Drosophila phototransduction is a model for signaling cascades that culminate in the activation of transient receptor potential (TRP) cation channels. TRP and TRPL are the canonical TRP (TRPC) channels that are regulated by light stimulation of rhodopsin and engagement of Galpha(q) and phospholipase Cbeta (PLC). Lipid metabolite(s) generated downstream of PLC are essential for the activation of the TRPC channels in photoreceptor cells. We sought to identify the key lipids produced subsequent to PLC stimulation that contribute to channel activation. Here, using genetics, lipid analysis, and Ca(2+) imaging, we found that light increased the amount of an abundant endocannabinoid, 2-linoleoyl glycerol (2-LG), in vivo. The increase in 2-LG amounts depended on the PLC and diacylglycerol lipase encoded by norpA and inaE, respectively. This endocannabinoid facilitated TRPC-dependent Ca(2+) influx in a heterologous expression system and in dissociated ommatidia from compound eyes. Moreover, 2-LG and mechanical stimulation cooperatively activated TRPC channels in ommatidia. We propose that 2-LG is a physiologically relevant endocannabinoid that activates TRPC channels in photoreceptor cells.
Title: Elevated Levels of Arachidonic Acid-Derived Lipids Including Prostaglandins and Endocannabinoids Are Present Throughout ABHD12 Knockout Brains: Novel Insights Into the Neurodegenerative Phenotype Leishman E, Mackie K, Bradshaw HB Ref: Front Mol Neurosci, 12:142, 2019 : PubMed
Derived from arachidonic acid (AA), the endogenous cannabinoid (eCB) 2-arachidonoyl glycerol (2-AG) is a substrate for alpha/beta hydrolase domain-12 (ABHD12). Loss-of-function mutations of ABHD12 are associated with the neurodegenerative disorder polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cataract (PHARC). ABHD12 knockout (KO) mice show PHARC-like behaviors in older adulthood. Here, we test the hypothesis that ABHD12 deletion age-dependently regulates bioactive lipids in the CNS. Lipidomics analysis of the brainstem, cerebellum, cortex, hippocampus, hypothalamus, midbrain, striatum and thalamus from male young (3-4 months) and older (7 months) adult ABHD12 KO and age-matched wild-type (WT) mice was performed on over 80 lipids via HPLC/MS/MS, including eCBs, lipoamines, 2-acyl glycerols, free fatty acids, and prostaglandins (PGs). Aging and ABHD12 deletion drove widespread changes in the CNS lipidome; however, the effects of ABHD12 deletion were similar between old and young mice, meaning that many alterations in the lipidome precede PHARC-like symptoms. AA-derived lipids were particularly sensitive to ABHD12 deletion. 2-AG increased in the striatum, hippocampus, cerebellum, thalamus, midbrain, and brainstem, whereas the eCB N-arachidonoyl ethanolamine (AEA) increased in all 8 brain regions, along with at least 2-PGs. Aging also had a widespread effect on the lipidome and more age-related changes in bioactive lipids were found in ABHD12 KO mice than WT suggesting that ABHD12 deletion exacerbates the effects of age. The most robust effects of aging (independent of genotype) across the CNS were decreases in N-acyl GABAs and N-acyl glycines. In conclusion, levels of bioactive lipids are dynamic throughout adulthood and deleting ABHD12 disrupts the wider lipidome, modulating multiple AA-derived lipids with potential consequences for neuropathology.
Title: Lipidomics: A Corrective Lens for Enzyme Myopia Bradshaw HB, Leishman E Ref: Methods Enzymol, 593:123, 2017 : PubMed
Classifications and characterizations of specific proteins, such as enzymes, not only allow us to understand biosynthetic and metabolic pathways but they also help to drive our understanding of protein structure and function. How those characterizations are evaluated, however, may change our interpretations and lead us into broader and novel directions in research. Here, we will make the argument that using lipidomics as a tool for characterizing enzymatic function over more traditional toolkit options allows for these types of revelations. Using lipidomics techniques on specific brain regions with a series of enzyme knockout and disease models, we have generated a novel set of analyses from which to view protein function. Through these data, we have demonstrated that NAPE-PLD, MAG lipase, and FAAH all have broader roles throughout the brain than previously thought. Much like the data on how the extinction of specific species within an ecosystem has unpredicted outcomes, so too does the elimination of these enzymes affect the brain lipidome. From a purely biochemical standpoint, it is a fascinating story of how one change in a system can have exponential effects; however, from a drug-target standpoint, it may prove to be a cautionary tale.
BACKGROUND AND PURPOSE: Neuropathic pain is commonly treated with GABA analogues, steroids or non-steroidal anti-inflammatory drugs (NSAIDs). NSAIDs inhibit one or more COX isozymes but chronic COX inhibition paradoxically increases gastrointestinal inflammation and risk of unwanted cardiovascular events. The cannabinoids also have analgesic and anti-inflammatory properties and reduce neuropathic pain in animal models. The present study investigated the analgesic effects of inhibiting both monoacylglycerol lipase (MAGL) and COX enzymes, using low doses of both inhibitors. EXPERIMENTAL APPROACH: Mice subjected to chronic constriction injury (CCI) were tested for mechanical and cold allodynia after administration of the MAGL inhibitor, JZL184, or the non-selective COX inhibitor diclofenac. Then, both drugs were co-administered at fixed dose proportions of 1:3, 1:1 and 3:1, based on their ED50 values. PGs, endocannabinoids and related lipids were quantified in lumbar spinal cord. KEY RESULTS: Combining low doses of JZL184 and diclofenac synergistically attenuated mechanical allodynia and additively reduced cold allodynia. The cannabinoid CB1 receptor antagonist, rimonabant, but not the CB2 receptor antagonist, SR144528, blocked the analgesic effects of the JZL184 and diclofenac combination on mechanical allodynia, implying that CB1 receptors were primarily responsible for the anti-allodynia. Diclofenac alone and with JZL184 significantly reduced PGE2 and PGF2alpha in lumbar spinal cord tissue, whereas JZL184 alone caused significant increases in the endocannabinoid metabolite, N-arachidonoyl glycine. CONCLUSIONS AND IMPLICATIONS: Combining COX and MAGL inhibition is a promising therapeutic approach for reducing neuropathic pain with minimal side effects.
