Misgeld Lab - Publications
Nanoresolution real-time 3D orbital tracking for studying mitochondrial trafficking in vertebrate axons in vivo
Authors : Fabian Wehnekamp, Gabriela Plucińska, Rachel Thong Thomas Misgeld, Don C Lamb | 10 Jun 2019
We present the development and in vivo application of a feedback-based tracking microscope to follow individual mitochondria in sensory neurons of zebrafish larvae with nanometer precision and millisecond temporal resolution. By combining various technical improvements, we tracked individual mitochondria with unprecedented spatiotemporal resolution over distances of >100 mm. Using these nanoscopic trajectory data, we discriminated five motional states: a fast and a slow directional motion state in both the anterograde and retrograde directions and a stationary state. The transition pattern revealed that, after a pause, mitochondria predominantly persist in the original direction of travel, while transient changes of direction often exhibited longer pauses. Moreover, mitochondria in the vicinity of a second, stationary mitochondria displayed an increased probability to pause. The capability of following and optically manipulating a single organelle with high spatiotemporal resolution in a living organism offers a new approach to elucidating their function in its complete physiological context.
Calcium Influx through Plasma-Membrane Nanoruptures Drives Axon Degeneration in a Model of Multiple Sclerosis
Authors : Maarten E. Witte, Adrian-Minh Schumacher, Christoph F. Mahler, Jan P. Bewersdorf, Jonas Lehmitz, Alexander Scheiter, Paula Sánchez, Philip R. Williams, Oliver Griesbeck, Ronald Naumann, Thomas Misgeld, Martin Kerschensteiner | 20 Feb 2019
Axon loss determines persistent disability in multiple sclerosis patients. Here, we use in vivo calcium imaging in a multiple sclerosis model to show that cytoplasmic calcium levels determine the choice between axon loss and survival. We rule out the endoplasmic reticulum, glutamate excitotoxicity, and the reversal of the sodium-calcium exchanger as sources of intra-axonal calcium accumulation and instead identify nanoscale ruptures of the axonal plasma membrane as the critical path of calcium entry.
Authors : Alessio Colombo, Hung-En Hsia, Mengzhe Wang, Peer-Hendrik Kuhn, Monika S Brill, Paolo Canevazzi, Regina Feederle, Carla Taveggia, Thomas Misgeld, Stefan F Lichtenthaler | 19 Feb 2018
Death receptor 6 (DR6) is an orphan member of the TNF receptor superfamily and controls cell death and differentiation in a cellautonomous manner in different cell types. Here, we report an additional non-cell-autonomous function for DR6 in the peripheral nervous system (PNS). DR6-knockout (DR6 KO) mice showed precocious myelination in the PNS. Using an in vitro myelination assay, we demonstrate that neuronal DR6 acts in trans on Schwann cells (SCs) and reduces SC proliferation and myelination independently of its cytoplasmic death domain. Mechanistically, DR6 was found to be cleaved in neurons by “a disintegrin and metalloprotease 10” (ADAM10), releasing the soluble DR6 ectodomain (sDR6). Notably, in the in vitro myelination assay, sDR6 was sufficient to rescue the DR6 KO phenotype. Thus, in addition to the cell-autonomous receptor function of full-length DR6, the proteolytically released sDR6 can unexpectedly also act as a paracrine signaling factor in the PNS in a non-cell-autonomous manner during SC proliferation and myelination. This new mode of DR6 signaling will be relevant in future attempts to target DR6 in disease settings.
Authors : Katerina Akassoglou, Mario Merlini, Victoria A. Rafalski, Raquel Real, Liang Liang, Yunju Jin, Sarah E. Dougherty, Vincenzo De Paola, David J. Linden, Thomas Misgeld, Binhai Zheng | 8 Nov 2017
In vivo opticalimaging has emerged as a powerfultool with whichto study cellular responsestoinjury and diseaseinthemammalian CNS. Important new insights have emerged regarding axonal degeneration and regeneration, glial responses and neuroinflammation, changes in the neurovascular unit, and, more recently, neural transplantations. Accompanying a 2017 SfN Mini-Symposium, here, we discuss selected recent advances in understandingthe neuronal, glial, and other cellular responsesto CNS injury and disease with in vivo imaging of the rodent brain or spinal cord. We anticipate that in vivo optical imaging will continue to be at the forefront of breakthrough discoveries of fundamental mechanisms and therapies for CNS injury and disease.
Authors : Thomas Misgeld, Thomas L. Schwarz | 1 Nov 2017
Neurons have more extended and complex shapes than other cells and consequently face a greater challenge in distributing and maintaining mitochondria throughout their arbors. Neurons can last a lifetime, but proteins turn over rapidly. Mitochondria, therefore, need constant rejuvenation no matter how far they are from the soma. Axonal transport of mitochondria and mitochondrial fission and fusion contribute to this rejuvenation, but local protein synthesis is also likely. Maintenance of a healthy mitochondrial population also requires the clearance of damaged proteins and organelles. This involves degradation of individual proteins, sequestration in mitochondria-derived vesicles, organelle degradation by mitophagy and macroautophagy, and in some cases transfer to glial cells. Both long-range transport and local processing are thus at work in achieving neuronal mitostasis—the maintenance of an appropriately distributed pool of healthy mitochondria for the duration of a neuron’s life. Accordingly, defects in the processes that support mitostasis are significant contributors to neurodegenerative disorders.
Authors : Fargol Mazaheri, Nicolas Snaidero, Gernot Kleinberger, Charlotte Madore, Anna Daria, Georg Werner, Susanne Krasemann, Sebastian Bultmann, ..., Sabina Tahirovic, Martin Kerschensteiner, Thomas Misgeld, Oleg Butovsky, Christian Haas | 8 May 2017
Sequence variations in the triggering receptor expressed on myeloid cells 2 (TREM2) have been linked to an increased risk for neurodegenerative disorders such as Alzheimer’s disease and frontotemporal lobar degeneration. In the brain, TREM2 is predominantly expressed in microglia. Several disease-associated TREM2 variants result in a loss of function by reducing microglial phagocytosis, impairing lipid sensing, preventing binding of lipoproteins and affecting shielding of amyloid plaques. We here investigate the consequences of TREM2 loss of function on the microglia transcriptome. Among the differentially expressed messenger RNAs in wildtype and Trem2/ microglia, gene clusters are identified which represent gene functions in chemotaxis, migration and mobility. Functional analyses confirm that loss of TREM2 impairs appropriate microglial responses to injury and signals that normally evoke chemotaxis on multiple levels. In an ex vivo organotypic brain slice assay, absence of TREM2 reduces the distance migrated by microglia. Moreover, migration towards defined chemo-attractants is reduced upon ablation of TREM2 and can be rescued by TREM2 reexpression. In vivo, microglia lacking TREM2 migrate less towards injected apoptotic neurons, and outgrowth of microglial processes towards sites of laser-induced focal CNS damage in the somatosensory cortex is slowed. The apparent lack of chemotactic stimulation upon depletion of TREM2 is consistent with a stable expression profile of genes characterizing the homoeostatic signature of microglia.
Authors : Sylvia Heink, Nir Yogev, Christoph Garbers, Marina Herwerth, Lilian Aly, Christiane Gasperi, ..., Thomas Misgeld, Thomas F Wunderlich, Juan Hidalgo, Mohamed Oukka, Stefan Rose-John, Marc Schmidt-Supprian, Ari Waisman, Thomas Korn | 28 Nov 2016
The cellular sources of interleukin 6 (IL-6) that are relevant for differentiation of the TH17 subset of helper T cells remain unclear. Here we used a novel strategy for the conditional deletion of distinct IL-6-producing cell types to show that dendritic cells (DCs) positive for the signaling regulator Sirpa were essential for the generation of pathogenic TH17 cells. Using their IL-6 receptor a-chain (IL-6Ra), Sirpa+ DCs trans-presented IL-6 to T cells during the process of cognate interaction. While ambient IL-6 was sufficient to suppress the induction of expression of the transcription factor Foxp3 in T cells, trans-presentation of IL-6 by DC-bound IL-6Ra (called ‘IL-6 cluster signaling’ here) was needed to prevent premature induction of interferon-g (IFN-g) expression in T cells and to generate pathogenic TH17 cells in vivo. Our findings should guide therapeutic approaches for the treatment of TH17-cell-mediated autoimmune diseases.
Branch-Specific Microtubule Destabilization Mediates Axon Branch Loss during Neuromuscular Synapse Elimination
Authors : Monika S. Brill, Tatjana Kleele, Laura Ruschkies, Mengzhe Wang, Natalia A. Marahori, Miriam S. Reuter, Torben J. Hausrat, Emily Weigand, ..., Andrea Ahles, Stefan Engelhardt, Derron L. Bishop, Matthias Kneussel, Thomas Misgeld | 23 Nov 2016
Developmental axon remodeling is characterized by the selective removal of branches from axon arbors. The mechanisms that underlie such branch loss are largely unknown. Additionally, how neuronal resources are specifically assigned to the branches of remodeling arbors is not understood. Here we show that axon branch loss at the developing mouse neuromuscular junction is mediated by branch-specific microtubule severing, which results in local disassembly of the microtubule cytoskeleton and loss of axonal transport in branches that will subsequently dismantle. Accordingly, pharmacological microtubule stabilization delays neuromuscular synapse elimination. This branch-specific disassembly of the cytoskeleton appears to be mediated by the microtubulesevering enzyme spastin, which is dysfunctional in some forms of upper motor neuron disease. Our results demonstrate a physiological role for a neurodegeneration-associated modulator of the cytoskeleton, reveal unexpected cell biology of branch-specific axon plasticity and underscore the mechanistic similarities of axon loss in development and disease.
Myelinosome formation represents an early stage of oligodendrocyte damage in multiple sclerosis and its animal model
Authors : Elisa Romanelli, Doron Merkler, Aleksandra Mezydlo, Marie-Theres Weil, Martin S. Weber, ..., Karl-Klaus Conzelmann, Imke Metz, Wolfgang Brück, Matthew Routh, Mikael Simons, Derron Bishop, Thomas Misgeld, Martin Kerschensteiner | 16 Nov 2016
Oligodendrocyte damage is a central event in the pathogenesis of the common neuroinflammatory condition, multiple sclerosis (MS). Where and how oligodendrocyte damage is initiated in MS is not completely understood. Here, we use a combination of light and electron microscopy techniques to provide a dynamic and highly resolved view of oligodendrocyte damage in neuroinflammatory lesions. We show that both in MS and in its animal model structural damage is initiated at the myelin sheaths and only later spreads to the oligodendrocyte cell body. Early myelin damage itself is characterized by the formation of local myelin out-foldings—‘myelinosomes’—, which are surrounded by phagocyte processes and promoted in their formation by anti-myelin antibodies and complement. The presence of myelinosomes in actively demyelinating MS lesions suggests that oligodendrocyte damage follows a similar pattern in the human disease, where targeting demyelination by therapeutic interventions remains a major open challenge.
Authors : Peter Engerer, Caroline Fecher, Thomas Misgeld | Sep 2016
Seeing is believing, but many subcellular structures are simply too small and densely packed to be resolved with light microscopes. Over the past two decades, ever-more sophisticated techniques for ‘super-resolution’ microscopy have succeeded in surpassing the capabilities of conventional microscopy. And last year, a new technology called expansion microscopy achieved the precision of super-resolution imaging not by optical tricks but by a radically different approach in which the biological specimen is physically enlarged through chemical treatments. In this issue and in a recent issue of Nature Methods, three groups, Tillberg et al., Ku et al., and Chozinski et al., improve on this method with simplified protocols that use off-the-shelf chemicals and are compatible with standard immunofluorescence techniques and genetically encoded fluorophores. The new protocols should broaden the applications of expansion microscopy and bring it within reach of almost any laboratory.
Seminal past publications of the lab
Multiparametric optical analysis of mitochondrial redox signals during neuronal physiology and pathology in vivo
Authors : Michael O Breckwoldt, Franz M J Pfister, Peter M Bradley, Petar Marinković, Philip R Williams, ..., Markus Schwarzländer, Leanne Godinho, Florence M Bareyre, Tobias P Dick, Martin Kerschensteiner, Thomas Misgeld | 20 Apr 2014
Mitochondrial redox signals have a central role in neuronal physiology and disease. Here we describe a new optical approach to measure fast redox signals with singleorganelle resolution in living mice that express genetically encoded redox biosensors in their neuronal mitochondria. Moreover, we demonstrate how parallel measurements with several biosensors can integrate these redox signals into a comprehensive characterization of mitochondrial function. This approach revealed that axonal mitochondria undergo spontaneous ‘contractions’ that are accompanied by reversible redox changes. These contractions are amplified by neuronal activity and acute or chronic neuronal insults. Multiparametric imaging reveals that contractions constitute respiratory chain–dependent episodes of depolarization coinciding with matrix alkalinization, followed by uncoupling. In contrast, permanent mitochondrial damage after spinal cord injury depends on calcium influx and mitochondrial permeability transition. Thus, our approach allows us to identify heterogeneity among physiological and pathological redox signals, correlate such signals to functional and structural organelle dynamics and dissect the underlying mechanisms.
Authors : Catherine Diamante Sorbara, Naomi Elizabeth Wagner, Anne Ladwig, Ivana Nikic, Doron Merkler, Tatjana Kleele, ..., Ronald Naumann, Leanne Godinho, Florence Martine Bareyre, Derron Bishop, Thomas Misgeld, Martin Kerschensteiner | 17 Dec 2014
Impaired axonal transport can contribute to axon degeneration and has been described in many neurodegenerative diseases. Multiple sclerosis (MS) is a common neuroinflammatory disease, which is characterized by progressive axon degeneration— whether, when, and how axonal transport is affected in this condition is unknown. Here we used in vivo two-photon imaging to directly assay transport of organelles and the stability of microtubule tracks in individual spinal axons in mouse models of MS. We found widespread transport deficits, which preceded structural alterations of axons, cargos, or microtubules and could be reversed by acute antiinflammatory interventions or redox scavenging. Our study shows that acute neuroinflammation induces a pervasive state of reversible axonal dysfunction, which coincides with acute disease symptoms. Moreover, perpetuated transport dysfunction, as we found in a model of progressive MS, led to reduced distal organelle supply and could thus contribute to axonal dystrophy in advanced stages of the disease.
Athors : Philip R. Williams, Bogdan-Nicolae Marincu, Catherine D. Sorbara, Christoph F. Mahler, Adrian-Minh Schumacher, Oliver Griesbeck, Martin Kerschensteiner, Thomas Misgeld | 2014
Therapeutic strategies for spinal cord injury (SCI) commonly focus on regenerating disconnected axons. An alternative approach would be to maintain continuity of damaged axons, especially after contusion. The viability of such neuropreservative strategies depends on the degree to which initially injured axons can recover. Here we use morphological and molecular in vivo imaging after contusion SCI in mice to show that injured axons persist in a metastable state for hours. Intra-axonal calcium dynamics influence fate, but the outcome is not invariably destructive in that many axons with calcium elevations recover homeostasis without intervention. Calcium enters axons primarily through mechanopores. Spontaneous pore resealing allows calcium levels to normalize and axons to survive long term. Axon loss can be halted by blocking calcium influx or calpain, even with delayed initiation. Our data identify an inherent self-preservation process in contused axons and a window of opportunity for rescuing connectivity after nontransecting SCI.
Authors : Tatjana Kleele, Petar Marinković, Philip R. Williams, Sina Stern, Emily E. Weigand, Peter Engerer, ..., Frank Bradke, Derron Bishop, Jochen Herms, Arthur Konnerth, Martin Kerschensteiner, Leanne Godinho, Thomas Misgeld | 12 Sep 2014
Microtubule dynamics in neurons play critical roles in physiology, injury and disease and determine microtubule orientation, the cell biological correlate of neurite polarization. Several microtubule binding proteins, including end-binding protein 3 (EB3), specifically bind to the growing plus tip of microtubules. In the past, fluorescently tagged end-binding proteins have revealed microtubule dynamics in vitro and in non-mammalian model organisms. Here, we devise an imaging assay based on transgenic mice expressing yellow fluorescent proteintagged EB3 to study microtubules in intact mammalian neurites. Our approach allows measurement of microtubule dynamics in vivo and ex vivo in peripheral nervous system and central nervous system neurites under physiological conditions and after exposure to microtubule-modifying drugs. We find an increase in dynamic microtubules after injury and in neurodegenerative disease states, before axons show morphological indications of degeneration or regrowth. Thus increased microtubule dynamics might serve as a general indicator of neurite remodelling in health and disease.
Axonal transport deficits and degeneration can evolve independently in mouse models of amyotrophic lateral sclerosis
Authors : Petar Marinković, Miriam S. Reutera, Monika S. Brilla, Leanne Godinhoa, Martin Kerschensteinerb, Thomas Misgeld | 31 Jan 2012
Axonal transport deficits have been reported in many neurodegenerative conditions and are widely assumed to be an immediate causative step of axon and synapse loss. By imaging changes in axonal morphology and organelle transport over time in several animal models of amyotrophic lateral sclerosis (ALS), we now find that deficits in axonal transport of organelles (mitochondria, endosomes) and axon degeneration can evolve independently. This conclusion rests on the following results: (i) Axons can survive despite long-lasting transport deficits: In the SODG93A model of ALS, transport deficits are detected soon after birth, months before the onset of axon degeneration. (ii) Transport deficits are not necessary for axon degeneration: In the SODG85R model of ALS, motor axons degenerate, but transport is unaffected. (iii) Axon transport deficits are not sufficient to cause immediate degeneration: In mice that overexpress wild-type superoxide dismutase-1 (SODWT), axons show chronic transport deficits, but survive. These data suggest that disturbances of organelle transport are not a necessary step in the emergence of motor neuron degeneration.
Authors : Derron Bishop, Ivana Nikić, Mary Brinkoetter, Sharmon Knecht, Stephanie Potz, Martin Kerschensteiner, Thomas Misgeld | 5 Jun 2011
The correlation of light and electron microscopy of complex tissues remains a major challenge. Here we report near-infrared branding (NIRB), which facilitates such correlation by using a pulsed, near-infrared laser to create defined fiducial marks in three dimensions in fixed tissue. As these marks are fluorescent and can be photo-oxidized to generate electron contrast, they can guide re-identification of previously imaged structures as small as dendritic spines by electron microscopy.
A reversible form of axon damage in experimental autoimmune encephalomyelitis and multiple sclerosis
Authors : Ivana Nikić, Doron Merkler, Catherine Sorbara, Mary Brinkoetter, Mario Kreutzfeldt, Florence M Bareyre, Wolfgang Brück, Derron Bishop, Thomas Misgeld, Martin Kerschensteiner | 27 Mar 2011
In multiple sclerosis, a common inflammatory disease of the central nervous system, immune-mediated axon damage is responsible for permanent neurological deficits. How axon damage is initiated is not known. Here we use in vivo imaging to identify a previously undescribed variant of axon damage in a mouse model of multiple sclerosis. This process, termed ‘focal axonal degeneration’ (FAD), is characterized by sequential stages, beginning with focal swellings and progressing to axon fragmentation. Notably, most swollen axons persist unchanged for several days, and some recover spontaneously. Early stages of FAD can be observed in axons with intact myelin sheaths. Thus, contrary to the classical view, demyelination—a hallmark of multiple sclerosis—is not a prerequisite for axon damage. Instead, focal intra-axonal mitochondrial pathology is the earliest ultrastructural sign of damage, and it precedes changes in axon morphology. Molecular imaging and pharmacological experiments show that macrophage-derived reactive oxygen and nitrogen species (ROS and RNS) can trigger mitochondrial pathology and initiate FAD. Indeed, neutralization of ROS and RNS rescues axons that have already entered the degenerative process. Finally, axonal changes consistent with FAD can be detected in acute human multiple sclerosis lesions. In summary, our data suggest that inflammatory axon damage might be spontaneously reversible and thus a potential target for therapy.
Authors : Thomas Misgeld, Martin Kerschensteiner, Florence M Bareyre, Robert W Burgess, Jeff W Lichtman | 10 Jun 2007
Neuronal mitochondria regulate synaptic physiology and cellular survival, and disruption of their function or transport causes neurological disease. We present a fluorescence method to selectively image mitochondrial dynamics in the mouse nervous system, in both live mice and acute explants. We show that axon damage and recovery lead to early and sustained changes in anterograde and retrograde transport. In vivo imaging of mitochondria will be a useful tool to analyze this essential organelle.
Authors : Thomas Misgeld, Martin Kerschensteiner | Jun 2006
In vivo microscopy is an exciting tool for neurological research because it can reveal how single cells respond to damage of the nervous system. This helps us to understand how diseases unfold and how therapies work. Here, we review the optical imaging techniques used to visualize the different parts of the nervous system, and how they have provided fresh insights into the aetiology and therapeutics of neurological diseases. We focus our discussion on five areas of neuropathology (trauma, degeneration, ischaemia, inflammation and seizures) in which in vivo microscopy has had the greatest impact. We discuss the challenging issues in the field, and argue that the convergence of new optical and non-optical methods will be necessary to overcome these challenges.
Authors : Martin Kerschensteiner, Martin E Schwab, Jeff W Lichtman, Thomas Misgeld | 10 Apr 2005
The poor response of central axons to transection underlies the bleak prognosis following spinal cord injury. Here, we monitor individual fluorescent axons in the spinal cords of living transgenic mice over several days after spinal injury. We find that within 30 min after trauma, axons die back hundreds of micrometers. This acute form of axonal degeneration is similar in mechanism to the more delayed Wallerian degeneration of the disconnected distal axon, but acute degeneration affects the proximal and distal axon ends equally. In vivo imaging further shows that many axons attempt regeneration within 6–24 h after lesion. This growth response, although robust, seems to fail as a result of the inability of axons to navigate in the proper direction. These results suggest that time-lapse imaging of spinal cord injury may provide a powerful analytical tool for assessing the pathogenesis of spinal cord injury and for evaluating therapies that enhance regeneration.