Effects of STAU1/staufen1 on autophagy in neurodegenerative diseases

ABSTRACT

The double-stranded RNA-binding protein, STAU1 (staufen double-stranded RNA binding protein 1) is a multifunctional protein that localizes to stress granules (SGs). We had previously found that STAU1 is overabundant in fibroblast cell lines from patients with spinocerebellar ataxia type 2 (SCA2) or amyotrophic lateral sclerosis (ALS)-frontotemporal dementia (FTD) as well as in animal models of these diseases. STAU1 overabundance is post-transcriptional and associated with MTOR hyperactivation and links SG formation with macroautophagy/autophagy. Reducing STAU1 levels in mice with ALS mutations normalizes MTOR activity and autophagy-related marker proteins. We also see increased STAU1 levels in HEK293 cells expressing C9orf72-relevant dipeptide repeats (DPRs), and DPRs are not observed in cells where STAU1 is targeted by RNAi. Overexpression of STAU1 in HEK293 cells increases MTOR translation by directly interacting with the MTOR mRNA 5ʹUTR, activating downstream targets and impairing autophagic flux. STAU1 may constitute a novel target to modulate MTOR activity and autophagy and for the treatment of neurodegenerative diseases.

Our work on RNA-binding proteins and autophagy began more than 30 years ago with studies to find the mutation causing SCA2, a rare neurological disease. SCA2 has adult-onset and affects primarily cerebellar neurons, but also other parts of the nervous system such as dopaminergic, cortical and primary and secondary motor neurons. Using a positional and candidate gene cloning approach, we identified a CAG DNA repeat expansion mutation in the ATXN2 gene as the cause of SCA2.

Success in targeting the ATXN2 mRNA with antisense oligonucleotides (ASOs) in preclinical models and now a human phase 1 study (clinicaltrials.gov NCT04494256), prompted us to define ATXN2 interactors by co-immunoprecipitation and mass spectroscopy. One of the most intriguing interactors was STAU1. stau (staufen) was identified by the Nusslein-Volhard group in a fly random mutagenesis screen as a protein necessary to establish RNA gradients in the developing embryo. It binds double-stranded RNA and is involved in the processes of mRNA localization and translation. stau deficiency in the fly results in a lack of offspring and is part of the “grandchildless” protein family named after royal lines like the Staufers that ceased to have offspring. STAU1 duplicated in evolution and is present in two copies in mammals.

When we verified the ATXN2-STAU1 interaction at endogenous concentrations in cells expressing polyQ-expanded mutant ATXN2, we observed that STAU1 is greatly overabundant at the protein, but not at the mRNA level. STAU1 overabundance is the result of reduced degradation via autophagy. Next, we used HEK293 cells and subjected them to endoplasmic reticulum (ER), metal, thermal or oxidative stress, all of which induce STAU1 overabundance, MTOR elevation, and increased phosphorylation of MTOR targets [1].

STAU1 binds MTOR mRNA enhancing translation

As STAU1 overabundance in wild-type cells without endogenous or exogenous stressors increases MTOR protein, we queried whether there is a direct link between STAU1 and autophagy regulation. Indeed, we found that STAU1 binds to the 5ʹUTR of MTOR mRNA in a sequence-specific fashion and that this interaction results in enhanced translation of MTOR mRNA. This in turn results in a change of cellular markers consistent with reduced autophagy such as an increase in SQSTM1/p62, p-RPS6KB/S6K, and LC3-II.

STAU1-depleted cells are resistant to poly-GA aggregates and MTOR activation

Expansion of an intronic G₄C₂ repeat in the C9orf72 gene results in ALS or FTD. A hallmark of C9orf72 disease is the translation of DPRs initiated by a mechanism designated repeat-associated non-ATG/RAN translation. When we deplete STAU1 from HEK293 cells expressing C9orf72-relevant DPRs, the presence of DPRs, and MTOR activation are greatly reduced. Further studies will need to clarify whether this is due to reduced translation by STAU1 interference or due to improvement of autophagic flux.

Reduced STAU1 levels ameliorate autophagy function across animal disease models

Next, we asked whether reduction of overabundant STAU1 improves autophagic flux in rodent models of neurodegeneration. A mouse with a Stau1 loss-of-function allele allowed us to determine the effect of Stau1 reduction using genetic interaction with two well-established mouse models of human diseases, the Pcp2-ATXN2^Q127 SCA2 mouse, and the Thy1-TARDBP/TDP-43 ALS-FTD mouse. We tested restoration of autophagy by measuring the levels of MTOR, p-MTOR, p-RPS6KB/S6K, SQSTM1/p62, and LC3-II by quantitative western blot analysis. Lowering Stau1 copy number in wild-type animals does not alter autophagy marker proteins in an appreciable way. In contrast, reducing pathologically elevated STAU1 in SCA2 mice improves markers of autophagy.

The mechanistic relationship between STAU1 and autophagy may be more complex than STAU1 solely enhancing MTOR translation. STAU1 also controls RNA degradation through a process called STAU1-mediated-mRNA decay that may act on mRNAs modifying autophagic flux, resulting in a more nuanced control of autophagy in response to a number of stressors. As STAU1 also functions in microtubule-dependent mRNA transport, overabundance and altered mRNA transport in neuronal processes may change autophagy at or near synapses. A model for STAU1 in neurodegeneration is shown in Figure 1.

Figure 1.

STAU1 in neurodegeneration. Mutations in ATXN2, TARDBP/TDP-43, and C9orf72 stimulate low-complexity domain interactions and RNP granule accumulations that are normally cleared by autophagy. In the pathogenic state, reduced autophagic clearance leads to accumulating stress granules including STAU1 overabundance that directly leads to MTOR translational activation further inhibiting autophagy simultaneous with UPR activation and STAU1-mediated-mRNA decay (SMD). STAU1 elevation is predicted to alter mRNA nuclear translocation and mRNA dendritic trafficking, processes that also involve TARDBP. UPR: unfolded protein response; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; p-EIF2A/eIF2α: phospho-eukaryotic translation initiation factor 2A; ATXN2: ataxin 2; TARDBP/TDP-43: TAR DNA binding protein.

Finally, STAU1 may be a valid target for mRNA-directed therapies. At the moment, reduction of STAU1 via siRNA or artificial miRNAs can normalize markers of reduced autophagic flux in cellular models of TARDBP/TDP-43 and C9orf72 disease; genetic interaction using Stau1^−/− mice changes autophagy markers in vivo. Next steps will test a series of ASOs targeting STAU1 in various models of neurodegeneration with the ultimate aim to develop a therapeutic for neurodegenerative diseases characterized by STAU1-overabundance.

Funding Statement

This work was supported by National Institutes of Neurological Disorders and Stroke (NINDS) grants R37NS033123, R56NS33123, and R35NS127253 to SMP, R01NS097903 to DRS, and R21NS103009, R21NS081182, and R61NS124965 to DRS and SMP. DRS was also supported by a Harrington Discovery Institute Rare Disease Scholar award.

Disclosure statement

No potential conflict of interest was reported by the authors.