Degeneration of basal forebrain cholinergic neurons (BFCNs) contributes to cognitive dysfunction in Alzheimer's disease (AD) and Down's syndrome (DS). We used Ts65Dn and Ts1Cje mouse models of DS to show that the increased dose of the amyloid precursor protein gene, App, acts to markedly decrease NGF retrograde transport and cause degeneration of BFCNs. NGF transport was also decreased in mice expressing wild-type human APP or a familial AD-linked mutant APP; while significant, the decreases were less marked and there was no evident degeneration of BFCNs. Because of evidence suggesting that the NGF transport defect was intra-axonal, we explored within cholinergic axons the status of early endosomes (EEs). NGF-containing EEs were enlarged in Ts65Dn mice and their App content was increased. Our study thus provides evidence for a pathogenic mechanism for DS in which increased expression of App, in the context of trisomy, causes abnormal transport of NGF and cholinergic neurodegeneration.

Stem cell transplantation promises new hope for the treatment of stroke although significant questions remain about how the grafted cells elicit their effects. One hypothesis is that transplanted stem cells enhance endogenous repair mechanisms activated after cerebral ischaemia. Recognizing that bilateral reorganization of surviving circuits is associated with recovery after stroke, we investigated the ability of transplanted human neural progenitor cells to enhance this structural plasticity. Our results show the first evidence that human neural progenitor cell treatment can significantly increase dendritic plasticity in both the ipsi- and contralesional cortex and this coincides with stem cell-induced functional recovery. Moreover, stem cell-grafted rats demonstrated increased corticocortical, corticostriatal, corticothalamic and corticospinal axonal rewiring from the contralesional side; with the transcallosal and corticospinal axonal sprouting correlating with functional recovery. Furthermore, we demonstrate that axonal transport, which is critical for both proper axonal function and axonal sprouting, is inhibited by stroke and that this is rescued by the stem cell treatment, thus identifying another novel potential mechanism of action of transplanted cells. Finally, we established in vitro co-culture assays in which these stem cells mimicked the effects observed in vivo. Through immunodepletion studies, we identified vascular endothelial growth factor, thrombospondins 1 and 2, and slit as mediators partially responsible for stem cell-induced effects on dendritic sprouting, axonal plasticity and axonal transport in vitro. Thus, we postulate that human neural progenitor cells aid recovery after stroke through secretion of factors that enhance brain repair and plasticity.

This communication describes a novel protein labeling method that uses a single amino-acid tag -N-terminal cysteine residue -and small-molecule probes carrying the cyanobenzothiazole unit for specific labeling of proteins in vitro and at the surface of live cells. This simple ligation reaction proceeds with a high degree of specificity in physiological conditions, and should offer an important alternative to currently available protein labeling methods. Graphical abstract KeywordsProtein Labeling; Condensation; Terminal Cysteine; Chemical ligation; Live-cell Imaging Site-specific labeling of proteins with molecular tags enables direct visualization of protein dynamics, localization and interactions in single living cells and is a powerful tool for studying structure and function of proteins. [1] Proteins of interest can be labeled by genetic fusions to fluorescent proteins, or chemical reactions with fluorescent dyes. Chemical labeling often employs a receptor protein, for example, a mutant of human O 6 -alkylguanine-* Fax: (+1) 650-736-7925, ; Email: jrao@stanford.edu, Homepage: http://raolab.stanford.edu Supporting information for this article is available on the WWW under http://www.angewandte.org or from the author. HHS Public Access Author ManuscriptAuthor Manuscript Author ManuscriptAuthor Manuscript DNA transferase, [2a] and E. coli dihydrofolate reductase, [2b] that binds to or reacts with its fluorescently tagged ligand. [2] Alternatively, smaller tags such as short peptides can be labeled by selective binding to fluorogenic dyes [3] or by enzyme-catalyzed ligation to fluorescent probes. [4] Water-compatible chemical reactions can also be applied to protein labeling, such as the Staudinger reaction between the azides and triphenylphosphines, [5] the Huisgen cycloaddition or "Click chemistry" between the azides and alkynes, [6] the reaction between aldehydes (or ketones) and aminooxy containing reagents (or hydrazides). [7] Herein, we describe a water-compatible condensation reaction for labeling terminal cysteine residues on proteins in vitro and at the cell surface.N-terminal cysteine has been frequently used in protein engineering for site-specific labeling and modification. [8] Thioesters are commonly used in a ligation reaction with terminal cysteines, which proceeds through thioester and S-to N-acyl exchanges. [9] This native chemical ligation reaction has been successfully applied to protein semi-synthesis and labeling. [10] Our method to label the terminal cysteine on a protein is based on the condensation of 2-cyanobenzothiazole (CBT) and D-cysteine, a reaction used at the last step of the synthesis of D-luciferin a common substrate for firefly luciferase (reaction 1 in Scheme 1). [11] This reaction can proceed smoothly in aqueous solutions. We hypothesized that CBT could react with the terminal cysteine on a protein. If a fluorophore is conjugated to the CBT motif, this reaction should ligate a fluorescent label specifically to the terminal cysteine of the protein (reaction...

Superresolution imaging techniques based on sequential imaging of sparse subsets of single molecules require fluorophores whose emission can be photoactivated or photoswitched. Because typical organic fluorophores can emit significantly more photons than average fluorescent proteins, organic fluorophores have a potential advantage in superresolution imaging schemes, but targeting to specific cellular proteins must be provided. We report the design and application of HaloTag-based target-specific azido DCDHFs, a class of photoactivatable push-pull fluorogens which produce bright fluorescent labels suitable for single-molecule superresolution imaging in live bacterial and fixed mammalian cells.Recently, sequential imaging of sparse subsets of photoactivatable/photoswitchable singlemolecule fluorophores has enabled optical imaging beyond the diffraction limit (DL), providing insight into the sub-diffraction world (e.g. PALM, FPALM, STORM). 1-3 These single-molecule superresolution (SR) techniques have provided the impetus for development of new controllable fluorophores with large numbers of emitted photons N, because the achievable resolution scales as . 4 Most previous SR experiments in living cells 5 have used photocontrollable fluorescent proteins. 6-9 However, despite having the advantage of being target-specific, fluorescent proteins on average provide 10-fold fewer photons before photobleaching than good organic fluorophores. 10,11 Small organic fluorophores have the additional benefit of synthetic design flexibility for tuning target specificity, spectral wavelength, solubility, and other desired properties. Therefore, targeted bright organic Here we present a target-specific photoactivatable organic fluorophore for use inside living and fixed cells, 3, based on the commercial HaloTag targeting approach. [20][21][22] This method requires a genetic fusion to the HaloEnzyme (HaloEnz), which forms a covalent linkage to the HaloTag substrate, thus labeling the protein of interest (i.e. a protein-HaloEnzHaloTag-fluorophore covalent unit). Specifically, we present: (i) the basic photophysical properties of a new targeted photoactivatable probe; (ii) proof-of-principle labeling of known structures in fixed and living mammalian cells validated by co-staining with antibodies or co-transfection with fluorescent proteins; (iii) specific SR imaging of microtubules in a mammalian cell with quantification of resolution enhancement; (iv) demonstration of targeted labeling in living bacteria with diffraction-limited imaging; and finally, (v) SR imaging of poorly understood structures inside living bacteria.As molecules with bright emission for single-molecule imaging, dicyanomethylenedihydrofuran (DCDHF) push-pull fluorophores emit millions of photons before photobleaching, and can enter living cells. 15,23 Recently, we reported a photoactivatable DCDHF fluorogen based on photocaging the fluorescence by replacing the amine donor with a poorly-donating but photolabile azide, which can then be converted back to an am...