The Synaptic Ribbon Is a Site of Phosphatidic Acid Generation in Ribbon Synapses

Abstract

Ribbon synapses continuously transmit graded membrane potential changes into changes of synaptic vesicle exocytosis and rely on intense synaptic membrane trafficking. The synaptic ribbon is considered central to this process. In the present study we asked whether tonically active ribbon synapses are associated with the generation of certain lipids, specifically the highly active signaling phospholipid phosphatidic acid (PA). Using PA-sensor proteins, we demonstrate that PA is enriched at mouse retinal ribbon synapses in close vicinity to the synaptic ribbon in situ. As shown by heterologous expression, RIBEYE, a main component of synaptic ribbons, is responsible for PA binding at synaptic ribbons. Furthermore, RIBEYE is directly involved in the synthesis of PA. Using various independent substrate binding and enzyme assays, we demonstrate that the B domain of RIBEYE possesses lysophosphatidic acid (LPA) acyltransferase (LPAAT) activity, which leads to the generation of PA from LPA. Since an LPAAT-deficient RIBEYE mutant does not recruit PA-binding proteins to artificial synaptic ribbons, whereas wild-type RIBEYE supports PA binding, we conclude that the LPAAT activity of the RIBEYE(B) domain is a physiologically relevant source of PA generation at the synaptic ribbon. We propose that PA generated at synaptic ribbons likely facilitates synaptic vesicle trafficking.

Phosphatidic acid and neurotransmission

“…This enzyme appears to play an important role in ribbon synapses (Schwarz, Natarajan, 2011). These synapses are found in certain sensory neurons requiring large amount of vesicle recycling in a fast and efficient manner; photoreceptor cells, retinal bipolar cells, and inner ear hair cells.…”

“…This protein has both a structural and enzymatic role. Its enzymatic activity was shown to be an LPAAT generating PtdOH important for exocytosis at these synapses (Schwarz, Natarajan, 2011). We note that endophilin I, a protein involved in endocytosis that interacts with synaptojanin and dynamin, was once believed to possess LPAAT activity (Schmidt et al, 1999).…”

“…The observations that PLD and DGK (Davletov and Montecucco, 2010, Humeau, Vitale, 2001, Liu et al, 2005, Rohrbough and Broadie, 2005, Waring et al, 1999)(Almena and Merida, 2011, Kanoh et al, 2002, Merida et al, 2008, van Blitterswijk and Houssa, 2000)) play roles in neurotransmitter release strongly implicate this class of enzymes in modulating the levels of PtdOH in neurons (Cremona, Di, 1999b, Davletov and Montecucco, 2010, Goldschmidt, Tu-Sekine, 2016, Huttner and Schmidt, 2000, Lauwers, Goodchild, 2016, Lim and Wenk, 2009, Puchkov and Haucke, 2013, Rohrbough and Broadie, 2005, Schwarz, Natarajan, 2011, Tu-Sekine, Goldschmidt, 2015, Tu-Sekine and Raben, 2011, Yuan, Liu, 2015). Much has been written about the regulation of PLDs and has been the subject of numerous other reviews (Bruntz et al, 2014, Exton, 2002a, b, Klein, 2005, Lauwers, Goodchild, 2016, Lee, Kang, 1997, Lee et al, 2000, Sarri et al, 1998, Selvy et al, 2011, Vitale et al, 2005, Waring, Drappatz, 1999, Wenk and De Camilli, 2004).…”

“…As a result of the research to examine the role of lipid involvement in neurotransmission, it is now clear that phosphoinositides (PtdIns), diacylglycerol (DAG), and phosphatidic acid (PtdOH), play key roles (Davletov and Montecucco, 2010, Lauwers, Goodchild, 2016, Lim and Wenk, 2009, Rohrbough and Broadie, 2005, Schwarz et al, 2011, Tu-Sekine, Goldschmidt, 2015, Tu-Sekine and Raben, 2011, Yuan et al, 2015). Given their popularity, it is understandable that PtdIns(4,5)P 2 and PtdIns(3,4,5)P 3 have received a great deal of attention (see (Cremona et al, 1999a, Davletov and Montecucco, 2010, Di Paolo and De Camilli, 2006, Di Paolo et al, 2004, Lauwers, Goodchild, 2016, Lim and Wenk, 2009, Martin, 2015, Micheva et al, 2001, Puchkov and Haucke, 2013).…”

“…While not receiving a lot of the initial attention, there are now compelling data implicating PtdOH in modulating exocytosis and endocytosis (Antonescu et al, 2010, Burger et al, 2000, Chasserot-Golaz et al, 2010, Lim and Wenk, 2009, Paillart et al, 2003, Puchkov and Haucke, 2013, Rohrbough and Broadie, 2005, Schwarz, Natarajan, 2011, Takei et al, 1998, Xie et al, 2015) although its precise role is not well defined. This lipid could play roles by altering membrane architecture necessary for membrane fusion (see (Lauwers, Goodchild, 2016, Rohrbough and Broadie, 2005)), mediating membrane penetration of proteins such as dynamin (Burger, Demel, 2000, Takei, Haucke, 1998); (see (Doherty and McMahon, 2009, Ferguson and De Camilli, 2012, Kokotos and Cousin, 2015)), serving as a precursor for other important SV cycling lipids such as PtdIns(4,5)P 2 , or activating enzymes such as a PI kinase (Jenkins et al, 1994, Moritz et al, 1992) needed for the synthesis of this lipid (Thieman et al, 2009, Volpicelli-Daley et al, 2010).…”