bims-glecem 2023-04-09 papers

Issue of 2023–04–09
five papers selected by
Dipsikha Biswas, Københavns Universitet

Mol Ther Methods Clin Dev. 2023 Jun 08. 29 108-119

Genome editing using Staphylococcus aureus Cas9 in a canine model of glycogen storage disease Ia.

Benjamin Arnson, Hye Ri Kang, Elizabeth D Brooks, Dorothy Gheorghiu, Ekaterina Ilich, David Courtney, Jeffrey I Everitt, Bryan R Cullen, Dwight D Koeberl.

Glycogen storage disease type Ia (GSD Ia) is the inherited deficiency of glucose-6-phosphatase (G6Pase), associated with life-threatening hypoglycemia and long-term complications, including hepatocellular carcinoma formation. Gene replacement therapy fails to stably reverse G6Pase deficiency. We attempted genome editing using two adeno-associated virus vectors, one that expressed Staphylococcus aureus Cas9 protein and a second containing a donor transgene encoding G6Pase, in a dog model for GSD Ia. We demonstrated donor transgene integration in the liver of three adult-treated dogs accompanied by stable G6Pase expression and correction of hypoglycemia during fasting. Two puppies with GSD Ia were treated by genome editing that achieved donor transgene integration in the liver. Integration frequency ranged from 0.5% to 1% for all dogs. In adult-treated dogs, anti-SaCas9 antibodies were detected before genome editing, reflecting prior exposure to S. aureus. Nuclease activity was low, as reflected by a low percentage of indel formation at the predicted site of SaCas9 cutting that indicated double-stranded breaks followed by non-homologous end-joining. Thus, genome editing can integrate a therapeutic transgene in the liver of a large animal model, either early or later in life, and further development is warranted to provide a more stable treatment for GSD Ia.

Keywords: CRISPR-Cas9; adeno-associated virus vectors; genome editing; glucose-6-phosphatase; glycogen storage disease; inherited disorder of metabolism; von Gierke disease

DOI: https://doi.org/10.1016/j.omtm.2023.03.001

Bioorg Med Chem Lett. 2023 Mar 31. pii: S0960-894X(23)00141-5. [Epub ahead of print] 129263

Development of [18F]Thiazolylacylaminopyridine-Based Glycogen Synthase Kinase-3β Ligands for Positron Emission Tomography Imaging.

Jianhua Jia, Lan Yi, Zhu Xia, Meixian Yang, Dachuan Qiu, Zhenghuan Zhao, Zhiping Peng.

Glycogen synthase kinase-3β (GSK-3β) regulates numerous of CNS-specific signaling pathways, and is particularly implicated in various pathogenetic mechanisms of Alzheimer's disease (AD). A noninvasive method for detecting GSK-3β in AD brains via positron emission tomography (PET) imaging could enhance the understanding of AD pathogenesis and aid in the development of AD therapeutic drugs. In this study, an array of fluorinated thiazolyl acylaminopyridines (FTAAP) targeting GSK-3β were designed and synthesized. These compounds showed moderate to high affinities (IC50 = 6.0 - 426 nM) for GSK-3β in vitro. A potential GSK-3β tracer, [18F]8, was successfully radiolabeled. [18F]8 had unsatisfactory initial brain uptake despite its suitable lipophilicity, molecular size and good stability. Further structural refinement of the lead compound is needed to develop promising [18F]-labeled radiotracers for the detection of GSK-3β in AD brains.

Keywords: Alzheimer’s disease; Glycogen synthase kinase-3β; PET imaging agent; [(18)F]Thiazolylacylaminopyridines

DOI: https://doi.org/10.1016/j.bmcl.2023.129263

J Biol Chem. 2023 Apr 01. pii: S0021-9258(23)00311-3. [Epub ahead of print] 104669

Cyp26a1 supports post-natal retinoic acid homeostasis and glucoregulatory control.

Hong Sik Yoo, Michael A Cockrum, Joseph L Napoli.

Considerable evidence confirms the importance of Cyp26a1 to all-trans-retinoic acid (RA) homeostasis during embryogenesis. In contrast, despite its presence in post-natal liver as a potential major RA catabolizing enzyme, and its acute sensitivity to induction by RA, some data suggested that Cyp26a1 contributes only marginally to endogenous RA homeostasis postnatally. We report re-evaluation of a conditional Cyp26a1 knockdown in the postnatal mouse. The current results show that Cyp26a1 mRNA in wild-type (WT) mouse liver increases 16-fold upon re-feeding after a fast, accompanied by an increased rate of RA elimination and a 41% decrease in the RA concentration. In contrast, Cyp26a1 mRNA in the re-fed homozygotic knockdown reached only 2% of its extent in WT during re-feeding, accompanied by a slower rate of RA catabolism and no decrease in liver RA, relative to fasting. Re-fed homozygous knockdown mice also had decreased Akt1 and 2 phosphorylation and pyruvate dehydrogenase kinase 4 (Pdk4) mRNA, and increased glucokinase (Gck) mRNA, glycogen phosphorylase (Pygl) phosphorylation, and serum glucose, relative to WT. Fasted homozygous knockdown mice had increased glucagon/insulin relative to WT. These data indicate that Cyp26a1 participates prominently in moderating the post-natal liver concentration of endogenous RA, and contributes essentially to glucoregulatory control.

Keywords: Corticosterone; Cyp26a1; glucagon; glucoregulation; insulin; retinoic acid

DOI: https://doi.org/10.1016/j.jbc.2023.104669

Am J Physiol Endocrinol Metab. 2023 Apr 05.

Human skeletal muscle methylome after low carbohydrate energy balanced exercise.

Piotr P Gorski, Daniel C Turner, Juma Iraki, James P Morton, Adam P Sharples, José L Areta.

We aimed to investigate the human skeletal muscle (SkM) DNA methylome after exercise in low carbohydrate (CHO) energy balance (with high fat) compared with exercise in low-CHO energy deficit (with low fat) conditions. The objective to identify novel epigenetically regulated genes and pathways associated with 'train-low sleep-low' paradigms. The sleep-low conditions included 9 males that cycled to deplete muscle glycogen while reaching a set energy expenditure. Post-exercise, low-CHO meals (protein-matched) completely replaced (using high-fat) or only partially replaced (low-fat) the energy expended. The following morning resting baseline biopsies were taken and the participants then undertook 75 minutes of cycling exercise, with skeletal muscle biopsies collected 30 minutes and 3.5 hours post exercise. Discovery of genome-wide DNA methylation was undertaken using Illumina EPIC arrays and targeted gene expression analysis was conducted by RT-qPCR. At baseline participants under energy balance (high fat) demonstrated a predominantly hypermethylated (60%) profile across the genome compared to energy deficit-low fat conditions. However, post exercise performed in energy balance (with high fat) elicited a more prominent hypomethylation signature 30 minutes post-exercise in gene regulatory regions important for transcription (CpG islands within promoter regions) compared with exercise in energy deficit (with low fat) conditions. Such hypomethylation was enriched within pathways related to: IL6-JAK-STAT signalling, metabolic processes, p53 / cell cycle and oxidative / fatty acid metabolism. Hypomethylation within the promoter regions of genes: HDAC2, MECR, IGF2 and c13orf16 were associated with significant increases in gene expression in the post-exercise period in energy balance compared with energy deficit. Furthermore, histone deacetylase, HDAC11 was oppositely regulated at the gene expression level compared with HDAC2, where HDAC11 was hypomethylated yet increased in energy deficit compared with energy balance conditions. Overall, we identify some novel epigenetically regulated genes associated with train-low sleep-low paradigms.

Keywords: HDAC; exercise; low CHO; methylome

DOI: https://doi.org/10.1152/ajpendo.00029.2023

Mol Biotechnol. 2023 Apr 06.

Single-Cell Sequencing Data Analysis Unveiled HDAC1 as the Therapeutic Target for Chronic Pancreatitis.

Jie Yang, Rui Li.

Chronic pancreatitis (CP) as a progressive inflammatory disorder, remains untreatable. The novel treatment strategy for CP is imperative. We attempted to explore the therapeutic biomarkers for CP. The single-cell sequencing data were retrieved from Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) in idiopathic CP were identified, followed by function and pathway annotation, and PPI network established. DEGs of interest were verified in human tissue samples. The function of candidate biomarker was determined in the murine model with CP. A total of 208 genes were specially differentially expressed in idiopathic patients. Functional enrichment analysis showed DEGs were mainly enriched in glycogen catabolic process, RNA splicing, and glucagon signaling pathway. A PPI network centered on HDAC1 was constructed. HDAC1 was overexpressed in CP patients. The murine model with CP was induced by repetitive cerulein treatment. Silencing sh-HDAC1 treatment reversed cerulein-induced inflammatory cells accumulation, high expression of TGF-β1, and collagen 1 in pancreas in vivo. HDAC1 might be served as potential biomarker for CP. The present study provided insights into the molecular mechanism of CP that may be useful in further investigations.

Keywords: CP mice model; Chronic pancreatitis; HDAC1; Protein–protein interaction network; Single-cell sequencing

DOI: https://doi.org/10.1007/s12033-023-00718-x