spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online 27 February 2007
doi: 10.1242/jcs.03401

Journal of Cell Science 120, 1134-1141 (2007)
Published by The Company of Biologists 2007
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Related articles in JCS
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tóth, M. L.
Right arrow Articles by Vellai, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tóth, M. L.
Right arrow Articles by Vellai, T.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

Influence of autophagy genes on ion-channel-dependent neuronal degeneration in Caenorhabditis elegans

Márton L. Tóth1, Péter Simon1, Attila L. Kovács2 and Tibor Vellai1,*

1 Department of Genetics, Eötvös Loránd University, Budapest, H-1117, Hungary
2 Department of Anatomy, Cell and Developmental Biology, Eötvös Loránd University, Budapest, H-1117, Hungary


Figure 1
View larger version (59K):
[in this window]
[in a new window]

  		Fig. 1. Gain-of-function mutations in the mec-4 gene cause a degenerative state of touch receptor neurons in C. elegans. (A) Schematic representation of the position of cell bodies of the six touch receptor neurons that express pmec-4::GFP. (B) Expression of pmec-4::GFP at the L2 larval stage in a wild-type animal. Arrows show the six touch receptor neurons. (C) Expression of pmec-4::GFP at the L2 stage in a mec-4(u231) mutant. Cell death was inferred when glowing touch receptor neurons were missing from their normal positions. Arrows indicate residual pmec-4::GFP expression. (D) Nomarski image shows the morphology of a vacuolated PLM neuron (arrow) undergoing necrosis in a mec-4(u231) mutant L1 larva. Bars, 100 µm.

 

Figure 2
View larger version (41K):
[in this window]
[in a new window]

  		Fig. 2. Inactivation of the autophagy genes bec-1, unc-51 and lgg-1 suppresses ion-channel-dependent neuronal degeneration in C. elegans. (A) Number of vacuolated (dying) touch receptor neurons at the late L1 larval stage per 100 mec-4(u231) mutant animals with reduced autophagy gene activity. bec-1(–); Ex[bec-1(+)]refers to bec-1 mutant genotype bearing a non-integrated, extrachromosomal array (see Materials and Methods). For homozygous bec-1 mutants, n=50, P<0.001; for lgg-1(RNAi) animals, n=200, P<0.001; for other genotypes, n=200; P<0.0001 (unpaired t-tests). (B) Percentage of pmec-4::GFP-positive touch receptor neurons at young adulthood in mec-4(u231) mutants with reduced autophagy gene activity. In contrast to panel A, this panel indicates survival rate (%) of cells. For bec-1 mutants, n=50, P<0.001; for lgg-1(RNAi) animals, n=285, P<0.001; for other genotypes, n=200, P<0.0001 (unpaired t-tests). As a control, the expression of mec-4::GFP was assayed in bec-1(ok691) and unc-51(e369) single mutants as well as in lgg-1(RNAi) animals. Number of GFP-positive cells: unc-51(e369) mutants, 5.38 (n=263); bec-1(ok691) mutants, 5.47 (n=37); lgg-1(RNAi) animals, 5.67 (n=168); wild-type animals, 5.6 (n=493). These data show that inactivation of bec-1 and unc-51 only slightly influenced the development of touch receptor neurons, whereas lgg-1 RNAi treatment did not affect it at all (it is notable that lgg-1 RNAi completely abolished the expression of an lgg-1::gfp reporter). (C) Expression of a translational fusion pmec-4::MEC-4::GFP reporter in a PLM cell in a wild-type (left) and in an affected arrested bec-1(RNAi) (right) larva. In the latter, the expression intensity and number of GFP punctae were comparable with the wild type. (D) Number of vacuolated PVC interneurons at the L1 stage per 100 deg-1(u506) animals with reduced autophagy gene activity. For homozygous bec-1 mutants, n=50, P<0.001; for lgg-1(RNAi) animals, n=300, P<0.01; for other genotypes, n=300, P<0.0001 (unpaired t-tests). (E) Number of vacuolated IL1 sensory neurons and PVC interneurons at the L1 stage per 100 deg-3(u662) mutants with reduced autophagy gene activity. For lgg-1(RNAi) animals, n=300, P<0.018; for other genotypes, n=300, P<0.0001; (unpaired t-tests). In RNAi experiments, control animals were fed with E. coli HT115 expressing the empty vector under RNAi-inducing conditions. Data are mean ± s.e.m.

 

Figure 3
View larger version (34K):
[in this window]
[in a new window]

  		Fig. 3. UNC-51 and BEC-1 deficiency suppresses touch insensitivity in mec-4(u231) and deg-1(u506) mutants. Ex, Ex[pbec-1::BEC-1::GFP + rol-6(su1006)] genotype. Because unc-51 mutant animals are paralyzed, we scored their ability to shift the anterior or posterior body region upon touching. For each double mutant genotype n=150, P<0.0001 (unpaired t-test). Data are mean ± s.e.m.

 

Figure 4
View larger version (53K):
[in this window]
[in a new window]

  		Fig. 4. BEC-1 and LGG-1 are expressed in the nervous system. (A) A non-functional translational fusion BEC-1::GFP reporter accumulates in the PVM of a wild-type animal. Both cell body and processes (arrowhead) of this cell are GFP positive. VNC, ventral nerve cord. (B) BEC-1 expression in the nerve ring (bracket). (C) LGG-1::GFP, which lacks the C terminus of LGG-1 (see Materials and Methods), is expressed in PLML. (D) LGG-1::GFP accumulation in the ventral nerve cord. (E) CeTor, which encodes a kinase that downregulates autophagy, is expressed in all neurons. Bracket indicates the nerve ring. (F-H) Nomarski, fluorescence and merged images, respectively, showing the expression of a non-integrated, translational fusion (functional) autophagosome marker GFP::LGG-1 (Meléndez et al., 2003Go) in a PVC interneuron of a deg-3(u662) animal. The GFP-positive punctate areas are inferred to label preautophagosomal and autophagosomal structures (Meléndez et al., 2003Go). In this specimen, the PVC interneuron is at the early stage of degeneration. Note that GFP becomes gradually diluted as degeneration progresses. The nearly exclusive expression of GFP in PVC is due to a mosaicism of the non-integrated marker.

 

Figure 5
View larger version (26K):
[in this window]
[in a new window]

  		Fig. 5. BEC-1 and UNC-51 interfere with neurotoxin-induced necrotic cell death. (A) Expression of pdat-1::GFP in the head dopamine neurons in a wild-type hermaphrodite. (B) pdat-1::GFP expression in a wild-type hermaphrodite exposed to the neurotoxin 6-OHDA. 6-OHDA treatment results in severe degeneration of CEP and ADE dopamine neurons, revealed by the loss of processes and/or cell bodies. (C) pdat-1::GFP expression in the head dopamine neurons of an unc-51(e1189) mutant hermaphrodite. Although in some individuals the running of the processes is slightly affected, GFP expression is similar to that found in the wild type. (D) pdat-1::GFP expression in the head dopamine neurons in an unc-51(e1189) mutant hermaphrodite exposed to 6-OHDA. (E) UNC-51 and BEC-1 deficiency suppresses 6-OHDA sensitivity of the head dopamine neurons. The percentage of animals with intact CEP and ADE neurons (both cell bodies and processes) was scored. Data are mean ± s.e.m. Empty columns represent control animals, filled columns indicate animals exposed to 6-OHDA. n=100, P<0.01 (unpaired t-test).

 

Figure 6
View larger version (21K):
[in this window]
[in a new window]

  		Fig. 6. Influence of nutrient signaling on degenerin-induced neurotoxicity. Survival rate (%) of the six touch receptor neurons at the young adult stage in mec-4(u231) mutants with reduced CeTOR activity or exposed to starvation for 1 day at the L1 larval stage. Number of mec-4::GFP-positive cells was scored. Note that the percentage of GFP-positive cells was significantly higher in 11-day-old adults well fed during development (20.4%) than in young adults deprived of food for 1 day at the L1 stage (6.2%). Thus the effects of starvation but not starvation regimens extending development by 1 day, resulted in an increased loss of neurons. Control (OP50) animals were fed with E. coli OP50. Control RNAi (HT115) animals were fed with E. coli HT115 expressing the empty vector under RNAi-inducing conditions. let-60 encodes a component of the Ras signaling pathway. n=300, P<0.0001 (unpaired t-test). Data are mean ± s.e.m.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2007