Phospholipase D1-promoted release of tissue plasminogen activator facilitates neurite outgrowth

Abstract

Temporal lobe epilepsy (TLE) is the most common form of epilepsy, affecting approximately 1-2% of the population. Seizure events resulting from TLE are characterized by aberrant hippocampal mossy fiber sprouting and plastic responses that affect brain function. Seizure susceptibility is modulated by the enzyme tissue plasminogen activator (tPA), the normal physiological role of which includes promotion of synaptic reorganization in the mossy fiber pathway by initiating a proteolytic cascade that cleaves extracellular matrix components and influences neurite extension. tPA is concentrated at and selectively secreted from growth cones during excitatory events. However, the mechanisms underlying tPA release during seizure-induced synaptogenesis are not well understood. We examine here potential roles for the signaling enzyme phospholipase D1 (PLD1), which promotes regulated exocytosis in non-CNS cell types, and which we previously demonstrated increases in expression in hippocampal neurons during seizure-induced mossy fiber sprouting. We now show that overexpression of wild-type PLD1 in cultured neurons promotes tPA release and tPA-dependent neurite extension, whereas overexpression of an inactive PLD1 allele or pharmacological inhibition of PLD1 inhibits tPA release. Similarly, viral delivery of wild-type PLD1 into the hippocampus facilitates tPA secretion and mossy fiber sprouting in a seizure-inducing model, whereas the inactive PLD1 allele inhibits tPA release and elicits blunted and abnormal mossy fiber extension similar to that observed for tPA-/- mice. Together, these findings secretion and thus mossy fiber extension in the setting of elevated suggest that PLD1 functions endogenously to regulate tPA-/- neuronal stimulation, such as that seen in TLE.

Figure 1.

Expression of EGFP-tagged PLD1 and its catalytically inactive mutant PLD1(K898R) in mouse primary hippocampal neurons. Cultured C57BL/6 mouse hippocampal neurons were infected with viral vectors encoding EGFP, EGFP-PLD1, or EGFP-PLD1(K898R). Ten hours later, total cell lysates were collected, and equal amounts (4 μg) of protein were separated on 8% SDS-PAGE. PLD1 was detected by quantitative Western blot using a polyclonal anti-PLD1 antibody. a, Representative Western blot; equal loading of proteins was confirmed by anti-actin Western blotting (b). c, Quantification of PLD1 expression normalized to actin. Neurons do express endogenous PLD1 protein that migrates faster (121 kDa) than EGFP-PLD1 (144 kDa); however, it was not detected in this Western blot because of the small amount of protein loaded on the gel as per optimal conditions for detecting the overexpressed fusion proteins. Experiments were performed in quadruplicate. ^**p < 0.01 compared with EGFP-expressing control cells.

Figure 2.

PLD1 facilitates agonist-stimulated tPA secretion from hippocampal neurons in an activity-dependent manner. Cultured C57BL/6 mouse hippocampal neurons were infected with Sinbis viral vectors as described above and then challenged with 50 μm forskolin (a) or 10 μm Ca²⁺ ionophore A23187 (b, c) for different time intervals. A total amount of 0.5% 1-butanol or 0.5% 3-butanol was additionally present in some wells. Neurons in a and b were overexpressing either wild-type or mutant PLD1; neurons in c were expressing only endogenous levels of PLD1. Conditioned medium was collected and analyzed by ELISA for tPA content. The amount of secreted tPA in the culture medium was normalized to the total protein concentration. Experiments were performed in triplicate and analyzed by ANOVA. ^*p < 0.05; ^**p < 0.01 compared with EGFP-expressing control cells.

Figure 3.

PLD1 promotes neurite extension from cultured wild-type mouse hippocampal neurons. Two days after being plated in culture, dissociated wild-type and tPA^-/- mouse hippocampal neurons were infected with pseudovirions SIN-EGFP, SIN-EGFP-PLD1, or SIN-EGFP-PLD1(K898R). Successfully infected cells were identified via the presence of EGFP fluorescence. Twenty-four hours after infection, measurement of neurite length was performed on captured images of fixed cells using SPOT RT software. a, Quantification of the neurite length of wild-type (WT) and tPA^-/- neurons. Neurite length is 51 ± 13 μm in neurons expressing EGFP, 69 ± 21 μm in neurons expressing EGFP-PLD1, and 53 ± 17 μm in neurons expressing the inactive PLD1 allele. b, Representative images of individual wild-type neurons infected with the respective virions. The lengths of the neurites emanating from these neurons are shown. Sixty neurons from each condition were used to generate the pooled measurement and were analyzed by ANOVA. ^**p < 0.01 compared with EGFP-expressing control cells.

Figure 4.

Expression of recombinant PLD1 in vivo at day 15 after intrahippocampal delivery of Sindbis pseudovirions. Recombinant Sindbis viral vectors encoding EGFP, EGFP-PLD1, or EGFP-PLD1(K898R) were delivered into the DG via mini-osmotic pumps in wild-type mice. Expression of the GFPs was examined in coronal brain sections of animals killed at day 15 (b). Parallel brain sections were stained with cresyl violet to illustrate the locations of fluorescent signals inside the hippocampi (a). EGFP fluorescence is detected focally around the site of infusion (b, arrowhead) and diffuses to a limited distance (b, arrows). The expression is confined to neurons as judged by costaining with the neuronal marker NeuN (b, red).

Figure 5.

The presence of newly formed MF sprouts without overt neurodegeneration in the hippocampus. At day 15 after unilateral intra-amygdaloid kainate injection, cresyl violet stain shows intact neuronal layers throughout the hippocampus (a, injected side indicated). Punctate GAP-43 stain indicates the presence of newly formed MF sprouts in the ipsilateral CA3 area (b, arrows), whereas only background staining was seen in the contralateral CA3 area (c). or, Stratum oriens; pyr, stratum pyramidale; rad, stratum radiatum; luc, stratum lacunosum. Scale bars: a, 350 μm; b, c, 50 μm.

Figure 6.

Dramatic changes in CA3 MF sprouting at day 15 with neuronal overexpression of wild-type and catalytically inactive PLD1. Wild-type and tPA^-/- mice were given injections of 1.5 nmol of kainate into the amygdala unilaterally. In parallel, viral vectors encoding PLD1 [wild type (d-f); tPA^-/- (d′-f′)] or its catalytically inactive mutant [PLD1(K898R)] [wild type (g-i); tPA^-/- (g′-i′)] were delivered into the DG on the kainate-injected side via mini-osmotic pumps (labeled as ipsilateral). Infusion of EGFP vector served as a negative control [wild type (a-c); tPA^-/- (a′-c′)]. Mice were killed at day 15, and the MF was visualized using Timm silver staining. MF sprouts are indicated by arrows or asterisks.

Figure 7.

Gain- and loss-of-function changes, respectively, in DG and CA3 MF sprouting at day 15 as a consequence of CNS overexpression of wild-type and catalytically inactive PLD1. Higher-magnification images of selected regions in Figure 6 are shown. MF sprouts and boutons are indicated by red arrows. Blue arrows denote the abnormal laminar band of Timm staining observed in wild-type mice expressing inactive PLD1 and in tPA^-/- mice at the border separating the molecular and granular cell layers.

Figure 8.

Regulation of tPA secretion by PLD1 as a consequence of kainate challenge at day 7. In situ zymography was performed on 30 μm coronal brain sections from wild-type mice killed at day 7 after unilateral viral infusion and kainate injection (labeled as ipsilateral; the untreated side is marked as contralateral). The grayscale image was converted to pseudocolor for better visualization of the zymography images, and the intensity scale bar is shown on the right. The blue area outlined by arrows indicates the presence of tPA activity. An enhanced level of secreted tPA was detected on the side with elevated neuronal expression of active PLD1 (c, arrows) compared with either the contralateral hippocampus of the same mouse (d, arrows) or that of the SIN-EGFP-infused hippocampus (a, arrows). In contrast, tPA activity was diminished in the hippocampus expressing inactive PLD1 (e, arrows) compared with either the contralateral side (f, arrows indicating basal tPA activity) or control animals (a, arrows).