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. Author manuscript; available in PMC: 2015 Apr 18.
Published in final edited form as: Neuroscience. 2014 Jan 31;265:253–262. doi: 10.1016/j.neuroscience.2014.01.045

RanBP9 overexpression reduces dendritic arbor and spine density

Hongjie Wang 1, Meagan Lewsadder 1, Eric Dorn 1, Shaohua Xu 2, Madepalli K Lakshmana 1,*
PMCID: PMC3972333  NIHMSID: NIHMS562216  PMID: 24486966

Abstract

RanBP9 is a multi-domain scaffolding protein known to integrate extracellular signaling with intracellular targets. We previously demonstrated that RanBP9 enhances Aβ generation and amyloid plaque burden which results in loss of specific pre- and postsynaptic proteins in vivo in a transgenic mouse model. Additionally, we showed that the levels of spinophilin, a marker of dendritic spines were inversely proportional to the RanBP9 protein levels within the synaptosomes isolated from AD brains. In the present study, we found reduced dendritic intersections within the layer 6 pyramidal neurons of the cortex as well as hippocampus of RanBP9 transgenic mice compared to age-matched wild-type (WT) controls at 12-months of age but not at 6 months. Similarly, the dendritic spine numbers were reduced in the cortex at only 12-months of age by 30% (p<0.01), but not at 6 months. In the hippocampus also the spine densities were reduced at 12-months of age (38%, p<0.01) in the RanBP9 transgenic mice. Interestingly, the levels of phosphorylated form of cofilin, an actin binding protein that plays crucial role in the regulation of spine numbers were significantly decreased in the cortical synaptosomes at only 12 months of age by 26% (p<0.01). In the hippocampal synaptosomes, the decrease in cofilin levels were 36% (p<0.01) at 12-months of age. Thus dendritic arbor and spine density were directly correlated to the levels of phosphorylated form of cofilin in the RanBP9 transgenic mice. Similarly, cortical synaptosomes showed a 20% (p<0.01) reduction in the levels of spinophilin in the RanBP9 transgenic mice. These results provided the physical basis for the loss of synaptic proteins by RanBP9 and most importantly it also explains the impaired spatial learning and memory skills previously observed in the RanBP9 transgenic mice.

Keywords: RanBP9, cofilin, dendritic arbor, spine density, transgenic mice, Golgi staining

INTRODUCTION

Neuronal morphology is crucial to our understanding of information processing and communication in the brain because neuronal shape is directly related to the computations performed by the neuron (Spruston, 2008). The two most important morphological characteristics of neurons are dendritic arbor structure and dendritic spine density. The shape, size, and complexity of dendritic trees can modulate action potential propagation (Vetter et al., 2001) and influence the firing pattern of a neuron (Mainen and Sejnowski, 1996). Similarly, the shape and the number of dendritic spines play important roles in synaptic plasticity. Increasing evidence indicates that deficient structural neuronal network connectivity is a major, if not primary, cause of several neurodegenerative disorders including Alzheimer’s disease (AD) (Knobloch and Mansuy, 2008), Huntington’s disease (HD) (Spires et al., 2004) and Parkinson’s disease (PD) (Day et al., 2006). Moreover, changes in the structure and function of dendritic spines contribute to several physiological processes including synaptic transmission and learning and memory (Kennedy et al., 2005; Tada and Sheng, 2006). Therefore identification of molecules that inadvertently contributes to loss of dendritic arbor and spine density is crucial in understanding their role in neurodegenerative diseases.

We previously demonstrated that RanBP9 forms a multi-protein complex with amyloid precursor protein (APP), low-density lipoprotein receptor-related protein (LRP) and β-site APP cleaving enzyme (BACE1), thereby regulate Aβ generation (Lakshmana et al., 2009). Consistent with our report, RanBP9 was recently found to be within the clusters of RNA transcript pairs associated with markers of AD progression (Arefin et al., 2012), supporting our idea that RanBP9 might play a critical role in the pathogenesis of AD. Our subsequent investigations using transgenic mice overexpressing RanBP9 confirmed that RanBP9 in fact regulates Aβ generation and amyloid plaque burden in vivo in the mouse brain. More importantly, RanBP9 overexpression led to decreased levels of specific presynaptic and postsynaptic proteins in the brain, whereas RanBP9 null mice showed increased levels of synaptic proteins (Lakshmana et al., 2012; Woo et al., 2012a). These data taken together with demonstrations by others that RanBP9 interaction with plexin-A coordinates semaphorin3A signaling controlling axonal outgrowth (Togashi et al., 2006) and also the demonstration that RanBP9 regulates BDNF-mediated neuronal morphology and survival through the MAPK and Akt pathways (Yin et al., 2010), all suggest essential role for RanBP9 at the synapses. Also our finding that RanBP9 is present throughout the neuron including neurites in the primary neuronal cultures and within the whole dendritic network in the adult brain (Lakshmana et al., 2012), followed by our demonstration that RanBP9 levels are significantly increased in the brains of patients with AD (Lakshmana et al., 2010), as well as APP transgenic mice (Woo et al., 2012a; Wang et al., 2013) strongly implicates RanBP9 to play pivotal role in the regulation of synaptic density. More recent work from our laboratory demonstrated an excellent correlation between RanBP9 protein levels at the synapses and the loss of spinophilin, a marker of spines, in a brain region-specific manner due to defects in the mitochondrial bioenergetics (Palavicini et al., 2013). Interestingly, in the same study we found normalized levels of RanBP9 protein relatively more in the synaptosomes than the whole homogenate or cytosolic fractions in the human brain (Palavicini et al., 2013). Thus the enriched presence of RanBP9 at the synapses is further evidence that RanBP9 has a crucial role in the regulation of spines and synapses.

Here we further extended our studies and demonstrated that RanBP9 overexpression in the transgenic mice leads to significant reductions in the dendritic arbor and spine density in both the hippocampus and the cortical brain regions in an age-dependent manner. Furthermore, phosphorylated form of cofilin, a filamentous-actin (F-actin) severing protein that increases the turnover of F-actin was highly reduced in the synaptosomes which in turn correlated well with spine density. Thus, we have now provided the physical basis for loss of pre- and postsynaptic proteins by RanBP9.

EXPERIMENTAL PROCEDURES

Chemicals and antibodies

Ethanol (cat # E7023) and Xylene (cat # 2476) were purchased from Sigma-Aldrich (St. Louis, USA). Tissue-Tek O.C.T. compound (cat # 4583) was purchased from Sakura FineTek USA Inc. (Torrance, CA, USA). Anti-flag-tag antibody (M2, cat # F3165) was purchased from Sigma (St. Louis, USA). Polyclonal phospho-cofilin antibody (cat # 3311), polyclonal spinophilin antibody (cat # 9061S) and polyclonal LC3 antibody (cat# 4599) were purchased from Cell Signaling (Danvers, MA, USA). Monoclonal ant-drebrin antibody (cat # D029-3) was purchased from MBL international corporation (Woburn, MA, USA). Anti-TFEB polyclonal antibody (Cat # LS-C118813) was purchased from LifeSpan Biosciences, Inc. (Seattle, WA, USA). Polyclonal anti-TGF-β1 antibody (cat # NBP1-67698) and caspase 3 antibody (cat # NB100-56708) were purchased from Novus biologicals (Littleton, CO, USA). Mouse monoclonal antibody against beta-actin (cat # A00702) and polyclonal antibody against lamin-A were purchased from Genscript USA Inc. (Piscataway, NJ, USA). Secondary antibodies such as peroxidase-conjugated AffiniPure goat anti-mouse (Code # 115-035-146) and ant-rabbit (code # 111-035-144) IgGs were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA).

Mice

All animal experiments were carried out based on ARRIVE guidelines and in strict accordance with the National Institute of Health’s ‘Guide for the Care and Use of Animals’ and as approved by the Torrey Pines Institute’s Animal Care and Use Committee (IACUC). Generation of RanBP9 transgenic mice have been described previously (Lakshmana et al., 2012). The RanBP9 specific primers used in the polymerase chain reaction (PCR) is as follows. The forward primer is 5′ – gcc acg cat cca ata cca g -3′, and the reverse primer is 5 – tgc ctg gat ttt ggt tct c – 3′. Positive mice were then backcrossed with native C57Bl/6 mice and the colonies were expanded. RanaBP9 transgenic line 629 which expressed the transgene in most brain regions was used in this study. All mice were backcrossed to maintain in the C57Bl/6 background, expanded and genotyped and used at the specified ages. To avoid the influence of gender, only male mice were used for both WT and RanBP9-Tg genotypes.

The mice were fed with ad libitum food and water all the time. The food is the irradiated global rodent chow from Harlan. The mice were maintained in a 12-hour light/dark cycle at a temperature of 21–23°C and a humidity of 55±10. After weaning, mice were kept in home cages comprising single sex, single genotype and groups of only 5 mice per cage. All of the mice lived in an enhanced environment with increased amounts of bedding and nesting materials.

Golgi staining

We used the FD Rapid Golgi Stain kit (FD Neurotechnologies) to perform Golgi staining following manufacturer’s protocol. Briefly, the mice were euthanized and the brains were removed rapidly and cut in to small blocks of about 10 mm. The tissue blocks were then rinsed briefly in double distilled water to remove blood from the surface. The tissues were immersed in the impregnation solution made by mixing equal volumes of solutions A and B and changed the solution after 24 h and stored for two weeks in the dark. The tissues were then transferred to solution C, replaced the solution again after 24 h and stored at 4° C in the dark for a week. The tissues were rapidly frozen in Tissue-Tek solution to prevent ice crystal formation which might damage the sections. The brain was oriented such that the plane of sectioning was perpendicular to the base of the brain and then serial sections were cut in a rostral to caudal direction at about 120 μm thickness in a cryostat at −21°C. The sections were then mounted on gelatin-coated microscopic slides with a drop of solution C and excess solution was removed with a Pasteur pipette and the slides were dried naturally at room temperature.

Images were acquired in a fluorescent microscope (Axio Examiner D1) using bright light. For quantification of dendritic intersections, images of pyramidal neurons from layer 6 of cortex and the CA1 region of the hippocampus were captured by selecting well-stained neurons randomly at 40x magnification with water immersion and for the analysis of dendritic spine density images were acquired randomly at 100x magnification with oil immersion. The automated quantitation of dendritic intersections was done by Sholl analysis by installing Sholl analysis plugin in the Image J application folder. This plugin automates the task of doing Sholl analysis on a neuron. Its algorithm is based on how Sholl analysis is done manually by creating a series of concentric circles around the soma of the neuron, and counts how many times the neuron intersects with the circumference of these circles. The images were first converted in to 8-bit grayscale images. Thresholding was done to maintain similar background and noise on all neurons. The pixels were converted in to microns from the scale of approximately 0.16873607 μm/pixel for 40x magnification images and 0.067060678 μm/pixel for 100x magnification images. The number of intersections of dendrites was calculated with concentric spheres positioned at radial intervals of 2 μm. The dendritic morphology and spine quantification were done by a blinded analyzer. The criteria used for analyzing neurons are as follows: the pyramidal neurons had to be fully impregnated and located either in the layer 6 of cortex or CA1 region of hippocampus without truncated branches and the soma located centrally within the 120 μm section depth. The criteria for spines included impregnation intensity allowing visibility of spines, a low level of background, spines counted only on dendrites starting at more than 85 μm distal to the soma and after the first branch point.

Isolation of synaptosomes

To isolate synaptosomes, mice were euthanized under isoflurane anesthesia and cortical and hippocampal tissues from RanBP9 transgenic (TG) and age-matched WT mice were weighed and dounced in a grinder using Syn-PER synaptic protein extraction reagent (cat # 87793) purchased from Thermo Scientific (Rockford, IL, USA). Immediately before use protease inhibitor mixture for mammalian cells from Sigma (cat # P8340) was added to the Syn-PER reagent. The homogenate was centrifuged at 2000 g for 10 minutes to remove cell debris. The resulting supernatant was centrifuged at 15,000 g for 20 minutes. The supernatant formed the cytosolic fraction and the synaptosome pellet was gently resuspended in Syn-PER synaptic protein extraction reagent. The amount of total proteins in the homogenate, cytosolic fraction and synaptosomes were measured by BCA method and compared. The quality of synaptosome preparation was verified by immunoblotting for two cytosolic proteins (TGFβ and LC3), two nuclear proteins (laminA and transcription factor, EB, (TFEB)) and two synaptic proteins (spinophilin and drebrin A).

Immunoblotting

Total protein concentrations of synaptosomes were measured by BCA method (Pierce Biotechnology Inc., Rockford, USA). Equal amounts of proteins were loaded into each well and subjected to SDS-PAGE electrophoresis. The proteins were then transferred onto PVDF membranes, blocked with 5% milk and incubated overnight with primary antibodies followed by one hour incubation with HRP-conjugated secondary antibodies. The protein signals were detected using Super Signal West Pico Chemiluminescent substrate (Pierce, USA).

Immunohistochemistry for caspase 3

RanBP9 transgenic and age-matched WT control mice were deeply anesthetized using isoflurane and perfused with 4% paraformaldehyde (PFA) in PBS. The brains were removed quickly and immersed again in PFA solution with gentle rocking at 4° C for 24 hours. The rest of the immunohistochemical staining procedure was exactly as published from our laboratory (Palavicini et al., 2013). Images were acquired by a laser-scanning confocal microscope (Nikon 90i C1 SHS, Melles Griot laser system). The images were deconvoluted, filtered and analyzed with Image-Pro Plus 3D Suite software.

Transmission electron microscopy (TEM)

To prepare samples for TEM analysis, 3 μl of synaptosomes were applied on to a TEM grid and allowed to incubate for 3–4 minutes. Excess liquid was removed with the edge of a kim wipe. The sample was washed with 30–40 μl of deionized water and stained with 4–5 μl of 2.5% uranyl acetate. The grid was washed with 30–40 μl of deionized water and dried for 10 minutes before analyzing under TEM. Ultrastructure of synaptosomes was imaged with a Joel 1010 TEM (Peabody, MA). Images were captured with a Hamamatsu (Bridgewater, MA) digital camera by using AMT (Danvers, MA) software.

Statistical analysis

Immunoblot signal for phospho-cofilin and spinophilin were quantified using Image J software. Cofilin and spinophilin levels in WT and RanBP9 transgenic mice were analyzed by Student’s t-test. The differences in the number of spines in the WT versus TG mice were analyzed by Student’s t-test using Instat3 software (GraphPad Software, San Diego, CA, USA). We used two-tailed p value assuming populations may have different standard errors. The differences in the number of dendritic intersections versus the radial distance from soma in the WT versus TG mice were analyzed by one-way analysis of variance (ANOVA) followed by the post-hoc test. The data presented are mean ± SEM. The data were considered significant only if the p<0.05, * indicates p<0.05, and **, p<0.01, ***, p<0.001.

RESULTS

RanBP9 overexpression leads to age-dependent reduction in dendritic arbor in the pyramidal neurons of cortex and the hippocampus

In order to understand the role of RanBP9 in synaptic damage, we generated RanBP9 transgenic mice by cloning 3x-flag-RanBP9 cDNA in the mouse thy-1 gene cassette in the pTSC21K plasmid as described previously (Lakshmana et al., 2012). We used thy-1 promoter to restrict RanBP9 expression to the postnatal/adult brain only so that any adverse effect of RanBP9 during embryonic development may be prevented. It is well known that the degree of complexity of dendritic trees can modulate action potential propagation (Vetter et al., 2001) and influence the intrinsic firing pattern of a neuron (Mainen and Sejnowski, 1996). Particularly, the action potential propagation is strongly influenced by the number of dendritic branching points and the rate of increase in dendritic membrane area (Vetter et al., 2001). In order to understand the physical basis for loss of synaptic proteins as well as learning deficits in the RanBP9 transgenic mice (Lakshmana et al., 2012; Woo et al., 2012a; Palavicini et al., 2013), we first directly quantified the numbers of dendritic intersections in the layer 6 pyramidal neurons of cortex and the CA1 region of hippocampus, the two most vulnerable brain regions in AD. We analyzed 30 Golgi-stained neurons per age per genotype of mice. Thus a total of 120 neurons were analyzed in the WT mice and another 120 neurons from the RanBP9 transgenic mice were analyzed. We performed Sholl analysis of Golgi-stained neurons by measuring the number of dendrites that cross circles at different radial distances from the cell body. The Sholl analysis plugin for Image J automates the task of doing Sholl analysis on a neuron by creating a series of concentric circles around the soma of the neuron and counts how many times the neuron intersects with the circumferences of these circles. Thus Sholl analysis provides unbiased and automated information on the dendritic branching patterns of neurons.

Analysis of dendritic arbor structure in the RanBP9 transgenic mice revealed a visible effect of RanBP9 on the pyramidal neurons of layer 6 cortex at 12-months of age but had no effect at 6-months of age (Fig. 1A & B). Statistical analysis revealed significant reductions in those dendritic intersections originating roughly between 30 μm and 60 μm from the soma of cortical neurons. Quantitative data in the hippocampus suggested that small reductions in the dendritic complexity of pyramidal neurons in the CA1 region can be observed even at 6-months of age at about 60 μm from soma in the RanBP9 transgenic mice (TG) compared to age-matched wild-type (WT) mice (Fig. 2A & B), though it was not statistically significant. However, more robust and statistically significant reductions were seen in the hippocampus in 12-month old mice starting from 15 μm and extending as far as 70 μm from soma. Thus hippocampus is relatively more vulnerable brain region in terms of loss of dendritic branches and complexity by RanBP9 overexpression (Fig. 2A & B).

Figure 1.

Figure 1

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RanBP9 overexpression reduces dendritic intersections in the pyramidal neurons of layer 6 of cortex at only12-months of age but not at 6 months. A, Representative photomicrographs of Golgi-stained cortical pyramidal neurons shown for 6- and 12-months- old mice overexpressing RanBP9 (TG) and age-matched wild-type (WT) controls. B, Sholl analysis of Golgi-stained neurons by Image J software. The ordinate represent the distance from soma in μm and the abscissa represent number of dendritic intersections that cross along the concentric circles at defined distance from soma. Significant differences in the dendritic arbor were observed only in those dendritic branches that originate approximately between 30 to 60 μm from soma as indicated by asterisks. *, p<0.01 in RanBP9 transgenic mice compared to WT mice by ANOVA followed by post hoc test. Scale bar indicates 25 μm. The data are mean ± SEM, n=6 for each of RanBP9 TG and WT mice.

Figure 2.

Figure 2

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RanBP9overexpression reduces dendritic intersections in the pyramidal neurons of CA1 region of the hippocampus at only 12-months of age but not at 6 months. A, Representative photomicrographs of Golgi-stained hippocampal pyramidal neurons shown for 6- and 12-months- old mice overexpressing RanBP9 (TG) and age-matched wild-type (WT) controls. B, Sholl analysis of Golgi-stained neurons by Image J software. The ordinate represent the distance from soma in μm and the abscissa represent number of dendritic intersections that cross along the concentric circles at defined distance from soma. Significant differences in the dendritic arbor were observed only in those dendritic branches that originate approximately between 15 to 70 μm from soma in the 12-month-old mice as indicated by asterisks. *, p<0.01 in RanBP9 transgenic mice compared to WT mice ANOVA followed by post hoc test. Scale bar indicates 25 μm The data are mean ± SEM, n=6 for each of RanBP9 TG and WT mice.

RanBP9 overexpression reduces number of dendritic spines in the pyramidal neurons of cortex and the hippocampus

It is now widely accepted that dendritic spines are anatomical specializations on neuronal cells that form distinct compartments that isolate input from different synapses and are crucial for excitatory synaptic transmission. Therefore, like dendritic arbor, the number of spines can have a great impact on the neuronal function. Given the role of RanBP9 in reducing synaptic proteins such as PSD95 and spinophilin (Lakshmana et al., 2012; Palavicini et al., 2013), we hypothesized that RanBP9 would also significantly reduce the number of spines. This is especially true because spinophilin which is a marker of spines is significantly reduced in RanBP9 overexpressing APΔE9 transgenic mice (Palavicini et al., 2013). Similar to dendritic arbor, spine density was not altered at 6-months of age in the pyramidal neurons of layer 6 cortex of RanBP9 transgenic mice compared to WT mice (Fig. 3A & B). However, at 12-months of age spine density was significantly reduced by 29% (p<0.01) in the RanBP9 mice compared to WT mice (Fig. 3A & B). In the hippocampus, similar to cortex, spine density was not altered in the pyramidal neurons of CA1 region at 6-months of age. However, at 12-months the reduction was 38% (p<0.001) in the RanBP9 transgenic mice versus WT controls (Fig. 4A & B). Although endogenous versus exogenous expression of RanBP9 was 1:1 in the cortex and only 1:0.8 in the hippocampus (Palavicini et al., 2013), more robust reduction in the spine density in the hippocampus (38%) compared to cortex (29%), clearly suggest that hippocampus is more vulnerable to the effect of RanBP9. Thus age- and brain region-specific effect of RanBP9 on the spine density within the pyramidal neurons was confirmed.

Figure 3.

Figure 3

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Reduced spine density in the layer 6 of cortical pyramidal neurons of brains from 12-month-old mice overexpressing RanBP9. A, Representative examples of Golgi-stained cortical pyramidal neurons showing dendritic segments at 100x magnifications to display spines in the 6- and 12-months-old mice overexpressing RanBP9 (TG) and age-matched wild-type (WT) controls. B, Semi-automated quantitation of spine numbers per 10 μm dendritic segment by image J software was subjected to statistical analysis. **, p<0.01 in RanBP9 TG mice versus WT mice by Student’s t-test. The data are mean ± SEM, n= 6 for each of RanBP9 TG and WT mice.

Figure 4.

Figure 4

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Reduced spine density in the CA1 region of the hippocampal pyramidal neurons of brains from 6- and 12-month-old mice overexpressing RanBP9. A, Representative examples of Golgi-stained hippocampal neurons showing dendritic segments at 100x magnifications to display spines in the 6- and 12-months-old mice overexpressing RanBP9 (TG) and age-matched wild-type (WT) controls. B, Semiautomated quantitation of spine numbers per 10 μm dendritic segment by image J software was subjected to statistical analysis. ***, p<0.001 in RanBP9 TG mice versus WT mice by Student’s t-test. The data are mean ± SEM, n= 6 for each of RanBP9 TG and WT mice.

RanBP9 overexpression decreases levels of phosphorylated form of cofilin in the synaptosomes of cortex and hippocampus

Dendritic spines are the postsynaptic sites of most excitatory synapses in the brain and are highly enriched in polymerized F-actin which drives the formation and maintenance of mature spines. Cofilin is an F-actin-severing protein that increases the turnover of F-actin by severing the filaments and creating new barbed ends for F-actin growth (Moriyama et al., 1990; Yahara et al., 1996; Carlier et al. 1997; Lappalainen and Drubin, 1997; Rosenblatt et al., 1997). We recently showed that RanBP9 dephosphorylates cofilin in primary hippocampal neurons (Woo et al., 2012a). Since cofilin is a key regulator of actin dynamics and because dendritic spines are rich in actin molecules which provide shape and structure to the spines, we wanted to assess whether reduced spine density in the RanBP9 transgenic mice is due to changes in the levels of phosphorylated cofilin protein.

Synaptosomes consist of presynaptic terminals attached to postsynaptic dendritic spines that are pinched off from the adjoining dendritic shaft, suggesting that they can also serve as a model to study dendritic spines in isolation. Therefore we isolated and quantified cofilin protein levels in synaptosomes instead of whole brain homogenates which might provide overall changes in the neuron and is likely to dilute the effects of transgene. Brain extracts were prepared as cytosolic (C), homogenate (H) and synaptosomal (S) fractions by centrifugation. We determined the purity of synaptosomes by two independent methods. We first qualitatively looked for two proteins in each of C, H and S fractions. Cytosolic proteins such as TGF-β and LC3 were almost completely absent in the S fractions, but as expected were present in both the C and H fractions (Fig. 5A, left panels). Similarly nuclear proteins such as lamin-A and transcription factor, EB (TFEB) were completely absent in the S and C fractions, though substantial amounts of these protein could be detected in the H fractions (Fig. 5A, middle panels). Finally, we could detect enriched amounts of two synaptic proteins, drebrin A and spinophilin in the synaptosomal fractions relative to H or C fractions (Fig. 5A, right panels). As such, the synaptosomes can be used to reflect changes in protein levels in the spines.

Figure 5.

Figure 5

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Characterization of synaptosomes by biochemical and morphological methods. A, The purity of the synaptosomes prepared from mouse brains was verified by immunoblotting the cytosolic (C), homogenate (H) and synaptosomal (S) fractions for two cytosolic proteins (TGFβ and LC3), two nuclear proteins (lamin-A and transcription factor, EB (TFEB)) and two synaptic marker proteins (drebrin A and spinophilin). Please note enrichment of synaptic proteins and the absence of nuclear proteins or cytosolic proteins in the S fractions, attesting to the purity of synaptosomes. B, Transmission electron microscopy (TEM) images at 20,000 magnification showing intact synaptosomes. Arrows indicate the preservation of postsynaptic densities at the synapses.

To determine whether synaptosomal architecture is preserved in our preparation by another independent method, we used transmission electron microscopy (TEM) to examine the synaptosomes at the ultrastructural level. As shown in Fig. 5B, we observed synaptosomes with intact tightly opposed pre- and post-synaptic elements held in close proximity with each other. The postsynaptic density observed as dark and thick layer are shown (arrows in Fig. 5B), which represents the pinched-off dendritic spines. The plasma membrane of most of the synaptosomes appeared continuous suggesting that the cytoplasmic contents inside the synaptosomes are not perturbed. Thus we confirmed the integrity of our synaptosome preparations by both biochemical and morphological methods.

Next, we quantified phosphorylated form of cofilin in the synaptosomes isolated from the cortical and hippocampal brain tissues and compared between WT and RanBP9 TG mice. Similar to changes in the dendritic intersections as well as spine density, cofilin levels in the cortical synaptosomes isolated from 6-month old RanBP9 transgenic mice were not significantly altered (only 10% reduction) when compared to control mice (Fig. 6A). At 12 months, however RanBP9 transgenic mice showed a reduction of cofilin protein by 26% (p<0.05) in the cortical synaptosomes (Fig. 6A & B). Hippocampus also did not show significant reductions (only 11%) in the cofilin levels at 6-months of age. By 12-months of age, the reduction was 36% (p<0.01) in the RanBP9 TG mice compared to WT controls (Fig. 6A & B). Thus the reduction in the levels of phosphorylated form of cofilin was consistent with changes in the dendritic arbor and spine density.

Figure 6.

Figure 6

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RanBP9 overexpression decreases phosphorylated form of cofilin protein levels in the synaptosomes of cortex and hippocampus. A, Cortical and hippocampal synaptosomes from RanBP9 transgenic (TG) and age-matched wild-type (WT) control mice prepared from 6- and 12-month old mice were subjected to SDS-PAGE electrophoresis and probed with anti-phospho-cofilin antibody to detect phosphorylated form of cofilin. Immunoblotting using flag specific monoclonal antibody detected flag-tagged exogenous RanBP9 in the TG mice but not in WT mice. Actin was detected as a loading control. B, Image J quantitation did not reveal significant changes in the levels of cofilin in the cortex at 6-months of age but by 12-months the levels were reduced significantly by 26%. Similarly, in the hippocampus cofilin levels were significantly reduced only at 12-months (36%). **, p<0.01 in RanBP9 TG mice versus WT control mice by Student’s t-test. Data are mean ± SEM, n=5 for each of TG and WT mice.

RanBP9 overexpression decreases spinophilin levels in the cortical synaptosomes

We previously demonstrated that RanBP9 overexpression in the APΔE9 mice significantly reduced spinophilin levels in the synaptosomes (Palavicini et al., 2013). However it is not clear whether RanBP9 transgenic mice also show decreased spinophilin protein in the synaptosomes. Decreased spine density in the RanBP9 transgenic mice observed in the present study also prompted us to quantify spinophilin levels in the synaptosomes. Consistent with changes in cofilin levels, synaptosomes isolated from cortex did not show any alteration in spinophilin protein at 6-months of age (Fig. 7A). At 12-months, however RanBP9 transgenic mice showed a 20% reduction (p<0.01) when compared to synaptosomes prepared from WT mice (Fig. 7A & B). Thus although the extent of reduction in spinophilin levels is lower than that of cofilin levels in the synaptosomes, a decreased trend for both proteins in synaptosomes is consistent with reduced spine density.

Figure 7.

Figure 7

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RanBP9 overexpression decreases spinophilin protein levels in the synaptosomes of cortex. A, Cortical synaptosomes from RanBP9 transgenic (TG) and age-matched wild-type (WT) control mice prepared from 6- and 12-month old mice were subjected to SDS-PAGE electrophoresis and probed with anti-spinophilin antibody to detect spinophilin protein. Immunoblotting using flag specific monoclonal antibody detected flag-tagged exogenous RanBP9 in the TG mice but not in WT mice. Actin was detected as a loading control. B, Image J quantitation did not reveal significant changes in the levels of spinophilin in the cortex at 6-months of age but by 12-months the levels were reduced significantly by 20%. **, p<0.01 in RanBP9 TG mice versus WT mice by Student’s t-test. Data are mean ± SEM, n=5 for each of TG and WT mice.

RanBP9 overexpression does not alter activated caspase 3-positive cells

The presence of activated caspase 3 is an indicator of neurodegeneration in the brain. We stained for activated caspase 3 by immunohistochemistry using an antibody which specifically recognizes activated form of caspase 3. At both 6- and 12-months of age we could see only few cells stained for activated caspase 3 in the cortex as well as hippocampus of both the WT and RanBP9 transgenic mice (Fig. 8), suggesting that reduced spine density as well as spinophilin and cofilin protein levels are unlikely due to neurodegeneration.

Figure 8.

Figure 8

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Cells positively stained for caspase 3 in the cortex and hippocampus in the WT and RanBP9 TG mice. Representative brain sections from cortex and hippocampus stained with anti-caspase 3 (red) and counter-stained with DAPI (blue). Only few Caspase 3 positive cells (red) were observed in both the RanBP9 TG mice and the WT mice at 6- and 12-months of age.

DISCUSSION

Here we report that RanBP9 overexpression in mice results in age-dependent reductions in the dendritic arbor and spine density in the pyramidal neurons of layer 6 of cortex and the CA1 region of hippocampal brain regions. It is interesting to note that the reductions in dendritic intersections as well as spine density were similarly altered in an age- and brain region-specific manner. In addition, the reduced spine density in the synaptosomes by RanBP9 was directly correlated with the reduced protein levels of phosphorylated cofilin as well as spinophilin. These results are consistent with several properties of RanBP9 demonstrated previously by others and from our laboratory.

We recently demonstrated by both immunohistochemistry and immunoblots that RanBP9 overexpression in the APΔE9 transgenic mice decreases the levels of spinophilin, a marker of spines in the cortex and hippocampus at 12-months of age (Palavicini et al., 2013). We also showed that reduced spinophilin levels were accompanied by reduced mitochondrial activity in the synaptosomes, suggesting that the loss of spinophilin is due to defects in mitochondrial bioenergetics. The present finding of reduced spine density by RanBP9 is consistent with decreased spinophilin in the synaptosomes in the same brain regions. Thus our recent demonstration of loss of spinophilin (Palavicini et al., 2013) and other pre- and post-synaptic proteins by RanBP9 (Lakshmana et al., 2012; Woo et al., 2012a) can now be directly attributed to loss of dendritic intersections and spines. A large body of accumulating data points to the dendritic spines as the principal signaling hub responsible for transducing excitatory synaptic transmission and for the expression of postsynaptic plasticity such as long-term potentiation (LTP). LTP in turn is considered the physical basis for learning and memory. Therefore loss of spines can also explain impaired spatial learning skills demonstrated previously from our laboratory using both a T maze paradigm (Palavicini et al., 2013) and Morris water maze (Woo et al., 2012a) in mice overexpressing RanBP9. But it is important to remember that spines can undergo structural changes within minutes and the spine density estimated in the present study reports only steady state levels at the time. This also explains why there was significant reduction in the spine density at 12 months but not at 6-months of age. It is also possible that even in the absence of significant alterations in the spine numbers, existing spines may not be functionally efficient in signal processing, compromising neuronal plasticity. As RanBP9 is enriched in the complex neuritic processes in the cultured primary neurons and in the dendritic processes in the neurons of adult brain (Lakshmana et al., 2012), RanBP9 is expected to play pivotal role at the synapses. Moreover, RanBP9 is a ligand for Rho-GEF (Bowman et al., 2008) and as Rho family GTPases are essential regulators of actin polymerization (Luo, 2002) as well as formation of dendritic spines (Zhang et al., 2005; Tolias et al., 2007; Xie et al., 2007; Saneyoshi et al., 2008; Wegner et al., 2008), RanBP9 is also likely to play crucial role in the regulation of spine density. More recent studies demonstrated that even in the adult brain, Rho GTPases play pivotal role in the plasticity of dendritic spines (Martino et al., 2013). The actin cytoskeleton has long been suspected to be crucial in controlling the development and stability of dendritic spines as dendritic spines are highly enriched in actin. Actin cytoskeleton is mainly regulated by Rho GTPases that includes Rho, Rac, and cdc42 subfamilies (Etienne-Manneville and Hall, 2002) which can regulate activity-dependent structural plasticity by which dendritic spines are produced and modified. Thus as a Rho GTPase ligand, RanBP9 might contribute to the loss of dendritic intersections and spines, which can account for the loss of hippocampal-dependent learning and memory skills in mice overexpressing RanBP9. In fact, RanBP9 overexpression results in drastic reductions in the growth and branching of neurites from dorsal root ganglion (DRG) neurons (Togashi et al., 2006) and cerebellar neurons (Cheng et al., 2005).

Another most important observation in the present study is the direct correlation between loss of spines as well as dendritic intersections and the reduced levels of phosphorylated form of cofilin in the synaptosomes derived from the hippocampus and cortical brain regions. But what is not clear from this study is whether reduced spine density is the cause or consequence of changes in cofilin levels. Primary neuronal cultures transfected with RanBP9 gene also resulted in decreased levels of phosphorylated form of cofilin as determined by immunoblots, though non phosphorylated form of cofilin i.e., total cofilin levels were increased (Woo et al., 2012a). Our present results of decreased cofilin levels in the synaptosomes are consistent with these data and confirm the direct and positive relationship between phosphorylated cofilin levels and loss of spines. This is also consistent with the finding that both LTP and long-term depression (LTD) are impaired in mice lacking cofilin with associated learning deficits in spatial, aversive and reward learning (Rust et al., 2010). As spine dynamics are completely dependent on the cytoskeletal network, a change in the levels of actin-remodeling protein such as cofilin is expected to alter spine numbers. Binding of cofilin with actin filaments increases the removal of actin monomers from the sharp end of the filaments leading to filament depolymerization which can alter the shape and size of spines. Although RanBP9 decreased the levels of phosphorylated cofilin, it is not clear how exactly RanBP9 influences the phosphorylation status of cofilin. An obvious mechanism may involve protein kinases and phosphatases. We previously showed that RanBP9 binds both low-density lipoprotein receptor-related protein (LRP) and β1-integrin and regulate their endocytosis (Woo et al., 2012b). The Src and LIM kinases are activated downstream of β1-integrins, and LIM kinase is known to control phosphorylation of cofilin (Bernard, 2007). Thus RanBP9 can indirectly influence the phosphorylation of cofilin and therefore actin dynamics, which in turn can alter spine density. Additionally, synaptosomes derived from RanBP9 transgenic mice showed reduced spinophilin. Since spinophilin is considered a marker of dendritic spines, reduced levels of spinophilin in the synaptosomes is also consistent with reduced spine density in the RanBP9 transgenic mice. Overall, these data suggest that reduced dendritic arbor and spine density in the RanBP9 transgenic mice may be due to decreased levels of constitutively active phosphorylated form of cofilin as well as spinophilin.

Highlights.

  1. RanBP9 overexpression decreases dendritic arbor in the cortex and hippocampus

  2. RanBP9 reduced spine density at 12-months of age but not at 6 months

  3. Decreased spine density correlated well with the reduced levels of phospho-cofilin

  4. Reduced spine density provided the physical basis for the loss of synaptic proteins

Acknowledgments

This work was supported by National Institute of Aging (NIA)/NIH grant numbers (1R03AG032064-01, M.K. Lakshmana), 1R01AG036859-01, M.K. Lakshmana). We are grateful to the staff at TPIMS vivarium for their support throughout this study.

Footnotes

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