Alzheimer's disease constitutes a rising threat to public health. Despite extensive research in cellular and animal models, identifying the pathogenic agent present in the human brain and showing that it confers key features of Alzheimer's disease has not been achieved. We extracted soluble amyloid-beta protein (Abeta) oligomers directly from the cerebral cortex of subjects with Alzheimer's disease. The oligomers potently inhibited long-term potentiation (LTP), enhanced long-term depression (LTD) and reduced dendritic spine density in normal rodent hippocampus. Soluble Abeta from Alzheimer's disease brain also disrupted the memory of a learned behavior in normal rats. These various effects were specifically attributable to Abeta dimers. Mechanistically, metabotropic glutamate receptors were required for the LTD enhancement, and N-methyl D-aspartate receptors were required for the spine loss. Co-administering antibodies to the Abeta N-terminus prevented the LTP and LTD deficits, whereas antibodies to the midregion or C-terminus were less effective. Insoluble amyloid plaque cores from Alzheimer's disease cortex did not impair LTP unless they were first solubilized to release Abeta dimers, suggesting that plaque cores are largely inactive but sequester Abeta dimers that are synaptotoxic. We conclude that soluble Abeta oligomers extracted from Alzheimer's disease brains potently impair synapse structure and function and that dimers are the smallest synaptotoxic species.

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With the advent of the “-omics” era, biological research has shifted from functionally analyzing single proteins to understanding how entire protein networks connect and adapt to environmental cues. Frequently, pathological processes are initiated by a malfunctioning protein network rather than a single protein. It is therefore crucial to investigate the regulation of proteins in the context of a pathway first and signaling network second. In this study, we demonstrate that a quantitative interaction proteomic approach, combining immunoprecipitation, in-solution digestion and label-free quantification mass spectrometry, provides data of high accuracy and depth. This protocol is applicable, both to tagged, exogenous and untagged, endogenous proteins. Furthermore, it is fast, reliable and, due to a label-free quantitation approach, allows the comparison of multiple conditions. We further show that we are able to generate data in a medium throughput fashion and that we can quantify dynamic interaction changes in signaling pathways in response to mitogenic stimuli, making our approach a suitable method to generate data for system biology approaches.

Hippo pathway downstream effectors Yap and Taz play key roles in cell proliferation and regeneration, regulating gene expression especially via Tead transcription factors. To investigate their role in skeletal muscle stem cells, we analyzed Taz in vivo and ex vivo in comparison with Yap. Small interfering RNA knockdown or retroviral‐mediated expression of wild‐type human or constitutively active TAZ mutants in satellite cells showed that TAZ promoted proliferation, a function shared with YAP. However, at later stages of myogenesis, TAZ also enhanced myogenic differentiation of myoblasts, whereas YAP inhibits such differentiation. Functionally, while muscle growth was mildly affected in Taz (gene Wwtr1 –/–) knockout mice, there were no overt effects on regeneration. Conversely, conditional knockout of Yap in satellite cells of Pax7Cre‐ERT2/+: Yapfl°x/fl°x:Rosa26Lacz mice produced a regeneration deficit. To identify potential mechanisms, microarray analysis showed many common TAZ/YAP target genes, but TAZ also regulates some genes independently of YAP, including myogenic genes such as Pax7, Myf5, and Myod1 (ArrayExpress–E‐MTAB‐5395). Proteomic analysis revealed many novel binding partners of TAZ/YAP in myogenic cells, but TAZ also interacts with proteins distinct from YAP that are often involved in myogenesis and aspects of cytoskeleton organization (ProteomeXchange–PXD005751). Neither TAZ nor YAP bind members of the Wnt destruction complex but both regulated expression of Wnt and Wnt‐cross talking genes with known roles in myogenesis. Finally, TAZ operates through Tead4 to enhance myogenic differentiation. In summary, Taz and Yap have overlapping functions in promoting myoblast proliferation but Taz then switches to enhance myogenic differentiation. Stem Cells 2017;35:1958–1972

VGLL proteins are transcriptional co-factors that bind TEAD family transcription factors to regulate events ranging from wing development in fly, to muscle fibre composition and immune function in mice. Here, we characterise Vgll3 in skeletal muscle. We found that mouse Vgll3 was expressed at low levels in healthy muscle but that its levels increased during hypertrophy or regeneration; in humans, VGLL3 was highly expressed in tissues from patients with various muscle diseases, such as in dystrophic muscle and alveolar rhabdomyosarcoma. Interaction proteomics revealed that VGLL3 bound TEAD1, TEAD3 and TEAD4 in myoblasts and/or myotubes. However, there was no interaction with proteins from major regulatory systems such as the Hippo kinase cascade, unlike what is found for the TEAD co-factors YAP (encoded by YAP1 ) and TAZ (encoded by WWTR1 ). Vgll3 overexpression reduced the activity of the Hippo negative-feedback loop, affecting expression of muscle-regulating genes including Myf5 , Pitx2 and Pitx3 , and genes encoding certain Wnts and IGFBPs. VGLL3 mainly repressed gene expression, regulating similar genes to those regulated by YAP and TAZ. siRNA-mediated Vgll3 knockdown suppressed myoblast proliferation, whereas Vgll3 overexpression strongly promoted myogenic differentiation. However, skeletal muscle was overtly normal in Vgll3 -null mice, presumably due to feedback signalling and/or redundancy. This work identifies VGLL3 as a transcriptional co-factor operating with the Hippo signal transduction network to control myogenesis.