Mutations in α-synuclein, TDP-43 and tau prolong protein half-life through diminished degradation by lysosomal proteases

Abstract

Background Autosomal dominant mutations in α-synuclein, TDP-43 and tau are thought to predispose to neurodegeneration by enhancing protein aggregation. While a subset of α-synuclein, TDP-43 and tau mutations has been shown to increase the structural propensity of these proteins toward self-association, rates of aggregation are also highly dependent on protein steady state concentrations, which are in large part regulated by their rates of lysosomal degradation. Previous studies have shown that lysosomal proteases operate precisely and not indiscriminately, cleaving their substrates at very specific linear amino acid sequences. With this knowledge, we hypothesized that certain coding mutations in α-synuclein, TDP-43 and tau may lead to increased protein steady state concentrations and eventual aggregation by an alternative mechanism, that is, through disrupting lysosomal protease cleavage recognition motifs and subsequently conferring protease resistance to these proteins. Results To test this possibility, we first generated comprehensive proteolysis maps containing all of the potential lysosomal protease cleavage sites for α-synuclein, TDP-43 and tau. In silico analyses of these maps indicated that certain mutations would diminish cathepsin cleavage, a prediction we confirmed utilizing in vitro protease assays. We then validated these findings in cell models and induced neurons, demonstrating that mutant forms of α-synuclein, TDP-43 and tau are degraded less efficiently than wild type despite being imported into lysosomes at similar rates. Conclusions Together, this study provides evidence that pathogenic mutations in the N-terminal domain of α-synuclein (G51D, A53T), low complexity domain of TDP-43 (A315T, Q331K, M337V) and R1 and R2 domains of tau (K257T, N279K, S305N) directly impair their own lysosomal degradation, altering protein homeostasis and increasing cellular protein concentrations by extending the degradation half-lives of these proteins. These results also point to novel, shared, alternative mechanism by which different forms of neurodegeneration, including synucleinopathies, TDP-43 proteinopathies and tauopathies, may arise. Importantly, they also provide a roadmap for how the upregulation of particular lysosomal proteases could be targeted as potential therapeutics for human neurodegenerative disease.

Lysosomal proteomics reveals mechanisms of neuronal apoE4-associated lysosomal dysfunction

“…Video S1). Nonetheless, these findings support previous notions of tau interfacing with the autophagic/lysosomal system 84–92 , highlighting that a minor fraction of hyperphosphorylated tau could directly affect lysosomal function.…”

The Parkinson’s disease risk gene cathepsin B promotes fibrillar alpha-synuclein clearance, lysosomal function and glucocerebrosidase activity in dopaminergic neurons

“…While CA074me is selective for catB, it has been reported to inhibit other cathepsins, albeit at concentrations greater than those used in this study 63,64 . RNA sequencing of our iPSC-derived DA neurons indicated that 12 cathepsin family members were expressed at the RNA level (data not shown), including CTSD and CTSL which were previously found to cleave α-syn in vitro [18][19][20] . To determine how individual cathepsin species contribute to fibrillar α-syn clearance in a cellular context we used CRISPR-interference (CRISPRi) to generate RPE1 cell lines in which CTSB, CTSD, CTSL or α-syn (SNCA) were stably repressed (denoted CTSBi, CTSDi, CTSLi and SYNi respectively) as well CRISPR-activation (CRISPRa) to upregulate CTSB (CTSBa) (Fig 4A ,B).…”

“…One potential mechanism by which CTSB variants may influence PD risk is through the ability of catB to cleave and degrade α-syn [18][19][20] . However, while several cathepsins appear capable of cleaving α-syn in vitro, CTSB alone stand out as a genetic risk factor for PD.…”

“…One potential mechanism linking catB to PD is through its ability to cleave both monomeric and aggregated forms of α-syn, which has been demonstrated in vitro [18][19][20] . However, while this could argue for a protective role of catB against synucleinopathy, the α-syn truncations produced by in vitro catB cleavage exhibit an increased propensity to aggregate 21 and although lysosome function is essential for degradation of fibrillar α-syn 22 , it has also been suggested that catB activity contributes to α-syn toxicity in some cellular models 23 .…”

Precision Proteoform Design for 4R Tau Isoform Selective Templated Aggregation

“…The specific induction and passaging of these aggregates confirms a prion-like mechanism of tau spread capable of precise templating the donor structure. The peptide corresponds to a relatively “cleavage poor” region of tau 58 possibly related to the two histidines which is the only amino acid with a pKa within the physiological range and will make the peptide uniquely suited to be protonated inside the acidic lysosome. While mainly of interest as a heuristic probe, the resistance of the peptide to proteolysis under physiologic conditions may generate a similar peptide in vivo .…”