Introduction
Combination antiretroviral therapy (cART) has caused a dramatic decline in human immunodeficiency virus (HIV)-associated dementia and mortality. However, the overall prevalence of more moderate motor and cognitive deficits, collectively termed HIV-associated neurocognitive disorders (HAND), has remained similar in cART treated patients (Robertson et al.
2007; McArthur et al.
2010; Heaton et al.
2011). In resource-limited countries, where estimates are that only 36 % of the HIV-positive population receives cART, the severity and/or prevalence of neurocognitive disorders is higher (Nakasujja et al.
2005; Wong et al.
2007; Joint United Nations Programme on HIV/AIDS
2010a; Heaton et al.
2010; Tozzi et al.
2005). HAND occurs even in aviremic, cART-treated individuals, suggesting that many anti-retroviral regimens fail to reverse neurological damage, even while lengthening survival (Heaton et al.
2010,
2011; McArthur et al.
2010).
Approximately half of the 40 million persons infected with HIV-1 worldwide are women (Joint United Nations Programme on HIV/AIDS
2010a). The incidence of HIV is higher in men compared with women in all ethnic groups in the US (Joint United Nations Programme on HIV/AIDS
2010b; Centers for Disease Control and Prevention
2009) and Europe (European Centre for Disease Prevention and Control/WHO Regional Office for Europe
2011), and most studies on HAND epidemiology in western countries focus on males. Studies both pre- and post-cART have reported no difference in rate of HIV-associated neurocognitive complications between sexes in the United States (Robertson et al.
2004; Bouwman et al.
1998). In other studies, the pre- and post-cART risk of similar complications in both developed and resource-limited regions was reportedly higher in females (Wojna et al.
2006; Chiesi et al.
1996; Gupta et al.
2007; Hestad et al.
2012). In contrast, Joska (Joska et al.
2011) found the male genotype to be associated with HIV-related cognitive dysfunction, and Liu (Liu et al.
1996) reported that male HIV
+ injection drug abusers had higher cognitive impairment rates. A recent survey of HIV status among patients in the National Epidemiological Survey on Alcohol and Related Conditions found HIV
+ males but not HIV
+ females had higher rates of mood, anxiety, and personality disorders than their same-sex HIV
− counterparts, even when adjusted for socio-demographic factors (Lopes et al.
2012). Disparities between the sexes in health care services and access to HIV treatment may be confounds, as globally, HIV-infected women are more likely to live in poverty, and have lower literacy levels, higher injection drug abuse rates, and poorer mental health. These considerations might significantly influence either CNS vulnerability to HIV, or a diagnosis of neurocognitive disability (Basso and Bornstein
2000; Farinpour et al.
2003; Maki and Martin-Thormeyer
2009).
We utilized a mouse model with conditional expression of HIV-1 Tat1–86 in the CNS to test whether biological sex can influence HIV-related motor and cognitive outcomes. Intriguingly, males showed more impairment in motor, learning, and anxiety tests. Since multiple brain regions are undoubtedly involved in such varied deficits, we specifically examined striatum, which is clearly impacted by HIV and is also involved as a coordinating center in multiple motor, memory, and motivational/emotional tasks and behaviors. At a cellular level, Tat induction significantly altered non-neuronal populations in both sexes. Importantly, males had greater increases in astroglia and activated microglia, and increased TUNEL+ neurons. Dendritic spine density was decreased more significantly in Tat+ males, and both inhibitory and excitatory pre- and post-synaptic proteins were more significantly altered.
The substantial changes in synaptic organization observed in male mice after chronic HIV-1 Tat exposure may make males more vulnerable to motor and social/cognitive behavioral impairment. We propose that HIV-1 Tat differentially affects aspects of inflammation and glial reactivity/remodeling in males and females and that these factors may underlie sex differences in synaptic reorganization and behavioral impairment in Tat transgenic mice, and perhaps also in neuroAIDS patients.
Materials and methods
Animal studies were approved by the Institutional Animal Care and Use Committee at Virginia Commonwealth University.
Animals
Doxycycline (DOX)-inducible HIV-1
IIIB Tat
1–86 transgenic mice were generated as described (Hauser et al.
2009; Fitting et al.
2010b,
2013; Bruce-Keller et al.
2008). Since
tat transgene activity is controlled by the glial fibrillary acidic protein (GFAP) promoter, Tat protein expression in the CNS is limited to astroglia. All mice were genotyped to confirm the presence of
tat and
rtTA transgenes. Chronic CNS Tat expression was induced by feeding chow containing DOX (Harlan Laboratories, Inc., Indianapolis, IN; 6 g/kg) to mice starting at 3 months of age for a 12-week period. Control Tat
− mice were also fed DOX-chow to control for off-target drug effects. In some behavior studies (grip strength, open field test, light/dark box test), other mice of the same age and genotype received normal chow through the experimental period so that data could be normalized. All mice were fed ad libitum. Mice were habituated to the study room for 60 min before all behavior tests. GFAP transcription is sensitive to levels of many hormones and growth factors, yet basal levels of GFAP transcription using such promoters were reported to be roughly similar in males and females in several studies (Cordeau et al.
2008; Cho et al.
2009; Lundkvist et al.
1999). Doxycycline pharmacokinetics in males and females are also similar (Binh et al.
2009).
Rotarod assay
An accelerating rotarod assay was used to evaluate motor performance/coordination and motor memory (Shiotsuki et al.
2010; Brooks and Dunnett
2009; Liu et al.
2010; Jones and Roberts
1968), which are adversely affected in HAND (Tozzi et al.
2005; Goodkin et al.
1997; Simioni et al.
2010). All mice were naïve to the apparatus until the first test day. The Rotamex-5 treadmill (Columbus Instruments, Columbus, OH) consists of a rubber-surfaced, 3.0-cm diameter cylindrical treadmill connected to a computer-controlled stepper motor-driven drum. For each trial, mice were placed in individual compartments; the rod was gradually accelerated from 1 to 40 rpm, in 1 rpm increases per 15 s. Acceleration continued until 40 rpm was reached or the animal fell from the rod. Falls were detected by sensors at the bottom of each compartment. Rotarod testing began after 1 week DOX treatment, and was performed once per week for 4 weeks (13–16 weeks of age). After a 4-week break, testing was resumed during the third month of DOX treatment (21–24 weeks of age).
Forelimb grip strength test
Forelimb grip strength was measured as a second indication of potential motor deficits (Chatillon
® DFE II grip strength meter, Ametek Test and Calibration Instruments, Largo, FL). Mice were held near the tail base and lowered toward the apparatus until the bar was gripped firmly with both forepaws. The mouse was then gently, but steadily, pulled away from the bar until both forepaws released. Peak force disturbance was automatically registered in grams-force (gf). Each mouse was tested five times in quick succession; the strongest measurement was taken as the score (reviewed by Crabbe et al.
2003). Grip strength was normalized by body weight (g) for statistical comparison.
Open-field and light/dark box testing
Open field and light/dark box tests are based upon the rodent’s innate conflict between an aversion to exposed spaces and tendency to explore novel environments (Wallace et al.
2008; Cryan and Holmes
2005). The open field test measures changes in overall locomotion and may indicate states such as depression/anxiety that reduce willingness to explore. The test apparatus is a box (30 × 30 × 15 cm) divided into nine squares (Med Associates Inc., St. Albans, VT), enclosed in a larger, sound-attenuating box equipped with overhead lighting and ventilation. In the open-field test, a mouse naïve to the test apparatus was placed at its center and habituated for 1 min. Activity over 10 min was then monitored by 16 infrared beam sensors along the
X‐
Y plane (Ramezani et al.
2011; Burger et al.
2005). In the light–dark box adaptation (Crawley and Goodwin
1980; Malmberg-Aiello et al.
2002), the test apparatus described above is separated into two equal compartments by a partition with an opening in the middle; a black plastic roof covers one compartment. Anxious animals tend to avoid exposure by spending more time in the dark (Bourin and Hascoet
2003). Each mouse was placed in the center of the lighted area (facing the darkened area) and allowed to explore the novel environment for 10 min. Activity was monitored as in the open field test. The number of squares crossed in both light and dark regions was recorded for 10 min. For both tests, data from 6-month-old mice that received 12 weeks of DOX treatment were normalized by scores from control age-matched groups fed normal chow.
Stereology
Mice were deeply anesthetized with isoflurane (Baxter, Deerfield, IL, USA) prior to perfusion with 4 % paraformaldehyde (pH 7.4, Sigma-Aldrich Co., St. Louis, MO) in phosphate-buffered saline (PBS). After perfusion, brains were immediately removed and post-fixed in fresh fixative overnight, hemisected, rinsed several times, and left overnight in 15 ml of PBS. The left brain halves were coronally sectioned at 50 μm and stored individually in cryoprotectant [30 % sucrose (w/v), 1 % polyvinylpyrrolidone (v/v), 30 % ethylene glycol (v/v) in 0.05 M phosphate buffer, pH 7.2, all from Sigma] at −20 °C until use. Free-floating sections containing the striatum were stained with Hoescht 33342 (1 µg/1 ml, 8 min, Molecular Probes Inc., Eugene, OR) for all unbiased stereological estimation. Sections were thoroughly rinsed and then mounted on gelatin-coated SuperFrost Plus slides (VWR Scientific, West Chester, PA) in ProLong Gold anti-fade reagent (Life Technologies, Grand Island, NY, USA), and then air-dried for ≥8 h in the dark. The total number of (Hoescht
+) cells in the striatum was estimated using the optical fractionator method (West et al.
1991); the Cavalieri principle was applied to measure striatal volume with assistance from a computerized stereology system (Stereologer, Stereology Resource Center, Chester, MD) (Mouton
2002). Every fifth section was selected from the total sections through the striatum and analyzed to estimate total cell number and striatal volume. Each reference space was outlined at low power (5×), and cells were counted using 100× magnification under oil-immersion. A guard volume of 2.0 μm was used during cell counting to avoid sectioning artifacts, including lost caps and uneven section surfaces. The slides were viewed on a Zeiss AxioObserver system with integrated Sony 3CCD Exwave HAD camera system (Carl Zeiss, Inc., Thornwood, NY).
Immunohistochemistry and quantification
Perfusion and preparation of tissue sections were performed as described for stereology. 10 μm frozen or 50 μm free-floating sections were permeabilized with 0.2 % Triton X-100 in phosphate-buffered saline containing 1 % bovine serum albumin (Sigma-Aldrich) for 30 min. To assess proportional numbers of specific cells in the striata, single- or double-label immunostaining was performed. Primary antibodies specific for neuron-specific enolase (NSE; 1:100, Abcam, Cambridge, MA), neuron nuclei (NeuN; 1:200, Millipore, Temecula, CA), oligodendrocyte transcription factor 2 (Olig2; 1:100, Immuno-Biological Laboratories, Minneapolis, MN), aldehyde dehydrogenase family 1 member L1 (ALDH1L1; 1:500, Abcam), ionized calcium binding adaptor molecule 1 (Iba-1; 1:200, Wako, Osaka, Japan), and 3-nitrotyrosine (3-NT, 1:100, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were applied to sections. For double immunostaining, individual primary antibodies were sequentially applied and incubated overnight at 4 °C in a humidified chamber, followed by host-matched fluorescent-conjugated secondary antibodies (1 h, room temperature; Life Technologies). Immunostained sections were then incubated with Hoechst 33342 dye (1 μg/ml, 8 min; Life Technologies) to identify nuclei, rinsed thoroughly, mounted in ProLong Gold anti-fade reagent (Life Technologies), and then air-dried for ≥8 h in the dark. To verify the proportion of neurons, oligodendrocytes, astrocytes and microglia in the striata, 300–350 Hoechst+ cells were selected randomly per striatum and assessed for NSE, Olig2, ALDH1L1, and Iba-1 expression, respectively (n = 5–7). To determine the relative activation state of microglia, an independent group of 200 Iba-1+ cells were randomly selected per striatum and assessed for 3-NT expression. Striata were examined under oil immersion at 100× using a Zeiss AxioObserver system with integrated MRm camera system.
Striatal neuron death
Neuron death was assessed by labeling for terminal deoxynucleotidyl transferase-mediated UTP nick end-labeling (TUNEL) followed by NeuN and Hoechst staining. A monoclonal antibody to NeuN and Hoechst 33342 (Life Technologies) were applied to 10 μm frozen coronal sections that included the striatum from all DOX treated groups as described above. TUNEL detection of in situ DNA fragmentation was performed in accordance with procedures in the In Situ Cell Death Detection Kit, TMR red (Roche Applied Science, Indianapolis, IN) in the same slides. To quantify active neuron death, the proportion of cells that were both TUNEL+ and NeuN+ was determined by counting at least 200 NeuN+ cells in adjacent fields in two different sections per animal (n = 6), at 63× magnification. To determine neuron death, 200 NeuN+ cells were randomly selected for assessment of TUNEL (n = 6).
Immunoblotting
In vivo astrocytic markers and pre-/postsynaptic vesicle-associated proteins were examined by immunoblotting in striatal samples from all DOX treated groups (n = 6). Striata were freshly harvested and homogenized (T-PER Reagent, Thermo Scientific, Pittsburgh, PA), including a protease inhibitor cocktail (Roche Applied Science). Homogenized tissue lysates were centrifuged and then stored at −80 °C until use. Protein concentration of each sample was measured using the BCA protein assay (Pierce, Rockford, IL). 20 μg of lysates were loaded per well onto 4–20 % Tris–HCl Ready Gels (Bio-Rad Laboratories, Hercules, CA), and Precision Plus Protein Dual Color Standards (Bio-Rad; MW range 10–250 kDa) were used to visualize protein transfer and determine molecular weight. Proteins were transferred to PVDF membranes (Bio-Rad). Antibodies to astrocyte (ALDH1L1, 1:1000, Abcam; GFAP, 1:1000, Millipore) and pre-/postsynaptic [synaptotagmin 2 (Syt2), 1:100, Zebrafish International Resource Center; synapsin (Syn), 1:1,000, Synaptic Systems; gephyrin (Geph), 1:1,000, Synaptic Systems,; post synaptic density protein 95 (PSD-95), 1:3,000, Affinity BioReagents] were used to probe the blots. Anti-GAPDH (1:2,500, Abcam) was used to normalize protein loading. Host-matched IRDye® Infrared Dyes-conjugated secondary antibodies (LI-COR Biotechnology, Lincoln, Nebraska) were applied to visualize each protein band. Protein bands were detected on an Odyssey® Infrared Imaging System and intensity was analyzed by Odyssey 2.0 software (LI-COR).
Morphology of striatal cells
Medium spiny neuron morphology was also assessed in tissues impregnated using a Golgi–Kopsch procedure (Hauser et al.
2009; Fitting et al.
2010b). Briefly, brain tissues were separately prepared from all groups after 10–16 weeks DOX administration (
n = 6). Dendrites were assessed in tissues subjected to a modified Golgi–Kopsch procedure that impregnates cell processes of random neurons and glia in their entirety (Hauser et al.
1989,
2009). After 4 % paraformaldehyde perfusion as described above, whole forebrains were isolated and immersed in 2 % potassium dichromate and 5 % glutaraldehyde (v/v) in the dark at room temperature, and silver-impregnated as previously published (Fitting et al.
2010b,
2013). The ratio of dichromate solution to tissue volume was ≥50:1. After 6 days, tissues were gently washed in ultrapure water (3 × 1 min), tissues were gently blotted to remove excessive dichromate solution, and placed in aqueous 0.75 % silver nitrate for 5 days in the dark (50:1 fluid to tissue volume ratio). Intact forebrains were serial-sectioned on a vibrating microtome (Leica VT1200S, Leica Biosystems, Nussloch, Germany) at 110 μm in the coronal plane, dehydrated through graded ethanols, cleared in xylene, and mounted in Permount (Fisher Scientific, Waltham, MA). Wooden tools were used in all tissue handling. Several criteria were used to select dendrites for quantification: (1) the cell must be fully impregnated throughout its entirety (partially/incompletely impregnated cells were not assessed); (2) the dendrite must be parallel to the plane of the section, as tilted/angled dendrites can foreshorten the dendrite and influence density; (3) the dendrite must be distinct from dendrites on other neurons. Dendrites were considered pathologic when they were either stunted or extremely thin, or had spines that were unevenly distributed or stunted (Fig.
7A, E, F) (Fitting et al.
2010b; Ferrante et al.
1991; McNeill et al.
1988). The density of Golgi-impregnated spines was assessed on third order dendrites (3 dendrites per neuron; 6 neurons per mouse;
n = 6 mice). In some neurons in male mice (but not females), all of the dendrites had gross decreases in spine density (≤5 spines per 10 mM) that were obvious even without counting (Fig.
7F). We purposely left these grossly abnormal neurons out of the measurements of average spine density so as not to unduly bias the results. Instead, those neurons were included in the counts of cells with pathologic dendrites (Fig.
7A).
Electron microscopy
Male transgenic mice exposed to Tat for 2.5 months (
n = 3) were perfused with 2 % paraformaldehyde/2 % glutaraldehyde in phosphate buffer (pH 7.4, Sigma-Aldrich Co.), and then post-fixed in 1 % osmium tetroxide (OsO
4) for 1 h. For dehydration, the fixed tissues were processed through graded ethanols and infiltrated overnight in EMbed 812 (EMS, Hatfield, PA). Tissue was embedded in EMbed 812 and polymerized at 60 °C for 1–2 days. Thin sections of 600–700 Å thickness were cut on a Leica EM UC6i ultramicrotome (Leica Microsystems), collected onto formvar-coated grids, and stained with 5 % uranyl acetate and Reynolds’s lead citrate (Reynolds
1963). Sections were observed with a JEOL JEM-1230 TEM (JEOL USA, Inc.) at 2,000–12,000× magnification and images were obtained using a Gatan Ultrascan 4000 digital camera (Gatan Inc., Pleasanton, CA) with DigitalMicrograph™ software (Gatan Inc.).
Statistical analysis
Analyses were done by one way, or main effect analysis of variance (ANOVA) followed by Duncan’s post hoc testing, and t test using Statistica 6.0 software (Statistica, Tulsa, OK). Student's t-test analyses were performed using GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA).
Discussion
In the post-cART era, neuroAIDS has become a chronic disease largely characterized by an increased prevalence of milder cognitive and motor disorders (HAND) with less encephalitis (Antinori et al.
2007; McArthur et al.
2010; Sacktor et al.
2001; Gonzalez-Scarano and Martin-Garcia
2005; Woods et al.
2009; Ellis et al.
2007; Wojna et al.
2006; Dore et al.
2003; Lawrence and Major
2002; Grant et al.
1995; Heaton et al.
1995). Although the subset of female HIV patients has expanded, the question of whether sex influences the occurrence and characteristics of neurocognitive dysfunction in HIV
+ patients is largely unexplored. Studies on HAND epidemiology in western countries, where the disease is more prevalent in men, focus largely on males. In more resource-limited settings, social variables between the sexes that affect health care access and quality make sex-related disease vulnerabilities difficult to determine. A recent study suggests that overall rates of neuropsychological impairment may not reflect the entire picture; HIV
+ men and women may be differentially impaired in certain attention/memory tasks yet show a similar overall decline (Failde-Garrido et al.
2008).
Inducible Tat-transgenic mice are a reasonable model for testing whether sex influences HAND development since they exhibit many neuropathologic and behavioral deficits seen in HIV patients with HAND, including gliosis and microglial activation (Bruce-Keller et al.
2008), dendritic abnormalities and reduced dendritic spine density (Fitting et al.
2010b,
2013; Hauser et al.
2009), disrupted hippocampal circuitry, changes in synaptic proteins (Fitting et al.
2013), and learning/behavioral deficits (Fitting et al.
2013; Carey et al.
2012). The transgenic line used here, developed by Dr. Avi Nath, appears to exhibit less CNS pathology than a similar line (Zou et al.
2007; Kim et al.
2003) and was chosen because the pathology has a slower onset more representative of that in HIV patients. While most in vivo studies have used HIV-1 Tat exposure times of ≤2 weeks, we chose a 3-month exposure as potentially more reflective of persistent human disease. In a variety of well-characterized animal behavior tests, chronic Tat exposure negatively affected motor, cognitive, and anxiety measures, in general agreement with outcomes after more acute exposure (Fitting et al.
2013; Carey et al.
2012,
2013), and mirroring cortical and subcortical functional deficits in HIV patients that affect both cognitive and motor skills (Woods et al.
2009; McArthur et al.
2010; Heaton et al.
2010). Importantly, our results also provide initial evidence of an underlying difference in male versus female vulnerability. In all instances where severity differed between sexes, Tat
+ male mice had poorer outcomes, while Tat
+ females were similar to control/Tat
− mice.
CNS tissue was evaluated to determine if cell populations were altered in a similar sex-specific manner, perhaps accounting for sex-specific behavioral changes. Since multiple brain regions are undoubtedly involved in these quite varied behavior deficits, we specifically examined the dorsal striatum, which is clearly impacted by HIV, and as the main input region for the basal ganglia is also involved in coordinating multiple behaviors. While the striatum is well known to control and modulate movements and tasks involving motor memory, striatal integration is also a key component in executive functions and decision making, and in processing motivational and emotional information relating to reward and anxiety (Voytek and Knight
2010; Balleine et al.
2007; Helfinstein et al.
2012). Unbiased stereological analyses indicated that Tat exposure did not affect either striatal volume or total striatal cell number in either sex. When individual populations of cells were quantified, there were effects of both sex and Tat exposure (Figs.
4,
5). Tat expression increased the percentage of apoptotic (TUNEL
+) neurons, with slightly greater effect on male brains. The increased neuron loss was not reflected in total NSE
+ numbers. We interpret this to suggest that neurons may only have started to die relatively recently, which is in keeping with our finding that TUNEL
+ striatal neurons were not increased after short-term Tat exposure in these mice (Bruce-Keller et al.
2008). Over time, if a low rate of neuron death continues in the absence of replacement, the neuron population will eventually be reduced by a significant level . Tat expression also reduced Olig2
+ oligodendroglia independent of sex, perhaps relating to reports that HIV-1 Tat reduces young oligodendrocyte proliferation in vitro (Hahn et al.
2012).
In the absence of neuron loss, what might account for observed behavioral deficits? Since we only examined total neurons, changes in subpopulations of striatal neurons affecting behavior may have been overlooked. Another possibility relates to changes in glial populations (Figs.
5,
6). Chronic exposure to Tat expanded the CNS population of ALDH1L1
+ astroglia, as previously observed for GFAP
+ astroglia after acute Tat exposure (Bruce-Keller et al.
2008). Cell counts and immunoblots generally concurred that astroglial expansion/astrogliosis was greater in males. As in more acute studies (Gupta et al.
2010; Hahn et al.
2010; Fitting et al.
2010c; Suzuki et al.
2011; Bruce-Keller et al.
2008), the microglial population also increased after chronic Tat exposure. Both sexes showed equivalent Iba-1
+ cell increases, but 3-NT, a nitrosative product indicative of microglial activation, was specifically elevated in males. Thus, both microglial activation and astrogliosis correlate with altered behavior in male Tat
+ brains. While not proving a causal relationship, the sustained, low levels of inflammation described in HIV
+ brains even after cART treatment do produce cumulative deficits (Heaton et al.
2010,
2011; Cohen et al.
2010; Gongvatana et al.
2013) and would predict this outcome. Additionally, or alternatively, the relative deficits in male behavior might derive from their more significant abnormalities in dendrite structure and levels of specific synaptic proteins (Figs.
7,
8). Chronic Tat exposure reduced the density of spines on striatal medium spiny neurons in both sexes, similar to other reports with Tat, gp120 or HIV (Sa et al.
2004; Fitting et al.
2010b; Viviani et al.
2006). The pattern of loss was remarkably similar to spine changes reported with cortical deafferentation (Cheng et al.
1997), suggesting interruption of normal cortical connections that account for over 80 % of striatal input. The fact that spine loss recovers after a deafferentation injury (Cheng et al.
1997) may be a hopeful sign for the recovery potential of HIV patients. Both spine loss and abnormal dendrite pathology were clearly higher in male mice, which also showed changes in proteins associated with both inhibitory (presynaptic Syt2, postsynaptic gephyrin) and excitatory (presynaptic synapsin, postsynaptic PSD95) synapses. In females, only the excitatory presynaptic protein synapsin was modestly reduced. While HIV exposure may reduce synaptodendritic efficacy in general (Masliah et al.
1997; Everall et al.
1999; Fitting et al.
2013), Tat interactions may specifically affect how efficiently inhibitory versus excitatory information is processed, at least in striatum (Figs.
7,
8) and hippocampus (Fitting et al.
2013). Sex-specific protein alterations may thus contribute to differences in behavioral outcomes. Notably, reversible changes in excitatory synaptic proteins have been reported in vitro (Shin and Thayer
2013; Shin et al.
2012). Changes in synaptic efficiency likely occur before neuron loss, since synaptic protein changes occurred in hippocampus after more acute (10 days) Tat exposure (Fitting et al.
2013).
An extensive literature, both epidemiological and experimental, suggests that males are more vulnerable to trauma and neurodegenerative events/diseases (de Lau and Breteler
2006; Van Den Eeden et al.
2003; Coronado et al.
2011; Mehal et al.
2013). The lower vulnerability of females may be related to the documented neuroprotective role of estrogen in trauma and neurodegenerative studies (Yune et al.
2008; Rau et al.
2003; Roof and Hall
2000; D’Astous et al.
2004; Hoffman et al.
2006; Tang et al.
1996), although recent work emphasizes that sex as an injury variable includes more than single hormone-single receptor interactions, and involves both genomic and non-genomic effects (Cheng and Hurn
2010; Herson et al.
2009). Although a preponderance of evidence suggests that some component of estrogen-mediated protection derives from down-regulation of dopamine transporter function in the striatum (Wallace et al.
2006; Disshon and Dluzen
1999; Murray et al.
2003), other mechanisms are likely involved in striatum and elsewhere, including expression of endogenous anti-oxidants such as paraoxonase 2 (Giordano et al.
2013) and glutathione (Kumar et al.
2011), as well as anti-oxidant enzyme systems (Kumar et al.
2011; Rao et al.
2011). Recent evidence also suggests that a component of estrogen-mediated neuroprotection may be due to promotion of adaptive responses within neurons, and that males and females can respond differently to locally produced steroids versus those produced or delivered systemically (Gillies and McArthur
2010). Estrogens or ER-β signaling has been shown to exert neuroprotective effects on several HIV models including gp120 injection (Corasaniti et al.
2005), and in vitro Tat or gp120 exposure (Russo et al.
2005; Adams et al.
2010,
2012; Wallace et al.
2006; Brooke et al.
1997) potentially by modulating the balance of protective and inflammatory chemokine/cytokines (Corasaniti et al.
2005; Kipp and Beyer
2009; Dhandapani et al.
2005; Bruce-Keller et al.
2007), or reducing oxidative changes in mitochondria (Simpkins et al.
2010). A cautionary note for all HIV studies is the potential for sex steroids, including progestins, to modulate infective processes (Asin et al.
2008; Zhang et al.
2008; Hel et al.
2010; Lee et al.
1997), which may complicate comparisons between epidemiological and experimental studies.
In conclusion, the data support the hypothesis that chronic exposure to HIV-1 Tat may play a significant role in the decline of cognitive and motor function in HIV patients, perhaps related to or driven by changes in specific cell populations. This study has examined cellular changes specifically within the striatum, but the behavioral deficits observed almost certainly will involve pathology in other CNS regions. Sex appears to be an important variable in predicting vulnerability to behavioral deficits, with males being significantly more affected in several standard rodent tests of motor and social/cognitive skills. Males also showed an enhanced state of glial activation and more evidence of synaptic damage, both of which have been related to cognitive and motor impairments in HIV patients. Importantly, Tat will be produced and released by residually infected CNS cells even when new infection is limited by cART (Johnson et al.
2013), suggesting its importance in determining the stability of CNS cell populations and motor and cognitive health during chronic infection.