Cross-platform proteomics signatures of extreme old age.

In previous work, we used a SomaLogic platform targeting approximately 5000 proteins to generate a serum protein signature of centenarians that we validated in independent studies that used the same technology. We set here to validate and possibly expand the results by profiling the serum proteome of a subset of individuals included in the original study using liquid chromatography tandem mass spectrometry (LC-MS/MS). Following pre-processing, the LC-MS/MS data provided quantification of 398 proteins, with only 266 proteins shared by both platforms. At 1% FDR statistical significance threshold, the analysis of LC-MS/MS data detected 44 proteins associated with extreme old age, including 23 of the original analysis. To identify proteins for which associations between expression and extreme-old age were conserved across platforms, we performed inter-study conservation testing of the 266 proteins quantified by both platforms using a method that accounts for the correlation between the results. From these tests, a total of 80 proteins reached 5% FDR statistical significance, and 26 of these proteins had concordant pattern of gene expression in whole blood generated in an independent set. This signature of 80 proteins points to blood coagulation, IGF signaling, extracellular matrix (ECM) organization, and complement cascade as important pathways whose protein level changes provide evidence for age-related adjustments that distinguish centenarians from younger individuals. The comparison with blood transcriptomics also highlights a possible role for neutrophil degranulation in aging.

Atp1a2 and Kcnj9 are candidate genes underlying oxycodone behavioral sensitivity and withdrawal in C57BL/6 substrains.

Opioid use disorder is heritable, yet its genetic etiology is largely unknown. Analysis of addiction model traits in rodents (e.g., opioid behavioral sensitivity and withdrawal) can facilitate genetic and mechanistic discovery. C57BL/6J and C57BL/6NJ substrains have extremely limited genetic diversity, yet can show reliable phenotypic diversity which together, can facilitate gene discovery. The C57BL/6NJ substrain was less sensitive to oxycodone (OXY)-induced locomotor activity compared to the C57BL/6J substrain. Quantitative trait locus (QTL) mapping in an F2 cross identified a distal chromosome 1 QTL explaining 7-12% of the variance in OXY locomotor sensitivity and anxiety-like withdrawal in the elevated plus maze. We identified a second QTL for withdrawal on chromosome 5 near the candidate gene Gabra2 (alpha-2 subunit of GABA-A receptor) explaining 9% of the variance. Next, we generated recombinant lines from an F2 founder spanning the distal chromosome 1 locus (163-181 Mb), captured the QTL for OXY sensitivity and withdrawal, and fine-mapped a 2.45-Mb region (170.16-172.61 Mb). There were five striatal cis-eQTL transcripts in this region (Pcp4l1, Ncstn, Atp1a2, Kcnj9, Igsf9), two of which were confirmed at the protein level (KCNJ9, ATP1A2). Kcnj9, a.k.a., GIRK3, codes for a potassium channel that is a major effector of mu opioid receptor signaling. Atp1a2 codes for a subunit of a Na+/K+ ATPase enzyme that regulates neuronal excitability and shows adaptations following chronic opioid administration. To summarize, we identified genetic sources of opioid behavioral differences in C57BL/6 substrains, two of the most widely and often interchangeably used substrains in opioid addiction research.

STING promotes homeostatic maintenance of tissues and confers longevity with aging.

Local immune processes within aging tissues are a significant driver of aging associated dysfunction, but tissue-autonomous pathways and cell types that modulate these responses remain poorly characterized. The cytosolic DNA sensing pathway, acting through cyclic GMP-AMP synthase (cGAS) and Stimulator of Interferon Genes (STING), is broadly expressed in tissues, and is poised to regulate local type I interferon (IFN-I)-dependent and independent inflammatory processes within tissues. Recent studies suggest that the cGAS/STING pathway may drive pathology in various in vitro and in vivo models of accelerated aging. To date, however, the role of the cGAS/STING pathway in physiological aging processes, in the absence of genetic drivers, has remained unexplored. This remains a relevant gap, as STING is ubiquitously expressed, implicated in multitudinous disorders, and loss of function polymorphisms of STING are highly prevalent in the human population (>50%). Here we reveal that, during physiological aging, STING-deficiency leads to a significant shortening of murine lifespan, increased pro-inflammatory serum cytokines and tissue infiltrates, as well as salient changes in histological composition and organization. We note that aging hearts, livers, and kidneys express distinct subsets of inflammatory, interferon-stimulated gene (ISG), and senescence genes, collectively comprising an immune fingerprint for each tissue. These distinctive patterns are largely imprinted by tissue-specific stromal and myeloid cells. Using cellular interaction network analyses, immunofluorescence, and histopathology data, we show that these immune fingerprints shape the tissue architecture and the landscape of cell-cell interactions in aging tissues. These age-associated immune fingerprints are grossly dysregulated with STING-deficiency, with key genes that define aging STING-sufficient tissues greatly diminished in the absence of STING. Changes in immune signatures are concomitant with a restructuring of the stromal and myeloid fractions, whereby cell:cell interactions are grossly altered and resulting in disorganization of tissue architecture in STING-deficient organs. This altered homeostasis in aging STING-deficient tissues is associated with a cross-tissue loss of homeostatic tissue-resident macrophage (TRM) populations in these tissues. Ex vivo analyses reveal that basal STING-signaling limits the susceptibility of TRMs to death-inducing stimuli and determines their in situ localization in tissue niches, thereby promoting tissue homeostasis. Collectively, these data upend the paradigm that cGAS/STING signaling is primarily pathological in aging and instead indicate that basal STING signaling sustains tissue function and supports organismal longevity. Critically, our study urges caution in the indiscriminate targeting of these pathways, which may result in unpredictable and pathological consequences for health during aging.

Cross-platform proteomics signatures of extreme old age.

In previous work we used a Somalogic platform targeting approximately 5000 proteins to generate a serum protein signature of centenarians that we validated in independent studies that used the same technology. We set here to validate and possibly expand the results by profiling the serum proteome of a subset of individuals included in the original study using liquid chromatography tandem mass spectrometry (LC-MS/MS). Following pre-processing, the LC-MS/MS data provided quantification of 398 proteins, with only 266 proteins shared by both platforms. At 1% FDR statistical significance threshold, the analysis of LC-MS/MS data detected 44 proteins associated with extreme old age, including 23 of the original analysis. To identify proteins for which associations between expression and extreme-old age were conserved across platforms, we performed inter-study conservation testing of the 266 proteins quantified by both platforms using a method that accounts for the correlation between the results. From these tests, a total of 80 proteins reached 5% FDR statistical significance, and 26 of these proteins had concordant pattern of gene expression in whole blood. This signature of 80 proteins points to blood coagulation, IGF signaling, extracellular matrix (ECM) organization, and complement cascade as important pathways whose protein level changes provide evidence for age-related adjustments that distinguish centenarians from younger individuals.

A Simple Strategy for Identifying Conserved Features across Non-independent Omics Studies.

False discovery is an ever-present concern in omics research, especially for burgeoning technologies with unvetted specificity of their biomolecular measurements, as such unknowns obscure the ability to characterize biologically informative features from studies performed with any single platform. Accordingly, performing replication studies of the same samples using different omics platforms is a viable strategy for identifying high-confidence molecular associations that are conserved across studies. However, an important caveat of replication studies that include the same samples is that they are inherently non-independent, leading to overestimation of conservation if studies are treated otherwise. Strategies for accounting for such inter-study dependencies have been proposed for meta-analysis methods that are devised to increase statistical power to detect molecular associations present in one-or-more studies but are not immediately suited for identifying conserved molecular associations across multiple studies. Here we present a unifying strategy for performing inter-study conservation analysis as an alternative to meta-analysis strategies for aggregating summary statistical results of shared features across complementary studies, while accounting for inter-study dependency. This method, which we refer to as "adjusted maximum p-value" (AdjMaxP), is easy to implement with both inter-study dependency and conservation estimated directly from the p-values from molecular feature-level association testing results from each study. Through simulation-based assessment we demonstrate that AdjMaxP's improved performance for accurately identifying conserved features over a related meta-analysis strategy for non-independent studies.

BAITING AND BANDING: EXPERT OPINION ON HOW BAIT TRAPPING MAY INFLUENCE THE OCCURRENCE OF HIGHLY PATHOGENIC AVIAN INFLUENZA (HPAI) AMONG DABBLING DUCKS.

A Eurasian lineage highly pathogenic avian influenza virus (HPAIV) of the clade 2.3.4.4b (Goose/Guangdong lineage) was detected in migratory bird populations in North America in December 2021, and it, along with its reassortants, have since caused wild and domestic bird outbreaks across the continent. Relative to previous outbreaks, HPAIV cases among wild birds in 2022 exhibited wider geographic extent within North America and higher levels of mortality, suggesting the potential for population-level impacts. Given the possible conservation implications of HPAIV in wild birds, natural resource managers have sought guidance on actions that may mitigate negative effects of disease among North American bird populations, including modification of existing management practices. Banding of waterfowl is a critical tool for population management for several harvested species in North America, but some banding techniques, such as bait trapping, can lead to increased congregation of waterfowl, potentially altering HPAIV transmission. We used an expert opinion exercise to assess how bait trapping of dabbling ducks in Canada may influence HPAIV transmission and wild bird health. The expert group found that it is moderately likely that bait trapping of dabbling ducks in wetlands will significantly increase the transmission of HPAIV among individual ducks, but there is a low probability that this will result in significant population-level effects on North American dabbling ducks. Considering the lack of empirical work studying how capture and handling methods may change transmission of HPAIV among waterfowl, as well as the importance of bait trapping for waterfowl management in North America, future work should focus on filling knowledge gaps pertaining to the influence of baiting on HPAIV occurrence to better inform banding procedures and management decision making.

ß-catenin/CBP activation of mTORC1 signaling promotes partial epithelial-mesenchymal states in head and neck cancer.

Head and neck cancers, which include oral squamous cell carcinoma (OSCC) as a major subsite, exhibit cellular plasticity that includes features of an epithelial-mesenchymal transition (EMT), referred to as partial-EMT (p-EMT). To identify molecular mechanisms contributing to OSCC plasticity, we performed a multiphase analysis of single cell RNA sequencing (scRNAseq) data from human OSCC. This included a multiresolution characterization of cancer cell subgroups to identify pathways and cell states that are heterogeneously represented, followed by casual inference analysis to elucidate activating and inhibitory relationships between these pathways and cell states. This approach revealed signaling networks associated with hierarchical cell state transitions, which notably included an association between ß-catenin-driven CREB-binding protein (CBP) activity and mTORC1 signaling. This network was associated with subpopulations of cancer cells that were enriched for markers of the p-EMT state and poor patient survival. Functional analyses revealed that ß-catenin/CBP induced mTORC1 activity in part through the transcriptional regulation of a raptor-interacting protein, chaperonin containing TCP1 subunit 5 (CCT5). Inhibition of ß-catenin-CBP activity through the use of the orally active small molecule, E7386, reduced the expression of CCT5 and mTORC1 activity in vitro, and inhibited p-EMT-associated markers and tumor development in a murine model of OSCC. Our study highlights the use of multiresolution network analyses of scRNAseq data to identify targetable signals for therapeutic benefit, thus defining an underappreciated association between ß-catenin/CBP and mTORC1 signaling in head and neck cancer plasticity.

Multi-modal profiling of peripheral blood cells across the human lifespan reveals distinct immune cell signatures of aging and longevity.

BACKGROUND: Age-related changes in immune cell composition and functionality are associated with multimorbidity and mortality. However, many centenarians delay the onset of aging-related disease suggesting the presence of elite immunity that remains highly functional at extreme old age. METHODS: To identify immune-specific patterns of aging and extreme human longevity, we analyzed novel single cell profiles from the peripheral blood mononuclear cells (PBMCs) of a random sample of 7 centenarians (mean age 106) and publicly available single cell RNA-sequencing (scRNA-seq) datasets that included an additional 7 centenarians as well as 52 people at younger ages (20-89 years). FINDINGS: The analysis confirmed known shifts in the ratio of lymphocytes to myeloid cells, and noncytotoxic to cytotoxic cell distributions with aging, but also identified significant shifts from CD4+ T cell to B cell populations in centenarians suggesting a history of exposure to natural and environmental immunogens. We validated several of these findings using flow cytometry analysis of the same samples. Our transcriptional analysis identified cell type signatures specific to exceptional longevity that included genes with age-related changes (e.g., increased expression of STK17A, a gene known to be involved in DNA damage response) as well as genes expressed uniquely in centenarians' PBMCs (e.g., S100A4, part of the S100 protein family studied in age-related disease and connected to longevity and metabolic regulation). INTERPRETATION: Collectively, these data suggest that centenarians harbor unique, highly functional immune systems that have successfully adapted to a history of insults allowing for the achievement of exceptional longevity. FUNDING: TK, SM, PS, GM, SA, TP are supported by NIH-NIAUH2AG064704 and U19AG023122. MM and PS are supported by NIHNIA Pepper center: P30 AG031679-10. This project is supported by the Flow Cytometry Core Facility at BUSM. FCCF is funded by the NIH Instrumentation grant: S10 OD021587.

Postpartum Psychosis.

PURPOSE OF REVIEW: Postpartum psychosis is a psychiatric emergency that can affect the health and life of mothers, infants, and families. Postpartum psychosis (PPP) is distinct from non-postpartum psychosis in many ways, and it is crucial to study and understand PPP to identify, treat, and possibly prevent this condition. We therefore sought to review the latest research findings about PPP with the intention of updating readers about the latest evidence base. RECENT FINDINGS: Multiple physiologic pathways have been implicated in the development of PPP, and further understanding these pathways may allow for early detection and treatment. Risk assessment and treatment should include consideration of the woman patient but also the mother-infant dyad and the larger family. It is our hope that this review of research updates in postpartum psychosis may inform clinical practice and promote specialized, evidence-based diagnosis, risk assessment, and treatment.

A quantitative trait variant in Gabra2 underlies increased methamphetamine stimulant sensitivity.

Psychostimulant (methamphetamine, cocaine) use disorders have a genetic component that remains mostly unknown. We conducted genome-wide quantitative trait locus (QTL) analysis of methamphetamine stimulant sensitivity. To facilitate gene identification, we employed a Reduced Complexity Cross between closely related C57BL/6 mouse substrains and examined maximum speed and distance traveled over 30 min following methamphetamine (2 mg/kg, i.p.). For maximum methamphetamine-induced speed following the second and third administration, we identified a single genome-wide significant QTL on chromosome 11 that peaked near the Cyfip2 locus (LOD = 3.5, 4.2; peak = 21 cM [36 Mb]). For methamphetamine-induced distance traveled following the first and second administration, we identified a genome-wide significant QTL on chromosome 5 that peaked near a functional intronic indel in Gabra2 coding for the alpha-2 subunit of the GABA-A receptor (LOD = 3.6-5.2; peak = 34-35 cM [66-67 Mb]). Striatal cis-expression QTL mapping corroborated Gabra2 as a functional candidate gene underlying methamphetamine-induced distance traveled. CRISPR/Cas9-mediated correction of the mutant intronic deletion on the C57BL/6J background to the wild-type C57BL/6NJ allele was sufficient to reduce methamphetamine-induced locomotor activity toward the wild-type C57BL/6NJ-like level, thus validating the quantitative trait variant (QTV). These studies show the power and efficiency of Reduced Complexity Crosses in identifying causal variants underlying complex traits. Functionally restoring Gabra2 expression decreased methamphetamine stimulant sensitivity and supports preclinical and human genetic studies implicating the GABA-A receptor in psychostimulant addiction-relevant traits. Importantly, our findings have major implications for studying psychostimulants in the C57BL/6J strain-the gold standard strain in biomedical research.

A Data-Driven Transcriptional Taxonomy of Adipogenic Chemicals to Identify White and Brite Adipogens.

BACKGROUND: Chemicals in disparate structural classes activate specific subsets of the transcriptional programs of peroxisome proliferator-activated receptor-γ (PPARγ) to generate adipocytes with distinct phenotypes. OBJECTIVES: Our objectives were to a) establish a novel classification method to predict PPARγ ligands and modifying chemicals; and b) create a taxonomy to group chemicals on the basis of their effects on PPARγ's transcriptome and downstream metabolic functions. We tested the hypothesis that environmental adipogens highly ranked by the taxonomy, but segregated from therapeutic PPARγ ligands, would induce white but not brite adipogenesis. METHODS: 3T3-L1 cells were differentiated in the presence of 76 chemicals (negative controls, nuclear receptor ligands known to influence adipocyte biology, potential environmental PPARγ ligands). Differentiation was assessed by measuring lipid accumulation. mRNA expression was determined by RNA-sequencing (RNA-Seq) and validated by reverse transcription-quantitative polymerase chain reaction. A novel classification model was developed using an amended random forest procedure. A subset of environmental contaminants identified as strong PPARγ agonists were analyzed by their effects on lipid handling, mitochondrial biogenesis, and cellular respiration in 3T3-L1 cells and human preadipocytes. RESULTS: We used lipid accumulation and RNA-Seq data to develop a classification system that a) identified PPARγ agonists; and b) sorted chemicals into likely white or brite adipogens. Expression of Cidec was the most efficacious indicator of strong PPARγ activation. 3T3-L1 cells treated with two known environmental PPARγ ligands, tetrabromobisphenol A and triphenyl phosphate, which sorted distinctly from therapeutic ligands, had higher expression of white adipocyte genes but no difference in Pgc1a and Ucp1 expression, and higher fatty acid uptake but not mitochondrial biogenesis. Moreover, cells treated with two chemicals identified as highly ranked PPARγ agonists, tonalide and quinoxyfen, induced white adipogenesis without the concomitant health-promoting characteristics of brite adipocytes in mouse and human preadipocytes. DISCUSSION: A novel classification procedure accurately identified environmental chemicals as PPARγ ligands distinct from known PPARγ-activating therapeutics. CONCLUSION: The computational and experimental framework has general applicability to the classification of as-yet uncharacterized chemicals. https://doi.org/10.1289/EHP6886.

Multi-resolution characterization of molecular taxonomies in bulk and single-cell transcriptomics data.

As high-throughput genomics assays become more efficient and cost effective, their utilization has become standard in large-scale biomedical projects. These studies are often explorative, in that relationships between samples are not explicitly defined a priori, but rather emerge from data-driven discovery and annotation of molecular subtypes, thereby informing hypotheses and independent evaluation. Here, we present K2Taxonomer, a novel unsupervised recursive partitioning algorithm and associated R package that utilize ensemble learning to identify robust subgroups in a 'taxonomy-like' structure. K2Taxonomer was devised to accommodate different data paradigms, and is suitable for the analysis of both bulk and single-cell transcriptomics, and other '-omics', data. For each of these data types, we demonstrate the power of K2Taxonomer to discover known relationships in both simulated and human tissue data. We conclude with a practical application on breast cancer tumor infiltrating lymphocyte (TIL) single-cell profiles, in which we identified co-expression of translational machinery genes as a dominant transcriptional program shared by T cells subtypes, associated with better prognosis in breast cancer tissue bulk expression data.

Mechanisms of altered skeletal muscle action potentials in the R6/2 mouse model of Huntington's disease.

Huntington's disease (HD) patients suffer from progressive and debilitating motor dysfunction for which only palliative treatment is currently available. Previously, we discovered reduced skeletal muscle Cl- channel (ClC-1) and inwardly rectifying K+ channel (Kir) currents in R6/2 HD transgenic mice. To further investigate the role of ClC-1 and Kir currents in HD skeletal muscle pathology, we measured the effect of reduced ClC-1 and Kir currents on action potential (AP) repetitive firing in R6/2 mice using a two-electrode current clamp. We found that R6/2 APs had a significantly lower peak amplitude, depolarized maximum repolarization, and prolonged decay time compared with wild type (WT). Of these differences, only the maximum repolarization was accounted for by the reduction in ClC-1 and Kir currents, indicating the presence of additional ion channel defects. We found that both KV1.5 and KV3.4 mRNA levels were significantly reduced in R6/2 skeletal muscle compared with WT, which explains the prolonged decay time of R6/2 APs. Overall, we found that APs in WT and R6/2 muscle significantly and progressively change during activity to maintain peak amplitude despite buildup of Na+ channel inactivation. Even with this resilience, the persistently reduced peak amplitude of R6/2 APs is expected to result in earlier fatigue and may help explain the motor impersistence experienced by HD patients. This work lays the foundation to link electrical changes to force generation defects in R6/2 HD mice and to examine the regulatory events controlling APs in WT muscle.

5' UTR variants in the quantitative trait gene Hnrnph1 support reduced 5' UTR usage and hnRNP H protein as a molecular mechanism underlying reduced methamphetamine sensitivity.

We previously identified a 210 kb region on chromosome 11 (50.37-50.58 Mb, mm10) containing two protein-coding genes (Hnrnph1, Rufy1) that was necessary for reduced methamphetamine-induced locomotor activity in C57BL/6J congenic mice harboring DBA/2J polymorphisms. Gene editing of a small deletion in the first coding exon supported Hnrnph1 as a quantitative trait gene. We have since shown that Hnrnph1 mutants also exhibit reduced methamphetamine-induced reward, reinforcement, and dopamine release. However, the quantitative trait variants (QTVs) that modulate Hnrnph1 function at the molecular level are not known. Nine single nucleotide polymorphisms and seven indels distinguish C57BL/6J from DBA/2J within Hnrnph1, including four variants within the 5' untranslated region (UTR). Here, we show that a 114 kb introgressed region containing Hnrnph1 and Rufy1 was sufficient to cause a decrease in MA-induced locomotor activity. Gene-level transcriptome analysis of striatal tissue from 114 kb congenics vs Hnrnph1 mutants identified a nearly perfect correlation of fold-change in expression for those differentially expressed genes that were common to both mouse lines, indicating functionally similar effects on the transcriptome and behavior. Exon-level analysis (including noncoding exons) revealed decreased 5' UTR usage of Hnrnph1 and immunoblot analysis identified a corresponding decrease in hnRNP H protein in 114 kb congenic mice. Molecular cloning of the Hnrnph1 5' UTR containing all four variants (but none of them individually) upstream of a reporter induced a decrease in reporter signal in both HEK293 and N2a cells, thus, identifying a set of QTVs underlying molecular regulation of Hnrnph1.

Assessment of a Highly Multiplexed RNA Sequencing Platform and Comparison to Existing High-Throughput Gene Expression Profiling Techniques.

The need to reduce per sample cost of RNA-seq profiling for scalable data generation has led to the emergence of highly multiplexed RNA-seq. These technologies utilize barcoding of cDNA sequences in order to combine multiple samples into a single sequencing lane to be separated during data processing. In this study, we report the performance of one such technique denoted as sparse full length sequencing (SFL), a ribosomal RNA depletion-based RNA sequencing approach that allows for the simultaneous sequencing of 96 samples and higher. We offer comparisons to well established single-sample techniques, including: full coverage Poly-A capture RNA-seq, microarrays, as well as another low-cost highly multiplexed technique known as 3' digital gene expression (3'DGE). Data was generated for a set of exposure experiments on immortalized human lung epithelial (AALE) cells in a two-by-two study design, in which samples received both genetic and chemical perturbations of known oncogenes/tumor suppressors and lung carcinogens. SFL demonstrated improved performance over 3'DGE in terms of coverage, power to detect differential gene expression, and biological recapitulation of patterns of differential gene expression from in vivo lung cancer mutation signatures.

C57BL/6 substrain differences in inflammatory and neuropathic nociception and genetic mapping of a major quantitative trait locus underlying acute thermal nociception.

Sensitivity to different pain modalities has a genetic basis that remains largely unknown. Employing closely related inbred mouse substrains can facilitate gene mapping of nociceptive behaviors in preclinical pain models. We previously reported enhanced sensitivity to acute thermal nociception in C57BL/6J (B6J) versus C57BL/6N (B6N) substrains. Here, we expanded on nociceptive phenotypes and observed an increase in formalin-induced inflammatory nociceptive behaviors and paw diameter in B6J versus B6N mice (Charles River Laboratories). No strain differences were observed in mechanical or thermal hypersensitivity or in edema following the Complete Freund's Adjuvant model of inflammatory pain, indicating specificity in the inflammatory nociceptive stimulus. In the chronic constrictive nerve injury, a model of neuropathic pain, no strain differences were observed in baseline mechanical threshold or in mechanical hypersensitivity up to one month post-chronic constrictive nerve injury. We replicated the enhanced thermal nociception in the 52.5°C hot plate test in B6J versus B6N mice from The Jackson Laboratory. Using a B6J × B6N-F2 cross (N = 164), we mapped a major quantitative trait locus underlying hot plate sensitivity to chromosome 7 that peaked at 26 Mb (log of the odds [LOD] = 3.81, p < 0.01; 8.74 Mb-36.50 Mb) that was more pronounced in males. Genes containing expression quantitative trait loci associated with the peak nociceptive marker that are implicated in pain and inflammation include Ryr1, Cyp2a5, Pou2f2, Clip3, Sirt2, Actn4, and Ltbp4 (false discovery rate < 0.05). Future studies involving positional cloning and gene editing will determine the quantitative trait gene(s) and potential pleiotropy of this locus across pain modalities.

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions.

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ex vivo preparation of the levator auris longus (LAL) is used because it is a thin muscle that provides easy visualization of the neuromuscular junction for microelectrode impalement at the motor endplate. This method allows for the recording of spontaneous miniature endplate potentials and currents (mEPPs and mEPCs), nerve-evoked endplate potentials and currents (EPPs and EPCs), as well as the membrane properties of the motor endplate. Results obtained from this method include the quantal content (QC), number of vesicle release sites (n), probability of vesicle release (prel), synaptic facilitation and depression, as well as the muscle membrane time constant (τm) and input resistance. Application of this technique to mouse models of human disease can highlight key pathologies in disease states and help identify novel treatment strategies. By fully voltage-clamping a single synapse, this method provides one of the most detailed analyses of synaptic transmission currently available.

MicroRNAs in CSF as prodromal biomarkers for Huntington disease in the PREDICT-HD study.

OBJECTIVE: To investigate the feasibility of microRNA (miRNA) levels in CSF as biomarkers for prodromal Huntington disease (HD). METHODS: miRNA levels were measured in CSF from 60 PREDICT-HD study participants using the HTG protocol. Using a CAG-Age Product score, 30 prodromal HD participants were selected based on estimated probability of imminent clinical diagnosis of HD (i.e., low, medium, high; n = 10/group). For comparison, participants already diagnosed (n = 15) and healthy controls (n = 15) were also selected. RESULTS: A total of 2,081 miRNAs were detected and 6 were significantly increased in the prodromal HD gene expansion carriers vs controls at false discovery rate q < 0.05 (miR-520f-3p, miR-135b-3p, miR-4317, miR-3928-5p, miR-8082, miR-140-5p). Evaluating the miRNA levels in each of the HD risk categories, all 6 revealed a pattern of increasing abundance from control to low risk, and from low risk to medium risk, which then leveled off from the medium to high risk and HD diagnosed groups. CONCLUSIONS: This study reports miRNAs as CSF biomarkers of prodromal and diagnosed HD. Importantly, miRNAs were detected in the prodromal HD groups furthest from diagnosis where treatments are likely to be most consequential and meaningful. The identification of potential biomarkers in the disease prodrome may prove useful in evaluating treatments that may postpone disease onset. CLINICALTRIALSGOV IDENTIFIER: NCT00051324.

Depressed Synaptic Transmission and Reduced Vesicle Release Sites in Huntington's Disease Neuromuscular Junctions.

Huntington's disease (HD) is a progressive and fatal degenerative disorder that results in debilitating cognitive and motor dysfunction. Most HD studies have focused on degeneration of the CNS. We previously discovered that skeletal muscle from transgenic R6/2 HD mice is hyperexcitable due to decreased chloride and potassium conductances. The progressive and early onset of these defects suggest a primary myopathy in HD. In this study, we examined the relationship between neuromuscular transmission and skeletal muscle hyperexcitability. We used an ex vivo preparation of the levator auris longus muscle from male and female late-stage R6/2 mice and age-matched wild-type controls. Immunostaining of the synapses and molecular analyses revealed no evidence of denervation. Physiologically, we recorded spontaneous miniature endplate currents (mEPCs) and nerve-evoked EPCs (eEPCs) under voltage-clamp, which, unlike current-clamp records, were independent of the changes in muscle membrane properties. We found a reduction in the number of vesicles released per action potential (quantal content) in R6/2 muscle, which analysis of eEPC variance and morphology indicate is caused by a reduction in the number of vesicle release sites (n) rather than a change in the probability of release (prel). Furthermore, analysis of high-frequency stimulation trains suggests an impairment in vesicle mobilization. The depressed neuromuscular transmission in R6/2 muscle may help compensate for the muscle hyperexcitability and contribute to motor impersistence.SIGNIFICANCE STATEMENT Recent evidence indicates that Huntington's disease (HD) is a multisystem disorder. Our examination of neuromuscular transmission in this study reveals defects in the motor nerve terminal that may compensate for the muscle hyperexcitability in HD. The technique we used eliminates the effects of the altered muscle membrane properties on synaptic currents and thus provides hitherto the most detailed analysis of synaptic transmission in HD. Clinically, the striking depression of neurotransmission we found may help explain the motor impersistence in HD patients. Therapies that target the highly accessible peripheral nerve and muscle system provide a promising new avenue to lessen the debilitating motor symptoms of HD.

The greater snow goose Anser caerulescens atlanticus: Managing an overabundant population.

Between the early 1900s and the 1990s, the greater snow goose Anser caerulescens atlanticus population grew from 3000 individuals to more than 700 000. Because of concerns about Arctic degradation of natural habitats through overgrazing, a working group recommended the stabilization of the population. Declared overabundant in 1998, special management actions were then implemented in Canada and the United States. Meanwhile, a cost-benefit socioeconomic analysis was performed to set a target population size. Discussions aiming towards attaining a common vision were undertaken with stakeholders at multiple levels. The implemented measures have had varying success; but population size has been generally stable since 1999. To be effective and meet social acceptance, management actions must have a scientific basis, result from a consensus among stakeholders, and include an efficient monitoring programme. In this paper, historical changes in population size and management decisions along with past and current challenges encountered are discussed.