pxn-1 and pxn-2 May Interact Negatively during Neuronal Development and Aging in C. elegans

Abstract

C. elegans has two functional peroxidasins (PXN), PXN-1 and PXN-2. PXN-2 is essential to consolidate the extracellular matrix during development and is suggested to interact with PXN-1 antagonistically. pxn-1 is involved in neuronal development and possibly maintenance; therefore, we investigated the relationship between pxn-1 and pxn-2 in neuronal development and in aging. During neuronal development, defects caused by pxn-1 overexpression were suppressed by overexpression of both pxn-1 and pxn-2. In neuronal aging process, pxn-1 mutants showed less age-related neuronal defects, such as neuronal outgrowth, neuronal wavy processes, and enhanced short-term memory performance. In addition, pxn-2 overexpressing animals retained an intact neuronal morphology when compared with age-matched controls. Consistent with these results, overexpression of both pxn-1 and pxn-2 restored the severe neuronal defects present with pxn-1 overexpression. These results implied that there is a negative relationship between pxn-1 and pxn-2 via pxn-1 regulating pxn-2. Therefore, pxn-1 may function in neuronal development and age-related neuronal maintenance through pxn-2.

Keywords: C. elegans; antagonistic relationship; neuronal aging; neuronal development; pxn-1; pxn-2.

Fig. 1.

pxn-1 and pxn-2 double overexpression suppresses the effect of pxn-1 overexpression in developing neurons. (A) Neuronal defects during development are presented as the percentage of the total number of neurons counted in overexpressing animals: control (juIs76 [Punc-25::gfp]), pxn-1 overexpression (Ppxn-1::pxn-1), pxn-2 overexpression (Ppxn-2::pxn-2), and pxn-1 and pxn-2 overexpression (Ppxn-1::pxn-1+Ppxn-2::pxn-2). (B) Neuronal defects during development are presented as a percentage in various ectopic overexpressing worms: Phs::gfp, pxn-1 overexpression (Phs::pxn-1), pxn-2 overexpression (Phs::pxn-2), and pxn-1 and pxn-2 over-expression (Phs::pxn-1+Phs::pxn-2). All experiments were performed in juIs76 background. n > 300 worms per line. Error bar = STD. *p < 0.01; Student’s t-test.

Fig. 2.

Neuronal defects increase with age. (A, B) A representative image of a mechanosensory neuron ALM, and PLM in a control worm (vector only in zdIs5) on day 1, young adult stage. (C–E) Representative ALM and PLM on day 10 (C) and on day 15 (D, E, respectively). The arrowheads indicate a wavy phenotype (C and E) and the arrows represent an outgrowth (C, D). Scale bar = 50 μm. (F, G) ALM outgrowth and PLM wavy neurons are presented as a percentage on days 1, 5, 10, and 15 in control, pxn-1 mutant (ok785), pxn-2 RNAi treated worms and pxn-1 treated with pxn-2 RNAi worms. All experiments were performed in zdIs5(Pmec-4::gfp) background. n > 300 worms per line. Error bar = STD. *p < 0.05; **p < 0.01; Student’s t-test.

Fig. 3.

pxn-1 overexpression exacerbates age-related neuronal defects. (A–C) ALM outgrowth, PLM wavy neurons, and PLM branching in each genotype are presented as a percentage on days 5 and 15 respectively in control (zdIs5), pxn-1 overexpressing, pxn-2 overexpressing, and pxn-1 and pxn-2 overexpressing worms. (D) A representative image of a mechanosensory neuron PLM in a pxn-1 overexpressing worm on day 5. Arrows and arrowheads represent wavy process and branching respectively. Open arrow represents a blebbing. Scale bar = 50 μm. n > 300 worms per line. Error bar = STD. *p < 0.05; **p < 0.01; Student’s t-test.

Fig. 4.

Age-related short term memory loss is attenuated in pxn-1 mutants. The massed learning index (LI) represents a measurement of short term memory and it is calculated by chemotaxis responses. The measurements are performed on day 1, 3, 5, and 7 in control (N2 with gfp RNAi), pxn-1 mutant, N2 with pxn-2 RNAi treated worms and pxn-1 treated with pxn-2 RNAi worms. N > 300 worms per line. Error bar = STD. *p < 0.05; **p < 0.01; Student’s t-test.