using-microfluidics-chips-for-live-imaging-study-injury-responses

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in <em>Drosophila</em> Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

University of Michigan

alpha2,8 Polysialic acid (PSA) is a carbohydrate attached to the glycoprotein backbone of the neural cell adhesion molecule (NCAM) and implicated in nervous system development and repair. Here, we investigated whether PSA can improve functional recovery after peripheral nerve lesion in adult mice. We applied a functional PSA mimicking peptide or a control peptide in a polyethylene cuff used to surgically reconnect the severed stumps of the femoral nerve before it bifurcates into the motor and sensory branches. Using video-based motion analysis to monitor motor recovery over a 3 month postoperative period, we observed a better functional outcome in the PSA mimetic-treated than in control mice receiving a control peptide or phosphate buffered saline. Retrograde tracing of regenerated motoneurons and morphometric analyses showed that motoneuron survival, motoneuron soma size and axonal diameters were not affected by treatment with the PSA mimetic. However, remyelination of regenerated axons distal to the injury site was considerably improved by the PSA mimetic indicating that effects on Schwann cells in the denervated nerve may underlie the functional effects seen in motor recovery. In line with this notion was the observation that the PSA mimetic enhanced the elongation of Schwann cell processes and Schwann cell proliferation in vitro, when compared with the control peptide. Moreover, Schwann cell proliferation in vivo was enhanced in both motor and sensory branches of the femoral nerve by application of the PSA mimetic. These effects were likely mediated by NCAM through its interaction with the fibroblast growth factor receptor (FGFR), since they were not observed when the PSA mimetic was applied to NCAM-deficient Schwann cells, and since application of two different FGFR inhibitors reduced process elongation from Schwann cells in vitro. Our results indicate the potential of PSA mimetics as therapeutic agents promoting motor recovery and myelination after peripheral nerve injury.

Polysialic acid glycomimetics promote myelination and functional recovery after peripheral nerve injury in mice.

Polysialic acid (PSA) is a large and highly negatively charged glycan that plays crucial roles in nervous system development and function in the adult. It has been suggested to facilitate cell migration, neurite outgrowth, and synaptic plasticity because its hydration volume could enhance flexibility of cell interactions. Evidence for receptors of PSA has so far been elusive. We now identified histone H1 as binding partner of PSA via a single-chain variable fragment antibody using an anti-idiotypic approach. Histone H1 directly binds to PSA as shown by ELISA. Surface biotinylation of cultured cerebellar neurons indicated an extracellular localization of histone H1. Immunostaining of live cerebellar neurons and Schwann cells confirmed that an extracellular pool of histone H1 colocalizes with PSA at the cell surface. Histone H1 was also detected in detergent-insoluble synaptosomal membrane subfractions and postsynaptic densities. When applied in vitro, histone H1 stimulated neuritogenesis, process formation and proliferation of Schwann cells, and migration of neural precursor cells via a PSA-dependent mechanism, further indicating that histone H1 is active extracellularly. These in vitro observations suggested an important functional role for the interaction between histone H1 and PSA not only for nervous system development but also for regeneration in the adult. Indeed, histone H1 improved functional recovery, axon regrowth, and precision of reinnervation of the motor branch in adult mice with femoral nerve injury. Our findings encourage investigations on the therapeutic potential of histone H1 in humans.

Functional role of the interaction between polysialic acid and extracellular histone H1.

With powerful genetics and a translucent cuticle, the Drosophila larva is an ideal model system for live imaging studies of neuronal cell biology and function. Here, we present an easy-to-use approach for high resolution live imaging in Drosophila using microfluidic chips. Two different designs allow for non-invasive and chemical-free immobilization of 3(rd) instar larvae over short (up to 1 hour) and long (up to 10 hours) time periods. We utilized these 'larva chips' to characterize several sub-cellular responses to axotomy which occur over a range of time scales in intact, unanaesthetized animals. These include waves of calcium which are induced within seconds of axotomy, and the intracellular transport of vesicles whose rate and flux within axons changes dramatically within 3 hours of axotomy. Axonal transport halts throughout the entire distal stump, but increases in the proximal stump. These responses precede the degeneration of the distal stump and regenerative sprouting of the proximal stump, which is initiated after a 7 hour period of dormancy and is associated with a dramatic increase in F-actin dynamics. In addition to allowing for the study of axonal regeneration in vivo, the larva chips can be utilized for a wide variety of in vivo imaging applications in Drosophila.

Microfluidic chips for in vivo imaging of cellular responses to neural injury in Drosophila larvae.

Axonal degeneration is a hallmark of many neuropathies, neurodegenerative diseases, and injuries. Here, using a Drosophila injury model, we have identified a highly conserved E3 ubiquitin ligase, Highwire (Hiw), as an important regulator of axonal and synaptic degeneration. Mutations in hiw strongly inhibit Wallerian degeneration in multiple neuron types and developmental stages. This new phenotype is mediated by a new downstream target of Hiw: the NAD+ biosynthetic enzyme nicotinamide mononucleotide adenyltransferase (Nmnat), which acts in parallel to a previously known target of Hiw, the Wallenda dileucine zipper kinase (Wnd/DLK) MAPKKK. Hiw promotes a rapid disappearance of Nmnat protein in the distal stump after injury. An increased level of Nmnat protein in hiw mutants is both required and sufficient to inhibit degeneration. Ectopically expressed mouse Nmnat2 is also subject to regulation by Hiw in distal axons and synapses. These findings implicate an important role for endogenous Nmnat and its regulation, via a conserved mechanism, in the initiation of axonal degeneration. Through independent regulation of Wnd/DLK, whose function is required for proximal axons to regenerate, Hiw plays a central role in coordinating both regenerative and degenerative responses to axonal injury.

The Highwire ubiquitin ligase promotes axonal degeneration by tuning levels of Nmnat protein.

Polysialic acid (PSA) is a homopolymeric glycan that plays crucial roles in the developing and adult nervous system. So far only a few PSA-binding proteins have been identified. Here, we identify myristoylated alanine-rich C kinase substrate (MARCKS) as novel PSA binding partner. Binding assays showed a direct interaction between PSA and a peptide comprising the effector domain of MARCKS (MARCKS-ED). Co-immunoprecipitation of PSA-carrying neural cell adhesion molecule (PSA-NCAM) with MARCKS and co-immunostaining of MARCKS and PSA at the cell membrane of hippocampal neurons confirm the interaction between PSA and MARCKS. Co-localization and an intimate interaction of PSA and MARCKS at the cell surface was seen by confocal microscopy and fluorescence resonance energy transfer (FRET) analysis after the addition of fluorescently labeled PSA or PSA-NCAM to live CHO cells or hippocampal neurons expressing MARCKS as a fusion protein with green fluorescent protein (GFP). Cross-linking experiments showed that extracellularly applied PSA or PSA-NCAM and intracellularly expressed MARCKS-GFP are in close contact, suggesting that PSA and MARCKS interact with each other at the plasma membrane from opposite sides. Insertion of PSA and MARCKS-ED peptide into lipid bilayers from opposite sides alters the electric properties of the bilayer confirming the notion that PSA and the effector domain of MARCKS interact at and/or within the plane of the membrane. The MARCKS-ED peptide abolished PSA-induced enhancement of neurite outgrowth from cultured hippocampal neurons indicating an important functional role for the interaction between MARCKS and PSA in the developing and adult nervous system.

Functional role of the interaction between polysialic acid and myristoylated alanine-rich C kinase substrate at the plasma membrane.

Chondroitin sulfate (CS) and dermatan sulfate (DS) proteoglycans are major components of the extracellular matrix implicated in neural development, plasticity and regeneration. While it is accepted that CS are major inhibitors of neural regeneration, the contributions of DS to regeneration have not been assessed. To enable a novel approach in studies on DS versus CS roles during development and regeneration, we generated a mouse deficient in the dermatan 4-O-sulfotransferase1 (Chst14(-/-)), a key enzyme in the synthesis of iduronic acid-containing modules found in DS but not CS. In wild-type mice, Chst14 is expressed at high levels in the skin and in the nervous system, and is enriched in astrocytes and Schwann cells. Ablation of Chst14, and the assumed failure to produce DS, resulted in smaller body mass, reduced fertility, kinked tail and increased skin fragility compared with wild-type (Chst14(+/+)) littermates, but brain weight and gross anatomy were unaffected. Neurons and Schwann cells from Chst14(-/-) mice formed longer processes in vitro, and Chst14(-/-) Schwann cells proliferated more than Chst14(+/+) Schwann cells. After femoral nerve transection/suture, functional recovery and axonal regrowth in Chst14(-/-) mice were initially accelerated but the final outcome 3months after injury was not better than that in Chst14(+/+) littermates. These results suggest that while Chst14 and its enzymatic products might be of limited importance for neural development, they may contribute to the regeneration-restricting environment in the adult mammalian nervous system.

Dermatan 4-O-sulfotransferase1 ablation accelerates peripheral nerve regeneration.

Polysialic acid (PSA) is a major regulator of cell-cell interactions in the developing nervous system and in neural plasticity in the adult. As a polyanionic molecule with high water-binding capacity, PSA increases the intercellular space generating permissive conditions for cell motility. PSA enhances stem cell migration and axon path finding and promotes repair in the lesioned peripheral and central nervous systems, thus contributing to regeneration. As a next step in developing an improved PSA-based approach to treat nervous system injuries, we searched for small organic compounds that mimic PSA and identified as a PSA mimetic 5-nonyloxytryptamine oxalate, described as a selective 5-hydroxytryptamine receptor 1B (5-HT1B ) agonist. Similar to PSA, 5-nonyloxytryptamine binds to the PSA-specific monoclonal antibody 735, enhances neurite outgrowth of cultured primary neurons and process formation of Schwann cells, protects neurons from oxidative stress, reduces migration of astrocytes and enhances myelination in vitro. Furthermore, nonyloxytryptamine treatment enhances expression of the neural cell adhesion molecule (NCAM) and its polysialylated form PSA-NCAM and reduces expression of the microtubule-associated protein MAP2 in cultured neuroblastoma cells. These results demonstrate that 5-nonyloxytryptamine mimics PSA and triggers PSA-mediated functions, thus contributing to the repertoire of molecules with the potential to improve recovery in acute and chronic injuries of the mammalian peripheral and central nervous systems. Polysialic acid (PSA) plays important roles in nervous system development, as well as synaptic plasticity and regeneration in the adult. 5-Nonyloxytryptamine oxalate (5-NOT) mimics PSA and triggers PSA-mediated functions in neurons and glial cells. 5-NOT stimulates neuritogenesis, myelination and Schwann cell migration. This study sets the basis to develop a PSA-mediated therapy of acute and chronic nervous system diseases.

Nonyloxytryptamine mimics polysialic acid and modulates neuronal and glial functions in cell culture.

Axons degenerate after injury and in neuropathies and disease via a self-destruction program whose mechanism is poorly understood. Axons that have lost connection to their cell bodies have altered electrical and synaptic activities, but whether such changes play a role in the axonal degeneration process is not clear. We have used a Drosophila model to study the Wallerian degeneration of motoneuron axons and their neuromuscular junction synapses. We found that degeneration of the distal nerve stump after a nerve crush is greatly delayed when there is increased potassium channel activity (by overexpression of two different potassium channels, Kir2.1 and dORKΔ-C) or decreased voltage-gated sodium channel activity (using mutations in the para sodium channel). Conversely, degeneration is accelerated when potassium channel activity is decreased (by expressing a dominant-negative mutation of Shaker). Despite the effect of altering voltage-gated sodium and potassium channel activity, recordings made after nerve crush demonstrated that the distal stump does not fire action potentials. Rather, a variety of lines of evidence suggest that the sodium and potassium channels manifest their effects upon degeneration through changes in the resting membrane potential, which in turn regulates the level of intracellular free calcium within the isolated distal axon.

Article	Year
Polysialic acid glycomimetics promote myelination and functional recovery after peripheral nerve injury in mice. Brain : a journal of neurology\| PubMed ID: 19454531	2009
Functional role of the interaction between polysialic acid and extracellular histone H1. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 20844135	2010
Microfluidic chips for in vivo imaging of cellular responses to neural injury in Drosophila larvae. PloS one\| PubMed ID: 22291895	2012
The Highwire ubiquitin ligase promotes axonal degeneration by tuning levels of Nmnat protein. PLoS biology\| PubMed ID: 23226106	2012
Functional role of the interaction between polysialic acid and myristoylated alanine-rich C kinase substrate at the plasma membrane. The Journal of biological chemistry\| PubMed ID: 23329829	2013
Dermatan 4-O-sulfotransferase1 ablation accelerates peripheral nerve regeneration. Experimental neurology\| PubMed ID: 23360803	2013
Nonyloxytryptamine mimics polysialic acid and modulates neuronal and glial functions in cell culture. Journal of neurochemistry\| PubMed ID: 23957498	2014
Sodium and potassium currents influence Wallerian degeneration of injured Drosophila axons. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 24285879	2013

Bibhudatta Mishra

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