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ABSTRACT: Joint degeneration observed in the rat monoiodoacetate (MIA) model of osteoarthritis shares many histological features with the clinical condition. The accompanying pain phenotype has seen the model widely used to investigate the pathophysiology of osteoarthritis pain, and for preclinical screening of analgesic compounds. We have investigated the pathophysiological sequellae of MIA used at low (1 mg) or high (2 mg) dose. Intra-articular 2 mg MIA induced expression of ATF-3, a sensitive marker for peripheral neuron stress/injury, in small and large diameter DRG cell profiles principally at levels L4 and 5 (levels predominated by neurones innervating the hindpaw) rather than L3. At the 7 day timepoint, ATF-3 signal was significantly smaller in 1 mg MIA treated animals than in the 2 mg treated group. 2 mg, but not 1 mg, intra-articular MIA was also associated with a significant reduction in intra-epidermal nerve fibre density in plantar hindpaw skin, and produced spinal cord dorsal and ventral horn microgliosis. The 2 mg treatment evoked mechanical pain-related hypersensitivity of the hindpaw that was significantly greater than the 1 mg treatment. MIA treatment produced weight bearing asymmetry and cold hypersensitivity which was similar at both doses. Additionally, while pregabalin significantly reduced deep dorsal horn evoked neuronal responses in animals treated with 2 mg MIA, this effect was much reduced or absent in the 1 mg or sham treated groups. These data demonstrate that intra-articular 2 mg MIA not only produces joint degeneration, but also evokes significant axonal injury to DRG cells including those innervating targets outside of the knee joint such as hindpaw skin. This significant neuropathic component needs to be taken into account when interpreting studies using this model, particularly at doses greater than 1 mg MIA.
PLoS ONE 01/2012; 7(3):e33730. · 4.09 Impact Factor
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ABSTRACT: Until recently, endocannabinoid (eCB) signalling was largely studied in the context of synaptic plasticity in the postnatal brain in the absence of detailed knowledge of the nature of the enzyme(s) responsible for the synthesis of the eCBs. However, the identification of two diacylglycerol lipases (DAGLα and DAGLβ) responsible for the synthesis of 2-arachidonoylglycerol (2-AG) has increased the understanding of where this eCB is synthesised in relationship to the expression of cannabinoid receptor (CB)1 and CB2. Furthermore, the generation of knockout animals for each enzyme has allowed for the direct testing of their importance for established and emerging eCB functions. Based on this, we now know that DAGLα is enriched in dendritic spines that appose CB1-positive synaptic terminals, and that 2-AG functions as a retrograde signal controlling synaptic strength throughout the nervous system. Consequently, we have built on the principle that expression of eCB components dictates function to identify additional physiological functions of this signalling cassette. A number of studies have now provided support for DAGL-dependent eCB signalling playing important roles in brain development and in cellular plasticity in the adult nervous system. In this article, we will review evidence based on the localisation of the enzymes, as well as from genetic and pharmacological studies, that show DAGL-dependent eCB signalling to play an important role in axonal growth and guidance during development, in retrograde synaptic signalling at mature synapses, and in the control of adult neurogenesis in the hippocampus and subventricular zone.
European Journal of Neuroscience 11/2011; 34(10):1634-46. · 3.63 Impact Factor
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ABSTRACT: Many persistent pain states (pain lasting for hours, days, or longer) are poorly treated because of the limitations of existing therapies. Analgesics such as nonsteroidal anti-inflammatory drugs and opioids often provide incomplete pain relief and prolonged use results in the development of severe side effects. Identification of the key mediators of various types of pain could improve such therapies. Here, we tested the hypothesis that hitherto unrecognized cytokines and chemokines might act as mediators in inflammatory pain. We used ultraviolet B (UVB) irradiation to induce persistent, abnormal sensitivity to pain in humans and rats. The expression of more than 90 different inflammatory mediators was measured in treated skin at the peak of UVB-induced hypersensitivity with custom-made polymerase chain reaction arrays. There was a significant positive correlation in the overall expression profiles between the two species. The expression of several genes [interleukin-1β (IL-1β), IL-6, and cyclooxygenase-2 (COX-2)], previously shown to contribute to pain hypersensitivity, was significantly increased after UVB exposure, and there was dysregulation of several chemokines (CCL2, CCL3, CCL4, CCL7, CCL11, CXCL1, CXCL2, CXCL4, CXCL7, and CXCL8). Among the genes measured, CXCL5 was induced to the greatest extent by UVB treatment in human skin; when injected into the skin of rats, CXCL5 recapitulated the mechanical hypersensitivity caused by UVB irradiation. This hypersensitivity was associated with the infiltration of neutrophils and macrophages into the dermis, and neutralizing the effects of CXCL5 attenuated the abnormal pain-like behavior. Our findings demonstrate that the chemokine CXCL5 is a peripheral mediator of UVB-induced inflammatory pain, likely in humans as well as rats.
Science translational medicine 07/2011; 3(90):90ra60. · 7.80 Impact Factor
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ABSTRACT: In the adult brain, neural stem cells proliferate within the subventricular zone before differentiating into migratory neuroblasts that travel along the rostral migratory stream (RMS) to populate the olfactory bulb with new neurons. Because neuroblasts have been shown to migrate to areas of brain injury, understanding the cues regulating this migration could be important for brain repair. Recent studies have highlighted an important role for endocannabinoid (eCB) signaling in the proliferation of the stem cell population, but it remained to be determined whether this pathway also played a role in cell migration. We now show that mouse migratory neuroblasts express cannabinoid receptors, diacylglycerol lipase α (DAGLα), the enzyme that synthesizes the endocannabinoid 2-arachidonoylglycerol (2-AG), and monoacylglycerol lipase, the enzyme responsible for its degradation. Using a scratch wound assay for a neural stem cell line and RMS explant cultures, we show that inhibition of DAGL activity or CB(1)/CB(2) receptors substantially decreases migration. In contrast, direct activation of cannabinoid receptors or preventing the breakdown of 2-AG increases migration. Detailed analysis of primary neuroblast migration by time-lapse imaging reveals that nucleokinesis, as well as the length and branching of the migratory processes are under dynamic control of the eCB system. Finally, similar effects are observed in vivo by analyzing the morphology of green fluorescent protein-labeled neuroblasts in brain slices from mice treated with CB(1) or CB(2) antagonists. These results describe a novel role for the endocannabinoid system in neuroblast migration in vivo, highlighting its importance in regulating an additional essential step in adult neurogenesis.
Journal of Neuroscience 03/2011; 31(11):4000-11. · 7.11 Impact Factor
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Ying Gao,
Dmitry V Vasilyev,
Maria Beatriz Goncalves,
Fiona V Howell, Carl Hobbs,
Melina Reisenberg,
Ru Shen,
Mei-Yi Zhang,
Brian W Strassle,
Peimin Lu, [......],
Kangwen Deng,
Evguenia V Kouranova,
Robert H Ring,
Garth T Whiteside,
Brian Bates,
Frank S Walsh,
Gareth Williams,
Menelas N Pangalos,
Tarek A Samad,
Patrick Doherty
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ABSTRACT: Endocannabinoids (eCBs) function as retrograde signaling molecules at synapses throughout the brain, regulate axonal growth and guidance during development, and drive adult neurogenesis. There remains a lack of genetic evidence as to the identity of the enzyme(s) responsible for the synthesis of eCBs in the brain. Diacylglycerol lipase-alpha (DAGLalpha) and -beta (DAGLbeta) synthesize 2-arachidonoyl-glycerol (2-AG), the most abundant eCB in the brain. However, their respective contribution to this and to eCB signaling has not been tested. In the present study, we show approximately 80% reductions in 2-AG levels in the brain and spinal cord in DAGLalpha(-/-) mice and a 50% reduction in the brain in DAGLbeta(-/-) mice. In contrast, DAGLbeta plays a more important role than DAGLalpha in regulating 2-AG levels in the liver, with a 90% reduction seen in DAGLbeta(-/-) mice. Levels of arachidonic acid decrease in parallel with 2-AG, suggesting that DAGL activity controls the steady-state levels of both lipids. In the hippocampus, the postsynaptic release of an eCB results in the transient suppression of GABA-mediated transmission at inhibitory synapses; we now show that this form of synaptic plasticity is completely lost in DAGLalpha(-/-) animals and relatively unaffected in DAGLbeta(-/-) animals. Finally, we show that the control of adult neurogenesis in the hippocampus and subventricular zone is compromised in the DAGLalpha(-/-) and/or DAGLbeta(-/-) mice. These findings provide the first evidence that DAGLalpha is the major biosynthetic enzyme for 2-AG in the nervous system and reveal an essential role for this enzyme in regulating retrograde synaptic plasticity and adult neurogenesis.
Journal of Neuroscience 02/2010; 30(6):2017-24. · 7.11 Impact Factor
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ABSTRACT: For a dentition representing the most basal extant gnathostomes, that of the shark can provide us with key insights into the evolution of vertebrate dentitions. To detail the pattern of odontogenesis, we have profiled the expression of sonic hedgehog, a key regulator of tooth induction. We find in the catshark (Scyliorhinus canicula) that intense shh expression first occurs in a bilaterally symmetrical pattern restricted to broad regions in each half of the dentition in the embryo jaw. As in the mouse, there follows a changing temporal pattern of shh spatial restriction corresponding to epithelial bands of left and right dental fields, but also a subfield for symphyseal teeth. Then, intense shh expression is restricted to loci coincident with a temporal series of teeth in iterative jaw positions. The developmental expression of shh reveals previously undetected timing within epithelial stages of tooth formation. Each locus at alternate, even then odd, jaw positions establishes precise sequential timing for successive replacement within each tooth family. Shh appears first in the central cusp, iteratively along the jaw, then reiteratively within each tooth for secondary cusps. This progressive, sequential restriction of shh is shared by toothed gnathostomes and conserved through 500 million years of evolution.
Proceedings of the Royal Society B: Biological Sciences 02/2009; 276(1660):1225-33. · 5.41 Impact Factor
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ABSTRACT: Huntington's disease (HD) is an inherited progressive neurodegenerative disorder caused by a CAG repeat expansion in the ubiquitously expressed HD gene resulting in an abnormally long polyglutamine repeat in the huntingtin protein. Polyglutamine inclusions are a hallmark of the neuropathology of HD. We have previously shown that inclusion pathology is also present in the peripheral tissues of the R6/2 mouse model of HD which expresses a small N-terminal fragment of mutant huntingtin. To determine whether this peripheral pathology is a consequence of the aberrant expression of this N-terminal fragment, we extend this analysis to the genetically precise knock-in mouse model of HD, HdhQ150, which expresses mutant mouse huntingtin.
We have previously standardized the CAG repeat size and strain background of the R6/2 and HdhQ150 knock-in mouse models and found that they develop a comparable and widespread neuropathology. To determine whether HdhQ150 knock-in mice also develop peripheral inclusion pathology, homozygous Hdh(Q150/Q150) mice were perfusion fixed at 22 months of age, and tissues were processed for histology and immunohistochemistry with the anti-huntingtin antibody S830. The peripheral inclusion pathology was almost identical to that found in R6/2 mice at 12 weeks of age with minor differences in inclusion abundance.
The highly comparable peripheral inclusion pathology that is present in both the R6/2 and HdhQ150 knock-in models of HD indicates that the presence of peripheral inclusions in R6/2 mice is not a consequence of the aberrant expression of an N-terminal huntingtin protein. It remains to be determined whether peripheral inclusions are a pathological feature of the human disease. Both mouse models carry CAG repeats that cause childhood disease in humans, and therefore, inclusion pathology may be a feature of the childhood rather than the adult forms of HD. It is important to establish the extent to which peripheral pathology causes the peripheral symptoms of HD from the perspective of a mechanistic understanding and future treatment options.
PLoS ONE 01/2009; 4(11):e8025. · 4.09 Impact Factor
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ABSTRACT: Endocannabinoids are retrograde neurotransmitters, which act upon the presynaptically located, G-protein coupled receptor CB1, to modulate synaptic transmission in the adult brain. Recently, however, a number of lines of evidence have suggested that endocannabinoid signalling may play an important role in early neuronal development. In this study, we show that the CB1 receptor has a wide expression pattern in the developing nervous system and that its expression follows neuronal differentiation in the embryo from the earliest stages. We also show that the enzymes involved in 2-AG synthesis are expressed in an overlapping manner at these stages. We further show that interfering with CB1 function using a pharmacological inhibitor causes problems in axon pathfinding and fasciculation. Similarly, CB1 gene knock down in the zebrafish by morpholino injection results in defects in axonal growth and fasciculation in these embryos. Thus CB1 function is required in the early embryo for axonal growth and fasciculation.
Molecular and Cellular Neuroscience 06/2008; 38(1):89-97. · 3.66 Impact Factor
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ABSTRACT: Phosphatidylinositol transfer proteins (PITPs) mediate the transfer of phosphatidylinositol (PtdIns) or phosphatidylcholine (PtdCho) between two membrane compartments, thereby regulating the interface between signalling, phosphoinositide (PI) metabolism and membrane traffic. Here, we show that PITPalpha is enriched in specific areas of the postnatal and adult brain, including the hippocampus and cerebellum. Overexpression of PITPalpha, but not PITPbeta or a PITPalpha mutant deficient in binding PtdIns, enhances laminin-dependent extension of axonal processes in hippocampal neurons, whereas knockdown of PITPalpha protein by siRNA suppresses laminin and BDNF-induced axonal growth. PITPalpha-mediated axonal outgrowth is sensitive to phosphoinositide 3-kinase (PI3K) inhibition and shows dependency on the Akt/GSK-3/CRMP-2 pathway. We conclude that PITPalpha controls the polarized extension of axonal processes through the provision of PtdIns for localized PI3K-dependent signalling.
Journal of Cell Science 04/2008; 121(Pt 6):796-803. · 6.11 Impact Factor
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Erika L F Holzbaur,
David S Howland,
Nicholas Weber,
Karen Wallace,
Yijin She,
Seung Kwak,
Lioudmilla A Tchistiakova,
Erin Murphy,
Joseph Hinson,
Riyez Karim,
Xiang Yang Tan,
Pamela Kelley,
Kevin C McGill,
Gareth Williams, Carl Hobbs,
Patrick Doherty,
Margaret M Zaleska,
Menelas N Pangalos,
Frank S Walsh
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ABSTRACT: Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease leading to motor neuron cell death, but recent studies suggest that non-neuronal cells may contribute to the pathological mechanisms involved. Myostatin is a negative regulator of muscle growth whose function can be inhibited using neutralizing antibodies. In this study, we used transgenic mouse and rat models of ALS to test whether treatment with anti-myostatin antibody slows muscle atrophy, motor neuron loss, or disease onset and progression. Significant increases in muscle mass and strength were observed in myostatin-antibody-treated SOD1(G93A) mice and rats prior to disease onset and during early-stage disease. By late stage disease, only diaphragm muscle remained significantly different in treated animals in comparison to untreated controls. Myostatin inhibition did not delay disease onset nor extend survival in either the SOD1(G93A) mouse or rat. Together, these results indicate that inhibition of myostatin does not protect against the onset and progression of motor neuron degenerative disease. However, the preservation of skeletal muscle during early-stage disease and improved diaphragm morphology and function maintained through late stage disease suggest that anti-myostatin therapy may promote some improved muscle function in ALS.
Neurobiology of Disease 10/2006; 23(3):697-707. · 5.40 Impact Factor
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Tiziana Bisogno,
Fiona Howell,
Gareth Williams,
Alberto Minassi,
Maria Grazia Cascio,
Alessia Ligresti,
Isabel Matias,
Aniello Schiano-Moriello,
Praveen Paul,
Emma-Jane Williams,
Uma Gangadharan, Carl Hobbs,
Vincenzo Di Marzo,
Patrick Doherty
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ABSTRACT: Diacylglycerol (DAG) lipase activity is required for axonal growth during development and for retrograde synaptic signaling at mature synapses. This enzyme synthesizes the endocannabinoid 2-arachidonoyl-glycerol (2-AG), and the CB1 cannabinoid receptor is also required for the above responses. We now report on the cloning and enzymatic characterization of the first specific sn-1 DAG lipases. Two closely related genes have been identified and their expression in cells correlated with 2-AG biosynthesis and release. The expression of both enzymes changes from axonal tracts in the embryo to dendritic fields in the adult, and this correlates with the developmental change in requirement for 2-AG synthesis from the pre- to the postsynaptic compartment. This switch provides a possible explanation for a fundamental change in endocannabinoid function during brain development. Identification of these enzymes may offer new therapeutic opportunities for a wide range of disorders.
The Journal of Cell Biology 12/2003; 163(3):463-8. · 10.26 Impact Factor
