Assessment of normal and mutant human presenilin function in Caenorhabditis elegans

Abstract

We provide evidence that normal human presenilins can substitute for Caenorhabditis elegans SEL-12 protein in functional assays in vivo. In addition, six familial Alzheimer disease-linked mutant human presenilins were tested and found to have reduced ability to rescue the sel-12 mutant phenotype, suggesting that they have lower than normal presenilin activity. A human presenilin 1 deletion variant that fails to be proteolytically processed and a mutant SEL-12 protein that lacks the C terminus display considerable activity in this assay, suggesting that neither presenilin proteolysis nor the C terminus is absolutely required for normal presenilin function. We also show that sel-12 is expressed in most neural and nonneural cell types in all developmental stages. The reduced activity of mutant presenilins and as yet unknown gain-of-function properties may be a contributing factor in the development of Alzheimer disease.

Figure 1

Rescue of the sel-12 Egl and abnormal vulva phenotypes by normal and mutant human presenilins. The data are shown for transgenic lines generated by injecting the construct being tested at a concentration of 20 μg/ml. Each line in the histogram represents data for an independent transgenic line; the number of hermaphrodites scored is shown above each line. The transgene is indicated on the horizontal axis. The percentage of Egl⁺ hermaphrodites is indicated on the vertical axis. Egl⁺ signifies robust egg-laying after 2 days; this criterion is very stringent and underestimates the degree of rescuing activity. The ability of PS1 point mutant proteins (data not shown) and the PS1ΔE9 mutant protein (see Fig. 2) to rescue sel-12(ar131) was further reduced when transgenic lines were generated by injecting DNA at a concentration of 2 μg/ml. Most PS1 mutations that cause Alzheimer disease affect amino acids that are identical in SEL-12. The N termini of PS1, PS2, and SEL-12 are not well conserved and are of different lengths. Therefore, for the mutations used herein, the amino acid corresponding to Met-146 in PS1 is Met-115 in SEL-12; PS1 His-163 is SEL-12 His-132; PS1 Ala-246 is SEL-12 Val-216; PS1 Leu-286 is SEL-12 Leu-255; PS1 Cys-410 is SEL-12 Cys-384. The ΔE9 mutation inhibits cleavage of PS1 (17); we note that SEL-12 is cleaved in a comparable position (24). Note that the sel-12 cDNA used (11) has a frameshift mutation, beginning at codon 413, resulting in the substitution of 31 amino acids C-terminal to the frameshift mutation by 49 amino acids.

Figure 2

Rescue of the sel-12 Egl phenotype by PS1 and PS1ΔE9 expressed from arrays formed at a concentration of 2 μg/ml. Each line in the histogram represents data for an independent transgenic line; the number of hermaphrodites scored is shown above each line. The transgene is indicated on the horizontal axis. The percentage of Egl⁺ hermaphrodites (see Fig. 1) is indicated on the vertical axis. At 2 μg/ml of injected DNA, expression from arrays or representation of the plasmid in the arrays may be reduced, accounting for the reduced activity of SEL-12 (transgenic line 3) and PS1 (transgenic line 5) compared with arrays generated at 20 μg/ml (Fig. 1).

Figure 3

Transgenic hermaphrodites expressing a sel-12::lacZ transgene. Expression is seen in neural and nonneural cells. (A) Adult. Large arrow indicates nerve ring; smaller arrows indicate muscle nuclei. (B) Adult. Arrows indicate ventral cord nuclei. (C) L3 larva. Arrows indicate nuclei of the vulval precursor cells P3.p–P8.p. (D). L2 larva. Arrows indicate the nuclei of the somatic gonadal cells Z1.ppp and Z4.aaa. sel-12 activity has been shown to influence the fates of P3.p–P8.p and Z1.ppp and Z4.aaa in sensitized genetic backgrounds (11). Compromised neural function associated with reduced activity has not yet been seen in the nerve ring or ventral cord, possibly because an appropriate sensitized genetic background has not been examined. Complete genotype: smg-1(r861) unc-54(r293); arIs17 [pRF4, pIB1Z17].