Heterogeneity of subcellular localization and electrophoretic mobility of survival motor neuron (SMN) protein in mammalian neural cells and tissues - PubMed (original) (raw)

Heterogeneity of subcellular localization and electrophoretic mobility of survival motor neuron (SMN) protein in mammalian neural cells and tissues

J W Francis et al. Proc Natl Acad Sci U S A. 1998.

Abstract

Spinal muscular atrophy is caused by defects in the survival motor neuron (SMN) gene. To better understand the patterns of expression of SMN in neuronal cells and tissues, we raised a polyclonal antibody (abSMN) against a synthetic oligopeptide from SMN exon 2. AbSMN immunostaining in neuroblastoma cells and mouse and human central nervous system (CNS) showed intense labeling of nuclear "gems," along with prominent nucleolar immunoreactivity in mouse and human CNS tissues. Strong cytoplasmic labeling was observed in the perikarya and proximal dendrites of human spinal motor neurons but not in their axons. Immunoblot analysis revealed a 34-kDa species in the insoluble protein fractions from human SY5Y neuroblastoma cells, embryonic mouse spinal cord cultures, and human CNS tissue. By contrast, a 38-kDa species was detected in the cytosolic fraction of SY5Y cells. We conclude that SMN protein is expressed prominently in both the cytoplasm and nucleus in multiple types of neurons in brain and spinal cord, a finding consistent with a role for SMN as a determinant of neuronal viability.

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Figures

Figure 1

Characterization of abSMN. (A) Immunoblots of E. coli expressed SMN fusion proteins and subcellular fractions of human neuroblastoma cells probed with abSMN (lanes 1–8) or chicken anti-human SMN antibody C3 (lanes 9–10). AbSMN reacts with two different fusion constructs expressing wild-type SMN (fusion proteins 1 and 2) but not with extracts from bacteria lacking the SMN cDNA insert or extracts from cells obtained before induction of protein expression. Subcellular fractions of SY5Y cells were probed by using the same abSMN antibody concentration. Lane 1: BL21DE3 bacteria, negative control (2 μg). Lane 2: BL21DE3 bacteria expressing fusion protein 1 (2 μg). Lane 3: DH5α bacteria, negative control (5 μg). Lane 4: DH5α bacteria expressing fusion protein 2 (5 μg). Lane 5: SY5Y cells, total cell lysate (20 μg). Lane 6: SY5Y cells, cytosolic fraction (20 μg). Lane 7: SY5Y cells, soluble nuclear extract (20 μg). Lane 8: SY5Y cells, insoluble nuclear material (20 μg). Lane 9: SY5Y cells, cytosolic fraction (20 μg). Lane 10: SY5Y cells, insoluble nuclear material (20 μg). (B) Immunocytochemical localization of SMN in human SY5Y neuroblastoma cells determined by using double-label indirect immunofluorescence. (i) Fixed cells viewed when using Hoffman modulation (Bar = 20 μm). (ii) abSMN (200 ng/ml) immunostaining demonstrates moderate cytoplasmic labeling and strong labeling of nuclear gems. (iii) Simultaneous immunostaining with monoclonal anti-p80-coilin antibody (1:150) shows that abSMN reactive gems are often closely associated with nuclear coiled bodies. (C) Preadsorption studies of abSMN. Detection of SMN protein by immunoblot analysis persists after preincubation of abSMN with an exon 4 oligopeptide (lanes 1–4), whereas preadsorption with the appropriate exon 2 peptide eliminates SMN immunoreactivity (lanes 5–8). Lanes 1 and 5: BL21DE3 bacteria, negative control (2 μg). Lanes 2 and 6: BL21DE3 bacteria, fusion protein 1 (2 μg). Lanes 3 and 7: SY5Y cells, cytosolic fraction (20 μg). Lanes 4 and 8: SY5Y cells, insoluble nuclear material (10 μg).

Figure 2

Immunocytochemical and Western blot analysis of SMN protein in dissociated cultures of mouse embryonic spinal cord. (A) Phase contrast micrograph of cultured cells after 7 days in vitro (Bar = 30 μm). (B) abSMN immunoreactivity is limited to neuronal cell bodies. (C) Concurrent labeling of cell shown in B with mouse anti-Islet 1 antibody indicates that SMN is present within spinal motor neurons. (D) Immunoblot of cell extracts prepared from spinal cord cultures. Samples (8 μg total protein) were probed with 125 ng/ml abSMN. Like SY5Y cells, mixed neuron/glia cultures from spinal cord demonstrated a 34-kDa band in the insoluble protein fraction. Lane 1: SY5Y cell extract, insoluble nuclear fraction. Lane 2: Ventral spinal cord culture, insoluble fraction. Lane 3: Dorsal spinal cord culture, insoluble fraction. Lane 4: Cortical glia culture, insoluble fraction. Lane 5: Ventral spinal cord culture, soluble fraction. Lane 6: Dorsal spinal cord culture, soluble fraction. Lane 7: Cortical glia culture, soluble fraction.

Figure 3

Immunohistochemical and Western blot analyses of SMN in human CNS tissues. (A) Pyramidal neuron in the neocortex shows strong SMN immunolabeling of cytoplasm and nuclear gems. (Bar = 5 μm.) (B) Preadsorption of abSMN with 100-fold excess of peptide abolishes staining. (Bar = 50 μm.) (C) Low-magnification view of anterior horn cells within ventral gray matter of adult lumbar spinal cord. The cell bodies and proximal dendrites of α-motor neurons are intensely immunoreactive. (Bar = 230 μm.) (D) High-power view of adult anterior horn cell. (Bar = 20 μm.) (E) SMN labeling of nucleoli in cerebellar Purkinje cells often appeared as a solid ring of immunoreactivity circumscribing the outer surface of the nucleolus. (Bar = 10 μm.) (F) Western blot analysis of low-speed pellet material prepared from homogenates of human cerebellar autopsy tissue. After transfer to nitrocellulose, tissue fractions (20 μg total protein) were probed with either abSMN (i) or chicken antibody C3 (ii). Lanes: 1, autopsy case 96017; 2, autopsy case 97029; 3, autopsy case 97051.

Figure 4

Immunohistochemical and Western blot analyses of selected CNS tissues from the adult mouse. (A) Vibratome section of cerebellum showing Purkinje cells in which abSMN-reactive gems appear to have a perinucleolar localization. No discernible staining of Purkinje cell cytoplasm was observed. (Bar = 8 μm.) (B) Frozen sections of lumbar spinal cord reveal strong abSMN immunoreactivity within gems and nucleoli of ventral horn motor neurons. In this preparation, gems were observed in contact with the nucleolus (i, arrow) or free in the nucleoplasm (ii) (Bar = 3 μm for both i and ii.) (C) Western blot of low-speed pellet material prepared from cerebellum. After transfer to nitrocellulose, tissue samples were probed with either abSMN (lane 1) or chicken antibody C3 (lane 2). In both cases a major 34-kDa band was detected.

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Heterogeneity of subcellular localization and electrophoretic mobility of survival motor neuron (SMN) protein in mammalian neural cells and tissues - PubMed (original) (raw)