Synaptic PRG-1 modulates excitatory transmission via lipid phosphate-mediated signaling

Abstract

Plasticity related gene-1 (PRG-1) is a brain-specific membrane protein related to lipid phosphate phosphatases, which acts in the hippocampus specifically at the excitatory synapse terminating on glutamatergic neurons. Deletion of prg-1 in mice leads to epileptic seizures and augmentation of EPSCs, but not IPSCs. In utero electroporation of PRG-1 into deficient animals revealed that PRG-1 modulates excitation at the synaptic junction. Mutation of the extracellular domain of PRG-1 crucial for its interaction with lysophosphatidic acid (LPA) abolished the ability to prevent hyperexcitability. As LPA application in vitro induced hyperexcitability in wild-type but not in LPA(2) receptor-deficient animals, and uptake of phospholipids is reduced in PRG-1-deficient neurons, we assessed PRG-1/LPA(2) receptor-deficient animals, and found that the pathophysiology observed in the PRG-1-deficient mice was fully reverted. Thus, we propose PRG-1 as an important player in the modulatory control of hippocampal excitability dependent on presynaptic LPA(2) receptor signaling.

Figure 1. PRG-1 Is a Neuronal, Synaptic Molecule Expressed by Glutamatergic Neurons

(A) PRG-1 expression in the adult hippocampus as shown by immunohistochemistry (the scale bar represents 250 μm on the left, and 50 μm on the right, respectively). Dendrites of CA1 pyramids exhibit high expression, with membrane-bound expression shown in higher magnification (insets, arrows). PRG-1 did not colocalize with glial markers such as GFAP (the scale bar represents 15 μm). (B) In situ hybridization of prg-1 colocalized with the neuronal marker NeuN but not with GFAP⁺ cells in the CA1 region (the scale bar represents 50 μm). (C) PRG-1/prg-1 was not expressed in interneurons as shown for GAD67, Parvalbumin (PV, arrows), Calbindin (CB, arrows) and Calretinin (CR) by immune stainings and in situ hybridization but was expressed in CB+ CA1 pyramids (arrowheads) (the scale bar represents 15 μm). (D) PRG-1 was mainly expressed on dendrites and colocalized with ProSAP2 and Homer, but not Gephyrin (the scale bar represents 2 μm). Subcellular fractionation of mouse cerebral cortex revealed PRG-1 within the PSD. RabGDI was slightly enriched in the SC+CSV fraction but undetectable in the PSD-95 enriched PSD fraction, whereas PRG-1 was detected in this fraction but not in the presynaptic vesicle fraction (SC+CSV). Ultrastructural analysis of PRG-1 expression shows preferential expression of PRG-1 at the postsynaptic density (the scale bar represents 200 nm).

Figure 2. PRG-1 KO Mice Show Epileptic Activity at the End of the Third Week of Life

(A) Preictal events at P20/P21 and ictal activity at P22 during in vivo recordings of a PRG-1 KO mouse (left column; right column: WT littermate). Video images show tonic-clonic seizures of the same KO mice at P22. (B) Ictal (red) and interictal (blue) periods during in vivo electrographic recording in a P21 PRG-1 KO mouse. (C) In vivo recording of a PRG-1 KO mouse with implanted electrodes at both hemispheres at P20–P22. Hypersynchronized activity and preictal events can be identified at the right hemisphere around P20/P21 (see arrows); generalized ictal activity is seen over both hemispheres at P22.

Figure 3. No Evident Structural Changes, but Pathological Network Properties in Hippocampal Slices

(A) Juvenile PRG-1 KO mice (P21) show no changes in morphology as shown by NeuN staining or PV+ interneuron distribution in the hippocampus. The scale bar represents 100 μm (left) and 50 μm (right). (B) Resting membrane potential, input resistance and action potential height of CA1 pyramidal cells did not vary significantly between WT and PRG-1 KO mice. (C) Hippocampal sharp wave (SPW)-ripples were expressed in PRG-1 KO slices. Upper traces: example data from CA3 in a WT (black trace) and KO slice (red trace). Below: magnified signals (see asterisks for respective locations in recordings), their band-pass-filtered derivative and SPW peak-triggered averages (±SEM), respectively, of 50 randomly selected SPWs. (D1) SPW-incidence was not affected in either condition. (D2) Averaged PSD functions (±SEM) reveal unchanged spectral frequency of ripple oscillation in WT and KO slices. Right: Unaltered ripple power in PRG-1^{−/ −}. (D3) Distribution of slices expressing different classes of hippocampal network oscillations: all tested slices expressed SPWs (100%); following carbachol administration, 75% of slices from WT and 62.5% of slices from PRG-1^{−/ −} animals displayed gamma activity. Interictal-like discharges were not observed in WT but frequently in KO slices (77.8%). Numbers indicate slice fractions displaying the respective electrographic events. (E) Example traces showing gamma oscillation from a WT slice (left) and seizure-like events in a KO slice (right) following carbachol administration, with magnified example (box).

Figure 4. Hyperexcitability in CA1 Neurons in PRG-1 KO Mice

(A) Extracellular fEPSPs were evoked by Schaffer collateral stimulation. fEPSP slope is plotted for WT, PRG-1^+/− and PRG-1^{−/ −}. Increased network excitability is seen for the PRG-1^{−/ −} mice (white) compared with WT mice (black). In WT, PRG-1^+/− and PRG-1^{−/ −} mice, the fEPSP slope increased linearly with increasing fiber volley amplitudes, indicating a gene-dose-dependent effect of PRG-1 reflected in the adjacent western blot of brain homogenates. (B) Evoked synaptic glutamatergic currents (at similar stimulation intensities and stimulation electrode positions) were enhanced for PRG-1 KO mice compared with WT mice. (C) A significantly higher mEPSC frequency was observed in PRG-1 KO CA1 pyramidal cells compared to WT cells (left panel), however the amplitude of these events did not significantly vary between the two groups (right panel). (D) Synaptically evoked IPSCs at different stimulation strengths did not differ between the WT and PRG-1 KO cells. (E) IV curves of inhibitory currents show no significant difference between the WT and PRG-1 KO cells. (F) mIPSC frequency (left panel) and amplitude (right panel) did not differ between PRG-1 KO and WT CA1 pyramidal cells. Data are represented as mean ± SEM, **p < 0.01.

Figure 5. PRG-1 Action in Single CA1 Neurons in Acute Slices

Using in utero electroporation prg-1 was deleted (GFP⁺) in conditional or re-expressed (GFP⁺) in constitutive PRG-1 KO mice in a subset of cells. (A) Left: In simultaneous recordings (upper image) from GFP⁺ (KO) and GFP⁻ (WT, arrow) CA1 pyramidal neurons (middle image) EPSC amplitude (experimental setting: lower image) was significantly increased in KO cells compared to WT cells. Right: In simultaneous recordings from GFP⁺ (PRG-1⁺) and GFP⁻ (KO) CA1 pyramidal neurons EPSC amplitude was significantly decreased in PRG-1-expressing cells compared to KO cells. (B) Left: mEPSCs were recorded from GFP⁺ (KO) and GFP⁻ (WT) cells. Significantly higher mEPSC frequency was observed in the PRG-1^{−/ −} cells compared to WT neurons. Arrow points to GFP⁺ cell, in which PRG-1 (red) has been deleted. Right: mEPSC frequency was significantly reduced in the GFP⁺ (PRG-1-expressing KO) cells compared to the GFP⁻ (KO) cells. GFP signal in neurons indicates PRG-1 expression, confirmed by PRG-1 (red) immunolabeling. Data are represented as mean ± SEM *p < 0.05, **p < 0.01. The scale bar represents 15 μm.

Figure 6. No Evidence for Changes in the Classical Molecular Machinery at the Synapse

(A) Immunofluorescence for presynaptic markers, such as synaptophysin (Syn), vesicular glutamate transporter 1 (VGlut1), vesicular GABA transporter (VGat) and glutamate decarboxylase 67 kDa isoform (GAD67), as well as postsynaptic markers, such as AMPA receptor subunits (GluR1, Glur2/3, GluR4) and the NMDA receptor subunit NR1, do not differ between PRG-1 KO and WT litters. The scale bar represents 50 μm. (B) Protein analysis of the PSD fraction prepared from PRG-1 KO and WT mice. 2.5 μg of purified PSD fractions were subjected to western blotting to estimate expression levels of NR1, NR2A/B, and GluR1, GluR2/3, GluR4, and PSD-95. Expression of the synaptic proteins were quantified and normalized by the loading control (b-actin) and to the WT. (C) Immune staining for VGluT1 and GluR2^ext on dendrites of CA1 pyramidal cells in WT and PRG-1 KO mice. Arrows indicate colocalization. Stereological analysis of synaptically localized GluR2 revealed no difference between WT (n = 5) and PRG-1 KO (n = 5). (D) The AMPA/NMDA ratio did not differ between the WT and PRG1-KO mice. (E) The holding current changes on AMPA (50 nM) wash-in did not differ significantly between the WT and PRG-1 KO mice. Data are represented as mean ± SEM.

Figure 7. PRG-1 Influences Phospholipid Signaling

(A) NBD-PA uptake experiments in primary neurons revealed significantly lower fluorescence intensity in KO cells compared to WT litter cells, indicating reduced lipid uptake (the scale bar represents 10 μM). (B) Mutated PRG-1 (H253K) lacking the ability to interact with phospholipids was expressed in a subset of cells (GFP⁺) in PRG-1 KO animals using in utero electroporation. mEPSC frequency at P21 was not reduced to WT levels in PRG-1 (H253K)-expressing neurons after electroporation of the WT prg-1 construct (see Figure 5). (C) mEPSC frequency rapidly increased in CA1 pyramidal neurons after application of 10 μM LPA, but not in LPA₂ receptor-deficient animals (LPA₂-R-KO). For the analysis, the mEPSC frequency of each analyzed neuron was averaged for the last four minutes under control conditions and in steady state after LPA treatment, and these values then compared. (D) 3D reconstruction of a presynaptical terminal positive for the LPA₂-R, the presynaptic glutamatergic marker VGlut1 and the active zone marker Munc13-1 (the scale bar represents 0.5 μm). (E) Ultrastructural analysis of the LPA₂-R localization revealed a strong DAB signal on the presynaptic side of an asymmetric, presumably glutamatergic synapse (arrowhead). Two adjacent axons display a positive signal underlining the presynaptic LPA2-R localization. In contrast, no LPA₂-R signal was found in symmetric, presumably inhibitory synapses (open arrowhead) (the scale bar represents 200 nm). (F) Normal network excitability is seen in PRG-1 KO/LPA₂-R KO ^{− /−} animals when compared to the PRG-1 KO mice. In contrast to PRG-1 KO mice, double KO animals showed fEPSP slopes similar to WT animals. mEPSC frequency in CA1 pyramidal neurons in hippocampus from double KO mice also returned to WT levels and did not show the increase seen in PRG-1^{−/ −} animals. (G) In vivo recordings of double KO animals did not display the hypersynchronized activity around P21 seen in PRG-1^{−/ −} mice of same age. Data are represented as mean ± SEM, **p < 0.01, ***p < 0.001.