GBE deficiency is a rare disorder with a very heterogeneous clinical presentation that appears to be determined in part by the degree of residual enzyme activity. Complete loss of activity in humans is lethal either in the third trimester of pregnancy or in infancy, while mutations that reduce enzyme activity cause juvenile or adult-onset disease. Juvenile onset disease typically exhibits liver and/or heart involvement, while adult-onset disease is mainly a neuromuscular disorder and may be misdiagnosed as amyotrophic lateral sclerosis, multiple sclerosis or Alzheimer disease (5
). Here we describe a mouse model of GSD IV that accurately recapitulates histological
this disease spectrum. Deletion of exon 7 eliminates enzyme activity in all tissues and thus is a model of Andersen disease. In contrast, decreasing the expression of the Gbe1
via transcriptional interference by the reverse-oriented PGK-Neomycin cassette leads to a hypomorphic allele with residual enzyme activity and later onset of disease, yet a pronounced accumulation of PG. Exon 7 of Gbe1
contains the c-terminus of the alpha amylase domain and the linker region that connects the alpha amylase domain to the glycosyl transferase domain. Since exon 7 encodes an in-frame 70-amino acid polypeptide, its deletion may potentially lead to an internally truncated protein. Unfortunately, our western blot result does not show the shorter predicted protein due to a cross-reacting band that obscures the predicted size. Removal of exon 7 in vivo
by FLPe-mediated recombination leads to Gbe1−/−
pups that die at birth. In utero
development appears unaffected by the mutation. While total glycogen content is reduced in all tissues, a significant amount of PG is present. This finding is in contrast to that reported recently by Lee et al
), where a nonsense mutation in Gbe1
was identified following random chemical mutagenesis. The authors reported little accumulation of PG, and attributed the embryonic lethality that was observed to a developmental heart defect (ventricular non-compaction) and fetal hydrops, findings we have not observed. This difference may reflect strain-specific differences, or, perhaps less likely, a second linked mutation present in the mutagenized strain studied. The absence of significant amounts of PG in the strain reported by Lee et al
. is in contrast to the observed accumulation of PG in postmortem human embryos harboring large homozygous deletions or point mutations that are predicted to eliminate GBE activity (16
). In these human cases, PG accumulated in vital organs such as the brain stem and the sympathetic and parasympathetic nervous system that control postnatal respiration.
Normally, glycogen is found in fetal skeletal muscle, liver and heart, as shown in the PAS staining of whole-mount wild-type E17.5 embryo sections. This demonstrates that there is fetal glycogen synthesis and thus GYS activity that would be required to initiate glycogen synthesis. This fetal glycogen is stored to be used during delivery and after birth in order to provide glucose until newborns begin feeding. With this in mind, we studied the GYS activity in embryos. GYS is under both hormonal and allosteric regulation by glucose-6 phosphate. Allosteric regulation is believed to supersede the hormonal inhibition mediated by phosphorylation, so as to protect the cell against very high intracellular glucose concentrations. High intracellular glucose causes glycogen accumulation irrespective of cellular energy status, as demonstrated in disorders of glycolysis, where, despite a metabolic block in glycolysis that causes an intracellular energy deficit, glycogen still accumulates (17
). We have found substantial GYS activity in Gbe1−/−
skeletal muscle and abundant GYS protein. However, GYS activity in Gbe1−/−
mice is considerably less responsive to glucose 6-phosphate-mediated activation in comparison to that of control mice, suggesting that there is a minimal requirement for GBE in the allosteric activation of GYS. Lack of glycogen in tissues in the absence of GYS has been reported previously (19
). Unlike GBE, GYS has two isoforms; liver-specific GYS2 and GYS1 that is expressed in other tissues. GYS2-deficient mice have near-normal development, whereas 90% of GYS1-deficient mice die after birth. Thus, given that there is a single Gbe1
mice mimic the course of GYS1-deficient mice, yet, like GYS2-deficient mice, also lack significant amounts of liver glycogen (but readily detectable PG). Hence, the early death of GBE-deficient mice is likely not solely a consequence of an absence of structurally normal glycogen in the liver. While we did not observe hydropic embryos or left ventricular non-compaction, the hearts of Gbe1−/−
mice appear to have disrupted myocardial architecture in association with widespread PG accumulation. Future studies in Gbe1−/−
mice will address the molecular basis for this structurally abnormal myocardium.
The PGK-Neomycin cassette used for positive selection in mouse embryonic stem (ES) cells alters the expression of Gbe1.
Both GBE activity assays and western blotting have shown that transcription of the Gbe1
gene has been reduced. This has generated a phenotype similar to the juvenile and adult forms of GSD IV. Point mutations that decrease enzyme activity such as Y329S and R545H cause amylopectinosis as a recessive trait. Thus, in order to manifest the disease, enzyme activity must be <50% (21
). However, there are rare case reports of manifesting heterozygotes, with clinical features occurring in the sixth decade of life (22
). In aged humans and animals, PG-like bodies known as corpora amylacea can be seen in white matter and axons, but the relationship to GBE activity remains unexplored (23
mice will be aged to determine whether a reduction in GBE can lead to the formation of PG bodies in the brain.
PG is also a component of the Lafora bodies that lead to progressive myoclonus epilepsy [Lafora disease (LD)]. Lafora disease is an autosomal recessive disorder that becomes symptomatic in teenage years, with progressive myoclonic epilepsy leading to death within a decade. LD are found in the pericarya of neurons and other cell types. The pathogenic mechanism of Lafora disease does not involve the glycogenolytic enzymes per se
, but is caused by the functional loss of regulatory enzymes that may indirectly affect glycogenolytic enzymes (27
). Two genes causing LD, EPM2A
), have been identified (28
). An animal model of LD has been described; however, the exact mechanism of PG formation is not fully understood (30
). Thus, breeding GBE-deficient mice to LD mice would explore the role of GBE in the pathogenesis of LD.
Gbe1neo/neo mice tolerate fasting and respond to a glucose bolus in a near normal manner, suggesting that storage of glucose as glycogen or PG can occur very efficiently, even with low GBE activity. However, there is a tissue-specific difference in the ability to degrade PG, as reflected by the persistence of muscle PG after fasting, in contrast to its depletion in liver. The loss of PG from the liver belies the common belief that PG is an inert indigestible molecule and suggests that differences between hepatic phosphorylase and myophosphorylase may account for this observation. We found that PG can be degraded in liver but not in other tissues. Further studies will be necessary to compare glycogen breakdown in muscle of Gbe1Neo/Neo mice not only after fasting, but also after exercise, and after a combination of the two conditions.
These results raise the possibility of treatment strategies that enhance the physiologic degradation of PG. Therefore, this mouse model will serve as a useful tool for examining the biology of PG formation and its degradation, and as a means for testing possible therapeutic approaches.