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. 2021 Jan-Jun:296:100150.
doi: 10.1074/jbc.RA120.015773. Epub 2020 Dec 10.

An inducible glycogen synthase-1 knockout halts but does not reverse Lafora disease progression in mice

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An inducible glycogen synthase-1 knockout halts but does not reverse Lafora disease progression in mice

Silvia Nitschke et al. J Biol Chem. 2021 Jan-Jun.

Abstract

Malstructured glycogen accumulates over time in Lafora disease (LD) and precipitates into Lafora bodies (LBs), leading to neurodegeneration and intractable fatal epilepsy. Constitutive reduction of glycogen synthase-1 (GYS1) activity prevents murine LD, but the effect of GYS1 reduction later in disease course is unknown. Our goal was to knock out Gys1 in laforin (Epm2a)-deficient LD mice after disease onset to determine whether LD can be halted in midcourse, or even reversed. We generated Epm2a-deficient LD mice with tamoxifen-inducible Cre-mediated Gys1 knockout. Tamoxifen was administered at 4 months and disease progression assessed at 12 months. We verified successful knockout at mRNA and protein levels using droplet digital PCR and Western blots. Glycogen determination and periodic acid-Schiff-diastase staining were used to analyze glycogen and LB accumulation. Immunohistochemistry using astrocytic (glial fibrillary acidic protein) and microglial (ionized calcium-binding adapter molecule 1) markers was performed to investigate neuroinflammation. In the disease-relevant organ, the brain, Gys1 mRNA levels were reduced by 85% and GYS1 protein depleted. Glycogen accumulation was halted at the 4-month level, while LB formation and neuroinflammation were significantly, though incompletely, prevented. Skeletal muscle analysis confirmed that Gys1 knockout inhibits glycogen and LB accumulation. However, tamoxifen-independent Cre recombination precluded determination of disease halting or reversal in this tissue. Our study shows that Gys1 knockdown is a powerful means to prevent LD progression, but this approach did not reduce brain glycogen or LBs to levels below those at the time of intervention. These data suggest that endogenous mechanisms to clear brain LBs are absent or, possibly, compromised in laforin-deficient murine LD.

Keywords: Lafora bodies; Lafora disease (Lafora progressive myoclonic epilepsy, MELF); glycogen; glycogen storage disease; glycogen synthase; neurodegenerative disease; neuroinflammation.

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Conflict of interest statement

Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.

Figures

Figure 1
Figure 1
Generation of Lafora disease (LD) model mice with tamoxifen (TAM)-inducible Gys1 knockout.A, overview of breeding strategy. B, schematic of study timeline. C, overview of experimental groups of mice. LKO, laforin (Epm2a) knockout; WT, wild-type. D, western blots depicting brain and muscle GYS1 protein levels with GAPDH as the loading control. Duplicates are biological replicates. CAG, chicken beta actin promoter/enhancer coupled with the cytomegalovirus immediate-early enhancer; En2 SA, mouse engrailed 2 splice acceptor sequence; ER, mutant estrogen receptor 1 ligand-binding domain; FRT, flippase recombination target; lacZ, reporter gene encoding β-galactosidase; loxP, Cre recombinase recognition site; neo, neomycin resistance gene; pA, SV40 polyadenylation signal.
Figure 2
Figure 2
Gys1 expression, glycogen, and Lafora body (LB) accumulation in the brain.A, Gys1 mRNA levels in the brain. Tfrc served as the reference gene. B, western blots depicting brain GYS1 protein levels with GAPDH as the loading control. Replicate lanes are from individual animals. C, total glycogen content in the brain. D, LB quantification in the hippocampus. Error bars indicate SD. Statistical significance (p < 0.05) is denoted by different letters, while lack of significance is reflected by at least one shared letter. Where applicable, subsets of experimental groups underwent secondary follow-up statistical testing (Fig. S1). E, representative images of the PASD-stained hippocampus. Scale bar: 50 μm. PASD, periodic acid–Schiff–diastase.
Figure 3
Figure 3
Reactive gliosis in the brain.AB, quantification of immunohistochemistry stain against GFAP (A) and IBA1 (B) in the hippocampus. Error bars indicate SD. Statistical significance (p < 0.05) is denoted by different letters, while lack of significance is reflected by at least one shared letter. Where applicable, subsets of experimental groups underwent secondary follow-up statistical testing (Fig. S3). C and D, representative images of GFAP (C) and IBA1 (D) immunohistochemistry in the hippocampus. Scale bars: 50 μm. GFAP, glial fibrillary acidic protein; IBA1, ionized calcium–binding adapter molecule 1.
Figure 4
Figure 4
Gys1 expression, glycogen, and Lafora body accumulation in the skeletal muscle.A, Gys1 mRNA levels in the muscle. Actb served as the reference gene. B, western blots depicting muscle GYS1 protein levels with GAPDH as the loading control. Replicate lanes are from individual animals. C, total glycogen content in the muscle. D, insoluble glycogen content in the muscle. Error bars indicate SD. Statistical significance (p < 0.05) is denoted by different letters, while lack of significance is reflected by at least one shared letter. Where applicable, subsets of experimental groups underwent secondary follow-up statistical testing (Fig. S5). EF, representative images of the PASD-stained muscle. Scale bars: 100 μm (E); 50 μm (F). PASD, periodic acid–Schiff–diastase.

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