Journal: Molecular neurodegeneration

Article Title: Phosphorylation of collapsin response mediator protein-2 disrupts neuronal maturation in a model of adult neurogenesis: Implications for neurodegenerative disorders.

doi: 10.1186/1750-1326-6-67

Figure Lengend Snippet: Figure 2 Down-regulation of CDK5 reduces CRMP2 phosphorylation in NPC-derived neural progeny. Differentiating NPCs were treated on day 2 with the pharmacological inhibitor Roscovitine (Rosco) or siRNA against CDK5 (siCDK5), followed by infection with p35-adenovirus. On day 4, NPC-derived neural progeny were fixed for immunofluorescence or lysed for immunoblot analysis. (A-C) Immunocytochemical analysis with an antibody against pSer522-CRMP2 showed low levels of endogenous immunoreactivity in uninfected, vehicle-treated cells (A), with a slight reduction in cells treated with Roscovitine (B) or siCDK5 (C). (D-F) NPC-derived neural progeny expressing p35 showed a notable increase in CRMP2 phosphorylation (D) that was reversed by treatment with Rosco (E) or siCDK5 (F). (G) Reversal of CRMP2 hyperphosphorylation with CDK5 down-regulation. (H) Immunoblot analysis of pSer522-CRMP2, total (t)CRMP2, CDK5, and actin as a loading control. (I) Semi-quantitative image analysis of levels of CRMP2 phosphorylation in NPCs infected with p35-adv with or without Rosco or siCDK5. * p < 0.05 compared to vehicle-treated controls by one-way ANOVA with post-hoc Dunnett’s test. # p < 0.05 compared to p35-expressing NPCs by one-way ANOVA with post-hoc Tukey-Kramer test. Scale bar = 10 μm.

Article Snippet: 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.

Techniques: Phospho-proteomics, Derivative Assay, Infection, Immunofluorescence, Western Blot, Expressing, Control

Journal: Molecular neurodegeneration

Article Title: Phosphorylation of collapsin response mediator protein-2 disrupts neuronal maturation in a model of adult neurogenesis: Implications for neurodegenerative disorders.

doi: 10.1186/1750-1326-6-67

Figure Lengend Snippet: Figure 3 Expression of S522A-CRMP2 mutant construct rescues neurite deficits in p35-expressing NPC-derived neural progeny. Differentiating NPCs were transfected on day 2 with wild-type (WT) human CRMP2 or mutated S522A-CRMP2 plasmids, followed by infection with p35 adenovirus. Cells were fixed on day 4 of differentiation with glutaraldehyde for b-tubulin immunofluorescence, or lysed for immunoblot analysis. (A-F) Immunocytochemical analysis of b-tubulin immunoreactivity showed reduced b-tubulin-positive neurites in NPC-derived neural progeny infected with p35-adv with or without co-expression of WT-CRMP2, and rescue with co-expression of S522A-CRMP2 plasmid. (G) Image analysis showing reduced b-tubulin-positive neurite lengths in p35-expressing and p35+WT-CRMP2 NPC-derived neural progeny that was recovered after co-expression of p35 and S522A-CRMP2. (H) Immunoblot analysis showing levels of CRMP2 phosphorylation in p35-expressing NPC-derived neural progeny transfected with S522A-CRMP2 or WT-CRMP2 plasmid. (I) Semi-quantitative image analysis of ratios of pSer522- CRMP2 (pCRMP2)/total CRMP2 (tCRMP2) levels in NPC-derived neural progeny expressing p35 with and without WT-CRMP2 or S522A-CRMP2 co- expression. * p < 0.05 compared to vehicle-treated controls by one-way ANOVA with post-hoc Dunnett’s test. # p < 0.05 compared to WT- CRMP2-transfected NPCs by one-way ANOVA with post-hoc Tukey-Kramer test. Scale bar = 25 μm, 10 μm (insets).

Article Snippet: 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.

Techniques: Expressing, Mutagenesis, Construct, Derivative Assay, Transfection, Infection, Immunofluorescence, Western Blot, Plasmid Preparation, Phospho-proteomics

Journal: Molecular neurodegeneration

Article Title: Phosphorylation of collapsin response mediator protein-2 disrupts neuronal maturation in a model of adult neurogenesis: Implications for neurodegenerative disorders.

doi: 10.1186/1750-1326-6-67

Figure Lengend Snippet: Figure 6 Increased CRMP2 phosphorylation in NPCs treated with HIV-gp120 protein, and rescue with CDK5 siRNA knockdown. Differentiating NPCs were transfected day 2 of differentiation with siRNA specific for CDK5 (siCDK5) or transfection reagent control, followed by treatment with recombinant HIV-gp120 protein (100 ng/mL) or vehicle control. Cells were fixed on day 4 of differentiation for double- immunolabeling analysis with antibodies against b-III Tubulin (immature neuronal marker) and pSer522-CRMP2 (pSer-CRMP2). (A-C) b-III Tubulin- positive NPC-derived neuronal progeny treated with vehicle control express background levels of pSer-CRMP2. (D-F) NPC-derived neuronal progeny treated with gp120 for 48 hrs display increased pSer-CRMP2 immunoreactivity in b-III Tubulin-positive cells. (G-I) b-III Tubulin-positive NPC-derived neuronal progeny treated with siCDK5 show low levels of pSer-CRMP2 immunoreactivity. (J-L) b-III Tubulin-positive NPC-derived neuronal progeny treated with siCDK5 and gp120 show background levels of pSer-CRMP2 immunoreactivity. (M, N) Semi-quantitative image analysis of b-III Tubulin (M) and pSer-CRMP2 (N) immunoreactivity. * p < 0.05 compared to vehicle-treated controls by one-way ANOVA with post-hoc Dunnett’s test. Scale bar = 20 μm.

Article Snippet: 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.

Techniques: Phospho-proteomics, Knockdown, Transfection, Control, Recombinant, Immunolabeling, Marker, Derivative Assay

Journal: Molecular neurodegeneration

Article Title: Phosphorylation of collapsin response mediator protein-2 disrupts neuronal maturation in a model of adult neurogenesis: Implications for neurodegenerative disorders.

doi: 10.1186/1750-1326-6-67

Figure Lengend Snippet: Figure 7 CRMP2 is hyperphosphorylated and dendritic development is impaired in primary neurons over-expressing p35 alone or in combination with HIV-gp120 protein treatment. Primary rat hippocampal neurons were infected two days after plating with p35-adv, followed by treatment with recombinant HIV-gp120 protein or vehicle control. Cells were fixed on day 4 of differentiation for double- immunolabeling analysis with antibodies against MAP2 (dendritic marker) and pSer522-CRMP2 (pSer-CRMP2). (A-C) MAP2-positive primary neurons treated with vehicle control express background levels of pSer-CRMP2. (D-F) Primary neurons infected with p35-adv display increased pSer-CRMP2 immunoreactivity and reduced MAP2-immunoreactive dendrites. (G-I) Primary neurons treated with recombinant HIV-gp120 display increased pSer-CRMP2 immunoreactivity and reduced MAP2-immunoreactive dendrites, with an accumulation of pSer-CRMP2 immunoreactivity in varicosities along the neuronal processes (arrows). (J-L) Primary neurons infected with p35-adv and treated with recombinant HIV-gp120 display increased pSer-CRMP2 immunoreactivity and extensive damage to MAP2-immunoreactive dendrites, with an abundant accumulation of pSer-CRMP2 immunoreactivity in varicosities along the neuronal processes (arrows). (M, N) Semi-quantitative image analysis of pSer-CRMP2 (M) and MAP2 (N) immunoreactivity. (O) Immunoblot analysis of total cell lysates showing increased CRMP2 phosphorylation in primary hippocampal neurons infected with p35-adv or treated with HIV-gp120 protein. (P) Semi-quantitative image analysis of fold change in CRMP2 phosphorylation at the CDK5 epitope (Ser522) in immunoblots from cells expressing p35-adv or treated with gp120. * p < 0.05 compared to vehicle-treated controls by one-way ANOVA with post-hoc Dunnett’s test. Scale bar = 20 μm.

Article Snippet: 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.

Techniques: Expressing, Infection, Recombinant, Control, Immunolabeling, Marker, Western Blot, Phospho-proteomics

Journal: Molecular neurodegeneration

Article Title: Phosphorylation of collapsin response mediator protein-2 disrupts neuronal maturation in a model of adult neurogenesis: Implications for neurodegenerative disorders.

doi: 10.1186/1750-1326-6-67

Figure Lengend Snippet: Figure 8 Patterns of CRMP2 immunoreactivity in the hippocampus of an HIV encephalitis brain and a gp120 transgenic mouse brain. Forty μm-thick vibratome sections from the brains of HIV encephalitis (HIVE) patients and gp120 transgenic (tg) mice were processed for double-immunolabeling analysis with antibodies against doublecortin (DCX) and CRMP2. (A-C) Intracellular CRMP2 immunoreactivity was detected throughout cells in the granular cell layer (GCL) and into the molecular layer (ML) of the hippocampal dentate gyrus in a representative section from the brain of a patient with HIVE. A subset of DCX-positive cells in the subgranular zone (SGZ) co-expressed CRMP2 (arrows). (D-F) Intracellular CRMP2 immunoreactivity was detected throughout cells in the granular cell layer (GCL) and into the molecular layer (ML) of the hippocampal dentate gyrus in a representative section from the brain of a gp120 tg mouse. A subset of DCX-positive cells in the subgranular zone (SGZ) co-expressed CRMP2 (arrows). Scale bar = 15 μm.

Article Snippet: 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.

Techniques: Transgenic Assay, Immunolabeling

Journal: Molecular neurodegeneration

Article Title: Phosphorylation of collapsin response mediator protein-2 disrupts neuronal maturation in a model of adult neurogenesis: Implications for neurodegenerative disorders.

doi: 10.1186/1750-1326-6-67

Figure Lengend Snippet: Figure 9 Increased CRMP2 expression and phosphorylation in the hippocampus of patients with HIV encephalitis. Forty μm-thick vibratome sections from the hippocampus of HIV+ control and HIV encephalitis (HIVE) patients were processed for immunolabeling analysis with antibodies against CRMP2 and pSer522-CRMP2 (pSer-CRMP2). Low power images show overall immunoreactivity throughout the hippocampus, including the CA1-4 regions, the dentate gyrus (DG) and the subgranular zone (SGZ), and high power images show more detail of the DG including the SGZ and granular cell layer (GCL). (A-D) Patterns of total CRMP2 immunoreactivity in the hippocampus from representative HIV+ non-encephalitis (control) and HIVE brains at low (A, C) and high (B, D) power. Numerous intensely immunoreactive cell profiles were detected in the SGZ (arrows), particularly in the brains of HIVE patients. (E) Semi-quantitative image analysis of CRMP2 immunoreactivity in the hippocampal DG of HIV patients. (F-I) Patterns of pSer-CRMP2 immunoreactivity in the hippocampus from representative HIV+ non-encephalitis (control) and HIVE brains at low (F, H) and high (G, I) power. (J) Semi-quantitative image analysis of pSer-CRMP2 immunoreactivity in the hippocampal DG of HIV patients. * p < 0.05 compared to HIV+ controls by unpaired two-tailed Student’s t-test (n = 8 per group). Scale bar = 200 μm (A,C,F,H); 30 μm (B,D,G,I).

Article Snippet: 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.

Techniques: Expressing, Phospho-proteomics, Control, Immunolabeling, Two Tailed Test

Journal: Molecular neurodegeneration

Article Title: Phosphorylation of collapsin response mediator protein-2 disrupts neuronal maturation in a model of adult neurogenesis: Implications for neurodegenerative disorders.

doi: 10.1186/1750-1326-6-67

Figure Lengend Snippet: Figure 10 Increased CRMP2 phosphorylation in gp120 transgenic mice is rescued with down-modulation of CDK5 expression. Forty μm-thick vibratome sections from the brains of 8-month old non-transgenic (nontg), gp120 transgenic (tg), CDK5 heterozygous-deficient (CDK5 +/-), and gp120 tg/CDK5+/- mice were processed for immunolabeling analysis with antibodies against CRMP2 and pSer522-CRMP2 (pSer-CRMP2). (A-D) Patterns of total CRMP2 immunoreactivity in the dentate gyrus from representative mouse brains showing a punctate expression pattern throughout the granular cell layer (GCL) and subgranular zone (SGZ), with background immunoreactivity detected in the molecular layer (ML). Intensely immunoreactive cell profiles were detected in the SGZ (arrows), particularly in the brains of gp120 tg mice. (E) Semi-quantitative image analysis of CRMP2 immunoreactivity in the hippocampal DG of nontg, gp120 tg, CDK5+/- and gp120 tg/CDK5+/- mice. (F-I) Patterns of pSer- CRMP2 immunoreactivity in the hippocampal DG from representative mouse brains showing a punctate expression pattern throughout the granular cell layer (GCL) and subgranular zone (SGZ), with background immunoreactivity detected in the molecular layer (ML). Intensely immunoreactive cell profiles were detected in the SGZ (arrows), particularly in the brains of gp120 tg mice. (J) Semi-quantitative image analysis of pSer-CRMP2 immunoreactivity in the hippocampus of nontg, gp120 tg, CDK5+/- and gp120 tg/CDK5+/- mice. * p < 0.05 compared to nontg controls by one-way ANOVA with post-hoc Dunnett’s test (n = 4 per group). Scale bar = 30 μm.

Article Snippet: 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.

Techniques: Phospho-proteomics, Transgenic Assay, Expressing, Immunolabeling

Journal: Neurobiology of Aging

Article Title: Combined age- and trauma-related proteomic changes in rat neocortex: a basis for brain vulnerability

doi: 10.1016/j.neurobiolaging.2011.09.029

Figure Lengend Snippet: Protein isoforms differentially expressed with age or traumatic brain injury

Article Snippet: Primary antibodies used were total CRMP2 (1:2000) and phospho-Thr514-CRMP2 (1:1000, Cell Signaling Technology, Danvers, MA); T-kininogen 1/2 (1:1000, sc-103887) and oxoglutarate dehydrogenase ( α KGD, 1:1000, sc-67238, Santa Cruz Biotechnology, Inc, Santa Cruz, CA).

Techniques: Protease Inhibitor

Journal: Neurobiology of Aging

Article Title: Combined age- and trauma-related proteomic changes in rat neocortex: a basis for brain vulnerability

doi: 10.1016/j.neurobiolaging.2011.09.029

Figure Lengend Snippet: Protein spot relative expression with age and brain injury: two-way analysis of variance

Article Snippet: Primary antibodies used were total CRMP2 (1:2000) and phospho-Thr514-CRMP2 (1:1000, Cell Signaling Technology, Danvers, MA); T-kininogen 1/2 (1:1000, sc-103887) and oxoglutarate dehydrogenase ( α KGD, 1:1000, sc-67238, Santa Cruz Biotechnology, Inc, Santa Cruz, CA).

Techniques: Expressing

Journal: Neurobiology of Aging

Article Title: Combined age- and trauma-related proteomic changes in rat neocortex: a basis for brain vulnerability

doi: 10.1016/j.neurobiolaging.2011.09.029

Figure Lengend Snippet: Age- and Injury-related CRMP2 Changes Differed With Brain Region and Epitope Detected by Immunoblot (A) Hippocampal CRMP2 decreased with injury in juvenile and adult, but not in geriatric brains. (B) Cortex CRMP2 increased in juvenile and adult, but not in geriatric brains. (C) Hippocampal phospho-Thr514-CRMP2 (pCRMP2, Thr514-CRMP2 phospho-specific antibody, Methods) appeared to decrease with injury (p < 0.05, all ages combined). (D) Cortex pCRMP2 increased in each age group compared to shams. Data normalized to β-actin in each sample and presented as mean ± SEM. Immunoblots (n = 4–5 per group) were arranged by age (all juvenile specimens on one gel, etc.) and repeated arranged by same treatment (shams of all ages on one gel, etc.); results were comparable for both approaches. No significant changes in the β–actin internal control were observed. Two-way ANOVAs [Hippocampus FCRMP2 = 11.12, p < 0.0001; FpCRMP2 = 6.40, p < 0.0002; Cortex FCRMP2 = 11.12, p < 0.0001; FpCRMP2 = 3.77, p < 0.004] were followed by one-way ANOVA with Dunnett post hoc tests; *p < 0.05 vs. Sham (within age group); §p < 0.05 vs. Adult (within treatment group).

Article Snippet: Primary antibodies used were total CRMP2 (1:2000) and phospho-Thr514-CRMP2 (1:1000, Cell Signaling Technology, Danvers, MA); T-kininogen 1/2 (1:1000, sc-103887) and oxoglutarate dehydrogenase ( α KGD, 1:1000, sc-67238, Santa Cruz Biotechnology, Inc, Santa Cruz, CA).

Techniques: Western Blot