Figure 6

Electroporation of SHP-2 shRNA construct into soleus muscle of adult mice leads to disassembly of NMJs. Adult mice soleus muscles were electroporated in vivo with a mixture of pSUPER.shSHP2 and NLS-GFP constructs (A, C), or pSUPER vector and NLS-GFP as control (B, D). After six weeks muscles were dissected, whole mounts of fibers prepared and stained with rhodamine-α-BTX and a mixture of neurofilament and synaptophysin antibodies followed by Cy5 secondary antibody. NMJs were analyzed by confocal microscopy, whereby successfully electroporated muscle fibers were identified by the expression of NLS-GFP in myonuclei. (A) Expression of pSUPER.shSHP2 results in NMJ disassembly in GFP-positive myofibers (closed arrowhead). NMJs of GFP-negative fibers remain intact (open arrowheads). (B) Expression of control pSUPER vector and NLS-GFP has no effect on the NMJs. A three-dimensional reconstruction of a confocal image of a muscle electroporated with pSUPER.shSHP2 and NLS-GFP (C), or control pSUPER vector and NLS-GFP (D) shows details of the NMJs. (C) A 3D view from inside the muscle illustrates that expression of SHP-2 shRNA leads to disassembly of the NMJ, loss of the usual morphology and pretzel shape, and a resulting fragmented appearance of the NMJ, with the nerve becoming visible through the pretzel remnants. (D) In control electroporated muscle fibers, GFP-positive synaptic nuclei are located below the intact pretzel-shaped accumulations of AChRs and no nerve is visible through the pretzel. (E) The number of intact and disassembled NMJs is shown for control (pSUPER) vector and pSUPER.shSHP2 electroporated muscle fibers, as percentage of the total number of NMJs analyzed (total of 25 endplates from 3 mice for pSUPER and 15 endplates from 5 mice for pSUPER.shSHP2). Only endplates with synapse-associated nuclei expressing NLS-GFP were analyzed. Scale bars, 30 μm in A, B; 10 μm in C, D.