Discussion
In summary, we demonstrate that the homozygous E122D mutation of ATG5, a unique mutation found in two human subjects with ataxia, results in reduced conjugation to ATG12 and in an overall decrease in autophagy activity. The homologous mutation in yeast also interferes with autophagy, and the ataxia phenotype was replicated in a fly model. Based on these results we propose that this ATG5 mutation, and the consequent disruption in autophagy activity, is the cause of the ataxic phenotype and disturbance of the cerebellum in the affected siblings. This hypothesis is in agreement with previously characterized mouse models, in which neuron-specific knockout of Atg5 results in ataxia-like phenotypes (Hara et al., 2006; Nishiyama et al., 2007). By contrast, mice with complete knockout of Atg5 die shortly after birth, demonstrating that autophagy is essential for mammalian survival (Kuma et al., 2004). Our results indicate that E122D is a partial loss-of-function allele that impairs but does not completely abolish ATG5 activity. Although the overall structure of the E122D mutant ATG5 superimposes well with the wild-type protein, the mutation causes a striking decrease in the level of ATG12–ATG5 conjugate that is formed when the C terminus of ATG12 is covalently linked to Lys130 of ATG5. We speculate that the E122D mutation causes subtle changes in the conformational dynamics that propagate to Lys130, which is less than 10 Å away, resulting in less ATG12–ATG5, which in turn leads to reduced LC3/Atg8 conjugation.
In neurons, which are among the cells most dependent on autophagy for tissue homeostasis (Button et al., 2015), the residual function of the E122D allele is inadequate, resulting in predominantly neurological symptoms in the two patients. Since homozygous mutations with complete loss-of-function have not been reported, we predict that individuals carrying such mutations, similar to Atg5-null mice (Kuma et al., 2004), might not be viable.
Autophagy is quickly gaining importance for its roles in preventing neurodegeneration. WDR45 is a redundant, non-core autophagy gene, one of four mammalian homologs to Atg18, and mutations in WDR45 cause SENDA, static encephalopathy of childhood with neurodegeneration in adulthood (Haack et al., 2012; Saitsu et al., 2013). Autophagy appears to be critical in ataxia, whether mutant proteins evade autophagy processes or normal autophagy is disrupted. Several ataxias are attributed to intranuclear or cytoplasmic aggregation of mutant proteins within the cell (Matilla-Duenas et al., 2014). These protein aggregates, in humans and in mouse models, not only evade autophagic sequestration but may even inhibit autophagy (Alves et al., 2014), or lead to reduction in autophagy available for other proteins due to saturation. Assessment of autophagy in patient cells may be used to refine and identify the genetic cause of a patient’s ataxia.
Further discovery of the role of autophagy in neurodegenerative diseases should be used to investigate therapies targeted at the autophagy process. Many drugs enhance autophagy and their effects on a multitude of neurodegenerative diseases have been studied (Sarkar et al., 2009). Recently more studies have been conducted assessing the value of autophagy enhancers in ataxia models and patients. Induction of autophagy through administration of Temsirolimus, a rapamycin ester, and lentiviral overexpression of BECN1 in SCA3 model mice increase autophagy and the clearance of mutant protein aggregates, and reduce the ataxic phenotype (Menzies et al., 2010; Nascimento-Ferreira et al., 2013). In a single patient trial, trehalose treatment of patient fibroblasts increased autophagy and alleviated cellular pathogenic features by improving mitochondrial morphology and reducing free radicals in the cell (Casarejos et al., 2014; Sarkar et al., 2007). Trehalose also showed success in trials involving models of SCA17 (Chen et al., 2015). Lithium, another inducer of autophagy, improved symptoms in a SCA1 mouse model (Watase et al., 2007), but did not slow or reduce symptoms in a treatment trial in SCA2 patients (Sacca et al., 2015). An autophagy enhancer may be an appropriate treatment to test in the presented subjects, as autophagic flux is attenuated, but not completely abrogated, by the ATG5E122D/E122D mutation.
This study’s finding of the pathogenic human E122D mutation in ATG5, a gene encoding part of the autophagy-controlling core machinery, is important and novel, but consistent with reports of neurodegenerative disorders in other autophagy-related genes (Frake et al., 2015). Our results suggest that other mutations in this and other ATG genes, which impair but do not completely abolish autophagy, may result in similar forms of ataxia, intellectual disability and developmental delay. This study exemplifies the utility of exome sequencing in the identification of rare disease-causing variants, and supports the role of impaired autophagy in neurodegenerative disease. In addition, we demonstrate the utility of a combined genetic, biochemical and cell biological analysis in multiple model systems to elucidate the underlying pathogenic mechanism of rare human diseases.