Peter S. Klein

GSK-3α regulates production of Alzheimer's disease amyloid-β peptides

Alzheimers disease is associated with increased production and aggregation of amyloid-β (Aβ) peptides. Aβ peptides are derived from the amyloid precursor protein (APP) by sequential proteolysis, catalysed by the aspartyl protease BACE, followed by presenilin-dependent γ-secretase cleavage. Presenilin interacts with nicastrin, APH-1 and PEN-2 (ref. 6), all of which are required for γ-secretase function. Presenilins also interact with α-catenin, β-catenin and glycogen synthase kinase-3β (GSK-3β), but a functional role for these proteins in γ-secretase activity has not been established. Here we show that therapeutic concentrations of lithium, a GSK-3 inhibitor, block the production of Aβ peptides by interfering with APP cleavage at the γ-secretase step, but do not inhibit Notch processing. Importantly, lithium also blocks the accumulation of Aβ peptides in the brains of mice that overproduce APP. The target of lithium in this setting is GSK-3α, which is required for maximal processing of APP. Since GSK-3 also phosphorylates tau protein, the principal component of neurofibrillary tangles, inhibition of GSK-3α offers a new approach to reduce the formation of both amyloid plaques and neurofibrillary tangles, two pathological hallmarks of Alzheimers disease.

Lithium Reduces Tau Phosphorylation by Inhibition of Glycogen Synthase Kinase-3

Lithium is one of the most widely used drugs for treating bipolar (manic-depressive) disorder. Despite its efficacy, the molecular mechanism underlying its action has not been elucidated. One recent study has proposed that lithium inhibits glycogen synthase kinase-3 and thereby affects multiple cellular functions. Because glycogen synthase kinase-3 regulates the phosphorylation of tau (microtubule-binding protein that forms paired helical filaments in neurons of the Alzheimer’s disease brain), we hypothesized that lithium could affect tau phosphorylation by inhibiting glycogen synthase kinase-3. Using cultured human NT2N neurons, we demonstrate that lithium reduces the phosphorylation of tau, enhances the binding of tau to microtubules, and promotes microtubule assembly through direct and reversible inhibition of glycogen synthase kinase-3. These results provide new insights into how lithium mediates its effects in the central nervous system, and these findings could be exploited to develop a novel intervention for Alzheimer’s disease.

Nuclear Receptor Rev-erbα Is a Critical Lithium-Sensitive Component of the Circadian Clock

Lithium is commonly used to treat bipolar disorder, which is associated with altered circadian rhythm. Lithium is a potent inhibitor of glycogen synthase kinase 3 (GSK3), which regulates circadian rhythm in several organisms. In experiments with cultured cells, we show here that GSK3β phosphorylates and stabilizes the orphan nuclear receptor Rev-erbα, a negative component of the circadian clock. Lithium treatment of cells leads to rapid proteasomal degradation of Rev-erbα and activation of clock gene Bmal1. A form of Rev-erbα that is insensitive to lithium interferes with the expression of circadian genes. Control of Rev-erbα protein stability is thus a critical component of the peripheral clock and a biological target of lithium therapy.

Glycogen synthase kinase-3beta haploinsufficiency mimics the behavioral and molecular effects of lithium.

Lithium is widely used to treat bipolar disorder, but its mechanism of action in this disorder is unknown. Several molecular targets of lithium have been identified, but these putative targets have not been shown to be responsible for the behavioral effects of lithium in vivo. A robust model for the effects of chronic lithium on behavior in mice would greatly facilitate the characterization of lithium action. We describe behaviors in mice that are robustly affected by chronic lithium. Remarkably, these lithium-sensitive behaviors are also observed in mice lacking one copy of the gene encoding glycogen synthase kinase-3β (Gsk-3β), a well established direct target of lithium. In addition, chronic lithium induces molecular changes consistent with inhibition of GSK-3 within regions of the brain that are paralleled in Gsk-3β+/- heterozygous mice. We also show that lithium therapy activates Wnt signaling in vivo, as measured by increased Wnt-dependent gene expression in the amygdala, hippocampus, and hypothalamus. These observations support a central role for GSK-3β in mediating behavioral responses to lithium.

Histone deacetylase is a target of valproic acid-mediated cellular differentiation

Valproic acid (VPA), a well-established therapy for seizures and bipolar disorder, has recently been shown to inhibit histone deacetylases (HDACs). Similar to more widely studied HDAC inhibitors, VPA can cause growth arrest and induce differentiation of transformed cells in culture. Whether this effect of VPA is through inhibition of HDACs or modulation of another target of VPA has not been tested. We have used a series of VPA analogs to establish a pharmacological profile for HDAC inhibition. We find that VPA and its analogs inhibit multiple HDACs from class I and class II (but not HDAC6 or HDAC10) with a characteristic order of potency in vitro. These analogs also induce hyperacetylation of core histones H3 and H4 in intact cells with an order of potency that parallels in vitro inhibition. VPA and VPA analogs induce differentiation in hematopoietic cell lines in a p21-dependent manner, and the order of potency for induction of differentiation parallels the potencies for inhibition in vitro, as well as for acetylation of histones associated with the p21 promoter, supporting the argument that differentiation caused by VPA is mediated through inhibition of HDACs. These findings provide additional evidence that VPA, a well-tolerated, orally administered drug with extensive clinical experience, may serve as an effective chemotherapeutic agent through targeting of HDACs.

Inhibitory Phosphorylation of Glycogen Synthase Kinase-3 (GSK-3) in Response to Lithium EVIDENCE FOR AUTOREGULATION OF GSK-3

Glycogen synthase kinase-3 (GSK-3) is a critical, negative regulator of diverse signaling pathways. Lithium is a direct inhibitor of GSK-3 and has been widely used to test the putative role of GSK-3 in multiple settings. However, lithium also inhibits other targets, including inositol monophosphatase and structurally related phosphomonoesterases, and thus additional approaches are needed to attribute a given biological effect of lithium to a specific target. For example, lithium is known to increase the inhibitory N-terminal phosphorylation of GSK-3, but the target of lithium responsible for this indirect regulation has not been identified. We have characterized a short peptide derived from the GSK-3 interaction domain of Axin that potently inhibits GSK-3 activity in vitro and in mammalian cells and robustly activates Wnt-dependent transcription, mimicking lithium action. We show here, using the GSK-3 interaction domain peptide, as well as small molecule inhibitors of GSK-3, that lithium induces GSK-3 N-terminal phosphorylation through direct inhibition of GSK-3 itself. Reduction of GSK-3 protein levels, either by RNA interference or by disruption of the mouse GSK-3β gene, causes increased N-terminal phosphorylation of GSK-3, confirming that GSK-3 regulates its own phosphorylation status. Finally, evidence is presented that N-terminal phosphorylation of GSK-3 can be regulated by the GSK-3-dependent protein phosphatase-1·inhibitor-2 complex.

Dishevelled phosphorylation, subcellular localization and multimerization regulate its role in early embryogenesis

Dishevelled (Dsh) induces a secondary axis and can translocate to the membrane when activated by Frizzleds; however, dominant‐negative approaches have not supported a role for Dsh in primary axis formation. We demonstrate that the Dsh protein is post‐translationally modified at the dorsal side of the embryo: timing and position of this regulation suggests a role of Dsh in dorsal–ventral patterning in Xenopus. To create functional links between these properties of Dsh we analyzed the influence of endogenous Frizzleds and the Dsh domain dependency for these characteristics. Xenopus Frizzleds phosphorylate and translocate Xdsh to the membrane irrespective of their differential ectopic axes inducing abilities, showing that translocation is insufficient for axis induction. Dsh deletion analysis revealed that axis inducing abilities did not segregate with Xdsh membrane association. The DIX region and a short stretch at the N‐terminus of the DEP domain are necessary for axis induction while the DEP region is required for Dsh membrane association and its phosphorylation. In addition, Dsh forms homomeric complexes in embryos suggesting that multimerization is important for its proper function.

Cellular Plasticity Cascades in the Pathophysiology and Treatment of Bipolar Disorder

Bipolar disorder (BPD) is characterized by recurrent episodes of disturbed affect including mania and depression as well as changes in psychovegetative function, cognitive performance, and general health. A growing body of data suggests that BPD arises from abnormalities in synaptic and neuronal plasticity cascades, leading to aberrant information processing in critical synapses and circuits. Thus, these illnesses can best be conceptualized as genetically influenced disorders of synapses and circuits rather than simply as deficits or excesses in individual neurotransmitters. In addition, commonly used mood-stabilizing drugs that are effective in treating BPD have been shown to target intracellular signaling pathways that control synaptic plasticity and cellular resilience. In this article we draw on clinical, preclinical, neuroimaging, and post-mortem data to discuss the neurobiology of BPD within a conceptual framework while highlighting the role of neuroplasticity in the pathophysiology and treatment of this disorder.

Hypomorphic mutations in PRF1, MUNC13-4, and STXBP2 are associated with adult-onset familial HLH

Familial hemophagocytic lymphohistiocytosis (HLH) is a rare primary immunodeficiency disorder characterized by defects in cell-mediated cytotoxicity that results in fever, hepatosplenomegaly, and cytopenias. Familial HLH is well recognized in children but rarely diagnosed in adults. We conducted a retrospective review of genetic and immunologic test results in patients who developed HLH in adulthood. Included in our study were 1531 patients with a clinical diagnosis of HLH; 175 patients were 18 years or older. Missense and splice-site sequence variants in PRF1, MUNC13-4, and STXBP2 were found in 25 (14%) of the adult patients. The A91V-PRF1 genotype was found in 12 of these patients (48%). The preponderance of hypomorphic mutations in familial HLH-causing genes correlates with the later-onset clinical symptoms and the more indolent course in adult patients. We conclude that late-onset familial HLH occurs more commonly than was suspected previously.

naked cuticle encodes an inducible antagonist of Wnt signalling.

During animal development, cells have to respond appropriately to localized secreted signals. Proper responses to Hedgehog, transforming growth factor-β, epidermal growth factor and fibroblast growth factor/Ras signals require cognate inducible antagonists such as Patched, Dad, Argos and Sprouty. Wnt signals are crucial in development and neoplasia. Here we show that naked cuticle (nkd), a Drosophila segment-polarity gene, encodes an inducible antagonist for the Wnt signal Wingless (Wg). In fly embryos and imaginal discs nkd transcription is induced by Wg. In embryos, decreased nkd function has an effect similar to excess Wg; at later stages such a decrease appears to have no effect. Conversely, overproduction of Nkd in Drosophila and misexpression of Nkd in the vertebrate Xenopus laevis result in phenotypes resembling those of loss of Wg/Wnt function. nkd encodes a protein with a single EF hand (a calcium-binding motif) that is most similar to the recoverin family of myristoyl switch proteins. Nkd may therefore link ion fluxes to the regulation of the potency, duration or distribution of Wnt signals. Signal-inducible feedback antagonists such as nkd may limit the effects of Wnt proteins in development and disease.