Exploring microtubule dynamics in Alzheimer's disease: Longitudinal assessment using [11C]MPC-6827 PET imaging in rodent models of Alzheimer's-related pathology.

INTRODUCTION: Microtubule (MT) stability is crucial for proper neuronal function. Understanding MT dysregulation is critical for connecting amyloid beta (Aß) and tau-based degenerative events and early changes in presymptomatic Alzheimer's disease (AD). Herein we present positron emission tomography (PET) imaging properties of our MT-PET radiotracer, [11C]MPC-6827, in multiple established AD mouse models. METHODS: Longitudinal PET, biodistribution, autoradiography, immunohistochemistry, and behavioral studies were conducted at multiple time points in APPswe/PSEN1dE9 (APP/PS1), P301S-PS19 (P301S), 5xFAD, and age-matched control mice. RESULTS: Longitudinal [11C]MPC-6827 brain imaging showed significant increases in APP/PS1, P301S, and 5xFAD mice compared to controls. Longitudinal MT-PET correlated positively with biodistribution, autoradiography, and immunohistochemistry results and negatively with behavior data. DISCUSSION: Our study demonstrated significant longitudinal [11C]MPC-6827 PET increases in multiple AD mouse models for the first time. Strong correlations between PET and biomarker data underscored the interplay of MT destabilization, amyloid, and tau pathology in AD. These results suggest [11C]MPC-6827 PET as a promising tool for monitoring MT dysregulation early in AD progression. HIGHLIGHTS: Longitudinal positron emission tomography (PET) imaging studies using [11C]MPC-6827 in multiple established Alzheimer's disease (AD) mouse models revealed an early onset of microtubule dysregulation, with significant changes in brain radiotracer uptake evident from 2 to 4 months of age. Intra-group analysis showed a progressive increase in microtubule dysregulation with increasing AD burden, supported by significant correlations between PET imaging data and biodistribution, autoradiography, and molecular pathological markers. [11C]MPC-6827 PET imaging demonstrated its efficacy in detecting early microtubule alterations preceding observable behavioral changes in AD mouse models, suggesting its potential for early AD imaging. The inclusion of the 5xFAD mouse model further elucidated the impact of amyloid beta (Aß) toxicity on inducing tau hyperphosphorylation-mediated microtubule dysregulation, highlighting the versatility of [11C]MPC-6827 in delineating various aspects of AD pathology. Our study provides immediate clarity on high uptake of the microtubule-based radiotracer in AD brains in a longitudinal setting, which directly informs clinical utility in Aß/tau-based studies.

The E592K variant of SF3B1 creates unique RNA missplicing and associates with high-risk MDS without ring sideroblasts.

Among the most common genetic alterations in the myelodysplastic syndromes (MDS) are mutations in the spliceosome gene SF3B1. Such mutations induce specific RNA missplicing events, directly promote ring sideroblast (RS) formation, and generally associate with more favorable prognosis. However, not all SF3B1 mutations are the same, and little is known about how distinct hotspots influence disease. Here we report that the E592K variant of SF3B1 associates with high-risk disease features in MDS, including a lack of RS, increased myeloblasts, a distinct co-mutation pattern, and a lack of the favorable survival seen with other SF3B1 mutations. Moreover, compared to other hotspot SF3B1 mutations, E592K induces a unique RNA missplicing pattern, retains an interaction with the splicing factor SUGP1, and preserves normal RNA splicing of the sideroblastic anemia genes TMEM14C and ABCB7. These data have implications for our understanding of the functional diversity of spliceosome mutations, as well as the pathobiology, classification, prognosis, and management of SF3B1-mutant MDS.

An in vitro CRISPR screen of cell-free DNA identifies apoptosis as the primary mediator of cell-free DNA release.

ABTRACT: Clinical circulating cell-free DNA (cfDNA) testing is now routine, however test accuracy remains limited. By understanding the life-cycle of cfDNA, we might identify opportunities to increase test performance. Here, we profile cfDNA release across a 24-cell line panel and utilize a cell-free CRISPR screen (cfCRISPR) to identify mediators of cfDNA release. Our panel outlines two distinct groups of cell lines: one which releases cfDNA fragmented similarly to clinical samples and purported as characteristic of apoptosis, and another which releases larger fragments associated with vesicular or necrotic DNA. Our cfCRISPR screens reveal that genes mediating cfDNA release are primarily involved with apoptosis, but also identify other subsets of genes such as RNA binding proteins as potential regulators of cfDNA release. We observe that both groups of cells lines identified primarily produce cfDNA through apoptosis. These results establish the utility of cfCRISPR, genetically validate apoptosis as a major mediator of DNA release in vitro, and implicate ways to improve cfDNA assays.

"You gotta tell the camera": Advancing children's engineering learning opportunities through tinkering and digital storytelling.

This study addressed whether combining tinkering with digital storytelling (i.e., narrating and reflecting about experiences to an imagined audience) can engender engineering learning opportunities. Eighty-four families with 5- to 10-year-old (M = 7.69) children (48% female children; 57% White, 11% Asian, 6% Black) watched a video introducing a tinkering activity and were randomly assigned either to a digital storytelling condition or a no digital storytelling condition during tinkering. After tinkering, families reflected on their tinkering experience and were randomly assigned to either engage in digital storytelling or not. Children in the digital storytelling condition during tinkering spoke most to an imagined audience during tinkering, talked most about engineering at reflection, and remembered the most information about the experience weeks later.

A retrospective cohort study of the anesthetic management of postpartum tubal ligation.

BACKGROUND: Neuraxial anesthesia with reactivation of a labor epidural catheter is commonly utilized for postpartum tubal ligations (PPTL), although the optimal anesthetic approach is unknown. We assessed institutional anesthesia practices for PPTL, and evaluated the failure rates of reactivation of labor epidural catheters, de novo spinal anesthesia, and spinal anesthesia after failed blocks. METHODS: We conducted a single-center retrospective cohort analysis of 300 consecutive patients who underwent a PPTL and 100 having spinal anesthesia for cesarean delivery. Anesthetic management data (existing labor epidural catheter reactivation, de novo spinal anesthesia or general anesthesia) were collected from electronic medical records. Anesthetic block failure rates were determined for each anesthetic technique. RESULTS: The failure rate was 15% for de novo spinal anesthesia and 23% after failed reactivation of a labor epidural catheter or spinal anesthesia. The epidural catheter reactivation failure rate was 35%. The failure rate of spinal anesthesia for cesarean delivery was 4%. Drug dosage, epidural catheter use in labor, time since epidural catheter placement or delivery, labor neuraxial technique (combined spinal-epidural, epidural), supplemental top-up doses during labor, and anesthesiologist experience did not predict neuraxial anesthesia failures. CONCLUSIONS: Our analysis revealed an unexpectedly high neuraxial anesthesia failure rate even when de novo spinal anesthesia was used for PPTL. The results are consistent with other institutions' recent findings, and are higher than spinal anesthesia failure rates associated with cesarean delivery. Further studies are required to determine optimal anesthesia dosing strategies, and to understand the mechanisms behind high neuraxial anesthesia failures for PPTL.

De novo GRIN variants in M3 helix associated with neurological disorders control channel gating of NMDA receptor.

N-methyl-D-aspartate receptors (NMDARs) are members of the glutamate receptor family and participate in excitatory postsynaptic transmission throughout the central nervous system. Genetic variants in GRIN genes encoding NMDAR subunits are associated with a spectrum of neurological disorders. The M3 transmembrane helices of the NMDAR couple directly to the agonist-binding domains and form a helical bundle crossing in the closed receptors that occludes the pore. The M3 functions as a transduction element whose conformational change couples ligand binding to opening of an ion conducting pore. In this study, we report the functional consequences of 48 de novo missense variants in GRIN1, GRIN2A, and GRIN2B that alter residues in the M3 transmembrane helix. These de novo variants were identified in children with neurological and neuropsychiatric disorders including epilepsy, developmental delay, intellectual disability, hypotonia and attention deficit hyperactivity disorder. All 48 variants in M3 for which comprehensive testing was completed produce a gain-of-function (28/48) compared to loss-of-function (9/48); 11 variants had an indeterminant phenotype. This supports the idea that a key structural feature of the M3 gate exists to stabilize the closed state so that agonist binding can drive channel opening. Given that most M3 variants enhance channel gating, we assessed the potency of FDA-approved NMDAR channel blockers on these variant receptors. These data provide new insight into the structure-function relationship of the NMDAR gate, and suggest that variants within the M3 transmembrane helix produce a gain-of-function.

Therapeutic potential of N-methyl-D-aspartate receptor modulators in psychiatry.

N-methyl-D-aspartate (NMDA) receptors mediate a slow component of excitatory synaptic transmission, are widely distributed throughout the central nervous system, and regulate synaptic plasticity. NMDA receptor modulators have long been considered as potential treatments for psychiatric disorders including depression and schizophrenia, neurodevelopmental disorders such as Rett Syndrome, and neurodegenerative conditions such as Alzheimer's disease. New interest in NMDA receptors as therapeutic targets has been spurred by the findings that certain inhibitors of NMDA receptors produce surprisingly rapid and robust antidepressant activity by a novel mechanism, the induction of changes in the brain that well outlast the presence of drug in the body. These findings are driving research into an entirely new paradigm for using NMDA receptor antagonists in a host of related conditions. At the same time positive allosteric modulators of NMDA receptors are being pursued for enhancing synaptic function in diseases that feature NMDA receptor hypofunction. While there is great promise, developing the therapeutic potential of NMDA receptor modulators must also navigate the potential significant risks posed by the use of such agents. We review here the emerging pharmacology of agents that target different NMDA receptor subtypes, offering new avenues for capturing the therapeutic potential of targeting this important receptor class.

Biomarker Responses to Acute Exercise and Relationship with Brain Blood Flow.

BACKGROUND: There is evidence that aerobic exercise is beneficial for brain health, but these effects are variable between individuals and the underlying mechanisms that modulate these benefits remain unclear. OBJECTIVE: We sought to characterize the acute physiological response of bioenergetic and neurotrophic blood biomarkers to exercise in cognitively healthy older adults, as well as relationships with brain blood flow. METHODS: We measured exercise-induced changes in lactate, which has been linked to brain blood flow, as well brain-derived neurotrophic factor (BDNF), a neurotrophin related to brain health. We further quantified changes in brain blood flow using arterial spin labeling. RESULTS: As expected, lactate and BDNF both changed with time post exercise. Intriguingly, there was a negative relationship between lactate response (area under the curve) and brain blood flow measured acutely following exercise. Finally, the BDNF response tracked strongly with change in platelet activation, providing evidence that platelet activation is an important mechanism for trophic-related exercise responses. CONCLUSIONS: Lactate and BDNF respond acutely to exercise, and the lactate response tracks with changes in brain blood flow. Further investigation into how these factors relate to brain health-related outcomes in exercise trials is warranted.

Clinical and functional consequences of GRIA variants in patients with neurological diseases.

AMPA receptors are members of the glutamate receptor family and mediate a fast component of excitatory synaptic transmission at virtually all central synapses. Thus, their functional characteristics are a critical determinant of brain function. We evaluate intolerance of each GRIA gene to genetic variation using 3DMTR and report here the functional consequences of 52 missense variants in GRIA1-4 identified in patients with various neurological disorders. These variants produce changes in agonist EC50, response time course, desensitization, and/or receptor surface expression. We predict that these functional and localization changes will have important consequences for circuit function, and therefore likely contribute to the patients' clinical phenotype. We evaluated the sensitivity of variant receptors to AMPAR-selective modulators including FDA-approved drugs to explore potential targeted therapeutic options.

Using 19F NMR to Investigate Cationic Carbon Dot Association with Per- and Polyfluoroalkyl Substances (PFAS).

There is much concern about per- and polyfluoroalkyl substances (PFAS) based on their environmental persistence and toxicity, resulting in an urgent need for remediation technologies. This study focused on determining if nanoscale polymeric carbon dots are a viable sorbent material for PFAS and developing fluorine nuclear magnetic resonance spectroscopy (19F NMR) methods to probe interactions between carbon dots and PFAS at the molecular scale. Positively charged carbon dots (PEI-CDs) were synthesized using branched polyethyleneimine to target anionic PFAS by promoting electrostatic interactions. PEI-CDs were exposed to perfluorooctanoic acid (PFOA) to assess their potential as a PFAS sorbent material. After exposure to PFOA, the average size of the PEI-CDs increased (1.6 ± 0.5 to 7.8 ± 1.8 nm) and the surface charge decreased (+38.6 ± 1.1 to +26.4 ± 0.8 mV), both of which are consistent with contaminant sorption. 19F NMR methods were developed to gain further insight into PEI-CD affinity toward PFAS without any complex sample preparation. Changes in PFOA peak intensity and chemical shift were monitored at various PEI-CD concentrations to establish binding curves and determine the chemical exchange regime. 19F NMR spectral analysis indicates slow-intermediate chemical exchange between PFOA and CDs, demonstrating a high-affinity interaction. The α-fluorine had the greatest change in chemical shift and highest affinity, suggesting electrostatic interactions are the dominant sorption mechanism. PEI-CDs demonstrated affinity for a wide range of analytes when exposed to a mixture of 24-PFAS, with a slight preference toward perfluoroalkyl sulfonates. Overall, this study shows that PEI-CDs are an effective PFAS sorbent material and establishes 19F NMR as a suitable method to screen for novel sorbent materials and elucidate interaction mechanisms.

Bovine oviductal organoids: a multi-omics approach to capture the cellular and extracellular molecular response of the oviduct to heat stress.

BACKGROUND: The mammalian oviduct is a complex, fibromuscular organ known for its role in orchestrating a series of timely and dynamic changes to suitably support early embryogenesis. Climate change-induced heat stress (HS) is one of the largest single stressors compromising reproductive function in humans and farm animals via systemic changes in the redox status of the maternal environment, adversely affecting fertilization and early embryonic development. Oviductal organoids represent a unique 3-dimensional, biomimetic model to study the physiology of the oviduct and its subsequent impact on embryo development under various environmental conditions. RESULTS: Our study is the first to demonstrate an innovative approach to understanding the cascade of molecular changes sustained by bovine oviductal organoids under HS and the subsequent maternal signals harnessed within their secreted extracellular vesicles (EVs). Transcriptomic analysis of oviductal organoids exposed to HS revealed 2,570 differentially expressed genes (1,222 up- and 1,348 downregulated), while EV-coupled miRNome analysis disclosed 18 miRNAs with significant differential expression (12 up- and 6 downregulated) in EVs from thermally stressed organoids compared to EVs released from organoids cultured under thermoneutral conditions. Genes activated in oviductal organoids in response to thermal stress, include: COX1, ACTB, CST6, TPT1, and HSPB1, while miR-1246, miR-148a, miR21-5p, miR-451, and miR-92a represent the top highly abundant EV-coupled miRNAs released in response to HS. Pathway analysis of genes enriched in organoids exposed to thermal stress showed the enrichment of endocrine resistance, cellular senescence, and notch signaling pathways. Similarly, EV-coupled miRNAs released from thermally stressed organoids showed their potential regulation of genes involved in cellular senescence, p53 signaling, and TGF-beta signaling pathways. CONCLUSIONS: In conclusion, the cellular and extracellular response of bovine oviductal organoids to in vitro HS conditions reveal the prospective impact of environmental HS on the physiology of the oviduct and the probable subsequent impacts on oocyte fertilization and early embryo development. Future studies elucidating the potential impact of HS-associated EVs from oviductal organoids on oocyte fertilization and preimplantation embryo development, would justify the use of an organoid model to optimally understand the oviduct-embryo communication under suboptimal environments.

Development of a risk score to increase detection of severe alpha-1 antitrypsin deficiency.

Background: Alpha-1 antitrypsin deficiency (AATD) is an under-recognised genetic cause of chronic obstructive lung disease, and many fewer cases than estimated have been identified. Can a reported respiratory and hepatic disease history from a large AATD testing database be used to stratify a person's risk of severe AATD? Methods: We analysed data extracted from the AATD National Detection Program. Demographics and medical history were evaluated to predict AATD PI*ZZ genotype. Logistic regression and integer programming models identified predictors and obtained risk scores. These were internally validated on a subset of the data. Results: Out of 301 343 subjects, 1529 (0.5%) had PI*ZZ genotype. Predictors of severe AATD were asthma, bronchitis, emphysema, allergies, bronchiectasis, family history of AATD, cirrhosis, hepatitis and history of abnormal liver function tests. The derived model establishes a subject's risk of severe AATD, and scores ≥0 had an estimated risk of 0.41%, sensitivity 84.62% and specificity 24.32%. A model simulating guideline recommendations had an estimated risk of 0.51% with a sensitivity of 37.98% and specificity 46.60%. By recommending screening for scores ≥0, we estimate that more subjects would be screened (75.7% versus 53.4%) and detected (84.6% versus 58.2%) compared to a guideline-simulated model. Conclusion: This medical history risk model is a useful predictive tool to detect subjects at greater risk of having severe AATD and improves sensitivity of detection. Scores <0 are at lower risk and may need not be screened; testing is recommended for scores ≥0 and consistent with current guidelines.

Differential responses of disease-related GRIN variants located in pore-forming M2 domain of N-methyl-D-aspartate receptor to FDA-approved inhibitors.

N-methyl-D-aspartate receptors (NMDAR), ionotropic glutamate receptors, mediate a slow component of excitatory synaptic transmission in the central nervous system and play a key role in normal brain function and development. Genetic variations in GRIN genes encoding NMDAR subunits that alter the receptor's functional characteristics are associated with a wide range of neurological and neuropsychiatric conditions. Pathological GRIN variants located in the M2 re-entrant loop lining the channel pore cause significant functional changes, the most consequential alteration being a reduction in voltage-dependent Mg2+ inhibition. Voltage-dependent Mg2+ block is a unique feature of NMDAR biology whereby channel activation requires both ligand binding and postsynaptic membrane depolarization. Thus, loss of NMDAR Mg2+ block will have a profound impact on synaptic function and plasticity. Here, we choose 11 missense variants within the GRIN1, GRIN2A, and GRIN2B genes that alter residues located in the M2 loop and significantly reduce Mg2+ inhibition. Each variant was evaluated for tolerance to genetic variation using the 3-dimensional structure and assessed for functional rescue pharmacology via electrophysiological recordings. Three FDA-approved NMDAR drugs-memantine, dextromethorphan, and ketamine-were chosen based on their ability to bind near the M2 re-entrant loop, potentially rectifying dysregulated NMDAR function by supplementing the reduced voltage-dependent Mg2+ block. These results provide insight of structural determinants of FDA-approved NMDAR drugs at their binding sites in the channel pore and may further define conditions necessary for the use of such agents as potential rescue pharmacology.

Rubredoxin Protein Scaffolds Sourced from Diverse Environmental Niches as an Artificial Hydrogenase Platform.

Nickel-substituted rubredoxin (NiRd) from Desulfovibrio desulfuricans has previously been shown to act as both a structural and functional mimic of the [NiFe] hydrogenase. However, improvements both in turnover frequency and overpotential are needed to rival the native [NiFe] hydrogenase enzymes. Characterization of a library of NiRd mutants with variations in the secondary coordination sphere suggested that protein dynamics played a substantial role in modulating activity. In this work, rubredoxin scaffolds were selected from diverse organisms to study the effects of distal sequence variation on catalytic activity. It was found that though electrochemical catalytic activity was only slightly impacted across the series, the Rd sequence from a psychrophilic organism exhibited substantially higher levels of solution-phase hydrogen production. Additionally, Eyring analyses suggest that catalytic activation properties relate to the growth temperature of the parent organism, implying that the general correlation between the parent organism environment and catalytic activity often seen in naturally occurring enzymes may also be observed in artificial enzymes. Selecting protein scaffolds from hosts that inhabit diverse environments, particularly low-temperature environments, represents an alternative approach for engineering artificial metalloenzymes.

Development of a Dihydroquinoline-Pyrazoline GluN2C/2D-Selective Negative Allosteric Modulator of the N-Methyl-d-aspartate Receptor.

Subunit-selective inhibition of N-methyl-d-aspartate receptors (NMDARs) is a promising therapeutic strategy for several neurological disorders, including epilepsy, Alzheimer's and Parkinson's disease, depression, and acute brain injury. We previously described the dihydroquinoline-pyrazoline (DQP) analogue 2a (DQP-26) as a potent NMDAR negative allosteric modulator with selectivity for GluN2C/D over GluN2A/B. However, moderate (<100-fold) subunit selectivity, inadequate cell-membrane permeability, and poor brain penetration complicated the use of 2a as an in vivo probe. In an effort to improve selectivity and the pharmacokinetic profile of the series, we performed additional structure-activity relationship studies of the succinate side chain and investigated the use of prodrugs to mask the pendant carboxylic acid. These efforts led to discovery of the analogue (S)-(-)-2i, also referred to as (S)-(-)-DQP-997-74, which exhibits >100- and >300-fold selectivity for GluN2C- and GluN2D-containing NMDARs (IC50 0.069 and 0.035 µM, respectively) compared to GluN2A- and GluN2B-containing receptors (IC50 5.2 and 16 µM, respectively) and has no effects on AMPA, kainate, or GluN1/GluN3 receptors. Compound (S)-(-)-2i is 5-fold more potent than (S)-2a. In addition, compound 2i shows a time-dependent enhancement of inhibitory actions at GluN2C- and GluN2D-containing NMDARs in the presence of the agonist glutamate, which could attenuate hypersynchronous activity driven by high-frequency excitatory synaptic transmission. Consistent with this finding, compound 2i significantly reduced the number of epileptic events in a murine model of tuberous sclerosis complex (TSC)-induced epilepsy that is associated with upregulation of the GluN2C subunit. Thus, 2i represents a robust tool for the GluN2C/D target validation. Esterification of the succinate carboxylate improved brain penetration, suggesting a strategy for therapeutic development of this series for NMDAR-associated neurological conditions.

De novo CLPTM1 variants with reduced GABAA R current response in patients with epilepsy.

OBJECTIVE: To investigate the clinical features and potential pathogenesis mechanism of de novo CLPTM1 variants associated with epilepsy. METHODS: Identify de novo genetic variants associated with epilepsy by reanalyzing trio-based whole-exome sequencing data. We analyzed the clinical characteristics of patients with these variants and performed functional in vitro studies in cells expressing mutant complementary DNA for these variants using whole-cell voltage-clamp current recordings and outside-out patch-clamp recordings from transiently transfected human embryonic kidney (HEK) cells. RESULTS: Two de novo missense variants related to epilepsy were identified in the CLPTM1 gene. Functional studies indicated that CLPTM1-p.R454H and CLPTM1-p.R568Q variants reduced the γ-aminobutyric acid A receptor (GABAA R) current response amplitude recorded under voltage clamp compared to the wild-type receptors. These variants also reduced the charge transfer and altered the time course of desensitization and deactivation following rapid removal of GABA. The surface expression of the GABAA R γ2 subunit from the CLPTM1-p.R568Q group was significantly reduced compared to CLPTM1-WT. SIGNIFICANCE: This is the first report of functionally relevant variants within the CLPTM1 gene. Patch-clamp recordings showed that these de novo CLPTM1 variants reduce GABAA R currents and charge transfer, which should promote excitation and hypersynchronous activity. This study may provide insights into the molecular mechanisms of the CLPTM1 variants underlying the patients' phenotypes, as well as for exploring potential therapeutic targets for epilepsy.

Production of Mare Chorionic Girdle Organoids That Secrete Equine Chorionic Gonadotropin.

The equine chorionic girdle is comprised of specialized invasive trophoblast cells that begin formation approximately 25 days after ovulation (day 0) and invade the endometrium to become endometrial cups. These specialized trophoblast cells transition from uninucleate to differentiated binucleate trophoblast cells that secrete the glycoprotein hormone equine chorionic gonadotropin (eCG; formerly known as pregnant mare serum gonadotropin or PMSG). This eCG has LH-like activity in the horse but variable LH- and FSH-like activity in other species and has been utilized for these properties both in vivo and in vitro. To produce eCG commercially, large volumes of whole blood must be collected from pregnant mares, which negatively impacts equine welfare due to repeated blood collections and the birth of an unwanted foal. Attempts to produce eCG in vitro using long-term culture of chorionic girdle explants have not been successful beyond 180 days, with peak eCG production at 30 days of culture. Organoids are three-dimensional cell clusters that self-organize and can remain genetically and phenotypically stable throughout long-term culture (i.e., months). Human trophoblast organoids have been reported to successfully produce human chorionic gonadotropin (hCG) and proliferate long-term (>1 year). The objective of this study was to evaluate whether organoids derived from equine chorionic girdle maintain physiological functionality. Here we show generation of chorionic girdle organoids for the first time and demonstrate in vitro production of eCG for up to 6 weeks in culture. Therefore, equine chorionic girdle organoids provide a physiologically representative 3D in vitro model for chorionic girdle development of early equine pregnancy.

Classification of missense variants in the N-methyl-d-aspartate receptor GRIN gene family as gain- or loss-of-function.

Advances in sequencing technology have generated a large amount of genetic data from patients with neurological conditions. These data have provided diagnosis of many rare diseases, including a number of pathogenic de novo missense variants in GRIN genes encoding N-methyl-d-aspartate receptors (NMDARs). To understand the ramifications for neurons and brain circuits affected by rare patient variants, functional analysis of the variant receptor is necessary in model systems. For NMDARs, this functional analysis needs to assess multiple properties in order to understand how variants could impact receptor function in neurons. One can then use these data to determine whether the overall actions will increase or decrease NMDAR-mediated charge transfer. Here, we describe an analytical and comprehensive framework by which to categorize GRIN variants as either gain-of-function (GoF) or loss-of-function (LoF) and apply this approach to GRIN2B variants identified in patients and the general population. This framework draws on results from six different assays that assess the impact of the variant on NMDAR sensitivity to agonists and endogenous modulators, trafficking to the plasma membrane, response time course and channel open probability. We propose to integrate data from multiple in vitro assays to arrive at a variant classification, and suggest threshold levels that guide confidence. The data supporting GoF and LoF determination are essential to assessing pathogenicity and patient stratification for clinical trials as personalized pharmacological and genetic agents that can enhance or reduce receptor function are advanced. This approach to functional variant classification can generalize to other disorders associated with missense variants.

Improved Decision Making for Water Lead Testing in U.S. Child Care Facilities Using Machine-Learned Bayesian Networks.

Tap water lead testing programs in the U.S. need improved methods for identifying high-risk facilities to optimize limited resources. In this study, machine-learned Bayesian network (BN) models were used to predict building-wide water lead risk in over 4,000 child care facilities in North Carolina according to maximum and 90th percentile lead levels from water lead concentrations at 22,943 taps. The performance of the BN models was compared to common alternative risk factors, or heuristics, used to inform water lead testing programs among child care facilities including building age, water source, and Head Start program status. The BN models identified a range of variables associated with building-wide water lead, with facilities that serve low-income families, rely on groundwater, and have more taps exhibiting greater risk. Models predicting the probability of a single tap exceeding each target concentration performed better than models predicting facilities with clustered high-risk taps. The BN models' Fß-scores outperformed each of the alternative heuristics by 118-213%. This represents up to a 60% increase in the number of high-risk facilities that could be identified and up to a 49% decrease in the number of samples that would need to be collected by using BN model-informed sampling compared to using simple heuristics. Overall, this study demonstrates the value of machine-learning approaches for identifying high water lead risk that could improve lead testing programs nationwide.

Functional effects of disease-associated variants reveal that the S1-M1 linker of the NMDA receptor critically controls channel opening.

The short pre-M1 helix within the S1-M1 linker (also referred to as the pre-M1 linker) between the agonist-binding domain (ABD, S1) and the M1 transmembrane helix of the NMDA receptor (NMDAR) is devoid of missense variants within the healthy population but is a locus for de novo pathogenic variants associated with neurological disorders. Several de novo variants within this helix have been identified in patients presenting early in life with intellectual disability, developmental delay, and/or epilepsy. In this study, we evaluated functional properties for twenty variants within the pre-M1 linker in GRIN1, GRIN2A, and GRIN2B genes, including six novel missense variants. The effects of pre-M1 variants on agonist potency, sensitivity to endogenous allosteric modulators, response time course, channel open probability, and surface expression were assessed. Our data indicated that virtually all of the variants evaluated altered channel function, and multiple variants had profound functional consequences, which may contribute to the neurological conditions in the patients harboring the variants in this region. These data strongly suggest that the residues within the pre-M1 helix play a key role in channel gating and are highly intolerant to genetic variation.