Injury-induced HDAC5 nuclear export is essential for axon regeneration.

Reactivation of a silent transcriptional program is a critical step in successful axon regeneration following injury. Yet how such a program is unlocked after injury remains largely unexplored. We found that axon injury in peripheral sensory neurons elicits a back-propagating calcium wave that invades the soma and causes nuclear export of HDAC5 in a PKCµ-dependent manner. Injury-induced HDAC5 nuclear export enhances histone acetylation to activate a proregenerative gene-expression program. HDAC5 nuclear export is required for axon regeneration, as expression of a nuclear-trapped HDAC5 mutant prevents axon regeneration, whereas enhancing HDAC5 nuclear export promotes axon regeneration in vitro and in vivo. Components of this HDAC5 pathway failed to be activated in a model of central nervous system injury. These studies reveal a signaling mechanism from the axon injury site to the soma that controls neuronal growth competence and suggest a role for HDAC5 as a transcriptional switch controlling axon regeneration.

FK506-binding protein-like and FK506-binding protein 8 regulate dual leucine zipper kinase degradation and neuronal responses to axon injury.

The dual leucine zipper kinase (DLK) is a key regulator of axon regeneration and degeneration in response to neuronal injury; however, regulatory mechanisms of the DLK function via its interacting proteins are largely unknown. To better understand the molecular mechanism of DLK function, we performed yeast two-hybrid screening analysis and identified FK506-binding protein-like (FKBPL, also known as WAF-1/CIP1 stabilizing protein 39) as a DLK-binding protein. FKBPL binds to the kinase domain of DLK and inhibits its kinase activity. In addition, FKBPL induces DLK protein degradation through ubiquitin-dependent pathways. We further assessed other members in the FKBP protein family and found that FK506-binding protein 8 (FKBP8) also induced DLK degradation. We identified the lysine 271 residue in the kinase domain as a major site of DLK ubiquitination and SUMO3 conjugation and was thus responsible for regulating FKBP8-mediated proteasomal degradation that was inhibited by the substitution of the lysine 271 to arginine. FKBP8-mediated degradation of DLK is mediated by autophagy pathway because knockdown of Atg5 inhibited DLK destabilization. We show that in vivo overexpression of FKBP8 delayed the progression of axon degeneration and suppressed neuronal death after axotomy in sciatic and optic nerves. Taken together, this study identified FKBPL and FKBP8 as novel DLK-interacting proteins that regulate DLK stability via the ubiquitin-proteasome and lysosomal protein degradation pathways.

The stem cell marker Prom1 promotes axon regeneration by down-regulating cholesterol synthesis via Smad signaling.

Axon regeneration is regulated by a neuron-intrinsic transcriptional program that is suppressed during development but that can be reactivated following peripheral nerve injury. Here we identify Prom1, which encodes the stem cell marker prominin-1, as a regulator of the axon regeneration program. Prom1 expression is developmentally down-regulated, and the genetic deletion of Prom1 in mice inhibits axon regeneration in dorsal root ganglion (DRG) cultures and in the sciatic nerve, revealing the neuronal role of Prom1 in injury-induced regeneration. Elevating prominin-1 levels in cultured DRG neurons or in mice via adeno-associated virus-mediated gene delivery enhances axon regeneration in vitro and in vivo, allowing outgrowth on an inhibitory substrate. Prom1 overexpression induces the consistent down-regulation of cholesterol metabolism-associated genes and a reduction in cellular cholesterol levels in a Smad pathway-dependent manner, which promotes axonal regrowth. We find that prominin-1 interacts with the type I TGF-ß receptor ALK4, and that they synergistically induce phosphorylation of Smad2. These results suggest that Prom1 and cholesterol metabolism pathways are possible therapeutic targets for the promotion of neural recovery after injury.

ßPix Guanine Nucleotide Exchange Factor Regulates Regeneration of Injured Peripheral Axons.

Axon regeneration is essential for successful recovery after peripheral nerve injury. Although growth cone reformation and axonal extension are crucial steps in axonal regeneration, the regulatory mechanisms underlying these dynamic processes are poorly understood. Here, we identify ßPix (Arhgef7), the guanine nucleotide exchange factor for Rac1 GTPase, as a regulator of axonal regeneration. After sciatic nerve injury in mice, the expression levels of ßPix increase significantly in nerve segments containing regenerating axons. In regrowing axons, ßPix is localized in the peripheral domain of the growth cone. Using ßPix neuronal isoform knockout (NIKO) mice in which the neuronal isoforms of ßPix are specifically removed, we demonstrate that ßPix promotes neurite outgrowth in cultured dorsal root ganglion neurons and in vivo axon regeneration after sciatic nerve crush injury. Activation of cJun and STAT3 in the cell bodies is not affected in ßPix NIKO mice, supporting the local action of ßPix in regenerating axons. Finally, inhibiting Src, a kinase previously identified as an activator of the ßPix neuronal isoform, causes axon outgrowth defects in vitro, like those found in the ßPix NIKO neurons. Altogether, these data indicate that ßPix plays an important role in axonal regrowth during peripheral nerve regeneration.

A JUN N-terminal kinase inhibitor induces ectodomain shedding of the cancer-associated membrane protease Prss14/epithin via protein kinase CßII.

Serine protease 14 (Prss14)/epithin is a transmembrane serine protease that plays essential roles in tumor progression and metastasis and therefore is a promising target for managing cancer. Prss14/epithin shedding may underlie its activity in cancer and worsen outcomes; accordingly, a detailed understanding of the molecular mechanisms in Prss14/epithin shedding may inform the design of future cancer therapies. On the basis of our previous observation that an activator of PKC, phorbol 12-myristate 13-acetate (PMA), induces Prss14/epithin shedding, here we further investigated the intracellular signaling pathway involved in this process. While using mitogen-activated protein kinase inhibitors to investigate possible effectors of downstream PKC signaling, we unexpectedly found that an inhibitor of c-Jun N-terminal kinase (JNK), SP600125, induces Prss14/epithin shedding even in the absence of PMA. SP600125-induced shedding, like that stimulated by PMA, was mediated by tumor necrosis factor-α-converting enzyme. In contrast, a JNK activator, anisomycin, partially abolished the effects of SP600125 on Prss14/epithin shedding. Moreover, the results from loss-of-function experiments with specific inhibitors, short hairpin RNA-mediated knockdown, and overexpression of dominant-negative PKCßII variants indicated that PKCßII is a major player in JNK inhibition- and PMA-mediated Prss14/epithin shedding. SP600125 increased phosphorylation of PKCßII and tumor necrosis factor-α-converting enzyme and induced their translocation into the plasma membrane. Finally, in vitro cell invasion experiments and bioinformatics analysis of data in The Cancer Genome Atlas breast cancer database revealed that JNK and PKCßII are important for Prss14/epithin-mediated cancer progression. These results provide important information regarding strategies against tumor metastasis.

Intramembrane proteolysis of an extracellular serine protease, epithin/PRSS14, enables its intracellular nuclear function.

BACKGROUND: Epithin/PRSS14, a type II transmembrane serine protease, is an emerging target of cancer therapy because of its critical roles in tumor progression and metastasis. In many circumstances, the protease, through its ectodomain shedding, exists as a soluble form and performs its proteolytic functions in extracellular environments increasing cellular invasiveness. The seemingly functional integrity of the soluble form raises the question of why the protease is initially made as a membrane-associated protein. RESULTS: In this report, we show that the epithin/PRSS14 intracellular domain (EICD) can be released from the membrane by the action of signal peptide peptidase-like 2b (SPPL2b) after ectodomain shedding. The EICD preferentially localizes in the nucleus and can enhance migration, invasion, and metastasis of epithelial cancer when heterologously expressed. Unbiased RNA-seq analysis and subsequent antibody arrays showed that EICD could control the gene expression of chemokines involved in cell motility, by increasing their promoter activities. Finally, bioinformatics analysis provided evidence for the clinical significance of the intramembrane proteolysis of epithin/PRSS14 by revealing that the poor survival of estrogen receptor (ER)-negative breast cancer patients with high epithin/PRSS14 expression is further worsened by high levels of SPPL2b. CONCLUSIONS: These results show that ectodomain shedding of epithin/PRSS14 can initiate a unique and synchronized bidirectional signal for cancer metastasis: extracellularly broadening proteolytic modification of the surrounding environment and intracellularly reprogramming the transcriptome for metastatic conversion. Clinically, this study also suggests that the intracellular function of epithin/PRSS14 should be considered for targeting this protease for anti-cancer treatment.

Experimental Model Systems for Understanding Human Axonal Injury Responses.

Neurons are structurally unique and have dendrites and axons that are vulnerable to injury. Some neurons in the peripheral nervous system (PNS) can regenerate their axons after injuries. However, most neurons in the central nervous system (CNS) fail to do so, resulting in irreversible neurological disorders. To understand the mechanisms of axon regeneration, various experimental models have been utilized in vivo and in vitro. Here, we collate the key experimental models that revealed the important mechanisms regulating axon regeneration and degeneration in different systems. We also discuss the advantages of experimenting with the rodent model, considering the application of these findings in understanding human diseases and for developing therapeutic methods.

DLK regulates a distinctive transcriptional regeneration program after peripheral nerve injury.

Following damage to a peripheral nerve, injury signaling pathways converge in the cell body to generate transcriptional changes that support axon regeneration. Here, we demonstrate that dual leucine zipper kinase (DLK), a central regulator of injury responses including axon regeneration and neuronal apoptosis, is required for the induction of the pro-regenerative transcriptional program in response to peripheral nerve injury. Using a sensory neuron-conditional DLK knockout mouse model, we show a time course for the dependency of gene expression changes on the DLK pathway after sciatic nerve injury. Gene ontology analysis reveals that DLK-dependent gene sets are enriched for specific functional annotations such as ion transport and immune response. A series of comparative analyses shows that the DLK-dependent transcriptional program is distinct from that promoted by the importin-dependent retrograde signaling pathway, while it is partially shared between PNS and CNS injury responses. We suggest that DLK-dependency might provide a selective filter for regeneration-associated genes among the injury-responsive transcriptome.

Independent role for presynaptic FMRP revealed by an FMR1 missense mutation associated with intellectual disability and seizures.

Fragile X syndrome (FXS) results in intellectual disability (ID) most often caused by silencing of the fragile X mental retardation 1 (FMR1) gene. The resulting absence of fragile X mental retardation protein 1 (FMRP) leads to both pre- and postsynaptic defects, yet whether the pre- and postsynaptic functions of FMRP are independent and have distinct roles in FXS neuropathology remain poorly understood. Here, we demonstrate an independent presynaptic function for FMRP through the study of an ID patient with an FMR1 missense mutation. This mutation, c.413G > A (R138Q), preserves FMRP's canonical functions in RNA binding and translational regulation, which are traditionally associated with postsynaptic compartments. However, neuronally driven expression of the mutant FMRP is unable to rescue structural defects at the neuromuscular junction in fragile x mental retardation 1 (dfmr1)-deficient Drosophila, suggesting a presynaptic-specific impairment. Furthermore, mutant FMRP loses the ability to rescue presynaptic action potential (AP) broadening in Fmr1 KO mice. The R138Q mutation also disrupts FMRP's interaction with the large-conductance calcium-activated potassium (BK) channels that modulate AP width. These results reveal a presynaptic- and translation-independent function of FMRP that is linked to a specific subset of FXS phenotypes.

Disruption of TACE-filamin interaction can inhibit TACE-mediated ectodomain shedding.

Ectodomain shedding regulates functions of many membrane proteins through the cleavage of their juxtamembrane region mainly by a disintegrin and metalloproteinase family proteinases. Tumor necrosis factor-alpha converting enzyme (TACE) is known to be responsible for phorbol myristate acetate (PMA)-induced shedding of various membrane proteins. How PMA regulates TACE-dependent shedding and how TACE exhibits substrate specificity without proteolysis of other membrane proteins are questionable. Here, we show that TACE can interact with an actin-binding protein, filamin, through 20th filamin repeat. We found that the interaction between TACE and filamin was increased by PMA treatment. In addition, loss of filamin or specific disruption of TACE-filamin interaction inhibited ectodomain shedding of representative TACE substrates, CD44 and amyloid protein precursor. From these data, we suggest that filamin may work as a scaffold that can recruit TACE and its substrates in a PMA-dependent manner to achieve substrate specificity for TACE.

Filamin A is required in injured axons for HDAC5 activity and axon regeneration.

Microtubule dynamics are important for axon growth during development as well as axon regeneration after injury. We have previously identified HDAC5 as an injury-regulated tubulin deacetylase that functions at the injury site to promote axon regeneration. However, the mechanisms involved in the spatial control of HDAC5 activity remain poorly understood. Here we reveal that HDAC5 interacts with the actin binding protein filamin A via its C-terminal domain. Filamin A plays critical roles in HDAC5-dependent tubulin deacetylation because, in cells lacking filamin A, the levels of acetylated tubulin are elevated markedly. We found that nerve injury increases filamin A axonal expression in a protein synthesis-dependent manner. Reducing filamin A levels or interfering with the interaction between HDAC5 and filamin A prevents injury-induced tubulin deacetylation as well as HDAC5 localization at the injured axon tips. In addition, neurons lacking filamin A display reduced axon regeneration. Our findings suggest a model in which filamin A local translation following axon injury controls localized HDAC5 activity to promote axon regeneration.

Tubulin-tyrosine Ligase (TTL)-mediated Increase in Tyrosinated α-Tubulin in Injured Axons Is Required for Retrograde Injury Signaling and Axon Regeneration.

Injured peripheral neurons successfully activate a pro-regenerative program to enable axon regeneration and functional recovery. The microtubule-dependent retrograde transport of injury signals from the lesion site in the axon back to the cell soma stimulates the increased growth capacity of injured neurons. However, the mechanisms initiating this retrograde transport remain poorly understood. Here we show that tubulin-tyrosine ligase (TTL) is required to increase the levels of tyrosinated α-tubulin at the axon injury site and plays an important role in injury signaling. Preventing the injury-induced increase in tyrosinated α-tubulin by knocking down TTL impairs retrograde organelle transport and delays activation of the pro-regenerative transcription factor c-Jun. In the absence of TTL, axon regeneration is reduced severely. We propose a model in which TTL increases the levels of tyrosinated α-tubulin locally at the injury site to facilitate the retrograde transport of injury signals that are required to activate a pro-regenerative program.

HDAC5 is a novel injury-regulated tubulin deacetylase controlling axon regeneration.

Axon regeneration is an essential process to rebuild functional connections between injured neurons and their targets. Regenerative axonal growth requires alterations in axonal microtubule dynamics, but the signalling mechanisms involved remain incompletely understood. Our results reveal that axon injury induces a gradient of tubulin deacetylation, which is required for axon regeneration both in vitro and in vivo. This injury-induced tubulin deacetylation is specific to peripheral neurons and fails to occur in central neurons. We found that tubulin deacetylation is initiated by calcium influx at the site of injury, and requires protein kinase C-mediated activation of the histone deacetylase 5 (HDAC5). Our findings identify HDAC5 as a novel injury-regulated tubulin deacetylase that plays an essential role in growth cone dynamics and axon regeneration. In addition, our results suggest a mechanism for the spatial control of tubulin modifications that is required for axon regeneration.

Syntaxin13 expression is regulated by mammalian target of rapamycin (mTOR) in injured neurons to promote axon regeneration.

Injured peripheral neurons successfully activate intrinsic signaling pathways to enable axon regeneration. We have previously shown that dorsal root ganglia (DRG) neurons activate the mammalian target of rapamycin (mTOR) pathway following injury and that this activity enhances their axon growth capacity. mTOR plays a critical role in protein synthesis, but the mTOR-dependent proteins enhancing the regenerative capacity of DRG neurons remain unknown. To identify proteins whose expression is regulated by injury in an mTOR-dependent manner, we analyzed the protein composition of DRGs from mice in which we genetically activated mTOR and from mice with or without a prior nerve injury. Quantitative label-free mass spectrometry analyses revealed that the injury effects were correlated with mTOR activation. We identified a member of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family of proteins, syntaxin13, whose expression was increased by injury in an mTOR-dependent manner. Increased syntaxin13 levels in injured nerves resulted from local protein synthesis and not axonal transport. Finally, knockdown of syntaxin13 in cultured DRG neurons prevented axon growth and regeneration. Together, these data suggest that syntaxin13 translation is regulated by mTOR in injured neurons to promote axon regeneration.

SCG10 is a JNK target in the axonal degeneration pathway.

Axons actively self-destruct following genetic, mechanical, metabolic, and toxic insults, but the mechanism of axonal degeneration is poorly understood. The JNK pathway promotes axonal degeneration shortly after axonal injury, hours before irreversible axon fragmentation ensues. Inhibition of JNK activity during this period delays axonal degeneration, but critical JNK substrates that facilitate axon degeneration are unknown. Here we show that superior cervical ganglion 10 (SCG10), an axonal JNK substrate, is lost rapidly from mouse dorsal root ganglion axons following axotomy. SCG10 loss precedes axon fragmentation and occurs selectively in the axon segments distal to transection that are destined to degenerate. Rapid SCG10 loss after injury requires JNK activity. The JNK phosphorylation sites on SCG10 are required for its rapid degradation, suggesting that direct JNK phosphorylation targets SCG10 for degradation. We present a mechanism for the selective loss of SCG10 distal to the injury site. In healthy axons, SCG10 undergoes rapid JNK-dependent degradation and is replenished by fast axonal transport. Injury blocks axonal transport and the delivery of SCG10, leading to the selective loss of the labile SCG10 distal to the injury site. SCG10 loss is functionally important: Knocking down SCG10 accelerates axon fragmentation, whereas experimentally maintaining SCG10 after injury promotes mitochondrial movement and delays axonal degeneration. Taken together, these data support the model that SCG10 is an axonal-maintenance factor whose loss is permissive for execution of the injury-induced axonal degeneration program.

Comparison of overlapping (OP) and adjacent thumb positions (AP) for cardiac compressions using the encircling method in infants.

OBJECTIVES: The aim of this manikin study was to compare the efficiency between overlapping (OP) and adjacent thumb positions (AP) for cardiac compressions using the encircling method in infants. METHODS: The study conducted from December 2010 to August 2011 involved 48 volunteers who were students in the emergency medical technician course. The authors let volunteers practice OP and AP as a crossover design. The authors monitored the simulated mean arterial pressure (MAP) generated during a 5-min chest compression. The fatigue level of the volunteers after the chest compression was evaluated with the Likert scale. RESULTS: There were no significant differences in MAP between the dominant hand and the non-dominant hand as the lower thumb of OP. Significant differences were observed in simulated systolic blood pressure, MAP and simulated pulse pressure between OP and AP at 1, 2, 3, 4 and 5 min. There were no significant differences among the changes in heart rate, respiratory rate and end-tidal CO(2) during a 5-min chest compression by OP and AP. The Likert scale scores (1 no fatigue to 5 = extreme fatigue) during the 5-min chest compressions were higher in AP than in OP at 2, 3 and 5 min. CONCLUSION: Higher intrathoracic pressures were achieved by OP in this study. However, further studies are needed to validate these effects of overlapping thumbs technique in infant cardiopulmonary resuscitation, not manikin.

Precise base editing without unintended indels in human cells and mouse primary myoblasts.

Base editors are powerful tools for making precise single-nucleotide changes in the genome. However, they can lead to unintended insertions and deletions at the target sites, which is a significant limitation for clinical applications. In this study, we aimed to eliminate unwanted indels at the target sites caused by various evolved base editors. Accordingly, we applied dead Cas9 instead of nickase Cas9 in the base editors to induce accurate substitutions without indels. Additionally, we tested the use of chromatin-modulating peptides in the base editors to improve nucleotide conversion efficiency. We found that using both dead Cas9 and chromatin-modulating peptides in base editing improved the nucleotide substitution efficiency without unintended indel mutations at the desired target sites in human cell lines and mouse primary myoblasts. Furthermore, the proposed scheme had fewer off-target effects than conventional base editors at the DNA level. These results indicate that the suggested approach is promising for the development of more accurate and safer base editing techniques for use in clinical applications.

Potential roles of stem cell marker genes in axon regeneration.

Axon regeneration is orchestrated by many genes that are differentially expressed in response to injury. Through a comparative analysis of gene expression profiling, injury-responsive genes that are potential targets for understanding the mechanisms underlying regeneration have been revealed. As the efficiency of axon regeneration in both the peripheral and central nervous systems can be manipulated, we suggest that identifying regeneration-associated genes is a promising approach for developing therapeutic applications in vivo. Here, we review the possible roles of stem cell marker- or stemness-related genes in axon regeneration to gain a better understanding of the regeneration mechanism and to identify targets that can enhance regenerative capacity.

Comparative gene expression profiling reveals the mechanisms of axon regeneration.

Axons are vulnerable to injury, potentially leading to degeneration or neuronal death. While neurons in the central nervous system fail to regenerate, neurons in the peripheral nervous system are known to regenerate. Since it has been shown that injury-response signal transduction is mediated by gene expression changes, expression profiling is a useful tool to understand the molecular mechanisms of regeneration. Axon regeneration is regulated by injury-responsive genes induced in both neurons and their surrounding non-neuronal cells. Thus, an experimental setup for the comparative analysis between regenerative and nonregenerative conditions is essential to identify ideal targets for the promotion of regeneration-associated genes and to understand the mechanisms of axon regeneration. Here, we review the original research that shows the key factors regulating axon regeneration, in particular by using comparative gene expression profiling in diverse systems.