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

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

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

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

Die Integrität von basalen Vorderhirn-Neuronen setzt eine permanente Expression von NkX2-1 voraus: Chance im Verständnis der humanen Haploinsuffizienz

Coordinated movements require the caudate-putamen and the globus pallidus, two nuclei belonging to the basal ganglia, to be intact and functioning properly. Many neurons populating these regions derive from the medial ganglionic eminence (MGE), a transient structure that expresses the transcription factor Nkx2-1 during prenatal development. Nkx2-1 synthesis is essential for the determination of the cell fate and the migration of cortical interneurons, as well as several classes of neurons of the ventral forebrain. Interestingly, heterozygous mutation of the NKX2-1 gene in humans has been described as causing an unusual disorder, from the first year of life onwards, which is mainly characterized by disturbances of motor abilities and delayed speech development. These symptoms suggest that both prenatal and postnatal lack of NKX2-1 could be responsible for the motor dysfunction observed in these patients. So far, data obtained from rodents have been unable to explain convincingly the symptoms of human NKX2-1-haploinsufficiency. Interestingly, postnatal conditional mutation of the Nkx2-1 gene in mature neurons led to hypothalamic dysfunctions such as delayed puberty and reduced reproductive capacity, but no impairments regarding motor coordination or cognitive functions were detected in these mutants. This study shows for the first time which neurons are affected by NKX2-1 haploinsufficiency and it demonstrates that the development of these cells is heavily impaired following prenatal Nkx2-1 deletion in GAD67-expressing cells. As shown for cholinergic neurons, permanent Nkx2-1 expression is essential for the integrity of the cholinergic projection systems. In line with this, predominately prenatal, but also postnatal inactivation of Nkx2-1 in these neurons is accompanied by impairments of the spatial memory and learning. The disturbed motor abilities as observed for NKX2-1-haploinsufficient patients could be explained by the deficits of the intra-striatal neuronal loops and GABAergic projection neurons of the lateral globus pallidus. This assumption is further supported by the observation that the same cell types express NKX2-1 in the adult human basal ganglia. It remains to be elucidated why no memory and learning deficits have been described for the patients so far.Die Koordination komplexer Bewegungen haengt von der Integritaet und Funktion des Striatum und Globus Pallidus, zwei Basalganglienkerne. In der Entwicklung stammen diese Regionen von der mediale ganglionaere Eminenz (MGE), aus der mehrere Neurone migrieren. Die MGE exprimiert den Traskriptionsfaktor Nkx2-1, dessen Synthese ist notwendig fuer die Spezifikation und Migration von striatalen und kortikalen Interneuronen und mehereren anderen basalen Vorderhirn-Zellen. Besonders, beim Menschen fuehrt heterozygote NKX2-1 Mutationen zu einer ungewoehnlich Erkrankung, der fangt in Einjaehrigen, mit complexen Bewegungstoerungen und verzoegerte Sprachentwicklung. Diese Symptome daraufhin hinweisen, dass sowohl praenatal als auch postnatal Fehlen von NKX2-1 fuer die Bewegungstoerung einstehen koennten. Bis jetzt, die Studien an Maeuse- und Rattenmodelle koennten nicht erklaeren die menschliche NKX2-1-Haploinsuffizienz Symptome.Vor allem, obwohl postnatal konditionaelle Mutation im Nkx2-1 Gen in differenzierten Neuronen zu Funktionstoerungen des Hypothalamus wie verpaetetd Pubertaet und reduzierte Reproduktionskapazitaet fuehrt, keine Bewegungstoerungen oder Fehler des kognitives Funktions sind entdeckbar in diese Mutanten. Erstmals, zeigt diese Dissertation welche Neurone wegen NKX2-1 Haploinsuffizienz fehlerhaft sind. Ausserdem die zeigt dass die Entwicklung dieser Zellen nach der praenatal Nkx2-1 Mutation in GABAerge Neurone stark beeintraegtikt ist. Wie aus der cholinerge Neuronen ersichtlich, permanent Nkx2-1 Expression ist notwendig fuer die Integritaet des Systems. Folglich, vorwiegend praenatal, aber auch postnatal Inactivierung des Nkx2-1 Gen in diesen Neuronen ist begleitet von kognitive Beeintraechtigungen (spatial Gedaechtnis und Lernen). Die Bewegungstoerungen der NKX2-1-haploinsuffizienten Patienten finden eine Erklaerung in der Fehlen der intra-striatalen neuronal â loopsâ und Globus Pallidus GABAerge Projektionsneurone. Die Voraussetzung wird further untermauern von der Beachtung dass die gleiche Zelltypen NKX2-1 in erwachsene menschliche Basalganglien exprimieren. Bislang es bleibt unentdekt weil keine Gedaechtnis- oder Lernstoerungen erkannt werden

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

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

Phospholipase C epsilon (PLC epsilon) has been characterized as a direct effector of Ras in vitro and in cellular systems; however, the role of PLC epsilon in tumorigenesis and its link to Ras in this context remain unclear. To assess the role of PLC epsilon 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 PLC epsilon. Although significant differences were not seen in the LSL-KrasG12D nonsmall cell lung carcinoma model, down-regulation of PLC epsilon was found in animal tumors and in cellular systems following expression of the oncogenic Ras. An inhibitory impact of PLC epsilon 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 PLC epsilon 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

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.status: publishe

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

Abstract 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

Cell-Intrinsic Control of Interneuron Migration Drives Cortical Morphogenesis

International audienceGraphical Abstract Highlights d CCP1 controls MLCK activity by processing its polyglutamate carboxy-terminal d CCP1 controls the stereotypic two-stroke cycle of cortical interneuron migration d Asynchronous pausing during interneuron migration controls cortical invasion d Interneuron cortical invasion modulates generation of age-matched projection neuron