Rebecca M. Nisbet

The role of exosomes in the processing of proteins associated with neurodegenerative diseases

Exosomes are small membranous vesicles secreted by a number of cell types and can be isolated from conditioned cell media or bodily fluids such as urine and plasma. Exosome biogenesis involves the inward budding of multivesicular bodies (MVB) to form intraluminal vesicles (ILV). When fused with the plasma membrane, the MVB releases the vesicles into the extracellular environment as exosomes. Proposed functions of these vesicles include roles in cell–cell signalling, removal of unwanted proteins, and the transfer of pathogens between cells, such as HIV-1. Another such pathogen which exploits this pathway is the prion, the infectious particle responsible for the transmissible neurodegenerative diseases such as Creutzfeldt-Jakob disease (CJD) of humans or bovine spongiform encephalopathy (BSE) of cattle. Interestingly, this work is mirrored by studies on another protein involved in neurodegenerative disease, the amyloid precursor protein (APP) which is associated with Alzheimer’s disease (AD). Recent work has found APP proteolytic fragments in association with exosomes, suggesting a common pathway previously unknown for proteins associated with neurodegenerative diseases. This review will be discussing the current literature regarding the role of exosomes in secretion of the proteins, PrP and APP, and the subsequent implications for neurodegenerative disease.

Inhibition of γ-secretase causes increased secretion of amyloid precursor protein C-terminal fragments in association with exosomes

Alzheimers disease (AD) is the most common form of dementia and is associated with the deposition of the 39‐ to 43‐amino acid β‐amyloid peptide (Aβ) in the brain. C‐terminal fragments (CTFs) of amyloid precursor protein (APP) can accumulate in endosomally derived multivesicular bodies (MVBs). These intracellular structures contain intraluminal vesicles that are released from the cell as exosomes when the MVB fuses with the plasma membrane. Here we have investigated the role of exosomes in the processing of APP and show that these vesicles contain APP‐ CTFs, as well as Aβ. In addition, inhibition of γ‐secre‐ tase results in a significant increase in the amount of α‐ and β‐secretase cleavage, further increasing the amount of APP‐CTFs contained within these exosomes. We identify several key members of the secretase family of proteases (BACE, PS1, PS2, and ADAM10) to be localized in exosomes, suggesting they may be a previously unidentified site of APP cleavage. These results provide further evidence for a novel pathway in which APP fragments are released from cells and have implications for the analysis of APP processing and diagnostics for Alzheimers disease.—Sharples, R. A., Vella, L. J., Nisbet, R. M., Naylor, R., Perez, K., Barnham, K. J., Masters, C. L., Hill, A. F. Inhibition of γ‐secretase causes increased secretion of amyloid precursor protein C‐terminal fragments in association with exosomes. FASEB J. 22, 1469–1478 (2008)

Tau aggregation and its interplay with amyloid-β.

Neurofibrillary tangles and amyloid plaques constitute the hallmark brain lesions of Alzheimer’s disease (AD) patients. Tangles are composed of fibrillar aggregates of the microtubule-associated protein tau, and plaques comprise fibrillar forms of a proteolytic cleavage product, amyloid-β (Aβ). Although plaques and tangles are the end-stage lesions in AD, small oligomers of Aβ and tau are now receiving increased attention as they are shown to correlate best with neurotoxicity. One key question of debate, however, is which of these pathologies appears first and hence is upstream in the pathocascade. Studies suggest that there is an intense crosstalk between the two molecules and, based on work in animal models, there is increasing evidence that Aβ, at least in part, exerts its toxicity via tau, with the Src kinase Fyn playing a crucial role in this process. In other experimental paradigms, Aβ and tau have been found to exert both separate and synergistic modes of toxicity. The challenge, however, is to integrate these different scenarios into a coherent picture. Furthermore, the ability of therapeutic interventions targeting just one of these molecules, to successfully neutralize the toxicity of the other, needs to be ascertained to improve current therapeutic strategies, such as immunotherapy, for the treatment of AD. Although this article is not intended to provide a comprehensive review of the currently pursued therapeutic strategies, we will discuss what has been achieved by immunotherapy and, in particular, how the inherent limitations of this approach can possibly be overcome by novel strategies that involve single-chain antibodies.

Engineering of an anti‐epidermal growth factor receptor antibody to single chain format and labeling by sortase A‐mediated protein ligation

Sortase‐mediated protein ligation is a biological covalent conjugation system developed from the enzymatic cell wall display mechanism found in Staphylococcus aureus. This three‐component system requires: (i) purified Sortase A (SrtA) enzyme; (ii) a substrate containing the LPXTG peptide recognition sequence; and (iii) an oligo‐glycine acceptor molecule. We describe cloning of the single‐chain antibody sc528, which binds to the extracellular domain of the epidermal growth factor receptor (EGFR), from the parental monoclonal antibody and incorporation of a LPETGG tag sequence. Utilizing recombinant SrtA, we demonstrate successful incorporation of biotin from GGG‐biotin onto sc528. EGFR is an important cancer target and is over‐expressed in human tumor tissues and cancer lines, such as the A431 epithelial carcinoma cells. SrtA‐biotinylated sc528 specifically bound EGFR expressed on A431 cells, but not negative control lines. Similarly, when sc528 was labeled with fluorescein we observed antigen‐specific labeling. The ability to introduce functionality into recombinant antibodies in a controlled, site‐specific manner has applications in experimental, diagnostic, and potentially clinical settings. For example, we demonstrate addition of all three reaction components in situ within a biosensor flow cell, resulting in oriented covalent capture and presentation of sc528, and determination of precise affinities for the antibody–receptor interaction. Biotechnol. Bioeng. 2012; 109:1461–1470.

Ultrasound treatment of neurological diseases — current and emerging applications

Like cardiovascular disease and cancer, neurological disorders present an increasing challenge for an ageing population. Whereas nonpharmacological procedures are routine for eliminating cancer tissue or opening a blocked artery, the focus in neurological disease remains on pharmacological interventions. Setbacks in clinical trials and the obstacle of access to the brain for drug delivery and surgery have highlighted the potential for therapeutic use of ultrasound in neurological diseases, and the technology has proved useful for inducing focused lesions, clearing protein aggregates, facilitating drug uptake, and modulating neuronal function. In this Review, we discuss milestones in the development of therapeutic ultrasound, from the first steps in the 1950s to recent improvements in technology. We provide an overview of the principles of diagnostic and therapeutic ultrasound, for surgery and transient opening of the blood–brain barrier, and its application in clinical trials of stroke, Parkinson disease and chronic pain. We discuss the promising outcomes of safety and feasibility studies in preclinical models, including rodents, pigs and macaques, and efficacy studies in models of Alzheimer disease. We also consider the challenges faced on the road to clinical translation.

Conformation Sensors that Distinguish Monomeric Proteins from Oligomers in Live Cells

Proteins prone to misfolding form large macroscopic deposits in many neurodegenerative diseases. Yet the in situ aggregation kinetics remains poorly understood because of an inability to demarcate precursor oligomers from monomers. We developed a strategy for mapping the localization of soluble oligomers and monomers directly in live cells. Sensors for mutant huntingtin, which forms aggregates in Huntingtons disease, were made by introducing a tetracysteine motif into huntingtin that becomes occluded from binding biarsenical fluorophores in oligomers, but not monomers. Up to 70% of the diffusely distributed huntingtin molecules appeared as submicroscopic oligomers in individual neuroblastoma cells expressing mutant huntingtin. We anticipate the sensors to enable insight into cellular mechanisms mediated by oligomers and monomers and for the approach to be adaptable more generally in the study of protein self-association.

Conformational detection of prion protein with biarsenical labeling and FlAsH fluorescence.

Prion diseases are associated with the misfolding of the host-encoded cellular prion protein (PrP(C)) into a disease associated form (PrP(Sc)). Recombinant PrP can be refolded into either an alpha-helical rich conformation (alpha-PrP) resembling PrP(C) or a beta-sheet rich, protease resistant form similar to PrP(Sc). Here, we generated tetracysteine tagged recombinant PrP, folded this into alpha- or beta-PrP and determined the levels of FlAsH fluorescence. Insertion of the tetracysteine tag at three different sites within the 91-111 epitope readily distinguished beta-PrP from alpha-PrP upon FlAsH labeling. Labelling of tetracysteine tagged PrP in the alpha-helical form showed minimal fluorescence, whereas labeling of tagged PrP in the beta-sheet form showed high fluorescence indicating that this region is exposed upon conversion. This highlights a region of PrP that can be implicated in the development of diagnostics and is a novel, protease free mechanism for distinguishing PrP(Sc) from PrP(C). This technique may also be applied to any protein that undergoes conformational change and/or misfolding such as those involved in other neurodegenerative disorders including Alzheimers, Huntingtons and Parkinsons diseases.

Residues Surrounding the Glycosylphosphatidylinositol Anchor Attachment Site of PrP Modulate Prion Infection: Insight from the Resistance of Rabbits to Prion Disease

ABSTRACT Prion diseases are a group of transmissible, invariably fatal neurodegenerative diseases that affect both humans and animals. According to the protein-only hypothesis, the infectious agent is a prion (proteinaceous infectious particle) that is composed primarily of PrPSc, the disease-associated isoform of the cellular prion protein, PrP. PrPSc arises from the conformational change of the normal, glycosylphosphatidylinositol (GPI)-anchored protein, PrPC. The mechanism by which this process occurs, however, remains enigmatic. Rabbits are one of a small number of mammalian species reported to be resistant to prion infection. Sequence analysis of rabbit PrP revealed that its C-terminal amino acids differ from those of PrP from other mammals and may affect the anchoring of rabbit PrP through its GPI anchor. Using a cell culture model, this study investigated the effect of the rabbit PrP-specific C-terminal amino acids on the addition of the GPI anchor to PrPC, PrPC localization, and PrPSc formation. The incorporation of rabbit-specific C-terminal PrP residues into mouse PrP did not affect the addition of a GPI anchor or the localization of PrP. However, these residues did inhibit PrPSc formation, suggesting that these rabbit-specific residues interfere with a C-terminal PrPSc interaction site.

Combined effects of scanning ultrasound and a tau-specific single chain antibody in a tau transgenic mouse model

One of the greatest challenges for the treatment of neurodegenerative disease is crossing the blood-brain barrier. Nisbet et al. demonstrate that non-invasive scanning ultrasound increases the delivery of tau-specific single-chain antibody fragments across the blood-brain barrier and into neurons of tau transgenic mice, reducing anxiety-like behaviour and tau pathology.

Co-immunoprecipitation with Tau Isoform-specific Antibodies Reveals Distinct Protein Interactions and Highlights a Putative Role for 2N Tau in Disease

Alternative splicing generates multiple isoforms of the microtubule-associated protein Tau, but little is known about their specific function. In the adult mouse brain, three Tau isoforms are expressed that contain either 0, 1, or 2 N-terminal inserts (0N, 1N, and 2N). We generated Tau isoform-specific antibodies and performed co-immunoprecipitations followed by tandem mass tag multiplexed quantitative mass spectrometry. We identified novel Tau-interacting proteins of which one-half comprised membrane-bound proteins, localized to the plasma membrane, mitochondria, and other organelles. Tau was also found to interact with proteins involved in presynaptic signal transduction. MetaCore analysis revealed one major Tau interaction cluster that contained 33 Tau pulldown proteins. To explore the pathways in which these proteins are involved, we conducted an ingenuity pathway analysis that revealed two significant overlapping pathways, “cell-to-cell signaling and interaction” and “neurological disease.” The functional enrichment tool DAVID showed that in particular the 2N Tau-interacting proteins were specifically associated with neurological disease. Finally, for a subset of Tau interactions (apolipoprotein A1 (apoA1), apoE, mitochondrial creatine kinase U-type, β-synuclein, synaptogyrin-3, synaptophysin, syntaxin 1B, synaptotagmin, and synapsin 1), we performed reverse co-immunoprecipitations, confirming the preferential interaction of specific isoforms. For example, apoA1 displayed a 5-fold preference for the interaction with 2N, whereas β-synuclein showed preference for 0N. Remarkably, a reverse immunoprecipitation with apoA1 detected only the 2N isoform. This highlights distinct protein interactions of the different Tau isoforms, suggesting that they execute different functions in brain tissue.