DYNLRB1 is essential for dynein mediated transport and neuronal survival

The cytoplasmic dynein motor complex transports essential signals and organelles from the cell periphery to the perinuclear region, hence is critical for the survival and function of highly polarized cells such as neurons. Dynein Light Chain Roadblock-Type 1 (DYNLRB1) is thought to be an accessory subunit required for specific cargos, but here we show that it is essential for general dynein-mediated transport and sensory neuron survival. Homozygous Dynlrb1 null mice are not viable and die during early embryonic development. Furthermore, heterozygous or adult knockdown animals display reduced neuronal growth, and selective depletion of Dynlrb1 in proprioceptive neurons compromises their survival. Conditional depletion of Dynlrb1 in sensory neurons causes deficits in several signaling pathways, including beta-catenin subcellular localization, and a severe impairment in the axonal transport of both lysosomes and retrograde signaling endosomes. Hence, DYNLRB1 is an essential component of the dynein complex, and given dynein\u27s critical functions in neuronal physiology, DYNLRB1 could have a prominent role in the etiology of human neurodegenerative diseases

Enhanced axon outgrowth and improved long-distance axon regeneration in Sprouty2 deficient mice

Sproutyproteine sind Negativfeedbackinhibitoren des Tyrosinkinaserezeptor-signalweges. Es wurde bereits gezeigt, dass die Herabregulation der Sprouty2-Expression axonales Längenwachstum in Kulturen von zentralen und peripheren Neuronen fördert. In dieser Dissertationsschrift wurden Mäuse mit globaler Sprouty2-Genabschaltung mit Fokus auf axonalen Wachstum in vitro und in vivo analysiert. Adulte, artifiziell kultivierte Neurone aus Sprouty2-defizienten sensorischen Ganglia zeigten eine stärkere Aktivierung der "Extracellular-Regulated Kinase" und vermehrtes axonales Wachstum. Wobei bei Neuronen mit heterozygotem Sprouty2+/--Genotyp vorwiegend axonales Längenwachstum auftrat, während sich bei Neuronen mit homozygotem Sprouty2-/--Genotyp mehr axonale Verästelungen zeigten. Nach artifizieller Ischiasnervquetschung, erholten sich Sprouty2+/- motorisch schneller, was sich jedoch nicht in sensorischen Testverfahren bestätigen ließ. (Sprouty2-/--Mäuse vertrugen die für die tierchirurgischen Prozeduren notwendige Anästhesie nicht.) Wir führen die verbesserte Leistung im "Rotarod"-Test auf eine höhere Anzahl von myelinisierten Fasern im regenerierenden Ischiasnerv, eine höhere Anzahl von motorischen Endplatten und erhöhte Mengen der mRNA von GAP-43, einem nachgeordneten Zielgen der "Extracellular-Regulated Kinase", zurück. Andererseits zeigten Sprouty2-/--Mäuse erhöhte mechano-sensorische Aktivität im Von Frey-Test, die von erhöhter Innervation der Epidermis, einer vermehrten Anzahl von unmyelinisierten Fasern und mehr IB4-positiven Neuronen im Spinalganglion begleitet ist. Die vorliegenden Resultate bestätigen die funktionale Bedeutung des Tyrosinkinaserezeptorensignalweges für axonales Wachstum während der biologischen Entwicklung und der Regeneration verletzter Nerven. Außerdem legen diese ErgebnisseSprouty proteins are negative feedback inhibitors of receptor tyrosine kinase signaling. Down-regulation of Sprouty2 has been demonstrated to promote elongative axon growth of cultured peripheral and central neurons. In this thesis, we analyzed Sprouty2 global knockout mice with respect to axon outgrowth in vitro and peripheral axon regeneration in vivo. Adult neurons dissociated from Sprouty2 deficient sensory ganglia reveal stronger ERK activation and enhanced axon outgrowth with prominent axon elongation of heterozygous Sprouty2+/- neurons, whereas homozygous Sprouty2-/- neurons exhibit an axonal branching phenotype. Following sciatic nerve crush, Sprouty2+/- mice recover faster in motor but not in sensory testing paradigms (Sprouty2-/- mice do not tolerate anesthesia required for nerve surgery). We attribute the improvement in the rotarod test to higher numbers of myelinated fibers in the regenerating sciatic nerve, higher densities of motor endplates in hind limb muscles and increased levels of GAP-43, a downstream target of extracellular regulated kinase signaling. On the other hand, homozygous Sprouty2-/- mice reveal enhanced mechanosensory function (von Frey test) that is accompanied by an increased innervation of the epidermis, elevated numbers of unmyelinated axons and more IB4-positive neurons in dorsal root ganglia. The present results corroborate the functional significance of receptor tyrosine kinase signaling inhibitors for axon outgrowth during development and nerve regeneration and propose Sprouty2 as a novel potential target for pharmacological inhibition to accelerate long-distance axon elongation in injured peripheral nerves.Letizia MarvaldiAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersInnsbruck, Med. Univ., Diss., 2014OeBB(VLID)13612

Inhibition of calpains fails to improve regeneration through a peripheral nerve conduit

Intramuscular injection of the calpain inhibitor leupeptin promotes peripheral nerve regeneration in primates (Badalamente et al., 1989 [13]), and direct positive effects of leupeptin on axon outgrowth were observed in vitro (Hausott et al., 2012 [12]). In this study, we applied leupeptin (2mg/ml) directly to collagen-filled nerve conduits in the rat sciatic nerve transection model. Analysis of myelinated axons and retrogradely labeled motoneurons as well as functional 'CatWalk' video analysis did not reveal significant differences between vehicle controls and leupeptin treated animals. Therefore, leupeptin does not improve nerve regeneration via protease inhibition in regrowing axons or in surrounding Schwann cells following a single application to a peripheral nerve conduit suggesting indirect effects on motor endplate integrity if applied systemically

Locally translated mTOR controls axonal local translation in nerve injury

How is protein synthesis initiated locally in neurons? We found that mTOR (mechanistic target of rapamycin) was activated and then up-regulated in injured axons, owing to local translation of mTOR messenger RNA (mRNA). This mRNA was transported into axons by the cell size-regulating RNA-binding protein nucleolin. Furthermore, mTOR controlled local translation in injured axons. This included regulation of its own translation and that of retrograde injury signaling molecules such as importin β1 and STAT3 (signal transducer and activator of transcription 3). Deletion of the mTOR 3' untranslated region (3'UTR) in mice reduced mTOR in axons and decreased local translation after nerve injury. Both pharmacological inhibition of mTOR in axons and deletion of the mTOR 3'UTR decreased proprioceptive neuronal survival after nerve injury. Thus, mRNA localization enables spatiotemporal control of mTOR pathways regulating local translation and long-range intracellular signaling