Differential expression and dendritic transcript localization of Shank family members: identification of a dendritic targeting element in the 3′ untranslated region of Shank1 mRNA

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Abstract

Shank proteins are scaffolding proteins in the postsynaptic density of excitatory synapses in the mammalian brain. In situ hybridization revealed that Shank1/SSTRIP and Shank2/ProSAP1 mRNAs are widely expressed early in postnatal brain development whereas Shank3/ProSAP2 expression increases during postnatal development especially in the cerebellum and thalamus. Shank1 and Shank3 (but not Shank2) mRNAs are present in the molecular layers of the hippocampus, consistent with a dendritic transcript localization. Shank1 and Shank2 transcripts are detectable in the dendritic fields of Purkinje cells, whereas Shank3 mRNA is restricted to cerebellar granule cells. The appearance of dendritic Shank mRNAs in cerebellar Purkinje cells coincides with the onset of dendrite formation. Expression of reporter transcripts in hippocampal neurons identifies a 200-nucleotide dendritic targeting element (DTE) in the Shank1 mRNA. The widespread presence of Shank mRNAs in dendrites suggests a role for local synthesis of Shanks in response to stimuli that induce alterations in synaptic morphology.

Introduction

The activity-dependent modification of synaptic transmission is believed to be a key event in processes such as learning and memory. A long-lasting modification of synaptic strength requires protein synthesis (Kang and Schuman, 1996), suggesting that newly synthesized proteins contribute to the modification of synaptic efficacy. The observation of polyribosomes beneath synapses at the base of dendritic spines (Steward and Levy, 1982) indicates that protein synthesis may occur locally in dendrites under the control of the nearby synapse. This concept implies that mRNAs are present in dendrites, and that dendritically synthesized proteins can mediate activity-dependent structural and functional modifications of the postsynaptic apparatus. To date, the number of mRNAs that has been bona fide identified in dendrites is limited. One notable example is the mRNA encoding the α-subunit of the calcium/calmodulin-dependent protein kinase II (αCaMKII; Burgin et al., 1990). Recently, it was shown in mutant mice that dendritic targeting of αCaMKII transcripts in dendrites is required for the maintenance of long-term potentiation and specific learning tasks (Miller et al., 2002). In the case of other dendritically localized transcripts such as those coding for microtubule-associated protein 2 (Garner et al., 1988), dendrin (Herb et al., 1997), and arg3.1/arc Link et al., 1995, Lyford et al., 1995, it is not yet clear how extrasomatic protein synthesis might affect synaptic function.

Here we report the cellular and subcellular distribution of mRNAs coding for the three members of the Shank family of postsynaptic proteins (Boeckers et al., 2002). Shank1–3 (Naisbitt et al., 1999), also identified as somatostatin receptor interacting protein (SSTRIP; Zitzer et al., 1999), proline-rich synapse-associated protein 1 (ProSAP1; Boeckers et al., 1999a) or cortactin binding protein 1 (Du et al., 1998), and ProSAP2 (Boeckers et al., 1999b) or synamon (Yao et al., 1999) consist of multiple protein interaction domains, which are linked directly or indirectly to postsynaptic N-methyl-d-aspartate receptors and metabotropic glutamate receptors Boeckers et al., 1999b, Naisbitt et al., 1999, Tu et al., 1999, the calcium-independent receptor for α-latrotoxin Kreienkamp et al., 2000, Tobaben et al., 2000 or somatostatin receptor subtype 2 (Zitzer et al., 1999). Moreover, interactions with the actin-associated proteins cortactin (Du et al., 1998) and α-fodrin (Boeckers et al., 2001) suggest that Shanks provide the interface between synaptic cell surface receptors and the actin-based cytoskeleton.

Shank gene expression patterns have not been characterized in much detail in the mammalian brain. In particular, it is unknown if individual brain structures express multiple (or all) Shanks, or if expression of individual Shank genes is restricted to distinct neuronal populations. By in situ hybridization, we have analyzed the regional pattern of expression of individual Shank genes during the postnatal development of the rat brain. Besides the previously reported presence of Shank1 transcripts in hippocampal pyramidal cell dendrites (Zitzer et al., 1999), we find a prominent dendritic localization of Shank mRNAs in cerebellar Purkinje cells (Shank1 and Shank2) and in the hippocampus (Shank1 and Shank3). In addition, we identify a dendritic targeting element (DTE) in the 3′ untranslated region (3′ UTR) of Shank1 mRNAs that mediates efficient dendritic trafficking of chimeric reporter transcripts in primary neurons.

Section snippets

Shank mRNA distribution in postnatal rat brain

The distribution of Shank transcripts in the rat brain was analyzed at various postnatal developmental stages by in situ hybridization using isoform-specific 35S-labeled oligonucleotides. Probes recognize all known mRNA variants transcribed from a particular Shank gene. At each stage, analysis was performed on consecutive horizontal sections to allow for a direct comparison of expression patterns (Fig. 1A, see also Table 1). All three Shank mRNAs are detected in many brain regions the first day

Discussion

In this study, significant regional differences in the expression pattern of members from the Shank gene family (Boeckers et al., 2002) were found in the developing and adult rat brain. For instance, Shank1 is the only family member present in the hypothalamus, but it is not found in the caudate putamen where Shank2 and Shank3 genes are expressed. In the cerebellum, Shank3 expression is limited to granule cells while Shank1 and Shank2 are prominently found only in Purkinje cells. Thus, neurons

In situ hybridization on rat brain sections

Oligonucleotides (sequences listed in Table 2; MWG-Biotech, Ebersberg, Germany) were end-labeled with α-35S-UTP using terminal transferase (Boeckers et al., 1999a). Rat brains were frozen in isopentane at −40°C, horizontal sections (20 μm) were cut with a cryostat, mounted on Superfrost Plus slides (Menzel, Braunschweig, Germany), and stored at −70°C until use. Hybridization conditions were described previously (Boeckers et al., 1999a). For each developmental stage, two or more sections of at

Acknowledgements

We thank Arne Blichenberg for help with the establishment of the neuronal expression system, Hans-Hinrich Hönck, Gisela Gaede, Sylvia Loheide, and Annelie Ahle for excellent technical assistance, and Heike Zitzer and Irm Hermanns-Borgmeyer for help with initial in situ hybridizations. Financial support from the Deutsche Forschungsgemeinschaft (Bo1718/1-1 to T.M.B. and H.-J.K.; in part: SFB426/A1 to E.D.G.; Kr1879/2-2 to M.R.K.; Ki488/2-6 to S.K.) and the EU (contract QLG3-CT-1999-00908 to D.R.)

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These authors equally contributed to this work.

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