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TLE1 in cortical and cerebellar neurons
Molecular Brain Research 140 (2005) 106 – 110 www.elsevier.com/locate/molbrainres
Short Communication
Dendritic localization of the transcriptional co-repressor Groucho/TLE1 in cortical and cerebellar neurons Lee Stewart, Stefano Stifani* Center for Neuronal Survival, Montreal Neurological Institute, Montreal, Quebec, Canada H3A 2B4 Accepted 27 June 2005 Available online 2 August 2005
Abstract In the present study we show that the transcription factor Groucho/TLE1 (TLE1) is expressed in virtually all major cortical subdivisions, hippocampus, amygdala, and thalamus, as well as in the cerebellum of the adult rat brain. In both neocortex and subcortical structures, TLE1 expression was mostly localized to neurons. In addition to the expected nuclear localization, TLE1 immunoreactivity was also detected in apical dendritic shafts of neocortical layer III and V pyramidal cells and in Purkinje cell dendrites. These results demonstrate that TLE1 expression occurs in the mature nervous system and suggest that this protein may perform new functions outside of the nucleus in selected cortical and cerebellar neurons. D 2005 Elsevier B.V. All rights reserved. Theme: Development and regeneration Topic: Cerebral cortex and limbic system Keywords: Cerebellum; Dendrite; Groucho; Hippocampus; Neocortex; Transducin-like enhancer of split 1
The Drosophila protein Groucho and its mammalian homologues, referred to as Transducin-like enhancer of split (TLE) 1 to 4 [16], are general transcriptional co-repressors that lack intrinsic DNA-binding ability but can be recruited to different genes through interactions with DNA-binding proteins [3,10]. Groucho/TLE proteins (TLEs) are expressed in a variety of tissues, play important roles in numerous developmental pathways, including neurogenesis [20], and are correlated with adult forms of disease [15]. To date, the majority of studies on TLE proteins have focused on their roles in cell fate determination during embryogenesis and few have examined their expression in the mature central nervous system (CNS). During mouse cortical development, it was shown that TLE1 is expressed in both neural progenitor cells and post-mitotic neurons in the outer layers of the cortical plate, whereas TLE4 is
* Corresponding author. Fax: +1 514 398 1319. E-mail address: [email protected] (S. Stifani). 0169-328X/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.molbrainres.2005.06.016
expressed by post-mitotic neurons of the inner layers [19]. Consistent with these observations, Petersen and coworkers [13] have recently shown that TLE4 is expressed in layer VI, but also in more superficial layers (II, III) of the adult mouse cortex. It has also been reported that TLE3 transcripts are expressed in the adult rat hippocampus [6]. In contrast, less is known about the expression pattern and possible functional role of TLE1 in the adult brain. In the present study, our primary objective was to characterize the expression of TLE1 in cortical and subcortical regions of the adult rat brain. To that end, we performed immunohistochemical studies using a panel of different antibodies previously shown to recognize TLE1 [7,10,11,19]. Adult male Sprague –Dawley rats were anesthetized with sodium pentobarbital and transcardially perfused with 4% paraformaldehyde. The brains were removed, cryoprotected in 30% sucrose, and sectioned on a cryostat. All animal procedures followed the guidelines of the Canadian Council for Animal Care.
L. Stewart, S. Stifani / Molecular Brain Research 140 (2005) 106 – 110
Three previously characterized antibodies were used for immunohistochemical studies: a rabbit polyclonal against the SP domain of TLE1 (Fanti-TLE1_) [10,19,20], a rabbit polyclonal against TLE1 phosphorylated at serine239 within the CcN domain (Fanti-(pS239)TLE1_) [10], and a rat monoclonal against the highly conserved TLE WD40 repeat domain (this antibody recognizes all four members of the TLE family and has been termed FpanTLE_) [7,11,16]. For immunohistochemistry, tissue sections were first incubated in 0.2% H2O2 in phosphate-buffered saline containing 0.2% Triton X-100 (PB-Tx) for 20 min, then in 10% normal goat serum (NGS) in PB-Tx for 2 h at room temperature. Sections were incubated with anti-TLE1 (1:500 in 1% NGS), anti-p(S239)TLE1 (1:500), or panTLE (1:200) antibodies overnight at 4 -C. Double labeling was performed using antibodies against MAP2 (monoclonal, 1:100; Sigma), glutamate transporter EAAC-1 (monoclonal, 1:100; Chemicon), SMI-32 (monoclonal, 1:500; Sternberger Monoclonals), NeuN (monoclonal, 1:100; Chemicon), and GFAP (monoclonal, 1:200; Sigma). After several washes in PB-Tx, sections were incubated in the appropriate fluorescein isothiocyanate (FITC)-conjugated or carboxymethylindocyanin-3 (Cy3)-conjugated secondary antibodies (1:200 in 1% NGS; Jackson ImmunoResearch). The 31 (FStandard_) Series of filters (Chroma Technology Corp.) was utilized for fluorescence microscopy. For control experiments, the primary antibodies were omitted (data not shown). Immunohistochemistry was performed on coronal brain sections at the levels of (in the rostrocaudal axis) primary motor cortex (bregma +1.70 mm), septum (+0.20 mm), primary somatosensory cortex and dorsal hippocampus ( 3.30 mm), primary visual cortex and optic tectum ( 5.80 mm), and entorhinal cortex ( 7.30 mm) according to the atlas of Paxinos and Watson [12]. The structures examined at each anatomical level are listed in Table 1. All images were captured with a DVC black and white camera mounted on a Zeiss Axioskop 2 fluorescence microscope. Grayscale images were digitally assigned to the appropriate red (Cy3) or green (FITC) channels using Northern Eclipse software (Empix). Using a previously characterized anti-TLE1 antibody [10,19,20], nuclear TLE1 expression was detected within most of the adult brain regions examined and was primarily co-localized with the neuronal marker NeuN, indicating that the majority of TLE1+ cells were neurons (Table 1; Fig. 1B, panel vi; and data not shown). TLE1 expression appeared to be mostly confined to forebrain structures, such as the hippocampus, cortex, thalamus, and amygdala (Figs. 1A and D, Table 1). TLE1 expression was observed in caudal regions of neocortex (i.e., visual and retrosplenial cortices) but was absent in subcortical structures caudal to the thalamus (i.e., substantia nigra, tectum, pons). Nuclear TLE1 expression was also detected in the cerebellum where it was primarily restricted to the Purkinje cell layer (Fig. 1C, panel i) with few labeled cells in the granule cell layer.
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Table 1 Distribution of TLE1 expression in the adult rat brain Bregma (mm)
Area
TLE1-ir
+1.70
Primary motor cortex (M1) Piriform cortex Cingulate cortex Caudate nucleus Claustrum Nucleus accumbens Medial septum Lateral septum Ventral pallidum Primary somatosensory cortex (S1) Primary auditory cortex CA1 CA2 CA3 CA4 (hilus) Dentate gyrus Ventral posterior thalamus Lateral dorsal thalamus Mediodorsal thalamus Basolateral amygdala Primary visual cortex (V1) Retrosplenial cortex Periaqueductal grey Substantia nigra reticulata Superior colliculus (deep grey layer) Subiculum Medial geniculate Medial entorhinal cortex Lateral entorhinal cortex Pedunculopontine tegmentum Inferior colliculus
+ + +
+0.20
3.30
5.80
7.30
Abbreviation: ir, immunoreactivity. +/ ( ) of nuclear TLE1 expression.
+
+ + + + + + + + + + + + +
+ +
indicates presence (+) or absence
Unexpectedly, anti-TLE1 immunoreactivity was detected in the apical dendrites of a large number of layer V, and to a lesser extent layer III, pyramidal cells in nearly all major subdivisions of the neocortex (Figs. 1A and B, panel i). TLE1 expression appeared to be prominent among type 1 pyramidal neurons, as indicated by co-expression with both glutamate transporter EAAC-1, a general pyramidal cell marker protein, and non-phosphorylated neurofilament proteins recognized by the SMI-32 antibody [18] (Fig. 1B, panels iv and v). To confirm the specificity of this dendritic immunoreactivity, similar studies were performed with a previously characterized anti-(pS239)TLE1 antibody that recognizes TLE1 phosphorylated at serine239 [10]. In layer V of visual cortex, anti-TLE1 and anti-(pS239)TLE1 immunoreactivities were both present within groups of apical dendrites resembling dendritic bundles [17] (Fig. 1B, panels i and ii). These dendritic bundles extended dorsally to superficial layers forming ‘‘tufts’’ near the cortical surface. Importantly, we observed an overlapping dendritic staining with a panTLE monoclonal antibody that recognizes all four mammalian TLE family members [7,10,11,16] (Fig. 1B, panel iii), further indicating the specificity of the observed immunoreactivity. At the cortical level shown in Fig. 1B (panel iii), nuclear and dendritic TLE1 expression was absent in superficial cortical
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L. Stewart, S. Stifani / Molecular Brain Research 140 (2005) 106 – 110
Fig. 1. Nuclear and dendritic TLE1 expression in adult rat brain. (A) Immunohistochemical detection of TLE1 expression in motor cortex of the adult rat using the anti-TLE1 antibody. Note the presence of TLE1-immunoreactive soma and dendrites in layers V and II/III. (B) In visual cortex, TLE1 expression was detected in apical dendritic bundles formed by layer V pyramidal cells (panel i) as indicated by EAAC-1 and SMI-32 co-expression (panels iv and v). Unlike motor cortex, however, nuclear TLE1 expression was largely absent in shallow visual cortex. Nuclear TLE1 expression in the layer V pyramidal cells was confirmed by co-expression with the neuronal marker NeuN (panel vi). A similar dendritic immunoreactivity was also observed in layer V apical dendrites with the anti-(pS239)TLE1 antibody (panel ii). Double-labeling studies with rabbit anti-TLE1 and rat panTLE antibodies revealed overlapping layer V dendritic staining in visual cortex (panel iii); additional panTLE-immunoreactive structures present in superficial layers likely represent different TLE isoforms. (C) In the cerebellum, TLE1 showed robust co-expression with MAP2 in Purkinje cell dendrites, with a few TLE1+ nuclei scattered in the subjacent granule cell layer (panels i – iii). (D) In the hippocampus, nuclear TLE1 expression was seen throughout the pyramidal cell layer including the hilus (data not shown) as well as in the outer blade of the dentate gyrus, which was characterized by a ‘‘patch’’ of TLE1+ granule cells. The representative anatomical levels for each of the above labeling studies are shown in inset: (a) motor cortex; (b) visual cortex; (c) cerebellum; and (d) hippocampus and dentate gyrus [12]. Abbreviations: M1, primary motor cortex; I – VI, cortical layers; V1, primary visual cortex; PC, Purkinje cell layer; GCL, granule cell layer; PFl, paraflocculus; sm, stratum moleculare; DG, dentate gyrus.
layers as compared with more rostral regions such as motor cortex (Fig. 1A). Thus, the panTLE immunoreactivity detected in shallow visual cortex, in addition to layer V apical dendrites, likely represents the expression of different TLE isoforms (Fig. 1B, panel iii). Nuclear localization of TLE1 in layer V pyramidal cells was confirmed by coexpression with NeuN (Fig. 1B, panel vi). In the cerebellum,
TLE1 was co-expressed with MAP2 in Purkinje cell dendrites (Fig. 1C, panels i –iii). Despite its presence in the nuclei of granule cells in the dentate gyrus, TLE1 expression did not exhibit any overlap with MAP2 in the stratum moleculare (Fig. 1D, panels i –iii). The main findings of this study are that (1) nuclear TLE1 expression occurs throughout the adult rat forebrain,
L. Stewart, S. Stifani / Molecular Brain Research 140 (2005) 106 – 110
primarily in neurons; (2) TLE1 immunoreactivity is detected in the apical dendrites of layer III and V pyramidal neurons in selected cortical regions; and (3) TLE1 expression is equally abundant in the nuclei and dendrites of cerebellar Purkinje cells. To the best of our knowledge, this study is the first to characterize the expression of TLE1 in the adult rat brain. Furthermore, these findings also provide the first evidence that TLE1, a transcriptional regulator, may have a functional role outside of the nucleus. Similar to TLE3 and TLE4, the expression of TLE1 in the adult rat brain suggests a role for this protein in the mature nervous system. The observation that nuclear TLE1 expression is widespread throughout the forebrain is consistent with its role in the regulation of gene transcription [3,7,10]. However, given its robust expression in dendrites it is likely that TLE1 also has functional roles distinct from that as a transcriptional regulator. Dendritic TLE1 expression may be a salient feature of neurons involved with complex brain functions, such as visual discrimination in primary visual cortex or motor learning in the cerebellum. For example, TLE1+ dendritic bundles may correspond to cortical architecture referred to as output columns [17], which have been implicated in higher cortical information processing and neurological disease [9]. Feldman and colleagues [6] characterized TLE3 gene expression in the hippocampus following epileptic seizures and suggested that seizure-induced increases in TLE3 expression may relate to the functional plasticity of neurons. Indeed, sustained excitatory stimulation can up-regulate the expression of multiple genes necessary for enhancing synaptic efficacy [1]. But before changes in gene expression that coincide with neuronal plasticity can occur, the transport of proteins from dendrites to the nucleus is often required to regulate gene activity in response to physiological stimuli. Among those proteins, transcription factors are likely to play a pivotal role in regulatory pathways between distal regions of the cell and the nucleus [2,5]. For a transcription factor potentially involved in a dendritic signaling pathway one would expect to observe a punctate expression pattern [4]. By contrast, the distribution of TLE1 immunoreactivity observed in the present study appears uniform throughout the dendritic tree. Thus, it is plausible that TLE1 could play an alternative role in the maintenance of dendritic structure and morphology, perhaps through interactions with cytoskeletal proteins such as h-actin [8]. The C-terminal region of TLEs contains a well-defined structure, the WD40 repeat domain, which contains seven repeats of ¨40 amino acids, each harboring the sequence tryptophan – aspartate [14]. This structure contributes to the interaction of TLE proteins with numerous transcription factors. It is possible that the presence of a WD40 domain confers upon TLEs an even larger capacity for protein – protein interactions. This may enable TLE1 to perform functions outside of the nucleus by binding to proteins that are not typically associated with transcriptional regulatory pathways. Further studies are necessary to discern the exact
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functional role of TLE1 in neocortical dendrites and elsewhere in the mature CNS. In summary, the present results show that TLE1 exhibits a unique nuclear and dendritic expression pattern in the adult rat brain and is primarily restricted to forebrain structures, with the exception of the cerebellum. Most studies of TLE proteins have focused on their roles in cell fate determination during embryogenesis. We propose that TLE1 may have a novel non-nuclear function in the adult brain, in addition to its role as a transcriptional co-repressor in both the mature and developing CNS.
Acknowledgments The authors thank Dr. B. Jones for the gift of the SMI-32 antibody. This work was supported by grants from the Canadian Institutes of Health Research (MOP-13957 and MGC-14971) to S.S. L.S. is the recipient of a J. Timmins Costello Postdoctoral Fellowship, and S.S. is a Senior Scholar of the Fonds de la Recherche en Sante du Quebec.
References [1] A.J. Becker, J. Chen, A. Zien, D. Sochivko, S. Normann, J. Schramm, C.E. Elger, O.D. Wiestler, I. Blumcke, Correlated stageand subfield-associated hippocampal gene expression patterns in experimental and human temporal lobe epilepsy, Eur. J. Neurosci. 18 (10) (2003) 2792 – 2802. [2] A.H. Brivanlou, J.E. Darnell, Signal transduction and the control of gene expression, Science 295 (2002) 813 – 818. [3] G. Chen, A.J. Courey, Groucho/TLE family proteins and transcriptional repression, Gene 249 (2000) 1 – 16. [4] P. Crino, K. Khodakhah, K. Becker, S. Ginsberg, S. Hemby, J. Eberwine, Presence and phosphorylation of transcription factors in developing dendrites, Proc. Natl. Acad. Sci. U. S. A. 95 (5) (1998) 2313 – 2318. [5] J. Eberwine, C. Job, J.E. Kacharmina, K. Miyashiro, S. Therianos, Transcription factors in dendrites: dendritic imprinting of the cellular nucleus, Results Probl. Cell Differ. 34 (2001) 57 – 68. [6] J.D. Feldman, L. Vician, M. Crispino, W. Hoe, M. Baudry, H.R. Herschman, rTLE3, a newly identified transducin-like enhancer of split, is induced by depolarization in brain, J. Neurochem. 74 (5) (2000) 1838 – 1847. [7] D. Grbavec, R. Lo, Y. Liu, A. Greenfield, S. Stifani, Groucho/transducin-like enhancer of split (TLE) family members interact with the yeast transcriptional co-repressor SSN6 and mammalian SSN6-related proteins: implications for evolutionary conservation of transcription repression mechanisms, Biochem. J. 337 (1999) 13 – 17. [8] B.G. Ju, D. Solum, E.J. Song, K.J. Lee, D.W. Rose, C.K. Glass, M.G. Rosenfeld, Activating the PARP-1 sensor component of the Groucho/TLE1 corepressor complex mediates a CaMKinase IIdeltadependent neurogenic gene activation pathway, Cell 119 (6) (2004) 815 – 829. [9] D. LaBerge, Attention, consciousness, and electrical wave activity within the cortical column, Int. J. Psychophysiol. 43 (2001) 5 – 24. [10] H.N. Nuthall, K. Joachim, S. Stifani, Phosphorylation of serine 239 of Groucho/TLE1 by protein kinase CK2 is important for inhibition of neuronal differentiation, Mol. Cell. Biol. 24 (19) (2004) 8395 – 8407. [11] A. Palaparti, A. Baratz, S. Stifani, The Groucho/Transducin-like enhancer of split transcriptional repressors interact with the genetically
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[12] [13]
[14]
[15] [16]
L. Stewart, S. Stifani / Molecular Brain Research 140 (2005) 106 – 110 defined amino-terminal silencing domain of histone H3, J. Biol. Chem. 272 (1997) 26604 – 26610. G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, Second Edition, Academic Press, Orlando, 1986. P.H. Petersen, K. Zou, S. Krauss, W. Zhong, Continuing role for mouse Numb and Numbl in maintaining progenitor cells during cortical neurogenesis, Nat. Neurosci. 7 (8) (2004) 803 – 811. L.M. Pickles, S.M. Roe, E.J. Hemingway, S. Stifani, L.H. Pearl, Crystal structure of the C-terminal WD40 repeat domain of the Groucho/TLE1 transcriptional corepressor, Structure (Camb.) 10 (6) (2002) 751 – 761. P. Polakis, Wnt signaling and cancer, Genes Dev. 14 (2000) 1837 – 1851. S. Stifani, C.M. Blaumueller, N.J. Redhead, R.E. Hill, S. ArtavanisTsakonas, Human homologs of a Drosophila enhancer of split gene product define a novel family of nuclear proteins, Nat. Genet. 2 (1992) 119 – 127.
[17] A.E. Vercelli, D. Garbossa, R. Curtetti, G.M. Innocenti, Somatodendritic minicolumns of output neurons in the rat visual cortex, Eur. J. Neurosci. 20 (2004) 495 – 502. [18] C.C.J. Voelker, N. Garin, J.S.H. Taylor, B.H. Ga¨hwiler, J.-P. Hornung, Z. Molnar, Selective neurofilament (SMI-32, FNP-7 and N200) expression in subpopulations of Layer V pyramidal cells in vivo and in vitro, Cereb. Cortex 14 (2004) 1276 – 1286. [19] J. Yao, Y. Liu, J. Husain, R. Lo, A. Palaparti, J. Henderson, S. Stifani, Combinatorial expression patterns of individual TLE proteins during cell determination and differentiation suggests non-redundant functions for mammalian homologues of Drosophila Groucho, Dev. Growth Differ. 40 (1998) 133 – 146. [20] J. Yao, Y. Liu, R. Lo, I. Tretjakoff, A. Peterson, S. Stifani, Disrupted development of the cerebral hemispheres in transgenic mice expressing the mammalian Groucho homologue transducin-like-enhancer of split 1 in postmitotic neurons, Mech. Dev. 93 (2000) 105 – 115.
Short Communication
Dendritic localization of the transcriptional co-repressor Groucho/TLE1 in cortical and cerebellar neurons Lee Stewart, Stefano Stifani* Center for Neuronal Survival, Montreal Neurological Institute, Montreal, Quebec, Canada H3A 2B4 Accepted 27 June 2005 Available online 2 August 2005
Abstract In the present study we show that the transcription factor Groucho/TLE1 (TLE1) is expressed in virtually all major cortical subdivisions, hippocampus, amygdala, and thalamus, as well as in the cerebellum of the adult rat brain. In both neocortex and subcortical structures, TLE1 expression was mostly localized to neurons. In addition to the expected nuclear localization, TLE1 immunoreactivity was also detected in apical dendritic shafts of neocortical layer III and V pyramidal cells and in Purkinje cell dendrites. These results demonstrate that TLE1 expression occurs in the mature nervous system and suggest that this protein may perform new functions outside of the nucleus in selected cortical and cerebellar neurons. D 2005 Elsevier B.V. All rights reserved. Theme: Development and regeneration Topic: Cerebral cortex and limbic system Keywords: Cerebellum; Dendrite; Groucho; Hippocampus; Neocortex; Transducin-like enhancer of split 1
The Drosophila protein Groucho and its mammalian homologues, referred to as Transducin-like enhancer of split (TLE) 1 to 4 [16], are general transcriptional co-repressors that lack intrinsic DNA-binding ability but can be recruited to different genes through interactions with DNA-binding proteins [3,10]. Groucho/TLE proteins (TLEs) are expressed in a variety of tissues, play important roles in numerous developmental pathways, including neurogenesis [20], and are correlated with adult forms of disease [15]. To date, the majority of studies on TLE proteins have focused on their roles in cell fate determination during embryogenesis and few have examined their expression in the mature central nervous system (CNS). During mouse cortical development, it was shown that TLE1 is expressed in both neural progenitor cells and post-mitotic neurons in the outer layers of the cortical plate, whereas TLE4 is
* Corresponding author. Fax: +1 514 398 1319. E-mail address: [email protected] (S. Stifani). 0169-328X/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.molbrainres.2005.06.016
expressed by post-mitotic neurons of the inner layers [19]. Consistent with these observations, Petersen and coworkers [13] have recently shown that TLE4 is expressed in layer VI, but also in more superficial layers (II, III) of the adult mouse cortex. It has also been reported that TLE3 transcripts are expressed in the adult rat hippocampus [6]. In contrast, less is known about the expression pattern and possible functional role of TLE1 in the adult brain. In the present study, our primary objective was to characterize the expression of TLE1 in cortical and subcortical regions of the adult rat brain. To that end, we performed immunohistochemical studies using a panel of different antibodies previously shown to recognize TLE1 [7,10,11,19]. Adult male Sprague –Dawley rats were anesthetized with sodium pentobarbital and transcardially perfused with 4% paraformaldehyde. The brains were removed, cryoprotected in 30% sucrose, and sectioned on a cryostat. All animal procedures followed the guidelines of the Canadian Council for Animal Care.
L. Stewart, S. Stifani / Molecular Brain Research 140 (2005) 106 – 110
Three previously characterized antibodies were used for immunohistochemical studies: a rabbit polyclonal against the SP domain of TLE1 (Fanti-TLE1_) [10,19,20], a rabbit polyclonal against TLE1 phosphorylated at serine239 within the CcN domain (Fanti-(pS239)TLE1_) [10], and a rat monoclonal against the highly conserved TLE WD40 repeat domain (this antibody recognizes all four members of the TLE family and has been termed FpanTLE_) [7,11,16]. For immunohistochemistry, tissue sections were first incubated in 0.2% H2O2 in phosphate-buffered saline containing 0.2% Triton X-100 (PB-Tx) for 20 min, then in 10% normal goat serum (NGS) in PB-Tx for 2 h at room temperature. Sections were incubated with anti-TLE1 (1:500 in 1% NGS), anti-p(S239)TLE1 (1:500), or panTLE (1:200) antibodies overnight at 4 -C. Double labeling was performed using antibodies against MAP2 (monoclonal, 1:100; Sigma), glutamate transporter EAAC-1 (monoclonal, 1:100; Chemicon), SMI-32 (monoclonal, 1:500; Sternberger Monoclonals), NeuN (monoclonal, 1:100; Chemicon), and GFAP (monoclonal, 1:200; Sigma). After several washes in PB-Tx, sections were incubated in the appropriate fluorescein isothiocyanate (FITC)-conjugated or carboxymethylindocyanin-3 (Cy3)-conjugated secondary antibodies (1:200 in 1% NGS; Jackson ImmunoResearch). The 31 (FStandard_) Series of filters (Chroma Technology Corp.) was utilized for fluorescence microscopy. For control experiments, the primary antibodies were omitted (data not shown). Immunohistochemistry was performed on coronal brain sections at the levels of (in the rostrocaudal axis) primary motor cortex (bregma +1.70 mm), septum (+0.20 mm), primary somatosensory cortex and dorsal hippocampus ( 3.30 mm), primary visual cortex and optic tectum ( 5.80 mm), and entorhinal cortex ( 7.30 mm) according to the atlas of Paxinos and Watson [12]. The structures examined at each anatomical level are listed in Table 1. All images were captured with a DVC black and white camera mounted on a Zeiss Axioskop 2 fluorescence microscope. Grayscale images were digitally assigned to the appropriate red (Cy3) or green (FITC) channels using Northern Eclipse software (Empix). Using a previously characterized anti-TLE1 antibody [10,19,20], nuclear TLE1 expression was detected within most of the adult brain regions examined and was primarily co-localized with the neuronal marker NeuN, indicating that the majority of TLE1+ cells were neurons (Table 1; Fig. 1B, panel vi; and data not shown). TLE1 expression appeared to be mostly confined to forebrain structures, such as the hippocampus, cortex, thalamus, and amygdala (Figs. 1A and D, Table 1). TLE1 expression was observed in caudal regions of neocortex (i.e., visual and retrosplenial cortices) but was absent in subcortical structures caudal to the thalamus (i.e., substantia nigra, tectum, pons). Nuclear TLE1 expression was also detected in the cerebellum where it was primarily restricted to the Purkinje cell layer (Fig. 1C, panel i) with few labeled cells in the granule cell layer.
107
Table 1 Distribution of TLE1 expression in the adult rat brain Bregma (mm)
Area
TLE1-ir
+1.70
Primary motor cortex (M1) Piriform cortex Cingulate cortex Caudate nucleus Claustrum Nucleus accumbens Medial septum Lateral septum Ventral pallidum Primary somatosensory cortex (S1) Primary auditory cortex CA1 CA2 CA3 CA4 (hilus) Dentate gyrus Ventral posterior thalamus Lateral dorsal thalamus Mediodorsal thalamus Basolateral amygdala Primary visual cortex (V1) Retrosplenial cortex Periaqueductal grey Substantia nigra reticulata Superior colliculus (deep grey layer) Subiculum Medial geniculate Medial entorhinal cortex Lateral entorhinal cortex Pedunculopontine tegmentum Inferior colliculus
+ + +
+0.20
3.30
5.80
7.30
Abbreviation: ir, immunoreactivity. +/ ( ) of nuclear TLE1 expression.
+
+ + + + + + + + + + + + +
+ +
indicates presence (+) or absence
Unexpectedly, anti-TLE1 immunoreactivity was detected in the apical dendrites of a large number of layer V, and to a lesser extent layer III, pyramidal cells in nearly all major subdivisions of the neocortex (Figs. 1A and B, panel i). TLE1 expression appeared to be prominent among type 1 pyramidal neurons, as indicated by co-expression with both glutamate transporter EAAC-1, a general pyramidal cell marker protein, and non-phosphorylated neurofilament proteins recognized by the SMI-32 antibody [18] (Fig. 1B, panels iv and v). To confirm the specificity of this dendritic immunoreactivity, similar studies were performed with a previously characterized anti-(pS239)TLE1 antibody that recognizes TLE1 phosphorylated at serine239 [10]. In layer V of visual cortex, anti-TLE1 and anti-(pS239)TLE1 immunoreactivities were both present within groups of apical dendrites resembling dendritic bundles [17] (Fig. 1B, panels i and ii). These dendritic bundles extended dorsally to superficial layers forming ‘‘tufts’’ near the cortical surface. Importantly, we observed an overlapping dendritic staining with a panTLE monoclonal antibody that recognizes all four mammalian TLE family members [7,10,11,16] (Fig. 1B, panel iii), further indicating the specificity of the observed immunoreactivity. At the cortical level shown in Fig. 1B (panel iii), nuclear and dendritic TLE1 expression was absent in superficial cortical
108
L. Stewart, S. Stifani / Molecular Brain Research 140 (2005) 106 – 110
Fig. 1. Nuclear and dendritic TLE1 expression in adult rat brain. (A) Immunohistochemical detection of TLE1 expression in motor cortex of the adult rat using the anti-TLE1 antibody. Note the presence of TLE1-immunoreactive soma and dendrites in layers V and II/III. (B) In visual cortex, TLE1 expression was detected in apical dendritic bundles formed by layer V pyramidal cells (panel i) as indicated by EAAC-1 and SMI-32 co-expression (panels iv and v). Unlike motor cortex, however, nuclear TLE1 expression was largely absent in shallow visual cortex. Nuclear TLE1 expression in the layer V pyramidal cells was confirmed by co-expression with the neuronal marker NeuN (panel vi). A similar dendritic immunoreactivity was also observed in layer V apical dendrites with the anti-(pS239)TLE1 antibody (panel ii). Double-labeling studies with rabbit anti-TLE1 and rat panTLE antibodies revealed overlapping layer V dendritic staining in visual cortex (panel iii); additional panTLE-immunoreactive structures present in superficial layers likely represent different TLE isoforms. (C) In the cerebellum, TLE1 showed robust co-expression with MAP2 in Purkinje cell dendrites, with a few TLE1+ nuclei scattered in the subjacent granule cell layer (panels i – iii). (D) In the hippocampus, nuclear TLE1 expression was seen throughout the pyramidal cell layer including the hilus (data not shown) as well as in the outer blade of the dentate gyrus, which was characterized by a ‘‘patch’’ of TLE1+ granule cells. The representative anatomical levels for each of the above labeling studies are shown in inset: (a) motor cortex; (b) visual cortex; (c) cerebellum; and (d) hippocampus and dentate gyrus [12]. Abbreviations: M1, primary motor cortex; I – VI, cortical layers; V1, primary visual cortex; PC, Purkinje cell layer; GCL, granule cell layer; PFl, paraflocculus; sm, stratum moleculare; DG, dentate gyrus.
layers as compared with more rostral regions such as motor cortex (Fig. 1A). Thus, the panTLE immunoreactivity detected in shallow visual cortex, in addition to layer V apical dendrites, likely represents the expression of different TLE isoforms (Fig. 1B, panel iii). Nuclear localization of TLE1 in layer V pyramidal cells was confirmed by coexpression with NeuN (Fig. 1B, panel vi). In the cerebellum,
TLE1 was co-expressed with MAP2 in Purkinje cell dendrites (Fig. 1C, panels i –iii). Despite its presence in the nuclei of granule cells in the dentate gyrus, TLE1 expression did not exhibit any overlap with MAP2 in the stratum moleculare (Fig. 1D, panels i –iii). The main findings of this study are that (1) nuclear TLE1 expression occurs throughout the adult rat forebrain,
L. Stewart, S. Stifani / Molecular Brain Research 140 (2005) 106 – 110
primarily in neurons; (2) TLE1 immunoreactivity is detected in the apical dendrites of layer III and V pyramidal neurons in selected cortical regions; and (3) TLE1 expression is equally abundant in the nuclei and dendrites of cerebellar Purkinje cells. To the best of our knowledge, this study is the first to characterize the expression of TLE1 in the adult rat brain. Furthermore, these findings also provide the first evidence that TLE1, a transcriptional regulator, may have a functional role outside of the nucleus. Similar to TLE3 and TLE4, the expression of TLE1 in the adult rat brain suggests a role for this protein in the mature nervous system. The observation that nuclear TLE1 expression is widespread throughout the forebrain is consistent with its role in the regulation of gene transcription [3,7,10]. However, given its robust expression in dendrites it is likely that TLE1 also has functional roles distinct from that as a transcriptional regulator. Dendritic TLE1 expression may be a salient feature of neurons involved with complex brain functions, such as visual discrimination in primary visual cortex or motor learning in the cerebellum. For example, TLE1+ dendritic bundles may correspond to cortical architecture referred to as output columns [17], which have been implicated in higher cortical information processing and neurological disease [9]. Feldman and colleagues [6] characterized TLE3 gene expression in the hippocampus following epileptic seizures and suggested that seizure-induced increases in TLE3 expression may relate to the functional plasticity of neurons. Indeed, sustained excitatory stimulation can up-regulate the expression of multiple genes necessary for enhancing synaptic efficacy [1]. But before changes in gene expression that coincide with neuronal plasticity can occur, the transport of proteins from dendrites to the nucleus is often required to regulate gene activity in response to physiological stimuli. Among those proteins, transcription factors are likely to play a pivotal role in regulatory pathways between distal regions of the cell and the nucleus [2,5]. For a transcription factor potentially involved in a dendritic signaling pathway one would expect to observe a punctate expression pattern [4]. By contrast, the distribution of TLE1 immunoreactivity observed in the present study appears uniform throughout the dendritic tree. Thus, it is plausible that TLE1 could play an alternative role in the maintenance of dendritic structure and morphology, perhaps through interactions with cytoskeletal proteins such as h-actin [8]. The C-terminal region of TLEs contains a well-defined structure, the WD40 repeat domain, which contains seven repeats of ¨40 amino acids, each harboring the sequence tryptophan – aspartate [14]. This structure contributes to the interaction of TLE proteins with numerous transcription factors. It is possible that the presence of a WD40 domain confers upon TLEs an even larger capacity for protein – protein interactions. This may enable TLE1 to perform functions outside of the nucleus by binding to proteins that are not typically associated with transcriptional regulatory pathways. Further studies are necessary to discern the exact
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functional role of TLE1 in neocortical dendrites and elsewhere in the mature CNS. In summary, the present results show that TLE1 exhibits a unique nuclear and dendritic expression pattern in the adult rat brain and is primarily restricted to forebrain structures, with the exception of the cerebellum. Most studies of TLE proteins have focused on their roles in cell fate determination during embryogenesis. We propose that TLE1 may have a novel non-nuclear function in the adult brain, in addition to its role as a transcriptional co-repressor in both the mature and developing CNS.
Acknowledgments The authors thank Dr. B. Jones for the gift of the SMI-32 antibody. This work was supported by grants from the Canadian Institutes of Health Research (MOP-13957 and MGC-14971) to S.S. L.S. is the recipient of a J. Timmins Costello Postdoctoral Fellowship, and S.S. is a Senior Scholar of the Fonds de la Recherche en Sante du Quebec.
References [1] A.J. Becker, J. Chen, A. Zien, D. Sochivko, S. Normann, J. Schramm, C.E. Elger, O.D. Wiestler, I. Blumcke, Correlated stageand subfield-associated hippocampal gene expression patterns in experimental and human temporal lobe epilepsy, Eur. J. Neurosci. 18 (10) (2003) 2792 – 2802. [2] A.H. Brivanlou, J.E. Darnell, Signal transduction and the control of gene expression, Science 295 (2002) 813 – 818. [3] G. Chen, A.J. Courey, Groucho/TLE family proteins and transcriptional repression, Gene 249 (2000) 1 – 16. [4] P. Crino, K. Khodakhah, K. Becker, S. Ginsberg, S. Hemby, J. Eberwine, Presence and phosphorylation of transcription factors in developing dendrites, Proc. Natl. Acad. Sci. U. S. A. 95 (5) (1998) 2313 – 2318. [5] J. Eberwine, C. Job, J.E. Kacharmina, K. Miyashiro, S. Therianos, Transcription factors in dendrites: dendritic imprinting of the cellular nucleus, Results Probl. Cell Differ. 34 (2001) 57 – 68. [6] J.D. Feldman, L. Vician, M. Crispino, W. Hoe, M. Baudry, H.R. Herschman, rTLE3, a newly identified transducin-like enhancer of split, is induced by depolarization in brain, J. Neurochem. 74 (5) (2000) 1838 – 1847. [7] D. Grbavec, R. Lo, Y. Liu, A. Greenfield, S. Stifani, Groucho/transducin-like enhancer of split (TLE) family members interact with the yeast transcriptional co-repressor SSN6 and mammalian SSN6-related proteins: implications for evolutionary conservation of transcription repression mechanisms, Biochem. J. 337 (1999) 13 – 17. [8] B.G. Ju, D. Solum, E.J. Song, K.J. Lee, D.W. Rose, C.K. Glass, M.G. Rosenfeld, Activating the PARP-1 sensor component of the Groucho/TLE1 corepressor complex mediates a CaMKinase IIdeltadependent neurogenic gene activation pathway, Cell 119 (6) (2004) 815 – 829. [9] D. LaBerge, Attention, consciousness, and electrical wave activity within the cortical column, Int. J. Psychophysiol. 43 (2001) 5 – 24. [10] H.N. Nuthall, K. Joachim, S. Stifani, Phosphorylation of serine 239 of Groucho/TLE1 by protein kinase CK2 is important for inhibition of neuronal differentiation, Mol. Cell. Biol. 24 (19) (2004) 8395 – 8407. [11] A. Palaparti, A. Baratz, S. Stifani, The Groucho/Transducin-like enhancer of split transcriptional repressors interact with the genetically
110
[12] [13]
[14]
[15] [16]
L. Stewart, S. Stifani / Molecular Brain Research 140 (2005) 106 – 110 defined amino-terminal silencing domain of histone H3, J. Biol. Chem. 272 (1997) 26604 – 26610. G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, Second Edition, Academic Press, Orlando, 1986. P.H. Petersen, K. Zou, S. Krauss, W. Zhong, Continuing role for mouse Numb and Numbl in maintaining progenitor cells during cortical neurogenesis, Nat. Neurosci. 7 (8) (2004) 803 – 811. L.M. Pickles, S.M. Roe, E.J. Hemingway, S. Stifani, L.H. Pearl, Crystal structure of the C-terminal WD40 repeat domain of the Groucho/TLE1 transcriptional corepressor, Structure (Camb.) 10 (6) (2002) 751 – 761. P. Polakis, Wnt signaling and cancer, Genes Dev. 14 (2000) 1837 – 1851. S. Stifani, C.M. Blaumueller, N.J. Redhead, R.E. Hill, S. ArtavanisTsakonas, Human homologs of a Drosophila enhancer of split gene product define a novel family of nuclear proteins, Nat. Genet. 2 (1992) 119 – 127.
[17] A.E. Vercelli, D. Garbossa, R. Curtetti, G.M. Innocenti, Somatodendritic minicolumns of output neurons in the rat visual cortex, Eur. J. Neurosci. 20 (2004) 495 – 502. [18] C.C.J. Voelker, N. Garin, J.S.H. Taylor, B.H. Ga¨hwiler, J.-P. Hornung, Z. Molnar, Selective neurofilament (SMI-32, FNP-7 and N200) expression in subpopulations of Layer V pyramidal cells in vivo and in vitro, Cereb. Cortex 14 (2004) 1276 – 1286. [19] J. Yao, Y. Liu, J. Husain, R. Lo, A. Palaparti, J. Henderson, S. Stifani, Combinatorial expression patterns of individual TLE proteins during cell determination and differentiation suggests non-redundant functions for mammalian homologues of Drosophila Groucho, Dev. Growth Differ. 40 (1998) 133 – 146. [20] J. Yao, Y. Liu, R. Lo, I. Tretjakoff, A. Peterson, S. Stifani, Disrupted development of the cerebral hemispheres in transgenic mice expressing the mammalian Groucho homologue transducin-like-enhancer of split 1 in postmitotic neurons, Mech. Dev. 93 (2000) 105 – 115.