The hereditary spastic paraplegia-related enzyme DDHD2 is a principal brain triglyceride lipase

Complex hereditary spastic paraplegia (HSP) is a genetic disorder that causes lower limb spasticity and weakness and intellectual disability. Deleterious mutations in the poorly characterized serine hydrolase DDHD2 are a causative basis for recessive complex HSP. DDHD2 exhibits phospholipase activity in vitro, but its endogenous substrates and biochemical functions remain unknown. Here, we report the development of DDHD2^−/− mice and a selective, in vivo-active DDHD2 inhibitor and their use in combination with mass spectrometry-based lipidomics to discover that DDHD2 regulates brain triglycerides (triacylglycerols, or TAGs). DDHD2^−/− mice show age-dependent TAG elevations in the central nervous system, but not in several peripheral tissues. Large lipid droplets accumulated in DDHD2^−/− brains and were localized primarily to the intracellular compartments of neurons. These metabolic changes were accompanied by impairments in motor and cognitive function. Recombinant DDHD2 displays TAG hydrolase activity, and TAGs accumulated in the brains of wild-type mice treated subchronically with a selective DDHD2 inhibitor. These findings, taken together, indicate that the central nervous system possesses a specialized pathway for metabolizing TAGs, disruption of which leads to massive lipid accumulation in neurons and complex HSP syndrome.Determining the genetic basis for rare hereditary human diseases has benefited from advances in DNA sequencing technologies (1). As a greater number of disease-causing mutations are mapped, however, it is also becoming apparent that many of the affected genes code for poorly characterized proteins. Assigning biochemical and cellular functions to these proteins is critical to achieve a deeper mechanistic understanding of human genetic disorders and for identifying potential treatment strategies.Hereditary spastic paraplegia (HSP) is a genetically heterogeneous neurologic syndrome marked by spasticity and lower extremity weakness (2). Many genetic types of HSP have been identified and are numbered according to their order of discovery [spastic paraplegia (SPG) 1-72] (2, 3). Of these genetic variants, more than 40 have been mapped to causative mutations in protein-coding genes. HSP genes code for a wide range of proteins that do not conform to a single sequence- or function-related class. A subset of HSP genes, including PNPLA6 (or neuropathy-target esterase) (SPG39) (4), DDHD1 (SPG28) (5), and DDHD2 (SPG54) (3, 6–8), code for serine hydrolases. These enzymes have been designated as (lyso)phospholipases based on in vitro substrate assays (9–11), but their endogenous substrates and physiological functions remain poorly understood. The mutational landscape that affects these lipid hydrolases to cause recessive HSP is complex but collectively represents a mix of null and putatively null and/or functional mutations. Moreover, the type of HSP appears to differ in each case, with DDHD1 mutations causing uncomplicated HSP, whereas PNPLA6 and DDHD2 mutations lead to complex forms of the disease that exhibit additional phenotypes including, in the case of DDHD2, intellectual disability. Human subjects with DDHD2 mutations also displayed evidence of brain lipid accumulation as detected by cerebral magnetic resonance spectroscopy (6). Both rodent and human DDHD2 enzymes are highly expressed in the brain compared with most peripheral tissues (6, 9); however, the specific lipids regulated by DDHD2 in the central nervous system (CNS) have not yet been identified.Determining the metabolic function of DDHD2 in the brain is an important step toward understanding how mutations in this enzyme promote complex HSP and for identifying possible therapeutic strategies for the disease. Toward this end, we report herein the generation and characterization of DDHD2^−/− mice and a selective DDHD2 inhibitor. DDHD2^−/− mice exhibit defects in movement and cognitive function. Mass spectrometry (MS)-based lipidomics (12, 13) revealed a striking and selective elevation in triglycerides (triacylglycerols, or TAGs) throughout the CNS, but not in peripheral tissues, of DDHD2^−/− mice. This metabolic change correlated with pervasive lipid droplet (LD) accumulation in neuronal cell bodies of DDHD2^−/− mice. Biochemical assays confirmed that DDHD2 possesses TAG hydrolase activity. Finally, wild-type mice treated subchronically with a DDHD2 inhibitor also exhibited significant elevations in CNS TAGs. These data, taken together, indicate that DDHD2 is a principal TAG hydrolase of the mammalian brain and point to deregulation of this pathway as a major contributory factor to complex HSP.     

Inhibition of monoacylglycerol lipase reduces nicotine withdrawal

Background and Purpose

Abrupt discontinuation of nicotine, the main psychoactive component in tobacco, induces a withdrawal syndrome in nicotine-dependent animals, consisting of somatic and affective signs, avoidance of which contributes to drug maintenance. While blockade of fatty acid amide hydrolase, the primary catabolic enzyme of the endocannabinoid arachidonoylethanolamine (anandamide), exacerbates withdrawal responses in nicotine-dependent mice, the role of monoacylglycerol lipase (MAGL), the main hydrolytic enzyme of a second endocannabinoid 2-arachidonylglycerol (2-AG), in nicotine withdrawal remains unexplored.

Experimental Approach

To evaluate the role of MAGL enzyme inhibition in nicotine withdrawal, we initially performed a genetic correlation approach using the BXD recombinant inbred mouse panel. We then assessed nicotine withdrawal intensity in the mouse after treatment with the selective MAGL inhibitor, JZL184, and after genetic deletion of the enzyme. Lastly, we assessed the association between genotypes and smoking withdrawal phenotypes in two human data sets.

Key Results

BXD mice displayed significant positive correlations between basal MAGL mRNA expression and nicotine withdrawal responses, consistent with the idea that increased 2-AG brain levels may attenuate withdrawal responses. Strikingly, the MAGL inhibitor, JZL184, dose-dependently reduced somatic and aversive withdrawal signs, which was blocked by rimonabant, indicating a CB₁ receptor-dependent mechanism. MAGL-knockout mice also showed attenuated nicotine withdrawal. Lastly, genetic analyses in humans revealed associations of the MAGL gene with smoking withdrawal in humans.

Conclusions and Implications

Overall, our findings suggest that MAGL inhibition maybe a promising target for treatment of nicotine dependence.     

Blockade of endocannabinoid hydrolytic enzymes attenuates precipitated opioid withdrawal symptoms in mice

Δ(9)-Tetrahydrocannbinol (THC), the primary active constituent of Cannabis sativa, has long been known to reduce opioid withdrawal symptoms. Although THC produces most of its pharmacological actions through the activation of CB(1) and CB(2) cannabinoid receptors, the role these receptors play in reducing the variety of opioid withdrawal symptoms remains unknown. The endogenous cannabinoids, N-arachidonoylethanolamine (anandamide; AEA) and 2-arachidonylglycerol (2-AG), activate both cannabinoid receptors but are rapidly metabolized by fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively. The objective of this study was to test whether increasing AEA or 2-AG, via inhibition of their respective hydrolytic enzymes, reduces naloxone-precipitated morphine withdrawal symptoms in in vivo and in vitro models of opioid dependence. Morphine-dependent mice challenged with naloxone reliably displayed a profound withdrawal syndrome, consisting of jumping, paw tremors, diarrhea, and weight loss. THC and the MAGL inhibitor 4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184) dose dependently reduced the intensity of most measures through the activation of CB(1) receptors. JZL184 also attenuated spontaneous withdrawal signs in morphine-dependent mice. The FAAH inhibitor N-(pyridin-3-yl)-4-(3-(5-(trifluoromethyl)pyridin-2-yloxy)benzyl)-piperdine-1-carboxamide (PF-3845) reduced the intensity of naloxone-precipitated jumps and paw flutters through the activation of CB(1) receptors but did not ameliorate incidence of diarrhea or weight loss. In the final series of experiments, we investigated whether JZL184 or PF-3845 would attenuate naloxone-precipitated contractions in morphine-dependent ilea. Both enzyme inhibitors attenuated the intensity of naloxone-induced contractions, although this model does not account mechanistically for the autonomic withdrawal responses (i.e., diarrhea) observed in vivo. These results indicate that endocannabinoid catabolic enzymes are promising targets to treat opioid dependence.     

骨质疏松性椎体骨折模型的建立

目的:建立大鼠的骨质疏松性椎体骨折模型,探讨骨折愈合程度与X射线、骨结构和力学性能的相互关系,以期能为临床治疗提供科学的指导和理论依据。方法:实验于2005-07/2006-07在解放军兰州军区总医院骨研所完成。实验动物:选择雌性SPF级8个月龄SD大鼠54只。实验分组:采用随机数字法将大鼠分为2组:骨质疏松组和对照组,每组27只。实验干预:骨质疏松组经双背侧手术切除卵巢,对照组行伪手术。术后3个月,所有动物麻醉下,采用L5椎体手术开窗刮除术区内松质骨方法建立人工椎体骨折模型。实验评估:于术后1,2,4,6,8,12周观察两组大鼠腰椎影像学、骨组织切片组织学与受累椎体力学性能。结果:54只SD大鼠全部进入结果分析。①影像学观察:术后两组X射线片示L5椎体有一骨折缺损透光区。对照组在术后6周时原透光区与周围骨质无明显差别,而骨质疏松组原透光区仍清晰可见,于8周时无明显差别。②组织学观察:两组软骨细胞在骨愈合1周时出现,形成软骨岛,但骨质疏松组软骨细胞每高倍视野数量明显少于对照组,另外,软骨细胞改建成成熟骨细胞,骨小梁形成数量,胶原纤维排列与对照组比较有显著性差异。③力学性能:在骨质愈合6～12周,L5椎体的最大载荷、弹性模量、最大应力明显低于同期对照组,差异有显著性意义(P<0.05)。结论:骨质疏松性椎体骨折SD大鼠模型,符合动物模型标准,可用于研究新骨形成与正常骨质结构关系,观察骨质疏松性椎体骨折愈合机制,并证明骨质疏松性松质骨骨折修复过程中,骨折愈合质量降低。     

Early postoperative hyperglycaemia is not a risk factor for infectious complications and prolonged in-hospital stay in patients undergoing oesophagectomy: a retrospective analysis of a prospective trial

Introduction  

Treating hyperglycaemia in hospitalized patients has proven to be beneficial, particularly in those with obstructive vascular disease. In a cohort of patients undergoing resection for oesophageal carcinoma (a group of patients with severe surgical stress but a low prevalence of vascular disease), we investigated whether early postoperative hyperglycaemia is associated with increased incidence of infectious complications and prolonged in-hospital stay.     

Fatty acid amide hydrolase ablation promotes ectopic lipid storage and insulin resistance due to centrally mediated hypothyroidism

Fatty acid amide hydrolase (FAAH) knockout mice are prone to excess energy storage and adiposity, whereas mutations in FAAH are associated with obesity in humans. However, the molecular mechanism by which FAAH affects energy expenditure (EE) remains unknown. Here we show that reduced energy expenditure in FAAH(-/-) mice could be attributed to decreased circulating triiodothyronine and thyroxine concentrations secondary to reduced mRNA expression of both pituitary thyroid-stimulating hormone and hypothalamic thyrotropin-releasing hormone. These reductions in the hypothalamic-pituitary-thyroid axis were associated with activation of hypothalamic peroxisome proliferating-activated receptor γ (PPARγ), and increased hypothalamic deiodinase 2 expression. Infusion of NAEs (anandamide and palmitoylethanolamide) recapitulated increases in PPARγ-mediated decreases in EE. FAAH(-/-) mice were also prone to diet-induced hepatic insulin resistance, which could be attributed to increased hepatic diacylglycerol content and protein kinase Cε activation. Our data indicate that FAAH deletion, and the resulting increases in NAEs, predispose mice to ectopic lipid storage and hepatic insulin resistance by promoting centrally mediated hypothyroidism.     

Reverse LISS plating for intertrochanteric Hip Fractures in elderly patients

Background  

Fractures of the intertrochanteric hip are common and the treatment of unstable fractures generally requires an operative approach. In elderly patients, osteoporosis makes internal fixation problematic and frequently contributes to failed fixation and poor clinical results. We have attempted to apply the Less Invasive Stabilization System (LISS) in reverse position for the repair of intertrochanteric hip fractures in elderly patients with osteoporotic bones. A retrospective review is presented of the cases of 28 elderly patients with stable and unstable fractures of the intertrochanteric hip treated using the reverse LISS.