Elena Nikulina

Myelin-associated glycoprotein interacts with the Nogo66 receptor to inhibit neurite outgrowth.

Myelin inhibitors of axonal regeneration, like Nogo and MAG, block regrowth after injury to the adult CNS. While a GPI-linked receptor for Nogo (NgR) has been identified, MAGs receptor is unknown. We show that MAG inhibits regeneration by interaction with NgR. Binding of and inhibition by MAG are lost if neuronal GPI-linked proteins are cleaved. Binding of MAG to NgR-expressing cells is GPI dependent and sialic acid independent. Conversely, NgR binds to MAG-expressing cells. MAG, but not a truncated MAG that binds neurons but does not inhibit regeneration, precipitates NgR from NgR-expressing cells, DRG, and cerebellar neurons. Importantly, NgR antibody, soluble NgR, or dominant-negative NgR each prevent inhibition of neurite outgrowth by MAG. Also, MAG and Nogo66 compete for binding to NgR. These results suggest redundancy in myelin inhibitors and indicate therapies for CNS injuries.

Activated CREB Is Sufficient to Overcome Inhibitors in Myelin and Promote Spinal Axon Regeneration In Vivo

Inhibitors in myelin play a major role in preventing spontaneous axonal regeneration after CNS injury. Elevation of cAMP overcomes this inhibition, in a transcription-dependent manner, through the upregulation of Arginase I (Arg I) and increased synthesis of polyamines. Here, we show that the cAMP effect requires activation of the transcription factor cAMP response element binding protein (CREB) to overcome myelin inhibitors; a dominant-negative CREB abolishes the effect, and neurons expressing a constitutively active form of CREB are not inhibited. Activation of CREB is also required for cAMP to upregulate Arg I, and the ability of constitutively active CREB to overcome inhibition is blocked by an inhibitor of polyamine synthesis. Finally, expression of constitutively active CREB in DRG neurons is sufficient to promote regeneration of subsequently lesioned dorsal column axons. These results indicate that CREB plays a central role in overcoming myelin inhibitors and so encourages regeneration in vivo.

Soluble myelin-associated glycoprotein released from damaged white matter inhibits axonal regeneration.

The adult, mammalian CNS does not regenerate after injury largely because of a glial scar and inhibitors of regeneration in myelin. To date, two myelin inhibitors, myelin-associated glycoprotein (MAG) and Nogo, both transmembrane proteins, have been identified. No secreted inhibitors of regeneration have been described. However, a proteolytic fragment of MAG (dMAG), consisting of the entire extracellular domain, is readily released from myelin and is found in vivo. Here, we show, first, that a soluble, chimeric form of MAG (MAG-Fc), when secreted from CHO cells in a collagen gel and hence in the absence of a fixed substrate, inhibits/deflects neurite outgrowth from P6 dorsal root ganglion (DRG) neurons. This inhibition was blocked when a MAG monoclonal antibody was included in the gel and a control chimera sialoadhesin-Fc (Sn-Fc), which, like MAG, binds neurons in a sialic acid-dependent manner but does not inhibit axonal growth, had no effect. Using the same assay system we showed that factors secreted from damaged white matter inhibited/deflected neurite outgrowth. This inhibition was neutralized when a MAG monoclonal antibody was included in the gel and there was no inhibition when white matter from a MAG knockout mouse was used. Factors secreted from damaged white matter from wild-type mice had no effect on neurite outgrowth from E18 DRG neurons. These results show that factors secreted from damaged white matter inhibit axonal regeneration and that the majority of inhibitory activity can be accounted for by dMAG. Thus, released dMAG is likely to play an important role in preventing regeneration, immediately after injury before the glial scar forms.

Rolipram promotes functional recovery after contusive thoracic spinal cord injury in rats.

Numerous animal model studies in the past decade have demonstrated that pharmacological elevation of cyclic AMP (cAMP) alone, or in combination with other treatments, can promote axonal regeneration after spinal cord injury. Elevation of cAMP via the phosphodiesterase 4 (PDE4) inhibitor, rolipram, decreases neuronal sensitivity to myelin inhibitors, increases growth potential and is neuroprotective. Roliprams ability to cross the blood-brain barrier makes it a practical and promising treatment for CNS regeneration. However, several studies have questioned the efficacy of rolipram when given alone. The purpose of this investigation was to determine the effects of continuous administration of rolipram, given alone for 2 weeks, following a moderate T10 contusion injury in rat. Functional recovery was evaluated using the 21-point Basso, Beattie and Bresnahan (BBB) locomotor recovery scale and the beam walk. We used three-dimensional (3D) instrumented gait analysis to allow detailed assessment and quantification of hindlimb motion. The amount of the damaged tissue and spared white matter was estimated stereologically. Our results show that administration of rolipram following acute spinal cord contusion results in improved motor performance at each time-point. Dynamic assessment of foot motion during treadmill walking revealed a significantly decreased external rotation during the entire step cycle after 8 weeks in rolipram-treated animals. Stereological analysis revealed no significant differences in lesion volume and length. By contrast, spared white matter was significantly higher in the group treated with rolipram. Our results suggest a therapeutic role for rolipram delivered alone following acute SCI.

Soluble Adenylyl Cyclase Is Necessary and Sufficient to Overcome the Block of Axonal Growth by Myelin-Associated Factors

Neurons in the CNS do not regenerate following injury; regeneration is blocked by inhibitory proteins in myelin, such as myelin-associated glycoprotein (MAG). Elevating neuronal levels of the second messenger cAMP overcomes this blocked axonal outgrowth. One way to elevate cAMP is pretreating neurons with neurotrophins, such as brain-derived neurotrophic factor (BDNF). However, pleiotropic effects and poor bioavailability make exogenous administration of neurotrophins in vivo problematic; therefore, alternative targets must be considered. In neurons, two families of adenylyl cyclases synthesize cAMP, transmembrane adenylyl cyclases (tmACs), and soluble adenylyl cyclase (sAC). Here, we demonstrate that sAC is the essential source of cAMP for BDNF to overcome MAG-dependent inhibition of neurite outgrowth. Elevating sAC in rat and mouse neurons is sufficient to induce neurite outgrowth on myelin in vitro and promotes regeneration in vivo. These results suggest that stimulators of sAC might represent a novel therapeutic strategy to promote axonal growth and regeneration.

Metallothionein-I/II Promotes Axonal Regeneration in the Central Nervous System.

Background: MT-I/II are zinc-binding proteins that are also neuro-protective. Results: MT-I/II can overcome myelin-mediated inhibition in vitro and in vivo. MT-I/II-deficient mice have reduced spinal axon regeneration. Conclusion: MT-I/II are required for the conditioning lesion effect and can promote axonal regeneration in the injured CNS. Significance: MT-I/II have therapeutic potential for the treatment of spinal cord injury. The adult CNS does not spontaneously regenerate after injury, due in large part to myelin-associated inhibitors such as myelin-associated glycoprotein (MAG), Nogo-A, and oligodendrocyte-myelin glycoprotein. All three inhibitors can interact with either the Nogo receptor complex or paired immunoglobulin-like receptor B. A conditioning lesion of the sciatic nerve allows the central processes of dorsal root ganglion (DRG) neurons to spontaneously regenerate in vivo after a dorsal column lesion. After a conditioning lesion, DRG neurons are no longer inhibited by myelin, and this effect is cyclic AMP (cAMP)- and transcription-dependent. Using a microarray analysis, we identified several genes that are up-regulated both in adult DRGs after a conditioning lesion and in DRG neurons treated with cAMP analogues. One gene that was up-regulated under both conditions is metallothionein (MT)-I. We show here that treatment with two closely related isoforms of MT (MT-I/II) can overcome the inhibitory effects of both myelin and MAG for cortical, hippocampal, and DRG neurons. Intrathecal delivery of MT-I/II to adult DRGs also promotes neurite outgrowth in the presence of MAG. Adult DRGs from MT-I/II-deficient mice extend significantly shorter processes on MAG compared with wild-type DRG neurons, and regeneration of dorsal column axons does not occur after a conditioning lesion in MT-I/II-deficient mice. Furthermore, a single intravitreal injection of MT-I/II after optic nerve crush promotes axonal regeneration. Mechanistically, MT-I/II ability to overcome MAG-mediated inhibition is transcription-dependent, and MT-I/II can block the proteolytic activity of α-secretase and the activation of PKC and Rho in response to soluble MAG.

Ancient DNA reveals the Arctic origin of Viking Age cod from Haithabu, Germany

Significance A rich archaeological record of fish-bone remains testifies to the millennia-long human exploitation of the natural resources of the oceans. In Europe, historical evidence demonstrates that an extensive international industry developed during the Middle Ages that exported preserved cod from the Lofoten Archipelago, northern Norway, to expanding urban centers around the North and Baltic Sea regions. The early origins of this iconic exchange, however, have long been debated. We genetically trace the ancestry of Viking Age fish from mainland Europe to the North East Arctic cod population that supports the modern Lofoten fisheries. This application of genome-wide analyses from ancient fish bone reveals an early origin of what became an economically important trade, with implications for archaeology and environmental history. Knowledge of the range and chronology of historic trade and long-distance transport of natural resources is essential for determining the impacts of past human activities on marine environments. However, the specific biological sources of imported fauna are often difficult to identify, in particular if species have a wide spatial distribution and lack clear osteological or isotopic differentiation between populations. Here, we report that ancient fish-bone remains, despite being porous, brittle, and light, provide an excellent source of endogenous DNA (15–46%) of sufficient quality for whole-genome reconstruction. By comparing ancient sequence data to that of modern specimens, we determine the biological origin of 15 Viking Age (800–1066 CE) and subsequent medieval (1066–1280 CE) Atlantic cod (Gadus morhua) specimens from excavation sites in Germany, Norway, and the United Kingdom. Archaeological context indicates that one of these sites was a fishing settlement for the procurement of local catches, whereas the other localities were centers of trade. Fish from the trade sites show a mixed ancestry and are statistically differentiated from local fish populations. Moreover, Viking Age samples from Haithabu, Germany, are traced back to the North East Arctic Atlantic cod population that has supported the Lofoten fisheries of Norway for centuries. Our results resolve a long-standing controversial hypothesis and indicate that the marine resources of the North Atlantic Ocean were used to sustain an international demand for protein as far back as the Viking Age.

Activated Protein C blocks the inhibitory effect on neurite outgrowth by extracellular histones that mediates its inhibition through a retrograde YB-1 signal

Axonal regeneration in the mature CNS is limited by inhibitory factors within the extracellular environment. In this study, we show that histones H3 and H4 inhibit neurite outgrowth when applied to cortical neurons in vitro, and that this effect can be reversed by the addition of activated protein C (APC). Elevated levels of histones H3 and H4 were also detected in cerebrospinal fluid following CNS injury, and treatment with APC significantly increased axonal regeneration. Mechanistically, we have determined that histones activate a retrograde signaling cascade that results in phosphorylation of the transcription factor YB-1, and that neurite outgrowth is impaired when YB-1 is overexpressed in cortical neurons. These findings identify extracellular histones as a new class of growth-inhibiting molecules within the injured CNS.Axonal regeneration in the mature CNS is limited by extracellular inhibitory factors. Triple knockout mice lacking the major myelin-associated inhibitors do not display spontaneous regeneration after injury, indicating the presence of other inhibitors. Searching for such inhibitors we have detected elevated levels of histone H3 in human cerebrospinal fluid (CSF) 24 hours after spinal cord injury. Following dorsal column lesions in mice and optic nerve crushes in rats, elevated levels of extracellular histone H3 were detected at the injury site. Similar to myelin-associated inhibitors, these extracellular histones induced growth cone collapse and inhibited neurite outgrowth. Histones mediate inhibition through the transcription factor YB-1 and Toll-like receptor 2, and these effects are independent of the Nogo receptor. Histone-mediated inhibition can be reversed by the addition of activated protein C (APC) in vitro, and APC treatment promotes axonal regeneration in the crushed optic nerve in vivo. These findings identify extracellular histones as a new class of nerve regeneration-inhibiting molecules within the injured CNS. One sentence summary Proteins typically associated with chromatin structure play an unexpected role in limiting axonal regeneration after injury.

Making motor axons grow

Centre for Neuroscience and Florey Neuroscience Institutes, The University of Melbourne, Australia Within the vertebrate central nervous system (CNS) the efficiency and speed of action potentials relies on myelin, a specialized membrane structure formed by oligodendrocytes. Proper establishment and maintenance of myelin is essential for the functioning of the CNS during both development and adult life, as evidenced by diseases such as the leukodystrophies and Multiple Sclerosis. In addition, ongoing myelination in the adult is thought to contribute to neural plasticity and learning. The regulation of myelination is thus a vital feature of both developmental and adult CNS processes. Recently, a number of regulatory mechanisms controlling myelination have been identified. At the extracellular level, these include a series of ligands expressed by neurons and astrocytes as well as neuronal activity itself. Within the oligodendrocyte lineage, recent advances have identified transcriptional and epigenetic pathways that are vital for the generation of myelinating cells. A significant current challenge to the field is to understand how these different levels of regulation interact with and influence each other. We have recently identified a previously uncharacterized transcription factor, Myelin Gene Regulatory Factor (MRF), which is specifically expressed within the brain by myelinating oligodendrocytes. MRF is vital for the process of CNS myelination during development; conditional knockout mice lacking MRF within the oligodendrocyte lineage generate oligodendrocyte progenitors and pre-myelinating oligodendrocytes however these oligodendrocytes are unable to express myelin genes or myelinate adjacent axons. Conversely, forced expression of MRF causes precocious expression of myelin proteins. We have demonstrated via ChIPSeq experiments that MRF acts directly at promoter and enhancer regions of genes encoding the protein components of myelin, as well as cytoskeletal, lipid metabolism and junctional proteins that underpin the myelination process. These findings establish MRF as a central point in the molecular events regulating myelination from which we may be able to understand how the other levels of regulation converge and interact.