Pharmacogenetic analysis reveals a post-developmental role for Rac GTPases in Caenorhabditis elegans GABAergic neurotransmission

Abstract

The nerve-cell cytoskeleton is essential for the regulation of intrinsic neuronal activity. For example, neuronal migration defects are associated with microtubule regulators, such as LIS1 and dynein, as well as with actin regulators, including Rac GTPases and integrins, and have been thought to underlie epileptic seizures in patients with cortical malformations. However, it is plausible that post-developmental functions of specific cytoskeletal regulators contribute to the more transient nature of aberrant neuronal activity and could be masked by developmental anomalies. Accordingly, our previous results have illuminated functional roles, distinct from developmental contributions, for Caenorhabditis elegans orthologs of LIS1 and dynein in GABAergic synaptic vesicle transport. Here, we report that C. elegans with function-altering mutations in canonical Rac GTPase-signaling-pathway members demonstrated a robust behavioral response to a GABA(A) receptor antagonist, pentylenetetrazole. Rac mutants also exhibited hypersensitivity to an acetylcholinesterase inhibitor, aldicarb, uncovering deficiencies in inhibitory neurotransmission. RNA interference targeting Rac hypomorphs revealed synergistic interactions between the dynein motor complex and some, but not all, members of Rac-signaling pathways. These genetic interactions are consistent with putative Rac-dependent regulation of actin and microtubule networks and suggest that some cytoskeletal regulators cooperate to uniquely govern neuronal synchrony through dynein-mediated GABAergic vesicle transport in C. elegans.

Figure 1.—

Rac GTPase and canonical Rac regulator mutant anterior convulsions are commensurate with increasing concentrations of PTZ. (A and B) The response level (the percentage of young adult worms convulsing per total sample size; n = 30 for each of three independent experiments) of various C. elegans strains is depicted for each PTZ concentration, ranging from 0 to 10 mg/ml. N2 wild-type worms (black squares) did not exhibit anterior convulsions at any tested concentration of PTZ. Conversely, representative GABA (black diamonds) and general synaptic transmission (black circles) mutants did exhibit PTZ-induced anterior convulsions. (A) Rac gain-of-function (blue and green circles), but not loss-of-function mutants (collectively shown by gray circles), demonstrated PTZ-induced anterior convulsions. (B) Rac regulator mutants also displayed PTZ-induced anterior convulsions. Although both unc-73 Rac GEF mutants were hypersensitive to PTZ, the weakest mutant tested, e936 (brown circles), was more sensitive to PTZ than a stronger unc-73 Rac GEF mutant, rh40 (purple circles). The strongest mig-15 mutant tested, rh326 (blue circles), was more sensitive to PTZ than a weaker mig-15 mutant, rh148 (blue circles), but was not significantly different from a mig-15 mutant, rh80 (gray circles), of intermediate strength. Hypomorphic alpha integrin (ina-1) mutants, gm39 (green circles) and gm144 (gray squares), were hypersensitive to PTZ. Of these two mutants, gm39, thought to be the weakest, exhibited the greatest sensitivity, as 97.8 ± 3.8% convulsed on 10 mg/ml PTZ, whereas 25.6 ± 3.8% of gm144 worms convulsed at the same concentration. Each data point represents mean ± SD.

Figure 2.—

Still-frame images demonstrating C. elegans mutant strains with anterior convulsions in response to 10 mg/ml PTZ. The still images are representative frames from movies (25 frames/sec), which are available in the supporting information. The black lines represent stationary reference points for visualization of anterior movements in relation to time (“s” indicates seconds). Anterior is to the left in all images where lines are placed perpendicularly to the original position of each worm's nose. The convulsions of a general synaptic transmission mutant, rab-3(y251), mimic those of a Rac gain-of-function mutant, ced-10(n3246), and the Rac regulator mutants, mig-15(rh80) and ina-1(gm39). Bar, 100 μm.

Figure 3.—

Dynein motor complex and canonical Rac-signaling pathway mutants have increased neuromuscular excitability, as revealed by hypersensitivity to aldicarb. (A–C) The response level (the percentage of young adult worms paralyzed per total sample size; n = 30 for each of three independent experiments) of various C. elegans strains is depicted for each 30-min time point over a 3-hr exposure to 0.5 mm aldicarb. Paralysis of N2 wild-type worms (black squares) was commensurate with the time of aldicarb exposure. A representative GABA mutant (unc-25, black diamonds) was hypersensitive to aldicarb, whereas a general synaptic vesicle transport mutant (rab-3, black circles) was resistant to aldicarb when compared to wild type. (A) A hypomorphic dhc-1 mutant, js121 (orange circles), demonstrated hypersensitivity to aldicarb. Likewise, a predicted lis-1 null mutant (blue circles) exhibited robust aldicarb hypersensitivity, despite also carrying a mutation (unc-32) that confers resistance to aldicarb in isolation (green circles). (B) Rac loss-of-function mutants (collectively shown by gray circles) exhibited wild-type aldicarb sensitivities. However, Rac gain-of-function mutants (blue and green circles) and transgenic worms, which express either constitutively active mig-2(G16V) (purple circles) or rac-2(G12V) (brown circles) under the control of the neuron-specific unc-115 promoter (P_unc-115), were hypersensitive to aldicarb. (C) Rac regulator mutants also displayed hypersensitivity to aldicarb. A higher percentage of mutants with a weak Rac GEF allele, unc-73(e936) (brown circles), was paralyzed at 1 hr of aldicarb exposure, compared with mutants in a stronger Rac GEF allele, unc-73(rh40) (purple circles) at the same time. Conversely, significantly fewer mutants with the weakest mig-15 allele, rh148 (dark blue circles), were paralyzed after 1 hr of aldicarb exposure, compared to the percentage of mutants with the strongest mig-15 allele, rh326 (dark blue circles), or to the percentage of mutants with a mig-15 allele of intermediate strength, rh80 (gray circles). Likewise, there was a differential paralysis observed between ina-1(gm39) (green circles) and ina-1(gm144) (gray squares) mutants after 1 hr of aldicarb exposure. Notably, very few N2 wild-type worms were paralyzed after 1 hr of aldicarb exposure. All dynein motor complex, Rac gain-of-function, and regulator mutants were hypersensitive at 60 and 90 min. Two independently generated transgenic lines, which overexpress an ina-1 cDNA transgene under the control of the GABAergic neuron-specific unc-47 promoter (P_unc-47), were resistant to aldicarb (light blue circles). Each data point represents mean ± SD. Trends in sensitivity are shown in pale yellow. (D) The response level (the percentage of young adult worms paralyzed per total sample size; n = 30 for each of three independent experiments) of various C. elegans Rac-signaling mutants with (+) or without (−) an ina-1 cDNA transgene (Tg) under the control of P_unc-47 is depicted for 90 min of aldicarb exposure. Averaged results from two independently generated P_unc-47∷ina-1 transgenic lines are shown. GABAergic neuron overexpression of ina-1 did not significantly affect aldicarb sensitivity of wild-type (WT) worms at this time point. Similarly, P_unc-47∷ina-1 failed to alter aldicarb sensitivity of either the Rac gain-of-function mutants n3246 or gm103 or the strongest Rac-deficient GEF mutant, unc-73(rh40). Yet, GABAergic neuron overexpression of ina-1 significantly reduced aldicarb hypersensitivity of weaker ina-1(gm144), mig-15(rh148), and unc-73(e936) mutants. Each data point represents mean ± SD.

Figure 4.—

Rac GTPase and canonical Rac regulator mutants exhibit synaptic vesicle misaccumulations, but not architectural breaks, in GABAergic D-type motor neurons of ventral nerve cords. (A) The percentage of axonal GFP gaps (the percentage of young adult worms with gaps per total sample size; n = 30 for each of three independent experiments) in GABAergic D-type motor neurons of ventral nerve cords (VNCs) in various C. elegans strains. Soluble GFP expression showed no architectural breaks in the VNC axons of wild-type (WT) or Rac-signaling-pathway mutant young adult oxIs12 (P_unc-47∷GFP) worms (dark gray bars). Yet, Rac-signaling mutants, except for hypomorphic mig-15(rh148) mutants, had misaccumulated synaptic vesicles, as revealed by gaps in GABAergic neuron-specific expression of a synaptobrevin-1 (SNB-1) and GFP translational fusion protein (light gray bars). Results from oxIs12-bearing Rac-signaling mutants were standardized against wild-type oxIs12 worms. Likewise, results from juIs1-bearing Rac-signaling mutants were standardized against wild-type juIs1 (P_unc-25∷SNB-1∷GFP) worms. Each data point represents mean ± SD. *P < 0.05; Fisher's exact test. The ced-10(n3246) mutants with juIs1 are heterozygous for the dominant n3246 allele, whereas ced-10(n3246) mutants with oxIs12 are homozygous for the dominant n3246 allele. (B) A representative wild-type juIs1 young adult hermaphrodite exhibited no axonal SNB-1∷GFP gaps, unlike an unc-73(e936) homozygote, which was deficient in Rac activation. (C) Soluble GFP expression revealed no significant architectural breaks in the VNC axons of wild-type, Rac gf, or Rac regulator mutant L1 larval oxIs12 (P_unc-47∷GFP) worms. GABAergic neuron-specific expression of a SNB-1 and GFP translational fusion protein in L1 larval GABAergic D-type motor neurons showed significant percentages of synaptic vesicle misaccumulations, as demonstrated by SNB-1∷GFP gaps, in Rac gf, ina-1(gm144), ina-1(gm39), unc-73(e936), and unc-73(rh40) mutants. No axonal GFP gaps were observed in the VNCs of mig-15(rh148) mutant L1 larvae. Results from oxIs12-bearing Rac signaling mutants were standardized against wild-type oxIs12 worms at the same developmental stage. Likewise, results from juIs1-bearing Rac signaling mutants were standardized against wild-type juIs1 (P_unc-25∷SNB-1∷GFP) worms at the same developmental stage. Each data point represents mean ± SD. *P < 0.05; Fisher's exact test. Asterisks for P_unc-25∷SNB-1∷GFP data indicate significant differences in percentages of axonal GFP gaps, compared to the wild-type juIs1 background and P_unc-47∷GFP data for the same mutant, suggesting that synaptic vesicle transport defects (SNB-1∷GFP gaps) occur independently of architectural defects (soluble GFP gaps). Bar, 5 μm.

Figure 5.—

Lactose-induced RNAi feeding against canonical Rac-signaling pathway members results in synaptic vesicle misaccumulations, but not architectural breaks, in GABAergic D-type motor neurons of ventral nerve cords. The percentage of axonal GFP gaps (the percentage of young adult worms with gaps per total sample size; n = 30 for each of three to five independent experiments) in GABAergic D-type motor neurons of ventral nerve cords (VNCs) of various RNAi treatments. Soluble GFP expression showed no architectural breaks in the VNC axons of young adult oxIs12 (P_unc-47∷GFP) worms with mock (α-synuclein) RNAi or RNAi against Rac-signaling-pathway members (dark gray bars). Yet, RNAi against Rac-signaling-pathway members, except for mig-15, resulted in misaccumulated synaptic vesicles, as revealed by gaps in GABAergic neuron-specific expression of a synaptobrevin-1 (SNB-1) and GFP translational fusion protein (light gray bars). Combinatorial RNAi was used against two of three triply redundant Racs, ced-10 and rac-2. Results from mock RNAi against oxIs12 worms were used to standardize other results with oxIs12 worms. Likewise, results from mock RNAi against juIs1 (P_unc-25∷SNB-1∷GFP) worms were used to standardize other results with juIs1 worms. Each data point represents mean ± SD. *P < 0.05; Fisher's exact test.

Figure 6.—

RNAi feeding and pharmacological assays with Rac GTPase mutant backgrounds reveal synergistic genetic interactions with the dynein motor complex and canonical Rac regulators. (A and B) The response level (the percentage of young adult worms paralyzed per total sample size; n = 30 for each of three to five independent experiments) of various RNAi treatments after 60 min of exposure to aldicarb (A) or PTZ (B). (A) Like mock (α-synuclein) RNAi, RNAi against triply redundant Rac, ced-10, or rac-2 was not sufficient to enhance aldicarb sensitivity of N2 wild-type worms (dark gray bars). Conversely, combinatorial RNAi against ced-10 (medium gray bars) and rac-2 (light gray bars) was sufficient to enhance aldicarb sensitivity of wild-type worms. Similar enhancements of N2 sensitivity to aldicarb were observed with RNAi against the canonical Rac regulators, ina-1, mig-15, pes-7, or unc-73. Combinatorial RNAi against the functionally redundant PAK orthologs, pak-1 and max-2, or RNAi against the dynein motor complex members, bicd-1 and lis-1, was also sufficient to increase N2 sensitivity to aldicarb. RNAi against most of these Rac-signaling-pathway or dynein motor complex members also enhanced aldicarb sensitivities of either or both Rac gain-of-function mutants, ced-10(n3246) and mig-2(gm103). RNAi against either ced-10 or rac-2 was not sufficient to enhance the aldicarb sensitivity of either Rac gain-of-function mutant, while pes-7(RNAi) enhanced aldicarb sensitivity of mig-2(gm103), but not of ced-10(n3246). (B) The same synergistic genetic interactions, which were uncovered with aldicarb exposure, were confirmed with PTZ exposure. Yet, RNAi against wild-type worms was not sufficient to yield convulsions. Each data point represents mean ± SD. *P < 0.05; Fisher's exact test. Asterisks indicate enhancement, compared to mock RNAi against a given genotypic background. Red outlines around bars indicate synergism, in which RNAi against a mutant background results in greater drug sensitivity than the sum of the same RNAi treatment against a wild-type background and mock RNAi against the same mutant.

Figure 7.—

RNAi feeding and pharmacological assays with hypomorphic Rac regulator mutant backgrounds reveal synergistic genetic interactions with the dynein motor complex and canonical Rac regulators. (A and B) The response level (the percentage of young adult worms paralyzed per total sample size; n = 30 for each of three to five independent experiments) of various RNAi treatments after 60 min of exposure to aldicarb (A) or PTZ (B). All RNAi treatments against N2 wild-type worms are repeated from Figure 6 for convenience (dark gray bars). As denoted by “X”, lethality resulted from ina-1(RNAi) against ina-1(gm144) mutants, precluding analysis. (A) RNAi against most of the Rac-signaling-pathway or dynein motor complex members enhanced aldicarb sensitivities of either or both Rac regulator mutants, ina-1(gm144) (medium gray bars) and mig-15(rh148) (light gray bars). Unlike combinatorial RNAi against both genes, RNAi against the redundant Racs ced-10 or rac-2 was not sufficient to enhance the aldicarb sensitivity of either Rac regulator mutant. RNAi against pes-7 enhanced aldicarb sensitivity of mig-15(rh148), but not of ina-1(gm144), whereas unc-73(RNAi) enhanced aldicarb sensitivity of ina-1(gm144), but not of mig-15(rh148). (B) The same synergistic genetic interactions that were uncovered with aldicarb were confirmed with PTZ. Unlike the results from aldicarb exposure, RNAi against either ced-10 or rac-2 was sufficient to enhance PTZ-induced anterior convulsions of ina-1(gm144) mutants, but not of mig-15(rh148). PTZ exposure also did not reveal synergistic genetic interactions between mig-15 and lis-1, pes-7, or the PAK orthologs pak-1 and max-2. Yet, unc-73(RNAi) enhanced mig-15(rh148) convulsions, revealing interactions that were not apparent from aldicarb exposure. Each data point represents mean ± SD. *P < 0.05; Fisher's exact test. Asterisks indicate enhancement, compared to mock RNAi against a given genotypic background. Red outlines around bars indicate synergism, in which RNAi against a mutant background results in greater drug sensitivity than the sum of the same RNAi treatment against a wild-type background and mock RNAi against the same mutant.

Figure 8.—

Model depicting a potential role for a Rac GTPase-signaling pathway in dynein-mediated synaptic vesicle transport in en passant C. elegans GABAergic motor axons. Triply redundant Racs (CED-10, MIG-2, and RAC-2) and their activator, UNC-73 (Trio), may transmit extracellular signals from a Nck-interacting kinase (MIG-15), α-integrin (INA-1), and β-integrin (PAT-3) complex to a PAK-mediated interface between actin and microtubule networks. Redundant worm PAK orthologs (PAK-1 and MAX-2) may promote dynein motility through physical interactions with dynein motor complex subunits and/or actin. BICD-1 may regulate vesicle transport via physical interactions with dynein. LIS-1 may also regulate dynein motor activity through direct physical interactions with dynein motor complex subunits and/or Racs. These interactions may be involved in coordinating the binding of GABAergic synaptic vesicles at microtubule plus ends and subsequent retrograde transport. Disrupting these interactions could also lead to anterograde transport defects, due to the putative interdependency of dynein and kinesin, and to diminished levels of inhibitory GABA secretion. Blue arrows indicate that physical and genetic interactions have been shown. Orange arrows indicate that only a genetic interaction has been observed. Microtubules are shown in green, while F-actin is shown in brown. GABA is colored purple.