scholarly journals An in vivo drug repurposing screen and transcriptional analyses reveals the serotonin pathway and GSK3 as major therapeutic targets for NGLY1 deficiency

2021 ◽  
Author(s):  
Kevin A. Hope ◽  
Alexys R. Berman ◽  
Randall T. Peterson ◽  
Clement Y Chow
Keyword(s):  
Rare Disease ◽  
Glycogen Synthase ◽  
Genetic Modifier ◽  
Drug Repurposing ◽  
Dopamine Neurons ◽  
Patient Specific ◽  
Global Developmental Delay ◽  
Loss Of Function ◽  
Serotonin Pathway

NGLY1 deficiency, a rare disease with no effective treatment, is caused by autosomal recessive, loss-of-function mutations in the N-glycanase 1 (NGLY1) gene and is characterized by global developmental delay, hypotonia, alacrima, and seizures. We used an adult Drosophila model of NGLY1 deficiency to conduct an in vivo, unbiased, small molecule, repurposing screen of FDA-approved drugs to identify therapeutic compounds. Seventeen molecules rescued lethality in a patient-specific NGLY1 deficiency model, including multiple serotonin and dopamine modulators. Exclusive dNGLY1 expression in serotonin and dopamine neurons, in an otherwise dNGLY1-null fly, was sufficient to rescue lethality. Further, genetic modifier and transcriptomic data supports the importance of serotonin signaling in NGLY1 deficiency. Connectivity Map analysis identified glycogen synthase kinase 3 (GSK3) inhibition as a potential therapeutic mechanism for NGLY1 deficiency, which we experimentally validated with TWS119 and lithium. Strikingly, GSK3 inhibitors and a serotonin modulator rescued size defects in dNGLY1 deficient larvae upon proteasome inhibition, suggesting that these compounds act through NRF1, a transcription factor that regulates proteasome expression. This study reveals the importance of the serotonin pathway in NGLY1 deficiency, and serotonin modulators or GSK3 inhibitors may be effective therapeutics for this rare disease.

scholarly journals Propofol attenuates lung ischemia/reperfusion injury though the involvement of the MALAT1/microRNA-144/GSK3β axis

2021 ◽  
Vol 27 (1) ◽  
Author(s):  
Jian-Ping Zhang ◽  
Wei-Jing Zhang ◽  
Miao Yang ◽  
Hua Fang
Keyword(s):  
Cell Apoptosis ◽  
Ischemia Reperfusion Injury ◽  
Glycogen Synthase ◽  
Ischemia Reperfusion ◽  
Inflammatory Factors ◽  
Therapeutic Effects ◽  
Protective Effects ◽  
Loss Of Function

Abstract Background Propofol, an intravenous anesthetic, was proven to protect against lung ischemia/reperfusion (I/R) injury. However, the detailed mechanism of Propofol in lung I/R injury is still elusive. This study was designed to explore the therapeutic effects of Propofol, both in vivo and in vitro, on lung I/R injury and the underlying mechanisms related to metastasis-associated lung adenocarcinoma transcript 1 (MALAT1)/microRNA-144 (miR-144)/glycogen synthase kinase-3β (GSK3β). Methods C57BL/6 mice were used to establish a lung I/R injury model while pulmonary microvascular endothelial cells (PMVECs) were constructed as hypoxia/reperfusion (H/R) cellular model, both of which were performed with Propofol treatment. Gain- or loss-of-function approaches were subsequently employed, followed by observation of cell apoptosis in lung tissues and evaluation of proliferative and apoptotic capabilities in H/R cells. Meanwhile, the inflammatory factors, autophagosomes, and autophagy-related proteins were measured. Results Our experimental data revealed that Propofol treatment could decrease the elevated expression of MALAT1 following I/R injury or H/R induction, indicating its protection against lung I/R injury. Additionally, overexpressing MALAT1 or GSK3β promoted the activation of autophagosomes, proinflammatory factor release, and cell apoptosis, suggesting that overexpressing MALAT1 or GSK3β may reverse the protective effects of Propofol against lung I/R injury. MALAT1 was identified to negatively regulate miR-144 to upregulate the GSK3β expression. Conclusion Overall, our study demonstrated that Propofol played a protective role in lung I/R injury by suppressing autophagy and decreasing release of inflammatory factors, with the possible involvement of the MALAT1/miR-144/GSK3β axis.

Abstract 14532: Computational Drug Repurposing Identifies a Piperlongumine Analog That Controls a Gstp1-iscu Pathogenic Axis in Pulmonary Hypertension

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Jimin Yang
Keyword(s):  
Drug Repurposing ◽  
Gene Clusters ◽  
Loss Of Function ◽  
Sequencing Data ◽  
Glutathione S Transferase ◽  
Computational Screening ◽  
Therapeutic Development ◽  
Pulmonary Arterial

Background and Hypothesis: Pulmonary arterial hypertension (PAH) is an incurable vascular disease for which chemotherapies are being considered for therapeutic development. There is no method reported to date for effective computational screening of these drugs for this disease. Big data analyses that leverage the molecular parallels between cancer and PH may define novel pathogenic mechanisms and facilitate repurposing of chemotherapies for PAH. More specifically, while functional deficiency of the iron-sulfur (Fe-S) biogenesis gene ISCU and oxidative metabolism in human pulmonary arterial endothelial cells (PAECs) is known to drive PAH, the pathogenic regulation of ISCU is not fully defined, and no tailored drugs have been identified to bolster ISCU activity. Methods and Results: We applied a computational algorithm EDDY (Evaluating Differential DependencY), which analyzes RNA sequencing data from 810 cancer cell lines exposed to 368 small molecules, in order to identify chemotherapeutics that depended upon rewired PH-related gene clusters. The top ranked drug was a piperlongumine (PL) analog (BRD2889) that was predicted to extensively rewire dependencies across PH gene clusters, mediated by ISCU. In vitro, coupling gain- and loss-of-function analyses of GSTP1 with BRD2889 exposure in PAECs, we found that BRD2889 inhibits glutathione S-transferase P1 (GSTP1), an enzyme which in turn catalyzes ISCU glutathionylation and increases its stability in hypoxia. Consequently, BRD2889 and GSTP1 knockdown phenocopy one another by increasing Fe-S-dependent Complex I activity and mitochondrial oxygen consumption while ameliorating pathogenic apoptosis. Consistent with these computational and in vitro results, in a mouse model of PAH (IL-6 transgenic mice in hypoxia), BRD2889 improved hemodynamic and molecular disease manifestations in vivo. Conclusions: Using a novel computational platform, we identified a coordinated connection between BRD342289 and GSTP1-ISCU axis, crucial to PAEC metabolism. This study offers insight to fundamental PH pathobiology and sets the stage for accelerated repurposing of chemotherapies such as BRD342289 in PH.

scholarly journals A homozygous loss-of-function CAMK2A mutation causes growth delay, frequent seizures and severe intellectual disability

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Poh Hui Chia ◽  
Franklin Lei Zhong ◽  
Shinsuke Niwa ◽  
Carine Bonnard ◽  
Kagistia Hana Utami ◽  
...  
Keyword(s):  
Intellectual Disability ◽  
Germline Mutation ◽  
Growth Delay ◽  
Global Developmental Delay ◽  
Loss Of Function ◽  
C Elegans ◽  
Calcium Calmodulin Dependent ◽  
Severe Intellectual Disability

Calcium/calmodulin-dependent protein kinase II (CAMK2) plays fundamental roles in synaptic plasticity that underlies learning and memory. Here, we describe a new recessive neurodevelopmental syndrome with global developmental delay, seizures and intellectual disability. Using linkage analysis and exome sequencing, we found that this disease maps to chromosome 5q31.1-q34 and is caused by a biallelic germline mutation in CAMK2A. The missense mutation, p.His477Tyr is located in the CAMK2A association domain that is critical for its function and localization. Biochemically, the p.His477Tyr mutant is defective in self-oligomerization and unable to assemble into the multimeric holoenzyme.In vivo, CAMK2AH477Y failed to rescue neuronal defects in C. elegans lacking unc-43, the ortholog of human CAMK2A. In vitro, neurons derived from patient iPSCs displayed profound synaptic defects. Together, our data demonstrate that a recessive germline mutation in CAMK2A leads to neurodevelopmental defects in humans and suggest that dysfunctional CAMK2 paralogs may contribute to other neurological disorders.

scholarly journals In vivo bioassay to test the pathogenicity of missense human AIP variants

2018 ◽  
Vol 55 (8) ◽  
pp. 522-529 ◽  
Author(s):  
Elena Daniela Aflorei ◽  
Benjamin Klapholz ◽  
Chenghao Chen ◽  
Serban Radian ◽  
Anca Neluta Dragu ◽  
...  
Keyword(s):  
Genetic Counselling ◽  
Fruit Fly ◽  
Positive Control ◽  
Patient Specific ◽  
Loss Of Function ◽  
Interacting Protein ◽  
Missense Variants ◽  
Variants Of Unknown Significance ◽  
Pathogenic Variants

BackgroundHeterozygous germline loss-of-function mutations in the aryl hydrocarbon receptor-interacting protein gene (AIP) predispose to childhood-onset pituitary tumours. The pathogenicity of missense variants may pose difficulties for genetic counselling and family follow-up.ObjectiveTo develop an in vivo system to test the pathogenicity of human AIP mutations using the fruit fly Drosophila melanogaster.MethodsWe generated a null mutant of the Drosophila AIP orthologue, CG1847, a gene located on the Xchromosome, which displayed lethality at larval stage in hemizygous knockout male mutants (CG1847exon1_3). We tested human missense variants of ‘unknown significance’, with ‘pathogenic’ variants as positive control.ResultsWe found that human AIP can functionally substitute for CG1847, as heterologous overexpression of human AIP rescued male CG1847exon1_3 lethality, while a truncated version of AIP did not restore viability. Flies harbouring patient-specific missense AIP variants (p.C238Y, p.I13N, p.W73R and p.G272D) failed to rescue CG1847exon1_3 mutants, while seven variants (p.R16H, p.Q164R, p.E293V, p.A299V, p.R304Q, p.R314W and p.R325Q) showed rescue, supporting a non-pathogenic role for these latter variants corresponding to prevalence and clinical data.ConclusionOur in vivo model represents a valuable tool to characterise putative disease-causing human AIP variants and assist the genetic counselling and management of families carrying AIP variants.

scholarly journals Neurodegeneration caused by LRRK2-G2019S requires Rab10 in select dopaminergic neurons

2019 ◽  
Author(s):  
Stavroula Petridi ◽  
C. Adam Middleton ◽  
Alison Fellgett ◽  
Laura Covill ◽  
Amy Stewart ◽  
...  
Keyword(s):  
Dopaminergic Neurons ◽  
Dopamine Neurons ◽  
Rab Gtpases ◽  
Loss Of Function ◽  
Knock Out ◽  
Electrophysiological Responses ◽  
Lrrk2 G2019s ◽  
Inherited Mutations

AbstractInherited mutations in the LRRK2 protein are the commonest known cause of Parkinson’s, but the molecular link from increased kinase activity to pathological neurodegeneration remains to be determined.In vitro(biochemical and cell culture) assays led to the hypothesis that several Rab GTPases might be LRRK2 substrates. Here we show that Rab10 potently modifiesLRRK2-G2019Smediated electrophysiological responses in anin vivoscreen, in which each Rab was overexpressed inDrosophiladopaminergic neurons. We therefore tested the effect ofRab10loss of function on threeLRRK2-G2019Sphenotypes (vision, movement and sleep) that rely on dopaminergic circuits in both flies and mammals. The knock-out of Rab10in vivofully rescues the reduced responses induced by dopaminergicLRRK2-G2019Sin visual and motor (reaching, proboscis extension) assays, but the sleep phenotype is unaffected. We show that Rab10 is expressed in dopaminergic (tyrosine hydroxylase positive) neurons controlling vision and proboscis movement, but undetectable in those controlling sleep, indicating that anatomical and physiological patterns of Rab10 are related. Our results support the idea that LRRK2 phosphorylates separate targets in distinct neurons and confirm that one degenerative pathway starts with Rab10. Although Rab3 is another putative substrate of LRRK2, it shows no synergy with G2019S and localises to a different subset of dopaminergic neurons from Rab10. We propose that variations inRabexpression may contribute to differences in the rate of neurodegeneration seen in different dopaminergic nuclei in Parkinson’s.Significance StatementA key question in Parkinson’s is why dopamine neurons die particularly fast in some parts of thesubstantia nigra. We focused on the commonest Parkinson’s-related mutation, LRRK2-G2019S.In vitroassays suggested that neurodegeneration may start by LRRK2-G2019S increasing phosphorylation of Rab10. We found Rab10 in fly dopamine neurons in visual and motor pathways, but not in the sleep system. Rab10 knock-out rescues G2019S-induced visual and movement degeneration, leaving sleep dysfunction unaffected. Thus, LRRK2 activates at least two pathways, one Rab10-dependent, leading to neurodegenerationin vivo. Rab3 is found in a different subset of dopaminergic neurons and shows no synergy withLRRK2-G2019S. We propose that variations inRabexpression contribute to differences in neurodegeneration seen in Parkinson’s.

The mutant type 1 protein phosphatase encoded by glc7-1 from Saccharomyces cerevisiae fails to interact productively with the GAC1-encoded regulatory subunit

1994 ◽  
Vol 14 (2) ◽  
pp. 896-905
Author(s):  
J S Stuart ◽  
D L Frederick ◽  
C M Varner ◽  
K Tatchell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Phosphatase ◽  
Glycogen Synthase ◽  
Regulatory Subunit ◽  
Loss Of Function ◽  
Mutant Type ◽  
Glycogen Accumulation

Loss-of-function gac1 mutants of Saccharomyces cerevisiae fail to accumulate normal levels of glycogen because of low glycogen synthase activity. Increased dosage of GAC1 results in increased activity of glycogen synthase and a corresponding hyperaccumulation of glycogen. The glycogen accumulation phenotype of gac1 is similar to that of glc7-1, a type 1 protein phosphatase mutant. We have partially characterized the GAC1 gene product (Gac1p) and show that levels of Gac1p increase during growth with the same kinetics as glycogen accumulation. Gac1p is phosphorylated in vivo and is hyperphosphorylated in a glc7-1 mutant. Gac1p and the type 1 protein phosphatase directly interact in vitro, as assayed by coimmunoprecipitation, and in vivo, as determined by the dihybrid assay described elsewhere (S. Fields and O.-k. Song, Nature [London] 340:245-246, 1989). The interaction between Gac1p and the glc7-1-encoded form of the type 1 protein phosphatase is defective, as assayed by either immunoprecipitation or the dihybrid assay. Increased dosage of GAC1 partially suppresses the glycogen defect of glc7-1. Collectively, our data support the hypotheses that GAC1 encodes a regulatory subunit of type 1 protein phosphatase and that the glycogen accumulation defect of glc7-1 is due at least in part to the inability of the mutant phosphatase to interact with its regulatory subunit.

scholarly journals The mutant type 1 protein phosphatase encoded by glc7-1 from Saccharomyces cerevisiae fails to interact productively with the GAC1-encoded regulatory subunit.

1994 ◽  
Vol 14 (2) ◽  
pp. 896-905 ◽  
Author(s):  
J S Stuart ◽  
D L Frederick ◽  
C M Varner ◽  
K Tatchell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Phosphatase ◽  
Glycogen Synthase ◽  
Regulatory Subunit ◽  
Loss Of Function ◽  
Mutant Type ◽  
Glycogen Accumulation

Loss-of-function gac1 mutants of Saccharomyces cerevisiae fail to accumulate normal levels of glycogen because of low glycogen synthase activity. Increased dosage of GAC1 results in increased activity of glycogen synthase and a corresponding hyperaccumulation of glycogen. The glycogen accumulation phenotype of gac1 is similar to that of glc7-1, a type 1 protein phosphatase mutant. We have partially characterized the GAC1 gene product (Gac1p) and show that levels of Gac1p increase during growth with the same kinetics as glycogen accumulation. Gac1p is phosphorylated in vivo and is hyperphosphorylated in a glc7-1 mutant. Gac1p and the type 1 protein phosphatase directly interact in vitro, as assayed by coimmunoprecipitation, and in vivo, as determined by the dihybrid assay described elsewhere (S. Fields and O.-k. Song, Nature [London] 340:245-246, 1989). The interaction between Gac1p and the glc7-1-encoded form of the type 1 protein phosphatase is defective, as assayed by either immunoprecipitation or the dihybrid assay. Increased dosage of GAC1 partially suppresses the glycogen defect of glc7-1. Collectively, our data support the hypotheses that GAC1 encodes a regulatory subunit of type 1 protein phosphatase and that the glycogen accumulation defect of glc7-1 is due at least in part to the inability of the mutant phosphatase to interact with its regulatory subunit.

scholarly journals Survival effect of PDGF-CC rescues neurons from apoptosis in both brain and retina by regulating GSK3β phosphorylation

2010 ◽  
Vol 207 (4) ◽  
pp. 867-880 ◽  
Author(s):  
Zhongshu Tang ◽  
Pachiappan Arjunan ◽  
Chunsik Lee ◽  
Yang Li ◽  
Anil Kumar ◽  
...  
Keyword(s):  
Neuronal Death ◽  
Glycogen Synthase ◽  
Neuroprotective Effect ◽  
Neutralizing Antibody ◽  
Neuronal Injury ◽  
Loss Of Function ◽  
Neuronal Tissues ◽  
Or Gene ◽  
6 Hydroxydopamine

Platelet-derived growth factor CC (PDGF-CC) is the third member of the PDGF family discovered after more than two decades of studies on the original members of the family, PDGF-AA and PDGF-BB. The biological function of PDGF-CC remains largely to be explored. We report a novel finding that PDGF-CC is a potent neuroprotective factor that acts by modulating glycogen synthase kinase 3β (GSK3β) activity. In several different animal models of neuronal injury, such as axotomy-induced neuronal death, neurotoxin-induced neuronal injury, 6-hydroxydopamine–induced Parkinson’s dopaminergic neuronal death, and ischemia-induced stroke, PDGF-CC protein or gene delivery protected different types of neurons from apoptosis in both the retina and brain. On the other hand, loss-of-function assays using PDGF-C null mice, neutralizing antibody, or short hairpin RNA showed that PDGF-CC deficiency/inhibition exacerbated neuronal death in different neuronal tissues in vivo. Mechanistically, we revealed that the neuroprotective effect of PDGF-CC was achieved by regulating GSK3β phosphorylation and expression. Our data demonstrate that PDGF-CC is critically required for neuronal survival and may potentially be used to treat neurodegenerative diseases. Inhibition of the PDGF-CC–PDGF receptor pathway for different clinical purposes should be conducted with caution to preserve normal neuronal functions.

Targeting Tau Hyperphosphorylation via Kinase Inhibition: Strategy to Address Alzheimer's Disease

2020 ◽  
Vol 20 (12) ◽  
pp. 1059-1073 ◽  
Author(s):  
Ahmad Abu Turab Naqvi ◽  
Gulam Mustafa Hasan ◽  
Md. Imtaiyaz Hassan
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Tau Protein ◽  
Glycogen Synthase ◽  
Paired Helical Filaments ◽  
Tau Hyperphosphorylation ◽  
Microtubule Stabilization ◽  
Extracellular Signal

Microtubule-associated protein tau is involved in the tubulin binding leading to microtubule stabilization in neuronal cells which is essential for stabilization of neuron cytoskeleton. The regulation of tau activity is accommodated by several kinases which phosphorylate tau protein on specific sites. In pathological conditions, abnormal activity of tau kinases such as glycogen synthase kinase-3 β (GSK3β), cyclin-dependent kinase 5 (CDK5), c-Jun N-terminal kinases (JNKs), extracellular signal-regulated kinase 1 and 2 (ERK1/2) and microtubule affinity regulating kinase (MARK) lead to tau hyperphosphorylation. Hyperphosphorylation of tau protein leads to aggregation of tau into paired helical filaments like structures which are major constituents of neurofibrillary tangles, a hallmark of Alzheimer’s disease. In this review, we discuss various tau protein kinases and their association with tau hyperphosphorylation. We also discuss various strategies and the advancements made in the area of Alzheimer's disease drug development by designing effective and specific inhibitors for such kinases using traditional in vitro/in vivo methods and state of the art in silico techniques.

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