Targeted protein degradation systems to enhance Wnt signaling.

Molecules that facilitate targeted protein degradation (TPD) offer great promise as novel therapeutics. The human hepatic lectin asialoglycoprotein receptor (ASGR) is selectively expressed on hepatocytes. We have previously engineered an anti-ASGR1 antibody-mutant RSPO2 (RSPO2RA) fusion protein (called SWEETS) to drive tissue-specific degradation of ZNRF3/RNF43 E3 ubiquitin ligases, which achieved hepatocyte-specific enhanced Wnt signaling, proliferation, and restored liver function in mouse models, and an antibody-RSPO2RA fusion molecule is currently in human clinical trials. In the current study, we identified two new ASGR1- and ASGR1/2-specific antibodies, 8M24 and 8G8. High-resolution crystal structures of ASGR1:8M24 and ASGR2:8G8 complexes revealed that these antibodies bind to distinct epitopes on opposing sides of ASGR, away from the substrate-binding site. Both antibodies enhanced Wnt activity when assembled as SWEETS molecules with RSPO2RA through specific effects sequestering E3 ligases. In addition, 8M24-RSPO2RA and 8G8-RSPO2RA efficiently downregulate ASGR1 through TPD mechanisms. These results demonstrate the possibility of combining different therapeutic effects and degradation mechanisms in a single molecule.

An Unbiased View of Neural Networks: More than Meets the Eye.

In this issue of Neuron, Macé et al. (2018) use whole-brain functional ultrasound imaging in mice to unveil the circuits involved reflexive eye movements. They separated the sensory and motor networks and discovered that certain eye movements robustly suppress the amygdala.

The Ancient Origins of Neural Substrates for Land Walking.

Walking is the predominant locomotor behavior expressed by land-dwelling vertebrates, but it is unknown when the neural circuits that are essential for limb control first appeared. Certain fish species display walking-like behaviors, raising the possibility that the underlying circuitry originated in primitive marine vertebrates. We show that the neural substrates of bipedalism are present in the little skate Leucoraja erinacea, whose common ancestor with tetrapods existed ∼420 million years ago. Leucoraja exhibits core features of tetrapod locomotor gaits, including left-right alternation and reciprocal extension-flexion of the pelvic fins. Leucoraja also deploys a remarkably conserved Hox transcription factor-dependent program that is essential for selective innervation of fin/limb muscle. This network encodes peripheral connectivity modules that are distinct from those used in axial muscle-based swimming and has apparently been diminished in most modern fish. These findings indicate that the circuits that are essential for walking evolved through adaptation of a genetic regulatory network shared by all vertebrates with paired appendages. VIDEO ABSTRACT.

Parallel Pbx-Dependent Pathways Govern the Coalescence and Fate of Motor Columns.

The clustering of neurons sharing similar functional properties and connectivity is a common organizational feature of vertebrate nervous systems. Within motor networks, spinal motor neurons (MNs) segregate into longitudinally arrayed subtypes, establishing a central somatotopic map of peripheral target innervation. MN organization and connectivity relies on Hox transcription factors expressed along the rostrocaudal axis; however, the developmental mechanisms governing the orderly arrangement of MNs are largely unknown. We show that Pbx genes, which encode Hox cofactors, are essential for the segregation and clustering of neurons within motor columns. In the absence of Pbx1 and Pbx3 function, Hox-dependent programs are lost and the remaining MN subtypes are unclustered and disordered. Identification of Pbx gene targets revealed an unexpected and apparently Hox-independent role in defining molecular features of dorsally projecting medial motor column (MMC) neurons. These results indicate Pbx genes act in parallel genetic pathways to orchestrate neuronal subtype differentiation, connectivity, and organization.

Mir-17∼92 Governs Motor Neuron Subtype Survival by Mediating Nuclear PTEN.

Motor neurons (MNs) are unique because they project their axons outside of the CNS to innervate the peripheral muscles. Limb-innervating lateral motor column MNs (LMC-MNs) travel substantially to innervate distal limb mesenchyme. How LMC-MNs fine-tune the balance between survival and apoptosis while wiring the sensorimotor circuit en route remains unclear. Here, we show that the mir-17∼92 cluster is enriched in embryonic stem cell (ESC)-derived LMC-MNs and that conditional mir-17∼92 deletion in MNs results in the death of LMC-MNs in vitro and in vivo. mir-17∼92 overexpression rescues MNs from apoptosis, which occurs spontaneously during embryonic development. PTEN is a primary target of mir-17∼92 responsible for LMC-MN degeneration. Additionally, mir-17∼92 directly targets components of E3 ubiquitin ligases, affecting PTEN subcellular localization through monoubiquitination. This miRNA-mediated regulation modulates both target expression and target subcellular localization, providing LMC-MNs with an intricate defensive mechanism that controls their survival.

Evolution of patterning systems and circuit elements for locomotion.

Evolutionary modifications in nervous systems enabled organisms to adapt to their specific environments and underlie the remarkable diversity of behaviors expressed by animals. Resolving the pathways that shaped and modified neural circuits during evolution remains a significant challenge. Comparative studies have revealed a surprising conservation in the intrinsic signaling systems involved in early patterning of bilaterian nervous systems but also raise the question of how neural circuit compositions and architectures evolved within specific animal lineages. In this review, we discuss the mechanisms that contributed to the emergence and diversity of animal nervous systems, focusing on the circuits governing vertebrate locomotion.

Sorting of human mesenchymal stem cells by applying optimally designed microfluidic chip filtration.

Human bone marrow-derived mesenchymal stem cells (hMSCs) consist of heterogeneous subpopulations with different multipotent properties: small and large cells with high and low multipotency, respectively. Accordingly, sorting out a target subpopulation from the others is very important to increase the effectiveness of cell-based therapy. We performed flow-based sorting of hMSCs by using optimally designed microfluidic chips based on the hydrodynamic filtration (HDF) principle. The chip was designed with the parameters rigorously determined by the complete analysis of laminar flow for flow fraction and complicated networks of main and multi-branched channels for hMSCs sorting into three subpopulations: small (<25 µm), medium (25-40 µm), and large (>40 µm) cells. By focusing with a proper ratio between main and side flows, cells migrate toward the sidewall due to a virtual boundary of fluid layers and enter the branch channels. This opens the possibility of sorting stem cells rapidly without damage. Over 86% recovery was achieved for each population of cells with complete purity in small cells, but the sorting efficiency of cells is slightly lower than that of rigid model particles, due to the effect of cell deformation. Finally, we confirmed that our method could successfully fractionate the three subpopulations of hMSCs by analyzing the surface marker expressions of cells from each outlet.

Long-term outcomes of infrainguinal bypass surgery for patients with diabetes mellitus and tissue loss.

PURPOSE: To determine the long-term outcomes of patients with diabetes mellitus (DM) and tissue loss who have undergone infrainguinal bypass surgery (IBS). METHODS: We retrospectively reviewed the medical records of 91 patients with DM and tissue loss who underwent IBS between July 2003 and December 2013. We determined the rates of overall survival (OS), amputation-free survival (AFS), limb salvage (LS), and graft patency (GP). In addition, we evaluated data to identify risk factors that affected long-term outcomes. RESULTS: The mean age of patients was 66 ± 8 years, and 78 patients (85.7%) were men. The locations of tissue loss were toe on 76 limbs (71.6%), heel on 6 limbs (5.7%) and others on 24 limbs (22.6%). Single lesions were found in 81 limbs (76.4%). According to categorization by distal anastomosis artery, there were 57 popliteal (53.8%) and 49 infrapopliteal bypasses (46.2%). Among infrapopliteal bypasses, 5 cases (10.2%) were sequential bypasses. The OS at 1, 3, and 5 years was 90.5%, 70.9%, and 44.2%, respectively. At 1, 3, and 5 years, the LS was 92.1%, 88.9%, 88.9%, respectively; and AFS was 84.4%, 67.6%, 45.7%, respectively. At 1, 3, and 5 years, the GP was 84.8%, 74.5%, and 69.8%, respectively. Renal failure was a negative predictor for OS, and female gender was a negative predictor for GP. CONCLUSION: IBS for patients with DM and tissue loss led to acceptable OS, AFS, LS, and GP. Active revascularization for patients with DM and tissue loss can reduce the risk of major amputation.

Therapeutic outcomes and thromboembolic events after treatment of acute arterial thromboembolism of the upper extremity.

BACKGROUND: To investigate short- and long-term outcomes of patients with acute arterial thromboembolism of upper extremity, focusing on postoperative thromboembolic events by etiology. METHODS: Hospital records of 53 patients (average age 70 years; males 49%) with acute arterial thromboembolism of upper extremity treated between June 1993 and December 2013 were retrospectively reviewed, evaluating patient characteristics and clinical outcomes, both short and long term. Subjects were stratified as those with (group I, 34) and without (group II, 19) atrial fibrillation as underlying cause. RESULTS: Two patients received anticoagulation alone as conservative treatment. The remainder (n = 51) underwent surgical revascularization. Symptoms resolved in 51 patients (96%) except 2 patients with postoperative reocclusion, and there was no need of amputation in all patients. Overall inpatient mortality was 5.6% (3/53). Patients of group I suffered most of the recurrent thromboembolic events (group I: 17 events, 14 patients; group II: 2 events, 2 patients) recorded during follow-up (mean duration 56.8 ± 62.2 months). Respective event-free survival rates at 1, 3, and 5 years differed significantly by group: 77%, 44%, and 44% for group I; 100%, 100%, and 83% for group II (P = 0.004). Among 14 patients with recurrent embolic events in group I, half of the patients were not receiving anticoagulants; however, anticoagulant cessation was generally arbitrary. Overall survival rates at 1, 3, and 5 years were 82%, 69%, and 52% for group I and 84%, 78%, and 70% for group II (P = 0.21). CONCLUSIONS: In this study, surgical treatment of acute arterial thromboembolism of upper extremity was largely successful. Especially in patients with atrial fibrillation, adequate long-term anticoagulation is indicated as prophylaxis, given the high rates of recurrent thromboembolic events.

Ankyrin repeat-rich membrane spanning (ARMS)/Kidins220 scaffold protein regulates neuroblastoma cell proliferation through p21.

Cell proliferation is tightly controlled by the cell-cycle regulatory proteins, primarily by cyclins and cyclin-dependent kinases (CDKs) in the G1 phase. The ankyrin repeat-rich membrane spanning (ARMS) scaffold protein, also known as kinase D-interacting substrate of 220 kDa (Kidins 220), has been previously identified as a prominent downstream target of neurotrophin and ephrin receptors. Many studies have reported that ARMS/Kidins220 acts as a major signaling platform in organizing the signaling complex to regulate various cellular responses in the nervous and vascular systems. However, the role of ARMS/Kidins220 in cell proliferation and cell-cycle progression has never been investigated. Here we report that knockdown of ARMS/Kidins220 inhibits mouse neuroblastoma cell proliferation by inducing slowdown of cell cycle in the G1 phase. This effect is mediated by the upregulation of a CDK inhibitor p21, which causes the decrease in cyclin D1 and CDK4 protein levels and subsequent reduction of pRb hyperphosphorylation. Our results suggest a new role of ARMS/Kidins220 as a signaling platform to regulate tumor cell proliferation in response to the extracellular stimuli.

Evolving Hox activity profiles govern diversity in locomotor systems.

The emergence of limb-driven locomotor behaviors was a key event in the evolution of vertebrates and fostered the transition from aquatic to terrestrial life. We show that the generation of limb-projecting lateral motor column (LMC) neurons in mice relies on a transcriptional autoregulatory module initiated via transient activity of multiple genes within the HoxA and HoxC clusters. Repression of this module at thoracic levels restricts expression of LMC determinants, thus dictating LMC position relative to the limbs. This suppression is mediated by a key regulatory domain that is specifically found in the Hoxc9 proteins of appendage-bearing vertebrates. The profile of Hoxc9 expression inversely correlates with LMC position in land vertebrates and likely accounts for the absence of LMC neurons in limbless species such as snakes. Thus, modulation of both Hoxc9 protein function and Hoxc9 gene expression likely contributed to evolutionary transitions between undulatory and ambulatory motor circuit connectivity programs.

Central Transposition of the Cephalic Vein in Patients with Brachiocephalic Arteriovenous Fistula and Cephalic Arch Stenosis.

PURPOSE: Our study aims to evaluate to evaluate clinical outcomes after cephalic vein transposition (CVT) to the axilla in patients with brachiocephalic arteriovenous fistula (BC-AVF) and cephalic arch stenosis (CAS). MATERIALS AND METHODS: Hospital records of 13 patients (median age, 61 years; males, 54%) who received CVT to the proximal basilic/axillary vein due to either dysfunction (n=2) or thrombosis (n=11) between January 2010 and February 2014 were retrospectively reviewed. RESULTS: Operation was performed under local anesthesia in all cases. There was no technical failure. Concomitant inflow procedure (banding or aneurysmorrhaphy) was performed in 5 patients (38%). During follow-up (1 to 50 months, median 17 months), 3 patients died with functioning AVF and one was successfully transplanted. Two patients suffered from recurrent symptomatic stenosis of AVF and received percutaneous balloon angioplasty. Another 2 patients experienced AVF occlusion treated with interposition graft and manual fragmentation. Overall primary, assisted primary, and secondary patency rates were 77.5%, 92.3%, and 100% at 6 months and 66.1%, 92.3%, and 100% at 1 year, respectively. CONCLUSION: Although most patients presented with BC-AVF occlusion, technical success and access patency rates after CVT were favorable compared with historical data for interventional treatment. CVT should be considered as an appropriate option in selected patients with CAS.

Open Repair of Ruptured Huge Aorto-Iliac Aneurysm: Warning of Colon Ischemia.

A giant abdominal aortic aneurysm (AAA) renders surgical treatment much more difficult by deforming the proximal infrarenal aortic neck (shortened length and disturbed angulation), by altering the iliac arteries (marked tortuosity and aneurysmal dilatation), and by displacing abdominal organs. Because the retroperitoneal rupture of giant AAA makes the mesentery more elongated and deformed, compromising its blood flow and thus increasing the risk of mesenteric ischemia such as colon ischemia. We describe here the surgical repair of a large infrarenal AAA with a ruptured huge left common iliac artery aneurysm of 13.5 cm in diameter, accompanied by colostomy due to colon ischemia which occurred during the operation. We discuss the pathophysiology and preventive strategy of colon ischemia during ruptured giant AAA repair.

BMI1 represses Ink4a/Arf and Hox genes to regulate stem cells in the rodent incisor.

The polycomb group gene Bmi1 is required for maintenance of adult stem cells in many organs. Inactivation of Bmi1 leads to impaired stem cell self-renewal due to deregulated gene expression. One critical target of BMI1 is Ink4a/Arf, which encodes the cell-cycle inhibitors p16(Ink4a) and p19(Arf). However, deletion of Ink4a/Arf only partially rescues Bmi1-null phenotypes, indicating that other important targets of BMI1 exist. Here, using the continuously growing mouse incisor as a model system, we report that Bmi1 is expressed by incisor stem cells and that deletion of Bmi1 resulted in fewer stem cells, perturbed gene expression and defective enamel production. Transcriptional profiling revealed that Hox expression is normally repressed by BMI1 in the adult, and functional assays demonstrated that BMI1-mediated repression of Hox genes preserves the undifferentiated state of stem cells. As Hox gene upregulation has also been reported in other systems when Bmi1 is inactivated, our findings point to a general mechanism whereby BMI1-mediated repression of Hox genes is required for the maintenance of adult stem cells and for prevention of inappropriate differentiation.

Genetic and functional modularity of Hox activities in the specification of limb-innervating motor neurons.

A critical step in the assembly of the neural circuits that control tetrapod locomotion is the specification of the lateral motor column (LMC), a diverse motor neuron population targeting limb musculature. Hox6 paralog group genes have been implicated as key determinants of LMC fate at forelimb levels of the spinal cord, through their ability to promote expression of the LMC-restricted genes Foxp1 and Raldh2 and to suppress thoracic fates through exclusion of Hoxc9. The specific roles and mechanisms of Hox6 gene function in LMC neurons, however, are not known. We show that Hox6 genes are critical for diverse facets of LMC identity and define motifs required for their in vivo specificities. Although Hox6 genes are necessary for generating the appropriate number of LMC neurons, they are not absolutely required for the induction of forelimb LMC molecular determinants. In the absence of Hox6 activity, LMC identity appears to be preserved through a diverse array of Hox5-Hox8 paralogs, which are sufficient to reprogram thoracic motor neurons to an LMC fate. In contrast to the apparently permissive Hox inputs to early LMC gene programs, individual Hox genes, such as Hoxc6, have specific roles in promoting motor neuron pool diversity within the LMC. Dissection of motifs required for Hox in vivo specificities reveals that either cross-repressive interactions or cooperativity with Pbx cofactors are sufficient to induce LMC identity, with the N-terminus capable of promoting columnar, but not pool, identity when transferred to a heterologous homeodomain. These results indicate that Hox proteins orchestrate diverse aspects of cell fate specification through both the convergent regulation of gene programs regulated by many paralogs and also more restricted actions encoded through specificity determinants in the N-terminus.

Citrus unshiu peel extract ameliorates hyperglycemia and hepatic steatosis by altering inflammation and hepatic glucose- and lipid-regulating enzymes in db/db mice.

Insulin resistance in Type 2 diabetes leads to hepatic steatosis that can accompanied by progressive inflammation of the liver. Citrus unshiu peel is a rich source of citrus flavonoids that possess anti-inflammatory, anti-diabetic and lipid-lowering effects. However, the ability of citrus unshiu peel ethanol extract (CPE) to improve hyperglycemia, adiposity and hepatic steatosis in Type 2 diabetes is unknown. Thus, we evaluated the effects of CPE on markers for glucose, lipid metabolism and inflammation in Type 2 diabetic mice. Male C57BL/KsJ-db/db mice were fed a normal diet with CPE (2 g/100 g diet) or rosiglitazone (0.001 g/100 g diet) for 6 weeks. Mice supplemented with the CPE showed a significant decrease in body weight gain, body fat mass and blood glucose level. The antihyperglycemic effect of CPE appeared to be partially mediated through the inhibition of hepatic gluconeogenic phosphoenolpyruvate carboxykinase mRNA expression and its activity and through the induction of insulin/glucagon secretion. CPE also ameliorated hepatic steatosis and hypertriglyceridemia via the inhibition of gene expression and activities of the lipogenic enzymes and the activation of fatty acid oxidation in the liver. These beneficial effects of CPE may be related to increased levels of anti-inflammatory adiponectin and interleukin (IL)-10, and decreased levels of pro-inflammatory markers (IL-6, monocyte chemotactic protein-1, interferon-γ and tumor necrosis factor-α) in the plasma or liver. Taken together, we suggest that CPE has the potential to improve both hyperglycemia and hepatic steatosis in Type 2 diabetes.

Global control of motor neuron topography mediated by the repressive actions of a single hox gene.

In the developing spinal cord, regional and combinatorial activities of Hox transcription factors are critical in controlling motor neuron fates along the rostrocaudal axis, exemplified by the precise pattern of limb innervation by more than fifty Hox-dependent motor pools. The mechanisms by which motor neuron diversity is constrained to limb levels are, however, not well understood. We show that a single Hox gene, Hoxc9, has an essential role in organizing the motor system through global repressive activities. Hoxc9 is required for the generation of thoracic motor columns, and in its absence, neurons acquire the fates of limb-innervating populations. Unexpectedly, multiple Hox genes are derepressed in Hoxc9 mutants, leading to motor pool disorganization and alterations in the connections by thoracic and forelimb-level subtypes. Genome-wide analysis of Hoxc9 binding suggests that this mode of repression is mediated by direct interactions with Hox regulatory elements, independent of chromatin marks typically associated with repressed Hox genes.

Human mesenchymal stem cell-derived Schwann cell-like cells exhibit neurotrophic effects, via distinct growth factor production, in a model of spinal cord injury.

Human bone marrow-derived mesenchymal stem cells (hMSCs) are considered a desirable cell source for autologous cell transplantation therapy to treat nervous system injury due to their ability to differentiate into specific cell types and render the tissue microenvironment more favorable for tissue repair by secreting various growth factors. To potentiate their possible trophic effect, hMSCs were induced without genetic modification to adopt characteristics of Schwann cells (SCs), which provide trophic support for regenerating axons. The induced hMSCs (shMSCs) adopted a SC-like morphology and expressed SC-specific proteins including the p75 neurotrophin receptor, which correlated with cell-cycle exit. In addition, shMSCs secreted higher amounts of several growth factors, such as hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) when compared with uninduced hMSCs. Coculture of shMSCs with Neuro2A cells significantly increased neurite outgrowth and cell proliferation but decreased cell death. Transplantation of shMSCs in an ex vivo model of spinal cord injury dramatically enhanced axonal outgrowth, which was mediated by HGF and VEGF secretion and also decreased cell death. These results demonstrate that shMSCs could serve as an endogenous source of neurotrophic growth factors to facilitate axonal regeneration while at the same time protecting the resident cells at the site of tissue injury. We propose that these induced hMSCs without genetic modification are useful for autologous cell therapy to treat nervous system injury.

Mechanism of macrophage activation induced by beta-glucan produced from Paenibacillus polymyxa JB115.

Beta-glucans are heterogeneous groups of glucose polymers found in the cell walls of fungi, plants and some bacteria. Our previous report showed that a novel beta-1,3/1,6-glucan produced from Paenibacillus (P.) polymyxa JB115 can induce nitric oxide (NO) production in RAW264.7 cells. In the present study, the beta-glucan significantly increased luciferase activity in cells transfected with NFkappaB or AP1, but not STAT1, reporter vector DNA, which contain their binding promoter site. All specific NFkappaB and MAPKs pathway inhibitors (pyrrolidine dithiocarbamate, AG490, U0126, SB203580 and SP600125) remarkably attenuated NO production induced by the beta-glucan. Furthermore, Western blot analysis revealed that the stimulation of Raw264.7 cells by beta-glucan induced phosphorylation of IkappaB and the consequent translocation of NFkappaB into the nucleus. Meanwhile, phosphorylation of ERK1/2, JNK/SAPK and p38 MAPKs in cytoplasm were also confirmed. All these results indicated that beta-glucan from P. polymyxa JB115 activates macrophages through MAPKs and NFkappaB signaling pathway.

Regulation of the Drosophila apoptosome through feedback inhibition.

Apoptosis is induced by caspases, which are members of the cysteine protease family. Caspases are synthesized as inactive zymogens and initiator caspases first gain activity by associating with an oligomeric complex of their adaptor proteins, such as the apoptosome. Activated initiator caspases subsequently cleave and activate effector caspases. Although such a proteolytic cascade would predict that a small number of active caspases could irreversibly amplify caspase activity and trigger apoptosis, many cells can maintain moderate levels of caspase activity to perform non-apoptotic roles in cellular differentiation, shape change and migration. Here we show that the Drosophila melanogaster apoptosome engages in a feedback inhibitory loop, which moderates its activation level in vivo. Specifically, the adaptor protein Apaf-1 lowers the level of its associated initiator caspase Dronc, without triggering apoptosis. Conversely, Dronc lowers Apaf-1 protein levels. This mutual suppression depends on the catalytic site of Dronc and a caspase cleavage site within Apaf-1. Moreover, the Drosophila inhibitor of apoptosis protein 1 (Diap1) is required for this process. We speculate that this feedback inhibition allows cells to regulate the degree of caspase activation for apoptotic and non-apoptotic purposes.