Neuronal progenitor cell and primary neuronal cell culture and in vitro modeling of abnormal CDK5 activation in progenitor cell and mature neuron populations
Adult rat hippocampal (ARH) NPCs (Millipore, Temecula, CA) were cultured routinely for expansion essentially as previously described [49
] with some modifications. Briefly, cells were grown for expansion in DMEM/F12 media (Mediatech, Manassas, VA) containing B27 supplement, 1X L-glutamine and 1X antibiotic-antimycotic (all from Invitrogen, Carlsbad, CA). For induction of neuronal differentiation, cells were plated onto poly-ornithine/laminin (Sigma-Aldrich, St. Louis, MO) coated plates or coverslips and transferred the next day to differentiation media containing N2 supplement (Invitrogen), 1 μM all-trans retinoic acid (Sigma-Aldrich), 5 μM forskolin (Sigma-Aldrich) and 1% FBS. Cells were differentiated for four days, and fresh differentiation media was added at day 2. It should be noted that this differentiation procedure generates heterogeneous cultures, and therefore we refer to the cells derived from the differentiation process as "NPC-derived neural progeny."
To induce CDK5/p35 activity in vitro, cells were infected on day 2 of differentiation with adenovirus expressing human p35 or GFP control (Vector Biolabs, Philadelphia, PA) at a multiplicity of infection (MOI) of 30. Additional control experiments were performed with cells transfected using BPfectin according to the manufacturer's guidelines (Biopioneer, San Diego, CA) with a plasmid expressing myc-tagged p35 (Addgene plasmid 1347, deposited by Dr. Li-Huei Tsai, Picower Institute, Cambridge, MA). Two days after infection or transfection with p35, cells were processed for immunoblot analysis with total cell lysates or immunocytochemical analysis with fixed cells on coverslips.
Similar experiments were performed in rat hippocampal primary neurons (ScienCell Research Laboratories, Carlsbad, CA) cultured on poly-lysine-coated plates and glass coverslips according to the supplier's recommendations in Neuronal Medium (ScienCell). For induction of CDK5/p35 activity, cells were infected two days after plating with p35-adv at an MOI of 100. A higher MOI was selected for the primary neuronal experiments because these cells were plated at a lower density than NPCs in culture. Two days after infection, cells were prepared for immunoblot or immunocytochemical analyses. A subset of experiments were performed with NPCs and primary hippocampal neurons where uninfected cells or cells infected p35-adv were exposed to recombinant gp120 protein for 24-48 hrs (100-200 ng/mL, LAV IIIB, Protein Sciences Corp., Meriden, CT) to model HIV protein associated neurotoxicity in vitro. At the conclusion of the in vitro experiments, cells were lysed for immunoblot analysis, or cells on coverslips were fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for immunocytochemical analysis.
Site-directed mutagenesis and generation of hCRMP2 mutant constructs
The wild-type hCRMP2/pCMV6-XL4 plasmid DNA (Origene, Rockville, MD) was maintained in E. coli TOP10 cells (dam+/dcm+ strain). Using plasmid DNA isolated from the E. coli strain as a template, site-directed mutagenesis of hCRMP2 was carried out using a QuickChange® Lightning Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) with the primers 5'-ctcggccaagacggctcctgccaagcag-3' (sense) and 5'-ctgcttggcaggagccgtcttggccgag-3' (antisense) for S522A single point mutation. The PCR reaction was performed according to the manufacturer's instructions, with 10-min extension cycles at 68°C. In order to remove the parental DNA template, PCR products were subjected to DpnI restriction enzyme digestion reaction and then directly transformed into E. coli TOP10 cells. After selection on LB medium supplemented with ampicillin, plasmid DNA was extracted from positive transformants using a QIAprep Spin Miniprep kit (Qiagen) according to the manufacturer's instructions. Purified plasmids were subjected to DNA sequencing with hCRMP2 specific primers: 5'-atcaaggcaaggagcaggct-3' (sense), and 5'-aatgttgtcatcaatctgagcacca-3' (antisense). After sequence confirmation, the pCMV6-XL4 vector constructs containing either the wild-type hCRMP2, or the S522A construct were used for transformation and amplification in XL10 E. coli cells (Stratagene).
Cell culture treatments--Pharmacological treatments, and siRNA and CRMP2 plasmid DNA transfection
For inhibition of CDK5 activity, cells were treated at day 2 of differentiation with either the pharmacological CDK5 inhibitor Roscovitine (1-10 μM, Calbiochem, San Diego, CA) or transfected with siRNA against CDK5, CRMP2, or control non-targeting fluorescent-tagged siRNA (37.5-150 ng, 5 nM final concentration, Qiagen, Valencia, CA) using the HiPerfect transfection reagent (Qiagen) according to the manufacturer's protocol. For each target, at least two different siRNAs were tested, and the one with the highest efficacy was selected for subsequent experiments.
For overexpression of wild-type or mutant human (h)CRMP2 in NPCs, cells were differentiated from day 0 in medium without antibiotics, and transfected on day 2 of differentiation (6 hrs prior to virus infection) with pCMV6-XL4 plasmids hCRMP2-WT, or hCRMP2-S522A, or pCMV-GFP control. Transfection was performed using Lipofectamine 2000 transfection reagent (Invitrogen) at a concentration of 2.5 μL/mL according to the manufacturer's instructions. For transfection of cells in 6-well plates, 4.0 μg plasmid DNA was applied per well, and for transfection of cells on coverslips in 6 cm dishes, 8.0 μg plasmid DNA was applied per dish. Six hrs after transfection, fresh differentiation medium without antibiotics was applied with or without viral vectors. Cells were then lysed for biochemical analyses or fixed for immunocytochemical analysis. For disruption of microtubules, cells were treated with nocodazole (5 μg/mL, Sigma-Aldrich) for 3 hrs. Then, cultures were washed with differentiation media three times, followed by incubation in fresh media for 10 mins, 20 mins or 30 mins. Cells were then fixed with glutaraldehyde for tubulin immunofluorescence and neurite outgrowth analysis.
Neurite outgrowth studies
A subset of cultured cells on coverslips were fixed in a solution containing glutaraldehyde, which better preserves the cytoskeleton for optimal visualization of neurites using immunofluorescence with an antibody against β-Tubulin. For this purpose, a fixation procedure was used essentially as previously described by Desai and Mitchison [50
]. Briefly, media was gently aspirated from cells growing on glass coverslips, and then cells were extracted for 30 sec in cytoskeletal buffer (CB, 80 mM PIPES pH 6.8, 1 mM MgCl2
, 4 mM EGTA) containing 0.5% freshly added Triton-X 100. Glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA) was immediately added to the CB on the coverslips at a final concentration of 0.5%. Coverslips were incubated for 10 min at 37°C. Fixative was then removed and a freshly-prepared solution of 0.1% NaBH4
in PBS was added and samples were incubated for 7 min at room temperature to quench free glutaraldehyde. Coverslips were washed at least 3 times in PBS to remove the NaBH4
, and samples were then processed for β-Tubulin immunofluorescence analysis.
For β-Tubulin immunofluorescence and neurite outgrowth studies, NPC-derived neural progeny growing on coverslips were fixed with glutaraldehyde and incubated with a mouse monoclonal primary anti-β-Tubulin antibody (1:250, Clone B2.1, Sigma-Aldrich) for 1 hr at room temperature and detected with FITC-conjugated secondary antibodies (1:75, Vector Laboratories). β-Tubulin-labeled coverslips were mounted under glass coverslips with ProLong Gold antifade reagent with DAPI (Invitrogen) and imaged with a fluorescent digital microscopy (Olympus). For analysis of neurite outgrowth of NPC-derived neural progeny immunolabeled with β-Tubulin, neurites were traced and lengths were measured using the ImageJ Program (NIH, Bethesda, MD) with the NeuronJ Plugin [51
Live-cell staining and imaging of polymerized tubulin in NPC-derived neural progeny
For staining of polymerized tubulin in live cells, NPC-derived neural progeny treated with vehicle control or p35-adv were grown on glass coverslips in 12-well plates as described above, and incubated with Tubulin Tracker Green (Invitrogen) according to the manufacturer's guidelines. Briefly, at day 4 of differentiation, media was removed from the cultures, and replaced with 1 mL of warm HBSS (containing Ca, Mg, and 4 mM HEPES buffer [HBSS/HEPES buffer]) with 150 nM Tubulin Tracker reagent. Plates were incubated for an additional 10 mins at 37°C, and then washed three times in warm HBSS/HEPES buffer. Coverslips were rapidly transferred to slides and imaged within 5 mins on a digital fluorescent Olympus microscope.
Electron microscopy analysis
Briefly, as previously described [52
], NPCs were plated in 35 mm dishes with a coverslip in the bottom (MatTek, Ashland, MA) and infected with p35-adv as described in the cell culture conditions. After 4 days of differentiation, cells were fixed in 1% glutaraldehyde in media, then fixed in osmium tetraoxide and embedded in epon araldite. Once the resin hardened, blocks with the cells were detached from the coverslips and mounted for sectioning with an ultramicrotome (Leica, Germany). Grids were analyzed with a Zeiss OM 10 electron microscope as previously described [53
HIV cases and neuropathological assessment
For the present study HIV+ cases with and without encephalitis were selected from a cohort of 43 HIV+ subjects from the HIV Neurobehavioral Research Center (HNRC) and California NeuroAIDS Tissue Network (CNTN) at the University of California, San Diego. Subjects were excluded in these analyses if they had a history of CNS opportunistic infections or non-HIV-related developmental, neurologic, psychiatric, or metabolic conditions that might affect CNS functioning (e.g., loss of consciousness exceeding 30 minutes, psychosis, substance dependence). A total of 16 age-matched cases were identified with and without encephalitis (n = 8 per group), and without other complications, for inclusion in the present study (Table ). All cases had neuromedical and neuropsychological examinations within a median of 12 months before death. Most patients died as a result of acute bronchopneumonia or septicemia, and autopsy was performed within 24 hrs of death (Table ). Autopsy findings were consistent with AIDS, and the associated pathology was most frequently due to systemic CMV, Kaposi sarcoma, and liver disease. In all cases, neuropathological assessment was performed in paraffin sections from the frontal, parietal, and temporal cortices, and the hippocampus, basal ganglia and brainstem stained with H&E, or immunolabeled with antibodies against p24 and glial fibrillary acidic protein (GFAP, marker of astrogliosis) [54
]. The diagnosis of HIVE was based on the presence of microglial nodules, astrogliosis, HIV-p24 positive cells, and myelin pallor. Formalin-fixed sections and frozen brain samples were obtained from the hippocampus of HIV and HIVE cases for biochemical analysis. Brain tissue from HIV-infected subjects without evidence of neuroinflammation provides a close control for the systemic effects of HIV infection in the absence of neurodegenerative changes, and previous studies have shown that the CDK5 pathway is dysregulated specifically in cases with encephalitis compared to HIV-infected non-encephalitis cases [8
]. For these reasons, and due to the scarcity of tissue samples available from age-matched non-HIV infected control subjects, HIV-positive cases without neuroinflammatory changes were used for comparison with cases with HIVE.
Generation of GFAP-gp120 tg mice and crosses with CDK5-deficient mice
For studies of CDK5 activation in an animal model of HIV-protein mediated neurotoxicity, tg mice expressing high levels of gp120 under the control of the GFAP promoter were used [57
]. These mice develop neurodegeneration accompanied by astrogliosis, microgliosis [57
], and memory deficits in the water maze [58
]. To study the effects of genetic CDK5 inhibition on CRMP2 expression and phosphorylation in vivo
, CDK5 heterozygous-deficient mice (CDK5+/-
] were crossed with the GFAP-gp120 tg mice as previously described [8
]. Full ablation of both copies of CDK5 (CDK5-/-
) causes severe neurodevelopmental alterations, so in order to study CDK5 knockdown in the adult mouse brain, the CDK5+/-
animals were used as a model of reduced CDK5 activity. For in vivo
studies, brain sections from 8-month old nontg, CDK5+/-
, gp120 tg, or gp120 tg/CDK5+/-
crossed mice (n = 4 mice per group) were used for biochemical analysis of CRMP2 expression and phosphorylation.
In accordance with NIH guidelines for the humane treatment of animals, mice were anesthetized with chloral hydrate and flush-perfused transcardially with 0.9% saline. Brains were removed and divided sagittally. One hemibrain was post-fixed in phosphate-buffered 4% paraformaldehyde at 4°C for 48 hrs and sectioned at 40 μm with a Vibratome 2000, while the other hemibrain was snap frozen and stored at -70°C for protein analysis. All experiments described were approved by the animal subjects committee at the University of California at San Diego (UCSD) and were performed according to NIH recommendations for animal use.
For immunoblot analysis, adherent cells in culture or mouse or human brain samples (0.1 g) were lysed in buffer composed of 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA (TNE) containing 1% Triton-X 100, homogenized using a microgrinder, and centrifuged at 10,000 rpm for 10 minutes to clear insoluble material. The supernatant was harvested, and protein content in the total cell lysates was determined using the bicinchoninic acid protein assay kit (Pierce, Rockford, IL). Then, 20 μg of each sample was separated by gel electrophoresis on 4-12% Bis-Tris gels (Invitrogen) and blotted onto 0.45 μm PVDF membranes (Millipore, Temecula, CA). All immunoblots were incubated in primary antibodies diluted in 5% BSA in PBS-Tween (PBS-T) overnight at 4°C. Immunoblots were probed with rabbit polyclonal antibodies against phosphorylated (pSer522) CRMP2 (1:1000, ECM Biosciences, Versailles, KY), phosphorylated (pThr514) CRMP2 (1:1000, Cell Signaling Technology, Danvers, MA), phosphorylated (pThr555) CRMP2 (1:1000, ECM Biosciences), total CRMP2 (1:1000, Millipore), p35/p25 (1:500, C-19, SantaCruz Biotechnology, Santa Cruz, CA), CDK5 (1:500, C-8, SantaCruz Biotechnology), or GFP (1:1000, Millipore); or mouse monoclonal antibodies against β-III Tubulin (1:5000, clone Tuj1, Covance), β-Tubulin (1:1000, clone B2.1, Sigma-Aldrich), or GFAP (1:1000, Millipore).
For immunoblot analysis with a panel of additional phosphorylated or total CRMP proteins, blots were probed with antibodies described in Table at a dilution of 1:1000 for all antibodies. All immunoblots were stripped and probed with an antibody against actin (C4 clone, Millipore) as a loading control as previously described [53
]. After incubation with primary antibodies, blots were incubated for 45 mins at room temperature in secondary antibodies diluted in 5% non-fat milk with 1% BSA in PBS-T. Blots were developed with enhanced chemiluminescence (Perkin-Elmer, Waltham, MA), and images were obtained and semi-quantitative analysis was performed using the VersaDoc gel imaging system and Quantity One software (Bio-Rad, Hercules, CA).
Immunocytochemistry and image analysis
For immunocytochemical analysis, briefly, as previously described [60
], cells on coverslips were fixed in 4% paraformaldehyde (PFA) in PBS and washed with Tris buffered saline (TBS, pH 7.4). For single-label immunostaining, coverslips were pre-treated in 3% H2
, blocked with 10% serum (Vector Laboratories, Burlingame, CA), and incubated with a rabbit polyclonal primary antibody against phospho-CRMP2 (Ser522, 1:1500, ECM Biosciences) diluted in PBS-T, and detected with the Tyramide Signal Amplification™-Direct (Red) system (NEN Life Sciences, Boston, MA). Immunolabeled coverslips were mounted under glass coverslips with ProLong Gold antifade reagent with DAPI (Invitrogen) and imaged with a Zeiss 63X (N.A. 1.4) objective on an Axiovert 35 microscope (Zeiss, Germany) with an attached MRC1024 laser scanning confocal microscope system (BioRad) [61
]. All samples were processed simultaneously under the same conditions and the experiments were performed twice to assess reproducibility. To confirm the specificity of primary antibodies, control experiments were performed where sections were incubated overnight in the absence of primary antibody (deleted) or primary antibody pre-incubated with blocking peptide.
For immunocytochemical analysis in human or mouse brain tissue, briefly as previously described [61
] vibratome sections from the hippocampus (40 μm thick) of the HIV patients or from nontg, gp120 tg or CDK5+/-
mice were incubated with antibodies against phospho-CRMP2 (Ser522, 1:300, ECM Biosciences) or CRMP2 (1:300, Millipore). Primary antibody incubation was followed by incubation with secondary biotinylated IgG, then avidin-HRP and diaminobenzidine (DAB) detection as previously described [8
]. Immunostained sections were imaged with a digital Olympus microscope and assessment of levels of immunoreactivity was performed utilizing the Image-Pro Plus program (Media Cybernetics, Silver Spring, MD).
For double-labeling analysis, coverslips or brain sections were incubated with a rabbit polyclonal primary antibody against phospho-CRMP2 (Ser522, 1:1500, ECM Biosciences) or CRMP2 (1:1500, Millipore) detected with Tyramide Red. The next day samples were co-labeled with mouse monoclonal antibodies against β-III Tubulin (1:250, Tuj1 clone, Covance) or MAP2 (1:100, Millipore), or a goat polyclonal antibody against the immature neuronal marker DCX (1:100, Santa Cruz Biotechnology), detected with FITC-conjugated secondary antibodies (1:75, Vector Laboratories). Samples were mounted and imaged as described above for single-labeling immunofluorescence analysis. For each sample a total of three sections (10 digital images per section at 400×) were analyzed in order to estimate the average number of immunolabeled cells per unit area (mm2) and the average intensity of the immunostaining (corrected optical density).
All experiments were performed blind coded and in triplicate. Values in the figures are expressed as means ± SEM. To determine the statistical significance, values were compared by unpaired two-tailed Student's t-test or by one-way ANOVA with post-hoc Dunnett's test when comparing differences to controls, or by one-way ANOVA with post-hoc Tukey-Kramer test when comparisons were made among groups. The differences were considered to be significant if p values were less than 0.05.