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Platelet-derived growth factor-BB (PDGF-BB) has been reported to provide tropic support for neurons in the central nervous system (CNS). However, whether PDGF-BB regulates neurogenesis especially in the context of HIV-associated neurological disorder (HAND) and drug abuse, remains largely unknown. In this study we demonstrate that pre-treatment of rat hippocampal neuronal progenitor cells (NPCs) with PDGF-BB restored proliferation that had been impaired by HIV Tat & cocaine via the cognate receptors. We identify the essential role of transient receptor potential canonical (TRPC) channels in PDGF-BB-mediated proliferation. Parallel but distinct ERK/CREB, PI3K/Akt signaling pathways with downstream activation of mTOR/4E-BP & p70S6K and NF-kB were critical for proliferation. Blocking TRPC 1 channel suppressed PDGF-mediated proliferation as well as PDGF-BB-induced ERK/CREB and mTOR/4E-BP & p70S6K activation thereby underscoring its role in this process. In vivo relevance of these findings was further corroborated in Tat transgenic mice wherein hippocampal injection of recombinant rAAV2-PDGF-B restored impaired NPC proliferation that was induced by Tat & cocaine. Together these data underpin the role of TRPC 1 channel as a novel target that regulates cell proliferation-mediated by PDGF-BB with implications for therapeutic intervention for reversal of impaired neurogenesis inflicted by Tat and cocaine.
HIV-associated neurological disorders (HAND) comprise a range of symptomatology with varying degrees of HIV-related neuropsychiatric impairments. Drugs of abuse have been known to accelerate the incidence and progression of HAND (Gurwell et al., 2001; Aksenov et al., 2006). Psychostimulant cocaine that is widely abused by HIV+ individuals has been suggested to worsen HIV progression in the brain. Although the advent of anti-retroviral therapy (ART) has decreased the incidence of HAND, its prevalence is actually on a rise (Gonzalez-Scarano and Martin-Garcia, 2005). Mounting evidence indicates that brains of patients with HIV-associated CNS disease exhibit not only neuronal damage/loss, but also exhibit fewer adult neural stem/progenitor cells (NPCs) in the dentate gyrus of the hippocampus. Such a defect could account for increased prevalence of behavioral deficits observed in patients with HAND in the post-ART era.
New dentate granule cells are continuously generated from neural progenitor cells and are integrated into the existing hippocampal circuitry in the adult mammalian brain by a process termed as neurogenesis (Venkatesan et al., 2007). Adult hippocampal neurogenesis is regulated by a variety of physiological and pathological stimulii. It has been reported that HIV transactivating protein Tat, that is both released by infected cells as well as taken up by neighboring cells, is known to impair neurogenesis (Mishra et al., 2010). In addition to this protein, cocaine can also negatively affect the self-renewal capacity of the hippocampus by diminishing the proliferative rate of NPCs (Yamaguchi et al., 2004; Yamaguchi et al., 2005; Hu et al., 2006). These findings raise the possibility that cognitive dysfunction in the setting of HIV infection and drug abuse may, in part, be attributed to impairment of hippocampal neurogenesis.
Neurotrophic family of growth factors plays key roles in maintaining neuronal homeostasis via regulation of neurogenesis (Almeida et al., 2005; Mohapel et al., 2005). Growth factor PDGF is composed of a family of five dimeric ligands that act via two receptor tyrosine kinases, PDGF-αR & -βR (Li et al., 2000; Bergsten et al., 2001; Heldin et al., 2002). PDGF-BB has been implicated as a crucial factor in the developing postnatal rat brains(Smits et al., 1991) and has also been implicated in reversing neuronal toxicity (Yao et al., 2009a). Based on the role of PDGF-BB in the developing CNS, and its increased expression in the subventricular zone during peak embryonic proliferative and migration periods (Sasahara et al., 1992), PDGF-BB can be envisioned as a promising candidate for stimulating neurogenesis.
Transient receptor potential canonical (TRPC) channels are Ca2+-permeable, nonselective cation channels formed by homomeric or heteromeric complexes of TRPC protein (Jia et al., 2007). Seven mammalian TRPC proteins (TRPC1-7) have been identified (Vazquez et al., 2004). Among these subtypes, TRPC1 has been shown to be involved in bFGF-induced self-renewal of embryonic rat NPCs (Fiorio Pla et al., 2005). Whether TRPC functions to modulate PDGF-BB-mediated neurogenesis in NPCs remains an enigma.
In the current study, we show direct evidence that PDGF-BB/PDGF-R axis in NPCs may contribute to the NPC proliferation via a previously unidentified role of TRPC 1.
Recombinant PDGF-BB was purchased from R&D Systems (Minneapolis, MN, USA) and Tat1-72 was obtained from UK College of Medicine, Lexington, KY. The specific PI3-kinase inhibitor LY294002, L-, N-, P-type voltage-dependent calcium channel (VDCC) blocker calcicludine, PLC inhibitor U73122 and MEK1/2 inhibitor U0126 were purchased from Calbiochem (San Diego, CA). Tyrosine kinase inhibitor STI 571 was obtained from Novartis, Basel, Switzerland. Cocaine, TRPC blocker SKF96365, T-type VDCC blocker NiCl2, and IKK2 inhibitor SC514 was purchased from Sigma Chemicals (St. Louis, MO). H89 and 2-ApB were obtained from Tocris (Park Ellisville, MO, USA). Anti-TRPC1,4, 5 & 6 antibodies were purchased from Alomone lab (Jerusalem, Israel). The primary antibodies used were: p- PDGF-αR & -βR/ PDGF-αR & -β R, p-ERK/ERK, p-CREB/CREB, p-Akt/Akt, p65NF-kB, Histone, p-mTOR/mTOR, p-p70S6K/p70S6K, p-4E-BP/4E-BP, p-S6 ribosomal protein/S6 ribosomal protein (Cell Signaling, 1:200), and β-actin (Sigma, 1:4000).
Pregnant rats were purchased from (Charles River Laboratories, Inc., Wilmington, MA). All of animals were housed under conditions of constant temperature and humidity on a 12-h light, 12-h dark cycle, with lights on at 0700 h. Food and water were available ad libitum. All animal procedures were performed according to the protocols approved by the Institutional Animal Care and Use Committee of the University of Nebraska Medical Center. Tat transgenic mouse is a doxycycline-inducible and brain-specific mouse model developed by Drs. Avindra Nath and Kurt Hauser. This is a well-characterized rodent model containing a Tat gene driven by GFAP and is induced by doxycylcline.
NPCs derived from the hippocampus of embryonic day18 (E18) fetus were cultured in substrate-free tissue culture T75 flasks as reported by Tian et al.. After 4–7 days, NPCs formed neurospheres and were dissociated with Trypsin-EDTA for 20 min at 37°C and plated on poly-D-lysine pre-coated plates and were used if found more than 90% nestin+ (a marker for progenitor cells). Based upon our earlier studies, NPCs were treated with varying concentrations of PDGF-BB (20, 50 & 100 ng/ml), HIV-1 Tat (50, 100 & 200 ng/ml) and cocaine (1, 10 & 100μM). Treatment of NPCs with pharmacological inhibitors (STI-571: 1μM; SKF96365: 20μM; EGTA: 2 mM; 2ApB: 100 μM; Xest-C: 1 μM; U73122: 1 μM; U73343: 1 μM ; U0126: 20μM; LY294002: 20μM; Rapamycin: 1μM) involved pretreating cells with the respective inhibitors for 1h followed by exposure with Tat & cocaine and/or PDGF-BB. Forty eight hours later, cells were examined for cell proliferation.
Cell proliferation was measured by CyQUANT cell proliferation assay according to the manufacture’s protocol (Invitrogen). NPCs dissociated from neurosphere were seeded in 96-well plates at a density of 104 cells/well for 2 days and were pre-treated with PDGF-BB for 1hr followed by subsequent treatment with Tat and cocaine for 48 hrs. Then, 200 μl of the CyQUANT® GR dye/cell-lysis buffer was added into each well and incubated in the CO2 incubator for 15 min. Fluorescence intensity of each well was obtained using a Dynatech MR5000 plate counter at excitation and emission wavelengths of 480 and 520 nm, respectively.
To assess the cytotoxicity of cocaine and/or Tat, cells were lysed and assessed for LDH released into the culture medium using a nonradioactive cytotoxicity assay kit (CytoTox 96, Promega) (Peng et al., 2008).
The primers for rat PDGF-αR & βR were obtained from SABiosciences. Total RNA was extracted with Trizol reagent (Invitrogen, Carlsbad, CS) according to the manufacturer’s instructions and our previous report for the expression of TRPC channels (Yao et al., 2009b; Yao et al., 2009a).
Treated cells were lysed using the Mammalian Cell Lysis kit (Sigma, St. Louis, MO, USA) and the NE-PER Nuclear and Cytoplasmic Extraction kit (Pierce, Rockford, IL, USA). Equal amounts of the proteins were electrophoresed in a sodium dodecyl sulfate-polyacrylamide gel (12%) under reducing conditions followed by transfer to PVDF membranes. The blots were blocked with 5% non-fat dry milk in phosphate buffered saline as described previously (Yao et al., 2009b; Yao et al., 2009a).
For immunocytochemistry, NPCs were plated on cover slips, fixed with 4% paraformaldehyde and permeabilized with 0.3% Triton X-100 in PBS. For BrdU detection, the fixed cells were incubated in 2N HCl with 0.3% Triton X-100 followed by neutralization with 0.1M boric acid (pH 8.0). Cells were then incubated with a blocking buffer followed by incubation with rat anti-Nestin (1:100; Millpore, Billerica, MA) and rabbit anti-PDGF-αR & -βR (1:500; Cell Signalling,Danvers, MA), BrdU (1:200, Santa Cruz Biotechnology, Santa Cruz, CA) antibodies overnight at 4°C. Secondary AlexaFluor 488 goat anti-rat IgG and AlexaFluor 569 goat anti-rabbit IgG, was added at a 1:500 dilution for 2h to detect Nestin and PDGF-αR & βR, followed by mounting of cells with Vectashield onto glass slides (Vector Laboratories, Burlingame, CA). Fluorescent images were acquired at RT on a Zeiss Observer. A Z1 inverted microscope was used; images were processed using AxioVs 40 Version 126.96.36.199 software (Carl Zeiss MicroImaging GmbH). Photographs were acquired using an AxioCam MRm digital camera.
siRNA targeted against PDGF-αR & βR, TRPC1, 4, 5 and 6 were obtained from Thermo Scientific Dharmacon RNAi Technologies (Accell SMART Pool) and transfected using the rat stem cell Nucleofector Kit (Amaxa, Gaithersburg, MD) according to the manufacturer’s instructions. The sequences of these siRNAs are listed in Table 1. Briefly, dissociated cells from neurospheres were resuspended in the transfection medium, mixed with respective siRNAs (100 nM), and electroporated following which cells were quickly centrifuged, resuspended and plated. The knockdown efficiency was determined by western blotting of extracts from transfected cultures 96 h post siRNA delivery. Cells were treated with PDGF-BB for proliferation or WB analyses at 96 h following siRNA delivery.
The sections were denatured by incubation in 50% formamide in 2xSSC at 65°C. After rinses with PBS, sections were incubated with 2N HCl at 37°C, washed and treated with 0.1M boric acid (pH 8.5) followed by incubation with H2O2 for 10 minutes. After blocking sections were incubated with mouse anti-BrdU (1:200) overnight at 4°C. Next day, sections were washed followed by incubation with biotinylated goat anti-mouse immunoglobulin G (1:200) in immunoblocking buffer at RT for 1hr, and incubated with an avidin-biotin-peroxidase kit for 1h. Horseradish peroxidase reaction product was visualized with nickel-enhanced DAB peroxidase substrate kit. Every 6th section from each animal was used to quantify BrdU-positive cells in the subgranular zone and hillus of the dentate gyrus bilaterally. The number of BrdU-positive cells per section was calculated and multiplied by 6 to obtain the total number of cells per dentate gyrus per mouse as reported by Garza et al.
The rodent GFP PCR product was cloned into rAAV2-MCS-WPRE vector. The vector was packaged in AAV-293 cells (derived from HEK293 cells to produce higher viral titers) by CaPO4 transient transfection of the vector plasmid & virion production plasmids (Stratagene AAV-helperfree kit), followed by lysis of cells to recover replication-deficient virions that were purified by affinity column (ViraTrap AAV purification kit, GeneMega Inc). Recombinant AAV2 was titered for total particles by serial-dilution transfection in HEK293 and verified by quantitative-competitive PCR assay. rAAV2-GFP was then transduced into NPCs at the MOI (2×105) followed by monitoring for PDGF-BB expression by imaging and WB analyses.
Ten-week-old HIV Tat Tg mice were divided into four groups of 24 mice with each group (n=6; male) treated with either: 1) Saline; 2) Doxycycline+Cocaine; 3) Doxycycline+Cocaine+rAAV2-PDGF-B or 4) Doxycycline+Cocaine+rAAV2-GFP. In the group of AAV2-PDGF-B and AAV2-GFP, rAAV2 was microinjected into the hippocampus of mice (1μl of 109 viral genomes/μl) using the microinjection parameters (coordinates 2.06 mm behind the bregma, 2.0 mm lateral from the sagittal midline at a depth of 1.7 mm to skull surface). To reduce the volume of inoculation and increase the precision, a 10 μl Hamilton syringe with a 33G needle was utilized for intra-hippocampal injections as described. One month later, mice were administrated doxycycline feed and cocaine injection daily for additional 14 days. To evaluate the effect of rAAV2-PDGF-B on cell proliferation, groups of 24 mice were divided as described above, injected with BrdU and assessed for proliferation.
Data were expressed as mean ± SD. Significance of differences between control and samples treated with various drugs was determined by one-way ANOVA followed by post hoc least significant difference (LSD) test. Values of p < 0.05 were taken as statistically significant
To assess the effect of Tatand cocaine on NPC proliferation, NPCs were exposed to different concentrations of cocaine (1, 10 & 100 μM) or Tat (50,100 & 200ng/ml) and assessed for cell proliferation. Cocaine concentrations were based on the levels found in the plasma of human volunteers (Van Dyke et al., 1976; Kalasinsky et al., 2000; Stephens et al., 2004). As shown in Figs 1A-B, there was a concentration-dependent reduction of proliferation in presence of both Tat & cocaine. For the physiological relevance of cocaine concentration, 10 μM cocaine and 200 ng/ml Tat was therefore chosen for all our further studies. Treatment with heat-inactivated or mutant Tat had no effect on proliferation.
Next, we assessed the effect of PDGF-BB on proliferation of NPCs. PDGF-BB at all the concentrations tested resulted in significant increase in proliferation of NPCs (Fig. 1C). We next examined the effect of PDGF-BB on Tat & cocaine-mediated inhibition of proliferation. Tat alone decreased NPC proliferation by 20.3%, cocaine alone decreased it by 21.2%, and in conjunction both caused a statistically significant decrease in proliferation (almost 35%-Fig. 1D). Pre-treatment of NPCs with PDGF-BB followed by exposure of cells to Tat & cocaine, however, resulted in restoration of proliferation. These findings were further validated by immunostaining using the anti-BrdU antibody (Fig. 1E).
In order to test whether cocaine and/or Tat also affect survival of NPCs, MTT assay was employed. As shown in Figure 1F, both cocaine and Tat significantly decreased NPC survival. To exclude the possibility of the cytotoxic effects of either cocaine or Tat, exposed cells were assessed for toxicity using the lactate dehydrogenase assay. Treatment of NPCs with either agent failed to demonstrate increased cytotoxicity (Fig. 1G). Furthermore, TUNEL analysis, as an indicator of cell death revealed that cocaine and Tat did not induce cytoxicity (Fig. 1H & I). Taken together these findings suggested that decreased cell proliferation induced by cocaine/Tat was not due to the cytotoxic effects.
Since PDGF-BB mediates signaling through its receptors PDGF-αR and -βR, we first sought to examine the expression of these receptors in NPCs. While NPCs expressed both types of receptors as evidenced by RT-PCR and WB analyses (Fig. 2A &B), there was higher abundance of PDGF-αR, which was confirmed by immunocytochemistry. PDGF-αR & -βR immunoreactivity co-localized with Nestin+ cells in NPCs (Fig. 2C). Furthermore, exogenous PDGF-BB rapidly phosphorylated PDGF-αR & -βR in NPCs as evidenced by immunoprecipitation analysis (Fig. 2D). Intriguingly, pre-treatment of NPCs with the tyrosine kinase receptor antagonist STI-571 abolished PDGF-BB-mediated increase in proliferation, thus confirming the role of PDGF-BB/PDGF-R axis in this process (Fig. 2E).
It is well recognized that STI-571 is not a specific antagonist for either PDGF-αR/-βR, since it can inhibit other tyrosine kinases as well. As an alternative approach we thus sought to knock down the respective receptor expression in NPCs using the siRNA strategy. PDGF-αR & -βR siRNAs abrogated the respective expression of the two receptors in NPCs (Fig. 2F), while also partially abrogating PDGF-BB-mediated increased proliferation of NPCs (Fig. 2G).
In order to study the effects of PDGF-BB on the intracellular Ca2+ level, we measured Ca2+ influx using Fluo-4/AM imaging. Exposure of NPCs to PDGF-BB triggered rapid and sustained intracellular Ca2+ elevation in NPCs (Fig. 3A). This response was inhibited in NPCs pretreated with the pan TRPC inhibitor SKF96365, thus underscoring its role (Fig. 3B).
Based on the premise that Ca2+ mediates proliferation and that TRPC functions as Ca2+ influx channels, it was rationalized that Ca2+ influx through TRPC channels is a pre-requisite for increased NPC proliferation mediated by PDGF-BB. NPCs were pretreated with the TRPC blocker and PDGF-BB and subsequently assessed for proliferation in the presence of Tat& cocaine. Pretreatment of NPCs with SKF96365 resulted in failure of PDGF-BB to rescue Tat & cocaine-mediated inhibition of proliferation (Fig. 3C). Further validation of the role of extracellular Ca2+ influx in PDGF-BB mediated proliferation of NPCs was supported by pretreating the cells with EGTA, which resulted in suppression of NPC proliferation mediated by PDGF-BB (Fig. 3D).
PDGF-BB upon activating its receptor is known to stimulate phospholipase C (PLC), resulting in inositol triphosphate (IP3)-dependent release of Ca2+ from intracellular stores as well from extracellular sources. Interference of the PLC-IP3 pathway thus ought to suppress the increased NPC proliferation exerted by PDGF-BB. As expected, pretreatment of NPCs with the PLC inhibitor-U73122 but not its inactive analog-U73343, abolished PDGF-mediated increase in proliferation in Tat and cocaine treated NPCs. The effect of PDGF-BB on proliferation was also dependent on IP3R activation, since exposure of NPCs to antagonists specific for IP3 receptor, 2-ApB and Xest-C resulted in suppression of PDGF-BB-mediated increase in proliferation (Fig. 3D). All of inhibitors alone failed to inhibit cell proliferation significantly.
Since TRPC family comprises of 7 subtypes it was important to first investigate the expression pattern of these subtypes in NPCs. NPCs expressed TRPC1, 4, 5 & 6 but not 2, 3 or 7 (Fig. 3E). To ascertain the TRPC subtype(s) critical for PDGF-BB-mediated increased NPC proliferation, each of the subtypes was individually down-regulated using the specific siRNAs, followed by assessment of proliferation. siRNA against TRPC1, 4, 5 & 6 suppressed the expression of the respective TRPC subtype as expected (Fig. 3F) however, TRPC 1 but not any of the other TRPC siRNAs alleviated PDGF-BB-mediated proliferation of NPCs (Fig. 3G). To specifically confirm the role of TRPC1 in PDGF-BB-mediated cell proliferation, we took the approach of knocking-down the three TRPC channels together while leaving only one TRPC effective. As shown in Figure 3H, only expression of TRPC1 but not TRPC 4,5, and 6, was essential for PDGF-BB-mediated cell proliferation.
The activation of TRPC channels can be regulated via multiple mechanisms including hydrolysis of phosphoinositide(Kwon et al., 2007). A recent study provides evidence for regulation of TRPC-dependent Ca2+ influx by PI(3,4,5)P3. Consistent with previous findings (Henle et al., 2011), pre-treatment of NPCs with PI3K inhibitor-LY294002 significantly attenuated Ca2+ entry induced by PDGF-BB (Fig. 4A). To assess the role of PI3K in specifically regulating TRPC-dependent Ca2+ influx, NPCs were also pretreated with the other voltage-dependent calcium channel (VDCC) blockers (calcicludine [200 nM; L-, N-, P-type VDCC blocker] and NiCl2 [50 μM;T-type VDCC; Sigma-Aldrich) to specifically isolate TRPC-dependent Ca2+ influx from voltage-dependent Ca2+ influx, followed by treatment of cells with the PI3K inhibitor. As shown in Figure 4B, VDCC blocker decreased PDGF-BB-induced Ca2+ influx, and this was further decreased in cells treated with LY294002, thereby underpinning the role PI3K in TRPC-dependent Ca2+ influx.
MAPK pathway has been demonstrated to play a crucial role in NPC proliferation (Learish et al., 2000). It was therefore of interest to examine the effect of PDGF-BB on ERK and its downstream transcription factor CREB in NPCs. Exposure of NPCs to PDGF-BB resulted in sustained and time-dependent activation of ERK/CREB (Figs.5A& 6A). Having determined the role of TRPC channels in PDGF-BB-mediated cell proliferation, we next wanted to examine the role of these channels in PDGF-BB-mediated activation of ERK/CREB. Pre-treatment of NPCs with SKF96365 significantly attenuated PDGF-BB-induced ERK/CREB phosphorylation, suggesting thereby that PDGF-BB-mediated activation of ERK/CREB involved TRPC (Figs.5B&6B). The functional role of PDGF-BB-induced ERK/CREB activation was further corroborated using proliferation assays, wherein PDGF-BB failed to exert proliferative effect in cells pre-treated with ERK inhibitor (Fig. 5C), DN-MEK (Fig. 5D) or PKA inhibitor (Fig. 6C). Similar to the role of TRPC1 in PDGF-BB-mediated proliferation, transfection of NPCs with siRNA for TRPC1 resulted in abrogation of PDGF-mediated activation of ERK/CREB (Figs 5E & 6D). To specifically confirm the role of TRPC in PDGF-BB-mediated ERK phosphorylation, we again sought the approach of knocking-down the three TRPC channels together while leaving only one TRPC effective. Expression of TRPC1 but not TRPC 4, 5, and 6, was essential for PDGF-BB-mediated phosphorylation of ERK (Fig. 5F) and CREB (Fig. 6E).
In addition to the activation of MEK/ERK pathway, Akt pathway also is known to play a critical role in proliferation(Peltier et al., 2007). To examine the role of this pathway in PDGF-BB-mediated proliferation, cell lysates were examined for phosphorylation of Akt. Following exposure of NPCs to PDGF-BB there was an enhanced and sustained activation of Akt and its downstream mediator NF-kB (Figs.7A & 8A). Intriguingly, pre-treatment of NPCs with SKF96365 did not attenuate PDGF-BB-induced Akt/NF-kB activation (Figs.7B & 8B). The functional role of PDGF-BB-induced Akt/NF-kB activation in mediating cell proliferation was corroborated in cells pre-treated with either PI3K (Fig. 7C) or Ikk2 (Fig. 8C) inhibitors. The role of Akt and NF-kB in PDGF-BB-mediated cell proliferation was further validated in cells transduced with either a DN-Akt (Fig. 7D) or mutant NF-kB (Fig. 8D) construct. Unlike the involvement of TRPC1 in the activation of the ERK/CREB pathway, PDGF-BB-mediated activation of Akt/NF-kB while critical for NPC proliferation, did not involve TRPC1 as evidenced by the failure of siRNA to suppress activation of the latter pathway (Figs.7E & 8E). To specifically confirm the role of TRPC in PDGF-BB-mediated activation of A Akt/NF-kB, cells were simultaneously knocked-down for the three TRPC channels together while leaving only one TRPC effective. None of the combinations of TRPC knowck down had any effect on PDGF-BB-mediated activation of Akt (Fig. 7F) or NF-kB (Fig. 8F). These findings thus lead us to speculate that PDGF-BB-mediated activation of Akt/NF-κB pathway was independent of TRPC activation. Furthermore, treatment of NPCs with LY294002 attenuated ERK phosphorylation induced by PDGF-BB, however, reciprocal inhibition of MEK by U0126 failed to exert any effect onAkt phosphorylation induced by PDGF-BB (Fig. 7G).
Intracellular Ca2+ elevations have been implicated in the activation of ERK as well as the mammalian target of rapamycin (mTOR) pathways (Xu et al., 2011). Based on the activation of ERK pathway by PDGF-BB, we next sought to examine whether the mTOR/4E-BP & p70S6K pathways played a role in this process. mTOR and its downstream mediators 4E-BP & p70S6K/S6 ribosomal protein (S6) were phosphorylated time-dependently in the presence of PDGF-BB (Fig. 9A). Functional implication of PDGF-BB-induced mTOR activation in NPC proliferation was further corroborated using proliferation assays, wherein PDGF-BB failed to induce proliferation in NPCs pre-treated with mTOR inhibitor rapamycin (Fig. 9B). These findings underpinned the role of mTOR in PDGF-BB-mediated NPC proliferation.
To further unravel the role of TRPC channels in PDGF-BB-mediated activation of mTOR/4E-BP & p70S6K pathway, NPCs were pretreated with the TRPC blocker and assessed for expression of the activation mediators. Pre-treatment of cells with SKF96365 markedly attenuated PDGF-BB-induced phosphorylation of mTOR/4E-BP & p70S6K (Fig. 9C).
Since TRPC was involved in both ERK and mTOR activation, we next wanted to assess the relationship between ERK and mTOR. Intriguingly, pharmacological blocking of ERK activation by U0126 inhibited PDGF-BB-mediated mTOR/4E-BP & p70S6K phosphorylation. In concordance with the reported finding by Bodine et. al., our results also confirmed the link between Akt and mTOR/4E-BP & p70S6K phosphorylation, as evidenced by the fact that PI3K inhibitor significantly inhibited mTOR/4E-BP & p70S6K phosphorylation induced by PDGF-BB (data not shown). To confirm the role of specific TRPC channels in mTOR pathway, NPCs were transfected with the respective TRPC siRNAs and assessed for expression of the activated signaling mediators. NPCs transfected with TRPC1 siRNA exhibited attenuation of mTOR/4E-BP & p70S6K phosphorylation induced by PDGF-BB (Figs. 9D–F). These results implicate the role of TRPC1 channel in PDGF-BB mediated activation of mTOR/p-P70S6/4E-BP.
To examine the relevance of PDGF-BB-mediated proliferation of NPCs in vivo, we assessed the proliferative potential of microinjected rAAV2-PDGF-B in the hippocampi of Tat transgenic mice that were administered cocaine for 14 days. Having determined the successful expression of the rAAV2-PDGF-B constructs (Figs. 10A–C), the next step was to test the efficacy of rAAV2-GFP/PDGF-B transduction in vivo. Expression of GFP was largely restricted within the hippocampus (Fig. 10D) and as expected, there was enhanced expression of PDGF-BB in the hippocampi of rAAV2-PDGF-B injected mice compared with mice injected AAV2-GFP (Fig. 10E). Four weeks following adenoviral construct injections (to ensure optimal expression of the transgene; unpublished observations); mice were put on a doxycycline feed (for induction of Tat in the CNS) and also administered cocaine daily for 14 days followed by assessment of BrdU positive cells in the hippocampii. There was decreased proliferation in doxycycline-fed, cocaine-injected mice as evidenced by decreased BrdU positive cells compared with controls (saline injected and normal fed diet) (Fig. 10F). Decreased proliferation induced by Tat and cocaine was ameliorated in mice pre-treated with AAV2-PDGF-B (quantified in Fig. 10G), thereby suggesting the role of PDGF-BB in restoring impaired NPC proliferation.
It is well-recognized that new dentate granule cells are continuously generated from neural progenitor cells and are integrated into the existing hippocampal circuitry in the adult mammalian brain through an orchestrated process termed adult neurogenesis(Venkatesan et al., 2007). Neurogenesis is regulated by a variety of physiological as well as pathological stimulii. Both viral protein as well as drugs of abuse such as cocaine can negatively affect the self-renewal capacity of the hippocampus by diminishing the proliferative rate of NPCs(Yamaguchi et al., 2004; Yamaguchi et al., 2005; Hu et al., 2006). These findings thus raise the possibility that cognitive impairment due to comorbid conditions such as HIV infection and drug abuse may, in part, be attributed to impaired hippocampal neurogenesis. Intriguingly, patients with HAND manifest fewer adult NPCs in the dentate gyrus compared with either the non-infected or HIV+ subjects without dementia as reported by Krathwohl et al.
PDGF is composed of a family of five dimeric ligands assembled from four gene products (PDGF A-D) that act via two receptor tyrosine kinases, PDGF-αR & -βR(Li et al., 2000; Bergsten et al., 2001; Heldin et al., 2002). Intriguingly, PDGF-BB has been implicated as a crucial factor in the developing postnatal rat brains(Smits et al., 1991). Furthermore, PDGF-BB pretreatment is known to induce striatal neurogenesis in a Parkinsonian rat model of 6-hydroxydopamine lesions (Mohapel et al., 2005). The novel finding of this report is that PDGF-BB via binding to its cognate receptors was able to rescue Tat & cocaine-mediated impairment of NPC proliferation both in vitro and in vivo.
Another novel finding of this study is the role of TRPC 1 in PDGF-BB-mediated increased proliferation against Tat & cocaine, thereby lending credence to previous reports indicating the involvement of TRPC signaling in neurogenesis(Fiorio Pla et al., 2005; Weick et al., 2009). Consistent with the previous reports demonstrating the co-localization of TRPC (1, 4, 5 & 6) with the progenitor cells in hippocampus (De March et al., 2006; Martorana et al., 2006), our findings also provide evidence that all these subtypes were expressed in NPCs. The present study identified a novel molecular target-TRPC1 underlying the restoration of PDGF-BB-mediated neurogenesis. These findings are in agreement with a previous study indicating that bFGF-mediated Ca2+ increase and cell proliferation were significantly reduced in TRPC1 knock out animals(Fiorio Pla et al., 2005).
PDGF is known to exerts its action by triggering [Ca2+]i transients in neuronal precursor cells(Cuddon et al., 2008), however, the mechanism of action remains less clear. Herein we report that PDGF-BB induced [Ca2+]i elevations through activation of TRPC1. The mammalian TRPC channels can be activated by G-protein-coupled receptors and RTKs(Clapham, 2003). PDGF-R belongs to the receptor tyrosine kinase family and is known to activate PLC, leading to hydrolysis of PIP2 into membrane-bound diacyglycerol and soluble IP3. Generation of IP3 results in IP3 receptor-mediated release of Ca2+ from intracellular stores as well as Ca2+ influx extracellularly (White et al., 2005). In addition to the role of IP3 in regulating activation of TRPC, Lu et al have also shown that activation of PI3K leads to generation of PIP3-mediated Ca2+ influx via the TRPC channels in platelets, Jurkat and mast cells(Ching et al., 2001). A key finding here is that PDGF-BB-induced influx of Ca2+ via TRPC activation involved PI3K. As was evident from our findings PDGF-BB-mediated activation of PI3K involved PI(3,4,5)P3 dependent activation of TRPC1 resulting in influx of Ca2+ and ensuing NPC proliferation.
Using both pharmacological and genetic approaches, we demonstrated activation of parallel but distinct cell signaling pathways including ERK/CREB, Akt/ mTOR/4E-BP & p70S6K, and Akt/NF-kB in PDGF-BB-mediated proliferation. Interestingly, blocking TRPC1 resulted in suppression of PDGF-BB-induced ERK/CREB and mTOR/4E-BP & p70S6K activation, but had no impact on Akt/NF-kB activation. The findings reported here on PDGF-BB-mediated activation of ERK/CREB pathway are consistent with several lines of published reports specifically, the study wherein activation of TRPC3 and TRPC6 by the growth factor BDNF resulted in stimulation of two signaling pathways: Ca2+/Ras/MEK/ERK and Ca2+/CaM/CaMK, intersecting at CREB(Jia et al., 2007; Yao et al., 2009b; Yao et al., 2009a).
In addition to activation of mTOR/4E-BP & p70S6K signaling pathway by PI3K, our findings also underpin the role of TRPC1 channel in activating this pathway. Our data however, is in disagreement with a report describing increased mTOR phosphorylation in lysates of heart tissue isolated from TRPC1 knock out (KO) mice following the transverse aortic constriction operation compared with the WT controls(Seth et al., 2009). This inconsistency could be due to a compensatory response in TRPC1 KO mouse model since mTOR may positively affect the protein translation of other TRPC channels or proteins involved in the TRPC/PI3K network(Vollenbroker et al., 2009). Our results suggest that ERK pathway, that lies downstream of TRPC, could be involved in the regulation of mTOR. We demonstrate a crosstalk between MEK/ERK and mTOR pathways as evidenced by the fact that MEK inhibitor was capable of inhibiting PDGF-BB-induced phosphorylation of mTOR/4E-BP & p70S6K. This is in agreement with a previous report indicating ERK pathway-mediated regulation of mTOR activation through TCS2 phosphorylation(Ma et al., 2005).
Another interesting finding herein was the observation that inhibition of PI3K/Akt resulted in reversal of PDGF-BB-mediated NPC proliferation thereby highlighting the potential role of this pathway. Role of Akt pathway was further confirmed using a loss of function approach by transfecting NPCs with a DN construct of Akt. Our results are in agreement with previous reports that PI3K/Akt transduces intracellular signals that regulate NPC proliferation (Peltier et al., 2007). Interestingly, unlike the TRPC-mediated activation of ERK/CREB, PDGF-BB-mediated activation of Akt and its downstream NF-kB was independent of TRPC1 channel. Activation of NF-κB plays a key role in enhancing NPC proliferation following exposure to a wide array of stimuli(Piotrowska et al., 2006; Kaus et al., 2010). Our findings are consistent with a report indicating Akt-mediated transduction of signal via activation of NF-κB in toll-like receptor-mediated modulation of adult hippocampal neurogenesis(Rolls et al., 2007).
To investigate the relevance of PDGF-BB-mediated cell proliferation in vivo, a genetic approach using the inducible Tat transgenic mice was employed. Microinjection of rAAV2-PDGF-B into the hippocampii of mice that were induced to express Tat in the CNS and that were also administered cocaine for 14 days, resulted in restoration of impaired NPC proliferation thus confirming the role of PDGF-BB in NPC proliferation. These findings are in agreement with the previous reports describing the role of PDGF-BB in induction of striatal neurogenesis in adult rats with 6-hydroxydopamine lesions(Mohapel et al., 2005).
In summary, activation of the PDGF-BB/PDGF-R axis resulted in stimulation of PI3K and PLC/IP3 R pathways leading to activation of TRPC channels, which in turn, resulted in Ca2+ influx, culminating in activation of ERK /CREB and mTOR/4E-BP & p70S6K (Fig. 11). Taken together our findings suggest that although the two pathways involved in PDGF-BB-mediated restoration of impaired proliferation of NPCs operate independent of each other, their combined actions are necessary for the observed proliferation potential of PDGF-BB. A better understanding of these molecular pathways could be critical for the development of therapeutic interventions aimed at targeting cognitive impairment observed in cocaine-abusing HIV-infected individuals.
This work was supported by grants DA024442 (SB), DA030285 (HY) and DA 033150 (SB and HY) from the National Institutes of Health.
Conflict of interest disclosure: The authors declare no competing financial interests.