Name: monitoring cell-to-cell transmission of prion-like protein aggregates in drosophila melanogaster
Uploaded: 2018-03-12T14:45:00
Description: Watch this Scientific Journal Video about Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster at JoVE.com

We thank members of the Kopito, Luo, and Pearce labs for many helpful discussions during development of these methods. We also thank Brian Temsamrit for critical reading of this manuscript. This work was supported by funds from University of the Sciences and the W.W. Smith Charitable Trusts.

1. Coupling Gal4- and QF-mediated Htt Transgene Expression in Drosophila
<ol>
	<li>Collect and/or generate transgenic Drosophila melanogaster lines containing tissue-specific Gal4 or QF &#34;drivers,&#34; as well as lines containing wild-type or mutant Htt transgenes downstream of the Gal4-UAS38 or QF-QUAS39. Ensure that the proteins that are expressed from these transgenes are fused to fluorescent proteins or epitope-tagged to allow for differentiation ...

     

As the numbers of patients suffering from neurodegenerative diseases continues to increase, there is an urgent need to increase the understanding of the molecular pathogenesis of these diseases so that better therapies can be developed. Here, we describe methods that allow for monitoring prion-like transmission of pathogenic protein aggregates between different cell types in the model organism, Drosophila melanogaster. We have recently used this methodology to demonstrate prion-like transmission of mutant Htt ag...

The prion hypothesis states that the infectious agent responsible for the transmissible spongiform encephalopathies (e.g., Creutzfeldt-Jakob disease in humans, scrapie in sheep, chronic wasting disease in deer and elk, and &#34;mad cow disease&#34; in cattle) is solely composed of protein and devoid of nucleic acids1. In prion diseases, the cellular prion protein (PrPC) assumes a non-native, stable fold (PrPSc) that is highly beta sheet-rich and can self-propagate by converting and recruiting monomeric PrPC molecules into stable amyloid aggregates. PrPSc aggregates use this self-replicating mechanism to spread between different cells in an organism and even between individual organisms2.
Protein misfolding and aggregation is also a central feature of most neurodegenerative diseases (Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), Huntington&#39;s disease (HD), and amyotrophic lateral sclerosis (ALS))3. Formation of intra- or extra-cellular aggregated protein assemblies in these diseases is closely associated with cytotoxicity4 and progresses along highly reproducible and disease-specific paths through the brain over time5,6. These patterns of spread suggest that pathogenic aggregates associated with these disorders have prion-like properties. Strong support now exists for prion-like transmission of aggregates associated with AD, PD, HD, and ALS - they spread from cell-to-cell and template the conformational change of monomeric forms of the same protein in previously unaffected cells7,8.
The majority of studies investigating prion-like spread of protein aggregates to date have been performed using mammalian cell culture models, where aggregates transfer into the cytoplasm of na&#239;ve cells from the extracellular space or from another cell&#39;s cytoplasm9,10,11,12,13,14,15, or by injecting aggregate-containing material into mouse brains and monitoring aggregate appearance outside of the injection site16,17,18,19,20,21,22,23. More recently, transgenic animals have been used to demonstrate that intracellular aggregates spread to other cells within intact brains24,25,26,27,28,29,30. Here, we describe a method for direct visualization of aggregate transfer between individual cells in the intact brain of Drosophila melanogaster. Drosophila models of HD/polyglutamine (polyQ) diseases were first developed nearly two decades ago31,32 and have provided many invaluable insights into the pathogenic mechanisms that underlie these disorders33. HD is an inherited neurodegenerative disorder caused by an autosomal dominant mutation in the gene that codes for the protein huntingtin (Htt)34. This mutation results in expansion of a polyQ stretch near Htt&#39;s N-terminus beyond a pathogenic threshold of ~ 37 glutamines, causing the protein to misfold and aggregate35,36. Wild-type Htt proteins containing &#60;37 glutamines in this stretch achieve their native fold, but can be induced to aggregate upon direct physical contact with a Htt aggregate &#34;seed&#34;12,27,37. We exploit this homotypic, nucleated aggregation of wild-type Htt as a readout for prion-like transfer and cytoplasmic entry of mutant Htt aggregates originating in other cells.
Determining the mechanisms by which prion-like aggregates travel between cells can lead to the identification of novel therapeutic targets for incurable neurodegenerative diseases. We take advantage of the rapid life cycle, ease of use, and genetic tractability of Drosophila melanogaster to define molecular mechanisms for cell-to-cell spread of mutant Htt aggregates. Our experimental strategy employs two binary expression systems available in Drosophila, the well-established Gal4-specific upstream activating sequence (Gal4-UAS) system38 and the recently-developed QF-QUAS system39. Coupling these two independent systems allows restricting expression of mutant and wild-type Htt transgenes to distinct cell populations within the same fly40. Using this approach, we examine prion-like spreading of mutant Htt by monitoring the redistribution of cytoplasmic wild-type Htt from its normally diffuse, soluble state to an aggregated state, a direct consequence of physical contact with a pre-formed mutant Htt aggregate &#34;seed.&#34; Conversion of wild-type Htt by mutant Htt can be confirmed using biochemical or biophysical techniques that report protein-protein interactions, such as fluorescence resonance energy transfer (FRET)9,27,41.
Importantly, we can also access a large number of genetic tools in Drosophila to identify genes and/or pathways that mediate prion-like spread of protein aggregates. We have recently used this approach to unveil a key role for the cell surface scavenger receptor, Draper42,43, in transferring mutant Htt aggregates from neuronal axons to nearby phagocytic glia in the Drosophila central nervous system (CNS)27. Thus, the genetic- and imaging-based approach that we describe here can reveal important basic biological information about a disease-relevant phenomenon in the simple-to-use but powerful model organism, Drosophila.

<table border="0" cellpadding="0" cellspacing="0" style="width:849px;" width="849">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Phosphate buffered saline (PBS), 10X, pH 7.4</td>
			<td style="width:123px;">ThermoFisher Scientific</td>
			<td style="width:344px;">AM9625</td>
			<td style="width:167px;">Dilute to 1X</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Triton X-100</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:344px;">T9284-1L</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Kimwipes</td>
			<td style="width:123px;">Thomas Scientific</td>
			<td style="width:344px;">2904F24</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">20% paraformaldehyde (PFA)</td>
			<td style="width:123px;">Electron Microscopy Sciences</td>
			<td style="width:344px;">15713-S</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Normal Goat Serum (NGS), filtered</td>
			<td style="width:123px;">Lampire Biological Laboratories</td>
			<td style="width:344px;">7332500</td>
			<td style="width:167px;">Aliquot and freeze upon receipt</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Chicken anti-GFP</td>
			<td style="width:123px;">Aves Labs</td>
			<td style="width:344px;">GFP-1020</td>
			<td style="width:167px;">Use at 1:500 dilution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Rabbit anti-DsRed</td>
			<td style="width:123px;">Clontech</td>
			<td style="width:344px;">632496</td>
			<td style="width:167px;">Use at 1:2000 dilution; can recognize DsRed-based fluorescent proteins (e.g. mCherry, mStrawberry, tdTomato, etc.)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Mouse anti-Bruchpilot</td>
			<td style="width:123px;">Developmental Studies Hybridoma Bank</td>
			<td style="width:344px;">nc82</td>
			<td style="width:167px;">Use at 1:100 dilution; will label active pre-synaptic structures thoughout the fly brain</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">FITC anti-chicken</td>
			<td style="width:123px;">ThermoFisher Scientific</td>
			<td style="width:344px;">SA1-7200</td>
			<td style="width:167px;">Use at 1:250 dilution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Alexa Fluor 568 anti-rabbit</td>
			<td style="width:123px;">Life Technologies</td>
			<td style="width:344px;">A11011</td>
			<td style="width:167px;">Use at 1:250 dilution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Alexa Fluor 647 anti-mouse antibody</td>
			<td style="width:123px;">Life Technologies</td>
			<td style="width:344px;">A21235</td>
			<td style="width:167px;">Use at 1:250 dilution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Slowfade Gold Antifade Reagent</td>
			<td style="width:123px;">Life Technologies</td>
			<td style="width:344px;">S36936</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Microscope Slides (25 x 75 x 1.0 mm)</td>
			<td style="width:123px;">Fisher Scientific</td>
			<td style="width:344px;">12-550-143</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Cover Glass (22 x 22 mm)</td>
			<td style="width:123px;">Globe Scientific</td>
			<td style="width:344px;">1404-15</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Dumont Biology Grade Forceps, Style 3</td>
			<td style="width:123px;">Ted Pella</td>
			<td style="width:344px;">503</td>
			<td style="width:167px;">use in non-dominant hand</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Dumont Biology Grade Forceps, Style 5</td>
			<td style="width:123px;">Ted Pella</td>
			<td style="width:344px;">505</td>
			<td style="width:167px;">use in dominant hand</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">LAS X image analysis software</td>
			<td style="width:123px;">Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Imaris image analysis software</td>
			<td style="width:123px;">Bitplane</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

monitoring cell-to-cell transmission of prion-like protein aggregates in drosophila melanogaster

Protein aggregation is a central feature of most neurodegenerative diseases, including Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), Huntington&#39;s disease (HD), and amyotrophic lateral sclerosis (ALS). Protein aggregates are closely associated with neuropathology in these diseases, although the exact mechanism by which aberrant protein aggregation disrupts normal cellular homeostasis is not known. Emerging data provide strong support for the hypothesis that pathogenic aggregates in AD, PD, HD, and ALS have many similarities to prions, which are protein-only infectious agents responsible for the transmissible spongiform encephalopathies. Prions self-replicate by templating the conversion of natively-folded versions of the same protein, causing spread of the aggregation phenotype. How prions and prion-like proteins in AD, PD, HD, and ALS move from one cell to another is currently an area of intense investigation. Here, a Drosophila melanogaster model that permits monitoring of prion-like, cell-to-cell transmission of mutant huntingtin (Htt) aggregates associated with HD is described. This model takes advantage of powerful tools for manipulating transgene expression in many different Drosophila tissues and utilizes a fluorescently-tagged cytoplasmic protein to directly report prion-like transfer of mutant Htt aggregates. Importantly, the approach we describe here can be used to identify novel genes and pathways that mediate spreading of protein aggregates between diverse cell types in vivo. Information gained from these studies will expand the limited understanding of the pathogenic mechanisms that underlie neurodegenerative diseases and reveal new opportunities for therapeutic intervention.

The methods described here produce robust data demonstrating prion-like transfer of Htt protein aggregates from one cell population to another in the intact fly CNS. Conversion of wild-type Htt from diffuse to punctate is observed by direct fluorescence of this YFP fusion protein in recipient glia as a result of HttQ91-mCherry expression in donor ORNs (Figure 2A-C and Figure 4A, B). Accurate repo...

Watch this Scientific Journal Video about Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster at JoVE.com

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Accumulating evidence supports the idea that pathogenic protein aggregates associated with neurodegenerative diseases spread between cells with prion-like properties. Here, we describe a method that enables visualization of cell-to-cell spreading of prion-like aggregates in the model organism, Drosophila melanogaster.

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Protein aggregation is a central feature of most neurodegenerative diseases, including Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), ...

The overall goal of this procedure is to monitor prion-like spreading of protein aggregates in vivo. Using transgenic flies that express an aggregation prone protein in a soluble version of the same protein in isolated cell populations. This method can help answer key questions in a cell biology in neurodegeneration fields, such as how protein aggregate mythology or other material transfers from cell to cell in the brain.The main advantage of this technique is that it allows for imaging of protein aggregate spreading from one cell site opossum to another in an intact central nervous system. The implications of this technique extend for therapy of Huntington's disease and other prion-like neurodegenerative diseases. Because aggregates spreading appears to be a common mechanism underlying the progression of these fatal disorders.Using standard culturing conditions, generate transgenic drosophila melanogaster lines containing tissue specific Gal4 or QF drivers. As well as lines containing wild-type or mutant huntingtin trans genes downstream of the Gal4-UAS or QF-QUAS. Collect the progeny of the desire genotype and aged them until they are ready to dissect.To prepare for the brain dissection, collect the solutions needed and chill them on ice, along with the glass multi-well dish, pipette tips, and one pair of forceps. Next, anesthetized the adult flies using carbon dioxide and transfer them into a well of the glass dish. Then, add about a half of a milliliter of PBST to the well with the flies and to an adjacent well.Next, under a dissecting microscope, focus on a single fly and illuminate it from both sides with gooseneck lights. Then position a folded tissue under the dish for cleaning the dissection tools. Now, using number three forceps in the non-dominant hand, transfer a single fly to the well containing PBST.Immobilize the fly by grabbing hold of its abdomen with the ventral side facing up and fully submerge the fly. Now, insert one tine of a number five forceps under the cuticle in the small space adjacent to the proboscis. Then secure the grip on the head by pinching the eye from both sides and pull the forceps apart to detach the head.Dispose of the fly body on the folded tissue while maintaining the head submerge in the PBST. Next, insert a tine of the number three forceps into the same space on the other side of the proboscis and gently pull apart the head cuticle to expose the brain. Before proceeding, discard the cuticle residue on a lab tissue.While removing cuticle, avoid directly contacting the brain so as not to damage it with a forceps. Now, remove the dissected brain from the PBST, either by grabbing a hold of an attached trachea or by aspirating it into the space between the forceps tips using capillary action. Then, transfer the fly brain into a microcentrifuge tube containing fixative solution on ice.Observe carefully to watch for the brain releasing from the forceps. Once all of the brains have been dissected, transfer the collection tube to a nutator at room temperature and wrap them for about five minutes. After the fixation period, remove the majority of the fixative solution using a p1000 pipette and discard it, being very careful to leave the brains in the tubes.This requires gentle suction and careful observation. Next, add one milliliter of fresh PBST to the brains. Cover up the tube and let it rock on the nutator for about a minute to wash off the remaining fixative.Then remove the solution and repeat this short wash step. Follow the two short washes with one five minute wash, then three 20 minute washes, and finally a single one hour wash. After the last wash, carefully remove most of the PBST and submerge the brains in 30 microliters of glycerol based antifade reagent.Then incubate the brains at 4 degree Celsius in the dark without movement for one to 24 hours. Later, remove the brains from the collection tube using a blunted pipette tip and transfer them to a microscope slide. Then gently orient the brains as needed for imaging using forceps.Multiple samples can be mounted on the same slide in separate rows. Next, remove the excess antifade reagent from the slide using the corner of a folded lab tissue. Do not let the tissue come into contact with the brains.Then leave the samples in the dark for five to 10 minutes to let the brains adhere to the slide. Next, take small pieces of broken cover glass and position them around the brains covering an area that is about 19 square millimeters, then gently lower a 22 square millimeter cover glass over the mosaic of brains and glass to make a bridge mount. Next, slowly dispense fresh antifade reagent under the cover slip so it fills in the empty spaces.Do this very carefully so that the brains and glass stay in place. Then seal the cover slip with nail polish. First, just apply it at the corners.Let the corner dabs dry five to 10 minutes before completing the seal along the edges. The brain should be imaged as soon as possible. Image the mounted brains using a confocal microscope equipped with a 40X or 63X oil objective to collect Z slices in the region of the brain where the trans genes are expressed.Then analyze the data by quantifying the puncta in the individual Z slices or alternatively after rendering the slices in three dimensions. To quantify mutant huntingtin aggregates which are well separated and have little background signal, open the confocal Z series in the 3D viewing mode. Then, use the analysis wizard to identify individual spots in a selective channel.In the settings, adjust the thresholding and filters to accurately represent all heterogeneously sized aggregates as individual objects in the image. Then, enable split objects under binary processing pre-filter to separate closely associated aggregates. Quantitative information about the objects identified by the software is reported under measurements.After counting the puncta, further characterize them in image analysis software. For example, take relevant measurements of the spots or surfaces to obtain aggregate diameter, volume or intensity information. Some wild-type huntingtin aggregates can be quantified by manually moving through the Z stack and counting green puncta that are distinguishable from the surrounding diffuse signal.Be careful to avoid counting single aggregates twice when they appear in more than one slice. Another analysis of interest is determining the frequency of co localization between huntingtin Q25-YFP and huntingtin Q91-mCherry aggregates. Do this using manual counting by moving slice by slice through a confocal Z stack.Careful selection of the genetic drivers and fluorescent protein trans gene fusions allows for clear detection of mutant and wild-type huntingtin proteins in non-overlapping cell populations. Conversion of wild-type huntingtin from diffused to punctate is observed by direct fluorescence of the YFP fusion protein in recipient glia as a result of the huntingtin Q91-mCherry expression in donor olfactory receptor neurons. Careful selection of the genetic drivers and fluorescent protein trans gene fusions allows for clear detection of mutant and wild-type huntingtin proteins in non-overlapping cell populations.Mutant and wild-type huntingtin aggregates can be quantified as punctate objects, either manually or by using image analysis software. The aggregates can be measured and characterized further as populations and for their frequency of co localization. Co localized puncta can be analyzed further by fluorescence energy resonance transfer or FRET to demonstrate close proximity of the fluorescent protein fusions.Once mastered, brain dissection and imaging can be done from start to finish in less than eight hours, if it is performed properly. While attempting this procedure, it's important to remember to carefully distinguish punctate aggregated objects from other fluorescent structures in the same tissue using appropriate controls. This procedure can be modified to include other methods such as gene knockdown in order to determine the molecular mechanisms for aggregate transfer between different cell types.Thanks and best of luck with your experiments.

Research

JoVE Journal

Neuroscience

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

A  growing number of genetically encoded tools are becoming available that allow non-invasive  manipulation of the neural activity of specific neurons in ...

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such ...

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by ...

Conditions Affecting Social Space in Drosophila melanogaster

Conditions Affecting Social Space in Drosophila melanogaster

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a ...

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense ...

Foraging Path-length Protocol for Drosophila melanogaster Larvae

Foraging Path-length Protocol for Drosophila melanogaster Larvae

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral ...

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of ...

Measuring and Altering Mating Drive in Male Drosophila melanogaster

Measuring and Altering Mating Drive in Male Drosophila melanogaster

Despite decades of investigation, the neuronal and molecular bases of motivational states remain mysterious. We have recently developed a novel, ...

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs ...