Diagnosing and staging epithelial ovarian cancer by serum glycoproteomic profiling.

BACKGROUND: There is a need for diagnostic tests for screening, triaging and staging of epithelial ovarian cancer (EOC). Glycoproteomics of blood samples has shown promise for biomarker discovery. METHODS: We applied glycoproteomics to serum of people with EOC or benign pelvic masses and healthy controls. A total of 653 analytes were quantified and assessed in multivariable models, which were tested in an independent cohort. Additionally, we analyzed glycosylation patterns in serum markers and in tissues. RESULTS: We identified a biomarker panel that distinguished benign lesions from EOC with sensitivity and specificity of 83.5% and 90.1% in the training set, and of 86.7 and 86.7% in the test set, respectively. ROC analysis demonstrated strong performance across a range of cutoffs. Fucosylated multi-antennary glycopeptide markers were higher in late-stage than in early-stage EOC. A comparable pattern was found in late-stage EOC tissues. CONCLUSIONS: Blood glycopeptide biomarkers have the potential to distinguish benign from malignant pelvic masses, and early- from late-stage EOC. Glycosylation of circulating and tumor tissue proteins may be related. This study supports the hypothesis that blood glycoproteomic profiling can be used for EOC diagnosis and staging and it warrants further clinical evaluation.

Mass Spectrometry Analysis of Glycopeptides Enriched by Anion Exchange-Mediated Methods Reveals PolyLacNAc-Extended N-Glycans in Integrins and Tetraspanins in Melanoma Cells.

Glycosylation is a key modulator of the functional state of proteins. Recent developments in large-scale analysis of intact glycopeptides have enabled the identification of numerous glycan structures that are relevant in pathophysiological processes. However, one motif found in N-glycans, poly-N-acetyllactosamine (polyLacNAc), still poses a substantial challenge to mass spectrometry-based glycoproteomic analysis due to its relatively low abundance and large size. In this work, we developed approaches for the systematic mapping of polyLacNAc-elongated N-glycans in melanoma cells. We first evaluated five anion exchange-based matrices for enriching intact glycopeptides and selected two materials that provided better overall enrichment efficiency. We then tested the robustness of the methodology by quantifying polyLacNAc-containing glycopeptides as well as changes in protein fucosylation and sialylation. Finally, we applied the optimal enrichment methods to discover glycopeptides containing polyLacNAc motifs in melanoma cells and found that integrins and tetraspanins are substantially modified with these structures. This study demonstrates the feasibility of glycoproteomic approaches for identification of glycoproteins with polyLacNAc motifs.

Inter- and transgenerational heritability of preconception chronic stress or alcohol exposure: Translational outcomes in brain and behavior.

Chronic stress and alcohol (ethanol) use are highly interrelated and can change an individual's behavior through molecular adaptations that do not change the DNA sequence, but instead change gene expression. A recent wealth of research has found that these nongenomic changes can be transmitted across generations, which could partially account for the "missing heritability" observed in genome-wide association studies of alcohol use disorder and other stress-related neuropsychiatric disorders. In this review, we summarize the molecular and behavioral outcomes of nongenomic inheritance of chronic stress and ethanol exposure and the germline mechanisms that could give rise to this heritability. In doing so, we outline the need for further research to: (1) Investigate individual germline mechanisms of paternal, maternal, and biparental nongenomic chronic stress- and ethanol-related inheritance; (2) Synthesize and dissect cross-generational chronic stress and ethanol exposure; (3) Determine cross-generational molecular outcomes of preconception ethanol exposure that contribute to alcohol-related disease risk, using cancer as an example. A detailed understanding of the cross-generational nongenomic effects of stress and/or ethanol will yield novel insight into the impact of ancestral perturbations on disease risk across generations and uncover actionable targets to improve human health.

Plasma glycoproteomic biomarkers identify metastatic melanoma patients with reduced clinical benefit from immune checkpoint inhibitor therapy.

The clinical success of immune-checkpoint inhibitors (ICI) in both resected and metastatic melanoma has confirmed the validity of therapeutic strategies that boost the immune system to counteract cancer. However, half of patients with metastatic disease treated with even the most aggressive regimen do not derive durable clinical benefit. Thus, there is a critical need for predictive biomarkers that can identify individuals who are unlikely to benefit with high accuracy so that these patients may be spared the toxicity of treatment without the likely benefit of response. Ideally, such an assay would have a fast turnaround time and minimal invasiveness. Here, we utilize a novel platform that combines mass spectrometry with an artificial intelligence-based data processing engine to interrogate the blood glycoproteome in melanoma patients before receiving ICI therapy. We identify 143 biomarkers that demonstrate a difference in expression between the patients who died within six months of starting ICI treatment and those who remained progression-free for three years. We then develop a glycoproteomic classifier that predicts benefit of immunotherapy (HR=2.7; p=0.026) and achieves a significant separation of patients in an independent cohort (HR=5.6; p=0.027). To understand how circulating glycoproteins may affect efficacy of treatment, we analyze the differences in glycosylation structure and discover a fucosylation signature in patients with shorter overall survival (OS). We then develop a fucosylation-based model that effectively stratifies patients (HR=3.5; p=0.0066). Together, our data demonstrate the utility of plasma glycoproteomics for biomarker discovery and prediction of ICI benefit in patients with metastatic melanoma and suggest that protein fucosylation may be a determinant of anti-tumor immunity.

Advancements in Genomic and Behavioral Neuroscience Analysis for the Study of Normal and Pathological Brain Function.

Psychiatric and neurological disorders are influenced by an undetermined number of genes and molecular pathways that may differ among afflicted individuals. Functionally testing and characterizing biological systems is essential to discovering the interrelationship among candidate genes and understanding the neurobiology of behavior. Recent advancements in genetic, genomic, and behavioral approaches are revolutionizing modern neuroscience. Although these tools are often used separately for independent experiments, combining these areas of research will provide a viable avenue for multidimensional studies on the brain. Herein we will briefly review some of the available tools that have been developed for characterizing novel cellular and animal models of human disease. A major challenge will be openly sharing resources and datasets to effectively integrate seemingly disparate types of information and how these systems impact human disorders. However, as these emerging technologies continue to be developed and adopted by the scientific community, they will bring about unprecedented opportunities in our understanding of molecular neuroscience and behavior.

A proximity labeling method for protein-protein interactions on cell membrane.

Antibodies targeting specific antigens are widely utilized in biological research to investigate protein interactions or to quantify target antigens. Here, we introduce antigen-antibody proximity labeling (AAPL), a novel method to map the antigen interaction sites as well as interactors of antibody-targeted proteins. As a proof of concept, AAPL was demonstrated using sodium/potassium transporting ATPase (ATP1A1) and epidermal growth factor receptor 2 (ERBB2)-specific antibodies that were modified with an Fe(iii) catalytic probe. Once bound to their target proteins, Fe(iii)-induced catalytic oxidation occurred in proximity of the antigen's epitope. Oxidative proteomic analysis was then used to determine the degree of oxidation, the site of oxidation within the targeted antigen, and the interacting proteins that were in close proximity to the targeted antigen. An AAPL score was generated for each protein yielding the specificity of the oxidation and proximity of the interacting protein to the target antigen. As a final demonstration of its utility, the AAPL approach was applied to map the interactors of liver-intestine-cadherin (CDH17) in colon cancer cells.

Developing guideline-based key performance indicators for recurrent miscarriage care: lessons from a multi-stage consensus process with a diverse stakeholder group.

BACKGROUND: Standardised care pathways tailored to women/couples who experience recurrent miscarriage are needed; however, clinical practice is inconsistent and poorly organised. In this paper, we outline our processes and experiences of developing guideline-based key performance indicators (KPIs) for recurrent miscarriage care with a diverse stakeholder group which will be used to evaluate national services. To date, such exercises have generally only involved clinicians, with the need for greater stakeholder involvement highlighted. METHODS: Our study involved six stages: (i) identification and synthesis of recommendations for recurrent miscarriage care through a systematic review of clinical practice guidelines; (ii) a two-round modified e-Delphi survey with stakeholders to develop consensus on recommendations and outcomes; (iii) four virtual meetings to develop this consensus further; (iv) development of a list of candidate KPIs; (v) survey to achieve consensus on the final suite of KPIs and a (vi) virtual meeting to agree on the final set of KPIs. Through participatory methods, participants provided feedback on the process of KPI development. RESULTS: From an initial list of 373 recommendations and 14 outcomes, 110 indicators were prioritised for inclusion in the final suite of KPIs: (i) structure of care (n = 20); (ii) counselling and supportive care (n = 7); (iii) investigations (n = 30); treatment (n = 34); outcomes (n = 19). Participants' feedback on the process comprised three main themes: accessibility, richness in diversity, streamlining the development process. CONCLUSIONS: It is important and feasible to develop guideline-based KPIs with a diverse stakeholder group. One hundred and ten KPIs were prioritised for inclusion in a suite of guideline-based KPIs for recurrent miscarriage care. Insights into our experiences may help others undertaking similar projects, particularly those undertaken in the absence of a clinical guideline and/or involving a range of stakeholders.

Women/couples who have recurrent/repeated miscarriages should receive care that meets their needs, through agreed care pathways. This is often not the case. Key performance indicators (KPIs) are measures of specific elements of care (structures, processes and/or outcomes), which can help us to judge the quality of care given. In this paper, we describe how we worked with women and men with lived experience, doctors, nurses, managers, and others, to develop and agree on a list KPIs for recurrent miscarriage care in Ireland. We will use these to check what services are doing across the country and what could be done better. Participants filled out surveys and took part in meetings to vote on and agree on what KPIs were important to include. They also shared their views and experiences of taking part in this work. Together, we developed 110 KPIs for recurrent miscarriage care. These include measures of how care is structured, counselling and supports, investigations and treatments provided, and health-related outcomes. Participants' valued the different views that people brought to discussions and what they learned. They suggested ways that the process could be made more participant-friendly. For example, being up-front about the time it would take, explaining medical terms more, and cutting down on the number of items to be rated in surveys. It is important and possible to develop KPIs with different groups, particularly those with lived experience. Learning from our study may help others who want to do similar projects, such as develop KPIs or guidelines for care.

Serum Glycoprotein Markers in Nonalcoholic Steatohepatitis and Hepatocellular Carcinoma.

Fatty liver disease progresses through stages of fat accumulation and inflammation to nonalcoholic steatohepatitis (NASH), fibrosis and cirrhosis, and eventually hepatocellular carcinoma (HCC). Currently available diagnostic tools for HCC lack sensitivity and specificity. In this study, we investigated the use of circulating serum glycoproteins to identify a panel of potential prognostic markers that may be indicative of progression from the healthy state to NASH and further to HCC. Serum samples were processed and analyzed using a novel high-throughput glycoproteomics platform. Our initial dataset contained healthy, NASH, and HCC serum samples. We analyzed 413 glycopeptides, representing 57 abundant serum proteins, and compared among the three phenotypes. We studied the normalized abundance of common glycoforms and found 40 glycopeptides with statistically significant differences in abundances in NASH and HCC compared to controls. Summary level relative abundances of core-fucosylated, sialylated, and branched glycans containing glycopeptides were higher in NASH and HCC as compared to controls. We replicated some of our findings in an independent set of samples of individuals with benign liver conditions and HCC. Our results may be of value in the management of liver diseases. Data generated in this work can be downloaded from MassIVE (https://massive.ucsd.edu) with identifier MSV000088809.

Differential Peripheral Blood Glycoprotein Profiles in Symptomatic and Asymptomatic COVID-19.

Glycosylation is the most common form of post-translational modification of proteins, critically affecting their structure and function. Using liquid chromatography and mass spectrometry for high-resolution site-specific quantification of glycopeptides coupled with high-throughput artificial intelligence-powered data processing, we analyzed differential protein glycoisoform distributions of 597 abundant serum glycopeptides and nonglycosylated peptides in 50 individuals who had been seriously ill with COVID-19 and in 22 individuals who had recovered after an asymptomatic course of COVID-19. As additional comparison reference phenotypes, we included 12 individuals with a history of infection with a common cold coronavirus, 16 patients with bacterial sepsis, and 15 healthy subjects without history of coronavirus exposure. We found statistically significant differences, at FDR < 0.05, for normalized abundances of 374 of the 597 peptides and glycopeptides interrogated between symptomatic and asymptomatic COVID-19 patients. Similar statistically significant differences were seen when comparing symptomatic COVID-19 patients to healthy controls (350 differentially abundant peptides and glycopeptides) and common cold coronavirus seropositive subjects (353 differentially abundant peptides and glycopeptides). Among healthy controls and sepsis patients, 326 peptides and glycopeptides were found to be differentially abundant, of which 277 overlapped with biomarkers that showed differential expression between symptomatic COVID-19 cases and healthy controls. Among symptomatic COVID-19 cases and sepsis patients, 101 glycopeptide and peptide biomarkers were found to be statistically significantly abundant. Using both supervised and unsupervised machine learning techniques, we found specific glycoprotein profiles to be strongly predictive of symptomatic COVID-19 infection. LASSO-regularized multivariable logistic regression and K-means clustering yielded accuracies of 100% in an independent test set and of 96% overall, respectively. Our findings are consistent with the interpretation that a majority of glycoprotein modifications observed which are shared among symptomatic COVID-19 and sepsis patients likely represent a generic consequence of a severe systemic immune and inflammatory state. However, there are glycoisoform changes that are specific and particular to severe COVID-19 infection. These may be representative of either COVID-19-specific consequences or susceptibility to or predisposition for a severe course of the disease. Our findings support the potential value of glycoproteomic biomarkers in the biomedical understanding and, potentially, the clinical management of serious acute infectious conditions.

Clinical practice guidelines for recurrent miscarriage in high-income countries: a systematic review.

Recurrent miscarriage affects 1-2% of women of reproductive age, depending on the definition used. A systematic review was conducted to identify, appraise and describe clinical practice guidelines (CPG) published since 2000 for the investigation, management, and/or follow-up of recurrent miscarriage within high-income countries. Six major databases, eight guideline repositories and the websites of 11 professional organizations were searched to identify potentially eligible studies. The quality of eligible CPG was assessed using the Appraisal of Guidelines for Research and Evaluation (AGREE II) Tool. A narrative synthesis was conducted to describe, compare and contrast the CPG and recommendations therein. Thirty-two CPG were included, from which 373 recommendations concerning first-trimester recurrent miscarriage were identified across four sub-categories: structure of care (42 recommendations, nine CPG), investigations (134 recommendations, 23 CPG), treatment (153 recommendations, 24 CPG), and counselling and supportive care (46 recommendations, nine CPG). Most CPG scored 'poor' on applicability (84%) and editorial independence (69%); and to a lesser extent stakeholder involvement (38%) and rigour of development (31%). Varying levels of consensus were found across CPG, with some conflicting recommendations. Greater efforts are required to improve the quality of evidence underpinning CPG, the rigour of their development and the inclusion of multi-disciplinary perspectives, including those with lived experience of recurrent miscarriage.

Reproductive Health Knowledge About Miscarriage: A Cross-Sectional Study of University Students.

OBJECTIVE: To assess university students' knowledge of reproductive health information about miscarriage. METHODS: A single-centre, cross-sectional study was carried out using an online survey at a higher education institution in the Republic of Ireland between April and May of 2016. A total of 746 university students' responses were analysed. RESULTS: Approximately 60% and 70% of college students correctly identified features of first and second trimester miscarriage. After adjusting for confounders, male students were two times more likely to have a poor knowledge of features of miscarriage compared to females (aOR 2.0, 95% CI 1.3-3.0 and aOR 1.7, 95% CI 1.1-2.6 for first and second trimester respectively). Poor knowledge of features of first trimester miscarriage was less common among older students and students who were married, cohabiting or in a relationship (aOR 0.4, 95% CI 0.2-0.6 and aOR 0.4, 95% CI 0.3-0.8 respectively). Students who studied Medicine and Health were more likely to identify any type of treatment for miscarriage compared to students who studied other disciplines. Students who studied Arts and Social Science were more likely to overestimate the percentage of miscarriages with an identified cause compared to students who studied Medicine and Health. CONCLUSION: Our results provide additional information about the gap of knowledge in regards to reproductive health information about miscarriage, specifically among university students.

Parents and clinicians: partners in perinatal bereavement research -experiences from the International Stillbirth Alliance Conference 2017.

In recent years, there has been a global call to reduce the numbers of preventable stillbirths and increase public awareness about the incidence and impact of pregnancy loss. The lived experiences of bereaved parents have much to contribute to developing the research agenda and clinical care in pregnancy loss. The multidisciplinary Pregnancy Loss Research Group (PLRG) based at the INFANT Centre at University College Cork and Cork University Maternity Hospital, has an established practice of active engagement and participation of patient members. This partnership provided the catalyst to model a similar collaborative approach between clinicians, researchers and bereaved parents when the PLRG was successful in their bid to host the International Stillbirth Alliance (ISA) annual conference in 2017. Over 400 hundred delegates from around the globe attended the conference, of which one quarter were bereaved parents. Establishing a culture of collaboration, support and mutual respect in the field of pregnancy loss, requires scientists, clinicians and parents to be brought together so each can be informed by the other in the efforts to prevent stillbirth and improve bereavement care. As part of ISA 2017 conference, a sub-committee of staff and parents was established to ensure that the voice of parents could contribute to the research agenda and developments in clinical and bereavement care. A creative workshop specifically for parents, followed by a parent assembly were organised to facilitate this. Remembrance activities, organised by the parent committee, were central to the conference and actively engaged in by parents, clinicians and researchers. This commentary, written collaboratively by a parent, a chaplain, a bereavement and loss specialist midwife and a consultant obstetrician, gives voice to this experience, identifying four key messages that arose from our reflection on the conference. These include; the value of active partnership between clinicians and patients, the use of creativity as a unifying expression of grief and as a means to facilitate learning, the value of collaboration with global stakeholders in raising awareness about stillbirth, and the importance of facilitating meaningful patient/public engagement in scientific research. The potential for education and learning opportunities are also explored, highlighting the connection between parents, researchers and clinicians as central stakeholders in the prevention of stillbirth and in improving bereavement care.

University students' awareness of causes and risk factors of miscarriage: a cross-sectional study.

BACKGROUND: Spontaneous miscarriage is the most common complication of pregnancy, occurring in up to 20% of pregnancies. Despite the prevalence of miscarriage, little is known regarding peoples' awareness and understanding of causes of pregnancy loss. The aim of this study was to explore university students' understanding of rates, causes and risk factors of miscarriage. METHODS: A cross-sectional study including university students. An online questionnaire was circulated to all students at the University College Cork using their university email accounts in April and May 2016. Main outcomes included identification of prevalence, weeks of gestation at which miscarriage occurs and causative risk factors for miscarriage. RESULTS: A sample of 746 students were included in the analysis. Only 20% (n = 149) of students correctly identified the prevalence of miscarriage, and almost 30% (n = 207) incorrectly believed that miscarriage occurs in less than 10% of pregnancies. Female were more likely to correctly identify the rate of miscarriage than men (21.8% versus 14.5%). However, men tended to underestimate the rate and females overestimate it. Students who did not know someone who had a miscarriage underestimated the rate of miscarriage, and those who were aware of some celebrities who had a miscarriage overestimated the rate. Almost 43% (n = 316) of students correctly identified fetal chromosomal abnormalities as the main cause of miscarriage. Females, older students, those from Medical and Health disciplines and those who were aware of a celebrity who had a miscarriage were more likely to identify chromosomal abnormalities as a main cause. However, more than 90% of the students believed that having a fall, consuming drugs or the medical condition of the mother was a causative risk factor for miscarriage. Finally, stress was identified as a risk factor more frequently than advanced maternal age or smoking. CONCLUSION: Although almost half of the participants identified chromosomal abnormalities as the main cause of miscarriage, there is still a lack of understanding about the prevalence and most important risk factors among university students. University represents an ideal opportunity for health promotion strategies to increase awareness of potential adverse outcomes in pregnancy.

Microglial repopulation resolves inflammation and promotes brain recovery after injury.

Microglia mediate chronic neuroinflammation following central nervous system (CNS) disease or injury, and in doing so, damage the local brain environment by impairing recovery and contributing to disease processes. Microglia are critically dependent on signaling through the colony-stimulating factor 1 receptor (CSF1R) and can be eliminated via administration of CSF1R inhibitors. Resolving chronic neuroinflammation represents a universal goal for CNS disorders, but long-term microglial elimination may not be amenable to clinical use. Notably, withdrawal of CSF1R inhibitors stimulates new microglia to fully repopulate the CNS, affording an opportunity to renew this cellular compartment. To that end, we have explored the effects of acute microglial elimination, followed by microglial repopulation, in a mouse model of extensive neuronal loss. Neuronal loss leads to a prolonged neuroinflammatory response, characterized by the presence of swollen microglia expressing CD68 and CD45, as well as elevated levels of cytokines, chemokines, complement, and other inflammatory signals. These collective responses are largely resolved by microglial repopulation. Furthermore, microglial repopulation promotes functional recovery in mice, with elevated plus maze performance matching that of uninjured mice, despite the loss of 80% of hippocampal neurons. Analyses of synaptic surrogates revealed increases in PSD95 and synaptophysin puncta with microglial repopulation, suggesting that these cells sculpt and regulate the synaptic landscape. Thus, our results show that short-term microglial elimination followed by repopulation may represent a clinically feasible and novel approach to resolve neuroinflammatory events and promote brain recovery.

Eliminating microglia in Alzheimer's mice prevents neuronal loss without modulating amyloid-ß pathology.

In addition to amyloid-ß plaque and tau neurofibrillary tangle deposition, neuroinflammation is considered a key feature of Alzheimer's disease pathology. Inflammation in Alzheimer's disease is characterized by the presence of reactive astrocytes and activated microglia surrounding amyloid plaques, implicating their role in disease pathogenesis. Microglia in the healthy adult mouse depend on colony-stimulating factor 1 receptor (CSF1R) signalling for survival, and pharmacological inhibition of this receptor results in rapid elimination of nearly all of the microglia in the central nervous system. In this study, we set out to determine if chronically activated microglia in the Alzheimer's disease brain are also dependent on CSF1R signalling, and if so, how these cells contribute to disease pathogenesis. Ten-month-old 5xfAD mice were treated with a selective CSF1R inhibitor for 1 month, resulting in the elimination of ∼80% of microglia. Chronic microglial elimination does not alter amyloid-ß levels or plaque load; however, it does rescue dendritic spine loss and prevent neuronal loss in 5xfAD mice, as well as reduce overall neuroinflammation. Importantly, behavioural testing revealed improvements in contextual memory. Collectively, these results demonstrate that microglia contribute to neuronal loss, as well as memory impairments in 5xfAD mice, but do not mediate or protect from amyloid pathology.

Elimination of Microglia Improves Functional Outcomes Following Extensive Neuronal Loss in the Hippocampus.

With severe injury or disease, microglia become chronically activated and damage the local brain environment, likely contributing to cognitive decline. We previously discovered that microglia are dependent on colony-stimulating factor 1 receptor (CSF1R) signaling for survival in the healthy adult brain, and we have exploited this dependence to determine whether such activated microglia contribute deleteriously to functional recovery following a neuronal lesion. Here, we induced a hippocampal lesion in mice for 25 d via neuronal expression of diphtheria toxin A-chain, producing both a neuroinflammatory reaction and behavioral alterations. Following the 25 d lesion, we administered PLX3397, a CSF1R inhibitor, for 30 d to eliminate microglia. This post-lesion treatment paradigm improved functional recovery on elevated plus maze and Morris water maze, concomitant with reductions in elevated proinflammatory molecules, as well as normalization of lesion-induced alterations in synaptophysin and PSD-95. Further exploration of the effects of microglia on synapses in a second cohort of mice revealed that dendritic spine densities are increased with long-term microglial elimination, providing evidence that microglia shape the synaptic landscape in the adult mouse brain. Furthermore, in these same animals, we determined that microglia play a protective role during lesioning, whereby neuronal loss was potentiated in the absence of these cells. Collectively, we demonstrate that microglia exert beneficial effects during a diphtheria toxin-induced neuronal lesion, but impede recovery following insult. SIGNIFICANCE STATEMENT: It remains unknown to what degree, and by what mechanisms, chronically activated microglia contribute to cognitive deficits associated with brain insults. We induced a genetic neuronal lesion in mice for 25 d and found activated microglia to increase inflammation, alter synaptic surrogates, and impede behavioral recovery. These lesion-associated deficits were ameliorated with subsequent microglial elimination, underscoring the importance of developing therapeutics aimed at eliminating/modulating chronic microglial activation. Additionally, we found long-term microglial depletion globally increases dendritic spines by ∼35% in the adult brain, indicating that microglia continue to sculpt the synaptic landscape in the postdevelopmental brain under homeostatic conditions. Microglial manipulation can therefore be used to investigate the utility of increasing dendritic spine numbers in postnatal conditions displaying synaptic aberrations.

Colony-stimulating factor 1 receptor signaling is necessary for microglia viability, unmasking a microglia progenitor cell in the adult brain.

The colony-stimulating factor 1 receptor (CSF1R) is a key regulator of myeloid lineage cells. Genetic loss of the CSF1R blocks the normal population of resident microglia in the brain that originates from the yolk sac during early development. However, the role of CSF1R signaling in microglial homeostasis in the adult brain is largely unknown. To this end, we tested the effects of selective CSF1R inhibitors on microglia in adult mice. Surprisingly, extensive treatment results in elimination of ∼99% of all microglia brain-wide, showing that microglia in the adult brain are physiologically dependent upon CSF1R signaling. Mice depleted of microglia show no behavioral or cognitive abnormalities, revealing that microglia are not necessary for these tasks. Finally, we discovered that the microglia-depleted brain completely repopulates with new microglia within 1 week of inhibitor cessation. Microglial repopulation throughout the CNS occurs through proliferation of nestin-positive cells that then differentiate into microglia.

Age-related downregulation of the CaV3.1 T-type calcium channel as a mediator of amyloid beta production.

Alzheimer's is a crippling neurodegenerative disease that largely affects aged individuals. Decades of research have highlighted age-related changes in calcium homeostasis that occur before and throughout the duration of the disease, and the contributions of such dysregulation to Alzheimer's disease pathogenesis. We report an age-related decrease in expression of the CaV3.1 T-type calcium channel at the level of messenger RNA and protein in both humans and mice that is exacerbated with the presence of Alzheimer's disease. Downregulating T-type calcium channels in N2a cells and the 3xTg-AD mouse model of Alzheimer's disease, by way of pharmacologic inhibition with NNC-55-0396, results in a rapid increase in amyloid beta production via reductions in non-amyloidogenic processing, whereas genetic overexpression of the channel in human embryonic kidney cells expressing amyloid precursor protein produces complementary effects. The age-related decline in CaV3.1 expression may therefore contribute to a pro-amyloidogenic environment in the aging brain and represents a novel opportunity to intervene in the course of Alzheimer's disease pathogenesis.

Ethanol-induced conditioned partner preference in female mice.

Drinking behavior and social context are intimately intertwined. Peer relations can promote drinking. Conversely, alcohol promotes social interaction. The present study tested female mice for ethanol-induced conditioned partner preference. Ovariectomized (OVX) C57Bl/6 females with chronic estradiol replacement (OVX+E) received saline or ethanol (1, 2 or 4 g/kg) ip and were paired 4 × for 30 min each with 1 of 2 stimulus females. The test female was paired with the CS- stimulus female following saline, and was paired with the CS+ female following ethanol. After pairing, we measured proximity of the test female to the CS+ and CS- females in a 10' test. In a second study, OVX and OVX+E females were tested for conditioned partner preference (CS+ vs. CS-) in response to 2.5 g/kg ethanol. In separate groups of mice, both test and stimulus females (IS+) received ethanol during pairing to determine if test mice develop conditioned partner preference for another intoxicated mouse. OVX+E test females showed conditioned partner preference for the CS+ female in response to ethanol at 2g/kg (change in preference score for CS+: +86.6 ± 30.0 s/10 min), but not at 0, 1 or 4 g/kg. At 2.5 g/kg ethanol, OVX+E females developed conditioned partner preference for either IS+ (+63.6 ± 24.0 s) or CS+ females (+93.8 ± 27.1 s). OVX test females demonstrated ethanol-induced conditioned partner preference only for the IS+ female (+153.8 ± 32.0 s). These data demonstrate that ethanol promotes social preference in female mice, and that estradiol enhances this effect.