Supplementation with Ashitaba (Angelica keiskei) Yellow Stem Exudate Prevents Aging-Induced Thrombotic Tendencies and Systemic Inflammation Without Affecting Body Weight Gain in Mice.

Angelica keiskei Koidzumi (Ashitaba) is a traditional folk medicine and health supplement in Japan. Ashitaba yellow stem exudate (AYE) contains abundant chalcones and thus has the potential to treat and prevent many pathological states such as cancer, inflammation, obesity, diabetics, thrombosis, and hypertension. Levels of plasminogen activator inhibitor 1 (PAI-1), a key regulator of the fibrinolytic system, increase with age in mouse plasma. Therefore, we aimed to determine the effects of AYE on plasma thrombotic parameters in aging mice. Long-term (52 weeks) AYE supplementation significantly decreased age-induced increases of PAI-1 in mouse plasma. Supplementation with AYE decreased levels of the acute-phase and fibrinolytic protein plasma plasminogen, and significantly decreased those of tumor necrosis factor α. These results suggested that continuous intake of AYE throughout life decreases age-induced systemic inflammation and prevents thrombotic tendencies without affecting body weight gain in aged mice. Our findings showed that supplementing diets with AYE might help to prevent thrombotic diseases in elderly individuals.

A mosaic adeno-associated virus vector as a versatile tool that exhibits high levels of transgene expression and neuron specificity in primate brain.

Recent emphasis has been placed on gene transduction mediated through recombinant adeno-associated virus (AAV) vector to manipulate activity of neurons and their circuitry in the primate brain. In the present study, we created a novel vector of which capsid was composed of capsid proteins derived from both of the AAV serotypes 1 and 2 (AAV1 and AAV2). Following the injection into the frontal cortex of macaque monkeys, this mosaic vector, termed AAV2.1 vector, was found to exhibit the excellence in transgene expression (for AAV1 vector) and neuron specificity (for AAV2 vector) simultaneously. To explore its applicability to chemogenetic manipulation and in vivo calcium imaging, the AAV2.1 vector expressing excitatory DREADDs or GCaMP was injected into the striatum or the visual cortex of macaque monkeys, respectively. Our results have defined that such vectors secure intense and stable expression of the target proteins and yield conspicuous modulation and imaging of neuronal activity.

Effects of the factor Xa inhibitor rivaroxaban on the differentiation of endothelial progenitor cells.

BACKGROUND: We evaluated the efficacy of the factor Xa inhibitor rivaroxaban on the differentiation ability of vascular endothelial progenitor cells (EPCs), which play roles in vascular injury repair and atherogenesis. Antithrombotic treatment in patients with atrial fibrillation undergoing percutaneous coronary intervention (PCI) is challenging, and current guidelines recommend oral anticoagulant monotherapy 1 year or more after PCI. However, biological evidence of the pharmacological effects of anticoagulants is insufficient. METHODS: EPC colony-forming assays were performed using peripheral blood-derived CD34-positive cells from healthy volunteers. Adhesion and tube formation of cultured EPCs were assessed in human umbilical cord-derived CD34-positive cells. Endothelial cell surface markers were assessed using flow cytometry, and Akt and endothelial nitric oxide synthase (eNOS) phosphorylation were examined using western blot analysis of EPCs. Adhesion, tube formation and endothelial cell surface marker expression was observed in EPCs transfected with small interfering RNA (siRNA) against protease-activated receptor (PAR)-2. Finally, EPC behaviors were assessed in patients with atrial fibrillation undergoing PCI in whom warfarin was changed to rivaroxaban. RESULTS: Rivaroxaban increased the number of large EPC colonies and increased the bioactivities of EPCs, including adhesion and tube formation. Rivaroxaban also increased vascular endothelial growth factor receptor (VEGFR)-1, VEGFR-2, Tie-2, and E-selectin expression as well as Akt and eNOS phosphorylation. PAR-2 knockdown increased the bioactivities of EPCs and endothelial cell surface marker expression. Patients in whom the number of large colonies increased after switching to rivaroxaban showed better vascular repair. CONCLUSIONS: Rivaroxaban increased the differentiation ability of EPCs, leading to potential advantages in the treatment of coronary artery disease.

BBB opening with focused ultrasound in nonhuman primates and Parkinson's disease patients: Targeted AAV vector delivery and PET imaging.

Intracerebral vector delivery in nonhuman primates has been a major challenge. We report successful blood-brain barrier opening and focal delivery of adeno-associated virus serotype 9 vectors into brain regions involved in Parkinson's disease using low-intensity focus ultrasound in adult macaque monkeys. Openings were well tolerated with generally no associated abnormal magnetic resonance imaging signals. Neuronal green fluorescent protein expression was observed specifically in regions with confirmed blood-brain barrier opening. Similar blood-brain barrier openings were safely demonstrated in three patients with Parkinson's disease. In these patients and in one monkey, blood-brain barrier opening was followed by 18F-Choline uptake in the putamen and midbrain regions based on positron emission tomography. This indicates focal and cellular binding of molecules that otherwise would not enter the brain parenchyma. The less-invasive nature of this methodology could facilitate focal viral vector delivery for gene therapy and might allow early and repeated interventions to treat neurodegenerative disorders.

Local transplantation of syngeneic adipose stromal vascular fraction ameliorates damaged anal sphincter function in a rat model of vaginal distension.

BACKGROUND: The adipose stromal vascular fraction contains abundant mesenchymal stem cells and is utilized for cell therapy of male stress urinary incontinence. The purpose of this paper was to explore the effect of local transplantation of the stromal vascular fraction on improvement of damaged anal sphincter function. METHODS: A rat model of vaginal distension was used as a model of damaged anal sphincter function. The adipose stromal vascular fraction was separated from the inguinal fat of syngeneic green fluorescent protein transgenic rats and delivered into the internal anal sphincter of vaginal distension rats. The maximum resting pressure was evaluated during insertion and withdrawal of the catheter at 4 or 10 days after vaginal distension treatment to estimate anal sphincter function. Green fluorescent protein-transfected human-adipose-derived mesenchymal stem cells were transplanted into the internal anal sphincter of nude rats. Hematoxylin-eosin and Masson trichrome staining were performed to evaluate tissue damage and collagen synthesis. Transplanted cells were identified using a green fluorescent protein antibody and a human-specific antibody. Activation of the transplanted human-ADSC was evaluated by quantitative RT-PCR RESULTS: The mean maximum resting pressure (during catheter withdrawal) of vaginal distension rats was significantly lower than that of control rats, and stromal vascular fraction injection normalized it 4 days after treatment (control: 5.66 ± 0.98, vaginal distension: 4.04 ± 1.28, vaginal distension + stromal vascular fraction: 5.92 ± 1.28 [mmHg, control versus vaginal distension: P = .039; vaginal distension versus vaginal distension + stromal vascular fraction: P = .007]). Histological examination showed that vaginal distension disrupted the internal anal sphincter, and the transplanted syngeneic stromal vascular fraction survived for 10 days. Transplanted xenogeneic human-adipose-derived mesenchymal stem cells survived in the internal anal sphincter of nude rats for 4 and 10 days. Genes related to extracellular remodeling were up-regulated in the transplanted human-adipose-derived mesenchymal stem cells CONCLUSION: Syngeneic and heterotopic transplanted adipose-derived mesenchymal stem cells engrafted in the internal anal sphincter and ameliorated damaged anal sphincter function in a rat model of vaginal distension.

Chemogenetic Disconnection between the Orbitofrontal Cortex and the Rostromedial Caudate Nucleus Disrupts Motivational Control of Goal-Directed Action.

The orbitofrontal cortex (OFC) and its major downstream target within the basal ganglia-the rostromedial caudate nucleus (rmCD)-are involved in reward-value processing and goal-directed behavior. However, a causal contribution of the pathway linking these two structures to goal-directed behavior has not been established. Using the chemogenetic technology of designer receptors exclusively activated by designer drugs with a crossed inactivation design, we functionally and reversibly disrupted interactions between the OFC and rmCD in two male macaque monkeys. We injected an adeno-associated virus vector expressing an inhibitory designer receptor, hM4Di, into the OFC and contralateral rmCD, the expression of which was visualized in vivo by positron emission tomography and confirmed by postmortem immunohistochemistry. Functional disconnection of the OFC and rmCD resulted in a significant and reproducible loss of sensitivity to the cued reward value for goal-directed action. This decreased sensitivity was most prominent when monkeys had accumulated a certain amount of reward. These results provide causal evidence that the interaction between the OFC and the rmCD is needed for motivational control of action on the basis of the relative reward value and internal drive. This finding extends the current understanding of the physiological basis of psychiatric disorders in which goal-directed behavior is affected, such as obsessive-compulsive disorder.SIGNIFICANCE STATEMENT In daily life, we routinely adjust the speed and accuracy of our actions on the basis of the value of expected reward. Abnormalities in these kinds of motivational adjustments might be related to behaviors seen in psychiatric disorders such as obsessive-compulsive disorder. In the current study, we show that the connection from the orbitofrontal cortex to the rostromedial caudate nucleus is essential for motivational control of action in monkeys. This finding expands our knowledge about how the primate brain controls motivation and behavior and provides a particular insight into disorders like obsessive-compulsive disorder in which altered connectivity between the orbitofrontal cortex and the striatum has been implicated.

Establishment of mouse model of inherited PIGO deficiency and therapeutic potential of AAV-based gene therapy.

Inherited glycosylphosphatidylinositol (GPI) deficiency (IGD) is caused by mutations in GPI biosynthesis genes. The mechanisms of its systemic, especially neurological, symptoms are not clarified and fundamental therapy has not been established. Here, we report establishment of mouse models of IGD caused by PIGO mutations as well as development of effective gene therapy. As the clinical manifestations of IGD are systemic and lifelong lasting, we treated the mice with adeno-associated virus for homology-independent knock-in as well as extra-chromosomal expression of Pigo cDNA. Significant amelioration of neuronal phenotypes and growth defect was achieved, opening a new avenue for curing IGDs.

Supervised machine learning algorithm identified KRT20, BATF and TP63 as biologically relevant biomarkers for bladder biopsy specimens from interstitial cystitis/bladder pain syndrome patients.

OBJECTIVES: This study was carried out to identify biomarkers that distinguish Hunner-type interstitial cystitis from non-Hunner-type interstitial cystitis patients. METHODS: Total ribonucleic acid was purified from 212 punch biopsy specimens of 89 individuals who were diagnosed as interstitial cystitis/bladder pain syndrome. To examine the expression profile of patients' bladder specimens, 68 urothelial master transcription factors and nine known markers (E-cadherin, cytokeratins, uroplakins and sonic hedgehog) were selected. To classify the biopsy samples, principal component analysis was carried out. A decision tree algorithm was adopted to identify critical determinants, in which 102 and 116 bladder specimens were used for learning and validation, respectively. RESULTS: Principal component analysis segregated tissues from Hunner-type and non-Hunner-type interstitial cystitis specimens in principal component axes 2 and 4. Principal components 2 and 4 contained urothelial stem/progenitor transcription factors and cytokeratins, respectively. A decision tree identified KRT20, BATF and TP63 to classify non-Hunner-type and Hunner-type interstitial cystitis specimens. KRT20 was lower in tissues from Hunner-type compared with non-Hunner-type interstitial cystitis specimens (P < 0.001). TP63 was lower in Hunner's lesions compared with adjacent mucosa from Hunner-type interstitial cystitis patients (P < 0.001). Blinded validation using additional biopsy specimens verified that the decision tree showed fairly precise concordance with cystoscopic diagnosis. CONCLUSION: KRT20, BATF and TP63 were identified as biologically relevant biomarkers to classify tissues from interstitial cystitis/bladder pain syndrome specimens. The biologically explainable determinants could contribute to defining the elusive interstitial cystitis/bladder pain syndrome pathogenesis.

Development of Non-alcoholic Steatohepatitis and Nodular Regenerative Hyperplasia in C57BL/6J Mice Fed a Fructose-containing Western Diet.

BACKGROUND/AIM: We generated a novel disease mouse model in which a fructose-containing western diet (FD) induces development of non-alcoholic steatohepatitis (NASH). MATERIALS AND METHODS: C57BL/6J mice were fed FD for 60 weeks and body weight and blood pressure were monitored. Plasma cholesterol level was measured at the end of the experiments. Histopathology of NASH was examined by hematoxylin and eosin staining, Masson-Trichrome staining, periodic acid-Schiff staining, and immunohistochemistry against a proliferation marker. Circadian gene expression levels were compared by sampling the livers in 4-h intervals, followed by quantitative RT-PCR analysis. RESULTS: FD-fed mice developed obesity, transient hypertension, hypercholesterolemia, and liver adiposity. The mice spontaneously developed hepatic nodules, which were diagnosed as non-neoplastic nodular regenerative hyperplasia. FD-fed mice had increased expression of growth factor genes and cirrhosis markers compared to control mice. Circadian expression of lipid metabolism genes was deregulated by FD intake. CONCLUSION: C57BL/6J mice fed FD developed non-alcoholic steatohepatitis and nodular regenerative hyperplasia over time.

Neuronal XRN1 is required for maintenance of whole-body metabolic homeostasis.

Control of mRNA stability and degradation is essential for appropriate gene expression, and its dysregulation causes various disorders, including cancer, neurodegenerative diseases, diabetes, and obesity. The 5'-3' exoribonuclease XRN1 executes the last step of RNA decay, but its physiological impact is not well understood. To address this, forebrain-specific Xrn1 conditional knockout mice (Xrn1-cKO) were generated, as Xrn1 null mice were embryonic lethal. Xrn1-cKO mice exhibited obesity with leptin resistance, hyperglycemia, hyperphagia, and decreased energy expenditure. Obesity resulted from dysregulated communication between the central nervous system and peripheral tissues. Moreover, expression of mRNAs encoding proteins that regulate appetite and energy expenditure was dysregulated in the hypothalamus of Xrn1-cKO mice. Therefore, we propose that XRN1 function in the hypothalamus is critical for maintenance of metabolic homeostasis.

Chemogenetic activation of nigrostriatal dopamine neurons in freely moving common marmosets.

To interrogate particular neuronal pathways in nonhuman primates under natural and stress-free conditions, we applied designer receptors exclusively activated by designer drugs (DREADDs) technology to common marmosets. We injected adeno-associated virus vectors expressing the excitatory DREADD hM3Dq into the unilateral substantia nigra (SN) in four marmosets. Using multi-tracer positron emission tomography imaging, we detected DREADD expression in vivo, which was confirmed in nigrostriatal dopamine neurons by immunohistochemistry, as well as by assessed activation of the SN following agonist administration. The marmosets rotated in a contralateral direction relative to the activated side 30-90 min after consuming food containing the highly potent DREADD agonist deschloroclozapine (DCZ) but not on the following days without DCZ. These results indicate that non-invasive and reversible DREADD manipulation will extend the utility of marmosets as a primate model for linking neuronal activity and natural behavior in various contexts.

Specific gene expression in unmyelinated dorsal root ganglion neurons in nonhuman primates by intra-nerve injection of AAV 6 vector.

Adeno-associated virus 6 (AAV6) has been proposed as a potential vector candidate for specific gene expression in pain-related dorsal root ganglion (DRG) neurons, but this has not been confirmed in nonhuman primates. The aim of our study was to analyze the transduction efficiency and target specificity of this viral vector in the common marmoset by comparing it with those in the rat. When green fluorescent protein-expressing serotype-6 vector was injected into the sciatic nerve, the efficiency of gene expression in DRG neurons was comparable in both species. We found that the serotype-6 vector was largely specific to the pain-related ganglion neurons in the marmoset, as well as in the rat, whereas the serotype-9 vector resulted in contrasting effects in the two species. Neither AAV6 nor AAV9 resulted in DRG toxicity when administered via the sciatic nerve, suggesting this as a safer route of sensory nerve transduction than the currently used intrathecal or intravenous administrative routes. Furthermore, the AAV6 vector could be an optimal serotype for gene therapy for human chronic pain that has a minimal effect on other somatosensory functions of DRG neurons.

Retrograde Transgene Expression via Neuron-Specific Lentiviral Vector Depends on Both Species and Input Projections.

For achieving retrograde gene transfer, we have so far developed two types of lentiviral vectors pseudotyped with fusion envelope glycoprotein, termed HiRet vector and NeuRet vector, consisting of distinct combinations of rabies virus and vesicular stomatitis virus glycoproteins. In the present study, we compared the patterns of retrograde transgene expression for the HiRet vs. NeuRet vectors by testing the cortical input system. These vectors were injected into the motor cortex in rats, marmosets, and macaques, and the distributions of retrograde labels were investigated in the cortex and thalamus. Our histological analysis revealed that the NeuRet vector generally exhibits a higher efficiency of retrograde gene transfer than the HiRet vector, though its capacity of retrograde transgene expression in the macaque brain is unexpectedly low, especially in terms of the intracortical connections, as compared to the rat and marmoset brains. It was also demonstrated that the NeuRet but not the HiRet vector displays sufficiently high neuron specificity and causes no marked inflammatory/immune responses at the vector injection sites in the primate (marmoset and macaque) brains. The present results indicate that the retrograde transgene efficiency of the NeuRet vector varies depending not only on the species but also on the input projections.

Surgical Injury and Ischemia Prime the Adipose Stromal Vascular Fraction and Increase Angiogenic Capacity in a Mouse Limb Ischemia Model.

The adipose-derived stromal vascular fraction (SVF) is an effective source for autologous cell transplantation. However, the quality and quantity of SVFs vary depending on the patient's age, complications, and other factors. In this study, we developed a method to reproducibly increase the cell number and improve the quality of adipose-derived SVFs by surgical procedures, which we term "wound repair priming." Subcutaneous fat from the inguinal region of BALB/c mice was surgically processed (primed) by mincing adipose parenchyma (injury) and ligating the subcutaneous fat-feeding artery (ischemia). SVFs were isolated on day 0, 1, 3, 5, or 7 after the priming procedures. Gene expression levels of the primed SVFs were measured via microarray and pathway analyses which were performed for differentially expressed genes. Changes in cellular compositions of primed SVFs were analyzed by flow cytometry. SVFs were transplanted into syngeneic ischemic hindlimbs to measure their angiogenic and regeneration potential. Hindlimb blood flow was measured using a laser Doppler blood perfusion imager, and capillary density was quantified by CD31 staining of ischemic tissues. Stabilization of HIF-1 alpha and VEGF-A synthesis in the SVFs were measured by fluorescent immunostaining and Western blotting, respectively. As a result, the number of SVFs per fat weight was increased significantly on day 7 after priming. Among the differentially expressed genes were innate immunity-related signals on both days 1 and 3 after priming. In primed SVFs, the CD45-positive blood mononuclear cell fraction decreased, and the CD31-CD45-double negative mesenchymal cell fraction increased on day 7. The F4/80-positive macrophage fraction was increased on days 1 and 7 after priming. There was a serial decrease in the mesenchymal-gated CD34-positive adipose progenitor fraction and mesenchymal-gated CD140A-positive/CD9-positive preadipocyte fraction on days 1 and 3. Transplantation of primed SVFs resulted in increased capillary density and augmented blood flow, improving regeneration of the ischemic limbs. HIF-1 alpha was stabilized in the primed cutaneous fat in situ, and VEGF-A synthesis of the primed SVFs was on a peak on 5 days after priming. Wound repair priming thus resulted in SVFs with increased number and augmented angiogenic potential.

Optogenetic recruitment of spinal reflex pathways from large-diameter primary afferents in non-transgenic rats transduced with AAV9/Channelrhodopsin 2.

KEY POINTS: We demonstrated optical activation of primary somatosensory afferents with high selectivity to fast-conducting fibres by means of adeno-associated virus 9 (AAV9)-mediated gene transduction in dorsal root ganglion (DRG) neurons. AVV9 expressing green fluorescent protein showed high selectivity and transduction efficiency for fast-conducting, large-sized DRG neurons. Compared with conventional electrical stimulation, optically elicited volleys in primary afferents had higher sensitivity with stimulus amplitude, but lower sensitivity with stimulus frequency. Optically elicited dorsal root volleys activated postsynaptic neurons in the segmental spinal pathway. This proposed technique will help establish the causal relationships between somatosensory afferent inputs and neural responses in the CNS as well as behavioural outcomes in higher mammals where transgenic animals are not available. ABSTRACT: Previously, fundamental structures and their mode of action in the spinal reflex circuit were determined by confirming their input-output relationship using electrophysiological techniques. In those experiments, the electrical stimulation of afferent fibres was used as a core element to identify different types of reflex pathways; however, a major disadvantage of this technique is its non-selectivity. In this study, we investigated the selective activation of large-diameter afferents by optogenetics combined with a virus vector transduction technique (injection via the sciatic nerve) in non-transgenic male Jcl:Wistar rats. We found that green fluorescent protein gene transduction of rat dorsal root ganglion (DRG) neurons with a preference for medium-to-large-sized cells was achieved using the adeno-associated virus 9 (AAV9) vector compared with the AAV6 vector (P = 0.021). Furthermore, the optical stimulation of Channelrhodopsin 2 (ChR2)-expressing DRG neurons (transduced by AAV9) produced compound action potentials in afferent nerves originating from fast-conducting nerve fibres. We also confirmed that physiological responses to different stimulus amplitudes were comparable between optogenetic and electrophysiological activation. However, compared with electrically elicited responses, the optically elicited responses had lower sensitivity with stimulus frequency. Finally, we showed that afferent volleys evoked by optical stimulation were sufficient to activate postsynaptic neurons in the spinal reflex arc. These results provide new ways for understanding the role of sensory afferent input to the central nervous system regarding behavioural control, especially when genetically manipulated animals are not available, such as higher mammals including non-human primates.

Cancer-associated fibroblasts show heterogeneous gene expression and induce vascular endothelial growth factor A (VEGFA) in response to environmental stimuli.

AIM: Cancer-associated fibroblasts (CAF) play a crucial role in angiogenesis in the complex tumor microenvironment. However, fibroblasts show extensive heterogeneity and their dynamic functions against stressors remain largely unknown. METHODS: We collected patient-derived CAF and carried out perturbation-based monitoring of the dynamic functions. Clinically relevant experimental stimuli were defined as follows: hypoxia, cisplatin, fluorouracil, coculture with cancer spheroids (interaction through paracrine signals). We selected 18 marker genes that encode components for fibroblast activation, intracellular communication, and extracellular matrix remodeling. Quantitative reverse transcription polymerase chain reaction was carried out for data collection and statistical analyses were carried out using SPSS software. RESULTS: Kruskal-Wallis multivariate analysis of variance showed that variations in expression of 11 marker genes were explained, in part, by a difference in tissue of origin. Friedman and two-sided Wilcoxon signed rank tests detected significant perturbations in expression of marker genes. Paracrine signal from cancer spheroids induced vascular endothelial growth factor A (VEGFA) in CAF but not in fetal lung fibroblasts. CONCLUSION: We have established perturbation-based monitoring of patients' CAF. Further data collection and individual patient follow up is ongoing to identify critical determinants of disease outcome.

Recruitment of calbindin into nigral dopamine neurons protects against MPTP-Induced parkinsonism.

BACKGROUND: Parkinson's disease is caused by dopamine deficiency in the striatum, which is a result of loss of dopamine neurons from the substantia nigra pars compacta. There is a consensus that a subpopulation of nigral dopamine neurons that expresses the calcium-binding protein calbindin is selectively invulnerable to parkinsonian insults. The objective of the present study was to test the hypothesis that dopamine neuron degeneration might be prevented by viral vector-mediated gene delivery of calbindin into the dopamine neurons that do not normally contain it. METHODS: A calbindin-expressing adenoviral vector was injected into the striatum of macaque monkeys to be conveyed to cell bodies of nigral dopamine neurons through retrograde axonal transport, or the calbindin-expressing lentiviral vector was injected into the nigra directly because of its predominant uptake from cell bodies and dendrites. The animals in which calbindin was successfully recruited into nigral dopamine neurons were administered systemically with MPTP. RESULTS: In the monkeys that had received unilateral vector injections, parkinsonian motor deficits, such as muscular rigidity and akinesia/bradykinesia, appeared predominantly in the limbs corresponding to the non-calbindin-recruited hemisphere after MPTP administration. Data obtained from tyrosine hydroxylase immunostaining and PET imaging for the dopamine transporter revealed that the nigrostriatal dopamine system was preserved better on the calbindin-recruited side. Conversely, on the non-calbindin-recruited control side, many more dopamine neurons expressed α-synuclein. CONCLUSIONS: The present results indicate that calbindin recruitment into nigral dopamine neurons protects against the onset of parkinsonian insults, thus providing a novel approach to PD prevention. © 2018 International Parkinson and Movement Disorder Society.

Enhancement of the transduction efficiency of a lentiviral vector for neuron-specific retrograde gene delivery through the point mutation of fusion glycoprotein type E.

BACKGROUND: Pseudotyping of a lentiviral vector with fusion glycoproteins composed of rabies virus glycoprotein (RVG) and vesicular stomatitis virus glycoprotein (VSVG) segments achieves high gene transfer efficiency through retrograde transport in the nervous system. In our previous study, we determined the junction of RVG/VSVG segments of glycoproteins that enhances the transduction efficiency of the neuron-specific retrograde gene transfer (NeuRet) vector (termed fusion glycoprotein type E or FuG-E). NEW METHOD: We aimed to optimize the amino acid residue at position 440 in the membrane-proximal region of FuG-E to improve the efficiency of retrograde gene transfer in the brain. RESULTS: We constructed variants of FuG-E with 18 kinds of single amino acid substitutions at residue 440 to generate lentiviral vectors pseudotyped with these variants, and tested in vivo gene transfer of the vectors in the mouse brain. The FuG-E (P440E) variant, in which proline was substituted by glutamate at residue 440 in FuG-E, showed the greatest retrograde gene transfer efficiency in the brain, bearing the property of the NeuRet vector. The FuG-E (P440E) pseudotype also displayed efficient retrograde gene transfer in the common marmoset brain. COMPARISON WITH EXISTING METHODS: The NeuRet vector with the FuG-E (P440E) variant demonstrated higher retrograde gene transfer efficiency into different neural pathways compared with the parental FuG-E vector. CONCLUSIONS: The FuG-E (P440E) pseudotype provides a powerful tool to investigate neural circuit mechanisms underlying various brain functions and for gene therapy trials of neurological and neurodegenerative diseases.

Expression profile of urothelial transcription factors in bladder biopsies with interstitial cystitis.

OBJECTIVES: To characterize interstitial cystitis pathology based on the expression profile of urothelial tissue-specific master transcription factors. METHODS: Bladder carcinoma cell lines derived from the urothelial stem cells (epithelial or mesenchymal) were used to identify candidate urothelial master transcription factors. Gene expression was measured with quantitative reverse transcription polymerase chain reaction. From the initial screening of 170 transcription factors (human homologs of Drosophila segmentation genes and known master transcription factors from a database), 28 transcription factors were selected. Subsequently, messenger ribonucleic acid from bladder biopsies of interstitial cystitis patients was purified, and gene expression levels of known urothelial marker genes and candidate master transcription factors were measured. Multivariate expression data were analyzed with spss software. RESULTS: Factor analysis decomposed the expression profile into four axes: principal axis 1 included retinoic acid receptors and 17 candidate master transcription factors. Principal axis 2 included KRT5 and five candidates. Principal axis 3 included transcription factor TP63 and two candidates. Principal axis 4 included SHH and two candidates. Principal component analysis segregated biopsies from Hunner's lesion in the principal component 1 (retinoic acid)/principal component 2 (SOX13)/principal component 3 (TP63) space. CONCLUSIONS: Urothelial master transcription factors could serve as novel diagnostic markers and potentially explain the molecular pathology of interstitial cystitis.

The use of an optimized chimeric envelope glycoprotein enhances the efficiency of retrograde gene transfer of a pseudotyped lentiviral vector in the primate brain.

Lentiviral vectors have been used not only for various basic research experiments, but also for a wide range of gene therapy trials in animal models. The development of a pseudotyped lentiviral vector with the property of retrograde infection allows us to introduce foreign genes into neurons that are localized in regions innervating the site of vector injection. Here, we report the efficiency of retrograde gene transfer of a recently developed FuG-E pseudotyped lentiviral vector in the primate brain by comparing its transduction pattern with that of the parental FuG-C pseudotyped vector. After injection of the FuG-E vector encoding green fluorescent protein (GFP) into the striatum of macaque monkeys, many GFP-immunoreactive neurons were found in regions projecting to the striatum, such as the cerebral cortex, thalamus, and substantia nigra. Quantitative analysis revealed that in all regions, the number of neurons retrogradely transduced with the FuG-E vector was larger than in the FuG-C vector injection case. It was also confirmed that the FuG-E vector displayed explicit neuronal specificity to the same extent as the FuG-C vector. This vector might promote approaches to pathway-selective gene manipulation and provide a powerful tool for effective gene therapy trials against neurological disorders through enhanced retrograde delivery.