Synaptic defects in type I spinal muscular atrophy in human development†
Rebeca Martínez-Hernández
Department of Genetics, Hospital de la Santa Creu i Sant Pau and IIB Sant Pau, Avda Sant Antoni Maria Claret 167, 08025 Barcelona, Spain
Search for more papers by this authorSara Bernal
Department of Genetics, Hospital de la Santa Creu i Sant Pau and IIB Sant Pau, Avda Sant Antoni Maria Claret 167, 08025 Barcelona, Spain
Search for more papers by this authorEva Also-Rallo
Department of Genetics, Hospital de la Santa Creu i Sant Pau and IIB Sant Pau, Avda Sant Antoni Maria Claret 167, 08025 Barcelona, Spain
Search for more papers by this authorLaura Alías
Department of Genetics, Hospital de la Santa Creu i Sant Pau and IIB Sant Pau, Avda Sant Antoni Maria Claret 167, 08025 Barcelona, Spain
Centro de Investigación Bíomédica en Red de Enfermedades Raras (CIBERER U705), Instituto de Salud Carlos III, Spain
Search for more papers by this authorMaJesús Barceló
Department of Genetics, Hospital de la Santa Creu i Sant Pau and IIB Sant Pau, Avda Sant Antoni Maria Claret 167, 08025 Barcelona, Spain
Centro de Investigación Bíomédica en Red de Enfermedades Raras (CIBERER U705), Instituto de Salud Carlos III, Spain
Search for more papers by this authorMarta Hereu
Unitat de Neurobiologia Cel·lular, Department of Experimental Medicine, Universitat de LLeida and IRBLLeida, LLeida, Spain
Search for more papers by this authorJosep E Esquerda
Unitat de Neurobiologia Cel·lular, Department of Experimental Medicine, Universitat de LLeida and IRBLLeida, LLeida, Spain
Search for more papers by this authorCorresponding Author
Eduardo F Tizzano
Department of Genetics, Hospital de la Santa Creu i Sant Pau and IIB Sant Pau, Avda Sant Antoni Maria Claret 167, 08025 Barcelona, Spain
Centro de Investigación Bíomédica en Red de Enfermedades Raras (CIBERER U705), Instituto de Salud Carlos III, Spain
Eduardo F Tizzano, Department of Genetics, Hospital de la Santa Creu i Sant Pau, Avda Sant Antoni Maria Claret 167, 08025 Barcelona, Spain. e-mail: [email protected]Search for more papers by this authorRebeca Martínez-Hernández
Department of Genetics, Hospital de la Santa Creu i Sant Pau and IIB Sant Pau, Avda Sant Antoni Maria Claret 167, 08025 Barcelona, Spain
Search for more papers by this authorSara Bernal
Department of Genetics, Hospital de la Santa Creu i Sant Pau and IIB Sant Pau, Avda Sant Antoni Maria Claret 167, 08025 Barcelona, Spain
Search for more papers by this authorEva Also-Rallo
Department of Genetics, Hospital de la Santa Creu i Sant Pau and IIB Sant Pau, Avda Sant Antoni Maria Claret 167, 08025 Barcelona, Spain
Search for more papers by this authorLaura Alías
Department of Genetics, Hospital de la Santa Creu i Sant Pau and IIB Sant Pau, Avda Sant Antoni Maria Claret 167, 08025 Barcelona, Spain
Centro de Investigación Bíomédica en Red de Enfermedades Raras (CIBERER U705), Instituto de Salud Carlos III, Spain
Search for more papers by this authorMaJesús Barceló
Department of Genetics, Hospital de la Santa Creu i Sant Pau and IIB Sant Pau, Avda Sant Antoni Maria Claret 167, 08025 Barcelona, Spain
Centro de Investigación Bíomédica en Red de Enfermedades Raras (CIBERER U705), Instituto de Salud Carlos III, Spain
Search for more papers by this authorMarta Hereu
Unitat de Neurobiologia Cel·lular, Department of Experimental Medicine, Universitat de LLeida and IRBLLeida, LLeida, Spain
Search for more papers by this authorJosep E Esquerda
Unitat de Neurobiologia Cel·lular, Department of Experimental Medicine, Universitat de LLeida and IRBLLeida, LLeida, Spain
Search for more papers by this authorCorresponding Author
Eduardo F Tizzano
Department of Genetics, Hospital de la Santa Creu i Sant Pau and IIB Sant Pau, Avda Sant Antoni Maria Claret 167, 08025 Barcelona, Spain
Centro de Investigación Bíomédica en Red de Enfermedades Raras (CIBERER U705), Instituto de Salud Carlos III, Spain
Eduardo F Tizzano, Department of Genetics, Hospital de la Santa Creu i Sant Pau, Avda Sant Antoni Maria Claret 167, 08025 Barcelona, Spain. e-mail: [email protected]Search for more papers by this authorNo conflicts of interest were declared.
Abstract
Childhood spinal muscular atrophy is an autosomal recessive neuromuscular disorder caused by alterations in the Survival Motor Neuron 1 gene that triggers degeneration of motor neurons within the spinal cord. Spinal muscular atrophy is the second most common severe hereditary disease of infancy and early childhood. In the most severe cases (type I), the disease appears in the first months of life, suggesting defects in fetal development. However, it is not yet known how motor neurons, neuromuscular junctions, and muscle interact in the neuropathology of the disease. We report the structure of presynaptic and postsynaptic apparatus of the neuromuscular junctions in control and spinal muscular atrophy prenatal and postnatal human samples. Qualitative and quantitative data from confocal and electron microscopy studies revealed changes in acetylcholine receptor clustering, abnormal preterminal accumulation of vesicles, and aberrant ultrastructure of nerve terminals in the motor endplates of prenatal type I spinal muscular atrophy samples. Fetuses predicted to develop milder type II disease had a similar appearance to controls. Postnatal muscle of type I spinal muscular atrophy patients showed persistence of the fetal subunit of acetylcholine receptors, suggesting a delay in maturation of neuromuscular junctions. We observed that pathology in the severe form of the disease starts in fetal development and that a defect in maintaining the initial innervation is an early finding of neuromuscular dysfunction. These results will improve our understanding of the spinal muscular atrophy pathogenesis and help to define targets for possible presymptomatic therapy for this disease. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Supporting Information
| Filename | Description |
|---|---|
| path4080-sup-0001-suppinfoFigureS1.tif7.8 MB | Supporting Information: Figure 1 Comparison of AChR clustering (AChRγ in red), innervation (NF in green), and synaptophysin expression (SyPhy in fuchsia) between controls (A-D) and SMA type I with two SMN2 copies (E-H) and one SMN2 copy (I-L). Arrow in A shows a non-innervated AChR cluster related with a secondary myotube.Arrowhead in J indicates accumulation of synaptic vesicles in the most distal part of preterminal nerves. Note that NF vanishes at the end of the terminal in panel K (arrow). Scale bar: 20 μm. |
| path4080-sup-0002-suppinfoFigureS2.tif10.4 MB | Supporting Information: Figure 2. Details of the different NMJ appearances observed in type I SMA fetuses at 12 weeks. Triple fluorescent-labelled NMJs were immunostained with AChRγ in red;SyPhy in fuchsia and NF in green. (A-D) “Normal-like” appearance. (E-H) “Early preterminal accumulation”.SyPhy remains in preterminal zones (arrow in F) that co-localize with NF. (I-L) “Preterminal accumulation”. A higher expression of SyPhy merged with nerve terminals is visualized (arrow in J). (M-P) “Early AChR disassembly”. Arrowhead in M indicates early disorganization of AChRs clustering followed by an early aggregation of presynaptic vesicles (N). Scale bar: 20 μm |
| path4080-sup-0003-suppinfoFigureS3.tif6.6 MB | Supporting Information: Figure 3. Comparison of fetal NMJ aspects in controls (A-D), SMA type II samples (E-H), and type I SMA (I-L) at lower magnification. Control and type II NMJ pattern show similar appearance. In type I SMA, the most significant findings include preterminal accumulation of vesicles revealed by SyPhy (in fuchsia) and postsynaptic disaggregation detected by AChRγ(in red). Scale bar: 100 μm |
| path4080-sup-0004-suppinfoFigureS4.tif8.4 MB | Supporting Information: Figure 4. Comparison of control (A-D) and SMA (E-L) NMJ in postnatal samples. Triple fluorescent-labelled NMJs were immunostained with AChRγ in red; SyPhy in fuchsia and NF in green. Note the absence of AChRγ (A) and the presence of SyPhy (B) at the end of the terminal in the control sample in comparison with the persistence of AChRγ (E and I) in SMA samples without traces of SyPhy (F and J). Scale bar: 20 μm. |
| path4080-sup-0005-suppinfoFigureS5.tif10.6 MB | Supporting Information: Figure 5. SMN expression in fetal samples.NMJs of control samples immunolabelled for SMN (in green), ?-bungarotoxin (in red), NF (in fuchsia), and DAPI. Nuclear gems and cytoplasmic dot-like structures (arrowheads in D and H) are present. No co-localization with AChRs was observed (see selected regions of white dotted lines in A, C, E, and G). Negative controls omitting the primary antibody are included (I for prenatal samples and J for postnatal samples). Scale bar: 10 μm |
| path4080-sup-0006-suppinfoFigureS6.tif9.7 MB | Supporting Information: Figure 6. SMN expression in postnatal samples.NMJs of control samples immunolabelled for SMN (in green), ?-bungarotoxin (in red), NF (in fuchsia), and DAPI. Nuclear gems are present (arrowheads in D and H). In some NMJs, positive SMN staining was detected co-localizing with ?-bungarotoxin(large arrows in selected regions by dotted lines in C andin image G), whilst in others, expression was undetectable (asterisk in C). Note in H SMN expression in NMJ at higher magnification (square). The negative control is included in Supplementary Figure 5. Scale bar: 10 μm |
Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
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