Hippocampal LTP triggers proteasome-mediated SPAR degradation in CA1 neurons


  • Ying Chen,

    1. Institutes of Brain Science, Fudan University, Shanghai 200032, People's Republic of China
    2. State Key Laboratory of Medical Neurobiology, Fudan University, Shanghai 200032, People's Republic of China
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  • Pingan Yuanxiang,

    1. Institutes of Brain Science, Fudan University, Shanghai 200032, People's Republic of China
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  • Thomas Knöpfel,

    1. Laboratory for Neuronal Circuit Dynamics, RIKEN Brain Science Institute, Wako-shi, Saitama, Japan
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  • Ulrich Thomas,

    1. Leibniz Institute for Neurobiology, Magdeburg 39118, Germany
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  • Thomas Behnisch

    Corresponding author
    1. Institutes of Brain Science, Fudan University, Shanghai 200032, People's Republic of China
    2. State Key Laboratory of Medical Neurobiology, Fudan University, Shanghai 200032, People's Republic of China
    • Institutes of Brain Science, Fudan University, 138 Yi Xue Yuan Road, Shanghai 200032, People's Republic of China
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Activity-dependent synaptic plasticity is associated with synaptic protein turnover involving the ubiquitin proteasome system (UPS) for protein degradation. In primary hippocampal cell culture, it has been shown that increased or decreased activity of synaptic transmission can regulate the amount of postsynaptic density (PSD) proteins via UPS. However, the specific spatio-temporal dynamic of PSD protein degradation after LTP induction and its downstream signaling pathways remains to be clarify. We used confocal microscopy to monitor levels of eGFP-tagged SPAR (spine-associated Rap GTPase activating protein) expressed in acute hippocampal slices and found that LTP induction triggered a UPS-dependent decay of eGFP-SPAR fluorescence. SPAR degradation was reduced upon inhibition of cyclin-dependent kinase 5 (CDK5) as well as by a protein synthesis inhibitor. Comparison of eGFP-tagged SPAR levels with those obtained in control experiments with eGFP revealed a protein synthesis-independent component of LTP-associated SPAR degradation. This second component required UPS and NMDA receptor activation but not CDK5. We conclude that LTP triggers a down regulation of SPAR by two complementary mechanisms, one of which has previously been described to mediate homeostatic plasticity. Synapse, 2012. © 2011 Wiley Periodicals, Inc.


Activity-dependent synaptic plasticity represents a cellular mechanism suitable for lasting adaptation of network activities during memory formation (Bliss and Collingridge,1993; Frey and Frey,2008; Malenka and Nicoll,1999; Matthies et al.,1990). The molecular and structural changes that cause these long-lasting functional changes in synaptic efficacy require remodeling of synaptic protein ensembles by regulated phosphorylation (Malinow et al.,1989; Roberson et al.,1999), protein translocation (Krucker et al.,2000), and turnover of proteins (Behnisch et al.,2004; Deadwyler et al.,1987; Frey et al.,1988, 1996; Mochida et al.,2001; Stanton and Sarvey,1984). Regulated turnover of proteins is accomplished by de novo protein synthesis (Bramham and Wells,2007; Hernandez and Abel,2008) as well as via targeted degradation through the ubiquitin-proteasome system (UPS) (Hegde,2004; Yi and Ehlers,2007). In fact, inhibition of either protein ubiquitination (Foley et al.,2000), proteasome activity (Lopez-Salon et al.,2001) or ubiquitin ligase (Jiang et al.,1998) leads to deficits in hippocampal LTP and behavioral performance. Moreover, we previously demonstrated that UPS-dependent protein degradation is a prerequisite for induction and input specificity of hippocampal LTP in the CA1 area (Cai et al.,2010; Karpova et al.,2006).

Postsynaptic proteins that are targeted by UPS include several prominent scaffolding molecules such as PSD-95, GKAP/SAPAP, ProSAP/Shank and AKAP (Ehlers,2003). Little is known, however, about the precise sequence of proteasome-dependent degradation events relative to the onset of synaptic stimulation and subsequent reorganization of the synapse. In several cases, activity-dependent protein degradation has emerged as a second order event related to the structural remodeling of synapses. For instance, degradation of SPAR—a key regulator of dendritic spine morphology that acts at the interface between the PSD and the actin-based cytoskeleton (Pak et al.,2001)—results from its phosphorylation by Polo-like kinase 2 (Plk2; also known as serum-induced kinase, SNK; Pak and Sheng,2003). Expression of the kinase largely depends on the level of synaptic activity (Kauselmann et al.,1999), implying that Plk2-induced degradation of SPAR by UPS represents a relatively late event.

The role of SPAR in activity-dependent synaptic plasticity is of great interest. SPAR appears to be selective for spiny neurons and is present in about 65% of the mature mushroom-shaped spines, inducing an increase in complexity of spine shape when overexpressed (Pak et al.,2001). In hippocampal neurons, SPAR is localized in dendritic spines and its over expression causes enlargement of spines, many of which adopt an irregular appearance associated with putative multiple synapses. Dominant negative SPAR constructs cause narrowing and elongation of spines. In addition, it was reported that the EphA4 receptor regulates neuronal morphology through SPAR-mediated inactivation of Rap GTPases (Richter et al.,2007) implicating Rap signaling in the regulation of postsynaptic structures.

Previous studies in primary cell culture systems, suggested that SPAR degradation induced by Plk2 contributes to homeostatic regulation of synaptic transmission. Homeostatic plasticity refers to changes in synaptic function and structure that are induced by prolonged increases or decreases of synaptic activity (Jakawich et al.,2010; Perez-Otano and Ehlers,2005) and which normalize the overall synaptic drive of neurons and thereby stabilizes network dynamics (Seeburg and Sheng,2008). Mechanisms associated with LTP are, in contrast, considered to occur at a much faster time scale.

It remains to be established whether activity-dependent SPAR degradation takes place during synaptic input-specific induction of LTP in CA1 neurons of acute hippocampal slices. Here, we employed a viral gene delivery system to express eGFP-tagged SPAR proteins in CA1 neurons. We combined electrophysiological recordings and confocal microscopy to monitor eGFP-SPAR fluorescence in apical dendrites of CA1 neurons while applying a LTP inducing stimulation paradigm. We found that tetanization induced a reduction of eGFP-SPAR fluorescence that was diminished by proteasome-, cyclin-dependent kinase 5 (CDK5)- and protein synthesis inhibitors. We conclude that tetanization induces CDK5-dependent phosphorylation of SPAR that, in turn, caused SPAR degradation by UPS. These results link LTP induction to mechanisms of activity-dependent structural remodeling of synapses.


CDK5cyclin dependent kinase 5

Plk2Polo-like kinase 2

fEPSPfield excitatory postsynaptic potential

HFShigh-frequency stimulation

late-LTPlate-phase LTP

LTPlong-term potentiation, NMDA N-methyl-D-aspartate.


Preparation of Semliki forest particles carrying an eGFP-SPAR fusion gene

A fragment encoding enhanced green fluorescence protein (eGFP) fused to spine-associated Rap GTPase activating protein (SPAR) was excised from a pCDNA3.1-based construct (kind gift by Dr. Christina Spilker, Leibniz Institute for Neurobiology, Magdeburg, Germany) through digestion with NheI and SmaI. The fragment was blunted by a fill-in reaction using Klenow polymerase and inserted into the SmaI restriction site of the pSFV1 expression vector. The recombinant pSFV-eGFP-SPAR plasmid, a pSFV-eGFP control plasmid and the pSFV-Helper2 plasmid (which encodes SFV structural proteins) were linearized and reverse-transcribed to yield RNAs for the generation of Semliki Forest particles as described by Ehrengruber et al. (1999). In brief, RNA derived from pSFV-eGFP-SPAR or pSFV-eGFP were co-transfected with RNA derived from pSFV-Helper2 into Human Embryonic Kidney 293 (HEK-293T) cells with Lipofectamine 2000 (Invitrogen). After 48 h, the culture medium containing budded particles was harvested, passed through 0.22 μm filters and stored at −80°C. Thirty minutes before conducting intrahippocampal injection, particles were activated by alpha-chymotrypsin (Sigma, US; CAS No. 9004-07-3; 500 mg/l) for 30 min at room temperature and the reaction inactivated by aprotinin (Sigma, US; CAS No 9087-70-1; 250 mg/l).

Ethics statement

Animal care and procedures were approved and conducted under established standards of the Institutes of Brain Science and State Key Laboratory of Medical Neurobiology of Fudan University, Shanghai, China.

Intrahippocampal virus injections

Wistar rats (2–3 weeks old) (Experimental Animal Center, Shanghai Medical School, Fudan University, People's Republic of China) were anesthetized with chloral hydrate (450 mg/kg, Sigma, MO) by intraperitoneal (i.p.) injection and then secured in a stereotaxic frame (Stoelting, Wood Dale, IL). Glass pipettes (Harvard Apparatus, Holliston, MA) with a tip length of about 10 mm and a tip diameter of about 30 μm were filled with activated virus particle containing solution and positioned in the hippocampus through a 0.5 mm diameter hole in the skull (vertical coordinates V: −4.0 mm). The injection coordinates for anterior–posterior (AP) and lateral (L) positions were −5.4 mm and ±5.2 mm, respectively. Pipettes were connected over a paraffin oil-filled plastic tube with a Hamilton syringe (CR-700-50, Hamilton Co., Höchst, Germany). A total of 1.5 μl of the particle containing solution was injected in three portions at 5 min intervals. The pipette was redrawn by 0.2 mm after each injection. The injection pipette remained at the final position for additional 10 min. This procedure was effective to transduce a high number of cells expressing eGFP or eGFP-SPAR (Fig. 1A).

Figure 1.

Expression pattern of eGFP-SPAR and eGFP in hippocampal neurons and their effects on fEPSP-potentiation. A: Intra-hippocampal injection of activated SFV particles for eGFP-SPAR or eGFP expression led to transduction of CA1, CA3, and dentate gyrus neurons as depicted with the low resolution non confocal fluorescence image. Scale bars: 40 μm. B: Confocal fluorescence imaging using 63× water objectives revealed that eGFP-SPAR was enriched in spine heads (white arrows), whereas eGFP was augmented in dendrites. Scale bars: 5 μm.

Hippocampal slices preparation

Acute hippocampal slices were prepared as described previously (Cai et al.,2010; Leutgeb et al.,2003, 2005). Briefly, 20–24 h after intra-hippocampal injection, rats were anesthetized using ether and the isolated brains were immersed in carbogenated ice-cold Gey's solution (composition in mM: 130 NaCl, 4.9 KCl, 1.5 CaCl2·2H2O, 0.3 MgSO4·7H2O, 11 MgCl2·6H2O, 0.23 KH2PO4, 0.8 Na2HPO4, 5 glucose, 25 HEPES, 22 NaHCO3, pH 7.32). Transverse hippocampal slices (300 μm) were cut (Vibratome 3000, St. Louis, MO) and immediately transferred to an interface type slice chamber to recover for at least 1 h at 32°C in carbogenated ACSF (containing in mM: 110 NaCl, 5 KCl, 2.5 CaCl2·2H2O, 1.5 MgSO4·7H2O, 1.24 KH2PO4, 10 glucose, 27.4 NaHCO3, pH 7.3).


Hippocampal slices were transferred to a submerged slice chamber and perfused constantly with carbogenated ACSF at 32°C. Field excitatory postsynaptic potentials (fEPSPs) were evoked by stimulation of Schaffer-collateral fibers with biphasic rectangular current pulses (200 μs/polarity) in a range of 15–30 μA through ACSF filled glass pipettes with a resistance of 0.5 MΩ. The tip of the stimulation electrodes was positioned in close proximity to clearly visible fluorescent dendrites (Fig. 2A1) (Jager et al.,2002). The fESPSs were recorded using ACSF filled pipettes and amplified by an AxonClamp 200B amplifier (Molecular Devices, Shanghai, China). The strength of synaptic transmission was estimated by calculating the initial slope of fEPSPs. Stimulation strength was adjusted to 40% of the maximum fEPSP-slope values. Responses to test stimuli were measured every minute or 5 min throughout the experiment. The averaged fEPSP-slope values were depicted in diagrams as Mean ± SEM. The recorded field potentials were digitized at a sample frequency of 10 kHz by Digidata 1400plus AD/DA converter (Molecular Devices, Shanghai, China).

Late-LTP was induced by a tetanization consisting of three 1-s trains at 100 Hz (Cai et al.,2010) every 6 min (Fig. 2A2).

Image acquisition and analysis of time-lapse recordings

The time course of fluorescence changes in GFP-expressing CA1 neurons was acquired by scanning of several optical planes using a confocal microscope (Olympus, Fluoview 1000, Shanghai Branch, China) mounted on an upright fixed stage microscope every 3 min. The distance between the first and the last plane was between 30 and 90 μm. Fluorescence intensity values (averages over regions of interest, ROIs, as indicated) were calculated for each time point according to the following mode of image processing: (i) averaging of the planes that encompass dendrites, (ii) crop of dendrite, (iii) alignment of averaged planes across time points, (iv) measuring of fluorescence intensities of ROIs located on dendrites (including their spines) as well as background. ROIs were placed around clearly visible and in focus dendritic segments (Fig. 2D). Dendrites were chosen that are clearly visible and localized at the same horizontal and vertical positions as the stimulation electrode (Fig. 2A1) (Jager et al., 1998,2002; Karpova et al.,2006). Fluorescence intensities were background corrected and expressed as percentage changes from baseline. Image processing and analysis were conducted using open source imaging software ImageJ3.5. Experiments with drug applications were interleaved with control measurements in the absence of drug. Control measurements are presented as one group.


Drugs, including AP5 (50 μM; Du et al.,2009), roscovitine (5 μM), anisomycin (30 μM; Sigma-Aldrich, Shanghai, China), MG132 (25 μM; Calbiochem, La Jolla, CA) (Cai et al.,2010; Craiu et al.,1997; Karpova et al.,2006) were bath-applied at concentrations as indicated. Stock solutions of MG132 in dimethyl sulfoxide (DMSO, Sigma-Aldrich, Shanghai, China) were stored at -20°C. Stock solutions were diluted prior to use to the desired working concentration in ACSF. To avoid precipitation of MG132, the stock solution was diluted slowly in freshly carbogenated ACSF and kept protected from light. The final DMSO concentration did not exceed 0.1%.

Statistical analysis

Data acquisition and analyses were carried out using Clampex 10.2 and Clampfit 10.2 (Molecular Devices, Shanghai, China) and SPSS that allow measurements of fEPSP-slope and the calculation of descriptive statistics. Data gained from electrophysiological or imaging experiments were normalized to baseline values and expressed in percentage as Mean ± SEM%. Comparisons of groups were done by using Mann–Whitney U-test. P values of ≤0.05 were considered as statistically significant. Numbers of slices corresponds to numbers of animals studied. Drug experiments were interleaved with drug-free controls.


To investigate LTP-related SPAR degradation and to monitor its activity-dependent degradation, SFV-based particles encoding eGFP-SPAR were injected into the hippocampus of 2–3 week old rats. Particles encoding eGFP served as a control. Fluorescence microscopic inspection of hippocampal slices from injected animals revealed substantial expression of eGFP-SPAR or eGFP within 20–24 h post injection. Moreover, eGFP-SPAR was enriched in spine heads whereas fluorescence of eGFP alone was uniformly distributed within spines and dendrites (Fig. 1B).

Synaptic activity-dependent attenuation of eGFP-SPAR fluorescence

To monitor the time course of activity-dependent SPAR degradation, we acquired the fluorescence of dendrites (including their spines) every 3 min (Fig. 2B). Over a period of 45 min eGFP-SPAR fluorescence decreased by about 20% (9th min: 95.2 ± 6.78%; 42nd min: 78.3 ± 4.46%). We attribute this decrease to photobleaching of eGFP. In contrast, eGFP-SPAR decreased by 70% in slices that were tetanized three times every 6 min to induce LTP (9th min: 91.7 ± 4.59%; 42nd min: 38.7 ± 5.55%; Fig. 2C).

Figure 2.

LTP induction and monitoring of tetanization induced eGFP-SPAR degradation in transduced hippocampal CA1 neurons. A: A single experiment is represented indicating (A1) transduced hippocampal CA1 neurons and that LTP is inducible in such acute hippocampal slice. A2: Time course of fEPSP-slopes after tetanization (gray filled diamonds) and no-tetanization (black filled squares). Arrows indicate time of the one second 100 Hz tetanizations. An analog trace of the first 150 ms of one second tetanization is shown as an insert. A3: Corresponding fEPSPS are depicted for time points 10 (light gray trace) and 40 min (black trace). The position of electrodes was not optimized for signal size but to be on the same Z-plane as the fluorescent dendrites (horizontal scale bar: 4 ms; the vertical one: 0.2 mV). B: In parallel to fEPSPs-recordings, time-lapse imaging was performed by scanning several focal planes every 3 min. The fluorescence intensity was measured from aligned stacks of averages for every clearly visual dendrite separately (D). Background values were obtained within dendrite-free areas and subtracted from fluorescence values. The single data points were normalized to before tetanization. The diagram in C depicts the change of fluorescence under conditions with and without tetanization (seven dendrites of three slices, gray filled diamonds and five dendrites of three slices, black filled squares, respectively). Arrows indicate time points of the one second 100 Hz tetanizations. Please note the accelerated reduction of eGFP-SPAR fluorescence after triple tetanization. D: Representative fluorescence images after stack averaging and alignment for time points 3, 24, and 45 min. A bracket indicates the interval of significant difference between tetanized and non-tetanized slices (*P ≤ 0.05). SPAR+no tet: recordings without tetanizations; SPAR: with tetanization. White horizontal scale bar: 5 μm. Str. rad: stratum radiatum.

To test if this additional decrease in eGFP-SPAR fluorescence is mediated through activation of the proteasome we bath-applied the proteasome inhibitor MG132 starting 20 min before tetanization (Karpova et al.,2006). Bath-application of MG132 reduced the fluorescence decrease significantly in comparison to drug free experiments (P ≤ 0.05; 9th min: 99.4 ± 3.33%; 42nd min: 60.3 ± 5.76%; Fig. 3A). We therefore ascribed the tetanization-induced additional decline in eGFP-SPAR fluorescence to activity and UPS-dependent SPAR degradation.

Figure 3.

Synaptic activity-dependent degradation of eGFP-SPAR. Diagrams summarizing normalized fluorescence changes of eGFP-SPAR under different drug treatments in comparison to drug-free experiments. A1: Dark gray diamonds indicate the time course of normalized eGFP-SPAR fluorescence intensity in response to tetanizations (seven dendrites of three slices). With gray triangles intensity of eGFP-SPAR under MG132 (25 μM) are shown (five dendrites of three slices). A2: Representative images indicate eGFP-SPAR fluorescence in hippocampal dendrites 20 h after in vivo injection of activated Semliki forest particles. Uniform fluorescence in dendrites and spines was observable. Region of interest (ROI) was placed along dendrite including spines. Representative fluorescence images after stack averaging and alignment are shown for 3, 24, and 45 min. B1: Black squares indicate the time course of fluorescence intensity whereas protein synthesis was inhibited by anisomycin (SPAR+ani, nine dendrites of five slices). B2: Representative fluorescence images are depicted. C1: With black filled circles the eGFP-SPAR fluorescence change under CDK5 inhibition is represented (five dendrites of three slices). C2: Representative fluorescence images are shown. Black horizontal brackets indicate interval of significant difference between groups (*P ≤ 0.05). Black vertical arrows are indicating the time of one second 100 Hz tetanizations. White horizontal scale bar: 5 μm.

It has been suggested that SPAR degradation requires protein synthesis. To study if protein synthesis is indeed required for SPAR degradation we inhibited RNA translation using anisomycin. Application of anisomycin (30 μM) significantly reduced the decrease of fluorescence after tetanization (P ≤ 0.05; 9th min: 94.4 ± 1.2%; 42nd min: 59.9 ± 3.56%; Figs. 3B1 and 3B2). Comparisons of drug-free and anisomycin-treated experiments revealed a significant difference 10 min after tetanization (Fig. 3B).

Previously it was suggested that CDK5 phosphorylates SPAR (Seeburg et al.,2008), however its involvement in tetanization-induced SPAR degradation in hippocampal slices has not yet been established. To clarify if phosphorylation by CDK5 is required for activity-dependent SPAR degradation we bath-applied the CDK5 inhibitor roscovitine during image acquisition. Bath application of roscovitine (5 μM) during tetanization reduced the eGFP-SPAR degradation (9th min: 95.9 ± 1.44%; 42nd min: 63.5 ± 5.41%). Differences in comparison to drug-free experiments reached significance as soon as 5 min after first tetanization (P ≤ 0.05, Fig. 3C).

Protein synthesis-independent eGFP-SPAR degradation

The results described so far showed that SPAR degradation depends, at least in part, on protein synthesis and CDK5-mediated phosphorylation. However, in the presence of protein synthesis inhibitor the fluorescence decay of eGFP-SPAR still differed from that of eGFP (P ≤ 0.05; eGFP: 9th min: 96.8 ± 0.99% and 42nd min: 79.1 ± 3.40%; Fig. 4A). This suggests that strong synaptic activity could enhance SPAR degradation in addition via a protein-synthesis independent signaling pathway. In line with this possibility, we observed that in the presence of anisomycin, bath application of the NMDA receptor blocker APV over the whole time of imaging caused significantly less degradation of eGFP-SPAR (9th min: 97.3 ± 0.62%; 42nd min: 81.37 ± 4.39%) than seen in APV-free experiments (P ≤ 0.05; Fig. 4B).

Figure 4.

Protein-synthesis independent eGFP-SPAR degradation. A: The diagram summarizes the time course of eGFP-SPAR fluorescence intensity (black filled squares) in comparison to eGFP (dark gray diamonds) while protein synthesis was inhibited (P ≤ 0.05; both: nine dendrites of five slices). B: Comparison of eGFP-SPAR fluorescence under protein synthesis inhibition alone and with NMDA-receptor blockage (black filled circles; six dendrites of two slices) indicates a significant difference after the second tetanization. C: No difference between fluorescence change of “anisomycin” and “anisomycin+roscovitine” group (gray filled circles; seven dendrites of three slices) were detected. D: Gray triangles represent the time course of fluorescence during proteasome inhibition with 25 μM MG132 and protein synthesis inhibition (eight dendrites of four slices) in comparison to anisomycin alone treated group (black filled squares). Black horizontal brackets indicate interval of significant difference between groups (P ≤ 0.05). Black vertical arrows are indicating the time of 1-s 100 Hz tetanizations. Representative fluorescence images for the 3, 24, and 45 min are presented on the right side of the diagrams. White horizontal scale bar indicates 5 μm. Representative fESPSP traces are shown for the different conditions for the time 10 (light gray), 15 (black trace), and 40 (dark gray trace) min on the very right (horizontal scale bar: 4 ms; the vertical one: 0.2 mV). Ani.: Anisomycin.

Importantly, when protein synthesis was blocked, the CDK5 inhibitor lost its effect on SPAR degradation (9th min: 98.5 ± 1.97%; 42nd min: 67.6 ± 9.01%; Fig. 4C) whereas inhibition of the proteasome still significantly delayed the fluorescence decrease after tetanization in comparison to the degradation under anisomycin (P ≤ 0.05; MG132: 9th min: 94.3 ± 1.2%; 42nd min: 74.82 ± 5.93%; Fig. 4D). These findings are consistent with the Plk2-CDK5 pathway being dependent on de novo protein synthesis but also imply the existence of a second, protein synthesis-independent pathway involved in UPS-mediated SPAR degradation.


The present results revealed that induction of LTP in acute hippocampal slices triggers a UPS-dependent degradation of SPAR via two complementary signaling pathways: (i) a protein synthesis-dependent route involving CDK5, which corresponds to a previously demonstrated mode of homeostatic plasticity during chronically elevated activity (Seeburg et al.,2008) and (ii) a protein-synthesis independent pathway that requires activation of NMDA receptors.

In a previous study we found that LTP induction triggered de novo synthesis and a net increase in the level of d2eGFP, a degradable reporter sensitive to UPS activity. This effect was reverted to a MG132-sensitive net decrease when protein synthesis was inhibited, demonstrating that in this case synthesis exceeds degradation (Karpova et al.,2006).

On a global level, LTP-related synaptic remodeling requires the well-balanced synthesis and degradation of proteins (Fonseca et al.,2006). Focusing on the turn-over of an individual synaptic protein, however, both processes may even act in synergy. This is particularly obvious in the case of SPAR. In the present study we found that even in the absence of protein synthesis inhibition, LTP did not increase but rather decreased eGFP-SPAR levels. Therefore, in the case of SPAR, degradation exceeds any potential increase in translational activity triggered by LTP induction. Moreover, SPAR degradation itself depends on de novo synthesis of a cofactor as indicated by the effect of anisomycin. This factor most likely is Plk2, a member of the Polo-like kinase family (Pak and Sheng,2003; Seeburg et al.,2005).

For example, it was found that Plk2 transcription was enhanced in response to prolonged epileptiform activity of neurons and mediated activity-dependent reduction in membrane excitability (Kauselmann et al.,1999). Expression of a dominant-negative Plk2 or suppression of Plk2 by RNA interference not only prevented down-regulation of membrane excitability during epileptiform activity, but also unmasked a potentiation in synaptic strength that prevented further induction of long-term potentiation (Seeburg and Sheng,2008). Moreover, it was shown that SPAR degradation induced by Plk2 is an effective homeostatic mechanism to maintain synaptic plasticity.

In addition, it was suggested that interaction of SPAR with other proteins requires phosphorylation. In particular, phosphorylation of SPAR on ser-1328 by CDK5 was shown to prime the binding of Plk2 to its substrate SPAR. Moreover, it was shown that inhibition of CDK5 by roscovitine increased SPAR protein levels in neurons (Seeburg et al.,2008; Seeburg and Sheng,2008). Accordingly, in our experiments inhibition of CDK5-mediated phosphorylation by roscovitine prevented activity-dependent SPAR proteolysis. Similar results were described in cell culture studies where CDK5 inhibition also prevented the decay. Moreover, CDK5 was shown to be involved in LTP induction. This implies that SPAR degradation requires a precise sequence of steps including phosphorylation and protein synthesis to become a target of proteasome mediated protein degradation.

The CDK5 and protein synthesis-independent component of tetanization-induced SPAR degradation has previously not been described. Interestingly, this mechanism required NMDA receptor activation and, hence, most likely a calcium-dependent signaling pathway. Our results are consistent with the idea, that a tight control of SPAR expression is crucial for learning and memory (Lu et al.,2009; Spilker and Kreutz,2010).

The present findings strongly suggest that the integrity of SPAR mediated homeostatic plasticity is required for normal LTP. More generally, our study supports the notion that LTP must be associated with some form of homeostatic plasticity to secure long-term balance and stability within a synaptic network.