Our data show that a lack of laforin decreases autophagy. This was assessed using a range of assays. In patient cells, MEFs and mice, we observed decreased autophagosome formation when laforin activity was lost, as assessed by LC3-II blotting. This was associated with decreased clearance of long-lived proteins via autophagy and the accumulation of ubiquitinated proteins and the endogenous autophagy substrate, p62. The impaired autophagy associated with the lack of laforin correlated with increased activity of the autophagy inhibitor mTOR. In agreement with these observations, it has been recently shown that laforin-deficient mice have higher levels of the autophagy substrate tau (33
) in their brains (34
). Consistent with these data, we found that laforin overexpression induced autophagy and enhanced autophagy substrate clearance only in autophagy-competent cells. This was assessed by measuring the formation of autophagosomes by LC3-II western blotting, by immunofluorescence quantification of EGFP-LC3 vesicles (autophagosomes) and by assessing the accumulation of mutant huntingtin exon 1, an autophagy substrate, in wild-type and autophagy-incompetent (atg5−/−
) cells. Thus, the ability of laforin to stimulate autophagy is not saturated in cells.
The accumulation of ubiquitinated proteins is a well-described phenomenon that occurs in autophagy-compromised tissues (25
). This may occur if some proteins are specifically targeted to autophagy via ubiquitination and subsequent binding to adaptor proteins that link to the autophagic machinery (35
). However, another reason for the accumulation of ubiquitinated proteins may be because autophagy inhibition results in a secondary reduction in flux through the ubiquitin-proteasome system. This phenomenon is driven by the accumulation of p62, which appears to impair the delivery of ubiquitinated proteins to the proteasome (37
). Thus, it is possible that the reduced rates of autophagy in LD may have effects on the other major intracellular proteolytic system, the ubiquitin proteasome pathway.
The effects of laforin on autophagy are mTOR-dependent, as no effects are seen in tsc2−/−
cells, and it is well known that TSC2 is a negative regulator of mTOR. Some studies have suggested that laforin dephosphorylates GSK3-β at Ser 9, thereby activating this enzyme (38
). Lafora bodies are poorly branched polyglucosans, which could arise from an excess of glycogen synthase activity relative to glycogen branching enzyme activity. Glycogen synthase kinase 3 (GSK3) is the principal inhibitor of glycogen synthase. Thus, if laforin were a key GSK3 phosphatase, then one would expect that the loss of activity of this phosphatase would decrease GSK3 activity, increase glycogen synthase activity and thereby increase the formation of Lafora bodies. Unfortunately, this model is not supported by all studies, including analyses of Laforin null mice (34
The exact mechanism for the laforin effect on autophagy is still elusive and may remain so for some time pending identification of authentic substrates. It is quite possible that laforin may have many substrates (such as other phosphatases), particularly since it is a dual specificity phosphatase that can hydrolyze phosphotyrosine as well as phosphoserine/threonine substrates in vitro
(reviewed in 16
). Thus the identification of the specific substrates relevant to autophagy will be the challenge. However, the consequences of its lack of activity in LD patients are easier to predict. Previous analyses of mice that lack laforin revealed progressive changes in the properties and structure of glycogen that paralleled the formation of Lafora bodies. One of the features observed was a progressive accumulation of glycogen, which also became more phosphorylated and insoluble (18
). Since Lafora bodies are composed of poorly branched, water-insoluble, glycogen-like polymers that are also decorated with antiubiquitin antibodies, we believe that it is likely that decreased autophagy caused by laforin deficiency will enhance their accumulation, given that autophagy compromise leads to the accumulation of glycogen, ubiquitinated proteins and aggregate-prone proteins (27
) (Fig. ). Indeed, it appears that glycogen autophagy is regulated via the mTOR signalling pathway, which is perturbed by the loss of laforin activity. In addition to enhancing formation of Lafora bodies, which may be a toxic entity in LD, autophagy deficiency also increases cellular susceptibility to various toxic insults, which may further exacerbate the problem (40
Figure 7. Schematic representation of regulation of autophagy by laforin and the disease consequences. Wild-type laforin induces autophagy by inhibiting mTOR in a TSC2-dependent manner. Loss of laforin in LD activates mTOR and inhibits autophagy. An impairment (more ...)
The situation we have described has similarities and differences when compared with lysosomal storage diseases associated with hydrolase deficiencies. Both sets of conditions exhibit impaired autophagic flux and the accumulation of various substrates in the affected tissues. However, there are important differences. In lysosomal storage diseases, autophagy is impaired not by reduced autophagosome synthesis, as occurs in laforin deficiency, but due to a partial block in the delivery of autophagosomes to lysosomes (22
). Indeed, lysosomal storage diseases are frequently characterized by the accumulation of autophagosomes (22
). Also, in lysosomal storage diseases, it is likely that there is an initial loss of lysosomal enzyme function that then leads to impaired autophagic flux, while in laforin deficiency the impaired autophagic flux may be a direct consequence of enzyme activity, since we saw increased autophagy with laforin overexpression. Currently, we do not know how early the autophagy defect is in the pathogenic cascade of LD. While it is likely that the Lafora bodies contribute to pathology, our data do raise the possibility that autophagy compromise may be an important driver of pathology in its own right (independent of the Lafora bodies), but also that the formation of these inclusions may be enhanced as a consequence of impaired clearance.