Polyglucosan body structure in Lafora disease

Abstract

Abnormal carbohydrate structures known as polyglucosan bodies (PGBs) are associated with neurodegenerative disorders, glycogen storage diseases (GSDs), and aging. A hallmark of the GSD Lafora disease (LD), a fatal childhood epilepsy caused by recessive mutations in the EPM2A or EPM2B genes, are cytoplasmic PGBs known as Lafora bodies (LBs). LBs result from aberrant glycogen metabolism and drive disease progression. They are abundant in brain, muscle and heart of LD patients and Epm2a-/and Epm2b-/mice. LBs and PGBs are histologically reminiscent of starch, semicrystalline carbohydrates synthesized for glucose storage in plants. In this study, we define LB architecture, tissue-specific differences, and dynamics. We propose a model for how small polyglucosans aggregate to form LBs. LBs are very similar to PGBs of aging and other neurological disorders, and so these studies have direct relevance to the general understanding of PGB structure and formation.

Central Nervous System Delivery and Biodistribution Analysis of an Antibody–Enzyme Fusion for the Treatment of Lafora Disease

“…This study focuses on VAL-0417, a 3E10-based cell penetrating AEF that utilizes pancreatic amylase to degrade LBs and is efficacious in preclinical LD mouse models. 35 We developed an ELISA for VAL-0417 and provided initial preclinical data demonstrating VAL-0417 delivery into WT and LD mice.…”

“…Generation of VAL-0417: VAL-0417 was designed by Valerion Therapeutics (Concord, MA) and produced and purified by Proteos (Kalamazoo, MI) as previously described. 35 A gene fusion of the codon optimized genes for human IgG2A 3E10 Fab heavy chain followed by a linker and AMY2A, and the 3E10 kappa light chain were synthesized and cloned into a proprietary version of the pTT5 plasmid (Canadian National Research Centre). This plasmid was transfected into HEK293-6E cells, which grew for 5 days before VAL-0417 was purified using pre-packed CaptureSelect IgG-CH1 affinity columns (Thermo Fisher).…”

“…Lafora bodies were purified using established methods. 35,36 Briefly, Epm2a−/− mouse brains were harvested and homogenized in lysis buffer (100 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM CaCl 2 , 0.5% NaN 3 ) and then centrifuged for 10 min at 10,000 xg at 4°C. The pellet was collected and resuspended in lysis buffer with 0.2% SDS and 0.4% mg/ml proteinase K and digested overnight at 37°C.…”

“…34 Recent work demonstrated that VAL-0417 degrades LBs in vitro and in vivo. 35 As a key tool to allow monitoring and iterative design of a dosage protocol, we developed an indirect sandwich enzyme-linked immunosorbent assay (ELISA) to specifically detect and monitor VAL-0417. The assay utilizes a capture antibody specific for the DNA binding region of VAL-0417 (anti-3E10 Fab) and allows for orthogonal detection of the fused amylase.…”

Generation and characterization of a laforin nanobody inhibitor

“…The architecture and depth of this active site allow laforin to dephosphorylate glycogen 15,17,18 . The laforin CBM belongs to CBM family 20 and allows laforin to bind glycogen as well as other complex carbohydrates in vitro and in vivo, like LBs, amylopectin, and maltodextrins 8,18,19 . Key structural aspects of laforin that promote glucan-specific phosphatase activity are: closely integrated CBM-DSP domains, an anti-parallel dimerization, and a signature DSP sequence within the active site channel 15 .…”