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To address this controversy regarding the expression and localization of PLD1 and PLD2 in the mammalian brain, we generated endogenously-expressed, epitope-tagged PLD1 and PLD2 knockin mice. Using neuron cultures and brain slices from these mice, we were able to demonstrate the expression of both PLD1 and PLD2 in mature neurons, with PLD2 expression being much higher in glial cells throughout development and into maturity. Additionally, we observed the presence of PLD1 exclusively in the mouse retina, in the synaptic plexiform layers. Taken together, this is the first study to use PLD knockin mice to examine the expression of PLD1 and PLD2 in neuronal and glial cultures, as well as in the brain and retina. Our results clearly demonstrate that PLDs are differentially expressed throughout the central nervous system.
Materials & MethodsReagentsKnockin MiceAll animal protocols were approved by the Johns Hopkins University Animal Care and Use Committee. To generate myc-PLD1 knockin and HA-PLD2 knockin mouse lines, CRISPR-Cas9 was used to insert tags into the N-terminal region of the protein sequence. Three tandem myc tags were inserted into exon 2 (the first protein-coding exon) of PLD1, and three tandem HA tags were inserted into exon 2 (the first protein-coding exon) of PLD2. Both insertion points were upstream of the PX domain within each protein. Pronuclear injection of one-cell C57BL/6J embryos (Jackson Laboratories, 000664) was performed by the JHU Transgenic Core using standard microinjection techniques (26Nagy A, Gertsenstein M, Vintersten K, Behringer R. (2003) Manipulating the Mouse Genome, A Laboratory Manual 3rd ed. Cold Spring Harbor Press.
) using a mix of Cas9 protein (30ng/ul, PNABio), tracrRNA (0.6μM, IDT), PLD1 crRNA (0.3μM, IDT), PLD2 crRNA (0.3μM, IDT), PLD1 ssDNA oligo (5ng/ul, IDT) and PLD2 ssDNA oligo (5ng/ul, IDT) diluted in RNAse free injection buffer (10 mM Tris-HCl, pH 7.4, 0.25 mM EDTA). See table below for sequences. Injected embryos were transferred into the oviducts of pseudopregnant ICR females (Envigo, 030) using previously described techniques (26Nagy A, Gertsenstein M, Vintersten K, Behringer R. (2003) Manipulating the Mouse Genome, A Laboratory Manual 3rd ed. Cold Spring Harbor Press.
). Resulting mice were then backcrossed to wild-type C57BL/6J mice and heterozygotes were crossed to produce mice homozygous for the inserted tags.Harvesting brain tissueTo confirm the presence of myc and HA tags in our PLD1 and PLD2 knockin mouse lines, respectively, WT, myc-PLD1, and HA-PLD2 mice were sacrificed at postnatal day 21 (P21) and brains were removed. Whole brain tissue was homogenized with a glass homogenizer in homogenization buffer (0.32M sucrose, 4mM Hepes, pH7.4) until solution was uniform. The homogenate was transferred to Eppendorf tubes and centrifuged at 1000 x g for 10 mins at 4C. The supernatant (S1) was collected and transferred to a new tube and kept at -20C until further analysis.
Western blotting analysisSDS-PAGE was performed with Bolt 8% Bis-Tris mini protein gels. S1 from whole brain lysates were diluted with water and 4x Laemmli sample buffer containing 10% β-mercapthoethanol and boiled for 5 min at 100C before loading on gels. Following electrophoresis, proteins were transferred to nitrocellulose membranes with the Bio-Rad Mini Trans-Blot system and blocked with 3% bovine serum albumin (BSA) for 1 hr. Blots were incubated with primary antibodies diluted in 3% BSA in phosphate-buffered saline containing 0.1% Triton-X (PBST) on a 4C rocker overnight. The next day, blots were washed 3x with PBST and incubated with LICOR IRDye-conjugated secondary antibodies for 1 hr at RT. Blots were then washed 3x with PBST before infrared imaging using an Odyssey Imaging System.
Neuron CultureTo prepare mouse primary neuron cultures, brains were removed from P0 pups. Cortices were incubated in dissection media with papain and DNase for 25min at 37C and then fully dissociated with gentle trituration. Cells were then plated on poly-L-lysine (PLL)-coated 18mm glass coverslips placed in the wells of a 12well plate in Neurobasal Plus medium supplemented with 5% horse serum, 50U/ml penicillin, 50U/ml streptomycin, 2mM Glutamax and 2% b27 Plus at 500k cells/well. Neurons were maintained in a 37C incubator (5%CO2). At DIV4, neurons were fed with serum-reduced (2% horse serum) media supplemented with fluoro-deoxyuridine (FDU) to prevent the proliferation of glial cells. Subsequently, neurons were fed every 4 days with Neurobasal Plus medium supplemented with 50 U/ml penicillin, 50 U/ml streptomycin, 2mM Glutamax, and 2% b27 Plus.
Culture ImmunofluorescenceTo perform immunofluorescence experiments on mouse neurons plated on glass coverslips as described above, the coverslips were first washed 1x with PBS and then incubated in parafix (4% paraformaldehyde, 4% sucrose in PBS) for 15 min at room temperature (RT). Cells were washed 3x with PBS and then permeabilized for 10 min with 0.25% Triton-X in PBS. They were then washed 3x with PBS and incubated with 10% BSA at 37C for 1 hr to reduce non-specific antibody binding. Coverslips were then incubated with primary antibodies diluted in 3% BSA overnight at 4C. The following day, coverslips were washed 3x with PBS and incubated with secondary antibodies (goat conjugated Alexa Fluor 647, 568, or 488) diluted in 3% BSA in PBST for 1 hr at RT. They were then washed 3x with PBS and mounted on glass slides with Fluoromount-G and stored at 4C. Images were obtained with an LSM 880-laser scanning confocal microscope (Zeiss) and images were analyzed with ImageJ and Fiji.
Brain slice immunofluorescenceWT (P25), myc-PLD1 (P26), and HA-PLD2 (P32) mice were deeply anaesthetized and transcardially perfused with PBS followed by 4% PFA, each ice cold. Brains were post-fixed in 4% PFA for 4 hr at 4C and then washed 3x with PBS. Brains were kept at 4C in PBS until slicing. Brains were sliced into 60μm sections on a vibratome (VT-1000, Leica) and kept in PBS until staining. For staining, slices were washed 3x with PBS before permeabilizing with 0.3% Triton-X in PBST for 20 min at RT. Slices were then incubated with 5% normal goat serum (NGS), 0.15% Triton-X in PBST for 1 hr at RT to reduce background staining. Primary antibodies were diluted in 5% NGS, 0.15% Triton-X in PBST and incubated with slices overnight on a rocker at 4C. The next day, slices were washed 5x with PBS and incubated with secondary antibodies diluted in 5% NGS, 0.15% Triton-X in PBST overnight on a rocker at 4C. Finally, slices were washed 5x with PBS and mounted on glass slides with Epredia Lab Vision PermaFluor aqueous mounting medium. Images were obtained with an LSM 880-laser scanning confocal microscope (Zeiss) and images were analyzed with ImageJ and Fiji.
Retinal slice immunofluorescenceMouse retinal slices from WT, myc-PLD1 and HA-PLD2 mice were prepared according to established protocols (27Large-scale phenotypic drug screen identifies neuroprotectants in zebrafish and mouse models of retinitis pigmentosa.). Briefly, mice were anesthetized using isoflurane and decapitated. Both eyes were explanted and incubated in 4% PFA for 30 min. Next, the cornea and lens were removed and eye cups were incubated in 4% PFA for 2 hr on ice. Eye cups were washed carefully with 1x PBS and then soaked in 15% sucrose (w/v) in 1x PBS for 3 hr, then incubated in 30% sucrose (w/v) in 1x PBS overnight. Tissue was embedded in OCT (optimal cutting temperature) compound (TIssueTek) and frozen on dry ice. Slices of 12-14 μm thickness were sectioned using a Leica CM3050S cryostat and were subsequently mounted on Superfrost Plus slides (Fisher Scientific).To perform retinal staining experiments, slides containing WT, myc-PLD1 and HA-PLD2 retinal slices were first washed in 1x PBS three times for 5 min each. Tissue was permeabilized by incubation with 0.25% Triton-X in 1x PBS for 5 min at RT. To limit nonspecific staining, slides were incubated in 0.1% Triton-X in 1x PBS + 5% normal goat serum for 1 hr at RT. Subsequently, slides were incubated with myc/HA, vGlut1, and PKCα primary antibodies diluted in 0.1% Triton-X in 1x PBS + 5% normal goat serum overnight at 4C. The next day, slides were washed 3x 15 min each with 1x PBS. Slides were then incubated with secondary antibodies (goat conjugated Alexa Fluor 647, 568, or 488) diluted in 0.1% Triton-X in 1x PBS + 5% normal goat serum for 1 hr at RT in the dark. Slides were then washed 3x 15 min each with 1x PBS and glass slides were placed on top with Epredia Lab Vision PermaFluor aqueous mounting medium. Images were obtained with an LSM 880-laser scanning confocal microscope (Zeiss) and images were analyzed with ImageJ and Fiji.
ElectroretinogramsElectroretinograms were recorded from PLD1 KO and WT mice using the Celeris full-field ERG system from Diagnosys (model D430) with an internal platform heater to maintain body temperature. Mice were dark adapted overnight, and anesthetized with 10 mg/kg Ketamine/1mg/kg xylazine under dim red light conditions. Pupils were dilated using 0.5% tropicamide eye drops (AKORN Inc., Lake Forest, IL), and GenTeal eye gel (0.3% Hypomellose, Alcon) was applied on both eyes before placing the electrodes. ERG responses were obtained using the opposite eye electrode as a reference and an electrode placed into the haunch as a ground electrode. For scotopic measurements, single flash recordings were performed at light intensities of 0.01, 0.1, 1 cd.s/m2 of white light with no illumination between flashes. Following 10 minutes of light adaptation (3 cd.s/m2), we obtained photopic flash recordings at light intensities of 3 and 10 cd.s/m2 with a background intensity of .1 cd/m2. Averages of 5 (scotopic) or 10 (photopic) sweeps were computed and analyzed using Espion V6 software from Diagnosys. The a-wave amplitude measured from stimulus onset to the trough of the a-wave and the b-wave amplitude ranging from the trough of the a-wave to the peak of the b-wave. Data was exported to Microsoft Excel, and a- and B-wave values from the left and right eyes were averaged. Data were compiled, plotted and analyzed in GraphPad Prism.
PLD AssayPLD activity was assessed with the EnzyChrom Phospholipase D Assay Kit (BioAssay Systems EPPD-100). A standard curve was prepared with 4 defined amounts of choline diluted in dH20. Known concentrations of WT, myc-PLD1, and HA-PLD2 whole brain lysate were combined with a master mix of assay buffer, purified PLD enzyme, dye reagent, and phosphatidylcholine substrate. Reactions were mixed and incubated for 10 mins at 37C and 570nm optical density readings were recorded at 10, 15, and 30 mins.
DiscussionIn an effort to provide a clear understanding of PLD expression and localization in the mammalian brain, we generated the first endogenously-expressed, epitope-tagged PLD1 and PLD2 knockin mouse lines. Our studies demonstrate that PLD1 and PLD2 are both localized synaptically in neurons in vitro, using cultured neurons, and in vivo, using brain slices. PLD1 expression is predominantly in neurons, while PLD2 expression is robust in glial cells. In addition to tissue from the central nervous system (CNS), we also examined retinal slices from our knockin mice. These data indicate that PLD1 alone is expressed abundantly in the synaptic plexiform layer. However, analysis of cellular connectivity in the retina via ERG recordings revealed no differences between WT and PLD1 KO mice, suggesting PLD1 is not required for efficient neurotransmission within these cells. Altogether, these studies permit a more definitive understanding of the expression of PLD isoforms in neuronal and glial cells. Additionally, the generation of the myc-PLD1 and HA-PLD2 knockin mouse lines will be beneficial to a wide range of studies regarding PLDs, from their anti-apoptotic role in cancer (32Phospholipase D and its essential role in cancer., 33Bruntz R. Lindsley C. Brown H.A. Phospholipase D signaling pathways and phosphatidic acid as therapeutic target in cancer.), to their potential as a therapeutic target for Alzheimer’s disease (34Krishnan B. Kayed R. Taglialatela G. Elevated phospholipase D isoform 1 in Alzheimer’s disease patients’ hippocampus: relevance to synaptic dysfunction and memory deficits., 45Phospholipase D1 corrects impaired βAPP trafficking and neurite outgrowth in familial Alzheimer’s disease-linked presenilin-1 mutant neurons., 46Phospholipase D2 ablation ameliorates Alzheimer’s disease-linked synaptic dysfunction and cognitive deficits.).The results in this report will impact a range of other topics regarding functional roles of PLDs. Although PLD-mediated production of PtdOH is implicated in exocytosis, functional studies of PLD1 in neurons have also revealed a role for PLD1 in dendritic branching. Studies in rat hippocampal cultures concluded that overexpression of PLD1 led to a decrease in the complexity of dendritic arborization, while inhibition of PLD1 caused increased dendritic branching (24Zhu Y. Kang K. Zhang Y. Qi C. Li G. Yin D. Wang Y. PLD1 negatively regulates dendritic branching.). Further, it was recently found that PLD1 interacts with PKD1 in neurons and positively regulates dendritic spine morphogenesis (35Li W. Luo L. Hu Z. Lyu T. Cen C. Wang Y. PLD1 promotes dendritic spine morphogenesis via activating PKD1.). Our data that PLD1 is expressed in neurons throughout development, into maturity, and is expressed at the synapse supports these findings. Myc-PLD1 cortical cultures display PLD1 expression robustly throughout dendrites. In contrast to these functional studies, the literature lacks concrete evidence about PLD1’s potential role in synaptic vesicle cycling. Its localization at synapses identified in our studies contributes to the hypothesis that PLD1 modulates synaptic vesicle exocytosis, also supported by the 2001 study that found the injection of catalytically inactive PLD1 led to a decrease in acetylcholine release (29Humeau Y. Vitale N. Chasserot-Golaz S. Dupont J. Du G. Frohman M. Bader M. Poulain B. A role for phospholipase D in neurotransmitter release.). While this is still an exciting finding, it is important to recall that conceptually, the injection of dominant-negative proteins into neurons can have a wide range of unexpected effects (36Chen X. Rzhetskaya M. Kareva T. Bland R. During M. Tank A.W. Kholodilov N. Burke R. Antiapoptotic and trophic effects of dominant-negative forms of dual leucine zipper kinase in dopamine neurons of the substantia nigra in vivo., 37Peakman M. Colby C. Perrotti L. Tekumalla P. Carle T. Ulery P. Chao J. Duman C. Steffen C. Monteggia L. Allen M. Stock J. Duman R. McNeish J. Barrot M. Self D. Nestler E. Schaeffer E. Inducible, brain region-specific expression of a dominant negative mutant of c-Jun in transgenic mice decreases sensitivity to cocaine.). The results from this study (29Humeau Y. Vitale N. Chasserot-Golaz S. Dupont J. Du G. Frohman M. Bader M. Poulain B. A role for phospholipase D in neurotransmitter release.) also suggest that PLD1 affects the number of available vesicle release sites and not nece
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