The hypermorphic PLCγ2 S707Y variant dysregulates microglial cell function - Insight into PLCγ2 activation in brain health and disease, and opportunities for therapeutic modulation.

Phospholipase C-gamma 2 (PLCγ2) is highly expressed in hematopoietic and immune cells, where it is a key signalling node enabling diverse cellular functions. Within the periphery, gain-of-function (GOF) PLCγ2 variants, such as the strongly hypermorphic S707Y, cause severe immune dysregulation. The milder hypermorphic mutation PLCγ2 P522R increases longevity and confers protection in central nervous system (CNS) neurodegenerative disorders, implicating PLCγ2 as a novel therapeutic target for treating these CNS indications. Currently, nothing is known about what consequences strong PLCγ2 GOF has on CNS functionality, and more precisely on the specific biological functions of microglia. Using the PLCγ2 S707Y variant as a model of chronic activation we investigated the functional consequences of strong PLCγ2 GOF on human microglia. PLCγ2 S707Y expressing human inducible pluripotent stem cells (hiPSC)-derived microglia exhibited hypermorphic enzymatic activity under both basal and stimulated conditions, compared to PLCγ2 wild type. Despite the increase in PLCγ2 enzymatic activity, the PLCγ2 S707Y hiPSC-derived microglia display diminished functionality for key microglial processes including phagocytosis and cytokine secretion upon inflammatory challenge. RNA sequencing revealed a downregulation of genes related to innate immunity and response, providing molecular support for the phenotype observed. Our data suggests that chronic activation of PLCγ2 elicits a detrimental phenotype that is contributing to unfavourable CNS functions, and informs on the therapeutic window for targeting PLCγ2 in the CNS. Drug candidates targeting PLCγ2 will need to precisely mimic the effects of the PLCγ2 P522R variant on microglial function, but not those of the PLCγ2 S707Y variant.

PLCG2-associated immune dysregulation (PLAID) comprises broad and distinct clinical presentations related to functional classes of genetic variants.

BACKGROUND: Pathogenic variants of phospholipase C gamma 2 (PLCG2) cause 2 related forms of autosomal-dominant immune dysregulation (ID), PLCγ2-associated antibody deficiency and immune dysregulation (PLAID) and autoinflammatory PLAID (APLAID). Since describing these conditions, many PLCG2 variants of uncertain significance have been identified by clinical sequencing of patients with diverse features of ID. OBJECTIVE: We sought to functionally classify PLCG2 variants and explore known and novel genotype-function-phenotype relationships. METHODS: Clinical data from patients with PLCG2 variants were obtained via standardized questionnaire. PLCG2 variants were generated by mutagenesis of enhanced green fluorescent protein (EGFP)-PLCG2 plasmid, which was overexpressed in Plcg2-deficient DT-40 B cells. B-cell receptor-induced calcium flux and extracellular signal-regulated kinase phosphorylation were assayed by flow cytometry. In some cases, stimulation-induced calcium flux was also measured in primary patient cells. RESULTS: Three-fourths of PLCG2 variants produced functional alteration of B-cell activation, in vitro. Thirteen variants led to gain of function (GOF); however, most functional variants defined a new class of PLCG2 mutation, monoallelic loss of function (LOF). Susceptibility to infection and autoinflammation were common with both GOF and LOF variants, whereas a new phenotypic cluster consisting of humoral immune deficiency, autoinflammation, susceptibility to herpesvirus infection, and natural killer cell dysfunction was observed in association with multiple heterozygous LOF variants detected in both familial and sporadic cases. In some cases, PLCG2 variants produced greater effects in natural killer cells than in B cells. CONCLUSIONS: This work expands the genotypic and phenotypic associations with functional variation in PLCG2, including a novel form of ID in carriers of heterozygous loss of PLCG2 function. It also demonstrates the need for more diverse assays for assessing the impact of PLCG2 variants on human disease.

Human PLCG2 haploinsufficiency results in a novel natural killer cell immunodeficiency.

BACKGROUND: Although most individuals effectively control herpesvirus infections, some suffer from severe and/or recurrent infections. A subset of these patients possess defects in natural killer (NK) cells, lymphocytes that recognize and lyse herpesvirus-infected cells; however, the genetic etiology is rarely diagnosed. PLCG2 encodes a signaling protein in NK-cell and B-cell signaling. Dominant-negative or gain-of-function variants in PLCG2 cause cold urticaria, antibody deficiency, and autoinflammation. However, loss-of-function variants and haploinsufficiency have not been reported to date. OBJECTIVES: The investigators aimed to identify the genetic cause of NK-cell immunodeficiency in 2 families and herein describe the functional consequences of 2 novel loss-of-function variants in PLCG2. METHODS: The investigators employed whole-exome sequencing in conjunction with mass cytometry, microscopy, functional assays, and a mouse model of PLCG2 haploinsufficiency to investigate 2 families with NK-cell immunodeficiency. RESULTS: The investigators identified novel heterozygous variants in PLCG2 in 2 families with severe and/or recurrent herpesvirus infections. In vitro studies demonstrated that these variants were loss of function due to haploinsufficiency with impaired NK-cell calcium flux and cytotoxicity. In contrast to previous PLCG2 variants, B-cell function remained intact. Plcg2+/- mice also displayed impaired NK-cell function with preserved B-cell function, phenocopying human disease. CONCLUSIONS: PLCG2 haploinsufficiency represents a distinct syndrome from previous variants characterized by NK-cell immunodeficiency with herpesvirus susceptibility, expanding the spectrum of PLCG2-related disease.

Scaffold protein SH3BP2 signalosome is pivotal for immune activation in nephrotic syndrome.

Despite clinical use of immunosuppressive agents, the immunopathogenesis of minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS) remains unclear. Src homology 3-binding protein 2 (SH3BP2), a scaffold protein, forms an immune signaling complex (signalosome) with 17 other proteins, including phospholipase Cγ2 (PLCγ2) and Rho-guanine nucleotide exchange factor VAV2 (VAV2). Bioinformatic analysis of human glomerular transcriptome (Nephrotic Syndrome Study Network cohort) revealed upregulated SH3BP2 in MCD and FSGS. The SH3BP2 signalosome score and downstream MyD88, TRIF, and NFATc1 were significantly upregulated in MCD and FSGS. Immune pathway activation scores for Toll-like receptors, cytokine-cytokine receptor, and NOD-like receptors were increased in FSGS. Lower SH3BP2 signalosome score was associated with MCD, higher estimated glomerular filtration rate, and remission. Further work using Sh3bp2KI/KI transgenic mice with a gain-in-function mutation showed ~6-fold and ~25-fold increases in albuminuria at 4 and 12 weeks, respectively. Decreased serum albumin and unchanged serum creatinine were observed at 12 weeks. Sh3bp2KI/KI kidney morphology appeared normal except for increased mesangial cellularity and patchy foot process fusion without electron-dense deposits. SH3BP2 co-immunoprecipitated with PLCγ2 and VAV2 in human podocytes, underscoring the importance of SH3BP2 in immune activation. SH3BP2 and its binding partners may determine the immune activation pathways resulting in podocyte injury leading to loss of the glomerular filtration barrier.

Plaque attack by microglial PLCγ2.

PLCγ2 is genetically linked to Alzheimer's disease (AD), but it is unclear how PLCγ2 contributes to pathology. Tsai et al. demonstrate that AD-associated PLCG2 variants bidirectionally orchestrate microglial responses to plaques and impact neural function in an AD mouse model. This positions PLCγ2 as a key microglial signaling node and shows that targeting PLCγ2 could have therapeutic benefits in AD.

Endothelial VEGFR2-PLCγ signaling regulates vascular permeability and antitumor immunity through eNOS/Src.

Endothelial phospholipase Cγ (PLCγ) is essential for vascular development; however, its role in healthy, mature, or pathological vessels is unexplored. Here, we show that PLCγ was prominently expressed in vessels of several human cancer forms, notably in renal cell carcinoma (RCC). High PLCγ expression in clear cell RCC correlated with angiogenic activity and poor prognosis, while low expression correlated with immune cell activation. PLCγ was induced downstream of vascular endothelial growth factor receptor 2 (VEGFR2) phosphosite Y1173 (pY1173). Heterozygous Vegfr2Y1173F/+ mice or mice lacking endothelial PLCγ (Plcg1iECKO) exhibited a stabilized endothelial barrier and diminished vascular leakage. Barrier stabilization was accompanied by decreased expression of immunosuppressive cytokines, reduced infiltration of B cells, helper T cells and regulatory T cells, and improved response to chemo- and immunotherapy. Mechanistically, pY1173/PLCγ signaling induced Ca2+/protein kinase C-dependent activation of endothelial nitric oxide synthase (eNOS), required for tyrosine nitration and activation of Src. Src-induced phosphorylation of VE-cadherin at Y685 was accompanied by disintegration of endothelial junctions. This pY1173/PLCγ/eNOS/Src pathway was detected in both healthy and tumor vessels in Vegfr2Y1173F/+ mice, which displayed decreased activation of PLCγ and eNOS and suppressed vascular leakage. Thus, we believe that we have identified a clinically relevant endothelial PLCγ pathway downstream of VEGFR2 pY1173, which destabilizes the endothelial barrier and results in loss of antitumor immunity.

A gain-of-function variation in PLCG1 causes a new immune dysregulation disease.

BACKGROUND: Phospholipase C (PLC) γ1 is a critical enzyme regulating nuclear factor-κB (NF-κB), extracellular signal-related kinase, mitogen-activated protein kinase, and nuclear factor of activated T cells signaling pathways, yet germline PLCG1 mutation in human disease has not been reported. OBJECTIVE: We aimed to investigate the molecular pathogenesis of a PLCG1 activating variant in a patient with immune dysregulation. METHODS: Whole exome sequencing was used to identify the patient's pathogenic variants. Bulk RNA sequencing, single-cell RNA sequencing, quantitative PCR, cytometry by time of flight, immunoblotting, flow cytometry, luciferase assay, IP-One ELISA, calcium flux assay, and cytokine measurements in patient PBMCs and T cells and COS-7 and Jurkat cell lines were used to define inflammatory signatures and assess the impact of the PLCG1 variant on protein function and immune signaling. RESULTS: We identified a novel and de novo heterozygous PLCG1 variant, p.S1021F, in a patient presenting with early-onset immune dysregulation disease. We demonstrated that the S1021F variant is a gain-of-function variant, leading to increased inositol-1,4,5-trisphosphate production, intracellular Ca2+ release, and increased phosphorylation of extracellular signal-related kinase, p65, and p38. The transcriptome and protein expression at the single-cell level revealed exacerbated inflammatory responses in the patient's T cells and monocytes. The PLCG1 activating variant resulted in enhanced NF-κB and type II interferon pathways in T cells, and hyperactivated NF-κB and type I interferon pathways in monocytes. Treatment with either PLCγ1 inhibitor or Janus kinase inhibitor reversed the upregulated gene expression profile in vitro. CONCLUSIONS: Our study highlights the critical role of PLCγ1 in maintaining immune homeostasis. We illustrate immune dysregulation as a consequence of PLCγ1 activation and provide insight into therapeutic targeting of PLCγ1.

Carriers of the p.P522R variant in PLCγ2 have a slightly more responsive immune system.

BACKGROUND: The rs72824905 single-nucleotide polymorphism in the PLCG2 gene, encoding the p.P522R residue change in Phospholipase C gamma 2 (PLCγ2), associates with protection against several dementia subtypes and with increased likelihood of longevity. Cell lines and animal models indicated that p.P522R is a functional hypermorph. We aimed to confirm this in human circulating peripheral immune cells. METHODS: We compared effects of p.P522R on immune system function between carriers and non-carriers (aged 59-103y), using in-depth immunophenotyping, functional B-cell and myeloid cell assays, and in vivo SARS-CoV-2 vaccination. RESULTS: In line with expectations, p.P522R impacts immune cell function only slightly, but it does so across a wide array of immune cell types. Upon B-cell stimulation, we observed increased PLCγ2 phosphorylation and calcium release, suggesting increased B-cell sensitivity upon antigen recognition. Further, p.P522R-carriers had higher numbers of CD20++CD21-CD24+ naive B cells and IgG1+ memory B cells. In myeloid cells, normalized ROS production was higher upon PLCγ2-dependent stimulation. On classical monocytes, CD33 levels were elevated. Furthermore, carriers expressed lower levels of allergy-related FcεRI on several immune cell subsets. Nevertheless, carriers and non-carriers had similar serological responses to SARS-CoV-2 vaccination. CONCLUSION: The immune system from p.P522R-carriers is slightly more responsive to stimulation than in non-carriers.

A novel fluorogenic reporter substrate for 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase gamma-2 (PLCγ2): Application to high-throughput screening for activators to treat Alzheimer's disease.

A rare coding variant in PLCγ2 (P522R) expressed in microglia induces a mild activation of enzymatic activity when compared to wild-type. This mutation is reported to be protective against the cognitive decline associated with late-onset Alzheimer's disease (LOAD) and therefore, activation of wild-type PLCγ2 has been suggested as a potential therapeutic target for the prevention and treatment of LOAD. Additionally, PLCγ2 has been associated with other diseases such as cancer and some autoimmune disorders where mutations with much greater increases in PLCγ2 activity have been identified. Here, pharmacological inhibition may provide a therapeutic effect. In order to facilitate our investigation of the activity of PLCγ2, we developed an optimized fluorogenic substrate to monitor enzymatic activity in aqueous solution. This was accomplished by first exploring the spectral properties of various "turn-on" fluorophores. The most promising turn-on fluorophore was incorporated into a water-soluble PLCγ2 reporter substrate, which we named C8CF3-coumarin. The ability of PLCγ2 to enzymatically process C8CF3-coumarin was confirmed, and the kinetics of the reaction were determined. Reaction conditions were optimized to identify small molecule activators, and a pilot screen of the Library of Pharmacologically Active Compounds 1280 (LOPAC1280) was performed with the goal of identifying small molecule activators of PLCγ2. The optimized screening conditions allowed identification of potential PLCγ2 activators and inhibitors, thus demonstrating the feasibility of this approach for high-throughput screening.

Clinical and immunological features of an APLAID patient caused by a novel mutation in PLCG2.

Background: The APLAID syndrome is a rare primary immunodeficiency caused by gain-of-function mutations in the PLCG2 gene. We present a 7-year-old APLAID patient who has recurrent blistering skin lesions, skin infections in the perineum, a rectal perineal fistula, and inflammatory bowel disease. Methods: To determine the genetic cause of our patient, WES and bioinformatics analysis were performed. Flow cytometry was used for phenotyping immune cell populations in peripheral blood. Cytokines released into plasma were analyzed using protein chip technology. The PBMCs of patient and a healthy child were subjected to single-cell RNA-sequencing analysis. Results: The patient carried a novel de novo missense mutation c.2534T>C in exon 24 of the PLCG2 gene that causes a leucine to serine amino acid substitution (p.Leu845Ser). Bioinformatics analysis revealed that this mutation had a negative impact on the structure of the PLCγ2 protein, which is highly conserved in many other species. Immunophenotyping by flow cytometry revealed that in addition to the typical decrease in circulating memory B cells, the levels of myeloid dendritic cells (mDCs) in the children's peripheral blood were significantly lower, as were the CD4+ effector T cells induced by their activation. Single-cell sequencing revealed that the proportion of different types of cells in the peripheral blood of the APLAID patient changed. Conclusions: We present the first case of APLAID with severely reduced myeloid dendritic cells carrying a novel PLCG2 mutation, and conducted a comprehensive analysis of immunological features in the ALPAID patient, which has not been mentioned in previous reports. This study expands the spectrum of APLAID-associated immunophenotype and genotype. The detailed immune analyses in this patient may provide a basis for the development of targeted therapies for this severe autoinflammatory disease.

A frequent PLCγ1 mutation in adult T-cell leukemia/lymphoma determines functional properties of the malignant cells.

BACKGROUND: Development of adult T-cell leukemia/lymphoma (ATL) involves human T-cell leukemia virus type 1 (HTLV-1) infection and accumulation of somatic mutations. The most frequently mutated gene in ATL (36 % of cases) is phospholipase C gamma1 (PLCG1). PLCG1 is also frequently mutated in other T-cell lymphomas. However, the functional consequences of the PLCG1 mutations in cancer cells have not been characterized. METHODS: We compared the activity of the wild-type PLCγ1 with that of a mutant carrying a hot-spot mutation of PLCγ1 (S345F) observed in ATL, both in cells and in cell-free assays. To analyse the impact of the mutation on cellular properties, we quantified cellular proliferation, aggregation, chemotaxis and apoptosis by live cell-imaging in an S345F+ ATL-derived cell line (KK1) and a KK1 cell line in which we reverted the mutation to the wild-type sequence using CRISPR/Cas9 and homology-directed repair. FINDINGS: The PLCγ1 S345F mutation results in an increase of basal PLC activity in vitro and in different cell types. This higher basal activity is further enhanced by upstream signalling. Reversion of the S345F mutation in the KK1 cell line resulted in reduction of the PLC activity, lower rates of proliferation and aggregation, and a marked reduction in chemotaxis towards CCL22. The PLCγ1-pathway inhibitors ibrutinib and ritonavir reduced both the PLC activity and the tested functions of KK1 cells. INTERPRETATION: Consistent with observations from clinical studies, our data provide direct evidence that activated variants of the PLCγ1 enzyme contribute to the properties of the malignant T-cell clone in ATL. FUNDING: MRC (UK) Project Grant (P028160).

Loss of phospholipase Cγ1 suppresses hepatocellular carcinogenesis through blockade of STAT3-mediated cancer development.

Phospholipase C gamma 1 (PLCγ1) plays an oncogenic role in several cancers, alongside its usual physiological roles. Despite studies aimed at identifying the effect of PLCγ1 on tumors, the pathogenic role of PLCγ1 in the tumorigenesis and development of hepatocellular carcinoma (HCC) remains unknown. To investigate the function of PLCγ1 in HCC, we generated hepatocyte-specific PLCγ1 conditional knockout (PLCγ1f/f ; Alb-Cre) mice and induced HCC with diethylnitrosamine (DEN). Here, we identified that hepatocyte-specific PLCγ1 deletion effectively prevented DEN-induced HCC in mice. PLCγ1f/f ; Alb-Cre mice showed reduced tumor burden and tumor progression, as well as a decreased incidence of HCC and less marked proliferative and inflammatory responses. We also showed that oncogenic phenotypes such as repressed apoptosis, and promoted proliferation, cell cycle progression and migration, were induced by PLCγ1. In terms of molecular mechanism, PLCγ1 regulated the activation of signal transducer and activator of transcription 3 (STAT3) signaling. Moreover, PLCγ1 expression is elevated in human HCC and correlates with a poor prognosis in patients with HCC. Our results suggest that PLCγ1 promotes the pathogenic progression of HCC, and PLCγ1/STAT3 axis was identified as a potential therapeutic target pathway for HCC.

Methylation-mediated silencing of PTPRD induces pulmonary hypertension by promoting pulmonary arterial smooth muscle cell migration via the PDGFRB/PLCγ1 axis.

OBJECTIVE: Pulmonary hypertension is a lethal disease characterized by pulmonary vascular remodeling and is mediated by abnormal proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs). Platelet-derived growth factor BB (PDGF-BB) is the most potent mitogen for PASMCs and is involved in vascular remodeling in pulmonary hypertension development. Therefore, the objective of our study is to identify novel mechanisms underlying vascular remodeling in pulmonary hypertension. METHODS: We explored the effects and mechanisms of PTPRD downregulation in PASMCs and PTPRD knockdown rats in pulmonary hypertension induced by hypoxia. RESULTS: We demonstrated that PTPRD is dramatically downregulated in PDGF-BB-treated PASMCs, pulmonary arteries from pulmonary hypertension rats, and blood and pulmonary arteries from lung specimens of patients with hypoxic pulmonary arterial hypertension (HPAH) and idiopathic PAH (iPAH). Subsequently, we found that PTPRD was downregulated by promoter methylation via DNMT1. Moreover, we found that PTPRD knockdown altered cell morphology and migration in PASMCs via modulating focal adhesion and cell cytoskeleton. We have demonstrated that the increase in cell migration is mediated by the PDGFRB/PLCγ1 pathway. Furthermore, under hypoxic condition, we observed significant pulmonary arterial remodeling and exacerbation of pulmonary hypertension in heterozygous PTPRD knock-out rats compared with the wild-type group. We also demonstrated that HET group treated with chronic hypoxia have higher expression and activity of PLCγ1 in the pulmonary arteries compared with wild-type group. CONCLUSION: We propose that PTPRD likely plays an important role in the process of pulmonary vascular remodeling and development of pulmonary hypertension in vivo .

Heterozygous expression of the Alzheimer's disease-protective PLCγ2 P522R variant enhances Aß clearance while preserving synapses.

BACKGROUND: A rare coding variant, P522R, in the phospholipase C gamma 2 (PLCG2) gene has been identified as protective against late-onset Alzheimer's disease (AD), but the mechanism is unknown. PLCG2 is exclusively expressed in microglia within the central nervous system, and altered microglial function has been implicated in the progression of AD. METHODS: Healthy control hiPSCs were CRISPR edited to generate cells heterozygous and homozygous for the PLCG2P522R variant. Microglia derived from these hiPSC's were used to investigate the impact of PLCγ2P522R on disease relevant processes, specifically microglial capacity to take up amyloid beta (Aß) and synapses. Targeted qPCR assessment was conducted to explore expression changes in core AD linked and microglial genes, and mitochondrial function was assessed using an Agilent Seahorse assay. RESULTS: Heterozygous expression of the P522R variant resulted in increased microglial clearance of Aß, while preserving synapses. This was associated with the upregulation of a number of genes, including the anti-inflammatory cytokine Il-10, and the synapse-linked CX3CR1, as well as alterations in mitochondrial function, and increased cellular motility. The protective capacity of PLCγ2P522R appeared crucially dependent on (gene) 'dose', as cells homozygous for the variant showed reduced synapse preservation, and a differential gene expression profile relative to heterozygous cells. CONCLUSION: These findings suggest that PLCγ2P522R may result in increased surveillance by microglia, and prime them towards an anti-inflammatory state, with an increased capacity to respond to increasing energy demands, but highlights the delicate balance of this system, with increasing PLCγ2P522R 'dose' resulting in reduced beneficial impacts.

Kinase-deficient BTK mutants confer ibrutinib resistance through activation of the kinase HCK.

The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib irreversibly binds BTK at Cys481, inhibiting its kinase activity and thus blocking transduction of B cell receptor (BCR) signaling. Although ibrutinib is durably effective in patients with B cell malignancies, many patients still develop ibrutinib-resistant disease. Resistance can arise because of mutations at the ibrutinib-binding site in BTK. Here, we characterized the mechanism by which two BTK mutations, C481F and C481Y, may lead to ibrutinib resistance. Both mutants lacked detectable kinase activity in in vitro kinase assays. Structural modeling suggested that bulky Phe and Tyr side chains at position 481 sterically hinder access to the ATP-binding pocket in BTK, contributing to loss of kinase activity. Nonetheless, BCR signaling still propagated through BTK C481F and C481Y mutants to downstream effectors, the phospholipase PLCγ2 and the transcription factor NF-κB. This maintenance of BCR signaling was partially achieved through the physical recruitment and kinase-independent activation of hematopoietic cell kinase (HCK). Upon BCR activation, BTK C481F or C481Y was phosphorylated by Src family kinases at Tyr551, which then bound to the SH2 domain of HCK. Modeling suggested that this binding disrupted an intramolecular autoinhibitory interaction in HCK. Activated HCK subsequently phosphorylated PLCγ2, which propagated BCR signaling and promoted clonogenic cell proliferation. This kinase-independent mechanism could inform therapeutic approaches to CLL bearing either the C481F or C481Y BTK mutants.

A kinetic model of phospholipase C-γ1 linking structure-based insights to dynamics of enzyme autoinhibition and activation.

Phospholipase C-γ1 (PLC-γ1) is a receptor-proximal enzyme that promotes signal transduction through PKC in mammalian cells. Because of the complexity of PLC-γ1 regulation, a two-state (inactive/active) model does not account for the intricacy of activation and inactivation steps at the plasma membrane. Here, we introduce a structure-based kinetic model of PLC-γ1, considering interactions of its regulatory Src homology 2 (SH2) domains and perturbation of those dynamics upon phosphorylation of Tyr783, a hallmark of activation. For PLC-γ1 phosphorylation to dramatically enhance enzyme activation as observed, we found that high intramolecular affinity of the C-terminal SH2 (cSH2) domain-pTyr783 interaction is critical, but this affinity need not outcompete the autoinhibitory interaction of the cSH2 domain. Under conditions for which steady-state PLC-γ1 activity is sensitive to the rate of Tyr783 phosphorylation, maintenance of the active state is surprisingly insensitive to the phosphorylation rate, since pTyr783 is well protected by the cSH2 domain while the enzyme is active. In contrast, maintenance of enzyme activity is sensitive to the rate of PLC-γ1 membrane (re)binding. Accordingly, we found that hypothetical PLC-γ1 mutations that either weaken autoinhibition or strengthen membrane binding influence the activation kinetics differently, which could inform the characterization of oncogenic variants. Finally, we used this newly informed kinetic scheme to refine a spatial model of PLC/PKC polarization during chemotaxis. The refined model showed improved stability of the polarized pattern while corroborating previous qualitative predictions. As demonstrated here for PLC-γ1, this approach may be adapted to model the dynamics of other receptor- and membrane-proximal enzymes.

Structural analysis of TrkA mutations in patients with congenital insensitivity to pain reveals PLCγ as an analgesic drug target.

Chronic pain is a major health issue, and the search for new analgesics has become increasingly important because of the addictive properties and unwanted side effects of opioids. To explore potentially new drug targets, we investigated mutations in the NTRK1 gene found in individuals with congenital insensitivity to pain with anhidrosis (CIPA). NTRK1 encodes tropomyosin receptor kinase A (TrkA), the receptor for nerve growth factor (NGF) and that contributes to nociception. Molecular modeling and biochemical analysis identified mutations that decreased the interaction between TrkA and one of its substrates and signaling effectors, phospholipase Cγ (PLCγ). We developed a cell-permeable phosphopeptide derived from TrkA (TAT-pQYP) that bound the Src homology domain 2 (SH2) of PLCγ. In HEK-293T cells, TAT-pQYP inhibited the binding of heterologously expressed TrkA to PLCγ and decreased NGF-induced, TrkA-mediated PLCγ activation and signaling. In mice, intraplantar administration of TAT-pQYP decreased mechanical sensitivity in an inflammatory pain model, suggesting that targeting this interaction may be analgesic. The findings demonstrate a strategy to identify new targets for pain relief by analyzing the signaling pathways that are perturbed in CIPA.

Endothelial Phospholipase Cγ2 Improves Outcomes of Diabetic Ischemic Limb Rescue Following VEGF Therapy.

Therapeutic vascular endothelial growth factor (VEGF) replenishment has met with limited success for the management of critical limb-threatening ischemia. To improve outcomes of VEGF therapy, we applied single-cell RNA sequencing (scRNA-seq) technology to study the endothelial cells of the human diabetic skin. Single-cell suspensions were generated from the human skin followed by cDNA preparation using the Chromium Next GEM Single-cell 3' Kit v3.1. Using appropriate quality control measures, 36,487 cells were chosen for downstream analysis. scRNA-seq studies identified that although VEGF signaling was not significantly altered in diabetic versus nondiabetic skin, phospholipase Cγ2 (PLCγ2) was downregulated. The significance of PLCγ2 in VEGF-mediated increase in endothelial cell metabolism and function was assessed in cultured human microvascular endothelial cells. In these cells, VEGF enhanced mitochondrial function, as indicated by elevation in oxygen consumption rate and extracellular acidification rate. The VEGF-dependent increase in cell metabolism was blunted in response to PLCγ2 inhibition. Follow-up rescue studies therefore focused on understanding the significance of VEGF therapy in presence or absence of endothelial PLCγ2 in type 1 (streptozotocin-injected) and type 2 (db/db) diabetic ischemic tissue. Nonviral topical tissue nanotransfection technology (TNT) delivery of CDH5 promoter-driven PLCγ2 open reading frame promoted the rescue of hindlimb ischemia in diabetic mice. Improvement of blood flow was also associated with higher abundance of VWF+/CD31+ and VWF+/SMA+ immunohistochemical staining. TNT-based gene delivery was not associated with tissue edema, a commonly noted complication associated with proangiogenic gene therapies. Taken together, our study demonstrates that TNT-mediated delivery of endothelial PLCγ2, as part of combination gene therapy, is effective in diabetic ischemic limb rescue.

PLCG2 is associated with the inflammatory response and is induced by amyloid plaques in Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is characterized by robust microgliosis and phenotypic changes that accompany disease pathogenesis. Accumulating evidence from genetic studies suggests the importance of phospholipase C γ 2 (PLCG2) in late-onset AD (LOAD) pathophysiology. However, the role of PLCG2 in AD is still poorly understood. METHODS: Using bulk RNA-Seq (N=1249) data from the Accelerating Medicines Partnership-Alzheimer's Disease Consortium (AMP-AD), we investigated whether PLCG2 expression increased in the brains of LOAD patients. We also evaluated the relationship between PLCG2 expression levels, amyloid plaque density, and expression levels of microglia specific markers (AIF1 and TMEM119). Finally, we investigated the longitudinal changes of PLCG2 expression in the 5xFAD mouse model of AD. To further understand the role of PLCG2 in different signaling pathways, differential gene expression and co-expression network analyses were performed using bulk RNA-Seq and microglial single-cell RNA-Seq data. To substantiate the human analyses, we performed differential gene expression analysis on wild-type (WT) and inactivated Plcg2 mice and used immunostaining to determine if the differentially expressed genes/pathways were altered by microglial cell coverage or morphology. RESULTS: We observed significant upregulation of PLCG2 expression in three brain regions of LOAD patients and significant positive correlation of PLCG2 expression with amyloid plaque density. These findings in the human brain were validated in the 5xFAD amyloid mouse model, which showed disease progression-dependent increases in Plcg2 expression associated with amyloid pathology. Of note, increased Plcg2 expression levels in 5xFAD mice were abolished by reducing microglia. Furthermore, using bulk RNA-Seq data, we performed differential expression analysis by comparing cognitively normal older adults (CN) with 75th percentile (high) and 25th percentile (low) PLCG2 gene expression levels to identify pathways related to inflammation and the inflammatory response. The findings in the human brain were validated by differential expression analyses between WT and plcg2 inactivated mice. PLCG2 co-expression network analysis of microglial single-cell RNA-Seq data identified pathways related to the inflammatory response including regulation of I-kappaB/NF-kappa B signaling and response to lipopolysaccharide. CONCLUSIONS: Our results provide further evidence that PLCG2 plays an important role in AD pathophysiology and may be a potential target for microglia-targeted AD therapies.