Neuroserpin regulates neurite outgrowth in nerve growth factor-treated PC12 cells

Neuroserpin regulates neurite outgrowth in nerve growth

factor-treated PC12 cells

Parmjeet K. Parmar,* Leigh C. Coates,* John F. Pearson,� Rena M. Hill* and Nigel P. Birch*

*Molecular Neuroendocrinology Laboratory, School of Biological Sciences, The University of Auckland, Auckland, New Zealand

�Department of Statistics, The University of Auckland, Auckland, New Zealand

Abstract

Neuroserpin is a serine protease inhibitor widely expressed in

the developing and adult nervous systems and implicated in

the regulation of proteases involved in processes such as

synaptic plasticity, neuronal migration and axogenesis. We

have analysed the effect of neuroserpin on growth factor-

induced neurite outgrowth in PC12 cells. We show that small

changes in neuroserpin expression result in changes to the

number of cells extending neurites and total neurite length

following NGF treatment. Increased expression of neuroserpin

resulted in a decrease in the number of cells extending

neurites and a reduction in total free neurite length whereas

reduced levels of neuroserpin led to a small increase in the

number of neurite extending cells and a significant increase in

total free neurite length compared to the parent cell line.

Neuroserpin also altered the response of PC12 cells to bFGF

and EGF treatment. Neuroserpin was localised to dense cored

secretory vesicles in PC12 cells but was unable to complex

with its likely enzyme target, tissue plasminogen activator at

the acidic pH found in these vesicles. These data suggest that

modulation of neuroserpin levels at the extending neurite

growth cone may play an important role in regulating axonal

growth.

Keywords: brain, plasminogen activators, protease inhibi-

tors, serpin.

J. Neurochem. (2002) 82, 1406–1415.

Proteolysis and protease inhibitors play important roles in

developmental, memory forming and pathological events in

the nervous system. Over the last decade considerable

research has been undertaken on several plasticity-related

serine proteases and their inhibitors (Seeds et al. 1992;

Turgeon and Houenou 1997; Shiosaka and Yoshida 2000).

The serine protease tissue plasminogen activator (tPA) has

been implicated in neuronal development, neuronal plasticity

and neuronal death. Plasminogen activator activity is

released at the growth cone of the extending neurite

(Krystosek and Seeds 1981a,b; Moonen et al. 1982) and

associated with axon elongation (Pittman et al. 1989;

Baranes et al. 1998; Siconolfi and Seeds 2001a,b) and

mossy fibre sprouting in the hippocampus in vivo following

seizure activity in mice (Wu et al. 2000). Some of tPAs

effects on axon growth are facilitated by tPA-mediated

degradation of the extracellular matrix components (Monard

1988; Pittman et al. 1989; Sumi et al. 1992; Wu et al. 2000).

A role for tPA in learning related synaptic plasticity in the

adult is supported by the observations that tPA mRNA and

protein levels are increased by long-term potentiation (LTP)

(Qian et al. 1993; Seeds et al. 1995). tPA knockout mice

show a selective defect in late phase-LTP and have a defect

in spatial learning (Baranes et al. 1998) and transgenic mice

overexpressing tPA have increased and prolonged hippo-

campal LTP and improved performance in spatial orientation

learning tasks (Madani et al. 1999).

The serine protease inhibitor neuroserpin is likely to be a

key regulator of plasminogen activator activity in the nervous

system. Neuroserpin was first characterised as a protein

secreted from dorsal root ganglia axons of chicken embryos

and subsequently shown to be expressed in nervous and

endocrine tissues (Stoeckli et al. 1989; Osterwalder et al.

1996; Hastings et al. 1997; Krueger et al. 1997; Hill et al.

2000). Neuroserpin inhibits the serine proteases tissue

plasminogen activator (tPA), urokinase-type plasminogen

Received February 2, 2002; revised manuscript received June 5, 2002;

accepted June 19, 2002.

Address correspondence and reprint requests to Dr Nigel P. Birch,

School of Biological Sciences, The University of Auckland, Private Bag

92019, Auckland, New Zealand. E-mail: [email protected]

Abbreviations used: bFGF, basic fibroblast growth factor; EGF, epi-

dermal growth factor; LTP, long-term potentiation; PC12, phaeochrom-

ocytoma 12; sctPA, single chain tissue plasminogen activator; tPA, tissue

plasminogen activator.

Journal of Neurochemistry, 2002, 82, 1406–1415

1406 � 2002 International Society for Neurochemistry, Journal of Neurochemistry, 82, 1406–1415

activator and trypsin, in vitro (Hastings et al. 1997; Krueger

et al. 1997; Osterwalder et al. 1998). The physiological role

of neuroserpin in the nervous system is unclear. During

development neuroserpin is expressed in neuronal precursors

of most CNS regions in the mouse immediately after

becoming post-mitotic and migrating from the ventricular

zones (Krueger et al. 1997).

As extension and remodelling of neurites play essential

roles in development and neuronal plasticity we investigated

a role for neuroserpin in neurite extension in PC12 cells.

Cultured PC12 pheochromocytoma cells are a clonal cell line

derived from a phaeochromocytoma of the rat adrenal

medulla and have been used extensively as an in vitro model

system for investigations of neuronal differentiation. PC12

cells respond to the neurotrophin nerve growth factor (NGF)

by differentiating into a sympathetic neuron phenotype and

extending axon-like processes called neurites (Greene and

Tischler 1976; Greene and Kaplan 1995). In this study we

have investigated the effect of neuroserpin on growth factor-

induced neurite outgrowth in PC12 cells.

Experimental procedures

Proteins and reagents

PC12 cells were obtained from Dr T. Martin (University of

Wisconsin at Madison, WI, USA). Epidermal Growth Factor

(EGF; murine natural epidermal growth factor from male mouse

submaxillary glands), nerve growth factor (NGF 2.5S, from male

mouse submaxillary glands) and human recombinant basic fibro-

blast growth factor (bFGF) were purchased from Life Technologies

(NY, USA). Recombinant tPA (Actylyse) was kindly donated by

Boehringer Ingelheim (NZ) Ltd (Auckland, New Zealand). The

transferrin receptor and secretogranin-1 antibodies were generously

donated by Dr Ian Trowbridge (The Salk Institute, California, USA)

and Dr Weiland Huttner (University of Heidelberg, Germany),

respectively. The synaptophysin (p38) antibody was purchased from

the Pierce Chemical Company (USA).

Preparation of PC12-Neuroserpin cell lines and analysis

of neuroserpin expression levels by western blotting

PC12 cells were grown in DMEM/5% fetal calf serum/10% horse

serum/100 U/mL penicillin, 100 lg/mL streptomycin, 4 mM gluta-

mine. PC12 cells were plated at the density of 2 · 105/well in a

6 well plate and transfected with rNS-2 cDNA which had been

subcloned into the mammalian expression vector pcDNA3.1

(Invitrogen Corp, USA) in both sense and antisense orientations.

Transfections were carried out using FugeneTM 6 (Roche Bio-

chemicals, IN, USA) and stable cell lines selected using the

antibiotic G418 at a final concentration of 500 lg/mL. Clonal cell

lines were established and maintained at a G418 concentration of

250 lg/mL. Viability was assessed in PC12, s10 and as5 cell lines

using a standard MTT (3[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-

tetrazolium bromide) assay. There were no significant differences

in the ratios of absorbance values at 0 days (�24 h after plating,

prior to NGF treatment) and 4 days after NGF treatment between

the cell lines (data not shown). Neuroserpin levels were measured in

PC12 cells, two neuroserpin sense (s10 and s26) and two neuroserpin

antisense (as5 and as12) cell lines by western blotting. Cells were

lysed in a high salt lysis buffer (50 mM HEPES, 150 mM NaCl,

10 mM EDTA, 1% Triton X-100, 10 mM sodium pyrophosphate,

100 mM sodium fluoride, 2 mM sodium orthovanadate, 1 · complete

mini-protease inhibitor cocktail (Roche Biochemicals, IN, USA),

1 lM Pepstatin A, pH 7.5. Protein concentrations were determined

using the Bio-Rad DC protein assay (CA, USA). 20 lg of protein

from each cell line was resolved on 10% SDS polyacrylamide gels

(Laemmli 1970) transferred to a nitrocellulose membrane (Schleicher

and Schuell, Dassel, Germany) and probed with affinity purified anti-

neuroserpin antibodies as described previously (Hill et al. 2000).

Immunoreactive neuroserpin was visualised by enhanced chemilu-

minescence with SuperSignal West Femto Maximum Sensitivity

substrate (Pierce Chemical Company, Rockford, IL, USA) and

images were collected with a Fuji LAS 1000 digital camera system

and quantitated using Image Gauge software (v3.3 Fuji Film Science

Laboratory, Tokyo, Japan).

Neurite outgrowth assays

Neurite outgrowth of PC12 cells and PC12 cell lines with reduced or

increased levels of neuroserpin was measured following treatment of

cells with nerve growth factor (NGF; 50 ng/mL), basic fibroblast

growth factor (bFGF; 25 ng/mL) and epidermal growth factor (EGF;

25 ng/mL). 3 · 104 cells were plated per well in 6 well plates for

NGF and bFGF treatments and 1.5 · 104 cells per well for EGF

treatment. As we found PC12 cells would attach to culture ware

(Falcon) and extend neurites in the absence of collagen, we

undertook all neurite extension studies on uncoated plastic ware.

After approximately 24 h cells were incubated in a serum-free

medium (Opti-MEM, Life Technologies Inc. NY, USA) for 1 h to

synchronize the cells in the Go state (Greene 1978; Rudkin et al.

1989) before adding PC12 growth medium supplemented with each

growth factor and 1% BSA. Cells were grown in NGF and bFGF for

4 days and EGF for 8 days and then photographed using a Zeiss

Axiovert S100 inverted microscope with phase-contrast optics. For

free neurite length measurements, data was collected from neurites

that did not contact other neurites or other cell bodies. Cells were

plated at relatively low cell densities to minimise neurite contact.

Nevertheless some neurites, particularly in neuroserpin antisense

cell lines, were excluded from measurement, meaning that total free

neurite lengths in these cell lines are likely to be underestimated.

Neurite length was quantitated using Analytical Imaging Station

imaging software (AIS v3.0, Imaging Research Inc., Canada). Data

was collected from between 60 and 70 cells for each cell line with

each treatment. For statistical analyses the neurite lengths from

individual cells were summed. To test the significance of differences

observed in total free neurite lengths we performed analyses of

variance (ANOVA) on the transformed (natural log) total free neurite

length.

Subcellular fractionation of PC12 cells on a sucrose density

gradient and western blot analysis using compartment-specific

antibodies

A crude PC12 post-nuclear fraction was layered onto a linear

sucrose gradient (0.6 M)1.6 M) buffered in 10 mM HEPES, pH 7.0

and centrifuged at 99 000 g for 16 h. Fractions (1 mL) were

Neuroserpin regulates neurite growth 1407

� 2002 International Society for Neurochemistry, Journal of Neurochemistry, 82, 1406–1415

collected from the bottom of the tube and samples prepared for

western blot analysis. Proteins in each fraction were solubilised with

1% (w/v) sodium deoxycholate and then precipitated with trichlo-

roacetic acid at a final concentration of 6% (v/v). Samples were

fractionated on 10% SDS–PAGE gels and western blots were

probed with antisera specific for neuroserpin, secretogranin-1,

transferrin receptor and synaptophysin (p38).

Neuroserpin immunocytochemistry

A neuroserpin-green fluorescent protein (NS-GFP) construct was

prepared by ligating the rat neuroserpin cDNA (amplified by PCR

to include the appropriate restriction sites and remove the stop

codon) into the pEGFP-N1 plasmid (BD Biosciences, Clontech,

Palo Alto, CA, USA). Recombinant plasmids were identified by

restriction fragment analysis and the amplified neuroserpin frag-

ment checked by sequence analysis. PC12 cells were plated onto

collagen-coated coverslips, treated for 8 days with 100 ng/mL

NGF 7S (Invitrogen) and then transfected with the NS-GFP

construct using Lipofectamine 2000 as described by the manufac-

turer (Invitrogen, Auckland, New Zealand). The following day

cells were fixed with 3% paraformaldehyde/PBS for 15 min on ice,

rinsed with PBS and mounted on microscope slides in the anti-fade

reagent Citifluor AF1 (Alltech, Citifluor Ltd., London, UK).

Images were collected on a Leica TCS 4D Confocal Scanning

Microscope. AtT20 cells, plated onto poly-L-lysine-coated covers-

lips, were transfected with pcDNA3.1-neuroserpin (Hill et al.

2000) using Lipofectamine 2000 as described by the manufacturer

(Invitrogen) and cultured for a further two days. Cells were fixed

with 3% paraformaldehyde/PBS for 15 min on ice, rinsed with

PBS and then processed for immunocytochemistry and confocal

microscopy following incubation with affinity-purified NS-PEP

antiserum (1 lg/mL) (Hill et al. 2000) and goat antirabbit IgG

Alexa fluor 488 (Molecular Probes, 1 : 400).

Complexation between recombinant tPA and neuroserpin,

in vitro

[35S]-labelled neuroserpin was synthesized using the Promega

TNTTM. T7 Coupled Reticulocyte Lysate System as described by

the manufacturer. Complexation reactions containing radiolabelled

neuroserpin (1 lL) and recombinant tPA (4 ng) were performed in

20 lL reactions containing 133 mM Tris-HCl, pH 7.4, 133 mM

NaCl for 30 min at 30�C. Reducing load buffer was added and the

samples denatured by heating to 95�C for 3 min. Proteins were

separated on 10% SDS–PAGE gels (30%T, 2.6% C) under

reducing conditions, and radiolabelled neuroserpin and neuroserpin

complexes were visualised using a Fuji FLA2000 phosphorimager.

The pH dependency of complexation was analysed using purified

rat neuroserpin from Drosophila S2 overexpressing cells (Hill

et al. 2001). Recombinant neuroserpin (�200 ng) was incubated

with recombinant tPA (100 ng) in 100 mM buffer (Tris-acetate,

pH 4.5–5.0; MES-HCl, pH 5.5–6.5; HEPES, pH 7.0; Tris-HCl,

pH 7.5), 200 mM NaCl, in a final volume of 30 lL for 30 min at

30�C. Reducing load buffer was added and the samples denatured

by heating to 95�C for 3 min. Proteins were separated on 10%

SDS–PAGE gels (30% T, 2.6% C) under reducing conditions,

electroblotted onto nitrocellulose membranes and probed with an

antiserum prepared in our laboratory to recombinant human tPA

and able to recognize the tPA : neuroserpin complex.

Results

Neuroserpin expression regulates NGF-induced

neurite outgrowth in PC12 cells

Stable PC12 cell lines under and overexpressing rat neuro-

serpin were established. The response of these cell lines to

NGF was assessed to determine if neuroserpin played a role

in the extension of neurites following growth factor-induced

differentiation of PC12 cells into a sympathetic neuron

phenotype. Neuroserpin antisense cell lines showed substan-

tially increased neurite extension following treatment with

NGF compared to the parent cell line. Similar results were

seen in several independently isolated cell lines suggesting

that the effect was not due to clonal variability of PC12 cells

or site of gene insertion – photomicrographs of four

independent cell lines are shown (Fig. 1a–d). In contrast,

the sense cell lines all showed markedly reduced neurite

extension compared to parent PC12 cells – photomicrographs

of four independent cell lines are shown (Fig. 1e-h). In the

absence of NGF neither the parent, sense or antisense cell

lines showed any significant neurite outgrowth (Fig. 1j–l and

data not shown). NGF treatment resulted in 83% of wild-type

PC12 cells extending neurites. In the neuroserpin over-

expressing cell lines, neuroserpin expression led to an �23%

drop (p ¼ 0.00002) in the proportion of cells extending

neurites (Fig. 2a). In contrast, in neuroserpin antisense cell

lines there was a small, although not statistically significant,

increase in the proportion of cells extending neurites

(Fig. 2a).

We investigated the relationship between neuroserpin

expression and neurite outgrowth in more detail by measur-

ing the total neurite length in parent PC12 cells and in two of

the sense and two of the antisense PC12-neuroserpin cell

lines. As expected from the visual appearance of the cells, the

neuroserpin overexpressing cell lines showed significantly

less neurite extension compared to the parental cell line,

while neuroserpin antisense cell lines showed significantly

longer neurite extension. Figure 2(b) shows dot plots of the

log total free neurite length for each cell line with confidence

intervals for the mean, with overall confidence of 95%. These

intervals between the antisense, parent and sense cell lines do

not overlap and clearly show that there are significant

differences between the mean neurite lengths. An ANOVA

performed on the log of the total free neurite lengths showed

a highly significant difference (p < 0.00005) between the

mean total free neurite lengths of the parent, sense and

antisense cell lines. The 95% confidence interval for the

mean neurite length of parent PC12 cells is 68.7 ± 12.9 lm.

Overexpression of neuroserpin resulted in a reduction in

NGF-induced neurite outgrowth. The 95% confidence

interval for the mean neurite lengths of PC12s10 and

PC12s26 cells is 23.7 ± 4.5 lm and 33.8 ± 4.1 lm, respec-

tively. In comparison, PC12 cell lines with reduced

1408 P. K. Parmar et al.


neuroserpin levels showed increased neurite outgrowth with

95% confidence intervals for the mean neurite lengths of

123.0 ± 18.1 lm and 99.9 ± 15.2 lm for PC12as5 and

PC12as12 cells, respectively. Only PC12as12 and PC12as5

showed no significant differences between their mean neurite

lengths at p ¼ 0.05.

Based on these results we looked to see if there was a

correlation between levels of neuroserpin and the extent of

neurite outgrowth. Immunoreactive neuroserpin levels were

determined by quantitative western blotting. Relatively low

levels of neuroserpin were detected in parent PC12 cells

(Fig. 3a) making measurements of reductions in these

levels difficult. However, we found that both neuroserpin

antisense cell lines had slightly lower levels of immuno-

reactive neuroserpin than untransfected PC12 cells. The

two neuroserpin-overexpressing cell lines were chosen on

the basis of differing levels of neuroserpin expression.

Neuroserpin s10 cells expressed neuroserpin at levels

�5-fold greater than the parent whereas the PC12s26 cell

line had only slightly increased expression (Fig. 3a). A

plot of the mean log total neurite length against the

median immunoreactive neuroserpin level indicated a

correlation between decreases in neuroserpin levels and

increased total free neurite length (Fig. 3b). The data

indicates a strong negative linear relationship over neuro-

serpin levels between 58 and 134 arbitrary units

(R2 ¼ 0.999, Pvalue ¼ 0.0002). At high neuroserpin levels

(PC12s10), the inhibitory effect of neuroserpin on neurite

extension appeared maximal, suggesting a threshold level

of neuroserpin expression for inhibition of neurite exten-

sion. This observation is consistent with the appearance of

the neuroserpin-sense cell lines, which showed very little

neurite extension in all the sense cell lines (Fig. 1e–h).

However, it should also be noted that the total free neurite

length of the PC12s10 cells, which had the highest levels

of neuroserpin expression, was less than that seen in

PC12s26 cells (Fig. 2b).

Neuroserpin also alters the neurite outgrowth response

of PC12 cells treated with EGF and bFGF

Treatment of PC12 cells with bFGF resulted in 74% of cells

extending neurites. Increased neuroserpin expression led to a

�33% drop in the number of cells extending neurites

following bFGF treatment whereas a reduction in neuroser-

pin expression showed no significant change in the number

of neurite-extending cells compared to the parental cell line

(Fig. 4a). Parent, neuroserpin-sense and neuroserpin-anti-

sense cell lines were also treated with EGF. As EGF has been

shown to be a weak stimulator of neurite outgrowth in PC12

cells, all EGF treatments were for 8 days. Incubation of

parent PC12 cells with EGF resulted in 41% of parent PC12

cells extending neurites. Increased neuroserpin expression

led to a small decrease in the number of cells extending

neurites that was not statistically significant. EGF treatment

of PC12as5 cells resulted in a statistically significant �14%

increase in the number of neurite-extending cells compared

Fig. 1 Neuroserpin expression regulates NGF-induced neurite out-

growth in PC12 cells. PC12 cells and four PC12 cell lines stably

transformed with an antisense (as) or sense (s) rat neuroserpin cDNA

were treated with NGF for 4 days and photographed using an inverted

microscope with phase-contrast optics (400 · magnification). Panels

a–d, PC12 neuroserpin antisense cell lines as4, as5, as11 and as12,

respectively. Panels e–h, PC12 neuroserpin sense cell lines s6, s10,

s16 and s26, respectively. Panel I, parent PC12 cells treated with NGF.

Panels j–l show parent PC12 cells (j), PC12s10 (k) and PC12as5 (l)

cells that have been grown for 4 days in the absence of NGF (NO

NGF). Scale bar ¼ 100 lm.



with the parent cell line (Fig. 4b). We also analysed the effect

of bFGF and EGF on neurite growth (Figs 4c and d). Similar

to the results seen with NGF, PC12as5 cells treated with

bFGF have longer total free neurite length than PC12 cells,

which in turn have longer mean total free neurite length than

PC12s10 cells (mean total free neurite length ± 95%

confidence interval; PC12, 92.0 ± 16.1 lm; PC12as5,

153.7 ± 28.8 lm; PC12s10, 42.6 ± 6.2 lm). In contrast,

overexpression of neuroserpin did not significantly reduce

the total free neurite length of PC12s10 cells treated with

EGF compared to parental PC12 cells (mean total free

neurite length ± 95% confidence interval; PC12, 52.7 ±

6.7 lm; PC12s10, 46.4 ± 6.8 lm. However, PC12as5 cells

do show increased total free neurite length following EGF

treatment (PC12as5, 140.2 ± 25.8 lm), similar to the results

seen for bFGF and NGF.

Neuroserpin is found in dense cored secretory vesicles

in PC12 cells but is unlikely to complex with sctPA

under the acidic intravesicular environment found

in these vesicles

To identify the intracellular localization of neuroserpin in

parent PC12 cells, a crude post-nuclear sample was frac-

tionated using a sucrose density gradient and analysed by

western blot analysis. The location of immunoreactive

neuroserpin was compared to several intracellular protein

markers (Fig. 5). Neuroserpin immunoreactivity was detec-

ted in fractions 5–7. This distribution colocalised with the

distribution of secretogranin-1. Secretogranin-1 has been

shown to be exclusively distributed to dense cored secretory

vesicles in PC12 cells. No neuroserpin immunoreactivity

colocalised with the peak fractions for endosomes (transfer-

rin receptor antibody, TfR), or synaptic vesicles (p38

antibody). TfR and p38 immunoreactivity were found in

lower density fractions. We also assessed the intracellular

distribution of neuroserpin by immunocytochemistry. As

levels of endogenous neuroserpin in PC12 cells were very

Fig. 2 Neuroserpin expression regulates the proportion of PC12 cells

extending neurites and total free neurite length. (a) the change in the

proportion of sense (s) or antisense (as) cells extending neurites

compared to the parent cell line, following treatment with NGF. p ¼ the

proportion of cells extending neurites and pparent ¼ the proportion of

parent PC12 cells extending neurites. Data was collected from three

independent experiments; Error bar, standard error of the mean. (b)

the total free neurite lengths from each cell line were summed and the

log of the data presented as a dot plot. Each dot represents the total

free neurite length of one cell. Data from 60 to 70 cells was collected

from three independent experiments. For each cell line a confidence

interval for the mean is shown, with an overall confidence of 95% (+).

Fig. 3 Analysis of neuroserpin expression in PC12 cell lines and

relationship to total free neurite outgrowth. (a) immunoreactive neu-

roserpin was visualised by western blotting in PC12 cells (P) and

PC12 cell lines stably transformed with an antisense (as5 and as12) or

sense (s26, s10) rat neuroserpin cDNA. The image is a representative

result from three independent experiments. (b) the relationship

between the mean log free neurite length for parent PC12 cells (p) and

cell lines with reduced (as5 and as12) and increased (s10, s26) levels

of neuroserpin and relative levels of immunoreactive neuroserpin is

presented. Data points for individual neuroserpin levels determined

from three independent experiments are shown. For total free neurite

length data, a confidence interval for the mean is shown, with an

overall confidence of 95% (+) for each cell line. The data omitting s10

fits the regression line log (total free neurite length) ¼ 5.6–0.016 *

median (neuroserpin level).



low and our PC12 overexpressing neuroserpin cell lines

inhibit neurite outgrowth we assessed the intracellular

distribution of chimeric neuroserpin-green fluorescent pro-

tein (NS-GFP) in PC12 cells pretreated with NGF. Punctate

fluorescence accumulated in the growth cone of neurite tips

(Fig. 6a). Punctate labelling was also seen in the region of

the cell body typically occupied by the Golgi, further

supporting trafficking of GFP-NS to the secretory pathway.

We also localised immunoreactive neuroserpin in transiently

transfected AtT20 cells as neuroserpin overexpression in this

cell line stimulates neurite outgrowth (Hill et al. 2000). We

found the growth cones labelled strongly for immunoreactive

neuroserpin (Fig. 6b) consistent with storage of neuroserpin

in regulated secretory granules.

As tPA has been localized to regulated secretory vesicles

we investigated whether tPA could form an inhibitory

complex with neuroserpin in the acidic environment found

in these vesicles, estimated to be between pH 5.0 and 5.5

(Loh et al. 1984). Recombinant neuroserpin and tPA were

combined and complex formation monitored between pH 4.5

and 7.5 using a tPA polyclonal antibody. Complexation

between single chain tPA (sctPA) and neuroserpin was seen

between pH 7.0 and 7.5 (Fig. 7a). A second immunoreactive

band with a molecular weight of �80 kDa, which probably

represent complexation between the reactive chain of the two

chain form of tPA and neuroserpin, was also detected. The

neuroserpin antibody (NS-PEP) was raised to the carboxy

terminus of neuroserpin and will not detect neuroserpin-tPA

complexes. Therefore, we confirmed the identity of these

bands by performing complexation experiments using radio-

labelled neuroserpin (Fig. 7b). Two radiolabelled complexes

were formed when [35S]-neuroserpin was combined with

tPA, with similar molecular weights to the complexes

detected by the anti-tPA antibody. The small differences in

size of the complexes can be attributed to glycosylation of

the neuroserpin expressed in Drosophila S2 cells. These

results indicate that the complexes detected with the tPA

antibody contain neuroserpin and are consistent with previ-

ous reports identifying single chain and two chain tPA-serpin

complexes (Pennica et al. 1983; Hastings et al. 1997;

Osterwalder et al. 1998). Immunoprecipitation analysis of

Fig. 4 Neuroserpin expression regulates bFGF- and EGF-induced

neurite outgrowth in PC12 cells. (a and b), the change in the proportion

of sense (s) or antisense (as) cells extending neurites compared to the

parent cell line, following treatment with bFGF (a) or EGF (b). p ¼ the

proportion of cells extending neurites and pparent ¼ the proportion of

untransfected PC12 cells extending neurites. Error bar, SEM. (c and d)

the total free neurite lengths from as5 (O), parent (D) or s10 (+) cells

were summed and the log of the data presented as a dot plot. (c)

bFGF, (d) EGF. Data was collected from 60 to 70 cells from two

independent experiments. For each cell line a confidence interval for

the mean is shown, with an overall confidence of 95% (+).

Fig. 5 Neuroserpin is found in dense cored secretory vesicles in

PC12 cells. (a) A PC12 cell post-nuclear supernatant was fractionated

on a sucrose density gradient and fractions analysed for neuroserpin

(NS) and for markers of dense cored secretory vesicles (secreto-

granin, SG-1), endosomes (transferrin receptor, TfR) and synaptic

vesicles (synaptophysin, p38) by western blotting.

Fig. 6 Neuroserpin accumulates in the growth cones of PC12 and

AtT20 cells. (a) PC12 cells were pretreated with NGF and then trans-

fected with a neuroserpin-green fluorescent protein construct. The

following day cells were fixed in 3% paraformaldehyde and green

fluorescent protein imaged using confocal microscopy. The arrow head

indicates a neurite tip. Scale bar ¼ 20 lm. (b) AtT20 cells were

transiently transfected with a rat neuroserpin cDNA. Neuroserpin

immunoreactivity was visualised two days after transfection, using

affinity-purified NS-pep antibody and goat anti-rabbit IgG labelled with

Alexa Fluor 488. Arrow heads indicate neurite tips. Scale bar ¼ 20 lm.



PC12 cells demonstrated that only the single chain form of

tPA was present in cell lysates (data not shown) indicating

that this latter complex would not be formed in PC12 cells.

Discussion

Our data show that PC12 cells synthesize neuroserpin and

that changes in levels of neuroserpin alters neurite outgrowth

following treatment with NGF. Photomicrographs clearly

indicate major differences in the extent of neurite outgrowth

in PC12 cells transfected with sense or antisense neuroserpin

constructs (Fig. 1). Similar results were seen in multiple

independently isolated stably transfected cell lines suggesting

that the effect was not due to clonal variability of PC12 cells

or the site of gene insertion. Measurements of neurite lengths

confirmed the photomicrograph results with cells overex-

pressing neuroserpin having a mean total free neurite length

�58% less than the parental PC12 cell line and cells with

reduced levels of neuroserpin having a mean total free

neurite length �160% greater than the parental cells.

We extended these results to look for a correlation between

levels of neuroserpin and the extent of neurite outgrowth. In

our initial screen of neuroserpin expression levels in stably

transfected PC12 cell lines we deliberately chose two

neuroserpin sense cell lines with significantly different levels

of neuroserpin expression. This screen also revealed that all

the neuroserpin antisense cell lines showed only small

reductions in neuroserpin expression compared to the

parental PC12 cell line (Fig. 3 and data not shown). This

may indicate that neuroserpin antisense PC12 cell lines with

very low levels of neuroserpin are unable to survive. While

our inability to isolate neuroserpin antisense PC12 cell lines

with very low levels of neuroserpin limited the range of our

correlative analysis, our results strongly supported a negative

linear relationship between neuroserpin levels and total free

neurite length, i.e. increases in neuroserpin levels resulted in

decreases in total free neurite length. The linear relationship

did not extend to PC12s10 cells, which expressed very high

levels of neuroserpin. Our data suggests that at these

neuroserpin levels, the inhibitory effect of neuroserpin on

neurite extension is maximal, suggesting a threshold level of

neuroserpin expression for inhibition of neurite extension.

Inspection of the photomicrographs of these cells suggests

why this is the case. Both overexpressing cell lines had very

short neurite projections. While these gave a relatively long

total free neurite length measurement when summed, the

length of individual neurites were often less than the cell

diameter, leaving very little opportunity for further measure-

ment of reductions in neurite length. We also observed that

the neurite projections in the sense cell lines were often

thicker suggesting structural differences from the more usual

neurites seen following NGF treatment of parental or

antisense PC12 cells. While our data indicate a threshold

effect it is also important to note that both sense cell lines

which had significantly different levels of neuroserpin

expression also had total free neurite lengths that were

significantly different from each other and the parent PC12

cell line. This result supported the underlying hypothesis that

increased neuroserpin expression reduces neurite extension.

Further support for a concentration-dependent effect of

neuroserpin on neurite outgrowth comes from the observa-

tion that the two neuroserpin antisense cell lines which had

similar neuroserpin levels were the only two cell lines that

did not have significantly different total free neurite lengths.

We also looked at the effect of NGF on levels of endogenous

neuroserpin. Western blot analysis showed no significant

change in levels of immunoreactive neuroserpin (data not

shown). However small differences in the expression of

endogenous neuroserpin in PC12 cells would be difficult to

detect. Further insight into the molecular mechanisms of

neuroserpin action will help assess any direct effects of NGF

on neuroserpin expression.

Similar changes in total free neurite length were seen in

PC12 cells with altered neuroserpin levels following treat-

ment with bFGF, which is known to activate a similar

signalling pathway to NGF (Neufeld et al. 1987; Pollock

et al. 1990; Corbit et al. 1999). Reduction of neuroserpin

levels also led to increased neurite outgrowth in EGF-treated

Fig. 7 Neuroserpin does not complex with sctPA under the low pH

conditions found in dense cored secretory vesicles. (a) recombinant

tPA was incubated with recombinant neuroserpin at varying pHs and

free tPA and SDS-stable tPA : neuroserpin complexes detected using

a rabbit anti-tPA antibody and western blotting. The data are repre-

sentative of results from three independent experiments. (b) radiola-

belled neuroserpin was incubated in the presence or absence of

recombinant tPA. Free neuroserpin (NS), tPA cleaved neuroserpin

and neuroserpin complexed with single chain tPA (NS-sctPA) and two

chain tPA (NS-tctPA) were detected using a Fuji FLA-2000 phos-

phorimager following SDS–PAGE.



PC12 cells. EGF’s effects on PC12 cells are principally

mitogenic (Huff et al. 1981; Chao 1992; Bonni and Green-

berg 1997) and longer incubation times were needed with

this growth factor to observe neurite growth. Thus, while

EGF and bFGF activate different signalling cascades (Corbit

et al. 1999; Corbit et al. 2000) they may utilise a common

signalling specificity towards some of the genes involved in

neurite extension.

Although the molecular mechanisms which mediate

neurite outgrowth in PC12 cells are far from clear, they are

likely to include direct or indirect modulation of the

interaction between the extending growth cone and the

extracellular matrix. Neuroserpin may regulate neurite out-

growth by regulating the local degradation of the extracel-

lular matrix by proteolytic enzymes such as tPA. tPA has

been shown to degrade extracellular matrix proteins involved

in neurite outgrowth (Carbonetto et al. 1983; Carbonetto

et al. 1988; Alexander and Werb 1989; Seeds et al. 1990;

Romanic and Madri 1994; Chen and Strickland 1997) and

neuroserpin can inhibit tPA in vitro (Hastings et al. 1997;

Osterwalder et al. 1998). PC12 cells expressing high levels

of tPA have been found to regenerate neurites within three-

dimensional gels of Matrigel to a greater extent than control

cells (Pittman and DiBenedetto 1995). More recently, tPA

has been found to induce mossy fibre sprouting in the

hippocampus in vivo following seizure activity in mice (Wu

et al. 2000) and tPA levels were shown to increase after

sciatic nerve crush during the period of peripheral nerve

regeneration (Siconolfi and Seeds 2001b). These data, which

suggest tPA may play an important role in nerve regeneration

in vivo, were supported by a study in knock-out mice

showing that plasminogen activators, including tPA, were

necessary for timely functional recovery by regenerating

peripheral nerves (Siconolfi and Seeds 2001a). The proteo-

lytic activity of tPA is likely to be regulated by the action of

specific proteolytic inhibitors, including neuroserpin. In the

sciatic nerve crush model of nerve regeneration, investigators

found no significant changes in plasminogen activator

inhibitor 1 activity between crush and sham after the injury

(Siconolfi and Seeds 2001b). It will be interesting to see if

neuroserpin expression changes in this model.

The increased neurite outgrowth seen in NGF-treated

PC12 cell lines with reduced levels of neuroserpin expression

contrasts with an earlier study from our group which found

that overexpression of neuroserpin led to increased �neurite�outgrowth in AtT20 cells (Hill et al. 2000). That both PC12

and AtT20 cells respond to altered neuroserpin levels by

extending neurites suggests this serpin plays a key role in

regulating changes in cell adhesion required for axon

elongation. However, the different levels of neuroserpin

required to achieve neurite extension in these two cell lines

may indicate differences in the mechanism(s) regulating cell

adhesion. Neuroserpin may stimulate neurite outgrowth by

regulating the activity of tPA or other matrix-degrading

proteases as already discussed, or by modulating cell

adhesion by a mechanism independent of its function as a

protease inhibitor. There is accumulating evidence that the

serpin Type 1 plasminogen activator inhibitor (PAI-1) can

regulate adhesion of cells to the adhesive glycoprotein

vitronection through a mechanism likely to involve integrins,

urokinase-type plasminogen activator and the urokinase-

type-plasminogen activator receptor (Stefansson and Law-

rence 1996; Dear and Medcalf 1998; Stefansson et al. 1998).

It is generally accepted that the synthesis of molecules

postulated to play a role in processes such as axon outgrowth

and synaptogenesis is regulated by neuronal activity. Both

neuroserpin and tPA mRNA levels are increased following a

depolarization stimulus (Berger et al. 1999). Many proteins

known to mediate synaptic plasticity also show regulated

secretion. We have shown that neuroserpin is colocalized

with secretogranin-1 in dense cored secretory granules of the

regulated secretory pathway in PC12 cells, and that neuro-

serpin is found in the neurite tips of both PC12 cells and

AtT20 cells. This intracellular location was consistent with

our previous work that identified immunoreactive neuroser-

pin in purified secretory vesicles from the pituitary and

adrenal glands (Hill et al. 2000). tPA has also been found to

target to the regulated secretory pathway in PC12 cells and

shown to undergo calcium-dependent secretion (Gualandris

et al. 1996; Parmer et al. 1997). Therefore, both neuroserpin

and tPA undergo activity-dependent synthesis and secretion

consistent with their postulated functions in neuronal plas-

ticity. As both proteins were sorted to the regulated secretory

pathway complexation could occur in the acidic environment

found in these vesicles. Our data indicate that neuroserpin

and single chain tPA are unlikely to form a complex within

the secretory vesicle suggesting that complexation occurs

extracellularly. They also suggest that the intragranular ratio

of tPA to neuroserpin is likely to play an important role in

determining the activity of nerve cell-derived tPA at the local

environment of the synapse. In this regard it is interesting to

note the different time courses of transcriptional regulation of

the neuroserpin and tPA genes in mouse hippocampal cell

cultures treated with elevated extracellular KCl or KCl and

NGF (Berger et al. 1999) suggesting a mechanism for

temporal regulation of proteolytic activity at the synapse.

In summary, our results identify neuroserpin as playing a

key role in regulating neurite outgrowth in PC12 cells. It will

be important to determine whether a similar role can be

attributed to neuroserpin in vivo. Neuroserpin is expressed in

the developing mouse nervous system during neuronal

migration, axon outgrowth and synaptogenesis (Krueger

et al. 1997). As maximal levels of neuroserpin expression

were seen after axogenesis, it has been suggested that

neuroserpin is more likely to be involved in regulating serine

protease activity in later developmental processes such as

synaptogenesis or refinement of synaptic connectivity.

However, our results suggest that neuroserpin may play a



role in axogenesis, possibly involving lower levels

of neuroserpin than is required for later developmental

events.

Acknowledgements

We thank David Christie for helpful comments on the manuscript,

Ian Trowbridge and Weiland Huttner for generous gifts of antibodies

and Boehringer Ingelheim (New Zealand) for providing the

recombinant tPA and David Palmer for help with the PC12

subcellular fractionation experiments. This work was supported by

grants from The Health Research Council of New Zealand and the

University of Auckland to NPB.

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Neuroserpin regulates neurite outgrowth in nerve growth factor-treated PC12 cells