Crowd4Discovery update: February 2013

March 05, 2013

Last time I checked in with an update at the end of January, the Crowd4Discovery/C4D project was still booting up in Professor Dave Sulzer’s lab at Columbia University Medical Center (CUMC). Lead experimentalist and resident electron microscopist Danny Korostyshevsky (@badomens on Twitter) started getting his feet wet in the Sulzer Lab two weeks into the new year. That transition period involved Danny getting used to working with the primary neuronal culture techniques employed in the Sulzer lab. Also, joining the new lab involved safety training sessions and orientations for laboratory animal research.

 

In terms of the science, February was about the appetizers leading up to the experimental main course: autoradiography. Danny has prepared the first draft of an autoradiography protocol, which we will continue to flesh out. Protocols, raw data and ultimately processed data generated by the C4D project can be viewed and downloaded on Figshare, our open data repository of choice; all project-related content will be tagged “Crowd4Discovery.” And in the spirit of Open Science, we will be soliciting feedback from experts on comment threads and in G+ hangouts. (Skype dates for C4D donors are also available upon request!)

 

In this update, I will discuss experimental appetizers. Since the beginning of last month, Danny has been working on experiments that leverage published experimental observations of where hydrophobic weak base drugs – a.k.a. cationic amphipaths – accumulate inside cells. Using vital stains that localize to specific compartments inside drug-treated versus untreated primary neuronal cell cultures derived from mouse ventral midbrain, we hope to gain preliminary insights into where amphetamines accumulate.

 

Recent work from Professor Jeff Krise’s lab at the University of Kansas shows that human fibroblast cell lines treated with psychoactive cationic amphipaths for  extended periods have greater capacity to take up a vital dye that collects in lysosomes. In other words, drug-treated cells adapt to drug exposure by increasing their total lysosomal volume. For the cognoscenti, please see Krise’s paper here.

 

In mid-February we “figshared” some raw data from vital dye labeling experiments with untreated versus amphetamine-treated cells. Danny stained for an autophagy protein called LC3 and a pH-sensitive dye called pHrodo, which labels acidic compartments called endosomes (the red puncta in the featured image above). There are many reasons why we care about autophagy and its evolutionarily conserved role as a cellular drug response. Based on the yeast work my lab did at Princeton, we know that cationic amphipaths related to amphetamines, namely antidepressants, trigger autophagy.

 

And that’s where the multilamellar bodies (MLBs) I discussed in the late January C4D update come in. MLBs are a mystery. Are they pit stops on the autophagy pathway? Are MLBs structural variants of other compartments, like endosomes? We suspect that MLBs form independently of autophagy, though they may ultimately be substrates for autophagic clearance. Under this scenario, MLBs are altered compartments formed by asymmetric drug accumulation in organellar membranes — basically the bilayer couple model extended to acidic intracellular compartments. To test the role of autophagy in MLB formation in response to drug treatment, we will eventually take advantage of an ATG7 mutant mouse created in the Sulzer Lab that specifically lacks the ability to perform autophagy but only in dopamine-positive cells that express DAT, which is the transporter that actively concentrates amphetamines.

 

Based on preliminary observations thus far, there doesn’t appear to be co-localization between LC3 and pHrodo, but Danny is currently doing this experiment more carefully with a confocal rig, and with quantification of co-localization. One interesting possibility is MLBs may contain amphetamines and LC3, but don’t stain with pHrodo because amphetamine is a weak base that neutralizes acidic compartments in which it accumulates. To explore this possibility, we will examine co-localization between LC3 and another vital dye called monodansylcadaverine, or MDC, which is known to accumulate in “autophagic vacuoles.” These experiments are now underway, so more on the autophagy story in the weeks ahead.

 

Finally, we will be ordering radioactive amphetamines soon, at which point we’ll have much more to say about autoradiography. And that will also be a good time to post our first open budget report, because so far our only expenditure has been on the MakerBot Replicator2 3D printer we purchased in mid-December.

 

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