Human Molecular Genetics, 2000, Vol. 9, No. 1 47-56
© 2000 Oxford University Press
Subcellular localization and axonal transport of the survival motor neuron (SMN) protein in the developing rat spinal cord
Molecular Neuroanatomy Laboratory, Experimental Neurophysiology Department, Neurological Institute ‘C. Besta’, via Celoria 11, 20133 Milano, Italy, 1Institute of Pharmacological Sciences, University of Milano, via Balzaretti 9, 20133 Milano, Italy and 2Department of General Physiology and Biochemistry, University of Milano, via Celoria 26, 20133 Milano, Italy
Received 22 October 1999; Revised and Accepted 29 October 1999.
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ABSTRACT |
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The subcellular localization of the survival motor neuron (SMN) protein, encoded by the spinal muscular atrophy determining gene, was investigated in motor neurons of the developing and adult rat spinal cord by light and electron microscopy immunocytochemistry. The experiments were carried out with a panel of anti-SMN antibodies, all recognizing an SMN-specific protein band at 39 kDa in HeLa cells and rat spinal cord protein extracts. SMN protein expression decreased during postnatal spinal cord development, but it remained unchanged in distribution and intensity in motor neurons at all ages examined. SMN protein was mainly organized in immunoreactive aggregates sparse in the nucleoplasm and cytoplasm of both mature and developing motor neurons, and it was more rarely localized within the endoplasmic reticulum and in apposition to the external mitochondrial membrane. Most strikingly, the SMN protein was found in association with cytoskeletal elements in spinal dendrites and axons, where it was particularly evident during postnatal development. The present findings suggest that SMN protein may be transported via axoplasmic flow in maturing neurons. Given the RNA-binding activity of SMN, the SMN protein could be involved in the transport of specific mRNAs in axons and dendrites of motor neurons. The reduced transport of specific mRNAs within motor neurons during development could play a role in the motoneuronal degeneration and impaired axonal sprouting observed in spinal muscular atrophy.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The autosomal recessive condition spinal muscular atrophy (SMA) is a neuromuscular disorder of varying clinical severity, primarily characterized by degeneration of brainstem and spinal motor neurons. SMA is divided into three major clinical types (types I, II and III) on the basis of age at onset and clinical course (1). All clinical types of SMA map to a genomic region on chromosome 5q13 where two candidate genes have been reported: the survival motor neuron (SMN) gene and the neuronal apoptosis inhibitory protein (NAIP) gene (2,3). The latter is homozygously deleted in ~50% of type I SMA patients, and it seems to mark the extent of deletion (4–6). In contrast, the SMN gene is disrupted by deletion or gene conversion in >95% of analyzed SMA patients of all grades of clinical severity (2,7,8), and additional different types of mutation, including missense point mutations, have been reported more recently (9–15). These findings conclusively identify this gene as the SMA-determining gene.
Two SMN genes are present on 5q13 in humans: the telomeric gene (tSMN, for telomeric SMN), which is the SMA-determining gene, and the centromeric analog gene (cSMN). Both genes are expressed in functionally active proteins of 294 amino acid residues with no homologies with any known protein (6,16). The cSMN gene is rarely missing in unaffected subjects (2–5%) and in some cases of sporadic adult-onset lower motor neuron disease (17). In contrast to the genomic situation in humans, a single copy of the SMN gene is present in mice and rats (18–20).
The SMN protein is localized in the cytoplasm and newly identified nuclear structures of cultured cells called gems (for gemini of coiled bodies) (21). Gems are associated with coiled bodies and they are probably related to RNA metabolism. The SMN protein interacts with a newly identified protein, SIP1, and the two proteins co-localize in gems (22). The SMN–SIP1 complex is involved in the biogenesis of spliceosomal small nuclear ribonucleoproteins (snRNPs) (23), thus supporting the hypothesis that gems are specifically related to the processing of pre-mRNAs. On the other hand, recent data also suggested a specific association of SMN with Bcl-2, an anti-apoptotic protein that needs to be bound to membranes to exert its activity (24,25). In diseased human tissues, the SMN protein is clearly reduced in type I and II SMA patients (6,16). Moreover, nuclear SMN in gems is lacking in spinal cord sections from a single type I SMA patient (16), and it is reduced in cultured fibroblasts from SMA patients according to the severity of the disease (6).
Despite the above-mentioned data, the pathogenic mechanisms resulting in SMA remain to be solved. Knock-out mice with homozygous SMN disruption are characterized by massive cell death during early embryonic stages, indicating that SMN protein has a fundamental role in cellular survival (26). An apparent contradiction exists between the pathological hallmark of SMA, i.e. motor neuron degeneration, and the supposed housekeeping function of the SMN protein. We previously demonstrated that the SMN protein was strongly expressed in brainstem and spinal motor neurons of rats and monkeys (20). To gain further insights into the role of the SMN protein in motor neurons, the present study investigated the subcellular localization of SMN protein in the developing and adult rat spinal cord. This was achieved by light and electron microscopy immunocytochemistry (ICC) with a panel of different anti-SMN antibodies.
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RESULTS |
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Characterization of anti-SMN antibodies
The anti-SMN antibodies were characterized by western blot analysis and immunoabsorption tests. Figure 1A shows a representative western blot of protein extracts from HeLa cells labeled by the different anti-SMN antibodies used. A single strongly reactive band of ~38–39 kDa was immunodetected by antibodies #1137, #854 and TL, whereas two reactive bands, the 38–39 kDa band and an additional band at ~36 kDa, were recognized by antibody #846 (Fig. 1A). In spinal cord extracts from postnatal day (P) 15 rats, antibodies #854 and #846 recognized an immunoreactive band of ~39 kDa in the total homogenate and cellular membrane fraction, and an additional band of ~36 kDa was recognized in the cellular membrane fraction by antibody #846 (Fig. 1B). No SMN-specific bands were detected in the soluble fraction (data not shown). To test for the specificity of the observed immunoreactive bands, immunoabsorption experiments were performed by incubating the primary antibodies #854 and #846 with the corresponding antigenic peptide. The immunoreactive bands were completely abolished by pre-incubation of the antigenic peptide in the protein extracts from P15 rat spinal cords (Fig. 1B). Similar results were obtained in HeLa cells (data not shown). The specificity of the antibodies was further confirmed by in situ immunoabsorption experiments on spinal cord sections. Again, the immunostaining was abolished by incubating the antibody with the corresponding antigenic peptide (Fig. 1C).
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Light microscopy ICC: SMN localization in the spinal cord
The anatomical distribution of the SMN protein was first investigated in the rat spinal cord during postnatal development (at P1, P8 and P15) and adulthood by means of light microscopy ICC (Figs 2 and 3). Consistent findings were obtained at different cervical and thoracic levels. A progressive reduction of SMN expression was observed from P1 to adulthood (Figs 2 and 3). At P1, SMN immunoreactivity in the spinal cord was intense and evenly distributed through dorsal and ventral spinal laminae (Fig. 2A and D). SMN immunoreactivity was mainly localized in the nuclei of most neurons of the dorsal and intermediate spinal laminae, whereas it was particularly intense in the cytoplasm of lamina IX motor neurons (Fig. 2A and D). In the ventral horn, moreover, many thread-like processes entering the ventral funiculus, possibly representing axons from lamina IX motor neurons, were intensely SMN immunoreactive (Fig. 3A). In the white matter, intense SMN immunoreactivity was present in the cuneate tract medial to the dorsal horn (Fig. 2A and D). At P8 and P15, SMN immunoreactivity was slightly decreased in comparison with that observed at P1, but it was still intense in lamina IX motor neurons and neuropil (Figs 2B and E and 3C). SMN-immunoreactive thread-like processes were still evident leaving the ventral horn and entering the dorsal horn through the dorsal root (Fig. 3B). In the white matter, SMN labeling was particularly intense in the corticospinal tract and the gracile funicle (Fig. 3D). In adulthood, SMN labeling was mainly confined to the motor neuron compartment (Figs 2C and F and 3E), and in a selected neuronal cell population, like neurons in the lateral spinal nucleus and the intermediolateral cell column (20). The pattern of cellular SMN labeling in motor neurons did not change in adulthood in comparison with postnatal developmental stages, but different intensities of motor neuron staining were evident (Fig. 3C and E). The SMN labeling of fiber tracts in the white matter was no longer evident (Fig. 2C and F), but the neuropil labeling of laminae I, II and III was slightly more intense, possibly for the SMN immunoreactivity of axon terminals in the superficial laminae (Fig. 3F).
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Electron microscopy ICC: ultrastructural localization of the SMN protein
The spinal localization of the SMN protein was then verified at the electron microscope level at the same developmental stages (P1, P8 and P15) and in adulthood. The experiments were performed by means of pre-embedding ICC with either conventional 3-3'-diaminobenzidine (DAB) reaction or silver intensification of nanogold particles bound to the secondary antibodies.
In spinal motor neurons, the pattern of SMN immunoreactivity did not change during postnatal development and in adulthood (Fig. 4). Intense SMN immunoreactivity was observed at all considered ages, in both the cytoplasm and the nucleus (Fig. 4). Within the nucleus, the SMN immunoreactivity was made up by aggregates of silver particles or DAB reaction product (Figs 4 and 5A). The immunoreactive aggregates were mainly sparse in the nucleoplasm, but they were also localized along the internal part of the nuclear membrane, or in close apposition to the nucleolus and the associated coiled bodies (Figs 4 and 5A). Intense SMN labeling was also present in the cytoplasm. Again, SMN-immunoreactive aggregates were mainly sparse in the cytoplasm. However, a few of them were also associated with cytoplasmic organelles, like the endoplasmic reticulum and the Golgi apparatus, and along the outer surface of the nuclear membrane (Figs 4 and 5B and C). SMN-immunoreactive aggregates were also associated with the mitochondria (Fig. 5D). SMN immunoreactivity was found in close apposition to the external mitochondrial membrane in experiments performed with all employed antibodies (Fig. 5C and D). Moreover, in experiments with antibody #1137, the electron-dense reaction product was also associated with the internal mitochondrial membrane.
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In addition to the SMN staining of motor neurons, two important features were confirmed by the ultrastructural analysis. First, strong SMN immunoreactivity was consistently present in dendritic and axonal profiles (Fig. 6). In lamina IX neuropils, SMN staining was present in dendrites of different size, in apposition to the external mitochondrial and plasma membranes and to microtubules (Fig. 6A–D). Labeled myelinated and unmyelinated axons, with immunoreactivity associated with cytoskeletal elements, and axon terminals, containing reaction product associated with mitochondria and synaptic vesicles, were also found (Fig. 6E and F). No immunoreactivity was associated with pre- or post-synaptic specializations. Secondly, intense SMN immunoreactivity was present in axonal fiber tracts of the spinal white matter during postnatal development. In the dorsal corticospinal tract (Fig. 6G), some myelinated and unmyelinated fibers were intensely stained at P8 and P15. In contrast, no SMN labeling was found in the corticospinal tract, as well as in other ascending or descending fiber tracts, of adult rats.
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Outside the motor neuron compartment, the SMN immunoreactivity in the more superficial laminae was mainly, although not exclusively, confined to the nucleus. The immunoreactive aggregates were again mainly sparse in the nucleoplasm and rarely associated with the nucleolus (data not shown). In agreement with that observed at the light microscope level, the nuclear SMN immunoreactivity in the superficial laminae of the adult rat was clearly less intense if compared with that of early postnatal stages. Finally, at all ages considered, the SMN immunoreactivity was mainly neuron specific, since the identifiable astrocytic and oligodendrocytic somata were devoid of labeling (Figs 4B and 6G).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The present experiments investigated the cellular and subcellular localization of the SMN protein during the development of the rat spinal cord. They provided evidence that SMN expression declined during spinal cord development but that it remained unchanged as localization and staining intensity in motor neurons during ontogenesis and adulthood. In addition, the present data demonstrated that SMN protein was mainly organized in molecular aggregates sparse in the nucleoplasm and cytoplasm of motor neurons, and more rarely associated with the membranes of subcellular organelles. Finally, these experiments pointed out the existence of an axoplasmic transport of the SMN protein that was particularly relevant during neuronal development.
Antibody specificity
Several lines of evidence demonstrated the specificity of the antibodies employed in the present experiments. First, the regional, cellular and subcellular distribution of the SMN immunoreactivity was mostly identical with the different antibodies used. Secondly, the same strongly immunoreactive band of ~38–39 kDa was recognized by the different anti-SMN antibodies in western blot experiments in both HeLa cell and rat spinal cord extracts. The existence of an additional SMN-specific lower band of ~36 kDa raises the possibility of post-translational modifications or differently spliced forms of the SMN protein. We are currently investigating the differential expression of the two SMN bands during the pre- and postnatal development of the spinal cord. Thirdly, the SMN-immunoreactive bands in western blot experiments and the SMN immunoreactivity in spinal cord neurons were consistently blocked by pre-incubation of the antibodies with the antigenic peptide. Taken together, these data demonstrate that the immunoreactivity reported here corresponds to the cellular and subcellular distribution of the SMN protein in the spinal cord.
SMN protein expression during spinal cord development
Our light microscopy immunocytochemical observations demonstrate an overall decrease of SMN protein expression from early postnatal stages to adulthood. These findings are in good agreement with previously reported western blot data demonstrating a progressive developmental downregulation of the protein from prenatal and early postnatal periods to adulthood in rat brain, spinal cord and non-neural tissues (27), and in human skeletal muscle, heart and brain (28). They are also consistent with the time course of SMA, which may be clinically evident in the most severe cases at birth and even during the late phases of pregnancy.
It should be noted, however, that although the SMN staining in the spinal cord decreased from P1 to adulthood, the pattern and intensity of SMN staining in motor neurons, i.e. the very cells whose degeneration is responsible for the clinical phenotype of SMA, were similar at all ages examined. This pattern of SMN expression is consistent with recent data on spinal motor neurons in adult mice and rats (27,29). The stability of SMN expression in motor neurons suggests that motor neurons are strictly depending on high levels of the protein for their function and survival not only during early phases of development. Our data also demonstrate strong SMN expression in the dendritic and axonal compartments of motor neurons. In axons, SMN expression was particularly evident during development and decreased during central nervous system maturation, as demonstrated by the strong SMN immunoreactivity in the ventral root (at P1), dorsal root (P1–P15), cuneate and gracile funicles (P1–P15) and dorsal corticospinal tract (P8–P15) that was no longer evident during adulthood. These data clearly suggest that the SMN protein is actively transported in different cellular compartments, and that this transport is particularly relevant during neuronal ontogenesis (see below).
Subcellular localization of SMN protein
The subcellular distribution of SMN in motor neurons suggests diverse cellular roles for this protein. The predominant localization was in immunoreactive aggregates that were sparse in the nucleoplasm and cytoplasm and not specifically associated with subcellular organelles. This finding supports the hypothesis that SMN is associated with multimeric protein complexes (22), as also suggested by recent data in fetal skeletal muscle demonstrating a specific SMN enrichment in the heavy sedimenting particulate fraction that could not be dissociated by detergent (28). This subcellular localization of SMN is also consistent with the reported interaction of SMN with SIP1 and snRNP proteins for spliceosomal biogenesis and pre-mRNA splicing (22,23,30).
On the other hand, our findings also demonstrate that in motor neurons SMN is partly associated with the membranes of subcellular organelles. Although not quantitatively predominant, the close apposition of the SMN immunoreactivity to subcellular organelles was consistently found in both developing and mature motor neurons, thus suggesting that the SMN association with membranes could be important for motor neuron function. The association of the SMN protein with the nuclear membrane has already been reported, and interpreted as suggestive for a role of SMN in the nucleocytoplasmic transport of the snRNPs (31). The localization of the SMN protein within the rough endoplasmic reticulum and Golgi apparatus is not quantitatively relevant. It may simply reflect that the SMN protein may undergo post-translational modifications, which may account, at least in part, for the already reported different molecular forms of SMN (27,32).
The SMN association with mitochondria was not reported previously, and no specific sequences targeting SMN to mitochondria have been described so far. However, SMN immunoreactivity was consistently observed in association with mitochondria during development and adulthood, in spinal cords fixed with different concentrations of glutaraldehyde, and with different antibodies directed against different antigenic epitopes of the SMN protein. These data argue against the possibility of a fortuitous finding or localization related to the chemical fixation of spinal cord tissues. It should be noted that previous data demonstrated a synergistic activity of SMN and Bcl-2 in preventing Bax-induced or Fas-mediated apoptosis in cultured HeLa cells (24), and that the subcellular localization of SMN reported here shares similarities with that of Bcl-2, since Bcl-2 is mainly associated with mitochondria and nuclear membrane (25,33–35). The association between SMN and Bcl-2 in motor neurons should, however, be further exploited in co-localization experiments by means of confocal microscopy.
The subcellular localization of SMN has recently been addressed in ultrastructural immunogold experiments with a different anti-SMN antibody (31). The data by Bechade et al. (31) are consistent with the present findings in demonstrating SMN labeling in both nuclear and cytoplasmic compartments of motor neurons, and SMN labeling in apposition to microtubules. They also provided a nice demonstration of the SMN localization in coiled bodies and at the cytoplasmic side of nuclear pores (31). In contrast to the present data, however, SMN labeling was not found in Golgi apparatus and in apposition to mitochondria, and localized over, and not in close apposition to, coiled bodies. It is possible, given the association of SMN with itself (36), SIP1 and the Sm proteins (22), and Bcl-2 (24), possibly in multimeric protein complexes, that SMN antigenic sites may be differentially recognized by the different SMN-specific antibodies in different phases of cellular metabolism or localization. Therefore, minor differences in the specific SMN epitopes recognized by the different SMN antibodies may account for fine differences in ultrastructural localization.
SMN localization in axonal and dendritic compartments: relevance for SMA pathogenesis
An additional striking and previously unreported finding was the SMN localization in the axonal compartment. The SMN immunoreactivity of axonal fiber tracts during early postnatal development was confirmed at the ultrastructural level by the SMN staining in axons of the corticospinal tract at P8 and P15. These findings suggest the idea that SMN is actively transported through axoplasmic flow in a particular phase of neuronal development. The SMN protein and the associated protein SIP1 have been proposed to possess a housekeeping function in the biogenesis of the spliceosomal snRNPs (22,23), and in the splicing of pre-mRNAs (30). Moreover, a role for SMN in regulating gene expression has recently been proposed (37). Consistent with these data, mice with homozygous SMN deletion display a lethal phenotype during early embryogenesis (26). However, the specific pathogenic mechanisms leading to the selective motoneuronal degeneration that characterizes SMA remain to be elucidated. The axoplasmic transport of the SMN protein suggested by the present data could be relevant in gaining insights into the pathogenesis of SMA. The SMN association with microtubules in axons and dendrites demonstrated here, together with the direct RNA-binding activity of SMN (38), raise the possibility that this protein could play a role in the transport of mRNAs in dendritic and axonal compartments. Given the length and the complexity of the dendritic and axonal arborization of motor neurons, the SMN transport from the perikarya of motor neurons to distal dendrites and terminal endings of the neuromuscular junction could be critical for motor neuron development and survival, in particular during the ontogenetic phase in which they find their target and shape their complex geometrical structure. Reduction of SMN protein levels could result in lack of transport of specific mRNAs to the distal axonal endings and to the neuromuscular junction. This hypothesis could explain the decreased ability to sprout of surviving motor neurons in SMA (39) and the axonal neuropathy already reported in selected SMA cases (40). In addition, the reduced SMN transport could impair the interaction between muscle and nerve, which has been already suggested as a possible cause of SMA (41–43).
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MATERIALS AND METHODS |
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Production of anti-SMN antibodies
An oligopeptide of 17 amino acids, corresponding to residues 4–20 of the N-terminal region of the rat SMN protein sequence, was synthesized by Neosystem (Strasbourg, France). The peptide was coupled to ovalbumin, serving as an immunogenic carrier, via a tyrosine residue to promote glutaraldehyde-mediated coupling to the carrier. Two rabbits were first immunized with an injection of the antigen–carrier conjugate, followed by two subsequent boosts, one every month. The two antisera, #846 and #854, were collected from a final bleeding. They were used for western blot analysis and ICC, along with a commercially available monoclonal antibody directed against residues 14–174 of a human SMN fusion protein (antibody TL; Transduction Laboratories, Lexington, KY), and a previously characterized anti-SMN antibody (antibody #1137) (20).
Western blot analysis
HeLa cells and rat spinal cord protein extracts were used for immunoblot analyses. Approximately 2 x 106 HeLa cells were trypsinized, pelleted, resuspended in 10 mM PK buffer (phosphate–potassium buffer pH 7.2 with 10 mM EDTA and 150 mM NaCl), sonicated, solubilized with 0.25% SDS and 2% Triton X-100 in the presence of a mixture of protease inhibitors, and centrifuged at 100 000 g for 30 min at 4°C. Sprague–Dawley rats were decapitated at P15. Spinal cords were quickly removed (in <1 min), immediately frozen on dry ice or by immersion in liquid nitrogen, and stored at –80°C until processing. For subcellular fractionation, spinal cords were homogenized (Teflon–glass potter, 10 strokes, 700 r.p.m.) in 4 vols of buffer containing 20 mM HEPES, 1 mM DTT, 1 mM EGTA, 10 mM EDTA, 0.1 mM phenylmethylsulfonylfluoride at pH 7.4, in the presence of a complete set of protease inhibitors (Complete; Boehringer Mannheim, Mannheim, Germany). The homogenate was centrifuged at 1000 g for 10 min. The supernatant was then centrifuged at 100 000 g for 45 min and the resulting pellet and supernatant were regarded as cellular membrane and soluble fractions, respectively. Proteins obtained in HeLa extracts or spinal subcellular fractions were separated by SDS–PAGE (11% acrylamide) and electroblotted onto nitrocellulose papers for 45–60 min at 180 mA. The latter were blocked with 10% no-fat milk in Tris-buffered saline (TBS), and then incubated for 1 h at room temperature with the anti-SMN primary antibodies #846 (diluted 1:500–1:1000), #854 (1:500–1:1000), TL (1:20 000) or #1137 (1:500) in 3% no-fat milk in TBS. In one set of experiments, the #846 and #854 antibodies were pre-incubated with 40 µg/µl of the antigenic peptide for 2 h at room temperature to test the antibody specificity (immunoabsorption test). After extensive rinsing in TBS/Tween, the nitrocellulose papers were then incubated with horseradish peroxidase-conjugated secondary antibodies [goat anti-rabbit, for polyclonal antibodies, dilution 1:5000 (Sigma); goat anti-mouse, for monoclonal antibody TL, dilution 1:10 000 (Kierkegaard and Perry Laboratories)] and the antigen–antibody complex revealed by enhanced chemiluminescence (ECL; Amersham International, Little Chalfont, UK).
Light and electron microscopy ICC
Twelve adult and postnatal [P1 (defined as the day of birth), P8 and P15] Sprague–Dawley rats were perfused under deep chloral hydrate (1 ml/100 g body wt of a 4% solution) anesthesia with 1% paraformaldehyde followed by 4% paraformaldehyde or 4% paraformaldehyde and 0.1–0.5% glutaraldehyde in phosphate buffer at pH 7.4. Spinal cords were dissected out and post-fixed in 4% paraformaldehyde. After perfusion and post-fixation, they were cut with a vibratome in 50 µm thick coronal sections, and collected in phosphate-buffered saline at pH 7.4 (PBS). For light microscopy ICC, sections were pre-treated with 1% H2O2 in PBS to remove the endogenous peroxidase activity, rinsed in PBS, and incubated with 10% normal goat serum (NGS) or normal horse serum (NHS) and 0.2% Triton X-100 for 60 min to mask non-specific absorption sites. Sections were then incubated overnight at 4°C with the anti-SMN antibodies [diluted 1:2000–1:5000 (#846, #854 and #1137 antibodies) and 1:1000–1:2500 (TL antibody) in 1% NGS or NHS and 0.2% Triton X-100]. After rinsing for 60 min in PBS, the sections were incubated with a 1:200 dilution of biotinylated goat anti-rabbit (GAR) or horse anti-mouse (HAM) IgG (Vector Laboratories, Burlingame, CA) for 75 min, rinsed for 60 min in PBS, and then incubated with the ABC Elite complex (Vector Laboratories) for 75 min. Peroxidase staining was achieved by incubating the sections in 0.075% DAB and 0.002% H2O2 in 50 mM Tris buffer at pH 7.6. Sections were then mounted on slides, dried, dehydrated and coverslipped with DPX. The sections adjacent to the immunoreacted ones were counterstained with 0.1% thionin.
For electron microscopy ICC, the free-floating sections were processed with either pre-embedding immunoperoxidase or immunogold procedures. For pre-embedding immunoperoxidase, the sections were permeabilized by freeze–thawing at –80°C (44), rinsed in PBS, treated with 1% H2O2 in PBS, and incubated with 10% NGS or NHS to mask non-specific absorption sites. Sections were then incubated with the primary anti-SMN antibodies, the secondary antibodies, and the ABC complex as specified above. Peroxidase staining was achieved by incubating the sections in 0.048% DAB, 0.024% CoCl2, 0.019% NAS and 0.003% H2O2 in 50 mM phosphate buffer (PB) at pH 7.5. For pre-embedding immunogold, the sections were permeabilized as described above, incubated for 30 min in 0.8% bovine serum albumin (BSA), 0.1% gelatin and 1% NGS or NHS in PBS, and rinsed for 15 min in washing buffer (WB: 0.8% BSA and 0.1% gelatin in PBS). Sections were then incubated overnight or for 48 h at 4°C in the primary anti-SMN antibodies diluted in 0.8% BSA, 0.1% gelatin and 1% NGS in PBS. After extensive rinses in WB, sections were incubated for 2 h or overnight in secondary antisera (GAR or HAM) conjugated to 1 nm gold particles (Amersham) diluted 1:50 in 0.8% BSA, 0.1% gelatin and 1% NGS in PBS. Sections were then rinsed in WB and in PBS, silver intensified with IntenSe M (Amersham) according to the manufacturer’s instructions, rinsed in PB, and fixed for 10 min in 2% glutaraldehyde in 0.1 M PB. Some of the sections were gold toned according to the protocol of Trembleau et al. (45). All sections were then osmicated in 1% OsO4, dehydrated, and flat embedded in Epon–Spurr. Thin sections were counterstained with lead citrate and examined and photographed with a Jeol T8 or Zeiss 901 electron microscope.
In situ immunoabsorption test
The diluted #846 and #854 antibodies (1:2000 in PBS–1% NGS) were pre-incubated for 2 h with 25 and 50 µM (31.6 and 63.3 µg/ml) free antigenic peptide before use. Vibratome sections were incubated overnight at 4°C with either control or immunoabsorbed primary antibodies. Sections were then parallel processed with a standard immunoperoxidase procedure as specified above.
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ACKNOWLEDGEMENTS |
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This research was supported by grants from the Italian Ministry of Health and Telethon grant no. 1053 (to G.B.).
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +39 02 2394266; Fax: +39 02 70600775; Email: battaglia.besta@interbusiness.it or battaglia@istituto-besta.it
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REFERENCES |
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