Assessing for prenatal risk factors associated with infant neurologic morbidity using a multivariate analysis.

OBJECTIVE: To characterize the biochemical and demographic profiles of pregnant people with maternal immune activation (MIA) and identify the prenatal characteristics associated with neurologic morbidity in offspring. STUDY DESIGN: This was a retrospective cohort study of 602 mother-infant dyads with births between 2009 and 2010 in California. Multivariable logistic regression was used to build a MIA vulnerability profile including mid-pregnancy biochemical markers and maternal demographic characteristics, and its relationship with infant neurologic morbidity was examined. RESULTS: Of the 602 mother-infant dyads, 80 mothers and 61 infants had diagnoses suggestive of MIA and neurologic morbidity, respectively. Our model, including two demographic and seven biochemical characteristics, identified mothers with MIA with good performance (AUC:0.814; 95% CI:0.7-0.8). Three demographic and five inflammatory markers together identified 80% of infants with neurological morbidity (AUC:0.802, 95% CI:0.7-0.8). CONCLUSION: Inflammatory environment in mothers with pre-existing risk factors like obesity, poverty, and prematurity renders offspring more susceptible to neurologic morbidities.

Cytokine responses to SARS-COV2 infection in mother-infant dyads: a systematic review and meta-analysis.

Background: The COVID-19 pandemic has affected a significant number of pregnant women worldwide, but studies on immune responses have presented conflicting results. This study aims to systematically review cytokine profiles in pregnant women with SARS-CoV-2 infection and their infants to evaluate immune responses and potential transplacental transfer of cytokines. Materials and methods: A comprehensive search of 4 databases was conducted to identify relevant studies. Inclusion criteria included studies measuring individual cytokines in pregnant women and/or their neonates. Studies were evaluated for quality, and data were extracted for analysis. Meta-analyses were performed using the random-effects model. Results: Seventeen studies met the inclusion criteria, including data from 748 pregnant women and 287 infants. More than three of these studies evaluated data of 20 cytokines in maternal serum, and data of 10 cytokines was available from cord blood samples. Only the serum level of CXCL10 was significantly up-regulated in SARS-CoV-2 positive pregnant women (n = 339) compared to SARS-CoV-2 negative pregnant women (n = 409). Subset analysis of maternal samples (n = 183) collected during the acute phase of COVID-19 infection showed elevated CXCL10 and IFN-γ. No significant differences in cytokine levels were found between cord blood samples collected from infants born to mothers with (n = 97) and without (n = 190) COVID-19 during gestation. Subset analysis of cord blood samples collected during the acute phase of maternal infection was limited by insufficient data. The heterogeneity among the studies was substantial. Conclusion: The findings suggest that maternal cytokines responses to SARS-CoV-2 infection during pregnancy are not significantly dysregulated, except for CXCL10 and IFN-γ during the acute phase of illness. No evidence of increased cytokine levels in cord blood samples was observed, although this could be impacted by the time period between initial maternal infection and cord blood collection. These results provide some reassurance to parents and healthcare providers but should be interpreted cautiously due to study variations and limitations.

A cross-species proteomic map reveals neoteny of human synapse development.

The molecular mechanisms and evolutionary changes accompanying synapse development are still poorly understood1,2. Here we generate a cross-species proteomic map of synapse development in the human, macaque and mouse neocortex. By tracking the changes of more than 1,000 postsynaptic density (PSD) proteins from midgestation to young adulthood, we find that PSD maturation in humans separates into three major phases that are dominated by distinct pathways. Cross-species comparisons reveal that human PSDs mature about two to three times slower than those of other species and contain higher levels of Rho guanine nucleotide exchange factors (RhoGEFs) in the perinatal period. Enhancement of RhoGEF signalling in human neurons delays morphological maturation of dendritic spines and functional maturation of synapses, potentially contributing to the neotenic traits of human brain development. In addition, PSD proteins can be divided into four modules that exert stage- and cell-type-specific functions, possibly explaining their differential associations with cognitive functions and diseases. Our proteomic map of synapse development provides a blueprint for studying the molecular basis and evolutionary changes of synapse maturation.

Collagen VI Is a Gi-Biased Ligand of the Adhesion GPCR GPR126/ADGRG6.

GPR126/ADGRG6, a member of the adhesion G-protein-coupled receptor family, balances cell differentiation and proliferation through fine-tuning of intracellular cAMP levels, which is achieved through coupling to Gs and Gi proteins. While GPR126-mediated cAMP increase has been proven to be essential for differentiation of Schwann cells, adipocytes and osteoblasts, Gi-signaling of the receptor was found to propagate breast cancer cell proliferation. Extracellular ligands or mechanical forces can modulate GPR126 activity but require an intact encrypted agonist sequence, coined the Stachel. Even though coupling to Gi can be seen for constitutively active truncated receptor versions of GPR126 as well as with a peptide agonist derived from the Stachel sequence, all known N-terminal modulators have so far only been shown to modulate Gs coupling. Here, we identified collagen VI as the first extracellular matrix ligand of GPR126 that induces Gi signaling at the receptor, which shows that N-terminal binding partners can mediate selective G protein signaling cascades that are masked by fully active truncated receptor variants.

GPR56 S4 variant is required for microglia-mediated synaptic pruning.

ADGRG1 (also called GPR56) plays critical roles in brain development and wiring, including cortical lamination, central nervous system (CNS) myelination, and developmental synaptic refinement. However, the underlying mechanism(s) in mediating such diverse functions is not fully understood. Here, we investigate the function of one specific alternative splicing isoform, the GPR56 splice variant 4 (S4), to test the hypothesis that alternative splicing variants of GPR56 in part support its different functions. We created a new transgenic mouse line, Gpr56∆S4 , using CRISPR/Cas9, in which GPR56 S4 was deleted. Detailed phenotype analyses show that Gpr56∆S4 mice manifest no deficits in cortical architecture and CNS myelination compared to controls. Excitingly, they present significantly increased synapse densities, decreased synapse engulfment by microglia, and impaired eye-segregation. Taken together, our findings support that the GPR56 S4 variant is dispensable for cortical development and CNS myelination but is essential for microglia-mediated synaptic pruning.

Microglial GPR56 is the molecular target of maternal immune activation-induced parvalbumin-positive interneuron deficits.

Parvalbumin-positive (PV+) interneurons play a critical role in maintaining circuit rhythm in the brain, and their reduction is implicated in autism spectrum disorders. Animal studies demonstrate that maternal immune activation (MIA) leads to reduced PV+ interneurons in the somatosensory cortex and autism-like behaviors. However, the underlying molecular mechanisms remain largely unknown. Here, we show that MIA down-regulates microglial Gpr56 expression in fetal brains in an interleukin-17a-dependent manner and that conditional deletion of microglial Gpr56 [Gpr56 conditional knockout (cKO)] mimics MIA-induced PV+ interneuron defects and autism-like behaviors in offspring. We further demonstrate that elevated microglial tumor necrosis factor-α expression is the underlying mechanism by which MIA and Gpr56 cKO impair interneuron generation. Genetically restoring Gpr56 expression in microglia ameliorates PV+ interneuron deficits and autism-like behaviors in MIA offspring. Together, our study demonstrates that microglial GPR56 plays an important role in PV+ interneuron development and serves as a salient target of MIA-induced neurodevelopmental disorders.

Oligodendrocyte Development and Implication in Perinatal White Matter Injury.

Perinatal white matter injury (WMI) is the most common brain injury in premature infants and can lead to life-long neurological deficits such as cerebral palsy. Preterm birth is typically accompanied by inflammation and hypoxic-ischemic events. Such perinatal insults negatively impact maturation of oligodendrocytes (OLs) and cause myelination failure. At present, no treatment options are clinically available to prevent or cure WMI. Given that arrested OL maturation plays a central role in the etiology of perinatal WMI, an increased interest has emerged regarding the functional restoration of these cells as potential therapeutic strategy. Cell transplantation and promoting endogenous oligodendrocyte function are two potential options to address this major unmet need. In this review, we highlight the underlying pathophysiology of WMI with a specific focus on OL biology and their implication for the development of new therapeutic targets.

The Inhibitory Receptor GPR56 (Adgrg1) Is Specifically Expressed by Tissue-Resident Memory T Cells in Mice But Dispensable for Their Differentiation and Function In Vivo.

Tissue-resident memory T (TRM) cells with potent antiviral and antibacterial functions protect the epithelial and mucosal surfaces of our bodies against infection with pathogens. The strong proinflammatory activities of TRM cells suggest requirement for expression of inhibitory molecules to restrain these memory T cells under steady state conditions. We previously identified the adhesion G protein-coupled receptor GPR56 as an inhibitory receptor of human cytotoxic lymphocytes that regulates their cytotoxic effector functions. Here, we explored the expression pattern, expression regulation, and function of GPR56 on pathogen-specific CD8+ T cells using two infection models. We observed that GPR56 is expressed on TRM cells during acute infection and is upregulated by the TRM cell-inducing cytokine TGF-ß and the TRM cell-associated transcription factor Hobit. However, GPR56 appeared dispensable for CD8+ T-cell differentiation and function upon acute infection with LCMV as well as Listeria monocytogenes. Thus, TRM cells specifically acquire the inhibitory receptor GPR56, but the impact of this receptor on TRM cells after acute infection does not appear essential to regulate effector functions of TRM cells.

The association of COVID-19 infection in pregnancy with preterm birth: A retrospective cohort study in California.

INTRODUCTION: Our understanding of the association between coronavirus disease 19 (COVID-19) and preterm or early term birth among racially and ethnically diverse populations and people with chronic medical conditions is limited. METHODS: We determined the association between COVID-19 and preterm (PTB) birth among live births documented by California Vital Statistics birth certificates between July 2020 and January 2021 (n=240,147). We used best obstetric estimate of gestational age to classify births as very preterm (VPTB, <32 weeks), PTB (< 37 weeks), early term (37 and 38 weeks), and term (39-44 weeks), as each confer independent risks to infant health and development. Separately, we calculated the joint effects of COVID-19 diagnosis, hypertension, diabetes, and obesity on PTB and VPTB. FINDINGS: COVID-19 diagnoses on birth certificates increased for all racial/ethnic groups between July 2020 and January 2021 and were highest for American Indian/Alaska Native (12.9%), Native Hawaiian/Pacific Islander (11.4%), and Latinx (10.3%) birthing people. COVID-19 diagnosis was associated with an increased risk of VPTB (aRR 1.6, 95% CI [1.4, 1.9]), PTB (aRR 1.4, 95% CI [1.3, 1.4]), and early term birth (aRR 1.1, 95% CI [1.1, 1.2]). There was no effect modification of the overall association by race/ethnicity or insurance status. COVID-19 diagnosis was associated with elevated risk of PTB in people with hypertension, diabetes, and/or obesity. INTERPRETATION: In a large population-based study, COVID-19 diagnosis increased the risk of VPTB, PTB, and early term birth, particularly among people with medical comorbidities. Considering increased circulation of COVID-19 variants, preventative measures, including vaccination, should be prioritized for birthing persons. FUNDING: UCSF-Kaiser Department of Research Building Interdisciplinary Research Careers in Women's Health Program (BIRCWH) National Institute of Child Health and Human Development (NICHD) and the Office of Research on Women's Health (ORWH) [K12 HD052163] and the California Preterm Birth Initiative, funded by Marc and Lynn Benioff.

Human microglia states are conserved across experimental models and regulate neural stem cell responses in chimeric organoids.

Microglia are resident macrophages in the brain that emerge in early development and respond to the local environment by altering their molecular and phenotypic states. Fundamental questions about microglia diversity and function during development remain unanswered because we lack experimental strategies to interrogate their interactions with other cell types and responses to perturbations ex vivo. We compared human microglia states across culture models, including cultured primary and pluripotent stem cell-derived microglia. We developed a "report card" of gene expression signatures across these distinct models to facilitate characterization of their responses across experimental models, perturbations, and disease conditions. Xenotransplantation of human microglia into cerebral organoids allowed us to characterize key transcriptional programs of developing microglia in vitro and reveal that microglia induce transcriptional changes in neural stem cells and decrease interferon signaling response genes. Microglia additionally accelerate the emergence of synchronized oscillatory network activity in brain organoids by modulating synaptic density.

Phospholipid-flippase chaperone CDC50A is required for synapse maintenance by regulating phosphatidylserine exposure.

Synaptic refinement is a critical physiological process that removes excess synapses to establish and maintain functional neuronal circuits. Recent studies have shown that focal exposure of phosphatidylserine (PS) on synapses acts as an "eat me" signal to mediate synaptic pruning. However, the molecular mechanism underlying PS externalization at synapses remains elusive. Here, we find that murine CDC50A, a chaperone of phospholipid flippases, localizes to synapses, and that its expression depends on neuronal activity. Cdc50a knockdown leads to phosphatidylserine exposure at synapses and subsequent erroneous synapse removal by microglia partly via the GPR56 pathway. Taken together, our data support that CDC50A safeguards synapse maintenance by regulating focal phosphatidylserine exposure at synapses.

Association of Maternal Immune Activation during Pregnancy and Neurologic Outcomes in Offspring.

OBJECTIVE: To evaluate neurologic morbidity among offspring during their first year of life in association with prenatal maternal immune activation (MIA), using an inclusive definition. STUDY DESIGN: This retrospective cohort study included singletons born in California between 2011 and 2017. MIA was defined by International Classification of Diseases diagnosis of infection, autoimmune disorder, allergy, asthma, atherosclerosis, or malignancy during pregnancy. Neurologic morbidity in infants was defined by International Classification of Diseases diagnosis of intraventricular hemorrhage, periventricular leukomalacia, seizures, abnormal neurologic examination, or abnormal neurologic imaging. Outcomes of delayed developmental milestones during the first year of life were also explored. Risk of neurologic morbidity in offspring was approximated for women with and without MIA using log link binary regression. RESULTS: Demographic characteristics among 3 004 166 mother-infant dyads with or without MIA were similar in both groups. Rate of preterm delivery in mothers with MIA (9.4%) was significantly higher than those without MIA (5.6%). Infants of mothers with MIA were more likely to experience neurologic morbidities across all gestational ages. Adjusted relative risk (95% CI) in the exposed infants was 2.0 (1.9-2.1) for abnormal neurologic examination; 1.6 (1.5-1.7) for seizures, and 1.6 (1.4-1.8) for periventricular leukomalacia. CONCLUSIONS: Our results demonstrate that MIA during pregnancy may be associated with considerably higher risk of neurologic morbidity in offspring.

Unexpected redundancy of Gpr56 and Gpr97 during hematopoietic cell development and differentiation.

Integrated molecular signals regulate cell fate decisions in the embryonic aortic endothelium to drive hematopoietic stem cell (HSC) generation during development. The G-protein-coupled receptor 56 (Gpr56, also called Adgrg1) is the most highly upregulated receptor gene in cells that take on hematopoietic fate and is expressed by adult bone marrow HSCs. Despite the requirement for Gpr56 in hematopoietic stem/progenitor cell (HS/PC) generation in zebrafish embryos and the highly upregulated expression of GPR56 in treatment-resistant leukemic patients, its function in normal mammalian hematopoiesis remains unclear. Here, we examine the role of Gpr56 in HS/PC development in Gpr56 conditional knockout (cKO) mouse embryos and Gpr knockout (KO) embryonic stem cell (ESC) hematopoietic differentiation cultures. Our results show a bias toward myeloid differentiation of Gpr56 cKO fetal liver HSCs and an increased definitive myeloid progenitor cell frequency in Gpr56KO ESC differentiation cultures. Surprisingly, we find that mouse Gpr97 can rescue Gpr56 morphant zebrafish hematopoietic generation, and that Gpr97 expression is upregulated in mouse Gpr56 deletion models. When both Gpr56 and Gpr97 are deleted in ESCs, no or few hematopoietic PCs (HPCs) are generated upon ESC differentiation. Together, our results reveal novel and redundant functions for these 2 G-protein coupled receptors in normal mammalian hematopoietic cell development and differentiation.

Cell type-specific evaluation of ADGRG1/GPR56 function in developmental central nervous system myelination.

Myelination of axons in the central nervous system (CNS) is a concerted effort between many cell types, resulting in significant cross-talk and communication among cells. Adhesion G protein-coupled receptor ADGRG1 (GPR56) is expressed in all major glial cells and regulates a wide variety of physiological processes by mediating cell-cell and cell-matrix communications. Previous literature has demonstrated the requirement of ADGRG1 in oligodendrocyte precursor cells (OPCs) during developmental myelination. However, it is unknown if ADGRG1 is responsible for myelin formation in a cell-type-specific manner. To that end, here we profiled myelin status in response to deletion of Adgrg1 specifically in OPCs, microglia, astrocytes, and neurons. Interestingly, we find that knocking out Adgrg1 in OPCs significantly decreases OPC proliferation and reduced number of myelinated axons. However, deleting Adgrg1 in microglia, astrocytes, and neurons does not impact developmental myelination. These data support an autonomous functional role for Adgrg1 in OPCs related to myelination.

Inhibition of lipopolysaccharide-induced inflammation via the protective role of T regulatory cells in the fetal liver in a late-pregnancy preterm mouse model.

OBJECTIVES: This study intended to explore the effect of T regulatory cells (Tregs) in the perinatal liver against LPS-induced inflammation in a preterm birth mouse model. Moreover, the role of adoptive Tregs on the inflammatory response induced by LPS was also studied. METHODS: Female BALB/C mice were injected intraperitoneally (IP) with LPS dissolved in normal saline solution at a dose of 50 µg/kg. Spleens from pregnant mice were used to obtain Tregs. The expression of Forkhead family transcription factor-3 (Foxp3), Interleukin-6 (IL-6), Toll-like receptor-4 (TLR-4), and Heme oxygenase-1 (HO-1) were assessed from fetal liver tissues by polymerase chain reaction and western blotting. RESULTS: LPS administered to mice induced an inflammatory response in the perinatal liver, and this inflammatory response was negatively regulated by Tregs in the experimental group. Maternal-fetal tolerance was maintained by Tregs. Transmission of Tregs was estimated in different experimental groups based on the mRNA expression of TLR-4, IL-6, HO-1, and Foxp3. CONCLUSIONS: After analysis of the experimental data, it was determined that Tregs exhibited regulatory potential against LPS-induced inflammatory response. Further, it was concluded that the transmission of Tregs improved the mother's immune tolerance against LPS-induced inflammation in the fetal liver.

GPR56/ADGRG1 is a platelet collagen-responsive GPCR and hemostatic sensor of shear force.

Circulating platelets roll along exposed collagen at vessel injury sites and respond with filipodia protrusion, shape change, and surface area expansion to facilitate platelet adhesion and plug formation. Various glycoproteins were considered to be both collagen responders and mediators of platelet adhesion, yet the signaling kinetics emanating from these receptors do not fully account for the rapid platelet cytoskeletal changes that occur in blood flow. We found the free N-terminal fragment of the adhesion G protein-coupled receptor (GPCR) GPR56 in human plasma and report that GPR56 is the platelet receptor that transduces signals from collagen and blood flow-induced shear force to activate G protein 13 signaling for platelet shape change. Gpr56-/- mice have prolonged bleeding, defective platelet plug formation, and delayed thrombotic occlusion. Human and mouse blood perfusion studies demonstrated GPR56 and shear-force dependence of platelet adhesion to immobilized collagen. Our work places GPR56 as an initial collagen responder and shear-force transducer that is essential for platelet shape change during hemostasis.

A splicing isoform of GPR56 mediates microglial synaptic refinement via phosphatidylserine binding.

Developmental synaptic remodeling is important for the formation of precise neural circuitry, and its disruption has been linked to neurodevelopmental disorders such as autism and schizophrenia. Microglia prune synapses, but integration of this synapse pruning with overlapping and concurrent neurodevelopmental processes, remains elusive. Adhesion G protein-coupled receptor ADGRG1/GPR56 controls multiple aspects of brain development in a cell type-specific manner: In neural progenitor cells, GPR56 regulates cortical lamination, whereas in oligodendrocyte progenitor cells, GPR56 controls developmental myelination and myelin repair. Here, we show that microglial GPR56 maintains appropriate synaptic numbers in several brain regions in a time- and circuit-dependent fashion. Phosphatidylserine (PS) on presynaptic elements binds GPR56 in a domain-specific manner, and microglia-specific deletion of Gpr56 leads to increased synapses as a result of reduced microglial engulfment of PS+ presynaptic inputs. Remarkably, a particular alternatively spliced isoform of GPR56 is selectively required for microglia-mediated synaptic pruning. Our present data provide a ligand- and isoform-specific mechanism underlying microglial GPR56-mediated synapse pruning in the context of complex neurodevelopmental processes.

Inhibition of lipopolysaccharide-induced inflammation via the protective role of T regulatory cells in the fetal liver in a late-pregnancy preterm mouse model

OBJECTIVES: This study intended to explore the effect of T regulatory cells (Tregs) in the perinatal liver against LPS-induced inflammation in a preterm birth mouse model. Moreover, the role of adoptive Tregs on the inflammatory response induced by LPS was also studied. METHODS: Female BALB/C mice were injected intraperitoneally (IP) with LPS dissolved in normal saline solution at a dose of 50 µg/kg. Spleens from pregnant mice were used to obtain Tregs. The expression of Forkhead family transcription factor-3 (Foxp3), Interleukin-6 (IL-6), Toll-like receptor-4 (TLR-4), and Heme oxygenase-1 (HO-1) were assessed from fetal liver tissues by polymerase chain reaction and western blotting. RESULTS: LPS administered to mice induced an inflammatory response in the perinatal liver, and this inflammatory response was negatively regulated by Tregs in the experimental group. Maternal-fetal tolerance was maintained by Tregs. Transmission of Tregs was estimated in different experimental groups based on the mRNA expression of TLR-4, IL-6, HO-1, and Foxp3. CONCLUSIONS: After analysis of the experimental data, it was determined that Tregs exhibited regulatory potential against LPS-induced inflammatory response. Further, it was concluded that the transmission of Tregs improved the mother's immune tolerance against LPS-induced inflammation in the fetal liver.

GAIN domain-mediated cleavage is required for activation of G protein-coupled receptor 56 (GPR56) by its natural ligands and a small-molecule agonist.

Adhesion G protein-coupled receptors (aGPCRs) represent a distinct family of GPCRs that regulate several developmental and physiological processes. Most aGPCRs undergo GPCR autoproteolysis-inducing domain-mediated protein cleavage, which produces a cryptic tethered agonist (termed Stachel (stinger)), and cleavage-dependent and -independent aGPCR signaling mechanisms have been described. aGPCR G1 (ADGRG1 or G protein-coupled receptor 56 (GPR56)) has pleiotropic functions in the development of multiple organ systems, which has broad implications for human diseases. To date, two natural GPR56 ligands, collagen III and tissue transglutaminase (TG2), and one small-molecule agonist, 3-α-acetoxydihydrodeoxygedunin (3-α-DOG), have been identified, in addition to a synthetic peptide, P19, that contains seven amino acids of the native Stachel sequence. However, the mechanisms by which these natural and small-molecule agonists signal through GPR56 remain unknown. Here we engineered a noncleavable receptor variant that retains signaling competence via the P19 peptide. We demonstrate that both natural and small-molecule agonists can activate only cleaved GPR56. Interestingly, TG2 required both receptor cleavage and the presence of a matrix protein, laminin, to activate GPR56, whereas collagen III and 3-α-DOG signaled without any cofactors. On the other hand, both TG2/laminin and collagen III activate the receptor by dissociating the N-terminal fragment from its C-terminal fragment, enabling activation by the Stachel sequence, whereas P19 and 3-α-DOG initiate downstream signaling without disengaging the N-terminal fragment from its C-terminal fragment. These findings deepen our understanding of how GPR56 signals via natural ligands, and a small-molecule agonist may be broadly applicable to other aGPCR family members.

The expanding functional roles and signaling mechanisms of adhesion G protein-coupled receptors.

The adhesion class of G protein-coupled receptors (GPCRs) is the second largest family of GPCRs (33 members in humans). Adhesion GPCRs (aGPCRs) are defined by a large extracellular N-terminal region that is linked to a C-terminal seven transmembrane (7TM) domain via a GPCR-autoproteolysis inducing (GAIN) domain containing a GPCR proteolytic site (GPS). Most aGPCRs undergo autoproteolysis at the GPS motif, but the cleaved fragments stay closely associated, with the N-terminal fragment (NTF) bound to the 7TM of the C-terminal fragment (CTF). The NTFs of most aGPCRs contain domains known to be involved in cell-cell adhesion, while the CTFs are involved in classical G protein signaling, as well as other intracellular signaling. In this workshop report, we review the most recent findings on the biology, signaling mechanisms, and physiological functions of aGPCRs.