Repulsive Sema3E-Plexin-D1 signaling coordinates both axonal extension and steering via activating an autoregulatory factor, Mtss1.

Axon guidance molecules are critical for neuronal pathfinding because they regulate directionality and growth pace during nervous system development. However, the molecular mechanisms coordinating proper axonal extension and turning are poorly understood. Here, metastasis suppressor 1 (Mtss1), a membrane protrusion protein, ensured axonal extension while sensitizing axons to the Semaphorin 3E (Sema3E)-Plexin-D1 repulsive cue. Sema3E-Plexin-D1 signaling enhanced Mtss1 expression in projecting striatonigral neurons. Mtss1 localized to the neurite axonal side and regulated neurite outgrowth in cultured neurons. Mtss1 also aided Plexin-D1 trafficking to the growth cone, where it signaled a repulsive cue to Sema3E. Mtss1 ablation reduced neurite extension and growth cone collapse in cultured neurons. Mtss1-knockout mice exhibited fewer striatonigral projections and irregular axonal routes, and these defects were recapitulated in Plxnd1- or Sema3e-knockout mice. These findings demonstrate that repulsive axon guidance activates an exquisite autoregulatory program coordinating both axonal extension and steering during neuronal pathfinding.

Impaired neuronal activity as a potential factor contributing to the underdeveloped cerebrovasculature in a young Parkinson's disease mouse model.

Misfolding of α-synuclein (α-Syn) in the brain causes cellular dysfunction, leading to cell death in a group of neurons, and consequently causes the progression of Parkinson's disease (PD). Although many studies have demonstrated the pathological connections between vascular dysfunction and neurodegenerative diseases, it remains unclear how neuronal accumulation of α-Syn affects the structural and functional aspects of the cerebrovasculature to accelerate early disease progression. Here, we demonstrated the effect of aberrant α-Syn expression on the brain vasculature using a PD mouse model expressing a familial mutant form of human α-Syn selectively in neuronal cells. We showed that young PD mice have an underdeveloped cerebrovasculature without significant α-Syn accumulation in the vasculature. During the early phase of PD, toxic α-Syn was selectively increased in neuronal cells, while endothelial cell proliferation was decreased in the absence of vascular cell death or neuroinflammation. Instead, we observed altered neuronal activation and minor changes in the activity-dependent gene expression in brain endothelial cells (ECs) in young PD mice. These findings demonstrated that neuronal expression of mutant α-Syn in the early stage of PD induces abnormal neuronal activity and contributes to vascular patterning defects, which could be associated with a reduced angiogenic potential of ECs.

Optimized protocol for translatome analysis of mouse brain endothelial cells.

Brain endothelial cells (BECs) are important conduits that deliver oxygen and nutrients, protect parenchyma cells from toxins, and drain wastes to maintain brain homeostasis. Impairment of BECs has been implicated in diverse neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Therefore, molecular analysis of BECs is important for understanding the molecular pathogenesis of these neurological diseases. Even though many transcriptome analyses for BECs have been developed, mRNA levels do not necessarily correlate with the levels of actively translated proteins. Translatome analysis using RiboTag mice, in which Rpl22, a ribosomal component, is tagged by the hemagglutinin epitope under Cre recombinase activation, could serve as an excellent tool that overcomes these caveats. However, implementation of this technique is limited by high noise-to-signal ratios as well as the low yield of mRNAs from BECs, which limits bulk gene expression analysis. In this study, we established a protocol to isolate highly pure mRNAs from BECs in the cortex of eight- to twelve-week-old male Tie2-Cre; Rpl22HA/HA mice by using a cell strainer to trap blood vessels prior to immunoprecipitation. According to the results of RT-PCR, the specificity of the mRNA pools isolated by our protocol was much higher than that of the pools isolated by the standard protocol. We were also able to generate a high-quality cDNA library for RNA-seq with the small amount of mRNA isolated with our protocol. Thus, this optimized method will be useful for future studies of BECs at the molecular level.

Vascular Sema3E-Plexin-D1 Signaling Reactivation Promotes Post-stroke Recovery through VEGF Downregulation in Mice.

Post-stroke vascular remodeling, including angiogenesis, facilitates functional recovery. Proper vascular repair is important for efficient post-stroke recovery; however, the underlying mechanisms coordinating the diverse signaling pathways involved in vascular remodeling remain largely unknown. Recently, axon guidance molecules were revealed as key players in injured vessel remodeling. One such molecule, Semaphorin 3E (Sema3E), and its receptor, Plexin-D1, control vascular development by regulating vascular endothelial growth factor (VEGF) signaling. In this study, using a mouse model of transient brain infarction, we aimed to investigate whether Sema3E-Plexin-D1 signaling was involved in cerebrovascular remodeling after ischemic injury. We found that ischemic damage rapidly induced Sema3e expression in the neurons of peri-infarct regions, followed by Plexin-D1 upregulation in remodeling vessels. Interestingly, Plexin-D1 reemergence was concurrent with brain vessels entering an active angiogenic process. In line with this, Plxnd1 ablation worsened neurological deficits, infarct volume, neuronal survival rate, and blood flow recovery. Furthermore, reduced and abnormal vascular morphogenesis was caused by aberrantly increased VEGF signaling. In Plxnd1 knockout mice, we observed significant extravasation of intravenously administered tracers in the brain parenchyma, junctional protein downregulation, and mislocalization in regenerating vessels. This suggested that the absence of Sema3E-Plexin-D1 signaling is associated with blood-brain barrier (BBB) impairment. Finally, the abnormal behavioral performance, aberrant vascular phenotype, and BBB breakdown defects in Plxnd1 knockout mice were restored following the inhibition of VEGF signaling during vascular remodeling. These findings demonstrate that Sema3E-Plexin-D1 signaling can promote functional recovery by downregulating VEGF signaling in the injured adult brain.

Rho Guanine Nucleotide Exchange Factor 4 (Arhgef4) Deficiency Enhances Spatial and Object Recognition Memory.

Guanine nucleotide exchange factors (GEFs) play multiple functional roles in neurons. In a previous study, we reported that Arhgef4 (Rho guanine nucleotide exchange factor 4) functioned as a negative regulator of the excitatory synaptic function by sequestering postsynaptic density protein 95 (PSD-95). However, the role of Arhgef4 in behavior has not been examined. We performed comprehensive behavioral tests in knockout (KO) mice to investigate of the effects of Arhgef4 deficiency. We found that the expressed PSD-95 particle size was significantly increased in hippocampal neuronal cultures from Arhgef4 KO mice, which is consistent with the previous in vitro findings. Arhgef4 KO mice exhibited general motor activity and anxiety-like behavior comparable to those of the wild type littermates. However, spatial memory and object recognition memory were significantly enhanced in the Arhgef4 KO mice. Taken together, these data confirm the role of Arhgef4 as a negative synaptic regulator at the behavioral level.

17ß-Estradiol Inhibits Lysophosphatidylcholine-Induced Apoptosis in Cultured Vascular Smooth Muscle Cells.

OBJECTIVES: Coronary heart disease (CHD) risk increases in women after menopause, but menopausal hormone therapy (MHT) helps prevent CHD if started early after menopause. To explore the mechanism underlying the direct vascular actions of estrogen, the effects of 17ß-estradiol (E2) on apoptosis of vascular smooth muscle cells (VSMCs) induced with lysophosphatidylcholine (lysoPC), an active component of oxidized low-density lipoprotein, were investigated in the present study. METHODS: VSMCs were isolated from rat aortas. Apoptosis and protein expression of caspases were assessed using propidium iodide staining and Western blot analysis, respectively. Intracellular formation of reactive oxygen species (ROS) was examined using dichlorofluorescein diacetate, a cell-permeable oxidation-sensitive probe, and quantitated with flow cytometry. Nuclear factor-κB (NF-κB) activation was determined after transfection with a reporter plasmid containing the luciferase reporter gene. RESULTS: After pre-treatment for 24 hours, 17ß-E2 suppressed lysoPC-induced (15 µM) apoptotic cell death in a dose-dependent manner with statistical significance at near physiological concentration. 17ß-E2 (10⁻6 M) also increased protein levels of caspase-9 and -8 precursors and decreased the active form of caspase-3. Western blot analysis using subcellular fractions showed that 17ß-E2 decreased mitochondrial Bax levels and concomitantly increased cytosolic Bax expression. Furthermore, intracellular production of ROS and NF-κB-mediated transcriptional activity were reduced with 17ß-E2. In addition, estrogen effects on apoptosis were partially blocked by ICI 182,780, a specific estrogen receptor antagonist. CONCLUSIONS: In cultured VSMCs treated with lysoPC, 17ß-E2 reduced apoptotic cell death by down-regulating both extrinsic and intrinsic apoptosis pathways, contributing to the preventive action of MHT against CHD.

Effects of 17ß-Estradiol on the Plasminogen Activator System in Vascular Smooth Muscle Cells Treated with Lysophophatidylcholine.

OBJECTIVES: When administered soon after menopause, hormone therapy can prevent coronary heart diseases in women. To explore the mechanism underlying the cardioprotective actions of estrogen, we investigated the effects of 17ß-estradiol (17ß-E2) on the plasminogen activator system using cultured vascular smooth muscle cells (VSMCs). METHODS: VSMCs were isolated from rat aortas. Protein expression of plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) were evaluated using Western blotting and enzyme-linked immunosorbent assay, respectively. The enzyme activity of PAI-1 in a conditioned medium was assessed via reverse fibrin overlay zymography and that of t-PA was assessed via fibrin overlay zymography. Gene expression was quantified using real-time reverse transcription-polymerase chain reaction. RESULTS: Following pre-treatment for 24 hours, 17ß-E2 suppressed both protein expression and enzyme activity of PAI-1 stimulated by lysophosphatidylcholine (lysoPC) in a significant and dose-dependent manner at a near physiological concentration. Moreover, 17ß-E2 (10⁻7 M) inhibited PAI-1 gene expression, and ICI 182,780-a specific estrogen receptor antagonist-blocked the effects of 17ß-E2 on the PAI-1 protein. 17ß-E2 did not affect t-PA secretion but significantly enhanced free t-PA activity through reduced binding to PAI-1. Furthermore, 17ß-E2 suppressed intracellular reactive oxygen species production and nuclear factor-κB-mediated transcription. CONCLUSIONS: In VSMCs stimulated with lysoPC, 17ß-E2 reduced PAI-1 expression through a non-receptor-mediated mechanism via antioxidant activity as well as a receptor-mediated mechanism; however, it did not alter t-PA secretion. Of note, 17ß-E2 suppressed PAI-1 activity and concurrently enhanced t-PA activity, suggesting a beneficial influence on fibrinolysis.

Improvement of a slimming cream's efficacy using a novel fabric as a transdermal drug delivery system: An in vivo and in vitro study.

Penetration of any compound into the body from the outside is prevented primarily by the corneal layer of the epidermis. The only way to circumvent the properties of the corneal layer is to disrupt it. Currently, transdermal systems can currently only deliver drugs that are of low molecular weight. The purpose of the present study was to assess the improvement of the slimming cream's efficacy using a novel fabric, with the aim of developing an improved method for transdermal drug delivery. The current study was conducted on four groups of guinea pigs. The control group was untreated, whereas the test groups were treated with either slimming cream and no fabric, slimming cream with 100% cotton fabric or slimming cream with the novel fabric. Ultrasound and microscopic histological analysis were used to assess animals. The results demonstrated that compared with the other groups, the novel fabric group demonstrated the greatest reductions in fat layer thickness, adipocyte size and number and proliferator-activated receptor-γ levels in adipose tissue. Furthermore, the novel fabric also enhanced the transdermal delivery of rhodamine B base and caffeine penetration compared with the control fabric (3.18-fold). In conclusion, the results of the present study demonstrated that the novel fabric can potentially be used to enhance transdermal drug delivery.

Sequestration of synaptic proteins by alpha-synuclein aggregates leading to neurotoxicity is inhibited by small peptide.

α-Synuclein (α-syn) is a major component of Lewy bodies found in synucleinopathies including Parkinson's disease (PD) and Dementia with Lewy Bodies (DLB). Under the pathological conditions, α-syn tends to generate a diverse form of aggregates showing toxicity to neuronal cells and able to transmit across cells. However, mechanisms by which α-syn aggregates affect cytotoxicity in neurons have not been fully elucidated. Here we report that α-syn aggregates preferentially sequester specific synaptic proteins such as vesicle-associated membrane protein 2 (VAMP2) and synaptosomal-associated protein 25 (SNAP25) through direct binding which is resistant to SDS. The sequestration effect of α-syn aggregates was shown in a cell-free system, cultured primary neurons, and PD mouse model. Furthermore, we identified a specific blocking peptide derived from VAMP2 which partially inhibited the sequestration by α-syn aggregates and contributed to reduced neurotoxicity. These results provide a mechanism of neurotoxicity mediated by α-syn aggregates and suggest that the blocking peptide interfering with the pathological role of α-syn aggregates could be useful for designing a potential therapeutic drug for the treatment of PD.

Astrocyte-derived lipocalin-2 mediates hippocampal damage and cognitive deficits in experimental models of vascular dementia.

Lipocalin-2 (LCN2) has diverse functions in multiple pathophysiological conditions; however, its pathogenic role in vascular dementia (VaD) is unknown. Here, we investigated the role of LCN2 in VaD using rodent models of global cerebral ischemia and hypoperfusion with cognitive impairment and neuroinflammation. Mice subjected to transient bilateral common carotid artery occlusion (tBCCAo) for 50 min showed neuronal death and gliosis in the hippocampus at 7 days post-tBCCAo. LCN2 expression was observed predominantly in the hippocampal astrocytes, whereas its receptor was mainly detected in neurons, microglia, and astrocytes. Furthermore, Lcn2-deficient mice, compared with wild-type animals, showed significantly weaker CA1 neuronal loss, cognitive decline, white matter damage, blood-brain barrier permeability, glial activation, and proinflammatory cytokine production in the hippocampus after tBCCAo. Lcn2 deficiency also attenuated hippocampal neuronal death and cognitive decline at 30 days after unilateral common carotid artery occlusion (UCCAo). Furthermore, intracerebroventricular (i.c.v) injection of recombinant LCN2 protein elicited CA1-neuronal death and a cognitive deficit. Our studies using cultured glia and hippocampal neurons supported the decisive role of LCN2 in hippocampal neurotoxicity and microglial activation, and the role of the HIF-1α-LCN2-VEGFA axis of astrocytes in vascular injury. Additionally, plasma levels of LCN2 were significantly higher in patients with VaD than in the healthy control subjects. These results indicate that hippocampal damage and cognitive impairment are mediated by LCN2 secreted from reactive astrocytes in VaD.

Multi-polydioxanone (PDO) scaffold for forehead wrinkle correction: A pilot study.

BACKGROUND: Forehead wrinkles are the result of contracture of the frontalis muscle and the skin aging process. Currently, hyaluronic acid filler and botulinum toxin are the main materials used for correction of these wrinkles. In addition, polydioxanone (PDO) thread has also been applied for this treatment. OBJECTIVE: In order to evaluate the efficacy and safety of multi-PDO scaffold in animal and human skin, we tested PDO insertion in rat and mini-pig models and human volunteers with forehead wrinkles. METHODS: A stent-shaped multi-PDO scaffold was inserted under the panniculus carnosus of rat dorsal skin and the subcutaneous layer of mini-pig dorsal skin and forehead wrinkles in three human volunteers. RESULTS: Histological analysis at 12 weeks revealed evidence of de novo collagen synthesis, which was consistent with clinical results on photo evaluation. CONCLUSION: Stent-shaped multi-PDO scaffolds may be another effective and safe treatment modality for reduction of forehead wrinkles.

Effects of highly concentrated hyaluronic acid filler on nasolabial fold correction: A 24-month extension study.

BACKGROUND: A previous 6-month study using a more highly concentrated novel hyaluronic acid (HA) filler, PP-501-B, found nasolabial fold (NLF) improvements with increased tolerability. OBJECTIVE: We investigated the long-term efficacy, durability and safety of PP-501-B in the correction of NLFs. METHODS: Subjects completing the initial six-month study were enrolled in this 24-month, randomized, multicenter, double-blind, split-face, extension study. The injection areas and treatment procedures were identical to those of the initial study: each subject was injected with PP-501-B in one NLF and with Restylane Perlane (Q-med) in the contralateral NLF. We reassessed wrinkle improvement using the five-point Wrinkle Severity Rating Scale (WSRS) and changes in the Global Aesthetic Improvement Scale at 12, 18 and 24 months after the initial treatment. RESULTS: Of the 81 patients enrolled, 72 completed the study. The WSRS score significantly decreased from baseline throughout the follow-up period after retreatment with both fillers. There was no significant difference in the WSRS scores between the two fillers at 24 months. Both fillers were well tolerated with no severe complications or adverse reactions. CONCLUSION: The new HA filler PP-501-B is safe and effective in the long term for the correction of moderate-to-severe NLFs, even after a second treatment.

1,213 Cases of Treatment of Facial Acne Using Indocyanine Green and Intense Pulsed Light in Asian Skin.

BACKGROUND: Photodynamic therapy (PDT) has been used for acne, with various combinations of photosensitizers and light sources. OBJECTIVE: We evaluated the effectiveness and safety of indocyanine green (ICG) and intense pulsed light (IPL) in the treatment of acne. MATERIALS AND METHODS: A total of 1,213 patients with facial acne were retrospectively reviewed. Patients received three or five treatments of ICG and IPL at two-week intervals. Clinical response to treatment was assessed by comparing pre- and posttreatment clinical photographs and patient satisfaction scores. RESULTS: Marked to excellent improvement was noted in 483 of 1,213 (39.8%) patients, while minimal to moderate improvement was achieved in the remaining 730 (60.2%) patients. Patient satisfaction scores revealed that 197 (16.3%) of 1,213 patients were highly satisfied, 887 (73.1%) were somewhat satisfied, and 129 (10.6%) were unsatisfied. There were no significant side effects. CONCLUSION: These results suggest that PDT with ICG and IPL can be effectively and safely used in the treatment of acne.

Neuropilin-1 functions as a VEGFR2 co-receptor to guide developmental angiogenesis independent of ligand binding.

During development, tissue repair, and tumor growth, most blood vessel networks are generated through angiogenesis. Vascular endothelial growth factor (VEGF) is a key regulator of this process and currently both VEGF and its receptors, VEGFR1, VEGFR2, and Neuropilin1 (NRP1), are targeted in therapeutic strategies for vascular disease and cancer. NRP1 is essential for vascular morphogenesis, but how NRP1 functions to guide vascular development has not been completely elucidated. In this study, we generated a mouse line harboring a point mutation in the endogenous Nrp1 locus that selectively abolishes VEGF-NRP1 binding (Nrp1(VEGF-)). Nrp1(VEGF-) mutants survive to adulthood with normal vasculature revealing that NRP1 functions independent of VEGF-NRP1 binding during developmental angiogenesis. Moreover, we found that Nrp1-deficient vessels have reduced VEGFR2 surface expression in vivo demonstrating that NRP1 regulates its co-receptor, VEGFR2. Given the resources invested in NRP1-targeted anti-angiogenesis therapies, our results will be integral for developing strategies to re-build vasculature in disease.

Midbrain dopamine neurons sustain inhibitory transmission using plasma membrane uptake of GABA, not synthesis.

Synaptic transmission between midbrain dopamine neurons and target neurons in the striatum is essential for the selection and reinforcement of movements. Recent evidence indicates that nigrostriatal dopamine neurons inhibit striatal projection neurons by releasing a neurotransmitter that activates GABAA receptors. Here, we demonstrate that this phenomenon extends to mesolimbic afferents, and confirm that the released neurotransmitter is GABA. However, the GABA synthetic enzymes GAD65 and GAD67 are not detected in midbrain dopamine neurons. Instead, these cells express the membrane GABA transporters mGAT1 (Slc6a1) and mGAT4 (Slc6a11) and inhibition of these transporters prevents GABA co-release. These findings therefore indicate that GABA co-release is a general feature of midbrain dopaminergic neurons that relies on GABA uptake from the extracellular milieu as opposed to de novo synthesis. This atypical mechanism may confer dopaminergic neurons the flexibility to differentially control GABAergic transmission in a target-dependent manner across their extensive axonal arbors.DOI: http://dx.doi.org/10.7554/eLife.01936.001.

Establishment of neurovascular congruency in the mouse whisker system by an independent patterning mechanism.

Nerves and vessels often run parallel to one another, a phenomenon that reflects their functional interdependency. Previous studies have suggested that neurovascular congruency in planar tissues such as skin is established through a "one-patterns-the-other" model, in which either the nervous system or the vascular system precedes developmentally and then instructs the other system to form using its established architecture as a template. Here, we find that, in tissues with complex three-dimensional structures such as the mouse whisker system, neurovascular congruency does not follow the previous model but rather is established via a mechanism in which nerves and vessels are patterned independently. Given the diversity of neurovascular structures in different tissues, guidance signals emanating from a central organizer in the specific target tissue may act as an important mechanism to establish neurovascular congruency patterns that facilitate unique target tissue function.