Phosphoinositide Research Articles

Evidence for Altered Phosphoinositide Signaling-Associated Molecules in the Postmortem Prefrontal Cortex of Patients with Schizophrenia.

Phosphoinositides (PIs) play important roles in the structure and function of the brain. Associations between PIs and the pathophysiology of schizophrenia have been studied. However, the significance of the PI metabolic pathway in the pathology of schizophrenia is unknown. We examined the expression of PI signaling-associated proteins in the postmortem brain of schizophrenia patients. Protein expression levels of phosphatidylinositol 4-phosphate 5-kinase type-1 gamma (PIP5K1C), phosphatidylinositol 4-kinase alpha (PIK4CA, also known as PIK4A), phosphatase and tensin homolog deleted from chromosome 10 (PTEN), protein kinase B (Akt), and glycogen synthase kinase 3β (GSK3β) were measured using enzyme-linked immunosorbent assays and multiplex fluorescent bead-based immunoassays of the prefrontal cortex (PFC) of postmortem samples from 23 schizophrenia patients and 47 normal controls. We also examined the association between PIK4CA expression and its genetic variants in the same brain samples. PIK4CA expression was lower, whereas Akt expression was higher, in the PFC of schizophrenia patients than in that of controls; PIP5K1C, PTEN, and GSK3β expression was not different. No single-nucleotide polymorphism significantly affected protein expression. We identified molecules involved in the pathology of schizophrenia via this lipid metabolic pathway. These results suggest that PIK4CA is involved in the mechanism underlying the pathogenesis of schizophrenia and is a potential novel therapeutic target.

Phosphoinositide Recognition Sites Are Blocked by Metabolite Attachment.

Membrane readers take part in trafficking and signaling processes by localizing proteins to organelle surfaces and transducing molecular information. They accomplish this by engaging phosphoinositides (PIs), a class of lipid molecules which are found in different proportions in various cellular membranes. The prototypes are the PX domains, which exhibit a range of specificities for PIs. Our meta-analysis indicates that recognition of membranes by PX domains is specifically controlled by modification of lysine and arginine residues including acetylation, hydroxyisobutyrylation, glycation, malonylation, methylation and succinylation of sidechains that normally bind headgroups of phospholipids including organelle-specific PI signals. Such metabolite-modulated residues in lipid binding elements are named MET-stops here to highlight their roles as erasers of membrane reader functions. These modifications are concentrated in the membrane binding sites of half of all 49 PX domains in the human proteome and correlate with phosphoregulatory sites, as mapped using the Membrane Optimal Docking Area (MODA) algorithm. As these motifs are mutated and modified in various cancers and the responsible enzymes serve as potential drug targets, the discovery of MET-stops as a widespread inhibitory mechanism may aid in the development of diagnostics and therapeutics aimed at the readers, writers and erasers of the PI code.

The phosphoinositide code is read by a plethora of protein domains

Introduction The proteins that decipher nucleic acid- and protein-based information are well known, however, those that read membrane-encoded information remain understudied. Here we report 70 different human, microbial and viral protein folds that recognize phosphoinositides (PIs), comprising the readers of a vast membrane code. Areas covered Membrane recognition is best understood for FYVE, PH and PX domains, which exemplify hundreds of PI code readers. Comparable lipid interaction mechanisms may be mediated by kinases, adjacent C1 and C2 domains, trafficking arrestin, GAT and VHS modules, membrane-perturbing annexin, BAR, CHMP, ENTH, HEAT, syntaxin and Tubby helical bundles, multipurpose FERM, EH, MATH, PHD, PDZ, PROPPIN, PTB and SH2 domains, as well as systems that regulate receptors, GTPases and actin filaments, transfer lipids and assembled bacterial and viral particles. Expert opinion The elucidation of how membranes are recognized has extended the genetic code to the PI code. Novel discoveries include PIP-stop and MET-stop residues to which phosphates and metabolites are attached to block phosphatidylinositol phosphate (PIP) recognition, memteins as functional membrane protein apparatuses, and lipidons as lipid “codons” recognized by membrane readers. At least 5% of the human proteome senses such membrane signals and allows eukaryotic organelles and pathogens to operate and replicate.

Structures and Dynamics of Native-State Transmembrane Protein Targets and Bound Lipids.

Membrane proteins work within asymmetric bilayers of lipid molecules that are critical for their biological structures, dynamics and interactions. These properties are lost when detergents dislodge lipids, ligands and subunits, but are maintained in native nanodiscs formed using styrene maleic acid (SMA) and diisobutylene maleic acid (DIBMA) copolymers. These amphipathic polymers allow extraction of multicomponent complexes of post-translationally modified membrane-bound proteins directly from organ homogenates or membranes from diverse types of cells and organelles. Here, we review the structures and mechanisms of transmembrane targets and their interactions with lipids including phosphoinositides (PIs), as resolved using nanodisc systems and methods including cryo-electron microscopy (cryo-EM) and X-ray diffraction (XRD). We focus on therapeutic targets including several G protein-coupled receptors (GPCRs), as well as ion channels and transporters that are driving the development of next-generation native nanodiscs. The design of new synthetic polymers and complementary biophysical tools bodes well for the future of drug discovery and structural biology of native membrane:protein assemblies (memteins).

NPC1 regulates the distribution of phosphatidylinositol 4‐kinases at Golgi and lysosomal membranes

Cholesterol and phosphoinositides (PI) are two critically important lipids that are found in cellular membranes and dysregulated in many disorders. Therefore, uncovering molecular pathways connecting these essential lipids may offer new therapeutic insights. We report that loss of function of lysosomal Niemann‐Pick Type C1 (NPC1) cholesterol transporter, which leads to neurodegenerative NPC disease, initiates a signaling cascade that alters the cholesterol/phosphatidylinositol 4‐phosphate (PtdIns4P) countertransport cycle between Golgi‐endoplasmic reticulum (ER), as well as lysosome‐ER membrane contact sites (MCS). Central to these disruptions is increased recruitment of phosphatidylinositol 4‐kinases—PI4KIIα and PI4KIIIβ—which boosts PtdIns4P metabolism at Golgi and lysosomal membranes. Aberrantly increased PtdIns4P levels elevate constitutive anterograde secretion from the Golgi complex, and mTORC1 recruitment to lysosomes. NPC1 disease mutations phenocopy the transporter loss of function and can be rescued by inhibition or knockdown of either key phosphoinositide enzymes or their recruiting partners. In summary, we show that the lysosomal NPC1 cholesterol transporter tunes the molecular content of Golgi and lysosome MCS to regulate intracellular trafficking and growth signaling in health and disease.

Superresolution Microscopy Reveals Distinct Phosphoinositide Subdomains Within the Cilia Transition Zone.

Primary cilia are evolutionary conserved microtubule-based organelles that protrude from the surface of most mammalian cells. Phosphoinositides (PI) are membrane-associated signaling lipids that regulate numerous cellular events via the recruitment of lipid-binding effectors. The temporal and spatial membrane distribution of phosphoinositides is regulated by phosphoinositide kinases and phosphatases. Recently phosphoinositide signaling and turnover has been observed at primary cilia. However, the precise localization of the phosphoinositides to specific ciliary subdomains remains undefined. Here we use superresolution microscopy (2D stimulated emission depletion microscopy) to map phosphoinositide distribution at the cilia transition zone. PI(3,4,5)P3 and PI(4,5)P2 localized to distinct subregions of the transition zone in a ring-shape at the inner transition zone membrane. Interestingly, the PI(3,4,5)P3 subdomain was more distal within the transition zone relative to PtdIns(4,5)P2. The phosphoinositide effector kinase pAKT(S473) localized in close proximity to these phosphoinositides. The inositol polyphosphate 5-phosphatase, INPP5E, degrades transition zone phosphoinositides, however, studies of fixed cells have reported recombinant INPP5E localizes to the ciliary axoneme, distant from its substrates. Notably, here using live cell imaging and optimized fixation/permeabilization protocols INPP5E was found concentrated at the cilia base, in a distribution characteristic of the transition zone in a ring-shaped domain of similar dimensions to the phosphoinositides. Collectively, this superresolution map places the phosphoinositides in situ with the transition zone proteins and reveals that INPP5E also likely localizes to a subdomain of the transition zone membrane, where it is optimally situated to control local phosphoinositide metabolism.

The Leishmania donovani LDBPK_220120.1 Gene Encodes for an Atypical Dual Specificity Lipid-Like Phosphatase Expressed in Promastigotes and Amastigotes; Substrate Specificity, Intracellular Localizations, and Putative Role(s).

The intracellular protozoan parasites of the Leishmania genus are responsible for Leishmaniases, vector borne diseases with a wide range of clinical manifestations. Leishmania (L.) donovani causes visceral leishmaniasis (kala azar), the most severe of these diseases. Along their biological cycle, Leishmania parasites undergo distinct developmental transitions including metacyclogenesis and differentiation of metacyclic promastigotes (MPs) to amastigotes. Metacyclogenesis inside the phlebotomine sandfly host’s midgut converts the procyclic dividing promastigotes to non-dividing infective MPs eventually injected into the skin of mammalian hosts and phagocytosed by macrophages where the MPs are converted inside modified phagolysosomes to the intracellular amastigotes. These developmental transitions involve dramatic changes in cell size and shape and reformatting of the flagellum requiring thus membrane and cytoskeleton remodeling in which phosphoinositide (PI) signaling and metabolism must play central roles. This study reports on the LDBPK_220120.1 gene, the L. donovani ortholog of LmjF.22.0250 from L. major that encodes a phosphatase from the “Atypical Lipid Phosphatases” (ALPs) enzyme family. We confirmed the expression of the LDBPK_220120.1 gene product in both L. donovani promastigotes and axenic amastigotes and showed that it behaves in vitro as a Dual Specificity P-Tyr and monophosphorylated [PI(3)P and PI(4)P] PI phosphatase and therefore named it LdTyrPIP_22 (Leishmaniad onovani Tyrosine PI Phosphatase, gene locus at chromosome 22). By immunofluorescence confocal microscopy we localized the LdTyrPIP_22 in several intracellular sites in the cell body of L. donovani promastigotes and amastigotes and in the flagellum. A temperature and pH shift from 25°C to 37°C and from pH 7 to 5.5, induced a pronounced recruitment of LdTyrPIP_22 epitopes to the flagellar pocket and a redistribution around the nucleus. These results suggest possible role(s) for this P-Tyr/PI phosphatase in the regulation of processes initiated or upregulated by this temperature/pH shift that contribute to the developmental transition from MPs to amastigotes inside the mammalian host macrophages.

Structural Determinants for Light-Dependent Membrane Binding of a Photoswitchable Polybasic Domain.

OptoPB is an optogenetic tool engineered by fusion of the phosphoinositide (PI)-binding polybasic domain of Rit1 (Rit-PB) to a photoreactive light-oxygen-voltage (LOV) domain. OptoPB selectively and reversibly binds the plasma membrane (PM) under blue light excitation, and in the dark, it releases back to the cytoplasm. However, the molecular mechanism of optical regulation and lipid recognition is still unclear. Here using nuclear magnetic resonance (NMR) spectroscopy, liposome pulldown assay, and surface plasmon resonance (SPR), we find that OptoPB binds to membrane mimetics containing di- or triphosphorylated phosphatidylinositols, particularly phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), an acidic phospholipid predominantly located in the eukaryotic PM. In the dark, steric hindrance prevented this protein-membrane interaction, while 470 nm blue light illumination activated it. NMR titration and site-directed mutagenesis revealed that both cationic and hydrophobic Rit-PB residues are essential to the membrane interaction, indicating that OptoPB binds the membrane via a specific PI(4,5)P2-dependent mechanism.

Inositol Phospholipid Interactions with Inward Rectifier Potassium Channels and Their Role in Disease

Ion channels are frequently modulated by specific protein-lipid interactions in the phospholipid bilayer. The activation of human inward rectifying potassium (hKir) channels by phosphoinositides (PI) has been well characterised in biophysical studies. Here, we provide an extensive application of a coarse-grained molecular dynamics free-energy perturbation (CG-MD-FEP) methodology to capture the energetics of binding of PI lipids to hKir channels. By using either a single- or multi-step protocol, we establish a consistent value for the binding of PIP2 to hKir channels, relative to the binding of the bulk phosphatidylcholine phospholipid and in the presence of anionic phospholipid such as phosphatidylserine (PS). By perturbing amino acid side chains on hKir6.2, we show that the neonatal diabetes mutation E179K has increased PIP2 affinity, while the congenital hyperinsulinism mutation K67N results in a reduced affinity. This is in good agreement with electrophysiological studies showing the E179K mutant exhibits a reduction in neomycin sensitivity, which implies that PIP2 binds more tightly to E179K mutant channels. We conclude that our application provides a valuable method to compare affinities between lipid species, and for annotating amino acid side chains within lipid-binding pockets.

Label-Free Quantification of Phosphoinositides in Drosophila by Mass Spectrometry.

Phosphoinositides (PIs), the seven phosphorylated derivatives of phosphatidylinositol, are recognized as key molecules in the control of multiple molecular events in eukaryotic cells. Within cells, PIs are low-abundance lipids making their detection and quantification challenging. While many methods that allow radiolabeling and quantification of PIs in the context of cultured cells are available, these are not useful in the context of in vivo animal models where cell and developmental processes are best studied. In this chapter, we describe radionuclide-free, mass spectrometry-based methods for the detection and quantification of PIs from Drosophila tissues in vivo. The use of these methods should facilitate the discovery of novel modes by which PIs regulate cellular and developmental processes in complex metazoans.

YPIBP: A repository for phosphoinositide-binding proteins in yeast

Phosphoinositides (PIs) are a family of eight lipids consisting of phosphatidylinositol (PtdIns) and its seven phosphorylated forms. PIs have important regulatory functions in the cell including lipid signaling, protein transport, and membrane trafficking. Yeast has been recognized as a eukaryotic model system to study lipid-protein interactions. Hundreds of yeast PI-binding proteins have been identified, but this research knowledge remains scattered. Besides, the complete PI-binding spectrum and potential PI-binding domains have not been interlinked. No comprehensive databases are available to support the lipid-protein interaction research on phosphoinositides. Here we constructed the first knowledgebase of Yeast Phosphoinositide-Binding Proteins (YPIBP), a repository consisting of 679 PI-binding proteins collected from high-throughput proteome-array and lipid-array studies, QuickGO, and a rigorous literature mining. The YPIBP also contains protein domain information in categories of lipid-binding domains, lipid-related domains and other domains. The YPIBP provides search and browse modes along with two enrichment analyses (PI-binding enrichment analysis and domain enrichment analysis). An interactive visualization is given to summarize the PI-domain-protein interactome. Finally, three case studies were given to demonstrate the utility of YPIBP. The YPIBP knowledgebase consolidates the present knowledge and provides new insights of the PI-binding proteins by bringing comprehensive and in-depth interaction network of the PI-binding proteins. YPIBP is available at http://cosbi7.ee.ncku.edu.tw/YPIBP/.

A genome engineering resource to uncover principles of cellular organization and tissue architecture by lipid signaling.

Phosphoinositides (PI) are key regulators of cellular organization in eukaryotes and genes that tune PI signaling are implicated in human disease mechanisms. Biochemical analyses and studies in cultured cells have identified a large number of proteins that can mediate PI signaling. However, the role of such proteins in regulating cellular processes in vivo and development in metazoans remains to be understood. Here, we describe a set of CRISPR-based genome engineering tools that allow the manipulation of each of these proteins with spatial and temporal control during metazoan development. We demonstrate the use of these reagents to deplete a set of 103 proteins individually in the Drosophila eye and identify several new molecules that control eye development. Our work demonstrates the power of this resource in uncovering the molecular basis of tissue homeostasis during normal development and in human disease biology.

Location-dependent role of phospholipase C signaling in the brain: Physiology and pathology.

Phosphoinositide-specific phospholipases C (PI-PLCs) are a class of enzymes involved in the phosphatidylinositol metabolism, which is implicated in the activation of several signaling pathways and which controls several cellular processes. The scientific community has long accepted the existence of a nuclear phosphoinositide (PI) metabolism, independent from the cytoplasmic one, critical in nuclear function control. Indeed, nuclear PIs are involved in many activities, such as cell cycle regulation, cell proliferation, cell differentiation, membrane transport, gene expression and cytoskeletal dynamics. There are several types of PIs and enzymes implicated in brain activities and among these enzymes, PI-PLCs contribute to a specific and complex network in the developing nervous system. Moreover, considering the abundant presence of PI-PLCβ1, PI-PLCγ1 and PI-PLCβ4 in the brain, a specific role for each PLC subtype has been suggested in the control of neuronal activity, which is important for synapse function, development and other mechanisms. The focus of this review is to describe the latest research about the involvement of PI-PLC signaling in the nervous system, both physiologically and in pathological conditions. Indeed, PI-PLC signaling imbalance seems to be also linked to several brain disorders including epilepsy, movement and behavior disorders, neurodegenerative diseases and, in addition, some PI-PLC subtypes could become potential novel signature genes for high-grade gliomas.

Evaluating inositol phospholipid interactions with inward rectifier potassium channels and characterising their role in disease

Membrane proteins are frequently modulated by specific protein-lipid interactions. The activation of human inward rectifying potassium (hKir) channels by phosphoinositides (PI) has been well characterised. Here, we apply a coarse-grained molecular dynamics free-energy perturbation (CG-FEP) protocol to capture the energetics of binding of PI lipids to hKir channels. By using either a single- or multi-step approach, we establish a consistent value for the binding of PIP2 to hKir channels, relative to the binding of the bulk phosphatidylcholine phospholipid. Furthermore, by perturbing amino acid side chains on hKir6.2, we show that the neonatal diabetes mutation E179K increases PIP2 affinity, while the congenital hyperinsulinism mutation K67N results in a reduced affinity. We show good agreement with electrophysiological data where E179K exhibits a reduction in neomycin sensitivity, implying that PIP2 binds more tightly E179K channels. This illustrates the application of CG-FEP to compare affinities between lipid species, and for annotating amino acid residues.

Phosphatidylinositol 4-kinase β is required for the ciliogenesis of zebrafish otic vesicle

The primary cilium, an important microtubule-based organelle, protrudes from nearly all the vertebrate cells. The motility of cilia is necessary for various developmental and physiological processes. Phosphoinositides (PIs) and its metabolite, PtdIns(4,5)P2, have been revealed to contribute to cilia assembly and disassembly. As an important kinase of the PI pathway and signaling, phosphatidylinositol 4-kinase β (PI4KB) is the one of the most extensively studied phosphatidylinositol 4-kinase isoform. However, its potential roles in organ development remain to be characterized. To investigate the developmental role of Pi4kb, especially its function on zebrafish ciliogenesis, we generated pi4kb deletion mutants using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 technique. The homozygous pi4kb mutants exhibit an absence of primary cilia in the inner ear, neuromasts, and pronephric ducts accompanied by severe edema in the eyes and other organs. Moreover, smaller otic vesicle, malformed semicircular canals, and the insensitivity on sound stimulation were characteristics of pi4kb mutants. At the protein level, both in vivo and in vitro analyses revealed that synthesis of Pi4p was greatly reduced owing to the loss of Pi4kb. In addition, the expression of the Pi4kb-binding partner of neuronal calcium sensor-1, as well as the phosphorylation of phosphatidylinositol-4-phosphate downstream effecter of Akt, was significantly inhibited in pi4kb mutants. Taken together, our work uncovers a novel role of Pi4kb in zebrafish inner ear development and the functional formation of hearing ability by determining hair cell ciliogenesis.

Subcellular Localization Relevance and Cancer-Associated Mechanisms of Diacylglycerol Kinases.

An increasing number of reports suggests a significant involvement of the phosphoinositide (PI) cycle in cancer development and progression. Diacylglycerol kinases (DGKs) are very active in the PI cycle. They are a family of ten members that convert diacylglycerol (DAG) into phosphatidic acid (PA), two-second messengers with versatile cellular functions. Notably, some DGK isoforms, such as DGKα, have been reported to possess promising therapeutic potential in cancer therapy. However, further studies are needed in order to better comprehend their involvement in cancer. In this review, we highlight that DGKs are an essential component of the PI cycle that localize within several subcellular compartments, including the nucleus and plasma membrane, together with their PI substrates and that they are involved in mediating major cancer cell mechanisms such as growth and metastasis. DGKs control cancer cell survival, proliferation, and angiogenesis by regulating Akt/mTOR and MAPK/ERK pathways. In addition, some DGKs control cancer cell migration by regulating the activities of the Rho GTPases Rac1 and RhoA.

Risk1, a Phosphatidylinositol 3-Kinase Effector, Promotes Rickettsia typhi Intracellular Survival.

To establish a habitable intracellular niche, various pathogenic bacteria secrete effectors that target intracellular trafficking and modulate phosphoinositide (PI) metabolism. Murine typhus, caused by the obligate intracellular bacterium Rickettsia typhi, remains a severe disease in humans. However, the mechanisms by which R. typhi effector molecules contribute to internalization by induced phagocytosis and subsequent phagosomal escape into the cytosol to facilitate the intracellular growth of the bacteria remain ill-defined. Here, we characterize a new molecule, Risk1, as a phosphatidylinositol 3-kinase (PI3K) secreted effector and the first bacterial secretory kinase with both class I and III PI3K activities. Inactivation of Risk1 PI3K activities reduced the phosphorylation of phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 3,4,5-trisphosphate within the host, which consequently diminished host colonization by R. typhi During infection, Risk1 targets the Rab5-EEA1-phosphatidylinositol 3-phosphate [PI(3)P] signaling axis to promote bacterial phagosomal escape. Subsequently, R. typhi undergoes ubiquitination and induces host autophagy; however, maturation to autolysosomes is subverted to support intracellular growth. Intriguingly, only enzymatically active Risk1 binds the Beclin-1 core complex and contributes to R. typhi-induced autophagosome formation. In sum, our data suggest that Risk1, with dual class I and class III PI3K activities, alters host PI metabolism and consequently subverts intracellular trafficking to facilitate intracellular growth of R. typhiIMPORTANCERickettsia species are Gram-negative obligate intracellular bacteria that infect a wide range of eukaryotes and vertebrates. In particular, human body louse-borne Rickettsia prowazekii and flea-borne Rickettsia typhi have historically plagued humankind and continue to reemerge globally. The unavailability of vaccines and limited effectiveness of antibiotics late in infection place lethality rates up to 30%, highlighting the need to elucidate the mechanisms of Rickettsia pathogenicity in greater detail. Here, we characterize a new effector, Risk1, as a secreted phosphatidylinositol 3-kinase (PI3K) with unique dual class I and class III activities. Risk1 is required for host colonization, and its vacuolar phosphatidylinositol 3-phosphate generation modulates endosomal trafficking to arrest autophagosomal maturation. Collectively, Risk1 facilitates R. typhi growth by altering phosphoinositide metabolism and subverting intracellular trafficking.

Phosphoinositide Lipids in Ocular Tissues.

Inositol phospholipids play an important role in cell physiology. The inositol head groups are reversibly phosphorylated to produce seven distinct phosphorylated inositides, commonly referred to as phosphoinositides (PIs). These seven PIs are dynamically interconverted from one PI to another by the action of PI kinases and PI phosphatases. The PI signals regulate a wide variety of cellular functions, including organelle distinction, vesicular transport, cytoskeletal organization, nuclear events, regulation of ion channels, cell signaling, and host–pathogen interactions. Most of the studies of PIs in ocular tissues are based on the PI enzymes and PI phosphatases. In this study, we examined the PI levels in the cornea, retinal pigment epithelium (RPE), and retina using PI-binding protein as probes. We have examined the lipids PI(3)P, PI(4)P, PI(3,4)P2, PI(4,5)P2, and PI(3,4,5)P3, and each is present in the cornea, RPE, and retina. Alterations in the levels of these PIs in mouse models of retinal disease and corneal infections have been reported, and the results of our study will help in the management of anomalous phosphoinositide metabolism in ocular tissues.

Dopamine, a key factor of mitochondrial damage and neuronal toxicity on rotenone exposure and also parkinsonic motor dysfunction—Impact of asiaticoside with a probable vesicular involvement

Persuasive evidence propose that the toxicity of dopamine in parkinsonism and the loss of dopaminergic neurons are the earliest events during the pathogenesis of Parkinson’s disease (PD). In our earlier study, Asiaticoside (AS), a triterpenoid saponin isolated from Centella asiatica was shown to exert a neuroprotective effect against hemiparkinsonism, purportedly due to phosphoinositides (PI)-assisted cytodynamics and synaptic function. Here, we evaluate AS in the modulation of dopamine (DA), mitochondrial integrity and neurite variations in vitro and motor dysfunctions in vivo. PC12 cells challenged with rotenone-(ROT) (0.1 μM/mL) were exposed to AS and l-DOPA (10 mM and 20 μM/mL respectively). The protein expressions of Bax and Bcl-2 that regulate cell death were assessed following neurite length assays. Rats were distributed into 6 groups (6 rats/group): Sham, Vehicle controls, ROT-infused (6 μg/μl/kg), AS- treated (50 mg/kg/day), Drug control, and ROT + L-DOPA-treated (6 mg/kg/day) groups. At the end of the experimental period, the rats were sacrificed after performing motor behavioral analysis, and the striatum was dissected out. The contents of synaptic vesicular and cytosolic DA were analyzed. Further, the levels of striatal PI were also measured. ROT had caused significant reduction in the neurite outgrowth in the exposed PC12 cells while the tested concentrations of AS and l-DOPA can exert their protective effect on the stunted neurite growth. The levels of Bax, Bcl-2, and cytochrome c which were significantly disturbed by ROT, could also be affected by AS thereby suggesting its effect on neurons. AS treatment caused an improved motor performance, vesicular and cytosolic DA, and striatal PI. These pre-clinical findings force us to speculate that AS could be a potential drug candidate in combating ROT-induced variations that are possibly precipitated by varied vesicular trafficking of DA.

Phosphoinositide-Dependent Signaling in Cancer: A Focus on Phospholipase C Isozymes.

Phosphoinositides (PI) form just a minor portion of the total phospholipid content in cells but are significantly involved in cancer development and progression. In several cancer types, phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] play significant roles in regulating survival, proliferation, invasion, and growth of cancer cells. Phosphoinositide-specific phospholipase C (PLC) catalyze the generation of the essential second messengers diacylglycerol (DAG) and inositol 1,4,5 trisphosphate (InsP3) by hydrolyzing PtdIns(4,5)P2. DAG and InsP3 regulate Protein Kinase C (PKC) activation and the release of calcium ions (Ca2+) into the cytosol, respectively. This event leads to the control of several important biological processes implicated in cancer. PLCs have been extensively studied in cancer but their regulatory roles in the oncogenic process are not fully understood. This review aims to provide up-to-date knowledge on the involvement of PLCs in cancer. We focus specifically on PLCβ, PLCγ, PLCδ, and PLCε isoforms due to the numerous evidence of their involvement in various cancer types.