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Role of Tissue Plasminogen Activator Receptor LRP in Hippocampal Long-Term Potentiation

Zhuo, Min, Holtzman, David M., Li, Yonghe, Osaka, Hiroshi, DeMaro, Joe, Jacquin, Mark, Bu, Guojun·2000·The Journal of Neuroscience
DOI 10.1523/jneurosci.20-02-00542.2000
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Abstract

The low-density lipoprotein (LDL) receptor-related protein (LRP) is a multifunctional endocytic receptor that is expressed abundantly in neurons of the CNS. Both LRP and several of its ligands, including tissue plasminogen activator (tPA), apolipoprotein E/lipoproteins, α 2-macroglobulin, and the β-amyloid precursor protein, have been implicated in various neuronal functions and in the pathogenesis of Alzheimer's disease. It has been reported that induction of tPA expression may contribute to activity-dependent synaptic plasticity in the hippocampus and cerebellum. In addition, long-term potentiation (LTP) is significantly decreased in mice lacking tPA. Here we demonstrate that tPA receptor LRP is abundantly expressed in hippocampal neurons and participates in hippocampal LTP. Perfusion of hippocampal slices with receptor-associated protein (RAP), an antagonist for ligand interactions with LRP, significantly reduced late-phase LTP (L-LTP). In addition, RAP also blocked the enhancing effect of synaptic potentiation by exogenous tPA in hippocampal slices prepared from tPA knock-out mice. Metabolic labeling and ligand binding analyses showed that both tPA and LRP are synthesized by hippocampal neurons and that LRP is the major cell surface receptor that binds tPA. Finally, we found that tPA binding to LRP in hippocampal neurons enhances the activity of cyclic AMP-dependent protein kinase, a key molecule that is known to be involved in L-LTP. Taken together, our results demonstrate that interactions between tPA and cell surface LRP are important for hippocampal L-LTP.

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Figure 1 LRP is important for hippocampal L-LTP. A , L-LTP is abolished by RAP pretreatment (0.5 m M, closed squares ). L-LTP recorded with buffer alone is shown with open squares . Four-train stimulation is marked with arrowheads . Inset , Traces of EPSPs indicating that 40 min after the induction of L-LTP, hippocampal potentials with RAP treatment have returned to those of baseline. B , RAPat a lower concentration (50 nM) produces a smaller inhibition. C , RAP (0.5 m M, closed squares ) does not affect E-LTP induced by one-train stimulation. E-LTP recorded with buffer alone is shown with open squares . D , Synaptic responses to a paired-pulse stimulation at different intervals (25, 50, 75, and 100 msec) are not affected by RAP (0.5 m M, closed squares ). Open squares are recordings with buffer alone. E , RAP does not affect NMDA receptormediated EPSPs. F , The input-output curves of NMDA receptormediated EPSPs in control medium ( open squares ) and medium containing RAP (0.5 m M, closed squares ).
Figure 1 LRP is important for hippocampal L-LTP. A , L-LTP is abolished by RAP pretreatment (0.5 m M, closed squares ). L-LTP recorded with buffer alone is shown with open squares . Four-train stimulation is marked with arrowheads . Inset , Traces of EPSPs indicating that 40 min after the induction of L-LTP, hippocampal potentials with RAP treatment have returned to those of baseline. B , RAPat a lower concentration (50 nM) produces a smaller inhibition. C , RAP (0.5 m M, closed squares ) does not affect E-LTP induced by one-train stimulation. E-LTP recorded with buffer alone is shown with open squares . D , Synaptic responses to a paired-pulse stimulation at different intervals (25, 50, 75, and 100 msec) are not affected by RAP (0.5 m M, closed squares ). Open squares are recordings with buffer alone. E , RAP does not affect NMDA receptormediated EPSPs. F , The input-output curves of NMDA receptormediated EPSPs in control medium ( open squares ) and medium containing RAP (0.5 m M, closed squares ).CC-BY-NC-SA · From the PDF
Figure 2 tPA-induced synaptic potentiation is blocked by RAP in tPA knock-out mice. A , Traces of EPSPs before and 60 min after 50 nM tPA application in the absence ( left ) or presence of 0.5 m M RAP ( right ). B , RAP pretreatment (0.5 m M, open squares ) blocked synaptic potentiation induced by tPA. EPSPs in the absence of RAP pretreatment are indicated by filled squares . Application of 50 nM tPA is indicated by a bar .
Figure 2 tPA-induced synaptic potentiation is blocked by RAP in tPA knock-out mice. A , Traces of EPSPs before and 60 min after 50 nM tPA application in the absence ( left ) or presence of 0.5 m M RAP ( right ). B , RAP pretreatment (0.5 m M, open squares ) blocked synaptic potentiation induced by tPA. EPSPs in the absence of RAP pretreatment are indicated by filled squares . Application of 50 nM tPA is indicated by a bar .CC-BY-NC-SA · From the PDF
Figure 3 RAP does not interact directly with tPA. A , RAP (1 m g) and tPA (1 m g) were incubated in ACSF for 1 hr at 30°C. The incubation mixture was then divided and incubated with normal rabbit IgG ( NR ), anti-tPA IgG, or anti-RAP IgG, followed with immunoprecipitation ( IP ) without detergent. The immunoprecipitated materials were then immunoblotted ( IB ) with either anti-tPA IgG or anti-RAP IgG. B , RAP-Sepharose was incubated with 125 I-tPA (5 nM) in ACSF for 1 hr at 30°C. After washing, both supernatant ( S ) and pellet ( P ) were analyzed via SDS-PAGE.
Figure 3 RAP does not interact directly with tPA. A , RAP (1 m g) and tPA (1 m g) were incubated in ACSF for 1 hr at 30°C. The incubation mixture was then divided and incubated with normal rabbit IgG ( NR ), anti-tPA IgG, or anti-RAP IgG, followed with immunoprecipitation ( IP ) without detergent. The immunoprecipitated materials were then immunoblotted ( IB ) with either anti-tPA IgG or anti-RAP IgG. B , RAP-Sepharose was incubated with 125 I-tPA (5 nM) in ACSF for 1 hr at 30°C. After washing, both supernatant ( S ) and pellet ( P ) were analyzed via SDS-PAGE.CC-BY-NC-SA · From the PDF
Figure 4 LRP is the major endocytic receptor for tPA expressed in hippocampal neurons. A , Fresh hippocampal slices are prepared and used for binding analysis of 125 I-tPA (5 nM) in the absence or presence of unlabeled tPA (500 nM) or RAP (500 nM). The experiment was performed in triplicate, with error bars representing SEM. RAP inhibits 125 I-tPA binding to hippocampal slices to the same extent as unlabeled tPA, suggesting that binding of tPA to hippocampal slices is mediated entirely via LRP. B , Hippocampal neurons were prepared and plated in 24-well plates. Binding of 125 I-tPA (5 nM) in the absence or presence of unlabeled RAP (500 nM) was performed at 4°C for 2 hr. Some plates were then warmed up to 37°C for either 5 or 10 min before returning to 4°C. Cell monolayers were then treated with 0.25% (w/v) Pronase for 30 min at 4°C to remove remaining cell surface-bound ligands. Radioactivity associated with cell pellets represents internalized ligand, whereas radioactivity in the supernatant fraction represents surface-bound ligand. Data represent LRP-mediated endocytosis after subtraction of those in the presence of excess RAP. The experiment was performed in triplicate, error bars representing SEM.
Figure 4 LRP is the major endocytic receptor for tPA expressed in hippocampal neurons. A , Fresh hippocampal slices are prepared and used for binding analysis of 125 I-tPA (5 nM) in the absence or presence of unlabeled tPA (500 nM) or RAP (500 nM). The experiment was performed in triplicate, with error bars representing SEM. RAP inhibits 125 I-tPA binding to hippocampal slices to the same extent as unlabeled tPA, suggesting that binding of tPA to hippocampal slices is mediated entirely via LRP. B , Hippocampal neurons were prepared and plated in 24-well plates. Binding of 125 I-tPA (5 nM) in the absence or presence of unlabeled RAP (500 nM) was performed at 4°C for 2 hr. Some plates were then warmed up to 37°C for either 5 or 10 min before returning to 4°C. Cell monolayers were then treated with 0.25% (w/v) Pronase for 30 min at 4°C to remove remaining cell surface-bound ligands. Radioactivity associated with cell pellets represents internalized ligand, whereas radioactivity in the supernatant fraction represents surface-bound ligand. Data represent LRP-mediated endocytosis after subtraction of those in the presence of excess RAP. The experiment was performed in triplicate, error bars representing SEM.CC-BY-NC-SA · From the PDF
Image 5
CC-BY-NC-SA · From the PDF
Figure 5 tPA and LRP expression in hippocampal neurons and activation of PKA. A , Primary cultured hippocampal neurons from E17 mouse embryos were metabolically labeled with [ 35 S]cysteine for 4 hr. Both cell lysates ( C ) and overlying media ( M ) were immunoprecipitated with either anti-tPA antibody or anti-LRP antibody, followed by analysis via SDS-PAGE (7.5% acrylamide) and autoradiography. LRP is abundantly expressed in these neurons, whereas tPA is synthesized and secreted by these neurons. A band corresponding to the migration of tPA/PAI-1 complexes is also detected. The molecular size markers are given in kilodaltons. B , Binding of tPA to LRP enhances PKA activity. Primary hippocampal neurons were incubated without or with tPA (50 nM) or tPA (50 nM) and RAP (500 nM) at 37°C for 20 min. Cell lysates were then assayed for PKA activity. Results are averages of triplicate determinations, error bars representing SEM. * p , 0.05 compared with no treatment control.
Figure 5 tPA and LRP expression in hippocampal neurons and activation of PKA. A , Primary cultured hippocampal neurons from E17 mouse embryos were metabolically labeled with [ 35 S]cysteine for 4 hr. Both cell lysates ( C ) and overlying media ( M ) were immunoprecipitated with either anti-tPA antibody or anti-LRP antibody, followed by analysis via SDS-PAGE (7.5% acrylamide) and autoradiography. LRP is abundantly expressed in these neurons, whereas tPA is synthesized and secreted by these neurons. A band corresponding to the migration of tPA/PAI-1 complexes is also detected. The molecular size markers are given in kilodaltons. B , Binding of tPA to LRP enhances PKA activity. Primary hippocampal neurons were incubated without or with tPA (50 nM) or tPA (50 nM) and RAP (500 nM) at 37°C for 20 min. Cell lysates were then assayed for PKA activity. Results are averages of triplicate determinations, error bars representing SEM. * p , 0.05 compared with no treatment control.CC-BY-NC-SA · From the PDF
Image 7
CC-BY-NC-SA · From the PDF
Figure 6 Schematic model of tPA/LTP pathway in L-LTP. See Discussion.
Figure 6 Schematic model of tPA/LTP pathway in L-LTP. See Discussion.CC-BY-NC-SA · From the PDF

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Metadata

Authors
Zhuo, M., Holtzman, D. M., Li, Y., Osaka, H., DeMaro, J., Jacquin, M., & Bu, G.
Venue
The Journal of Neuroscience
Publisher
Society for Neuroscience
Published
2000-01-15
Volume
20
Issue
2
Pages
542-549
ISSN
  • 0270-6474
  • 1529-2401
DOI
10.1523/jneurosci.20-02-00542.2000
Cited by
258
References
6
Language
en
Licence
creativecommons.org/licenses/by-nc-sa/4.0/ (vor, 182 d)

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