Age-related neuroendocrine, cognitive, and behavioral co-morbidities are promoted by HIV-1 Tat expression in male mice

Introduction

In the U.S. ~1.2 million people are living with human immunodeficiency virus type-1 (HIV-1) with men accounting for the majority of cases (~75%; [1]). Although combined antiretroviral therapy (cART) has reduced HIV-associated morbidity and mortality [2–4], patients continue to experience neurological and neuropsychiatric symptoms, collectively called “neuroHIV” [5–8]. This is likely due in part to efflux of some cART regimen from the central nervous system (CNS), high variance in brain accumulation of cART, and non-uniform distribution throughout the brain, all resulting in a lack of linear HIV RNA reduction in the CNS [9–11]. HIV is widely recognized as a disease-driver of aging as age-related disorders present prematurely and/or with greater severity in HIV-infected populations [12–15]. Actions of HIV in the CNS may contribute to premature aging partly by promoting secondary hypogonadism (i.e., hypothalamic and/or pituitary hormonal dysregulation) which is observed in 16% to 25% of young adult or middle-aged HIV-infected men [15–19]. In support, HIV⁺ men transition sooner to andropause [18, 19], characterized by lower total and/or free testosterone (T) seen in patients aged 20–39 years old [16–18, 20], greater circulating sex hormone-binding globulin [16, 17], and greater estradiol (E₂)-to-T ratios [18] versus healthy age-matched men [21–23]. In the post-cART era, neurotoxic viral proteins persist within the CNS and contribute to mild and moderate forms of neuroHIV [24, 25], yet their contributions to neuroendocrine dysfunction and/or age-related neuroHIV remain unclear and understudied.

One HIV protein secreted from CNS reservoirs (predominantly microglia) that has been well-characterized is the trans-activator of transcription (Tat) [26–28]. Tat is present in the cerebrospinal fluid of cART-treated patients and even when patients achieve complete viral suppression peripherally, reservoirs in the brain continue to secrete Tat [24, 25, 29, 30]. Furthermore, anti-Tat antibodies are associated with reduced cognitive deficits among people living with HIV (PLWH) [31]. These data support the potential role of Tat in the pathophysiology of HIV-associated neurological disorders [24, 25, 29, 30]. Given its persistence in the CNS, Tat may contribute to neuroendocrine dysfunction observed in PLWH, partly due to its well-characterized mitotoxic effects. Tat promotes mitochondrial bioenergetic dysregulation, depolarization, and mitophagy, all of which are known to instigate and exacerbate cellular dysfunction in advanced age [32–37]. Tat-induced mitochondrial dysfunction could also negatively impact neuroendocrine function as mitochondria are the rate-limiting organelle for all steroidogenesis. Indeed, Tat alters lipid bioavailability, limits necessary steroid substrates including cholesterol [38, 39], and promotes the accumulation of ceramides which are inhibitors of steroid-synthesizing enzymes [40]. Using transgenic mice that conditionally express Tat protein, we have observed disruption of the neuroendocrine system, including the hypothalamic-pituitary-adrenal (HPA) and -gonadal (HPG) axes in young adult mice as well as HPG dysregulation in middle-aged female mice [36, 41, 42]. Tat-mediated alterations in steroid hormones may contribute to neurotoxicity. In support, exogenous treatment with T, E₂, progesterone (P₄), and 5α-pregnan-3α-ol-20-one (a.k.a., allopregnanolone or alloP) ameliorate Tat-mediated neurotoxicity in cell culture [42–45] and exogenous administration of P₄ or alloP to Tat-transgenic mice attenuates some neuroHIV-like symptomatology [36, 45, 46]. These steroids, particularly alloP, are found to reduce neurodegeneration in several neurological disease states including Alzheimer’s disease [47, 48]. As such, gonadal hormones may be neuroprotective in middle-aged PLWH. However, the functional effects of Tat on the neuroendocrine milieu of aging males is unknown.

Herein, we investigate the combined effects of aging and HIV-1 Tat expression on the development of neuroHIV-like sequelae in young adult (6–8 months) and middle-aged (11–13 months) male mice to determine whether Tat precipitates age-related dysfunction. We hypothesized that conditional Tat expression in transgenic male mice [Tat(+)] would accelerate the development of (i.e., catalyze earlier presentation) or accentuate (i.e., increase magnitude) age-related affective, cognitive, neuromuscular, and neuropathic pain symptomatology compared to age-matched controls [Tat(–)] (Figure 1). We further expected that Tat would alter the production of circulating and central steroids (T, E₂, P₄, corticosterone, or alloP) concurrent with behavioral impairment.

Figure 1. Time-course of behavioral testing. In week 1, Tat protein expression was induced in young adult (6–8 mos. old) and middle-aged (11–13 mos. old) male mice via doxycycline administration (30 mg/kg, i.p. for 5 d) followed by two days of doxycycline washout. In week 2, mice were assessed for affective-like behavior in the open field (OF) and elevated plus-maze (EPM). In weeks 3 and 4, spatial memory performance was assessed in a radial arm water maze (RAWM). In week 5, neuromuscular function was assessed by grip strength and nociceptive/analgesic responding was assessed via the electronic-Von Frey and thermal probe tests.

Results

Aging and Tat expression altered steroidogenesis in male mice

Aging and Tat expression altered circulating gonadal and adrenal steroid hormones. Older age and Tat expression interacted to influence circulating T [F(1,24) = 5.46, p < 0.05; Figure 2A]. Middle-aged Tat(−) mice exhibited greater T than did their respective young adult Tat(−) controls (p = 0.05; Figure 2A, see §) or their respective middle-aged Tat(+) counterparts (p = 0.007; Figure 2A, see ^). Among young adult mice, circulating E₂ was greater in Tat(+) males compared to all other groups [F(1,23) = 34.98, p < 0.05; Figure 2B; see †]. Moreover, the calculated E₂:T ratio (a measure of aromatization) was greatest in young adult Tat(+) mice compared to any other group [F(1,20) = 10.39, p < 0.05], [E₂:T ratio: Tat(−)_{Young Adult} = 0 ± 0 ng/mL, Tat(+)_{Young Adult} = 0.06 ± 0.01 ng/mL, Tat(−)_Middle-aged = 0.02 ± 0.02 ng/mL, Tat(+)_Middle-aged = 0.01 ± 0.01 ng/mL]. E₂ and the E₂:T ratio were notably increased with middle-age; however, this did not reach statistical significance (p = 0.09, n.s.). Circulating P₄ also interacted with age and Tat expression [F(1,27) = 13.16, p < 0.05; Figure 2C]. Middle-aged Tat(−) mice demonstrated the greatest circulating P₄ concentrations of all groups (p < 0.0001 – 0.04; Figure 2C, see ^) and young adult Tat(+) mice demonstrated greater P₄ than their age-matched Tat(−) controls (p = 0.02; Figure 2C, see *. Corticosterone was highest among middle-aged Tat(+) mice versus all other groups [F(1,23) = 4.69, p < 0.05; Figure 2D, see †].

Figure 2. Circulating steroids (ng/mL) among young adult and middle-aged HIV-1 Tat-transgenic male mice [Tat(+)] or their non-Tat-expressing age-matched counterparts [Tat(−)]. (A) Testosterone, (B) estradiol, (C) progesterone, and (D) corticosterone detected in serum. ^§indicated group differs from young adult Tat(−) controls. ^{^}significant interaction wherein indicated group differs from respective Tat(−) controls. ^†indicated group differs from all other groups. ^*main effect for Tat(+) mice to differ from Tat(−) controls, (two-way ANOVA, p < 0.05).

Older age and Tat expression also interacted to influence brain alloP content. We previously observed that Tat expression increased alloP protein content in the whole brain of young male mice, without changes in serum [36]. However, regional analysis of Tat-provoked alloP in the brain has not been conducted heretofore. In hippocampus, older age [F(1,22) = 6.21, p < 0.05; Figure 3A, see †] or Tat expression [F(1,22) = 14.12, p < 0.05; Figure 3A, see *] increased alloP content. In midbrain, age and Tat expression interacted to increase alloP content in middle-aged Tat(+) mice to a greater extent than that seen in all other groups [F(1,24) = 13.71, p < 0.05, Figure 3B, see ‡]. No significant differences were observed in alloP content within the frontal cortex (Figure 3C).

Figure 3. Central steroid allopregnanolone (ng/g) among young adult and middle-aged HIV-1 Tat-transgenic male mice [Tat(+)] or their non-Tat-expressing age-matched counterparts [Tat(−)]. (A) Hippocampal, (B) midbrain, and (C) frontal cortex allopregnanolone (alloP). ^*main effect for Tat(−) mice to differ from Tat(−) controls. ^†main effect for middle-aged mice to differ from young adults. ^‡indicated group differs from all other groups, (two-way ANOVA, p < 0.05).

Aging or Tat increased anxiety-like behavior among male mice

Aging and Tat expression promoted anxiety-like behavior. In the open field, there were main effects for age [F(1,35) = 9.14, p < 0.05; Figure 4A, see †] and Tat expression [F(1,35) = 5.29, p < 0.05; Figure 4A, see *], both of which increased anxiety-like behavior and reduced time spent in the center of the open field compared to young adult or Tat(−) counterparts, respectively. As well, there was an interaction wherein age or Tat genotype increased anxiety-like behavior [F(1,36) = 5.86, p < 0.05; Figure 4B]. Both middle-aged mice and Tat(+) young adult mice made fewer entries into the brightly-lit center of the open field compared to young adult Tat(−) controls (p = 0.001–0.027; Figure 4B, see ^). In the elevated plus-maze, we observed a main effect for Tat [F(1,38) = 13.13, p < 0.05; Figure 4C, see *] to increase the latency to enter the open arms compared to Tat(−) controls. As well, Tat(+) mice demonstrated a notable decrease in the proportion of open arm time compared to Tat(−) mice; albeit, this did not reach statistical significance (p = 0.09; Figure 4D). Notably, motor differences were detected for the distance (cm) [F(1,36) = 16.82, p < 0.05; Table 1] and velocity (cm/s) [F(1,36) = 16.85, p < 0.05; Table 1] traveled in the open field. Middle-aged or Tat(+) mice demonstrated motor deficits and travelled shorter distances (p = 0.0001–0.042), and at lower velocities (p < 0.0001–0.041), than did young adult Tat(−) mice. Within the middle-aged group, Tat(−) males travelled shorter distances (p = 0.03) and at slower velocities than did Tat(+) mice (p = 0.03). No differences were observed in the total arm entries made in the elevated plus-maze (Table 1).

Figure 4. (A–D) Anxiety-like behavior in the open field and elevated plus-maze and (A’–D’) simple linear regressions between circulating and central steroid hormones among young adult and middle-aged HIV-1 Tat-transgenic male mice [Tat(+)] or their non-Tat-expressing age-matched counterparts [Tat(−)]. (A) Time (s) spent in the brightly-lit center of an open field. (B) Numbers of entries made into the center of an open field. (C) Latency (s) to enter the open arms of an elevated plus-maze. (D) The proportional time spent in the open arms of an elevated plus-maze. Regressions between (A’) circulating corticosterone and center time, (B’) hippocampal allopregnanolone and center entries, (C’) circulating corticosterone and latency to enter open arms, and (D’) circulating T and proportional open arm time. ^*main effect for Tat genotype wherein Tat(+) mice to differ from Tat(−) mice. ^†main effect for middle-aged mice to differ from young adult mice. ^{^}significant interaction wherein indicated group differs from young adult Tat(−) controls. Regression lines (solid) are depicted with 95% confidence intervals (dotted), (two-way ANOVA, p < 0.05).

Table 1. Motor behavior (open field and elevated plus maze), raw open arm time (elevated plus maze), grip strength, and proportional body weight and peripheral organ wet weights (mean ± SEM) among HIV-1 Tat transgenic [Tat(+)] young adult and middle-aged male mice and age-matched non-Tat-expressing controls [Tat(−)].

	Young Adult		Middle-Aged
	Tat(−) n = 9	Tat(+) n = 9–12	Tat(−) n = 12	Tat(+) n = 10
Open Field
Distance (cm)	1580.40 ± 164.05	1127.45 ± 147.46^	907.72 ± 99^	1242.34 ± 79.64^*
Velocity (cm/s)	5.28 ± 0.55	3.76 ± 0.49^	3.03 ± 0.33^	4.14 ± 0.27^
Elevated Plus Maze
Open Arm Time (s)	13.10 ± 4.96	11.72 ± 3.75	15.34 ± 4.41	3.86 ± 2.10
Total Arm Entries	29.78 ± 3.15	25.58 ± 3.53	25.00 ± 2.66	31.23 ± 4.80
Grip Strength
Forelimbs alone	0.50 ± 0.02	0.35 ± 0.01^	0.35 ± 0.02^	0.28 ± 0.01^§
Forelimbs and hindlimbs	0.62 ± 0.04	0.50 ± 0.01*	0.54 ± 0.01*#	0.41 ± 0.02*#
Body and Organ Weight
Body Weight (g)	31.76 ± 0.96	32.20 ± 1.29	41.92 ± 1.98‡	30.50 ± 0.90
Brain weight (%)	1.40 ± 0.06	1.40 ± 0.08	1.14 ± 0.12	1.49 ± 0.10
Heart weight (%)	0.50 ± 0.02	0.71 ± 0.07^	0.64 ± 0.07	1.49 ± 0.10†
Liver weight (%)	4.48 ± 0.17	4.32 ± 0.33	5.14 ± 0.62	4.92 ± 0.04
Spleen weight (%)	0.28 ± 0.02	0.22 ± 0.04	0.26 ± 0.03	0.62 ± 0.12‡
*indicates a main effect for Tat(+) mice to differ from Tat(−) mice, irrespective of age. #indicates a main effect for middle-aged mice to differ from young adults, irrespective of genotype. ^indicates a difference from young adult Tat(−) controls. †indicates a difference from young adult Tat(+) mice. §indicates a difference from young adult Tat(+) mice and middle-aged Tat(−) controls.‡indicates a difference from all other groups, p < 0.05.

Affective-like behavior correlated with steroid fluctuations. In the open field, circulating corticosterone (Figure 4A’; Table 2) and frontal cortex alloP (Table 2) were negatively correlated with the time spent in the center of the open field. Similarly, circulating P₄ (Table 2) and hippocampal alloP (Figure 4B’; Table 2) were negatively correlated with open field central entries. P₄ also negatively correlated with the velocity travelled (Table 2). In the elevated plus-maze, circulating corticosterone (Figure 4C’; Table 2) and alloP, within all brain regions examined (i.e., hippocampus, midbrain, frontal cortex; Table 2), positively correlated with the latency to enter the open arms. T levels were also positively correlated with the proportion of time spent on the open arms (Figure 4D’; Table 2).

Table 2. Simple linear regressions between circulating or central steroid hormones and behavioral assessments among HIV-1 Tat transgenic [Tat(+)] young adult and middle-aged male mice and age-matched non-Tat-expressing controls [Tat(−)].

Dependent Variable	Independent Variable	β	t-value (df)	r	R2	F-value (df)	p-value
Open field
Center time	Corticosterone	40.34	2.41 (23)	−0.46	0.21	5.82 (1,23)	0.025
	Frontal cortex alloP	57.21	3.29 (23)	−0.57	0.33	10.80 (1,23)	0.003
Center entries	Progesterone	11.31	2.65 (26)	0.47	0.22	7.00 (1,26)	0.01
	Hippocampal alloP	15.64	2.34 (20)	−0.47	0.22	5.50 (1,20)	0.031
	Frontal cortex alloP	13.84	2.06 (22)	−0.41	0.17	4.23 (1,22)	0.05
Velocity	Progesterone	4.54	2.24 (27)	−0.40	0.16	5.01 (1,27)	0.03
Distance	Progesterone	1360.40	2.25 (27)	−0.40	0.16	5.05 (1,27)	0.03
Elevated plus maze
Latency to open arm	Corticosterone	−58.35	5.72 (24)	−0.76	0.58	32.74 (1,25)	<0.0001
	Hippocampal alloP	−126.87	3.89 (24)	0.62	0.39	15.12 (1,25)	0.001
	Midbrain alloP	−63.86	2.87 (26)	0.49	0.24	8.24 (1,27)	0.01
	Frontal cortex alloP	−66.32	2.47 (26)	0.44	0.20	6.12 (1,27)	0.02
% Open arm time	Testosterone	3.35	2.10 (25)	0.38	0.15	4.29 (1,26)	0.05
RAWM
Reference memory error	Estradiol	5.10	2.56 (24)	0.29	0.08	6.53 (1,74)	0.013
Velocity	Progesterone	7.52	4.72 (28)	0.46	0.21	22.23 (1,86)	<0.0001
	Estradiol	8.12	3.74 (24)	0.40	0.16	14.00 (1,74)	0.0004
	Hippocampal alloP	6.30	3.26 (23)	0.35	0.12	10.65 (1,77)	0.002
	Testosterone	8.26	3.10 (25)	0.33	0.11	9.61 (1,80)	0.003
	Corticosterone	8.01	2.47 (24)	0.28	0.08	6.12 (1,74)	0.02
Distance	Testosterone	365.30	2.11 (24)	0.23	0.05	4.44 (1,80)	0.04
Grip strength
% forelimbs alone	Progesterone	1.33	2.04 (26)	−0.37	0.14	4.17 (1,27)	0.05
% Forelimbs and hindlimbs together	Corticosterone	1.82	−2.03 (21)	−0.41	0.17	4.14 (1,22)	0.05
Nociceptive-like behavior
Thermal hyperalgesia	Hippocampal alloP	42.89	2.54 (23)	0.47	0.22	6.45 (1,24)	0.02
Body and Organs Weight
Brain weight (%)	Testosterone	1.43	2.23 (24)	0.41	0.17	5.00 (1,25)	0.04

Aging or Tat reduced cognitive performance in male mice

Older age and Tat expression interacted to influence latencies to escape a radial arm water maze [F(2,76) = 3.42, p < 0.05; Table 3]. Young adult, Tat(−) mice demonstrated longer latencies to escape than young adult Tat(+) mice (day 2; p = 0.004) and middle-aged groups (day 3; p < 0.001–0.005). Age and Tat expression also interacted to alter the proportional performance improvements from day 1 [F(1,35) = 9.46, p < 0.05; Figure 5A]. Unexpectedly, young adult Tat(−) mice demonstrated less improvement than did young adult Tat(+) (p = 0.022; Figure 5A; see ^) and middle-aged Tat(−) mice (p = 0.018; Figure 5A; see ^). Middle-aged Tat(+) mice demonstrated less improvement than did their age-matched, control counterparts (p = 0.004; Figure 5A; see ‡) or young adult Tat(+) mice (p = 0.006; Figure 5A; see ‡). When assessing the total errors made, age and Tat expression interacted [F(1,76) = 17.95, p < 0.05; Figure 5B]. Among young adult mice, Tat expression increased the number of errors made (p < 0.0001; Figure 5B, see ^). Older age also increased the number of errors made among Tat(−) controls, compared to their young adult counterparts (p < 0.0001; Figure 5B, see ^). Contrary to expectation, middle-aged Tat(+) mice made fewer errors than did their respective Tat(−) controls or their young adult Tat(+) counterparts (p = 0.001–0.006; Figure 5B; see ‡). This may have been related to differences in swim speed. Among the middle-aged group, Tat(+) mice swam faster than their age-matched, or young-adult, Tat(−) counterparts only on day 1 (p < 0.0001–0.002; Figure 5C, see *). Middle-aged mice were faster than their young adult controls on days 2 (p < 0.0001–0.03; Figure 5C, see ||) and 3 (p < 0.0001; Figure 5C, see ^). Fewer errors made by middle-aged Tat(+) mice may be a consequence of increased anxiety and faster latencies to escape (Table 3). In support, despite observing no difference in the distance travelled (Table 3), a significant interaction between age, Tat expression, and day of testing was observed for swim speed [F(2,78) = 5.82, p < 0.05; Figure 5C]. Among young adults, Tat(+) mice swam faster than their Tat(−) counterparts over all three days (p < 0.0001–0.0003; Figure 5C, see ^).

Table 3. Latency to escape (s) and distance travelled (cm) in a radial arm water maze among HIV-1 Tat transgenic [Tat(+)] young adult and middle-aged male mice and age-matched non-Tat-expressing controls [Tat(−)].

Latency to Escape (s)	Young adult		Middle-aged
	Tat(−) n = 8–9	Tat(+) n = 12	Tat(−) n = 12	Tat(+) n = 10
Day 1	47 ± 6	36 ± 3	49 ± 6	37 ± 4
Day 2	49 ± 8	27 ± 2*	35 ± 6	37 ± 4
Day 3	50 ± 5	25 ± 3^	31 ± 6^	33 ± 4^
Distance (cm)
Day 1	372 ± 31	386 ± 29	410 ± 30	387 ± 30
Day 2	318 ± 32	320 ± 27	334 ± 37	380 ± 31
Day 3	349 ± 48	271 ± 23	307 ± 29	298 ± 31
*indicates an effect for young adult Tat(+) mice to differ from young adult Tat(−) mice. ^indicates a difference from young adult Tat(−) controls. p < 0.05.

Figure 5. (A, B) Spatial memory performance and (C) swim speed in a radial arm water maze and (B’, C’’) simple linear regressions for circulating and central steroid hormones among young adult and middle-aged HIV-1 Tat-transgenic male mice [Tat(+)] or their non-Tat-expressing age-matched counterparts [Tat(−)]. (A) Proportion of mice that exhibited improvement from day one performance (latency to escape). (B) Total frequency of errors. (C) Swim speed (cm/s). Simple linear regressions between (B’) circulating estradiol and frequency of errors, (C’) circulating corticosterone and swim speed, and (C’’) progesterone and swim speed. ^*main effect for Tat(+) mice to differ from Tat(−) mice. ^{^}interaction effect wherein indicated group differs from young adult Tat(−) controls. ^||indicates middle-aged differs from young-adult groups. ^‡indicated middle-aged Tat(+) groups differs from middle-aged Tat(−) and young adult Tat(+) mice. Regression lines (solid) are depicted with 95% confidence intervals (dotted), (repeated measure ANOVA, p < 0.05).

Radial arm water maze performance correlated with circulating and central steroid levels. Circulating E₂ was positively correlated with the total reference memory errors (Figure 5B’; Table 2). Corticosterone (Figure 5C’; Table 2), P₄ (Figure 5C’’; Table 2), E₂ (Table 2), hippocampal alloP (Table 2), and T (Table 2) were positively correlated with swim speed. T was negatively correlated with the total distance travelled (Table 2).

Aging and Tat reduced neuromuscular strength among male mice

Advanced age and Tat interacted to reduce grip strength when assessed either by forelimbs alone (normalized to body weight) [F(1,37) = 13.68, p < 0.05; Figure 6A] or by forelimbs and hindlimbs together (normalized to body weight) [F(1,36) = 7.00, p < 0.05; Figure 6B]. In either measure, middle-aged or Tat(+) mice were weaker than young Tat(−) controls (p < 0.0001–0.004, Figure 6A, 6B, see ^). As well, middle-aged Tat(−) mice were weaker than young adult Tat(+) mice (p = 0.02–0.03, Figure 6A, 6B, see †). Grip strength assessment without body weight normalization yielded comparable effects (Table 1). There was an interaction between age and Tat exposure when assessing forelimbs alone [F(1,37) = 9.24, p < 0.05; Table 1] such that all groups were weaker than young adult Tat(−) controls (p < 0.0001). As well, there was a “stair-step” effect wherein middle-aged Tat(+) mice were weaker than their own middle-aged Tat(−) controls (p = 0.002) or young adult Tat(+) mice (p = 0.002). There were also main effects for age [F(1,37) = 12.14, p < 0.05; Table 1] or Tat exposure [F(1,37) = 25.64, p < 0.05; Table 1] to reduce combined forelimb and hindlimb grip strength.

Figure 6. (A, B) Grip strength, (C) mechanical allodynia, and (D) thermal hyperalgesia and (B’ and D’) simple linear regression for circulating and central steroid hormones among young adult and middle-aged HIV-1 Tat-transgenic male mice [Tat(+)] or their non-Tat-expressing age-matched counterparts [Tat(−)]. Grip strength threshold for (A) forelimbs alone or (B) forelimbs and hindlimbs together. (C) Paw-withdrawal threshold (g) in an electronic Von Frey test. (D) Paw-withdrawal latency (s) in a thermal probe test. Simple linear regressions between (B’) circulating corticosterone and forelimbs and hindlimbs together or (D’) hippocampal allopregnanolone and paw-withdrawal latency. ^*main effect for Tat(+) mice to differ from Tat(−) mice. ^†main effect for middle-aged mice to differ from young adults. ^{^}significant interaction wherein indicated group differs from young adult Tat(−) controls. Regression lines (solid) are depicted with 95% confidence intervals (dotted), (two-way ANOVA, p ≤ 0.05).

Grip strength was also correlated with circulating steroids. Corticosterone was negatively correlated with neuromuscular function as assessed by the forelimbs and hindlimbs together (Figure 6B’; Table 2) and P₄ negatively correlated with neuromuscular function as assessed by the forelimbs alone (Table 2).

Aging and Tat altered nociceptive tolerance in male mice

Older age and Tat expression interacted to influence the threshold for paw withdrawal in response to a mechanical stimulus [F(1,37) = 5.87, p < 0.05; Figure 6C]. Unexpectedly, middle-aged Tat(−) and young adult Tat(+) mice exhibited a greater mechanical threshold than did young adult Tat(−) controls (p = 0.02–0.03; Figure 6C, see ^). No differences were observed in thermal hyperalgesia among middle-aged or Tat-exposed mice (Figure 6D); however, hippocampal alloP was positively correlated with the latency to paw withdrawal from a thermal stimulus (Figure 6D’; Table 2).

Aging and Tat influenced body and peripheral organ weight

Aging and Tat expression interacted to influence body weight [F(1,37) = 17.00, p < 0.05; Table 1] such that middle-aged Tat(−) mice were heavier than all other groups (p = 0.0001–0.004). There was also an interaction effect between age and Tat to increase proportional heart wet weights [F(1,25) = 8.41, p < 0.05; Table 1] wherein young adult Tat(+) mice had greater proportional mass than their age-matched Tat(−) counterparts (p = 0.011) or middle-aged Tat(+) mice (p = 0.04). The proportional spleen wet weight [F(1,24) = 12.33, p < 0.05; Table 1] was greatest in middle-aged Tat(+) mice compared to all other groups (p = 0.0001–0.0003). We did not observe differences in the proportional weights of brain or liver (Table 1). However, circulating T was positively correlated with proportional brain wet weight (Table 2).

Abbreviations

HIV-1: human immunodeficiency virus type-1; cART: combined antiretroviral therapy; T: testosterone; E₂: estradiol; Tat: trans-activator of transcription; HPA: hypothalamic-pituitary-adrenal; HPG: hypothalamic-pituitary-gonadal; P₄: progestogen; a.k.a., allopregnanolone or alloP: 5α-pregnan-3α-ol-20-one; eVF: electronic Von Frey; RAWM: radial arm water maze; ELISA: enzyme-linked immunosorbent assay.

Author Contributions

JJP, NMA, and MJK conceptualized the project. ANQ, JJP, NMA, and MJK garnered funding for the project. NMA oversaw behavioral work conducted in the UM Neuropharmacology Core. ANQ, JJP, and NMA analyzed the data. ANQ and FM collected data. ANQ first drafted and revised the manuscript. All authors revised the manuscript and approved the final version.

Acknowledgments

We appreciate the technical support from the University of Mississippi Neuropharmacology and DMPK Cores.

Conflicts of Interest

The authors declare no conflicts of interest related to this study.

Funding

This work was supported by funds from the University of Mississippi (Graduate Student Council award to ANQ) and the National Institutes of Health: R00 DA039791 (JJP), R01 DA052851 (JJP and NMA), R01 DA039044 (MJK), and an administrative supplement from award P30 GM122733 (pilot project to JJP and MJK).

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