Identification of the growth cone as a probe and driver of neuronal migration in the injured brain.

Axonal growth cones mediate axonal guidance and growth regulation. We show that migrating neurons in mice possess a growth cone at the tip of their leading process, similar to that of axons, in terms of the cytoskeletal dynamics and functional responsivity through protein tyrosine phosphatase receptor type sigma (PTPσ). Migrating-neuron growth cones respond to chondroitin sulfate (CS) through PTPσ and collapse, which leads to inhibition of neuronal migration. In the presence of CS, the growth cones can revert to their extended morphology when their leading filopodia interact with heparan sulfate (HS), thus re-enabling neuronal migration. Implantation of an HS-containing biomaterial in the CS-rich injured cortex promotes the extension of the growth cone and improve the migration and regeneration of neurons, thereby enabling functional recovery. Thus, the growth cone of migrating neurons is responsive to extracellular environments and acts as a primary regulator of neuronal migration.

Very-long-chain fatty acids are crucial to neuronal polarity by providing sphingolipids to lipid rafts.

Fatty acids have long been considered essential to brain development; however, the involvement of their synthesis in nervous system formation is unclear. We generate mice with knockout of GPSN2, an enzyme for synthesis of very-long-chain fatty acids (VLCFAs) and investigate the effects. Both GPSN2-/- and GPSN2+/- mice show abnormal neuronal networks as a result of impaired neuronal polarity determination. Lipidomics of GPSN2-/- embryos reveal that ceramide synthesis is specifically inhibited depending on FA length; namely, VLCFA-containing ceramide is reduced. We demonstrate that lipid rafts are highly enriched in growth cones and that GPSN2+/- neurons lose gangliosides in their membranes. Application of C24:0 ceramide, but not C16:0 ceramide or C24:0 phosphatidylcholine, to GPSN2+/- neurons rescues both neuronal polarity determination and lipid-raft density in the growth cone. Taken together, our results indicate that VLCFA synthesis contributes to physiological neuronal development in brain network formation, in particular neuronal polarity determination through the formation of lipid rafts.

JNK1-Dependent Phosphorylation of GAP-43 Serine 142 is a Novel Molecular Marker for Axonal Growth.

Mammalian axon growth has mechanistic similarities with axon regeneration. The growth cone is an important structure that is involved in both processes, and GAP-43 (growth associated protein-43 kDa) is believed to be the classical molecular marker. Previously, we used growth cone phosphoproteomics to demonstrate that S96 and T172 of GAP-43 in rodents are highly phosphorylated sites that are phosphorylated by c-jun N-terminal protein kinase (JNK). We also revealed that phosphorylated (p)S96 and pT172 antibodies recognize growing axons in the developing brain and regenerating axons in adult peripheral nerves. In rodents, S142 is another putative JNK-dependent phosphorylation site that is modified at a lower frequency than S96 and T172. Here, we characterized this site using a pS142-specific antibody. We confirmed that pS142 was detected by co-expressing mouse GAP-43 and JNK1. pS142 antibody labeled growth cones and growing axons in developing mouse neurons. pS142 was sustained until at least nine weeks after birth in mouse brains. The pS142 antibody could detect regenerating axons following sciatic nerve injury in adult mice. Comparison of amino acid sequences indicated that rodent S142 corresponds to human S151, which is predicted to be a substrate of the MAPK family, which includes JNK. Thus, we confirmed that the pS142 antibody recognized human phospho-GAP-43 using activated JNK1, and also that its immunostaining pattern in neurons differentiated from human induced pluripotent cells was similar to those observed in mice. These results indicate that the S142 residue is phosphorylated by JNK1 and that the pS142 antibody is a new candidate molecular marker for axonal growth in both rodents and human.

Preceding direct oral anticoagulant administration reduces the severity of stroke in patients with atrial fibrillation - K-PLUS registry.

BACKGROUND: Stroke severity can be mitigated by preceding anticoagulant administration in acute ischemic stroke patients with atrial fibrillation (AF). We investigated if such mitigative effects are different between warfarin and direct oral anticoagulants (DOACs). MATERIAL AND METHODS: We collected data from a regional multicenter stroke registry. Ischemic stroke or transient ischemic attack patients with AF were included. Background characteristics, National Institutes of Health Stroke Scale (NIHSS) score on admission, lesion characteristics, and in-hospital death were analyzed according to preceding antithrombotic agents at onset. RESULTS: A total of 2173 patients had AF; 628 were prescribed warfarin, 272 DOACs, 429 antiplatelets alone, and 844 no antithrombotics. The NIHSS score on admission was lowest in the DOACs group compared to the other groups. In neuroimaging analysis, small ischemic lesions were observed more frequently in the DOACs group, while large ischemic lesions were less frequent in this group. When the no antithrombotics group was used as a reference, the adjusted odds ratio for moderate to severe stroke was 0.56 (95% confidence interval, 0.40-0.78) in the DOACs group, while it was 0.98 (0.77-1.24) in the warfarin group and 0.94 (0.72-1.22) in the antiplatelets group. In-hospital mortality was lowest in the DOACs group compared to the other groups. CONCLUSION: Preceding DOAC administration might mitigate the severity of stroke in AF patients more strongly than other antithrombotics, possibly leading to a better outcome in patients with stroke.

Pre-Hospital Delay in Patients with Acute Ischemic Stroke in a Multicenter Stroke Registry: K-PLUS.

PURPOSE: There is scant data related to prehospital delay in cases of acute ischemic stroke from multicenter studies conducted after change of the therapeutic window of intravenous tissue plasminogen activator (iv-tPA) administration to within 4.5 h of onset. We investigated factors causing prehospital delay and their associations with clinical outcomes using data from a regional multicenter stroke registry. METHODS: Data from the multicenter regional stroke registry were analyzed. Patients admitted within 24 h of the last known well time were categorized according to whether their admission was early (≤ 4 h; n = 2350) or delayed (> 4 h; n = 2752). We then compared patients' backgrounds and outcomes between the two groups. RESULTS: Five-thousand, one-hundred two patients presented at hospitals within 24 h of onset. On multivariate analysis, atrial fibrillation, higher NIHSS score on admission, anterior circulation stroke, detection of symptoms immediately after onset, and emergency system use were positively associated with early admission, whereas modified Rankin Scale (mRS) score before onset, onset at home, diabetes, current smoking, dementia and symptom detection between 00:00 and 06:00 h were negatively associated. Early admission was associated with mRS scores of 0-2 at discharge independent of backgrounds, stroke severity, and thrombolytic therapy (odds ratio, 1.56; 95% confidence interval, 1.32-1.84). CONCLUSIONS: Certain patient factors relating to prehospital delay, such as lack of awareness of onset or non-cardioembolic etiology, are crucial but often inevitable. However, earlier admission was associated mRS scores of 0-2 independent of other factors. This study may help to plan educational activities to general population or public awareness campaigns.

Phosphorylation sites of microtubule-associated protein 1B (MAP 1B) are involved in axon growth and regeneration.

The growth cone is a specialized structure that forms at the tip of extending axons in developing and regenerating neurons. This structure is essential for accurate synaptogenesis at developmental stages, and is also involved in plasticity-dependent synaptogenesis and axon regeneration in the mature brain. Thus, understanding the molecular mechanisms utilized by growth cones is indispensable to understanding neuronal network formation and rearrangement. Phosphorylation is the most important and commonly utilized protein modification in signal transduction. We previously identified microtubule-associated protein 1B (MAP 1B) as the most frequently phosphorylated protein among ~ 1200 phosphorylated proteins. MAP 1B has more than 10 phosphorylation sites that were present more than 50 times among these 1200 proteins. Here, we produced phospho-specific antibodies against phosphorylated serines at positions 25 and 1201 of MAP 1B that specifically recognize growing axons both in cultured neurons and in vivo in various regions of the embryonic brain. Following sciatic nerve injury, immunoreactivity with each antibody increased compared to the sham operated group. Experiments with transected and sutured nerves revealed that regenerating axons were specifically recognized by these antibodies. These results suggest that these MAP 1B phosphorylation sites are specifically involved in axon growth and that phospho-specific antibodies against MAP 1B are useful markers of growing/regenerating axons.

Growth Cone Phosphoproteomics Reveals that GAP-43 Phosphorylated by JNK Is a Marker of Axon Growth and Regeneration.

Neuronal growth cones are essential for nerve growth and regeneration, as well as for the formation and rearrangement of the neural network. To elucidate phosphorylation-dependent signaling pathways and establish useful molecular markers for axon growth and regeneration, we performed a phosphoproteomics study of mammalian growth cones, which identified >30,000 phosphopeptides of ∼1,200 proteins. The phosphorylation sites were highly proline directed and primarily MAPK dependent, owing to the activation of JNK, suggesting that proteins that undergo proline-directed phosphorylation mediate nerve growth in the mammalian brain. Bioinformatics analysis revealed that phosphoproteins were enriched in microtubules and the cortical cytoskeleton. The most frequently phosphorylated site was S96 of GAP-43 (growth-associated protein 43-kDa), a vertebrate-specific protein involved in axon growth. This previously uncharacterized phosphorylation site was JNK dependent. S96 phosphorylation was specifically detected in growing and regenerating axons as the most frequent target of JNK signaling; thus it represents a promising new molecular marker for mammalian axonal growth and regeneration.

Glycoprotein M6a as a signaling transducer in neuronal lipid rafts.

Neuronal development is composed of the complex steps which involve many signaling proteins. On the other hand, there are many proteins highly expressed in the differentiated neurons at developmental stages, but of which physiological functions are not precisely known so far. Glycoprotein 6a (GPM6a) currently belongs to such proteins. This protein has four-transmembrane domains and is a member of proteolipid protein family. Recently, we demonstrated that GPM6a is highly concentrated in lipid rafts of the developing neuron with its palmitoylation, and that this molecule is involved in rapid determination of the neuronal polarity, in response to laminin, an extracellular matrix protein (Honda et al., J Neurosci 37: 4046-64 [2017]). Since lipid rafts are membrane domains enriched in sphingolipids and cholesterol, have lower fluidity than other membrane areas and are believed to be signaling "hot-spots", and here, we discuss the functions of this protein in neuronal lipid raft signaling for neuronal development.

Extracellular Signals Induce Glycoprotein M6a Clustering of Lipid Rafts and Associated Signaling Molecules.

Lipid raft domains, where sphingolipids and cholesterol are enriched, concentrate signaling molecules. To examine how signaling protein complexes are clustered in rafts, we focused on the functions of glycoprotein M6a (GPM6a), which is expressed at a high concentration in developing mouse neurons. Using imaging of lipid rafts, we found that GPM6a congregated in rafts in a GPM6a palmitoylation-dependent manner, thereby contributing to lipid raft clustering. In addition, we found that signaling proteins downstream of GPM6a, such as Rufy3, Rap2, and Tiam2/STEF, accumulated in lipid rafts in a GPM6a-dependent manner and were essential for laminin-dependent polarity during neurite formation in neuronal development. In utero RNAi targeting of GPM6a resulted in abnormally polarized neurons with multiple neurites. These results demonstrate that GPM6a induces the clustering of lipid rafts, which supports the raft aggregation of its associated downstream molecules for acceleration of neuronal polarity determination. Therefore, GPM6a acts as a signal transducer that responds to extracellular signals.SIGNIFICANCE STATEMENT Lipid raft domains, where sphingolipids and cholesterol are enriched, concentrate signaling molecules. We focused on glycoprotein M6a (GPM6a), which is expressed at a high concentration in developing neurons. Using imaging of lipid rafts, we found that GPM6a congregated in rafts in a palmitoylation-dependent manner, thereby contributing to lipid raft clustering. In addition, we found that signaling proteins downstream of GPM6a accumulated in lipid rafts in a GPM6a-dependent manner and were essential for laminin-dependent polarity during neurite formation. In utero RNAi targeting of GPM6a resulted in abnormally polarized neurons with multiple neurites. These results demonstrate that GPM6a induces the clustering of lipid rafts, which supports the raft aggregation of its associated downstream molecules for acceleration of polarity determination. Therefore, GPM6a acts as a signal transducer that responds to extracellular signals.

Intratumoral hemorrhage in a patient with malignant meningioma under anticoagulant therapy.

We report the case of an elderly woman with malignant meningioma and atrial fibrillation who started taking anticoagulants after an ischemic stroke and subsequently developed intratumoral hemorrhage. Further studies are required to confirm whether a particular anticoagulant agent is suitable for patients with brain tumor and atrial fibrillation.

The four-transmembrane protein IP39 of Euglena forms strands by a trimeric unit repeat.

Euglenoid flagellates have striped surface structures comprising pellicles, which allow the cell shape to vary from rigid to flexible during the characteristic movement of the flagellates. In Euglena gracilis, the pellicular strip membranes are covered with paracrystalline arrays of a major integral membrane protein, IP39, a putative four-membrane-spanning protein with the conserved sequence motif of the PMP-22/EMP/MP20/Claudin superfamily. Here we report the three-dimensional structure of Euglena IP39 determined by electron crystallography. Two-dimensional crystals of IP39 appear to form a striated pattern of antiparallel double-rows in which trimeric IP39 units are longitudinally polymerised, resulting in continuously extending zigzag-shaped lines. Structural analysis revealed an asymmetric molecular arrangement in the trimer, and suggested that at least four different interactions between neighbouring protomers are involved. A combination of such multiple interactions would be important for linear strand formation of membrane proteins in a lipid bilayer.

[Intravenous recombinant tissue plasminogen activator therapy in a 14-week pregnant woman with embolic stroke due to protein S deficiency].

When cerebral infarction develops during pregnancy, treatment without adverse effects must be considered not only for the mother but also for the fetus. Because pregnant women were excluded from many clinical trials, clear treatment guidance for them is not shown in the package inserts or guidelines of many drugs. We report the case of a 35-year-old woman (gravida 3, para 2) who developed sudden onset of left visual field defect, left hemiparesis, and dysesthesia over the left forearm during her fourth month of pregnancy. Brain diffusion-weighted MRI showed high intensity areas in the right occipital lobe, and magnetic resonance angiography revealed an occlusion of the right posterior cerebral artery. She was treated with an intravenous injection of recombinant tissue plasminogen activator 2 h 55 min after symptom onset, and the visual field and sensorimotor deficits improved. MRA obtained 3 days after the onset showed recanalization of the right posterior cerebral artery. We also conducted electrocardiography, neck vascular ultrasound, cardiovascular ultrasound, transcranial Doppler recordings from the temple area, and laboratory examinations for complete blood count, biochemistry, coagulation factors, endocrine secretion, and autoantibodies. Reduced protein S activity (35%) along with high intensity transient signals on transcranial Doppler indicated microemboli to be the embolic source. All other tests were negative. Anticoagulation therapy was initiated to prevent recurrence. She was initially given intravenous heparin, and then switched to warfarin therapy at 15 weeks of gestation. The patient delivered a healthy infant via caesarean section. Although reports and experiences of thrombolytic therapy with injection of recombinant tissue plasminogen activator during pregnancy remain scant, this therapy might be carefully used, especially after due consideration and understanding of the risks and benefits for both mother and fetus.

[Non-taking oral antithrombotic agents in patients with ischemic stroke].

BACKGROUND: In clinical practice, secondary prevention in patients with ischemic stroke (IS) needs to be continued permanently; however, antithrombotic agents are sometimes stopped by clinicians or the patients themselves. The rate of non-taking oral antithrombotic agents was evaluated in IS patients. METHODS: 266 consecutive patients (154 men and 112 women; age, 73.6 +/- 11.5 years) with first-ever acute IS were studied. Patients with transient ischemic attack (TIA) were also included. Emboligenic heart diseases, frequency of past stroke, oral antithrombotic agent use just before IS, and secondary prevention were evaluated. RESULTS: The number of past strokes was 0 in 182 cases (68.4%), 1 in 66 cases (24.8%), 2 in 14 cases (5.3%), 3 in 3 cases (1.1%), and 9 in 1 case (0.4%; 3 times with stroke, and 6 times with TIA). There were 42 cases (15.8%) with TIA, 47 (17.7%) with lacunar infarction, 69 (25.9%) with atherothrombotic infarction, 62 (23.3%) with cardioembolic infarction, 23 (8.7%) with other types of infarction, and 23 (8.7%) with stroke of unknown etiology. Although 15-26% of patients with their first IS had taken antithrombotic agents just before IS, about 40% of the patients with a previous IS history were not taking antithrombotic agents just before their recurrent IS. CONCLUSION: About 40% of the patients with recurrent IS were not taking antithrombotic agents at the time of their recurrent IS; had they been taking antithrombotic agents at the time, the recurrent IS might have been prevented. Clinicians must recognize the importance of antithrombotic agents in patients with IS, and patients must continue to take antithrombotic agents permanently.

Dual role of nitric oxide in gastric hypersecretion in the distended stomach: inhibition of acid secretion and stimulation of pepsinongen secretion.

AIMS: We investigate the role of nitric oxide (NO) in the hypersecretion of acid and pepsinogen induced by stomach distension. MAIN METHOD: The rat stomach was distended by instillation of saline through an acute fistula under urethane anesthesia. KEY FINDINGS: Both secretions of acid and pepsinogen were increased by the distension depending on the volume of saline introduced, and responses were attenuated by bilateral cervical vagotomy or prior administration of atropine. N(G)-nitro-l-arginine methyl ester (L-NAME) had a dual effect on these responses, causing an increase in the acid response and a decrease in the pepsin response, both in an l-arginine-sensitive manner. Distension of the stomach increased the luminal NO release; this response was suppressed by vagotomy and L-NAME. Intragastric application of FK409, a NO donor, dose-dependently increased pepsinogen secretion while decreasing acid secretion in the stomach without distension. However, serosal application of both FK409 and 8-bromo-guanosine cyclic 3', 5'-monophosphate (8-Br-cGMP) stimulated the secretion of pepsinogen in isolated mouse stomachs in vitro. The stimulatory effect of FK409 on pepsinogen secretion was totally abolished by LY83583, a guanylate cyclase inhibitor. SIGNIFICANCE: Distension of the stomach increases both acid and pepsinogen secretion through a vagal-cholinergic pathway in addition to the luminal release of NO, and NO affects these responses in opposite ways, suppressing the acid response while enhancing the pepsin response, both mediated by a guanylate cyclase/cGMP pathway.

[Upper airway obstruction with bilateral vocal cord paralysis secondary to ischemic stroke: report of two cases].

We present herein two patients with bilateral vocal cord paralysis that occurred during the subacute phase of brain infarction. Patients were a 73-year-old man and an 82-year-old woman who suffered from infarction of the basilar artery and the right middle cerebral artery, respectively. The former was diagnosed as atherothrombotic infarction, but the patient experienced repeated aggravation. The latter was diagnosed as cardioembolic infarction. After both patients took clopidogrel and warfarin for the secondary prevention of stroke, upper airway obstruction developed at Day 29 and Day 19, respectively. Vocal cords in Case 1 did not show any movement on laryngoscopy, and were fixed together nearly closed. In Case 2, vocal cords were again almost fixed together. Bilateral vocal cord paralysis is a common complication of cervical operations, but is rare after ischemic stroke. As patients who have suffered from bilateral vocal cord paralysis are often facing death, we must be careful of wheezing with ischemic stroke.

[Late-onset hemorrhagic infarction in patients with patent foramen ovale: reports of two cases].

We presented here two patients with hemorrhagic infarction occurred during subacute phase of brain embolism. The patients were 71-year-old and 73-year-old men who suffered from brain infarction of the left posterior cerebral artery and right middle cerebral artery territory, respectively. Both of them were diagnosed as having cryptogenic stroke and patent foramen ovale. After transferred to rehabilitation hospitals taking aspirin for a secondary prevention of stroke, they developed hemorrhagic infarction at day 17 and day 19, respectively. Their blood pressure remained within normal range throughout acute and subacute phase. Although most of hemorrhagic infarction occurs within 24 hours of stroke onset, some patients develop symptomatic hemorrhagic infarction even after 10 days. We need to be careful about late-onset hemorrhagic infarction, because many patients are now transferred early to rehabilitation hospitals to facilitate dedicated systematic rehabilitation.

[Brief report: stroke in multiple myeloma patient treated with thalidomide].

We presented a patient suffered from stroke related to thalidomide therapy. The patient was a 74-year-old man who had about two-year history of multiple myeloma and treated with 100 mg of oral thalidomide daily. He was diagnosed as having cryptogenic stroke attributable to patent foramen ovale, when he admitted to our hospital with sudden onset left-side hemiparesis. Antiplatelet and neuroprotective therapies were commenced along with the use of elastic stocking to prevent further embolic event. Then, warfarin was selected as secondary prevention to reduce the risk of paradoxical embolism during thalidomide therapy. Although the risk of deep vein thrombosis on thalidomide therapy has been well documented, only a few cases have been noted documenting the risk of stroke during thalidomide therapy. We need to be careful about the risk of deep vein thrombosis on thalidomide therapy, even as monotherapy, and consider using anticoagulant therapy while prescribing thalidomide.

Increased susceptibility of small intestine to NSAID-provoked ulceration in rats with adjuvant-induced arthritis: involvement of enhanced expression of TLR4.

NSAIDs damage the small intestine as well as the stomach as adverse effects. We previously reported that the gastric ulcerogenic response to NSAIDs was markedly increased in arthritic rats. The present study was designed to examine the intestinal ulcerogenic property of indomethacin in adjuvant-induced arthritic rats in comparison with normal animals. Arthritis was induced in male Dark Agouti rats by injection of Freund's complete adjuvant into the right hindfoot. Two weeks later, indomethacin was given orally and the intestine was examined for lesions at several time points after indomethacin. Indomethacin produced intestinal lesions in both normal and arthritic rats, but in the latter, the ulcerogenic response occurred much earlier and the severity was markedly enhanced. Aminoguanidine, an inhibitor of iNOS, significantly suppressed the damage, yet the efficacy differed in normal and arthritic rats, depending on the dose schedule; the effect of post-administration (6 h after) was greater than that of pre-administration (0.5 h before) in normal rats, whereas that of post-administration was less than that of pre-administration in arthritic rats. The expression of iNOS and TLR4 in the intestine was enhanced in arthritic rats as compared with normal rats. These results suggest that the intestinal ulcerogenic response to indomethacin is markedly aggravated in arthritic rats. Notably, the onset of the ulceration was much earlier in arthritic rats than normal rats. These phenomena may be accounted for by the upregulation of iNOS/NO through the increased expression of TLR4 in the small intestine of arthritic rats.