A preview of Crowd4Discovery experiments
The goal of Crowd4Discovery (C4D) is to find out where amphetamines accumulate inside mouse brain cells by combining a proven drug-detection technique called autoradiography with a powerful electron microscope to visualize drug molecules. As I will flesh out below, our hypothesis is that amphetamines accumulate in abnormal intracellular compartments that form as a result of drug incorporation in the lipid membranes of living cells. This accumulation may affect ancient cellular processes called endocytosis and secretion that are essential to normal brain cell function, and may be a key to unlocking the mystery of how amphetamines actually work in the human brain.
The C4D project will proceed in two phases. In Phase One, we will optimize autoradiography and electron microscopy for mouse brain cells that will be grown in the lab and exposed to radiolabeled amphetamines. In Phase Two, we will extend these validated protocols to examine postmortem brain samples from mice treated with radiolabeled amphetamines.
As of last week, lead project experimentalist Danny Korostyshevsky was officially on board and settling in the Sulzer Lab of Columbia University Medical School, where experiments will begin in earnest soon. Consider this post a prelude to what we, the C4D team, hope will be an open, interactive and sustained scientific colloquy in the weeks and months ahead.
Let me back up for a minute and explain why we’re so interested in determining where amphetamines accumulate at a molecular level. Conventional wisdom in psychopharmacology insists that amphetamines trigger the release of dopamine from brain cells by blocking the dopamine reuptake transporter protein, DAT, and that all the ensuing cellular effects of amphetamines, including toxicity, can be traced back to direct drug action on DAT.
However, I blogged last Fall in the run-up to our crowdfunding campaign that C4D team member David Sulzer first showed over 20 years ago that both dopamine and amphetamines accumulate inside synaptic vesicles because of a chemical attraction of positively-charged (“basic”) molecules for negatively-charged (“acidic”) environments, turning the DAT-centric model on its head. And the cellular effects of amphetamines don’t stop there.
Amphetamines are more hydrophobic than their chemical cousin dopamine, and this affinity for lipids drives drug accumulation in cell membranes. Since the 1970s, many labs, including the Sulzer Lab, showed that cultured mammalian cells or tissue slices removed from whole animals that were treated with hydrophobic weak bases like amphetamines contained abnormal structures called multilamellar bodies (MLBs), while untreated samples were free of MLBs.
My former lab at Princeton even observed MLBs in yeast cells treated with the antidepressant Zoloft, which is more hydrophobic than amphetamines, proving that these structures form in the absence of classical protein transporter drug targets, and therefore are a conserved cellular drug response exhibited by all eukaryotes:
There are two possibilities: either amphetamines are directly associated with MLBs or precursor structures that are ultimately incorporated into MLBs, or amphetamines accumulate somewhere else entirely and MLBs are a sequela of altered cellular lipid homeostasis. While we make preparations for autoradiography, we will attempt to distinguish between those two possibilities. MLBs appear to be intermediates of the autophagy pathway, an ancient cellular recycling program that my former Princeton lab showed is activated by Zoloft accumulation in yeast cells.
The Sulzer Lab has recently described a transgenic mouse in which the autophagy pathway has been genetically disrupted only in brain cells that express dopamine and DAT, which actively concentrate amphetamines. We will see if amphetamines induce the formation of MLBs in cultured neurons derived from this autophagy mutant mouse. If the answer is yes, then autoradiography will confirm the association. But if MLBs don’t form without functioning autophagy, the drug molecules still have to be somewhere and autoradiography will reveal their hiding place.
Please stay tuned for three upcoming developments: (1) release of our first open budget report; (2) posting of autoradiography protocols online for feedback; (3) our first online lab meeting, which will be open to C4D supporters and the general public.
As always, your comments are welcome!