- Email: [email protected]
Involvement of the Snk-SPAR pathway in glutamate-induced excitotoxicity in cultured hippocampal neurons
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Involvement of the Snk-SPAR pathway in glutamate-induced excitotoxicity in cultured hippocampal neurons Lan-Xiang Wu a , Chang-Kai Sun a,⁎, Yu-Mei Zhang a , Ming Fan b , Jing Xu a , Hui Ma a , Jian Zhang a a
Institute for Brain Disorders, Dalian Medical University, 465 Zhong Shan Road, Shahekou District, Dalian 116027, China Institute of Basic Medical Science, Academy of Military Medical Sciences, Beijing 100850, China
b
A R T I C LE I N FO
AB S T R A C T
Article history:
The serum-induced kinase (Snk)–spine-associated Rap GTPase-activating protein (SPAR)
Accepted 20 June 2007
signaling pathway is reported as a new molecular mechanism in activity-dependent
Available online 27 July 2007
remodeling of synapses. However, the relationship between Snk-SPAR pathway and glutamate-induced excitotoxicity is not well understood. We report here that in cultured
Keywords:
hippocampal neurons, glutamate stimulation induces the activation of Snk-SPAR pathway,
Serum-induced kinase
and leads to a loss of mature dendritic spines. The time-dependent changes in Snk and SPAR
Spine-associated Rap
expression after glutamate exposure are also elucidated. Furthermore, the activation of Snk-
GTPase-activating protein
SPAR pathway induced by glutamate treatment can be blocked by an NMDA receptor
Dendritic spine
antagonist, MK801. These results demonstrate that Snk-SPAR pathway may play a pivotal
Glutamate
role in glutamate-induced exicitotoxic damage in CNS through regulating the stability of
MK801
synapse. © 2007 Elsevier B.V. All rights reserved.
1.
Introduction
Dendritic spines are small postsynaptic protrusions that receive over 90% of the excitatory glutamatergic inputs in the CNS (Harris and Kater 1994). “Dysgenesis” of dendritic spine is a common feature of the microstructural pathology that occurs in profound mental retardation (Marin-Padilla, 1972; Purpura, 1974), and the distribution and structure of dendritic spine are altered in many diseases, such as temporal lobe epilepsy, Huntington's disease, and acquired immunodeficiency syndrome-related dementia (Halpain et al., 1998; Fiala et al., 2002; Carlisle and Kennedy, 2005). In glutamatergic excitatory synaptic transmission, glutamate is released from presynaptic membrane and activates glutamate receptors which are mainly localized in postsyn-
aptic density (PSD). There are also many signaling proteins, cytoskeletal proteins, and ion channels in PSD, any changes in these molecular can influence the morphology and function of dendritic spine obviously (Allison et al., 2000; Schubert and Dotti, 2007). SPAR, spine-associated Rap GTPase-activating protein, associates with actin cytoskeleton and forms a complex with PSD-95 and NMDA receptors in dendritic spine. PSD-95 and NMDA receptor have been reported to play an important role in synaptic remodeling (Charych et al., 2006; Shi and Ethell, 2006), which makes SPAR an attractive candidate for mediating activity-dependent remodeling of synapses (Pak et al., 2001; Segal, 2001). In 2003, Pak and Sheng revealed that serum-induced kinase (Snk) induced by neuronal activity could lead to synapse loss through inducing the degradation of SPAR. The Snk-SPAR signaling pathway may be
⁎ Corresponding author. E-mail address: [email protected] (C.-K. Sun). Abbreviations: Snk, serum-induced kinase; SPAR, spine-associated Rap GTPase-activating protein; NMDA, N-methyl-D-aspartate 0006-8993/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2007.06.082
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lation could induce the activation of Snk-SPAR pathway, which could be blocked by non-competitive antagonist of NMDA receptor, MK801. The present study provides a new insight into the molecular mechanism of glutamate-induced excitotoxicity.
2.
Results
2.1. Glutamate-induced cell damage in cultured hippocampal neurons Fig. 1 – The release of LDH activity in cultured hippocampal neurons illustrating the neurotoxic effects of glutamate and the neuroprotective effects of the non-competitive antagonist of NMDA receptor, MK801. Data are shown as mean ± SEM of six measurements. *P b 0.05, **P b 0.01, ***P b 0.001, compared with control group.
In order to study the kinetics of glutamate cytotoxicity, LDH release assay was performed. The result indicated that glutamate toxicity varied during the course of the cultures. We also showed the neuroprotective effect of MK801 on glutamate-induced neuronal damage (Fig. 1).
2.2. Snk and Spar mRNA levels are regulated by glutamate treatment in cultured hippocampal neurons a homeostatic regulatory process that destabilizes synaptic connections in response to neuronal activity (Pak and Sheng, 2003; Meyer and Brose, 2003). The purpose of the present study was to investigate the role of Snk-SPAR pathway in glutamate-induced excitotoxic damage in cultured hippocampal neurons. Glutamate stimu-
Using semiquantitative RT-PCR, we first examined the effect of glutamate on Snk and SPAR mRNA levels in cultured hippocampal neurons. The corresponding transcripts had sizes of 665 bp for Snk, 483 bp for SPAR, and 843 bp for β-actin. Incubation of hippocampal neurons with 100 μM glutamate and 10 μM
Fig. 2 – The changes in Snk and SPAR mRNA expression following glutamate stimulation in cultured hippocampal neurons. (A) Total RNA was isolated from normal neurons (con), different time points of neurons after 100 μM glutamate and 10 μM glycine stimulation for 10 min (1 h, 3 h, 6 h, 9 h, and 12 h). (C) 6 h after glutamate stimulation, Snk and SPAR mRNA transcripts were detected in normal neurons (con), and glutamate-treated neurons with (MK801 + Glu) or without (Glu) MK801. These data were statistically analyzed by Student's t test (B) and one-way ANOVA (D) respectively. Error bars represent the mean ± SEM for five to six samples per group. *p b 0.05, **p b 0.01, ***p b 0.005 compared with Snk mRNA level in control group; #p b 0.05, ## p b 0.01, ###p b 0.005 compared with SPAR mRNA level in control group; ^^p b 0.01 compared with Snk mRNA level in glutamate-treated group; OOp b 0.01 compared with SPAR mRNA level in glutamate-treated group.
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glycine for 10 min induced time-dependent changes in Snk and SPAR mRNA expression. Our results showed that in control neurons, Snk mRNA was barely detectable, but 1 h after glutamate exposure, the mRNA level of Snk was increased ∼ 1.8-fold compared with the basal level (n = 6, p b 0.01). At 6 h, Snk mRNA level reached its highest peak, which was increased ∼ 4-fold compared with the basal level (n = 6, p b 0.005), then returned to baseline by 12 h. In control neurons, SPAR mRNA was highly detectable, but after glutamate insult, the tendency of change in SPAR mRNA level was contrary to Snk (Figs. 2A, B). Pretreated neurons with 10 μM MK801 before glutamate exposure could dramatically block the changes in Snk and SPAR mRNA expression induced by glutamate: 6 h after glutamate stimulation, compared with glutamate-treated
group, MK801 could decrease Snk mRNA level for about 41% (n = 5, p b 0.01), and increase SPAR mRNA level for about 36% (n = 5, p b 0.01) (Figs. 2C, D).
2.3. Snk and Spar protein levels are regulated by glutamate treatment in cultured hippocampal neurons The changes in Snk and SPAR protein levels were evaluated by Western blot analysis. Identical with mRNA level, in control hippocampal neurons, Snk protein was barely detectable by Western blot, but SPAR protein was highly detectable. 3 h after glutamate stimulation, Snk protein level began to increase (n = 6, p b 0.05). It reached the highest peak by 36 h, at this time point, the level of Snk protein was increased ∼ 3.3-fold
Fig. 3 – The changes in Snk and SPAR protein expression following glutamate stimulation in cultured hippocampal neurons. (A) Lysates of 18DIV hippocampal neurons of control, 3 h, 6 h, 12 h, 18 h, 24 h, 36 h, 48 h, and 72 h after glutamate insult were analyzed by immunoblot assay with Snk and SPAR antibodies. (C) 36 h after glutamate stimulation, Snk and SPAR proteins were detected in normal neurons (con), and glutamate-treated neurons with (MK801 + Glu) or without (Glu) MK801. These data were statistically analyzed by Student's t test (B) and one-way ANOVA (D) respectively. Error bars represent the mean ± SEM for five to six samples per group. *p b 0.05, **p b 0.01, ***p b 0.005 compared with Snk level in control group; #p b 0.05, ##p b 0.01, ### p b 0.005 compared with SPAR level in control group; ^p b 0.05 compared with Snk level in glutamate-treated group; OOp b 0.01 compared with SPAR level in glutamate-treated group.
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compared with the basal level (n = 6, p b 0.005), and then gradually decreased thereafter. The level of SPAR protein began to decrease after glutamate treatment, and at 36 h, SPAR protein was barely detectable (n = 6, p b 0.005), and then gradually increased thereafter (Figs. 3A, B). In agreement with RT-PCR, 36 h after glutamate stimulation, MK801 could decrease Snk protein level (n = 5, p b 0.05) and increase SPAR protein level (n = 5, p b 0.01) markedly compared with glutamate-treated group (Figs. 3C, D).
2.4. Effect of glutamate treatment on the distribution of Snk, SPAR and PSD-95 in hippocampal neurons In this experiment, we found little staining of Snk in unstimulated neurons by immunocytochemistry. After
41
glutamate stimulation, Snk immunoreactivity increased specifically in the cell body, and also enriched in dendrite. The same glutamate-treated neurons were immunostained for SPAR and PSD-95. In unstimulated cells, SPAR and PSD95 were evenly distributed along dendrites. Glutamatetreated cells showing high levels of Snk in dendrites exhibited a loss of both SPAR and PSD-95 in the same regions (Fig. 4). So, elimination of SPAR by Snk is associated with the loss of the PSD marker PSD-95. Because SPAR is a large scaffold protein of the PSD with the ability to regulate the actin cytoskeleton, it is quite plausible that SPAR degradation would lead indirectly to dismantling of the PSD and depletion of PSD-95. Our results also showed that MK801 could block these changes induced by glutamate insult.
Fig. 4 – Glutamate treatment induces Snk and leads to loss of SPAR and PSD-95. Unstimulated hippocampal cultures (DIV18) (A and D) or cultured received stimulation of 100 μM glutamate and 10 μM glycine for 10 min with (C and F) or without (B and E) 10 μM MK801 pretreatment were double-stained for Snk (green) and either SPAR or PSD-95 (red), as indicated, with merge in color. Scale bar, 20 μm.
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Fig. 5 – Representative multipolar neurons expressing GFP which were incubated in the absence (A) and presence (B) of glutamate, or pretreated with MK801 before glutamate stimulation (C). Panels D–F are enlargements of rectangles in panels A–C, respectively. Scale bars are 40 μm for panels A–C; and 10 μm for panels D–F.
2.5. Effect of glutamate treatment on the morphology and number of dendritic spine in hippocampal neurons We used GFP labeling to observe the effect of glutamate treatment on the morphology and number of dentritic spine in cultured hippocampal neurons. Cultured hippocampal neurons were transfected with GFP vectors at 6 DIV, and dendritic spines were observed at 20 DIV (36 h after glutamate exposure), because at 36 h after glutamate treatment, the level of Snk protein was highest while the level of SPAR protein was lowest in neurons. Our results revealed that by 20 DIV, GFP was still highly expressed in the hippocampal neurons, and the majority of the spines were mushroom-shaped spines (Fig. 5). Compared with control group, glutamate-treated neurons showed a dramatic decrease in the density of mushroomshaped spines (Fig. 6A, n = 20 neurons per group, pb 0.01) and a corresponding increase in immature filopodia (Fig. 6A, n= 20 neurons per group, pb 0.01). We also observed that glutamatetreated neurons showed an increase in length of dendritic protrusions (Fig. 6B). Compared with control group, the average dendritic protrusion length in glutamate-treated neurons increased to 1.87±0.08 μm from 1.07±0.04 μm (n=20 neurons per
group, p b 0.005). Some varicosities were also observed on dendritic shafts. MK801 pretreatment could dramatically block the decrease in the density of mushroom-shaped spines (n=20 neurons per group, p b 0.05 compared with glutamate-treated group) and the increase in length of dendritic protrusions (1.19 ±0.05 μm, n=20 neurons per group, p b 0.01 compared with glutamate-treated group) induced by glutamate stimulation. Because the dendritic spine analysis was carried out on twodimensional projections of apical dendrites, many necks of mushroom spines could potentially be occluded by the apical dendrite's shaft, making them appear to be stubby spines. But since experimental and control groups were treated identically, and spine lengths do not differ between groups, this underestimation should not affect the outcome of comparisons of density between groups.
3.
Discussion
Dendritic spines are remarkably diverse in size and shape, ranging from plump mushroom-shaped spines to thin fingerlike filopodia. During development, dendritic protrusions
Fig. 6 – Quantitative analysis of glutamate effects on dendritic spines. (A) Glutamate treatment induces loss of mushroom-shaped spines and increased filopodia. These data were statistically analyzed by one-way ANOVA. *p b 0.05, **p b 0.01 compared with number of mushroom-shaped spines in control group; #p b 0.05, ##p b 0.01 compared with number of filopodia in control group; ^ p b 0.05 compared with number of mushroom-shaped spines in glutamate-treated group; Op b 0.05 compared with number of filopodia in glutamate-treated group. (B) Frequency distribution of dendritic protrusion lengths in neurons.
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develop from long, highly motile filopodia and stubby protrusions into more mature mushroom-shaped spines, each with an excitatory synapse on the tip (Fiala et al., 1998; Spires et al., 2005; Huang et al., 2006; Grossman et al., 2006). In the 17-21 DIV hippocampal neurons, ∼ 60% of the dendritic spines on pyramidal cells are mushroom-shaped (Parnass et al., 2000). Shape of a spine head can change within a few seconds, which is determined by the architecture of its actin cytoskeleton (Carlisle and Kennedy, 2005; Matus, 2000; Matus et al., 2000). Actin is highly enriched in spine as compared with the surrounding neuropil, but the arrangement and content of it are closely correlated to the activity of glutamate receptors. Excessive stimulation of glutamate receptors can destroy the ionic balance in neurons, decrease actin number, and develop dendritic varicosities or swelling (Halpain et al., 1998; Matus et al., 2000; Fischer et al., 2000). SPAR is a Rap-specific GTPase-activating protein (RapGAP). In dendritic spine, SPAR forms a complex with the postsynaptic scaffolding protein PSD-95 and NMDA receptor, and influences their downstream signaling targets. SPAR also reorganizes the actin cytoskeleton and recruits PSD-95 to F-actin, the predominant cytoskeleton in dendritic spines. So, SPAR can act as a bridging molecule between the PSD-95 complex and F-actin, and then control spine shape by regulating actin arrangement. SPAR appears to be selective for spiny neurons and is present in about 65% of the mature mushroom-shaped spines, inducing an increase in complexity of spine shape when overexpressed (Segal, 2001; Ehlers, 2002; Pak et al., 2001). Snk belongs to a family of serine/threonine-specific pololike kinases that are involved in cell cycle control. But recent investigation revealed that unlike other family numbers, Snk seemed to lack a prominent role in the cell cycles, and might play an important role in the process of synaptic remodeling after neuronal activity (Seeburg et al., 2005). In rat hippocampus following pentylenetetrazole (PTZ)-induced seizures, the mRNA and protein levels of Snk were increased dramatically (Kauselmann et al., 1999). In 2003, Pak and his colleagues identified Snk as a new binding partner for SPAR, and interestingly, overexpression
43
of Snk caused dramatic down-regulation of recombinant SPAR in cultured COS cells and of endogenous SPAR in hippocampal neurons. They also showed that Snk-induced depletion of SPAR appeared to depend on the degradation of SPAR by the ubiquitin-proteasome pathway. This is the SnkSPAR signaling pathway (Pak and Sheng, 2003; Meyer and Brose, 2003). There are three main findings in the present study. First, we report that stimulation of hippocampal neuron cultures with 100 μM glutamate and 10 μM glycine for 10 min successfully induces the activation of Snk-SPAR signaling pathway. Second, we present the time-dependent changes in Snk and SPAR expression in cultured hippocampal neurons induced by glutamate treatment. Finally, we demonstrate that MK801, a non-competitive antagonist of N-methyl-D-aspartate (NMDA) receptor, can block the activation of Snk-SPAR pathway induced by glutamate stimulation, and protect the dendritic spines from glutamate-induced injury. We can presume that excessive accumulation of glutamate can over-activate the NMDA receptors, and in this circumstance, Snk is expressed. Snk can associate with and phosphorylate SPAR. An ubiquitin ligase recognizes phosphorylated SPAR and modifies it with ubiquitin. SPAR is then degraded via the proteasome pathway (Fig. 7). After NMDA receptor is activated, MK801 can block the ion channel of it, reduce neuronal activity, subsequently block the activation of Snk-SPAR signaling pathway, and protect the dendritic spines against glutamate-induced injury. Taken together, Snk-SPAR signaling pathway may be a negative feedback mechanism during the excitotoxic procedure induced by glutamate, it may plays an important role in CNS neuronal excitotoxic damage.
4.
Experimental procedures
4.1.
Hippocampal neuron culture
Hippocampal tissues were derived from newborn Sprague– Dawley rats and incubated with 0.125% trypsin for 30 min at 37 °C. The tissues were then dissociated by trituration through
Fig. 7 – NMDA receptor, Snk, SPAR and spine destabilization. (A) In dendritic spine, SPAR (red) forms a complex with PSD-95 (green) and NMDA receptor (blue), and associates with actin cytoskeleton (brown). (B) After the NMDA receptor is activated by glutamate (purple), Snk (orange) is expressed. Snk associates with and phosphorylates (P) SPAR. (C) An ubiquitin ligase (yellow) recognizes phosphorylated SPAR and modifies it with ubiquitin (Ub). (D) SPAR is then degraded via the proteasome pathway. (E) The dendritic spine loses PSD-95 and Possibly other protein components, and changes its shape to become a long, thin protrusion.
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a fire-polished Pasteur pipette. Cells were plated at a density of 1 × 106 cells/cm2 in 3.5 mm dishes or 2 × 105 cells/cm2 on glass coverslips coated with poly-L-lysine in plating medium containing 78% DMEM, 10% fetal bovine serum, 10% horse serum, 25 μM glutamine and 1% antibiotic solution (Invitrogen). One day after plating, the culture medium was switched into a maintenance medium which contained 90% DMEM, 5% horse serum, 1% N2, 2% B27, 25 μM glutamine and 1% antibiotic solution. All these cultures were maintained at 37 °C, under a humidified atmosphere of 95% air and 5% CO2. Subsequent media replacement occurred twice a week by replacing 50% medium with fresh maintaining medium. After 5 days, nonneuronal cell division was halted by exposure to 10 μM cytosine arabinoside for 48 h.
4.2.
Treatment
Hippocampal neurons, cultured for 18 DIV, attained a mature morphology with highly branched dendrites bearing numerous dendritic spines. For glutamate treatment, 18 DIV rat hippocampal neurons were treated with 100 μM glutamate and 10 μM glycine for 10 min, then the exposure was terminated by returning cultures to normal maintaining medium. In experimental cultures, neurons were pretreated with 10 μM MK801 for 2 min before glutamate exposure. In control group, cultures were treated with equivalent volume of 0.9% saline.
4.3.
LDH release
For LDH release assay, hippocampal neurons were cultured in 24-well plates for 18 days. The medium was then changed and the effect of 100 μM glutamate and 10 μM glycine on LDH release was measured for up to 72 h using Cytotoxicity Detection Kit (LDH) (Roche).
4.4.
RNA isolation and RT-PCR
Total RNA was extracted from cells grown on 3.5 mm according to the TRIzol reagent protocol (Invitrogen). The resulting RNA was DNase-treated using RQ1 DNase (Promega). The quality and concentration of the RNA were confirmed by spectrophotometry and electrophoresis on ethidium bromide-stained agarose gels. RT-PCR amplification was performed using the Takara RNA PCR Kit (AMV) Ver 3.0 (Takara Biotechnology) and oligo-dT primers according to the manufacturer's instructions. cDNA was prepared from 500 ng of total RNA, and amplified with 20 μM forward primer, and 20 μM reverse primer. Primer pairs were, respectively, 5′-GCT CCT GCG GAC TAT CAC CTA-3′ and 5′-CCA AGC CGA AGT CTC CAA CC-3′ for Snk; 5′-GCC TGA AGG GTT TGG AGT GAG C-3′ and 5′-CCT GAA CGA GGT GAG GAT GTG GA-3′ for SPAR; 5′-TGA TCT TCA TGG TGC TAG GAG CC-3′ and 5′-GGT ATG GGT CAG AAG GAC TCC-3′ for β-actin. For Snk and β-actin, PCR was performed by denaturing at 94 °C for 2 min, followed by 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 2 min. For Spar, PCR was performed by denaturing at 94 °C for 2 min, followed by 35 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 2 min. Eight microliters of the PCR reaction was loaded in parallel with the DNA Marker DL2000 (Takara Biotechnology) on a 2% Agarose gel containing ethidium bromide. The β-actin served as a control for the quality and quantity of cDNA.
4.5.
Western blot analysis
For Western blot analysis, the cells were seeded in 3.5-mm dishes at a density of 1 × 106 cells/cm2. Cells were washed with cold PBS and incubated in ice-cold lysis buffer containing of 50 mM Tris buffer, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 0.1% SDS, 1 mM PMSF for 30 min, and the neurons were harvest by cell scraper. Then the cell lysates were centrifuged at 12,000×g for 15 min, and the concentration of protein in the supernatant was determined by the BCA protein assay (Nanjing Jiancheng Bioengineering Institute). Samples were resolved by 12% SDS-PAGE were transferred to PVDF membranes, and blocked with 5% nonfat dry milk for 1 h at room temperature. The membranes were then incubated overnight at 4 °C in polyclonal goat anti-Snk antibody (1:500; Santa Cruz Biotechnology), polyclonal goat anti-SPAR antibody (1:500; Santa Cruz Biotechnology), monoclonal mouse anti-β-actin antibody (1:4000, Sigma). AP-conjugated secondary antibodies were used, and membranes were treated with chromogenic substrate. Protein levels were expressed as a ratio to β-actin.
4.6.
Immunofluorescence microscopy
Double immunofluorescence staining for Snk and SPAR was performed as described (Wang and Larsson, 1985). In short, cells cultured on glass coverslips were washed with PBS, fixed in 4% paraformaldehyde for 20 min and permeabilized in phosphate-buffered saline (PBS) containing 0.2% Triton X-100 for 10 min. A blocking solution of 10% horse serum in PBS was applied for 30 min, followed by overnight incubation at 4 °C in PBS containing 0.15% horse serum plus the goat anti-SPAR primary antibody (1:100). Following several washes, TRITCconjugated donkey anti-goat IgG (1:400; ZSGB-Bio) was applied for 1 h at room temperature. After denaturation of free anti-IgG binding sites by formaldehyde vapor treatment at 80 °C for 4 h, the cells were incubated overnight at 4 °C in PBS containing 0.15% horse serum plus the goat anti-Snk primary antibody (1:100), then the FITC-conjugated donkey anti-goat IgG (1:400, ZSGB-Bio) was applied for 1 h at room temperature. Double immunofluorescence staining for Snk and PSD-95 was performed as follows: the fixed neurons were treated with 0.2% Triton X-100 and blocked with 10% horse serum, then were incubated with goat anti-Snk antibody (1:100) and rabbit anti-PSD-95 antibody (1:100, Santa Cruz Biotechnology) at 4 °C overnight, after several washes, FITCconjugated donkey anti-goat IgG (1:400, ZSGB-Bio) and TRITCconjugated donkey anti-rabbit IgG (1:400, ZSGB-Bio) were applied for 1 h at room temperature. After several more washes cells were microscope slides using a fluorescence mounting medium (Molecular Probes) and visualized with a fluorescence microscopy.
4.7.
Plasmid and transfection
The GFP expression plasmid (pgz21äxz) was a gift from Dr. Kenneth M. Yamada. Plasmid DNA was transformed into E. coli DH5α competent cells and highly purified covalently closed circular plasmid DNA was isolated by plasmid purification kit (Biodev Technology) according to the manufacturer's instructions. For the transfection assays, the cells
BR A IN RE S E A RCH 1 1 68 ( 20 0 7 ) 3 8 –4 5
were seeded at a density of 2 × 105 cells per well in 24-well plates. Transfection was performed in 7 DIV. One day before transfection, placed the cells per well in 500 μl of growth medium without antibiotics. For each well, prepare DNALipofectamine 2000 complexes as follows: dilute 0.1 μg DNA in 50 μl of Opti-MEM I Reduced Serum Medium, and then 2 μl of Lipofectamine 2000 was combined with 50 μl of Opti-MEM I Reduced Serum Medium and allowed to sit at room temperature for 5 min. These two solutions were then combined and complexed for 20 min at room temperature. After 20 min of complexing, the Opti-MEM I/DNA/Lipofectamine 2000 mixture was added to the culture at 100 μl per well and the cells were incubated at 37 °C in a humidified incubator with 5% CO2 for 4 h, then the cells were placed in the previous culture medium.
Acknowledgment This work was supported by the National Natural Science Funds, Approval No. 30400143 and No. 30470682.
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Involvement of the Snk-SPAR pathway in glutamate-induced excitotoxicity in cultured hippocampal neurons Lan-Xiang Wu a , Chang-Kai Sun a,⁎, Yu-Mei Zhang a , Ming Fan b , Jing Xu a , Hui Ma a , Jian Zhang a a
Institute for Brain Disorders, Dalian Medical University, 465 Zhong Shan Road, Shahekou District, Dalian 116027, China Institute of Basic Medical Science, Academy of Military Medical Sciences, Beijing 100850, China
b
A R T I C LE I N FO
AB S T R A C T
Article history:
The serum-induced kinase (Snk)–spine-associated Rap GTPase-activating protein (SPAR)
Accepted 20 June 2007
signaling pathway is reported as a new molecular mechanism in activity-dependent
Available online 27 July 2007
remodeling of synapses. However, the relationship between Snk-SPAR pathway and glutamate-induced excitotoxicity is not well understood. We report here that in cultured
Keywords:
hippocampal neurons, glutamate stimulation induces the activation of Snk-SPAR pathway,
Serum-induced kinase
and leads to a loss of mature dendritic spines. The time-dependent changes in Snk and SPAR
Spine-associated Rap
expression after glutamate exposure are also elucidated. Furthermore, the activation of Snk-
GTPase-activating protein
SPAR pathway induced by glutamate treatment can be blocked by an NMDA receptor
Dendritic spine
antagonist, MK801. These results demonstrate that Snk-SPAR pathway may play a pivotal
Glutamate
role in glutamate-induced exicitotoxic damage in CNS through regulating the stability of
MK801
synapse. © 2007 Elsevier B.V. All rights reserved.
1.
Introduction
Dendritic spines are small postsynaptic protrusions that receive over 90% of the excitatory glutamatergic inputs in the CNS (Harris and Kater 1994). “Dysgenesis” of dendritic spine is a common feature of the microstructural pathology that occurs in profound mental retardation (Marin-Padilla, 1972; Purpura, 1974), and the distribution and structure of dendritic spine are altered in many diseases, such as temporal lobe epilepsy, Huntington's disease, and acquired immunodeficiency syndrome-related dementia (Halpain et al., 1998; Fiala et al., 2002; Carlisle and Kennedy, 2005). In glutamatergic excitatory synaptic transmission, glutamate is released from presynaptic membrane and activates glutamate receptors which are mainly localized in postsyn-
aptic density (PSD). There are also many signaling proteins, cytoskeletal proteins, and ion channels in PSD, any changes in these molecular can influence the morphology and function of dendritic spine obviously (Allison et al., 2000; Schubert and Dotti, 2007). SPAR, spine-associated Rap GTPase-activating protein, associates with actin cytoskeleton and forms a complex with PSD-95 and NMDA receptors in dendritic spine. PSD-95 and NMDA receptor have been reported to play an important role in synaptic remodeling (Charych et al., 2006; Shi and Ethell, 2006), which makes SPAR an attractive candidate for mediating activity-dependent remodeling of synapses (Pak et al., 2001; Segal, 2001). In 2003, Pak and Sheng revealed that serum-induced kinase (Snk) induced by neuronal activity could lead to synapse loss through inducing the degradation of SPAR. The Snk-SPAR signaling pathway may be
⁎ Corresponding author. E-mail address: [email protected] (C.-K. Sun). Abbreviations: Snk, serum-induced kinase; SPAR, spine-associated Rap GTPase-activating protein; NMDA, N-methyl-D-aspartate 0006-8993/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2007.06.082
BR A IN RE S E A RCH 1 1 68 ( 20 0 7 ) 3 8 –4 5
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lation could induce the activation of Snk-SPAR pathway, which could be blocked by non-competitive antagonist of NMDA receptor, MK801. The present study provides a new insight into the molecular mechanism of glutamate-induced excitotoxicity.
2.
Results
2.1. Glutamate-induced cell damage in cultured hippocampal neurons Fig. 1 – The release of LDH activity in cultured hippocampal neurons illustrating the neurotoxic effects of glutamate and the neuroprotective effects of the non-competitive antagonist of NMDA receptor, MK801. Data are shown as mean ± SEM of six measurements. *P b 0.05, **P b 0.01, ***P b 0.001, compared with control group.
In order to study the kinetics of glutamate cytotoxicity, LDH release assay was performed. The result indicated that glutamate toxicity varied during the course of the cultures. We also showed the neuroprotective effect of MK801 on glutamate-induced neuronal damage (Fig. 1).
2.2. Snk and Spar mRNA levels are regulated by glutamate treatment in cultured hippocampal neurons a homeostatic regulatory process that destabilizes synaptic connections in response to neuronal activity (Pak and Sheng, 2003; Meyer and Brose, 2003). The purpose of the present study was to investigate the role of Snk-SPAR pathway in glutamate-induced excitotoxic damage in cultured hippocampal neurons. Glutamate stimu-
Using semiquantitative RT-PCR, we first examined the effect of glutamate on Snk and SPAR mRNA levels in cultured hippocampal neurons. The corresponding transcripts had sizes of 665 bp for Snk, 483 bp for SPAR, and 843 bp for β-actin. Incubation of hippocampal neurons with 100 μM glutamate and 10 μM
Fig. 2 – The changes in Snk and SPAR mRNA expression following glutamate stimulation in cultured hippocampal neurons. (A) Total RNA was isolated from normal neurons (con), different time points of neurons after 100 μM glutamate and 10 μM glycine stimulation for 10 min (1 h, 3 h, 6 h, 9 h, and 12 h). (C) 6 h after glutamate stimulation, Snk and SPAR mRNA transcripts were detected in normal neurons (con), and glutamate-treated neurons with (MK801 + Glu) or without (Glu) MK801. These data were statistically analyzed by Student's t test (B) and one-way ANOVA (D) respectively. Error bars represent the mean ± SEM for five to six samples per group. *p b 0.05, **p b 0.01, ***p b 0.005 compared with Snk mRNA level in control group; #p b 0.05, ## p b 0.01, ###p b 0.005 compared with SPAR mRNA level in control group; ^^p b 0.01 compared with Snk mRNA level in glutamate-treated group; OOp b 0.01 compared with SPAR mRNA level in glutamate-treated group.
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glycine for 10 min induced time-dependent changes in Snk and SPAR mRNA expression. Our results showed that in control neurons, Snk mRNA was barely detectable, but 1 h after glutamate exposure, the mRNA level of Snk was increased ∼ 1.8-fold compared with the basal level (n = 6, p b 0.01). At 6 h, Snk mRNA level reached its highest peak, which was increased ∼ 4-fold compared with the basal level (n = 6, p b 0.005), then returned to baseline by 12 h. In control neurons, SPAR mRNA was highly detectable, but after glutamate insult, the tendency of change in SPAR mRNA level was contrary to Snk (Figs. 2A, B). Pretreated neurons with 10 μM MK801 before glutamate exposure could dramatically block the changes in Snk and SPAR mRNA expression induced by glutamate: 6 h after glutamate stimulation, compared with glutamate-treated
group, MK801 could decrease Snk mRNA level for about 41% (n = 5, p b 0.01), and increase SPAR mRNA level for about 36% (n = 5, p b 0.01) (Figs. 2C, D).
2.3. Snk and Spar protein levels are regulated by glutamate treatment in cultured hippocampal neurons The changes in Snk and SPAR protein levels were evaluated by Western blot analysis. Identical with mRNA level, in control hippocampal neurons, Snk protein was barely detectable by Western blot, but SPAR protein was highly detectable. 3 h after glutamate stimulation, Snk protein level began to increase (n = 6, p b 0.05). It reached the highest peak by 36 h, at this time point, the level of Snk protein was increased ∼ 3.3-fold
Fig. 3 – The changes in Snk and SPAR protein expression following glutamate stimulation in cultured hippocampal neurons. (A) Lysates of 18DIV hippocampal neurons of control, 3 h, 6 h, 12 h, 18 h, 24 h, 36 h, 48 h, and 72 h after glutamate insult were analyzed by immunoblot assay with Snk and SPAR antibodies. (C) 36 h after glutamate stimulation, Snk and SPAR proteins were detected in normal neurons (con), and glutamate-treated neurons with (MK801 + Glu) or without (Glu) MK801. These data were statistically analyzed by Student's t test (B) and one-way ANOVA (D) respectively. Error bars represent the mean ± SEM for five to six samples per group. *p b 0.05, **p b 0.01, ***p b 0.005 compared with Snk level in control group; #p b 0.05, ##p b 0.01, ### p b 0.005 compared with SPAR level in control group; ^p b 0.05 compared with Snk level in glutamate-treated group; OOp b 0.01 compared with SPAR level in glutamate-treated group.
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compared with the basal level (n = 6, p b 0.005), and then gradually decreased thereafter. The level of SPAR protein began to decrease after glutamate treatment, and at 36 h, SPAR protein was barely detectable (n = 6, p b 0.005), and then gradually increased thereafter (Figs. 3A, B). In agreement with RT-PCR, 36 h after glutamate stimulation, MK801 could decrease Snk protein level (n = 5, p b 0.05) and increase SPAR protein level (n = 5, p b 0.01) markedly compared with glutamate-treated group (Figs. 3C, D).
2.4. Effect of glutamate treatment on the distribution of Snk, SPAR and PSD-95 in hippocampal neurons In this experiment, we found little staining of Snk in unstimulated neurons by immunocytochemistry. After
41
glutamate stimulation, Snk immunoreactivity increased specifically in the cell body, and also enriched in dendrite. The same glutamate-treated neurons were immunostained for SPAR and PSD-95. In unstimulated cells, SPAR and PSD95 were evenly distributed along dendrites. Glutamatetreated cells showing high levels of Snk in dendrites exhibited a loss of both SPAR and PSD-95 in the same regions (Fig. 4). So, elimination of SPAR by Snk is associated with the loss of the PSD marker PSD-95. Because SPAR is a large scaffold protein of the PSD with the ability to regulate the actin cytoskeleton, it is quite plausible that SPAR degradation would lead indirectly to dismantling of the PSD and depletion of PSD-95. Our results also showed that MK801 could block these changes induced by glutamate insult.
Fig. 4 – Glutamate treatment induces Snk and leads to loss of SPAR and PSD-95. Unstimulated hippocampal cultures (DIV18) (A and D) or cultured received stimulation of 100 μM glutamate and 10 μM glycine for 10 min with (C and F) or without (B and E) 10 μM MK801 pretreatment were double-stained for Snk (green) and either SPAR or PSD-95 (red), as indicated, with merge in color. Scale bar, 20 μm.
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Fig. 5 – Representative multipolar neurons expressing GFP which were incubated in the absence (A) and presence (B) of glutamate, or pretreated with MK801 before glutamate stimulation (C). Panels D–F are enlargements of rectangles in panels A–C, respectively. Scale bars are 40 μm for panels A–C; and 10 μm for panels D–F.
2.5. Effect of glutamate treatment on the morphology and number of dendritic spine in hippocampal neurons We used GFP labeling to observe the effect of glutamate treatment on the morphology and number of dentritic spine in cultured hippocampal neurons. Cultured hippocampal neurons were transfected with GFP vectors at 6 DIV, and dendritic spines were observed at 20 DIV (36 h after glutamate exposure), because at 36 h after glutamate treatment, the level of Snk protein was highest while the level of SPAR protein was lowest in neurons. Our results revealed that by 20 DIV, GFP was still highly expressed in the hippocampal neurons, and the majority of the spines were mushroom-shaped spines (Fig. 5). Compared with control group, glutamate-treated neurons showed a dramatic decrease in the density of mushroomshaped spines (Fig. 6A, n = 20 neurons per group, pb 0.01) and a corresponding increase in immature filopodia (Fig. 6A, n= 20 neurons per group, pb 0.01). We also observed that glutamatetreated neurons showed an increase in length of dendritic protrusions (Fig. 6B). Compared with control group, the average dendritic protrusion length in glutamate-treated neurons increased to 1.87±0.08 μm from 1.07±0.04 μm (n=20 neurons per
group, p b 0.005). Some varicosities were also observed on dendritic shafts. MK801 pretreatment could dramatically block the decrease in the density of mushroom-shaped spines (n=20 neurons per group, p b 0.05 compared with glutamate-treated group) and the increase in length of dendritic protrusions (1.19 ±0.05 μm, n=20 neurons per group, p b 0.01 compared with glutamate-treated group) induced by glutamate stimulation. Because the dendritic spine analysis was carried out on twodimensional projections of apical dendrites, many necks of mushroom spines could potentially be occluded by the apical dendrite's shaft, making them appear to be stubby spines. But since experimental and control groups were treated identically, and spine lengths do not differ between groups, this underestimation should not affect the outcome of comparisons of density between groups.
3.
Discussion
Dendritic spines are remarkably diverse in size and shape, ranging from plump mushroom-shaped spines to thin fingerlike filopodia. During development, dendritic protrusions
Fig. 6 – Quantitative analysis of glutamate effects on dendritic spines. (A) Glutamate treatment induces loss of mushroom-shaped spines and increased filopodia. These data were statistically analyzed by one-way ANOVA. *p b 0.05, **p b 0.01 compared with number of mushroom-shaped spines in control group; #p b 0.05, ##p b 0.01 compared with number of filopodia in control group; ^ p b 0.05 compared with number of mushroom-shaped spines in glutamate-treated group; Op b 0.05 compared with number of filopodia in glutamate-treated group. (B) Frequency distribution of dendritic protrusion lengths in neurons.
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develop from long, highly motile filopodia and stubby protrusions into more mature mushroom-shaped spines, each with an excitatory synapse on the tip (Fiala et al., 1998; Spires et al., 2005; Huang et al., 2006; Grossman et al., 2006). In the 17-21 DIV hippocampal neurons, ∼ 60% of the dendritic spines on pyramidal cells are mushroom-shaped (Parnass et al., 2000). Shape of a spine head can change within a few seconds, which is determined by the architecture of its actin cytoskeleton (Carlisle and Kennedy, 2005; Matus, 2000; Matus et al., 2000). Actin is highly enriched in spine as compared with the surrounding neuropil, but the arrangement and content of it are closely correlated to the activity of glutamate receptors. Excessive stimulation of glutamate receptors can destroy the ionic balance in neurons, decrease actin number, and develop dendritic varicosities or swelling (Halpain et al., 1998; Matus et al., 2000; Fischer et al., 2000). SPAR is a Rap-specific GTPase-activating protein (RapGAP). In dendritic spine, SPAR forms a complex with the postsynaptic scaffolding protein PSD-95 and NMDA receptor, and influences their downstream signaling targets. SPAR also reorganizes the actin cytoskeleton and recruits PSD-95 to F-actin, the predominant cytoskeleton in dendritic spines. So, SPAR can act as a bridging molecule between the PSD-95 complex and F-actin, and then control spine shape by regulating actin arrangement. SPAR appears to be selective for spiny neurons and is present in about 65% of the mature mushroom-shaped spines, inducing an increase in complexity of spine shape when overexpressed (Segal, 2001; Ehlers, 2002; Pak et al., 2001). Snk belongs to a family of serine/threonine-specific pololike kinases that are involved in cell cycle control. But recent investigation revealed that unlike other family numbers, Snk seemed to lack a prominent role in the cell cycles, and might play an important role in the process of synaptic remodeling after neuronal activity (Seeburg et al., 2005). In rat hippocampus following pentylenetetrazole (PTZ)-induced seizures, the mRNA and protein levels of Snk were increased dramatically (Kauselmann et al., 1999). In 2003, Pak and his colleagues identified Snk as a new binding partner for SPAR, and interestingly, overexpression
43
of Snk caused dramatic down-regulation of recombinant SPAR in cultured COS cells and of endogenous SPAR in hippocampal neurons. They also showed that Snk-induced depletion of SPAR appeared to depend on the degradation of SPAR by the ubiquitin-proteasome pathway. This is the SnkSPAR signaling pathway (Pak and Sheng, 2003; Meyer and Brose, 2003). There are three main findings in the present study. First, we report that stimulation of hippocampal neuron cultures with 100 μM glutamate and 10 μM glycine for 10 min successfully induces the activation of Snk-SPAR signaling pathway. Second, we present the time-dependent changes in Snk and SPAR expression in cultured hippocampal neurons induced by glutamate treatment. Finally, we demonstrate that MK801, a non-competitive antagonist of N-methyl-D-aspartate (NMDA) receptor, can block the activation of Snk-SPAR pathway induced by glutamate stimulation, and protect the dendritic spines from glutamate-induced injury. We can presume that excessive accumulation of glutamate can over-activate the NMDA receptors, and in this circumstance, Snk is expressed. Snk can associate with and phosphorylate SPAR. An ubiquitin ligase recognizes phosphorylated SPAR and modifies it with ubiquitin. SPAR is then degraded via the proteasome pathway (Fig. 7). After NMDA receptor is activated, MK801 can block the ion channel of it, reduce neuronal activity, subsequently block the activation of Snk-SPAR signaling pathway, and protect the dendritic spines against glutamate-induced injury. Taken together, Snk-SPAR signaling pathway may be a negative feedback mechanism during the excitotoxic procedure induced by glutamate, it may plays an important role in CNS neuronal excitotoxic damage.
4.
Experimental procedures
4.1.
Hippocampal neuron culture
Hippocampal tissues were derived from newborn Sprague– Dawley rats and incubated with 0.125% trypsin for 30 min at 37 °C. The tissues were then dissociated by trituration through
Fig. 7 – NMDA receptor, Snk, SPAR and spine destabilization. (A) In dendritic spine, SPAR (red) forms a complex with PSD-95 (green) and NMDA receptor (blue), and associates with actin cytoskeleton (brown). (B) After the NMDA receptor is activated by glutamate (purple), Snk (orange) is expressed. Snk associates with and phosphorylates (P) SPAR. (C) An ubiquitin ligase (yellow) recognizes phosphorylated SPAR and modifies it with ubiquitin (Ub). (D) SPAR is then degraded via the proteasome pathway. (E) The dendritic spine loses PSD-95 and Possibly other protein components, and changes its shape to become a long, thin protrusion.
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a fire-polished Pasteur pipette. Cells were plated at a density of 1 × 106 cells/cm2 in 3.5 mm dishes or 2 × 105 cells/cm2 on glass coverslips coated with poly-L-lysine in plating medium containing 78% DMEM, 10% fetal bovine serum, 10% horse serum, 25 μM glutamine and 1% antibiotic solution (Invitrogen). One day after plating, the culture medium was switched into a maintenance medium which contained 90% DMEM, 5% horse serum, 1% N2, 2% B27, 25 μM glutamine and 1% antibiotic solution. All these cultures were maintained at 37 °C, under a humidified atmosphere of 95% air and 5% CO2. Subsequent media replacement occurred twice a week by replacing 50% medium with fresh maintaining medium. After 5 days, nonneuronal cell division was halted by exposure to 10 μM cytosine arabinoside for 48 h.
4.2.
Treatment
Hippocampal neurons, cultured for 18 DIV, attained a mature morphology with highly branched dendrites bearing numerous dendritic spines. For glutamate treatment, 18 DIV rat hippocampal neurons were treated with 100 μM glutamate and 10 μM glycine for 10 min, then the exposure was terminated by returning cultures to normal maintaining medium. In experimental cultures, neurons were pretreated with 10 μM MK801 for 2 min before glutamate exposure. In control group, cultures were treated with equivalent volume of 0.9% saline.
4.3.
LDH release
For LDH release assay, hippocampal neurons were cultured in 24-well plates for 18 days. The medium was then changed and the effect of 100 μM glutamate and 10 μM glycine on LDH release was measured for up to 72 h using Cytotoxicity Detection Kit (LDH) (Roche).
4.4.
RNA isolation and RT-PCR
Total RNA was extracted from cells grown on 3.5 mm according to the TRIzol reagent protocol (Invitrogen). The resulting RNA was DNase-treated using RQ1 DNase (Promega). The quality and concentration of the RNA were confirmed by spectrophotometry and electrophoresis on ethidium bromide-stained agarose gels. RT-PCR amplification was performed using the Takara RNA PCR Kit (AMV) Ver 3.0 (Takara Biotechnology) and oligo-dT primers according to the manufacturer's instructions. cDNA was prepared from 500 ng of total RNA, and amplified with 20 μM forward primer, and 20 μM reverse primer. Primer pairs were, respectively, 5′-GCT CCT GCG GAC TAT CAC CTA-3′ and 5′-CCA AGC CGA AGT CTC CAA CC-3′ for Snk; 5′-GCC TGA AGG GTT TGG AGT GAG C-3′ and 5′-CCT GAA CGA GGT GAG GAT GTG GA-3′ for SPAR; 5′-TGA TCT TCA TGG TGC TAG GAG CC-3′ and 5′-GGT ATG GGT CAG AAG GAC TCC-3′ for β-actin. For Snk and β-actin, PCR was performed by denaturing at 94 °C for 2 min, followed by 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 2 min. For Spar, PCR was performed by denaturing at 94 °C for 2 min, followed by 35 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 2 min. Eight microliters of the PCR reaction was loaded in parallel with the DNA Marker DL2000 (Takara Biotechnology) on a 2% Agarose gel containing ethidium bromide. The β-actin served as a control for the quality and quantity of cDNA.
4.5.
Western blot analysis
For Western blot analysis, the cells were seeded in 3.5-mm dishes at a density of 1 × 106 cells/cm2. Cells were washed with cold PBS and incubated in ice-cold lysis buffer containing of 50 mM Tris buffer, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 0.1% SDS, 1 mM PMSF for 30 min, and the neurons were harvest by cell scraper. Then the cell lysates were centrifuged at 12,000×g for 15 min, and the concentration of protein in the supernatant was determined by the BCA protein assay (Nanjing Jiancheng Bioengineering Institute). Samples were resolved by 12% SDS-PAGE were transferred to PVDF membranes, and blocked with 5% nonfat dry milk for 1 h at room temperature. The membranes were then incubated overnight at 4 °C in polyclonal goat anti-Snk antibody (1:500; Santa Cruz Biotechnology), polyclonal goat anti-SPAR antibody (1:500; Santa Cruz Biotechnology), monoclonal mouse anti-β-actin antibody (1:4000, Sigma). AP-conjugated secondary antibodies were used, and membranes were treated with chromogenic substrate. Protein levels were expressed as a ratio to β-actin.
4.6.
Immunofluorescence microscopy
Double immunofluorescence staining for Snk and SPAR was performed as described (Wang and Larsson, 1985). In short, cells cultured on glass coverslips were washed with PBS, fixed in 4% paraformaldehyde for 20 min and permeabilized in phosphate-buffered saline (PBS) containing 0.2% Triton X-100 for 10 min. A blocking solution of 10% horse serum in PBS was applied for 30 min, followed by overnight incubation at 4 °C in PBS containing 0.15% horse serum plus the goat anti-SPAR primary antibody (1:100). Following several washes, TRITCconjugated donkey anti-goat IgG (1:400; ZSGB-Bio) was applied for 1 h at room temperature. After denaturation of free anti-IgG binding sites by formaldehyde vapor treatment at 80 °C for 4 h, the cells were incubated overnight at 4 °C in PBS containing 0.15% horse serum plus the goat anti-Snk primary antibody (1:100), then the FITC-conjugated donkey anti-goat IgG (1:400, ZSGB-Bio) was applied for 1 h at room temperature. Double immunofluorescence staining for Snk and PSD-95 was performed as follows: the fixed neurons were treated with 0.2% Triton X-100 and blocked with 10% horse serum, then were incubated with goat anti-Snk antibody (1:100) and rabbit anti-PSD-95 antibody (1:100, Santa Cruz Biotechnology) at 4 °C overnight, after several washes, FITCconjugated donkey anti-goat IgG (1:400, ZSGB-Bio) and TRITCconjugated donkey anti-rabbit IgG (1:400, ZSGB-Bio) were applied for 1 h at room temperature. After several more washes cells were microscope slides using a fluorescence mounting medium (Molecular Probes) and visualized with a fluorescence microscopy.
4.7.
Plasmid and transfection
The GFP expression plasmid (pgz21äxz) was a gift from Dr. Kenneth M. Yamada. Plasmid DNA was transformed into E. coli DH5α competent cells and highly purified covalently closed circular plasmid DNA was isolated by plasmid purification kit (Biodev Technology) according to the manufacturer's instructions. For the transfection assays, the cells
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were seeded at a density of 2 × 105 cells per well in 24-well plates. Transfection was performed in 7 DIV. One day before transfection, placed the cells per well in 500 μl of growth medium without antibiotics. For each well, prepare DNALipofectamine 2000 complexes as follows: dilute 0.1 μg DNA in 50 μl of Opti-MEM I Reduced Serum Medium, and then 2 μl of Lipofectamine 2000 was combined with 50 μl of Opti-MEM I Reduced Serum Medium and allowed to sit at room temperature for 5 min. These two solutions were then combined and complexed for 20 min at room temperature. After 20 min of complexing, the Opti-MEM I/DNA/Lipofectamine 2000 mixture was added to the culture at 100 μl per well and the cells were incubated at 37 °C in a humidified incubator with 5% CO2 for 4 h, then the cells were placed in the previous culture medium.
Acknowledgment This work was supported by the National Natural Science Funds, Approval No. 30400143 and No. 30470682.
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