User:Ramz Aziz/IP3 Pathway

The IP3 pathway is a highly abundant pathway observed in cellular signal transduction. Like many others, the IP3 pathway begins with the binding of a ligand to a receptor (e.g. GPCR, RTK), but upon receptor binding, this pathway has the unique feature of activating the enzyme phospholipase C (PLC), which cleaves a specific phospholipid ester bond in phosphatidylinositol 4,5-biphosphate (PIP2) into inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG). The IP3 then acts as a second messenger to trigger the release of calcium from intracellular stores, which then activate a cascade of other more complex cellular events. The IP3 pathway has been found widely throughout the human body with a particularly high abundance in the brain. Consequently, the IP3 pathway is believed to be actively involved normal neural functioning, and damage to this pathway is implicated in various neurological disorders.

Discovery
The first breakthrough that allowed researchers to discover the cellular events that occur during IP3 signalling was in 1953. Mabel R. Hokin and Lowell E. Hokin were investigating what they thought was an increase in the incorporation of 32P into RNA caused by the acetylcholine-induced stimulation of pancreatic slices at the University of Sheffield in the United Kingdom. However, before they were able to purify the RNA, they moved to McGill University in Montréal, Quebec, Canada to which they transferred all of their non-purified radiolabelled samples. When they tried to continue their experiments, they discovered that as they purified the RNA, the radioactivity was lost and that most of the radioactivity was found in the phospholipid components. This was a surprising discovery at the time because up to this point phospholipids were believed to only be inert structural components of cellular membranes. Hokin and Hokin went on in 1955 to further characterize how hormone stimulation increases the incorporation of 32P into phosphoinositide. This later became known as the IP effect or IP response. It was then shown that the hormonal stimulation was causing the hydrolysis phosphatidylinositol 4,5-bisphosphate PIP2 into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG).

The next insight into the IP3 signaling pathway came in 1975 when Bob Michell proposed in a review that lipid hydrolysis was responsible for Ca2+ signalling. Mitchell made this conclusion from pharmacological observations in which he observed that both cAMP and Ca2+ both seemed to function as second messengers, but receptors that seemed to use Ca2+ also showed the IP response. This observation would then later go on to heavily impact the direction of Michael J. Berridge's research on the role of calcium in cells. In 1980, Yasutomi Nishizuka, who in 1977 discovered protein kinase C (PKC), published a paper showing that DAG, the other product of the IP effect, functions as a second messenger and activates PKC.

Finally, in 1983, 30 years after Hokin and Hokin first suggested that phospholipids might play an important role in cellular functioning, Michael J. Berridge showed that IP3 was in fact a second messenger and played a pivotal role in cellular signaling. Berridge and his colleagues worked on permeabilized pancreatic acinar cells and, through a series of experiments and controls, they were able to establish that IP3 indeed mobilizes calcium from the endoplasmic reticulum.

Phospholipase C-β Activation via G Protein Coupled Receptors
The IP3 pathway can be quite varied; the following is a summary of the major steps involved in a commonly found IP3 pathway that is coupled to G-protein coupled receptors and results in the release of Ca2+ from intracellular stores (e.g. smooth ER, sarcoplasmic reticulum).

1. The extracellular signaling agent (ligand) binds to a GPCR, activating the heterotrimeric G protein.

2. The αq subunit of the q family of G proteins activates the effector PI-PLCβ.

3. Carbon-4 of PI becomes phosphorylated by PI4K, producing PIP.

4. Carbon-5 of PIP becomes phosphorylated by PIP5K, producing PIP2.

5. PI-PLCβ catalyzes the hydrolysis of PIP2 into DAG and IP3.

6. DAG remains membrane bound, which recruits and activates PKC. PKC then activates the ERK ½ (extracellular Signal Regulated Kinase – 1/2 )  pathway, which promotes cell survival and transcription factor activation.

7. IP3, formed by the activation of the PI-PLCβ, departs from the inner-membrane and diffuses into the cytosol

8. IP3 binds to the specific IP3R located on the smooth ER. The IP3R contains 3 major domains: the inositol triphosphate binding domain located near the N terminus (which is responsible for the isoform-specific IP3 binding affinity); a coupling domain in the middle; and a transmembrane spanning domain located near the C terminus. The IP3R‘s tetramer structure allows it to function as a ligand-gated channel that regulates the release of Ca2+ from the endoplasmic reticulum.

9. Binding of the IP3 to the IP3R allosterically and dynamically changes the form of the receptor, opening the Ca2+ channel. This activates the release of Ca2+ from stores within the ER to the cytoplasm. This results in waves of cytoplasmic Ca2+ (Ca2+ induced Ca2+ release) released throughout the cell, which, in turn, acts as second messenger and activates calmodulin and other Ca2+ binding proteins.

Phospholipase C-γ Activation via Receptor Tyrosine Kinases
In addition, PLCγ can also be activated indirectly by receptor tyrosine kinases (RTK). This is caused by the downstream activation of Shp-2 from RTK. This leads Shp-2 to dephosphorylate binding sites of the protein that maintain the inhibitory C-terminus Src kinase’s (CSK) colocalization with a Src family protein. This results in the activation of Src which, in turn, activates PLC. The pathway continues as described above with the phospholipase cleaving PIP2 into IP3 and DAG

Activation
The production of IP3/DAG is modulated by the activity of the isozymes of phosphatidylinositol-specific phospholipase C. Therefore, the pathway is susceptible to change with alterations in PLC activity. PLC can be influenced directly by molecules that target its activity specifically (i.e. binding directly to PLC) or it can by affected indirectly by molecules that influence the activities of receptor complexes upstream from PLC. Such upstream regulation may include receptor tyrosine kinases (RTK) as well as GPCR coupled to Gq.

Inactivation
The down-regulation of IP3 may also occur via several routes. IP3 specific enzymes may catalyze the phosphorylation or dephosphorylation of IP3 through direct interaction with IP3. In addition, there are also indirect mechanisms of inactivation wherein further increases in the amount of IP3 are blocked by regulators of PLC.

Role in Muscle Function and Tissue Repair
Since the IP3 pathway is concerned with the release of Ca2+ from intracellular stores, it serves several vital functions in various regions of the human body. Muscle fibres primarily utilize calcium for contraction and are dependent on extracellular and intracellular calcium stores. Smooth and cardiac muscle are both rich with IP3 receptors, which facilitate slow waves that build up to spikes and a subsequent action potential. Hence, manipulation of IP3 in this regard can have effects on myocytes and cardiac muscle irregularities. In skeletal muscle, calcium promotes contraction through the ryanodine receptor (RyR) and dihydropyridine receptors (DHPR). Accordingly, irregularities of these muscle fibres as well as Purkinje fibers (as reflected in their role in heart rhythms) have been linked to abnormally functioning IP3 pathways.

In addition, tissue repair, chiefly cardiac myogenesis initiation, has also been suggested to be related to Ca2+ level abnormalities. Glycosaminoglycans (GAG) have been shown to play a significant role in the regulation of growth factors and cell differentiation. Synthetic GAGs like RGTAs are strongly believed to play a role in skeletal muscle regeneration after damage. More specifically, OTR4120 (a synthetic GAG), interacts with basic Fibroblast Growth Factor (bFGF) thereby facilitating myoblast differentiation into myotubes. Moreover, through in vitro studies, GAGs were shown to induce the release of intracellular calcium concentration in SolD7 myoblasts. GAG mimitec OTR4120 (artificially constructed GAG) brought about a marked increase in free cytosolic Ca2+ concentration in pre-fusing myoblasts, which can lead to a reduction in apoptosis of actively differentiating myoblasts or fibroblasts. This development has raised questions about the manipulation of the IP3 pathway to facilitate muscle repair, and to reduce oxidative stress. As a result, cell-based therapeutics of muscle degeneration disorders could perhaps take advantage of the myoblasts influenced by these GAG mimitecs, which themselves manipulate the IP3 mechanism for calcium release. Generally, most mechanisms that induce an increase in in intracellular Ca2+ concentration are expected to initiate myoblast differentiation. More specifically, OTR4120 has been applied to pre-fusing myoblasts, which themselves result in an increase in intracellular calcium concentrations via the IP3 pathway.

Furthermore, studies of IP3R1 deficiencies at the parallel fiber (PF) Purkinje cell synapse in the cerebellum, which is responsible for motor learning, indicate an absence of long term depression (LTD) in the cerebellum. These results were further confirmed via function blocking antibodies against IP3 R1 which were also shown to inhibit LTD.

Importance in Oxidative Stress and Alzheimer's Disease
Both Alzheimer’s and Parkinson’s diseases exhibit oxidative damage in neurons, which have led scientists to believe they are a causal factor of aging. More specifically, in a study involving Drosophila melanogaster, overexpression of Drosophila inositol triphosphate kinase I (D-IP3 RI), which is homologous to the IP3 kinase in mammals, has led to the argument that the overexpression of the D-IP3 R1 can lead to reduced oxidative damage. The chief agent of this improvement is the reduction of the IP3 level, which leads to a reduction in Ca2+ release from the intracellular stores, thereby minimzing the ill-effects brought about by the presence of reactive oxygen species (ROS) such as H2O2, which increase intracellular free calcium concentrations. Moreover, the defense mechanisms against ROSs, such as the superoxide dismutase (SOD) and catalase (CAT) enzymes, have been shown to be independent of the IP3 -mediated reduction in oxidative damage; hence, the modulation of calcium has been shown to have promising results in preventing oxidative stress. From a clinical perspective, this manipulation of the IP3 -dependent calcium release via the use of homolog kinases in humans could possibly address ailments in which oxidative damage are prevalent, most particularly in Alzheimer’s disease.

Recent studies involving Alzheimer’s disease (AD) have identified erroneous calcium signalling as having a profound relation to said pathology. Normally, intracellular calcium signals are coupled to effectors in a delicate balance that ensure a healthy physiological state. In Alzheimer’s disease, however, alterations of intracellular calcium levels (thought to have been caused by the irregular functioning of the IP3 pathway) typically result in a variety of symptoms, including beta amyloid plaques, hyperphosphorylation of tau, cell death and synaptic dysfunction. These manifestations of AD have been shown to be greatly influenced by improper calcium signalling brought about by errors in the calcium release mechanism.

A primary root cause of this excess release of Ca2+ can be attributed to the Presenilin proteins. Studies have shown that Alzheimer afflicted cells possess a mutation in PS1 (Presenilin 1) and PS2 (Presenilin 2) cells. Mutation in these cells has been strongly linked to the up-regulation of RyR, in addition to the prolonged activation of the IP3 evoked calcium response. This relation was strongly depicted in a study done with mice possessing mutant PS1 and excessive calcium release. The excess of calcium was attributed to the RyR, as blocking these receptors with dantrolene or ryanodine brought the IP3 –evoked Ca2+ response back down to normal levels. It has also been demonstrated via clinical studies that the mutation of PS1 and PS2 can lead to exaggerated Ca2+ release, and said mutation can be identified in a much abbreviated or timely manner when compared to the typical histological markers and symptomatic cognitive decline most commonly attributed with Alzheimer's.

Hence, while low levels of Ca2+ can cause impaired functioning, an excess of cytosolic Ca2+ are correlated with apoptosis and cell death as previously discussed. These results are further supported by the demonstration of excessive Ca2+ release from fibroblasts obtained from patients suffering from Alzheimer’s disease in which the IP3 receptors were stimulated.

Studies utilizing whole-cell patch clamp recording and real-time Ca2+ imaging in the brain involving mutant incidences of PS1 have quantified a three-fold increase in the IP3 dependent release of calcium from the ER. More recently, studies have introduced the use of triple transgenic mice, or mice experiencing mutations in their PS1, APP and tau genes. These three genes are primarily concerned with the manifestation and development of Alzheimer’s; mutant APP has been shown to be responsible for the beta amyloid plaques, while the hyperphosphorylation of tau has led to the incidences of neurofibrillary tangles.

Importance in Other Neurological Disorders
Altered calcium homeostasis has also been linked to depression, affective disorders, epilepsy in addition to Alzheimer’s disease.

A significant effect of abnormal IP3 functioning is exhibited in epileptic seizures observed in test mice who possessed a significant absence of IP3 R1. In a healthy cell, CICR arising from the activation of IP3 receptors and RyR is terminated when a threshold level of cytosolic Ca2+ is reached. Neurons experiencing the effect of neurodegeneration generally lack this termination mechanism ,and an abnormally high level of Ca2+ is found in these cells. Furthermore, excess amounts of cytochrome C were also shown to bind to IP3 receptors on the ER membrane. As a result, calcium release was stimulated from the ER, while the IP3R inhibition mechanism initiated in a high concentration of cytosolic Ca2+ was simultaneously blocked. To further exacerbate this excessive Ca2+ release, cytochrome C is further reinforced by the release of calcium. Since greater and greater levels of Ca2+ are inevitably reached due to the positive feedback loop of cytochrome C (and the subsequent sustained stimulation of IP3), extremely high levels of Ca2+ are pooled in the cytosol, bringing about cell death and synaptic dysfunction.

With respect to microglia activity after trauma or brain injury, studies have suggested the role of Tumor Necrosis Factor (TNF) in manipulating the release of Ca2+. TNF is prone to stimulation by nicotine, which acts upon the α7 nAChRs of microglia; these receptors are, in turn, coupled to the activation of PLC and Ca2+ from IP3 modulated ER calcium reserves. The role of TNF (and subsequent manipulation of the IP3 ) has been shown to occur in Alzheimer’s and Parkinson’s disease with neuronal inflammation and the mass activation of microglia.

Bipolar affective disorder (BPAD) has also shown to have strong links with malfunctioning calcium release; calcium channel blockers such as verapamil and nimodipine have been used to treat patients suffering from BPAD and depression. Several antipsychotic drugs, including clozapine, fluspiriline and haloperidol have been shown to be directly related to the impediment of the IP3 induced calcium release in a dose-dependent fashion. These drugs typically target the pre-synaptic/post-synaptic specializations in neurons. Historically, the treatment of manic depression has involved the use of the above drugs, in addition to other calcium channel blockers which have manipulated the IP3 pathway. In terms of the treatment of bipolar disorder, errors in IP3 -dependent calcium regulation have been shown to be responsible for abnormally high levels of Ca2+ in the platelets and lymphocytes of bipolar patients.

In addition, altered serotonin concentrations have been found in the brains of patients suffering from affective disorder and those exhibiting suicidal behaviour. These serotonin receptors are related to the phosphoinositide system, responsible for the release of IP3 and DAG. Numerous studies have demonstrated altered phosphoinositide signalling systems as one of the causal factors in neuropsychological disorders such as schizophrenia and affective disorders. Such alterations can include a drastic increase in the formation of protein kinase C in platelets in the brain and in exceptionally high levels of IP3 receptor proteins and IP3 binding sites.