Methamphetamine (METH) is currently one of the most abused drugs in the United States. Through its neurotoxic actions, chronic exposure results in brain damage, including striatal dopaminergic terminal degeneration, among other injuries. Zhu et al. (2016) used a chronic escalating dosage exposure model to examine proteome changes in rat brains as a way to characterize structural and functional responses to METH.1
Addiction to METH results in brain changes that are often not apparent immediately. Regions affected include the limbic system, with long-lasting damage resulting in adaptive changes and structural modification. Since there is often a delayed response in symptoms of the damage arising, it can be difficult to adequately treat and support METH addicts. Zhu and coauthors sought to examine changes brought about by two weeks of METH treatment in rats, looking at alterations in the proteome of the hippocampus as representative of the limbic system. They also chose to examine olfactory bulb tissue, since it has connections within the hippocampus. There is also strong evidence of this interconnectedness in Alzheimer’s disease, for which loss of smell can be an early diagnostic sign,2 and from other studies showing that olfactory bulb abnormalities exist with major depressive disorder in drug addiction.3
Male Sprague Dawley rats (n = 5) received intraperitoneal METH injections with daily doses increasing throughout the study period. At two weeks, the team decapitated the rats and dissected the brain tissue, which they froze immediately in liquid nitrogen, storing it at −80°C until assay. Following standard protein extraction protocols, the researchers digested the tissue samples with trypsin and then characterized the peptides by liquid chromatography–tandem mass spectrometry (LC-MS/MS), conducting separation on an Acclaim PepMap RSLC column, followed by analysis through a Dionex UltiMate 3000 Nano LC system coupled with an LTQ Orbitrap Velos mass spectrometer (all Thermo Scientific). The team examined spectral data using Proteome Discoverer software revision 1.2 (Thermo Scientific) to search the Swiss-Prot database (Rattus).
The initial shotgun proteomics analysis identified 1,829 peptides (1,628 METH; 1,481 controls) corresponding to 302 proteins (301 METH; 298 controls) from the hippocampal extracts. Of these, the team found that hippocampal extracts contained four proteins unique to the METH group and one protein unique to the control group. LC-MS/MS characterized 332 proteins (1,415 peptides) from olfactory bulb extracts, with three unique to the METH group and one unique to controls.
Semi-quantitative assessment found differential abundance of 42 proteins in hippocampal tissue (31 increased with METH treatment, and 11 decreased) and 25 proteins in olfactory bulb extracts (14 increased; 11 decreased). Principal component analysis suggested that METH treatment affects the hippocampus more than the olfactory bulb in this model of drug addiction in rats.
From these data, Zhu et al. created a multiple reaction monitoring assay targeted to quantify proteome changes within the two brain tissues. Targeting 79 peptides (42 proteins) in hippocampal tissue and 46 peptides (25 proteins) in olfactory bulb extracts, the team discovered that 13 proteins increased with METH treatment (seven hippocampal; six olfactory bulb) whereas five decreased (four hippocampal; one olfactory bulb). Following systems biology analysis using PANTHER and Pathway Studio 8, the researchers found that chronic METH exposure affects catalytic activity most with proteins involved in cell death, inflammation, oxidation and apoptosis. Cell pathways showing the most disturbances include cellular communication and systems processes.
Although this study shows the effect of chronic METH exposure in rats, indications that the drug alters the brain tissue proteome could open up new therapeutic options for supporting and treating addicts in the future.
1. Zhu, R., et al. (2016) “The effect of chronic methamphetamine exposure on the hippocampal and olfactory bulb neuroproteomes of rats,” PLoS One 11(4), doi:10.1371/journal.pone.0151034.
2. Daulatzai, M.A. (2015) “Olfactory dysfunction: Its early temporal relationship and neural correlates in the pathogenesis of Alzheimer’s disease,” Journal of Neural Transmission, 122(10) (pp. 1475–1497), doi: 10.1007/s00702-015-1404-6.
3. Zuloaga D.G., Jacobskind, J.S., and Raber, J. (2015) “Methamphetamine and the hypothalamic-pituitary-adrenal axis,” Frontiers in Neuroscience, 9(178), doi: 10.3389/fnins.2015.00178.