Glycogen synthase downregulation rescues the amylopectinosis of murine RBCK1 deficiency

Abstract

Longer glucan chains tend to precipitate. Glycogen, by far the largest mammalian glucan and the largest molecule in the cytosol with up to 55,000 glucoses, does not, due to a highly regularly branched spherical structure that allows it to be perfused with cytosol. Aberrant construction of glycogen leads it to precipitate, accumulate into polyglucosan bodies (PBs) that resemble plant starch amylopectin, and cause disease. This pathology, amylopectinosis, is caused by mutations in a series of single genes whose functions are under active study toward understanding the mechanisms of proper glycogen construction. Concurrently, we are characterizing the physicochemical particularities of glycogen and polyglucosans associated with each gene. These genes include GBE1, EPM2A and EPM2B, which respectively encode the glycogen branching enzyme, the glycogen phosphatase laforin and the laforin-interacting E3 ubiquitin ligase malin for which an unequivocal function is not yet known. Mutations in GBE1 cause a motor neuron disease (Adult Polyglucosan Body Disease, APBD), and mutations in EPM2A or EPM2B a fatal progressive myoclonus epilepsy (Lafora disease, LD). RBCK1 deficiency (RD) causes an amylopectinosis with fatal skeletal and cardiac myopathy (Polyglucosan Body Myopathy 1, PGBM1, OMIM# 615895). RBCK1 is a component of the linear ubiquitin chain assembly complex (LUBAC), with unique functions including generating linear ubiquitin chains and ubiquitinating hydroxyl (versus canonical amine) residues, including of glycogen. In a mouse model we now show (1) that the amylopectinosis of RD like in APBD and LD affects the brain, (2) that RD glycogen like in APBD and LD has overlong branches, (3) that unlike APBD but like LD, RD glycogen is hyperphosphorylated, and finally (4) that unlike laforin-deficient LD but like malin-deficient LD RD’s glycogen hyperphosphorylation is limited to precipitated polyglucosans. In summary, the fundamental glycogen pathology of RD recapitulates that of malin-deficient LD. Additionally, we uncover sex and genetic background effects in RD on organ and brain region specific amylopectinoses, and in the brain on consequent neuroinflammation and behavioral deficits. Finally, we exploit the portion of the basic glycogen pathology that is common to APBD, both forms of LD, and RD, namely overlong branches, to show that a unified approach based on downregulating glycogen synthase, the enzyme that elongates glycogen branches, can rescue all four diseases.

AAV-Mediated Artificial miRNA Reduces Pathogenic Polyglucosan Bodies and Neuroinflammation in Adult Polyglucosan Body and Lafora Disease Mouse Models

“…In any event, the abnormal glycogen in these diseases all but certainly has overextended branches driving it to precipitation, and glycogen synthase downregulation should mitigate the problem as in LD and APBD. In fact, our recent results already confirm this for RBCK1 deficiency [ 53 , 54 ]. An important point to note is that the present GYS1 downregulating therapy or others are expected to slow or halt PB formation but not remove existing ones.…”

HOIL‐1 ubiquitin ligase activity targets unbranched glucosaccharides and is required to prevent polyglucosan accumulation

“…Polyglucosan accumulates in the brain and heart of HOIL-1 [C458S] mice HOIL-1 knockout (KO) mice exhibit early embryonic lethality (Fujita et al, 2018;Peltzer et al, 2018), but HOIL-1 "KO" mice expressing low levels of a truncated protein comprising the N-terminal ubiquitin-like (UBL) and Npl4 zinc finger (NZF) domains, but lacking the C-terminal region including the catalytic RBR domain (Fujita et al, 2018), accumulate polyglucosan in their brain, spinal cord and heart (MacDuff et al, 2015;Nitschke et al, 2022). Because HOIL-1 stabilises its binding partner HOIP, the expression of HOIP and Sharpin is also greatly reduced in these HOIL-1 "KO" mice.…”

“…However, HOIL‐1 deficiency in humans also leads to cardiomyopathy and death from heart failure in early adulthood (Boisson et al , 2012 ; Nilsson et al , 2013 ; Wang et al , 2013 ; Fanin et al , 2015 ; Krenn et al , 2018 ; Phadke et al , 2020 ), which is unrelated to the immune defects, and arises from the progressive accumulation of toxic polyglucosan bodies in cardiac muscle and other tissues, such as the brain, with some patients also displaying cognitive impairment (Phadke et al , 2020 ; Chen et al , 2021 ). Mice expressing low levels of HOIL‐1 (Fujita et al , 2018 ) also form toxic polyglucosan bodies in cardiac muscle (MacDuff et al , 2015 ), brain and spinal cord (Nitschke et al , 2022 ), but it is the brain that is affected predominantly in mice, the animals displaying defects in learning, memory and motor coordination (Nitschke et al , 2022 ).…”

Non-lysine ubiquitylation: Doing things differently

“…One of these, Lafora Disease, is reminiscent of HOIL-1 deficiency and is also caused by mutations in genes not encoding the classical enzymes of glycogen synthesis. Notably, both Lafora Disease and HOIL-1 deficiency lead to hyperphosphorylation of glycogen at the C6 hydroxy group, something not observed in adult polyglucosan body disease caused by mutations in GBE1 (Glycogen Branching Enzyme 1) ( Tagliabracci et al, 2008 ; Turnbull et al, 2010 ; DePaoli-Roach et al, 2015 ; Nitschke et al, 2017 ; Sullivan et al, 2019 ; Nitschke et al, 2022 ). This observation reveals an intriguing interplay between glycogen branching, ubiquitylation, and phosphorylation at the C6 position while also suggesting that HOIL-1 may operate as part of a system that intersects or parallels that which malfunctions to cause Lafora Disease.…”