Live imaging reveals the cellular events downstream of SARM1 activation
- Washington University School of Medicine, United States
- Genetics, Washington University School of Medicine, United States
- Genetics, Hope Center for Neurological Disorders, Washington University School of Medicine, United States
- Developmental Biology, Needleman Center for Neurometabolism and Axonal Therapeutics, Washington University School of Medicine, United States
Figures
Figure 1 with 1 supplement
SARM1 enzymatic activity regulates mitochondrial movement and calcium homeostasis in injured axons.
(A) Representative kymograph of injured SARM1 KO axons expressing either GFP, SARM1, or SARM1.E642A (E642A). For imaging mitochondria movement, MitoDsRed lentivirus was transduced in all …
Figure 1—figure supplement 1
Expression level of SARM1.WT and SARM1.E642A.
Figure 2 with 1 supplement
The role of calcium in axon degeneration.
(A) Pre-incubation of MPTP inhibitor (1, 10, or 100 µM CsA) did not significantly prevent the degeneration of wild-type axons after axon injury. Axon degeneration is defined as a degeneration index >…
Figure 2—figure supplement 1
EGTA efficiently blocks calcium influx.
However, it is toxic to mitochondria.
Figure 3 with 1 supplement
Live single axon imaging enables temporal dissection of cellular events in injured axons.
(A) Schematic diagram of laser axotomy in cultured embryonic DRG neurons. GCaMP6 and mRuby3 were expressed to observe calcium fluctuations and axonal morphology. (B) Snapshots of an injured …
Figure 3—figure supplement 1
DRG neuron culture for single axon imaging.
Figure 4 with 1 supplement
Mitochondrial dysfunction precedes calcium influx in injured axons.
(A) (Top) Experimental design for observing calcium influx (GCaMP6) and mitochondria movement (MitoDR) after axon injury. MitoDR images were acquired every 5 s (for 300 s, 60 frames) followed by …
Figure 4—figure supplement 1
Assessing mitochondrial potential and calcium influx in injured axons.
(Left) Differentiation of TMRM and GCaMP6 measurements in Figure 4E. The differentiation is defined as the percentage change from the previous measurement. The percentage change (y-axis) of both …
Figure 5 with 2 supplements
ATP levels drop before mitochondria stop in injured axons.
(A) (Left) Experimental design for imaging changes to ATP (PercevalHR) and mitochondrial movement (MitoDR) after axonal injury. Prior to axotomy, baseline PercevalHR intensity and mitochondrial …
Figure 5—figure supplement 1
Validation of PercevalHR.
(Left) Representative images of either DMSO or 50 µm CCCP treated DIV7 axons at baseline and 2 hr after treatment. Lentivirus of PercevalHR was transduced at DIV2 (Right). Fluorescent signal of …
Figure 5—figure supplement 2
Axonal ATP level in WT and SARM1 KO after axotomy.
Figure 6
Calcium influx disrupts membrane integrity.
(A) (Left) Snapshots of representative live axon images for GCaMP6, mRuby3, and Alex647-conjugated Annexin-V at baseline, and 4.78 and 6.28 hr after axotomy. (Right) Representative single axon …
Figure 7 with 1 supplement
Model of axon degeneration.
Figure 7—figure supplement 1
Mitochondria accumulate in axonal swellings in injured axons.
(Top) Representative images of injured (a) and uninjured (b) axons labeled with GFP and MitoDR. (Bottom) Pixel intensity of each axon is plotted. Note that most axonal swellings overlap with …
Videos
Video 1
Time lapse imaging of injured single axon.
Additional files
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Transparent reporting form
- https://cdn.elifesciences.org/articles/71148/elife-71148-transrepform1-v2.docx
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Source data 1
Data for figures.
- https://cdn.elifesciences.org/articles/71148/elife-71148-supp1-v2.zip
