Analysis of cpg2 Exon/Intron Structure and Transcript Distribution
A partial cpg2
cDNA isolated in a screen for activity-regulated genes (Nedivi et al., 1993
) was used as a probe to obtain a full-length cpg2
clone from a rat dentate gyrus cDNA library (Nedivi et al., 1996
). The full-length cDNA contains a 2.8 kb open reading frame that predicts a protein of 941 amino acids and is flanked by a 266 nucleotide 5′ untranslated region (UTR) and a 2.3 kb 3′UTR (Nedivi et al., 1996
). When using the cpg2
cDNA sequence for database searches, we identified the 18 exons of the cpg2
coding region as corresponding to exons 16 to 33 of the syne-1
gene, with the syne-1
exon 34 contained in the cpg2
3′UTR (). A Northern blot of total RNA from rat cerebral cortex probed with a segment of the cpg2
coding sequence showed a single 5.9 kb transcript (), the approximate size of the longest cpg2
cDNA clone. Inspection of the cpg2
3′UTR revealed the likely sites of syne-1
early transcription and translation termination that generate the cpg2
transcript. The cpg2
3′UTR contains an unspliced intron between exons 33 and 34 of the syne
-1 gene. Exon 34 is followed by a noncanonical polyadenylation hexamer, conserved in rats and humans, which likely serves as the site of cpg2
transcription termination (). The unspliced intron between exons 33 and 34 contains a stop codon, seven codons downstream of the end of exon 33, which serves as the translation termination site.
cpg2 Is a Brain-Specific Splice Variant of syne-1
To determine whether the original cpg2 cDNA contains the complete 5′UTR, we used 5′RACE to map the cpg2 mRNA start site and isolated two separate bands (). Subcloning and sequencing these bands identified two 5′ ends to the cpg2 transcript. One transcript (cpg2) corresponds to the original cpg2 cDNA isolated in the screen. A second transcript (cpg2b; accession number AY597251) contains an additional exon at the 5′ end of the cpg2 coding region () encoding 24 N-terminal residues with no known structural domains. To determine the tissue distribution of the two cpg2 transcripts, we performed RT-PCR using primers that amplify the cpg2 transcripts but not other syne-1 splice variants (see ). Both transcripts were amplified only from brain cDNA, showing that cpg2 and cpg2b are brain-specific splice variants of syne-1 (). Using a probe against the shared cpg2 and cpg2b 3′UTR that only recognized the cpg2 transcripts, in situ hybridization of a sagittal section from an adult rat brain showed that they are expressed in the cerebral cortex, hippocampus, striatum, and cerebellum of adult rats (). Thus, the two cpg2 transcripts are brain-specific splice variants of the syne-1 gene that are predominately expressed in brain regions associated with long-term potentiation (LTP) or LTD, electrophysiological paradigms of synaptic plasticity.
CPG2 Localizes to the Postsynaptic Site of Excitatory Synapses
Consistent with its derivation from the Syne-1 separator region, the CPG2 protein contains predicted structural or protein interaction motifs including two spectrin repeats and several coiled coils (). To gain insight into CPG2 function, we first determined its localization within neurons. To this end, we generated a monoclonal antibody against a peptide from CPG2. When used to probe a Western blot containing protein extracts from rat cerebral cortex, this antibody recognized a doublet band of the predicted size of CPG2 (). Protein extracts from 293T cells transfected with the cpg2 cDNA showed the same-sized doublet band (), suggesting that the in vivo doublet does not correspond to the translation of the cpg2 and cpg2b transcripts but rather results from posttranslational modification of CPG2 or leaky translation initiation of cpg2.
CPG2 Is Specifically Localized to the Postsynaptic Side of Excitatory Synapses
Immunostaining of cultured hippocampal neurons with the anti-CPG2 monoclonal antibody showed a punctate staining pattern on dendrites (). To further confirm the specificity of the anti-CPG2 monoclonal antibody, neurons were infected with a lentivirus expressing a CPG2-GFP fusion protein and then stained with the anti-CPG2 antibody. The CPG2-GFP signal showed an overlapping distribution with anti-CPG2 antibody labeling of CPG2 (), demonstrating that the CPG2 antibody recognizes endogenous CPG2 protein and the CPG2-GFP protein and results in no other detectable signal in neurons. We observed no labeling when neurons were stained with an antibody against the Syne-1 C terminus previously shown to recognize the Syne-1 protein in muscle cells (Apel et al., 2000
) (data not shown). Given the Western and immunocytochemistry data, we suggest that the immunostaining patterns seen with the anti-CPG2 monoclonal antibody result from staining of the CPG2 protein and not the product of another syne-1
Since the punctate distribution of CPG2 in hippocampal neurons was indicative of a synaptic localization, we tested this possibility by infecting neurons with a GFP-expressing lentivirus and double staining for CPG2 and synapsin I. We found that CPG2 was localized apposed to most synapsin I-positive presynaptic terminals (89% ± 1%; n > 500 synapses, 5 neurons), often on dendritic spines (). These results suggest that CPG2 localizes to the postsynaptic component of a subset of synapses that are often located on dendritic spines. To determine the type of synapses containing CPG2, GFP-filled neurons were double labeled for CPG2 and the inhibitory synapse marker GAD65 or the excitatory synapse marker PSD-95. CPG2 rarely colocalized with GAD65-positive synapses (15% ± 2%; n > 500 synapses, 5 neurons) () but was consistently observed at PSD-95-positive synapses (94% ± 1%; n > 500 synapses, 5 neurons) (). Additionally, CPG2 was not found at PSD-95-positive excitatory synapses on aspiny presumptive inhibitory neurons (data not shown). Thus, CPG2 is specifically localized to the postsynaptic side of excitatory synapses on glutamatergic neurons.
CPG2 Localizes to an Endocytic Zone of Excitatory Synapses
Close examination of the CPG2 distribution reveals that it does not colocalize with PSD-95 within spines, suggesting that CPG2 is not part of the PSD and localizes to a separate spine domain (see ). To characterize CPG2's localization in relation to the PSD and the spine cytoskeleton, we stained neurons for actin filaments, CPG2, and PSD-95. High-magnification images of individual spines from these triple-labeled neurons show little overlap in the distribution of the three proteins, as CPG2 was often localized segregated from the PSD and actin filaments in the spine head ().
CPG2 Localizes to a Subdomain of Excitatory Synapses
CPG2's localization was further resolved using immunoEM. In preembedding anti-CPG2 immunoEM, approximately 47% of the labeling was associated with distal dendrites/dendritic spines, 47% was associated with cytosol/endoplasmic reticulum in the perikaryon or proximal dendrites, and 6% was associated with PSDs. No immunoreactivity was detected in presynaptic terminals. Of 100 synaptic membrane specializations that showed CPG2 labeling, 100 were asymmetrical, excitatory synapses. No symmetrical, inhibitory synapses were identified in association with CPG2 immunoreactivity. At excitatory synapses, CPG2 was localized beneath the PSD predominantly to the more lateral aspects of the PSD, both in vivo in dendritic spine () and shaft (data not shown) synapses and in vitro in spine () and shaft () synapses from cultured hippocampal neurons. We used postembedding immunoEM against CPG2 and the NR1 NMDA receptor subunit on adjacent serial sections to define further the localization of CPG2 relative to the PSD (). Of 32 CPG2 immunoreactive postsynaptic zones, 21 were also immunoreactive for NR1, and the average distance between immunogold particles representing CPG2 immunoreactivity and the postsynaptic membrane thickening was 35 ± 8 nm. We also found CPG2 in dendritic spines positively labeled for the GluR2 AMPA receptor subunit using immunogold labeling of alternate sections (). Thus, the immunoEM confirms the immunofluorescence data and demonstrates that in vivo CPG2 is concentrated in the postsynaptic site of excitatory synapses and is localized to a distinct synaptic subdomain, lateral to and beneath the PSD.
The localization of CPG2 within spines is similar to that of a clathrin-GFP fusion protein previously shown to colocalize with endogenous clathrin in neurons and to mark an endocytic zone of excitatory synapses (Blanpied et al., 2002
). To determine if CPG2 is localized to this zone, we generated a lentivirus that expresses a clathrin light chain A1-GFP fusion sequence. In neurons infected with this virus, clathrin-GFP was distributed in the dendritic shaft and within dendritic spines labeled for CPG2 and PSD-95 (). High-magnification views of individual spines from the clathrin-GFP-infected cells show that CPG2 and clathrin-GFP colocalize in spines to a site lateral to the PSD (). Within spines, CPG2 did not overlap with dynamin 3, recently shown to be localized to dendritic spines (Gray et al., 2003
) (data not shown). These results are consistent with localization of dynamin 3 to the PSD and not to the endocytic zone. The colocalization studies together with the immunoEM data strongly suggest that CPG2 is localized to an endocytic zone within spines that is distinct from the postsynaptic density.
CPG2 Localizes to an Endocytic Zone of Excitatory Synapses
If the region of CPG2 and clathrin colocalization is an endocytic zone, CPG2 should be associated with clathrin-coated pits and/or vesicles. shows an immunoEM micrograph of a synapse from a cultured neuron where CPG2 is localized near a clathrin-coated pit separated from the PSD. shows CPG2 in the vicinity of a clathrin-coated vesicle. Of 18 asymmetric synapses that contained a clathrin-coated pit or vesicle, all 18 contained CPG2 near (within 40 nm) these structures. Double labeling using preembedding HRP staining for CPG2 and postembedding immunogold for the NR1 subunit of the NMDA receptor shows that at least some of these CPG2-associated vesicles traffic glutamate receptors (). Thus, the immunoEM staining pattern is consistent with our immunofluorescence finding that CPG2 is colocalized with clathrin in the postsynaptic endocytic zone and suggests that it may be involved in the endocytosis of glutamate receptors.
CPG2 Levels Affect Dendritic Spine Size
CPG2's localization to the postsynaptic endocytic zone suggests that it may regulate the internalization of surface membrane via clathrin-mediated endocytosis. Therefore, we hypothesized that altering CPG2 levels may affect the size of dendritic spines. To test this hypothesis, we manipulated CPG2 protein levels using overexpression or RNA interference (RNAi) and examined the effects on spine morphology.
For CPG2 overexpression, we generated a lentivirus expressing a CPG2-GFP fusion protein driven by the human ubiquitin-C promoter (cpg2-gfp) that increases CPG2 levels within dendritic spines of infected neurons by 2- to 3-fold, as assessed by immunostaining (data not shown). To knock down CPG2 levels in neurons, we developed a lentivirus vector for RNAi delivery. We first generated a plasmid expressing a cpg2-specific small hairpin RNA (shRNA) driven by the mouse U6 promoter, that could effectively knock down exogenous cpg2-containing mRNA transcripts in 293T cells (data not shown; see Experimental Procedures for details). This cpg2-shRNA sequence and the U6 promoter were subcloned into the pFUGW lentivirus transfer vector upstream of the human ubiquitin-C promoter that drives expression of GFP (). A mutated cpg2-shRNA (Mcpg2-shRNA) lentivirus was generated as a negative control. Western blots of protein extracts from high-density cortical neurons infected with the cpg2-shRNA lentivirus showed a 90% ± 1% reduction (n = 5 cultures) in CPG2 levels relative to Mcpg2-shRNA-infected neurons (). In contrast, there were no changes in levels of GluR1, NR1, or β-tubulin (). Control and knockdown cultures showed comparable levels of GFP expression, demonstrating a similar infection rate. CPG2 knockdown was also evident at the synaptic level. Neurons infected with the control Mcpg2-shRNA virus showed a normal CPG2 subcellular distribution pattern (), whereas neurons infected with cpg2-shRNA showed a reduction in CPG2 levels but no apparent change in the levels or distribution of PSD-95 (), synapsin (), or actin filaments (data not shown). In addition, a lentivirus delivering a shRNA that knocks down the levels of a different candidate plasticity gene, cpg15, had no effect upon CPG2 levels (U. Putz and E.N., unpublished data). Thus, cpg2-shRNA delivered by a lentivirus vector effectively knocks down endogenous CPG2 levels in cultured neurons.
CPG2 Protein Levels Affect Dendritic Spine Size
We measured spine head area from control and knockdown neurons of similar size and morphology to determine if manipulating CPG2 protein levels leads to changes in spine morphology. shows individual spines from neurons infected with Mcpg2-shRNA, cpg2-shRNA, and cpg2-gfp. We found that neurons infected with cpg2-shRNA had significantly smaller dendritic spine heads (−18% ± 2.4%; p < 0.0005; n = 10 cells per group, 100 spines per cell) than Mcpg2-shRNA-infected neurons, while cpg2-gfp-infected neurons had larger spine heads (+7% ± 2.1%; p < 0.05; n = 10 cells per group, 100 spines per cell) (). In addition, measurement of spine length from these neurons showed that dendritic spines from CPG2 knockdown neurons were 12% ± 2% (p < 0.0001; n = 10 cells per group, 100 spines per cell) shorter than those from control neurons. The localization of CPG2 to the synaptic endocytic zone and its effect on dendritic spine size raise the possibility that CPG2 may play a role in regulating membrane removal from the spine surface via vesicle-mediated endocytosis.
CPG2 Knockdown Disrupts Constitutive Glutamate Receptor Internalization
If CPG2 regulates the removal of synaptic proteins from the spine surface through vesicle-mediated endocytosis, then altering its levels may also affect the number of clathrin-coated pits and vesicles seen within spines. Using electron microscopy, we analyzed the number of synapse-associated clathrin-coated pits and vesicles in cultured hippocampal neurons infected with either the Mcpg2-shRNA or the cpg2-shRNA lentivirus. shows examples of spine and shaft synapses from Mcpg2-shRNA (top)- and cpg2-shRNA (bottom)-infected cultures. The number of synapse-associated clathrin-coated pits and vesicles was increased nearly 4-fold in the cpg2-shRNA-treated cultures (0.95 ± 0.18 pits or vesicles per synapse; n = 28 neurons) as compared to the Mcpg2-shRNA-treated controls (0.25 ± 0.06 pits or vesicles per synapse; n = 35 neurons; p < 0.0002) (). There were no obvious effects of CPG2 knockdown on presynaptic terminals. Thus, CPG2 knockdown increases the number of postsynaptic clathrin-coated pits and vesicles in the vicinity of synapses.
CPG2 Knockdown Disrupts Constitutive Glutamate Receptor Internalization
To confirm that the clathrin-coated pits and vesicles that accumulate in response to CPG2 knockdown contain synaptic proteins, we performed preembedding immunoEM for NR1 on cpg2-shRNA-treated cultures. In RNAi-treated cultures, clathrin-coated vesicles near synapses stained positively for NR1 (). In RNAi-treated cultures, 41% ± 11% of coated pits or vesicles stained for NR1, as opposed to only 0.5% ± 0.5% percent in control cultures (n = 12 neurons; p < 0.01). No staining was seen on similar vesicles from unstained cultures (). These data suggest that CPG2 regulates endocytosis of clathrin-coated pits that traffic glutamate receptors.
There are two potential mechanisms by which CPG2 knockdown can increase the number of postsynaptic clathrin-coated vesicles that traffic glutamate receptors: either it can increase the rate of their formation, or it can decrease the rate of their removal. To understand the mechanism of CPG2's regulation of glutamate receptor internalization, we performed a biochemical internalization assay. Surface proteins from cortical cultures infected with Mcpg2-shRNA
were biotinylated with a reversible biotin and kept at 4°C to block internalization or moved to 37°C for 30 min to allow for receptor internalization (in the presence of a proteasome inhibitor). Remaining surface biotin was removed with a cell-impermeable cleavage buffer. Biotinylated proteins were then isolated, probed on a Western blot, and compared with calibration controls. In Mcpg2-shRNA
-infected cultures, 6.2% ± 0.8% of surface NMDA receptors and 8.2% ± 0.6% of surface AMPA receptors were internalized after 30 min at 37°C (), similar to what has been previously reported for glutamate receptor internalization (Ehlers, 2000
; Lin et al., 2000
). In cpg2-shRNA
-infected cultures, AMPA and NMDA receptor internalization was reduced to 2.7% ± 0.8% (p < 0.005; n = 3) and 1.7% ± 0.5% (p < 0.0003; n = 4), respectively (). In contrast, there was no significant change in the internalization of insulin receptors in the cpg2-shRNA
-treated neurons (4.3% ± 0.4%) versus Mcpg2-shRNA
-treated neurons (5.4% ± 0.8%; n = 4; p = 0.4) (). The internalization of AMPA and NMDA receptors was reduced by 66% ± 11% (p < 0.02) and 73% ± 5% (p < 0.002), respectively, in cpg2-shRNA
-infected neurons (). These results demonstrate that the cpg2-shRNA
blockade was specific to the internalization of synaptic proteins. The reduction in glutamate receptor internalization was not a result of a general reduction in GluR2 or NR1 levels, as total GluR2 and NR1 levels remained constant in Mcpg2-shRNA
- and cpg2-shRNA
-infected neurons (see ). These data indicate that CPG2 is necessary for normal constitutive glutamate receptor internalization. Since CPG2 knockdown resulted in an accumulation of clathrin-coated pits and vesicles but disrupted rather than enhanced glutamate receptor internalization, CPG2 is likely involved in the clearance of internalized vesicles from the synapses rather than the formation of these vesicles (see Discussion).
If CPG2 knockdown inhibits the internalization of glutamate receptors, one might expect an accumulation of surface receptors. To examine the amount of surface AMPA and NMDA receptors, we performed a surface protein biotinylation assay. Surface proteins from control or CPG2 knockdown neurons were biotinylated, separated from total protein using Neutravidin, then run on SDS-PAGE gels side by side with total protein from the same cells and probed on Western blots with antibodies against GluR2 or NR1. We then compared the ratios of surface receptors to total receptors in cpg2-shRNA-treated and control cultures. Knockdown of CPG2 resulted in a significant increase in the surface to total protein ratios for both GluR2 (+25% ± 10%; n = 5; p < 0.05) and NR1 (+20% ± 7%; n = 4; p < 0.05) (), demonstrating that CPG2 knockdown does result in an increase in surface receptors. Since biotinylation assays measure the amount of both synaptic and extrasynaptic receptors, we further examined the levels of surface AMPA receptors by staining fixed, unperme-abilized control and knockdown neurons with an antibody against an extracellular epitope of the GluR1 AMPA receptor subunit. When we quantified the intensity of GluR1 punctae, we found that there was a similar increase in synaptic GluR1 levels in CPG2 knockdown neurons (+18% ± 5%; p < 0.05; n = 8 to 10 cells per group) (). Thus, CPG2 knockdown increases the levels of synaptic glutamate receptors, supporting the conclusion that CPG2 plays a role in their internalization.
CPG2 Knockdown Disrupts Activity-Induced Internalization of AMPA Receptors
CPG2 Knockdown Disrupts Activity-Induced AMPA Receptor Internalization
LTD is thought to occur in part by the rapid clathrin-mediated internalization of AMPA receptors following the activation of a specific set of signaling pathways (Beattie et al., 2000
; Lin et al., 2000
; Man et al., 2000
; Snyder et al., 2001
; Wang and Linden, 2000
). To see if CPG2 plays a potential role in LTD, we examined the effect of CPG2 knockdown on the activity-induced internalization of AMPA receptors. Control and CPG2 knockdown neurons were incubated with the anti-GluR1 antibody and then in control media or media plus NMDA, followed by fixation and secondary staining for surface GluR1. The intensity of GluR1 punctae was then quantified. In control neurons, there was a 40% ± 5% (p < 0.000001; n = 8 to 10 cells per group) reduction in surface, synaptic AMPA receptors following NMDA treatment. In contrast, CPG2 knockdown inhibited the NMDA-induced internalization of surface AMPA receptors (−8% ± 3%; p > 0.1; n = 8 cells per group) (). Thus, CPG2 is critical for the activity-dependent internalization of AMPA receptors and may be a crucial component of the LTD pathway.