Exosomes are microvesicles of endosomal origin that are secreted, and their contents (proteins, lipids, DNA, or microRNAs) can alter the physiological states of recipient cells. We demonstrated that phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor suppressor protein normally localized in the cytoplasm and nucleus, was secreted in exosomes. Secreted PTEN was internalized by recipient cells with resultant functional activity, which resulted in reduced phosphorylation of the serine and threonine kinase Akt and reduced cellular proliferation. PTEN secretion in exosomes required Ndfip1, an adaptor protein for members of the Nedd4 family of E3 ubiquitin ligases. Without Ndfip1, neither Nedd4-1 nor Nedd4-2 promoted the recruitment of PTEN into exosomes. In addition, lysine 13 within PTEN, which is required for its ubiquitination by Nedd4-1, was required for exosomal transport of PTEN. These results implicate Ndfip1 as a molecular regulator of the exosomal export of PTEN, with consequences for non-cell-autonomous PTEN activity. Thus, we suggest that the ability of PTEN to exert phosphatase activity beyond the cell in which it is produced has implications for PTEN function during development, health, and disease.

Exosomes represent an attractive vehicle for the delivery of biomolecules. However, mechanisms for loading functional molecules into exosomes are relatively unexplored. Here we report the use of the evolutionarily conserved late-domain (L-domain) pathway as a mechanism for loading exogenous proteins into exosomes. We demonstrate that labeling of a target protein, Cre recombinase, with a WW tag leads to recognition by the L-domain-containing protein Ndfip1, resulting in ubiquitination and loading into exosomes. Our results show that Ndfip1 expression acts as a molecular switch for exosomal packaging of WW-Cre that can be suppressed using the exosome inhibitor GW4869. When taken up by floxed reporter cells, exosomes containing WW-Cre were capable of inducing DNA recombination, indicating functional delivery of the protein to recipient cells. Engineered exosomes were administered to the brain of transgenic reporter mice using the nasal route to test for intracellular protein delivery in vivo. This resulted in the transport of engineered exosomes predominantly to recipient neurons in a number of brain regions, including the olfactory bulb, cortex, striatum, hippocampus, and cerebellum. The ability to engineer exosomes to deliver biologically active proteins across the blood-brain barrier represents an important step for the development of therapeutics to treat brain diseases.

Edited by Paul FraserMore than 30 years ago, two unexpected findings were discovered that challenged conventional thinking in biology. The first was the identification of a misfolded protein with transmissible properties associated with a group of neurodegenerative diseases known as transmissible spongiform encephalopathies. The second was the discovery of a new pathway used for the extracellular release of biomolecules, including extracellular vesicles called exosomes. Two decades later, the convergence of these pathways was shown when exosomes were found to play a significant role in both the transmission and propagation of protein aggregates in disease. Recent research has now revealed that the majority of proteins involved in neurodegenerative diseases are transported in exosomes, and that external stresses due to age-related impairment of protein quality control mechanisms can promote the transcellular flux of these proteins in exosomes. Significantly, exosomes provide an environment that can induce the conformational conversion of native proteins into aggregates that can be transmitted to otherwise aggregate-free cells in the brain. Here we review the current roles of exosomes in the pathology of neurodegenerative diseases.Exosomes are released into the extracellular environment by the majority of cell types in the body. Originally identified to be involved in the non-degradative removal of the transferrin receptor during the maturation process of reticulocytes (1, 2), exosomes have now also been recognized as an important communication and signaling pathway in the body in both normal and disease settings. Exosomes differ from other extracellular vesicles (EVs) 3 based on the secretion pathway used and the size of the vesicle released. Unlike other EVs that can bud from the plasma membrane, such as microvesicles (3), exosomes are created from intraluminal vesicles that form within multivesicular bodies (MVBs, or multivesicular endosomes). The subsequent fusion of the MVB at the plasma membrane releases these vesicles into the extracellular milieu where they are known as exosomes (Fig. 1). This secretion process results in a large number of exosomes being released in the body, with estimates of 3 ϫ 10 6 exosomes per microliter of blood serum. Recent evidence has highlighted the importance of exosomes both for cellular communication and in the delivery of biomolecules.The function of exosomes differs depending on the cell type from which they originate. Initial in vivo studies identified that exosomes derived from dendritic cells could express MHC class II molecules to promote an immune response (4). Since then, exosomes have been found to function in angiogenesis, inflammation, morphogen transportation, and programmed cell death (5). The richest area of exosome research, however, has come from disease studies, in particular the cancer field. Recent evidence has highlighted a role for exosomes to promote metastasis and regulate tumor immune response (6). Of particular interest is the ability of tumors to rel...

Recognition of the large secreted protein Slit by receptors of the Robo family provides fundamental signals in axon guidance and other developmental processes. In Drosophila, Slit–Robo signalling regulates midline crossing and the lateral position of longitudinal axon tracts. We report the functional dissection of Drosophila Slit, using structure analysis, site‐directed mutagenesis and in vitro assays. The N‐terminal region of Slit consists of a tandem array of four independently folded leucine‐rich repeat (LRR) domains, connected by disulphide‐tethered linkers. All three Drosophila Robos were found to compete for a single highly conserved site on the concave face of the second LRR domain of Slit. We also found that this domain is sufficient for biological activity in a chemotaxis assay. Other Slit activities may require Slit dimerisation mediated by the fourth LRR domain. Our results show that a small portion of Slit is able to induce Robo signalling and indicate that the distinct functions of Drosophila Robos are encoded in their divergent cytosolic domains.

Slit is a large secreted protein that provides important guidance cues in the developing nervous system and in other organs. Signaling by Slit requires two receptors, Robo transmembrane proteins and heparan sulfate (HS) proteoglycans. How HS controls Slit-Robo signaling is unclear. Here we show that the second leucine-rich repeat domain (D2) of Slit, which mediates binding to Robo receptors, also contains a functionally important binding site for heparin, a highly sulfated variant of HS. Heparin markedly enhances the affinity of the Slit-Robo interaction in a solid-phase binding assay. Analytical gel filtration chromatography demonstrates that Slit D2 associates with a soluble Robo fragment and a heparin-derived oligosaccharide to form a ternary complex. Retinal growth cone collapse triggered by Slit D2 requires cell surface HS or exogenously added heparin. Mutation of conserved basic residues in the C-terminal cap region of Slit D2 reduces heparin binding and abolishes biological activity. We conclude that heparin/HS is an integral component of the minimal Slit-Robo signaling complex and serves to stabilize the relatively weak Slit-Robo interaction.Slits are large secreted leucine-rich repeat (LRR) 6 proteins with multiple roles in cell signaling and adhesion. They have well established and evolutionarily conserved functions as guidance cues in the developing nervous system (1, 2), but Slits are also important in the development of the vasculature (3) and other organs (4). The first class of Slit receptors to be identified were Robo family members, which are transmembrane proteins with an extracellular domain resembling cell adhesion molecules and a large cytosolic signaling domain (1, 2). Biochemical studies have defined the domains mediating the SlitRobo interaction (5, 6), as well as some of the components of the signaling cascade downstream of Robo activation (7, 8), but how binding of Slit to Robo receptors conveys a signal across the cell membrane remains unknown.The first indication that there might exist a second Slit receptor came from the observation that heparan sulfate (HS) was required for the repellent activity of Slit in vitro (9) and in vivo (10). The identity of this receptor was revealed by recent genetic studies in invertebrates, which showed that Slit signaling requires Robo to be co-expressed on the same cell with the HS proteoglycan syndecan (11-13). Syndecan is a membranespanning protein to which are covalently attached several HS chains, consisting of repeating sulfated disaccharide units (14). Heparin is a member of the HS family that is more highly and uniformly sulfated than other HS. Johnson et al. (12) showed that both Slit and Robo can be co-immunoprecipitated with syndecan, suggesting the presence of a ternary (or higher order) complex at the neuronal cell membrane. However, the composition and functional relevance of this putative ternary complex was not established. Because the Slit distribution was found to be altered in syndecan-deficient embryos, HS may also be required for...

The ability to remove unwanted proteins is an important cellular feature. Classically, this involves the enzymatic addition of ubiquitin moieties followed by degradation in the proteasome. Nedd4 proteins are ubiquitin ligases important not only for protein degradation, but also for protein trafficking. Nedd4 proteins can bind to target proteins either by themselves or through adaptor protein Ndfip1 (Nedd4 family-interacting protein 1). An alternative mechanism for protein removal and trafficking is provided by exosomes, which are small vesicles (50 -90-nm diameter) originating from late endosomes and multivesicular bodies (MVBs). Exosomes provide a rapid means of shedding obsolete proteins and also for cell to cell communication. In the present work, we show that Ndfip1 is detectable in exosomes secreted from transfected cells and also from primary neurons. Compared with control, Ndfip1 increases exosome secretion from transfected cells. Furthermore, while Nedd4, Nedd4-2, and Itch are normally absent from exosomes, expression of Ndfip1 results in recruitment of all three Nedd4 proteins into exosomes. Together, these results suggest that Ndfip1 is important for protein trafficking via exosomes, and provides a mechanism for cargoing passenger proteins such as Nedd4 family proteins. Given the positive roles of Ndfip1/Nedd4 in improving neuronal survival during brain injury, it is possible that exosome secretion provides a novel route for rapid sequestration and removal of proteins during stress.The ability to dispose of unwanted proteins is an important function during cellular homeostasis in health or disease. The best studied pathway for disposing of unwanted proteins involves the addition of ubiquitin chains to target proteins followed by degradation of the complex in the proteasome. A major class of enzymes involved in target recognition in this pathway is the E3 ligases with HECT (homology to the E6-associated protein C terminus domain) domains (1). Nedd4 and Nedd4-2 are archetypal members of this family, with ability to bind and ubiquitinate proteins containing PPXY motifs (2, 3). In the nervous system, Nedd4-mediated ubiquitination is required for down-regulating voltage-gated K ϩ and Na ϩ channels (4, 5), axon-guidance proteins (6), and TrkA neurotrophin receptor (7). In addition, Nedd4 family proteins (e.g. Nedd4, Nedd4-2, and Itch) can also ubiquitinate target proteins that are bound to Nedd4 adaptors. One such adaptor is Ndfip1 (Nedd4 family-interacting protein 1) originally identified in a screen for Nedd4-binding partners (8, 9). Ndfip1 contains three transmembrane domains and is localized in the Golgi and postGolgi vesicles. Through their transmembrane domains, Ndfip1 can bind membrane proteins and in doing so, either recruit or inhibit the interaction of membrane proteins with cytosolic Nedd4. In the brain, both Ndfip1 and Nedd4 are co-expressed in the same neuron, and their interaction has been shown to be crucial for increasing the survival of cortical neurons during injury (10).Besides proteasomal...

PTEN nuclear entry driven by ubiquitination is mediated by the ligase-interacting protein Ndfip1 and is essential for neuronal survival in mice after cerebral ischemia.

No abstract