The association of neurodevelopmental abnormalities, congenital heart and renal defects in a tuberous sclerosis complex patient cohort.

BACKGROUND: Tuberous sclerosis complex (TSC) is a rare multi-system genetic disorder characterised by the presence of benign tumours throughout multiple organs including the brain, kidneys, heart, liver, eyes, lungs and skin, in addition to neurological and neuropsychiatric complications. Intracardiac tumour (rhabdomyoma), neurodevelopmental disorders (NDDs) and kidney disorders (KD) are common manifestations of TSC and have been linked with TSC1 and TSC2 loss-of-function mutations independently, but the dynamic relationship between these organ manifestations remains unexplored. Therefore, this study aims to characterise the nature of the relationship specifically between these three organs' manifestations in TSC1 and TSC2 mutation patients. METHODS: Clinical data gathered from TSC patients across South Wales registered with Cardiff and Vale University Health Board (CAV UHB) between 1990 and 2020 were analysed retrospectively to evaluate abnormalities in the heart, brain and kidney development. TSC-related abnormalities such as tumour prevalence, location and size were analysed for each organ in addition to neuropsychiatric involvement and were compared between TSC1 and TSC2 mutant genotypes. Lastly, statistical co-occurrence between organ manifestations co-morbidity was quantified, and trajectories of disease progression throughout organs were modelled. RESULTS: This study found a significantly greater mutational frequency at the TSC2 locus in the cohort in comparison to TSC1. An equal proportion of male and female patients were observed in this group and by meta-analysis of previous studies. No significant difference in characterisation of heart involvement was observed between TSC1 and TSC2 patients. Brain involvement was seen with increased severity in TSC2 patients, characterised by a greater prevalence of cortical tubers and communication disorders. Renal pathology was further enhanced in TSC2 patients, marked by increased bilateral angiomyolipoma prevalence. Furthermore, co-occurrence of NDDs and KDs was the most positively correlated out of investigated manifestations, regardless of genotype. Analysis of disease trajectories revealed a more diverse clinical outcome for TSC2 patients: however, a chronological association of rhabdomyoma, NDD and KD was most frequently observed for TSC1 patients. CONCLUSIONS: This study marks the first empirical investigation of the co-morbidity between congenital heart defects (CHD), NDDs, and KDs in TSC1 and TSC2 patients. This remains a unique first step towards the characterisation of the dynamic role between genetics, heart function, brain function and kidney function during the early development in the context of TSC.

Transcriptional programs regulating neuronal differentiation are disrupted in DLG2 knockout human embryonic stem cells and enriched for schizophrenia and related disorders risk variants.

Coordinated programs of gene expression drive brain development. It is unclear which transcriptional programs, in which cell-types, are affected in neuropsychiatric disorders such as schizophrenia. Here we integrate human genetics with transcriptomic data from differentiation of human embryonic stem cells into cortical excitatory neurons. We identify transcriptional programs expressed during early neurogenesis in vitro and in human foetal cortex that are down-regulated in DLG2-/- lines. Down-regulation impacted neuronal differentiation and maturation, impairing migration, morphology and action potential generation. Genetic variation in these programs is associated with neuropsychiatric disorders and cognitive function, with associated variants predominantly concentrated in loss-of-function intolerant genes. Neurogenic programs also overlap schizophrenia GWAS enrichment previously identified in mature excitatory neurons, suggesting that pathways active during prenatal cortical development may also be associated with mature neuronal dysfunction. Our data from human embryonic stem cells, when combined with analysis of available foetal cortical gene expression data, de novo rare variants and GWAS statistics for neuropsychiatric disorders and cognition, reveal a convergence on transcriptional programs regulating excitatory cortical neurogenesis.

Neuron-Glia Interactions Increase Neuronal Phenotypes in Tuberous Sclerosis Complex Patient iPSC-Derived Models.

Tuberous sclerosis complex (TSC) is a rare neurodevelopmental disorder resulting from autosomal dominant mutations in the TSC1 or TSC2 genes, leading to a hyperactivated mammalian target of rapamycin (mTOR) pathway, and gray and white matter defects in the brain. To study the involvement of neuron-glia interactions in TSC phenotypes, we generated TSC patient induced pluripotent stem cell (iPSC)-derived cortical neuronal and oligodendrocyte (OL) cultures. TSC neuron mono-cultures showed increased network activity, as measured by calcium transients and action potential firing, and increased dendritic branching. However, in co-cultures with OLs, neuronal defects became more apparent, showing cellular hypertrophy and increased axonal density. In addition, TSC neuron-OL co-cultures showed increased OL cell proliferation and decreased OL maturation. Pharmacological intervention with the mTOR regulator rapamycin suppressed these defects. Our patient iPSC-based model, therefore, shows a complex cellular TSC phenotype arising from the interaction of neuronal and glial cells and provides a platform for TSC disease modeling and drug development.

Curcumin and derivatives function through protein phosphatase 2A and presenilin orthologues in Dictyostelium discoideum.

Natural compounds often have complex molecular structures and unknown molecular targets. These characteristics make them difficult to analyse using a classical pharmacological approach. Curcumin, the main curcuminoid of turmeric, is a complex molecule possessing wide-ranging biological activities, cellular mechanisms and roles in potential therapeutic treatment, including Alzheimer's disease and cancer. Here, we investigate the physiological effects and molecular targets of curcumin in Dictyostelium discoideum We show that curcumin exerts acute effects on cell behaviour, reduces cell growth and slows multicellular development. We employed a range of structurally related compounds to show the distinct role of different structural groups in curcumin's effects on cell behaviour, growth and development, highlighting active moieties in cell function, and showing that these cellular effects are unrelated to the well-known antioxidant activity of curcumin. Molecular mechanisms underlying the effect of curcumin and one synthetic analogue (EF24) were then investigated to identify a curcumin-resistant mutant lacking the protein phosphatase 2A regulatory subunit (PsrA) and an EF24-resistant mutant lacking the presenilin 1 orthologue (PsenB). Using in silico docking analysis, we then showed that curcumin might function through direct binding to a key regulatory region of PsrA. These findings reveal novel cellular and molecular mechanisms for the function of curcumin and related compounds.

Prolyl oligopeptidase, inositol phosphate signalling and lithium sensitivity.

Inhibition of prolyl oligopeptidase (PO) elevates inositol phosphate (IP) signalling and reduces cell sensitivity to lithium (Li+). This review discusses recent evidence that shows PO acts via the multiple inositol polyphosphate phosphatase (MIPP) to regulate gene expression. As a consequence, PO inhibition causes both a transient, rapid increase in I(1,4,5)P(3) and a long-term elevation of IP signalling. This pathway is evolutionary conserved, being present in both the social amoeba Dictyostelium and human cell systems, and has potential implications for mental health.

Glycogen synthase kinase-3 is required for efficient Dictyostelium chemotaxis.

Glycogen synthase kinase-3 (GSK3) is a highly conserved protein kinase that is involved in several important cell signaling pathways and is associated with a range of medical conditions. Previous studies indicated a major role of the Dictyostelium homologue of GSK3 (gskA) in cell fate determination during morphogenesis of the fruiting body; however, transcriptomic and proteomic studies have suggested that GSK3 regulates gene expression much earlier during Dictyostelium development. To investigate a potential earlier role of GskA, we examined the effects of loss of gskA on cell aggregation. We find that cells lacking gskA exhibit poor chemotaxis toward cAMP and folate. Mutants fail to activate two important regulatory signaling pathways, mediated by phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) and target of rapamycin complex 2 (TORC2), which in combination are required for chemotaxis and cAMP signaling. These results indicate that GskA is required during early stages of Dictyostelium development, in which it is necessary for both chemotaxis and cell signaling.

Genetic control of lithium sensitivity and regulation of inositol biosynthetic genes.

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.

PtdIns(3,4,5)P(3) and inositol depletion as a cellular target of mood stabilizers.

Lithium (Li(+)) is the mood stabilizer most frequently used in the treatment of bipolar mood disorder; however, its therapeutic mechanism is unknown. In the 1980s, Berridge and colleagues proposed that Li(+) treatment acts via inhibition of IMPase (inositol monophosphatase) to deplete the cellular concentration of myo-inositol. Inositol depletion is also seen with the alternative mood stabilizers VPA (valproic acid) and CBZ (carbamazepine), suggesting a common therapeutic action. All three drugs cause changes in neuronal cell morphology and cell chemotaxis; however, it is unclear how reduced cellular inositol modulates these changes in cell behaviour. It is often assumed that reduced inositol suppresses Ins(1,4,5)P(3), a major intracellular signal molecule, but there are other important phosphoinostide-based signal molecules in the cell. In the present paper, we discuss evidence that Li(+) has a substantial effect on PtdIns(3,4,5)P(3), an important signal molecule within the nervous system. As seen for Ins(1,4,5)P(3) signalling, suppression of PtdIns(3,4,5)P(3) signalling also occurs via an inositol-depletion mechanism. This has implications for the cellular mechanisms controlling phosphoinositide signalling, and offers insight into the genetics underlying risk of bipolar mood disorder.

The mood stabiliser lithium suppresses PIP3 signalling in Dictyostelium and human cells.

Bipolar mood disorder (manic depression) is a major psychiatric disorder whose molecular origins are unknown. Mood stabilisers offer patients both acute and prophylactic treatment, and experimentally, they provide a means to probe the underlying biology of the disorder. Lithium and other mood stabilisers deplete intracellular inositol and it has been proposed that bipolar mood disorder arises from aberrant inositol (1,4,5)-trisphosphate [IP(3), also known as Ins(1,4,5)P(3)] signalling. However, there is no definitive evidence to support this or any other proposed target; a problem exacerbated by a lack of good cellular models. Phosphatidylinositol (3,4,5)-trisphosphate [PIP(3), also known as PtdIns(3,4,5)P(3)] is a prominent intracellular signal molecule within the central nervous system (CNS) that regulates neuronal survival, connectivity and synaptic function. By using the genetically tractable organism Dictyostelium, we show that lithium suppresses PIP(3)-mediated signalling. These effects extend to the human neutrophil cell line HL60. Mechanistically, we show that lithium attenuates phosphoinositide synthesis and that its effects can be reversed by overexpression of inositol monophosphatase (IMPase), consistent with the inositol-depletion hypothesis. These results demonstrate a lithium target that is compatible with our current knowledge of the genetic predisposition for bipolar disorder. They also suggest that lithium therapy might be beneficial for other diseases caused by elevated PIP(3) signalling.

Use of the Dictyostelium stalk cell assay to monitor GSK-3 regulation.

GSK-3 activity mediates cAMP repression of stalk induction of cells in low-density monolayer culture. The lower the GSK-3 activity the greater the percentage of stalk cells formed. This protocol describes a robust and quantitative method utilizing an adapted stalk cell monolayer assay to measure GSK-3 activation.

Dephosphorylation of 2,3-bisphosphoglycerate by MIPP expands the regulatory capacity of the Rapoport-Luebering glycolytic shunt.

The Rapoport-Luebering glycolytic bypass comprises evolutionarily conserved reactions that generate and dephosphorylate 2,3-bisphosphoglycerate (2,3-BPG). For >30 years, these reactions have been considered the responsibility of a single enzyme, the 2,3-BPG synthase/2-phosphatase (BPGM). Here, we show that Dictyostelium, birds, and mammals contain an additional 2,3-BPG phosphatase that, unlike BPGM, removes the 3-phosphate. This discovery reveals that the glycolytic pathway can bypass the formation of 3-phosphoglycerate, which is a precursor for serine biosynthesis and an activator of AMP-activated protein kinase. Our 2,3-BPG phosphatase activity is encoded by the previously identified gene for multiple inositol polyphosphate phosphatase (MIPP1), which we now show to have dual substrate specificity. By genetically manipulating Mipp1 expression in Dictyostelium, we demonstrated that this enzyme provides physiologically relevant regulation of cellular 2,3-BPG content. Mammalian erythrocytes possess the highest content of 2,3-BPG, which controls oxygen binding to hemoglobin. We determined that total MIPP1 activity in erythrocytes at 37 degrees C is 0.6 mmol 2,3-BPG hydrolyzed per liter of cells per h, matching previously published estimates of the phosphatase activity of BPGM. MIPP1 is active at 4 degrees C, revealing a clinically significant contribution to 2,3-BPG loss during the storage of erythrocytes for transfusion. Hydrolysis of 2,3-BPG by human MIPP1 is sensitive to physiologic alkalosis; activity decreases 50% when pH rises from 7.0 to 7.4. This phenomenon provides a homeostatic mechanism for elevating 2,3-BPG levels, thereby enhancing oxygen release to tissues. Our data indicate greater biological significance of the Rapoport-Luebering shunt than previously considered.

Phospholipase C regulation of phosphatidylinositol 3,4,5-trisphosphate-mediated chemotaxis.

Generation of a phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)] gradient within the plasma membrane is important for cell polarization and chemotaxis in many eukaryotic cells. The gradient is produced by the combined activity of phosphatidylinositol 3-kinase (PI3K) to increase PI(3,4,5)P(3) on the membrane nearest the polarizing signal and PI(3,4,5)P(3) dephosphorylation by phosphatase and tensin homolog deleted on chromosome ten (PTEN) elsewhere. Common to both of these enzymes is the lipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)], which is not only the substrate of PI3K and product of PTEN but also important for membrane binding of PTEN. Consequently, regulation of phospholipase C (PLC) activity, which hydrolyzes PI(4,5)P(2), could have important consequences for PI(3,4,5)P(3) localization. We investigate the role of PLC in PI(3,4,5)P(3)-mediated chemotaxis in Dictyostelium. plc-null cells are resistant to the PI3K inhibitor LY294002 and produce little PI(3,4,5)P(3) after cAMP stimulation, as monitored by the PI(3,4,5)P(3)-specific pleckstrin homology (PH)-domain of CRAC (PH(CRAC)GFP). In contrast, PLC overexpression elevates PI(3,4,5)P(3) and impairs chemotaxis in a similar way to loss of pten. PI3K localization at the leading edge of plc-null cells is unaltered, but dissociation of PTEN from the membrane is strongly reduced in both gradient and uniform stimulation with cAMP. These results indicate that local activation of PLC can control PTEN localization and suggest a novel mechanism to regulate the internal PI(3,4,5)P(3) gradient.

Use of a penetratin-linked peptide in Dictyostelium.

The plasma membrane is an effective barrier to most macromolecules and hydrophilic molecules. Remarkably, a class of positively charged cell-penetrating peptides (CPPs) has been discovered that can translocate themselves and associated cargoes into the cytoplasm. These have been used to carry oligopeptide- and oligonucleotide-based inhibitors into mammalian cells. A recent report indicates that the same CPPs are internalized by plant protoplasts, suggesting that this may be a universal phenomenon. We report here that the prototypical CPP, penetratin, enters cells of the free-living amoebae Dictyostelium discoideum. To investigate the functionality of this technology, we fused the penetratin sequence to PKI, a peptide inhibitor of the cAMP-dependent protein kinase (PKA). Consistent with its PKA inhibitory action, Penetratin-PKI blocked aggregation in wild-type cells and, at appropriate concentrations, rescued the phenotype of a Dictyostelium mutant that has constitutively high PKA activity. This technology offers an effective method for delivery of oligopeptides and oligonucleotides into Dictyostelium.

cAMP-induced degradation of cyclin D3 through association with GSK-3beta.

In this study we report a new mechanism whereby cyclic AMP (cAMP) regulates the cell-cycle machinery. We demonstrate that elevation of intracellular levels of cAMP promotes degradation of cyclin D3 in proteasomes, and that this occurs via glycogen synthase kinase-3beta (GSK-3beta)-mediated phosphorylation of cyclin D3 at Thr-283. Elevation of cAMP did not change the subcellular distribution of either cyclin D3 or GSK-3beta. However, cAMP promoted the interaction between cyclin D3 and GSK-3beta both in vitro and in vivo, indicating that GSK-3beta-mediated phosphorylation of cyclin D3 might require the association between the two proteins. These results demonstrate how cAMP enhances degradation of cyclin D3. Furthermore, we provide evidence for a novel mechanism by which GSK-3beta might phosphorylate unprimed substrates in vivo.

The interaction of glycogen synthase kinase-3 (GSK-3) with the cell cycle.

GSK-3 is a multifunctional protein kinase known to play a pivotal role in the regulation of metabolism, the cytoskeleton and gene expression. It also interacts with the cell cycle in a number of ways. GSK-3 forms part of both Wnt and Hh signalling pathways and hence controls expression of a number of cell cycle regulatory genes. Prominent among these is cyclin D1. GSK-3 also phosphorylates cyclin D1 to promote its nuclear export and subsequent degradation. In this chapter we examine how GSK-3 mediates these effects and consider how therapeutic strategies may be developed to specifically target these pathways.

Glycogen synthase kinase-3 inhibition by lithium and beryllium suggests the presence of two magnesium binding sites.

Lithium inhibits (Li(+)) glycogen synthase kinase-3 (GSK-3) by competition for magnesium (Mg(2+)), but not ATP or substrate. Here, we show that the group II metal ion beryllium (Be(2+)) is a potent inhibitor of GSK-3 and competes for both Mg(2+) and ATP. Be(2+) also inhibits the related protein kinase cdc2 at similar potency, but not MAP kinase 2. To compare the actions of Li(+) and Be(2+) on GSK-3, we have devised a novel dual inhibition analysis. When Be(2+) and ADP are present together each interferes with the action of the other, indicating that both agents inhibit GSK-3 at the ATP binding site. In contrast, Li(+) exerts no interference with ADP inhibition or vice versa. We find, however, that Li(+) and Be(2+) do interfere with each other. These results suggest that Be(2+) competes for two distinct Mg(2+) binding sites: one is Li(+)-sensitive and the other, which is Li(+)-insensitive, binds the Mg:ATP complex.