Extracellular Vesicles Isolated from Human Induced Pluripotent Stem Cell-Derived Neurons Contain a Transcriptional Network.

Healthy brain function is mediated by several complementary signalling pathways, many of which are driven by extracellular vesicles (EVs). EVs are heterogeneous in both size and cargo and are constitutively released from cells into the extracellular milieu. They are subsequently trafficked to recipient cells, whereupon their entry can modify the cellular phenotype. Here, in order to further analyse the mRNA and protein cargo of neuronal EVs, we isolated EVs by size exclusion chromatography from human induced pluripotent stem cell (iPSC)-derived neurons. Electron microscopy and dynamic light scattering revealed that the isolated EVs had a diameter of 30-100 nm. Transcriptomic and proteomics analyses of the EVs and neurons identified key molecules enriched in the EVs involved in cell surface interaction (integrins and collagens), internalisation pathways (clathrin- and caveolin-dependent), downstream signalling pathways (phospholipases, integrin-linked kinase and MAPKs), and long-term impacts on cellular development and maintenance. Overall, we show that key signalling networks and mechanisms are enriched in EVs isolated from human iPSC-derived neurons.

The cellular expression and proteolytic processing of the amyloid precursor protein is independent of TDP-43.

Alzheimer's disease (AD) is a neurodegenerative condition, of which one of the cardinal pathological hallmarks is the extracellular accumulation of amyloid ß (Aß) peptides. These peptides are generated via proteolysis of the amyloid precursor protein (APP), in a manner dependent on the ß-secretase, BACE1 and the multicomponent γ-secretase complex. Recent data also suggest a contributory role in AD of transactive response DNA binding protein 43 (TDP-43). There is little insight into a possible mechanism linking TDP-43 and APP processing. To this end, we used cultured human neuronal cells to investigate the ability of TDP-43 to interact with APP and modulate its proteolytic processing. Immunocytochemistry showed TDP-43 to be spatially segregated from both the extranuclear APP holoprotein and its nuclear C-terminal fragment. The latter (APP intracellular domain) was shown to predominantly localise to nucleoli, from which TDP-43 was excluded. Furthermore, neither overexpression of each of the APP isoforms nor siRNA-mediated knockdown of APP had any effect on TDP-43 expression. Doxycycline-stimulated overexpression of TDP-43 was explored in an inducible cell line. Overexpression of TDP-43 had no effect on expression of the APP holoprotein, nor any of the key proteins involved in its proteolysis. Furthermore, increased TDP-43 expression had no effect on BACE1 enzymatic activity or immunoreactivity of Aß1-40, Aß1-42 or the Aß1-40:Aß1-42 ratio. Also, siRNA-mediated knockdown of TDP-43 had no effect on BACE1 immunoreactivity. Taken together, these data indicate that TDP-43 function and/or dysfunction in AD is likely independent from dysregulation of APP expression and proteolytic processing and Aß generation.

Proteolytic shedding of the prion protein via activation of metallopeptidase ADAM10 reduces cellular binding and toxicity of amyloid-ß oligomers.

The cellular prion protein (PrPC) is a key neuronal receptor for ß-amyloid oligomers (AßO), mediating their neurotoxicity, which contributes to the neurodegeneration in Alzheimer's disease (AD). Similarly to the amyloid precursor protein (APP), PrPC is proteolytically cleaved from the cell surface by a disintegrin and metalloprotease, ADAM10. We hypothesized that ADAM10-modulated PrPC shedding would alter the cellular binding and cytotoxicity of AßO. Here, we found that in human neuroblastoma cells, activation of ADAM10 with the muscarinic agonist carbachol promotes PrPC shedding and reduces the binding of AßO to the cell surface, which could be blocked with an ADAM10 inhibitor. Conversely, siRNA-mediated ADAM10 knockdown reduced PrPC shedding and increased AßO binding, which was blocked by the PrPC-specific antibody 6D11. The retinoic acid receptor analog acitretin, which up-regulates ADAM10, also promoted PrPC shedding and decreased AßO binding in the neuroblastoma cells and in human induced pluripotent stem cell (iPSC)-derived cortical neurons. Pretreatment with acitretin abolished activation of Fyn kinase and prevented an increase in reactive oxygen species caused by AßO binding to PrPC Besides blocking AßO binding and toxicity, acitretin also increased the nonamyloidogenic processing of APP. However, in the iPSC-derived neurons, Aß and other amyloidogenic processing products did not exhibit a reciprocal decrease upon acitretin treatment. These results indicate that by promoting the shedding of PrPC in human neurons, ADAM10 activation prevents the binding and cytotoxicity of AßO, revealing a potential therapeutic benefit of ADAM10 activation in AD.

Intermittent High-Dose Scheduling of AZD8835, a Novel Selective Inhibitor of PI3Kα and PI3Kδ, Demonstrates Treatment Strategies for PIK3CA-Dependent Breast Cancers.

The PIK3CA gene, encoding the p110α catalytic unit of PI3Kα, is one of the most frequently mutated oncogenes in human cancer. Hence, PI3Kα is a target subject to intensive efforts in identifying inhibitors and evaluating their therapeutic potential. Here, we report studies with a novel PI3K inhibitor, AZD8835, currently in phase I clinical evaluation. AZD8835 is a potent inhibitor of PI3Kα and PI3Kδ with selectivity versus PI3Kß, PI3Kγ, and other kinases that preferentially inhibited growth in cells with mutant PIK3CA status, such as in estrogen receptor-positive (ER(+)) breast cancer cell lines BT474, MCF7, and T47D (sub-µmol/L GI50s). Consistent with this, AZD8835 demonstrated antitumor efficacy in corresponding breast cancer xenograft models when dosed continuously. In addition, an alternative approach of intermittent high-dose scheduling (IHDS) was explored given our observations that higher exposures achieved greater pathway inhibition and induced apoptosis. Indeed, using IHDS, monotherapy AZD8835 was able to induce tumor xenograft regression. Furthermore, AZD8835 IHDS in combination with other targeted therapeutic agents further enhanced antitumor activity (up to 92% regression). Combination partners were prioritized on the basis of our mechanistic insights demonstrating signaling pathway cross-talk, with a focus on targeting interdependent ER and/or CDK4/6 pathways or alternatively a node (mTOR) in the PI3K-pathway, approaches with demonstrated clinical benefit in ER(+) breast cancer patients. In summary, AZD8835 IHDS delivers strong antitumor efficacy in a range of combination settings and provides a promising alternative to continuous dosing to optimize the therapeutic index in patients. Such schedules merit clinical evaluation. Mol Cancer Ther; 15(5); 877-89. ©2016 AACR.