Annabel E. Smith

Aggregates of the protein tau are proposed to drive pathogenesis in neurodegenerative diseases. Tau can be targeted by using passively transferred antibodies (Abs), but the mechanisms of Ab protection are incompletely understood. In this work, we used a variety of cell and animal model systems and showed that the cytosolic Ab receptor and E3 ligase TRIM21 (T21) could play a role in Ab protection against tau pathology. Tau-Ab complexes were internalized to the cytosol of neurons, which enabled T21 engagement and protection against seeded aggregation. Ab-mediated protection against tau pathology was lost in mice that lacked T21. Thus, the cytosolic compartment provides a site of immunotherapeutic protection, which may help in the design of Ab-based therapies in neurodegenerative disease.

Selective degradation of pathological protein aggregates while sparing monomeric forms is of major therapeutic interest. The E3 ligase tripartite motif-containing protein 21 (TRIM21) degrades antibody-bound proteins in an assembly state-specific manner due to the requirement of TRIM21 RING domain clustering for activation, yet effective targeting of intracellular assemblies remains challenging. Here, we fused the RING domain of TRIM21 to a target-specific nanobody to create intracellularly expressed constructs capable of selectively degrading assembled proteins. We evaluated this approach against green fluorescent protein-tagged histone 2B (H2B-GFP) and tau, a protein that undergoes pathological aggregation in Alzheimer's and other neurodegenerative diseases. RING-nanobody degraders prevented or reversed tau aggregation in culture and in vivo, with minimal impact on monomeric tau. This approach may have therapeutic potential for the many disorders driven by intracellular protein aggregation.

Assemblies of tau can transit between neurons, seeding aggregation in a prion-like manner. To accomplish this, tau must cross cell-limiting membranes, a process that is poorly understood. Here, we establish assays for the study of tau entry into the cytosol as a phenomenon distinct from uptake, in real time, and at physiological concentrations. The entry pathway of tau is cell type specific and, in neurons, highly sensitive to cholesterol. Depletion of the cholesterol transporter Niemann-Pick type C1 or extraction of membrane cholesterol renders neurons highly permissive to tau entry and potentiates seeding even at low levels of exogenous tau assemblies. Conversely, cholesterol supplementation reduces entry and almost completely blocks seeded aggregation. Our findings establish entry as a rate-limiting step to seeded aggregation and demonstrate that dysregulated cholesterol, a feature of several neurodegenerative diseases, potentiates tau aggregation by promoting entry of tau assemblies into the cell interior.

Tau is a soluble protein interacting with tubulin to stabilize microtubules. However, under pathological conditions, it becomes hyperphosphorylated and aggregates, a process that can be induced by treating cells with exogenously added tau fibrils. Here, we employ single-molecule localization microscopy to resolve the aggregate species formed in early stages of seeded tau aggregation. We report that entry of sufficient tau assemblies into the cytosol induces the self-replication of small tau aggregates, with a doubling time of 5 h inside HEK cells and 1 day in murine primary neurons, which then grow into fibrils. Seeding occurs in the vicinity of the microtubule cytoskeleton, is accelerated by the proteasome, and results in release of small assemblies into the media. In the absence of seeding, cells still spontaneously form small aggregates at lower levels. Overall, our work provides a quantitative picture of the early stages of templated seeded tau aggregation in cells.

High consumption of colorful fruits and vegetables correlates with low dementia risk, but the exact molecules and the underlying biological mechanisms governing their bioactive profiles are largely unknown. Using a 10-year observational cohort study coupled with an AI-driven systems pharmacology platform, we identified a natural triterpenoid compound found in colorful fruits and vegetables, α-Amyrin (αA), as a therapeutic candidate for Alzheimer's disease (AD). The efficacy of αA in treating the symptoms of AD, such as Tau tangles, damaged mitochondria, and memory loss, was examined using cross-species models; αA retained memory in AD-like animal models while also strongly inhibiting Tau pathology, especially p-Tau217, in a cellular 'Tau seeding' system and in Tau[P301S] mice, followed by validation using a human 3D microfluidic system. At molecular level, αA is a robust mitochondrial regulator, enhancing mitochondrial stress resilience and activation of mitophagy. Mechanistically, αA inhibits dual leucine zipper kinase (DLK), leading to the inhibition of DLK-Sterile Alpha and TIR Motif Containing 1 (SARM1)-dependent neurodegeneration; this inhibition frees unc-51 Like Autophagy Activating Kinase 1 (ULK1) from the ULK1-SARM1 complex, allowing it to participate in autophagy/mitophagy. αA also shows strong translational potential with a 10.1 h half-life and the ability to cross the blood-brain barrier. Our results indicate that αA may act as a mitochondrial guardian against AD via modulating the DLK-SARM1-ULK1-autophagy/mitophagy axis while further preclinical and clinical studies are warranted.