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.

Designed switch from covalent to non-covalent inhibitors of carboxylesterase Notum activity.

N-Acyl indolines 4 are potent, non-covalent Notum inhibitors developed from a covalent virtual screening hit 2a. The lead compounds were simple to synthesise, achieved excellent potency in a biochemical Notum-OPTS assay and restored Wnt signalling in a cell-based TCF/LEF reporter assay. Multiple high resolution X-ray structures established a common binding mode of these inhibitors with the indoline bound centred in the palmiteolate pocket with key interactions being aromatic stacking and a water mediated hydrogen bond to the oxyanion hole. These N-acyl indolines 4 will be useful tools for use in vitro studies to investigate the role of Notum in disease models, especially when paired with a structurally related covalent inhibitor (e.g. 4w and 2a). Overall, this study highlights the designed switch from covalent to non-covalent Notum inhibitors and so illustrates a complementary approach for hit generation and target inhibition.

A primary rodent triculture model to investigate the role of glia-neuron crosstalk in regulation of neuronal activity.

Neuroinflammation and hyperexcitability have been implicated in the pathogenesis of neurodegenerative disease, and new models are required to investigate the cellular crosstalk involved in these processes. We developed an approach to generate a quantitative and reproducible triculture system that is suitable for pharmacological studies. While primary rat cells were previously grown in a coculture medium formulated to support only neurons and astrocytes, we now optimised a protocol to generate tricultures containing neurons, astrocytes and microglia by culturing in a medium designed to support all three cell types and adding exogenous microglia to cocultures. Immunocytochemistry was used to confirm the intended cell types were present. The percentage of ramified microglia in the tricultures decreases as the number of microglia present increases. Multi-electrode array recordings indicate that microglia in the triculture model suppress neuronal activity in a dose-dependent manner. Neurons in both cocultures and tricultures are responsive to the potassium channel blocker 4-aminopyridine, suggesting that neurons remained viable and functional in the triculture model. Furthermore, suppressed neuronal activity in tricultures correlates with decreased densities of dendritic spines and of the postsynaptic protein Homer1 along dendrites, indicative of a direct or indirect effect of microglia on synapse function. We thus present a functional triculture model, which, due to its more complete cellular composition, is a more relevant model than standard cocultures. The model can be used to probe glia-neuron interactions and subsequently aid the development of assays for drug discovery, using neuronal excitability as a functional endpoint.

Fate mapping reveals mixed embryonic origin and unique developmental codes of mouse forebrain septal neurons.

The septum is a key structure at the core of the forebrain that integrates inputs and relays information to other brain areas to support cognition and behaviours such as feeding and locomotion. Underlying these functions is a rich diversity of neuronal types and an intricate complexity of wiring across and within the septal region. We currently have very little understanding of how septal neuronal diversity emerges during development. Using transgenic mice expressing Cre in different subsets of telencephalic precursors we explored the origins of the three main neuronal types of the septal complex: GABAergic, cholinergic and glutamatergic neurons. We find that septal neurons originate from distinct neuroepithelial domains of the developing septum and are born at different embryonic time points. An exception to this is the GABAergic medial septal Parvalbumin-expressing population which is generated outside the septum from surrounding germinal zones. We identify the transcription factor BSX as being expressed in the developing glutamatergic neuron population. Embryonic elimination of BSX in the septum results in a reduction of septal glutamatergic cell numbers and a consequent deficit in locomotion. Further refinement of septal neuron diversity is needed to understand the multiple roles of septal neurons and their contribution to distinct behaviours.

MTG8 interacts with LHX6 to specify cortical interneuron subtype identity.

Cortical interneurons originating in the embryonic medial ganglionic eminence (MGE) diverge into a range of different subtypes found in the adult mouse cerebral cortex. The mechanisms underlying this divergence and the timing when subtype identity is set up remain unclear. We identify the highly conserved transcriptional co-factor MTG8 as being pivotal in the development of a large subset of MGE cortical interneurons that co-expresses Somatostatin (SST) and Neuropeptide Y (NPY). MTG8 interacts with the pan-MGE transcription factor LHX6 and together the two factors are sufficient to promote expression of critical cortical interneuron subtype identity genes. The SST-NPY cortical interneuron fate is initiated early, well before interneurons migrate into the cortex, demonstrating an early onset specification program. Our findings suggest that transcriptional co-factors and modifiers of generic lineage specification programs may hold the key to the emergence of cortical interneuron heterogeneity from the embryonic telencephalic germinal zones.

Virtual Screening Directly Identifies New Fragment-Sized Inhibitors of Carboxylesterase Notum with Nanomolar Activity.

Notum is a negative regulator of Wnt signaling acting through the hydrolysis of a palmitoleoylate ester, which is required for Wnt activity. Inhibitors of Notum could be of use in diseases where dysfunctional Notum activity is an underlying cause. A docking-based virtual screen (VS) of a large commercial library was used to shortlist 952 compounds for experimental validation as inhibitors of Notum. The VS was successful with 31 compounds having an IC50 < 500 nM. A critical selection process was then applied with two clusters and two singletons (1-4d) selected for hit validation. Optimization of 4d guided by structural biology identified potent inhibitors of Notum activity that restored Wnt/ß-catenin signaling in cell-based models. The [1,2,4]triazolo[4,3-b]pyradizin-3(2H)-one series 4 represent a new chemical class of Notum inhibitors and the first to be discovered by a VS campaign. These results demonstrate the value of VS with well-designed docking models based on X-ray structures.

PLCγ2 regulates TREM2 signalling and integrin-mediated adhesion and migration of human iPSC-derived macrophages.

Human genetic studies have linked rare coding variants in microglial genes, such as TREM2, and more recently PLCG2 to Alzheimer's disease (AD) pathology. The P522R variant in PLCG2 has been shown to confer protection for AD and to result in a subtle increase in enzymatic activity. PLCγ2 is a key component of intracellular signal transduction networks and induces Ca2+ signals downstream of many myeloid cell surface receptors, including TREM2. To explore the relationship between PLCγ2 and TREM2 and the role of PLCγ2 in regulating immune cell function, we generated human induced pluripotent stem cell (iPSC)- derived macrophages from isogenic lines with homozygous PLCG2 knockout (Ko). Stimulating TREM2 signalling using a polyclonal antibody revealed a complete lack of calcium flux and IP1 accumulation in PLCγ2 Ko cells, demonstrating a non-redundant role of PLCγ2 in calcium release downstream of TREM2. Loss of PLCγ2 led to broad changes in expression of several macrophage surface markers and phenotype, including reduced phagocytic activity and survival, while LPS-induced secretion of the inflammatory cytokines TNFα and IL-6 was unaffected. We identified additional deficits in PLCγ2- deficient cells that compromised cellular adhesion and migration. Thus, PLCγ2 is key in enabling divergent cellular functions and might be a promising target to increase beneficial microglial functions.

Transient developmental imbalance of cortical interneuron subtypes presages long-term changes in behavior.

Cortical GABAergic interneurons are generated in large numbers in the ganglionic eminences and migrate into the cerebral cortex during embryogenesis. At early postnatal stages, during neuronal circuit maturation, autonomous and activity-dependent mechanisms operate within the cortex to adjust cell numbers by eliminating naturally occurring neuron excess. Here, we show that when cortical interneurons are generated in aberrantly high numbers-due to a defect in precursor cell proliferation during embryogenesis-extra parvalbumin interneurons persist in the postnatal mouse cortex during critical periods of cortical network maturation. Even though cell numbers are subsequently normalized, behavioral abnormalities remain in adulthood. This suggests that timely clearance of excess cortical interneurons is critical for correct functional maturation of circuits that drive adult behavior.

TREM2/PLCγ2 signalling in immune cells: function, structural insight, and potential therapeutic modulation.

The central role of the resident innate immune cells of the brain (microglia) in neurodegeneration has become clear over the past few years largely through genome-wide association studies (GWAS), and has rapidly become an active area of research. However, a mechanistic understanding (gene to function) has lagged behind. That is now beginning to change, as exemplified by a number of recent exciting and important reports that provide insight into the function of two key gene products - TREM2 (Triggering Receptor Expressed On Myeloid Cells 2) and PLCγ2 (Phospholipase C gamma2) - in microglia, and their role in neurodegenerative disorders. In this review we explore and discuss these recent advances and the opportunities that they may provide for the development of new therapies.

Hippocampal CA1 Somatostatin Interneurons Originate in the Embryonic MGE/POA.

Oriens lacunosum-moleculare (O-LM) interneurons constitute 40% of hippocampal interneurons expressing Somatostatin (SST). Recent evidence has indicated a dual origin for these cells in the medial and caudal ganglionic eminences (MGE and CGE), with expression of Htr3a as a distinguishing factor. This is strikingly different from cortical SST interneurons that have a single origin within the MGE/preoptic area (POA). We reassessed the origin of hippocampal SST interneurons using a range of genetic lineage-tracing mice combined with single-cell transcriptomic analysis. We find a common origin for all hippocampal SST interneurons in NKX2-1-expressing progenitors of the telencephalic neuroepithelium and an MGE/POA-like transcriptomic signature for all SST clusters. This suggests that functional heterogeneity within the SST CA1 population cannot be attributed to a differential MGE/CGE genetic origin.

Single-Cell Quantification of mRNA Expression in The Human Brain.

RNA analysis at the cellular resolution in the human brain is challenging. Here, we describe an optimised approach for detecting single RNA transcripts in a cell-type specific manner in frozen human brain tissue using multiplexed fluorescent RNAscope probes. We developed a new robust analytical approach for RNAscope quantification. Our method shows that low RNA integrity does not significantly affect RNAscope signal, recapitulates bulk RNA analysis and provides spatial context to transcriptomic analysis of human post-mortem brain at single-cell resolution. In summary, our optimised method allows the usage of frozen human samples from brain banks to perform quantitative RNAscope analysis.

Alzheimer's disease phospholipase C-gamma-2 (PLCG2) protective variant is a functional hypermorph.

BACKGROUND: Recent Genome Wide Association Studies (GWAS) have identified novel rare coding variants in immune genes associated with late onset Alzheimer's disease (LOAD). Amongst these, a polymorphism in phospholipase C-gamma 2 (PLCG2) P522R has been reported to be protective against LOAD. PLC enzymes are key elements in signal transmission networks and are potentially druggable targets. PLCG2 is highly expressed in the hematopoietic system. Hypermorphic mutations in PLCG2 in humans have been reported to cause autoinflammation and immune disorders, suggesting a key role for this enzyme in the regulation of immune cell function. METHODS: We assessed PLCG2 distribution in human and mouse brain tissue via immunohistochemistry and in situ hybridization. We transfected heterologous cell systems (COS7 and HEK293T cells) to determine the effect of the P522R AD-associated variant on enzymatic function using various orthogonal assays, including a radioactive assay, IP-One ELISA, and calcium assays. RESULTS: PLCG2 expression is restricted primarily to microglia and granule cells of the dentate gyrus. Plcg2 mRNA is maintained in plaque-associated microglia in the cerebral tissue of an AD mouse model. Functional analysis of the p.P522R variant demonstrated a small hypermorphic effect of the mutation on enzyme function. CONCLUSIONS: The PLCG2 P522R variant is protective against AD. We show that PLCG2 is expressed in brain microglia, and the p.P522R polymorphism weakly increases enzyme function. These data suggest that activation of PLCγ2 and not inhibition could be therapeutically beneficial in AD. PLCγ2 is therefore a potential target for modulating microglia function in AD, and a small molecule drug that weakly activates PLCγ2 may be one potential therapeutic approach.

Classes and continua of hippocampal CA1 inhibitory neurons revealed by single-cell transcriptomics.

Understanding any brain circuit will require a categorization of its constituent neurons. In hippocampal area CA1, at least 23 classes of GABAergic neuron have been proposed to date. However, this list may be incomplete; additionally, it is unclear whether discrete classes are sufficient to describe the diversity of cortical inhibitory neurons or whether continuous modes of variability are also required. We studied the transcriptomes of 3,663 CA1 inhibitory cells, revealing 10 major GABAergic groups that divided into 49 fine-scale clusters. All previously described and several novel cell classes were identified, with three previously described classes unexpectedly found to be identical. A division into discrete classes, however, was not sufficient to describe the diversity of these cells, as continuous variation also occurred between and within classes. Latent factor analysis revealed that a single continuous variable could predict the expression levels of several genes, which correlated similarly with it across multiple cell types. Analysis of the genes correlating with this variable suggested it reflects a range from metabolically highly active faster-spiking cells that proximally target pyramidal cells to slower-spiking cells targeting distal dendrites or interneurons. These results elucidate the complexity of inhibitory neurons in one of the simplest cortical structures and show that characterizing these cells requires continuous modes of variation as well as discrete cell classes.

Cell-Intrinsic Control of Interneuron Migration Drives Cortical Morphogenesis.

Interneurons navigate along multiple tangential paths to settle into appropriate cortical layers. They undergo a saltatory migration paced by intermittent nuclear jumps whose regulation relies on interplay between extracellular cues and genetic-encoded information. It remains unclear how cycles of pause and movement are coordinated at the molecular level. Post-translational modification of proteins contributes to cell migration regulation. The present study uncovers that carboxypeptidase 1, which promotes post-translational protein deglutamylation, controls the pausing of migrating cortical interneurons. Moreover, we demonstrate that pausing during migration attenuates movement simultaneity at the population level, thereby controlling the flow of interneurons invading the cortex. Interfering with the regulation of pausing not only affects the size of the cortical interneuron cohort but also impairs the generation of age-matched projection neurons of the upper layers.

NKX2-1 Is Required in the Embryonic Septum for Cholinergic System Development, Learning, and Memory.

The transcription factor NKX2-1 is best known for its role in the specification of subsets of cortical, striatal, and pallidal neurons. We demonstrate through genetic fate mapping and intersectional focal septal deletion that NKX2-1 is selectively required in the embryonic septal neuroepithelium for the development of cholinergic septohippocampal projection neurons and large subsets of basal forebrain cholinergic neurons. In the absence of NKX2-1, these neurons fail to develop, causing alterations in hippocampal theta rhythms and severe deficiencies in learning and memory. Our results demonstrate that learning and memory are dependent on NKX2-1 function in the embryonic septum and suggest that cognitive deficiencies that are sometimes associated with pathogenic mutations in NKX2-1 in humans may be a direct consequence of loss of NKX2-1 function.

Tumor suppressor role of phospholipase C epsilon in Ras-triggered cancers.

Phospholipase Cε (PLCε) has been characterized as a direct effector of Ras in vitro and in cellular systems; however, the role of PLCε in tumorigenesis and its link to Ras in this context remain unclear. To assess the role of PLCε in Ras-driven cancers, we generated two new mouse strains: one carrying a targeted deletion of Plce (Plce(-/-)) and the other carrying mutant alleles of Plce unable to bind to Ras (Plce(RAm/RAm)). The Plce(-/-) and, to a lesser degree, Plce(RAm/RAm) transgenic mice exhibited increased susceptibility to tumor formation in the two-stage skin carcinogenesis protocol, revealing a tumor suppressor function for this PLC. This result also suggests that in this context Ras binding in part regulates functions of PLCε. Although significant differences were not seen in the LSL-Kras(G12D) nonsmall cell lung carcinoma model, down-regulation of PLCε was found in animal tumors and in cellular systems following expression of the oncogenic Ras. An inhibitory impact of PLCε on cell growth requires intact lipase activity and is likely mediated by protein kinase C enzymes. Further cellular studies suggest involvement of histone deacetylase in the mechanism of PLCε down-regulation. Taken together, our results show a previously unidentified tumor suppressor role for this PLC in animal models and, together with observations of marked down-regulation in colorectal, lung, and skin tumors, suggest its use as a biological marker in cancer.

Genetic programs controlling cortical interneuron fate.

The origins of cortical interneurons in rodents have been localized to the embryonic subcortical telencephalon where distinct neuroepithelial precursors generate defined interneuron subsets. A swathe of research activity aimed at identifying molecular determinants of subtype identity has uncovered a number of transcription factors that function at different stages of interneuron development. Pathways that lead to the acquisition of mature interneuron traits are therefore beginning to emerge. As genetic programs are influenced by external factors the search continues not only into genetic determinants but also extrinsic influences and the interplay between the two in cell fate specification.

Adult neural stem cells in distinct microdomains generate previously unknown interneuron types.

Throughout life, neural stem cells (NSCs) in different domains of the ventricular-subventricular zone (V-SVZ) of the adult rodent brain generate several subtypes of interneurons that regulate the function of the olfactory bulb. The full extent of diversity among adult NSCs and their progeny is not known. Here, we report the generation of at least four previously unknown olfactory bulb interneuron subtypes that are produced in finely patterned progenitor domains in the anterior ventral V-SVZ of both the neonatal and adult mouse brain. Progenitors of these interneurons are responsive to sonic hedgehog and are organized into microdomains that correlate with the expression domains of the Nkx6.2 and Zic family of transcription factors. This work reveals an unexpected degree of complexity in the specification and patterning of NSCs in the postnatal mouse brain.

Multiple embryonic origins of nitric oxide synthase-expressing GABAergic neurons of the neocortex.

CORTICAL GABAERGIC INTERNEURONS IN RODENTS ORIGINATE IN THREE SUBCORTICAL REGIONS: the medial ganglionic eminence (MGE), the lateral/caudal ganglionic eminence (LGE/CGE), and the preoptic area (POA). Each of these neuroepithelial precursor domains contributes different interneuron subtypes to the cortex. Neuronal NOS (nNOS)-expressing neurons represent a heterogenous population of cortical interneurons. We examined the development of these cells in the mouse embryonic cortex and their abundance and distribution in adult animals. Using genetic lineage tracing in transgenic mice we find that nNOS type I cells originate only in the MGE whereas type II cells have a triple origin in the MGE, LGE/CGE, and POA. The two populations are born at different times during development, occupy different layers in the adult cortex and have distinct neurochemical profiles. nNOS neurons are more numerous in the adult cortex than previously reported and constitute a significant proportion of the cortical interneuron population. Our data suggest that the heterogeneity of nNOS neurons in the cortex can be attributed to their multiple embryonic origins which likely impose distinct genetic specification programs.

The integrity of cholinergic basal forebrain neurons depends on expression of Nkx2-1.

The transcription factor Nkx2-1 belongs to the homeobox-encoding family of proteins that have essential functions in prenatal brain development. Nkx2-1 is required for the specification of cortical interneurons and several neuronal subtypes of the ventral forebrain. Moreover, this transcription factor is involved in migratory processes by regulating the expression of guidance molecules. Interestingly, Nkx2-1 expression was recently detected in the mouse brain at postnatal stages. Using two transgenic mouse lines that allow prenatal or postnatal cell type-specific deletion of Nkx2-1, we show that continuous expression of the transcription factor is essential for the maturation and maintenance of cholinergic basal forebrain neurons in mice. Notably, prenatal deletion of Nkx2-1 in GAD67-expressing neurons leads to a nearly complete loss of cholinergic neurons and parvalbumin-containing GABAergic neurons in the basal forebrain. We also show that postnatal mutation of Nkx2-1 in choline acetyltransferase-expressing cells causes a striking reduction in their number. These degenerative changes are accompanied by partial denervation of their target structures and results in a discrete impairment of spatial memory.