Background
During aging and in the progression of neurodegenerative conditions such as Alzheimer's disease (AD) and HIV-associated neurocognitive disorders, synaptic plasticity and neuronal integrity are disturbed [
1‐
3]. Although the precise mechanisms leading to neurodegeneration in these conditions remain unclear, some common signaling factors have been identified that contribute to the pathogenesis of multiple neurodegenerative processes. One important signaling molecule that may represent a common denominator in several neurodegenerative disorders is cyclin-dependent kinase-5 (CDK5). Previous studies have revealed that dysregulation of CDK5 and its activators p35 and p25 contribute to the abnormal accumulation of hyperphosphorylated CDK5 substrates and eventual mature neuronal cell death in AD, HIV-associated neuroinflammatory conditions such as HIV encephalitis (HIVE), and prion-related disorders such as scrapie [
4‐
6]. Furthermore, previous studies have shown that levels of CDK5 are increased in the brains of AD [
7] and HIVE [
8] patients, and in scrapie-infected hamsters [
6].
In addition to the alterations in synaptic plasticity in mature neurons in these disorders, recent studies have uncovered evidence suggesting that the pathogenic process in humans and animal models of AD and HIV in the brain might include dysregulation of adult neurogenesis [
9‐
14]. This suggests that neurodegeneration may be characterized by not only a loss of mature neurons but also by a decrease in the generation of new neurons in the neurogenic niches of the adult brain. These cell populations that could be targeted include neural progenitor cells (NPCs) in the subventricular zone (SVZ) and in the dentate gyrus (DG) of the hippocampus. Mechanisms of neurogenesis in the fetal brain have been extensively studied, however less is known about the signaling pathways regulating neurogenesis in the adult nervous system and their role in neurodegenerative disorders.
It is clear that the abnormal activation of CDK5 via calpain-mediated cleavage of p35 into the more stable p25 fragment contributes to the pathogenesis of neurodegenerative conditions such as AD and HIVE [
4‐
6,
8], however, previous studies have also demonstrated that physiological CDK5 activity is essential for adult neurogenesis [
15,
16]. Thus, it is possible that abnormal activation of CDK5 and aberrant phosphorylation of its physiological substrates might have detrimental effects on cells residing in the neurogenic niches of the adult brain, and deficits in neurogenesis associated with neurodegeneration might be related to alterations in CDK5 in NPCs. In support of this possibility, we have previously shown that abnormal CDK5 activation impairs neurite outgrowth and neuronal maturation in an
in vitro model of adult neurogenesis, and in a mouse model of AD-like neurodegeneration and impaired neurogenesis [
17]. However, the downstream regulators mediating CDK5-associated defective neurogenesis are unknown.
In this context, CDK5 may mediate alterations in neurogenesis in AD and HIVE via aberrant phosphorylation of its substrates, which include cytoskeletal (neurofilaments, nestin) [
18] and synaptic proteins (e.g. synapsin) [
19], among others. It is possible that CDK5 substrates implicated in toxicity to mature neuronal populations, such as tau, might be involved, however another possibility is that alternative downstream substrates of CDK5 might modulate neurogenesis and neuronal maturation in the adult hippocampus. Elucidating the signaling pathways and downstream molecular targets involved in the dysregulation of neurogenesis is important to fully understand the mechanisms of neuroplasticity in neurodegenerative disorders.
A subset of physiological substrates of CDK5 includes proteins that have been implicated in neurogenesis (doublecortin, nestin) or neuronal development (collapsin-response mediator protein-2 [CRMP2]). Interestingly, in previous reports, CDK5-mediated phosphorylation of CRMP2 has been linked to neurodegeneration in AD [
20‐
22], and the known role of this protein in growth cone collapse and neuronal development makes it an intriguing candidate for regulating neurogenesis in neurodegenerative disorders. Notably, several recent proteomics-based studies in the brains of human HIV patients have detected differential expression of CRMP2 in human cases with HIVE or HIV-associated dementia [
23,
24]. Since the role of CRMP2 phosphorylation has not been previously examined in the adult neurogenic niche, or in HIV-associated neurodegenerative disorders (HAND), we sought to better understand the potential function of this protein in the molecular mechanisms involved in CDK5-mediated dysregulation of adult neurogenesis using an
in vitro model of CDK5 activation in adult hippocampal NPCs [
17], and
in vivo in the brains of patients with HIV and in an animal model of HIV protein neurotoxicity. Here we demonstrate that aberrant phosphorylation of collapsin-response mediator protein-2 (CRMP2) is a critical downstream event contributing to impaired neurite outgrowth in an
in vitro model of abnormal activation of CDK5 in adult neurogenesis. Using biochemical techniques with genetic and pharmacological manipulation of the p35/CDK5/CRMP2 signaling pathway, we examined the effects of abnormal activation of CDK5 on neurite outgrowth and CRMP2 phosphorylation in NPC-derived neural progeny. We further investigated the contribution of CDK5-mediated hyperphosphorylation of CRMP2 in neurodegenerative processes
in vivo by evaluating expression and phosphorylation of CRMP2 in the neurogenic niche of human brains with HIVE and in gp120 transgenic (tg) mice. These results reveal a role for CRMP2 in impaired neuronal maturation mediated by abnormal CDK5 activation, and identify a potential downstream functional mechanism involving microtubule destabilization that could contribute to impaired neuronal maturation during adult neurogenesis in neurodegenerative conditions.
Discussion
Abnormal CDK5 activity has been shown to contribute to neurotoxicity during the pathogenesis of several neurodegenerative disorders, including HIV neurotoxicity, AD, and prion-related encephalopathies. In the progression of these disorders, activation of CDK5 may trigger a cascade of hyperphosphorylation of downstream targets, and subsequent modulation of critical cellular functions. Most studies have focused on the role of tau hyperphosphorylation in mediating the neurodegenerative effects of CDK5 and other aberrantly activated kinases in mature neurons [
8,
32,
33]. However, considering the myriad substrates phosphorylated by CDK5, other downstream targets may be involved, particularly in distinct functions such as neurogenesis and neuronal maturation. In this context, we report that dysregulation of the growth cone signaling protein CRMP2 by abnormal CDK5 activation in an
in vitro model of adult neurogenesis contributes to defective neurite outgrowth during neuronal maturation. Down-regulation of CDK5 activity with the pharmacological inhibitor Roscovitine or siRNA knockdown, or expression of a non-phosphorylatable CRMP2 mutant construct (S522A) rescued the neurite defects associated with abnormal activation of CDK5 in NPC-derived neural progeny.
In support of a role for CRMP2 in neurodegenerative pathways, previous studies have revealed that CRMP2 is hyperphosphorylated as a result of abnormal CDK5 activity in the brains of AD patients and in a transgenic mouse model of AD [
20]. In AD brains, phosphorylated CRMP2 associates with damaged neurites and neurofibrillary tangles [
21,
22,
34], and accumulates in neurons surrounding cortical amyloid plaques [
35]. More recently, several proteomics-based studies in the brains of human HIV patients have identified differential expression of CRMP2 in cases with HIVE or HIV-associated dementia [
23,
24]. The present study reveals that levels of total and phosphorylated (Ser522) CRMP2 were significantly increased in neurogenic sites of the hippocampus from HIVE patients compared to non-encephalitic HIV+ controls, and similar results were observed in a mouse model of HIV-gp120 neurotoxicity. While the present study was focused on the effects of CDK5 activation and CRMP2 alterations in the neurogenic region of the hippocampus, it is of interest that we observed cells throughout the SGZ and the granular cell layer displaying increased CRMP2 immunoreactivity and phosphorylation, particularly in the gp120 tg mouse model. This suggests that mature neuronal cell populations in addition to NPCs may be vulnerable to CDK5-mediated abnormal CRMP2 phosphorylation. Our results in primary hippocampal neurons also support this possibility, however future studies in other mature neuronal cell types and alternative brain regions (e.g. cortex) will be necessary to elucidate whether CDK5-mediated alterations in CRMP2 is a common pathological feature of HIV-associated neurodegeneration.
In the tg mouse model, hippocampal CRMP2 hyperphosphorylation was reversed with genetic down-modulation of CDK5. Since CDK5 activity and phosphorylation of other substrates in mature neurons is increased in models of HIV neurotoxicity [
8], under these conditions, heterozygous CDK5 deficiency may be protective by normalizing the activity of CDK5 via moderate reduction of CDK5 expression levels. In support of this possibility, we have previously demonstrated that heterozygous CDK5 deficiency reverses hyperphosphorylation of tau in mature neurons in the frontal cortex of gp120 tg mice [
8], and that CDK5 knock-down rescues neurogenic deficits in an animal model of AD [
17]. The present study suggests that hyperphosphorylation of CRMP2 may be an important feature of HIV-associated neurodegeneration, and identifies a potential link between abnormal activation of CDK5 and impaired neurogenesis that has been observed in animal models of HIV neurotoxicity [
36], however future studies will be necessary to elucidate whether CRMP2 hyperphosphorylation plays a direct causative role in neurogenic or neurodegenerative alterations in the pathogenesis of HAND.
Given that CRMP2 is a known substrate of CDK5, along with the recent evidence that CDK5 and CRMP2 are dysregulated in HIVE [
8,
23,
24], activation of CDK5 and subsequent hyperphosphorylation of CRMP2 may be an important mediator of neurodegeneration in HIV infection. The results of the present study also reveal a new potential role for CRMP2 alterations in the mechanisms of defective adult neurogenesis in neurodegenerative disorders such as HIVE. This is in line with previous studies that have shown defective neurogenesis in HIV patients and in animal models of HIV neurotoxicity (gp120 tg and others) [
13,
36,
37].
Although the physiological function of CRMP2 has been associated with neuronal development and neurite outgrowth [
38,
39], the effects of abnormal CDK5-directed CRMP2 hyperphosphorylation in adult neurogenesis have not been previously investigated. CRMP2 (also known as Dpysl2) is a signaling protein that has been shown to play a role in growth cone collapse and axon development [
40]. It is a member of a group of related proteins including CRMP1 and CRMP4, all of which are highly abundant in the brain. CRMP2 has no known enzymatic activity [
41], however its C-terminal region is highly Ser and Thr-rich, and is targeted for phosphorylation by a number of kinases, including CDK5, GSK3β [
28,
42], Rho kinase [
43,
44], and Fyn kinase [
26], among others. Phosphorylation of CRMP2 has been reported for at least five amino acid residues in the C-terminal region, namely Thr509, Thr514, Ser518, Ser522 and Thr555. Phosphorylation by CDK5 at Ser522 acts as a priming site for subsequent phosphorylation by GSK3β at Thr514 [
25,
28], while other residues can be phosphorylated independently of CDK5 activity. It is possible that manipulation of phosphorylation at residues other than Ser522 of CRMP2 might also regulate neurite outgrowth, however because CDK5 is the only kinase that has been reported to phosphorylate the Ser522 residue, and since phosphorylation at this site acts a gatekeeper that regulates subsequent phosphorylation at other epitopes, we focused our studies on evaluating the specific role of phosphorylation at the Ser522 residue.
In support of a pivotal role for CDK5 in the phosphorylation of CRMP2, a previous study demonstrated that CRMP2 was not phosphorylated in
cdk5-/- embryonic brain using an antibody that recognizes several phosphorylated epitopes of CRMP2 (Thr509, Ser518, and Ser522) [
27]. Furthermore, the association of CRMP2 with tubulin was enhanced in
cdk5-/- brains compared to wild-type brain, supporting the role of CDK5-mediated phosphorylation of CRMP2 in modulating CRMP2-tubulin interactions [
27]. The functional effects of phosphorylation at the other Ser/Thr residues of CRMP2 is not entirely clear, however previous studies have shown that some of these post-translational modifications dramatically modulate protein-protein interactions between CRMP2 and its binding partners, including tubulin heterodimers [
30]. Of interest, previous studies have also shown that CDK5 and p35 can associate with microtubules [
45,
46], and CDK5 substrates include a number of microtubule-associated proteins.
The results of the present study show that abnormal activation of CDK5 and subsequent hyperphosphorylation of CRMP2 may have detrimental effects on structural elements of developing neurons, such as microtubules and cytoskeletal organization. In support of this possibility, we showed that the alterations in neurite outgrowth and CRMP2 phosphorylation in p35-overexpressing NPC-derived neural progeny were accompanied by disrupted microtubule organization compared to controls. Live-cell imaging of polymerized tubulin, and ultrastructural analysis of microtubule structure in NPC-derived neural progeny revealed diffuse, mesh-like microtubule distribution under conditions of p35-mediated abnormal CDK5 activation. This was in stark contrast to more robust microtubules that were observed under normal differentiation conditions. Furthermore, control NPC-derived neural progeny displayed rapid re-polymerization of microtubules following chemical disruption with the microtubule-destabilization agent, nocodazole, while cultures expressing p35 showed only minimal re-polymerization even after further incubation post-washout. These observations are in line with previous studies suggesting that the mechanisms downstream of abnormal CDK5 activation may involve the destabilization of microtubules [
47] and that CRMP2 plays a role in regulating microtubule dynamics [
48]. Together, this supports a role for CDK5-mediated CRMP2 hyperphosphorylation in the mechanisms of defective adult neurogenesis in neurodegenerative conditions such as HIVE.
Methods
Neuronal progenitor cell and primary neuronal cell culture and in vitromodeling 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 MgCl
2, 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% NaBH
4 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 NaBH
4, 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
2). 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
2). 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,
55]. 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,
56]. 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
+/-) [
59] 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.
Tissue processing
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.
Immunoblot analysis
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
1 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% H
2O
2, 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).
Statistical Analysis
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.
Acknowledgements
This work was supported by NIH Grants AG022074, AG018440, NS057096, AG010435, AG005131, AG011385, MH062962, MH076681, MH045294, MH059745, MH058164, DA026306, and California NeuroAIDS Tissue Network U01 MH083506. The HIV Neurobehavioral Research Center (HNRC) is supported by Center award MH062512 from NIMH. The authors also wish to thank Dr. Paula Desplats for critical discussion and valuable suggestions during the manuscript revision process.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
LC participated in the conception, design and coordination of the study, carried out the in vitro experiments and biochemical assays, and drafted the manuscript. RR participated in the immunoblot studies, carried out the immunohistochemical studies in human and mouse brain sections, and provided critical review of the manuscript. CP carried out the immunocytochemical assays for in vitro experiments. WD provided human brain tissue samples and prepared tissue samples for immunoblot analysis. MTM carried out the electron microscopy studies. CA participated in the design of the study and provided critical review of the manuscript. ER carried out the breeding and crosses of transgenic mouse lines. EM conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.