Targeted protein degradation using intracellular antibodies and its application to neurodegenerative disease.

Antibodies mediate the majority of their effects in the extracellular domain, or in intracellular compartments isolated from the cytosol. Under a growing list of circumstances, however, antibodies are found to gain access to the cytoplasm. Cytosolic immune complexes are bound by the atypical antibody receptor TRIM21, which mediates the rapid degradation of the immune complexes at the proteasome. These discoveries have informed the development of TRIM-Away, a technique to selectively deplete proteins using delivery of antibodies into cells. A range of related approaches that elicit selective protein degradation using intracellular constructs linking antibody fragments to degradative effector functions have also been developed. These methods hold promise for inducing the degradation of proteins as both research tools and as a novel therapeutic approach. Protein aggregates are a pathophysiological feature of neurodegenerative diseases and are considered to have a causal role in pathology. Immunotherapy is emerging as a promising route towards their selective targeting, and a role of antibodies in the cytosol has been demonstrated in cell-based assays. This review will explore the mechanisms by which therapeutic antibodies engage and eliminate intracellularly aggregated proteins. We will discuss how future developments in intracellular antibody technology may enhance the therapeutic potential of such antibody-derived therapies.

Inhibition of TBC1D5 activates Rab7a and can enhance the function of the retromer cargo-selective complex.

The retromer complex is a vital component of the endosomal protein sorting machinery necessary for sorting into both the endosome-to-Golgi retrieval pathway and also the endosome-to-cell-surface recycling pathway. Retromer mediates cargo selection through a trimeric complex comprising VPS35, VPS29 and VPS26, which is recruited to endosomes by binding to Rab7a and Snx3. Retromer function is linked to two distinct neurodegenerative diseases, Parkinson's disease and Alzheimer's disease and modulating retromer function has been proposed as an avenue to explore for a putative therapy in these conditions. We hypothesised that activating Rab7a to promote the recruitment of retromer to endosomes could positively modulate its activity. Here, we show that inhibition of the GTPase activating protein TBC1D5 can enhance Rab7a activation and lead to a gain of function for retromer.

Analysis of novel endosome-to-Golgi retrieval genes reveals a role for PLD3 in regulating endosomal protein sorting and amyloid precursor protein processing.

The processing of amyloid precursor protein (APP) to the neurotoxic pro-aggregatory Aß peptide is controlled by the mechanisms that govern the trafficking and localisation of APP. We hypothesised that genes involved in endosomal protein sorting could play an important role in regulating APP processing and, therefore, analysed ~ 40 novel endosome-to-Golgi retrieval genes previously identified in a genome-wide siRNA screen. We report that phospholipase D3 (PLD3), a type II membrane protein, functions in endosomal protein sorting and plays an important role in regulating APP processing. PLD3 co-localises with APP in endosomes and loss of PLD3 function results in reduced endosomal tubules, impaired trafficking of several membrane proteins and reduced association of sortilin-like 1 with APP.

Cytosolic antibody receptor TRIM21 is required for effective tau immunotherapy in mouse models.

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.

Target-induced clustering activates Trim-Away of pathogens and proteins.

Trim-Away is a recently developed technology that exploits off-the-shelf antibodies and the RING E3 ligase and cytosolic antibody receptor TRIM21 to carry out rapid protein depletion. How TRIM21 is catalytically activated upon target engagement, either during its normal immune function or when repurposed for targeted protein degradation, is unknown. Here we show that a mechanism of target-induced clustering triggers intermolecular dimerization of the RING domain to switch on the ubiquitination activity of TRIM21 and induce virus neutralization or drive Trim-Away. We harness this mechanism for selective degradation of disease-causing huntingtin protein containing long polyglutamine tracts and expand the Trim-Away toolbox with highly active TRIM21-nanobody chimeras that can also be controlled optogenetically. This work provides a mechanism for cellular activation of TRIM RING ligases and has implications for targeted protein degradation technologies.

Tau assemblies do not behave like independently acting prion-like particles in mouse neural tissue.

A fundamental property of infectious agents is their particulate nature: infectivity arises from independently-acting particles rather than as a result of collective action. Assemblies of the protein tau can exhibit seeding behaviour, potentially underlying the apparent spread of tau aggregation in many neurodegenerative diseases. Here we ask whether tau assemblies share with classical pathogens the characteristic of particulate behaviour. We used organotypic hippocampal slice cultures from P301S tau transgenic mice in order to precisely control the concentration of extracellular tau assemblies in neural tissue. Whilst untreated slices displayed no overt signs of pathology, exposure to recombinant tau assemblies could result in the formation of intraneuronal, hyperphosphorylated tau structures. However, seeding ability of tau assemblies did not titrate in a one-hit manner in neural tissue. The results suggest that seeding behaviour of tau arises at high concentrations, with implications for the interpretation of high-dose intracranial challenge experiments and the possible contribution of seeded aggregation to human disease.

The Role of Antibodies and Their Receptors in Protection Against Ordered Protein Assembly in Neurodegeneration.

Ordered assemblies of proteins are found in the postmortem brains of sufferers of several neurodegenerative diseases. The cytoplasmic microtubule associated protein tau and alpha-synuclein (αS) are found in an assembled state in Alzheimer's disease and Parkinson's disease, respectively. An accumulating body of evidence suggests a "prion-like" mechanism of spread of these assemblies through the diseased brain. Under this hypothesis, assembled variants of these proteins promote the conversion of native proteins to the assembled state. This likely inflicts pathology on cells of the brain through a toxic gain-of-function mechanism. Experiments in animal models of tau and αS pathology have demonstrated that the passive transfer of anti-tau or anti-αS antibodies induces a reduction in the levels of assembled proteins. This is further accompanied by improvements in neurological function and preservation of brain volume. Immunotherapy is therefore considered one of the brightest hopes as a therapeutic avenue in an area currently without disease-modifying therapy. Following a series of disappointing clinical trials targeting beta-amyloid, a peptide that accumulates in the extracellular spaces of the AD brain, attention is turning to active and passive immunotherapies that target tau and αS. However, there are several remaining uncertainties concerning the mechanism by which antibodies afford protection against self-propagating protein conformations. This review will discuss current understanding of how antibodies and their receptors can be brought to bear on proteins involved in neurodegeneration. Parallels will be made to antibody-mediated protection against classical viral infections. Common mechanisms that may contribute to protection against self-propagating protein conformations include blocking the entry of protein "seeds" to cells, clearance of immune complexes by microglia, and the intracellular protein degradation pathway initiated by cytoplasmic antibodies via the Fc receptor TRIM21. As with anti-viral immunity, protective mechanisms may be accompanied by the activation of immune signaling pathways and we will discuss the suitability of such activation in the neurological setting.

Retromer-mediated endosomal protein sorting: The role of unstructured domains.

The retromer complex is a key element of the endosomal protein sorting machinery that is conserved through evolution and has been shown to play a role in diseases such as Alzheimer's disease and Parkinson's disease. Through sorting various membrane proteins (cargo), the function of retromer complex has been linked to physiological processes such as lysosome biogenesis, autophagy, down regulation of signalling receptors and cell spreading. The cargo-selective trimer of retromer recognises membrane proteins and sorts them into two distinct pathways; endosome-to-Golgi retrieval and endosome-to-cell surface recycling and additionally the cargo-selective trimer functions as a hub to recruit accessory proteins to endosomes where they may regulate and/or facilitate retromer-mediated endosomal proteins sorting. Unstructured domains present in cargo proteins or accessory factors play key roles in both these aspects of retromer function and will be discussed in this review.

The immune system GTPase GIMAP6 interacts with the Atg8 homologue GABARAPL2 and is recruited to autophagosomes.

The GIMAPs (GTPases of the immunity-associated proteins) are a family of small GTPases expressed prominently in the immune systems of mammals and other vertebrates. In mammals, studies of mutant or genetically-modified rodents have indicated important roles for the GIMAP GTPases in the development and survival of lymphocytes. No clear picture has yet emerged, however, of the molecular mechanisms by which they perform their function(s). Using biotin tag-affinity purification we identified a major, and highly specific, interaction between the human cytosolic family member GIMAP6 and GABARAPL2, one of the mammalian homologues of the yeast autophagy protein Atg8. Chemical cross-linking studies performed on Jurkat T cells, which express both GIMAP6 and GABARAPL2 endogenously, indicated that the two proteins in these cells readily associate with one another in the cytosol under normal conditions. The GIMAP6-GABARAPL2 interaction was disrupted by deletion of the last 10 amino acids of GIMAP6. The N-terminal region of GIMAP6, however, which includes a putative Atg8-family interacting motif, was not required. Over-expression of GIMAP6 resulted in increased levels of endogenous GABARAPL2 in cells. After culture of cells in starvation medium, GIMAP6 was found to localise in punctate structures with both GABARAPL2 and the autophagosomal marker MAP1LC3B, indicating that GIMAP6 re-locates to autophagosomes on starvation. Consistent with this finding, we have demonstrated that starvation of Jurkat T cells results in the degradation of GIMAP6. Whilst these findings raise the possibility that the GIMAPs play roles in the regulation of autophagy, we have been unable to demonstrate an effect of GIMAP6 over-expression on autophagic flux.