HIV-1 TAT protein enhances sensitization to methamphetamine by affecting dopaminergic function

Abstract

Methamphetamine abuse is common among humans with immunodeficiency virus (HIV). The HIV-1 regulatory protein TAT induces dysfunction of mesolimbic dopaminergic systems which may result in impaired reward processes and contribute to methamphetamine abuse. These studies investigated the impact of TAT expression on methamphetamine-induced locomotor sensitization, underlying changes in dopamine function and adenosine receptors in mesolimbic brain areas and neuroinflammation (microgliosis). Transgenic mice with doxycycline-induced TAT protein expression in the brain were tested for locomotor activity in response to repeated methamphetamine injections and methamphetamine challenge after a 7-day abstinence period. Dopamine function in the nucleus accumbens (Acb) was determined using high performance liquid chromatography. Expression of dopamine and/or adenosine A receptors (ADORA) in the Acb and caudate putamen (CPu) was assessed using RT-PCR and immunohistochemistry analyses. Microarrays with pathway analyses assessed dopamine and adenosine signaling in the CPu. Activity-dependent neurotransmitter switching of a reserve pool of non-dopaminergic neurons to a dopaminergic phenotype in the ventral tegmental area (VTA) was determined by immunohistochemistry and quantified with stereology. TAT expression enhanced methamphetamine-induced sensitization. TAT expression alone decreased striatal dopamine (D1, D2, D4, D5) and ADORA1A receptor expression, while increasing ADORA2A receptors expression. Moreover, TAT expression combined with methamphetamine exposure was associated with increased adenosine A receptors (ADORA1A) expression and increased recruitment of dopamine neurons in the VTA. TAT expression and methamphetamine exposure induced microglia activation with the largest effect after combined exposure. Our findings suggest that dopamine-adenosine receptor interactions and reserve pool neuronal recruitment may represent potential targets to develop new treatments for methamphetamine abuse in individuals with HIV.

Keywords: Adenosine receptors; Brain neurochemistry; Dopamine receptors; Gene expression microarrays; HPLC; Locomotor activity; Mice; Neurotransmitter respecification; TAT expression.

Figure 1. Effects of TAT protein expression on locomotor activity during repeated methamphetamine administration

Mice were treated daily with saline (SAL) or 2 mg/kg methamphetamine (METH; striped bars) and the total distance travelled (cm) over 30 min was assessed. Methamphetamine exposure significantly increased locomotor activity compared with saline at all days of testing. Methamphetamine-induced increases in locomotor activity were larger on Day 2 than Day 1, and on Day 3 compared to Day 2 (P< 0.001). No differences between TAT− and TAT+ mice were observed on the distance travelled after saline or methamphetamine. Data are expressed as Mean ± SEM (n=19–23). *** p < 0.001. ^# p < 0.001 compared to saline treatment on the corresponding day of testing.

Figure 2. Effects of TAT protein expression and methamphetamine exposure on the sensitized locomotor response

Locomotor responses to challenge with saline (A) or methamphetamine (B) in saline (SAL; circles) or methamphetamine (METH; squares)-exposed mice. Exposure to methamphetamine significantly increased the distance travelled in mice after both saline (A) and methamphetamine challenge (B). In response to the METH challenge, METH-exposed TAT+ mice travelled significantly more than METH-exposed TAT− mice (B), suggesting enhanced methamphetamine sensitization. Data are expressed as Mean ± SEM (SAL challenge: n=7–9, METH challenge n=11–14). * p < 0.05, ** p < 0.01, *** p < 0.001 between TAT− and TAT+ mice exposed to methamphetamine during the acquisition phase. ^### p < 0.001 between mice exposed to saline or methamphetamine during the acquisition phase.

Figure 3. Gene networks associated with the dopaminergic system

Gene changes induced by TAT expression in the brain after exposure to saline (SAL) or methamphetamine (METH) (n=5) in mice challenged with METH. A) Differences between TAT− and TAT+ mice exposed to SAL and challenged with METH. B) Differences between METH and SAL exposure in TAT− mice challenged with METH. C) Differences between METH and SAL exposure in TAT+ mice challenged with METH. D) Differences between TAT− and TAT+ mice exposed to METH and challenged with METH. Orange line connectors represent genes with shared protein domains or pathway interactions, and gray line connectors represent genes that are co-expressed or co-localized. Green colored shapes represent down regulated genes and Red colored shapes represents upregulated genes. Gray colored circles represent genes in the identified network that were not represented in the Agilent gene array platform. Squares represent p < 0.05 between two assigned groups.

Figure 4. Caudate putamen dopamine receptors expression and IBA-1 expression

Immunohistochemistry on paraffin embedded sections was utilized examine the protein distribution and levels of dopamine receptor D1 (A, B, C, D), dopamine receptor D2 (E, F, G, H), as well as of IBA-1 (I, J, K, L) in SAL TAT− (A, E, I), SAL TAT+ (B, F, J), METH TAT− (C, G, K), and METH TAT+ (D, H, L) mice. Representative positive cells in the 40× magnification images were labeled with a black arrow. (M) Normalized intensity density was calculated in ImageJ. Data are expressed as Mean ± SEM (n=5). * p < 0.05, ** p < 0.01, *** p < 0.001

Figure 5. Nucleus accumbens dopamine and adenosine receptor expression

Effects of TAT protein expression on nucleus accumbens dopamine receptor (DRD; A) and adenosine receptor (ADORA; B) expression in response to methamphetamine challenge after exposure to saline (SAL) or methamphetamine (METH). TAT expression, regardless of methamphetamine exposure decreased the expression of all DRDs (A). Methamphetamine exposure, regardless of TAT expression, decreased the expression of the ADORAs (B). TAT expression increased levels of ADORA2A and prevented the reduction of ADORA1 by methamphetamine exposure. Data are expressed as Mean ± SEM (n=5). * p < 0.05, ** p < 0.01, *** p < 0.001

Figure 6. Recruitment of reserve pool neurons to a dopaminergic phenotype

A. Horizontal midbrain section immunostained for tyrosine hydroxylase (TH), showing the substantia nigra pars compacta (SNc) and ventral tegmental subregions (perinigral, PN; parabrachial pigmented, PBP). B–C. Representative images of the VTA sectioned through the PBP of a TAT− SAL mouse (B) and a TAT+ METH mouse (C); black arrows indicate TH+ neurons. D. Graph showing the effects of TAT protein expression (TAT− and TAT+) on (TH)-positive cell number (mean cell count per hemi section) after prior exposure to saline or to methamphetamine. Both TAT expression and prior methamphetamine exposure tended to increase TH-positive cell numbers in the PBP with combined TAT expression and prior methamphetamine exposure producing the greatest number of TH-positive cells. Data are expressed as Mean ± SEM (n=4). Scale bars: a, 100 µm; b-c, 10 µm). * p < 0.05, ** p < 0.01.