Kalrn promoter usage and isoform expression respond to chronic cocaine exposure
© Mains et al; licensee BioMed Central Ltd. 2011
Received: 2 November 2010
Accepted: 17 February 2011
Published: 17 February 2011
The long-term effects of cocaine on behavior are accompanied by structural changes in excitatory glutamatergic synapses onto the medium spiny neurons of the striatum. The Kalrn gene encodes several functionally distinct isoforms; these multidomain guanine nucleotide exchange factors (GEFs) contain additional domains known to interact with phosphatidylinositides as well as with a number of different proteins. Through their activation of Rho proteins and their interactions with other proteins, the different Kalirin isoforms affect cytoskeletal organization. Chronic exposure of adult male rodents to cocaine increases levels of Kalirin 7 in the striatum. When exposed chronically to cocaine, mice lacking Kalirin 7, the major adult isoform, fail to show an increase in dendritic spine density in the nucleus accumbens, show diminished place preference for cocaine, and exhibit increased locomotor activity in response to cocaine.
The use of alternate promoters and 3'-terminal exons of the mouse Kalrn gene were investigated using real-time quantitative polymerase chain reaction. While the two most distal full-length Kalrn promoters are used equally in the prefrontal cortex, the more proximal of these promoters accounts for most of the transcripts expressed in the nucleus accumbens. The 3'-terminal exon unique to the Kalirin 7 isoform accounts for a greater percentage of the Kalrn transcripts in prefrontal cortex than in nucleus accumbens. Western blot analyses confirmed these differences. Chronic cocaine treatment increases usage of the promoter encoding the Δ-Kalirin isoforms but does not alter full-length Kalirin promoter usage. Usage of the 3'-terminal exon unique to Kalirin 7 increases following chronic cocaine exposure.
Kalrn promoter and 3'-terminal exon utilization are region-specific. In the nucleus accumbens, cocaine-mediated alterations in promoter usage and 3'-terminal exon usage favor expression of Kalirin 7 and Δ-Kalirin 7. The Δ-isoform, which lacks a Sec14p domain and four of the nine spectrin-like repeats found in full-length Kalirin isoforms, increases spine headsize without increasing dendritic spine numbers. Thus cocaine-mediated changes in alternative splicing of the Kalrn gene may contribute importantly to the behavioral, morphological and biochemical responses observed.
Despite the expression of multiple Rho GDP/GTP exchange factors (GEFs) in the nervous system, the Kalrn gene, which encodes multiple isoforms of Kalirin, plays an essential, non-redundant role. Linkage analyses identified Kalrn as a major risk factor for coronary artery disease and stroke [1–3], as well as for schizophrenia and adult attention-deficit/hyperactivity disorder [4–6]. In addition, Kalirin interacts with many different proteins including deleted in schizophrenia 1 (DISC1), a major risk factor for schizophrenia, the inducible form of nitric oxide synthase (iNOS; NOS2), Huntingtin-associated protein 1 (HAP1) and peptidylglycine α-amidating monooxygenase, an essential enzyme for neuropeptide biosynthesis [7–11]. Chronic cocaine treatment increases levels of Kal7 protein in the mouse and rat striatum . Estrogen treatment of ovariectomized female rats increases Kalirin expression , and Alzheimer's Disease is accompanied by a dramatic loss of Kal7 in the hippocampus . Hippocampal Kalirin mRNA is elevated at 4 h following a kainate-induced seizure and remains elevated for a month .
Kalirin has been implicated in many functions. Kalirin is one of a small number of RhoGEFs localized to the postsynaptic density (PSD) . In the adult brain, Kalirin 7 (Kal7) is the predominant isoform and is almost exclusively localized to the PSD [17, 18]. Overexpression of Kal7 leads to increases in dendritic spine number in cultured hippocampal and cortical pyramidal neurons [19–21]. Conversely, a specific reduction of Kal7 in primary neurons causes a dramatic decrease in spine density [13, 21]. Studies on Kalirin have focused on cortex and hippocampus, where Kal7 has a clear role in spine formation and maintenance.
With many functional domains, the different Kalirin isoforms are capable of integrating inputs from a variety of surface molecules (ephrins, N-cadherin, Trk receptors), phosphatidylinositides, cytoskeletal components, PDZ domain proteins like spinophilin and PSD-95, plus signaling molecules and growth factors [18, 22–25]. Kal7 is also crucial for the expression of normal long-term potentiation in CA1 hippocampal pyramidal neurons .
The rat Kalrn gene encompasses more than 65 exons spanning 600 kb of the rat genome, with at least six promoters and five 3'-terminal exons [26–28]. Alternative splicing yields multiple functionally different isoforms of rat Kalirin. We recently developed a mutant mouse specifically lacking the Kal7 isoform (Kal7KO) ; the 3'-terminal exon that encodes a PDZ binding motif known to interact with PSD-95 and AF-6 was eliminated [18, 25]. Kal7KO mice show decreased anxiety-like behavior, impaired acquisition of a passive avoidance task, and abnormal behavioral and morphological responses to chronic cocaine [12, 17]. A total Kalirin knockout mouse (T-KalKO) mice, in which exons 27-28 in the first GEF domain were replaced by a neomycin resistance gene, was also developed . T-KalKO mice also display a number of neurological phenotypes. Use of these mouse models to clarify the role of Kalirin proteins in synaptic plasticity requires better characterization of the major products of the mouse Kalrn gene.
Given the essential role of Kal7 in multiple cocaine-elicited behaviors as well as the interactions of Kalirin with cyclin dependent kinase 5 (Cdk5) and the NR2B subunit of the NMDA receptor [12, 17, 18, 30], proteins important for these behaviors, we chose to examine more closely the effects of cocaine on the expression of different Kalirin isoforms in the striatum and, more specifically, in the ventral striatum or nucleus accumbens. In order to identify the striatal cell types expressing Kal7, we took advantage of mice expressing GFP under control of the D1-dopamine receptor promoter.
Cocaine treatment of mice
Mice were kept in the University of Connecticut Health Center animal facility on a 12 h light/dark cycle (lights on 7:00 am to 7:00 pm) and handled in accordance with University of Connecticut Health Center Institutional Animal Care and Use Committee guidelines. Adult male C57BL/6 mice (Jackson Laboratories; 2-5 months old) were treated daily with cocaine (20 mg/kg/day, i.p.) or saline for 7 days  and sacrificed 24 h after the final injection. When tested, the mice exhibited the expected locomotor sensitization to chronic cocaine administration (not shown) . Mice expressing green fluorescent protein (GFP) under the control of the D1-dopamine receptor promoter were from the University of Missouri Mutant Mouse Regional Resource Center (Tg(Drd1a-EGFP)X60Gsat/Mmmh). These mice were bred at least seven generations into C57Bl/6 (Jackson Labs) before use.
Quantitative polymerase chain reaction (Q-PCR)
RNA was prepared from freshly dissected tissues with Trizol (Invitrogen) following the manufacturer's instructions, except that the isopropanol precipitation step was lengthened to overnight at -20°C and there were 2 ethanol washes instead of one. The cDNA samples were prepared using Superscript II (Invitrogen) and random primers, or iScript (BioRad) with random primers; a 5 min 65°C step was added to the iScript protocol before chilling and adding the enzyme. While GAPDH and actin samples were unaltered by the choice of random priming or oligo-dT priming, Kalirin transcripts, which are much longer, appeared to be 10-50-fold less abundant compared to GAPDH when using oligo-dT priming instead of random priming. Pairwise statistical comparisons used the t-test for two samples assuming unequal variance and the two-tailed value is reported.
Primers used for quantitative polymerase chain reaction
Kalirin A promoter
Kalirin B promoter
Kalirin C promoter
Kalirin D promoter
All primer pairs were chosen to keep the melt temperature (Tm) between 58 and 62°C (calculated using http://www.basic.northwestern.edu/biotools/oligocalc.html) and products in the 135 ± 20 nt size range. Primers were designed to delineate the unique 5' end of ΔKalirins (AF229255.1), the spectrin domains common to all forms of Kalirin with nine spectrin-like repeats (U88157.1), and sequences unique to the 3' ends of Kal7 (AF230644.1), Kal8 (U88157.1), Kal9 (AF232668.1), and Kal12 (NM_032062.2); rat sequences were used to identify the corresponding mouse sequences, which all appear as Ensembl sequences at http://www.ensembl.org/Mus_musculus/Info/. Additional primer pairs identifying transcripts which extend beyond a unique end (e.g. Exon 33 to Exon 34, Exon 38-39, Exon 51-52) instead of terminating in a specific 3' end, corroborated the major findings (not shown).
Putative mouse Kalrn mRNA initiation exons A, B, C and D were identified by homology to the rat and human Kalrn initiation exons [27, 28], as described here. Mouse Kalrn exons A, B, and C were identified by comparing the known rat sequences (A: Genbank U88157.1; B: U88156.1; C: AF230644.1) with the mouse genome (July 2007 [NCBI37/mm9]) at http://genome.ucsc.edu/cgi-bin/. The putative mouse Kalrn D initiation exon sequence was from XM_001001454.1, and the putative mouse ΔKalrn initiation exon sequence was from ENSMUST00000114949. The A, B, and C sequences all fit the current Ensembl sequences, and C is also a Refseq entry: NM_177357.
Adult male Drd1a-GFP mice were anesthetized with ketamine/xylazine and perfused transcardially with saline followed by 4% paraformaldehyde as described . Brains from two mice were postfixed in 4% paraformaldehyde and then incubated in 25% sucrose in phosphate buffered saline containing 0.02% NaN3. Coronal sections (12 μm) through the striatum and nucleus accumbens were collected on slides and incubated with rat monoclonal antibody to GFP (Nacalai USA, Elkton MD) and affinity-purified rabbit polyclonal antibody to the C-terminus of Kal7 [17, 18]. Anatomical boundaries were determined as in our previous studies using standard atlases [31, 32]. Confocal images were acquired on a Zeiss LSM 510 microscope as described [13, 21]. Neurons expressing GFP and/or Kal7 were identified using MetaMorph; three sections, each of which contained ~40 neurons, were analyzed from each animal for each brain region. Since the minimum distance between sections analyzed using the same antiserum was 84 μm, the same neuron was not counted twice.
Subcellular fractionation and Western blot analysis
Adult male mouse nucleus accumbens, dorsal striatum and cortical punches were sonicated and boiled in 1% SDS, 50 mM Tris [pH 7.4], 5 mM EDTA, 50 mM NaF, 2 mM sodium orthovanadate, 1 mM PMSF and protease inhibitor cocktail . For subcellular fractionation studies, whole striatum was homogenized, fractionated by differential centrifugation followed by sucrose gradient centrifugation and extraction with TX-100, and subjected to Western blot analysis. Where indicated, blots were quantified using Gene Tools (Syngene, Frederick, MD) [12, 17, 33]. Gels were loaded with equal protein using a Bicinchoninic Acid Assay and bovine serum albumin standard to determine protein concentrations (Pierce, Rockford, IL). Commercially available mouse monoclonal antibodies were used: βIII tubulin (TUJ1; Covance), NR2B (clone N59/20; NeuroMab), Cdk5 (Santa Cruz Biotechnology) and Rac1 (Transduction Labs; also 23A8, Millipore).
Kal7 is expressed in the major neuronal cell types in nucleus accumbens and dorsal striatum
Usage of promoters encoding full-length mouse Kalrn is region-specific
Usage of ΔKalrn promoter and alternate 3'-terminal exons is region-specific
Expression of transcripts encoding full-length vs. Δ-isoforms of Kalirin in mouse prefrontal cortex and nucleus accumbens was compared using Q-PCR with the indicated primers (Figure 4A); these regions were analyzed because both play essential roles in the response to cocaine and their patterns of promoter usage differed. Transcripts encoding the Δ-isoforms of Kalirin were more prevalent in prefrontal cortex than in nucleus accumbens (Figure 4B). Based on using primer pairs that spanned exons 10 and 11 (Figure 4A), transcripts encoding full-length isoforms of Kalirin were expressed at similar levels in these two brain regions.
Primers specific to the 3'-terminal exons unique to the different Kalrn isoforms (Figures 2, 4A) were used to look for differences between prefrontal cortex and nucleus accumbens (Figure 4C). Q-PCR analysis revealed much higher levels of transcripts encoding the Kal7-specific exon in prefrontal cortex than in nucleus accumbens. These transcripts could encode full-length Kal7 or ΔKal7. The Kal7-specific exon encodes a PDZ-binding domain known to interact with PSD-95 and spinophilin . Transcripts encoding Kal8, Kal9 and Kal12 were detected at much lower levels in both tissues, with no tissue-specific differences. The isoforms of Kalirin that include both GEF1 and GEF2 (Kal9, Kal12) account for a larger fraction of the Kalirin transcripts in the nucleus accumbens than in the prefrontal cortex.
Kal7 is the major Kalirin protein in the dorsal striatum and nucleus accumbens
To confirm the identity of the bands identified as Kal7 and ΔKal7, antiserum specific to the unique COOH-terminus of Kal7 was used (Figure 5, top right). Levels of both Kal7 and ΔKal7 were higher in cortex than in striatum or nucleus accumbens, with a clear signal for ΔKal7 detected only in cortex. As observed in the rat [26, 28], levels of ΔKal7 protein were significantly lower than predicted based on utilization of the ΔKalrn promoter (Figure 4B).
Kalirin function is altered when Thr1590 is phosphorylated by cyclin dependent kinase 5 (Cdk5), and levels of both Cdk5 and NR2B are decreased in PSDs prepared from Kal7KO animals [17, 30]. We therefore compared levels of NR2B and Cdk5 in these three brain regions (Figure 5); similar levels of both proteins were detected in all three regions. We also examined levels of Rac1, one of the major substrates of Kal7, in the same brain regions; again, no dramatic differences were observed (Figure 5).
Striatal PSDs are enriched in Kal7
Since Rac1 is one of the key substrates of Kal7, its distribution was also monitored (Figure 6). Although attached to the membrane by a geranylgeranyl tail, most of the Rac1 remained associated with the TX-100 washed PSD fraction. Very little Rac1 was removed by the TX-100 extraction step, leaving the TX-100 soluble fraction (TxS) depleted of Rac1.
Chronic cocaine treatment alters promoter and 3'-terminal exon expression
The effect of chronic cocaine on usage of different Kalrn 3'-terminal exons was next evaluated (Figure 8C). In the nucleus accumbens, the number of Kalirin transcripts generated using the Kal7-specific 3'-terminal exon increased following chronic treatment with cocaine. No significant change was seen in the level of transcripts encoding Kal8 or Kal12 and levels of Kal9 fell slightly (Figure 8C).
Effect of chronic cocaine on expression of Kalirin isoforms in striatal neurons
Many of the effects of cocaine involve the nucleus accumbens, or ventral striatum. Since preparation of purified PSDs from this relatively small region would require the use of multiple animals, we evaluated the effects of chronic cocaine on total lysates. Western blot analyses of nucleus accumbens extracts with the Kal-spectrin antibody revealed a 60% increase in Kal7 protein levels (Figure 9C); Kal9 levels were unchanged following cocaine treatment and Kal12 levels were too low for accurate quantification.
Discussion and Conclusions
Tissue-specific use of full-length Kalrn Promoters
The pattern of Kalrn promoter usage differs in NAc and prefrontal cortex; the C-promoter accounts for most of the full-length transcripts in the NAc while the B- and C-promoters contribute equally in the prefrontal cortex. The rat Kalrn A promoter was the first one identified and came from a cDNA library prepared from P21 rat hippocampus and cortex following electroconvulsive shock . Subsequent studies demonstrated that the Kalrn A promoter is a minor contributor in the adult rat, mouse and human tissues examined [27, 28]. The Kalrn D promoter is also a minor contributor in adult brain, but is the dominant Kalrn promoter in the pituitary (not shown). The Duet promoter, which produces transcripts that lack all of Kal7, is rarely used in the rat or human brain  and was not examined in this study. Interestingly, the intron sizes shown for mouse in Figure 1 are virtually identical to those reported for rat and human .
The N-terminal sequences encoded by the mouse and rat Kalrn B promoters are identical, as are the N-terminal sequences encoded by the mouse and rat Kalrn C promoters. Most of the full-length Kalirin isoforms in the NAc will begin with the more hydrophobic N-terminal sequence encoded by the Kalrn C promoter . The functional consequences of this difference have not yet been explored, but the proximity of this short sequence to the Sec14p domain, which is known to bind phosphatidylinositol-3-phosphate , suggests that future studies might identify an important role for the N-terminus of Kalirin.
Cocaine regulation of ΔKalrn promoter and Kal7 3'-terminal exon usage
Chronic cocaine treatment increased the prevalence of transcripts generated using the ΔKalrn promoter (Figure 8B). Neither the level of full-length Kalirin transcripts nor usage of the Kalrn B and C promoters (Figure 8A) was altered by this treatment. Chronic cocaine treatment is known to increase expression of several transcription factors, including CREB, ΔFosB, MEF2 and Sp1, in the NAc [45, 47, 49–51]. Our own analyses demonstrated an increase in transcripts encoding CREB, MEF2 and Sp1 in the NAc of mice treated with this particular regimen of cocaine .
Chronic treatment with cocaine also increased the level of transcripts that include the Kal7-specific 3'-terminal exon (Figure 8). Inclusion of this 3'-terminal exon results in the production of transcripts encoding Kal7 or ΔKal7 and precludes the production of Kal8, Kal9 or Kal12. Since chronic cocaine treatment had no effect on the level of full-length Kalirin transcripts (Figure 8B), an increase in the splicing events that include the Kal7-specific 3'-terminal exon would be associated with diminished usage of other 3'-terminal exons. While usage of the Kal8- and Kal12-specific exons was unaltered, usage of the Kal9-specific 3'-terminal exon declined slightly.
The effects of cocaine treatment on alternative splicing have received little attention. However, acute exposure to cocaine is known to alter splicing of transcripts encoded by the rat BDNF gene . BDNF, acting through its TrkB receptor, plays an important role in the behavioral response to cocaine and enhances responsiveness to glutamate [57–59]. Cocaine- and activity-regulated alternative splicing of the Homer family of scaffolding proteins also affects synaptic signaling [43, 60]. For Kalrn, splicing events that lead to the inclusion of different 3'-terminal exons produce proteins with distinctly different functions. Since the GEF2 region of Kalirin can be activated upon binding Gαq , leading to activation of RhoA, even a slight shift in the ratio of isoforms with both GEF domains vs GEF1 alone should be of significance. Cocaine-induced alternative splicing of Kalrn transcripts may be important in the biochemical and/or behavioral response to cocaine.
Kalirin expression in the striatum
The primary site of action of cocaine is the nucleus accumbens and the rest of the striatum [47, 62, 63]. Kal7 is expressed in almost all of the neurons in the dorsal striatum and nucleus accumbens that express the D1 receptor, but its expression is not limited to this population of neurons. Since medium spiny neurons expressing the D1 dopamine receptor and the D2 dopamine receptor receive different excitatory glutamatergic inputs and are part of very different functional pathways , examining the role of Kal7 in the effects of cocaine on both neuronal populations will be important. Whether expression of high levels of Kal7 in giant aspiny striatal cholinergic interneurons [36, 63] plays an important role in the actions of cocaine will require additional studies.
Based on Q-PCR analysis, expression of Kalirin in the NAc is lower than in hippocampus or prefrontal cortex. Consistent with a role for Kalirin in spine formation, dendritic spine density on NAc medium spiny neurons is lower than on hippocampal CA1 pyramidal neurons [12, 24]. In addition, PSDs purified from mouse striatum contained substantially lower levels of Kal7 than PSDs from cortex or hippocampus (Figure 7A). While the typical cortical or hippocampal PSD is estimated to have 9-12 molecules of Kal7, the typical striatal PSD would have only five.
Western blot analysis revealed an increase in levels of Kal7 in lysates of NAc and in purified striatal PSDs following chronic cocaine treatment (Figure 9). Many factors contribute to steady state protein levels, but the cocaine-stimulated increase in usage of the Kal7-specific 3'-terminal exon, coupled with maintained usage of the full-length Kalrn C promoter could account for the observed change. No cocaine-mediated alteration in Kal9 protein levels was observed (Figure 9B), despite a decrease in usage of the Kal9 specific 3'-terminal exon (Figure 8C). While ΔKal7 is detectable in mouse cortex, it is far less prevalent than predicted by promoter usage. The presence of four start codons followed by in-frame stop codons in the 5'-untranslated region of mouse ΔKal7 may limit successful translation , and increased turnover ΔKal7, a soluble, cytosolic protein, may contribute to this discrepancy.
A transient, cocaine-mediated change in levels of Kal7 vs. ΔKal7 would be expected to have functional significance. While expression of exogenous Kal7 increases spine density in cortical and hippocampal pyramidal neurons and even in interneurons [13, 21, 24], expression of exogenous ΔKal7 does not. Expression of ΔKal7 does increase spine head size  and the cocaine-mediated increase in ΔKalrn promoter usage may contribute to the increase in spine width known to occur following cocaine administration [12, 24]. Kal7 binding to its partners might be disrupted by ΔKal7, much as Homer-1a disrupts the clusters of proteins formed with the longer constitutive splice variants of the Homer family .
Initial studies on Kal7KO mice confirmed a role for Kal7/ΔKal7 in nervous system function [12, 17]. A slight decrease in linear spine density on CA1 hippocampal pyramidal neurons is accompanied by a decrease in spontaneous EPSP frequency and deficient hippocampal LTP . In contrast, spine density is normal in the Kal7KO NAc under basal conditions . Although chronic administration of cocaine increases spine density on the medium spiny neurons in the NAc [35, 42, 65], this response is absent in Kal7KO mice . The behavioral responses of Kal7KO mice to chronic cocaine are also altered . Levels of both Cdk5 and NR2B, which play essential roles in the structural and behavioral responses to cocaine [17, 30, 41, 66–69], are diminished in PSDs prepared from the cortices of Kal7KO mice. A similar relationship between Kal7 and Cdk5 and NR2B in the NAc may contribute to the behavioral deficits observed.
Although the Na+-dependent plasma membrane dopamine transporter is the immediate target of cocaine, the effects of chronic exposure to cocaine are complex, wide-spread and long-lasting. Since no effective treatment is available, it is essential to develop a better understanding of the molecular, cellular and system-wide changes that lead to addiction. Structural changes at many of the synapses involved in addiction are thought to underlie these long-lived changes. Since dendritic spine morphology is largely controlled by the actin cytoskeleton, attention has turned to the pathways through which cocaine could alter the cytoskeleton. Small GTP binding proteins of the Rho family play an important role in this process and are activated by RhoGEFs and inactivated by Rho GTPase Activating Proteins. There are ~60 RhoGEFs in the human genome, and about a dozen are found in significant amounts at the PSD . We have focused on one of these RhoGEFs, Kalirin, because it is essential for normal synaptic function and mice engineered to lack its major adult isoform exhibit altered responses to cocaine [12, 17].
Thanks go to Darlene D'Amato for her help with Western blots and Chris Mazzone for help with mouse husbandry. This work was supported by grant DA-15464 from the National Institute Drug Abuse, National Institutes of Health.
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