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There is a great need for pharmacological approaches to enhance neural progenitor cell (NPC) function particularly in neuroinflammatory diseases with failed neuroregeneration. In diseases such as multiple sclerosis and stroke, T cell infiltration occurs in periventricular zones where NPC are located and is associated with irreversible neuronal loss. We studied the effect of T cell activation on NPC functions. NPC proliferation and neuronal differentiation was impaired by granzyme B (GrB) released by the T cells. GrB mediated its effects by the activation of a Gi protein-coupled receptor leading to decreased intracellular levels of cyclic AMP and subsequent expression of the voltage-dependent potassium channel, Kv1.3. Importantly, blocking channel activity with margatoxin or blocking its expression reversed the inhibitory effects of GrB on NPC. We have thus identified a novel pathway in neurogenesis. The increased expression of Kv1.3 in pathological conditions makes it a novel target for promoting neurorestoration.
The incidence of neurodegenerative disorders continues to rise. However, currently available neuroprotective therapies have uniformly shown either no or at best marginal benefit in these diseases, hence the focus is shifting towards the development of neurorestorative therapies. The possibility that endogenous or transplanted neural progenitor cells (NPC) can replace lost brain tissue has raised enormous hope amongst the public and the scientific community. NPC are present in the adult brain and they retain the ability to proliferate and differentiate into neurons and glial cells(Arvidsson et al., 2002). Their therapeutic potential in neurodegenerative disorders is also being explored (Ikeda et al., 2005; Hayashi et al., 2006). Therefore, any damage to NPC may result in the inability to replace lost or injured neurons and could be a contributing factor to the brain atrophy. It is thus critical to understand the subcellular environment in which these cells reside.
T cells play an important role in inflammatory associated neurodegenerative diseases. It was recently demonstrated that blockade of the infiltration of T cells into the brain reduced infarct/reperfusion-induced brain infarction and accompanying neurological deficits (Shichita et al., 2009). In neuroinflammatory diseases such as multiple sclerosis, infiltrates of activated T cells are present in the periventricular zones where the NPC are localized (Nait-Oumesmar et al., 2007; Matsushita et al., 2008). Multiple sclerosis is also accompanied with progressive brain atrophy suggesting a failure of the regenerative process (Chard et al., 2004; Furby et al., 2008). Despite the recognition that if NPC were to be implanted in patients with multiple sclerosis, they would be subjected to an inflammatory environment, there seems to be great enthusiasm in exploring this as a therapeutic option in these patients (Duncan et al., 2008). While some studies have shown that NPC can attenuate T cell activation through its anti-inflammatory effect (Pluchino et al., 2005; Uccelli et al., 2006), it remains unknown whether activated T cells may affect NPC function.
In multiple sclerosis, CD8+ T cells predominate in the inflammatory infiltrate in both acute and chronic lesions. In these patients, clonal expansion of these cells may occur and they demonstrate the presence of cytotoxic granules (Friese and Fugger, 2005; Goverman et al., 2005; Friese et al., 2008). Other neuroinflammatory diseases such as Rasmussen's syndrome and CNS-immune reconstitution syndrome associated with human immunodeficiency virus infection are also associated with infiltration of CD8+ T cells and brain atrophy but have not been as well studied(Venkataramana et al., 2006; Schwab et al., 2009). Hence we sought to determine whether activated T cells could impact NPC function. We observed that granzyme B (GrB) was a key factor that inhibited NPC proliferation and differentiation. The underlying mechanisms involve activation of pertussis toxin (PTX)-sensitive Gi-coupled receptors and increased expression of voltage dependent potassium channel, Kv1.3 expression in NPC. Since there is increased expression of Kv1.3 in activated T cells as well and antagonists of this receptor can block T cell activation(Hu et al., 2007), Kv1.3 antagonists may play a dual role by controlling T cell activation and enhancing NPC function.
Cell culture medium and supplements were purchased from Invitrogen (Carlsbad, CA) if not specifically described. Human NPC were cultured from human fetal brain specimens of 7-8 weeks gestation in accordance with NIH guidelines and following approval by the Institutional Review Board at Johns Hopkins University. The tissues were then triturated after removing meninges and blood vessels. After centrifugation at 1000 rpm, cells were resuspended in DMEM/F12 media [containing 8 mM glucose, 1× N2 supplement, 1% antibiotics, 0.1% albumin (Sigma, St. Louis, MO), human fibroblast growth factor-beta (hFGFb; 20 ng/mL) and human epidermal growth factor (hEGF) (20 ng/mL)] and plated onto poly-D-lysine (Sigma) coated T 25 cm2 tissue culture flasks. When cell cultures reached 60% confluence, they were subcultured by treatment with 0.0125% trypsin (Sigma) and plated at a density of 2×104 cells/ml onto poly-D-lysine coated 96 well plates or cover slips in 24 well plates. Medium was replaced every other day. NPC cultures were ready for experiments 4-5 days after replating and >98% of the cells expressed the neural stem cell marker, nestin while <1% of the cells expressed GFAP (a marker for astrocyte) or beta-III tubulin (a neuronal cell marker) as determined by immunocytochemistry. Cell proliferation was assessed by incubating the cells with BrdU labeling reagent (1:100, ZYMED, San Francisco, CA) for 6 hours followed by immunostaining for BrdU. When subjected to differentiation media by replacing hEGF and hFGFb containing medium with fresh medium containing 1% of fetal bovine serum (FBS; Gemini, West Sacramento, CA), the NPC were capable of differentiating into neurons (10-50%) and astrocytes (50-90%) after 4-7 days (Suppl Figure 1).
CD8+ T cells were isolated from peripheral blood mononuclear cells of healthy human adult donors. CD8+ T cells were isolated by negative selection using MACS beads (Miltenyi Biotec, Bergisch Gladbach, Germany) and then incubated at 37° C in Iscove's modified Dulbecco's medium with 5% pooled human serum and activated by placing on plates coated with 1 ug/mL of anti-CD3 and 1 ug/mL of anti-CD28 for 72 hr in culture. Culture supernatants (sups) were then collected and incubated (1:20 dilution) with NPC. To deplete GrB from T cell sups, the sups were incubated 1 : 1 with pre-swollen protein G sepharose (GE Healthcare, Chalfont St. Giles, UK) for 2 h at 4°C, a step taken to eliminate proteins in the lysate which may bind non-specifically to the protein G. The sups were subsequently spun and incubated at 4°C overnight on a rotary table with a monoclonal anti-GrB (Millipore, Billerica, MA) or an isotype-matched mouse IgG2a negative control antibody (Millipore). This mix was then incubated for 2 h with protein G sepharose and filtered through a column. The flow through was used to treat NPC cultures. All incubations (antibody and protein G) were performed on a rotary table at 4°C, and all centrifugations were performed using a desktop Eppendorf centrifuge at 4°C for 5 min at maximum speed (9000 g).
Cells were fixed in 4% paraformaldehyde and permeabilized by incubation in 0.5% TritonX100 in PBS (PBS-T) for 20 min. For BrdU staining, the cells were also denatured in 2N HCl at 37°C for 30 min, and rinsed twice in 0.1 M sodium borate (pH 8.5), followed by the process described above. Cells were immunostained using monoclonal anti-BrdU (Sigma, 1:1000), monoclonal anti- beta-III tubulin (1:1000; Promega, Madison, WI), rabbit anti-GFAP (1:1000; Sigma), rabbit anti-active caspase-3 (1:1000; Sigma), monoclonal anti-nestin (1:1000; gifted by Dr. Eugene Major, NIH, Bethesda, MD) monoclonal anti-CD68 (1:100; Invitrogen), rabbit anti-Oct4(1: 500, Cemines Biosystems, Golden, CO) followed by corresponding secondary antibodies (anti-rabbit Alexa Flour 488, 1:400 and anti-mouse Alexa Flour 597, 1: 400, Invitrogen) and DAPI nuclear staining. Images were acquired on a Zeiss LSM 510 META multiphoton confocal system (Carl Zeiss).
GrB levels were measured by ELISA in tissue culture supernatants and in CSF by ELISA according to manufacturers instructions (Cell Sciences, Canton, MA). Briefly, peripheral blood mononuclear cells were obtained from healthy adult donors. CD8+ T cells were isolated by a standard protocol using a bead depletion method (Miltenyl Biotec). 2×105 cells were seeded per well in a 96-well round bottom plate. Cells were then stimulated with anti CD3 and ant-CD28 Dynabeads according to the manufactures protocol omitting recombinant IL-2 (Invitrogen) for 72 hours at 37°C and 5% carbon dioxide. Cell supernatants were collected and stored at -20 °C until analyzed for GrB levels. CSF samples were obtained from the Johns Hopkins CSF repository following approval by the Institutional Review Board. Control samples were from 10 patients with headaches due to pseudotumor cerebri. Another 10 patients had clinically definite remitting relapsing multiple sclerosis.
To monitor effect of NPC proliferation, cultures were treated with T cell sups (1:20) and GrB (4 nM, Calbiochem, San Diego, CA) for 24 hrs in proliferation media and then analyzed as described above. To determine effects on neuronal differentiation the NPC cultures were similarly treated in differentiating media for 4-7 days and the differentiating cell types were quantified by immunostaining. Beta-tubulin positive neurons and total DAPI positive cells were counted in 9 predetermined fields in each of the two cover slips in every treatment. The percentage of beta-tubulin positive cells was used as an index for neurogenesis.
Cytotoxicity was evaluated by using CellQuanti-Blue™ Cell Viability Assay Kit (BioAssay Systems, Hayward, CA) according to manufacturer's instructions. Briefly, NPC were cultured in maintaining media in 96-well plates and used for experiments at ~60% confluence. After adding GrB (1-4nM), the cells were cultured for 24 hr. CellQuanti-blue solution (10 ul/well) was added and the cells were incubated for 1 hour. The fluorescence was quantified at an excitation wave length of 530 nm and emission wave length of 590 nm using a fluorescence plate reader. Apoptosis was also evaluated by calculating the percentage of active caspase-3 positive NPC after 24 hr or 7 days of GrB (1-10 nM) treatment by using immunostaining.
NPC were seeded in 24 well plates until the cells reached 90% confluence. Cultures were then treated with GrB (1- 4 nM) in maintaining media for 30 min. After removing the media, the cells were treated with 100 μl of 0.1 M HCl and 1% Triton X-100 for 10 min to achieve cell lysis. The lysates were centrifuged at 600 g for 10 min at room temperature and the supernatants were used for the cyclic AMP assay using a competitive ELISA kit (Endogen, Rockford, IL) according to manufacturer's directions.
Kv1.3 expression was determined by quantitative real time polymerase chain reaction (PCR). NPC were treated with GrB for 3 hr and total RNA was extracted using Qiagen RNeasy mini kit (Qiagen, Valencia, CA). The RNA samples were treated with DNase I (Invitrogen) for 15 min and then used for first strand cDNA synthesis using Superscript III first strand cDNA kit (Invitrogen). Real time PCR was performed using ABI PRISM ® 7000 Sequence Detection System (Applied Biosystem, Foster City, CA) according to manufacturer's instructions, using a human Kv1.3 detection kit (SAbioscience, Frederick, MD). GADPH was used as an internal control. The standard curves were plotted for both Kv1.3 and GADPH in each plate using serially diluted preset samples. The ratio of Kv1.3/GADPH for every sample was first calculated and results expressed as fold changes compared to control. Kv1.3 expression was also monitored in NPC cells by immunocytochemistry and western blot using anti-Kv1.3 polyclonal serum (1: 100, Alomone Labs, Jerusalem, Israel). Dual staining for nestin was also performed in immunocytochemistry as described above.
Human NPC were maintained in proliferating medium in 24 well plates for approximately one week at which time they reached ~60% confluence and were used for transfection of siRNA. Kv1.3 siRNA was chemically synthesized, stabilized and fluorescent labeled by Dharmacon Inc (Chicago, IL). Briefly, 1 μl of transfectamine (Invitrogen) was added to Opti-MEM I reduced serum medium to a final volume of 50 μl. 0.2 μl of siRNA was added to Opti-MEM I to a final volume of 50 μl and incubated for 5 min. The siRNA and transfectamine solutions were combined and mixed by gently pipetting and incubated at room temperature for 20 min. Cells were washed with Opti-MEM I, and 400 ul of fresh Opti-MEM I was added to each well. The transfection agent/siRNA complex was added dropwise onto the cells and incubated for 24 hr at 37°C. Select wells were treated with 4 nM GrB + 1% serum in DMEM/F12. Gene expression was studied by real-time PCR and cell proliferation and differentiation were determined as described above.
Eight weeks old female Sprague-Dawley rats purchased from Charles River Laboratories (Raleigh, NC) were randomly divided into three groups for stereotaxic injection in the hippocampal dentate gyrus (DG). Group I (n=6) received 1 ug of recombinant GrB in 1 ul phosphate buffered saline (PBS). Group II (n=5) received 10 ng of margatoxin (MgTX, Alomone Labs) and 1 ug of recombinant GrB together in 1 ul PBS. Group III (n=7) received vehicle as a control. After 7 days, the rats received bromodeoxyuridine (BrdU, 100 mg/kg, Sigma) intraperitoneal injection two hours before being euthanized. The brains were collected and post-fixed in 4% paraformaldehyde for 24 hr at 4°C and then incubated in 30% sucrose solution. Serial coronal frozen sections of 40 μm thickness were made from the hippocampus and DG. Every sixth section (240-μm interval) was selected from each animal and processed for immunohistochemistry. Briefly, sections were permeabilized by incubation in 0.5% PBS-T for 20 min, then treated in 50% formamide-2× standard saline citrate (SCC) at 65°C for 2 hr, denatured in 2N HCl at 37°C for 30 min, and rinsed twice in 0.1 M sodium borate (pH 8.5) at room temperature. Afterwards, the sections were incubated overnight at 4°C with rat anti-BrdU (1:1000, Accurate, Westbury, NY), mouse anti-NeuN (1:500, Millipore, Billerica, MA) or mouse monoclonal anti-Kv1.3 (clone L23/27, 1:100; NeuroMab Facility, University of California, Davis, CA). Sections were washed in PBS-T and incubated with fluorescent conjugated secondary antibodies (Cy3 AffiniPure donkey anti-rat, 1:250, Jackson Immuno Research, West Grove, PA; anti-mouse Alexa Flour 488, 1:400, Invitrogen) for 1 hr at room temperature. Samples were also incubated in 4′,6-diamidino-2-phenylindole (DAPI, 1:5000, Sigma) for contrast nuclear staining. Images were acquired on a Zeiss LSM 510 META multiphoton confocal system using a multi-track configuration. All morphological analyses were performed on blind-coded slides. To analyze cell proliferation in the DG, BrdU immunopositive cells were counted in the subgranular zone. The reference volume was determined by tracing the granule cell layer of the hippocampal DG.
For experiments that required immunostaining, the total number of cells and immunolabelled cells were counted in nine pre-determined fields per cover slip or slide using a fluorescence microscope with a ×20 objective. Three cover slips/slides were counted in each group. At least three independent experiments were performed. Means and SEM were calculated for each treatment group. Statistical analysis was performed using PRISM version 3.0. Differences were tested using either one-way ANOVA followed by Bonferroni's test for multiple comparisons or Student's t test for two group comparisons. Two-tailed p-values of less than 0.05 were considered significant.
The effect of activated T cells on neurogenesis was studied by exposing cultured human NPC in either maintaining or differentiating media to culture supernatants (sups) from purified CD8+ T cells that had been activated with anti-CD3 and anti-CD28 antibodies. Cell proliferation was studied by monitoring bromodeoxyuridine (BrdU) incorporation in the NPC by immunostaining. NPC differentiation was studied by immunostaining the cells cultured in the differentiating media for neuronal marker, beta-III tubulin and astroglial marker, glial fibrillary acidic protein (GFAP). Sups from activated CD8+ T cells inhibited NPC proliferation (Figure 1A) and neuronal differentiation (Figure 1Bi) but increased astroglial differentiation (Figure 1Bii) significantly compared to control sups. To determine the role of GrB in activated T cell-induced inhibition of NPC neurogenesis, we immunodepleted GrB from the activated T cell sups by using a monoclonal antibody. Immunodepletion of GrB completely blocked activated T cell-induced inhibition of NPC proliferation and neuronal differentiation while depletion using a non-relevant isotope control antibody showed no effect. GrB levels in the supernatants were 3.2+1.6 ng/ml (mean ± SEM) or ~100pM. To further confirm the role of GrB-induced inhibition on NPC neurogenesis, we used recombinant GrB (1-4 nM) to treat NPC cultures. Cytoquanti-blue assay was used to determine the cell numbers after 24 hours of treatment with GrB. GrB treatment decreased the NPC cell numbers in a concentration-dependent manner with a significant inhibition at 4 nM (Figure 1C). Further, immunostaining for active caspase-3 showed that treatment with GrB at 10 nM for 24 hr and 7 days only induced modest apoptosis in NPC (Figure 1E and F) while BrdU immunostaining showed that GrB at 4 nM significantly reduced BrdU incorporation (Figure 1D), indicating its effect on the cell number was mainly through inhibition of cell proliferation. These observations indicate that activated T cells release soluble factors to inhibit NPC proliferation and neuron generation but increase astroglial differentiation. GrB is the main factor released from activated T cells that induces these effects.
In a previous study, we found that GrB signals neuronal cells, at least in part, through a PTX sensitive Giα coupled receptor (Wang et al., 2006), hence we determined if PTX could impact the GrB-induced inhibition of NPC proliferation observed herein. We treated NPC with 100 ng/ml PTX 1 hour prior to GrB (4 nM) treatment. As shown in Figure 2A, PTX treatment significantly attenuated GrB-mediated inhibition of NPC proliferation as determined by the amount of BrdU incorporation (p<0.05), suggesting a role for Giα/Go coupled receptors in the GrB-mediated inhibition of NPC proliferation. Further, NPC cultures were treated with GrB (1-4 nM) for 15 min and intracellular cAMP level was determined by using an ELISA kit. As shown in Figure 2B, GrB treatment induced a significant decrease in the intracellular cAMP level in NPC by 15 min in a dose-dependent manner (p<0.05). This observation is consistent with the ability of GrB to act on a PTX sensitive receptor on NPC.
Kv1.3 channel has been previously implicated in neurogenesis (Liebau et al., 2006). We thus studied the effect of GrB on Kv1.3 expression in NPC. The NPC cultures were treated with GrB (4 nM) for 1-3 hrs and Kv1.3 expression was monitored by using real-time PCR. GrB treatment increased Kv1.3 expression in a time-dependent manner, which peaked at 2-3 hrs (Figure 3A). To determine if the increase in transcripts was also accompanied by a corresponding increase in protein, we immunostained NPC for Kv1.3 following treatment with GrB. NPC showed moderate staining in the untreated control cells, while GrB treatment for 24 hours interestingly showed increased staining in a subpopulation of the NPC (Figure 3B). The GrB-induced Kv1.3 was also confirmed by western-blot analysis which showed a dose-dependent increase in Kv1.3 protein (Figure 3C).
To determine the effect of blocking Kv1.3 channel on GrB-mediated inhibition of NPC neurogenesis, we pretreated NPC with a Kv1.3 blocker, margatoxin (MgTX, 10 nM) 1 h prior to treatment with either GrB or sups from activated T cells. Neurogenesis was quantified by immunostaining after 7 days. It showed that MgTX significantly attenuated both, GrB (Figure 4A) and activated T cell sups-mediated (Figure 4B) inhibition of NPC neurogenesis. To further confirm the role of Kv1.3 in inhibiting NPC neurogenesis we delivered siRNA to the NPC and first confirmed that the siRNA could specifically block Kv1.3 expression. No effect of negative control siRNA was seen. Next, the siRNA treated cells were exposed to GrB and NPC neurogenesis was quantified as described above. Similar to MgTX, siRNA to Kv1.3 also attenuated GrB-mediated inhibition of NPC neurogenesis (Figure 4C and D).
To determine the effect of GrB on NPC proliferation in vivo and the role of Kv1.3 activation in mediating these effects, rats were stereotaxically injected in the dentate gyrus (DG) with GrB alone, MgTX and GrB or vehicle control. After 7 days, the rats received BrdU and the brains were processed for immunohistochemistry. GrB significantly decreased BrdU positive cells compared to control, while MgTX attenuated the decrease completely (Figure 5 A and B). GrB treatment increased the expression of Kv1.3 on NPC (Figure 5C).
To determine if the effect of GrB on Giα/Go coupled receptors and Kv1.3 channel expression was linked or independent of one another we pretreated NPC cultures with 100 ng/ml PTX 1 hour prior to GrB (4nM) treatment and monitored Kv1.3 expression after 3 hrs. PTX pretreatment significantly blocked Kv1.3 expression (Figure 6) suggesting that the increased expression of the Kv1.3 channel is regulated by the stimulation of a Giα/Go coupled receptor by GrB.
To determine the pathological relevance of GrB mediated effects we measured GrB levels in CSF of patients with multiple sclerosis (mean ± SE = 7.1 ± 0.2 pg/ml) and found that they had significantly elevated levels compared to patients with headaches (mean ± SE = 4.1 ± 0.4 pg/ml) (Figure 7).
In neuroinflammatory diseases such as multiple sclerosis, T cells play a key pathogenic role. While their role in mediating damage to myelin has been extensively studied, it has only recently been realized that activated T cells can directly injure neurons as well. For example, the extent of axonal damage is directly related to the numbers of infiltrated T cells in multiple sclerosis plaques (Kuhlmann et al., 2002). In vitro studies have also shown that activated T cells can induce direct neuronal damage, through both cell contact-dependent (Giuliani et al., 2003) and – independent pathways(Wang et al., 2006). In the present study, we observed that patients with multiple sclerosis had higher levels of GrB compared to controls in their CSF. To investigate the neuropathological role of GrB we activated CD8+ T cells and found that GrB released extracellularly by these cells decreased NPC proliferation and neurogenesis. The mechanism involves activation of a G-protein coupled receptor, decrease in intracellular cAMP and activation of the Kv1.3 channel. We show that Kv1.3 may be an important therapeutic target.
The discovery that NPC are present in the adult brain and that these cells are capable of forming neurons and glial cells has raised hopes that neurorestorative therapy for a wide variety of neurological disorders may be within reach (McDonald and Wojtowicz, 2005; Goya et al., 2007; Hsu et al., 2007; Duncan et al., 2008). One approach has been to implant the NPC or stem cells into the nervous system. This has been successfully used in experimental systems of acute injury models, where the local environment promotes the differentiation of the stem cells or NPC to form neurons and even establish the meaningful connections with target cells(Joannides et al., 2007; Niranjan et al., 2007; Yan et al., 2007). Human fetal brain cells have also been implanted in patients with Parkinson's disease however, with limited success(Goya et al., 2007). This type of an approach in neurodegenerative and neuroinflammatory diseases poses unique challenges. It remains unknown if the microenvironment of glial cell activation or T cell infiltration is conducive or hostile to the NPC. It is possible that the brain atrophy associated with these diseases may be in part due to the failure of the endogenous NPC to replace the lost neurons. Hence understanding the effects of the microenvironment on the NPC would be important for the function of the endogenous NPC as well as conditions in which these cells may be implanted in the nervous system. Previous studies have shown that under physiological conditions, T cells may promote neurogenesis in the adult brain via interactions with microglia and release of growth factors such as insulin growth factor (Ziv and Schwartz, 2008a, b). In pathological conditions such as multiple sclerosis, which is the best studied of all neuroinflammatory diseases, however, the T cells are activated and inflammatory infiltrate contains cytotoxic T cells (Goverman et al., 2005; Friese et al., 2008). These cells are often present in the subventricular zones where the NPC are commonly found (Nait-Oumesmar et al., 2007; Wang et al., 2008). We thus determined if activated CD8+ T cells could impact the function of NPC. Since NPC have the ability to proliferate and renew themselves, we maintained the human NPC in culture conditions that promote proliferation of these cells without differentiation and found that the sups from the activated CD8+ T cells impaired the ability of these cells to proliferate but did not affect their survival. Similarly, when we cultured the NPC in differentiating media the sups inhibited their differentiation to neurons. These findings are consistent with observations that patients with multiple sclerosis when treated with natalizumab, which blocks T cell entry into the brain, had decreased brain atrophy (Miller et al., 2007).
One of the features that differentiate the activated CD8+ T cells from other T cells is the presence of cytotoxic granules, which contains several enzymatic proteins. The contents of these granules are released upon activation. Of these proteins, GrB is known to trigger apoptotic pathways, once it enters the cells (Trapani, 2001), however, it may also cause neuronal injury through interaction with cell surface receptors. Through a series of experiments in which we exposed the NPC to culture supernatants from which GrB had been removed and treated NPC directly with recombinant GrB, it was clear that GrB alone was sufficient to impair NPC function. Both culture supernatants and recombinant GrB activated similar pathways, since their effects could be blocked by similar pharmacological agents as discussed below. The effect of GrB on NPC proliferation was further confirmed by in vivo studies, in which injection of GrB into the dentate gyrus resulted in deceased proliferation of the NPC. Interestingly, GrB impaired the ability of NPC to differentiate to neurons but not to astrocytes. The amount of recombinant GrB needed to affect NPC function was about 10-40 fold greater than that required when produced by T cells. This is likely because the process of purification and storage of recombinant proteins often leads to some degradation. Further, recombinant GrB was produced in E. coli and hence would lack post-translational modifications, which would be present when released from human lymphocytes and could alter its potency.
Although subsets of CD4+ T cells can also express GrB (van Leeuwen et al., 2004), the release of GrB is more dominant in CD8+ T cells. GrB is a serine protease that can induce apoptosis by caspase activation after crossing the plasma membrane of target cells, usually with the help with perforin (Trapani, 2001). Interestingly, by using recombinant GrB to treat NPC cultures, we confirmed that GrB treatment significantly inhibited NPC proliferation and neuronal differentiation independent of perforin. The effects of GrB were PTX sensitive, suggesting the stimulation of the Giα coupled receptors (Tepe and Liggett, 2000). Giα coupled receptors are known to regulate adenylyl cyclase which in turn regulates cAMP levels. cAMP and cAMP-dependent protein kinase (PKA) play a pivotal role in regulating neurogenesis. Increase in intracellular cAMP and subsequent activation of PKA pathway have been reported to enhance the proliferation of retinal ganglion cells but inhibit their differentiation by regulating the cell-cycle exit (Masai et al., 2005). Activation of the cAMP signaling pathway and its transcriptional factor, cAMP response element-binding protein (CREB) play critical roles in neurite growth and neuronal differentiation induced by neurotrophic factors in a variety of conditions (Chen et al., 2005; Pan et al., 2005; Chu et al., 2006). We found that GrB decreased cAMP levels in NPC in a dose-responsive manner. This effect was mediated via interactions of GrB with Giα coupled receptors. This suggests that cAMP may be the critical second messenger in mediating GrB-induced inhibition in NSC neurogenesis. Thus, GrB may play a role in regulating neurogenesis by regulating cAMP-CREB signaling and subsequent transcription of CREB-target genes essential for neuronal differentiation.
K+ is the most dominant ion in the cytosol and plays a critical role in maintaining the cellular ion homeostasis and normal cell volume (Lang et al., 1998). As cell shrinkage is a hallmark of apoptosis, it is not surprising that enhancement of the plasma membrane permeability to K+ ions has been associated with an early response to apoptotic stimuli in a number of cell types including neurons (Yu et al., 1999). Additionally, outward delayed rectifier K+ current is present in differentiating NPC and the amplitude of the current can be significantly diminished by inhibiting caspases, indicating that activation of the delayed rectifier K+ current in differentiating stem cells is related to apoptosis (Hribar et al., 2004). In contrast to the outward delayed rectifier K+ current, an inward rectifying potassium channel was reported to be important in maintaining cytokine-induced primitive progenitor cell growth and differentiation (Shirihai et al., 1996). Kv1.3 is a Shaker-type delayed rectifier K+ channel found in human T-lymphocytes and microglia (Storey et al., 2003). It has been shown that Kv1.3 expressed both on the cell surface and mitochondrial membrane and its function may be related to mitochondrial dysfunction (Bock et al., 2002; Szabo et al., 2005). Blockade of Kv1.3 in T cells results in T-cell depolarization, inhibition of T-cell activation and the attenuation of immune responses in vivo. Also, blockade of Kv1.3 in microglia results in attenuated microglial activation and subsequent prevention of neurotoxicity (Fordyce et al., 2005). As both T cells and microglia play important roles in the pathogenesis of multiple sclerosis, it is likely that Kv1.3 could be used as a target in treatment of multiple sclerosis. Indeed, treatment with several Kv1.3 antagonists have been shown to be effective in ameliorating experimental allergic encephalomyelitis (EAE) (Beeton et al., 2001; Beeton et al., 2006). However, Kv1.3 also plays a role in modulating oligodendrocyte progenitor cells (OPC) proliferation and blockade in Kv1.3 inhibits OPC proliferation (Chittajallu et al., 2002). Thus, it is of interest to carefully delineate the effect of Kv1.3 on NPC function. We found that while there was a small basal level expression of Kv1.3 in NPC, treatment with GrB significantly increased the expression of Kv1.3 transcripts and protein. Furthermore, pretreatment with MgTX, a Kv1.3 blocker, and siRNA to Kv1.3 enhanced neurogenesis in both control and GrB-treated NPC, suggesting that Kv1.3 plays an important role in regulating NPC function. Kv1.3 thus represents a novel target that could be exploited to prevent toxicity in NPC from activated T cells and at the same time enhance their ability to proliferate and differentiate into neurons.
In conclusion, we showed that activated T cells release GrB, which inhibits neurogenesis via membrane-mediated interactions. GrB causes stimulation of Giα receptors, leading to a decrease in cAMP levels and increased expression of Kv1.3 channel on NPC. Importantly, we also found that blocking Kv1.3 pharmacologically leads to increased differentiation of the NPC into neurons, which clearly suggests that Kv1.3 may be a key molecule for pharmacological intervention in stem cell therapy. These observations may have important implications for T cell mediated neurodegenerative diseases.
This work was supported by grants from the Maryland Stem cell fund, National Multiple Sclerosis Society (TR 3760-A-3) and the National Institutes of Health (R01-NS056884; R01-NS41435). We thank Dr. George Chandy for helpful comments.