Dystrophin and its isoforms in a sympathetic ganglion of normal and dystrophic mdx mice:...

DYSTROPHIN AND ITS ISOFORMS IN A SYMPATHETICGANGLION OF NORMAL AND DYSTROPHIC mdx MICE:IMMUNOLOCALIZATION BY ELECTRON MICROSCOPY

AND BIOCHEMICAL CHARACTERIZATION

M. E. DE STEFANO,* M. L. ZACCARIA,* M. CAVALDESI,† T. C. PETRUCCI,†R. MEDORI‡ and P. PAGGI*§

*Dipartimento di Biologia Cellulare e dello Sviluppo, Universita ‘‘La Sapienza’’, P. le Aldo Moro 5,00185, Roma, Italy

†Istituto Superiore di Sanita, 00161, Roma, Italy

‡Lilly Deutschland, 61350, Bad-Homburg, Germany

Abstract––In normal mouse superior cervical ganglion, dystrophin immunoreactivity is present inganglionic neurons, satellite cells and Schwann cells. It is associated with several cytoplasmic organellesand specialized plasma membrane domains, including two types of structurally and functionally differentintercellular junctions: synapses, where it is located at postsynaptic densities, and adherens junctions.Dystrophin immunostaining can be ascribed to the 427,000 mol. wt full-length dystrophin, as well as to theseveral dystrophin isoforms present in superior cervical ganglion, as revealed by western immunoblots. Inmdx mouse superior cervical ganglion, which lacks the 427,000 mol. wt dystrophin, the unchanged patternof dystrophin immunolabelling observed at several subcellular structures indicates the presence ofdystrophin isoforms at these sites. Moreover, the absence of labelled adherens junctions indicates thepresence of full-length dystrophin at this type of junction in the normal mouse superior cervical ganglion.The lower number of immunopositive postsynaptic densities in mdx mouse superior cervical ganglion

than in normal mouse ganglion suggests the presence, in the latter, of postsynaptic densities withdifferently organized dystrophin cytoskeleton: some containing dystrophin isoforms alone or togetherwith 427,000 mol. wt dystrophin, and others containing 427,000 mol. wt dystrophin alone. ? 1997 IBRO.Published by Elsevier Science Ltd.

Key words: mouse superior cervical ganglion, dystrophin, dystrophin isoforms, cytoskeleton, mdx mouse,immunoelectron microscopy.

The functional properties of a neuron and of thecircuit to which it belongs are strictly related to thestructural and functional organization of specializedneuronal membrane domains. It has been shown thatcortical cytoskeletal proteins, such as spectrin, playan essential role in the organization and maintenanceof highly organized neuronal membrane regions(reviewed in Refs 2, 26). Less is known about the roleof dystrophin, a protein of the cortical cytoskeletonwith a high mol. wt that belongs to the spectrinsuperfamily.19 Encoded by a gene located on the Xchromosome,14 this protein is defective in the muscleand brain of patients affected by Duchenne musculardystrophy (DMD),3,17,28,29,42–44 as well as of geneti-cally dystrophic mice (mdx).23,37

The finding that dystrophin is associated with aglycoprotein complex, referred to as dystrophin-associated proteins (DAPs),11,40 at the sarcolemmalmembrane suggests that dystrophin may play a keyrole among the proteins involved in linking theextracellular matrix to the inner cell cytoskeleton.9,34

Moreover, in the brain, dystrophin is localized at thesynaptic regions24 and co-fractionates with post-synaptic densities,18,32 suggesting a role in thegenesis and maintenance of interneuronal synapticconnections.In contrast to muscle, the nervous system expresses

a variety of isoforms,6,7,10,21,25,31 all encoded by thedystrophin gene and generated through alternativesplicing or differential promoter usage.13,31 Both thepresence of multiple dystrophin isoforms and thelocalization of dystrophin at specialized neuronaldomains (postsynaptic densities) suggest that dys-trophin has different roles in neurons and in muscle.Certainly, an insight into the neuronal function ofdystrophin, alone or together with its isoforms, maycome from identification of the subcellular sites towhich dystrophin proteins are associated.

§To whom correspondence should be addressed.Abbreviations: BSA, bovine serum albumin; DAB, 3,3*-diaminobenzidine tetrahydrochloride; DAPs, dystrophin-associated proteins; DMD, Duchenne muscular dystro-phy; DMSO, dimethylsulphoxide; PAP, peroxidase–antiperoxidase; SCG, superior cervical ganglion; SDS,sodium dodecyl sulphate; SDS–PAGE, sodium dodecylsulphate–polyacrylamide gel electrophoresis.

Pergamon

Neuroscience Vol. 80, No. 2, pp. 613–624, 1997Copyright ? 1997 IBRO. Published by Elsevier Science Ltd

Printed in Great Britain. All rights reserved0306–4522/97 $17.00+0.00PII: S0306-4522(97)00003-1

613

To date, nothing is known about the presence ofdystrophin and its isoforms in sympathetic ganglia.Therefore, we chose to characterize and localize themat the subcellular level in the sympathetic superiorcervical ganglion (SCG) of normal and mdx dys-trophic mice. This ganglion contains a limited varietyof nerve cells originating from the neural crest:catecholaminergic neurons and satellite cells. Theneurons are synapsed by preganglionic cholinergicterminals and postganglionic adrenergic elementsthat impinge on both the somata and their largedendritic tree (reviewed in Refs 27, 45). The presenceof numerous synapses and mechanical junctions,such as the adherens junctions, may allow us toexamine the distribution of dystrophin and its iso-forms at various specialized membrane domains andto gain an insight into their possible functional role.

EXPERIMENTAL PROCEDURES

Animals

Normal (C57BL/10) and dystrophic (C57BL/10 mdx/mdx) young adult mice (18–20 g body weight) (CharlesRiver, Italia SPA, Calco, Italy) were used. The mice werehoused and handled in accordance with the guidelinesproposed by the European Communities Council Directive(86/609/EEC of 24 November 1986) and the AmericanSociety for Neuroscience.

Antibodies

Two polyclonal antisera, P6 and H12 (courtesy of Dr P.Strong, Hammersmith Hospital, London, U.K.), were used.These antibodies are directed against the C-terminal regionof the dystrophin rod domain and recognize the full-lengthdystrophin (427,000 mol. wt) and lower mol. wt dystrophinisoforms. All dystrophin isoforms share the C-terminalregion and a variable length of rod domain with thefull-length molecule (Fig. 1). Moreover, P6 and H12 anti-sera do not recognize utrophin,37 a protein with a highsequence homology with full-length dystrophin that is theputative autosomal gene product. A polyclonal antiserum(á-D2) against dystrophin from Torpedo electric organ(courtesy of Dr J. Cartaud, Centre National de la ResercheScientifique, Universite Paris, Paris, France) and a mouse

monoclonal antibody (DYS1) against the mid-rod domainof human dystrophin (Novocatsra, Newcastle upon Tyne,U.K.) were also used in the biochemical characterization ofthe dystrophin isoforms.

Immunocytochemistry

The animals were deeply anaesthetized with an i.p. injec-tion of chloral hydrate (400 mg/kg body wt) (Fluka) andperfused through the aorta with a variant of oxygenatedRinger’s solution, pH 7.3, followed by a fixative composedof 4% freshly depolymerized paraformaldehyde in 0.1 Mphosphate buffer, pH 7.4. Immunocytochemistry for dys-trophin was carried out with the peroxidase–antiperoxidase(PAP) procedure.

Light microscopy

After perfusion, the SCGs were dissected and cryopro-tected overnight with 30% sucrose in saline, at 4)C. Cryostatsections (15 µm thick) were cut and collected free floating in0.5 M Tris–HCl, pH 7.6. The sections were incubated for10 min in 10% methanol and 3% H2O2 in 0.5 M Tris–HCl toinactivate endogenous peroxidase, thoroughly rinsed inbuffer and incubated for 1 h at room temperature, with ablocking solution of 5% dry milk and 0.5% Triton X-100 in0.5 M Tris–HCl. Specimens were then incubated for 36 h at4)C with the primary antisera to dystrophin, diluted 1:100(P6) and 1:1000 (H12) in 1% dry milk, 0.2% Triton X-100 in0.5 M Tris–HCl. Since both H12 and P6 gave identicalresults in localizing dystrophin, in this paper we will notdiscriminate between the two antibodies. After rinsing with0.5 M Tris–HCl, the sections were incubated for 1 h in goatanti-rabbit IgG (Jackson Immunoresearch Laboratories,West Grove, PA, U.S.A.) diluted 1:100, rinsed again andincubated for 1 h in rabbit PAP (Jackson ImmunoresearchLaboratories) diluted 1:600. After rinsing in buffer, speci-mens were incubated in 0.05% 3,3*-diaminobenzidine tet-rahydrochloride (DAB) and 0.01% H2O2 in 0.5 M Tris–HClto reveal antibody binding sites, thoroughly rinsed again inbuffer and dry mounted.

Electron microscopy

Vibratome sections (40 µm thick) were cut and collectedfree floating in 0.1 M phosphate buffer. The immunolabel-ling steps were as specified above, except for the incubationtime of the primary antibodies, which was 16 h, and thebuffer used for rinsing and antibody diluent, which wasphosphate buffer. Moreover, Triton X-100 was never usedduring incubation, and penetration of the antibodies wasfacilitated by freeze–thaw. In brief, sections were cryo-protected with 10% dimethylsulphoxide (DMSO) and 1%glycerol in 0.5 M Tris–HCl (10 min at 4)C), followed by20% DMSO and 2% glycerol (2#10 min at 4)C), transferredto the bottom of an aluminium planchette, quickly frozen inliquid nitrogen-cooled isopentane and then slowly thawedfour times. After the DAB reaction, sections were osmi-cated, treated with uranyl acetate, dehydrated and flatembedded in Epon 812. Ultrathin sections (60–70 nm thick)were cut on an ultramicrotome. Some of the sections werelightly counterstained with 0.2% lead citrate, while otherswere left unstained to allow better identification of theimmunopositive elements.Owing to the limited penetration of immunoreagents

obtained with this pre-embedding immunostaining method,the observations reported in the Results section were madeat the surface of the Vibratome slices, well within the rangeof antibody penetration. This should reduce, though notcompletely avoid, the risk of false-negative findings.

Control experiments

Control experiments were performed either by incubatingganglionic sections with H12 antibody preabsorbed with a

Fig. 1. Schematic representation of full-length dystrophin(427,000 mol. wt) and its isoforms (Dp) at lower mol. wts,showing the predicted position of the epitopes for H12 andP6 antibodies (arrows). Dotted box, N-terminal region;dashed box, cysteine-rich region; blank box, C-terminal

region.

614 M. E. De Stefano et al.

dystrophin enriched fraction (prepared by alkaline extrac-tion of microsome rabbit skeletal muscle according to Sharpet al.36) or by omitting the primary antibody. The sectionswere then treated for light and electron microscopy asdescribed above. In all cases the sections were free ofimmunoreaction products.

Preparation of superior cervical ganglion extracts

Mice were anaesthetized with ether and killed by decapi-tation. The ganglia were removed, frozen and stored at"70)C until use. Subsequently, SCGs were homogenized in25 mM Tris–HCl buffer containing 1% sodium dodecylsulphate (SDS) and 3 mM urea, pH 7.4, with a ground-glassmicrohomogenizer kept in ice. The homogenates were thencentrifuged at 100,000 g for 15 min at 4)C. Supernatantswere then boiled for 2 min. A measured aliquot of superna-tants was used to determine the protein concentration.4

Other aliquots of the samples, containing seven SCGs andcorresponding to 150 µg of proteins, were analysed bysodium dodecyl sulphate–polyacrylamide gel electro-phoresis (SDS–PAGE) and immunoblot.

Electrophoresis and immunoblotting

Proteins were separated by SDS–PAGE20 on 5% acryl-amide gels. The mol. wt standards were: myosin 200,000,â-galactosidase 116,000, phosphorylase B 97,000 and bo-vine serum albumin (BSA) 66,000. Proteins were thentransferred to nitrocellulose for western blotting.41 Follow-ing incubation with the primary antibody, the reactivebands were visualized by an enhanced chemiluminescenceprocedure (Pierce, Rockford, IL, U.S.A.), using a 1:5000dilution of the peroxidase-conjugated anti-rabbit secondaryantibody and RPN3103 films (Amersham Life Science,Bucks, U.K.). Antibodies against dystrophin, P6 and H12were diluted 1:700 with 3% BSA in 0.15 M NaCl, 10 mMTris–HCl, pH 7.4. The polyclonal antiserum (á-D2) and themouse monoclonal antibody (DYS1) were diluted 1:1000and 1:15, respectively. The heavy microsome fraction, pre-pared from rabbit skeletal muscle according to Sharpet al.,36 was used as a positive control for dystrophin.

RESULTS

Antibody specificity

No differences in the immunostaining pattern werefound between SCG sections incubated with affinity-purified P6 and H12 antibodies, and those incubatedwith immune sera or purified IgG. Immunocyto-chemistry was therefore carried out using dilutedimmune sera. Gastrocnemius muscles from normaland mdx mice were used as controls for the presenceand absence of full-length dystrophin, respectively. Innormal animals, the immunoreaction product wasclearly and abundantly distributed along the perim-eter of skeletal muscles fibres (Fig. 2D), while in mdxmice it was almost completely absent (Fig. 2E). Onlya few revertant fibres, already described in mdxmice39 and DMD patients,37 showed faint andirregularly distributed immunoreactivity (Fig. 2E).

Immunolocalization of dystrophin and its isoformsby light microscopy in superior cervical ganglia andmuscle of normal and mdx mice

The SCG was preferentially cut longitudinally, inorder to orientate it on the glass slides so as to

facilitate recognition of its proximal and distal por-tions and afferent and efferent nerves. On cryostatsections, the immunoreaction product for dystrophinand/or its isoforms appeared as a homogeneousnon-granular precipitate mainly surrounding theperikarya of the ganglionic neurons (Fig. 2A,B, insetto Fig. 2A), although a faint immunoreactivity wasobserved in the neuronal cytoplasm at higher mag-nification (Fig. 2B, inset to Fig. 2A). Nerve fibres,both within the ganglion and along preganglionicand postganglionic nerves, were also immunostained(Fig. 2A,B). In dystrophic mice, contrary to theobservation in skeletal muscle (Fig. 2E), the antibodylabelling of SCG was the same as in normal mice(Fig. 2C). Considering the normal variability betweenone experiment and another, the intensity of labellingwas apparently unchanged. We therefore decided toperform an immunoelectron microscopy study.

Immunolocalization of dystrophin and its isoforms byelectron microscopy in normal mouse superior cervicalganglion

Localization at plasma membrane specializations:synapses and adherens junctions. Immunoreactivity ofdystrophin and dystrophin isoforms was widely dis-tributed throughout the ganglionic neurons. About60–65% of more than 100 observed postsynapticspecializations are immunopositive (Fig. 3A,B). Al-though our observations were conducted at the sur-face of the specimens, within the range of antibodypenetration, the risk of false negatives is still high,especially for structures as thin as these ganglionicpostsynaptic densities, and the above percentage mayrepresent an underestimate. Presynaptic boutonswere never labelled (Fig. 3A,B).Large interneuronal adherens junctions were

positive to dystrophin antibodies (Fig. 3C,D) (about30–35% of more than 100 observed). Only portions ofthe dense plaques that are typical of this type ofjunction were stained and often the immunoreactionproduct was not evenly distributed on both sides(Fig. 3C,D). Small adherens junctions, formed be-tween perineuronal satellite cell processes or betweensatellite cells and ganglionic neurons (Fig. 3E), werealso immunopositive. The immunoreaction productwas intense and sometimes extended for a shortlength inside the facing cytoplasms (Fig. 3E). Bothsynapses and adherens junctions were immuno-negative when the primary antibody was omitted(Fig. 3F,G), as well as when the sections were treatedwith the dystrophin antibody previously incubatedwith skeletal muscle dystrophin (not shown).

Localization at membrane and cytoplasmic do-mains. Small somatic spines were sporadicallyimmunopositive (Fig. 4A,B); the immunoreactionproduct either filled the spines entirely (Fig. 4A) orwas localized at points on the neck (Fig. 4B) or tip.Discrete patches of dystrophin immunoreactivity

were found beneath the area of the plasma membrane

Dystrophin in mouse superior cervical ganglion 615

of the neuronal soma (Fig. 4C) devoid of postsynap-tic densities and adherens junctions. Frequently,cytoplasmic organelles, such as cisterns of the endo-plasmic reticulum, were found close to theseaggregates (Fig. 4C). In the cytoplasm, the immuno-reaction product formed sparse and small aggregates,

probably associated with clusters of free ribosomes.Occasional cisterns of the rough endoplasmic reticu-lum (Fig. 4D) were immunopositive on their mem-branes but not in their lumina. Sometimes, cisterns ofthe Golgi apparatus were also immunolabelled onthe lateral budding portion (Fig. 4E). Numerous

Fig. 2. Immunolocalization of dystrophin in SCG and gastrocnemius muscle of normal and mdx mice(light microscopy, cryostat sections). (A) Normal mouse SCG: the immunoreaction product is distributedmainly along the perimeter of the neuronal cell bodies and their fibres (f). CST, cervical sympathetic trunk;ECN, external carotid nerve; ICN, internal carotid nerve. #100. Inset to (A), (B,C): High magnificationof neurons from normal (inset to A,B) and mdx (C) mouse SCG. Arrows point to perineuronalimmunopositivity. The neuronal cytoplasm is faintly stained; some of the neurons (n) and nerve fibres (f)are indicated. Inset,#250; B,C,#450. (D) Gastrocnemius muscle of normal mouse. The immunoreactionproduct is distributed evenly along the perimeter of all muscle fibres. #200. (E) Gastrocnemius muscle ofmdx mouse. The majority of muscle fibres are negative. Only two revertant fibres (asterisks) show faint

and irregular immunopositivity. #250.


Fig. 3. Electron micrographs showing dystrophin immunoreactivity in normal mouse SCG. (A,B) Twopreganglionic boutons (b) synapse on a dendritic spine (ds in A) and a dendritic shaft (d in B). Thepostsynaptic densities (arrows) are immunopositive. In (B), SC indicates an immunopositive process of aperineuronal satellite cell. #61,600. (C,D) Large adherens junctions, established between two neuronalperikarya (N) and two dendrites (d), show an uneven distribution of the immunoreaction product(arrowheads) associated with the intracytoplasmic dense plaques. In (C), the dendrite is quite large and liesparallel to the neuronal cell body. v, cluster of intracytoplasmic vesicles. #61,600. (E) A small adherensjunction (triangles), established between a heavily immunostained process of a perineuronal satellite cell(SC) and a dendrite (d), is immunopositive on both sides.#61,600. (F,G) Control tissue sections obtainedby omitting the primary antibody. (F) Immunonegative postsynaptic density (arrows). b, preganglionicbouton; ds, dendritic spine. #61,600. (G) Immunonegative large (arrowheads) and small (triangles)

adherens junctions. b, preganglionic bouton; d, dendrite; SC, satellite cell process. #61,600.


Fig. 4. Electron micrographs showing dystrophin immunoreactivity in normal mouse SCG. (A,B) Theimmunoreaction product in (A) fills the neuronal somatic spine (s), and in (B) forms a small aggregate(block arrow) at the neck of the spine (s). The perineuronal satellite cell processes (SC) are alsoimmunopositive. N, neuronal cell bodies; bl, basal laminae. #44,800. (C) An aggregate of dystrophinimmunoreaction product (block arrow) is located underneath the neuronal plasma membrane. Cisterns ofthe endoplasmic reticulum (er) are present in its vicinity. N, neuronal cell body. #41,800. (D) A portionof a cistern of the rough endoplasmic reticulum (rer) is immunopositive (open block arrows). (E)Dystrophin immunoreactivity (open block arrow) is associated with a lateral budding sac of the Golgiapparatus (g). #53,200. (F) A multivesicular body (mb) is strongly immunopositive. In its vicinity, twoother multivesicular bodies (mb1 and mb2) are, in contrast, immunonegative.#41,800. (G) The cytoplasmof these myelinating (SC1) and non-myelinating (SC2) Schwann cells is immunopositive. The myelinlamellae (m) are not immunostained. a, axons. #31,000. Inset: the outer rim of this myelinating Schwanncell cytoplasm (SC) is strongly immunopositive, while its myelin coat (m) is devoid of immunoreaction

product. #35,200.


multivesicular bodies were strongly immunolabelled(Fig. 4F).The perineuronal satellite cells were intensely im-

munopositive. The immunoreaction product entirelyfilled the long thin processes that surround the neu-ronal somata (Figs 3B,E, 4A,B) and their processes(Fig. 4G). The cytoplasmic distribution of dystrophinimmunoreactivity was similar to that described abovefor the ganglionic neurons.The cytoplasm of Schwann cells forming the

myelin sheath of large axons was dystrophin im-munopositive, while the myelin lamellae wereimmunonegative (Fig. 4G, inset to Fig. 4G).

Immunolocalization of dystrophin isoforms by electronmicroscopy in mdx mouse superior cervical ganglion

Differences in dystrophin immunoreactivity be-tween normal and mdx mice SCGs were not detect-able by light microscopy. At the ultrastructural level,however, two striking differences were observed: (i)some of the postsynaptic densities (about 20–25% ofmore than 100 observed) were still immunopositive(Fig. 5A; for an immunonegative postsynaptic den-

sity see Fig. 5B), thus revealing the presence ofdystrophin isoforms; and (ii) the adherens junctionswere always immunonegative (Fig. 5C).Immunopositive and immunonegative somatic

spines alternated along the neuronal perimeter (Fig.5D), as observed in normal animals. The neuronalcell bodies appeared less intensely stained than innormal ganglia, mostly on account of a reduction inthe peripheral (Fig. 5E) and intracytoplasmic patchesof immunoreaction product. The perineuronal satel-lite cells (Fig. 5D,E) and the myelinating and non-myelinating Schwann cells were intensely labelled(not shown, but see Fig. 4G).The subcellular distribution of dystrophin im-

munoreactivity in normal and mdx dystrophic miceis summarized in Table 1.

Biochemical characterization of dystrophin and itsisoforms in normal and mdx mouse superior cervicalganglia

SCG extracts from normal and mdx mice wereexamined by western immunoblot. P6 and H12 anti-bodies recognized several bands in the range of an

Fig. 5. Electron micrographs showing dystrophin immunoreactivity in mdx mouse SCG. (A) Apreganglionic bouton (b) synapses on the perikaryon of a principal neuron (N); the postsynaptic density(arrows) is strongly immunopositive. #61,600. (B) A preganglionic bouton (b) synapses on a dendriticshaft (d); the postsynaptic density (arrows) is not immunolabelled. #61,600. (C) Immunonegative largeadherens junction (arrowheads) established between two dendrites (d). #61,600. (D) Neuronal somaticspines (s1, s2 and s3). s1 is filled with dystrophin immunoreaction product, while s2 and s3 areimmunonegative. A process of a perineuronal satellite cell (SC) is strongly immunopositive. bl, basallamina. #44,800. (E) A faint patch of immunoreaction product is located under the neuronal plasmamembrane (block arrow). The process of a perineuronal satellite cell (SC) is also immunopositive.

N, neuron. #41,800.


apparent mol. wt of 68,000–140,000, with stronglabelling at 116,000 and 71,000 (Fig. 6A). In thefractions of heavy microsomes prepared from rabbitskeletal muscle, used as positive control (Fig. 6A), thesame antibodies recognized a band at 427,000 corre-sponding to full-length dystrophin and several bandsat lower mol. wts corresponding to dystrophin break-down products. When the same blots were exposedfor a longer time—with an unavoidable increase inthe nitrocellulose background—two more bands ofapparent mol. wts of 427,000 and 260,000 werevisible in the SCG extracts from normal mice, butonly the band at 260,000 in the SCG extracts frommdx mice (Fig. 6A*).The presence of bands at mol. wts of 68,000–

140,000 in extracts of mdx mouse SCG (Fig. 6A)indicates that neither these nor the 260,000 mol. wtband (Fig. 6A*) represent degradation productsof full-length dystrophin, but specific dystrophinisoforms.In the SCG extract from normal mice, the 427,000

mol. wt dystrophin was also revealed when thenitrocellulose blots were stained with DYS1, a mono-clonal antibody raised against the mid-rod domain ofhuman dystrophin (Fig. 6B), and with á-D2, a poly-clonal antibody directed against the dystrophin fromTorpedo electric organ (Fig. 6B,B*). Moreover, theá-D2 antibody stained all the other dystrophinisoforms recognized by P6 and H12 antibodies (Fig.6B).

DISCUSSION

The results show that dystrophin immunoreactivitywas present in neurons, satellite cells and Schwanncells of the mouse sympathetic SCG, associated withspecialized plasma membrane domains (postsynapticdensities and adherens junctions) and cytoplasmicorganelles. Western immunoblots revealed severaldystrophin isoforms in both normal and mdx mouseSCGs. These isoforms were located in the samesubcellular structures that showed immunostaining in

SCG of normal and mdx mouse, which lack 427,000mol. wt full-length dystrophin. Full-length dys-trophin alone was present at several postsynapticdensities and at all the adherens junctions labelled innormal mouse SCG.

Biochemical characterization of dystrophin and itsisoforms

Western immunoblots revealed the presence of thefull-length dystrophin and several dystrophin iso-forms at mol. wts of 260,000 and 68,000–140,000 innormal mouse SCG. The staining of full-length dys-trophin with the P6 and H12 antisera was lighterthan that of lower mol. wt bands. However, itspresence was confirmed by the polyclonal antiserumá-D2, which showed a staining pattern similar to thatof P6 and H12, and by the monoclonal antibodyDYS1. The 260,000 mol. wt band probably repre-sents the dystrophin isoform described in mouseretina10,30 and shown to be associated with a normalelectroretinogram.10

As expected,23,37 the absence of the 427,000 mol.wt band in mdx mouse SCG was the only differenceobserved between normal and mdx mouse SCGimmunoblots.The 116,000 mol. wt dystrophin isoform, described

in Schwann cells of the sciatic nerve,6,12 is probablyresponsible for the strong labelling of the wide bandin the 110,000–140,000 mol. wt range. Moreover,apo-dystrophin-1 (Dp 71 and its splice variant),which represents the major product of the DMD genein brain,21 may be responsible for the stained bandsaround 71,000. However, we cannot exclude thatthese bands are breakdown products of the 116,000mol. wt isoform.

Immunolocalization of dystrophin and its isoforms atpostsynaptic densities and adherens junctions

In the normal mouse SCG, only a percentage ofthe observed postsynaptic densities was immuno-labelled by dystrophin antibodies. This may be due to

Table 1. Dystrophin immunoreactivity at subcellular domains in superior cervical ganglia of normal and mdx mice

normal mdx

NeuronsPostsynaptic densities +++/" +/"""Adherens junctions +/""" """"Somatic spines ++/"" ++/""Plasma membrane regions ** *Cytoplasmic organelles ++/"" ++/""

Perineuronal satellite cellsPerikaryon and processes ++++ ++++

Schwann cellsCytoplasm ++++ ++++Myelin """" """"

Scores were attributed to the listed structures as follows: ++++, all positive; """", all negative; +++/", numerouspositive and few negative; +/""", few positive and numerous negative; ++/"", equal number of positive andnegative. The asterisks indicate the presence of numerous (**) and few (*) discrete submembrane clusters ofimmunoreactivity.


low antibody penetration, since aggressive methodsof membrane permeance were not used. Nevertheless,we cannot exclude the presence at these sites ofdystrophin-related proteins not recognized by ourantibodies, such as utrophin,38 the presence of whichhas been demonstrated at the neuromuscular junc-tion33 and at postsynaptic membranes of neuronaldendrites.16

In mdx mouse SCG a few postsynaptic densitieswere still immunopositive, indicating the presence atthese sites of dystrophin isoforms in both normal andmdxmouse SCGs. These isoforms may correspond tothe 110,000 and/or 120,000 mol. wt proteins, thepresence of which at brain postsynaptic densities wasdemonstrated by Kim et al.,18 although other iso-

forms may also be present. The smaller percentage oflabelled postsynaptic densities in mdx mouse SCGcompared with normal mouse suggests that the nor-mal SCG contains postsynaptic densities providedwith 427,000 mol. wt dystrophin.Yokota and Yamauchi44 have shown the presence

of preganglionic cholinergic terminals and post-ganglionic adrenergic elements on the soma of themouse SCG neurons. We were not able to correlate aparticular type of synapse to a specific stainingpattern, since the fixatives used for immunocyto-chemistry did not allow identification of the chemicalnature of the nerve terminals. In order to estabishthis correlation, further studies will be directed tolabel acetylcholine receptors or adrenergic receptors,

Fig. 6. Immunoblot analysis of SCG of normal (ctr) and mdx mice (mdx), using dystrophin polyclonalantibodies P6 and H12 (A,A*), the monoclonal antibody DYS1 and the Torpedo dystrophin polyclonalantibody á-D2 (B,B*). Samples were analysed by electrophoresis on 5% polyacrylamide slab gels. (A,B)Western blots of rabbit skeletal muscle microsome (M) and SCG extracts (G) from normal and mdx mice.Protein loading was approximately 70 µg for M lanes and 150 µg for G lanes. (A) A prominent smearedband, ranging between 112,000 and 140,000 mol. wt (asterisk), and three bands of about 87,000, 71,000and 68,000 mol. wt are present in the SCG of both normal and mdx mice. The 427,000 mol. wt dystrophinband in muscle microsomes is indicated (arrow). (A*) Same blots as in (A), but exposed for a longer time.The 427,000 (arrow) and 260,000 mol. wt bands (arrowhead) in SCG of normal mice are shown. Only the260,000 mol. wt band is present in SCG of mdx mice. (B) The DYS1 monoclonal antibody shows thepresence of the 427,000 mol. wt band (arrow) in both M and G. In G, the á-D2 polyclonal antibody stainstwo faint bands at 427,000 (arrow) and 260,000 mol. wt (arrowhead), a prominent smeared band rangingbetween 112,000 and 140,000 mol. wt (asterisk), and three bands at about 87,000, 71,000 and 68,000 mol.wt. (B*) Same immunoblot with á-D2 antibody as in (B), but exposed for a longer time; the two bands at427,000 and 260,000 mol. wt are clearly stained. In (A) and (B), mol. wt standards (/1000) are indicated

on the left-hand side.


in combination with antibody staining specific foreach dystrophin isoform. Moreover, an insight intothe specific role of dystrophin and its isoforms at theinterneuronal synapses of the mouse SCG might beobtained by interfering with the postsynaptic organ-ization through preganglionic and postganglionicaxotomy.The dystrophin immunoreactivity found at the

adherens junctions in normal mouse SCG suggeststhat dystrophin is a component, together with otheractin binding proteins such as spectrin, of themembrane skeleton underlying mechanical contacts(reviewed in Ref. 26). The lack of staining at theadherens junctions in mdx mouse SCG suggests thatin normal mouse SCG full-length dystrophin ispresent at these sites.The high quantity of negative large adherens junc-

tions observed in normal mouse SCG might be aconsequence of low antibody penetration. However,we cannot exclude the presence in the mouse SCG ofa subtype of adherens junction devoid of dystrophinand its isoforms, considering the finding ofdystrophin-immunonegative adherens junctions inboth cardiac and smooth muscle.5 Therefore, thepresence of both dystrophin-immunopositive anddystrophin-immunonegative adherens junctions maybe indicative of structural heterogeneity in thesecontacts.The uneven distribution of dystrophin immuno-

reactivity often observed at these junctions may bedue to technical reasons (different plane of cut alongthe junction) or, more interestingly, it may reflecta different molecular organization of membranedomains.The function of full-length dystrophin at synaptic

and non-synaptic contacts might be related to itsactin-binding properties. Dystrophin, by linking theinner cytoskeleton to the plasma membrane, mayprovide mechanical stability: (i) to the specializedpostsynaptic domains, being indirectly involved inthe organization of neurotransmitter receptors assuggested for Torpedo electrocytes;15 and (ii) to thecomplex protein structure of the adherens junctions.The function of dystrophin isoforms is still unknown.They lack the actin-binding and part of the spectrin-like domains, but all have in common with full-lengthdystrophin the cystein-rich and C-terminal regions13

through which they bind DAPs.40 The cystein-richand C-terminal domains are functionally important,as mutations in these regions lead to severe musculardystrophy pathology,35,22 as well as to alteredelectroretinogram and cognitive impairment.22

Dystrophin immunoreactivity at membrane andcytoplasmic domains

Dystrophin labelling at membrane and cytoplas-mic domains of both neurons and satellite cells didnot change significantly between normal and mdxmouse SCG, suggesting that the immunoreactivity

derives mostly from dystrophin isoforms. In normalanimals, full-length dystrophin and its isoforms mayco-localize at the same structures.Dystrophin immunopositivity in some somatic

spines suggests a different organization of the innercytoskeleton and/or a different functional state ofsimilar specialized regions of the neuronal plasmamembrane. It may also indicate a general role fordystrophin and its isoforms in stabilizing cell mem-branes, supporting the shape of the cell soma and itsprocesses, and regulating the lateral mobility ofmolecules (i.e. ionic channels) inside the membrane.Immunolabelling of clustered free ribosomes and

multivesicular bodies presumably represents the sitesof synthesis and degradation, respectively, for dys-trophin and its isoforms. Moreover, the immuno-labelling of restricted areas of rough endoplasmicreticulum cisterns and budding Golgi vesicles mayindicate that dystrophin proteins are either intrinsiccomponents of the complex protein coat of theseorganelles or part of the cytoplasmic cytoskeletonsurrounding them. This suggests that dystrophin andits isoforms may be implicated in the movementand/or immobilization of organelles and vesicleswithin the cytoplasmic matrix. A similar role hasbeen proposed for spectrin isoforms,1,8 the super-family to which dystrophin belongs.The intense immunolabelling of the perineuronal

satellite cells, as well as non-myelinating and myeli-nating Schwann cells, is in accord with the immuno-reactivity observed in glial25 and Schwann cells,6,12

where it is related to the presence of the 140,000 and116,000 mol. wt isoforms, respectively. Whetherthe 116,000 mol. wt isoform is also responsible forthe labelling observed in mouse SCG satellite cellsremains to be clarified.

CONCLUSIONS

This study shows that several dystrophin isoforms,including the full-length 427,000 mol. wt dystrophinare widely expressed in mouse SCG, associated withboth plasma membrane specializations and cytoplas-mic organelles. Full-length dystrophin is not re-stricted to postsynaptic densities, but is also presentat adherens junctions. Moreover, the heterogeneity indystrophin immunolabelling observed among thesynapses and in the mechanical contacts revealsthe presence in mouse SCG of subsets of postsynapticdensities and adherens junctions with differentlyorganized cortical cytoskeletons.

Acknowledgements—This work was supported by CNRfunds (Contributo di Ricerca 95.02374.CT04) to PP,Telethon funds to PP and RM (project no. 85) and to TCP(project no. 189) and by a Postdoctoral Research Fellow-ship from Istituto Pasteur-Fondazione Cenci Bolognetti toMEDS. The authors wish to thank Dr E. Mugnaini and DrG. Toschi for their critical review of the manuscript andMaurizio Zini for his technical assistance.


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(Accepted 17 December 1996)


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