Phosphoinositide kinases are diverse group of enzymes that perform the addition of phosphate group on the hydroxyl residues of myo-inositol ring of PIs. These kinases have substrate specificity for PIs, as well as specificity for their target hydroxyl residue on these PIs.

Phosphoinositide phosphatases are enzymes that catalyze removal of phosphate groups from the position 3,4 or 5 hydroxyl residues of myo-inositol ring of PIs. Just like PI kinases, these phosphatases also have substrate and catalytic site specificity. Here, phosphatases that recognize only PIP₂ or PIP₃ as their substrates are in focus.

Phospholipases are enzymes that hydrolyze phospholipids into its constituent fatty acids. The most relevant class of phospholipases to our discussion is phospholipase C (PLC) class of PI-specific enzymes, which cleave PIP₂ to generate diacylglycerol (DAG) and inositol-1,4,5-triphosphate (IP₃). DAG and IP₃ are important secondary messengers that are involved in protein kinase C (PKC) signaling, intra-neuronal calcium (Ca²⁺) signaling, and transcription, among other regulatory roles [6]. PI specific PLCs have been classified into six families (β, γ, δ, ε, ζ, and η; Figure 4). Alternative splicing reportedly leads to the generation of about 30 isozymes in mammals [95]. All PI specific isozymes have a homologues core consisting of a N-terminal PH domain, four EF domains, a triose phosphate isomerase (TIM) barrel domain (X, Y), and a C-terminal C2 domain [96, 97]. The subtle structural nuances and specific combinations of these domains can regulate distribution and function of each PLC isozyme. PLC enzymes are activated through either GPCRs (β, δ, and η), or RTKs (γ and ζ), or both (ε) [98]. Of note here is a suggested role for the nuclear PLC-β1 in PIP₂ hydrolysis, raising questions about the existence of PI signaling in the nucleus [99]. The isoforms PLC-δ (1,3, 4), PLC-β (1,4), PLC-γ (1), PLC-ε and PLC-η (1,2) are most relevant to nervous system and have been implicated in neurodegenerative disorders [100].

The cellular plasma membranes are heterogenous in nature and are organized into microdomains or ordered/disordered liquid phases, based on their lipid and protein constituents. In the last decade or so, it has been reported that composition of these microdomains dictates localization of PIs to specific regions of the plasma membranes [101]. An important regulator of this localization is reported to be cholesterol. Plasma membrane microdomains can be classified, on the basis of cholesterol abundance, into cholesterol-rich, liquid-ordered (L_o) raft domains or cholesterol-poor, liquid-disordered (L_d) non-raft domains. Recent literature suggests that PIP₂ is present in both domains, however, it gets hydrolyzed by PLC faster, and is also restored more rapidly in cholesterol-rich (L_o) domain [102]. This compartmentalization of PIP₂ signaling seems to be conserved as it has been reported in plants membranes as well [103]. In the context of neurodegenerative diseases, increasing cholesterol levels in the membranes led to PLC-mediated depletion of PIP₂ and an increase in AD-associated secretory amyloid β42 in cell lines [104]. PTEN phosphatase binding to PIP₂ is also reported to be increased in cholesterol-rich environments [105].

GPCRs or RTKs mediated activation of PLC isoforms leads to hydrolysis of PIP₂ into IP₃ and DAG [6]. IP₃ binds to IP₃ receptors (IP₃Rs) on the ER to release Ca²⁺. These IP₃Rs are found on nuclear envelope as well. In order for Ca²⁺ to release from ER into the cytosol, IP₃ first needs to bind to all four monomers of an IP₃R tetramer, causing a conformational change that allows Ca²⁺ to pass. This allowance is, however, transient. When Ca²⁺ levels rise above a certain level, this signaling becomes inhibitory through complex feedback interactions [106–108]. These IP₃ induced Ca²⁺ oscillations are found in multiple cell types. In brains, these oscillations regulate differentiation and proliferation [109]. IP₃ levels regulate steering of axonal growth cones, while Ca²⁺ transients specify if a neuron will be inhibitory or regulatory by way of regulating neurotransmitters release [110]. Low-frequency oscillations lead to release of excitatory neurotransmitters (acetylcholine, glutamate), while higher frequency oscillations lead to expression of inhibitory transmitters (glycine, γ-aminobutyric acid) [111]. These oscillations are important in generating brain rhythms for sleep/wake cycle, memory formation, and synaptic plasticity [112, 113]. Alterations in IP₃/Ca²⁺ signaling are associated with many neurological disorders like AD, ASD, epilepsy, schizophrenia among others, which are discussed in section Disease relevance.

DAG, the second product of PIP₂ hydrolysis, is most notably involved in PKC signaling. The PKC family of kinases have two DAG-binding copies of C1 domains, and in the case of conventional PKCs (cPKCs), an additional C2 domain to sense intracellular Ca²⁺ levels for full activation [114, 115]. The subcellular distribution of cPKCs is cytosolic under basal Ca²⁺ conditions, and it has now been shown that Ca²⁺ binding to C2 domain is sufficient and necessary for rapid membrane translocation. The DAG association with C1 domain of cPKC is important for its retention on the membrane [116]. Hence, the downstream signaling of both PIP₂ hydrolysis products is intricately connected and has critical roles to play in activation of cPKCs. The activation of cPKCs has been implicated in interfering with inhibitory binding of calmodulin at plasma membrane Ca²⁺-ATPases (PMCA); regulation of transient receptor potential (TRP), Na⁺/Ca²⁺ exchanger (NCX) and sodium proton exchangers (NHE) channels; generation of cyclic adenosine monophosphates (cAMPs); and activation of phospholipase D (PLD) and DAG kinases to coordinate phosphatidic acid (PA) signaling [117–121] (Figure 5). The PA signaling is known to play role in neurite growth associated cytoskeletal and membrane remodeling [122]. Alterations in PA signaling levels are associated with glioblastomas, intellectual disability, and neurodegenerative disorders, which are discussed later.

Canonically, the activation of RTKs or GPCRs by extracellular stimuli recruits PI3K to plasma membrane, where it catalyzes the phosphorylation of PIP₂, which generates PIP₃. This generation of PIP₃ leads to activation of AKT (also known as protein kinase B)/mTOR signaling pathway, which is a central regulator for cell growth, metabolism, protein translation, cytoskeletal organization, membrane trafficking and survival [123]. PTEN is a negative regulator of this pathway which keeps the activation of this pathway in check, by dephosphorylating PIP₃ into PIP₂ [124]. The formation of PIP₃ recruits PH domain containing proteins like PDK and AKT to the plasma membrane [125]. The PDK is reported to localize in cholesterol-rich membrane rafts [126]. This close proximity allows PDK to phosphorylate AKT at T308 [127]. Phosphorylation at T308 stabilizes and activates AKT.

For AKT to reach its maximal activity potential a further phosphorylation at S473 is needed [128, 129]. It is thought that first phosphorylation event at T308 primes AKT for second phosphorylation at S473, which in turn, stabilizes the first T308 phosphorylation. This idea is supported by the observations that T308 phosphorylation can occur without prior S473 phosphorylation, but not vice versa [130]. The S473 phosphorylation is brought about by mTOR complex 2 (mTORC2). This complex is assembled by PIP₃ binding to the PH-domain containing mSIN1, and thereby relieving its suppression on mTOR kinase activity and localizing it to plasma membrane as well [131]. mTORC2 complex also contains a scaffolding protein Rictor which regulates its assembly. mTORC2, as the name of its scaffolding protein suggests, is insensitive to acute rapamycin treatment and is known to regulate cytoskeletal rearrangements to support formation of new dendritic branches [129, 132].

Targeting Rictor to inhibit mTORC2 activity has been shown to result in inhibition of basal synaptic transmission and dendritic outgrowth in hippocampal neurons [133]. Another candidate for mediating this S473 phosphorylation on AKT is DNA-dependent protein kinase catalytic subunit (DNA-PKcs), but its role has been described mostly in the context of DNA damage in the nucleus [134]. Reports of PIP₃ being enriched at endosome and nuclear envelope also support the idea of localized phosphorylation and activation of AKT in intracellular compartments [135, 136]. In addition, three subtypes of AKT (AKT1, AKT2, AKT3) exist, with a high degree of homology in amino acid sequence and corresponding phosphorylation sites. Mostly, only AKT1 and AKT3 expression has been reported in hippocampus, while AKT2 expression is limited to cerebellum [137].

AKT targets many substrates which are at the nodes of different signaling pathways. One such target protein is GSK3 whose inhibitory phosphorylations of its targets are relieved by an inactivating phosphorylation by AKT. GSK3 regulates cell growth and development by regulating glycogen metabolism. It is also involved in Wnt/β-catenin signaling pathway, indicating its role in crosstalk between these two pathways [138, 139]. Another downstream effect of AKT activation is the assembly of mTORC1. mTORC1 is not a direct substrate of AKT. AKT mediated inhibitory phosphorylation on TSC relieves its inhibition of Rheb-GTP formation and leads to subsequent activation of mTORC1. In addition, mTORC1 complex assembly is facilitated by a scaffolding protein Raptor, whose suppression is relieved by AKT-mediated phosphorylation of PRAS40 [140–142] (Figure 5).

Broadly, mTORC1 is involved in processes of enhanced protein synthesis and growth through downstream effectors like S6 kinase; lipid synthesis through sterol responsive element binding protein (SRBEP); cellular stress responses through its negative regulator AMPK; and cell survival through autophagy and ubiquitin proteosome regulation [143–146]. In neuronal context, mTORC1 is activated through stimuli like brain-derived neurotrophic factor (BDNF), reelin, glutamate, gamma-aminobutyric acid (GABA), acetyl choline, and neuropeptides [147–149]. The stimulation of mTOR increases dendritic protein synthesis locally and contributes to synaptic and structural plasticity [150, 151]. mTOR activity is also needed for proper dendritic arborization, axonal branching, neuronal polarization, and autophagy mediated differentiation [152–154].

AKT also targets transcription factors of FOXO family. These transcription factors translocate from cytoplasm to nucleus to regulate processes like apoptosis and oxidative stress resistance. In the nervous system, acute FOXO activity is involved in age-dependent axonal degeneration, spine density and consolidation of memories [155, 156].

Apart from signaling through its downstream effectors, PIP₂ may directly bind to and interact with ion transporters and membrane channels to affect their activity [11]. The phospholipid composition of the intracellular compartmentshas been hypothesizedtoregulate the activity of membrane channelsinspace, until they arrive at their target membrane compartment with their signature phospholipid composition. Another hypothesis is about regulation of channels’ activity in time. Cells may respond dynamically to extracellular stimuli by way of changes in PI signaling mediating cell’s electrical and transport activity [157, 158]. This is an area of active investigation and the exact nature of PI binding to membrane channels has been difficult to determine. However, recent crystallography and mutagenesis studies have identified clusters of conserved amino acid residues on the channels that interact with phosphate group of PIs. These interactions might cause conformational changes to stabilize channel protein in a certain state, as observed with crystal structures of PIP₂ bound to potassium channels. The conformational change brought about by binding of PIP₂ is thought to lead to channel activation [159, 160].

In the context of potassium channels activity, PIP₂ seems to be the principal phospholipid regulating their activity. For voltage-gated potassium channels (Kv), the Kv7 or potassium voltage-gated channel subfamily Q (KCNQ) family is most relevant to neuronal excitability [161]. The PLC mediate depletion of PIP₂ reduces their current in a matter of seconds [159]. Direct application of PIP₂ experimentally, slows this rundown [160]. Structurally, basic residues on the C-terminal TM segment and calmodulin binding may be required for PIP₂ coupling to KCNQ channels [162]. Inwardly rectifying potassium channels (Kir) were the first to be identified as PIP₂ dependent [163]. All members of this family bind to PIP₂, but do so with different affinities (high affinity: Kir1, 2.1 and 4; low affinity: Kir2.3 and 3). High affinity channels were slowest to run down in response to PIP₂ depletion [164]. PIP₂ stabilizes the open state of these channels by binding to basic and hydrophobic residues in the N- and distal C-terminal [165].

Calcium activated potassium channels have also been shown to regulated by PIP₂ levels in the cell [166, 167]. For voltage-gated calcium channels (CaV), PIP₂ is required for opening in response to membrane potential changes. PIP₂ is hypothesized to bind to CaV as a segmented ligand bringing two parts together, and is competed for interaction by CaV β subunits whose expression governs the PIP₂ sensitivity of these channels [168]. Some of the TRP channels are also PIP₂ sensitive, i.e., PIP₂ promotes their opening by binding to basic residues on cytoplasmic linker and C-terminal [169, 170]. An example being cold-activated TRPM8 channel is that it does not respond to stimuli in the absence of PIP₂ [171]. The P2X receptor, CNG, and TMEM16-ANO1 are examples of some other channels that are also sensitive to PIP₂ binding [158].

Actin provides an architectural scaffold for the cell, and is an important regulator of cellular shape, trafficking, and migration [172]. This actin network is integral to the morphological remodeling of highly specialized cells like neurons. PIs regulate the activity of actin-binding proteins which are reported to control the initiation of processes like spinogenesis and dendritogenesis.

In general, PIP₂ inactivates actin-biding proteins that inhibit actin polymerization, while activating proteins that support polymerization. Gelsolin is an actin severing protein, whose binding with PIP₂ frees up the ends of actin filament for polymerization [173]. Interestingly, this binding is enhanced by calcium ions to promote actin polymerization [174]. The actin-related protein 2/3 (Arp2/3) complex which is responsible for branching of actin filaments is also activated through binding of PIP₂ with its activation protein, Wiskott-Aldrich syndrome protein (WASP) [175]. PIP₂ has also been shown to bind to other actin regulatory proteins like cofilin and profilin. The severing action of cofilin is inhibited by binding with PIP₂, while PIP₂-profilin interaction inhibits PLCγ-mediated hydrolysis of PIP₂ [176, 177].

The neuronal membrane branching/bending into dendrites, axons or spines is observed to protrude from specialized filopodia like structures which are filled with actin. PIP₂ is reported to mediate the formation of these filopodia. PIP₂ recruits the inverse bin-amphiphysin-rvs (I-BAR) protein missing-in-metastasis (MIM)/MTSS1 and Arp2/3-mediated actin assembly to nucleate the formation of protrusion that are pre-cursors to spinogenesis [178]. Nerve growth factor (NGF) treatment is known to increase the formation of patches that are precursors to the formation of axonal filopodia in a PI3K mediated activity dependent manner [179]. Localized microdomains of PIP₃ are also reported to be synchronous with formation of these precursor patches that are formed in response to NGF treatment [180]. Application of brain derived neurotrophic factor was reported to enhance PIP₃ localization to dendritic filopodia as well [181].

Mutations in genes encoding for proteins involved in PI synthesis and metabolism have been associated with ASD. Of the kinases involved in PIP₂ and PIP₃ synthesis, mutations in catalytic and regulatory isoforms of PI3K have been observed to be overrepresented when it comes to disorders affecting brain development [15]. In the context of dysregulation of catalytic subunits of PI3K, mutations in PIK3CA gene have been observed in clinical cases of cortical dysplasia and megalencephaly [182]. Another study described missense mutations in gene coding for p110α in a patient with autism and macrocephaly [183]. There is also strong evidence for overexpression of p110β catalytic subunit in some cases of autism [184]. This overexpression has been shown to be caused by chromosomal duplication.

More studies have linked a loss of Fragile X mental retardation protein (FMRP) in Fragile X syndrome (FXS) to p110β overexpression [185]. Since p110β mRNA binds to FMRP, this loss of FMRP is associated with increase in p110β expression. This increase has been observed in mouse models [185], as well as human patients cell lines [186, 187]. Dysregulation of another p110δ subunit of PI3K has also been observed in autism and schizophrenia [188, 189]. The location for gene coding for p110γ has been identified as a potential autism susceptible locus [190]. As for the PI3K regulatory subunits, mutations in p85β have been described to be associated with autism and megalencephaly [191]. So far, members of other classes of PI3K (class II and III) that are involved in generation of PIs other than PIP₂ and PIP₃ have not been shown to be strongly associated with the incidence of autism, further highlighting the importance of maintaining PIP₂ an PIP₃ balance for neuronal health.

In addition to PI3K, PI4K and PIPKs have also been shown to be altered in autism and other related disorders. Deleterious mutations in a regulator of PI4K have been identified in patients with autism [192]. PIPK isoform 3 has been found to be duplicated in patients with developmental delay and autism [193]. Among the phosphatases regulating PIP₂ and PIP₃ balance, PTEN stands out for its role in developmental delay, autism, and epilepsy [194–196]. In multiple animal model studies, PTEN loss has been shown to cause autism-like phenotypes and behavior [197–199]. PTEN loss associated phenotypes include anxiety, seizures, macrocephaly, and deficitsin neuronal migration, growth, electrical activity, and social behavior [197, 199–201].

Trisomy of the locus containing SYNJ1 phosphatase has been shown to result in enlarged endosomes in cell lines developed from patients with Down’s syndrome (DS) [202]. Other PI phosphatases have not been linked with autism but have been associated with neurodegeneration.

The PIP₂/PIP₃ balance in the intracellular environment is reported to be perturbed in neurodegenerative disorders like Alzheimer’s and Parkinson’s [203]. SYNJ1, a PI phosphatase regulating synaptic activity, was observed to be increased in autopsy of adult brains of DS and early-onset AD patients [204]. The excess of SYNJ1 was also found to contribute to memory deficits in mouse models of AD. Reducing this excess experimentally was found to accelerate clearance of Amyloid β (Aβ) and associated cognitive decline. In another study, restoring PIP₂ levels was sufficient to ameliorate such synaptic dysfunction [205, 206]. Presence of tau protein has also been detected in patients with SYNJ1 mutations [207]. Missense mutation in sac1 domain of SYNJ1 has also been found in patients with early onset PD [208]. These patients experienced tremors and some cortical atrophy as well. Loss-of-function mutations in SYNJ1 have been observed in patients with infantile epileptic encephalopathy [209, 210].

Single nucleotide polymorphism (SNP) in INPP5B phosphatase have been shown to be associated with sporadic amyotrophic lateral sclerosis (ALS) [211]. Loss of function mutations in chorein or vacuolar protein sorting-associated protein 13A (VPS13A) have been found in patients with a rare hereditary genetic disorder called chorea-acanthocytosis (ChAc) [212]. ChAc is characterized by progressive movement disorder, seizures, cognitive difficulties, and neurodegeneration-particularly in striatum [213–215]. The mechanism behind disease pathology has remained puzzling for a while. However, recent studies in neuronal cell cultures and animal models have hinted at a role for PI signaling in the development of ChAc. Chorein is reported to be involved in activation of p85 regulatory subunit of PI3K, with subsequent activation of several downstream kinases [215, 216]. Compromised cytoskeleton and cell-survival was observed in patient-derived neuronal cell cultures containing mutations in chorein [217]. Mutations in genes encoding for IP₃R, for example inositol 1,4,5-trisphosphate receptor type 1 (ITPR1), have been reported in infantile-onset nonprogressive spinocerebellar ataxia (SCA) [218]. SCA are degenerative disorders related to movement control. The dysfunction of IP₃R leads to aberrant calcium signaling in cerebellar neurons which are implicated in SCA pathogenesis [219]. Mutations in FAM126A gene leading to loss of hyccin/FAM126A protein, a scaffolding partner of PIKIIIα, causes disorders of progressive hypomyelination in central and peripheral nervous system [30]. This hypomyelination manifests in form of leukoencephalopathy known as hypomyelination and congenital cataract (HCC), with symptoms of cognitive deficits and neuropathy [220].