Axons degenerate in the absence of mitochondria in C. elegans

Abstract

Many neurodegenerative disorders are associated with mitochondrial defects [1-3]. Mitochondria can play an active role in degeneration by releasing reactive oxygen species and apoptotic factors [4-7]. Alternatively, mitochondria can protect axons from stress and insults, for example by buffering calcium [8]. Recent studies manipulating mitochondria lend support to both of these models [9-13]. Here, we identify a C. elegans mutant, ric-7, in which mitochondria are unable to exit the neuron cell bodies, similar to the kinesin-1/unc-116 mutant. When axons lacking mitochondria are cut with a laser, they rapidly degenerate. Some neurons even spontaneously degenerate in ric-7 mutants. Degeneration can be suppressed by forcing mitochondria into the axons of the mutants. The protective effect of mitochondria is also observed in the wild-type: a majority of axon fragments containing a mitochondrion survive axotomy, whereas those lacking mitochondria degenerate. Thus, mitochondria are not required for axon degeneration and serve a protective role in C. elegans axons.

Figure 1. Cloning of ric-7

(A) The mutation n2657 was mapped to the interval on chromosome V between lin-25 and unc-76. Cosmid F58E10 (purple bar) spans 41 kilobases in this interval and rescued ric-7 mutants as a transgene. Further experiments using PCR fragments indicated that rescuing activity was contained in an 18 kilobase (kb) fragment that contains two hypothetical genes, F58E10.1 and F58E10.7. (B) All five ric-7 alleles contain alterations in F58E10.1 exons (see Table S1). Arrows mark the locations of premature stops. ox134 is a large deletion affecting three genes.

Figure 2. RIC-7 co-localizes with mitochondria and is required for mitochondrial distribution in axons

(A) Tagged RIC-7 expressed in GABA neurons (Punc-47:RFP::RIC-7, oxEx1598) rescues ric-7 mutants and colocalizes with mitochrondria tagged with Tom20::GFP (‘mito::GFP’). Worms were imaged with the dorsal nerve cord facing the objective. See also Figure S2A,B. (B) Mitochondria tagged with Tom20::GFP are trapped in the cell body and absent from axons in ric-7 and kinesin-1/unc-116 mutants but not kinesin-3/unc-104 mutants. Arrowheads indicate cell bodies, and arrows point to axonal mitochondria. Asterisks indicate gut autofluorescence. See also Figure S2C,D. Worms were imaged with the ventral nerve cord facing the objective. All scale bars are 10 μm. (C) Average number of mitochondria per 50 μm in the ventral nerve cord of GABA motor neuron axons +/− SEM. Mitochondrial distribution is rescued cell autonomously in axons when RFP::RIC-7 is expressed from an array (‘RIC-7(+)’, oxEx1598). p < 0.0001 Kruskal-Wallis test with Dunn’s multiple comparisons to the wild type (black) and to ric-7(n2657) (red) *** p<0.001, * p<0.05. The number of animals is displayed below.

Figure 3. Axon degeneration is enhanced in the absence of mitochondria

(A) PVQ axons spontaneously degenerate in ric-7 mutants. The degenerating axons proceed through the following morpholigies; swollen, beaded, and finally truncated. The percentage of axons with a given morphology is depicted for each life stage of ric-7(zd152). The number of worms assayed is indicated. (B) In the first larval stage, PVQ axons are intact in ric-7(zd152) mutants. GFP is expressed in PVQ and head neurons (ASH, ASI) under the sra-6 promoter. The two PVQ cell bodies are in the tail; each extends an axon along the ventral nerve cord to the head. (C) During the fourth larval stage, the distal portion of the axon degenerates, beginning with swellings. (D) PVQ axons are typically truncated, frequently near the vulva, by the end of the fourth larval stage. The proximal half of the axons and the cell bodies remain intact. Scale bar = 100 μm. See also Figure S3A–E. (E) GABA motor neuron axons degenerate following laser axotomy. Arrowheads point to the cut sites in the image taken immediately after axotomy. There are three severed axons (numbered). (F) After 24 hrs of recovery worms were reimaged and the presence or absence of severed axons was scored. The wild-type panel is the same worm from panel E. Two of the three axons exhibit swellings but are still present. Severed motor neuron axons completely degenerate in ric-7 and kinesin-1/unc-116 mutants. Scale bar = 25 μm. (G) The average percent of axons that are still present 24 hrs after axotomy +/− SEM. More axons degenerate in mutants lacking mitochondria compared to the wild type. The kinesin-3/unc-104 mutant, which has mitochondria throughout their axons, degenerates at wild-type levels. p < 0.0001 Kruskal-Wallis test with Dunn’s multiple comparisons to the wild type, *** p<0.001, **p<0.01. See also Figure S3F–K.

Figure 4. Mitochondria mediate axon protection

(A) The presence of mitochondria protects axons from degeneration in a wild-type background. ALA axons are labeled by expressing membrane-bound GFP under the acr-5 promoter (‘axon::GFP’) and mitochondria are labelled by expressing mito:RFP under the ida-1 promoter. ALA axons were cut by laser axotomy into multiple segments in wild-type worms that either contained (A) or lacked (B) mitochondria. 24 hrs after axotomy, the axon segment with a mitochondrion was still intact (below), whereas the segment without a mitochondrion had degenerated. The mito::RFP images have been inverted and the contrast has been enhanced to increase the visibilty of the mitochondria (arrows). Asterisks indicate gut autofluorescence. Scale bar = 10 μm. (C) 72% of the axon fragments containing a mitochondrion were still present 24 hrs later, whereas only 26% of axon fragments without mitochondria perdured. Error bars represent the 95% confidence interval (n = 13 worms and 48 axon fragments, p= 0.0028, two-tailed Fisher’s Exact Test). (D) A transport chimera construct was created by fusing the cDNA for unc-116 (kinesin-1) to the tomm-7 gene, which encodes for the outer mitochondrial membrane protein Tom7. GFP flanked by linkers was inserted between the motor and the mitochondrial protein. The construct was expressed in GABA neurons under the unc-47 promoter. Below is a conceptual cartoon of the transport chimera protein. (E) Expression of the transport chimera ‘Kinesin::Tom7’ restores mitochondria in ric-7 mutant axons. See also Figure S4B. (F) The transport chimera suppresses the GABA neurotransmission defects in ric-7 mutants. In C. elegans GABA release is required for enteric muscle contractions (emc) following posterior body muscle contractions (pboc). In ric-7 mutants only 16% of pbocs are followed by an emc. This defect was rescued by expressing RIC-7(+) (oxEx1598[RFP::RIC-7]), or the transport chimera ‘Kinesin::Tom7’ in GABA neurons. (G) Restoring mitochondria to ric-7 mutant axons (magenta bar) also suppressed degeneration of GABA motor neuron axons following injury. The transport chimera has no effect in a wild-type background (purple bar). See also Figure S4C. The wild-type and ric-7 data are the same as in Figure 3G. For E–G, p < 0.0001 Kruskal-Wallis test with Dunn’s multiple comparisons to the wild type (black) and to ric-7(n2657) (red), *** p<0.001, **p<0.01.