Dietary fatty acids mediate the secondary messenger phosphatidylinositol for microglial phagocytosis and migration

Alzheimer’s disease is one of the neurodegenerative diseases, characterized by the accumulation of abnormal protein deposits, which disrupt the signal transduction in neurons and other glia cells. The pathological protein Tau and amyloid-β contributes to the disrupted microglial signaling pathways, actin cytoskeleton, and cellular receptor expression. The important secondary messenger lipids i.e., phosphatidylinositols are largely affected by protein deposits of amyloid-beta in Alzheimer’s disease. Phosphatidylinositols are the product of different phosphatidylinositol kinases and the state of phosphorylation at D3, D4, and D5 positions of inositol ring. PI 3, 4, 5-P3 involves in phagocytic cup formation and relates actin remodeling whereas PI 4, 5-P2-mediates the process of phagosomes formation and further fusion with early endosome. The necessary activation of actin-binding proteins such as Rac, WAVE complex, and ARP2/3 complex for the actin polymerization in the process of phagocytosis, migration is regulated and maintained by PI 3, 4, 5-P3 and PI 4, 5-P2. Dietary fatty acids depending on their ratio and types of intake influence secondary lipid messenger along with the cellular content of phaphatidylcholine and phosphatidylethanolamine. The deposited Aβ deposits and extracellular Tau seed disrupt levels of phosphatidylinositol and actin cytoskeletal changes that hamper microglia signaling pathways in AD. We hypothesize that being a lipid species intracellular levels of phosphatidylinositol would be regulated by dietary fatty acids. We keen to understand different types of phosphatidylinositol species levels in signaling events such as phagocytosis and actin remodeling owing to the exposure of various types of dietary fatty acids. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 22 July 2020 doi:10.20944/preprints202007.0532.v1 © 2020 by the author(s). Distributed under a Creative Commons CC BY license.

The amyloidogenic processing of amyloid precursor protein (APP) leads to the formation of insoluble monomer, dimer, oligomer, and aggregates of the amyloid peptide. The accumulation of amyloid plaques disrupts majorly the neurotransmission amongst the neuron, affects Tau pathology, and even contributes to excessive activation of glial cells. The insoluble amyloid oligomer found to be more neurotoxic to disrupt intracellular signaling [9]. Aβ binds to various cellular receptors to induce neurotoxicity via mitochondrial dysfunction and oxidative stress leading to excessive calcium influx introduces toxicity [10]. The soluble Aβ can interact with various receptors to activate downstream signaling pathways that produce reactive oxygen species, hyperphosphorylated Tau and induce inflammatory response in brain cells [1]. The phosphatidylinositols metabolism necessary for various intracellular signaling, which is affected by Aβ oligomer by activating SHIP2 via FcγRIIb receptor. The levels of important secondary messenger phosphatidylinositols have been disrupted that are involved in important cellular processes such as phagocytosis, migration, actin cytoskeleton remodeling. The Aβ induced affected metabolism of phosphatidylinositols challenge Tau hyperphosphorylation by various protein kinases [11]. Amyloid-β is also capable of disrupting the function of phosphatidylinositol-3 kinase (PI3K) an important enzyme in the conversion of Phosphatidylinositol 4, 5-diphosphate (PI 4, 5-P2) to phosphatidylinositol 3, 4, 5triphosphate and also involved in Akt-mTOR signaling pathway [12]. The soluble Aβ oligomer also affects PI3K/Akt/GSK-3β pathway majorly causing neuronal death and Tau hyperphosphorylation [13].
The disrupted PI3K signaling targets Tau hyperphosphorylation, which impart pathological condition in AD. Aggregated form of Aβ 25-35 significantly impair phosphatidylinositol related enzyme phospholipase C found in the brain during AD [14]. Apart from the involved enzymes in phosphatidylinositol signaling (PI), Aβ directly reduced levels of PI 4, 5-P2 phospholipid that regulate various neuronal functions [15].

Phosphatidylinositol influence actin remodeling
The phosphorylated derivatives of phosphatidylinositols (PI) are the important secondary messenger in the cell. The phosphorylation at D3, D4, D5 positions of the inositol ring decides the type of response and the location of the derivative inside the cell. Phosphatidylinositol 4, 5-diphosphate (PI 4, 5-P2) and phosphatidylinositol 3, 4, 5-triphosphate (PI 3, 4, 5-P3) are the main lipid-derivative form in process of phagosome formation and early endosome maturation in phagocytosis [16]. The secondary lipid mediators PI 4, 5-P2 and PI 3, 4, 5-P3 concentrate at the plasma membrane to initiate the cellular processes such as endocytosis, phagosome maturation, actin polymerization and migration [17]. The PI 4, 5-P2 regulates actin dynamics by interacting with actin-binding proteins such as ARP2/3 complex, capping protein, WASP family proteins, and other actin-binding proteins [18]. Along with activating actin-binding proteins, PI 3, 4, 5-P3 specifically regulates membrane ruffling via protein kinase A (PKA) activity ( Figure 1). PKA inhibition triggers a marked decrease in bulk accumulation of PI 3, 4, 5-P3 at membrane ruffles independent of Rac activation [19]. According to studies, the local synthesis of PI 4, 5-P2 specifically via PIPKα, which induces actin polymerization via ARP2/3 and increase local levels of PI 3, 4, 5-P3 for actin remodeling related to membrane ruffling [20]. After ruffling for endocytosis and phagosome formation the concentration kinetics of PI 4, 5-P2, and PI 3, 4, 5-P3 mechanistically linked to related actin remodeling. PI 3, 4, 5-P3 concentration sharply increases at the site of phagosomal cup formation and disappears once the phagosome has been sealed off from the plasma membrane. Whereas PI 4, 5-P2 levels significantly increased for circular ruffle formation subsequently decrease during endocytosis of foreign particle. The difference in levels of PI 4, 5-P2, and PI 3,4,5-P3 regulated by PI3K is mechanically important for actin remodeling and macropinosome formation [16,21]. The negatively charged lipid such as PI 3, 4, 5-P3 activates N-WASP, cdc42, which triggers ARP2/3-mediated F-actin polymerization for the podosomes formation. PI 3, 4, 5-P3 enriches membrane-associated actin regulation factors-1e (Myo1e) which links PI signaling to phagosome assembly [22]. The production PI 4, 5-P2 by the enzyme Phosphatidylinositol-5 kinase (PI5K) from PI 4-P is triggered at the cell membrane and overexpression of PI5K and reduced expression of phosphatase increase the levels of PI 4, 5-P2 important for rocketing of vesicles [23]. On the other hand the actin regulating protein binds PI 4, 5-P2 with a basic and hydrophobic amino acid. The interacting proteins include WASP superfamily protein, ARP2/3 complex, gelsoline family protein, and capping protein, which are also affected by surface density of PI 4, 5-P2. PI 4, 5-P2 levels in cell manage F-actin levels along with their association with actin polymerization while the levels of PI 4,5-P2 depends upon the regulation of enzymes required for the production [24]. However the pool of PI 4, 5-P2 in cells is largely affected by extracellular stimulus [18].
In AD, presence of extracellular Aβ oligomers decrease the levels of PI 4, 5-P2 and increases PI 3, 4, -P2 levels via SHIP-2 and causes hyperphosphorylation of Tau. The disrupted metabolism of PI due to Aβ affects function of actin cytoskeleton and neurotoxicity, which contributes to neurodegeneration, synaptic failure in AD. The maintenance of the metabolism of PI has become one of the therapeutic strategies for AD [11]. Tau is another important protein in AD apart from Aβ, the hyperphosphorylation of Tau carried by PI3K pathway including GSK-3β [25]. The Aβ-induced increase in PI 3, 4-P2 levels trigger Tau hyperphosphorylation in neurons. In addition, disruption in PTEN eventually increases Tau hyperphosphorylation along with decreasing PI 4,5-P2 levels [11]. The Aβ has induced the increased phospho-Tau intermediate in disease pathology and contributes to extracellular Tau seed.  Arp2/3 complex has been proven ( Figure 1) [28].

Phosphatidylinositol 4, 5-bisphosphate a regulator of actin remodeling
The recent studies also suggested the importance of different PI derivatives PI (3) P and PI (4) P in the early and late stages of phagosome maturation in phagocytosis [29]. The cholesterol and sphingolipid rich membrane rafts act as a site for PI 4, 5-P2 production, and membrane-associated actin polymerization via the WASP-ARP2/3 pathway [30]. The PI 4, 5-P2 is mostly located at the inner leaflet of the membrane and regulated by lipid raft and membrane curvature. The PI 4, 5-P2 accumulates at the aggregated lipid raft regions and mediates the signaling cascade related to receptor-mediated phagocytosis [31].
The oligomeric species of amyloid-beta induced decrease of PI 4, 5-P2, is depend upon the extracellular Ca 2+ dyshomeostasis [15]. From the recent studies it can be stated as PI 4, 5-P2 is an important player to impose the neuronal loss and disrupted signaling cascades and hence act as a therapeutic strategy to target in AD [32].

Phosphatidylinositol signaling in microglial migration
Microglia is an immune cell of the brain that has surveillant nature, which is supported by high migration rates of microglia and capacity to respond to chemotaxis response [33]. The basic actin cytoskeleton is necessary to regulate the processes such as migration and surveillant nature of microglia [4,34]. In AD, the accumulated abnormal proteins serve in the classical activation of microglia inducing proinflammatory response. The excessive pro-inflammatory response triggers neuroinflammation, which imparts the anti-inflammatory stage of microglia [3,4]. The plasma membrane and the underlined cortical actin network is very important for migration and phagocytosis. For the process of migration coordinated polymerization of actin filaments provides a protrusive force (lamellipodia) and thin filamentous protrusion to sense and direct the migration (filopodia) is necessary. The lamellipodia-dependent migration carried out by actin-rich protrusion lamellipodia and filopodia at leading ends. Filopodia sever as antennae of the cell which probe the environment and serve pioneer in migration [35]. Lamellipodia on the other hand produce due to coordinated actin polymerization carried by ARP2/3 complex activation [34]. The actin polymerization beneath the plasma membrane produces the protrusion that drives forward moment of cell at the leading end [36]. The membrane protrusion around the targets for phagocytosis also involved actin cytoskeleton regulation. Phosphatidylinositols induce migration by the lamellipodiadependent mechanism via inducing actin polymerization at leading ends and also provide directional clues during chemotaxis. Phosphatidylinositols regulate signaling by directly binding to actin-binding proteins and influence their activity [18].   P3 which creates an intracellular signaling gradient for chemotaxis [37]. The chemotactic receptors P2X, P2X4R, P2Y12R also showed activation of the PI3K pathway over stimulation by ATP/ADP and also by amyloid-beta [33]. PI derivatives hence regulate the migratory movement of cells, which is necessary to catch targets during phagocytosis ( Figure 2).

Dietary fatty acids govern phosphatidylinositol signaling
Dietary fatty acids known to induce changes in cell membrane compositions and structure, which has various effects on signal transduction pathways. Dietary fatty acids influence phosphatidylethanolamine (PE), phaphatidylcholine (PC) of the cell membrane to the most and to some extent phosphatidylinositols (PI) [38]. The omega-3 fatty acids supplement of DHA and EPA specifically influence species and levels of PI in the cell, which could consider as one of the important mechanism to regulate signaling pathways and avoid cardiac arrhythmias [39]. Omega-3 fatty acids found to inhibit the phospholipase C (PLC)mediated hydrolysis of PI 4, 5-P2. The hydrolyzed product inositol 1, 4, 5-triphosphate (IP3) along with diacylglycerol (DAG) have been found to induces leukotriene (LTB4) mediated inflammatory response in neutrophils [40]. The activity of PI 3, 4, 5-P3 depends upon types of fatty acids at sn-1 and sn-2 positions of phospholipids [41]. fatty acids tend to incorporate at the sn-2 position of glycerol backbone, that holds the tendency to change the species of phospholipid [41]. The acyl chain remodeling carried out by different enzymes might act as a mechanism that decides the particular PUFA chain at the sn-2 position of PI and also determines the downstream lipid signaling molecule [42]. Different fatty acids found to influence pool particular PI derivatives in the cell. Incorporation of fatty acids into cells has been found to increase particular PI derivatives. The disrupted metabolism of PI and related signaling cascade in AD could be monitored with dietary fatty acids sources.

Conclusion
Alzheimer's disease as the most common cause of dementia drags serious attention to the therapy. The