The cannabinoid CB₂ receptor agonist AM1241 enhances neurogenesis in GFAP/Gp120 transgenic mice displaying deficits in neurogenesis

Abstract

Background and purpose: HIV-1 glycoprotein Gp120 induces apoptosis in rodent and human neurons in vitro and in vivo. HIV-1/Gp120 is involved in the pathogenesis of HIV-associated dementia (HAD) and inhibits proliferation of adult neural progenitor cells (NPCs) in glial fibrillary acidic protein (GFAP)/Gp120 transgenic (Tg) mice. As cannabinoids exert neuroprotective effects in several model systems, we examined the protective effects of the CB₂ receptor agonist AM1241 on Gp120-mediated insults on neurogenesis.

Experimental approach: We assessed the effects of AM1241 on survival and apoptosis in cultures of human and murine NPCs with immunohistochemical and TUNEL techniques. Neurogenesis in the hippocampus of GFAP/Gp120 transgenic mice in vivo was also assessed by immunohistochemistry.

Key results: AM1241 inhibited in vitro Gp120-mediated neurotoxicity and apoptosis of primary human and murine NPCs and increased their survival. AM1241 also promoted differentiation of NPCs to neuronal cells. While GFAP/Gp120 Tg mice exhibited impaired neurogenesis, as indicated by reduction in BrdU⁺ cells and doublecortin⁺ (DCX⁺) cells, and a decrease in cells with proliferating cell nuclear antigen (PCNA), administration of AM1241 to GFAP/Gp120 Tg mice resulted in enhanced in vivo neurogenesis in the hippocampus as indicated by increase in neuroblasts, neuronal cells, BrdU⁺ cells and PCNA⁺ cells. Astrogliosis and gliogenesis were decreased in GFAP/Gp120 Tg mice treated with AM1241, compared with those treated with vehicle.

Conclusions and implications: The CB₂ receptor agonist rescued impaired neurogenesis caused by HIV-1/Gp120 insult. Thus, CB₂ receptor agonists may act as neuroprotective agents, restoring impaired neurogenesis in patients with HAD.

Keywords: CB2 agonist; GFAP/Gp120 transgenic mice; HIV-1 Gp120 protein; neural progenitor cells; neurogenesis.

Figure 1

CB₁ and CB₂ receptor expression in human NPCs. (A) RNA from human NPC cells was extracted and then cDNA and PCR amplification were performed CB₁ was amplified and yielded a product of 403 bp. CB₂ was amplified and yielded a product of 1100 bp. GAPDH was used as a positive control yielding a product of 292 bp. (B, C) Western blot analysis of CB₁ and CB₂ receptor expression. NPC cells were lysed and the lysates were separated by SDS-PAGE. Lysates of the SH-SY5Y human neuroblastoma cell line were used as a negative control for CB₂ receptor expression and as a positive control for CB₁ receptors.

Figure 2

Functional analysis of the effects of CB receptor agonists on human NPCs following exposure to HIV-1/Gp120. (A) Gp120 and AM1241 were used as indicated. hNPCs were grown in the presence of control vehicle; varying concentrations of Gp120 (as indicated); or Gp120 (1 nM) and varying concentrations of AM1241 (as indicated) for 48 h. The cells were monitored and analysed by microscopy after 48 h, using MTT assay. hNPC survival was increased on addition of AM1241 to the Gp120-treated cells. *P < 0.05, significant differences as indicated; n = 3. (B) Effects of Gp120 and CB receptor agonists on apoptosis of hNPC cells. Human NPCs (3x10⁴) were cultured on eight-well chamber slides coated with laminin and maintained in ReNcell NSC Medium with freshly added FGF and EGF. Cells were treated as follows: untreated, with Gp120, with CB receptor ligands and/or Gp120 for 48 h as indicated. (C) Effects of AM1241 on Gp120-induced apoptosis. Apoptosis was detected using TUNEL assay after 48 h. Nuclei were stained with DAPI. Negative control included cells treated with proteinase K and positive control included cells digested with DNase I. For quantification of cell death by the TUNEL assay, approximately 50 hNPCs were analysed in each experiment. The proportion of TUNEL-positive cells was increased significantly on addition of AM1241 to the Gp120-treated cells. (D) Effects of AM1241 (100 nM) and the CB₁ receptor agonist ACEA (1 μM) on apopotosis induced by Gp120 (1nM). *P < 0.05, significant differences as indicated; n = 3.

Figure 3

Effects of Gp120 and CB receptor agonists on hNPC differentiation. (A) Immunostaining analysis of positive neuronal cells derived from hNPC. hNPCs (3x10⁴) were cultured on an eight-well chamber slide, as described above The cells were either treated or untreated with AM1241 (100 nM) or the FAAH inhibitor URB597, in the presence or absence of 1 nM of Gp120. The medium was replaced every 2 days and the cultures were kept for 2 weeks. Differentiation was detected by immunofluorescence staining using MAP-2 antibody, a marker for neuronal cells. Confocal microscopy analysis was performed (using a Zeiss LAM 510Meta). This is a representative experiment out of three experiments. (B) Quantification of neuronal cells derived from hNPCs, staining positive for MAP-2 antibody in the presence or absence of AM1241 or URB597. hNPCs were treated as indicated in Figure 3A. MAP-2-positive cells were determined with an optical fluorescence microscope analysing approximately 100 hNPCs from each experiment.* P < 0.05, significantly different from cells treated with Gp120 alone; n = 3.

Figure 4

Expression of CB₁ and CB₂ receptors and functional analysis of neurogenesis in murine NPCs, derived from murine ES cells. (A) Differentiation of murine E14/GFP ES cells to EBs. (B) RT-PCR analysis of mES cells and neural progenitor cells derived from mES cells. Lane 1: murine ES cells; lane 2: neural progenitor cells derived from ES cells. (C) Western blot analysis of CB₁ and CB₂ in mES cells (lane 1) and neural progenitor cells derived from ES cells (lane 2). (D) Genotype screening and protein expression in CB₁ knockout and WT mice. The size of the cDNA fragments obtained by PCR for the WT mice was 1237 bp and 1088 bp for the CB₁ knockout mice. (E) Protein expression analysis by Western blot assay in brain total lysates derived from CB₁ knockout mice and WT mice. Brain tissues were obtained, lysed and analysed by Western blotting with specific antibodies against CB₁ receptors and tyrosine kinase C-terminal SRC kinase (CSK), as loading control. (F) Apoptosis: Gp120 was added to murine NPCs at 1nM for 48 h. Apoptotic cell death was determined by TUNEL assay. (G) Proliferation: murine NPCs were exposed to Gp120 (1 nM) or Gp120 (R5) (1 nM) or Gp120-denatured at 100°C for 15 min (1 nM) or control solution. Cells were grown in media containing low concentration of EGF and FGF (1 ng·mL⁻¹). After 7 days, the cells were fixed and immunostained with ki67. In addition, coverslips were counterstained with Hoechst 33342 (2 mg·mL⁻¹) to determine total cell numbers. To assess the percentage of cell proliferation, the number of ki67-positive cells as well as the Hoechst-stained cells were counted for five fields each per coverslip. (H) Survival: quantitation of murine NPCs following exposure to Gp120 for 48 h, untreated or treated with AM1241 (100 nM) in the presence or absence of CB₂ receptor antagonist AM630 (1 mM). Percentages of survival of NPCs were analysed using MTT assay. (I) Quantification of positive neuronal cells derived from murine NPCs stained with MAP-2 antibody in the presence or absence of CB₂ receptor agonist AM1241 and CB₂ receptor antagonist AM630, as indicated. In all panels (A–I), the data are presented as mean ± SEM (n = 3). *P < 0.05, significant differences as indicated.

Figure 5

Effects of AM1241 on neurogenesis in vivo. (A) GFAP/Gp120 Tg mice and their WT littermate control were either treated with vehicle control or with AM1241 (10 mg·kg⁻¹) for the first 10 days. No treatment was given for the following 10 days. Then, AM1241 or vehicle control was administered daily for the following 10 days. All mice then received BrdU (50 mg·kg⁻¹) by injection for 5 days, and the experiments were terminated after 30 days and brains were analysed for neurogenesis. (B) Analysis of neurogenesis in GFAP/GP120 transgenic mice and WT mice, treated with AM1241. (a–b) Immunohistological analysis of BrdU⁺ cells and quantitative analysis in the SGZ of BrdU⁺ cells in GFAP/Gp120 Tg mice treated with AM1241. *P < 0.001, significantly different from WT mice treated with AM1241; Student's t-test; n = 8 per group. (c–d) Immunocytochemical analysis of DCX⁺ and quantitative analysis of DCX⁺ cells using the disector method in the SGZ shows increased numbers of DCX⁺ neurons in GFAP/Gp120 Tg mice after AM1241 treatment, compared with vehicle control treatment. *P < 0.001. GFAP/Gp120 Tg with vehicle significantly different from WT mice with vehicle control; **P < 0.001, GFAP/Gp120 Tg with AM1241 significantly different from GFAP/Gp120 treated with vehicle; one-way anova with post hoc Dunnett's test; n = 8. (e–f) Immunocytochemical analysis of PCNA⁺ cells in GFAP/Gp120 Tg and WT mice treated with vehicle or AM1241. Quantitative analysis using the disector method in the SGZ showing the numbers of PCNA⁺ NPC cells. *P < 0.001, GFAP/Gp120 Tg treated with AM1241 significantly different from vehicle; one-way anova with post hoc Dunnett's test; (n = 8).

Figure 6

Effects of in vivo administration of AM1241 on NeuN⁺ cells and astrogliosis. (A–B) Immunohistological analysis of NeuN⁺ cells within the BrdU+ cell population and estimates of the numbers of NeuN⁺ neurons within the BrdU+ cells in the hippocampal dentate gyrus using the disector method. *P < 0.005, significant differences as indicated; one-way anova with post hoc Tukey Kramer test; n = 8. (C–D) Immunostaining and analysis of NPCs converting to astroglial cells. Conversion of NPCs to astroglial cells (GFAP immunoreactivity) within the BrdU+ cells in the hippocampal dentate gyrus. *P < 0.001, significant differences as indicated; one-way anova with post hoc Tukey Kramer test; n = 8.