections between like neurons were stronger than those between different neuronal types. These findings reveal a complex network of connections within the CeL and provide the foundations for future behavior-specific circuit analysis of this complex network.
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http://dx.doi.org/10.1523/ENEURO.0367-16.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5364643PMC
October 2017

Structural Perspectives on Axon Guidance.

Annu Rev Cell Dev Biol 2016 10 24;32:577-608. Epub 2016 Aug 24.

Max Planck Institute of Neurobiology, 82152 Munich-Martinsried, Germany; email:

Axon guidance relies on a combinatorial code of receptor and ligand interactions that direct adhesive/attractive and repulsive cellular responses. Recent structural data have revealed many of the molecular mechanisms that govern these interactions and enabled the design of sophisticated mutant tools to dissect their biological functions. Here, we discuss the structure/function relationships of four major classes of guidance cues (ephrins, semaphorins, slits, netrins) and examples of morphogens (Wnt, Shh) and of cell adhesion molecules (FLRT). These cell signaling systems rely on specific modes of receptor-ligand binding that are determined by selective binding sites; however, defined structure-encoded receptor promiscuity also enables cross talk between different receptor/ligand families and can also involve extracellular matrix components. A picture emerges in which a multitude of highly context-dependent structural assemblies determines the finely tuned cellular behavior required for nervous system development.
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http://dx.doi.org/10.1146/annurev-cellbio-111315-125008DOI Listing
October 2016

Exosomes mediate cell contact-independent ephrin-Eph signaling during axon guidance.

J Cell Biol 2016 07 27;214(1):35-44. Epub 2016 Jun 27.

Max Planck Institute of Neurobiology, 82152 Martinsried, Germany Munich Cluster for Systems Neurology (SyNergy), 80336 Munich, Germany

The cellular release of membranous vesicles known as extracellular vesicles (EVs) or exosomes represents a novel mode of intercellular communication. Eph receptor tyrosine kinases and their membrane-tethered ephrin ligands have very important roles in such biologically diverse processes as neuronal development, plasticity, and pathological diseases. Until now, it was thought that ephrin-Eph signaling requires direct cell contact. Although the biological functions of ephrin-Eph signaling are well understood, our mechanistic understanding remains modest. Here we report the release of EVs containing Ephs and ephrins by different cell types, a process requiring endosomal sorting complex required for transport (ESCRT) activity and regulated by neuronal activity. Treatment of cells with purified EphB2(+) EVs induces ephrinB1 reverse signaling and causes neuronal axon repulsion. These results indicate a novel mechanism of ephrin-Eph signaling independent of direct cell contact and proteolytic cleavage and suggest the participation of EphB2(+) EVs in neural development and synapse physiology.
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http://dx.doi.org/10.1083/jcb.201601085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4932373PMC
July 2016

Multimodal Eph/Ephrin signaling controls several phases of urogenital development.

Kidney Int 2016 08 22;90(2):373-388. Epub 2016 Jun 22.

Department of Neuroscience, Developmental Genetics, Uppsala University, Uppsala, Sweden.

A substantial portion of the human population is affected by urogenital birth defects resulting from a failure in ureter development. Although recent research suggests roles for several genes in facilitating the ureter/bladder connection, the underlying molecular mechanisms remain poorly understood. Signaling via Eph receptor tyrosine kinases is involved in several developmental processes. Here we report that impaired Eph/Ephrin signaling in genetically modified mice results in severe hydronephrosis caused by defective ureteric bud induction, ureter maturation, and translocation. Our data imply that ureter translocation requires apoptosis in the urogenital sinus and inhibition of proliferation in the common nephric duct. These processes were disturbed in EphA4/EphB2 compound knockout mice and were accompanied by decreased ERK-2 phosphorylation. Using a set of Eph, Ephrin, and signaling-deficient mutants, we found that during urogenital development, different modes of Eph/Ephrin signaling occur at several sites with EphrinB2 and EphrinA5 acting in concert. Thus, Eph/Ephrin signaling should be considered in the etiology of congenital kidney and urinary tract anomalies.
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http://dx.doi.org/10.1016/j.kint.2016.04.021DOI Listing
August 2016

Super-complexes of adhesion GPCRs and neural guidance receptors.

Nat Commun 2016 Apr 19;7:11184. Epub 2016 Apr 19.

Department of Biochemistry, Oxford University, Oxford OX1 3QU, UK.

Latrophilin adhesion-GPCRs (Lphn1-3 or ADGRL1-3) and Unc5 cell guidance receptors (Unc5A-D) interact with FLRT proteins (FLRT1-3), thereby promoting cell adhesion and repulsion, respectively. How the three proteins interact and function simultaneously is poorly understood. We show that Unc5D interacts with FLRT2 in cis, controlling cell adhesion in response to externally presented Lphn3. The ectodomains of the three proteins bind cooperatively. Crystal structures of the ternary complex formed by the extracellular domains reveal that Lphn3 dimerizes when bound to FLRT2:Unc5, resulting in a stoichiometry of 1:1:2 (FLRT2:Unc5D:Lphn3). This 1:1:2 complex further dimerizes to form a larger 'super-complex' (2:2:4), using a previously undescribed binding motif in the Unc5D TSP1 domain. Molecular dynamics simulations, point-directed mutagenesis and mass spectrometry demonstrate the stability and molecular properties of these complexes. Our data exemplify how receptors increase their functional repertoire by forming different context-dependent higher-order complexes.
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http://dx.doi.org/10.1038/ncomms11184DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4838878PMC
April 2016

Mechanisms of ephrin-Eph signalling in development, physiology and disease.

Nat Rev Mol Cell Biol 2016 Apr 21;17(4):240-56. Epub 2016 Jan 21.

Department of Molecules - Signaling - Development, Max Planck Institute of Neurobiology, Am Klopferspitz 18, 82152 Martinsried, Germany.

Eph receptor Tyr kinases and their membrane-tethered ligands, the ephrins, elicit short-distance cell-cell signalling and thus regulate many developmental processes at the interface between pattern formation and morphogenesis, including cell sorting and positioning, and the formation of segmented structures and ordered neural maps. Their roles extend into adulthood, when ephrin-Eph signalling regulates neuronal plasticity, homeostatic events and disease processes. Recently, new insights have been gained into the mechanisms of ephrin-Eph signalling in different cell types, and into the physiological importance of ephrin-Eph in different organs and in disease, raising questions for future research directions.
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http://dx.doi.org/10.1038/nrm.2015.16DOI Listing
April 2016

Cis and trans RET signaling control the survival and central projection growth of rapidly adapting mechanoreceptors.

Elife 2015 Apr 2;4:e06828. Epub 2015 Apr 2.

Department of Neuroscience, Perelman School of Medicine, University of Pennsylvania, Philadelphia, United States.

RET can be activated in cis or trans by its co-receptors and ligands in vitro, but the physiological roles of trans signaling are unclear. Rapidly adapting (RA) mechanoreceptors in dorsal root ganglia (DRGs) express Ret and the co-receptor Gfrα2 and depend on Ret for survival and central projection growth. Here, we show that Ret and Gfrα2 null mice display comparable early central projection deficits, but Gfrα2 null RA mechanoreceptors recover later. Loss of Gfrα1, the co-receptor implicated in activating RET in trans, causes no significant central projection or cell survival deficit, but Gfrα1;Gfrα2 double nulls phenocopy Ret nulls. Finally, we demonstrate that GFRα1 produced by neighboring DRG neurons activates RET in RA mechanoreceptors. Taken together, our results suggest that trans and cis RET signaling could function in the same developmental process and that the availability of both forms of activation likely enhances but not diversifies outcomes of RET signaling.
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http://dx.doi.org/10.7554/eLife.06828DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4408446PMC
April 2015

Structural basis of latrophilin-FLRT interaction.

Structure 2015 Apr 26;23(4):774-81. Epub 2015 Feb 26.

Department of Biochemistry, Oxford University, South Parks Road, Oxford OX1 3QU, UK. Electronic address:

Latrophilins, receptors for spider venom α-latrotoxin, are adhesion type G-protein-coupled receptors with emerging functions in synapse development. The N-terminal region binds the endogenous cell adhesion molecule FLRT, a major regulator of cortical and synapse development. We present crystallographic data for the mouse Latrophilin3 lectin and olfactomedin-like (Olf) domains, thereby revealing the Olf β-propeller fold and conserved calcium-binding site. We locate the FLRT-Latrophilin binding surfaces by a combination of sequence conservation analysis, point mutagenesis, and surface plasmon resonance experiments. In stripe assays, we show that wild-type Latrophilin3 and its high-affinity interactor FLRT2, but not the binding-impaired mutants we generated, promote HeLa cell adhesion. In contrast, cortical neurons expressing endogenous FLRTs are repelled by wild-type Latrophilin3 and not by the binding-impaired mutant. Taken together, we present molecular level insights into Latrophilin structure, its FLRT-binding mechanism, and a role for Latrophilin and FLRT that goes beyond a simply adhesive interaction.
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http://dx.doi.org/10.1016/j.str.2015.01.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4396693PMC
April 2015

FLRT structure: balancing repulsion and cell adhesion in cortical and vascular development.

Neuron 2014 Oct 22;84(2):370-85. Epub 2014 Oct 22.

Max Planck Institute of Neurobiology, Am Klopferspitz 18, 82152 Martinsried, Germany; Munich Cluster for Systems Neurology (SyNergy), 80336 Munich, Germany. Electronic address:

FLRTs are broadly expressed proteins with the unique property of acting as homophilic cell adhesion molecules and as heterophilic repulsive ligands of Unc5/Netrin receptors. How these functions direct cell behavior and the molecular mechanisms involved remain largely unclear. Here we use X-ray crystallography to reveal the distinct structural bases for FLRT-mediated cell adhesion and repulsion in neurons. We apply this knowledge to elucidate FLRT functions during cortical development. We show that FLRTs regulate both the radial migration of pyramidal neurons, as well as their tangential spread. Mechanistically, radial migration is controlled by repulsive FLRT2-Unc5D interactions, while spatial organization in the tangential axis involves adhesive FLRT-FLRT interactions. Further, we show that the fundamental mechanisms of FLRT adhesion and repulsion are conserved between neurons and vascular endothelial cells. Our results reveal FLRTs as powerful guidance factors with structurally encoded repulsive and adhesive surfaces.
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http://dx.doi.org/10.1016/j.neuron.2014.10.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4210639PMC
October 2014

EphA4 receptor shedding regulates spinal motor axon guidance.

Curr Biol 2014 Oct 25;24(20):2355-65. Epub 2014 Sep 25.

Department of Molecules-Signaling-Development, Max Planck Institute of Neurobiology, Am Klopferspitz 18, 82152 Martinsried, Germany; Munich Cluster for Systems Neurology (SyNergy), 80336 Munich, Germany. Electronic address:

Background: Proteolytic processing of axon guidance receptors modulates their expression and functions. Contact repulsion by membrane-associated ephrins and Eph receptors was proposed to be facilitated by ectodomain cleavage, but whether this phenomenon is required for axon guidance in vivo is unknown.

Results: In support of established models, we find that cleavage of EphA4 promotes cell-cell and growth cone-cell detachment in vitro. Unexpectedly, however, a cleavage resistant isoform of EphA4 is as effective as a wild-type EphA4 in redirecting motor axons in limbs. Mice in which EphA4 cleavage is genetically abolished have motor axon guidance defects, suggesting an important role of EphA4 cleavage in nonneuronal tissues such as the limb mesenchyme target of spinal motor neurons. Indeed, we find that blocking EphA4 cleavage increases expression of full-length EphA4 in limb mesenchyme, which-via cis-attenuation-apparently reduces the effective concentration of ephrinAs capable of triggering EphA4 forward signaling in the motor axons.

Conclusions: We propose that EphA4 cleavage is required to establish the concentration differential of active ephrins in the target tissue that is required for proper axon guidance. Our study reveals a novel mechanism to regulate guidance decision at an intermediate target based on the modulation of ligand availability by the proteolytic processing of the receptor.
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http://dx.doi.org/10.1016/j.cub.2014.08.028DOI Listing
October 2014

EphA4-mediated ipsilateral corticospinal tract misprojections are necessary for bilateral voluntary movements but not bilateral stereotypic locomotion.

J Neurosci 2014 Apr;34(15):5211-21

Department of Physiology, Pharmacology and Neuroscience, City College of the City University of New York, New York, New York 10031; Department of Molecules-Signaling-Development, Max Planck Institute of Neurobiology, D-82152 Martinsried, Germany, Munich Cluster for Systems Neurology (SyNergy), D-80336 Munich, Germany; and Department of Neuroscience, Science for Life Laboratory, University of Uppsala, 75124 Uppsala, Sweden.

In this study, we took advantage of the reported role of EphA4 in determining the contralateral spinal projection of the corticospinal tract (CST) to investigate the effects of ipsilateral misprojections on voluntary movements and stereotypic locomotion. Null EphA4 mutations produce robust ipsilateral CST misprojections, resulting in bilateral corticospinal tracts. We hypothesize that a unilateral voluntary limb movement, not a stereotypic locomotor movement, will become a bilateral movement in EphA4 knock-out mice with a bilateral CST. However, in EphA4 full knock-outs, spinal interneurons also develop bilateral misprojections. Aberrant bilateral spinal circuits could thus transform unilateral corticospinal control signals into bilateral movements. We therefore studied mice with conditional forebrain deletion of the EphA4 gene under control by Emx1, a gene expressed in the forebrain that affects the developing CST but spares brainstem motor pathways and spinal motor circuits. We examined two conditional knock-outs targeting forebrain EphA4 during performance of stereotypic locomotion and voluntary movement: adaptive locomotion over obstacles and exploratory reaching. We found that the conditional knock-outs used alternate stepping, not hopping, during overground locomotion, suggesting normal central pattern generator function and supporting our hypothesis of minimal CST involvement in the moment-to-moment control of stereotypic locomotion. In contrast, the conditional knock-outs showed bilateral voluntary movements under conditions when single limb movements are normally produced and, as a basis for this aberrant control, developed a bilateral motor map in motor cortex that is driven by the aberrant ipsilateral CST misprojections. Therefore, a specific change in CST connectivity is associated with and explains a change in voluntary movement.
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http://dx.doi.org/10.1523/JNEUROSCI.4848-13.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3983801PMC
April 2014

Genetic evidence for the adhesion protein IgSF9/Dasm1 to regulate inhibitory synapse development independent of its intracellular domain.

J Neurosci 2014 Mar;34(12):4187-99

Department of Molecules-Signaling-Development and Synaptic Receptor Trafficking Group, Max Planck Institute of Neurobiology, D-82152 Martinsried, Germany, German Mouse Clinic, Institute of Experimental Genetics, Helmholtz Zentrum München, German Research Center for Environment and Health, D-85764 Neuherberg, Germany, Department of Neurology, Friedrich-Baur-Institute, Ludwig-Maximilians-University, D-80336 Munich, Germany, Munich Cluster for Systems Neurology (SyNergy), D-80336 Munich, Germany, and Department of Physiology II, University of Bonn, D-53115 Bonn, Germany.

Normal brain function requires balanced development of excitatory and inhibitory synapses. An imbalance in synaptic transmission underlies many brain disorders such as epilepsy, schizophrenia, and autism. Compared with excitatory synapses, relatively little is known about the molecular control of inhibitory synapse development. We used a genetic approach in mice to identify the Ig superfamily member IgSF9/Dasm1 as a candidate homophilic synaptic adhesion protein that regulates inhibitory synapse development. IgSF9 is expressed in pyramidal cells and subsets of interneurons in the CA1 region of hippocampus. Electrophysiological recordings of acute hippocampal slices revealed that genetic inactivation of the IgSF9 gene resulted in fewer functional inhibitory synapses; however, the strength of the remaining synapses was unaltered. These physiological abnormalities were correlated with decreased expression of inhibitory synapse markers in IgSF9(-/-) mice, providing anatomical evidence for a reduction in inhibitory synapse numbers, whereas excitatory synapse development was normal. Surprisingly, knock-in mice expressing a mutant isoform of IgSF9 lacking the entire cytoplasmic domain (IgSF9(ΔC/ΔC) mice) had no defects in inhibitory synapse development, providing genetic evidence that IgSF9 regulates synapse development via ectodomain interactions rather than acting itself as a signaling receptor. Further, we found that IgSF9 mediated homotypic binding and cell aggregation, but failed to induce synapse formation, suggesting that IgSF9 acts as a cell adhesion molecule (CAM) to maintain synapses. Juvenile IgSF9(-/-) mice exhibited increased seizure susceptibility indicative of an imbalance in synaptic excitation and inhibition. These results provide genetic evidence for a specific role of IgSF9 in inhibitory synapse development/maintenance, presumably by its CAM-like activity.
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http://dx.doi.org/10.1523/JNEUROSCI.3671-13.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6608096PMC
March 2014

Ephrin signalling in the developing nervous system.

Curr Opin Neurobiol 2014 Aug 7;27:16-24. Epub 2014 Mar 7.

Institut de Recherches Cliniques de Montréal (IRCM), Montréal, QC, Canada H2W 1R7; Département de Médecine, Université de Montréal, Montréal, QC, Canada H3T 1J4; Division of Experimental Medicine, Departments of Biology, and, Anatomy and Cell Biology and Integrated Program in Neurosciences, McGill University, Montréal, QC, Canada H3A 1A3. Electronic address:

Ephrin ligands and their Eph receptors hold our attention since their link to axon guidance almost twenty years ago. Since then, they have been shown to be critical for short distance cell-cell interactions in the nervous system. The interest in their function has not abated, leading to ever-more sophisticated studies generating as many surprising answers about their function as new questions. We discuss recent insights into their functions in the developing nervous system, including neuronal progenitor sorting, stochastic cell migration, guidance of neuronal growth cones, topographic map formation, as well as synaptic plasticity.
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http://dx.doi.org/10.1016/j.conb.2014.02.006DOI Listing
August 2014

FLRT3 is a Robo1-interacting protein that determines Netrin-1 attraction in developing axons.

Curr Biol 2014 Mar 20;24(5):494-508. Epub 2014 Feb 20.

Instituto de Neurociencias de Alicante, CSIC and Universidad Miguel Hernández, 03550 Sant Joan d'Alacant, Spain. Electronic address:

Background: Guidance molecules are normally presented to cells in an overlapping fashion; however, little is known about how their signals are integrated to control the formation of neural circuits. In the thalamocortical system, the topographical sorting of distinct axonal subpopulations relies on the emergent cooperation between Slit1 and Netrin-1 guidance cues presented by intermediate cellular targets. However, the mechanism by which both cues interact to drive distinct axonal responses remains unknown.

Results: Here, we show that the attractive response to the guidance cue Netrin-1 is controlled by Slit/Robo1 signaling and by FLRT3, a novel coreceptor for Robo1. While thalamic axons lacking FLRT3 are insensitive to Netrin-1, thalamic axons containing FLRT3 can modulate their Netrin-1 responsiveness in a context-dependent manner. In the presence of Slit1, both Robo1 and FLRT3 receptors are required to induce Netrin-1 attraction by the upregulation of surface DCC through the activation of protein kinase A. Finally, the absence of FLRT3 produces defects in axon guidance in vivo.

Conclusions: These results highlight a novel mechanism by which interactions between limited numbers of axon guidance cues can multiply the responses in developing axons, as required for proper axonal tract formation in the mammalian brain.
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http://dx.doi.org/10.1016/j.cub.2014.01.042DOI Listing
March 2014

Ret rescues mitochondrial morphology and muscle degeneration of Drosophila Pink1 mutants.

EMBO J 2014 Feb 28;33(4):341-55. Epub 2014 Jan 28.

Molecules - Signaling - Development, Max Planck Institute of Neurobiology, Martinsried, Germany.

Parkinson's disease (PD)-associated Pink1 and Parkin proteins are believed to function in a common pathway controlling mitochondrial clearance and trafficking. Glial cell line-derived neurotrophic factor (GDNF) and its signaling receptor Ret are neuroprotective in toxin-based animal models of PD. However, the mechanism by which GDNF/Ret protects cells from degenerating remains unclear. We investigated whether the Drosophila homolog of Ret can rescue Pink1 and park mutant phenotypes. We report that a signaling active version of Ret (Ret(MEN₂B) rescues muscle degeneration, disintegration of mitochondria and ATP content of Pink1 mutants. Interestingly, corresponding phenotypes of park mutants were not rescued, suggesting that the phenotypes of Pink1 and park mutants have partially different origins. In human neuroblastoma cells, GDNF treatment rescues morphological defects of PINK1 knockdown, without inducing mitophagy or Parkin recruitment. GDNF also rescues bioenergetic deficits of PINK knockdown cells. Furthermore, overexpression of Ret(MEN₂B) significantly improves electron transport chain complex I function in Pink1 mutant Drosophila. These results provide a novel mechanism underlying Ret-mediated cell protection in a situation relevant for human PD.
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http://dx.doi.org/10.1002/embj.201284290DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3983680PMC
February 2014
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