Inhibition of Lithium-Sensitive Phosphatase BPNT-1 Causes Selective Neuronal Dysfunction in C. elegans

Abstract

Lithium has been a mainstay for the treatment of bipolar disorder, yet the molecular mechanisms underlying its action remain enigmatic. Bisphosphate 3'-nucleotidase (BPNT-1) is a lithium-sensitive phosphatase that catalyzes the breakdown of cytosolic 3'-phosphoadenosine 5'-phosphate (PAP), a byproduct of sulfation reactions utilizing the universal sulfate group donor 3'-phosphoadenosine 5'-phosphosulfate (PAPS) [1-3]. Loss of BPNT-1 leads to the toxic accumulation of PAP in yeast and non-neuronal cell types in mice [4, 5]. Intriguingly, BPNT-1 is expressed throughout the mammalian brain [4], and it has been hypothesized that inhibition of BPNT-1 could contribute to the effects of lithium on behavior [5]. Here, we show that loss of BPNT-1 in Caenorhabditis elegans results in the selective dysfunction of two neurons, the bilaterally symmetric pair of ASJ chemosensory neurons. As a result, BPNT-1 mutants are defective in behaviors dependent on the ASJ neurons, such as dauer exit and pathogen avoidance. Acute treatment with lithium also causes dysfunction of the ASJ neurons, and we show that this effect is reversible and mediated specifically through inhibition of BPNT-1. Finally, we show that the selective effect of lithium on the nervous system is due in part to the limited expression of the cytosolic sulfotransferase SSU-1 in the ASJ neuron pair. Our data suggest that lithium, through inhibition of BPNT-1 in the nervous system, can cause selective toxicity to specific neurons, resulting in corresponding effects on behavior of C. elegans.

Figure 1. Bisphosphate 3′-nucleotidase (BPNT-1) is required for the function of the ASJ neurons

(A) BPNT-1 catalyzes the breakdown of cytosolic 3′phosphoadenosine 5′-phosphate (PAP), a byproduct of sulfation reactions utilizing the universal sulfur donor 3′phosphoadenosine 5′-phosphosulfate (PAPS). (B) daf-7 expression pattern in animals exposed to P. aeruginosa for 16 h. Empty triangles indicate the ASJ neurons (posterior) when visible. The ASI neuron pair (anterior) is unaffected by loss of BPNT-1. All genotypes contain ksIs2[daf-7p::gfp]. (C) Maximum fluorescence of daf-7p::gfp in the ASJ neurons after 16 h exposure to P. aeruginosa. All genotypes contain ksIs2[daf-7p::gfp]. (D) Lawn occupancy of animals on P. aeruginosa after 20 h. All genotypes contain npr-1(215F). (E) Fluorescence microscopy of the ASJ cell body (dashed line) visualized with a red-fluorescent lipophilic dye (left). Area of the ASJ cell body divided into nuclear and cytoplasmic compartments (right). (F–G) Fraction of animals that have exited the dauer developmental diapause state. All genotypes contain daf-2(e1368). For all panels: *** P < 0.001, * P < 0.05 as determined by one-way ANOVA followed by Dunnett’s Multiple Comparison Test. n.s. = not significant. Error bars indicate standard deviation. See also Figures S1 and S2.

Figure 2. Lithium induces dysfunction of the ASJ neurons through inhibition of BPNT-1

(A, D, G) Maximum fluorescence of daf-28::gfp in the ASJ neurons. All genotypes contain mgIs40[daf-28p::nls-GFP]. (B) Maximum fluorescence of gpa-9::gfp in the ASJ neurons. All genotypes contain pkIs586[gpa-9p::GFP]. (C) Maximum fluorescence of trx-1::mCherry in the ASJ neurons. All genotypes contain qdEx133[trx-1p::mCherry]. (E) Fluorescence microscopy of the ASJ cell body (dashed line) visualized with a red-fluorescent lipophilic dye (left). Area of the ASJ cell body (right). All genotypes contain mgIs40[daf-28p::nls-GFP]. (F) Fraction of animals that have exited the dauer developmental diapause state. All genotypes contain daf-2(e1368). Dashed lines indicate addition of 15 mM LiCl. For all panels: Open circles indicate addition of 15 mM LiCl. *** P < 0.001, ** P < 0.01, * P < 0.05 as determined by one-way ANOVA followed by Dunnett’s Multiple Comparison Test. n.s. = not significant. Error bars indicate standard deviation. See also Figure S3.

Figure 3. Loss of BPNT-1 inhibits the ASJ neurons through accumulation of cytosolic PAP

(A) daf-7 expression pattern in animals exposed to P. aeruginosa for 16 h. Empty triangles indicate the ASJ neurons (posterior) when visible. All genotypes contain ksIs2[daf-7p::gfp]. (B) Maximum fluorescence of daf-7p::gfp in the ASJ neurons after 16 h exposure to P. aeruginosa. All genotypes contain ksIs2[daf-7p::gfp]. (C) Maximum fluorescence of daf-28::gfp in the ASJ neurons. All genotypes contain mgIs40[daf-28p::nls-GFP]. (D) Fluorescence microscopy of the ASJ cell body (dashed line) visualized with a red-fluorescent lipophilic dye (left). Area of the ASJ cell body (right). All genotypes contain mgIs40[daf-28p::nls-GFP]. For all panels: *** P < 0.001, ** P < 0.01, * P < 0.05 as determined by one-way ANOVA followed by Dunnett’s Multiple Comparison Test. n.s. = not significant. Error bars indicate standard deviation. See also Figure S3.

Figure 4. Lithium selectively inhibits the ASJ neurons of C. elegans through inhibition of BPNT-1

We hypothesize that inhibition of the cytosolic PAP phosphatase BPNT-1 by lithium or genetic mutation leads to a buildup of toxic PAP, due to ASJ-specific expression of the cytosolic sulfotransferase SSU-1. PAP, in turn, causes alterations in cell morphology, transcription, and behavioral outputs of the ASJ neurons. BPNT-1 can also degrade PAP transported into the cytosol from the Golgi, which may explain the synthetic lethality between BPNT-1 and the Golgi-resident PAP phosphatase GPAP-1.