The spinal muscular atrophy mouse model, SMAΔ7, displays altered axonal transport without global neurofilament alterations - PubMed (original) (raw)

The spinal muscular atrophy mouse model, SMAΔ7, displays altered axonal transport without global neurofilament alterations

Jeffrey M Dale et al. Acta Neuropathol. 2011 Sep.

Abstract

Spinal muscular atrophy (SMA) is a neurodegenerative disease resulting from decreased levels of survival motor neuron 1 (SMN1) protein. Reduced SMN1 levels are linked to pathology at neuromuscular junctions (NMJs), which includes decreased vesicle density and organization, decreased quantal release, increased endplate potential duration, and neurofilament (NF) accumulations. This work presents a first study towards defining molecular alterations that may lead to the development of NMJ pathology in SMA. Fast, anterograde transport of synaptic vesicle 2 (SV2-c) and synaptotagmin (Syt1) proteins was reduced 2 days prior to the observed decrease in synaptic vesicle density. Moreover, reduced accumulation of SV2-c or Syt1 was not due to reduced protein expression or reduced kinesin activity. Dynein levels were reduced at times that are consistent with NF accumulations at NMJs. Furthermore, NF distribution, from cell body to sciatic nerve, appeared normal in SMA∆7 mice. Taken together, these results suggest that reduced axonal transport may provide a mechanistic explanation for reduced synaptic vesicle density and concomitant synaptic transmission defects, while providing evidence that suggests NF accumulations result from local NMJ alterations to NFs.

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Conflict of interest statement

Conflict of interest None.

Figures

Fig. 1

Ectopic accumulation of phosphorylated NFs does not occur in affected tissues of SMAΔ7 mice. Lumbar (a) spinal cord sections were immunostained with an antibody that recognizes dephosphorylated NFs (SMI-32) to identify motor neuron cell bodies. Ectopically phosphorylated NFs were identified with an antibody that recognizes NFs in a phosphorylated-dependent manner (SMI-31). NF localization was analyzed at both P11 and P16. Accumulation of ectopically phosphorylated NFs (red) was not observed in lumbar (a) motor neuron cell bodies, marked by nuclear DAPI staining (blue), at either time point. Scale bar 50 μm. NF phosphorylation and expression was not altered in SMAΔ7 (S) (N = 3) when compared with wild type (w) (N = 3) animals (b) shown by ROD analysis (c). Spinal cord lysates were separated on a 7.5% SDS polyacrylamide gels from wild type and SMAΔ7 mice at three different disease stages. Lysates were immunoblotted with antibodies recognizing phosphorylated NFs (SMI-31), unphosphorylated NFs (SMI-32) and a NF-L control (DA2). Statistical significance was determined by a Student’s t test

Fig. 2

NF organization in proximal motor axons is unaffected in SMAΔ7 mice. Transmission electron micrographs of P12 motor axons derived from the fifth lumbar spinal root from wild type (top N = 9) and SMAΔ7 (bottom N = 7) mice used for nearest neighbor distances, NF densities, and microtubule densities (a). Black box identifies the region of the axon that is magnified to the right of each micrograph. Scale bar 500 nm. Distribution of nearest neighbor distances was slightly altered in SMAΔ7 mice. Each point represents the averaged distribution of axon diameters from the entire roots of at least seven mice for each genotype and age group. Distributions of nearest neighbor distances were analyzed for overall statistical differences using a Mann–Whitney U test. There was a statistically significant difference between nearest neighbor distances of SMAΔ7 versus wild-type mice (P < 0.001) (b). Densities of NFs (c) and microtubules (d) in SMAΔ7 and wild-type motor axons were unaltered. NF densities were analyzed for statistical significance by Student’s t test, and microtubule densities were analyzed by Mann–Whitney test

Fig. 3

Kinesin accumulates proximal and distal to the ligation in P7 animals. Sciatic nerves were immunoblotted with (+Kif5C) and without (−Kif5C) an anti-kinesin antibody (Kif5c) in both ligated and unligated sciatic nerves. The proximal and distal nerve segments are identified, and the approximate location of the ligature site is indicated in ligated and unligated nerves

Fig. 4

NF content is unaltered in the distal axon of SMAΔ7 mice. Sciatic nerves were ligated for 6 h, and equal length sections were dissected. Proximal (P) and distal (D) sciatic nerve lysates were separated on 7.5% SDS polyacrylamide gels from wild type (N = 3) and SMAΔ7 (N = 3) mice at two different disease stages. Lysates were immunoblotted with antibodies recognizing mouse NF-H (CPCA NF-H), NF-M (RMO44), NF-L (DA2) and a neuron-specific isoform of tubulin, βIII-tubulin (TUJ1). NF subunits and βIII-tubulin accumulated on the proximal side of the ligation in both SMAΔ7 and wild-type mice. There was no difference in the level of accumulation between SMAΔ7 and wild-type mice at P7 (a) or P11 (c) as determined by relative optical density (ROD) measurements (b, d). Statistical significance was determined by a Student’s t test. X axis: proximal side of ligation (LP), distal side of ligation (LD), proximal section of unligated nerve (CP), distal section of unligated nerve (CD). Simultaneously run coomassie stained gels were used as loading controls (a, c top)

Fig. 5

Fast, anterograde axonal transport is unaffected prior to the onset of observable phenotypes in SMAΔ7 mice. Sciatic nerves were ligated for 6 h in P7 wild type and SMAΔ7 littermates, and then harvested for analysis. Sciatic nerve lysates proximal and distal to the ligature were separated on 7.5% SDS polyacrylamide gels. Accumulation of synaptic vesicle proteins was analyzed (a). Based on ROD measurements (b), there was no difference in accumulation of SV2-c (N = 3) and Syt1 (N = 5) in SMAΔ7 mice. Accumulation of molecular motors that transport cargos in anterograde, Kif5c, and retrograde, dynein, directions was analyzed (c). There was no difference in the accumulation of Kif5c (N = 4) or dynein (N = 3) in P7 SMAΔ7 mice (d). Statistical significance was determined by a Student’s t test. X axis: proximal side of ligation (LP), distal side of ligation (LD), proximal section of unligated nerve (CP), distal section of unligated nerve (CD). The heavy (a, c top) subunit of NFs was used as a loading control

Fig. 6

Fast, anterograde transport is reduced prior to observed reductions in synaptic vesicle densities. Sciatic nerves were ligated for 6 h in P11 wild type and SMAΔ7 littermates, and then harvested for analysis. Sciatic nerve lysates proximal and distal to the ligature were separated on 7.5% SDS polyacrylamide gels. Accumulation of synaptic vesicle proteins was analyzed (a). Accumulation of SV2-c (N = 3; P < 0.001) (a) was reduced in P11 SMAΔ7 mice as determined by ROD measurements (b). In addition, distal levels of Syt1 (N = 4; P < 0.01) in ligated and unligated nerves were reduced in P11 SMAΔ7 mice (b). Accumulation and steady state levels of Kif5c (N = 3) appeared unaffected in SMAΔ7 mice whereas both accumulation and steady state levels of dynein (N = 4; P < 0.05) were reduced (c, d). Statistical significance was determined by a Student’s t test. X axis: proximal side of ligation (LP), distal side of ligation (LD), proximal section of unligated nerve (CP), distal section of unligated nerve (CD). The heavy (a, c top) subunit of NFs was used as a loading control

Fig. 7

Reduction in transport of synaptic vesicle proteins is not due to reduced expression in SMAΔ7 mice. Three differing amounts of total protein (5, 10, and 20 μg) from spinal cord lysates were separated on 7.5% SDS polyacrylamide gels from wild type and SMAΔ7 mice at P7 (a) and P11 (c). Lysates were immunoblotted with antibodies recognizing SV2-c and Syt1 (mAb48). At P7 (N = 3; P < 0.05) and P11 (N = 5; P < 0.05), the spinal cord levels of SV2-c (a, c middle) were increased in SMAΔ7 mice. Relative expression levels were analyzed by ROD measurements (b). However, despite the reduction in transport and steady state levels observed at P11 for Syt1, spinal cord levels were unaffected in SMAΔ7 mice at P7 (N = 3) and P11 (N = 5) (a, c lower). Statistical significance was determined by a Student’s t test. The heavy (a, c top) subunit of NFs was used as a loading control

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The spinal muscular atrophy mouse model, SMAΔ7, displays altered axonal transport without global neurofilament alterations - PubMed (original) (raw)