Methamphetamine-Induced Proteomic Changes Within the Neuroinflammatory TLR4 Pathway Persist After Long-Term Self-Administration in Rats.

In the United States, at least 1.6 million people use methamphetamine (METH) annually, with over half of these meeting criteria for a substance use disorder. Extended METH use causes a state of chronic neural inflammation, which can lead to lasting detrimental effects if left untreated. Due to the cognitive, behavioral, and financial toll of METH use disorders, further investigation into the mechanism behind the persistence of these effects is warranted. Our lab conducted a pilot behavioral and proteomics study using a rodent model of intravenous (i.v.) METH self-administration to simulate daily METH use. We aimed to identify key protein expression changes in specific brain regions affected by METH-induced inflammation as potential future therapeutic targets. We hypothesized that in a model emulating contingent, chronic METH usage and subsequent long-term washout, we would observe regional and molecular proteomic changes indicating the persistence of METH-induced neural inflammation. Male Sprague-Dawley rats (N = 8) were trained to acquire a METH dose of 0.1 mg/kg via lever-press, and after acquisition criteria were met, underwent dose-substitution testing with METH (0.001-0.32 mg/kg/inf) in daily, 2-hour sessions. This was followed by progressive ratio testing in which rats responded for saline or METH (0.010-0.1 mg/kg/inf) in 12-hour sessions (all rats had 3-4 months access to METH). After a 72-hour washout period post-METH exposure, a subset of the brains (n = 3) were sectioned into hippocampus, striatum, frontal cortex, and cerebellum regions for non-targeted proteomics analysis. Identification of a total of 6,892 proteins was performed using a MaxQuant (Max Planck Institute) database search against Rattus norvegicus (May 2020), and these data were examined via Qiagen's Ingenuity Pathway Analysis tool. Our results displayed upregulation of acute phase response signaling across the four brain regions studied, indicating a METH-induced inflammatory state had been achieved and maintained despite a 72-hour period free of METH self-administration. Four key pro-inflammatory upstream regulators were identified to be upregulated - OSM, inosine, JUN, and IL1B. Inosine is an anti-inflammatory agent. OSM, JUN, and IL1B are activated by the Toll-like Receptor 4 (TLR4) and downstream Peroxisome Proliferator-Activated Receptor-Gamma (PPAR-γ) pathways, each of which have been previously implicated to modulate inflammatory signals in other substances of abuse such as ethanol, cocaine, and heroin. METH has been previously noted to induce microglial activation via a stabilizing interaction with TLR4, though mitigating METH-induced neuroinflammation via modulating regulators downstream of this interaction remains an underdeveloped area of study. Our preliminary findings suggest that TLR4 and PPAR-γ may play a role as important regulators of METH-induced neural inflammatory changes at least 72-hours post-exposure, and are potential targets for METH use disorder therapies.