Plasticity-related gene 5 promotes spine formation in murine hippocampal neurons

Abstract

The transmembrane protein plasticity-related genes 3 and 5 (PRG3 and PRG5) increase filopodial formation in various cell lines, independently of Cdc42. However, information on the effects of PRG5 during neuronal development is sparse. Here, we present several lines of evidence for the involvement of PRG5 in the genesis and stabilization of dendritic spines. First, PRG5 was strongly expressed during mouse brain development from embryonic day 14 (E14), peaked around the time of birth, and remained stable at least until early adult stages (i.e. P30). Second, on a subcellular level, PRG5 expression shifted from an equal distribution along all neurites toward accumulation only along dendrites during hippocampal development in vitro. Third, overexpression of PRG5 in immature hippocampal neurons induced formation of spine-like structures ahead of time. Proper amino acid sequences in the extracellular domains (D1 to D3) of PRG5 were a prerequisite for trafficking and induction of spine-like structures, as shown by mutation analysis. Fourth, at stages when spines are present, knockdown of PRG5 reduced the number but not the length of protrusions. This was accompanied by a decrease in the number of excitatory synapses and, consequently, by a reduction of miniature excitatory postsynaptic current frequencies, although miniature excitatory postsynaptic current amplitudes remained similar. In turn, overexpressing PRG5 in mature neurons not only increased Homer-positive spine numbers but also augmented spine head diameters. Mechanistically, PRG5 interacts with phosphorylated phosphatidylinositols, phospholipids involved in dendritic spine formation by different lipid-protein assays. Taken together, our data propose that PRG5 promotes spine formation.

Keywords: Cell Biology; Cell Differentiation; Dendritic Spine; Membrane Protein; Phosphatidylinositol Phosphatase.

FIGURE 1.

PRG5 is mainly expressed in the brain. A, qRT-PCR of different mouse tissues (harvested at postnatal day 5 (P5)) revealed the most prominent PRG5 mRNA expression in the brain. Data were normalized to hypoxanthine phosphoribosyltransferase (HPRT). B, age-dependent changes in PRG5 expression levels revealed by qRT-PCR of mouse hippocampal tissue between E14 and P30. The highest PRG5 mRNA expression was detected around birth. Data were normalized to hypoxanthine phosphoribosyltransferase. C, PRG5 mRNA expression in neurons, astrocytes, and microglia normalized to GAPDH. qRT-PCR analysis reveals a robust neuronal expression of PRG5. D, Western blot analysis of membrane (m) and cytosolic (c) protein fractions demonstrate endogenous membrane expression of PRG5 in immature primary hippocampal neurons (DIV2). Anti-α-tubulin served as loading control. IB, immunoblot. E, specificity analysis of the anti-PRG5 antibody revealed no cross-reactivity to other PRG family members. Fusion proteins of several PRG family members with eGFP (PRG1, PRG2, PRG3, PRG4, and PRG5) were used in the Western blot analyses. The anti-PRG5 antibody detected protein exclusively in the PRG5 lane. F, Western blot of HEK-293 cells after transfection with two different PRG5 constructs (PRG5-FLAG and PRG5 N158Q). Native lysates (1st and 3rd lanes) or lysates treated with N-glycosidase F (2nd and 4th lanes) were probed with an anti-FLAG antibody. When WT PRG5 protein lysate treated with N-glycosidase F, a band is detected at a lower molecular weight compared with untreated lysate (1st and 2nd lanes). This points out that PRG5 is N-glycosylated. Mutation of asparagine 158 to glutamine (3rd and 4th lanes) shifts the PRG5 band to a lower molecular weight with and without treatment. Note that the mutant PRG5 protein band is not at the same height than the WT PRG5-treated band. G, confocal images of immature neurons endogenously expressing PRG5 (green) and anti-α-tubulin (red). Magnification of structures within the white frame shows a strong dotted PRG5 signal along the neurites. Scale bars for the upper images represent 10 μm and for the magnified sections (lower images) 4 μm, respectively.

FIGURE 2.

PRG5 induces spine-like structures in immature hippocampal neurons. A, hippocampal primary neurons transfected with PRG5-FLAG (left, green) and co-immunostained with anti-F-actin (middle, red). Magnification (white frame, lower images) shows that PRG5 overexpression induces spine-like structures along neurites. Arrowheads indicate PRG5 expression in the bulbous head. B, eGFP overexpressing hippocampal neurons have the typical morphology of immature neurons. There is no spine formation along the neurites. C and D, magnification of neurites from neurons transfected with PRG5-FLAG or eGFP and co-stained with anti-FLAG (green) and anti-α-tubulin (red). White arrowheads indicate heads of different spine-like structures induced by PRG5 overexpression in comparison with eGFP-transfected cells (C). Scale bars for the upper images in A and B represent 10 μm and for the magnified sections (lower images) in A and B, and 4 μm for C and D, respectively. E, verification of PRG5 shRNA and shRNA-resistant PRG5 construct by Western blot. PRG5-FLAG was reduced when compared with controls or with PRG5 shRNA-resistant protein levels. Ratios below the Western blot bands refer to densitometry measurements of the gray intensity of these bands in relation to the control band. Anti-β-actin was used as loading control and for normalization. Anti-GFP shows transfection efficiency. IB, immunoblot. F, quantification of shRNA-PRG5 efficiency. The shRNA-PRG5 (n = 94) expression reduced endogenous PRG5 expression more than one-third when compared with control (n = 112). G, PRG5 knockdown did not affect the numbers of protrusions/filopodia in immature neurons. Primary neurons were transfected with shRNA-PRG5 or controls at DIV1 and morphologically analyzed at DIV2. Numbers of protrusions/filopodia per 10 μm in immature neurons were counted. When co-transfected with PRG5, rescue plasmid (Resc.) protrusion/filopodia drastically increased. The mutations are silent so that the rescue plasmid is comparable with PRG5 overexpression. ***, p ≤ 0.0001.

FIGURE 3.

Residues within the extracellular loops are essential for PRG5-mediated spine-like structure. A, structure model based on the amino acid sequence of human PRG5. PRG5, like the LPPs, contains six putative transmembrane regions and intracellularly located C and N termini. The three mutated amino acids Ser-193, Glu-195, and Arg-241 are marked in red. Boxes are positioned at D1, D2, and D3 regions, respectively. B, alignment of the domain sequences from mouse PRGs with mouse LPP-1 (mPRG1 (Q7TMEO), mPRG3 (NP_848871), mPRG5 (AAS80161), and mLPP-1 (NP_032273)). In red are residues of conserved amino acids that have been shown to be important for the PRG3 induction of filopodia. Western blot analysis is shown of protein lysates from HEK-293 cells transfected with PRG5-FLAG mutants or empty vector control and detected with an anti-FLAG. α-Tubulin served as loading control. C, immature primary neurons (DIV2) transfected with mutants S193W, E195G, R241E, or E195H and co-stained with anti-FLAG and anti-F-actin. Neurons, overexpressing PRG5 S193W, R241E, or E195G, did not show any protrusions at the plasma membrane. On the contrary, overexpression of PRG5-FLAG E195H induced a considerable rearrangement of the plasma membrane, with a high number of spine-like structures. White arrowheads point to the spine-like structures. Scale bars represent 10 or 3 μm (higher magnification images), respectively.

FIGURE 4.

PRG5 contributes to regulation of spine density and morphology in mature hippocampal neurons. A, Western blot analysis of membrane protein lysates from primary neurons at DIV14 shows endogenous membrane expression of PRG5. As loading control α-tubulin was used. IB, immunoblot. B, confocal images of dendritic and axonal structures from hippocampal neurons (DIV14) stained with anti-PRG5 (green) and anti-MAP2 (red, dendrites) or anti-Tau1 (red, axons) revealed a clear co-localization of PRG5 and MAP2 compared with Tau1. Scale bars, 10 μm. C, analyses of the PRG5 abundance on MAP2- and Tau1-positive structures measured by fluorescence signal intensity. Fluorescence intensity of PRG5 in MAP2-positive structures (n = 12) is significantly higher compared with the one in Tau1-positive structures (n = 69) (p ≤0.0001). D, morphology analyses in mature primary neurons transfected either with PRG5-FLAG or PRG3-FLAG 1 to 2 days after transfection. Determination of the numbers of protrusions, number of Homer-positive spines, and the spine head diameter revealed a significant increase compared with PRG3 control. *, 0.01 ≤ p ≥ 0.05, and ***, 0.001 ≤ p ≥ 0.01.

FIGURE 5.

Alteration of PRG5 expression reduces functional synapses in mature hippocampal neurons. A, efficiency of shRNA-PRG5 in mature neurons. The shRNA-PRG5 (n = 201) expression reduced the endogenous PRG5 expression more than one-third when compared with control (n = 123). Such PRG5 knockdown in mature primary neurons reduced the numbers of protrusions (shRNA-PRG5 n = 274 versus shRNA-luciferase n = 205) but not their length (shRNA-PRG5 n = 90 versus shRNA-luciferase n = 102). B, quantitative analysis of excitatory synapses. Left, structures expressing anti-GFP, anti-VGlut1, as marker for presynaptic structures, and anti-GluR2, as marker for postsynaptic structures in close proximity, are regarded as synapses. Scale bar represents 2 μm. Right, population data indicate that down-regulating PRG5 reduces the number of excitatory synapses, compared with control (shRNA-luciferase). C, representative example of nontransfected (control) and transfected (shRNA-PRG5, upper row) or nontransfected (control) and transfected (shRNA-luciferase, lower row) primary cultured hippocampal neurons (DIV14–16). Current traces were recorded somatically under whole-cell patch clamp conditions at −60 mV holding potential. Negative deflections represent electrically and pharmacologically isolated mEPSCs. Note that shRNA-PRG5 and shRNA-luciferase were transfected in different culture dishes and that control represents recordings in respective culture dishes, suggesting that differences in control frequencies are due to culture specific variations. D, population data of frequencies and amplitudes of mEPSCs without (control, gray columns, n = 24) or with PRG5 silencing (shRNA-PRG5, white columns, n = 18, upper row) and from untransfected (control, gray columns, n = 19) primary neurons and shRNA-luciferase transfected primary neurons (shRNA-luciferase, white columns, n = 25, lower row). *, 0.01 ≤ p ≥ 0.05; **, 0.001 ≤ p ≥ 0.01; and ***, p ≤ 0.0001.

FIGURE 6.

PRG5 C terminus is essential for spine formation and binds phospholipids. A, Western blot analysis of protein lysates from HEK-293 cells transfected with PRG5-FLAG or PRG5-Δ C-terminal FLAG, both detected with an anti-FLAG. The structure model is based on amino acid sequence of human PRG5 and shows the truncation of PRG5. Removal of 37 amino acids at the C terminus led to an ∼10-kDa shift as shown by Western blotting. Note that the PRG5-Δ C-terminal FLAG construct showed almost only one prominent band. B, protein-lipid overlay assay using 5 μg/ml total protein lysate of HEK-293 cells overexpressed with PRG5-FLAG or PRG5-Δ C-terminal FLAG incubated with immobilized phosphatidylinositols and other lipids at 4 °C overnight. Membranes were immunoblotted with anti-FLAG antibody, and bound protein was visualized by ECL. PRG5 bound to phosphatidic acid (PA), phosphatidylserine (PS) and to all phosphorylated phosphatidylinositols (PtdInsP), but not to nonphosphorylated-phosphatidylinositol (Pins), phosphatidylcholine (PC), phosphatidylethanolamine (PE), lysophosphatidic acid (LPA), lysophosphocholine (LPC), or sphingosine 1-phosphate (S1P). In contrast, PRG5-Δ C terminus did not bind to any lipid of the strip except weakly to PtdInsP. C, binding specificity of these lipids was assessed by PIP Arrays^TM. The array experiments probed with PRG5-FLAG again showed signals for all PtdInsP. Interestingly, PRG5 bound preferentially to monophosphorylated headgroups, and position of phosphate on the inositol ring had only a minor effect on binding. The array probed with the PRG5-Δ C-terminal FLAG showed no signal, pointing to the C terminus as region of interaction. D, PIP pulldown assay for confirming PRG5-PtdInsP binding. As in lipid strip results (B), PRG5 bound to all PtdInsP. A weak signal is also present for PtdIns. E, hippocampal primary neurons transfected with PRG5-FLAG (upper row, left, green) or PRG5-Δ C-terminal FLAG (lower row, left green) and co-immunostained with anti-tubulin (middle red). PRG5 overexpression induced spine-like structures, whereas PRG5-Δ C-terminal FLAG overexpressing hippocampal neurons showed no alteration. Scale bar, 5 μm.