Gys1 antisense therapy rescues neuropathological bases of murine Lafora disease

Abstract

Lafora disease is a fatal progressive myoclonus epilepsy. At root, it is due to constant acquisition of branches that are too long in a subgroup of glycogen molecules, leading them to precipitate and accumulate into Lafora bodies, which drive a neuroinflammatory response and neurodegeneration. As a potential therapy, we aimed to downregulate glycogen synthase, the enzyme responsible for glycogen branch elongation, in mouse models of the disease. We synthesized an antisense oligonucleotide (Gys1-ASO) that targets the mRNA of the brain-expressed glycogen synthase 1 gene (Gys1). We administered Gys1-ASO by intracerebroventricular injection and analysed the pathological hallmarks of Lafora disease, namely glycogen accumulation, Lafora body formation, and neuroinflammation. Gys1-ASO prevented Lafora body formation in young mice that had not yet formed them. In older mice that already exhibited Lafora bodies, Gys1-ASO inhibited further accumulation, markedly preventing large Lafora bodies characteristic of advanced disease. Inhibition of Lafora body formation was associated with prevention of astrogliosis and strong trends towards correction of dysregulated expression of disease immune and neuroinflammatory markers. Lafora disease manifests gradually in previously healthy teenagers. Our work provides proof of principle that an antisense oligonucleotide targeting the GYS1 mRNA could prevent, and halt progression of, this catastrophic epilepsy.

Keywords: Lafora disease; antisense oligonucleotides; glycogen synthase; neuroinflammation; therapy.

Figure 1

Gys1-ASO, administered at 1 and 2 months, leads to reduced Gys1 mRNA and GYS1 protein levels and attenuates glycogen and Lafora body accumulation in Epm2a^−/− mice at 3 months. (A) Brain Gys1 mRNA relative expression levels in PBS-, Ctrl-ASO-, and Gys1-ASO-injected Epm2a^−/− mice. Ctrl-ASO, a no-target control ASO. (B) Brain GYS1 western blots with GAPDH as loading control. (C) Quantification of GYS1 western blots shown in B, normalized to GAPDH. (D) Brain total glycogen content. (E) Lafora body (LB) quantification in the hippocampus. (F) Representative images of PASD stained hippocampus. Scale bar = 50 µm. All data are presented as mean ± SEM. Significance levels are indicated as: *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

Figure 2

Later ASO administration, after Lafora disease onset, effectively slows disease progression in Epm2a^−/− mice without signs of reversal. (A) Experimental design of three different trials. PBS, Ctrl-ASO (no-target control ASO), or Gys1-ASO were injected and mice sacrificed at indicated time points. Untreated mice, sacrificed at time of first injection, served as baseline control. (B–E) Results from the 3–6 month trial (trial design in A), showing Gys1 mRNA levels in Epm2a^−/− (B), brain total glycogen content in Epm2a^−/− (C), and wild-type (WT) (D), and Lafora body (LB) quantification in the hippocampus of Epm2a^−/− mice (E). (F–H) Results from the 8–14 month trial, using 300 µg ASO for each injection (trial design in A), showing Gys1 mRNA levels (F), brain total glycogen content (G) and Lafora body quantification in the hippocampus (H) of Epm2a^−/− mice. (I and J) Results the from 8–14 month trial, using a higher dose of 500 µg ASO for each injection (trial design in A), showing brain total glycogen content (I) and Lafora body quantification in the hippocampus (J) of Epm2a^−/− mice. (K) Representative images of PASD stained hippocampus of Epm2a^−/− mice from the three different trials, explained in A. Scale bar = 50 µm. All data are presented as mean ± SEM. Significance levels are indicated as: *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

Figure 3

Long-term ASO treatment strongly prevents glycogen and Lafora body accumulation in Epm2a^−/− mice. (A) Brain total glycogen content. (B) Lafora body (LB) quantification in the hippocampus. (C) Representative images of PASD stained hippocampus. Scale bar = 50 µm. PBS, Ctrl-ASO (no-target control ASO), or Gys1-ASO were injected at 3, 6 and 9 months and brain tissue analysed at 12 months. Untreated mice, sacrificed and analysed at 3 months, served as a baseline control. Significance levels are indicated as: *P < 0.05, ***P < 0.001 and ****P < 0.0001. Asterisks in pink indicate significance levels compared with the corresponding wild-type (WT). (D) Lafora body size distribution in Epm2a^−/− mice. (E) Differential Lafora body size distribution (fold change compared to baseline) in Epm2a^−/− mice. (D and E) Top: P-values comparing Lafora body number between indicated experimental groups at different Lafora body size bins. All data are presented as mean ± SEM.

Figure 4

Long-term ASO treatment rescues astrogliosis in Epm2a^−/− mice. (A–E) Relative expression levels of Gys1 mRNA (A), and inflammatory and immune system response marker genes Lcn2 (B), Cxcl10 (C), Ccl5 (D), and C3 (E) analysed by qRT-PCR. (F) GFAP signal quantification in the hippocampus. (G) Representative immunohistochemistry (IHC) images of anti-GFAP in the hippocampus. Scale bar = 50 µm. PBS, Ctrl-ASO (no-target control ASO), or Gys1-ASO were injected at 3, 6 and 9 months and brain tissue analysed at 12 months. Untreated mice, sacrificed and analysed at 3 months, served as a baseline control. All data are presented as mean ± SEM. Significance levels are indicated as: *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. Asterisks in pink indicate significance levels compared with the corresponding wild-type (WT).