Regulation Of Inositol Biosynthesis And Cellular Consequences Of Inositol Depletion: Implications For The Mechanism Of Action Of Valproate

Inositol is a six-carbon cyclitol that is ubiquitous in biological systems. It is a precursor for the synthesis of numerous biologically important compounds, including inositol phosphates and phosphoinositides that are essential for cell function and viability. Inositol compounds play a role in membrane formation, gene regulation, signaling, regulation of ion channels, and membrane trafficking. Furthermore, inositol regulates hundreds of genes, including those involved in the biosynthesis of inositol and phospholipids. While transcriptional regulation of inositol biosynthesis has been extensively studied and well characterized, regulation of inositol biosynthesis at the enzymatic level has not been addressed. The current study shows that myo-inositol 3-phosphate synthase (MIPS), the enzyme that catalyzes the rate-limiting step in inositol biosynthesis, is a phosphoprotein. Mass spectrometry analysis identified five phosphosites, three of which are conserved in yeast and human MIPS. Analysis of phosphorylation-deficient and phosphomimetic site-mutants of both yeast and human MIPS indicated that the three conserved sites affect MIPS activity. Two of the phosphosites are inhibitory, and one is critical for activity. Previous studies have shown that valproate (VPA), a branched chain fatty acid that has been successfully used for the treatment of bipolar disorder, epilepsy, and migraine, causes inositol depletion by inhibiting MIPS in vivo but not in vitro, which suggests that inhibition is indirect. Elimination of the two inhibitory phosphosites caused an increase in MIPS activity, conferred a growth advantage, and partially rescued sensitivity to VPA, suggesting that VPA-mediated inositol depletion may result from phosphorylation of MIPS. Decreased IP3 signaling caused by Inositol depletion has been proposed as a mechanism that underlies the therapeutic effect of VPA. However, no direct correlation between altered IP3 signaling and the therapeutic effect has been established. Because of the versatility of inositol compounds, inositol depletion may have more far-reaching consequences that may account for the effect of VPA. This study showed that inositol depletion caused by VPA or by starvation of ino1Δ cells, which cannot synthesize inositol, perturbs vacuolar structure, decreases vacuolar ATPase (V-ATPase) proton pumping, and causes partial un-coupling of the V-ATPase. These perturbations were rescued by inositol supplementation. Furthermore, VPA compromised the synthesis of PI3,5P2, which is necessary for stabilization of the V-ATPase complex. Osmotic stress, known to increase PI3,5P2 levels, did not rescue the compromised PI3,5P2 levels, nor did it induce vacuolar fragmentation in VPA-treated cells, suggesting that perturbation of the V-ATPase is a consequence of altered PI3,5P2 homeostasis under inositol-limiting conditions. These findings identify novel consequences of inositol depletion and provide evidence for a previously unidentified link between inositol levels and the V-ATPase. To identify novel pathways and processes that are altered in response to VPA, a yeast cDNA library was screened for genes that increase sensitivity to VPA when overexpressed. One of the major categories identified was endocytic trafficking genes, which led to the hypothesis that VPA perturbs endocytosis. The study showed that VPA perturbs endocytosis in yeast and human cells. Evidence showed that the likely mechanism underlying decreased endocytosis by VPA is decreased PI4,5P2 levels. Taken together, my studies led to three major novel findings. First, I identified a regulatory mechanism of inositol biosynthesis characterized by phosphorylation of the rate-limiting enzyme myo-inositol 3-phosphate synthase (MIPS). Second, I demonstrated that the highly conserved vacuolar ATPase (V-ATPase) is a target of VPA. Third, my studies indicated that VPA-mediated inositol depletion perturbs endocytosis in both yeast and mammalian cells. These findings suggest new mechanisms that may underlie the therapeutic action of VPA, and identify potential targets that may be used for the development of more effective and safer drugs

The Role of Arab Media as an Educational Institutions in Showing the Mental Image of Islam among Jordanian Youth

The study aims at identifying the role of the Arab media as educational institutions in showing the mental image of Islam among young people in Jordan. The descriptive approach was used for its relevance to the purposes of the study. The study sample was chosen by a simple random method, which numbered (304) individuals from the Jordanian youth category. The results of the study showed that the Arab media, as educational institutions, played a medium-level role in showing the mental image of Islam among young people in Jordan. The results showed that there were statistically significant differences at the significance level (0.05) in the role of Arab media as educational institutions in showing the mental image of Islam among young people in Jordan due to the age variable. The study highlights the necessity of activating the role of both local and Arab media to show the mental image of Islam higher and better than it is

Altered Differentiation Potential of Gaucherâs Disease iPSC Neuronal Progenitors due to Wnt/β-Catenin Downregulation

Summary: Gaucherâs disease (GD) is an autosomal recessive disorder caused by mutations in the GBA1 gene, which encodes acid β-glucocerebrosidase (GCase). Severe GBA1 mutations cause neuropathology that manifests soon after birth, suggesting that GCase deficiency interferes with neuronal development. We found that neuronopathic GD induced pluripotent stem cell (iPSC)-derived neuronal progenitor cells (NPCs) exhibit developmental defects due to downregulation of canonical Wnt/β-catenin signaling and that GD iPSCsâ ability to differentiate to dopaminergic (DA) neurons was strikingly reduced due to early loss of DA progenitors. Incubation of the mutant cells with the Wnt activator CHIR99021 (CHIR) or with recombinant GCase restored Wnt/β-catenin signaling and rescued DA differentiation. We also found that GD NPCs exhibit lysosomal dysfunction, which may be involved in Wnt downregulation by mutant GCase. We conclude that neuronopathic mutations in GCase lead to neurodevelopmental abnormalities due to a critical requirement of this enzyme for canonical Wnt/β-catenin signaling at early stages of neurogenesis. : In this article, Feldman and colleagues describe a new mechanism linking severe GBA1 mutations to neurodevelopmental defects through Wnt/β-catenin downregulation. Using GD iPSCs as a model, the authors show that the ability of neuronopathic GD NPCs to differentiate to DA neurons is strikingly reduced due to early loss of DA progenitors and that lysosomal dysfunction may be directly involved in canonical Wnt downregulation. Keywords: Gaucherâs disease, GBA1, glucocerebrosidase, neuronal progenitors, dopaminergic development, iPSCs, Wnt/β-catenin, lysosomal storage disease, neurodegeneratio

The antiepileptic drug valproic acid and other medium-chain fatty acids acutely reduce phosphoinositide levels independently of inositol in Dictyostelium

Valproic acid (VPA) is the most widely prescribed epilepsy treatment worldwide, but its mechanism of action remains unclear. Our previous work identified a previously unknown effect of VPA in reducing phosphoinositide production in the simple model Dictyostelium followed by the transfer of data to a mammalian synaptic release model. In our current study, we show that the reduction in phosphoinositide [PtdInsP (also known as PIP) and PtdInsP2 (also known as PIP2)] production caused by VPA is acute and dose dependent, and that this effect occurs independently of phosphatidylinositol 3-kinase (PI3K) activity, inositol recycling and inositol synthesis. In characterising the structural requirements for this effect, we also identify a family of medium-chain fatty acids that show increased efficacy compared with VPA. Within the group of active compounds is a little-studied group previously associated with seizure control, and analysis of two of these compounds (nonanoic acid and 4-methyloctanoic acid) shows around a threefold enhanced potency compared with VPA for protection in an in vitro acute rat seizure model. Together, our data show that VPA and a newly identified group of medium-chain fatty acids reduce phosphoinositide levels independently of inositol regulation, and suggest the reinvestigation of these compounds as treatments for epilepsy