Identification of genetic modifiers of murine hepatic β-glucocerebrosidase activity.
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ABSTRACT:
SUBMITTER: Duran A
PROVIDER: S-EPMC8379285 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Gaucher disease is an autosomal recessive disorder caused by deficiency of the enzyme glucocerebrosidase. Although it is a monogenic disease, there is vast phenotypic heterogeneity, even among patients with the same genotype. MicroRNAs (miRNAs) are small non-coding RNAs involved in many biological processes and diseases. To determine whether miRNAs can affect glucocerebrosidase activity, we performed a screen of 875 different miRNA mimics. The screen was performed using Gaucher fibroblasts, and glucocerebrosidase activity was used as the initial outcome parameter. We found several miRNAs that either up- or down-regulated glucocerebrosidase activity. In follow-up assays, we confirmed that one specific miRNA (miR-127-5p) down-regulated both glucocerebrosidase activity and protein levels by down-regulation of LIMP-2, the receptor involved in proper trafficking of glucocerebrosidase from the endoplasmic reticulum to the lysosome. A conditioned media assay demonstrated that cells treated with this miRNA secreted glucocerebrosidase into the extracellular environment, supporting impaired LIMP-2 function. Two other miRNAs, miR-16-5p and miR-195-5p, were found to up-regulate glucocerebrosidase activity by greater than 40% and to enhance expression and protein levels of the enzyme. In conclusion, we show that miRNAs can alter glucocerebrosidase activity in patient cells, indicating that miRNAs can potentially act as modifiers in Gaucher disease.
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Project description:Mutations within the lysosomal enzyme β-glucocerebrosidase (GC) result in Gaucher disease and represent a major risk factor for developing Parkinson disease (PD). Loss of GC activity leads to accumulation of its substrate glucosylceramide and α-synuclein. Since lysosomal activity of GC is tightly linked to expression of its trafficking receptor, the lysosomal integral membrane protein type-2 (LIMP-2), we studied α-synuclein metabolism in LIMP-2-deficient mice. These mice showed an α-synuclein dosage-dependent phenotype, including severe neurological impairments and premature death. In LIMP-2-deficient brains a significant reduction in GC activity led to lipid storage, disturbed autophagic/lysosomal function, and α-synuclein accumulation mediating neurotoxicity of dopaminergic (DA) neurons, apoptotic cell death, and inflammation. Heterologous expression of LIMP-2 accelerated clearance of overexpressed α-synuclein, possibly through increasing lysosomal GC activity. In surviving DA neurons of human PD midbrain, LIMP-2 levels were increased, probably to compensate for lysosomal GC deficiency. Therefore, we suggest that manipulating LIMP-2 expression to increase lysosomal GC activity is a promising strategy for the treatment of synucleinopathies.
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Project description:Targeted therapies have demonstrated efficacy against specific subsets of molecularly defined cancers. Although most patients with lung cancer are stratified according to a single oncogenic driver, cancers harbouring identical activating genetic mutations show large variations in their responses to the same targeted therapy. The biology underlying this heterogeneity is not well understood, and the impact of co-existing genetic mutations, especially the loss of tumour suppressors, has not been fully explored. Here we use genetically engineered mouse models to conduct a 'co-clinical' trial that mirrors an ongoing human clinical trial in patients with KRAS-mutant lung cancers. This trial aims to determine if the MEK inhibitor selumetinib (AZD6244) increases the efficacy of docetaxel, a standard of care chemotherapy. Our studies demonstrate that concomitant loss of either p53 (also known as Tp53) or Lkb1 (also known as Stk11), two clinically relevant tumour suppressors, markedly impaired the response of Kras-mutant cancers to docetaxel monotherapy. We observed that the addition of selumetinib provided substantial benefit for mice with lung cancer caused by Kras and Kras and p53 mutations, but mice with Kras and Lkb1 mutations had primary resistance to this combination therapy. Pharmacodynamic studies, including positron-emission tomography (PET) and computed tomography (CT), identified biological markers in mice and patients that provide a rationale for the differential efficacy of these therapies in the different genotypes. These co-clinical results identify predictive genetic biomarkers that should be validated by interrogating samples from patients enrolled on the concurrent clinical trial. These studies also highlight the rationale for synchronous co-clinical trials, not only to anticipate the results of ongoing human clinical trials, but also to generate clinically relevant hypotheses that can inform the analysis and design of human studies.
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Project description:Cytosolic aggregation of the nuclear RNA-binding protein TDP-43 is a histopathologic signature of degenerating neurons in amyotrophic lateral sclerosis (ALS), and mutations in the TARDBP gene encoding TDP-43 cause dominantly inherited forms of this condition. To understand the relationship between TDP-43 misregulation and neurotoxicity, we and others have used Drosophila as a model system, in which overexpression of either wild-type TDP-43 or its ALS-associated mutants in neurons is sufficient to induce neurotoxicity, paralysis, and early death. Using microarrays, we have examined gene expression patterns that accompany TDP-43-induced neurotoxicity in the fly system. Constitutive expression of TDP-43 in the Drosophila compound eye elicited widespread gene expression changes, with strong upregulation of cell cycle regulatory genes and genes functioning in the Notch intercellular communication pathway. Inducible expression of TDP-43 specifically in neurons elicited significant expression differences in a more restricted set of genes. Genes that were upregulated in both paradigms included SpindleB and the Notch target Hey, which appeared to be a direct TDP-43 target. Mutations that diminished activity of Notch or disrupted the function of downstream Notch target genes extended the lifespan of TDP-43 transgenic flies, suggesting that Notch activation was deleterious in this model. Finally, we showed that mutation of the nucleoporin Nup50 increased the lifespan of TDP-43 transgenic flies, suggesting that nuclear events contribute to TDP-43-dependent neurotoxicity. The combined findings identified pathways whose deregulation might contribute to TDP-43-induced neurotoxicity in Drosophila.
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Project description:Sickle cell disease (SCD) and β-thalassemia, two monogenic diseases caused by mutations in the β-globin gene, affect millions of individuals worldwide. These hemoglobin disorders are characterized by extreme clinical heterogeneity, complicating patient management and treatment. A better understanding of this patient-to-patient clinical variability would dramatically improve care and might also guide the development of novel therapies. Studies of the natural history of these β-hemoglobinopathies have identified fetal hemoglobin levels and concomitant α-thalassemia as important modifiers of disease severity. Several small-scale studies have attempted to identify additional genetic modifiers of SCD and β-thalassemia, without much success. Fortunately, improved knowledge of the human genome and the development of new genomic tools, such as genome-wide genotyping arrays and next-generation DNA sequencers, offer new opportunities to use genetics to better understand the causes of the many complications observed in β-hemoglobinopathy patients. Here I discuss the most important factors to consider when planning an experiment to find associations between β-hemoglobinopathy-related complications and DNA sequence variants, with a focus on how to successfully perform a genome-wide association study. I also review the literature and explain why most published findings in the field of SCD modifier genetics are likely to be false-positive reports, with the goal to draw lessons allowing investigators to design better genetic experiments.
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Project description:BackgroundGenetic variation in the regulatory region of the human serotonin transporter gene (SLC6A4) has been shown to affect brain functionality and personality. However, large heterogeneity in its biological effects is observed, which is at least partially due to genetic modifiers. To gain insight into serotonin transporter (SERT)-specific genetic modifiers, we studied an intercross between the Wistar SERT-/- rat and the behaviorally and genetically divergent Brown Norway rat, and performed a QTL analysis.ResultsIn a cohort of >150 intercross SERT-/- and control (SERT+/+) rats we characterized 12 traits that were previously associated with SERT deficiency, including activity, exploratory pattern, cocaine-induced locomotor activity, and abdominal and subcutaneous fat. Using 325 genetic markers, 10 SERT-/--specific quantitative trait loci (QTLs) for parameters related to activity and exploratory pattern (Chr.1,9,11,14), and cocaine-induced anxiety and locomotor activity (Chr.5,8) were identified. No significant QTLs were found for fat parameters. Using in silico approaches we explored potential causal genes within modifier QTL regions and found interesting candidates, amongst others, the 5-HT1D receptor (Chr. 5), dopamine D2 receptor (Chr. 8), cannabinoid receptor 2 (Chr. 5), and genes involved in fetal development and plasticity (across chromosomes).ConclusionsWe anticipate that the SERT-/--specific QTLs may lead to the identification of new modulators of serotonergic signaling, which may be targets for pharmacogenetic and therapeutic approaches.
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Project description:Parkinson's disease (PD) is the most common cause of neurodegenerative movement disorder and the second most common cause of dementia. Genes are thought to have a stronger effect on age-at-onset of PD than on risk, yet there has been a phenomenal success in identifying risk loci but not age-at-onset modifiers. We conducted a genome-wide study for age-at-onset. We analysed familial and non-familial PD separately, per prior evidence for strong genetic effect on age-at-onset in familial PD. GWAS was conducted in 431 unrelated PD individuals with at least one affected relative (familial PD) and 1544 non-familial PD from the NeuroGenetics Research Consortium (NGRC); an additional 737 familial PD and 2363 non-familial PD were used for replication. In familial PD, two signals were detected and replicated robustly: one mapped to LHFPL2 on 5q14.1 (PNGRC = 3E-8, PReplication = 2E-5, PNGRC + Replication = 1E-11), the second mapped to TPM1 on 15q22.2 (PNGRC = 8E-9, PReplication = 2E-4, PNGRC + Replication = 9E-11). The variants that were associated with accelerated onset had low frequencies (<0.02). The LHFPL2 variant was associated with earlier onset by 12.33 [95% CI: 6.2; 18.45] years in NGRC, 8.03 [2.95; 13.11] years in replication, and 9.79 [5.88; 13.70] years in the combined data. The TPM1 variant was associated with earlier onset by 15.30 [8.10; 22.49] years in NGRC, 9.29 [1.79; 16.79] years in replication, and 12.42 [7.23; 17.61] years in the combined data. Neither LHFPL2 nor TPM1 was associated with age-at-onset in non-familial PD. LHFPL2 (function unknown) is overexpressed in brain tumours. TPM1 encodes a highly conserved protein that regulates muscle contraction, and is a tumour-suppressor gene.
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Project description:Despite remarkable progress in the identification of mutations that drive genetic disorders, progress in understanding the effect of genetic background on the penetrance and expressivity of causal alleles has been modest, in part because of the methodological challenges in identifying genetic modifiers. Nonetheless, the progressive discovery of modifier alleles has improved both our interpretative ability and our analytical tools to dissect such phenomena. In this review, we analyze the genetic properties and behaviors of modifiers as derived from studies in patient populations and model organisms and we highlight conceptual and technological tools used to overcome some of the challenges inherent in modifier mapping and cloning. Finally, we discuss how the identification of these modifiers has facilitated the elucidation of biological pathways and holds the potential to improve the clinical predictive value of primary causal mutations and to develop novel drug targets.
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Project description:Gaucher disease is a genetic disorder and the most common lysosomal disease caused by the deficiency of enzyme β-glucocerebrosidase (GCase). Although enzyme replacement therapy (ERT) is successfully applied using mannose-exposed conjugated glucocerebrosidase, the lower stability of the enzyme in blood demands periodic intravenous administration that adds to the high cost of treatment. In this work, the enzyme β-glucocerebrosidase was encapsulated inside virus-like nanoparticles (VLPs) from brome mosaic virus (BMV), and their surface was functionalized with mannose groups for targeting to macrophages. The VLP nanoreactors showed significant GCase catalytic activity. Moreover, the Michaelis-Menten constants for the free GCase enzyme (KM =0.29 mM) and the functionalized nanoreactors (KM =0.32 mM) were similar even after chemical modification. Importantly, the stability of enzymes under physiological conditions (pH 7.4, 37 °C) was enhanced by ≈11-fold after encapsulation; this is beneficial for obtaining a higher blood circulation half-life, which may decrease the cost of therapy by reducing the requirement of multiple intravenous injections. Finally, the mannose receptor targeted enzymatic nanoreactors showed enhanced internalization into macrophage cells. Thus, the catalytic activity and cell targeting suggest the potential of these nanoreactors in ERT of Gaucher's disease.
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Project description:Understanding the role of common polymorphisms in modulating the clinical phenotype when they co-occur with a disease-causing lesion is of critical importance in medical genetics. We explored the impact of apparently neutral common polymorphisms, using the gene encoding the urea cycle enzyme, ornithine transcarbamylase (OTC), as a model system. Distinct combinations of genetic backgrounds embracing two missense polymorphisms were created in cis with the pathogenic p.Arg40His replacement. In vitro enzymatic assays revealed that the polymorphic variants were able to modulate OTC activity both in the presence or absence of the pathogenic lesion. First, we found that the combination of the minor alleles of polymorphisms p.Lys46Arg and p.Gln270Arg significantly enhanced enzymatic activity in the wild-type protein. Second, enzymatic assays revealed that the minor allele of the p.Gln270Arg polymorphism was capable of ameliorating OTC activity when combined in cis with the pathogenic p.Arg40His replacement. Structural analysis predicted that the minor allele of the p.Gln270Arg polymorphism would serve to stabilize the OTC wild-type protein, thereby corroborating the results of the experimental assays. Our findings demonstrate the potential importance of cis-interactions between common polymorphic variants and pathogenic missense mutations and illustrate how standing genetic variation can modulate protein function.
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