Chronic, Low-Dose Methamphetamine Reveals Sexual Dimorphism of Memory Performance, Histopathology, and Gene Expression Affected by HIV-1 Tat Protein in a Transgenic Model of NeuroHIV

Abstract

Methamphetamine (METH) use is frequent among people with HIV (PWH) and appears to increase the risk of neuronal injury and neurocognitive impairment (NCI). This study explored in vivo the effects of a 12 week (long-term), low-dose METH regimen in a transgenic animal model of neuroHIV with inducible expression of HIV-1 transactivator of transcription (Tat). Seven months after transient Tat induction and five months after METH exposure ended, we detected behavioral changes in the Barnes maze (BM) spatial memory task in the Tat and METH groups but not the combined Tat + METH group. The novel object recognition (NOR) task revealed that Tat extinguished discrimination in female animals with and without METH, although METH alone slightly improved NOR. In contrast, in males, Tat, METH, and Tat + METH all compromised NOR. Neuropathological examination detected sex-dependent and brain region-specific changes of pre-synaptic terminals, neurites, and activation of astrocytes and microglia. RNA-sequencing and quantitative reverse transcription polymerase chain reaction indicated that METH and Tat significantly altered gene expression, including factors linked to Alzheimer's disease-like NCI. In summary, chronic low-dose METH exerts long-term effects on behavioral function, neuropathology, and mRNA expression, and modulates the effects of Tat, suggesting sex-dependent and -independent mechanisms may converge in HIV brain injury and NCI.

Keywords: HIV-1; Tat; methamphetamine; neurodegeneration; sexual dimorphism.

Figure 1

Schematic of overall experimental protocol. Mouse cohort consisted of two genotypes, GFAP-rtTA⁺/pTRE-Tat⁺ (40 mice) and GFAP-rtTA⁺/pTRE-Tat⁻ (43 mice) with 35 females and 48 males, 83 mice total. The 12-week METH regimen entailed the following steps: Week 1, starting at 0.5 mg/kg s.c., 1 × day, step-wise increase by 0.5 mg/kg with each injection over 5 days (Mon–Fri), followed by 11 weeks 1 × 2.5 mg/kg/day (Mon–Fri = 20+ days per month). Controls received vehicle (saline). All animals were treated with Doxycyclin (Dox).

Figure 2

Learning and spatial memory impairment is affected by Tat expression and METH treatment. A cohort of 83 mice was subjected to behavioral assessment, n = Ctl (male: 10, female: 9), Tat (male: 12, female: 7), METH (male: 11, female: 13), and Tat + METH (male: 15, female: 6). (A,B) LM testing shows that female animals’ locomotor activity remained unaffected by Tat and METH or combination of both agents. In male animals, exposure to METH increased locomotor activity. (C) BM test was performed to assess spatial learning and memory in mice. In male animals, Tat expression and METH exposure both reduced spatial memory performance. However, Tat and METH in combination ameliorated deficit in spatial memory, resulting in performance being indistinguishable from control. (D) In female animals, only those with Tat expression alone had reduced spatial memory performance. (E,F) NOR test was performed to examine short term memory. Sex-dependent deficits in short term memory were observed. (E) Tat expression and METH exposure alone and in combination equally diminished novel object recognition in male animals. (F) In female animals, only METH exposed animals displayed novel object recognition reaching a significance level of p ≤ 0.01 while the Ctl group showed a trend (Familiar vs. Novel Ctl p-value = 0.0708). Values are mean ± SEM, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001; ANOVA and Fisher’s PLSD post hoc test.

Figure 3

Immunofluorescence staining of cellular markers in cortex and hippocampus. Images of sagittal brain tissue sections stained for synaptophysin in (A) LIII of cortex and (B) CA1 of hippocampus. Images of tissue stained for MAP2 in (C) LIII of cortex and (D) CA1 of hippocampus. Images of tissue stained for GFAP in (E) total cortex and (F) CA1 of hippocampus. Images of tissue stained for Iba1 in (G) total cortex and (H) CA1 of hippocampus. Representative images taken from male animals are shown. Scale bar = 50 μm.

Figure 4

Quantitative results of histological analysis investigating neuronal injury, activation of astrocytes and microglia. (A) SYP⁺ neuropil percentage in cortex (LIII). (B) SYP⁺ neuropil percentage in hippocampus (CA1). (C) MAP2⁺ neuropil percentage in cortex (LIII). (D) MAP2⁺ neuropil percentage in hippocampus (CA1). (E) GFAP fluorescence in cortex (total). (F) GFAP fluorescence in hippocampus (CA1). (G) Count of Iba1⁺ cells normalized to area in cortex (total). (H) Count of Iba1⁺ cells normalized to area in hippocampus (CA1). Values are mean ± SEM, n = 4 males and 4 females per group (12 microscopic fields per animal), * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001; ANOVA and Fisher’s PLSD post hoc test. Details of the statistical analysis are presented in Supplementary Table S2.

Figure 5

Changes in RNA expression of genes involved in AD, APP processing pathway, and inflammation are observed in cortex. (A) RNA analysis (qRT-PCR) of App expression showed an increased expression in female METH and Tat + METH animals. (B) Sex-dependent expression of Bace1 was observed. (C) Again, we observed a sex-dependent modulation of gene expression in Adam10, with an increased expression in METH and Tat + METH females. (D) Expression of Gsap is decreased in Tat and Tat + METH females. (E) Efnb1 expression is increased overall in METH and Tat + METH animals. (F) Expression of Ephb2 is increase in male METH and Tat + METH animals compared to Ctl. (G) Lcn2 expression is unchanged. (H) Tau expression is also unchanged. Values are mean ± SEM, n = 4 males and 4 females per group, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001; ANOVA and Fisher’s PLSD post hoc test. Details of statistical analysis are presented in Supplementary Table S3.

Figure 6

Changes in gene expression of hippocampus analyzed by RNA-Seq, reporting top 10 differentially regulated genes based on p-value. (A) Gene expression in male Tat compared to male Ctl. (B) Gene expression in female Tat compared to female Ctl. (C) Gene expression in male METH compared to male Ctl. (D) Gene expression in female METH compared to female Ctl. (E) Gene expression in male Tat + METH compared to male Ctl. (F) Gene expression in female Tat + METH compared to female Ctl. n = 3 males and 3 females per group, analyzed by DESeq2 (p ≤ 0.05).

Figure 7

Changes in gene expression of the hippocampus analyzed by RNA-Seq, showing top 10 genes based on highest Log₂ FC. (A) Gene expression in male Tat compared to male Ctl. (B) Gene expression in female Tat compared to female Ctl. (C) Gene expression in male METH compared to male Ctl. (D) Gene expression in female METH compared to female Ctl. (E) Gene expression in male Tat + METH compared to male Ctl. (F) Gene expression in female Tat + METH compared to female Ctl. n = 3 males and 3 females per group, analyzed by DESeq2 (p ≤ 0.05).

Figure 8

Ingenuity Pathway Analysis (IPA) software (version 2.2.1) generated network pathways with differential gene expression and predicted activation/inhibition by utilizing RNA-seq data of hippocampus. One of the top networks for male Ctl vs. Tat showed increased expression of Fos in groups 1–3, with decreased expression in group 5 (Insert FOS). This network also showed decreased expression of Th in group 4 and 6 (Insert TH). Thus, both Fos and Th display sex-dependent regulation in key components of the dopaminergic neurotransmission system. Groups: (1) Male Ctl vs. Tat, (2) Male Ctl vs. METH, (3) Male Ctl vs. Tat + METH, (4) Female Ctl vs. Tat, (5) Female Ctl vs. METH, and (6) Female Ctl vs. Tat + METH. (IPA analysis set at p < 0.01).