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.

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-␣ (DAGL␣) and -␤ (DAGL␤) 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 ϳ80% reductions in 2-AG levels in the brain and spinal cord in DAGL␣ Ϫ/Ϫ mice and a 50% reduction in the brain in DAGL␤ Ϫ/Ϫ mice. In contrast, DAGL␤ plays a more important role than DAGL␣ in regulating 2-AG levels in the liver, with a 90% reduction seen in DAGL␤ Ϫ/Ϫ 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 GABAmediated transmission at inhibitory synapses; we now show that this form of synaptic plasticity is completely lost in DAGL␣ Ϫ/Ϫ animals and relatively unaffected in DAGL␤ Ϫ/Ϫ animals. Finally, we show that the control of adult neurogenesis in the hippocampus and subventricular zone is compromised in the DAGL␣ Ϫ/Ϫ and/or DAGL␤ Ϫ/Ϫ mice. These findings provide the first evidence that DAGL␣ 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.

Huntington's disease (HD) is one of a class of inherited progressive neurodegenerative disorders that are caused by a CAG/polyglutamine repeat expansion. We have previously generated mice that are transgenic for exon 1 of the HD gene carrying highly expanded CAG repeats which develop a progressive movement disorder and weight loss with similarities to HD. Neuronal inclusions composed of the exon 1 protein and ubiquitin are present in specific brain regions prior to onset of the phenotype, which in turn occurs long before specific neurodegeneration can be detected. In this report we have extended the search for polyglutamine inclusions to non-neuronal tissues. Outside the central nervous system (CNS), inclusions were identified in a variety of post-mitotic cells. This is consistent with a concentration-dependent nucleation and aggregation model of inclusion formation and indicates that brain-specific factors are not necessary for this process. To possibly gain insights into the wasting that is observed in the human disease, we have conducted a detailed analysis of the timing and progression of inclusion formation in skeletal muscle and an investigation into the cause of the severe muscle atrophy that occurs in the mouse model. The formation of inclusions in non-CNS tissues will be particularly useful with respect to in vivo monitoring of pharmaceutical agents selected for their ability to prevent polyglutamine aggregation in vitro, without the requirement that the agent can cross the blood-brain barrier in the first instance.

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The mechanisms of phagocytosis of Candida albicans by human vascular endothelial cells and subsequent endothelial cell injury were examined in vitro. Both live and killed C. albicans cells were phagocytized by endothelial cells. This organism specifically induced endothelial cell phagocytosis because neither Candida tropicalis nor Torulopsis glabrata was ingested. Endothelial cell microfilaments polymerized around C. albicans as the organisms were phagocytized. Cytochalasin D inhibited this polymerization of microfilaments around C. albicans and blocked phagocytosis. The blocking of actin depolymerization with phalloidin had no effect on microfilament condensation around the organism, indicating that the microfilaments surrounding C. albicans are formed from a pool of G-actin. Intact microtubules were also necessary for the phagocytosis of C. albicans, since the depolymerizing of endothelial cell microtubules with nocodazole prevented the condensation of actin filaments around the organisms and inhibited phagocytosis. In contrast, microtubule depolymerization was not required for microfilament function because the blocking of microtubule depolymerization with taxol had no effect on microfilament condensation around C. albicans. The phagocytosis of C. albicans was pivotal in the induction of endothelial cell damage, since the blocking of candidal internalization significantly reduced endothelial cell injury. Endothelial cells were not damaged by phagocytosis of dead organisms, indicating that injury was caused by a factor associated with viable organisms. Therefore, C. albicans is uniquely able to induce endothelial cell phagocytosis by comparison with non-albicans species of Candida. Furthermore, at least two components of the endothelial cytoskeleton, microfilaments and microtubules, are necessary for the phagocytosis of C. albicans.

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.

Novel antibodies have been generated by immunizing with bacterially expressed fragments of the repetitive motif of the Ki-67 gene. One such antibody, MIB1, recognizes a fixation and embedding resistant epitope on the Ki-67 protein if sections are previously microwaved in a citrate buffer. We have investigated the utility of this antibody as a marker of cell proliferation in archival material. The microwave technique is simple but requires careful monitoring since different tissues and fixatives require different irradiation times. Strong nuclear immunoreactivity was detected with all fixatives studied. Cytoplasmic staining was not identified. In a wide range of normal tissues the distribution and number of MIB1 immunoreactive cells matched that of cryostat sections stained with Ki-67. In nude mouse xenografts in which the growth fraction had been defined using a fraction of labelled mitosis method, the labelling index with MIB1 matched that previously determined for Ki-67 and correlated well with the growth fraction. Other markers of proliferation (e.g. proliferating cell nuclear antigen) have been shown to be expressed in DNA repair, thus we investigated expression of MIB1 immunoreactivity in situations of DNA repair in vivo--ultraviolet irradiated human skin. MIB1 staining correlated with semi-conservative DNA synthesis rather than excision repair DNA synthesis. Finally, the morphological and cell cycle distribution of MIB1 expression is identical to that of Ki-67. Thus, MIB1 represents a new anti-Ki-67 antibody which appears to be a robust marker of cell proliferation easily applicable to archival material.