The impact of agrin on the formation of orthogonal arrays of particles in cultured astrocytes from wild-type and agrin-null mice

B RA IN RE S EA RCH 1 36 7 (2 0 1 1 ) 2 –1 2

available at www.sciencedirect.com

www.elsevier.com/locate/brainres

Research Report

The impact of agrin on the formation of orthogonal arrays of particles in cultured astrocytes from wild-type and agrin-null mice Petra Fallier-Becker a , Jan Sperveslage a , Hartwig Wolburg a,⁎, Susan Noell b a

Institute of Pathology, University of Tübingen, Medical School, Tübingen, Germany Clinics of Neurosurgery, University of Tübingen, Medical School, Tübingen, Germany

b

A R T I C LE I N FO

AB S T R A C T

Article history:

Astrocytic endfeet membranes are studded with aquaporin-4 (AQP4) containing orthogonal

Accepted 26 September 2010

arrays of particles (OAP) which can be visualized exclusively by the freeze-fracturing method.

Available online 1 October 2010

They are predominantly expressed where the astroglial membrane is in contact with the superficial and perivascular basal lamina. This polarity seems to be essential for the integrity of

Keywords:

the blood-brain barrier (BBB). The basal lamina containing many extracellular matrix (ECM)

Blood-brain barrier

components such as collagen, laminin and heparansulfate proteoglycans like agrin is thought

Extracellular matrix

to influence this OAP-related polarity of astrocytes. Recently, we have shown that agrin, in

Cell culture

particular the neuronal isoform A4B8, is capable of influencing the formation of OAPs in

Freeze-fracturing

astrocytes when cultured in the presence of agrin-conditioned media. In this paper we wanted

Agrin-null mouse

to investigate whether coating with exogenous agrin compared to coating with other ECM components would induce OAP formation in astrocytes of the agrin-null mouse. For this purpose, we cultured astrocytes from agrin-null and wild-type mice on agrin- or ECM-coated surfaces. Immunofluorescent cytochemical staining of AQP4 indicated a higher AQP4 expression level in cultures with agrin- or ECM-coated than in cultures with uncoated surfaces, whereas western blot analyses and PCR showed no differences. α-Dystroglycan is thought to be a potential receptor of agrin and was immunostained in wild-type as well as in agrin-null astrocytes. In freeze-fracture replicas, we observed an increase in OAP density in astrocytes when growing on agrin- and ECM-coatings. These results concurred with other experiments in which changes in volume were measured following hypotonic stress, which supported the positive influence of exogenous agrin on AQP4 insertion into the membrane, on OAP formation and on water transport. © 2010 Elsevier B.V. All rights reserved.

⁎ Corresponding author. Institute of Pathology, University of Tübingen, Medical School, Liebermeisterstraße 8, D-72076 Tübingen, Germany. Fax: +49 7071294834. E-mail address: [email protected] (H. Wolburg). Abbreviations: AQP4, Aquaporin-4; BBB, Blood-brain barrier; ECM, Extracellular matrix; OAP, Orthogonal arrays of intramembranous particles 0006-8993/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2010.09.092

BR A IN RE S EA RCH 1 3 67 ( 20 1 1 ) 2 –1 2

3

Fig. 1 – Scheme of the agrin-coating method. Agrin producing HEK cells (red arrows) are grown to confluence (top). After lysis of HEK cells, surface is coated with agrin (middle). Astrocytes are seeded on agrin-coated surface (bottom).

1.

Introduction

Orthogonal arrays of particles (OAP) of astrocytes were discovered in the 1970s (Dermietzel, 1973) using freezefracture techniques. But it was not until 30 years later that it became clear that they contain the water channel protein AQP4 (Rash et al., 1998). A specific property of this water channel is the occurrence of different isoforms (Moe et al., 2008). The best examined isoforms are AQP4-M1 and AQP4M23 which exhibit different water transport capacities (Silberstein et al., 2004). Furman et al. (2003) investigated M1 and M23 transfected cells and found that the AQP4-M23 isoform was capable of forming huge lattices in the cell membrane, whereas AQP4-M1-transfectants showed only few and small ones. Only the transfection of a mixture of M1 and M23 resulted in the formation of orthogonal arrays which resembled the phenotype of arrays found in astrocytes. In the healthy brain, perivascular as well as superficial astrocytic endfeet membranes are studded with OAPs, whereas parenchymal astrocyte membranes only reveal few OAPs (Rohlmann et al., 1992). It has been proposed that the contact of membranes with the basal lamina is responsible for the OAP formation and therefore the OAP-related polarization of the astrocyte. Agrin as a component of the ECM is an extracellular heparansulfate proteoglycan (Tsen et al., 1995) which was originally identified as being capable of clustering acetylcholine receptors at the neuromuscular junction (McMahan, 1990; Bezakova and Ruegg, 2003). Barber and Lieth (1997) described the importance of agrin in the CNS for the integrity of the BBB showing that during chick and rat development, agrin accumu-

lates on brain microvessels by the time the vasculature becomes impermeable. The agrin molecule exists in different splice variants; the endothelial isoform A0B0 is released, whereas the neuronal isoform A4B8 is inserted in the cell membrane. Noell et al. (2009) showed that in the agrin-null mouse the OAP formation at the superficial endfoot membrane of astrocytes is clearly reduced, pointing to the role of agrin for the formation of OAPs from subunits. In the present freeze-fracture study we wanted to find out whether cultured astrocytes from the agrin-null mouse brain growing on agrin- and ECM-precoated surfaces (Fig. 1) are able to form OAPs. Furthermore, we studied the influence of exogenous agrin on AQP4 expression and on changes in cell volumes of agrin-null astrocytes during and after hypotonic challenge.

2.

Results

2.1.

Freeze-fracture experiments

In freeze-fracture replicas of cultured astrocytes, we generally found an increase of OAP densities using culture dishes coated with either ECM or exogenous agrin from HEK cells compared with astrocytes cultured without ECM or agrin. Wild-type and agrin-null astrocytes were cultured on different coatings and subsequently fixed for freeze-fracture analysis. In wild-type as well as in agrin-null astrocytes, the influence of agrin- or ECM-coating on the number and density of OAPs was substantial. In freeze-fracture replicas of control

4

B RA IN RE S EA RCH 1 36 7 (2 0 1 1 ) 2 –1 2

astrocytes cultured without agrin or ECM coating of tissue culture dishes (Fig. 2A), we found an average density of 5 OAPs/μm2 (Fig. 3). In agrin-null astrocytes (Fig. 2B) the density was 3 OAPs/μm2 (Fig. 3). In both the wild-type (Fig. 2C) and the agrin-null astrocytes (Fig. 2D) coating with agrin-isoform A4B8 resulted in an increase up to 15 OAPs/μm2 (Fig. 3), whereas wild-type astrocytes on agrin A0B0-coating (Fig. 2E) only showed 6 OAPs/μm2 (Fig. 3). However, agrin-null astrocytes (Fig. 2F) revealed 19 OAPs/μm2 on agrin A0B0coating (Fig. 3). The influence of ECM gel was even more pronounced: wild-type astrocytes reached an OAP density of 17 OAPs/μm2 (Figs. 2G, 3) and agrin-null cells a density of 25 OAPs/μm2 (Figs. 2H, 3). Taken together, coating experiments using agrin A4B8, agrin A0B0 and ECM-gel revealed a 5-, 6-, or 8-fold increase of OAPs in agrin-null astrocytes in comparison with wild-type astrocytes, respectively (Table 1).

2.2.

Immunostaining

Wild-type and agrin-null astrocytes were cultured on agrin- or ECM-coated coverslips and processed for immunostaining with an antibody against AQP4 as previously described for wild-type astrocytes (Noell et al., 2007). Here, we examined the immunoreactivity against AQP4 in agrin-null astrocytes in comparison with wild-type astrocytes. In wild-type (Fig. 4A) as well as in agrin-null astrocytes (Fig. 4B) cultured without coating, AQP4 staining was diffusely distributed within the cytoplasm. Culturing the cells on agrin showed AQP4 staining concentrated in membrane areas in all cultures (Figs. 4C–F). ECM coating revealed intensive AQP4 fluorescence in membranes of wild-type cells (Fig. 4G) and in agrin-null cells (Fig. 4H). Thus, as reported in the freeze-fracture paragraph, also immunostaining against AQP4 revealed a stronger immunoreactivity in agrin-null astrocytes when cultured on ECM-, agrin A4B8- and agrin A0B0-coating (Table 1). In order to address the question of whether the presence of αdystroglycan as an agrin-receptor is an indispensable precondition for the formation of OAPs, we stained wild-type (Fig. 5A) and agrin-null (Fig. 5B) astrocytes immunocytochemically for αdystroglycan. Both cultures were found to be immunopositive for α-dystroglycan. This finding explains the observation that even agrin-null astrocytes are able to form OAPs when cultured on exogenous agrin (Table 1).

2.3.

Western blotting

Western blot analysis showed that the AQP4-isoform M23band was more abundant than the AQP4-isoform M1 band independent of the coating. For western blot analysis, wildtype and agrin-null astrocytes were grown on ECM- and agrinisoform A0B0 and A4B8 coatings. Western blotting was performed with an AQP4 antibody and showed two distinct bands, the lower band (32 kDa) representing the AQP4-M23 isoform and the upper band (34 kDa) representing the AQP4M1 isoform. The western blot showed a stronger immuno-

reactivity for AQP4-M23 than for AQP4-M1 regardless of the treatment and origin of the cells (Figs. 6A, B; Table 1).

2.4.

PCR

PCR was used to prove the expression of both AQP4-isoforms M23 and M1 on RNA level independent of the presence of a coating and independent of the origin of the astrocytes (Fig. 7, Table 1). Agrin does not seem to influence the expression of the AQP4, neither on the mRNA nor on protein level.

2.5.

Cell volume measurements

In swelling experiments we found differences between the uncoated astrocyte cultures and the cultures which were coated with the neuronal agrin-isoform A4B8. Coating with the endothelial agrin-isoform A0B0 did not induce any alteration of water flow (Table 1). Cultured wild-type and agrin-null astrocytes were grown on coated and uncoated coverslips fitting into the flow-through chamber. Wild-type astrocytes grown on uncoated surfaces showed a volume increase of 8%, but in the presence of the agrinisoform A4B8 we found a significant volume increase of 21% (Fig. 8). In contrast, in the presence of agrin A0B0, no significant difference to the uncoated controls was observed. Comparable results were achieved with agrin-null astrocytes. Coating with agrin A4B8 resulted in a significant cell volume increase of 26% compared to the uncoated controls, which showed only 14% of volume increase. A0B0-coating, however, revealed no significant difference to uncoated control cells (9%, Fig. 8). Taken together, agrin-null astrocytes did not show a significant difference in water transport compared to wildtype astrocytes.

3.

Discussion

Agrin, a heparansulfate proteoglycan, is known to influence the differentiation of the motor endplate by clustering acetylcholine receptors (McMahan, 1990). Noell et al. (2007) were able to show comparable effects of agrin on the clustering of AQP4-containing OAPs in cultured astrocytes. In addition, Noell et al. (2009) detected a paucity of OAPs in the endfeet membranes in the agrin-null mouse, although the expression of the AQP4 protein was not down-regulated. This finding suggested a major role of agrin for the formation of OAPs from AQP4 molecules as seen in the freeze-fracture replica. In the present study we asked whether exogenously applied agrin isoforms (endothelial A0B0 and neuronal A4B8) and extracellular matrix gel from Engelbreth–Holm–Swarm mouse sarcoma are capable of influencing OAP formation of cultured agrin-null astrocytes. Freeze-fracture analysis showed an effect of ECM- and agrin-coating on the occurrence of AQP4-containing OAPs in agrin-null and wild-type astrocytes. The number of OAPs in the coated cultures was

Fig. 2 – Freeze-fracture replicas of cultured wild-type (A, C, E, G) and agrin-null astrocytes (B, D, F, H) growing on different coatings, as indicated by the labels. The number of OAPs (circles) increases depending on different coatings. Scale bars: 100 nm.

BR A IN RE S EA RCH 1 3 67 ( 20 1 1 ) 2 –1 2

5

6

B RA IN RE S EA RCH 1 36 7 (2 0 1 1 ) 2 –1 2

Fig. 3 – Statistical analysis (box-blots) of OAP densities in wild-type and agrin-null astrocytes growing on different coatings. The difference between the wild-type control cells and the wild-type cells growing on agrin-isoform A4B8 and on ECM gel is significant (p < 0.05). The difference in OAP density between the agrin-null control cells and the agrin-null cells growing on agrin-isoform A4B8 and A0B0 and on ECM gel is significant (p < 0.05).

Table 1 – Summary of results. Cell cultures Coating Freeze fracturing (number of OAPs/μm2) Immunofluorescence of AQP4 Immunofluorescence of α-dystroglycan Western blot Cell volume measurements (Intensity dF/dF0) PCR a

Wild-type astrocytes ECM 17 ++ n.d. M23>M1 n.d. M23, M1

Agrin-null astrocytes

Agrin A0B0 Agrin A4B8 Control 6 + n.d. M23>M1 7% M23, M1

15 ++ n.d. M23>M1 21% M23, M1

5 + + M23>M1 8% M23, M1

ECM 25 +++ n.d. M23>M1 n.d. M23, M1

Agrin A0B0 Agrin A4B8 Control 19 + n.d. M23>M1 9% M23, M1

15 +++ n.d. M23>M1 26% M23, M1

3 + + M23>M1 14% M23, M1

n.d. not done. a By trend, M23 was higher expressed than M1 in all samples.

significantly increased compared to the uncoated controls. Immunofluorescent cytochemical staining of AQP4 revealed higher AQP4 expression in the coated cultures than in the uncoated cultures. The intensity of immunofluorescence staining correlated with the findings that cultured murine astrocytes in media conditioned with the neuronal agrinisoform A4B8 and the endothelial agrin-isoform A0B0 revealed a stronger immunoreactivity within the cell membrane than untreated controls (Noell et al., 2007). The AQP4 molecule exists in different isoforms. The M1 isoform is 22 amino acids longer at the N-terminus than the M23 isoform (Jung et al., 1994). Furman et al. (2003) and Silberstein et al. (2004) showed that in cells transfected with AQP4-isoform M1 the membranes reveal very small or no OAPs, whereas the AQP4-M23 transfectants form huge OAP lattices. Transfection of the cells with both isoforms (M1 and M23) resulted in the formation of OAPs reminiscent of those in astrocytes in vivo. As we could only find small or no OAPs in the astrocyte membranes of the agrin-null mouse we expected that the AQP4-isoform M1 was most abundant in western blots. Surprisingly, this was not the case. All western blots revealed a higher abundance of the M23 bands compared with the M1

bands regardless of the coatings. In freeze-fracture replicas of astrocytes, cultured on agrin or ECM coatings, we always found an increased number of OAPs compared to those in control astrocytes grown on uncoated surfaces. Since we found more M23 than M1 in the western blot and by trend also in PCR analyses of all cultures, we propose that the coating of ECM or agrin is crucial for the formation of OAPs rather than the ratio of the AQP4-isoforms. Based on these results and the experiments of Furman et al. (2003) who used cells transfected with M23 and M1 AQP4-isoforms and Suzuki et al. (2008) who found that the size of AQP4-M23 up to AQP4-M17 is small enough to form OAPs, we assume that the presence of AQP4-M23 could represent the preconditions for forming OAPs. At the healthy BBB, the astrocytic endfoot membrane reveals an extremely high density of OAPs at the contact site of the membranes and the endothelial basal lamina consisting of collagen, laminin and heparansulfate proteoglycans like agrin, whereas only few OAPs are found at the parenchymal membranes that do not contact the basal lamina. Furthermore, it is known that under pathological conditions, the number of OAPs decreases at the endfoot membranes (Neuhaus, 1990) despite an upregulation of AQP4

Fig. 4 – Immunocytochemical staining of AQP4 in wild-type (A, C, E, G) and agrin-null astrocytes (B, D, F, H) growing on different coatings, as indicated. Scale bars: 20 μm.

BR A IN RE S EA RCH 1 3 67 ( 20 1 1 ) 2 –1 2

7

8

B RA IN RE S EA RCH 1 36 7 (2 0 1 1 ) 2 –1 2

Fig. 5 – Immunocytochemical staining of α-dystroglycan is positive in both wild-type (A) and in agrin-null (B) astrocytes. Scale bars: 10 μm.

expression (Saadoun et al., 2002; Warth et al., 2004). Rascher et al. (2002) showed a loss of agrin at multiple sites of a brain tumor. This suggests that agrin and other components of the ECM enhance the formation of OAPs and the polarity of astrocytic endfeet. Agrin cannot be the only factor participating in OAP formation, because agrin-null astrocyte cultures are able to form OAPs also in the absence of exogenous agrin (Fig. 2B,

control). Since agrin does not bind to AQP4 directly, but indirectly via the dystrophin–dystroglycan complex (Gee et al., 1994), α-dystroglycan may serve as a receptor for exogenous agrin in agrin-null astrocytes as well. Indeed, α-dystroglycan could be detected immunocytochemically in wild-type as well as in agrin-null astrocytes (compare Figs. 5A and B). Furthermore, immunocytochemical staining of AQP4 revealed a fluorescent signal in the cell membranes, which was more intensive in coated than in uncoated cultures. This observation was independent of the origin of the cells. The staining of all coated cultures showed the same pattern supporting the freezefracture results that exogenously applied ECM components are able to influence the occurrence of OAPs. To test the function of water transport, cell volume measurements of agrin-null and wild-type astrocytes were performed and revealed an increased swelling capacity of cells grown on agrin-A4B8 coating independent of their origin. Volume measurements of ECM-coated astrocytes could not be tested due to the experimental design. Nevertheless, we can speculate that the swelling behavior of astrocytes should be similar as shown by astrocytes grown on the endothelial agrin-isoform A0B0, because this agrin isoform is present in the ECM of the basal lamina in the brain. Taken together, we were not able to find a difference in water transport between wild-type and agrin-null astrocytes growing on A4B8 agrin.

4.

Fig. 6 – (A) Western blot showing the distribution of AQP4 in agrin-null-astrocytes and wild-type astrocytes grown on PLL (poly-L-lysine, control), ECM, agrin-isoform A0B0 and A4B8. The lower bands (32 kDa) represent the M23 isoform of AQP4, the upper bands (34 kDa) represent the AQP4-M1 isoform. (B) Statistical analysis of (A) including SEM showing relative amounts of AQP4-M23 and -M1.

Conclusions

Two preconditions must be fulfilled to form OAPs of a certain size and number: AQP4-M23 isoform must be expressed and enough agrin or ECM should be present. No OAPs should be expected if one of these components was absent or reduced. Noell et al. (2009) have shown that in the absence of agrin the astrocytic endfoot membranes in vivo contained a very small number of OAPs. However, the amount of AQP4-M23 was normal. In addition, α-dystroglycan as a binding partner of agrin might be important for the localization of the OAPs at the

BR A IN RE S EA RCH 1 3 67 ( 20 1 1 ) 2 –1 2

Fig. 7 – PCR analyses of astrocyte cultures. AQP4-M1 (upper bands) and AQP4-M23 (middle) and HPRT (lower bands) representing the load control.

endfoot membranes. Wolburg-Buchholz et al. (2009) were able to show that at sites of inflammatory cuffs during experimental autoimmune encephalomyelitis, dystroglycan was lost and the OAP-related polarity was reduced. This polarity reduction appears to involve the appearance of OAPs on membrane domains which normally did not contain them. Taken together, the size and number of OAPs formed in astrocytes depend on agrin containing ECM and the presence of AQP4M23 and dystroglycan in the astroglial membrane.

5.

Experimental procedures

5.1. Culture of murine brain wild-type- and agrin-null astrocytes All animal care and experimental protocols conformed to the University of Tübingen Animal Ethics Committee guidelines and the German legislation regulating the use of animals in research. The use of animals in this study

9

was minimized to the necessary numbers for quantitative and qualitative analyses. Murine brain astrocytes were separately isolated and cultured from individual E19.5 BALB/c mice embryos derived from the breeding of heterozygous agrin mice (Noell et al., 2009). Briefly, after removal of meninges, the tissues were digested with 0.1% trypsin and 0.02% EDTA (Lonza, Verviers, Belgium) in PBS for 15 min at room temperature. Then, the cell suspension was transferred into culture medium, centrifuged (400 ×g; 5 min), resuspended in culture medium and filtered through a 70 μm nylon mesh. Cells were seeded in T25 culture flasks (Corning, Heidelberg, Germany) and cultured in Dulbecco's modified Eagle's medium supplemented with 20% fetal calf serum, penicillin (10,000 units/ml), and streptomycin (10,000 μg/ml) under standard conditions up to passage two (all Lonza, Verviers, Belgium). All littermates used for culture of astrocytes, cyto- and histochemistry and freeze-fracturing were genotyped by PCR with lysates of tail biopsies. For coating experiments wild-type and agrin-null astrocytes were used in passage 3. After 7 days cell cultures were stopped and analyzed using freeze-fracture, immunostaining, western blotting, PCR and cell volume detection technique.

5.2.

Coating with agrin

Transfected HEK 293 cells producing the neuronal splicing variant A4B8 or the endothelial splicing variant A0B0 of agrin (kindly provided by Stephan Kröger, LMU Munich) were cultured in 0.01% poly-L-lysine (PLL)-precoated flasks and on coverslips under standard conditions. PLL was purchased from Sigma (Deisenhofen, Germany). After reaching confluency the cells were lysed with 0.5% Triton X-100 solution (15 min at 37 °C) and washed with PBS. Using an antibody against agrin (kindly provided by Stephan Kröger, LMU Munich), the equal distribution of agrin coating was demonstrated. Subsequently, wild-type or agrin-null astrocytes were seeded on precoated agrin (A0B0 and A4B8) surface and cultured for 3–7 days (Fig. 1).

Fig. 8 – Statistical analysis (box-blots) of cell volume measurements of cultured wt- and agrin-null astrocytes growing on different coatings.

10 5.3.

B RA IN RE S EA RCH 1 36 7 (2 0 1 1 ) 2 –1 2

Coating with ECM

Precooled culture flasks and coverslips were coated with ECM gel originated from Engelbreth–Holm–Swarm mouse sarcoma (Sigma, Deisenhofen, Germany). Wild-type and agrin-null astrocytes were seeded in the coated culture flasks and cultured for 3–7 days.

5.4.

Freeze-fracture experiments

Monolayers of cultured cells were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 2 h at room temperature. The specimens were then cryoprotected for freeze-fracturing in 30% glycerol and snap-frozen in nitrogen slush (−210 °C). Subsequently, they were fractured in a Balzer's freeze-fracture device (BAF400D; Balzers, Liechtenstein) at 5 × 10− 6 mbar and − 150 °C. The fracture faces were shadowed with platinum/carbon (3 nm, 45°) for contrast and carbon (30 nm, 90°) for stabilization of the replica. After removal of the cell material in 12% sodium hypochlorite, the replicas were rinsed in double-distilled water several times and mounted on Pioloform-coated copper grids. The replicas were observed by using a Zeiss EM10 electron microscope (Zeiss, Oberkochen, Germany). OAP densities were determined as OAPs per square micrometer at a magnification of 100,000:1 in 30 replicas of three different assays in each experiment. Statistical evaluation was performed by using the Wilcoxon/Kruskal– Wallis one-way analysis of variance (ANOVA) on ranks followed by Tukey–Kramer method for comparisons (JMP, Cary, NC, USA).

5.5.

Immunocytochemistry

Coverslip cultures of wild-type and agrin-null astrocytes were fixed in 4% paraformaldehyde for 15 min. Fixed astrocytes were incubated at 4 °C overnight with a rabbit polyclonal AQP4 antibody (Santa Cruz, Heidelberg, Germany) 1:100 diluted in 0.25% Triton X-100 and 1% DMSO in PBS. AQP4 was detected using a secondary goat anti-rabbit Cy3-labeled antibody (Dianova, Hamburg, Germany, 1 h at room temperature). Immunostaining of α-dystroglycan was performed using a rabbit polyclonal antibody (Santa Cruz, Heidelberg, Germany). As control for nonspecific staining or autofluorescence, the primary antibody was omitted. Nonspecific binding was blocked by incubation for 30 min in 4% normal goat serum and 1% BSA in PBS. Specimens were mounted in Mowiol (Calbiochem, Merck, Darmstadt, Germany). Fluorescence was visualized with a

LSM510 META confocal laser scanning microscope (Zeiss, Oberkochen, Germany) using a HeNe laser for excitation at 543 nm with appropriate filter sets and a 40× oil immersion objective (N.A. 1.3).

5.6.

Electrophoresis and immunoblotting

Confluent astrocytes were lysed and prepared for western blotting as described by Neely et al. (1999). Briefly, cell cultures were lysed with Laemmli-buffer, protein was measured using the method of Bradford. 5 μg total protein of each sample was used for electrophoresis with 12.5% SDSPAGE. The samples were blotted on a nitrocellulose membrane and stained with an antibody against AQP4 (Santa Cruz, Heidelberg, Germany) and a secondary antibody labeled with horseradish peroxidase (Sigma, Deisenhofen, Germany). Western blots were densitometrically quantified using ImageJ software (NIH, Bethesda, MA, USA; available at http://rsb.info.nih.gov/ij). Absolute optical density (OD) was normalized to the ODs of the corresponding bands of βtubulin loading control and expressed as relative abundance in arbitrary units. Each experiment was performed at least nine times.

5.7.

Reverse Transcriptase (RT)-PCR

RT-PCR analysis of AQP4-isoform M23, M1 and HPRT (hypoxanthine guanine phosphoribosyl transferase) mRNAs was performed as described previously (Ivanova et al., 2002). The HPRT gene has been reported as a constitutively expressed housekeeping gene (Frericks and Esser, 2008) and has been identified as the single best reference gene (De Kok et al., 2005). Total RNA was isolated using the peqGold RNApure extraction kit (Peqlab, Erlangen, Germany) according to the manufacturer's protocol. cDNA was synthesized from 1 μg of total RNA with 1 μl dNTPs (0.8 nM), 1 μl MMLV reverse transcriptase, 5 μl 5×-buffer, 1.5 μl hexanucleotide (10 pmol/μl, all reagents from Invitrogen, Karlsruhe, Germany) for 1 h at 37 °C followed by enzyme inactivation for 5 min at 95 °C. PCR was conducted with 1.5 μl of the RT reaction, 0.5 μl sense and antisense primers (10 pmol/μl), 1 μl dNTPs (5 mM), 10×-PCR buffer (+ 15 mM MgCl2) and 0.3 μl AmpliTaq Gold DNA Polymerase (Applied Biosystems, Foster City, California, USA). PCR conditions for M23-, M1-, and HPRTprimers were: 35 cycles of denaturation for 1 min at 95 °C, annealing for 1 min at 62 °C, extension for 1 min at 72 °C, followed by a final elongation step at 72 °C for 5 min (FlexCycler, Analytik Jena). Concurrent RT-PCR amplification of HPRT was carried out as an internal control for variations in the efficiencies

Table 2 – Primer sequences. Target mRNA (mouse) HPRT AQP4 M23 AQP4 M1

s as s as s as

Primer sequence (5′→ 3′)

Product size (bp)

Annealing (°C)

GCT GGT GAA AAG GAC CTC T CAC AGG ACT AGA ACA CCT GC GGA AGG CTA GGT TGG TGA CTT C TGG TGA CTC CCA ATC CTC CAA C CTC CCA GTG TAC TGG AGC CCG TGG TGA CTC CCA ATC CTC CAA C

250

62

460

62

510

62

BR A IN RE S EA RCH 1 3 67 ( 20 1 1 ) 2 –1 2

of RNA isolation and RT. The primer sequences are shown in Table 2. The PCR products were separated by electrophoresis on a 1.5% agarose gel stained with ethidium bromide and were analyzed by a UV transilluminator.

5.8.

Cell volume measurements

For volume measurements, cells were grown on round coverslips fitting into an incubation chamber as described previously (Noell et al., 2007). Briefly, cells were incubated in 5 μM calcein (Invitrogen, Karlsruhe, Germany) in culture medium at 37 °C for 30 min. The coverslips were then mounted in a closed flowthrough incubation chamber (LaCon, Staig, Germany). Cells were imaged with a Zeiss LSM 510 META confocal microscope (Zeiss, Oberkochen, Germany) via a 63× long-distance objective. The 488-nm argon laser line was used for excitation with appropriate filter sets, the laser intensity being kept low to avoid cell damage and bleaching during the experiment. To test for cellular volume changes, the following paradigm was used. To establish a base line, the chamber was perfused with isotonic Hanks' balanced salt solution (HBSS; 300 mOsm, Invitrogen, Karlsruhe, Germany) at a flow rate of 300 μl/min. After 180 s, the solution was switched to hypotonic (200 mOsm) HBSS for an additional 180 s and finally back to isotonic buffer for at least another 200 s. For cell volume changes, the increase or decrease in fluorescence intensity was correlated with cell shrinkage or swelling, respectively. Subsequent calculations were performed with Microsoft Excel (Microsoft, Seattle, USA). The fluorescence measurements were plotted as dF/F0, where F0 is the averaged initial fluorescence intensity and dF is the difference of the measured fluorescence to the initial fluorescence. The initial fluorescence values were compared with fluorescence minima (highest volume) and tested for statistical significance by applying the nonparametric Wilcoxon rank test (n> 30 cells were measured in each experimental group) (Table 1).

Acknowledgments Yeliz Donat-Krasnici and Ria Knittel are thanked for the skilful technical assistance in cell culturing, freeze-fracturing, and microscopy. We thank Drs. Markus Rüegg (Biozentrum, University of Basel, Switzerland) and Urban Deutsch (TKI, University of Bern, Switzerland) for providing us with the agrin-null mice. Dr. Stephan Kröger (LMU Munich, Germany) is thanked for providing agrin-transfected HEK cells. With support by the Deutsche Krebshilfe-Mildred Scheel-Stiftung to HW and SN (grant numbers 107686 and 109219).

REFERENCES

Barber, A.J., Lieth, E., 1997. Agrin accumulates in the brain microvascular basal lamina during development of the blood-brain barrier. Dev. Dyn. 208, 62–74. Bezakova, G., Ruegg, M.A., 2003. New insights into the roles of agrin. Nat. Rev. Mol. Cell Biol. 4, 295–308. De Kok, J.B., Roelofs, R.W., Giesendorf, B.A., Pennings, J.L., Waas, E.T., Feuth, T., Swinkels, D.W., Span, P.N., 2005. Normalization

11

of gene expression measurements in tumor tissues: comparison of 13 endogenous control genes. Lab. Invest. 85, 154–159. Dermietzel, R., 1973. Visualization by freeze-fracturing of regular structures in glial cell membranes. Naturwissenschaften 60, 208. Frericks, M., Esser, C., 2008. A toolbox of novel murine house-keeping genes identified by meta-analysis of large scale gene expression profiles. Biochim. Biophys. Acta 1779, 830–837. Furman, C.S., Gorelick-Feldman, D.A., Davidson, K.G., Yasumura, T., Neely, J.D., Agre, P., Rash, J.E., 2003. Aquaporin-4 square array assembly: opposing actions of M1 and M23 isoforms. Proc. Natl Acad. Sci. USA 100, 13609–13614. Gee, S.H., Montanaro, F., Lindenbaum, M.H., Carbonetto, S., 1994. Dystroglycan-alpha, a dystrophin-associated glycoprotein, is a functional agrin receptor. Cell 77, 675–686. Ivanova, T., Mendez, P., Garcia-Segura, L.M., Beyer, C., 2002. Rapid stimulation of the PI3-kinase/Akt signalling pathway in developing midbrain neurones by oestrogen. J. Neuroendocrinol. 14, 73–79. Jung, J.S., Bhat, R.V., Preston, G.M., Guggino, W.B., Baraban, J. M., Agre, P., 1994. Molecular characterization of an aquaporin cDNA from brain: candidate osmoreceptor and regulator of water balance. Proc. Natl Acad. Sci. USA 91, 13052–13056. McMahan, U.J., 1990. The agrin hypothesis. Cold Spring Harb. Symp. Quant. Biol. 55, 407–418. Moe, S.E., Sorbo, J.G., Sogaard, R., Zeuthen, T., Petter Ottersen, O., Holen, T., 2008. New isoforms of rat aquaporin-4. Genomics 91, 367–377. Neely, J.D., Christensen, B.M., Nielsen, S., Agre, P., 1999. Heterotetrameric composition of aquaporin-4 water channels. Biochemistry 38, 11156–11163. Neuhaus, J., 1990. Orthogonal arrays of particles in astroglial cells: quantitative analysis of their density, size, and correlation with intramembranous particles. Glia 3, 241–251. Noell, S., Fallier-Becker, P., Beyer, C., Kroger, S., Mack, A.F., Wolburg, H., 2007. Effects of agrin on the expression and distribution of the water channel protein aquaporin-4 and volume regulation in cultured astrocytes. Eur. J. Neurosci. 26, 2109–2118. Noell, S., Fallier-Becker, P., Deutsch, U., Mack, A.F., Wolburg, H., 2009. Agrin defines polarized distribution of orthogonal arrays of particles in astrocytes. Cell Tissue Res. 337, 185–195. Rascher, G., Fischmann, A., Kroger, S., Duffner, F., Grote, E.H., Wolburg, H., 2002. Extracellular matrix and the blood-brain barrier in glioblastoma multiforme: spatial segregation of tenascin and agrin. Acta Neuropathol. 104, 85–91. Rash, J.E., Yasumura, T., Hudson, C.S., Agre, P., Nielsen, S., 1998. Direct immunogold labeling of aquaporin-4 in square arrays of astrocyte and ependymocyte plasma membranes in rat brain and spinal cord. Proc. Natl Acad. Sci. USA 95, 11981–11986. Rohlmann, A., Gocht, A., Wolburg, H., 1992. Reactive astrocytes in myelin-deficient rat optic nerve reveal an altered distribution of orthogonal arrays of particles (OAP). Glia 5, 259–268. Saadoun, S., Papadopoulos, M.C., Davies, D.C., Krishna, S., Bell, B.A., 2002. Aquaporin-4 expression is increased in oedematous human brain tumours. J. Neurol. Neurosurg. Psychiatry 72, 262–265. Silberstein, C., Bouley, R., Huang, Y., Fang, P., Pastor-Soler, N., Brown, D., Van Hoek, A.N., 2004. Membrane organization and function of M1 and M23 isoforms of aquaporin-4 in epithelial cells. Am. J. Physiol. Ren. Physiol. 287, F501–F511. Suzuki, H., Nishikawa, K., Hiroaki, Y., Fujiyoshi, Y., 2008. Formation of aquaporin-4 arrays is inhibited by palmitoylation

12

B RA IN RE S EA RCH 1 36 7 (2 0 1 1 ) 2 –1 2

of N-terminal cysteine residues. Biochim. Biophys. Acta 1778, 1181–1189. Tsen, G., Halfter, W., Kroger, S., Cole, G.J., 1995. Agrin is a heparan sulfate proteoglycan. J. Biol. Chem. 270, 3392–3399. Warth, A., Kroger, S., Wolburg, H., 2004. Redistribution of aquaporin-4 in human glioblastoma correlates with loss of

agrin immunoreactivity from brain capillary basal laminae. Acta Neuropathol. 107, 311–318. Wolburg-Buchholz, K., Mack, A.F., Steiner, E., Pfeiffer, F., Engelhardt, B., Wolburg, H., 2009. Loss of astrocyte polarity marks blood-brain barrier impairment during experimental autoimmune encephalomyelitis. Acta Neuropathol. 118, 219–233.