Name: quantitative measurement of &#947;-secretase-mediated amyloid precursor protein and notch cleavage in cell-based luciferase reporter assay platforms
Uploaded: 2018-01-25T12:30:00
Description: Watch this Scientific Journal Video about Quantitative Measurement of Î³-Secretase-mediated Amyloid Precursor Protein and Notch Cleavage in Cell-based Luciferase Reporter Assay Platforms at JoVE.com

The authors thank the Core Facility of the Institute of Cellular and Organismic Biology, Academia Sinica, for technical support. This study was supported by the Ministry of Science and Technology, Taiwan (MOST 103-2320-B-001-016-MY3 to Y.-F. L.), the Program for Translational Innovation of Biopharmaceutical Development &#8211; Technology Supporting Platform Axis Scheme (NP7 to Y.-F.L.), and Academia Sinica (to Y.-F.L.).

1. Measurement of Luciferase Reporter Signals, which Correspond to &#947;-Secretase Cleavage of APP-C99 or N&#916;E
NOTE: Please refer to the previous publications for detailed descriptions of the generation of CG and NG cells20,21.
<ol>
	<li>Seed NG or CG cells (20, 000 cells/well) onto 96-well microplates at a final volume of 200 &#956;L/well in growth medium that is composed of Dulbecco&#39;s Modified Eagle Medium (DMEM) p...

<ol>
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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+of+small+molecular+(D)-leucinamides+as+potent,+Notch-sparing+gamma-secretase+modulators.">Discovery of small molecular (D)-leucinamides as potent, Notch-sparing gamma-secretase modulators.</a> Eur J Med Chem. 79, 143-151 (2014).</li><li>Liao, Y. F., Wang, B. J., Cheng, H. T., Kuo, L. H., Wolfe, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+necrosis+factor-alpha,+interleukin-1beta,+and+interferon-gamma+stimulate+gamma-secretase-mediated+cleavage+of+amyloid+precursor+protein+through+a+JNK-dependent+MAPK+pathway.">Tumor necrosis factor-alpha, interleukin-1beta, and interferon-gamma stimulate gamma-secretase-mediated cleavage of amyloid precursor protein through a JNK-dependent MAPK pathway.</a> J Biol Chem. 279 (47), 49523-49532 (2004).</li><li>Wang, B. 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The promising outcomes from a phase 1b trial of aducanumab immunotherapy for AD have firmly established the critical role of A&#946; in the pathogenesis of AD22 and suggest that anti-A&#946; approaches are still a viable strategy for the development of anti-AD drugs. Here, we describe cell-based assays for quantifying &#947;-secretase-catalyzed proteolysis of APP-C99 and N&#916;E using firefly luciferase reporter systems. APP-C99 and N&#916;E are the two most well-studied &#947;-secretase substrat...

Oligomeric forms of amyloid-&#946; (A&#946;) are believed to be the primary cause of neurodegeneration in the brains of patients suffering from Alzheimer&#39;s disease (AD)1. A&#946; peptides are produced by the stepwise cleavage of amyloid precursor protein (APP), first by &#946;-secretase and then by &#947;-secretase2. In the past decade, therapeutic approaches toward treatment of AD have focused on the prevention of A&#946; production3. The majority of studies have focused on either the augmentation of &#945;-secretase activity, which can preclude &#947;-secretase cleavage of APP and thereby decrease the production of A&#946;, or the inhibition of &#946;-and/or &#947;-secretase activities4. Unfortunately, non-selective inhibition of &#946;- or &#947;-secretases results in unavoidable side effects that are due to interference with the functioning of other physiological substrates of &#946;- and &#947;-secretase5,6. With regard to &#947;-secretase inhibitors, recent studies have reported the discovery of a number of chemical and genetic modulators that can regulate A&#946; production while exerting negligible effects on the essential &#947;-secretase-mediated processing of Notch7,8,9,10,11,12, however, successful therapeutics have not yet been developed. Thus, further systematic screens are warranted to discover novel genetic and chemical modifiers that may selectively modulate &#947;-secretase-mediated APP processing.
&#947;-Secretase is known to cleave more than 90 different membrane-anchored proteins. Among these substrates is Notch, whose activity is critical for cell fate determination and differentiation during development13. Selectively modulating &#947;-secretase-catalyzed APP processing in the absence of affecting Notch processing will be essential for minimizing Notch-related side effects of &#947;-secretase inhibitors, and this selectivity is thought to be a primary factor that will dictate the biological efficacy of potential &#947;-secretase modulators for the chronic treatment of AD. There have been a number of arylsulfonamide derivatives, such as GSI-953 (begacestat) and BMS-708163 (avagacestat), that have been found to exhibit potent and selective inhibition of &#947;-secretase14,15. A previous study has demonstrated that the differential inhibition of &#947;-secretase cleavage of APP and Notch can be observed using a quantitative ELISA-based assay in combination with in vivo validation using zebrafish16. Furthermore, cell-based assay paradigms have been established to address the potential substrate selectivity of &#947;-secretase toward APP and Notch17,18. Using protocols similar to those described herein, we have recently discovered a novel class of (D)-leucinamides that potently modulate &#947;-secretase cleavage of APP with Notch-sparing selectivity19. Together, this collection of studies provides a proof-of-concept supporting the notion that the substrate selectivity/availability of &#947;-secretase can be subject to chemical modulation and experimental verification. However, these assay platforms often rely on relatively low-throughput readouts and labor-intensive approaches that might not meet industrial standards for drug discovery programs.
We have recently generated cell-based luciferase reporter gene assays that can quantitatively determine the catalytic activity of &#947;-secretase toward two distinct substrates, the 99-amino acid C-terminal fragment of APP (APP-C99) and the extracellular domain-deleted Notch peptide (N&#916;E). Both APP-C99 and N&#916;E have been widely used as direct substrates of &#947;-secretase in various assays. To generate homogeneous and consistent luciferase reporter gene assays for the &#947;-secretase cleavage of either APP-C99 or N&#916;E, we generated one HEK-derived stable cell line (CG) that constitutively expresses a Gal4 promoter-driven firefly luciferase reporter (Gal4-Luc) and Gal4/VP16-tagged APP-C99 fusion protein (C99-GV). In addition, we generated another HEK-derived stable cell line (NG) that constitutively expresses Gal4-Luc and Gal4/VP16-tagged N&#916;E (N&#916;E-GV). Using these novel substrate-specific &#947;-secretase assays, we now provide a method to quantify and distinguish the catalytic activity of &#947;-secretase toward distinct substrates (APP-C99 in CG cells, or N&#916;E in NG cells). Moreover, the substrate-specific &#947;-secretase assays were designed to be conducive to high-content screening platforms. These newly generated &#947;-secretase assays could pave the way for the discovery of novel &#947;-secretase modulators that could improve cognitive function by suppressing A&#946; production without eliciting undesirable Notch inhibition for the next-generation treatment of AD.

<table>
	<tbody>
		<tr>
			<td>VictorLight luminescence plate reader</td>
			<td>PerkinElmer</td>
			<td>2030-0010</td>
			<td></td>
		</tr>
		<tr>
			<td>Class II, Type 2 Biological Safety Cabinet</td>
			<td>Thermo Scientific</td>
			<td>1300 Series A2</td>
			<td></td>
		</tr>
		<tr>
			<td>CL-387,785</td>
			<td>EMD Calbiochem</td>
			<td>233100-1MG</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPT</td>
			<td>Merck</td>
			<td>565770</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium (DMEM)</td>
			<td>Thermo Fisher</td>
			<td>12100046</td>
			<td>main compnent of growth medium</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Thermo Fisher</td>
			<td>16000044</td>
			<td></td>
		</tr>
		<tr>
			<td>Blasticidin</td>
			<td>Thermo Fisher</td>
			<td>A1113903</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeocin</td>
			<td>Thermo Fisher</td>
			<td>R25005</td>
			<td></td>
		</tr>
		<tr>
			<td>Hygromycin B</td>
			<td>Gibco</td>
			<td>10687010</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytomer</td>
			<td>Sigma-Aldrich</td>
			<td>BR717805 Aldrich</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well microplate</td>
			<td>Nunc</td>
			<td>156545</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetracycline</td>
			<td>Sigma</td>
			<td>T7660</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Steady-Glo luciferase assay reagent</td>
			<td>Promega</td>
			<td>E2510</td>
			<td></td>
		</tr>
		<tr>
			<td>Humidified CO2 incubator</td>
			<td>Revco</td>
			<td>Ultima II</td>
			<td></td>
		</tr>
		<tr>
			<td>T-REx293 cell line</td>
			<td>Invitrogen</td>
			<td>R71007</td>
			<td></td>
		</tr>
		<tr>
			<td>Wallac 1420 software version 3.0</td>
			<td>PerkinElmer</td>
			<td></td>
			<td>instrument control software of the luminescence microplate reader</td>
		</tr>
	</tbody>
</table>

quantitative measurement of &#947;-secretase-mediated amyloid precursor protein and notch cleavage in cell-based luciferase reporter assay platforms

We have developed a pair of cell-based reporter gene assays to quantitatively measure &#947;-secretase cleavage of distinct substrates. This manuscript describes procedures that may be used to monitor &#947;-secretase-mediated cleavage of either APP-C99 or Notch, using a Gal4 promoter-driven firefly luciferase reporter system. These assays were established by stably co-transfecting HEK293 cells with the Gal4-driven luciferase reporter gene and either the Gal4/VP16-tagged C-terminal fragment of APP (APP-C99; CG cells), or the Gal4/VP16-tagged Notch-&#916;E (N&#916;E; NG cells). Using these reporter assays in parallel, we have demonstrated that an ErbB2 inhibitor, CL-387,785, can preferentially suppress &#947;-secretase cleavage of APP-C99 in CG cells, but not N&#916;E in NG cells. The differential responses exhibited by the CG and NG cells, when treated with CL-387,785, represent a preferred characteristic for &#947;-secretase modulators, and these responses are in stark contrast to the pan-inhibition of &#947;-secretase induced by DAPT. Our studies provide direct evidence that &#947;-secretase activities toward different substrates can be differentiated in a cellular context. These new assays may therefore be useful tools in drug discovery for improved AD therapies.

Inhibition of ErbB2 by CL-387,785 can differentially promote the processing of APP-C99 by &#947;-secretase without affecting Notch cleavage 
To establish a cell-based assay that can quantitatively measure the proteolytic cleavage of a particular &#947;-secretase substrate, we generated the HEK293-derived CG cell line by stable co-transfection of a tetracycline-inducible C-terminally Gal4/VP16-tagged APP-C99 and a Gal4 promoter-driven...

Watch this Scientific Journal Video about Quantitative Measurement of Î³-Secretase-mediated Amyloid Precursor Protein and Notch Cleavage in Cell-based Luciferase Reporter Assay Platforms at JoVE.com

Quantitative Measurement of &amp;#947;-Secretase-mediated Amyloid Precursor Protein and Notch Cleavage in Cell-based Luciferase Reporter Assay Platforms

We have successfully generated two substrate-specific &#947;-secretase assays. Both cell-based assays presented here are designed to quantify &#947;-secretase enzymatic activities via the output of firefly luciferase reporters.

Quantitative Measurement of &#947;-Secretase-mediated Amyloid Precursor Protein and Notch Cleavage in Cell-based Luciferase Reporter Assay Platforms

We have developed a pair of cell-based reporter gene assays to quantitatively measure &#947;-secretase cleavage of distinct substrates. This manuscript ...

The overall goal of this quantitative cell-based reporter gene assay for gamma-secretase is to examine the differential effect of putative gamma-secretase modulators on gamma-secretase-mediated cleavage. This method can help to answer the key questions in the Alzheimer disease field, such as identification of novel gamma-secretase modulators for the treatment of Alzheimer disease. The advantage of this technique is that it is a highly efficient assay system, and it can easily identify gamma-secretase modulators.The implication of this technique extend toward therapy of Alzheimer disease through the potential discovery of anti-AD drugs with minimal side effects. This method can provide insight to the substrate selectivity of gamma-secretase. It also can be applied to the studies of other diseases, such as neuroblastoma.Individual new to this method may struggle with careful removal and addition of cell culture media to 96 microplate, as too much disturbance may complicate the final results. Begin this procedure with preparation of NG and CG cells, as referenced in the text protocol. Count NG or CG cells with a hemocytometer.Then, seed the cells at 20, 000 cells per well onto 96-well microplates at a final volume of 200 microliters per well in growth medium. Transfer the microplates to a humidified carbon dioxide incubator, and incubate at 37 degrees Celsius overnight. Following overnight incubation, remove 100 microliters of growth medium from each well.Add 100 microliters per well of growth medium containing two micrograms per milliliter of tetracycline with either 0.2%dimethyl sulfoxide, two micromolar CL-387, 785, two micromolar DAPT, or other related ErbB1/ErbB2 inhibitors. Incubate the treated cells and microplates at 37 degrees Celsius for 24 hours. Following incubation, remove 150 microliters per well of growth medium from the microplates.Then, add 50 microliters per well of luciferase assay reagent. Next, transfer the microplates to a luminescence microplate reader, and maintain them at room temperature for five minutes. Determine the firefly luciferase-emitted luminescence using a predefined program stored in the luminescence microplate reader.First, open the instrument control software. Then, from the Tool menu, select Luminometry. Under the Parameters setting, choose No Filter for emission filter, tick Normal for emission aperture, enter one for counting time, and click on OK to set up the luminescence reading.Under the Instrument control tab, scroll through the list of active protocols, and select the pre-stored protocol for reading luminescence. Under the Live display tab, define the scale from the pull-down menu, and select Logarithm or Linear for the type. Under the Temperature tab, select Off for plate heating.Finally, click the Start button to perform the luminescence reading. Export the results into a spreadsheet format using the Explorer embedded within the control software. Estimate background luminescence from CG or NG cells treated with growth medium that does not include tetracycline.Define a luminescence signal emitted by CG or NG cells treated with 0.1%DMSO in growth medium containing one microgram per milliliter tetracycline alone as 100%relative gamma-secretase activity. Finally, normalize luciferase signals from CG or NG cells that are cultured with growth medium containing tetracycline and either CL-387, 785 or DAPT to the luciferase signal emitted by DMSO-treated CG or NG cells. Shown here, are representative results demonstrating the quantitative measurements of gamma-secretase cleavage of APP-C99 in the CG cells.Cells were treated with one micromolar of CL-387, 785 or DAPT in the presence of one microgram per milliliter tetracycline at 37 degrees Celsius for 24 hours. The luminescence derived from gamma-secretase-mediated proteolysis of C99-GV was quantified after the addition of 100 microliters per well of luciferase assay reagent. The luciferase signal emitted by cells treated with vehicle alone was set as 100%relative luminescence.Representative results demonstrating the quantitative measurements of gamma-secretase cleavage of N-delta-E in the NG cells are shown here. Similarly, cells were treated with one micromolar of CL-387, 785 or DAPT in the presence of one microgram per milliliter tetracycline at 37 degrees Celsius for 24 hours. The luminescence derived from gamma-secretase-mediated proteolysis of N-delta-E-GV was quantified after the addition of 100 microliters per well of luciferase assay reagent.The luciferase signal emitted by cells treated with vehicle alone was set as 100%relative luminescence. Once mastered, this technique only requires a hands-on time of one to two hours, pending the number of compounds to exam. After its development, this technique paved the way for the researcher in the field of Alzheimer disease to explore genetic and chemical modifier of gamma-secretase in pursuit of novel therapeutics for Alzheimer disease.

quantitative-measurement-secretase-mediated-amyloid-precursor-protein

Research

JoVE Journal

Neuroscience

Selection of Aptamers for Amyloid &beta;-Protein, the Causative Agent of Alzheimer&#39;s Disease

Alzheimer's disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis difficult1. Definite AD diagnosis is achieved only postmortem, thus establishing presymptomatic, early diagnosis of AD is crucial for developing and administering effective therapies2,3.
Amyloid &beta;-protein (A&beta;) is central to AD pathogenesis. Soluble, oligomeric A&beta; assemblies are believed to affect neurotoxicity underlying synaptic dysfunction and neuron loss in AD4,5. Various forms of soluble A&beta; assemblies have been described, however, their interrelationships and relevance to AD etiology and pathogenesis are complex and not well understood6. Specific molecular recognition tools may unravel the relationships amongst A&beta; assemblies and facilitate detection and characterization of these assemblies early in the disease course before symptoms emerge. Molecular recognition commonly relies on antibodies. However, an alternative class of molecular recognition tools, aptamers, offers important advantages relative to antibodies7,8. Aptamers are oligonucleotides generated by in-vitro selection: systematic evolution of ligands by exponential enrichment (SELEX)9,10. SELEX is an iterative process that, similar to Darwinian evolution, allows selection, amplification, enrichment, and perpetuation of a property, e.g., avid, specific, ligand binding (aptamers) or catalytic activity (ribozymes and DNAzymes).
Despite emergence of aptamers as tools in modern biotechnology and medicine11, they have been underutilized in the amyloid field. Few RNA or ssDNA aptamers have been selected against various forms of prion proteins (PrP)12-16. An RNA aptamer generated against recombinant bovine PrP was shown to recognize bovine PrP-&beta;17, a soluble, oligomeric, &beta;-sheet-rich conformational variant of full-length PrP that forms amyloid fibrils18. Aptamers generated using monomeric and several forms of fibrillar &beta;2-microglobulin (&beta;2m) were found to bind fibrils of certain other amyloidogenic proteins besides &beta;2m fibrils19. Ylera et al. described RNA aptamers selected against immobilized monomeric A&beta;4020. Unexpectedly, these aptamers bound fibrillar A&beta;40. Altogether, these data raise several important questions. Why did aptamers selected against monomeric proteins recognize their polymeric forms? Could aptamers against monomeric and/or oligomeric forms of amyloidogenic proteins be obtained? To address these questions, we attempted to select aptamers for covalently-stabilized oligomeric A&beta;4021 generated using photo-induced cross-linking of unmodified proteins (PICUP)22,23. Similar to previous findings17,19,20, these aptamers reacted with fibrils of A&beta; and several other amyloidogenic proteins likely recognizing a potentially common amyloid structural aptatope21. Here, we present the SELEX methodology used in production of these aptamers21.

Selection of Aptamers for Amyloid &amp;#946;-Protein, the Causative Agent of Alzheimer&#039;s Disease

Aptamers are short ribo-/deoxyribo-oligonucleotides selected by in-vitro evolution methods based on affinity for a specific target. Aptamers are molecular recognition tools with versatile therapeutic, diagnostic, and research applications. We demonstrate methods for selection of aptamers for amyloid &beta;-protein, the causative agent of Alzheimer's disease.

Alzheimer's disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field

The discovery of neural stem and progenitor cells (collectively termed neural precursor cells) (NPCs) in the adult mammalian brain has led to a body of research aimed at utilizing the multipotent and proliferative properties of these cells for the development of neuroregenerative strategies. A critical step for the success of such strategies is the mobilization of NPCs toward a lesion site following exogenous transplantation or to enhance the response of the endogenous precursors that are found in the periventricular region of the CNS. Accordingly, it is essential to understand the mechanisms that promote, guide, and enhance NPC migration. Our work focuses on the utilization of direct current electric fields (dcEFs) to promote and direct NPC migration - a phenomenon known as galvanotaxis. Endogenous physiological electric fields function as critical cues for cell migration during normal development and wound repair. Pharmacological disruption of the trans-neural tube potential in axolotl embryos causes severe developmental malformations1. In the context of wound healing, the rate of repair of wounded cornea is directly correlated with the magnitude of the epithelial wound potential that arises after injury, as shown by pharmacological enhancement or disruption of this dcEF2-3. We have demonstrated that adult subependymal NPCs undergo rapid and directed cathodal migration in vitro when exposed to an externally applied dcEF. In this protocol we describe our lab's techniques for creating a simple and effective galvanotaxis assay for high-resolution, long-term observation of directed cell body translocation (migration) on a single-cell level. This assay would be suitable for investigating the mechanisms that regulate dcEF transduction into cellular motility through the use of transgenic or knockout mice, short interfering RNA, or specific receptor agonists/antagonists.

In this protocol we demonstrate how to construct custom chambers that permit the application of a direct current electric field to enable time-lapse imaging of adult brain derived neural precursor cell translocation during galvanotaxis.

The discovery of neural stem and progenitor  cells (collectively termed neural precursor cells) (NPCs) in the adult  mammalian brain has led to a body of ...

High-throughput Analysis of Mammalian Olfactory Receptors: Measurement of Receptor Activation via Luciferase Activity

Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors to recognize thousands of odorants. Odorant ligands have been published for fewer than 6% of human receptors1-11. This lack of data is due in part to difficulties functionally expressing these receptors in heterologous systems. Here, we describe a method for expressing the majority of the olfactory receptor family in Hana3A cells, followed by high-throughput assessment of olfactory receptor activation using a luciferase reporter assay. This assay can be used to (1) screen panels of odorants against panels of olfactory receptors; (2) confirm odorant/receptor interaction via dose response curves; and (3) compare receptor activation levels among receptor variants. In our sample data, 328 olfactory receptors were screened against 26 odorants. Odorant/receptor pairs with varying response scores were selected and tested in dose response. These data indicate that a screen is an effective method to enrich for odorant/receptor pairs that will pass a dose response experiment, i.e. receptors that have a bona fide response to an odorant. Therefore, this high-throughput luciferase assay is an effective method to characterize olfactory receptors&#8212;an essential step toward a model of odor coding in the mammalian olfactory system.

Olfactory receptor activation patterns encode odor identity, but the lack of published data identifying odorant ligands for mammalian olfactory receptors hinders the development of a comprehensive model of odor coding. This protocol describes a method to facilitate high-throughput identification of olfactory receptor ligands and quantification of receptor activation.

Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors ...

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval neuromuscular junction (NMJ) in Drosophila. Previous studies have shown that the postsynaptic distribution of Dlg at the larval NMJ overlaps with that of Hu-li tai shao (Hts), a homologue to the mammalian adducins. In addition, Dlg and Hts are observed to form a complex with each other based on co-immunoprecipitation experiments involving whole adult fly lysates. Due to the nature of these experiments, however, it was unknown whether this complex exists specifically at the NMJ during larval development.
Proximity Ligation Assay (PLA) is a recently developed technique used mostly in cell and tissue culture that can detect protein-protein interactions in situ. In this assay, samples are incubated with primary antibodies against the two proteins of interest using standard immunohistochemical procedures. The primary antibodies are then detected with a specially designed pair of oligonucleotide-conjugated secondary antibodies, termed PLA probes, which can be used to generate a signal only when the two probes have bound in close proximity to each other. Thus, proteins that are in a complex can be visualized. Here, it is demonstrated how PLA can be used to detect in situ protein-protein interactions at the Drosophila larval NMJ. The technique is performed on larval body wall muscle preparations to show that a complex between Dlg and Hts does indeed exist at the postsynaptic region of NMJs.

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

This protocol demonstrates how Proximity Ligation Assay can be used to detect in situ protein-protein interactions at the Drosophila larval neuromuscular junction. With this technique, Discs large and Hu-li tai shao are shown to form a complex at the postsynaptic region, an association previously identified through co-immunoprecipitation.

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Quantitative Measurement of Relative Retinoic Acid Levels in E8.5 Embryos and Neurosphere Cultures Using the F9 RARE-Lacz Cell-based Reporter Assay

Retinoic acid (RA) is an important developmental morphogen that coordinates anteroposterior and dorsoventral axis patterning, somitic differentiation, neurogenesis, patterning of the hindbrain and spinal cord, and the development of multiple organ systems. Due to its chemical nature as a small amphipathic lipid, direct detection and visualization of RA histologically remains technically impossible. Currently, methods used to infer the presence and localization of RA make use of reporter systems that detect the biological activity of RA. Most established reporter systems, both transgenic mice and cell lines, make use of the highly potent RA response element (RARE) upstream of the RAR-beta gene to drive RA-inducible expression of reporter genes, such as beta-galactosidase or luciferase. The transgenic RARE-LacZ mouse is useful in visualizing spatiotemporal changes in RA signaling especially during embryonic development. However, it does not directly measure overall RA levels. As a reporter system, the F9 RARE-LacZ cell line can be used in a variety of ways, from simple detection of RA to quantitative measurements of RA levels in tissue explants. Here we describe the quantitative determination of relative RA levels generated in embryos and neurosphere cultures using the F9 RARE-LacZ reporter cell line.

Methods to accurately measure retinoic acid (RA) levels in small amounts of tissue do not exist. This protocol describes the easy, quantitative measurement of relative RA levels in E8.5 embryos and neurospheres using an RA reporter cell line.

Retinoic acid (RA) is an important developmental morphogen that coordinates anteroposterior and dorsoventral axis patterning, somitic differentiation, ...

Modeling Amyloid-&#946;42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to synaptic degeneration, inflammation, neuronal death, and learning deficits. Humans cannot regenerate lost neurons in the case of AD in part due to impaired proliferative capacity of the neural stem/progenitor cells (NSPCs) and reduced neurogenesis. Therefore, efficient regenerative therapies should also enhance the proliferation and neurogenic capacity of NSPCs. Zebrafish (Danio rerio) is a regenerative organism, and we can learn the basic molecular programs with which we could design therapeutic approaches to tackle AD. For this reason, the generation of an AD-like model in zebrafish was necessary. Using our methodology, we can introduce synthetic derivatives of A&#946;42 peptide with tissue penetrating capability into the adult zebrafish brain, and analyze the disease pathology and the regenerative response. The advantage over the existing methods or animal models is that zebrafish can teach us how a vertebrate brain can naturally regenerate, and thus help us to treat human neurodegenerative diseases better by targeting endogenous NSPCs. Therefore, the amyloid-toxicity model established in the adult zebrafish brain may open new avenues for research in the field of neuroscience and clinical medicine. Additionally, the simple execution of this method allows for cost-effective and efficient experimental assessment. This manuscript describes the synthesis and injection of A&#946;42 peptides into zebrafish brain.

Modeling Amyloid-&amp;#946;42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

This protocol describes the synthesis, characterization, and injection of monomeric amyloid-&#946;42 peptides for generating amyloid toxicity in adult zebrafish to establish an Alzheimer&#39;s disease model, followed by histological analyses and detection of aggregations.

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (SOD1). The structural instability of SOD1 and the detection of SOD1-positive inclusions in familial-ALS patients supports a potential causal role for misfolded and/or aggregated SOD1 in ALS pathology. In this study, we describe the development of a cell-based assay designed to quantify the dynamics of SOD1 aggregation in living cells by high content screening approaches. Using lentiviral vectors, we generated stable cell lines expressing wild-type and mutant A4V SOD1 tagged with yellow fluorescent protein and found that both proteins were expressed in the cytosol without any sign of aggregation. Interestingly, only SOD1 A4V stably expressed in HEK-293, but not in U2OS or SH-SY5Y cell lines, formed aggregates upon proteasome inhibitor treatment. We show that it is possible to quantify aggregation based on dose-response analysis of various proteasome inhibitors, and to track aggregate-formation kinetics by time-lapse microscopy. Our approach introduces the possibility of quantifying the effect of ALS mutations on the role of SOD1 in aggregate formation as well as screening for small molecules that prevent SOD1 A4V aggregation.

We describe a method to quantify the aggregation of misfolded proteins. Our protocol details lentiviral induced stable cell line generation, automated confocal imaging, and image analysis of protein aggregates. As an illustrative application, we studied the effect of small molecules in promoting SOD1 aggregation in a time- and dose-dependent manner.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

Quantitative Autoradiographic Method for Determination of Regional Rates of Cerebral Protein Synthesis In Vivo

Protein synthesis is required for development and maintenance of neuronal function and is involved in adaptive changes in the nervous system. Moreover, it is thought that dysregulation of protein synthesis in the nervous system may be a core phenotype in some developmental disorders. Accurate measurement of rates of cerebral protein synthesis in animal models is important for understanding these disorders. The method that we have developed was designed to be applied to the study of awake, behaving animals. It is a quantitative autoradiographic method, so it can yield rates in all regions of the brain simultaneously. The method is based on the use of a tracer amino acid, L-[1-14C]-leucine, and a kinetic model of the behavior of L-leucine in the brain. We chose L-[1-14C]-leucine as the tracer because it does not lead to extraneous labeled metabolic products. It is either incorporated into protein or rapidly metabolized to yield 14CO2 which is diluted in a large pool of unlabeled CO2 in the brain. The method and the model also allow for the contribution of unlabeled leucine derived from tissue proteolysis to the tissue precursor pool for protein synthesis. The method has the spatial resolution to determine protein synthesis rates in cell and neuropil layers, as well as hypothalamic and cranial nerve nuclei. To obtain reliable and reproducible quantitative data, it is important to adhere to procedural details. Here we present the detailed procedures of the quantitative autoradiographic L-[1-14C]-leucine method for the determination of regional rates of protein synthesis in vivo.

Protein synthesis is&#160;a critical biological process for cells. In brain, it is required for adaptive changes. Measurement of rates of protein synthesis in the intact brain requires careful methodological considerations. Here we present the L-[1-14C]-leucine quantitative autoradiographic method for determination of regional rates of cerebral protein synthesis in vivo.

Protein synthesis is required for development and maintenance of neuronal function and is involved in adaptive changes in the nervous system. Moreover, it ...