Matrix metalloproteinase-dependent shedding of intercellular adhesion molecule-5 occurs with long-term potentiation

Abstract

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that can be released or activated in a neuronal activity dependent manner. Although pathologically elevated levels of MMPs may be synaptotoxic, physiologically appropriate levels of MMPs may instead enhance synaptic transmission. MMP inhibitors can block long term potentiation (LTP), and at least one family member can affect an increase in the volume of dendritic spines. While the mechanism by which MMPs affect these changes is not completely understood, one possibility is that the cleavage of specific synaptic cell adhesion molecules plays a role. In the present study, we have examined the ability of neuronal activity to stimulate rapid MMP dependent shedding of the intercellular adhesion molecule-5 (ICAM-5), a synaptic adhesion molecule that is thought to inhibit the maturation and enlargement of dendritic spines. Since such cleavage would likely occur within minutes if it were relevant to a process such as LTP, we focused on post stimulus time points of 30 min or less. We show that NMDA can stimulate rapid shedding of ICAM-5 from cortical neurons in dissociated cell cultures and that such shedding is diminished by pretreatment of cultures with inhibitors that target MMP-3 and -9, proteases thought to influence synaptic plasticity. Additional studies suggest that MMP mediated cleavage of ICAM-5 occurs at amino acid 780, so that the major portion of the ectodomain is released. Since reductions in ICAM-5 have been linked to changes in dendritic spine morphology that are associated with LTP, we also examined the possibility that MMP dependent ICAM-5 shedding occurs following high frequency tetanic stimulation of murine hippocampal slices. Results show that the shedding of ICAM-5 occurs in association with LTP, and that both LTP and the associated ICAM-5 shedding are reduced when slices are pretreated with an MMP inhibitor. Together, these findings suggest that neuronal activity is linked to the shedding of a molecule that may inhibit dendritic spine enlargement and that MMPs can affect this change. While further studies will be necessary to determine the extent to which cleavage of ICAM-5 in particular contributes to MMP dependent LTP, our data support an emerging body of literature suggesting that MMPs are critical mediators of synaptic plasticity.

Figure 1. NMDA mediates rapid shedding of ICAM-5

For figure 1a, dissociated neuronal cultures were treated for 30 minutes with medium or medium containing100 μM NMDA. Lysates were subsequently analyzed by Western blot with an antibody to the N terminal domain of ICAM-5. An NMDA associated reduction in full length ICAM-5 (arrow) can be appreciated. Figure 1b shows results from an experiment in which a novel C terminal antibody to ICAM-5 was tested for its ability to detect ICAM-5. Lysates from HEK cells transfected with 1 μg ICAM-5 or vector alone were analyzed by Western blot. A band at approximately 148 kDa, representing mature glycosylated ICAM-5 is detected in ICAM-5 transfectants but not in vector only transfectants (left most lanes as indicated), and detection of this band is eliminated when the Ab was first incubated with those peptides used to generate the antibody (1:10 ratio Ab to peptides). A lower molecular weight band representing non-glycosyalted ICAM-5 is also present in the ICAM-5 transfectants. Figure 1c shows representative results of an experiment, performed in triplicate, in which cultures were stimulated for 15 minutes with100 μM NMDA in the presence or absence of APV (50 μM), as indicated. Lysates were subsequently analyzed by Western blot with anti- C terminal ICAM-5. A putative16 kDa CTF can be appreciated in the NMDA stimulated culture lysates (arrowhead). Figure 1d shows a time course experiment in which NMDA was tested for its ability to generate the 16 kDa CTF. There is an increase in CTF immunoreactivity in lysates within 5 minutes of NMDA administration. Figure 1e is a graphic representation of ICAM-5 showing the transmembrane domain which spans amino acids 834-854, 9 IgC2 like domains (yellow), and the approximate regions to which peptides were generated (orange). The 9.5 kDa portion of the molecule representing the region extending from the intracytoplasmic C terminus to the extracellular side of the transmembrane region, and the 16 kDa portion that likely represents the putative 16 kDa CTF, are also indicated. Figure 1f shows NTFs in supernatants from NMDA treated cultures. Such fragments can be appreciated in 2/3 supernatants from cultures that were stimulated for only 5 minutes, and in 2/2 that were stimulated for 15 minutes.

Figure 2. Postsynaptic expression of ICAM-5

Figures 2a–c show neuronal immunostaining for ICAM-5, MAP2, and the sodium channel Nav1.2. While ICAM-5 and MAP2 immunoreactivity colocalize to some extent, ICAM-5 staining is less apparent along the process that shows immunoreactivity for the sodium channel. The scale bar represents 10 μm. Figure 2d shows that a GAD positive cell (arrow) is negative for ICAM-5. The scale bar again represents 10 μm. Figure 2e shows a higher power image demonstrating both F-actin and ICAM-5 staining. The latter can be appreciated along thin processes (arrow), consistent with its known expression along filopodia and the necks of thin spines. The scale bar represents 5 μm. Figures 2f–g shows a neuronal process immunostained for ICAM-5, PSD95 and synaptophysin (SYP). There is relatively little colocalization with PSD95 or SYP, markers of relatively mature synapses. Quantified results are shown in 2h. The difference between ICAM-5 that was colocalized with SYP or PSD95 and that which was not is significant (P< 0.0001, Student’s t test). The scale bar represents 10 μm.

Figure 2. Postsynaptic expression of ICAM-5

Figures 2a–c show neuronal immunostaining for ICAM-5, MAP2, and the sodium channel Nav1.2. While ICAM-5 and MAP2 immunoreactivity colocalize to some extent, ICAM-5 staining is less apparent along the process that shows immunoreactivity for the sodium channel. The scale bar represents 10 μm. Figure 2d shows that a GAD positive cell (arrow) is negative for ICAM-5. The scale bar again represents 10 μm. Figure 2e shows a higher power image demonstrating both F-actin and ICAM-5 staining. The latter can be appreciated along thin processes (arrow), consistent with its known expression along filopodia and the necks of thin spines. The scale bar represents 5 μm. Figures 2f–g shows a neuronal process immunostained for ICAM-5, PSD95 and synaptophysin (SYP). There is relatively little colocalization with PSD95 or SYP, markers of relatively mature synapses. Quantified results are shown in 2h. The difference between ICAM-5 that was colocalized with SYP or PSD95 and that which was not is significant (P< 0.0001, Student’s t test). The scale bar represents 10 μm.

Figure 3. MMPs mediate rapid NMDA dependent shedding of ICAM-5

Dissociated neuronal cultures were stimulated for 15 minutes with 100 μM NMDA, in the presence or absence of the NMDA receptor blocker APV or the MMP activity inhibitors NNGH or BiPS as indicated. Lysates were then prepared and analyzed by Western blot with an antibody to the C terminal domain of ICAM-5. As shown in Figure 3a, which is representative of 3 experiments, NMDA was again associated with generation of the 16 kDa CTF of ICAM-5 and this was inhibited by pretreatment of cells with 50 mM APV as well as by either 3.2 μM NNGH or 25 μM BiPS. Figure 3b represents densitometric analysis of results, for which differences between control and NMDA were significant (P< 0.05) while differences between control and other treatment groups were not. For figure 3c, neurons were stimulated for 30 minutes with 100 μM NMDA or 100 ng/ml MMP-3, as indicated, and lysates analyzed by Western blot with an antibody to the exodomain. A reduction in immunoreactivity can be appreciated with both NMDA and MMP-3. For figure 3d, neurons were stimulated for 30 minutes with 100 ng/ml MMP-7, as indicated, and lysates analyzed by Western blot with N or C terminal specific antibodies as noted. The major band at approximately 50 kDa seen with Ponceau staining is shown as a loading control. A reduction in ICAM-5 immunoreactivity can be appreciated with MMP-7.

Figure 4. In vitro digests of recombinant ICAM-5

Recombinant mouse ICAM-5 was incubated with select MMPs as indicated, and the digestion products resolved by electrophoresis. Following transfer to a PVDF membrane, specific products were isolated for N terminal sequencing (arrowheads). Products denoted by the arrowheads in the MMP-3 and -9 digests in particular were analyzed (4a and b). Both MMPs generated a fragment having the construct’s original N terminus (uppermost arrowhead), as well as a fragment showing a new N terminus beginning with amino acid 780 (lowermost arrowhead).

Figure 5. MMP-dependent shedding of ICAM-5 occurs during early LTP

Hippocampal slices were stimulated (two 1s trains of 100 Hz separated by 20s) in the presence or absence of the MMP inhibitor NNGH (10 μM). Recordings were taken from 5 –10 control and MMP inhibitor treated slices, and confirm that LTP occurred with this protocol (5a). Control EPSP traces were taken in the first 10 minutes of recording and post tetanus traces taken in the last 10 minutes. Input-output data comparing control and NNGH treated slices is shown in figure 5b. For figure 5c, lysates were prepared from a subset of slices 15 minutes following their stimulation, and compared to lysates from control slices that had been similarly maintained in ACSF but not stimulated. Lysates were subsequently tested for the 16 kDa C terminal cleavage product (arrowhead) by Western blot. Densitometric results are shown in figure 5d, and demonstrate that the density of the 16 kDa band is increased in association with LTP. This increase is abrogated when slices were pretreated with NNGH. The difference between control and LTP densitometric results were significant (P< 0.01) as were the differences between LTP and LTP/NNGH results (P< 0.05).