Dysregulated Purinergic Signalling in Fragile X Syndrome Cortical Astrocytes.

The symptoms of fragile X syndrome (FXS), caused by a single gene mutation to Fmr1, have been increasingly linked to disordered astrocyte signalling within the cerebral cortex. We have recently demonstrated that the purinergic signalling pathway, which utilizes nucleoside triphosphates and their metabolites to facilitate bidirectional glial and glial-neuronal interactions, is upregulated in cortical astrocytes derived from the Fmr1 knockout (KO) mouse model of FXS. Heightened Fmr1 KO P2Y purinergic receptor levels were correlated with prolonged intracellular calcium release, elevated synaptogenic protein secretion, and hyperactivity of developing circuits. However, due to the relative lack of sensitive and reproducible quantification methods available for measuring purines and pyrimidines, determining the abundance of these factors in Fmr1 KO astrocytes was limited. We therefore developed a hydrophilic interaction liquid chromatography protocol coupled with mass spectrometry to compare the abundance of intracellular and extracellular purinergic molecules between wildtype and Fmr1 KO mouse astrocytes. Significant differences in the concentrations of UDP, ATP, AMP, and adenosine intracellular stores were found within Fmr1 KO astrocytes relative to WT. The extracellular level of adenosine was also significantly elevated in Fmr1 KO astrocyte-conditioned media in comparison to media collected from WT astrocytes. Glycosylation of the astrocyte membrane-bound CD39 ectonucleotidase, which facilitates ligand breakdown following synaptic release, was also elevated in Fmr1 KO astrocyte cultures. Together, these differences demonstrated further dysregulation of the purinergic signalling system within Fmr1 KO cortical astrocytes, potentially leading to significant alterations in FXS purinergic receptor activation and cellular pathology.

P2X7 expression patterns in the developing Fmr1-knockout mouse hippocampus.

Fragile-X Syndrome (FXS) is the leading monogenetic cause of intellectual disability among children but remains without a cure. Using the Fmr1 KO mouse model of FXS, much work has been done to understand FXS hippocampus dysfunction. Purinergic signaling, where ATP and its metabolites are used as signaling molecules, participates in hippocampus development, but it is unknown if purinergic signaling is affected in the developing Fmr1 KO hippocampus. In our study, we characterized the purinergic receptor P2X7. We first found that P2X7 was reduced in Fmr1 KO whole hippocampus tissue at P14 and P21, corresponding to the periods of neurite outgrowth and synaptic refinement in the hippocampus. We then evaluated the cell-specific expression of P2X7 with immunofluorescence and found differences between WT and Fmr1 KO mice in P2X7 colocalization with hippocampal microglia and neurons. P2X7 colocalized more with microglia at P14 and P21, but there was a sex-specific reduction in P2X7 colocalization with neurons. In contrast, male mice at P14 and P21 showed reduced neuronal P2X7 colocalization compared to females, but only females showed reduced absolute neuronal P2X7 expression across the dorsal hippocampal formation. Together, our results suggest that P2X7 expression is altered during Fmr1-KO hippocampal development, potentially influencing several developmental processes in the Fmr1-KO hippocampus formation.

Cohort profile: DNA methylation in the Northern Ireland Cohort for the Longitudinal Study of Ageing (NICOLA) - recruitment and participant characteristics.

PURPOSE: Epigenetic modifications including DNA methylation (DNAm) are proposed mechanisms by which social or environmental exposures may influence health and behaviours as we age. The Northern Ireland Cohort for the Longitudinal Study of Ageing (NICOLA) DNAm cohort, established in 2013, is one of several worldwide, nationally representative prospective studies of ageing with biological samples from participants who consented to multiomic analysis. PARTICIPANTS: NICOLA recruited 8478 participants (8283 aged 50 years or older and 195 spouses or partners at the same address aged under 50 years). Computer-Assisted Personal Interviews, Self-Completion Questionnaires and detailed Health Assessments (HA) were completed. Of the 3471 (44.1%) participants who attended the HA in wave 1, which included venous blood sampling, 2000 were identified for the DNAm cohort. Following technical and data quality control checks, DNAm data are currently available for n=1870. FINDINGS TO DATE: There was no significant difference based on age, self-reported gender, education, employment, smoking or alcohol status and subjective health reports between the DNAm cohort and other HA attendees. Participants were more likely to be in the DNAm group if they lived with one other person (OR 1.26, 95% CI 1.07 to 1.49). The DNAm group had a lower proportion of depressed participants and those meeting criteria for post-traumatic stress disorder (11.7% and 4.4% vs 13.5% and 4.5%, respectively) categorised by objective assessment tools but this was not significant (OR 0.84, 95% CI 0.69 to 1.02 and OR 0.87, 95% CI 0.64 to 1.19). FUTURE PLANS: The deeply phenotyped DNAm cohort in NICOLA with planned prospective follow-up and additional multiomic data releases will increase the cohort's utility for research into ageing. The genomic and epigenetic data for the DNAm cohort has been deposited on the European Genome-Phenome Archive, increasing the profile of this cohort and data availability to researchers.

Evaluating urine volume and host depletion methods to enable genome-resolved metagenomics of the urobiome.

Background: The gut microbiome has emerged as a clear player in health and disease, in part by mediating host response to environment and lifestyle. The urobiome (microbiota of the urinary tract) likely functions similarly. However, efforts to characterize the urobiome and assess its functional potential have been limited due to technical challenges including low microbial biomass and high host cell shedding in urine. Here, to begin addressing these challenges, we evaluate urine sample volume (100 ml - 5 mL), and host DNA depletion methods and their effects on urobiome profiles in healthy dogs, which are a robust large animal model for the human urobiome. We collected urine from seven dogs and fractionated samples into aliquots. One set of samples was spiked with host (canine) cells to model a biologically relevant host cell burden in urine. Samples then underwent DNA extraction followed by 16S rRNA gene and shotgun metagenomic sequencing. We then assembled metagenome assembled genomes (MAGs) and compared microbial composition and diversity across groups. We tested six methods of DNA extraction: QIAamp BiOstic Bacteremia (no host depletion), QIAamp DNA Microbiome, Molzym MolYsis, NEBNext Microbiome DNA Enrichment, Zymo HostZERO, and Propidium Monoazide. Results: In relation to urine sample volume, 3 3.0 mL resulted in the most consistent urobiome profiling. In relation to host depletion, individual (dog) but not extraction method drove overall differences in microbial composition. DNA Microbiome yielded the greatest microbial diversity in 16S rRNA sequencing data and shotgun metagenomic sequencing data, and maximized MAG recovery while effectively depleting host DNA in host-spiked urine samples. As proof-of-principle, we then mined MAGs for core metabolic functions and environmental chemical metabolism. We identified long chain alkane utilization in two of the urine MAGs. Long chain alkanes are common pollutants that result from industrial combustion processes and end up in urine. Conclusions: This is the first study, to our knowledge, to demonstrate environmental chemical degradation potential in urine microbes through genome-resolved metagenomics. These findings provide guidelines for studying the urobiome in relation to sample volume and host depletion, and lay the foundation for future evaluation of urobiome function in relation to health and disease.

Local Health Equity Action Teams (LHEATS) as a Novel and Emerging Practice of the Communities Organizing to Promote Equity (COPE) Project in Kansas.

The Communities Organizing to Promote Equity (COPE) Project was implemented in 20 counties across Kansas to build capacity to address health equity by forming local health equity action teams (LHEATS), hiring and training community health workers, facilitating state-wide learning collaboratives, and tailoring communication strategies. We conducted interviews and focus groups with project stakeholders who identified pragmatic recommendations related to LHEAT formation and leadership, establishing trust, nurturing autonomy, and optimizing impact. Insights can improve future community-based health equity efforts. (Am J Public Health. Published online ahead of print August 28, 2024:e1-e5. https://doi.org/10.2105/AJPH.2024.307802).

Communities organizing to promote equity: engaging local communities in public health responses to health inequities exacerbated by COVID-19-protocol paper.

Background: The COVID-19 pandemic has disproportionately impacted rural and under-resourced urban communities in Kansas. The state's response to COVID-19 has relied on a highly decentralized and underfunded public health system, with 100 local health departments in the state, few of which had prior experience engaging local community coalitions in a coordinated response to a public health crisis. Methods: To improve the capacity for local community-driven responses to COVID-19 and other public health needs, the University of Kansas Medical Center, in partnership with the Kansas Department of Health and Environment, will launch Communities Organizing to Promote Equity (COPE) in 20 counties across Kansas. COPE will establish Local Health Equity Action Teams (LHEATs), coalitions comprised of community members and service providers, who work with COPE-hired community health workers (CHWs) recruited to represent the diversity of the communities they serve. CHWs in each county are tasked with addressing unmet social needs of residents and supporting their county's LHEAT. LHEATs are charged with implementing strategies to improve social determinants of health in their county. Monthly, LHEATs and CHWs from all 20 counties will come together as part of a learning collaborative to share strategies, foster innovation, and engage in peer problem-solving. These efforts will be supported by a multilevel communications strategy that will increase awareness of COPE activities and resources at the local level and successes across the state. Our mixed methods evaluation design will assess the processes and impact of COPE activities as well as barriers and facilitators to implementation using aspects of both the Consolidated Framework for Implementation Research (CFIR) and Reach, Effectiveness, Adoption, Implementation and Maintenance (RE-AIM) models. Discussion: This protocol is designed to expand community capacity to strategically partner with local public health and social service partners to prioritize and implement health equity efforts. COPE intentionally engages historically resilient communities and those living in underserved rural areas to inform pragmatic strategies to improve health equity.

A Novel Electronic Record System for Documentation and Efficient Workflow for Community Health Workers: Development and Usability Study.

BACKGROUND: The COVID-19 pandemic added to the decades of evidence that public health institutions are routinely stretched beyond their capacity. Community health workers (CHWs) can be a crucial extension of public health resources to address health inequities, but systems to document CHW efforts are often fragmented and prone to unneeded redundancy, errors, and inefficiency. OBJECTIVE: We sought to develop a more efficient data collection system for recording the wide range of community-based efforts performed by CHWs. METHODS: The Communities Organizing to Promote Equity (COPE) project is an initiative to address health disparities across Kansas, in part, through the deployment of CHWs. Our team iteratively designed and refined the features of a novel data collection system for CHWs. Pilot tests with CHWs occurred over several months to ensure that the functionality supported their daily use. Following implementation of the database, procedures were set to sustain the collection of feedback from CHWs, community partners, and organizations with similar systems to continually modify the database to meet the needs of users. A continuous quality improvement process was conducted monthly to evaluate CHW performance; feedback was exchanged at team and individual levels regarding the continuous quality improvement results and opportunities for improvement. Further, a 15-item feedback survey was distributed to all 33 COPE CHWs and supervisors for assessing the feasibility of database features, accessibility, and overall satisfaction. RESULTS: At launch, the database had 60 active users in 20 counties. Documented client interactions begin with needs assessments (modified versions of the Arizona Self-sufficiency Matrix and PRAPARE [Protocol for Responding to and Assessing Patient Assets, Risks, and Experiences]) and continue with the longitudinal tracking of progress toward goals. A user-specific automated alerts-based dashboard displays clients needing follow-up and upcoming events. The database contains over 55,000 documented encounters across more than 5079 clients. Available resources from over 2500 community organizations have been documented. Survey data indicated that 84% (27/32) of the respondents considered the overall navigation of the database as very easy. The majority of the respondents indicated they were overall very satisfied (14/32, 44%) or satisfied (15/32, 48%) with the database. Open-ended responses indicated the database features, documentation of community organizations and visual confirmation of consent form and data storage on a Health Insurance Portability and Accountability Act-compliant record system, improved client engagement, enrollment processes, and identification of resources. CONCLUSIONS: Our database extends beyond conventional electronic medical records and provides flexibility for ever-changing needs. The COPE database provides real-world data on CHW accomplishments, thereby improving the uniformity of data collection to enhance monitoring and evaluation. This database can serve as a model for community-based documentation systems and be adapted for use in other community settings.

Purinergic Signalling Mediates Aberrant Excitability of Developing Neuronal Circuits in the Fmr1 Knockout Mouse Model.

Neuronal hyperexcitability within developing cortical circuits is a common characteristic of several heritable neurodevelopmental disorders, including Fragile X Syndrome (FXS), intellectual disability and autism spectrum disorders (ASD). While this aberrant circuitry is typically studied from a neuron-centric perspective, glial cells secrete soluble factors that regulate both neurite extension and synaptogenesis during development. The nucleotide-mediated purinergic signalling system is particularly instrumental in facilitating these effects. We recently reported that within a FXS animal model, the Fmr1 KO mouse, the purinergic signalling system is upregulated in cortical astrocytes leading to altered secretion of synaptogenic and plasticity-related proteins. In this study, we examined whether elevated astrocyte purinergic signalling also impacts neuronal morphology and connectivity of Fmr1 KO cortical neurons. Here, we found that conditioned media from primary Fmr1 KO astrocytes was sufficient to enhance neurite extension and complexity of both wildtype and Fmr1 KO neurons to a similar degree as UTP-mediated outgrowth. Significantly enhanced firing was also observed in Fmr1 KO neuron-astrocyte co-cultures grown on microelectrode arrays but was associated with large deficits in firing synchrony. The selective P2Y2 purinergic receptor antagonist AR-C 118925XX effectively normalized much of the aberrant Fmr1 KO activity, designating P2Y2 as a potential therapeutic target in FXS. These results not only demonstrate the importance of astrocyte soluble factors in the development of neural circuitry, but also show that P2Y purinergic receptors play a distinct role in pathological FXS neuronal activity.

P2X7 regulates ependymo-radial glial cell proliferation in adult Danio rerio following spinal cord injury.

In contrast to mammals, zebrafish undergo successful neural regeneration following spinal cord injury. Spinal cord ependymo-radial glia (ERG) undergo injury-induced proliferation and neuronal differentiation to replace damaged cells and restore motor function. However, the molecular cues driving these processes remain elusive. Here, we demonstrate that the evolutionarily conserved P2X7 receptors are widely distributed on neurons and ERG within the zebrafish spinal cord. At the protein level, the P2X7 receptor expressed in zebrafish is a truncated splice variant of the full-length variant found in mammals. The protein expression of this 50 kDa isoform was significantly downregulated at 7 days post-injury (dpi) but returned to basal levels at 14 dpi when compared to naïve controls. Pharmacological activation of P2X7 following SCI resulted in a greater number of proliferating cells around the central canal by 7 dpi but did not affect neuronal differentiation at 14 dpi. Our findings suggest that unlike in mammals, P2X7 signaling may not play a maladaptive role following SCI in adult zebrafish and may also work to curb the proliferative response of ERG following injury.

A Phase I Trial of Allogeneic γδ T Lymphocytes From Haploidentical Donors in Patients With Refractory or Relapsed Acute Myeloid Leukemia.

Introduction We report the results of a phase I clinical trial NCT03790072 of an adoptive transfer of γδ T lymphocytes from haploidentical donors in patients with refractory/relapsed acute myeloid leukemia after lymphodepletion regimen. Patients and methods Healthy donor mononuclear cells collected by leukapheresis were consistently expanded to generate products of 109 to 1010 γδ T cells. Seven patients received donor-derived T cell product at doses of 106/kg (n = 3), 107/kg (n = 3), and 108/kg (n = 1). Results Four patients had bone marrow evaluation at day 28. One patient had a complete remission, one was classified as morphologic leukemia-free state, one had stable disease and one had no evidence of response. In one patient, there was evidence of disease control with repeat infusions up to 100 days after first dosing. There were no treatment-related serious adverse events or treatment-related Common Terminology Criteria for Adverse Events grade 3 or greater toxicities at any dose level. Allogeneic Vγ9Vδ2 T cell infusion was shown to be safe and feasible up to a cell dose of 108/kg. Discussion In agreement with previously published studies, the infusion of allogeneic Vγ9Vδ2 cells was safe. The contribution of lymphodepleting chemotherapy to responses seen cannot be ruled out. Main limitation of the study is the low number of patients and interruption due to COVID-19 pandemic. Conclusion These positive Phase 1 results support progression to phase II clinical trials.

Northern Ireland Cohort for the Longitudinal Study of Ageing (NICOLA): health assessment protocol, participant profile and patterns of participation.

BACKGROUND: The Northern Ireland Cohort for the Longitudinal Study of Ageing (NICOLA) is a prospective, longitudinal study of a representative cohort of older adults living in Northern Ireland, United Kingdom. Its aim is to explore the social, behavioural, economic and biological factors of ageing and how these factors change as people age. The study has been designed to maximize comparability with other international studies of ageing thereby facilitating cross-country comparisons. This paper provides an overview of the design and methodology of the health assessment which was carried out as part of Wave 1. METHODS: Three thousand, six hundred and fifty five community dwelling adults, aged 50 years and over participated in the health assessment as part of Wave 1 of NICOLA. The health assessment included a battery of measurements across various domains that addressed key indicators of ageing namely: physical function, vision and hearing, cognitive function, and cardiovascular health. This manuscript describes the scientific rationale for the choice of assessments, provides an overview of the core objective measures carried out in the health assessment and describes the differences in characteristics of participants who took part in the health assessment compared to those who did not take part. RESULTS: The manuscript highlights the importance of incorporating objective measures of health in population based studies as a means of complementing subjective measures and as a way to advance our understanding of the ageing process. The findings contextualize NICOLA as a data resource within Dementias Platform UK (DPUK), the Gateway to Global Ageing (G2G) and other existing networks of population based longitudinal studies of ageing. CONCLUSION: This manuscript can help inform design considerations for other population based studies of ageing and facilitate cross-country comparative analysis of key life-course factors affecting healthy ageing such as educational attainment, diet, the accumulation of chronic conditions (including Alzheimer's disease, dementia and cardiovascular disease) as well as welfare and retirement policies.

Single-cell transcriptomic analysis of neuroepithelial cells and other cell types of the gills of zebrafish (Danio rerio) exposed to hypoxia.

The fish gill is a multifunctional organ involved in numerous physiological processes, such as gas exchange and sensing of hypoxia by respiratory chemoreceptors, called neuroepithelial cells (NECs). Many studies have focused on zebrafish (Danio rerio) to investigate the structure, function and development of the gills, yet the transcriptomic profile of most gill cells remains obscure. We present the results of a comprehensive transcriptomic analysis of the gills of zebrafish using single-cell RNA sequencing (scRNA-seq). Gill cells from ETvmat2:EGFP zebrafish were individually labelled before scRNA-seq library construction using 10× Genomics Chromium technology. 12,819 cells were sequenced with an average depth of over 27,000 reads per cell. We identified a median of 485 genes per cell and 16 cell clusters, including NECs, neurons, pavement cells, endothelial cells and mitochondrion-rich cells. The identity of NECs was confirmed by expression of slc18a2, encoding the vesicular monoamine transporter, Vmat2. Highly differentially-expressed genes in NECs included tph1a, encoding tryptophan hydroxylase, sv2 (synaptic vesicle protein), and proteins implicated in O2 sensing (ndufa4l2a, cox8al and epas1a). In addition, NECs and neurons expressed genes encoding transmembrane receptors for serotonergic, cholinergic or dopaminergic neurotransmission. Differential expression analysis showed a clear shift in the transcriptome of NECs following 14 days of acclimation to hypoxia. NECs in the hypoxia group showed high expression of genes involved in cell cycle control and proliferation. The present article provides a complete cell atlas for the zebrafish gill and serves as a platform for future studies investigating the molecular biology and physiology of this organ.

Purinergic signaling systems across comparative models of spinal cord injury.

Within the last several decades, the scientific community has made substantial progress in elucidating the complex pathophysiology underlying spinal cord injury. However, despite the many advances using conventional mammalian models, both cellular and axonal regeneration following spinal cord injury have remained out of reach. In this sense, turning to non-mammalian, regenerative species presents a unique opportunity to identify pro-regenerative cues and characterize a spinal cord microenvironment permissive to re-growth. Among the signaling pathways hypothesized to be dysregulated during spinal cord injury is the purinergic signaling system. In addition to its well-known role as energy currency in cells, ATP and its metabolites are small molecule neurotransmitters that mediate many diverse cellular processes within the central nervous system. While our understanding of the roles of the purinergic system following spinal cord injury is limited, this signaling pathway has been implicated in all injury-induced secondary processes, including cellular death, inflammation, reactive gliosis, and neural regeneration. Given that the purinergic system is also evolutionarily conserved between mammalian and non-mammalian species, comparisons of these roles may provide important insights into conditions responsible for recovery success. Here, we compare the secondary processes between key model species and the influence of purinergic signaling in each context. As our understanding of this signaling system and pro-regenerative conditions continues to evolve, so does the potential for the development of novel therapeutic interventions for spinal cord injury.

Comparative genomic analysis of the first Ehrlichia canis detections in Australia.

Ehrlichia canis (Rickettsiales; Anaplasmataceae) is one of the most prevalent tick-borne pathogens of dogs globally. The bacterium infects monocytes and is the aetiological agent of canine monocytic ehrlichiosis. For many decades Australia was thought to be free of the pathogen, but this abruptly changed in May 2020 when E. canis was detected in several dogs from Kununurra, Western Australia. Subsequent surveillance activities found unexpectedly large scale spread of E. canis throughout much of northern Australia. To gain insight into the genetic relationships of the Australian strain and its potential origin, we undertook a genomic analysis of E. canis positive domestic dog and tick (Rhipicephalus linnaei) samples from the north of Western Australia, the far north of South Australia and the Northern Territory, covering thousands of square kilometres. We obtained complete E. canis genomes from each of the three states, plus an additional 16 partial genomes, substantially increasing publicly available E. canis genetic resources. The Australian E. canis genomes were highly conserved across large geographic distances. Outside of Australia, the genomes were most similar to E. canis YZ-1 from China, although few reference sequences were available. We analysed the variable trp36 gene to obtain greater phylogenetic signal, which demonstrated that the Australian E. canis belonged to the Taiwan genotype, comprised of samples from Taiwan, China, Thailand and Turkey. Taken together, our findings suggest that E. canis in Australia may have originated from Asia or the Middle East and spread throughout northern and central Australia following its introduction.

Mitochondrial bioenergetics of astrocytes in Fragile X syndrome: new perspectives on culture conditions and sex effects.

Fragile X syndrome (FXS) is a genetic disorder that is characterized by a range of cognitive and behavioral deficits, including mild-moderate intellectual disability. The disease is characterized by an X-linked mutation of the Fmr1 gene, which causes silencing of the gene coding for fragile X mental retardation protein (FMRP), a translational regulator integral for neurodevelopment. Mitochondrial dysfunction has been recently associated with FXS, with reports of increases in oxidative stress markers, reactive oxygen species, and lipid peroxidation being present in the brain tissue. Astrocytes, a prominent glial cell within the central nervous system (CNS), play a large role in regulating oxidative homeostasis within the developing brain and dysregulation of astrocyte redox balance in FXS, which may contribute to oxidative stress. Astrocyte function and mitochondrial bioenergetics are significantly influenced by oxygen availability and circulating sex hormones; yet, these parameters are rarely considered during in vitro experimentation. Given that the brain normally develops in a range of hypoxic conditions and FXS is a sex-linked genetic disorder, we investigated how different oxygen levels (normoxic vs. hypoxic) and biological sex affected mitochondrial bioenergetics of astrocytes in FXS. Our results demonstrate that both mitochondrial respiration capacity and reactive oxygen species emission are altered with Fmr1 deletion in astrocytes and these changes were dependent upon both sexual dimorphism and oxygen availability.

Converging purinergic and immune signaling pathways drive IL-6 secretion by Fragile X cortical astrocytes via STAT3.

The symptoms of Fragile X syndrome (FXS) are driven in part by abnormal glial-mediated function. FXS astrocytes release elevated levels of immune-related factors interleukin-6 (IL-6) and tenascin C (TNC), and also demonstrate increased purinergic signaling, a pathway linked to signaling factor release. Here, in cortical astrocytes from the Fmr1 knockout (KO) FXS mouse model, purinergic agonism enhanced TNC secretion and STAT3 phosphorylation, two processes linked to elevated IL-6 secretion in FXS, while STAT3 knockdown and TLR4 antagonism normalized Fmr1 KO IL-6 release. We therefore suggest that purinergic signaling and immune regulatory pathways converge to drive FXS cortical pro-inflammatory responses.

Astrocyte-mediated disruption of ROS homeostasis in Fragile X mouse model.

Astrocytes, glial cells within the brain, work to protect neurons during high levels of activity by maintaining oxidative homeostasis via regulation of energy supply and antioxidant systems. In recent years, mitochondrial dysfunction has been highlighted as an underlying factor of pathology in many neurological disorders. In animal studies of Fragile X Syndrome (FXS), the leading genetic cause of autism, higher levels of reactive oxygen species, lipid peroxidation, and protein oxidation within the brain indicates that mitochondria function is also altered in FXS. Despite their integral contribution to redox homeostasis within the CNS, the role of astrocytes on the occurrence or progression of neurodevelopmental disorders in this way is rarely considered. This study specifically examines changes to astrocyte mitochondrial function and antioxidant expression that may occur in FXS. Using the Fmr1 knockout (KO) mouse model, mitochondrial respiration and reactive oxygen species (ROS) emission were analyzed in primary cortical astrocytes. While mitochondrial respiration was similar between genotypes, ROS emission was significantly elevated in Fmr1 KO astrocytes. Notably, NADPH-oxidase 2 expression in Fmr1 KO astrocytes was also enhanced but only changes in catalase antioxidant enzyme expression were noted. Characterization of astrocyte factors involved in redox imbalance is invaluable to uncovering potential sources of oxidative stress in neurodevelopmental disorders and more specifically, the intercellular mechanisms that contribute to dysfunction in FXS.

Astrocyte-mediated purinergic signaling is upregulated in a mouse model of Fragile X syndrome.

Fragile X syndrome (FXS) is the leading monogenic cause of intellectual disability and autism spectrum disorders. With increasing investigation into the molecular mechanisms underlying FXS, there is growing evidence that perturbations in glial signaling are widely associated with neurological pathology. Purinergic signaling, which utilizes nucleoside triphosphates as signaling molecules, provides one of the most ubiquitous signaling systems for glial-neuronal and glial-glial crosstalk. Here, we sought to identify whether purinergic signaling is dysregulated within the FXS mouse cortex, and whether this dysregulation contributes to aberrant intercellular communication. In primary astrocyte cultures derived from the Fmr1 knockout (KO) mouse model of FXS, we found that application of exogenous ATP and UTP evoked elevated intracellular calcium responses compared to wildtype levels. Accordingly, purinergic P2Y2 and P2Y6 receptor expression was increased in Fmr1 KO astrocytes both in vitro and in acutely dissociated tissue, while P2Y antagonism via suramin prevented intracellular calcium elevations, suggesting a role for these receptors in aberrant FXS astrocyte activation. To investigate the impact of elevated purinergic signaling on astrocyte-mediated synaptogenesis, we quantified synaptogenic protein TSP-1, known to be regulated by P2Y activation. TSP-1 secretion and expression were both heightened in Fmr1 KO vs wildtype astrocytes following UTP application, while naïve TSP-1 cortical expression was also transiently elevated in vivo, indicating increased potential for excitatory TSP-1-mediated synaptogenesis in the FXS cortex. Together, our results demonstrate novel and significant purinergic signaling elevations in Fmr1 KO astrocytes, which may serve as a potential therapeutic target to mitigate the signaling aberrations observed in FXS.