Defective recognition of LC3B by mutant SQSTM1/p62 implicates impairment of autophagy as a pathogenic mechanism in ALS-FTLD

Abstract

Growing 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.

Keap1/Cullin3 Modulates p62/SQSTM1 Activity via UBA Domain Ubiquitination

“…For Cherry-eGFP-p62 assay, p62−/−MEFs were transfected with Cherry-eGFP-p62-WT, K420R, or T350A along with or without Myc-Keap1/V5-Cul3 for 24hrs. Cells were washed two times with PBS and mounted with Mowiol media containing DAPI without fixation to avoid pH disruption (Goode et al, 2016a). Pearson’s coefficient between red and green channel was measured by ImageJ.…”

NF‐κB activation in astrocytes drives a stage‐specific beneficial neuroimmunological response in ALS

“…In fact, levels of misfolded SOD1 in MNs, detected by the conformation‐specific antibody B8H10 (Gros‐Louis et al , ), were significantly lower in SOD1/IKK than in SOD1 littermates between P30 and P70 (fraction of high‐burden mSOD1 MN—see Materials and Methods—SOD1/IKK versus SOD1: 4.6 ± 1.2% versus 24.4 ± 3.65%; 8.5 ± 4.4% versus 38.6 ± 5.5%; 22.5 ± 5.5% versus 43.4 ± 4.7% at P30, P50, and P70, respectively; P < 0.05) but this difference disappeared at P90 (78.8 ± 8.5% versus 73.4 ± 8.2%; P > 0.05; Fig E and F). Likewise, the accumulation of disease‐related autophagy markers LC3A and p62 (Goode et al , ) was reduced in SOD1/IKK. LC3A bright MNs were fewer in SOD1/IKK than in SOD1 at P30 and at P50 (% LC3A bright MNs: 23.4 ± 2.2% versus 9.3 ± 3.8% at P30, 39.4 ± 4.5% versus 12.5 ± 3.9% in SOD1 versus SOD1/IKK, respectively; P < 0.05); this difference was reduced at P70 and was no longer significant at P90 (60.4 ± 10.2% versus 49.8 ± 13.4%; P > 0.05; Fig G and H).…”

FOXO1, a Potential Therapeutic Target, Regulates Autophagic Flux, Oxidative Stress, Mitochondrial Dysfunction, and Apoptosis in Human Cholangiocarcinoma QBC939 Cells

“…For this reason, autophagy inhibition has been regarded as a promising anticancer strategy to inhibit the multiple cellular processes. Impaired autophagy could influencing the energy metabolism, protein balance, oxidatice stress, and MtD [17, 28, 29]. Hence, knowing the molecular mechanism by which the autophagy is regulated could help us to find a potential therapeutic target to solve this urgent problem.…”