Figure 1.
Agrin is depleted from NMJs following conditional deletion in adults.
A. Schematic of the method used to delete agrin from subsets of motoneurons. In the SLICKA transgene, separate Thy1 regulatory elements drive expression of CreER and YFP. Following administration of tamoxifen, CreER is activated and the agrin gene is deleted in many YFP+ motoneurons. B. A YFP+ motor axon (green) in a triangularis sterni muscle. Unlabeled axons are stained with neurofilament and syntaptotagmin 2 (red) to mark all nerve terminals. Scale bar = 100 µm. Explain the red labeling of motor axons ***SLICK negative = Neurofilament/Syn??? C. Agrin at NMJs in control (agrn+/flox; SLICKA) and mutant (agrn−/flox; SLICKA) muscle at the indicated times after tamoxifen (TX) administration. Agrin levels are decreased slightly 1 month after TX and markedly by 3 months. Scale bar = 10 µm. TX = tamoxifen.
Figure 2.
Agrin loss leads to disassembly of the postsynaptic apparatus.
A–B. Triangularis sterni muscle from control (A) and conditional agrin knockout (B) animals 3 months after tamoxifen (TX) administration. TX administration to heterozygotes does not disrupt apposition of axons (green) to AChR-rich postsynaptic membrane (red), but many axons appose AChR-poor postsynaptic structure (arrows) in the mutant. Scale bar = 100 µm. TX = tamoxifen C–F Muscles from agrin conditional knockouts and controls 3 months after TX stained for laminin α4 (C), laminin α5 (D), rapsyn (E), and dystroglycan (F). In each case, mutant NMJs show markedly decreased levels of immunoreactivity. Scale bar = 10 µm.
Figure 3.
Agrin loss disrupts nerve terminal organization.
A. Variation in nerve terminal morphology at AChR-poor NMJs in agrin mutants. Some terminals appear relatively normal (*) while others show marked dystrophies (arrows). Scale bar = 20 µm. B. Range of presynaptic defects in agrin mutant nerve terminals (arrows). YFP, green. C–D. The terminals that are affected show low levels of agrin relative to those that are unaffected (C), and the percentage of terminals that are altered increases over time (D). n = at least 4 samples per group.
Figure 4.
NMJs From the same motor neuron are similarly affected following conditional agrin deletion.
A. Three pairs of nerve terminals from triangularis muscles of conditional agrin mutant 3 months after tamoxifen administration. In each case, both terminals arise from the same axon. Both are unaffected in one pair and both are affected in a second pair; in the third pair, one terminal is normal and the other is dystrophic. Scale bar = 20 µm. B. Quantification of nerve terminal pairs. n = 100 total pairs.
Figure 5.
Nerve degeneration and death results from long-term agrin deletion.
A. YFP labeled axons in the triangularis of control and agrin mutant mice 6 months after tamoxifen administration. Axons in the mutant show less branching than those in controls. Scale bar = 100 µm. B–C. Mutant nerve terminals show little to no presynaptic specialization (B) but rather often end in a single axon terminal bulb (C). Scale bar = 50 µm. YFP, green.
Figure 6.
Abnormal NMJs in laminin-α4 knockout mice.
A–B. Laminin-α4 is completely absent in the NMJs from 6 month old lama4−/− mice (A), and at this time point the NMJ has begun to atrophy (B). There are clear signs of axonal swellings (arrow) and denervation (star) in control 6 months old mice. Scale bar = 10 um.
Figure 7.
Deletion of laminin-α4 does not alter the levels or localization of other synaptic components.
Distribution and levels of β-dystroglycan (A), agrin (B) and rapsyn (C) in 6 month old control and lama4−/− NMJs. All three proteins remain concentrated in NMJs lacking laminin-α4. Scale bar = 10 um.
Figure 8.
Absence of laminin-α4 causes NMJ to age prematurely.
A. Axon terminals (left) and AChRs (middle panels) from diaphragm muscles of 6 month old lama4−/− mice. The age-associated abnormalities observed in mutants at 6 months of age were similar to those seen in two-year-old wild type mice. B. Prevalence of axonal and terminal changes in lama4−/− and wild type animals. At least 60 NMJs were analyzed for each genotype at each age. Scale Bar = 10 um.
Figure 9.
Molecular changes associated with aging neuromuscular junctions.
A. Levels of RNA encoding different agrin isoforms were compared in the spinal cords of young adult and 2 year old mice by quantitative PCR. B. Levels of laminin-α4, -α5 and- β2 RNA were compared in young adult and 2 year old tibialis anterior muscles using quantitative PCR. C–F. Immunostaining with antibodies to agrin and three laminin subunits in young adult and old NMJs. Levels of these proteins did not change significantly with age, but their distribution was altered (see D bottom panel; star = extrasynaptic; arrow = diffuse or lack of expression). Bars represent the SEM. Scale Bar = 10 um.