Genetic control of lithium sensitivity and regulation of inositol biosynthetic genes

Abstract

Lithium (Li(+)) is a common treatment for bipolar mood disorder, a major psychiatric illness with a lifetime prevalence of more than 1%. Risk of bipolar disorder is heavily influenced by genetic predisposition, but is a complex genetic trait and, to date, genetic studies have provided little insight into its molecular origins. An alternative approach is to investigate the genetics of Li(+) sensitivity. Using the social amoeba Dictyostelium, we previously identified prolyl oligopeptidase (PO) as a modulator of Li(+) sensitivity. In a link to the clinic, PO enzyme activity is altered in bipolar disorder patients. Further studies demonstrated that PO is a negative regulator of inositol(1,4,5)trisphosphate (IP(3)) synthesis, a Li(+) sensitive intracellular signal. However, it was unclear how PO could influence either Li(+) sensitivity or risk of bipolar disorder. Here we show that in both Dictyostelium and cultured human cells PO acts via Multiple Inositol Polyphosphate Phosphatase (Mipp1) to control gene expression. This reveals a novel, gene regulatory network that modulates inositol metabolism and Li(+) sensitivity. Among its targets is the inositol monophosphatase gene IMPA2, which has also been associated with risk of bipolar disorder in some family studies, and our observations offer a cellular signalling pathway in which PO activity and IMPA2 gene expression converge.

Figure 1. Loss of dpoA reduces Li⁺ sensitivity.

A, PO activity peaks at 8 hours of Dictyostelium development, and then declines to levels only slightly higher than in growing cells (0 hours of development). Error Bars: mean ± standard error of 3 independent experiments. Peak activity corresponds to streaming and mound developmental stages. B, Cell tracks of wild type and mutant cells in 7 mM LiCl (or control of 7 mM NaCl) during chemotaxis along a 1 nM/µm cAMP gradient (direction of arrow). D =  Directionality, S =  cell speed (µm/min). dpoA null mutants have a reduced Li⁺ sensitivity, showing higher Directionality and speed than wild type in Li⁺.

Figure 2. Mipp1 mediates the effects of PO inhibition.

A, Mipp1 enzyme prepared from Mipp1 over-expressing cells were incubated with 10 nmoles InsP₆ for the indicated times and analyzed by HPLC-MDD. I(1,4)P₂ was generated by contaminating IP₃ 5-phosphatase activity, and demonstrates that the product of Mipp1 is I(1,4,5)P₃. B, Mipp1 activity measured as the rate of IP₃ formation in extracts from wild type, mipp1 null mutant and Mipp1overexpressingcells. C, Cell tracks of wild type and mipp1 mutant cells in 7 mM LiCl (or control of 7 mM NaCl) during chemotaxis. mipp, mipp:dpoA and Mipp1 over-expressing mutants (Mipp1^oe) are Li⁺ hypersensitive compared to wild type. D  =  Directionality, S =  cell speed (µm/min). D, Vegetative wild type and mipp1 null cells were treated with either 1.2 mM Z-pro-L-prolinal or an equal volume of DMSO carrier as a control. Samples were then removed at the times indicated and IP₃ concentration measured. Values plotted as mean ± standard error of 4 independent experiments. * P<0.05, paired T-test. E, Mipp1 protein extracts were incubated with recombinant DpoA with or without the PO inhibitor Z-pro-L-prolinal. Mipp1 activity is measured by the production of IP₃ from IP₆. Values plotted as mean ± standard error of 3 independent experiments. ** P<0.01, paired T-test. Samples of Mipp1 protein are shown on Western (underneath).

Figure 3. Elevated expression of Mipp1 and IP₃ kinases alter lithium sensitivity.

A, Comparison of the I(1,3,4,5,6)P₅ and IP₆ concentration of wild type, dpoA null; mipp1 null and Mipp1 over-expressing cells. Values plotted (means ± standard error of 3 independent experiments) as percentage of the total IP content relative to wild type (*P<0.05, ** P<0.01 T-test). B, Cell tracks of wild type and mipp1 null mutant cells over-expressing IpkA1 and IpkB during chemotaxis in 7 mM LiCl. D =  Directionality, S =  cell speed (µm/min). Over-expression of IpkA1 and IpkB in wild type cells confers Li⁺ resistance, whereas Li⁺ hypersensitivity is maintained after over-expression in a mipp1 null mutant.

Figure 4. PO regulates gene expression.

A, Expression of ino1 in wild type, dpoA null cells and wild type cells following overnight treatment with 1.2 mM Z-pro-L-prolinal (PO inhibitor), measured by Northern analysis. rnlA expression was used as a loading control. The graph shows the expression of ino1 quantified on a phosphoimager (Biorad) and normalised to rnlA relative to wild type or DMSO carrier treated controls (mean ± standard error of 3 independent experiments). B, The expression of genes involved in inositol metabolism (Table S1). Gene expression in wild type cells following overnight treatment with Z-pro-L-prolinal or the equivalent amount of DMSO carrier, and dpoA null mutant cells was measured by qRT-PCR, and normalised to rnlA expression levels. Values plotted are relative to the carrier control cells (mean ± standard error of 3 independent experiments). * P<0.05, ** P<0.01, *** P<0.005, T-Test. C, mipp1 is required for the regulation of gene expression by PO. Expression of ino1 and impA1 in wild type and mipp1 null cells following overnight treatment with Z-pro-L-prolinal or the equivalent amount of DMSO carrier, was measured by qRT-PCR, and normalised to rnlA expression levels. Values plotted are relative to the carrier control wild type cells (mean ± standard error of at least 3 independent experiments). There is a significant difference in gene expression between wild type and mipp1 null mutant following PO inhibition (** P<0.01, T-Test). D, Expression levels of ino1, impA1 and mipp1 in wild type, mipp1 null mutant and cells over-expressing Mipp1, IpkA1 or IpkB. Expression was calculated relative to either wild type, or wild type cells transformed with the empty expression vector as appropriate. All samples were normalised to rnlA as a loading control and the values plotted are the mean ± standard error of 3 independent experiments (*P<0.05, ** P<0.01 T-test). ND  =  not determined.

Figure 5. Increased ino1 and impA1 gene expression confers Li⁺ resistance.

A, Cell tracks of wild type cells over-expressing ImpA1 alone or both ImpA1and ino1 undergoing chemotaxis in 7 mM NaCl or LiCl. D =  Directionality, S =  cell speed (µm/min). B, Cells of wild type cells over-expressing ImpA1 alone or both ImpA1and ino1were developed in the presence of 10 mM LiCl. Images are taken after 24 hours, bar  = 5 mm.

Figure 6. PO regulates expression of human IMPA1, IMPA2 and ISYNA1 genes.

A, A diagram to illustrate the gene regulatory network through which PO activity regulates expression of the IMPA1, IMPA2 and ISYNA1 genes. The gene regulatory network is separate from ligand-stimulated regulation of IP₃ signalling via phospholipase C (PLC), but interacts via changes in gene expression or interchange of soluble IP₃. * marks genes and enzyme activities associated with bipolar mood disorder. B, HEK293 cells were treated with either mipp1 specific or non-targetting siRNA for 24 hours with or without 130 µM Z-pro-L-prolinal (PO inhibitor). Gene expression was quantified by qRT-PCR using expression of B2M as a reference. Expression of IMPA1, IMPA2 and ISYNA1 genes was quantified as percentage relative change compared to control (DMSO carrier treated, non-targeted siRNA samples). * p<0.05, ** p<0.02, *** p<0.001.