Sensitive quantification of α-glucans in mouse tissues, cell cultures, and human cerebrospinal fluid

Abstract

The soluble α-polyglucan glycogen is a central metabolite enabling transient glucose storage to suit cellular energy needs. Glycogen storage diseases (GSDs) comprise over 15 entities caused by generalized or tissue-specific defects in enzymes of glycogen metabolism. In several, e.g. in Lafora disease caused by the absence of the glycogen phosphatase laforin or its interacting partner malin, degradation-resistant abnormally structured insoluble glycogen accumulates. Sensitive quantification methods for soluble and insoluble glycogen are critical to research, including therapeutic studies, in such diseases. This paper establishes methodological advancements relevant to glycogen metabolism investigations generally, and GSDs. Introducing a pre-extraction incubation method, we measure degradation-resistant glycogen in as little as 30 mg skeletal muscle or a single hippocampus from Lafora disease mouse models. The digestion-resistant glycogen correlates with the disease-pathogenic insoluble glycogen and can readily be detected in very young mice where glycogen accumulation has just begun. Secondly, we establish a high-sensitivity glucose assay with detection of ATP depletion, enabling 1) quantification of α-glucans in cell culture using a medium-throughput assay suitable for assessment of candidate glycogen synthesis inhibitors, and 2) discovery of α-glucan material in healthy human cerebrospinal fluid, establishing a novel methodological platform for biomarker analyses in Lafora disease and other GSDs.

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

“…Further analyses focused on the effect of TAM-induced and TAM-independent Gys1 knockout on skeletal muscle total and insoluble glycogen content. For the latter, we used a recently established method that utilizes endogenously expressed metabolic enzymes to digest accessible soluble glycogen completely, leaving only the insoluble glycogen, which is not degradable ( 24 ). This method allowed us to determine whether changes in total glycogen content were mostly due to changes in soluble glycogen, insoluble glycogen, or both.…”

Targeting Gys1 with AAV‐SaCas9 decreases pathogenic polyglucosan bodies and neuroinflammation in Adult Polyglucosan Body and Lafora disease mouse models

“…In oxygen and glucose deprived states, normal brain glycogen is very swiftly digested to generate glucose in an attempt to protect the brain [39, 40]. This is so rapid that within minutes of sacrifice normal murine brain glycogen all but disappears [37]. In the APBD and LD mouse models, brain glycogen is a mix of normally branched soluble and consumable glycogen and the ever-accumulating abnormally branched precipitated and digestion-resistant polyglucosans (PBs).…”

“…In the APBD and LD mouse models, brain glycogen is a mix of normally branched soluble and consumable glycogen and the ever-accumulating abnormally branched precipitated and digestion-resistant polyglucosans (PBs). Within 60 minutes from sacrifice, the former disappear while the latter remain and can be biochemically quantified as a measure of PBs [3, 37, 41]. This degradation-resistant glycogen was reduced by 40 to 60% in whole hemisphere extracts in AAV-Cas9 treated mice in the three genotypes (Figs.…”

“…Aliquot of frozen ground brain was left out to thaw at room temperature for 1 hour to allow soluble glycogen to degrade, leaving behind degradation-resistant, i.e. PB, glycogen [37]. The sample was then boiled in 30% KOH and glycogen precipitated in 70% ethanol with 57 mM sodium sulfate.…”

“…(Abs2 -Abs1) of glucose standards was used to derive glucose quantity in samples. Following normalization to fresh weight, glucose quantity represents degradation-resistant glycogen [37]. Table 1.…”