Spinal cord injury regulates circular RNA expression in axons.

Introduction: Neurons transport mRNA and translational machinery to axons for local translation. After spinal cord injury (SCI), de novo translation is assumed to enable neurorepair. Knowledge of the identity of axonal mRNAs that participate in neurorepair after SCI is limited. We sought to identify and understand how axonal RNAs play a role in axonal regeneration. Methods: We obtained preparations enriched in axonal mRNAs from control and SCI rats by digesting spinal cord tissue with cold-active protease (CAP). The digested samples were then centrifuged to obtain a supernatant that was used to identify mRNA expression. We identified differentially expressed genes (DEGS) after SCI and mapped them to various biological processes. We validated the DEGs by RT-qPCR and RNA-scope. Results: The supernatant fraction was highly enriched for mRNA from axons. Using Gene Ontology, the second most significant pathway for all DEGs was axonogenesis. Among the DEGs was Rims2, which is predominately a circular RNA (circRNA) in the CNS. We show that Rims2 RNA within spinal cord axons is circular. We found an additional 200 putative circRNAs in the axonal-enriched fraction. Knockdown in primary rat cortical neurons of the RNA editing enzyme ADAR1, which inhibits formation of circRNAs, significantly increased axonal outgrowth and increased the expression of circRims2. Using Rims2 as a prototype we used Circular RNA Interactome to predict miRNAs that bind to circRims2 also bind to the 3'UTR of GAP-43, PTEN or CREB1, all known regulators of axonal outgrowth. Axonally-translated GAP-43 supports axonal elongation and we detect GAP-43 mRNA in the rat axons by RNAscope. Discussion: By enriching for axonal RNA, we detect SCI induced DEGs, including circRNA such as Rims2. Ablation of ADAR1, the enzyme that regulates circRNA formation, promotes axonal outgrowth of cortical neurons. We developed a pathway model using Circular RNA Interactome that indicates that Rims2 through miRNAs can regulate the axonal translation GAP-43 to regulate axonal regeneration. We conclude that axonal regulatory pathways will play a role in neurorepair.

An anchor-tether 'hindered' HCN1 inhibitor is antihyperalgesic in a rat spared nerve injury neuropathic pain model.

BACKGROUND: Neuropathic pain impairs quality of life, is widely prevalent, and incurs significant costs. Current pharmacological therapies have poor/no efficacy and significant adverse effects; safe and effective alternatives are needed. Hyperpolarisation-activated cyclic nucleotide-regulated (HCN) channels are causally implicated in some forms of peripherally mediated neuropathic pain. Whilst 2,6-substituted phenols, such as 2,6-di-tert-butylphenol (26DTB-P), selectively inhibit HCN1 gating and are antihyperalgesic, the development of therapeutically tolerable, HCN-selective antihyperalgesics based on their inverse agonist activity requires that such drugs spare the cardiac isoforms and do not cross the blood-brain barrier. METHODS: In silico molecular dynamics simulation, in vitro electrophysiology, and in vivo rat spared nerve injury methods were used to test whether 'hindered' variants of 26DTB-P (wherein a hydrophilic 'anchor' is attached in the para-position of 26DTB-P via an acyl chain 'tether') had the desired properties. RESULTS: Molecular dynamics simulation showed that membrane penetration of hindered 26DTB-Ps is controlled by a tethered diol anchor without elimination of head group rotational freedom. In vitro and in vivo analysis showed that BP4L-18:1:1, a variant wherein a diol anchor is attached to 26DTB-P via an 18-carbon tether, is an HCN1 inverse agonist and an orally available antihyperalgesic. With a CNS multiparameter optimisation score of 2.25, a >100-fold lower drug load in the brain vs blood, and an absence of adverse cardiovascular or CNS effects, BP4L-18:1:1 was shown to be poorly CNS penetrant and cardiac sparing. CONCLUSIONS: These findings provide a proof-of-concept demonstration that anchor-tethered drugs are a new chemotype for treatment of disorders involving membrane targets.

Ribosomal S6 kinases determine intrinsic axonal regeneration capacity.

Why do adult mammalian central nervous system axons not regenerate, when peripheral axons do? Two studies in PLOS Biology point to the role of 2 related ribosomal S6 kinase family members in the differences in regeneration capacity between central and peripheral axons.

Nicotinamide Riboside Improves Enteric Neuropathy in Streptozocin-Induced Diabetic Rats Through Myenteric Plexus Neuroprotection.

BACKGROUND: Diabetes Mellitus causes a systemic oxidative stress due in part to the hyperglycemia and the reactive oxygen species generated. Up to 75% of diabetic patients present with an autonomic neuropathy affecting the Enteric Nervous System. Deficits in the human population are chronic dysmotilities with either increased (i.e., constipation) or decreased (i.e., diarrhea) total gastrointestinal transit times. These are recapitulated in the streptozocin-induced diabetic rat, which is a model of Type I Diabetes Mellitus. AIMS: Examine the effects that a precursor of nicotinamide adenosine dinucleotide (NAD), nicotinamide riboside (NR), had on the development of dysmotility in induced diabetic rats and if fecal microbiota transplant (FMT) could produce the same results. MATERIALS AND METHODS: Utilizing a 6-week treatment paradigm, NR was administered intraperitoneally every 48 h. Total gastrointestinal transit time was assessed weekly utilizing the carmine red method. Three weeks following hyperglycemic induction, FMT was performed between NR-treated animals and untreated animals. SIGNIFICANT RESULTS: There is improvement in overall gastrointestinal transit time with the use of NR. 16S microbiome sequencing demonstrated decreased alpha and beta diversity in induced diabetic rats without change in animals receiving FMT. Improvements in myenteric plexus ganglia density in small and large intestines in diabetic animals treated with NR were seen. CONCLUSIONS: NR treatment led to functional improvement in total gastrointestinal transit time in induced diabetic animals. This was associated with neuroprotection in the myenteric plexuses of both small and large intestines of induced diabetic rats. This represents an important first step in showing NR's benefit as a treatment for diabetic enteric neuropathy. Streptozocin-induced diabetic rats have improved transit times and increased myenteric plexus ganglia density when treated with intraperitoneal nicotinamide riboside.

Failure to Upregulate the RNA Binding Protein ZBP After Injury Leads to Impaired Regeneration in a Rodent Model of Diabetic Peripheral Neuropathy.

Most diabetes patients eventually suffer from peripheral nerve degeneration. Unfortunately, there is no treatment for the condition and its mechanisms are not well understood. There is, however, an emerging consensus that the inability of peripheral nerves to regenerate normally after injury contributes to the pathophysiology. We have previously shown that regeneration of peripheral axons requires local axonal translation of a pool of axonal mRNAs and that the levels and members of this axonal mRNA pool are altered in response to injury. Here, we show that following sciatic nerve injury in a streptozotocin rodent model of type I diabetes, this mobilization of RNAs into the injured axons is attenuated and correlates with decreased axonal regeneration. This failure of axonal RNA localization results from decreased levels of the RNA binding protein ZBP1. Over-expression of ZBP1 rescues the in vitro growth defect in injured dorsal root ganglion neurons from diabetic rodents. These results provide evidence that decreased neuronal responsiveness to injury in diabetes is due to a decreased ability to alter the pool of axonal mRNAs available for local translation, and may open new therapeutic opportunities for diabetic peripheral neuropathy.

Stressing out translation.

Integrated stress response activation drives neuropathy in Charcot-Marie-Tooth disease.

Myelin-associated glycoprotein inhibits neurite outgrowth through inactivation of the small GTPase Rap1.

Rap1 is a small GTPase that has been implicated in dendritic development and plasticity. In this study, we investigated the role of Rap1 in axonal growth and its activation in response to neurotrophins and myelin-associated inhibitors. We report that Rap1 is activated by brain-derived neurotrophic factor and that this activation can be blocked by myelin-associated glycoprotein (MAG) or central nervous system myelin, which also induced increases in Rap1GAP1 levels. In addition, we demonstrate that adenoviral overexpression of Rap1 enhances neurite outgrowth in the presence of MAG and myelin, while inhibition of Rap1 activity through overexpression of Rap1GAP1 blocks neurite outgrowth. These findings suggest that Rap1GAP1 negatively regulates neurite outgrowth, making it a potential therapeutic target to promote axonal regeneration.

Sialic Acid Is Required for Neuronal Inhibition by Soluble MAG but not for Membrane Bound MAG.

Myelin-Associated Glycoprotein (MAG), a major inhibitor of axonal growth, is a member of the immunoglobulin (Ig) super-family. Importantly, MAG (also known as Siglec-4) is a member of the Siglec family of proteins (sialic acid-binding, immunoglobulin-like lectins), MAG binds to complex gangliosides, specifically GD1a and/or GT1b. Therefore, it has been proposed as neuronal receptors for MAG inhibitory effect of axonal growth. Previously, we showed that MAG binds sialic acid through domain 1 at Arg118 and is able to inhibit axonal growth through domain 5. We developed a neurite outgrowth (NOG) assay, in which both wild type MAG and mutated MAG (MAG Arg118) are expressed on cells. In addition we also developed a soluble form NOG in which we utilized soluble MAG-Fc and mutated MAG (Arg118-Fc). Only MAG-Fc is able to inhibit NOG, but not mutated MAG (Arg118)-Fc that has been mutated at its sialic acid binding site. However, both forms of membrane bound MAG- and MAG (Arg118)- expressing cells still inhibit NOG. Here, we review various results from different groups regarding MAG's inhibition of axonal growth. Also, we propose a model in which the sialic acid binding is not necessary for the inhibition induced by the membrane form of MAG, but it is necessary for the soluble form of MAG. This finding highlights the importance of understanding the different mechanisms by which MAG inhibits NOG in both the soluble fragmented form and the membrane-bound form in myelin debris following CNS damage.

Cyclic AMP and Polyamines Overcome Inhibition by Myelin-Associated Glycoprotein through eIF5A-Mediated Increases in p35 Expression and Activation of Cdk5.

Inhibitory molecules associated with CNS myelin, such as myelin-associated glycoprotein (MAG), represent major obstacles to axonal regeneration following CNS injury. Our laboratory has shown that elevating levels of intracellular cAMP, via application of the nonhydrolyzable analog dibutyryl cAMP (dbcAMP), can block the inhibitory effects of MAG and myelin. We have also shown that elevation of cAMP results in upregulation of arginase I and increased polyamine synthesis. Treatment with putrescine or spermidine blocks myelin-mediated inhibition of neurite outgrowth, but the mechanism underlying this effect has not yet been elucidated. Here we show that cyclin-dependent kinase 5 (Cdk5) is required for dbcAMP and putrescine to overcome MAG-mediated inhibition. The ability of dbcAMP and putrescine to overcome inhibition by MAG is abolished in the presence of roscovitine, a Cdk inhibitor that has greater selectivity for Cdk5, and expression of dominant negative Cdk5 abolishes the ability of dbcAMP or putrescine to enhance neurite outgrowth in the presence of MAG. Importantly, dbcAMP and putrescine increase expression of p35, the neuron-specific activator of Cdk5, and rat DRG neurons transduced with HSV overexpressing p35 can overcome inhibition by MAG. The upregulation of p35 by putrescine is also reflected in increased localization of p35 to neurites and growth cones. Last, we show that putrescine upregulates p35 expression by serving as a substrate for hypusine modification of eIF5A, and that this hypusination is necessary for putrescine's ability to overcome inhibition by MAG. Our findings reveal a previously unknown mechanism by which polyamines may encourage regeneration after CNS injury.

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

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.

BDNF activates CaMKIV and PKA in parallel to block MAG-mediated inhibition of neurite outgrowth.

The environment of the adult CNS prevents axonal regeneration after injury. This inhibition of axonal regeneration can be blocked by elevating cAMP. Previously, we showed that the cAMP pathway can be activated via pre-treatment with neurotrophins and requires activation of several signaling pathways which converge at activation of the transcription factor, CREB. Here, we show that calcium/calmodulin-dependent kinase IV (CaMKIV) is necessary for the neurotrophin-induced phosphorylation of CREB and the block of myelin-mediated inhibition of axonal growth. Pharmacological inhibition of CaMKIV or over-expression of a dominant-negative mutant form of CaMKIV blocks the neurotrophin effect. Interestingly, CaMKIV activation is not necessary if cAMP levels is already elevated. Finally, calcium flux from intracellular stores is necessary for this CaMKIV signaling. These results demonstrate that CaMKIV is another player in the neurotrophin-induced signaling which leads to axonal regeneration and therefore, is a potential target for therapeutic intervention following injury to the adult CNS.

The inhibition site on myelin-associated glycoprotein is within Ig-domain 5 and is distinct from the sialic acid binding site.

Myelin-associated glycoprotein (MAG) is a potent inhibitor of axonal regeneration. It contains five Ig-like domains and is a sialic binding protein. Previously, we showed that the sialic acid binding site on MAG maps to arginine 118 in Ig domain 1 (Kelm et al., 1994). However, sialic acid binding was neither necessary nor sufficient for MAG to bring about inhibition of neurite outgrowth. Consistent with this, we now map a distinct inhibition site on MAG to Ig domain 5 (Ig-5). We show that when a truncated form of MAG missing Ig domains 1 and 2 is expressed by Chinese hamster ovary (CHO) cells, it does not bind sialic acid, but still inhibits neurite outgrowth almost as effectively as full-length MAG. To determine whether the inhibition site mapped to Ig-3, Ig-4, or Ig-5, we made chimeric molecules of various combinations of these three MAG Ig domains fused to Ig domains from another Ig family member, sialoadhesin (Sn), which also binds to sialic acid in the same linkage as MAG. The MAG-Sn molecules were expressed in CHO cells and all contained five Ig domains and were able to bind sialic acid. However, only the chimeric molecules containing MAG Ig-5 inhibited neurite outgrowth. Furthermore, peptides corresponding to sequences in MAG Ig-5, but not Ig-4 or Sn Ig-5, are able to block inhibition of neurite outgrowth by both wild-type MAG and CNS myelin. We conclude that the inhibition site on MAG is carried by Ig domain 5 and that this site is distinct from the sialic-acid binding site.

MAG induces regulated intramembrane proteolysis of the p75 neurotrophin receptor to inhibit neurite outgrowth.

The three known inhibitors of axonal regeneration present in myelin--MAG, Nogo, and OMgp--all interact with the same receptor complex to effect inhibition via protein kinase C (PKC)-dependent activation of the small GTPase Rho. The transducing component of this receptor complex is the p75 neurotrophin receptor. Here we show that MAG binding to cerebellar neurons induces alpha- and then gamma-secretase proteolytic cleavage of p75, in a protein kinase C-dependent manner, and that this cleavage is necessary for both activation of Rho and inhibition of neurite outgrowth.

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.

Neurotrophins elevate cAMP to reach a threshold required to overcome inhibition by MAG through extracellular signal-regulated kinase-dependent inhibition of phosphodiesterase.

Inhibitors of regeneration in myelin, such as myelin-associated glycoprotein (MAG), play an important role in preventing regeneration after CNS injury. Elevation of cAMP, either with dibutyryl-cAMP (db-cAMP) or by priming with a variety of neurotrophins, overcomes inhibition by MAG and myelin. However, activation of cAMP is not generally regarded as a signaling pathway for neurotrophins. Here we show that the NGF-like neurotrophins overcome inhibition by MAG by activating tyrosine kinase receptors. We also show that activation of extracellular signal-regulated kinase (Erk) by BDNF is required to overcome inhibition by MAG, and that activated Erk transiently inhibits phosphodiesterase 4 (PDE4), the enzyme that hydrolyzes cAMP. Inhibition of PDE4 then allows cAMP to increase and so initiates the pathway to overcome inhibition. Furthermore, we also show that basal levels of Erk activation and basal cAMP levels contribute to the effects of db-cAMP by pushing the combined levels of cAMP above a threshold required to overcome inhibition. Together, these results not only show how NGF-like neurotrophins can elevate cAMP and overcome inhibition but also point to a novel mechanism of cross talk in neurons from the Erk to the cAMP signaling pathways.

Arginase I and polyamines act downstream from cyclic AMP in overcoming inhibition of axonal growth MAG and myelin in vitro.

Elevation of cAMP can overcome myelin inhibitors to encourage regeneration of the CNS. We show that a consequence of elevated cAMP is the synthesis of polyamines, resulting from an up-regulation of Arginase I, a key enzyme in their synthesis. Inhibiting polyamine synthesis blocks the cAMP effect on regeneration. Either over-expression of Arginase I or exogenous polyamines can overcome inhibition by MAG and by myelin in general. While MAG/myelin support the growth of young DRG neurons, they become inhibitory as DRGs mature. Endogenous Arginase I levels are high in young DRGs but drop spontaneously at an age that coincides with the switch from promotion to inhibition by MAG/myelin. Over-expressing Arginase I in maturing DRGs blocks that switch. Arginase I and polyamines are more specific targets than cAMP for intervention to encourage regeneration after CNS injury.