Association of Dystrophin-Related Protein 2 (DRP2) with Postsynaptic Densities in Rat Brain

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Molecular and Cellular Neuroscience 16, 674 – 685 (2000) doi:10.1006/mcne.2000.0895, available online at http://www.idealibrary.com on

Association of Dystrophin-Related Protein 2 (DRP2) with Postsynaptic Densities in Rat Brain 1 Roland G. Roberts* ,† ,2 and Morgan Sheng* *Howard Hughes Medical Institute and Department of Neurobiology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts; and †Division of Medical & Molecular Genetics, GKT Medical School, Guy’s Hospital, London SE1 9RT, United Kingdom

The fundamental function of the membrane-associated cytoskeletal proteins dystrophin and utrophin remains unclear. To gain further insights into the dystrophin family of proteins, we have studied dystrophin-related protein 2 (DRP2), whose expression is largely confined to the vertebrate central nervous system. Both human and rat DRP2 are expressed from two alternative but neighboring transcriptional start sites and have simple transcript structures. Antibodies raised against DRP2 detect a characteristic quartet of bands (⬃100 –120 kDa) in Western blots of rat brain. The DRP2 protein is associated with brain membrane fractions and highly enriched in the postsynaptic density. Immunohistochemistry shows DRP2 to be widely distributed in a punctate pattern on neuronal dendrites and in neuropil, with particular concentration in regions of the brain involved in cholinergic synaptic transmission. Given the presence of utrophin in the cholinergic neuromuscular junction, and perturbations of cholinergic transmission in dystrophin-deficient nematodes, our findings may suggest a role for DRP2 in the organization of central cholinergic synapses.

INTRODUCTION Dystrophin is a large membrane-associated cytoskeletal protein whose disruption results in the human genetic disorder Duchenne muscular dystrophy. The loss of this protein, together with the consequent secondary loss or mislocalization of a large number of associated proteins, gives rise to a complex syndrome of

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Accession Nos. AF195787, AF195788, and AF195789. To whom correspondence should be addressed at Division of Medical & Molecular Genetics, 8th Floor, Guy’s Tower, Guy’s Hospital, London SE1 9RT, UK. Fax: ⫹44-(0)20-7955-4644. E-mail: [email protected]. 2

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progressive skeletal and cardiac myopathy, electroretinopathy, and mental retardation. Dystrophin itself is found at the muscle sarcolemma and in parts of the peripheral and central nervous systems (Byers et al., 1993; Lidov et al., 1990). A close relative, utrophin, is found at the neuromuscular and myotendinous junctions in muscle and cerebral perivascular membranes (Khurana et al., 1992); loss of utrophin results in subtle changes in postsynaptic folds of the neuromuscular junction (Deconinck et al., 1997; Grady et al., 1997). All invertebrate phyla examined also seem to have a single dystrophin-like protein (Roberts and Bobrow, 1998). Despite intensive research, the precise physiological roles of dystrophin and utrophin and many of the proteins of the dystrophin/utrophin-associated complex remain unclear. We recently identified (Roberts et al., 1996) a human Xq-linked gene that encodes dystrophin-related protein 2 (DRP2), a homologue of dystrophin whose transcript expression is largely restricted to the central nervous system of vertebrates (Dixon et al., 1997). DRP2 is much smaller than dystrophin, corresponding to the C-terminal one-fourth of the largest dystrophin isoform, thereby resembling the peripheral nervous system dystrophin isoform, Dp116 (Byers et al., 1993). DRP2 retains two of the spectrin-like repeats, together with putative binding sites for ␤-dystroglycan, the syntrophins, and the dystrobrevins. It is therefore reasonable to hypothesize that DRP2 performs functions that are similar to or overlapping with those of dystrophin, such as the interaction with the extracellular matrix via dystroglycan and perhaps with both voltage-gated sodium channels (Gee et al., 1998) and neuronal nitric oxide synthase (Brenman et al., 1996) via the syntrophins. Consistent with such an idea, such dystrophin-associated proteins 1044-7431/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

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as dystrobrevins, syntrophins, and dystroglycan are widely expressed in the brain (Blake et al., 1998; Go´recki et al., 1994, 1997; Mummery et al., 1996; Peters et al., 1997). A direct interaction with the cytoskeleton, mediated by an N-terminal actin-binding domain in dystrophin and utrophin, appears not to have a specific parallel in DRP2, though it is possible that the latter’s unique N-terminus somehow compensates for this. As an initial step toward characterization of the DRP2 protein, we cloned the rat DRP2 cDNA and established the transcriptional diversity of both human and rat DRP2. Polyclonal antibodies were raised against bacterial fusion proteins of N- and C-terminal DRP2 sequences and used to study native DRP2 distribution in rat brain. We report here that DRP2 is highly enriched in the postsynaptic density (PSD) and distributed in a punctate somatodendritic pattern in neurons in specific regions of the rat brain. We discuss this distribution with respect to possible functions of DRP2 as a component of central cholinergic synapses.

RESULTS Characterization of the Rat DRP2 Transcript In order to confirm the validity of extrapolations between species, we cloned the rat DRP2 cDNA (Accession No. AF195787) and compared its sequence with that of its human counterpart (Accession No. U43519). Notional translation of the coding region shows 95% identity between rat and human DRP2 (and ⬎98% between rat and mouse DRP2) over the 957-amino-acid reading frame (Fig. 1A). Much of the variability is in the N-terminal domain (only 88% identity over 100 amino acids) and in the spacer between the two C-terminal leucine heptad regions. The codon previously assumed (Roberts et al., 1996) to be the initiator ATG in the human cDNA (on the basis of its close adherence to the Kozak consensus) encodes leucine in the rat, so translation must initiate at one of the nearby in-frame ATG codons in this, and probably other, animals. For the purposes of numbering, we have assumed that the longest open reading frame (957 codons) is used. The 5⬘ ends of both human and rat transcripts were characterized by 5⬘RACE. In both organisms there were found to be two distinct populations of transcript (A and B), derived from different transcription initiation sites (Fig. 1B). The A-type (Accession No. AF195787) of both rat and human initiates at a point ⬃240 bp upstream of the B-type (Accession No. AF195788). Whereas the two human transcripts show a simple

relationship to each other, in rat A-type transcripts, the B-type start site is spliced out as part of a ⬃170-bp intron. In human and rat whole brain cDNA the two forms have roughly equal representation. Surprisingly, neither rat transcript contains sequences equivalent to the noncoding exon 2 of the human transcript, but aside from this difference the 5⬘-untranslated regions (UTRs) are extremely highly conserved (87% identity) in the 340 bp upstream of the start codon. Human genomic sequence adjacent to the putative transcriptional start sites (cosmid V210E9, Accession No. Z70280) shows that the DRP2 gene has an unconventional promoter which lacks both a consensus TATA box and the usual high CpG dinucleotide content. A further unusual feature is a long polypyrimidine stretch immediately before the A-type start site (101/ 107 nucleotides are T or C). Unlike the complex pattern of alternative splicing observed toward the 3⬘ end of human and rodent dystrophin transcripts (Bies et al., 1992; Feener et al., 1989), no equivalent alternative splicing of DRP2 transcripts was observed in multiple amplifications from rat or human brain cDNA. The only unusual feature was the retention of the 366-bp intron 7 (Accession No. AF195789) in ⬃50% of rat DRP2 transcripts (probably caused by an A3 T transversion at position ⫹3, a change which has been known to cause exon skipping and human genetic disease (Krawczak et al., 1992)). The intron does not have a continuous open reading frame. Another feature of transcription of the dystrophin gene is the use of multiple promoters; indeed the dystrophin gene uses an internal promoter in intron 55 to generate the DRP2-like isoform Dp116 in peripheral nerve (Byers et al., 1993). A more widely used promoter lies in intron 62 of the dystrophin gene and results in the production of the even shorter isoform Dp71, which starts within the WW domain (Blake et al., 1992; Hugnot et al., 1992; Lederfein et al., 1992). In order to test whether this promoter is conserved in the DRP2 gene, we performed 5⬘RACE using primers 3⬘ to this region. This failed to give evidence for a Dp71-like transcript encoding an equivalent N-terminally truncated isoform of DRP2. Preliminary Characterization of Antibodies Rabbit polyclonal antibodies were raised against fusion proteins of the N-terminal region (amino acids 4 –231) and C-terminal region (amino acids 706 –957) of DRP2 and affinity purified (see Experimental Procedures for details). N-terminal antibodies (DRP2A1, DRP2A2) performed less well than C-terminal antibodies in immunoblotting and immunostaining, barely rec-

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FIG. 1. The rat DRP2 protein and transcript. (A) Alignment of rat and human DRP2 protein sequences (Accession Nos. AF195787 and U43519). Identical amino acids are highlighted with gray. Boxes indicate protein features. (B) Schematic diagram of the relationship between the 5⬘ ends of human and rat DRP2 transcripts. Open boxes represent untranslated regions, while hatched boxes represent coding sequence. Exons are labeled E1, E2, and E3. Percentages indicate degrees of identity between the corresponding human and rat sequences at the nucleotide level.

Dystrophin-related Protein 2 in the Brain

ognizing 1 ng of the target N-terminal DRP2 fusion protein and yielding weak signals on Western blots and sections of rat brain (see below and data not shown). Although the results obtained with DRP2A1 and A2 were entirely consistent with those found using C-terminal antibodies (see for example Fig. 4 later), data shown in this paper were mainly obtained using the C-terminal antibodies (DRP2D1, DRP2D2), which showed greater sensitivity. As the C-terminal fusion protein used to generate DRP2D1/D2 antibodies shares 42 and 39% identity with the corresponding regions of dystrophin and utrophin, respectively, we tested the specificity and sensitivity of the DRP2D1 and DPR2D2 antibodies on Western blots of serially diluted glutathione S-transferase (GST) fusion proteins of the corresponding regions of dystrophin, utrophin, and DRP2 (DysD, UtrD, DRP2D; Fig. 2). The results show that under the conditions of Western blotting, the DRP2D antibodies are ⬃20-fold more sensitive for DRP2 than dystrophin and about 50-fold more sensitive for DRP2 than utrophin (shown for DRP2D1; DRP2D2 yielded similar results). Thus DRP2D antibodies are highly specific for DRP2 over its relatives. Moreover, DRP2D antibodies show high absolute sensitivity in that they can reliably detect less than 0.1 ng of DRP2D fusion protein on Western blots (Fig. 2, top panel). Predepletion of dystrophin-crossreacting species by passing the DRP2D antisera over a hexahistidine (H 6)-DysD column before affinity purification further reduced the cross-reactivity with dystrophin and utrophin to less than 1/100th that of DRP2 (Fig. 2, second panel), but reduced the absolute sensitivity of detection of DRP2. Western Blot Analysis of DRP2 in Rat Brain We immunoblotted subcellular fractions of rat brain homogenates with DRP2D1 and DRP2D2 antibodies. This reproducibly revealed a characteristic quartet of bands with apparent molecular weight of 100 –120 kDa, predominantly in the membrane/particulate fractions (Fig. 3A). Results with the N-terminal antibodies, though weaker in signal, were similar (data not shown). To confirm this apparent molecular weight, we compared the bands detected in brain with recombinant DRP2 heterologously expressed from the putative fulllength coding region of DRP2. Crude lysate of COS7 cells that had been transfected with a pGW1 expression construct containing the entire coding region of human DRP2 (but not of cells transfected with empty vector or a vector bearing an unrelated insert) gave a heterogeneous set of bands of similar molecular weight to those

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FIG. 2. Specificity and sensitivity of anti-DRP2 antibodies. Serial dilutions of bacterial lysates containing 10, 1, or 0.1 ng of GST fusion proteins of corresponding regions from human DRP2, utrophin, and dystrophin (normal muscle splice form) were electrophoresed, blotted, and probed with the antibodies indicated. The results for DRP2D1 (with and without predepletion on a dystrophin column) are shown (DRP2D2 gave very similar results), together with control probings with antibodies to dystrophin (MAB1694, Chemicon International) and glutathione S-transferase (Santa Cruz Biotechnology, Inc.).

observed in brain (Fig. 3A, first lane). Since multiple bands for DRP2 arose from a single cDNA in a heterologous expression system, and no alternative splicing of DRP2 was detected that might affect the coding region, we surmise that the multiple DRP2 bands observed in brain immunoblots are likely to result from alternative translational initiation, posttranslational modification, or partial degradation, rather than from alternative splicing. The concordance of results with independent antibodies raised to nonoverlapping immunogens, the comigration of the bands with heterologously expressed DRP2, and the correspondence with the predicted molecular weight of DRP2 based on primary structure (108 kDa) convinced us that the 100- to 120kDa bands in brain truly represent rat DRP2. Biochemical fractionation of whole rat brain followed

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FIG. 3. Western blot analysis of DRP2 in rat brain. (A) Subcellular fractionation of DRP2 in rat brain. Ten- or 20-␮g amounts of protein fractions from whole rat brain were electrophoresed and blotted, together with lysate from COS7 cells transfected with a human DRP2 expression construct (COS). Probing with antibody DRP2D2 reveals a quartet of bands copurifying with membrane fractions. (B) Regional distribution of DRP2 in rat brain. Crude membrane protein fractions (20 ␮g) from the indicated brain regions were blotted and probed with antibody DRP2D2. (C) Developmental profile of DRP2. Equal quantities (15 ␮g) of whole brain lysates from adult rat (Ad) or pups 1–27 days after birth were electrophoresed, blotted, and probed with DRP2D2 (top panel). The blot was stripped and reprobed with an antibody against glutamate receptor-interacting protein, GRIP (Wyszynski et al., 1998) (bottom panel) as a control for loading.

by Western blotting showed DRP2 to be almost exclusively confined to membrane fractions (Fig. 3A). Crude membrane fractions of grossly dissected rat brain re-

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gions were also immunoblotted with anti-DRP2 antibodies. This showed DRP2 protein to be expressed at roughly equal levels in each region (Fig. 3B), in agreement with previous in situ hybridization data indicating widespread expression of DRP2 mRNA in the CNS (Dixon et al., 1997). Many proteins show a pronounced change in abundance during development. To assess whether this was the case for DRP2, we probed a Western blot of a series of whole brain protein preparations from rats aged 1 day postnatal to adulthood (Fig. 3C, top panel). This shows a modest increase in levels of DRP2 from the first week, peaking around 3 weeks of age and declining in adulthood. Dystrophin and utrophin are enriched at postysnaptic sites in brain and muscle, respectively (Khurana et al., 1991; Kim et al., 1992; Matsumura et al., 1992). To test whether DRP2 might be localized to postsynaptic specializations of central neurons, we probed Western blots of purified fractions of PSDs from rat brain. DRP2 showed a striking enrichment in PSD fractions compared with whole brain homogenate (Fig. 4). The degree of enrichment of DRP2 in the PSD is comparable to that of PSD-95, a major component of the PSD that binds to NMDA-type glutamate receptors in central synapses (Cho et al., 1992; Kim et al., 1995) (Fig. 4, right-hand panel). The DRP2 associated with the PSD fraction is resistant to one extraction (PSD-I) or two extractions (PSD-II) by the detergent Triton X-100, but is partially lost after extraction with sarkosyl (PSD-III). In order to semiquantify the amount of DRP2 in PSDs, we compared the PSD signals of DRP2 against a calibration series of known quantities of the fusion protein GST– DRP2D in the same immunoblot (data not shown). Assuming that the human fusion protein and the rat endogenous protein are similarly immunoreactive to our DRP2D antibodies (perhaps reasonable, as they share 239 of 252 amino acids in the region used as an immunogen), this analysis suggested that DRP2 accounts for approximately 0.1% of the protein in PSD fraction II. Thus, DRP2 is less abundant in the PSD than PSD-95 or is present in a smaller subset of PSDs than PSD-95. Immunohistochemical Staining of Rat Brain Slices Immunostaining with DRP2D antibodies was used to determine the regional and subcellular distribution of DRP2 in rat brain. Dendritic or somatodendritic neuronal staining was seen in many brain areas, with a regional pattern strongly reminiscent of the distribution of the DRP2 transcript determined by in situ hybridiza-

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FIG. 4. Enrichment of DRP2 in postsynaptic density fractions. Two (whole brain, PSDI, PSDII) or 1 ␮g (PSDIII) of protein was Western blotted and probed with antibodies to the C-terminus (DRP2D1 and DRP2D2, undepleted or predepleted— dep— of dystrophin-cross-reacting species) or N-terminus (DRP2A1 and DRP2A2) of DRP2 or to PSD95 (right-hand panel (Kim et al., 1995)).

tion in mouse brain (Dixon et al., 1997). As with the in situ hybridization, no staining of white matter was observed. The DRP2D antibody staining was abolished by preincubation with a 10-fold excess of H 6-DRP2D fusion protein but not with an equal concentration of H 6-DysD, arguing that the staining pattern is specific for DRP2. However, a few preparations of antibody which showed higher cross-reactivity with dystrophin fusion proteins on Western blots also showed additional immunoreactivity with brain microvasculature (presumably due to dystrophin, since a pattern similar to this was obtained using a commercial monoclonal antibody against the C-terminal 17 amino acids of muscle dystrophin—Chemicon International MAB1694) (data not shown). The cerebral cortex was stained heavily throughout most layers (Fig. 5A). In layer IV the staining was lighter and clearly defined DRP2-labeled apical dendrites of layer V pyramidal neurons could be seen coursing through this layer to ramify in layers II/III (Fig. 5B). This pattern of immunostaining is reminiscent of ␣-actinin, an actin-binding protein with a known PSD localization (Wyszynski et al., 1997). High-resolution immunofluorescence confocal microscopy showed densely punctate DRP2 staining along these apical dendrites (often in a double “tramtrack” pattern; compare Fig. 3a in Wyszynski et al., 1997) as well as within the neuropil of the cortex (Figs. 5C and 5D). Cell bodies of cortical neurons were minimally stained, appearing as black “holes” surrounded by the intense immunoreactivity of the synaptic neuropil. Similar micropunctate staining was also observed for DRP2 on dendrites and in neuropil of other brain regions (Fig. 6). At the regional level, the hippocampus was particu-

larly heavily stained by DRP2 antibodies (Fig. 5E). Whereas in situ hybridization (Dixon et al., 1997) showed an intense signal in the pyramidal cell bodies of Ammon’s horn (CA1-4), the immunohistochemistry showed sparing of the cell body layer and strong labeling of the dendritic fields in the stratum oriens and stratum radiatum (Fig. 5E). The dentate gyrus was more weakly stained. The combination of somatic mRNA and dendritic protein in the same cell population suggests active targeting of the DRP2 protein from its site of translation in the cell body to the dendrites. In concordance with the in situ findings, there was general DRP2 antibody staining of the thalamus and hypothalamus, with a number of regions (e.g., medial habenula and reticular nucleus in the thalamus, posterior nucleus in the hypothalamus) showing especially strong signals. The medial habenula, which is strikingly bright in in situ images, was also intensely stained in a dense mesh of processes by anti-DRP2 antibodies (Fig. 5F). An example of staining in the posterior hypothalamic nucleus is also shown (Figs. 5G, 6E, and 6F). As in cortex and hippocampus, the neuronal cell bodies were relatively spared, contrasting with the dense immunoreactivity of surrounding processes and neuropil. In some cases, bright rings of DRP2 puncta outlining the neuronal somata could be seen in confocal images (e.g., Fig. 6E). Moderate staining for DRP2 was seen throughout the striatum (Fig. 6G). In double labeling studies, the general pattern of DRP2 staining by confocal microscopy was similar to that of PSD-95 in that both were characterized by punctate immunoreactivity within the synaptic neuropil and sparing of cell somata. However, the punctate staining for PSD-95 appeared to be of greater density than that of DRP2.

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Striking staining for DRP2 was observed in the glomeruli of the main olfactory bulb (Fig. 5H), with intense punctate labeling of processes confined to each glomerulus (Figs. 6A and 6B). Based on the anatomy of the olfactory bulb, and the strong hybridization signal for DRP2 mRNA in olfactory bulb periglomerular cell bodies (Dixon et al., 1997), we conclude that DRP2 immunoreactivity is concentrated in the dendrites of periglomerular cells. Such dendrites are known to project into glomeruli to form synapses with both olfactory nerve axon termini and dendrites of other periglomerular cells and mitral/tufted cells (Kosaka et al., 1998). The cerebellum stained relatively poorly for DRP2; punctate staining was visible in both molecular and granule cell layers (Figs. 6C and 6D). Larger puncta were distributed over or inside the cell bodies of Purkinje cells.

DISCUSSION DRP2 in the Brain

FIG. 5. Immunohistochemical localization of DRP2 in the rat brain. Dorsal is up in all images. (A, B, E–G) coronal sections, with medial to the left. (A) Low magnification view of a section of rat cerebral cortex, showing heavy staining of most layers, with somewhat lighter staining of layer IV. Note absence of staining in the corpus callosum. (B) Higher magnification of boxed region of cortex from (A), showing staining of apical dendrites. (C and D) High magnification confocal fluorescent images of similar areas, showing punctate staining of dendrites and neuropil. (E) Low magnification view of hippocampus showing strong DRP2 reactivity in the dendrites throughout the stratum oriens and stratum radiatum, with clear sparing of the pyramidal cell bodies. (F) Intense staining of processes in the medial habenula. (G) Posterior nucleus of the hypothalamus, showing intense staining of certain cell bodies and processes. (H) Low magnification image of olfactory bulb (sagittal section; right is anterior), showing intense staining of glomeruli. Arrowheads indicate glomer-

DRP2 transcripts have previously been characterized on the basis of a few cDNA clones and RT-PCR products (Roberts et al., 1996). As the related genes (dystrophin, utrophin, ␣- and ␤-dystrobrevin) are all subject to elaborate use of multiple promoters and/or alternative splicing, we felt it important to establish the repertoire of DRP2 transcripts. We found in the brain (the principal site of expression of DRP2) no evidence for alternative splicing or a Dp71-like internal promoter. All DRP2 transcripts appear to originate from two start sites in a single promoter region. Both the 5⬘UTR and the coding region are highly conserved between primates and rodents, implying that the function of DRP2 is important for survival and that extrapolations can be confidently made between these organisms. Antibodies raised against N- and C-terminal fusion proteins of DRP2 recognize in rat brain a characteristic set of polypeptides of around the expected molecular weight of DRP2. The heterogeneity of these bands probably arises from posttranslational modifications or par-

uli. Abbreviations: CA1, Ammon’s horn, CA1 field; cc, corpus callosum; Hc, hippocampus; so, stratum oriens; sr, stratum radiatum; DG, dentate gyrus; v3, third ventricle; MH, medial habenula; PH, posterior hypothalamic nucleus; Cx, cerebral cortex; OB, olfactory bulb; AOB, accessory olfactory bulb. Bar, 200 ␮m in A, E, G, and H; 40 ␮m in B–D and F.

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tial degradation. More interestingly, DRP2 is associated with membranes, highly enriched in PSD fractions and insoluble in nonionic detergents, suggesting that it is a component of the cytoskeleton at postsynaptic sites. Consistent with a possible role in postsynaptic structures, DRP2 expression levels increase during the first 3 weeks of postnatal brain development, when synaptogenesis is most active, as does PSD-95. A synaptic enrichment of DRP2 is also supported by immunohistochemistry. On the whole, staining was neuronal, punctate, and predominantly associated with major dendrites and gray matter neuropil. In a few areas, cell body staining was also observed, but mostly DRP2 immunoreactivity was sparse in neuronal somata where its mRNA is concentrated, again resembling PSD-95 in this regard. We are convinced of the authenticity of the DRP2 staining for the following reasons: (a) The same pattern was obtained with antibodies from different rabbits injected with the same immunogen. (b) Antibodies raised against a nonoverlapping N-terminal DRP2 immunogen gave a similar, albeit weaker, pattern. (c) The staining was abolished by preincubation of the antibody with DRP2 fusion protein, but not with the corresponding dystrophin fusion protein. (d) Most preparations of antibody showed high specificity on Western blots for DRP2 over its homologues dystrophin and utrophin and reacted strongly only with the characteristic DRP2 bands of 100 –120 kDa in brain. Those preparations that showed less discrimination among DRP2 relatives consistently stained the microvasculature, a feature of staining by a commercial dystrophinspecific antibody. (e) The expression pattern was entirely consistent at the cellular level with that obtained by in situ hybridization with two independent DRP2specific oligonucleotide probes (Dixon et al., 1997).

Candidate Components of the DRP2 Complex

FIG. 6. Confocal double immunofluorescent images of DRP2 staining. Images show staining with: A, C, E, G, anti-DRP2; B, D, H, pan-anti-PSD95/SAP; F, anti-PSD95. (A, B) An olfactory bulb glomerulus, showing DRP2 staining of processes within the glomerulus (gl) contrasting with staining for PSD95-family member(s) expressed in incoming olfactory nerve (ON). (C, D) A region of the cerebellum (sagittal section), centered on the Purkinje cell body layer. Asterisks indicate Purkinje cell nuclei; m and g, molecular and granule cell layers, respectively. (E, F) Part of the posterior hypothalamic nucleus,

Given the high level of similarity between dystrophin, utrophin, and DRP2 within the C-terminal domains, it might be expected that these three proteins perform somewhat analogous roles. All three are now known to be expressed in the brain in patterns which are partially overlapping. Dystrophin is most highly expressed in the cortex, cerebellum, and hippocampus

showing intense punctate staining around a subset of cell bodies. (G, H) Confocal images of a representative portion of the striatum. Bar, 20 ␮m.

682 (Go´recki et al., 1992; Lidov et al., 1990, 1993); dystrophin is also enriched in PSD fractions (Kim et al., 1992), like DRP2. Electron microscopic studies reveal dystrophin to be localized in somatic PSDs in Purkinje cells (Lidov et al., 1990). Utrophin, on the other hand, is found in perivascular astrocytes (at the surface apposed to the vascular endothelium) and at certain specialized membrane structures (the pia mater, choroid plexus, and ependymal linings (Khurana et al., 1992)). A number of proteins interact directly with the Cterminal domains of dystrophin and/or utrophin. These immediately become candidate interactors of DRP2. Dystroglycan is a transmembrane receptor for agrin and laminins (Ervasti and Campbell, 1993; Gee et al., 1994; Campanelli et al., 1994). Although DRP2 is highly similar to dystrophin in the region that interacts with ␤-dystroglycan (the WW domain and the subsequent 250 amino acids), expression of dystroglycan in the brain is relatively limited, with levels undetectable in the cortex (Go´recki et al., 1994). Mummery et al., however, found that a distinct protein, immunologically related to dystroglycan, was highly enriched in PSD fractions of rat brain (Mummery et al., 1996). It remains to be determined whether DRP2 binds to dystroglycan or related proteins. The syntrophins, of which there are five known types (␣1, ␤1, ␤2, ␥1, ␥2), are adaptor proteins containing plekstrin homology and PDZ domains that bind to a region of dystrophin/utrophin that is fairly well conserved in DRP2 (amino acids 730 –780 of DRP2). In support of a direct interaction between DRP2 and syntrophin, we isolated a clone encoding full-length rat ␤1-syntrophin as a specific DRP2-binding protein in a DRP2 yeast two-hybrid screen of a brain cDNA library (R. G. Roberts, unpublished observations). Since the syntrophin family as a group binds both to the dystrophins and to the distantly related dystrobrevins, we anticipate that all five syntrophins should also interact with DRP2. Indeed, all known members of the syntrophin family are expressed in the brain (Adams et al., 1995; Go´recki et al., 1997; Piluso et al., 2000). Further work will be needed, however, to confirm an association of DRP2 and syntrophins in vivo. Finally, the dystrobrevins, small (87-kDa) proteins distantly related to the dystrophin C-terminal domains, interact with dystrophin and utrophin via the first coiled coil domain (Sadoulet-Puccio et al., 1997), a region that is conserved in DRP2. The recently identified ␤-dystrobrevin (Blake et al., 1998; Peters et al., 1997) is highly expressed in brain (largely in neurons, as opposed to the vascular localization of ␣-dystrobrevin)

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and would be an additional plausible binding partner for DRP2. DRP2 and Cholinergic Signaling Members of the dystrophin family have been implicated in cholinergic synapse function. For instance, the vertebrate paralogue utrophin is highly enriched in the neuromuscular junction (Khurana et al., 1991; Matsumura et al., 1992) (a specialized cholinergic synapse) and is closely but indirectly associated with nicotinic acetylcholine receptors. In addition, loss of the nematode dystrophin-like protein dys-1 (the orthologue of the last common ancestor of dystrophin, utrophin, and DRP2) results in a movement disorder which seems to stem from a hypersensitivity to acetylcholine (Bessou et al., 1998). This raises the possibility that a role in cholinergic transmission is ancestral in the dystrophin family and that the sarcolemmal distribution of vertebrate dystrophin itself (which does not appear to have a cholinergic link) may be a more recently acquired trait. With this in mind, it is provocative that the distribution of DRP2 protein bears a striking resemblance to that of central markers of cholinergic transmission. A sensitive marker that has recently come into use is the vesicular acetylcholine transporter (VAChT) (see, for example, Gilmor et al., 1996). Antibodies to VAChT show heavy staining of the caudate putamen, medial habenula, reticular thalamic nucleus, olfactory bulb glomeruli, lateral septal nucleus, and cerebral cortex which is highly reminiscent of the staining described here for DRP2. The expression of nicotinic acetylcholine receptors is also very similar at both the regional level (see, for example, expression of ␣-3 and ␣-4 genes in the cortex, hypothalamus, and habenula (Goldman et al., 1987)) and at the subcellular level (e.g., the coexpression of nicotinic and muscarinic AChRs in apical dendrites of cortical pyramidal neurons (van der Zee et al., 1992)). Given these correlations, a role for DRP2 in the organization of central cholinergic synapses must be considered. Determining at the ultrastructural level whether DRP2 is localized in cholinergic synapses would be helpful in this regard.

EXPERIMENTAL METHODS Cloning of Rat DRP2 cDNA The rat DRP2 cDNA was cloned in four sections using primers based on the human and/or partial murine DRP2 sequence (Accession Nos. U43519, U43520;

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primer sequences available on request) to perform PCR, 5⬘RACE, and 3⬘RACE from Marathon rat brain cDNA (Clontech) according to the manufacturer’s instructions. In the case of 5⬘RACE, parallel reactions were performed using Marathon human brain cDNA. PCR products were cloned into T-tailed pBluescript II SK⫺ (Stratagene) or pCR2.1-TOPO (Invitrogen) and sequenced using dRhodamine dye-terminator methodology on an ABI Prism 377 automated sequencer. Generation of Fusion Proteins Fusion proteins were generated by inserting cDNA encoding relevant members of the dystrophin family into the vectors pRSETB (Invitrogen; H 6- fusions), pET32a(⫹) (Novagen; thioredoxin or Trx fusions), or pGEX4T-1 (Pharmacia Biotech; GST fusions). The GST fusions of the C-terminal regions of dystrophin, utrophin, and DRP2 (Fig. 2) contained residues 3422–3685 of muscletype dystrophin, 3179 –3433 of utrophin, and 706 –957 of DRP2. Fusion protein constructs were transformed into Escherichia coli bacterial host strain BL21(DE3) (Novagen) and induced with isopropyl-␤-d-thiogalactopyranoside. Where necessary, H 6 and Trx fusion proteins were purified from bacterial lysates via their hexahistidine tags by passage through a Ni–NTA–agarose column (Probond, Invitrogen) followed by elution with imidazole. Generation and Affinity Purification of Antibodies A purified hexahistidine-tagged fusion protein of the C-terminal 252 amino acids (residues 706 –957) of human DRP2 (H 6-DRP2D) was injected into two rabbits and sera were collected (DRP2D1, DRP2D2) by Animal Pharm Services, Inc. (Healdsburg, CA). Antibodies were affinity purified from sera using chromatography through a column of fusion protein covalently linked to a agarose support (Sulfolink, Pierce). In order to reduce cross-reactivity with dystrophin, some sera were predepleted by passing over a column to which the corresponding region of dystrophin (H 6-DysD) had been bound, before affinity purifying on the H 6-DRP2D column. Antibodies were also similarly generated against an N-terminal fragment (residues 4 –231) of human DRP2 (H 6-DRP2A) and affinity purified. Heterologous Cell Expression The construct pGW1-DRP2 was made, containing the entire coding region of human DRP2, together with 109 bp of 5⬘UTR and 15 bp of 3⬘UTR, inserted between the

HindIII and EcoRI sites of the mammalian expression vector pGW1-CMV. This was transfected into cultured COS7 cells using Lipofectamine (Life Technologies) according to the manufacturer’s instructions. After 48 h of growth the cells were harvested and lysed in SDS loading buffer. Subcellular Fractionation and Western Blotting Fractionation of rat brain homogenates was as previously described (Huttner, 1983). PSD purification and detergent extraction were performed as in Cho et al. (1992). For immunoblotting, proteins were boiled in SDS loading buffer and briefly centrifuged before loading onto a discontinuous SDS-containing 7.5% polyacrylamide gel in Tris/glycine buffer. After electrophoresis, gels were electroblotted onto Hybond–ECL nitrocellulose membrane (Amersham Life Sciences) using a Bio-Rad mini trans-blot apparatus. After visualization of proteins using Ponceau-S stain, membranes were blocked using 5% nonfat dried milk in Tris-buffered saline (TBS). Membranes were then probed with primary antiserum in TBS containing 2% bovine serum albumin and 2% horse serum. After washing, the membranes were probed with secondary antibody (donkey anti-rabbit Ig) conjugated to horseradish peroxidase (Amersham Life Sciences), and the signal was visualized using Renaissance Plus chemiluminescence reagent (NEN Life Science Products) and exposure to X-ray film. Some blots were subsequently stripped and then reprobed. Immunohistochemistry Adult rat brains were fixed by perfusion with 4% paraformaldehyde followed by postfixing overnight. Fifty-micrometer sections were cut with a Vibratome and placed in phosphate-buffered saline. For diaminobenzidene (DAB) staining, sections were pretreated with 1% hydrogen peroxide. Sections were then permeabilized with 0.1% Triton X-100, blocked with 3% horse serum and 0.1% bovine serum albumin, and incubated with primary antibody overnight. After washing, sections were incubated with either fluorescent secondary antibodies (Cy3-conjugated anti-rabbit Ig or FITC-conjugated anti-mouse Ig; Jackson ImmunoResearch) or, for DAB staining, Vectastain biotinylated anti-rabbit Ig (Vector Laboratories). Fluorescent sections were then mounted in Vectashield and visualized using Zeiss Axioscope and Bio-Rad confocal MRC 1000 microscopes. Other sections were further treated with Vectastain ABC complex then incubated with 0.05% DAB with

684 0.01% hydrogen peroxide. After rinsing, sections were dehydrated and mounted in Permount.

ACKNOWLEDGMENTS M.S. is Assistant Investigator of the Howard Hughes Medical Institute. R.G.R. was supported by an International Travel Fellowship from the Wellcome Trust, UK.

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