Recruitment of kappa-B motif-binding phosphoprotein (KBBP) to the promoter is required for GLT1 transcriptional activation

To evaluate how neurons regulate astroglial GLT1/EAAT2, we cloned a 2.5kb upstream promoter of human EAAT2 that shares high sequence homology (>70%) with GLT1 promoter from multiple species (Rothstein et al., 2005). To test whether this 2.5kb promoter behaves similarly as full-length (15kb) GLT1 genomic promoter in rodent astrocytes, a DsRed EAAT2 promoter reporter (2.5kb) (Figure 3A) was transfected into astrocyte cultures derived from BAC GLT1 eGFP transgenic mice (Regan et al., 2007). After transfection, newly prepared cortical neurons were plated on transfected astrocytes. As shown in Figure 3A, the presence of neurons markedly induced the expression of both DsRed and eGFP reporters in the same astrocyte, indicating that the 2.5kb promoter is functional in a rodent astrocyte environment and contains conserved cis-elements sufficient for neuron-derived activation signals.

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Figure 3

Recruitment of Kappa B-motif binding phosphoprotein (KBBP) to GLT1 promoter is required for its activation

A. A 2.5kb of human EAAT2 promoter is sufficient for neuronal stimulation in mouse astrocyte environment. The EAAT2 2.5kb promoter-DsRed reporter and GLT1 genomic promoter-eGFP reporter were both activated in astrocytes following co-culture with neurons identified by βIII-tubulin immunostaining. B. Identification of the regions essential for the basal activity of human EAAT2 promoter by serial deletion of human EAAT2 promoter (n=5–7). C. Promoter sequence from −958bp to −557bp is mainly responsible for neuron-dependent GLT1 promoter activation in primary astrocytes (n=4–8). □=astrocytes alone; ■=astrocytes and neurons. D. Mutagenesis of GGGTGGGTGT from −688bp to −679bp almost completely abolished basal and neuron-dependent GLT1 promoter activity in vitro (n=6–8). For luciferase assay, luciferase promoter reporters were first electroporated into cultured astrocytes and then freshly prepared neurons were plated on the top of transfected astrocytes. β-galactosidase was co-transfected for normalization. E. Mutagenesis of GGGTGGGTGT from −688bp to −679bp abolished GLT1 promoter activation (indicated by DsRed reporter expression) in astrocyte in vivo during early post-natal development. Astrocytes were identified by positive eGFP expression (20–30 astrocytes examined per serial section/mouse for total 4 mice each). F. Mutagenesis of cis-element GGGTGGGTGT abolished its specific binding to nuclear factors during GLT1 promoter activation in early post-natal development. G. Affinity purification by using wild type oligo GGGTGGGTGT but not the mutant oligo found unique nuclear protein from P21 mice cortex nuclear extracts. H. Trypsin digestion and LC/MS/MS identified Kappa B-motif binding phosphoprotein (KBBP) that specifically binds to GGGTGGGTGT. The red-colored amino acid sequences are small peptide sequences identified from LC/MS/MS that also match with KBBP sequence in NCBI.

To identify the major promoter regions responsible for neuron-dependent activation, luciferase promoter reporters were generated by serial deletion of the 2.5kb sequence and electroporated into P2 mouse primary astrocyte cultures. Sequence deletion from −921 to −279 (pGL958 vs. pGL316 in Figure 3B) sharply reduced both basal and neuron-dependent promoter activity (Figure 3B, 3C), with the most significant drop of activity from pGL958 to pGL557, indicating that the sequence between −921 to −520 contains primary elements for neuron-dependent GLT1 promoter activation. Multiple highly conserved transcription factor binding sites are present in the region from −921 to −520 by sequence analysis. Site-directed mutagenesis of pGL958 generated multiple mutant luciferase promoter reporters, which were examined in astrocyte cultures (Figure 3D). The 958bp-mutant 2 exhibited the greatest reduction in luciferase activity without altering co-transfected β-galactosidase activity, suggesting that the sequence mutated from −688 to −679 GGGTGGGTGT is essential for GLT1 promoter activity. Sequence alignment across ten species characterized GGGTGGGTGT as an evolutionally conserved site among mammals (supplementary Figure 3A). Notably, mutations of putative NF- B binding sites within this promoter region had no dramatic influence on neuron stimulated promoter activation (not shown).

The requirement of nucleotides −688 to −679 for GLT1 promoter activity was further tested in vivo. GLT1 mRNA transcription is strongly induced during early postnatal development in rodent brain (Schmitt et al., 1996; Sutherland et al., 1996), providing an in vivo model to test the GLT1 promoter activation. A DsRed reporter driven by the wild type 958bp or 958bp-mutant 2 EAAT2 promoters was delivered to brain of young mice using an adeno-associated virus (AAV) vector to examine in vivo reporter activation. AAV particles carrying the EAAT2 promoter-DsRed expression cassette were injected into the lateral cerebral ventricle of P0 pups of BAC GLT1 eGFP transgenic mice (Broekman et al., 2006). As shown in Figure 3E, confocal analysis of coronal brain sections of mice, 3 weeks after injection with 958bp-WT-DsRed containing AAV revealed expression of the DsRed reporter in astrocytes that also express eGFP reporter. However, no expression of DsRed was found from brain sections of mice injected with 958bp-mutant 2-DsRed containing AAV. Expression of the DsRed was also seen in nearby neurons for both AAV viruses (data not shown), suggesting that both viruses are effective in driving the DsRed reporter expression in vivo. The inability of 958bp-mutant 2 promoter to drive DsRed reporter expression in astrocytes in vivo confirmed the in vitro results that the sequence from −688 to −679, GGGTGGGTGT, is essential for neuron-dependant EAAT2 promoter activity.

The nuclear factors recruited to the GGGTGGGTGT sequence, were identified by performing a gel shift assay with nuclear extracts prepared from mouse cortex at developmental time points prior to GLT1 promoter activation (P2) and after strong promoter activation (P21). Wild type and mutant oligos (45mer) that only differ in the GGGTGGGTGT were synthesized and labeled with biotin. As shown in Figure 3F, increased specific binding with labeled WT oligo was found from P2 to P21 mice when the GLT1 promoter was strongly activated. The addition of extra-unlabeled WT oligo completely abolished the binding, suggesting that the binding is specific to this oligo. On the other hand, no specific binding with labeled mutant (MT) oligo was found, indicating the change of core sequence from GGGTGGGTGT to AAATGCCACT abolished the recruitment of specific nuclear proteins to this cis-element during GLT1 promoter activation. In addition, no other binding with labeled MT oligo was observed, suggesting that the replacement of GGGTGGGTGT with AAATGCCACT does not introduce non-specific binding with other nuclear proteins. This direct biochemical evidence of gel shift result, along with the genetic analysis in Figure 3D and 3E, demonstrates that the recruitment of nuclear factors to GGGTGGGTGT is essential for neuron-stimulated GLT1 promoter activity.

To identify the actual transcription factor binding to GLT1 regulatory site GGGTGGGTGT, nuclear extracts were affinity purified with the same oligos used in gel shift analysis. After purification, proteins bound to the WT or MT oligos were resolved on 4–12% PAGE gel and visualized by silver staining. A specific band with molecular weight between 50 to 75KDa was only found with labeled WT oligo but not with MT oligo (Figure 3G), confirming the results from gel shift analysis. Subsequent trypsin digestion and LC/MS/MS analysis of digested peptides (Figure 3H) revealed a nuclear protein, kappa-B motif-binding phosphoprotein (KBBP, gi|1083569), specifically binds to WT oligo.

KBBP was first identified in a T lymphoma cell line that binds to kappa B enhancer element (GGGGACTTTCC)(Ostrowski et al., 1994). It is almost 100% homologous with human heterogeneous nuclear ribonucleoprotein K (hnRNP K) protein (Ostrowski et al., 1994). HnRNP K protein plays diverse roles in transcription, RNA splicing, and signal transduction through its interaction with many protein partners, including protein kinases, transcriptional factors and DNA/RNA(Bomsztyk et al., 2004). Notably, although the original kappa B enhancer element GGGGACTTTCC share partial homology with the KBBP binding sequence GGGTGGGTGT on GLT1 promoter, it is sufficient to compete out GGGTGGGTGT induced specific binding with KBBP in a dose dependent manner (supplementary Figure 3B). In addition, further mutation of GGG to AAA on KBBP binding sequence GGGTGGGTGT also abolished GLT1 promoter activity (supplementary Figure 3C), suggesting that these nucleotides are critical for KBBP binding and GLT1 promoter activity. As the mouse homolog of human hnRNP K, KBBP protein may also participate in transcription or RNA splicing. Expression of KBBP in astrocytes in vivo was first examined by using an antibody against human hnRNP K. Cortical immunoreactivity for KBBP in both P2 and P21 BAC GLT1 eGFP mice shows neuronal and astroglial immunolocalization. Notably, KBBP expression was barely detectable in P2 astrocytes and dramatically increased in astroglia from P2 to P21, which is identical to the very large increase in astroglial GLT1 promoter activity (indicated by eGFP reporter intensity) in the same astroglia (Figure 4A). The significant correlation between increased expression of astroglial KBBP and GLT1 promoter/protein (Figure 4B) during early postnatal development suggests that KBBP may play an important role in transcriptional regulation of GLT1. Interestingly, KBBP expression in cultured astrocytes was also induced by neurons, coupled to the increase of GLT1 (Figure 4C); the coupled increases of KBBP and GLT1 are consistent with the same changes in astrocytes observed in vivo.

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Figure 4

KBBP is essential and sufficient for neuron-dependent transactivation of GLT1

A. Expression of KBBP protein in astroglia in vivo. KBBP protein expression was minimal in P2 astroglia (BAC GLT1 eGFP reporter, white arrows) and was markedly induced in cortical astrocytes during early post-natal development, along with astroglial BAC-GLT1 eGFP expression (white arrows). Scale bar, 10μm B. Quantitative correlation of KBBP expression levels and GLT1 promoter activity in astroglia during early postnatal development. Positive linear relationship of KBBP expression levels and eGFP intensity was found. The KBBP immunoreactivity and eGFP fluorescence intensity was quantified in Image J (20 astrocytes examined per serial section/mouse for total 4 mice) C. KBBP expression in astrocytes is induced by the presence of neuron. Neurons and astrocytes were co-cultured for one week before sample collected. Positive control: mice brain lysate. D. Development of shRNA that specifically silences KBBP expression. shRNA against KBBP results in over 90% less of KBBP expression in 3T3 NIH cells. E. Silencing of KBBP expression by shRNA reduced GLT1 expression in astrocyte on neuron-astrocyte co-culture MCP. shRNA expressing astrocytes were identified by co-expressed eGFP from shRNA construct. GLT1 expression and neuronal processes were visualized by GLT1 or βIII-tubulin immunostaining, respectively. Scale bar, 20μm. F. AAV mediated expression of KBBP antisense in vivo reduced GLT1 promoter activity during early post-natal development. AAV8-GFAP-KBBP-A/S and AAV8-GFAP-DsRed particles were co-injected into cerebral lateral ventricle of P0 pups of BAC GLT1 eGFP transgenic mice. AAV8-GFAP-luciferase-A/S serves as antisense control for AAV8-GFAP-KBBP-A/S. Antisense expressing astrocytes were identified by DsRed reporter expression (20–30 astrocytes examined per serial section/mouse for total 4 mice each). Scale bar, 50μm (one-way ANOVA with Bonferroni posthoc analysis, **P<0.01, *** P<0.001; mean ± SEM) G. Overexpression of KBBP in astrocytes increases GLT1 expression with the presence of neurons. L: luciferase overexpression; K: KBBP overexpression. Neurons were plated on transfected astrocyte cultures 2d post transfection and kept for one week.

Small interference RNA (siRNA) specifically against KBBP was developed that effectively reduced KBBP expression level to 10% of that in control (Figure 4D) (Moumen et al., 2005), which results in significantly reduced GLT1 expression in astrocytes on neuron-astrocyte co-culture MCP (Figure 4E). We further tested whether a decrease of KBBP expression in astrocytes also reduces GLT1 promoter activity in vivo. To selectively silence astroglial KBBP, AAV particles that carried an expression cassette with the KBBP gene in the antisense orientation driven by the GFAP promoter, along with AAV particles that carried a DsRed reporter expression cassette driven by the same promoter, were intraventricularly injected into new born (P0) BAC GLT1 eGFP transgenic mice. AAV particles carrying luciferase in the antisense orientation served as a control. KBBP antisense was highly effective at silencing KBBP expression in cultured astrocytes (Supplementary Figure 4C). As shown in Figure 4F, KBBP antisense, but not luciferase antisense, effectively silenced KBBP expression in vivo. GLT1 promoter activity in KBBP antisense expressing astrocytes, identified by DsRed, was significantly reduced as indicated by reduced eGFP intensity (Figure 4F-I&II, shown by yellow arrow) compared to that in untransduced astrocytes (shown by white arrow) while GLT1 promoter activity in luciferase expressing astrocytes was unaltered (Figure 4F-III&IV, shown in yellow arrow). In aggregate, the loss-of-function analysis of KBBP by RNAi and antisense in astrocytes, along with the promoter mutagenesis analysis, indicate that recruitment of KBBP to the GLT1 promoter is required for GLT1 transcriptional activation in vivo.

We next determined if over-expression of KBBP could concomitantly alter GLT1 expression in astrocyte cultures. KBBP and luciferase overexpression constructs were prepared and electroporated into cultured astrocytes. As shown in Figure 4G, overexpression of KBBP, but not luciferase, was sufficient to increase GLT1 expression in the presence of neurons, though overexpression of KBBP itself was not sufficient to induce the GLT1 expression. In addition, direct treatment of cultured astrocytes with DHPG (50μM), the selective agonist for mGluR 1/5, increases both GLT1 and KBBP mRNA levels (supplementary Figure 4A, 4B), indicating that mGluR 1/5 may play more dominant role in neuron-depednent GLT1 up-regulation. Although direct treatment of physiological GluR agonist glutamate failed to increase the GLT1 expression, this could be due to the quick uptake of extracellular glutamate by GLAST in cultured astorcytes. These results are consistent with our observations that astroglial KBBP and GLT1 are expressed in parallel in vivo during early post-natal and synaptic maturation. This hints that the maturation of synapse might influence astroglial gene activation and expression of GLT1. Conversely, an alteration of this pathway due to the loss of pre-synaptic influence of neurons on astrocytes could be an important event linking neural injury to astroglial synaptic function.

In vivo neuron/axon denervation leads to GLT1 transcriptional dysregulation through reduction of KBBP expression

In order to evaluate the in vivo effect of altered synaptic coupling of neurons to astroglia, we investigated the alterations of astroglial GLT1 gene activation and regulation by KBBP in response to synaptic disruption by using several in vivo models including acute axon denervation induced by corticospinal tract lesion, ricin-induced acute motor neuron degeneration and the chronic neurodegeneration ALS mouse model employing SOD1 G93A mice.

Corticospinal Tract Transection

Corticospinal tracts originating from glutamatergic upper motor neurons possess excitatory presynaptic terminals on spinal cord interneurons and lower motor neurons that are modulated by perisynaptic astroglial GluTs. This pathway allows direct in vivo assessment of astroglial transporter expression in response to the loss of axonal signals induced by spinal cord transection. Thoracic cord (segment 9) transection was performed three weeks after mice received bilateral injection of Fluororuby (FR) into motor cortex to label corticospinal tracts by anterograde FR transport. One week after transection, thin horizontal sections of spinal cord were prepared and FR signals were examined to assess axon degeneration. Above the surgical lesion, abundant FR labeled axons, typical for corticospinal tract innervation, were found bilaterally in spinal cord white matter, projecting to grey matter neurons at each segment (Figure 5A–I). FR labeled neurons were also observed in grey matter (Figure 5A–II). In contrast, FR signals (including labeled axons and grey matter neurons) were reduced in the cord well below the lesion site in lumbar spinal cord (Figure 5A–III). Quantitative analysis revealed that more than 70% of FR labeled corticospinal axons in lumbar cord were depleted as early as 7 days following spinal transection (Figure 5A). GLT1 protein expression in lumbar cord of lesioned mice was markedly reduced compared to that of control mice (Figure 5B). A pronounced loss of GLT1 immunostaining was also observed in coronal sections of lesioned lumbar cords from BAC GLT1 eGFP mice. GLT1 promoter activity, as reflected by eGFP fluorescence intensity in coronal sections of lumbar cord of lesioned BAC GLT1 eGFP mice, was focally reduced (Figure 5C, 5D) in single astrocytes. These sharp astroglial changes were localized to regions with diminished synaptic terminals, as identified by the loss of pre-synaptic terminal marker vesicle glutamate transporter 1 (VGluT1), VGluT2 (supplementary Figure 5A) and preservation of post-synaptic marker PSD95.

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Figure 5

Corticospinal tract presynaptic degeneration induced by spinal cord transection results in loss of GLT1 protein and promoter activity

A. Thoracic transection induces massive degeneration of descending axons. Descending corticospinal tracts were shown by anterograde transport of FR from motor cortex. Abundant FR labelled axons/neurons (black arrow) were observed in white/grey matter of spinal cord above the lesion site (I, Scale bar, 0.5mm). Quantitative analysis of FR-labelled axon length showed that 70% of axons degenerated in lesioned mice compared to sham control mice (n=5). Representative images with FR labeled axons/neurons are shown in II&III, scale bar, 20μm. B. Thoracic spinal cord transection leads to loss of GLT1 protein in lumbar spinal cord by both immunoblotting (n=3) and immunostaining. Scale bar, 20μm. C. Single astrocyte analysis in vivo revealed loss of GLT1 promoter activity in astrocytes surrounding degenerated axons/pre-synaptic terminals of lesioned BAC GLT1 eGFP mice. Pre-synaptic complexes were identified by double staining for pre-synaptic VGluT1 and post-synaptic PSD95. Scale bar, 20μm. D. Quantitative analysis of the eGFP fluorescence intensity in lumbar cord astrocytes of lesioned mice (n≥93). E. Axon degeneration decreases KBBP immunostaining intensity in lumbar cord astrocytes of lesioned BAC GLT1 eGFP mice. Scale bar, 20μm. F. Quantitative analysis of the KBBP immunostaining intensity in lumbar cord astrocytes of lesioned mice (n≥45) (***P<0.001, mean ± SEM).

Overall, the loss of KBBP in individual astroglia (Figure 5E, yellow arrow) correlated with a decrease in GLT1 promoter-reporter eGFP intensity in same cells in lesioned lumbar cord (Figure 5E), along with loss of GLT1 protein (Figure 5B). The astroglial changes all followed the loss of presynaptic terminals. These in vivo studies demonstrate that presynaptic terminals act to maintain astroglial KBBP expression, which in turn activates GLT1 expression. Furthermore, these data provide a pathway by which abnormal axon/pre-synaptic signaling can alter astroglial synaptic integrity.

Neurotoxin Lesion

Neurodegeneration could alter the physiologic axon/astrocyte communication required for astroglial synaptic maintenance. The neurotoxin ricin, an extremely potent protein synthesis inhibitor, has been well established as a model for selectively lesioning discrete populations of motor neurons, through retrograde transport. In order to examine the response of individual astroglia in vivo, to the focal loss of spinal motor neurons, and their recurrent axon collaterals, we injected selected peripheral nerves with the toxin. Femoral nerves (originated from lumbar cord 2–4) of BAC GLT1 eGFP mice were dipped into fluororuby (FR) solution or mixture of ricin and FR solution unilaterally so that the change of GLT1 promoter activity in response to motor neuron death could be directly assessed at the same segment. As shown in Figure 6A, motor neurons were clearly labeled with FR in the control side of L2-L4 cord sections but not in the ricin injected side due to degeneration of motor neurons one week following injection (magnified view in Figure 6C). Mice received unilateral ricin injection also developed clear paralysis only in ricin side (Supplementary video), suggesting that retrograde ricin indeed induced dramatic motor neuron degeneration. The focal, selective loss of motor neurons and their recurrent collaterals was apparent on surrounding astroglia- eGFP fluorescence intensity was 30% reduced in ricin lesion side compared to that of control side (Figure 6B, 6C, 6D). This reduction of eGFP intensity was not due to astroglial death, as abundant GFAP immunoreactivity was still found in the astroglia with reduced eGFP on the lesioned side of the spinal cord (Figure 6C). Quantitative single astrocyte analysis of confocal images of thin section of L2-L4 cord from BAC GLT1 eGFP mice administrated with unilateral ricin also found 50% reduced KBBP immunoreactivity in astroglia with reduced eGFP intensity (Figure 6E, 6F). Ricin-induced motor neuron degeneration abolished its signaling to surrounding astroglia, likely mediated by both motor neuron dendrites and axon collaterals, which suggests that neuronal inputs are necessary to maintain KBBP expression levels and subsequent transactivation of the GLT1 promoter.

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Figure 6

Motor neuron degeneration induced by neurotoxin ricin leads to reduced GLT1 promoter activity and KBBP expression

A. Visualization of motor neurons in ventral horn of lumbar spinal cord by fluoro-ruby (FR). Femoral nerves were exposed and dipped into FR solution (10%) to allow retrograde transport of FR into motor neurons. Scale bar, 50μm (insert scale bar, 100μm). B. Unilateral administration of ricin induced reduction of eGFP intensity. C. Thin coronal sections of L2-L4 cord of BAC GLT1 eGFP mice was prepared and immunostained with GFAP antibody one week following unilateral administration of ricin. Reduced eGFP intensity but not the GFAP immunoreactivity was found in ricin side. Scale bar, 100μm (50μm for magnified image). D. Fluorescence intensity of eGFP in individual astroglial from ricin and control sides were measured in Image J and plotted using Prism (20–25 astrocytes per serial section/mouse for total 10 mice) (*, P<0.05, mean ± SEM, Student’s t-test). E. Single astrocyte analysis in vivo revealed reduction of KBBP immunoreactivity in astroglia with reduced eGFP intensity. Thin coronal sections of L2-L4 cord of BAC GLT1 eGFP mice was prepared and immunostained with KBBP antibody one week following unilateral administration of ricin. Motor neurons were identified by FR in control side. F. KBBP immunoreactivity in astroglia with reduced eGFP intensity was measured in Image J and plotted in Prism (20–25 astrocytes per serial section/mouse for total 10 mice). Scale bar, 20μm. Yellow arrow: astroglia in control side; White arrow: astroglia in ricin side. (Student’s t-test: ***, P<0.001, mean ± SEM).

Supported by NIH: NS33958 (JDR), NS36465 (MBR, JDR); the Robert Packard Center for ALS Research; Muscular Dystrophy Association (YY).