|Home | About | Journals | Submit | Contact Us | Français|
HIV-associated neurologic disease (HAND) still causes significant morbidity, despite success reducing viral loads with combination antiretroviral therapy (cART). The dopamine (DA) system is particularly vulnerable in HAND. We hypothesize that early, “reversible” DAergic synaptic dysfunction occurs long before DAergic neuron loss. As such, aging HIV-infected individuals may be vulnerable to other age-related neurodegenerative diseases like Parkinson’s Disease (PD), underscoring the need to understand shared molecular targets in HAND and PD. Previously we reported that the neurotoxic HIV-1 transactivating factor (Tat) acutely disrupts mitochondrial and endoplasmic reticulum calcium homeostasis via ryanodine receptor (RyR) activation. Here we further report that Tat disrupts DA transporter (DAT) activity and function, resulting in increased plasma membrane (PM) DAT and increased DAT Vmax, without changes in Km or total DAT protein. Tat also increases calpain protease activity at the PM, demonstrated by TIRF microscopy of a cleavable fluorescent calpain substrate. Tat-increased PM DAT and calpain activity are blocked by the RyR antagonists ryanodine and dantrolene, the calpain inhibitor calpastatin, and by a specific inhibitor of GSK-3β. We conclude that Tat activates RyRs via a calcium and calpain mediated mechanism that upregulates DAT trafficking to the PM, and is independent of DAT protein synthesis, reinforcing the feasibility of RyR and GSK-3β inhibition as clinical therapeutic approaches for HAND. Finally we provide key translational relevance for these findings by highlighting published human data of increased DAT levels in striata of HAND patients, and demonstrating similar findings in Tat-expressing transgenic mice.
HAND remains a significant source of morbidity despite efficacious reduction of viral load by combination anti-retroviral therapy (cART). Onset of HAND correlates with virus load within the brain and cerebrospinal fluid, and with CD4 count. While cART has reduced the incidence of HAND, it cannot always prevent HAND (Masliah et al., 2000), or reduce HAND severity when administered to cART-naïve patients (Chang et al., 2003). With cART-treated HIV+ patients living longer, the prevalence of HAND continues to increase, highlighting the importance of understanding HAND pathogenetic mechanisms as they may synergize with, or increase susceptibility to, co-morbidly evolving neurologic disease in aging HIV+ populations, for instance PD.
The dopaminergic (DAergic) nigrostriatal pathway is notably afflicted in HAND, and 42.8% of HIV patients presenting with Parkinsonism show direct effects of HIV-1 on the basal ganglia (Mattos et al., 2002), with the greatest neuronal loss occurring in the DAergic basal ganglia and substantia nigra (SN) (Reyes et al., 1991). DA’s chemical structure renders it easily auto-oxidized into electrophilic quinone-type species, making DAergic neurons particularly susceptible to oxidative stress. This may result in selective vulnerability of DAergic neurons in diseases such as PD and HAND. However, postmortem HIV and HIVE brains can present with considerable evidence of DA synaptic dysfunction without much evidence of frank DAergic neuron loss (Gelman et al., 2006; Silvers et al., 2006), and HAND symptoms referable to basal ganglia deficits are improved with cART (Hersh et al., 2001). These findings suggest many early DAergic synaptic deficits in HAND are reversible and thus amenable to early therapeutic intervention.
Likewise, HIV secretory neurotoxins decrease synaptic density and increase vacuolation of dendritic spines in affected brain regions of HIV patients (Wiley et al., 1986; Masliah et al., 1997; Everall et al., 1999; Sa et al., 2004), and these synaptic alterations correlate far better with pre-mortem neuropsychologic assessments of cognitive function than does frank neuronal cell loss (Everall et al., 1994). While cART can ameliorate or even reverse the course of HAND in some HIV-infected persons (Gendelman et al., 1998), the severity of neurocognitive impairment at cART initiation is the strongest predictor of persistent neurological deficits despite ongoing cART (Tozzi et al., 2007). Work from our and others’ laboratories has shown that the phospholipid mediator platelet-activating factor (PAF) and Tat can induce reversible synaptic dysfunction prior to cell death (Perry et al., 2005b; Lu et al., 2007; Kim et al., 2008), and elevated levels of PAF may render “normal” physiologic synaptic activity excitotoxic (Bellizzi et al., 2005).
Together these results suggest HAND symptomatology may in part arise from reversible synaptic dysfunctions, which, if left unchecked, ultimately result in neuronal cell loss and permanent deficit (Perry et al., 2005a; Bellizzi et al., 2006). Herein we investigated whether DAergic synapses might exhibit similar reversible deficits in response to the HIV neurotoxin Tat, which has well-established neurotoxic effects on DAergic pathways. We report Tat significantly elevates plasma membrane-localized DAT and DAT uptake activity, by a GSK-3β regulated pathway that modulates RyR and calpain-associated control of DAT.
The recombinant HIV-1 Tat1–72 was generously provided by the laboratory of Dr. Avindra Nath (Johns Hopkins University), and by Dr. Phil Ray (University of Kentucky). In these experiments, we used heat-inactivated aliquots of the above Tat protein as the control for Tat, to control for non-specific effects (e.g. pyrogenic effects) not related to Tat’s functional protein activity, as previously described (Kim et al., 2008). In addition, no significant differences between heat-inactivated Tat control, and vehicle control, were found in our assays. Therefore, having established this in these and previous studies, in some cases vehicle control alone is displayed, where described.
For these studies, Tat was used at 120nM (~1 ug/ml) concentration unless otherwise described, a concentration that closely approximates (or is lower than) doses used in similar studies of DAergic, synaptic, and nervous system function, using equivalently prepared Tat (Orsini et al., 1996; Gurwell et al., 2001; Aksenova et al., 2006; Aprea et al., 2006; Wallace et al., 2006; Zhong et al., 2008; Zhu et al., 2009). We and others have determined that doses on this order of magnitude (50 – 500 nM) represent the minimum Tat doses required to elicit modest (10–20%) but statistically significant increases in cell death and synapse loss over extended 24 hour treatments (Maggirwar et al., 1999; Ramirez et al., 2001; Perry et al., 2005b; Aksenova et al., 2006, unpublished data), while at the same time eliciting acute intracellular metabolic effects without cell death over abbreviated 30 min – 1 hour treatments (Perry et al., 2005b; Wallace et al., 2006, and herein), thus making 120 nM Tat a suitable dose to model early effects of HAND on dopaminergic neurons.
Indeed, soluble Tat levels in HIV+ patient sera have also been measured up to 40 ng/mL (Westendorp et al., 1995; Xiao et al., 2000), despite that Tat’s interactions with endogenous glycosaminoglycans and heparin sulfates may lower its measurable concentration in vivo (Chang et al., 1997; Xiao et al., 2000). Therefore, Tat concentrations surrounding HIV-infected cells are likely much higher than sera concentrations (Hayashi et al., 2006). Moreover, recombinantly produced Tat – using a commonly employed, highly standardized, and reproducible production procedure – is considerably less potent than Tat released from Tat-expressing astrocytic cells (Li et al., 2008), suggesting that comparatively higher doses of this commonly used recombinant Tat protein are required to adequately mimic the effects of Tat constituitively produced in the HIV infected brain. Additionally, Tat’s strong affinity for other proteins and glass/plastic surfaces, its temperature sensitivity, and its susceptibility to oxidation, make it impossible to determine what proportion of the initial Tat dose actually reaches the experimental specimen, leading to underestimation of Tat functions in vitro (Nath et al., 2000). Finally, in the context of HIV infection, many of Tat’s effects may occur over longterm chronic exposures. Chronic low dose effects of any reagent in vivo, are often appropriately modeled in vitro by proportionately higher doses of that same reagent over more acute time frames. In this context, many studies have found Tat concentrations on the order of magnitude used herein, to be appropriate doses by which to model, translate, and evaluate in vivo effects of Tat in vitro. For the sum of these reasons, we use 120 nM Tat for these experiments herein. Despite these caveats, in many cases we were also able to achieve similar but sometimes less robust effects with considerably lower Tat doses (~12 nM/~100 ng/ml; data not shown), but elected to use the 120 nM dose throughout for consistency of the response.
PC12-hDAT cells were kindly provided by Dr. Haley Melikian (University of Massachusetts), and eGFP-hDAT construct was kindly provided by Dr. Susan Amara (University of Pittsburgh). B27 supplement (with and without anti-oxidants), Neurobasal media, and ASP+ were purchased from Invitrogen (Carlsbad, CA). The cell-permeable GSK Inhibitor VIII and calpastatin peptide (#208902) were purchased from Calbiochem/EMD Biosciences (San Diego, CA). Antibodies for western blots were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and Millipore/Chemicon (Billerica, MA). All other chemicals and reagents were purchased from Sigma (St. Louis, MO), except as noted.
Cultures of primary rat mesencephalic neurons containing SN and the DA-rich rostral mesencephalon were prepared from E12–13 rodent SN and rostral mesencephalon in accordance with a protocol modified from Brewer (1993; 1995) as follows. Briefly, primary neuronal cultures were harvested and prepared from embryonic day 12–13 Sprague-Dawley rat pups. The above areas were isolated from a litter of E12–13 rats and the meninges and extraneous tissue removed. Tissue was incubated in 2 mL of Ca2+/Mg2+ free Hank’s balanced salt solution (HBSS with 10 mM HEPES, pH 7.3) with gentamicin (50 μg/mL) and 0.25% trypsin for 15 min at 37°C. The cells were centrifuged at 1000 rpm for 5 min, washed twice with HBSS (with Ca2+/Mg2+), then dissociated in Neurobasal media supplemented with glutamate, gentamicin and B27 supplement plus antioxidants (Invitrogen, Carlsbad, CA) by 10 passages through a 0.9 mm bore pipette tip. Dissociated cells were counted using the trypan blue viability assay and were plated on poly-D-lysine coated cell culture plastic and incubated in a humidified atmosphere of 5% CO2/95% air at 37°C. In addition, cyclic AMP (cAMP) (1 mM) and ascorbic acid (100 uM) were added for the first three days of culture to improve yield and survival of DA neurons. Fetal bovine serum (FBS) (irradiated, defined FBS; Hyclone/Thermo, Waltham, MA) was added at 2% for the first 3 days also to improve DA neuron survival, then diluted out thereafter. Cultures were used for experiments at days in vitro (DIV) 11–14 unless otherwise noted.
PC-12 and PC12-hDAT cells were cultured as previously described (Daniels and Amara, 1999; Loder and Melikian, 2003), in Dulbecco’s Modified Eagle Medium (high glucose) with 10% fetal bovine serum (Hyclone, defined, gamma-irradiated), non-essential amino acids, and penicillin-streptomycin.
Changes in DA uptake by the DA transporter (DAT) were assessed in primary mesencephalic cultures in response to 120 nM Tat treatment, as previously described (Prochiantz et al., 1979; Dal Toso et al., 1988; Engele et al., 1989; Fiszman et al., 1991; Dalia et al., 1993; Bennett et al., 1998; Salum et al., 2008). Briefly, mesencephalic neurons in 12 or 24 well plates were incubated with 120 nM Tat under normal culture conditions for 24 hours; for the acute 15 and 30 minute treatments, DA uptake assay was performed during Tat administration. Background (i.e. DA uptake that was not specific to DAT) was determined by incubating both Control and Tat conditions with and without the specific DAT antagonist GBR-12909 (1 uM). Wells were then washed 2×3 minutes with pre-heated 37°C HBSS with Ca+ and Mg+, containing 100 uM ascorbic acid (to protect against DA toxicity) and 50 uM pargyline (monoamine oxidase inhibitor that prevents endogenous DA catabolism). Washes were removed and replaced with 50 nm tritiated (“hot”) DA (3H-DA) and 450 nM cold DA in the same wash buffer, and incubated at 37°C for 20 minutes. Our own (Figure 2A) and previous (Dal Toso et al., 1988; Engele et al., 1989; Bennett et al., 1998) studies have demonstrated DAT uptake activity in cell culture to be linear over this time period. Reactions were stopped in ice-cold wash buffer. Cells were then lysed on ice for 30 minutes with 1% SDS in the same ice cold wash buffer (250 ul/well for 24 well plate, 500 ul/well for 12 well plate), then triturated to lift cells. Equal amounts of lysate protein were added to 5 ml aqueous scintillation fluid and amount of 3H-DA uptake was read by scintillation counter. Readout was given in background corrected counts per minute (CCPM). For each condition, non-specific DA uptake (+ GBR condition) was subtracted from total DA uptake (−GBR condition), to yield specific DA uptake, which was then normalized to control and data expressed as normalized mean CCPM ± SEM for each condition. Mean CCPM (i.e. DAT specific DA uptake) was obtained from N=4 experiments, and significance determined by student t-test at P < .05.
For kinetic analysis of Tat-induced changes in DAT uptake activity, a fluorescent assay was adopted in the DAergic PC-12 cell line, stably transfected with the human DA transporter (PC12-hDAT) (kind gift of Dr. Haley Melikian; (Loder and Melikian, 2003)). In this assay, the fluorescence substrate 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (4-Di-1-ASP) (herein referred to as ASP+) (Invitrogen/Molecular Probes item# D288, Carlsbad, CA) was used. ASP+ is a fluorescent dye with high affinity for the DA transporter that has been extensively validated for use as a non-radioactive, fluorescent indicator of DA-transporter specific uptake activity (Schwartz et al., 2003; Mason et al., 2005).
This assay was performed essentially as the radioactive assay above, using ASP+ uptake in the presence of ± GBR-12909 to determine DAT-specific uptake activity. Briefly, PC12-hDAT cells were cultured as above then treated in replicate (4–8 replicates/condition) with control and Tat (120 nM). Following treatment, media was removed, and ASP+ added in HBSS buffer including 8 uM trypan blue as masking agent. Mean fluorescent units were read in a fluoromark plate reader (Biorad, Hercules, CA), and kinetic analysis performed by non-linear regression analysis using GraphPad (La Jolla, CA) Prism or Synergy (Reading, PA) Kaleidagraph software for calculation of Km and Vmax values.
Primary neuronal cultures or PC12-hDAT cells were treated as described in figure legends, and immunoblots were prepared and performed as described previously (Norman et al., 2007; 2008), with the following exceptions: DAT antibodies were found to be extremely sensitive to the effects of heating the lysates to 100°C, as this appeared to alter the conformation of critical epitopes sufficiently to prevent DAT antibody binding. Therefore the lysates were not heated to 100°C before loading the gel. Antibodies were used as described in the figure legends. For relative quantification of the signal intensity between bands, western blots were digitally photographed using a uniform-intensity illuminator and monochrome digital CCD (Interfocus Imaging Ltd, Linton, Cambridge, UK; formerly Image Research, Ontario, Canada). Bands were quantified by summing the total value of all pixels within the band area, using ImageJ, then normalized to control value for group analysis across multiple runs. Two replicates for each condition were run in separate lanes to ensure consistency of results.
Plasma membrane and cytosolic components were separated from total cellular protein using a PEG and Dextran T-500 based centrifugal gradient kit, according to the manufacturer’s directions, resulting in plasma membrane purity over 90% (Biovision Membrane Protein Extraction Kit, Mountain View, CA).
TIRF studies employed PC-12 cells cultured as above on glass-bottom Petri dishes (MatTek, Ashland, MA), then transfected with eGFP-hDAT DNA using the Lipofectamine 2000 (Invitrogen, Carlsbad, CA) protocol and returned to the incubator for 2 days to maximize expression. Cells were removed from the incubator, and placed in Hibernate (BrainBits, Springfield, IL) media (intended for pH equilibration in ambient CO2 levels) ± treatment for the imaging procedure. Under these conditions, cells are stable at ambient air CO2 concentrations for several hours. Upon treatment with control or Tat, cells were immediately taken to the microscope, a random field of eGFP-hDAT transfected PC-12 cells was obtained, and images under TIRFM were taken at a rate of 1 image (500 ms exposure)/30 second for 30 minutes using a Qimaging (Surrey, British Columbia, Canada) Retiga Exi cooled CCD camera and Metamorph (Molecular Devices/MDS Analytical Technologies, Sunnyvale, CA) software for automated image acquisition. Hardware included 488 laser excitation with Melles Griot (Carlsbad, CA) tunable argon laser, Olympus (Melville, NY) TIRF illuminator on an Olympus IX70 microscope with Olympus 60x 1.45NA TIRF objective, Chroma (Rockingham, VT) z488/10x clean-up filter and dichroic mirror, with laser illumination custom-synchronized with camera exposures via the camera’s I/O port. Using Metamorph software, fluorescence intensities were analyzed by placing 100-pixel square regions of interest (ROIs) in random locations throughout each of the tranfected cells under study, and averaging the mean signal over all ROIs for each image series, normalized to the starting fluorescence intensity value for each image series.
TIRF imaging of membrane calpain activity was performed in the same fashion, except that cells were pre-loaded for 30 minutes at room temperature with the cell-permeable fluorescent calpain substrate bis-(CBZ-L-alanyl-L-alanine amide) Rhodamine 110 (BCAA-R110) (10 uM) (Anaspec, San Jose, CA) in Ca2+-free Hibernate buffer containing 1 uM calpastatin (loading buffer was Ca2+-free + calpastatin to prevent premature cleavage of the BCAA-R110 probe prior to experimental treatment). Following BCAA-R110 loading, dye was replaced with Hibernate (with calcium) plus experimental treatment, and TIRF-imaged as above.
The raw data were analyzed from 3–5 independent experiments and expressed as the mean ± SEM. Where applicable, the percentage of control and percentage of control SEM for each treatment condition were calculated by dividing the raw means and raw SEM’s by the control condition raw mean. A Student’s t test using a two-tailed distribution and unequal variance was used to compare data. A probability of p<0.05 was considered statistically significant. Replicates for each condition were run in separate lanes to ensure consistency of results.
Tat-transgenic mice were kindly provided by Dr. Johnny He (Indiana University School of Medicine, Indianapolis, IN) (Kim et al., 2003). Six-week old Tat-transgenic mice with Tat under the control of a doxycycline-inducible GFAP promoter, or WT control mice, were treated with 80mg/kg i.p. of doxycycline in saline, once daily for 7 days. Tat-Tg and WT control animals were then sacrificed under anesthesia in accordance with IACUC guidelines, followed by cardiac perfusion with PBS, then 4% PFA chased with 0.05M sodium phosphate, brains were removed and post-fixed in 4% PFA overnight, followed by storage at 4°C in PBS. Forty-micron coronal sections from striatum were cut on a vibratome, followed by free-floating ICC procedure with anti-DAT (1:1000, Millipore (formerly Chemicon) MAB369) and anti-synaptophysin (1:250, Millipore MAB5258) antibodies. Following ICC, sections were mounted, and analyzed for DAT and synaptophysin expression levels in each region by computerized image capture and analysis. A total of ≥ 50 × 40 um coronal striatal sections were analyzed for each antibody, over multiple runs (2 brains/group). For each section, background was normalized, regions of interest (ROIs) were manually drawn around striatum, and mean fluorescent intensity for DAT and Synaptophysin labeling was calculated for each ROI. To normalize DAT content to total pre-synaptic volume for each slice, intensity data was further expressed as mean DAT intensity/mean Synaptophysin intensity for each ROI, and these values were then averaged over all sections ± SEM. Metamorph software was used for image acquisition and analysis.
Tat is excitatory and excitotoxic (Cheng et al., 1998; Haughey et al., 2001; Perry et al., 2005b), and toxic to DAergic populations and imparts changes in DAergic function (Bansal et al., 2000; Aksenov et al., 2001; Aksenova et al., 2006; Wallace et al., 2006; Silvers et al., 2007; Aksenov et al., 2008). Therefore we hypothesized that Tat might disrupt DAT activity, leading to permanent neuronal loss and neurologic deficit, consequent to unsustainable metabolic demands and/or enhanced auto-oxidative DA toxicity pre-synaptically, or over/under-stimulation of post-synaptic striatal connections. We found that treatment of mesencephalic neurons with 120 nM Tat robustly increased DAT-specific DA uptake versus control (Figure 1A). This effect was greatest acutely, as DA uptake was markedly elevated 4–5 fold following 30 minute Tat treatment (P < .02). After 24 hour Tat treatment, this effect had declined to a more modest two-fold increase in DA uptake (P < .04). These results suggest that Tat’s actions on DAT activity in vitro begin acutely, persist for at least 24 hours during prolonged Tat exposure, and may be in part responsible to Tat’s effects on DAergic systems in the context of HAND.
Increased DA uptake via DAT could be accomplished in several ways, including increased production of DAT protein (i.e. leading to increased membrane DAT levels), increased rate of DAT substrate turnover across the membrane (i.e. individual DAT molecules are “running” faster), or changes in DAT trafficking to/from the cell membrane without changes in total intracellular DAT levels. To address these possibilities, we first measured total cellular DAT protein in primary mesencephalic cultures at time points matching the DA uptake studies in Figure 1A. We found no increase in DAT protein at 30 minutes Tat treatment (Figure 1B), when DA uptake was most elevated (Figure 1A), suggesting that the four-to-five-fold increase in DA uptake 30 minutes after Tat addition was not due to increased translational expression of DAT. Therefore, we speculated that the increased DA uptake at this acute time point resulted from changes in DAT membrane trafficking independent of DAT protein levels. In contrast, after 24 hours Tat treatment, total DAT protein levels were increased roughly twofold (P < .04) (Figure 1B), which closely approximated the two-fold increase in DAT uptake at this same time point (Figure 1A), suggesting that with 24 hour continuous exposure to 120 nM Tat, increased DAT protein expression might be at least in part responsible for the increased DA uptake observed at this longer time point. In other words, increased protein translation may eventually “catch up” to acute synaptic DAT demands that, in the short term, are satisfied exclusively by altered DAT kinetics (e.g. changes in DAT trafficking and/or substrate turnover), rather than by a generalized increase in DAT protein expression.
Because both DA uptake and total DAT protein were elevated at 24 hours 120 nM Tat treatment (albeit a smaller increase in DA uptake, compared to 30 minutes), to avoid the potentially confounding variable of changes in DAT protein translation as they affect intracellular DAT pools, and because we wished to investigate changes in DAT trafficking/kinetics commensurate with the time scale of synaptic transmission and adaptability prior to changes in protein expression, the remainder of this report focuses on this acute 30 minute time point. We hypothesized that changes in DAT membrane trafficking, rather than increased DAT expression, were likely to satisfy these elevated near-term demands for DAT at the synapse.
To test this hypothesis further, next we investigated whether Tat was altering kinetics of DA uptake. For these and studies that follow, we used PC12 cells transiently or stably transfected with the human DA transporter (PC12-hDAT; kind gift of Dr. Haley Melikian, University of Massachusetts), a dopaminergic model system that provides ease of genetic manipulation, reproducibly high yields of hDAT+ cells, and that has been well-characterized for investigations of DAT membrane trafficking (Melikian and Buckley, 1999; Loder and Melikian, 2003). Moreover, this model system obviates technical limitations associated with obtaining yields of primary rodent DAergic neurons sufficient for kinetic analyses. To investigate kinetics of DA uptake in this model system, we employed a well-characterized non-radioactive DAT uptake activity assay. This assay relies on uptake of the fluorescent probe 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (4-Di-1-ASP) (Molecular Probes/Invitrogen #D-288, Carlsbad, CA) (ASP+), which has a high affinity for DAT (Schwartz et al., 2003; Mason et al., 2005). In our own experience, and in the experience of laboratories that have developed this as a non-isotopic alternative to measuring DA uptake (Schwartz et al., 2003; Mason et al., 2005), this assay faithfully replicates kinetics of DAT-mediated uptake, and a slightly modified version of this assay has since been made commercially available in “kit” form (Part#s: R8173, R8174, MDS Analytical Technologies, Sunnyvale, CA). In our experiments, non-specific uptake was determined in the presence of the specific DAT antagonist GBR-12909, and this value was subtracted from uptake in the minus GBR-12909 condition, to yield DAT specific uptake. As expected, GBR-12909 exhibited dose-dependent effects against ASP+ uptake in this model system, and un-transfected (hDAT-negative) PC12 cells showed significantly reduced ASP+ uptake versus their PC12-hDAT counterparts (data not shown).
Because we were particularly interested in investigating changes in DAT activity over acute time periods, before significant changes in protein translation occur (see Figure 1B), we pre-treated PC12-hDAT cells for 30 minutes at 37°C with either control or 120 nM Tat, followed immediately by the ASP+ assay (see Methods). From these studies, we found that 30 minutes of 120 nM Tat treatment significantly increased DAT uptake of ASP+, when compared to control. Figure 2A illustrates this effect, during a ~50 minute uptake experiment at a single ASP+ concentration (12 uM). DAT uptake activity is best modeled as analogous to enzyme activity (Schenk et al., 2005). Our data validated this assumption, exhibiting sigmoid shaped three-phase ASP+ accumulation versus time, best fit by fourth degree polynomial curves demonstrating linear DAT substrate uptake in the range of ~10–40 minutes (Figure 2A). In contrast to more reductionist model systems (e.g. synaptosomes), which may have more rapidly saturable DA uptake due to higher frequencies of DAT/substrate interactions, linear dopamine uptake over this time range has been commonly described in DAergic whole cell systems (Dal Toso et al., 1988; Engele et al., 1989; Bennett et al., 1998). Further kinetic analysis of this effect showed that Tat increased the maximal velocity (Vmax) of DAT ASP+ uptake versus control (5.7 versus 4.3 RFU/sec respectively; p < .016; two tailed paired t test), without changing the Michaelis constant (Km, or the substrate concentration that achieves half-maximal rate) (Figure 2B).
Given that DAT uptake activity is tightly regulated by DAT trafficking between intracellular pools and the plasma membrane (Melikian and Buckley, 1999; Buckley et al., 2000; Loder and Melikian, 2003; Melikian, 2004), we reasoned that the most likely explanation for increased Vmax without any change in Km, was due to increased concentration of DAT at the plasma membrane, which would in turn increase maximal velocity of DAT-mediated uptake. To test this hypothesis, we again pre-treated PC12-hDAT cells for 30 minutes at 37°C with either vehicle control or 120 nM Tat, then performed fractionated western blot analysis for DAT in total protein, cytoplasmic, and plasma membrane cellular subfractions. This analysis showed that following 30 minutes Tat treatment, there was a significant increase in plasma membrane DAT (~177% versus control, p < .05, two-tailed paired t test), as well as a smaller but also significant decrease in cytoplasmic DAT (~86% versus control, p < .02), findings consistent with a redistribution of DAT to the PM. Total protein, however, was not changed (Figure 3A). These effects were specific to functional Tat activity, as 120 nM heat-inactivated Tat, in contrast, did not demonstrate any ability to alter plasma membrane or cytoplasmic DAT levels when compared to vehicle control (Figure S1).
Several additional experiments provided further visual confirmation that membrane DAT was increased in Tat treated cultures versus controls. First we performed the same treatments in the PC12-hDAT cells, then immediately following treatment, fixed the cultures with 4% paraformaldehyde and performed immunocytochemistry for hDAT with AlexaFluor 488 as the secondary label (the exogenous human DAT expressed in these cells is DAT only, with no fluorescent fusion reporter protein, thus necessitating immunocytochemical detection), and viewed them under total internal reflection fluorescence microscopy (TIRFM). By reflecting a 488 nM laser-based excitation source at incident angle against the underside of the glass coverslip upon which the cells are cultured, TIRFM utilizes the resulting evanescent wave to effectively optically isolate fluorescent events within roughly 100 nm (Z-direction) of the basilar plasma membrane, thus allowing us to visualize only those DAT that are at or near the cytoplasmic border of the plasma membrane. Congruent with the immunoblot results in Figure 3A, membrane expression of DAT was significantly increased in the Tat-treated PC12-hDAT cultures, as evidenced with TIRFM visualization (Figure 3B).
Since our stably transfected hDAT-PC12 cells did not contain a fluorescent reporter protein, to observe these changes live, we transiently transfected the dopamine-producing PC-12 cell line with plasmid DNA encoding an enhanced green fluorescent protein-human DAT (eGFP-hDAT) fusion protein (kind gift of Dr. Susan Amara, University of Pittsburgh), and observed membrane activity of the eGFP-hDAT signal in real-time using TIRFM. In this model system, in response to 30minute treatment with 120 nM Tat, we also observed DAT levels increasing at the membrane in real time [compare Tat treatment (red trace) to heat-inactivated Tat Control (black trace), Figure 4.] These graphical results are further highlighted by representative TIRFM image series of these eGFP-hDAT transfected PC-12 cells, showing a rise in membrane eGFP-hDAT signal during 30 minute treatment with 120 nM Tat (Supplementary Movie 1), versus a slight decline in membrane eGFP-hDAT in 30 minute control treated cells (Supplementary Movie 2), as summarized in Figure 4. Images were captured at 2 frames/minute for 30 minutes (~61 frames total), then played back at ~ 7 fps. A frame-by-frame graph is overlaid on each movie, representing the normalized averaged eGFP-hDAT intensity value over the cell area for each frame.
Taken together, these results support a mechanism whereby Tat acutely increases DAT expression at the plasma membrane by affecting trafficking mechanisms rather than increasing intracellular DAT protein levels.
DAT activity conforms to Michaelis-Menten kinetics and can be modeled as follows (Equation 1) (Schenk et al., 2005):
Fitting our data using non-linear regression analysis of specific DAT uptake activity in PC12-hDAT cells, we found Tat treatment increased Vmax by a factor of ~1.33 (5.693 Tat versus 4.266 Control). However, DAT concentration at the membrane in these same cells increased by a factor of 1.77 in the Tat treated condition (Figure 3A). Since Vmax = κ3[DAT] (Equation 2), i.e. the product of the translocation constant (i.e. how quickly DAT transports each DA molecule across the membrane) and the concentration of DAT at the membrane, by substituting our relative values into this equation (units are omitted for simplicity):
Thus we can infer that in the Tat-treated cultures, while Vmax was increased, the translocation constantκ3 instead declined by about 25%. In other words, Tat slows down the rate at which DAT transports each molecule of substrate across the membrane, but Vmax is slightly increased overall because of (a compensatory?) increase in DAT concentration at the membrane. This observation may help explain differences between our observed Tat effects on DAT and those reported previously (Wallace et al., 2006; Zhu et al., 2009) (see Discussion).
Previously we have shown that ryanodine receptors mediate several intracellular effects of Tat by regulating calcium release from mitochondria and endoplasmic reticulum (Norman et al., 2007; 2008). Calpains, in turn, are ubiquitous Ca2+-activated proteases involved in numerous integral cell functions including proteolytic regulation of cytoskeletal dynamics, and which are also increasingly being implicated as key mediators of neurodegenerative disease (for review, see Liu et al., 2008). GSK-3β mediates Tat’s (Maggirwar et al., 1999; Sui et al., 2006b) and platelet-activating factor’s (Tong et al., 2001) neurotoxic effects in models of HAND, and furthermore we have shown that GSK-3β is recruited to membrane lipid rafts, and GSK-3β inhibition blocks specific phosphorylation of raft-associated Tau (Sui et al., 2006a). Membrane DAT trafficking may also be regulated at lipid rafts (Adkins et al., 2007). Finally, calpain cleaves GSK-3β to augment its kinase activity (Goni-Oliver et al., 2007). Therefore we wished to determine whether inhibition of: 1. Ryanodine receptor activity with the RyR antagonists ryanodine or dantrolene, or 2. Calpain activity by a cell permeable calpastatin peptide (Item #208902, Calbiochem/EMD Biosciences, San Diego, CA), or 3. GSK-3β kinase activity with the specific GSK-3β inhibitor N-(4-Methoxybenzyl)-N′-(5-nitro-1,3-thiazol-2-yl) urea (GSK-3β inhibitor VIII, Calbiochem/EMD Biosciences, San Diego, CA) – prevented the Tat-induced rise in plasma membrane DAT levels.
Again tracking the eGFP-hDAT signal at the plasma membrane with real-time TIRFM, we found that the increased membrane DAT levels induced by 30 minute treatment with 120 nM Tat, could be prevented by co-application of: 1. Ryanodine or dantrolene (25 uM); 2. A twenty-seven amino acid cell permeable peptide from exon 1B of the endogenous calpain inhibitor calpastatin (1 uM) (Maki et al., 1989; Eto et al., 1995) (Calbiochem/EMD #208902), or 3. The GSK-3β inhibitor N-(4-Methoxybenzyl)-N′-(5-nitro-1,3-thiazol-2-yl) urea (200 nM) (Calbiochem/EMD GSK-3β inhibitor VIII) (Figure 4). These results indicate that Tat-mediated activation of RyRs, with accompanying changes in calpain and GSK-3β activity, alter DAT trafficking kinetics at the plasma membrane.
Having shown RyR receptor antagonism, and calpain inhibition, prevent Tat-induced increases in membrane DAT (Figure 4), we hypothesized a mechanism whereby Tat activates RyRs to increase intracellular calcium (and see Norman et al., 2007; 2008), thus activating calpain proteases. We additionally hypothesized that GSK-3β activity is linked to calpain signaling to further regulate membrane DAT trafficking. Therefore, using the cell-permeable fluorescent calpain substrate bis-(CBZ-L-alanyl-L-alanine amide) Rhodamine 110 (BCAA-R110) (Anaspec, San Jose, CA), which has been validated as an indicator of calpain protease activity in live cells (Gitler and Spira, 1998), we measured calpain activity at the plasma membrane, under the same treatment conditions as in Figure 4 above. This substrate is non-fluorescent until cleaved by proteases at the amide bond, thus releasing the Rhodamine 110 fluorophore, rendering it available for excitation. Imaging this dye by TIRFM, under the same treatment conditions as in Figure 4, we show that 30 minute treatment of PC12 cells with 120 nM Tat increases calpain protease activity at the plasma membrane versus control cells (Figure 5). Furthermore, just as 25 uM dantrolene, 25 uM ryanodine, 1 uM cell permeable calpastatin peptide, and 200 nM N-(4-Methoxybenzyl)-N′-(5-nitro-1,3-thiazol-2-yl) urea restore plasma membrane DAT to control levels (Figure 4), these treatments also restore membrane calpain activity to control levels (Figure 5). Together these results suggest membrane-localized control of DAT trafficking by RyR– and GSK-3β–mediated activation of calpain proteases located at or near the plasma membrane.
Above we have presented data on Tat-induced changes in DAT trafficking and function during acute time frames in situ, because we wished to investigate changes in DAT kinetics commensurate with the time scale of synaptic transmission and adaptability prior to changes in protein expression, to better understand mechanisms for DAergic deficits prior to frank loss of synapses or neurons. However, it is important to also correlate these findings with in vivo evidence of DAergic dysfunction in the context of HAND.
Our in situ studies suggest that Tat increases membrane DAT levels and functional uptake via alterations in DAT trafficking behavior, without increased production of DAT. Perhaps the best translational in vivo correlate for these findings comes from observations made in post-mortem brain tissue from HAND patients. In these studies, Gelman et al. (2006) found significantly increased DAT expression in the striatum in post-mortem brain tissue with neuropathological confirmation of HIV encephalitis (HIVE) (see Figure 1 and Figure 3 in (Gelman et al., 2006)), which the authors concluded did not result from generalized increases in synaptic protein expression or synaptic density. Other studies found no significant change (with a slight downward trend) in DAT levels in substantia nigra of HAND patients versus controls (Silvers et al., 2006, Figure 2). Taken together, these studies suggest that in HAND, increased striatal DAT expression may result from increased translocation of DAT from mesencephalic neuronal cell bodies, to distal striatal pre-synaptic sites, without significant changes in total DAT protein expression – thus representing a potential clinically relevant in vivo correlate for our in situ findings of increased membrane DAT expression, caused by altered DAT trafficking rather than increased protein expression.
To further support these clinical findings, and our own in situ data, we next examined synaptic DAT levels in the striatum of Tat expressing transgenic mice. These mice express a doxycycline-inducible Tat gene driven by the glial fibrillary acidic protein (GFAP) promoter, for regulatable Tat expression by astrocytes (Kim et al., 2003). Tat is released from HIV-infected microglia, macrophages, and astrocytes in the CNS, and release of soluble neurotoxins from activated immune cells likely contributes significantly to the neuropathology of HAND (Chang et al., 1997; Nath and Geiger, 1998; Nath, 2002; Rumbaugh and Nath, 2006). These doxycycline-induced Tat-transgenic mice, in turn, develop behavioral changes and neurological abnormalities including tremor, ataxia, and cognitive and motor deficits, and exhibit several neuropathologies including astrocytosis, increased infiltration of activated monocytes, neuronal apoptosis, and synaptic damage (Kim et al., 2003) – all of which closely mirror HAND neuropathologic features. Thus these mice model at least some of the features of HAND, and we now further extend the translational relevance of this model system, with novel studies of Tat’s effects on DAergic synapses in these mice.
In these studies, we found significantly higher DAT levels in the striatum of doxycycline-induced Tat expressing mice (~35% increase relative to control), versus their non-Tat expressing control counterparts (Figure 6). This occurred despite a moderate decline in total pre-synaptic density in the Tat Tg mice (~87% versus WT control; p < .0004), as assessed by synaptophysin staining (a reliable method for quantifying total synaptic density; (Masliah et al., 1989; Calhoun et al., 1996)). Together these observations suggest that synaptic DAT levels are significantly elevated per synapse in these Tat Tg mice (Figure 6), findings which are correlative with the findings in post-mortem HAND striatum as discussed above (Gelman et al., 2006), and which we posit reflect increased anterograde DAT trafficking to striatal synapses in vivo independent of gross changes in DAT protein synthesis. Future real-time in vivo and ex vivo studies will seek to further confirm this hypothesis.
In total, these observations that Tat increases synaptic DAT in vitro and in vivo, with up-regulation of high affinity transporter capacity independent of nerve terminal density, suggest that exposure of DAergic nerve terminals to Tat may alter DAergic tone by: 1. Increasing pre-synaptic DA turnover, 2. Creating a post-synaptic undersupply of DA, and/or 3. Increasing oxidative stress in the nerve terminal.
cART controls active viral replication, but once proviral DNA forms, cART does not prevent production of early viral proteins Tat, Nef, and Rev in infected cells (Rumbaugh et al., 2008; Li et al., 2009). These proteins may be excreted or, like Tat, actively secreted from infected cells, including glia (Chang et al., 1997; Nath, 2002). Indeed, soluble Tat levels in HIV+ patient CSF and sera have been measured up to 40 ng/mL (Westendorp et al., 1995; Xiao et al., 2000). Tat is also detectable in AIDS brains with progressive multifocal leukoencephalopathy, including in uninfected cells, suggesting that Tat can propagate effects on uninfected “bystander” brain cells (Del Valle et al., 2000), which may initiate Tat’s concatenative effects on glial cell activation and neurotoxicity (Rumbaugh and Nath, 2006). Moreover, a recent study showed that up to 19% of astrocytes in HAD patient brains are HIV-infected, with frequency of astrocyte infection correlating with neuropathological severity and proximity to perivascular macrophages (Churchill et al., 2009), suggesting that the number of HIV+ cells in the brain is far more than originally anticipated, with direct consequences for HAND progression and severity. Together these factors likely contribute to viral proteins’ ongoing ability to cause HAND despite cART, and underpin our rationale for investigating Tat’s synapse-modifying abilities in the brain.
We demonstrate here that Tat alters DAergic function by previously unreported mechanisms. In rodent mesencephalic neurons and hDAT-transfected PC12 cells treated with 120 nM Tat, DAT-specific DA uptake was significantly elevated within 30 minutes, with increased membrane DAT levels caused by redistribution of intracellular DAT to the plasma membrane, and consequent increases in Vmax (Figures 1–4). However, there was no increase in DAT protein synthesis until 24 hours Tat exposure (Figure 1). Likewise, DAT was elevated at striatal synaptic terminals of doxycycline-induced Tat Tg mice versus controls (Figure 6), which correlates with similar findings in post-mortem tissue from HAND patients (Gelman et al., 2006).
Providing key translational congruence with our results, Gelman et al. (2006) found significantly increased striatal DAT expression (i.e. on the pre-synaptic afferent DA inputs from the SN), in postmortem brain tissue with neuropathological confirmation of HIV encephalitis (HIVE). Tyrosine hydroxylase (TH) expression, in contrast, was decreased, suggesting down-regulated DA production compensatory to increased DA uptake. Others found no differences in SN (i.e. cell body) DAT in HAND versus controls (Silvers et al., 2006). Another study found decreased DA content, but stable levels of the DA metabolite homovanillic acid (HVA), in post-mortem HAND brains versus uninfected controls (Kumar et al., 2009). Stable HVA despite reduced DA may reflect the balance of down-regulated DA production, compensatory to increased DA turnover, which leaves net DA breakdown (and thus HVA) unchanged. We hypothesize these post-mortem findings, congruent with our findings herein, suggest total cellular DAT content is not greatly changed in the DAergic SN neurons of HAND versus control (Silvers et al., 2006). We further speculate that synaptic DAT – i.e. translocation of DAT from the SN cell body to the striatal synaptic membrane – is increased in HAND. Indeed, much of total cellular DAT is normally located intracellularly, with regulation of functional DAT uptake activity accomplished largely by altering DAT membrane trafficking/recycling, rather than gross changes in protein expression (see Figures 2–4, and Melikian and Buckley, 1999). Although no model system by itself can completely capture the continuum of neurodegenerative disease or DAergic dysfunction encompassed by HAND, our in situ and in vivo data herein mesh well with existing post-mortem data.
The sum of this data, together with previous work demonstrating that the HIV mediators Tat and PAF increase neuronal metabolic and synaptic activity (Haughey et al., 2001; Bellizzi et al., 2005; Perry et al., 2005b; Lu et al., 2007), suggests DAergic tone may be increased in HAND. Bolstering this concept, PAF also increases membrane DAT by mechanisms similar to those shown here for Tat (unpublished data). We further posit that increased membrane DAT levels together with a slower DA translocation constant (k3) (see Results), may model pre-synaptic nerve terminals under bioenergetic stress – further evidenced by other Tat-induced impairments of synapses, mitochondria, and ER (Bellizzi et al., 2005; Perry et al., 2005b; Norman et al., 2007; 2008). Likewise, others found that 48 hours of 50 nM Tat1–72 reduced [3H]WIN 35428 ligand binding to DAT in rat midbrain neurons, yet without changing total DAT protein despite significant cell death (Aksenova et al., 2006). These findings remain consistent with a mechanism whereby Tat interferes with DAT function (as evidenced by impaired ligand binding), but relative DAT protein levels (per cell) are stable or even increased despite synapse loss or neuronal death. Also similar to our results, 30 minute treatment of rat striatal synaptosomes with 60 nM or 1 uM Tat did not change Km or total synaptosomal DAT levels (Wallace et al., 2006; Zhu et al., 2009). Yet contrasting with our results, Tat decreased DAT Vmax in this synaptosome model. However synaptosomes contain only ~7% of the total DAT content of DA neurons (Johnson et al., 2005), and lack the more distal (extra-synaptic) molecular machinery that also influences DAT membrane trafficking. Therefore the DAT translocation constant (κ3) impacts Vmax more heavily in synaptosomes than in whole cells – which may explain how Tat lowers Vmax in synaptosomes (Wallace et al., 2006; Zhu et al., 2009), but increases Vmax in intact cells (Figure 2). Indeed, increased membrane DAT may be a compensatory response to decreased transporting efficiency of individual DATs.
Increased synaptic DAT and DA turnover/reuptake (herein, and Gelman et al., 2006) seems paradoxical given that Parkinsonian-like symptoms in HAND (reviewed in Berger and Arendt, 2000; Koutsilieri et al., 2002; Tse et al., 2004), which can manifest despite cART (Mirsattari et al., 1998; Shimohata et al., 2006; Tisch and Brew, 2009), have previously been thought to result from decreased DAergic tone, i.e. loss of DAT-containing DAergic synapses. However, chronically increased DAT motility and DA uptake or DA turnover as our data suggests, may progressively damage DAergic nerve terminals due to unsustainable metabolic demands and enhanced auto-oxidative DA toxicity from excessive intracellular DA accumulation. This in turn may ultimately leading to degeneration of DAergic synapses and/or cell bodies (Reyes et al., 1991; Everall et al., 1995; Gelbard et al., 1995; Itoh et al., 2000). On the other hand, more recent analyses of post-mortem HIV+ brains (cART status was not reported), found significant alterations in DAergic markers without evidence of synapse or cell body loss (Gelman et al., 2006; Silvers et al., 2006). Functional alterations at DAergic synapses – for e.g., increased DA reuptake – might still cause Parksinonian symptoms by depriving post-synaptic striatal connections of excitatory stimuli. Further investigations are required to clarify the mechanisms by which DAergic symptoms develop in HAND, particularly in the current cART era, in which DAergic neuronal and synaptic dropout may be less common.
Together our current and previous (Norman et al., 2007; 2008) data suggest Tat activates RyRs to elevate intracellular calcium, thus activating calpain proteases, which in turn regulate membrane DAT levels (see data herein, and cartoon Figure 7). Calpain proteases are known to mediate post-synaptic physiology (Liu et al., 2008), but this is the first report implicating membrane-localized calpain protease activity in regulating membrane DAT (Figures 4–5). (Both the fluorescent probe for calpain activity and the calpain-blocking calpastatin peptide are cell permeable (Eto et al., 1995; Gitler and Spira, 1998, and results herein), thus allowing us to assess intracellular calpain activity.) Details of this mechanism require further investigation, but calpains critically regulate both cytoskeletal elements and α-synuclein (Dufty et al., 2007), either of which can regulate trafficking of DAT (Lee et al., 2001; Wersinger and Sidhu, 2003) or other transporters (Jeannotte and Sidhu, 2008).
GSK-3β inhibition prevented Tat-induced increases in membrane DAT and membrane calpain activity, highlighting mechanisms by which GSK-3β inhibitors may confer neuroprotective benefits for DAergic symptoms in HAND (Dewhurst et al., 2007; Ances et al., 2008). This observed blockade effect was unlikely to be due to non-specific effects of AR-A014418 on calpain cdk2/cdk5 pathways, as the IC50 of AR-A014418 on these pathways is > 100 uM (Bhat et al., 2003), whereas our studies used 200 nM. AR-A014418 also prevents DAergic cell death and loss of striatal DA in an in vivo rodent model of MPTP toxicity (Wang et al., 2007), further emphasizing potential overlaps in DAergic dysfunctions characterizing HAND and PD. How GSK-3β interacts with calpain signaling to achieve these effects remains unclear, however GSK-3β has been implicated as an upstream trigger of ER and UPR stress conditions (Huang et al., 2009), which may in turn activate calpain proteases. GSK-3β and calpain also share many ubiquitous substrates, including tau and β-catenin (Liu et al., 2008; Vosler et al., 2008). We hypothesize that when GSK-3β activity is inhibited, this increases substrate availability to calpain, thus competitively diminishing calpain’s effects on DAT kinetic parameters (Figure 7). Future studies will seek to further clarify these mechanisms by which RyR, calpain, and GSK-3β interact with DAT to promote synaptic dysfunction in HAND, potentially yielding additional therapeutic options for treatment of this and related neurologic diseases.
PC12-hDAT cells were treated with either vehicle control or 120 nM heat-inactivated Tat for 30 minutes, and analyzed by western blot, in identical fashion to Figure 3. In contrast to 120 nM Tat versus vehicle control treatments in Figure 3, no significant differences in plasma membrane or cytoplasmic DAT levels were found between vehicle control and 120 nM heat-inactivated Tat treated cells, demonstrating functional specificity of Tat’s effects on cytoplasmic and plasma membrane DAT levels. And see further demonstration of the specificity of Tat’s effects on DAT activity, versus heat-inactivated Tat, in Figures 4 and and55.
This work was supported by National Institute of Health grants R21MH084718 and R21DA030256 to SWP, R01NS054578 and R01NS066801 to SBM, and R01MH056838 and PO1MH64570 to HAG.
Competing interests: HAG and SBM hold a patent filing related to treatment of HAND using glycogen synthase kinase inhibitors.