G alphas-induced neurodegeneration in Caenorhabditis elegans

Abstract

We describe a genetic model for neurodegeneration in the nematode Caenorhabditis elegans. Constitutive activation of the GTP-binding protein Galphas induces neurodegeneration. Neuron loss occurs in two phases whereby affected cells undergo a swelling response in young larvae and subsequently die sometime during larval development. Different neural cell types vary greatly in their susceptibility to Galphas-induced cytotoxicity, ranging from 0 to 88% of cells affected. Mutations that prevent programmed cell death do not prevent Galphas-induced killing, suggesting that these deaths do not occur by apoptosis. Mutations in three genes protect against Galphas-induced cell deaths. The acy-1 gene is absolutely required for neurodegeneration, and the predicted ACY-1 protein is highly similar (40% identical) to mammalian adenylyl cyclases. Thus, Gs-induced neurodegeneration is mediated by the second messenger cAMP. Mutations in the unc-36 and eat-4 genes are partially neuroprotective, which indicates that endogenous signaling modulates the severity of the neurotoxic effects of Galphas. These experiments define an intracellular signaling cascade that triggers a necrotic form of neurodegeneration.

Fig. 1.

Neurodegenerations caused by activated Gα_s. A GTPase-defective rat Gα_s cDNA containing the HA epitope tag was coexpressed with the green fluorescent protein (GFP) in GLR-1-expressing neurons, as described in Materials and Methods. Neurodegeneration occurs in two phases. In young larvae (A, B) affected cells swell to several times their normal diameter. Swelling is apparent by the morphology of GFP-expressing cells (A) and by their appearance in bright-field optics (B) as enlarged and apparently vacuolated cells, often with an intact nucleus. The interneurons AVE and AVD are swollen, compared with neighboring unaffected cells (asterisks). Cytotoxicity is scored by examining adult animals for the presence of the PVC neuron. GFP-expressing PVC neurons typically are missing inα_s(gf)(C), but not inα_s(gf); acy-1(nu343)(D) adults. Expression of Gα_s was monitored in α_s(gf); acy-1(nu343) animals by staining with anti-HA antibodies (E). This panel shows a stained L1 larva. Although some differences in expression levels are apparent, typically 10 brightly staining neurons are seen in the lateral ganglion. Staining of a PVC neuron is shown for comparison.

Fig. 2.

ACY-1 encodes an adenylyl cyclase.A, Genetic and physical map position ofacy-1. B, Transgenes containing the cosmid F17C8 rescue the acy-1(nu327) mutant phenotype.C, The predicted amino acid sequence, as predicted by GENEFINDER (accession number Z35719Z35719) and confirmed by our RT-PCR analysis, of ACY-1 (top) is shown aligned to mouse adenylyl cyclase type 9 (bottom), the most highly related sequence (40% identical) in the database.Underlined sequences indicate predicted transmembrane domains. D, Predicted structure of theacy-1 gene (using the GENEFINDER algorithm) and of the GFP fusion construct (KP#107) are shown. Positions of theacy-1 mutations nu327,nu343, and nu329 are indicated (C, D).

Fig. 3.

ACY-1 is expressed in neurons and muscles. The KP#107 acy-1:: gfp fusion gene was expressed in transgenic animals, as described in Materials and Methods. ACY-1 appears to be expressed in most or all muscles and neurons.A, Expression in the two ventral rows of body muscles (arrows) and in the ventral cord neurons and neuropile (lines). B, Expression in the vulva muscles (arrowheads). Nearly all of the 302 neurons in the adult appear to express ACY-1. Cell bodies can be identified on the basis of the bright fluorescence in intracellular membranes (presumably the endoplasmic reticulum or Golgi apparatus). ACY-1 does not appear to be expressed in non-neural tissues, nor is it expressed in the pharynx.