Human Adipose Tissue-Derived Stem Cells Inhibit Coronary Artery Vasculitis in a Mouse Model of Kawasaki Disease.

BACKGROUND: Adipose tissue-derived mesenchymal stem cells (ADSCs) are used for the treatment of various diseases because of their rapid proliferation and high anti-inflammatory and tissue repair properties. Kawasaki disease is a systemic vasculitis with coronary arteritis and aneurysms occurring in pediatric patients. In this study, we examined serologically and pathologically whether the administration of human ADSCs (hADSCs) to a mouse model of Kawasaki disease could suppress vasculitis. METHODS: Candida albicans water-soluble fractions were intraperitoneally injected into DBA/2 mice for 5 consecutive days to generate a mouse model of Kawasaki disease. The model mice were intravenously administered hADSCs or phosphate-buffered saline (PBS). Serum samples collected on days 15 and 29 were used to compare cytokine levels. Mouse hearts dissected on day 29 were subjected to hematoxylin and eosin and immunohistological staining using Galectin-1 (Gal-1), a protein involved in cardiovascular homeostasis, and CD44, a cell-surface marker of hADSCs. RESULTS: Comparison of inflammation-related cytokines showed a significant decrease in IL-1α expression at day 15 (P<0.05) and IL-6 expression at day 29 (P<0.01) in the hADSCs-treated group compared to the PBS group. Evaluation by hematoxylin and eosin staining showed decreased inflammatory cell infiltration and a tendency towards increased Gal-1 expression in the hADSCs group. CD44 expression was not observed in both the groups. The survival curve showed that the hADSCs group had a significantly longer survival time (P<0.05). CONCLUSIONS: The present experimental results indicate that hADSCs have an early anti-inflammatory effect, and that Gal-1 may be involved in preventing inflammation and reducing tissue damage.

Avian adeno-associated virus as an anterograde transsynaptic vector.

BACKGROUND: Retrograde and anterograde transsynaptic viral vectors are useful tools for studying the input and output organization of neuronal circuitry, respectively. While retrograde transsynaptic viral vectors are widely used, viral vectors that show anterograde transsynaptic transduction are not common. NEW METHOD: We chose recombinant avian adeno-associated virus (A3V) carrying the mCherry gene and injected it into the eyeball, cochlear duct, and midbrain auditory center of chickens. We observed different survival times to examine the virus transcellular transport and the resulting mCherry expression. To confirm the transcellular transduction mode, we co-injected A3V and cholera toxin B subunit. RESULTS: Injecting A3V into the eyeball and cochlea labeled neurons in the visual and auditory pathways, respectively. Second-, and third-order labeling occurred approximately two and seven days, respectively, after injection into the midbrain. The distribution of labeled neurons strongly suggests that A3V transport is preferentially anterograde and transduces postsynaptic neurons. COMPARISON WITH EXISTING METHOD(S): A3V displays no extrasynaptic leakage and moderate speed of synapse passage, which is better than other viruses previously reported. Compared with AAV1&9, which have been shown to pass one synapse anterogradely, A3V passes several synapses in the anterograde direction. CONCLUSIONS: A3V would be a good tool to study the topographic organization of projection axons and their target neurons.

trans-Fatty acids promote p53-dependent apoptosis triggered by cisplatin-induced DNA interstrand crosslinks via the Nox-RIP1-ASK1-MAPK pathway.

trans-Fatty acids (TFAs) are food-derived fatty acids associated with various diseases including cardiovascular diseases. However, the underlying etiology is poorly understood. Here, we show a pro-apoptotic mechanism of TFAs such as elaidic acid (EA), in response to DNA interstrand crosslinks (ICLs) induced by cisplatin (CDDP). We previously reported that TFAs promote apoptosis induced by doxorubicin (Dox), a double strand break (DSB)-inducing agent, via a non-canonical apoptotic pathway independent of tumor suppressor p53 and apoptosis signal-regulating kinase (ASK1), a reactive oxygen species (ROS)-responsive kinase. However, here we found that in the case of CDDP-induced apoptosis, EA-mediated pro-apoptotic action was reversed by knockout of either p53 or ASK1, despite no increase in p53 apoptotic activity. Upon CDDP treatment, EA predominantly enhanced ROS generation, ASK1-p38/c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) pathway activation, and ultimately cell death, all of which were suppressed either by co-treatment of the NADPH oxidase (Nox) inhibitor Apocynin, or by knocking out its regulatory protein, receptor-interacting protein 1 (RIP1). These results demonstrate that in response to CDDP ICLs, TFAs promote p53-dependent apoptosis through the enhancement of the Nox-RIP1-ASK1-MAPK pathway activation, providing insight into the diverse pathogenetic mechanisms of TFAs according to the types of DNA damage.

trans-Fatty acids facilitate DNA damage-induced apoptosis through the mitochondrial JNK-Sab-ROS positive feedback loop.

trans-Fatty acids (TFAs) are unsaturated fatty acids that contain one or more carbon-carbon double bonds in trans configuration. Epidemiological evidence has linked TFA consumption with various disorders, including cardiovascular diseases. However, the underlying pathological mechanisms are largely unknown. Here, we show a novel toxic mechanism of TFAs triggered by DNA damage. We found that elaidic acid (EA) and linoelaidic acid, major TFAs produced during industrial food manufacturing (so-called as industrial TFAs), but not their corresponding cis isomers, facilitated apoptosis induced by doxorubicin. Consistently, EA enhanced UV-induced embryonic lethality in C. elegans worms. The pro-apoptotic action of EA was blocked by knocking down Sab, a c-Jun N-terminal kinase (JNK)-interacting protein localizing at mitochondrial outer membrane, which mediates mutual amplification of mitochondrial reactive oxygen species (ROS) generation and JNK activation. EA enhanced doxorubicin-induced mitochondrial ROS generation and JNK activation, both of which were suppressed by Sab knockdown and pharmacological inhibition of either mitochondrial ROS generation, JNK, or Src-homology 2 domain-containing protein tyrosine phosphatase 1 (SHP1) as a Sab-associated protein. These results demonstrate that in response to DNA damage, TFAs drive the mitochondrial JNK-Sab-ROS positive feedback loop and ultimately apoptosis, which may provide insight into the common pathogenetic mechanisms of diverse TFA-related disorders.

Interleukin-1beta Inhibition Attenuates Vasculitis in a Mouse Model of Kawasaki Disease.

BACKGROUND: Kawasaki disease (KD), a systemic vasculitis, is suspected to be related to abnormalities in innate immunity. Based on the important role of IL-1 signaling in innate immunity, we investigated the effects of an anti-IL-1ß antibody using a Candida albicans water-soluble fraction (CAWS)-induced mouse model of KD. METHODS: CAWS (0.5 mg/mouse) was injected intraperitoneally into 5-week-old DBA/2 mice on five consecutive days. An anti-Murine IL-1ß antibody (01BSUR) was administered at various doses (2.5, 5.0, and 10.0 mg/kg) and time points (2 days before, same day, and 2, 5, 7, and 14 days after CAWS administration). After 4 weeks, vasculitis in the aortic root was investigated histologically. Cytokines including IL-1ß, -6, -10, and TNF-α were also measured. RESULTS: Groups administered 01BSUR at all doses showed a significant reduction in the area of vasculitis. In addition, 01BSUR inhibited vasculitis until 7 days after CAWS administration. In the analysis of various time points, the level of IL-6 was lower in all groups compared to the CAWS only group, but the levels of IL-1ß, TNFα, and IL-10 were lower when 01BSUR was administered before CAWS. On the other hand, TNFα and IL-10 levels were restored when 01BSUR was administered after CAWS, suggesting that 01BSUR may have additional effects beyond blocking IL-1ß signaling. CONCLUSIONS: The anti-IL-1ß antibody significantly attenuated CAWS-induced vasculitis. The mechanism of inhibiting vasculitis is thought to include inhibition of the IL-1ß pathway and additional effects beyond blocking IL-1ß signaling.

Simultaneous visualization of extrinsic and intrinsic axon collaterals in Golgi-like detail for mouse corticothalamic and corticocortical cells: a double viral infection method.

Here we present a novel tracing technique to stain projection neurons in Golgi-like detail by double viral infection. We used retrograde lentiviral vectors and adeno-associated viral vectors (AAV) to drive "TET-ON/TET-OFF system" in neurons connecting two regions. Using this method, we successfully labeled the corticothalamic (CT) cells of the mouse somatosensory barrel field (S1BF) and motor cortex (M1) in their entirety. We also labeled contra- and ipsilaterally-projecting corticocortical (CC) cells of M1 by targeting contralateral M1 or ipsilateral S1 for retrograde infection. The strength of this method is that we can observe the morphology of specific projection neuron subtypes en masse. We found that the group of CT cells extends their dendrites and intrinsic axons extensively below but not within the thalamorecipient layer in both S1BF and M1, suggesting that the primary target of this cell type is not layer 4. We also found that both ipsi- and contralateral targeting CC cells in M1 commonly exhibit widespread collateral extensions to contralateral M1 (layers 1-6), bilateral S1 and S2 (layers 1, 5 and 6), perirhinal cortex (layers 1, 2/3, 5, and 6), striatum and claustrum. These findings not only strengthened the previous findings of single cell tracings but also extended them by enabling cross-area comparison of CT cells or comparison of CC cells of two different labeling.

Viral vector-mediated selective and reversible blockade of the pathway for visual orienting in mice.

Recently, by using a combination of two viral vectors, we developed a technique for pathway-selective and reversible synaptic transmission blockade, and successfully induced a behavioral deficit of dexterous hand movements in macaque monkeys by affecting a population of spinal interneurons. To explore the capacity of this technique to work in other pathways and species, and to obtain fundamental methodological information, we tried to block the crossed tecto-reticular pathway, which is known to control orienting responses to visual targets, in mice. A neuron-specific retrograde gene transfer vector with the gene encoding enhanced tetanus neurotoxin (eTeNT) tagged with enhanced green fluorescent protein (EGFP) under the control of a tetracycline responsive element was injected into the left medial pontine reticular formation. 7-17 days later, an adeno-associated viral vector with a highly efficient Tet-ON sequence, rtTAV16, was injected into the right superior colliculus. 5-9 weeks later, the daily administration of doxycycline (Dox) was initiated. Visual orienting responses toward the left side were impaired 1-4 days after Dox administration. Anti-GFP immunohistochemistry revealed that a number of neurons in the intermediate and deep layers of the right superior colliculus were positively stained, indicating eTeNT expression. After the termination of Dox administration, the anti-GFP staining returned to the baseline level within 28 days. A second round of Dox administration, starting from 28 days after the termination of the first Dox administration, resulted in the reappearance of the behavioral impairment. These findings showed that pathway-selective and reversible blockade of synaptic transmission also causes behavioral effects in rodents, and that the crossed tecto-reticular pathway clearly controls visual orienting behaviors.

Avian adeno-associated virus vector efficiently transduces neurons in the embryonic and post-embryonic chicken brain.

The domestic chicken is an attractive model system to explore the development and function of brain circuits. Electroporation-mediated and retrovirus (including lentivirus) vector-mediated gene transfer techniques have been widely used to introduce genetic material into chicken cells. However, it is still challenging to efficiently transduce chicken postmitotic neurons without harming the cells. To overcome this problem, we searched for a virus vector suitable for gene transfer into chicken neurons, and report here a novel recombinant virus vector derived from avian adeno-associated virus (A3V). A3V vector efficiently transduces neuronal cells, but not non-neuronal cells in the brain. A single A3V injection into a postembryonic chick brain allows gene expression selectively in neuronal cells within 24 hrs. Such rapid and neuron-specific gene transduction raises the possibility that A3V vector can be utilized for studies of memory formation in filial imprinting, which occurs during the early postnatal days. A3V injection into the neural tube near the ear vesicle at early embryonic stage resulted in persistent and robust gene expression until E20.5 in the auditory brainstem. We further devised an A3V-mediated tetracycline (Tet) dependent gene expression system as a tool for studying the auditory circuit, consisting of the nucleus magnocellularis (NM) and nucleus laminaris (NL), that primarily computes interaural time differences (ITDs). Using this Tet system, we can transduce NM neurons without affecting NL neurons. Thus, the A3V technology complements current gene transfer techniques in chicken studies and will contribute to better understanding of the functional organization of neural circuits.

Genetic dissection of the circuit for hand dexterity in primates.

It is generally accepted that the direct connection from the motor cortex to spinal motor neurons is responsible for dexterous hand movements in primates. However, the role of the 'phylogenetically older' indirect pathways from the motor cortex to motor neurons, mediated by spinal interneurons, remains elusive. Here we used a novel double-infection technique to interrupt the transmission through the propriospinal neurons (PNs), which act as a relay of the indirect pathway in macaque monkeys (Macaca fuscata and Macaca mulatta). The PNs were double infected by injection of a highly efficient retrograde gene-transfer vector into their target area and subsequent injection of adeno-associated viral vector at the location of cell somata. This method enabled reversible expression of green fluorescent protein (GFP)-tagged tetanus neurotoxin, thereby permitting the selective and temporal blockade of the motor cortex­PN­motor neuron pathway. This treatment impaired reach and grasp movements, revealing a critical role for the PN-mediated pathway in the control of hand dexterity. Anti-GFP immunohistochemistry visualized the cell bodies and axonal trajectories of the blocked PNs, which confirmed their anatomical connection to motor neurons. This pathway-selective and reversible technique for blocking neural transmission does not depend on cell-specific promoters or transgenic techniques, and is a new and powerful tool for functional dissection in system-level neuroscience studies.

miR-124a is required for hippocampal axogenesis and retinal cone survival through Lhx2 suppression.

MicroRNA-124a (miR-124a) is the most abundant microRNA expressed in the vertebrate CNS. Despite past investigations into the role of miR-124a, inconsistent results have left the in vivo function of miR-124a unclear. We examined the in vivo function of miR-124a by targeted disruption of Rncr3 (retinal non-coding RNA 3), the dominant source of miR-124a. Rncr3(-/-) mice exhibited abnormalities in the CNS, including small brain size, axonal mis-sprouting of dentate gyrus granule cells and retinal cone cell death. We found that Lhx2 is an in vivo target mRNA of miR-124a. We also observed that LHX2 downregulation by miR-124a is required for the prevention of apoptosis in the developing retina and proper axonal development of hippocampal neurons. These results suggest that miR-124a is essential for the maturation and survival of dentate gyrus neurons and retinal cones, as it represses Lhx2 translation.

Selective optical control of synaptic transmission in the subcortical visual pathway by activation of viral vector-expressed halorhodopsin.

The superficial layer of the superior colliculus (sSC) receives visual inputs via two different pathways: from the retina and the primary visual cortex. However, the functional significance of each input for the operation of the sSC circuit remains to be identified. As a first step toward understanding the functional role of each of these inputs, we developed an optogenetic method to specifically suppress the synaptic transmission in the retino-tectal pathway. We introduced enhanced halorhodopsin (eNpHR), a yellow light-sensitive, membrane-targeting chloride pump, into mouse retinal ganglion cells (RGCs) by intravitreously injecting an adeno-associated virus serotype-2 vector carrying the CMV-eNpHR-EYFP construct. Several weeks after the injection, whole-cell recordings made from sSC neurons in slice preparations revealed that yellow laser illumination of the eNpHR-expressing retino-tectal axons, putatively synapsing onto the recorded cells, effectively inhibited EPSCs evoked by electrical stimulation of the optic nerve layer. We also showed that sSC spike activities elicited by visual stimulation were significantly reduced by laser illumination of the sSC in anesthetized mice. These results indicate that photo-activation of eNpHR expressed in RGC axons enables selective blockade of retino-tectal synaptic transmission. The method established here can most likely be applied to a variety of brain regions for studying the function of individual inputs to these regions.

Verticilide, a new ryanodine-binding inhibitor, produced by Verticillium sp. FKI-1033.

A new ryanodine-binding inhibitor, verticilide, was isolated from the cultured broth of a fungus, Verticillium sp. FKI-1033. It is a 24-membered ring cyclic depsipeptide, its structure being elucidated as cyclo[(2R)-2-hydroxyheptanoyl-N-methyl- L-alanyl](4). Verticilide inhibited ryanodine binding to ryanodine receptors in the cockroach at an IC(50) value of 4.2 microM, whereas inhibition against mouse ryanodine receptors was weak (IC(50)=53.9 microM).