Abstract
ABSTRACTGrowing evidence implicates impairment of autophagy as a candidate pathogenic mechanism in the spectrum of neurodegenerative disorders which includes amyotrophic lateral sclerosis and frontotemporal lobar degeneration (ALS-FTLD). SQSTM1, which encodes the autophagy receptor SQSTM1/p62, is genetically associated with ALS-FTLD, although to date autophagy-relevant functional defects in disease-associated variants have not been described. A key protein-protein interaction in autophagy is the recognition of a lipid-anchored form of LC3 (LC3-II) within the phagophore membrane by SQSTM1, mediated through its LC3-interacting region (LIR), and notably some ALS-FTLD mutations map to this region. Here we show that although representing a conservative substitution and predicted to be benign, the ALS-associated L341V mutation of SQSTM1 is defective in recognition of LC3B. We place our observations on a firm quantitative footing by showing the L341V-mutant LIR is associated with a ∼3-fold reduction in LC3B binding affinity and using protein NMR we rationalize the structural basis for the effect. This functional deficit is realized in motor neuron-like cells, with the L341V mutant EGFP-mCherry-SQSTM1 less readily incorporated into acidic autophagic vesicles than the wild type. Our data supports a model in which the L341V mutation limits the critical step of SQSTM1 recruitment to the phagophore. The oligomeric nature of SQSTM1, which presents multiple LIRs to template growth of the phagophore, potentially gives rise to avidity effects which amplify the relatively modest impact of any single mutation on LC3B binding. Over the lifetime of a neuron, impaired autophagy could expose a vulnerability, which ultimately tips the balance from cell survival toward cell death.
Highlights
ALS and FTLD are devastating and invariably fatal neurodegenerative diseases thought to belong to the same clinicopathological spectrum of disorders
Protein affinity isolation assays in which interaction partners LC3B or ubiquitin covalently immobilized on beads were used to capture recombinant GST-SQSTM1 fusion proteins, followed by detection of bound protein by western blotting
The same assay has previously been used to demonstrate ubiquitinbinding defects associated with Paget disease of bone (PDB)-linked UBA domain mutations of SQSTM1.21 Consistent with its location within the SQSTM1 LC3interacting region (LIR), defined by the 338 to 341 WXXL motif, we found that the L341V mutant selectively reduced capture of GST-SQSTM1 by LC3B, relative to the wild type, whereas binding to ubiquitin was unaffected (Fig. 1 and Fig. S1)
Summary
ALS and FTLD are devastating and invariably fatal neurodegenerative diseases thought to belong to the same clinicopathological spectrum of disorders. Both sporadic and familial forms occur with more than 30 different genes linked to ALS-FTLD and showing different degrees of causality from major effects through to genetic predisposition. Mutations affecting the autophagy cargo receptors UBQLN2/ubiquilin-2, OPTN/optineurin and SQSTM1/p62 have been reported in ALS-FTLD families.[3] Very recently mutations affecting the gene encoding TBK1 (TANK-binding kinase 1), a kinase which targets both OPTN and SQSTM1 linking the proteins in a common pathway, have been identified as a cause of familial. ALS-FTLD.[4,5] Autophagy is a critical pathway for the removal of damaged and aggregation-prone proteins and organelles; for example, in the ALS context mediating the turnover of aggregated forms of the major pathological protein TARDBP (TAR DNA binding protein),[6] and is essential for neuronal survival.[7,8]
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