Functional characterisation of the amyotrophic lateral sclerosis risk locus GPX3/TNIP1

Background: Amyotrophic lateral sclerosis (ALS) is a complex, late-onset, neurodegenerative disease with a genetic contribution to disease liability. Genome-wide association studies (GWAS) have identified ten risk loci to date, including the TNIP1/GPX3 locus on chromosome five. Given association analysis data alone cannot determine the most plausible risk gene for this locus, we undertook a comprehensive suite of in silico, in vivo and in vitro studies to address this. // Methods: The Functional Mapping and Annotation (FUMA) pipeline and five tools (conditional and joint analysis (GCTA-COJO), Stratified Linkage Disequilibrium Score Regression (S-LDSC), Polygenic Priority Scoring (PoPS), Summary-based Mendelian Randomisation (SMR-HEIDI) and transcriptome-wide association study (TWAS) analyses) were used to perform bioinformatic integration of GWAS data (Ncases = 20,806, Ncontrols = 59,804) with ‘omics reference datasets including the blood (eQTLgen consortium N = 31,684) and brain (N = 2581). This was followed up by specific expression studies in ALS case-control cohorts (microarray Ntotal = 942, protein Ntotal = 300) and gene knockdown (KD) studies of human neuronal iPSC cells and zebrafish-morpholinos (MO). // Results: SMR analyses implicated both TNIP1 and GPX3 (p < 1.15 × 10−6), but there was no simple SNP/expression relationship. Integrating multiple datasets using PoPS supported GPX3 but not TNIP1. In vivo expression analyses from blood in ALS cases identified that lower GPX3 expression correlated with a more progressed disease (ALS functional rating score, p = 5.5 × 10−3, adjusted R2 = 0.042, Beffect = 27.4 ± 13.3 ng/ml/ALSFRS unit) with microarray and protein data suggesting lower expression with risk allele (recessive model p = 0.06, p = 0.02 respectively). Validation in vivo indicated gpx3 KD caused significant motor deficits in zebrafish-MO (mean difference vs. control ± 95% CI, vs. control, swim distance = 112 ± 28 mm, time = 1.29 ± 0.59 s, speed = 32.0 ± 2.53 mm/s, respectively, p for all < 0.0001), which were rescued with gpx3 expression, with no phenotype identified with tnip1 KD or gpx3 overexpression. // Conclusions: These results support GPX3 as a lead ALS risk gene in this locus, with more data needed to confirm/reject a role for TNIP1. This has implications for understanding disease mechanisms (GPX3 acts in the same pathway as SOD1, a well-established ALS-associated gene) and identifying new therapeutic approaches. Few previous examples of in-depth investigations of risk loci in ALS exist and a similar approach could be applied to investigate future expected GWAS findings

Delinquent Behavior of Dutch Rural Adolescents

This article compares Dutch rural and non-rural adolescents’ delinquent behavior and examines two social correlates of rural delinquency: communal social control and traditional rural culture. The analyses are based on cross-sectional data, containing 3,797 participants aged 13–18 (48.7% females). The analyses show that rural adolescents are only slightly less likely to engage in delinquent behavior. Furthermore, while rural adolescents are exposed more often to communal social control, this does not substantially reduce the likelihood that they engage in delinquent behavior. Concerning rural culture, marked differences appeared between rural and non-rural adolescents. First, alcohol use and the frequency of visiting pubs were more related to rural adolescents’ engagement in delinquent behavior. Second, the gender gap in delinquency is larger among rural adolescents: whereas rural boys did not differ significantly from non-rural boys, rural girls were significantly less likely to engage in delinquent behavior than non-rural girls. However, the magnitude of the effects of most indicators was rather low. To better account for the variety of rural spaces and cultures, it is recommended that future research into antisocial and criminal behavior of rural adolescents should adopt alternative measurements of rurality, instead of using an indicator of population density only

The Cumulative Effects of Polymorphisms in the DNA Mismatch Repair Genes and Tobacco Smoking in Oesophageal Cancer Risk

The DNA mismatch repair (MMR) enzymes repair errors in DNA that occur during normal DNA metabolism or are induced by certain cancer-contributing exposures. We assessed the association between 10 single-nucleotide polymorphisms (SNPs) in 5 MMR genes and oesophageal cancer risk in South Africans. Prior to genotyping, SNPs were selected from the HapMap database, based on their significantly different genotypic distributions between European ancestry populations and four HapMap populations of African origin. In the Mixed Ancestry group, the MSH3 rs26279 G/G versus A/A or A/G genotype was positively associated with cancer (OR = 2.71; 95% CI: 1.34–5.50). Similar associations were observed for PMS1 rs5742938 (GG versus AA or AG: OR = 1.73; 95% CI: 1.07–2.79) and MLH3 rs28756991 (AA or GA versus GG: OR = 2.07; 95% IC: 1.04–4.12). In Black individuals, however, no association between MMR polymorhisms and cancer risk was observed in individual SNP analysis. The interactions between MMR genes were evaluated using the model-based multifactor-dimensionality reduction approach, which showed a significant genetic interaction between SNPs in MSH2, MSH3 and PMS1 genes in Black and Mixed Ancestry subjects, respectively. The data also implies that pathogenesis of common polymorphisms in MMR genes is influenced by exposure to tobacco smoke. In conclusion, our findings suggest that common polymorphisms in MMR genes and/or their combined effects might be involved in the aetiology of oesophageal cancer

Perspectives on tracking data reuse across biodata resources

c The Author(s) 2024. Published by Oxford University Press.Motivation: Data reuse is a common and vital practice in molecular biology and enables the knowledge gathered over recent decades to drive discovery and innovation in the life sciences. Much of this knowledge has been collated into molecular biology databases, such as UniProtKB, and these resources derive enormous value from sharing data among themselves. However, quantifying and documenting this kind of data reuse remains a challenge. Results: The article reports on a one-day virtual workshop hosted by the UniProt Consortium in March 2023, attended by representatives from biodata resources, experts in data management, and NIH program managers. Workshop discussions focused on strategies for tracking data reuse, best practices for reusing data, and the challenges associated with data reuse and tracking. Surveys and discussions showed that data reuse is widespread, but critical information for reproducibility is sometimes lacking. Challenges include costs of tracking data reuse, tensions between tracking data and open sharing, restrictive licenses, and difficulties in tracking commercial data use. Recommendations that emerged from the discussion include: development of standardized formats for documenting data reuse, education about the obstacles posed by restrictive licenses, and continued recognition by funding agencies that data management is a critical activity that requires dedicated resources

Birth dating of midbrain dopamine neurons identifies A9 enriched tissue for transplantation into Parkinsonian mice

C1 - Journal Articles RefereedClinical trials have provided proof of principle that new dopamine neurons isolated from the developing ventral midbrain and transplanted into the denervated striatum can functionally integrate and alleviate symptoms in Parkinson's disease patients. However, extensive variability across patients has been observed, ranging from long-term motor improvement to the absence of symptomatic relief and development of dyskinesias. Heterogeneity of the donor tissue is likely to be a contributing factor in the variable outcomes. Dissections of ventral midbrain used for transplantation will variously contain progenitors for different dopamine neuron subtypes as well as different neurotransmitter phenotypes. The overall impact of the resulting graft will be determined by the functional contribution from these different cell types. The A9 substantia nigra pars compacta dopamine neurons, for example, are known to be particularly important for motor recovery in animal models. Serotonergic neurons, on the other hand, have been implicated in unwanted dyskinesias. Currently little knowledge exists on how variables such as donor age, which have not been controlled for in clinical trials, will impact on the final neuronal composition of fetal grafts. Here we performed a birth dating study to identify the time-course of neurogenesis within the various ventral midbrain dopamine subpopulations in an effort to identify A9-enriched donor tissue for transplantation. The results show that A9 neurons precede the birth of A10 ventral tegmental area dopamine neurons. Subsequent grafting of younger ventral midbrain donor tissue revealed significantly larger grafts containing more mitotic dopamine neuroblasts compared to older donor grafts. These grafts were enriched with A9 neurons and showed significantly greater innervation of the target dorso-lateral striatum and DA release. Younger donor grafts also contained significantly less serotonergic neurons. These findings demonstrate the importance of standardized methods to improve cell therapy for Parkinson's disease and have significant implications for the generation and selectivity of dopamine neurons from stem cell based sources

α-Amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor and RNA processing gene dysregulation are early determinants of selective motor neuron vulnerability in a mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis is a late-onset adult neurodegenerative disease, although there is mounting electrophysiological and pathological evidence from patients and animal models for a protracted preclinical period of motor neuron susceptibility and dysfunction, long before clinical diagnosis. The key molecular mechanisms linked to motor neuron vulnerability in amyotrophic lateral sclerosis have been extensively studied using transcriptional profiling in motor neurons isolated from adult mutant superoxide dismutase 1 mice. However, neonatal and embryonic motor neurons from mutant superoxide dismutase 1 mice show abnormal morphology and hyperexcitability, suggesting preceding transcriptional dysregulation. Here, we used RNA sequencing on motor neurons isolated from embryonic superoxide dismutase 1G93A mice to determine the earliest molecular mechanisms conferring neuronal susceptibility and dysfunction known in a mouse model of amyotrophic lateral sclerosis. Transgenic superoxide dismutase 1G93A mice expressing the spinal motor neuron homeobox HB9:green fluorescent protein reporter allowed unambiguous identification and isolation of motor neurons using fluorescence-activated cell sorting. Gene expression profiling of isolated motor neurons revealed transcriptional dysregulation in superoxide dismutase 1G93A mice as early as embryonic Day 12.5 with the majority of differentially expressed genes involved in RNA processing and α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate-mediated glutamate receptor signalling. We confirmed dysregulation of the α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor Subunit 2, at transcript and protein levels, in embryonic superoxide dismutase 1G93A mouse motor neurons and human motor neurons derived from amyotrophic lateral sclerosis patient induced pluripotent stem cells harbouring pathogenic superoxide dismutase 1 mutations. Mutant superoxide dismutase 1 induced pluripotent stem cell-derived motor neurons showed greater vulnerability to α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate-mediated excitotoxicity, consistent with α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor Subunit 2 downregulation. Thus, α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor Subunit 2 deficiency leading to enhanced α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor signalling, calcium influx, hyperexcitability, and chronic excitotoxicity is a very early and intrinsic property of spinal motor neurons that may trigger amyotrophic lateral sclerosis pathogenesis later in life. This study reinforces the concept of therapeutic targeting of hyperexcitability and excitotoxicity as potential disease-modifying approaches for amyotrophic lateral sclerosis

Role of Dopamine 2 Receptor in Impaired Drug-Cue Extinction in Adolescent Rats

Adolescent drug users display resistance to treatment such as cue exposure therapy (CET), as well as increased liability to relapse. The basis of CET is extinction learning, which involves dopamine signaling in the medial prefrontal cortex (mPFC). This system undergoes dramatic alterations during adolescence. Therefore, we investigated extinction of a cocaine-associated cue in adolescent and adult rats. While cocaine self-administration and lever-alone extinction were not different between the two ages, we observed that cue extinction reduced cue-induced reinstatement in adult but not adolescent rats. Infusion of the selective dopamine 2 receptor (D2R)-like agonist quinpirole into the infralimbic cortex (IL) of the mPFC prior to cue extinction significantly reduced cue-induced reinstatement in adolescents. This effect was replicated by acute systemic treatment with the atypical antipsychotic aripiprazole (Abilify), a partial D2R-like agonist. These data suggest that adolescents may be more susceptible to relapse due to a deficit in cue extinction learning, and highlight the significance of D2R signaling in the IL for cue extinction during adolescence. These findings inspire new tactics for improving adolescent CET, with aripiprazole representing an exciting potential pharmacological adjunct for behavioral therapy

Transcriptional Profiling of Xenogeneic Transplants: Examining Human Pluripotent Stem Cell-Derived Grafts in the Rodent Brain

Human pluripotent stem cells are a valuable resource for transplantation, yet our ability to profile xenografts is largely limited to low-throughput immunohistochemical analysis by difficulties in readily isolating grafts for transcriptomic and/or proteomic profiling. Here, we present a simple methodology utilizing differences in the RNA sequence between species to discriminate xenograft from host gene expression (using qPCR or RNA sequencing [RNA-seq]). To demonstrate the approach, we assessed grafts of undifferentiated human stem cells and neural progenitors in the rodent brain. Xenograft-specific qPCR provided sensitive detection of proliferative cells, and identified germ layer markers and appropriate neural maturation genes across the graft types. Xenograft-specific RNA-seq enabled profiling of the complete transcriptome and an unbiased characterization of graft composition. Such xenograft-specific profiling will be crucial for pre-clinical characterization of grafts and batch-testing of therapeutic cell preparations to ensure safety and functional predictability prior to translation