In the Tannenbaum lab (Tanlab), interest in arsenic (As) was introduced by a Pilot Project to Kun Lu when he was a Postdoc and it has continued through the research of Charlie Knutson. Both of these researchers were focused on the role of the microbiome in As biotransformation and toxicity, but initially no connection was seen to our favorite molecule, nitric oxide (NO). Serendipity enters the game. A Departmental Seminar by Joseph Graziano (Columbia University) brought to our attention that an epidemiological study on a group of people in Maine revealed a tentative connection between Autism and high levels of As from natural sources in drinking water. The original hypothesis was that As and its methylated forms would induce oxidative stress, and since the brain is rich in NO synthases, NO would contribute to the damaging free radical chemistry, and also could modify protein function through reversible S-nitrosation of cysteines (SNO), and irreversible modification of tyrosine to form 3-nitrotyrosine (NT).
Uthpala Seneviratne in Tanlab had developed a powerful new method for analysis of SNO-peptides and proteins called SNOTRAP which is the first method capable of imaging SNO-proteins. Guanyu Gong had successfully applied this method to cells in culture, and had also used IHC to detect NT- containing proteins. We were able to collaborate with Alexi Nott in the lab of Li-Huei Tsai to use mouse neurons in culture to test our As hypothesis. We treated the cultured neurons and glia with varying concentrations of arsenite for varying periods of time. Arsenite concentrations of 100nM to 1uM caused immediate large increases in SNO-proteins associated with increases of NT. The cause of the increase was shown to be related to an immediate release of calcium from the endoplasmic reticulum in neurons. We speculate that the released calcium bound to calmodulin associated with the neuronal NO synthase, leading to a large increase of NO synthesis, which then culminated in the events described above.
Shown in the panel is a micrograph of neurons, astrocytes and dendrites exhibiting both an excess of SNO-proteins. The As hypothesis of neuropathology lives on.
