Differential expression patterns of phospholipase D isoforms 1 and 2 in the mammalian brain and retina.

Phosphatidic acid is a key signaling molecule heavily implicated in exocytosis due to its protein-binding partners and propensity to induce negative membrane curvature. One phosphatidic acid-producing enzyme, phospholipase D (PLD), has also been implicated in neurotransmission. Unfortunately, due to the unreliability of reagents, there has been confusion in the literature regarding the expression of PLD isoforms in the mammalian brain which has hampered our understanding of their functional roles in neurons. To address this, we generated epitope-tagged PLD1 and PLD2 knockin mice using CRISPR/Cas9. Using these mice, we show that PLD1 and PLD2 are both localized at synapses by adulthood, with PLD2 expression being considerably higher in glial cells and PLD1 expression predominating in neurons. Interestingly, we observed that only PLD1 is expressed in the mouse retina, where it is found in the synaptic plexiform layers. These data provide critical information regarding the localization and potential role of PLDs in the central nervous system.

Identification of Synaptic DGKθ Interactors That Stimulate DGKθ Activity.

Lipids and their metabolic enzymes are a critical point of regulation for the membrane curvature required to induce membrane fusion during synaptic vesicle recycling. One such enzyme is diacylglycerol kinase θ (DGKθ), which produces phosphatidic acid (PtdOH) that generates negative membrane curvature. Synapses lacking DGKθ have significantly slower rates of endocytosis, implicating DGKθ as an endocytic regulator. Importantly, DGKθ kinase activity is required for this function. However, protein regulators of DGKθ's kinase activity in neurons have never been identified. In this study, we employed APEX2 proximity labeling and mass spectrometry to identify endogenous interactors of DGKθ in neurons and assayed their ability to modulate its kinase activity. Seven endogenous DGKθ interactors were identified and notably, synaptotagmin-1 (Syt1) increased DGKθ kinase activity 10-fold. This study is the first to validate endogenous DGKθ interactors at the mammalian synapse and suggests a coordinated role between DGKθ-produced PtdOH and Syt1 in synaptic vesicle recycling.

Roles of DGKs in neurons: Postsynaptic functions?

Diacylglycerol kinases (DGKs) contribute to an important part of intracellular signaling because, in addition to reducing diacylglycerol levels, they generate phosphatidic acid (PtdOH) Recent research has led to the discovery of ten mammalian DGK isoforms, all of which are found in the mammalian brain. Many of these isoforms have studied functions within the brain, while others lack such understanding in regards to neuronal roles, regulation, and structural dynamics. However, while previously a neuronal function for DGKθ was unknown, it was recently found that DGKθ is required for the regulation of synaptic vesicle endocytosis and work is currently being conducted to elucidate the mechanism behind this regulation. Here we will review some of the roles of all mammalian DGKs and hypothesize additional roles. We will address the topic of redundancy among the ten DGK isoforms and discuss the possibility that DGKθ, among other DGKs, may have unstudied postsynaptic functions. We also hypothesize that in addition to DGKθ's presynaptic endocytic role, DGKθ might also regulate the endocytosis of AMPA receptors and other postsynaptic membrane proteins.

Lipid Metabolism Crosstalk in the Brain: Glia and Neurons.

Until recently, glial cells have been considered mainly support cells for neurons in the mammalian brain. However, many studies have unveiled a variety of glial functions including electrolyte homeostasis, inflammation, synapse formation, metabolism, and the regulation of neurotransmission. The importance of these functions illuminates significant crosstalk between glial and neuronal cells. Importantly, it is known that astrocytes secrete signals that can modulate both presynaptic and postsynaptic function. It is also known that the lipid compositions of the pre- and post-synaptic membranes of neurons greatly impact functions such as vesicle fusion and receptor mobility. These data suggest an essential lipid-mediated communication between glial cells and neurons. Little is known, however, about how the lipid metabolism of both cell types may interact. In this review, we discuss neuronal and glial lipid metabolism and suggest how they might interact to impact neurotransmission.

Phosphatidic acid-producing enzymes regulating the synaptic vesicle cycle: Role for PLD?

In cortical and hippocampal neurons of the mammalian brain, the synaptic vesicle cycle is a series of steps that tightly regulate exo- and endocytosis of vesicles. Many proteins contribute to this regulation, but lipids have recently emerged as critical regulators as well. Of all the many lipid signaling molecules, phosphatidic acid is important to the physical processes of membrane fusion. Therefore, the lipid-metabolizing enzymes that produce phosphatidic acid are vital to the regulation of the cycle. Our lab is particularly interested in the potential regulatory mechanisms and neuronal roles of two phosphatidic acid-producing enzymes: diacylglycerol kinase theta (DGKθ) and phospholipase D (PLD). We recently discovered a regulatory role of DGKθ on evoked endocytosis (Goldschmidt et al., 2016). In addition to this enzyme, studies implicate PLD1 in neurotransmission, although its precise role is of some debate. Altogether, the production of phosphatidic acid by these enzymes offer an interesting and novel pathway for the regulation of the synaptic vesicle cycle.

Phosphatidic acid and neurotransmission.

Lipids play a vital role in the health and functioning of neurons and interest in the physiological role of neuronal lipids is certainly increasing. One neuronal function in which neuronal lipids appears to play key roles in neurotransmission. Our understanding of the role of lipids in the synaptic vesicle cycle and neurotransmitter release is becoming increasingly more important. Much of the initial research in this area has highlighted the major roles played by the phosphoinositides (PtdIns), diacylglycerol (DAG), and phosphatidic acid (PtdOH). Of these, PtdOH has not received as much attention as the other lipids although its role and metabolism appears to be extremely important. This lipid has been shown to play a role in modulating both exocytosis and endocytosis although its precise role in either process is not well defined. The currently evidence suggest this lipid likely participates in key processes by altering membrane architecture necessary for membrane fusion, mediating the penetration of membrane proteins, serving as a precursor for other important SV cycling lipids, or activating essential enzymes. In this review, we address the sources of PtdOH, the enzymes involved in its production, the regulation of these enzymes, and its potential roles in neurotransmission in the central nervous system.

Compensatory plasticity in the olfactory epithelium: age, timing, and reversibility.

Like other biological systems, olfaction responds "homeostatically" to enduring change in the stimulus environment. This adaptive mechanism, referred to as compensatory plasticity, has been studied almost exclusively in developing animals. Thus it is unknown if this phenomenon is limited to ontogenesis and irreversible, characteristics common to some other forms of plasticity. Here we explore the effects of odor deprivation on the adult mouse olfactory epithelium (OE) using nasal plugs to eliminate nasal airflow unilaterally. Plugs were in place for 2-6 wk after which electroolfactograms (EOGs) were recorded from the occluded and open sides of the nasal cavity. Mean EOG amplitudes were significantly greater on the occluded than on the open side. The duration of plugging did not affect the results, suggesting that maximal compensation occurs within 2 wk or less. The magnitude of the EOG difference between the open and occluded side in plugged mice was comparable to adults that had undergone surgical naris occlusion as neonates. When plugs were removed after 4 wk followed by 2 wk of recovery, mean EOG amplitudes were not significantly different between the always-open and previously plugged sides of the nasal cavity suggesting that this form of plasticity is reversible. Taken together, these results suggest that compensatory plasticity is a constitutive mechanism of olfactory receptor neurons that allows these cells to recalibrate their stimulus-response relationship to fit the statistics of their current odor environment.