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.

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			<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>
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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), ...

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