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.
