We are particularly grateful to Dr. Barry Ganetzky, in who&#39;s lab the genetic screen was performed, allowing the identification and characterization of brat as a neuroprotective gene. We thank Dr. Steven Robinow for kindly providing the collection of ENU-mutagenized flies used in the genetic screen presented in this article. We thank the members of Ganetzky lab, Drs. Grace Boekhoff-Falk and David Wassarman for helpful discussions throughout the duration of this project, Ling Ling Ho and Bob Kreber for technical assistance, Dr. Aki Ikeda for the use of his microtome facility at the University of Wisconsin and Dr. Kim Lackey and the Optical Analysis Facility at the University of Alabama.

<ol>
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The authors declare no conflicts of interest.

Forward genetic screens in Drosophila have been an effective approach to identify genes involved in different biological processes, including age-dependent neuroprotection5,23,24,25. Using this strategy, we were successful in identifying brat as a novel neuroprotective gene17.
One critical step in this protocol involves the proper orientation of heads (as described in Section 4.4.3.) for histology analysis. Additionally, markers of the line used for the meiotic mapping should not interfere with the phenotype of the new mutation (in this study: neurodegeneration). We recommended an initial test to confirm that the chosen markers do not cause neurodegeneration of their own. For instance, Jammed (J) causes wing blisters that could possibly interfere with climbing as is the case for amyloid precursor protein (APP)-overexpressing flies that also have blistered wings26. Lobe (L) leads to reduction in eye size; however, we do not observe central brain degeneration and this marker can be used to map central brain neurodegeneration. Pin and Sternoplural (Sp) are also appropriate to use, as brains of flies carrying these markers are comparable to brains of wild type controls.
One disadvantage of the forward genetic methodology in flies is the length of time required to narrow regions of DNA to the gene of interest3,5. The use of improved mapping strategies27 along with the availability of next generation sequencing technologies 28 should allow even easier identification of mutations associated with phenotypes of interest. Forward genetic screens can be labor-intensive. For instance, histology confirmation, which assays directly for neurodegeneration in the brain, alone requires a considerable amount of time. However, this approach will be much more difficult and expensive to perform in mammalian models, not necessarily allowing extensive genetic studies. Chemical mutagenesis screens are advantageous because the identification of point mutations could provide critical information about the function of affected genes&#39; products. The identification of a point mutation causing an amino acid change in a conserved domain of the Brat protein (Figure&#160;4B) illustrates the importance of the coiled-coil domain of this TRIM-NHL protein in neuroprotection18. The climbing assay, which measures locomotor behavior, is certainly advantageous by allowing rapid testing of large numbers of flies for defects in mobility, also often seen in human neurodegeneration25. This assay could also be performed in another screen setting such as genome-wide in vivo RNA interference (RNAi) screen that could be used to identify neuroprotective genes. Although we decreased the distance between the bottom of the test chamber and the passing line from 8 cm10 to 5 cm to set a stricter passing rate in the present study, low climbing rates did not mirror neurodegeneration for large number of lines. Neurodegeneration may become apparent after the onset of behavioral defects, meaning that flies may need to be aged for longer before vacuoles appear. Another possibility is that the locomotor defects we observe in certain lines are not due to defective neurological functioning but rather to muscle weakness or more general unfitness of the flies. Additionally, it is possible that we are missing potential candidates, in which climbing defects do not manifest at younger age (e.g., 10 days old) but rather become prominent later in life. This screening strategy could certainly be applied to identify age-dependent genes, thus eliminating genes associated with potential defects of neural development. The protocol presented here can be adjusted depending on the desired probability of candidate lines to contain the mutated gene involved in neuroprotection. Lowering the passing rate threshold directly is another means of achieving the same result. These candidates would theoretically exhibit greater neurodegeneration, which could save time and resources during confirmation and mapping.
In general, the protocol describes a straightforward way to screen for neurodegeneration and subsequently identify neuroprotective gene candidates. This genetic approach is not limited to the behavior and histological assays described here, and may also be used to screen for other phenotypes like temperature-sensitive (ts) paralysis, egg laying or fertility phenotypes, just to cite a few. For instance, Drosophila ts-paralytic mutants are known to be enriched for neurodegeneration29 and could represent an additional source for the identification of neuroprotective genes.

Neurons are for the most part post-mitotic and incapable of dividing1,2. In most animals, neuroprotective mechanisms exist to maintain these cells throughout the organism&#39;s lifespan, especially at old age when neurons are most vulnerable to damage. Genes underlying these mechanisms can be identified in mutants exhibiting neurodegeneration, a phenotypic indicator for the loss of neuroprotection, using a forward genetic protocol. Forward genetic screens using chemical mutagens such as ethyl methanesulfonate (EMS) or N-ethyl-N-nitrosourea (ENU) are particularly useful due to the random point mutations they induce, resulting in an inherently unbiased approach that has shed light on numerous gene functions in eukaryotic model organisms3,4,5 (in contrast, X-ray mutagenesis creates DNA breaks and can result in rearrangement rather than point mutations6).
The common fruit fly Drosophila melanogaster is an ideal subject for these screens due to its high quality, well annotated genome sequence, its long history as a model organism with highly developed genetic tools, and most significantly, its shared evolutionary history with humans7,8. A limiting factor in the applicability of this protocol is early lethality caused by the mutated genes, which would prevent testing at old age9. However, for non-lethal mutations, a climbing assay, which takes advantage of negative geotaxis, is a simple, although extensive, method of quantifying impaired motor functioning10. To exhibit sufficient locomotor reactivity, flies depend on neural functions to determine direction, sense its position, and coordinate movement. The inability of flies to sufficiently climb in response to stimuli can therefore indicate neurological defects11. Once a particular defective climbing phenotype is identified, further testing using a secondary screen such as histological analysis of brain tissue, can be used to identify neurodegeneration in climbing-defective flies. Subsequent gene mapping can then be used to reveal the genomic region on the chromosome carrying the defective neuroprotective gene of interest. To narrow down the chromosomal region of interest, meiotic mapping using mutant fly lines carrying dominant marker genes with known locations on the chromosome can be performed. The marker genes serve as a reference point for the mutation as the frequency of recombination between two loci provides a measurable distance that can be used to map the approximate location of a gene. Finally, crossing the mutant lines with lines carrying balanced deficiencies on the meiotically mapped chromosomal region of interest creates a complementation test in which the gene of interest can be verified if its known phenotype is expressed5. Polymorphic nucleotide sequences in the identified gene, possibly resulting in an altered amino acid sequences, can be evaluated by sequencing the gene and comparing it to the Drosophila genome sequence. Subsequent characterization of the gene of interest can include testing of additional mutant alleles, mutation rescue experiments and examination of additional phenotypes.

<table><tbody><tr><td>&lt;strong&gt;Major equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Fume hood for histology</td><td></td><td></td><td></td></tr><tr><td>Light Microscope</td><td>Nikon</td><td>Eclipe E100</td><td>Preferred objective for imaging is 20x</td></tr><tr><td>Imaging software</td><td>Nikon</td><td></td><td></td></tr><tr><td>Microscope Camera</td><td>Nikon</td><td></td><td></td></tr><tr><td>Thermal cycler</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Fly pushing and climbing assay&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>VWR&amp;reg; Drosophila Vial, Narrow</td><td>VWR</td><td>75813-160</td><td></td></tr><tr><td>VWR&amp;reg; General-Purpose Laboratory Labeling Tape</td><td>VWR</td><td>89097-912</td><td></td></tr><tr><td>Standard mouse pad</td><td></td><td></td><td></td></tr><tr><td>Stereoscope</td><td>Motic</td><td></td><td>Model SMZ-168</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; anesthesia station (Blowgun, foot valve, Ultimate Flypad)</td><td>Genesee Scientific</td><td>54-104, 59-121, 59-172</td><td>Doesn&amp;rsquo;t iinclude CO2 tank</td></tr><tr><td>Fine-Tip Brushes</td><td>SOLO HORTON BRUSHES, INC.</td><td></td><td></td></tr><tr><td>&lt;em&gt;Drosophila&lt;/em&gt; Incubator</td><td>VWR</td><td>89510-750</td><td></td></tr><tr><td>&lt;strong&gt;Gene mapping&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CantonS</td><td>Bloomington Drosophila Stock Center</td><td>9517</td><td></td></tr><tr><td>w&lt;sup&gt;1118&lt;/sup&gt;</td><td>Bloomington Drosophila Stock Center</td><td>5905</td><td></td></tr><tr><td>yw&amp;nbsp;</td><td>Bloomington Drosophila Stock Center</td><td>6599</td><td></td></tr><tr><td>&lt;em&gt;Drosophila&lt;/em&gt; line used for recombination mapping</td><td>Bloomington Drosophila Stock Center</td><td>3227</td><td>Genotype: wg[Sp-1] J[1] L[2] Pin[1]/CyO, P{ry[+t7.2]=ftz/lacB}E3</td></tr><tr><td>CyO/sno[Sco]&amp;nbsp;</td><td>Bloomington Drosophila Stock Center</td><td>2555</td><td>Drosophila balancer line used for recombination mapping</td></tr><tr><td>Deficiency Kit for chromosome 2L</td><td>Bloomington Drosophila Stock Center</td><td>DK2L</td><td>Cook et al., 2012</td></tr><tr><td>&lt;strong&gt;Histology analysis&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Ethanol, (100%)</td><td>Thermo Fischer Scientific</td><td>A4094</td><td></td></tr><tr><td>Chloroform</td><td>Thermo Fischer Scientific</td><td>C298-500</td><td></td></tr><tr><td>Glacial Acetic Acid</td><td>Thermo Fischer Scientific</td><td>A38-500</td><td></td></tr><tr><td>Fisherbrand&amp;trade;&amp;nbsp;Premium Microcentrifuge Tubes: 1.5 mL</td><td>Thermo Fischer Scientific</td><td>05-408-129</td><td></td></tr><tr><td>Histochoice clearing agent 1x</td><td>VWR Life Sciences</td><td>97060-934</td><td></td></tr><tr><td>Harris Hematoxylin</td><td>VWR</td><td>95057-858</td><td></td></tr><tr><td>Eosin</td><td>VWR</td><td>95057-848</td><td></td></tr><tr><td>Thermo Scientific&amp;trade;&amp;nbsp;Richard-Allan Scientific&amp;trade; Mounting Medium</td><td>Thermo Scientific&amp;trade; 4112</td><td>22-110-610</td><td>CyO/sna[Sco]</td></tr><tr><td>Unifrost Poly-L-Lysine microscope slides, 75 mm x 25 mm x 1 mm, EverMark Select Plus</td><td>Azer Scientific</td><td></td><td></td></tr><tr><td>Fisherbrand&amp;trade;&amp;nbsp;Cover Glasses: Rectangles</td><td>Fisherbrand</td><td>12-545M</td><td>Dimensions: 24 mm x 60 mm</td></tr><tr><td>Traceable timer</td><td>VWR</td><td></td><td></td></tr><tr><td>Slide Warmer</td><td>Barnstead International</td><td></td><td>model no. 26025</td></tr><tr><td>Slide tray and racks</td><td>DWK Life Sciences</td><td></td><td>Rack to hold 20 slides</td></tr><tr><td>Fisherbrand&amp;trade;&amp;nbsp;General-Purpose Extra-Long Forceps</td><td>Fisherbrand</td><td>10-316A</td><td></td></tr><tr><td>Kimwipes&amp;trade;</td><td>Kimberly-Clark&amp;trade; Professional&amp;nbsp;</td><td></td><td></td></tr><tr><td>6 inch Puritan applicators</td><td>Hardwood Products Company, Guilford, Maine</td><td>807-12</td><td></td></tr><tr><td>VWR&amp;reg; Razor Blades</td><td>VWR</td><td>55411-050</td><td></td></tr><tr><td>Tupperware or glass containers for histology liquids</td><td></td><td></td><td>16 + 1 for running water</td></tr><tr><td>High Profile Coated Microtome Blades</td><td>VWR</td><td>95057-834</td><td></td></tr><tr><td>Corning&amp;trade;&amp;nbsp;Round Ice Bucket with Lid, 4 L</td><td>Corning&amp;trade;</td><td></td><td></td></tr><tr><td>Beaker</td><td></td><td></td><td>Or other container for ice water and cassettes</td></tr><tr><td>Tissue Bath</td><td>Precision Scientific Company</td><td>66630</td><td></td></tr><tr><td>Microtome</td><td>Leica Biosystems</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Molecular analysis&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Wizard&amp;reg; SV Gel and PCR Clean-Up System</td><td>Promega</td><td>A9282</td><td></td></tr><tr><td>Ex Taq DNA polymerase</td><td>TaKaRa</td><td></td><td>5 U/&amp;mu;l</td></tr><tr><td>Invitrogen&amp;trade;&amp;nbsp;SYBR&amp;trade; Safe&amp;trade; DNA Gel Stain&amp;nbsp;</td><td>&amp;nbsp;Invitrogen&amp;trade;</td><td></td><td></td></tr><tr><td>UltraPure&amp;trade; Agarose</td><td>&amp;nbsp;Invitrogen&amp;trade;</td><td></td><td></td></tr><tr><td>1 Kb Plus DNA Ladder</td><td>&amp;nbsp;Invitrogen&amp;trade;</td><td></td><td></td></tr><tr><td>ApE-A plasmid Editor software</td><td></td><td></td><td>Available for free download</td></tr><tr><td>&lt;strong&gt;Statistical analysis&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>R software package</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Further analysis&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>y[1] w[*]; wg[Sp-1]/CyO; Dr[1]/TM3, Sb[1]</td><td>Bloomington Drosophila Stock Center</td><td>59967</td><td></td></tr></tbody></table>

in vivo forward genetic screen to identify novel neuroprotective genes in drosophila melanogaster

There is much to understand about the onset and progression of neurodegenerative diseases, including the underlying genes responsible. Forward genetic screening using chemical mutagens is a useful strategy for mapping mutant phenotypes to genes among Drosophila and other model organisms that share conserved cellular pathways with humans. If the mutated gene of interest is not lethal in early developmental stages of flies, a climbing assay can be conducted to screen for phenotypic indicators of decreased brain functioning, such as low climbing rates. Subsequently, secondary histological analysis of brain tissue can be performed in order to verify the neuroprotective function of the gene by scoring neurodegeneration phenotypes. Gene mapping strategies include meiotic and deficiency mapping that rely on these same assays can be followed by DNA sequencing to identify possible nucleotide changes in the gene of interest.

In this aerticle, we present the steps used to identify the gene brain tumor (brat) as playing a role in maintenance of neuronal integrity (e.g., neuroprotection) in adult flies17; a methodology that can be used to identify genes involved in neuroprotection. We used a forward genetic approach (the strategy is outlined in Figure 1A) to screen through a collection of chemically mutagenized flies using a climbing assay (the apparatus used for this assay is shown in Figure 1B). Among 235 homozygous lines, about 37% of tested lines exhibited a climbing pass rate below 50% when tested at the age of 10-12 days (Figure 1C). 58% of tested lines exhibited significantly different climbing behavior when their percent climbing pass rate (CPR) was compared to the mean climbing percent value of all tested lines using one-way ANOVA (Figure 1D). Subsequent histological screen on 51 of the lines exhibiting the lowest climbing pass rate revealed that 29 of these lines showed visible appearance of holes in the brain neuropil (ranging from mild to severe) indicative of neurodegeneration20 (Figure 2). Among the lines showing defective climbing behavior and severe neurodegeneration, we choose to map the mutation underlying the phenotype in line 867 (0% CPR and P value= 1.36e-14 based on one-way ANOVA). The neurodegeneration phenotype in 867 flies is recessive because the brains of heterozygous 867 flies that have been outcrossed to a wild type strain are comparable to these of controls (Figure 3A). Using histology, meiotic mapping situated the mutation in the 31-51 cytological locations, between the phenotypic markers J (Jammed) and L (Lobe) on the second Drosophila chromosome. Using the climbing assay, deficiency mapping between cytological locations 31 and 51 with lines from the Drosophila Deficiency Kit for chromosome 2L (DK2L)16 mapped the mutation in a region encompassing 118 genes (Figure 3B; where 867/+ serves as control for the statistical analysis). Examining the brain phenotype of 867 mutants crossed to additional deficiency lines (Figure 3C) confirmed that the mutation is contained in a region of the genome that includes the gene brat. A complete list of all additional genes contained within these deletions can be found in Flybase by clicking on the link associated with the deleted segment (e.g., for Df(2L)ED1272 the deleted segment is 37C5-38A2). Based on previous observations that brat mutants have supernumerary cells in their brains21, a phenotype also observed in 867 mutants, brat was selected for DNA sequencing analysis. Sanger sequencing of the brat gene containing the exon-coding region identified G/A nucleotide change at position 37,739 of the gene (Figure 4A), leading to glycine to glutamic acid (G/E) change at position 470 of the Brat protein17 (Figure 4B). Rescue experiments confirm that Brat plays a neuroprotective role, as the overexpression of the functional gene in the 867 line suppresses the neurodegeneration phenotype (Figure 5A). Moreover, the phenotype in 867 mutants worsens over time, suggesting that brat plays an age-dependent role in neuroprotection17 (Figure 5B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59720/59720fig1.jpg" /> 
Figure 1: Forward genetic screen to identify novel neuroprotective genes. (A)&#160;Screen strategy. (B)&#160;Climbing assay testing apparatus. (C)&#160;Climbing assay results for males from 235 homozygous ENU-mutagenized lines. Pie chart represents the proportions of tested fly lines that fall into 4 groups of climbing pass rates: 0% , 1-20% , 21-50% , and 51-100% . (D)&#160;Results from 1 way ANOVA statistical analysis in which mutant lines were compared to the mean climbing percent value of all tested lines. Represented is the number for two P value categories: P &#60; 0.05 (significant change) and P &#62; 0.05 (not significant change).&#160;
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59720/59720fig2.jpg" /> 
Figure 2: Secondary histology screen. H&#38;E-stained mid-brain sections illustrating, from left to right, no neurodegeneration, mild neurodegeneration, and confirmed neurodegeneration. A total of 51 homozygous lines were retested by histology after identification of candidates using the climbing assay. Arrows indicate the holes in the brain indicative of neurodegeneration. The region circled by the dotted lines shows the severe neurodegenration observed in line 867.&#160;
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59720/59720fig3.jpg" /> 
Figure 3: Identification of the neuroprotective gene brat following deficiency mapping using the climbing assay. (A)&#160;Shown are representative images of H&#38;E-stained mid-brain sections of 18-20 days old w1118 (Control), heterozygous 867 (867/+) and homozygous&#160;867 mutant flies. (B)&#160;The deficiency line Df(2L)ED1272 (BL_24116) does not complement the 867 mutant phenotype (black histogram) based on the climbing assay. Histological verification shows that Df(2L)ED1272 line doesn&#39;t complement the 867 neurodegeneration phenotype, whereas Df(2L)ED1315 (BL_9269) does. Graph represents mean and standard deviation of 5 climbing trials for each line. Asterisks indicate significance based on one-way ANOVA, in which the mean climbing percent value of each line was compared to the mean climbing percent value of line 867/+ (green histogram). * P &#60; 0.05, ** P &#60; 0.01 and ***P &#60; 0.001. (C)&#160;Schematic representation of additional deficiency lines showing non-complementation of the 867 phenotype by histology. This figure has been modified from Loewen et al.17; an article published under the Creative Commons Attribution 4.0 International License.&#160;
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59720/59720fig4.jpg" /> 
Figure 4: Point mutation in the brat gene leads to an amino acid change in the Coiled-coil domain of the Brat protein. (A)&#160;brat gene model and primers used for the amplification and sequencing of the coding region. (B)&#160;DNA sequencing of the brat locus identifies a nucleotide change that leads to an amino acid change in the Coiled Coil domain of the Brat protein. This figure has been modified from Loewen et al.17; an article published under the Creative Commons Attribution 4.0 International License.&#160;
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59720/59720fig5.jpg" /> 
Figure 5: Further analysis of brat mutants confirms its neuroprotective role in Drosophila. (A)&#160;Using the Gal-4&#62;UAS system22 , neuroblast-specific expression of the functional brat gene rescues the neurodegeneration phenotype in 867 mutants. (B)&#160;Age-dependent neurodegeneration is observed in 867 mutants carrying a reporter gene pcna-GFP. bratchs corresponds to the name of the brat allele in line 867, which was named cheesehead (chs). Shown are representative H&#38;E-stained midbrain sections of yw (control) and homozygous bratchs; pcna-GFP flies of the indicated ages. This figure has been modified from Loewen et al.17; an article published under the Creative Commons Attribution 4.0 International License.&#160;
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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			<td>Station</td>
			<td>Time</td>
			<td>Temperature</td>
			<td>Vacuum/Pressure</td>
			<td>Solution</td>
		</tr>
		<tr>
			<td>1</td>
			<td colspan="4">NO DELAY-QUICK START</td>
		</tr>
		<tr>
			<td>2</td>
			<td>OFF</td>
			<td>OFF</td>
			<td>OFF</td>
			<td></td>
		</tr>
		<tr>
			<td>3</td>
			<td>: 15</td>
			<td>37 &#176;C</td>
			<td>ON/ON</td>
			<td>80%EtOH</td>
		</tr>
		<tr>
			<td>4</td>
			<td>: 15</td>
			<td>37 &#176;C</td>
			<td>ON/ON</td>
			<td>95% EtOH</td>
		</tr>
		<tr>
			<td>5</td>
			<td>: 15</td>
			<td>37 &#176;C</td>
			<td>ON/ON</td>
			<td>100% EtOH</td>
		</tr>
		<tr>
			<td>6</td>
			<td>: 15</td>
			<td>37 &#176;C</td>
			<td>ON/ON</td>
			<td>100% EtOH</td>
		</tr>
		<tr>
			<td>7</td>
			<td>: 15</td>
			<td>37 &#176;C</td>
			<td>ON/ON</td>
			<td>100% EtOH</td>
		</tr>
		<tr>
			<td>8</td>
			<td>: 15</td>
			<td>37 &#176;C</td>
			<td>ON/ON</td>
			<td>Xylenes</td>
		</tr>
		<tr>
			<td>9</td>
			<td>: 15</td>
			<td>37 &#176;C</td>
			<td>ON/ON</td>
			<td>Xylenes</td>
		</tr>
		<tr>
			<td>10</td>
			<td>: 15</td>
			<td>37 &#176;C</td>
			<td>ON/ON</td>
			<td>Xylenes</td>
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		<tr>
			<td>11</td>
			<td>: 30</td>
			<td>58 &#176;C</td>
			<td>ON/ON</td>
			<td>Paraffin</td>
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		<tr>
			<td>12</td>
			<td>: 15</td>
			<td>58 &#176;C</td>
			<td>ON/ON</td>
			<td>Paraffin</td>
		</tr>
		<tr>
			<td>13</td>
			<td>OFF</td>
			<td>58 &#176;C</td>
			<td>OFF</td>
			<td>Paraffin</td>
		</tr>
		<tr>
			<td>14</td>
			<td>: 15</td>
			<td>58 &#176;C</td>
			<td>ON/ON</td>
			<td>Paraffin</td>
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</table>
Table 1: Recommended program settings for automated tissue processor. The steps in the order listed here can be used with an automated tissue processor for fixed heads.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Volume (&#956;L)</td>
		</tr>
		<tr>
			<td>10x ExTaq Buffer (Mg2+ plus) (20 mM)</td>
			<td>2.5</td>
		</tr>
		<tr>
			<td>Forward primer (10 &#956;M)</td>
			<td>2.5</td>
		</tr>
		<tr>
			<td>Reverse primer (10 &#956;M)</td>
			<td>2.5</td>
		</tr>
		<tr>
			<td>2.5 mM dNTPs</td>
			<td>2</td>
		</tr>
		<tr>
			<td>Template DNA</td>
			<td>2</td>
		</tr>
		<tr>
			<td>TaKaRa Ex Tq (5 U/&#956;L)&#160;</td>
			<td>0.25</td>
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		<tr>
			<td>Nuclease-free water</td>
			<td>13.25</td>
		</tr>
		<tr>
			<td>Total volume</td>
			<td>25</td>
		</tr>
	</tbody>
</table>
Table 2: Reagents used for PCR reaction. Reagents and respective volumes used in the PCR reaction to amplify the brat-coding region.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Step</td>
			<td>Temperature</td>
			<td>Time</td>
		</tr>
		<tr>
			<td rowspan="3">35 cycles</td>
			<td>95 &#176;C</td>
			<td>15 s</td>
		</tr>
		<tr>
			<td>55 &#176;C</td>
			<td>45 s</td>
		</tr>
		<tr>
			<td>72 &#176;C</td>
			<td>3 min&#160; 10 s</td>
		</tr>
		<tr>
			<td>Hold</td>
			<td>4 &#176;C</td>
			<td>&#8734;</td>
		</tr>
	</tbody>
</table>
Table 3: Thermal cycling conditions used for PCR. The steps in the order listed here can be used to program a thermal cycler using the reaction mix shown in Table2.

Watch this Scientific Journal Video about In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster at JoVE.com

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

We present a protocol using a forward genetic approach to screen for mutants exhibiting neurodegeneration in Drosophila melanogaster. It incorporates a climbing assay, histology analysis, gene mapping and DNA sequencing to ultimately identify novel genes related to the process of neuroprotection.

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

There is much to understand about the onset and progression of neurodegenerative diseases, including the underlying genes responsible. Forward genetic ...

in-vivo-forward-genetic-screen-to-identify-novel-neuroprotective

Nanyang Technological University

Research

JoVE Journal

Genetics

9.3K Views.  University of Alabama. Het belangrijkste doel van dit protocol is om de kracht van voorwaartse genetica te gebruiken om genen te identificeren die bij ongereguleerde leiden tot neurodegeneratie. Het belangrijkste voordeel van deze techniek is dat het een onbevooroordeelde en eenvoudige aanpak biedt voor de identificatie van neuroprotectieve genen die mogelijk moeilijker uit te voeren zijn in zoogdiermodellen. Deze techniek kan worden gebruikt om nieuwe genen te identificeren die betrokken zijn bij het proces van ne...

Video: In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human genetics research. Although a number of in silico algorithms have been developed to predict the pathogenicity of variants identified in these datasets, functional studies are critical to determining how specific genomic variants affect protein function, especially for missense variants. In the Undiagnosed Diseases Network (UDN) and other rare disease research consortia, model organisms (MO) including Drosophila, C. elegans, zebrafish, and mice are actively used to assess the function of putative human disease-causing variants. This protocol describes a method for the functional assessment of rare human variants used in the Model Organisms Screening Center Drosophila Core of the UDN. The workflow begins with gathering human and MO information from multiple public databases, using the MARRVEL web resource to assess whether the variant is likely to contribute to a patient&#39;s condition as well as design effective experiments based on available knowledge and resources. Next, genetic tools (e.g., T2A-GAL4 and UAS-human cDNA lines) are generated to assess the functions of variants of interest in Drosophila. Upon development of these reagents, two-pronged functional assays based on rescue and overexpression experiments can be performed to assess variant function. In the rescue branch, the endogenous fly genes are &#34;humanized&#34;&#160;by replacing the orthologous Drosophila gene with reference or variant human transgenes. In the overexpression branch, the reference and variant human proteins are exogenously driven in a variety of tissues. In both cases, any scorable phenotype (e.g., lethality, eye morphology, electrophysiology) can be used as a read-out, irrespective of the disease of interest. Differences observed between reference and variant alleles suggest a variant-specific effect, and thus likely pathogenicity. This protocol allows rapid, in vivo assessments of putative human disease-causing variants of genes with known and unknown functions.

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

The goal of this protocol is to outline the design and performance of in vivo experiments in Drosophila melanogaster to assess the functional consequences of rare gene variants associated with human diseases.

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human ...

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We developed a novel nanoinjection/electrophysiology assay that allows the detection of bioactive modulatory effects of compounds on the function of a neuronal circuit that mediates the escape response in Drosophila melanogaster3,4. Our in vivo assay, which uses the Drosophila Giant Fiber System (GFS, Figure 1) allows screening of different types of compounds, such as small molecules or peptides, and requires only minimal quantities to elicit an effect. In addition, the Drosophila GFS offers a large variety of potential molecular targets on neurons or muscles. The Giant Fibers (GFs) synapse electrically (Gap Junctions) as well as chemically (cholinergic) onto a Peripheral Synapsing Interneuron (PSI) and the Tergo Trochanteral Muscle neuron (TTMn)5. The PSI to DLMn (Dorsal Longitudinal Muscle neuron) connection is dependent on D&alpha;7 nicotinic acetylcholine receptors (nAChRs)6. Finally, the neuromuscular junctions (NMJ) of the TTMn and the DLMn with the jump (TTM) and flight muscles (DLM) are glutamatergic7-12. Here, we demonstrate how to inject nanoliter quantities of a compound, while obtaining electrophysiological intracellular recordings from the Giant Fiber System13 and how to monitor the effects of the compound on the function of this circuit. We show specificity of the assay with methyllycaconitine citrate (MLA), a nAChR antagonist, which disrupts the PSI to DLMn connection but not the GF to TTMn connection or the function of the NMJ at the jump or flight muscles.
Before beginning this video it is critical that you carefully watch and become familiar with the JoVE video titled "Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster " from Augustin et al7, as the video presented here is intended as an expansion to this existing technique. Here we use the electrophysiological recordings method and focus in detail only on the addition of the paired nanoinjections and monitoring technique.

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

A rapid in vivo assay to test for neuromodulatory compounds using the Giant Fiber System (GFS) of Drosophila melanogaster is described. Nanoinjections in the head of the animal along with electrophysiological recordings of the GFS can reveal bioactivity of compounds on neurons or muscles.

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We ...

Neuroscience

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Developmental Biology

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the fly as an experimental system include its genetic tractability, short life span and the possibility to observe and quantitatively analyze complex behaviors. The expression of disease-linked genes in specific neuronal populations of the Drosophila brain, can be used to model human neurodegenerative diseases such as Parkinson's and Alzheimer's 5.
Dopaminergic (DA) neurons are among the most vulnerable neuronal populations in the aging human brain. In Parkinson's disease (PD), the most common neurodegenerative movement disorder, the accelerated loss of DA neurons leads to a progressive and irreversible decline in locomotor function. In addition to age and exposure to environmental toxins, loss of DA neurons is exacerbated by specific mutations in the coding or promoter regions of several genes. The identification of such PD-associated alleles provides the experimental basis for the use of Drosophila as a model to study neurodegeneration of DA neurons in vivo. For example, the expression of the PD-linked human &alpha;-synuclein gene in Drosophila DA neurons recapitulates some features of the human disease, e.g. progressive loss of DA neurons and declining locomotor function 2. Accordingly, this model has been successfully used to identify potential therapeutic targets in PD 8.
Here we describe two assays that have commonly been used to study age-dependent neurodegeneration of DA neurons in Drosophila: a climbing assay based on the startle-induced negative geotaxis response and tyrosine hydroxylase immunostaining of whole adult brain mounts to monitor the number of DA neurons at different ages. In both cases, in vivo expression of UAS transgenes specifically in DA neurons can be achieved by using a tyrosine hydroxylase (TH) promoter-Gal4 driver line 3, 10.

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Here we describe two assays that have been established to study age-dependent neurodegeneration of dopaminergic (DA) neurons in Drosophila: the climbing/startle-induced negative geotaxis assay which allows to study the functional effects of DA neurons degeneration and the tyrosine hydroxylase immunostaining which is used to identify and count DA neurons in whole brain mounts.

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the ...

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous system and protects neural tissue from toxins and pathogens. Defects in the formation of this barrier have been correlated with neuropathies, and the breakdown of this barrier has been observed in many neurodegenerative diseases. Therefore, it is critical to identify the genes that regulate the formation and maintenance of the blood-brain barrier to identify potential therapeutic targets. In order to understand the exact roles these genes play in neural development, it is necessary to assay the effects of altered gene expression on the integrity of the blood-brain barrier. Many of the molecules that function in the establishment of the blood-brain barrier have been found to be conserved across eukaryotic species, including the fruit fly, Drosophila melanogaster. Fruit flies have proven to be an excellent model system for examining the molecular mechanisms regulating nervous system development and function. This protocol describes a step-by-step procedure to assay for blood-brain barrier integrity during the embryonic and larval stages of D. melanogaster development.

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Blood-brain barrier integrity is critical for nervous system function. In Drosophila melanogaster, the blood-brain barrier is formed by glial cells during late embryogenesis. This protocol describes methods to assay for blood-brain barrier formation and maintenance in D. melanogaster embryos and third instar larvae.

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous ...

Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and thus current therapeutic treatment strategies are not curative. Through the use of whole exome sequencing, an increasing number of genes have been identified to play a role in human locomotion. Despite identifying these genes, it is not known how these genes are crucial to normal locomotive functioning. Therefore, a reliable assay, which utilizes model organisms to elucidate the role of these genes in order to identify novel targets of therapeutic interest, is needed more than ever. We have designed a sensitized version of the negative geotaxis assay that allows for the detection of milder defects earlier and has the ability to evaluate these defects over time. The assay is performed in a glass graduated cylinder, which is sealed with a wax barrier film. By increasing the threshold distance to be climbed to 17.5 cm and increasing the experiment duration to 2 min&#160;we have observed a greater sensitivity in detecting mild mobility dysfunctions. The assay is cost effective and does not require extensive training to obtain highly reproducible results. This makes it an excellent technique for screening candidate drugs in Drosophila mutants with locomotion defects.

We present an optimized inexpensive and reliable negative geotaxis assay in Drosophila melanogaster as a model for neurodegenerative disorders. Being more sensitive to mild locomotor defects, this assay will help screen for potential genetic interactions and drug targets.

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and ...

Mass Histology to Quantify Neurodegeneration in Drosophila

Progressive neurodegenerative diseases like Alzheimer&#39;s disease (AD) or Parkinson&#39;s disease (PD) are an increasing threat to human health worldwide. Although mammalian models have provided important insights into the underlying mechanisms of pathogenicity, the complexity of mammalian systems together with their high costs are limiting their use. Therefore, the simple but well-established Drosophila model-system&#160;provides an alternative for investigating the molecular pathways that are affected in these diseases. Besides behavioral deficits, neurodegenerative diseases are characterized by histological phenotypes such as neuronal death and axonopathy. To quantify neuronal degeneration and to determine how it is affected by genetic and environmental factors, we use a histological approach that is based on measuring the vacuoles in adult fly brains. To minimize the effects of systematic error and to directly compare sections from control and experimental flies in one preparation, we use the &#39;collar&#39; method for paraffin sections. Neurodegeneration is then assessed by measuring the size and/or number of vacuoles that have developed in the fly brain. This can either be done by focusing on a specific region of interest or by analyzing the entire brain by obtaining serial sections that span the complete head. Therefore, this method allows one to measure not only severe degeneration but also relatively mild phenotypes that are only detectable in a few sections, as occurs during normal aging.

Mass Histology to Quantify Neurodegeneration in Drosophila

Drosophila is widely used as a model system to study neurodegeneration. This protocol describes a method by which degeneration, as determined by vacuole formation in the brain, can be quantified. It also minimizes effects due to the experimental procedure by processing and sectioning control and experimental flies as one sample.

Progressive neurodegenerative diseases like Alzheimer&#39;s disease (AD) or Parkinson&#39;s disease (PD) are an increasing threat to human health ...

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various intrinsic and extrinsic inhibitory factors involved in the restriction of axon regeneration have been identified. However, simply removing these inhibitory cues is insufficient for successful regeneration, indicating the existence of additional regulatory machinery. Drosophila melanogaster, the fruit fly, shares evolutionarily conserved genes and signaling pathways with vertebrates, including humans. Combining the powerful genetic toolbox of flies with two-photon laser axotomy/dendriotomy, we describe here the Drosophila sensory neuron &#8211; dendritic arborization (da) neuron injury model as a platform for systematically screening for novel regeneration regulators. Briefly, this paradigm includes a) the preparation of larvae, b) lesion induction to dendrite(s) or axon(s) using a two-photon laser, c) live confocal imaging post-injury and d) data analysis. Our model enables highly reproducible injury of single labeled neurons, axons, and dendrites of well-defined neuronal subtypes, in both the peripheral and central nervous system.

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

Here, we present a protocol using the Drosophila sensory neuron - dendritic arborization (da) neuron injury model, which combines in vivo live imaging, two-photon laser axotomy/dendriotomy, and the powerful fly genetic toolbox, as a platform for screening potential promoters and inhibitors of neuroregeneration.

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various ...

Medicine

Stimulating and Analyzing Adult Neurogenesis in the Drosophila Central Brain

The molecular and cellular mechanisms underlying neurogenesis in response to disease or injury are not well understood. However, understanding these mechanisms is crucial for developing neural regenerative therapies. Drosophila melanogaster is a leading model for studies of neural development but historically has not been exploited to investigate adult brain regeneration. This is primarily because the adult brain exhibits very low mitotic activity. Nonetheless, penetrating traumatic brain injury (PTBI) to the adult Drosophila central brain triggers the generation of new neurons and new glia. The powerful genetic tools available in Drosophila combined with the simple but rigorous injury protocol described here now make adult Drosophila brain a robust model for neural regeneration research. Provided here are detailed instructions for (1) penetrating injuries to the adult central brain and (2) dissection, immunohistochemistry, and imaging post-injury. These protocols yield highly reproducible results and will facilitate additional studies to dissect mechanisms underlying neural regeneration.

Stimulating and Analyzing Adult Neurogenesis in the Drosophila Central Brain

This article provides detailed protocols for inflicting Penetrating Traumatic Brain Injury (PTBI) to adult Drosophila and examining the resulting neurogenesis.

The molecular and cellular mechanisms underlying neurogenesis in response to disease or injury are not well understood. However, understanding these ...

Fly Climbing Assay: A Method to Test General Locomotion in Drosophila melanogaster

Source: Gevedon, O., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/59720/in-vivo-forward-genetic-screen-to-identify-novel-neuroprotective">In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster</a>. J. Vis. Exp. (2019).
This video describes a simple method to quantify fly locomotion and shows the climbing assay performed.&#160; The fly climbing assay is a general test of motor function which relies on a negative geotaxis response.

Source: Gevedon, O., et al. In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster. J. Vis. Exp. (2019).
This ...

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

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