REVIEW Matrix metalloproteinase-3 in the central nervous ... · addition, MMP-3 was also discovered intranuclearly, more speci cally in the nucleus of hepatocytes (Si-Tayeb et al

REVIEW Matrix metalloproteinase-3 in the central nervous

system: a look on the bright side

Inge Van Hove, Kim Lemmens, Sarah Van de Velde, Mieke Verslegers

and Lieve Moons

Laboratory of Neural Circuit Development and Regeneration, Animal Physiology and Neurobiology

Section, Department of Biology, KU Leuven, Leuven, Belgium

Abstract

Matrix metalloproteinases (MMPs) are a large family of

proteases involved in many cell-matrix and cell-cell signalling

processes through activation, inactivation or release of extra-

cellular matrix (ECM) and non-ECM molecules, such as

growth factors and receptors. Uncontrolled MMP activities

underlie the pathophysiology of many disorders. Also matrix

metalloproteinase-3 (MMP-3) or stromelysin-1 contributes to

several pathologies, such as cancer, asthma and rheumatoid

arthritis, and has also been associated with neurodegenera-

tive diseases like Alzheimer’s disease, Parkinson’s disease

and multiple sclerosis. However, based on defined MMP

spatiotemporal expression patterns, the identification of novel

candidate molecular targets and in vitro and in vivo studies, a

beneficial role for MMPs in CNS physiology and recovery is

emerging. The main purpose of this review is to shed light on

the recently identified roles of MMP-3 in normal brain devel-

opment and in plasticity and regeneration after CNS injury and

disease. As such, MMP-3 is correlated with neuronal migration

and neurite outgrowth and guidance in the developing CNS

and contributes to synaptic plasticity and learning in the adult

CNS. Moreover, a strict spatiotemporal MMP-3 up-regulation

in the injured or diseased CNS might support remyelination

and neuroprotection, as well as genesis and migration of stem

cells in the damaged brain.

Keywords: central nervous system, development, matrix

metalloproteinase-3, plasticity, regeneration, repair.

J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07900.x

Matrix metalloproteinases: an overview

Matrix metalloproteinases (MMPs) are proteolytic enzymes

that remodel the pericellular environment by degrading all

protein constituents of the extracellular matrix (ECM).

Besides, they also regulate many cell signalling pathways

and homeostatic systems by cleavage and release of various

guidance and adhesion molecules, receptors, growth factors,

cytokines, etc., through either activation or inactivation. The

MMP family, a subgroup of the metzincins, constitutes more

than 20 mammalian members, which are all Zn2+-dependent

endopeptidases. Based on their substrate specificity and

domain organization, MMPs are classified into collagenases,

gelatinases, stromelysins, membrane-type MMPs, matrilysins

and ‘other MMPs’ (Nagase et al. 2006).

MMP activities are kept under tight control. First of all,

MMP expression can be regulated at the transcriptional level

by growth factors, cytokines, chemokines, hormones, epige-

netic processes and cell–cell/cell–ECM interactions. Mem-

brane-trafficking and subsequent release of MMPs at the cell

surface can be regulated by SNARE proteins (Kean et al.

2009). MMPs are synthesized as proenzymes with a

‘cysteine switch’, the disruption of the interaction between

the cysteine residue in the propeptide and the Zn2+ ion in the

catalytic site, as a pre-requisite for activation. This disruption

can be achieved by organomercurial compounds, heavy

metals, denaturating agents or oxidants, as well as through

removal of the propeptide by proteases (Van Wart and

Birkedal-Hansen 1990). Once activated, MMPs are subjected

Received May 16, 2012; revised manuscript received July 11, 2012;

accepted July 27, 2012.

Address correspondence and reprint requests to Dr. Lieve Moons,

Research Group Neural Circuit Development and Regeneration, Animal

Physiology and Neurobiology Section, Department of Biology, KU

Leuven, Naamsestraat 61, Box 2464, B-3000 Leuven, Belgium.

E-mail: [email protected]

Abbreviations used: AD, Alzheimer’s disease; aNPC, adult neural

stem/progenitor cell; BDNF, brain-derived neurotrophic factor; CSPG,

chondroitin sulphate proteoglycan; ECM, extracellular matrix; FasL, Fas

ligand; IGF, insulin-like growth factor; MMP, matrix metalloproteinase;

NG2, neuron/glia-type 2; NGF, nerve growth factor; PD, Parkinson’s

disease; PNN, perineuronal net; TIMP, tissue inhibitor of metallo-

proteinases.

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Journal of Neurochemistry © 2012 International Society for Neurochemistry, J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07900.x 1

© 2012 The Authors

• JOURNAL OF NEUROCHEMISTRY | 2012 doi: 10.1111/j.1471-4159.2012.07900.x

J N C 7 9 0 0 B Dispatch: 14.8.12 Journal: CE: Sangeetha

Journal Name Manuscript No. Author Received: No. of pages: 14 PE: Priya R

REVIEW

to inhibition by endogenous inhibitors: a2-macroglobulin,

RECK1 and tissue inhibitors of metalloproteinases (TIMPs),

the latter also being under tight transcriptional control (Oh

et al. 2001; Nagase et al. 2006). The four TIMPs identified

so far bind MMPs non-covalently, thereby blocking their

activities. As such, MMP activity is largely determined by

the MMP/TIMP balance. However, next to gene transcrip-

tion, zymogen activation and the MMP/TIMP ratio, MMP

activity can also be regulated at the less characterized levels

of mRNA stability, translational control, for example,

acetylation, phosphorylation or S-nitrosylation, (auto)prote-

olysis and receptor-mediated endocytosis (Chakraborti et al.

2003; Nagase et al. 2006; Clark et al. 2008).

In normal resting adult tissues, in which protease activities

are well controlled, MMP levels are quite low. However,

when tissue remodelling or cell signalling is required in

developing and adult organisms, these proteinases are up-

regulated. Consequently, MMPs play an important role in

many physiological processes, for example, wound healing,

ovulation, blastocyst implantation, bone growth and angio-

genesis. Inflammatory stimuli increase MMP levels even

more and uncontrolled MMP activity is known to underlie a

variety of diseases for example, tumor invasion and metas-

tasis, rheumatoid arthritis, periodontal disease and athero-

sclerosis. MMP-over-expression also strengthens blood–

brain barrier dysfunction, demyelination, neuroinflammation

and neurotoxicity and as such underlies a range of neuro-

logical diseases, for example, multiple sclerosis, amyotrophic

lateral sclerosis, Alzheimer’s (AD) and Parkinson’s (PD)

disease (Yong 2005; Rosenberg 2009; Klein and Bischoff

2011). Therefore, MMP inhibition has been put forward as a

possible therapeutic strategy for treatment of multiple CNS

disorders.

Besides a proven detrimental role in neurological disor-

ders, evidence concerning a beneficial contribution of MMPs

in key physiological and regenerative events in CNS

disorders and injuries is emerging. Furthermore, as neuroin-

flammation does not only have detrimental consequences,

MMP up-regulation is believed to underlie reparative

functions in the CNS at well-defined places and time points

after an insult (Yong 2010). The currently ongoing identi-

fication of MMP targets will undoubtedly provide indications

for plausible mechanisms via which MMPs exert these

beneficial functions. Among the recently discovered MMP

substrates, many ECM proteins, adhesion molecules, chemo-

kines, receptors and growth factors are known to contribute

to neuronal processes such as migration, survival, synapto-

genesis and plasticity, suggesting a critical role for MMPs in

CNS development and regeneration. The use of (non-

selective) MMP inhibitors as drug therapy might conse-

quently impede neuroprotective or reparative functions of

MMPs.

Therefore, an in-depth knowledge of MMP biology in time

and space and the identification of their individual functions

and working mechanisms in both physiological and patho-

physiological conditions is required.

The stromelysin MMP-3

MMP-3 (EC 3.4.24.17) or stromelysin-1, which belongs

together with MMP-10 and MMP-11 to the stromelysin-

subgroup, has a rather simple MMP structure with a

hemopexin domain attached to the catalytic site by a hinge

region. Different molecules, like cytokines, reactive oxygen

species, growth factors and cell–cell/cell–ECM interactions

can trigger MMP-3 gene transcription, whereas MMP-3

mRNA transcripts are stabilized by phorbol esters and

epidermal growth factor (Delany and Brinckerhoff 1992;

Kim and Hwang 2011). Once released into the ECM, the

inactive MMP-3 pro-enzyme can be activated extracellularly

by the plasmin cascade signalling pathway (Nagase et al.

1990). Extracellularly, MMP-3 has a rather broad substrate

specificity. Besides degrading multiple ECM proteins, it can

also activate growth factors, cleave cell adhesion molecules,

chemokines, cytokines and various receptors. In addition,

MMP-3 is able to activate pro-MMP-1, -3, -7, -8, -9 and -13

and to hydrolyze some of its upstream activators, such as

plasminogen and urokinase-type plasminogen activator (Og-

ata et al. 1992; Arza et al. 2000; McCawley and Matrisian

2001). Besides this extracellular activity, MMP-3 is known to

act intracellularly. As such, MMP-3 is present in dopaminer-

gic neurons, where it can be intracellularly activated by the

serine protease HtrA2/Omi (Choi et al. 2008; Shin et al.

2012). Although MMP-3 contains a furin recognition site

(Cao et al. 2005) like MMP-11 and MT1-MMP (Pei and

Weiss 1996), cleavage by this serine protease does not result

in the catalytically active MMP-3 form (Choi et al. 2008).

Importantly, secreted MMP-3 can be extracellulary activated

and subsequently transported back into the cell, probably via

clathrin-dependent endocytosis mechanisms (Traub 2009). In

addition, MMP-3 was also discovered intranuclearly, more

specifically in the nucleus of hepatocytes (Si-Tayeb et al.

2006) and chondrocytes (Eguchi et al. 2008). The association

of MMP-3 with a RAN 2-binding protein, which is involved in

nuclear import, together with nuclear translocation signals in

the MMP-3 catalytic site, creates possibilities for efficient

transport into the nucleus (Eguchi et al. 2008; Cauwe and

Opdenakker 2010). These recent findings and the intracellular

and nuclear substrates discovered until now, suggest new

functions for MMP-3 in apoptotic processes, transcriptional

regulation, protein synthesis and cytoskeletal remodelling

(Cauwe and Opdenakker 2010). Both intra- and extracellular

activities of MMP-3 can be regulated by TIMPs, among which

the TIMP-1/MMP-3 ratio is the best characterized (Kim et al.

2010). An altered balance between TIMPs and MMP-3 is

known to impair wound healing (Bullard et al. 1999) and to

contribute to the development of , for example, arthritis

(Green et al. 2003) and cancer metastasis (Li et al. 1994).

Journal of Neurochemistry © 2012 International Society for Neurochemistry, J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07900.x

© 2012 The Authors

2 I. Van Hove et al.

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INGE

Note

(reversion-inducing cysteine-rich protein with kazal motifs)

A detrimental role for MMP-3 in the CNS

MMP-3 up-regulation in CNS pathologies implicates its

contribution to several neurodegenerative disorders. Indeed,

uncontrolled MMP-3 activity seems to aggravate many

neurodegenerative disorders by disrupting the blood–brain

barrier, by promoting demyelination and apoptosis or by

evoking additional inflammatory responses. In the patholog-

ical brain, MMP-3 is expressed by injured neurons, oligo-

dendrocytes, astrocytes, pericytes and reactive microglia/

macrophages. After ischemia, trauma or infections in the

CNS, inflammatory mediators, such as tumor necrosis factor-

a and interleukin-1b, induce the transcription factors activa-

tor protein-1 and nuclear factor kB, which subsequently bind

their analogous sites in the MMP-3 promoter region of

activated microglia, thereby inducing pro-MMP-3 transcrip-

tion. During the injury cascade, other proteases, such as

plasmin, are produced and can generate active MMP-3 in the

matrix (Rosenberg et al. 2001; Conant and Gottschall 2005;

Gurney et al. 2006; Rosenberg 2009).

Up-regulated MMP-3 levels and activity support neuroin-

flammation as well as apoptosis, two processes which both

importantly contribute to neurodegeneration. MMP-3 levels

are often increased in neuronal cells after various forms of

cellular stress, like endoplasmic reticulum stress, and may

ultimately cause apoptosis (Kim et al. 2010). Endoplasmic

reticulum stress is involved in the pathogenesis of various

neurodegenerative diseases such as PD and AD (Lindholm

et al. 2006). MMP-3 up-regulation in dying substantia nigra

cells in experimental models of PD (Sung et al. 2005; Kim

et al. 2007; Choi et al. 2008) and a decreased cell loss of

these nigral neurons after MMP-3 inhibition and in MMP-

3-deficient mice (Kim et al. 2007; Choi et al. 2008) has been

demonstrated. MMP-3 seems to contribute to the pathophys-

iology of PD by degrading the neuroprotective protein DJ-1,

thereby impeding its antioxidant function, and by intracell-

ulary cleaving the protein a-synuclein, colocalized with

MMP-3 in Lewy bodies in stressed dopaminergic cells,

thereby leading to the formation of toxic a-synuclein

aggregates (Choi et al. 2011a,b). Although the functional

relevance for MMP-3 in the pathogenesis of AD still needs to

be unraveled, MMP-3 expression has been observed in senile

plaques, as well as in plasma and CSF of postmortem brains

from AD patients. In addition, altered MMP-3 levels are

correlated with altered b-amyloid levels (Deb and Gottschall

1996; Yoshiyama et al. 2000).

MMP-3 has also been linked to multiple sclerosis and

demyelination. MMP-3 is able to degrade myelin basic

protein and is up-regulated in the brain, prior to the onset of

disease in a spontaneous demyelinating mouse model

(Chandler et al. 1995; D’Souza et al. 2002). Furthermore,

MMP-3 expression is observed in and around lesions and in

the blood of multiple sclerosis patients (Maeda and Sobel

1996; Kouwenhoven et al. 2001). An elaborate review

describing the involvement of MMP-3 in neurodegeneration

was recently published by Kim and Hwang (2011).

A beneficial role for MMP-3 in the developingand adult CNS

However, and despite the fact thatMMP-3-deficient mice have

no apparent developmental defects (Mudgett et al. 1998),

more in depth investigations are becoming to reveal crucial

physiological functions of MMP-3 within the developing and

recovering CNS. Importantly, MMP-3 cleaves a very wide

range of CNS substrates, which associate MMP-3 with

pathological processes, but also with diverse physiological

functions in the developing and normal adult brain, as well as

during recovery/regeneration after CNS insults (Table 1).

Moreover, we previously reported mild deficits in balance and

motor performance in developing and adult MMP-3-deficient

mice (Van Hove et al. 2012). Additional behavioral studies in

adult MMP-3-deficient animals also revealed gait abnormal-

ities and showed significantly higher variabilities in stride

length and paw angle and an increase in hind base distance in

comparison to wild-type littermates, amplifying the contribu-

tion of MMP-3 in the normal functioning of the CNS (Fig. 1).

With this review, we intend to stress that MMP-3 actions

in the brain are extending beyond their intensively studied

detrimental involvement in various neuropathologies. There-

fore, we summarize the current knowledge of MMP-3

functions in CNS physiology and recovery/regeneration in

the following paragraphs.

MMP-3 in the developing and healthy adult CNS

Expression pattern of MMP-3 in the embryonic CNS

A number of in vitro and in vivo studies showed MMP-3

expression in the embryonic rodent brain and spinal cord.

More specifically, immunostainings on embryonic day (E) 14

rat spinal cord neuronal cultures revealed MMP-3 expression

in neuronal dendrites, in somata and remarkably also in

nuclei after 20 days in culture (Pauly et al. 2008). In the rat

embryo at E15, the time point at which axonal outgrowth is

intensively ongoing in the central and peripheral nervous

system, widespread MMP-3 immunoreactivity was detected

in most brain areas and spinal cord commissural axons, but

also in peripheral cell bodies and axons, for example, in

motor neuron axons, in distal parts of peripheral nerves and

their surrounding mesenchymal cells and in dorsal root

ganglia containing sensory neuron cell bodies (Nordstrom

et al. 1995). Also, studies in E15 mouse brain revealed

intense MMP-3 protein staining, especially in neurons of the

ventricular zone and cortical plate and in growing axons of

the intermediate zone. In situ zymography confirmed MMP-3

activity in these neocortical areas (Gonthier et al. 2007).

Using western blotting, RT-PCR and immunohistochemistry

on E15 rat cortical cultures, MMP-3 expression was localized

© 2012 The Authors


Beneficial role of MMP-3 in the CNS 3

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in primary cortical neurons, but not in glia (Wetzel et al.

2003). However, a detailed study on E18 whole rat brain

isolated cell cultures identified MMP-3 protein in neurons

and mature oligodendrocytes, but not in oligodendrocyte

precursors, astrocytes and microglia (Sole et al. 2004). In the

late gestational fetal rabbit brain (E21 and E26), MMP-3

mRNA was detected in forebrain germinal matrix cells, the

source of cortical neurons and glial cells, in most neurons of

the diencephalon and in large neurons of the brainstem (Del

Bigio and Jacque 1995).

Overall, these data indicate that MMP-3 expression is

largely confined to neurons in the embryonic CNS and is

only associated with mature oligodendrocytes in the late

prenatal period.

Expression pattern of MMP-3 in the postnatal CNS

A widespread distribution of MMP-3 mRNA was detected at

various postnatal stages in the rabbit brain (Del Bigio and

Jacque 1995). Diencephalic neurons expressed MMP-3 until

postnatal day (P)2 and most cells of the cerebral cortex,

hippocampus and striatum showed MMP-3 mRNA expres-

sion until P10. From P10 on, MMP-3 was found in mature

olfactory bulb neurons, in neurons of the facial nucleus,

dorsal raphe, gigantocellular region and inferior olive. The

cerebellar external and internal granular layer expressed

MMP-3 until P20 (Del Bigio and Jacque 1995). Using whole

mouse brain extracts, only low and rather temporally

unchanged MMP-3 mRNA levels were found at postnatal

weeks 1, 3, 5 and 7 (Ulrich et al. 2006). However, real-time

PCR analysis in postnatal cerebellar extracts revealed a

restricted MMP-3 mRNA pattern with elevated expression

levels during the second postnatal week (Van Hove et al.

2012). Using a commercially available and widely used

polyclonal antibody for MMP-3 (Santa Cruz 3), we showed

MMP-3 protein expression in the developing mouse cere-

bellar cortex. Our immunostaining revealed a change in time

from a rather diffuse distribution of MMP-3 in the ECM

towards a more restricted staining in cell bodies, with most

prominent MMP-3 immunoreactivity in Purkinje cell somata

and dendrites and to a lesser extent in GABAergic interneu-

rons and in some granule cells of the external and internal

granular layer (Van Hove et al. 2012). This pattern resem-

bles the previously observed MMP-3 immunopattern,

obtained after using an antibody from Chemicon, in the

developing rat cerebellar cortex, except for an extra diffuse

MMP-3 immunopositivity around Bergmann glial fibers in

this species (Vaillant et al. 1999). Remarkably, when

performing MMP-3 immunostainings on postnatal mouse

cerebellar sections using a rather novel commercially avail-

able antibody (Epitomics 4), MMP-3 immunoreactivity could

only be detected in glial cells (see Fig. 1). Western blotting

on supernatant of isolated primary mouse cerebellar glial

cells, harvested from P8 pups, showed with both antibodies

Table 1 Candidate MMP-3 targets in the CNS. This table provides a

catogerized list of potential MMP-3 substrates identified in the CNS,

with corresponding references listed below

MMP-3 substrate Reference

Matrix

proteins

Agrin Sole et al. (2004)

Aggrecan Durigova et al. (2011)

Brevican/neurocan/

NG-2 phosphacan/

versican

Muir et al. (2002)

Collagens Okada et al. (1986)

Decorin Imai et al. (1997)

Elastin Galloway et al. (1983)

Entactin Alexander et al. (1996)

Laminin/fibronectin Okada et al. (1986)

Osteopontin Agnihotri et al. (2001)

Perlecan Whitelock et al. (1996)

SPARC Sage et al. (2003)

Tenascin Imai et al. (1994)

Vitronectin Imai et al. (1995)

Growth

factors

Pro-BDNF, pro-NGF Lee et al. (2001)

Pro-HB-EGF Suzuki et al. (1997)

VEGF Lee et al. (2005)

Proteases Plasminogen Lijnen et al. (1998)

Pro-MMP-1, -3, -7,

-8, -9, -13

Reviewed in McCawley

and Matrisian (2001)

uPA Ugwu et al. (1998)

Adhesion

molecules

E-Cadherin Fuchs et al. (2005)

Receptors NMDA-receptor Pauly et al. (2008)

Cytokines/

chemokines

MCP-1, -2, -3 McQuibban et al. (2002)11

Pro-IL-1b Schonbeck et al. (1998)12

Pro-TNF-a Gearing et al. (1995)

SDF-1a McQuibban et al. (2001)

Others a2-Antiplasmin Lijnen et al. (2001)

a1-Antichymotrypsin Mast et al. (1991)

Fas ligand Matsuno et al. (2001)

Fibrinogen/fibrin Bini et al. (1996)

IGFBP-1, -3 Fowlkes et al. (2004)

myelin basic protein D’Souza and

Moscarello (2006)

PAI-1 Lijnen et al. (2000)

Serum amyloid A Stix et al. (2001)

Substance P Stack et al. (1991)

t-Kininogen Sakamoto et al. (1996)

(BDNF, brain-derived neurotrophic factor; HB-EGF, heparin-binding

epidermal growth factor; IGFBP-3, insulin like growth factor binding

protein 3; IL-1b, interleukin-1b; MCP, monocyte chemoattractant

protein; MMP-3, matrix metalloproteinase-3; NG2, neuron/glia-type 2;

NGF, nerve growth factor; PAI-1, plasminogen-activator inhibitor 1;

SDF-1a, stromal cell-derived factor 1a; SPARC, secreted protein acidic

and rich in cystein; TNF-a, tumor necrosis factor a; uPA, urokinase-

type plasminogen activator; VEGF, vascular endothelial growth factor).


© 2012 The Authors


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(Santa Cruz and Epitomics), the presence of pro-MMP-3

(55 kDa). Both antibodies also revealed pro-MMP-3

(55 kDa) and active MMP-3 (47 kDa) in extracts of

postnatal mouse cerebellum. However, the Santa Cruz

antibody additionally labelled a profound band around

70 kDa in the cerebellar samples and a similar but weak

band in glial cell supernatant (see Fig. 1). These findings

might explain the discrepancy in MMP-3 immunopattern

(a) (b) (c)

(d) (e) (f) (g)

(h) (i)

Fig. 1 Gait dynamics in adult MMP-3-/- mice and MMP-3 protein

expression patterns in the postnatal mouse cerebellar cortex Using the

DigiGaitTM Imaging System (Mouse specifics, Inc.), mice were imaged

ventrally with a digital video camera while running on a motorized

transparant treadmill (belt speed = 22 cm/s). Digital paw prints were

generated and many gait parameters were determined using the

DigiGaitTM software as previously described (Hampton et al. 2004;

Vincelette et al. 2007). (a) A similar front base width, but a significantly

enlarged hind base width is observed in 8 to 10 week old MMP-3

deficient mice (�/�), as compared with wild-type animals (+/+)

(n � 7). (b, c) The coefficient of variation (CV) of stride length (b),

calculated from the equation: 100 9 standard deviation/mean value,

and the variability in paw placement angle (c) were found to be

persistently higher in MMP-3�/� animals, indicating abnormalities in

gait pattern as compared with wild-type mice (n � 7). (d) A fluores-

cent immunohistochemical double labeling for GFAP (green) and

MMP-3 (red), performed on cerebella of P8 pups using an antibody

against MMP-3 from Epitomics (1908-1, 1 : 200), clearly shows MMP-

3 colocalisation in Bergmann glial fibers (arrowhead) and other

astrocytic processes. (e) Localization of MMP-3 (Epitomics antibody)

using a diaminobenzidine (DAB) chromogenic staining confirms these

findings. (f) A fluorescent immunostaining for GFAP and MMP-3, using

a Santa Cruz antibody (sc-6839, 1 : 200) against MMP-3, shows no

colocalization with glial cells, but clear MMP-3 expression in the

Purkinje cell layer (PCL), in interneurons of the molecular layer (ML)

and the internal granular layer (IGL). (g) A DAB staining for MMP-3

(Santa Cruz antibody) confirms its expression in neuronal cells. (h, i)

Western blotting for MMP-3 was performed on equal amounts of

proteins from P8 cerebellar extracts and glial cell supernatant using

both antibodies and a 55-kDa rhMMP-3 as standard (R&D; WBC015).

Both antibodies [Epitomics antibody used at 1 : 200 (H); Santa Cruz

antibody used at 1 : 250 (i)] revealed the presence of pro- and active

MMP-3 in P8 cerebellar samples. Both antibodies also revealed pro-

MMP-3 expression in glial cell supernatant. (i) However, the Santa

Cruz antibody also labelled a strong band around 70 kDa in P8

cerebellar samples and a similar but weaker band in the glial cell

supernatant. Immunohistochemistry and western blotting were per-

formed as previously described (Van Hove et al. 2012). Scale bar:

100 lm. Data are represented as mean ± SEM (*p < 0.05,

**p < 0.01, Student’s t-test) (CV, coefficient of variation; var, variabil-

ity; LF, left front; RF, right front; LH, left hind; RH, right hind;

R, rhMMP-3; C, P8 cerebellar extracts; GL, glial cell supernatant).

COLOR

© 2012 The Authors



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obtained when using both antibodies on mouse postnatal

cerebella. However, the western blots clearly reveal MMP-3

expression in glial cells within the postnatal CNS. Of note,

additional studies reported glial MMP-3 expression in the

postnatal brain. MMP-3 mRNA was detected in subpial and

perivascular astroglia as well as in white matter glia, mostly

oligodendrocytes, prior to and during myelination of the

postnatal rabbit brain (Del Bigio and Jacque 1995). Further-

more, western blotting on cultured primary astrocytes and

Schwann cells, isolated respectively from neonatal rat

neocortex and sciatic nerves, also revealed MMP-3 expres-

sion by these cells (Muir et al. 2002). Overall, these findings

indicate the presence of MMP-3 in neurons as well as glial

cells in the postnatal CNS.

Expression pattern of MMP-3 in the adult CNS

In the adult healthy CNS, very low to undetectable MMP-3

(m)RNA and protein levels are observed (Pagenstecher et al.

1998; Rosenberg et al. 2001; Ulrich et al. 2005; Li et al.

2009). In the adult rabbit brain, MMP-3 mRNA expression is

confined to the dorsal raphe (Del Bigio and Jacque 1995).

Western blotting revealed pro-MMP-3 levels in adult rat and

mouse brain or isolated hippocampus, but active MMP-3

levels were low to undetectable (Meighan et al. 2006;

Wright et al. 2006; our findings). Immunohistochemistry in

the adult rat cerebellum showed MMP-3 protein expression

in interneurons of the molecular layer, Purkinje cell somata

and granule cells in the internal granular layer (Vaillant et al.

1999). MMP-3 immunoreactivity was also detected in the

adult rat hypothalamo-neurohypophysial system, more spe-

cifically in a punctate pattern at the dendrites and terminals of

arginine-vasopressin expressing magnocellular neurons

(Miyata et al. 2005). In the healthy elderly human brain

(age range from 71 to 93 years), diverse studies showed

perineuronal, neuronal and neuropil MMP-3 labelling in the

frontal cortex, but MMP-3 was not found in the parietal

cortex or hippocampus (Yoshiyama et al. 2000; Baig et al.

2008). MMP-3 expression was also detected in microvessels

and perivascular microglia in the healthy human brain

(Maeda and Sobel 1996). Of note, these MMP-3 immuno-

stainings do not distinguish between pro- and mature MMP-3

forms. However, active MMP-3 could be detected in CSF of

healthy subjects (Candelario-Jalil et al. 2011). Thus, the

reported findings indicate that MMP-3 expression is largely

confined to neurons and perivascular microglia in the healthy

CNS.

In general, the variations/discrepancies reported in MMP-3

expression patterns observed in the embryonic, postnatal or

adult CNS, might result from the use of various antibodies

targeting different epitopes and/or might be caused by

distinct MMP-3 post-translational modifications and interac-

tions, together with a time-, place- and species-specific

localization. Future localization and activity studies, using

diverse antibodies, MMP-3-eGFP5 reporter animals or

improved activity assays, should provide detailed MMP-3

spatiotemporal expression and activity patterns, thereby

enabling its functional characterization in various regions

of the CNS.

MMP-3 in neuronal patterning and axon outgrowth

during embryonic and postnatal CNS development

The previously described spatiotemporal expression patterns

suggest a role for MMP-3 during CNS development.

Furthermore, an involvement of MMP-3 in brain develop-

ment can also be deduced from the large list of MMP-3

targets present in the developing brain and known to be

associated with one or more brain developmental processes,

such as neurite outgrowth, neuronal migration, survival,

synaptogenesis and/or myelinogenesis (Tables 1 and 2) (also

see Fig. 2).

One study demonstrated a correlation between MMP-3

mRNA expression and the time course of neurite extension in

PC12 cells treated with nerve growth factor (NGF) (Machida

et al. 1989). Additional in vitro experiments revealed MMP-

3 immunoreactivity in the growth cones of NGF treated

PC12 cells and a reduced ability of neurites from PC12 cells,

expressing antisense MMP-3 mRNA, to penetrate a basal

lamina-like matrix (Nordstrom et al. 1995). Further in vitro

experiments on cultured E15 mouse cortical neurons showed

increased extracellular MMP-3 protein and activity levels

after Sema3C treatment and a deviating axonal orientation

towards the chemoattractant Sema3C in a growth cone

turning assay in the presence of a specific MMP-3 inhibitor

(Gonthier et al. 2007). Together, these data clearly suggest

an involvement of MMP-3 in axonal extension, growth cone

invasiveness and axon guidance.

Recently, MMP-3 was shown to be functionally involved

in the postnatal development of the mouse cerebellar cortex.

Detailed phenotyping of cerebellar development in MMP-3

deficient mice revealed a protracted tangential granule cell

migration and a delayed granule cell radial migration,

together with a disturbed Purkinje cell dendritogenesis.

These deficits were also associated with a subsequent

retarded GABA-ergic interneuron migration. Importantly, a

reduced size of the Purkinje cell dendritic trees was shown in

cerebella of adult MMP-3�/� mice and might, in part,

underlie the aberrant motor performance observed in these

animals (Van Hove et al. 2012). MMP-3 might possibly

exert its effects on postnatal cerebellar development via

activation of MMP-9 (Ogata et al. 1992), known or

suggested to be important for granule cell migration,

apoptosis and axonal outgrowth in the cerebellum (Vaillant

et al. 2003). However, although the mRNA and protein

expression profiles of MMP-3 and MMP-9 in the developing

cerebellum are quite comparable, MMP-3 deficiency does

not phenocopy the findings observed in MMP-9 deficient

cerebellar cortices (Vaillant et al. 1999, 2003), supporting

the idea that additional molecules and pathways contribute.


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Besides its effect on neurite outgrowth and neuronal

migration, MMP-3 deficiency also resulted in a delayed or

abrogated synapse formation between granule cells and

Purkinje cells in the mouse postnatal cerebellar cortex (Van

Hove et al. 2012). Remarkably, during cerebellar develop-

ment, MMP-3 mRNA and protein expression levels are high

when synaptogenesis between granule cells and Purkinje cell

dendrites peaks (Vaillant et al. 1999; Van Hove et al. 2012).

MMP-3 might influence the formation and function of

synapses in the CNS by cleaving molecules which are

directly or indirectly involved in synaptic functioning (Ethell

and Ethell 2007), for example, laminin (Okada et al. 1986),

tenascin (Imai et al. 1994) and pro-brain-derived neuro-

trophic factor (pro-BDNF) (Lee et al. 2001). Although a

functional implication of MMP-3 in developmental synapto-

genesis remains largely elusive, this hypothesis can be

strengthened by several studies demonstrating a supporting

role of MMP-3 in synapse remodelling after CNS injuries

(see below).

MMP-3 has also been suggested to contribute to postnatal

myelinogenesis (Del Bigio and Jacque 1995; Muir et al.

2002). This process requires matrix remodelling and ECM

modifications for proper adhesion as well as an increased

availability of growth factors for oligodendrocyte maturation

and survival, processes which can be achieved by MMPs. It

has been shown that MMP-9 is expressed on growing tips of

oligodendrocytes in vitro and during initiation of myelin

formation in the mouse optic nerve, thereby, MMP-9 is

known to facilitate process outgrowth and oligodendrocyte

maturation (Oh et al. 1999; Larsen et al. 2006). Moreover,

MMP-9, which can be activated by MMP-3, might regulate

developmental myelination by affecting insulin-like growth

factor (IGF) bioavailability (Larsen et al. 2006). MMP-3

expression has also been observed in oligodendrocytes and

Schwann cells (Del Bigio and Jacque 1995; Muir et al. 2002)

and is known to promote the release of IGF by degrading

IGF/IGF-binding protein complexes (Fowlkes et al. 1994,

2004). As a role for MMP-3 in developmental myelination

was only suggested based on expression patterns and

myelination-associated MMP-3 targets, there is an obvious

need for more in-depth research on the involvement of

MMP-3 in myelin formation. Although all the above findings

Fig. 2 Beneficial roles of MMP-3 in the developing and adult CNS. This

scheme depicts the various cell types expressing MMP-3 and its

contribution to important processes in the developing and adult CNS.

Previous expression studies in the developing and healthy adult CNS

and during recovery/regeneration in the adult CNS show MMP-3

expression in neurons, oligodendrocytes, astrocytes and perivascular

microglia. In the developing CNS, MMP-3 contributes to neurite

outgrowth, axon invasiveness, axon guidance and neuronal migration

(shown in green). In the healthy adult CNS, MMP-3 is known to support

synaptic plasticity, learning and memory (shown in red). Up-regulated

MMP-3 levels might underlie recovery/regeneration after adult CNS

injury by promoting remyelination, neuroprotection and neural stem cells

genesis and migration and might contribute to axonal regeneration and

synaptic plasticity in the damaged brain (shown in blue).

COLOR

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already indicate the importance of MMP-3 in CNS develop-

ment, further functional studies are required, complemented

with the search for MMP-3 target molecules and mechanisms

underlying the observations during CNS development.

MMP-3 in plasticity, learning and memory in

the adult healthy brain

A role for MMPs in synapse formation, maturation, plasticity

and long-term potentiation has been demonstrated or

suggested, in part based on the identification of MMP

substrates, known to support these processes (Nagy et al.

2006; Ethell and Ethell 2007; Wang et al. 2008; Bajor et al.

2012) (Table 1).

In the adult mammalian CNS, most neuronal somata and

their dendrites are enwrapped by perineuronal nets (PNNs).

These PNNs consist of several ECM molecules, including

the chondroitin sulphate proteoglycans (CSPGs) aggrecan,

neurocan, brevican, versican, phosphacan, and their binding

partners, tenascin-R, hyaluronan and link proteins. Cleavage

of these PNNs is necessary to allow synaptic plasticity in the

adult CNS (Carulli et al. 2006) and an involvement of

MMPs in the turnover of these PNN molecules, thereby

stimulating plasticity, is not unlikely (Howell and Gottschall

2012). It was already shown that MMP-3 can cleave all of

these CSPGs (Muir et al. 2002; Durigova et al. 2011) and

the observed perineuronal MMP-3 protein localization

indicates an association of MMP-3 with PNN remodelling

(Baig et al. 2008). In addition, murine slices of ventral

hippocampus showed an increase in amplitude of the field

excitatory post-synaptic potential after exposure to MMP-3.

Together with a prominent disruption of PNNs in slices

exposed to a specific MMP-3 inhibitor, these data suggest a

possible interaction between MMP-3 and PNN integrity in

the hippocampus (Gordon et al. 2009)6 .

In addition, studies performed in the PNS showed active

MMP-3 at the neuromuscular junction and reduced MMP-3

activity following denervation and after blocking nerve

activity at the neuromuscular junction. Moreover, MMP-3

has been associated with synaptic sprouting in the PNS and

MMP-3 is able to cleave synaptic basal lamina molecules,

including agrin, a transmembrane protein involved in

synapse differentiation and synaptic transmission (VanSaun

et al. 2003, 2007; Werle and VanSaun 2003; Sole et al.

2004). Although MMP-3 is also able to cleave the brain agrin

isoform (Sole et al. 2004), a functional correlation between

MMP-3 and agrin in brain plasticity has not been determined

yet.

Most studies investigating a possible involvement of

MMP-3 in CNS synaptic plasticity are performed in the

hippocampus. A transient increase in hippocampal MMP-3

mRNA and protein levels has been observed during the

active phase of spatial learning in a Morris water maze test

(Meighan et al. 2006). These changes in MMP-3 levels,

possibly as a consequence of NMDA receptor activation,

might consequently contribute to activity-dependent changes

in synapse morphology and physiology. Importantly,

NMDA-induced shedding of the synaptic adhesion molecule

ICAM-5 7is associated with dendritic spine maturation and

might be regulated, at least in part, by MMP-3 (Tian et al.

2007; Conant et al. 2010). Next to disrupting ECM mole-

cules, plausible actions of MMP-3 in changing synaptic

morphology and efficacy during learning, include (in)

activation and/or release of non-ECM molecules, interruption

of ECM-cell adhesion interactions and modification of the

cytoskeleton (Meighan et al. 2006). As shown in Table 2,

MMP-3 has indeed intracellular substrates known to be

involved in cytoskeletal organization. An influence of MMP-

3 on synaptic plasticity and memory consolidation was

further elucidated during passive avoidance conditioning, a

hippocampus-dependent associative memory task, where

MMP-3 levels transiently augmented (Olson et al. 2008).

In addition, learning deficits were observed after administra-

tion of a MMP-3 inhibitor prior to passive avoidance

training, suggesting a causal relationship between learning-

induced elevated MMP-3 expression in the hippocampus and

associative memory formation. MMP-3 levels were also

elevated in the hippocampus, prefrontal and piriform cortex

after habituation of the head-shake response. This increase in

MMP-3 expression, accompanied by elevated MMP-9

activity, indicates that both MMPs could mediate changes

in neural plasticity following habituation (Wright et al.

2006). Also, the observed punctate MMP-3 immunopattern

in magnocellular neuronal dendrites and terminals of the

hypothalamo-neurohypophysial system, which is probably

corresponding to neurosecretory granules, suggests an

involvement of MMP-3 in inducing structural plasticity

(Miyata et al. 2005). Importantly, synaptic plasticity implies

structural and functional alterations at the post-synaptic

membrane (Wheal et al. 1998) and MMP-3 can contribute to

the necessary structural alterations of clustered post-synaptic

NMDA receptors through activity-dependent shedding of the

NMDA receptor NR1 subunit, as shown in cultured spinal

Table 2 Cellular and nuclear MMP-3 substrates. This table lists the

intracellular and intranuclear MMP-3 substrates known to date, with

corresponding references

MMP-3 substrate Reference

Actin-related protein (Arp)

2/3 complex subunits

Cauwe et al. (2009)

a-Synuclein Sung et al. (2005)

DJ-1 Choi et al. (2011a)

Gelsolin Park et al. (2006)

Histidyl-tRNA synthetase Cauwe et al. (2009)

Nucleolin Cauwe et al. (2009)

TRENDIC Eguchi et al. (2008)

Tubulin a/b Cauwe et al. (2009)


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cord neurons (Pauly et al. 2008). Clearly, multiple reports

have indicated MMP-3 as a regulator of synaptic plasticity

and learning in the CNS and future studies will undoubtedly

reveal additional involvements in various brain regions in

defined conditions.

MMP-3 in the adult pathological CNS

MMP-3 in neuroprotection

Through proteolytic shedding of receptors, ligands and

signalling molecules at the cell surface, MMP activity might

mediate cell survival/cell death signaling pathways in various

cell types. One route initiating the caspase activation cascade

starts after binding of the membrane protein Fas ligand

(FasL) to the cell surface death receptor Fas, which are both

up-regulated following injury or cellular stress in the CNS

(Felderhoff-Mueser et al. 2000; Qiu et al. 2002). In vitro

experiments with human L5178Y FasL transfected cells

showed the ability of MMP-3 to cleave FasL from the cell

surface into its soluble form (Matsuno et al. 2001). Further-

more, in cultured cortical neurons, treated with the chemo-

therapeutic drug Doxorubicin or subjected to oxygen-glucose

deprivation, both known to evoke Fas-mediated apoptosis,

TIMP-3 was required for apoptotic death while addition of

exogenous active MMP-3 promoted neuroprotection by

reducing Fas–FasL interactions (Wetzel et al. 2008). As

the TIMP-3/MMP-3 balance seems to facilitate neuronal

apoptosis through net MMP-3 inhibition, FasL ectodomain

shedding by addition of MMP-3 or removal of TIMP-3 might

therefore support neuroprotection (Wetzel et al. 2003, 2008).

Alternatively, MMP-3 might also modulate neuroprotec-

tion via increasing the bioavailability of IGF-1 through

degradation of IGF/IGF-binding protein (Fowlkes et al.

1994, 2004) and by releasing and cleaving pro-NGF and

pro-BDNF (Lee et al. 2001; Cunningham et al. 2005)8 . Of

note, mature NGF and BDNF promote cell survival by

mainly activating Trk receptors while their proform rather

mediates cell apoptosis via activation of the p75NTR -receptor

(Lee et al. 2001; Teng et al. 2005). Overall, although there is

evidence that MMP-3 up-regulation might support neuro-

protection, still a lot remains to be discovered about its

involvement in this process.

MMP-3 in post-injury axonal growth

Although a role of MMP-3 in axonal regeneration remains

largely elusive, some reports do already provide a reasonable

indication for its involvement in such processes, albeit in the

PNS. After sciatic nerve crush, MMP-3 is up-regulated by

regenerating peripheral nerve fibers (Demestre et al. 2004;

Shubayev and Myers 2004). As developmental studies

showed MMP-3 expression in growing axons and axonal

growth cones as well as an involvement of MMP-3 in neurite

outgrowth and guidance (Nordstrom et al. 1995; Gonthier

et al. 2007), a possible role of MMP-3 in adult CNS axonal

outgrowth can be hypothetized. Furthermore, as MMP-3 is

able to digest all known axon-inhibitory CSPGs in the glial

scar, e.g. phosphacan, neurocan, versican, neuron/glia-type 2

(NG2) and brevican, up-regulated MMP-3 levels might

create a permissive environment promoting neurite out-

growth by removing the growth-inhibitory glial scar (Muir

et al. 2002; Pizzi and Crowe 2007). Of note, removal of the

inhibitory environment is not sufficient to promote axonal

regrowth; also regeneration has to be promoted! Regenera-

tive properties could be enhanced via production or activity

of intrinsic growth factors such as NGF (Silver and Miller

2004), a process to which MMP-3 might well contribute.

Overall, future research on MMP-3 in axonal growth should

be encouraged as MMP-3 seems a potential candidate

protease necessary to overcome the inhibitory environment

and to support axonal regrowth after CNS injuries.

MMP-3 in post-injury synapse formation

Degradation of CSPGs might not only support axonal

growth, but also synaptic reorganization. Two injury models

in rat, namely the traumatic brain injury model, characterized

by deafferentiation of target neurons which induces reactive

synaptogenesis and functional recovery (Steward 1989), and

the combined traumatic brain injury-bilateral entorhinal

cortical lesion model, generating additional neuroexcitation

but less synaptic recovery, were applied to study changes in

MMP-3 levels associated with trauma-induced synaptogen-

esis. MMP-3 mRNA, protein and activity levels were

elevated, not only during the degenerative period, but also

during the later regenerative phase characterized by rapid

synaptic plasticity. This injury-induced up-regulated MMP-3

expression was localized predominantly in reactive astro-

cytes within the denervated neuropil, likely participating in

removing debris and reshaping the extracellular environment

for efficient synapse reorganization (Kim et al. 2005; Falo

et al. 2006). Importantly, the spatially and temporally

coordinated MMP-3 expression and activity levels correlate

with increased expression of its target ECM substrates,

molecules necessary to prepare the local environment for

reactive synaptogenesis (Deller et al. 2001; Dityatev and

Schachner 2003).

As mentioned before, MMP-3 supports synaptic plasticity

and learning in the normal adult CNS. Consequently, it can

be hypothetized that the MMP-3 up-regulation, described

above, might underlie post-injury synaptic plasticity. Future

investigations addressing these possibilities might create

valuable findings to overcome the limited synaptic reorga-

nization after CNS injury.

MMP-3 in remyelination

An involvement of MMP-3 in remyelination during the

regenerative period after CNS injury was demonstrated

for the first time in the murine cuprizone-induced demyelin-

ation model. More specifically, not only during the early

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demyelination stage, but also during the stage of remyelina-

tion, MMP-3 was highly expressed in astrocytes of the

corpus callosum. Although the exact mechanisms remain

elusive, MMP-3 might be involved in remyelination via its

known involvement in releasing IGF, which is essential for

proliferation and differentiation of myelin-forming cells

(Dubois-Dalcq and Murray 2000; McCawley and Matrisian

2001; Fowlkes et al. 2004). In addition, MMP-3 might

support remyelination by removing and cleaving myelin

debris, which inhibits oligodendrocyte precursor cell differ-

entiation (D’Souza and Moscarello 2006; Kotter et al. 2006;

Skuljec et al. 2011). Also, accumulation of NG2 after axonal

injury not only creates an inhibitory environment for axonal

growth, but is also unfavorable for proper maturation of

oligodendrocytes (Larsen et al. 2003). Interestingly, NG2 is

one of the CSPGs known to be cleaved by MMP-3, and up-

regulated MMP-9 expression facilitates remyelination by

removal of NG2 (Muir et al. 2002; Larsen et al. 2003).

All the above findings already provide a promising

contribution of MMP-3 in CNS remyelination and warrant

future in depth investigations.

MMP-3 in adult neurogenesis and migration

In discrete regions of the normal adult brain, more exactly in

the subventricular and subgranular zone of the hippocampus,

neurogenesis remains ongoing in the adult stage. In response

to focal ischemic injury in the brain, proliferation of adult

neural stem/progenitor cells (aNPCs) is enhanced in the

subventricular zone and followed by migration of the

neuroblasts towards the ischemic area (Kokaia and Lindvall

2003). A recent in vitro study revealed an increase in MMP-3

levels in differentiated mouse aNPCs when migrating in

response to injury-induced chemokines. Importantly, block-

ing MMP-3 expression with specific siRNAs inhibited both

aNPC differentiation and chemokine-induced neuroblast

migration (Barkho et al. 2008). These data were confirmed

in a rodent focal ischemia model, showing migrating

neuroblasts expressing MMP-3 mRNA, thereby probably

allowing these cells to mediate a neurogenic response after

ischemic insults (Barkho et al. 2008).

Conclusion

Until now, research on MMP-3 in the CNS has mainly

focused on the involvement of this protease in pathological

conditions. However, in this review, we consider the

increasing number of reports that also indicate a beneficial

contribution of MMP-3 in developmental events as well as in

recovery processes in the CNS. The complex role of MMP-3

in amplifying or attenuating CNS disorders/injuries depends

on when and where MMP-3 is exactly up-regulated,

combined with the availability of its potential substrates.

Although a lot remains to be discovered, the existing

literature suggests that MMP-3 expression is strictly regu-

lated and involved in a wide variety of neuronal develop-

mental processes like neurite outgrowth, migration and

myelination. In the normal adult brain, elevated levels of

MMP-3 are shown to contribute to activity-related plasticity

and learning. Finally, in the injured or diseased CNS, a time-,

space- and cell type-specific coordinated MMP-3 expression

and activity seems to create dual functions for this enzyme:

in the acute phase after injury, MMP-3 might be associated

with CNS degeneration, while in the later regenerative

stages, up-regulated MMP-3 levels seem to contribute to

axonal sprouting, remyelination and reactive synaptogenesis

(as summarized in Fig. 2).

In conclusion, the current data available today already

indicate advantageous and critical roles for this protease in

the brain, supporting the need to reappraise the use of (non)

selective MMP inhibitors as drug therapy in neurological as

well as non-neurological disorders. Combined use of prote-

omic and genetic approaches, using conditional and cell-

specific transgenic animals, along with the generation of

specific synthetic and/or protein-based inhibitors for MMP-3,

will be important to gain more insight in the exact MMP-3

working mechanisms and the substrates involved, and should

enable the use of more specifically targeted therapeutic

strategies.

Acknowledgements

The authors have no conflict of interest. This work was supported by

grants from the Flemish Institute for the promotion of Scientific

Research (IWT), the Research Council of KU Leuven and the

Research Foundation Flanders (FWO).

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reference Gordon et al. (2009).

O n c e y o u h a v e A c r o b a t R e a d e r o p e n o n y o u r c o m p u t e r , c l i c k o n t h e C o m m e n t t a b a t t h e r i g h t o f t h e t o o l b a r :

S t r i k e s a l i n e t h r o u g h t e x t a n d o p e n s u p a t e x tb o x w h e r e r e p l a c e m e n t t e x t c a n b e e n t e r e d .‚ H i g h l i g h t a w o r d o r s e n t e n c e .‚ C l i c k o n t h e R e p l a c e ( I n s ) i c o n i n t h e A n n o t a t i o n ss e c t i o n .‚ T y p e t h e r e p l a c e m e n t t e x t i n t o t h e b l u e b o x t h a ta p p e a r s .

T h i s w i l l o p e n u p a p a n e l d o w n t h e r i g h t s i d e o f t h e d o c u m e n t . T h e m a j o r i t y o ft o o l s y o u w i l l u s e f o r a n n o t a t i n g y o u r p r o o f w i l l b e i n t h e A n n o t a t i o n s s e c t i o n ,p i c t u r e d o p p o s i t e . W e ’ v e p i c k e d o u t s o m e o f t h e s e t o o l s b e l o w :S t r i k e s a r e d l i n e t h r o u g h t e x t t h a t i s t o b ed e l e t e d .

‚ H i g h l i g h t a w o r d o r s e n t e n c e .‚ C l i c k o n t h e S t r i k e t h r o u g h ( D e l ) i c o n i n t h eA n n o t a t i o n s s e c t i o n .

H i g h l i g h t s t e x t i n y e l l o w a n d o p e n s u p a t e x tb o x w h e r e c o m m e n t s c a n b e e n t e r e d .‚ H i g h l i g h t t h e r e l e v a n t s e c t i o n o f t e x t .‚ C l i c k o n t h e A d d n o t e t o t e x t i c o n i n t h eA n n o t a t i o n s s e c t i o n .‚ T y p e i n s t r u c t i o n o n w h a t s h o u l d b e c h a n g e dr e g a r d i n g t h e t e x t i n t o t h e y e l l o w b o x t h a ta p p e a r s .

M a r k s a p o i n t i n t h e p r o o f w h e r e a c o m m e n tn e e d s t o b e h i g h l i g h t e d .‚ C l i c k o n t h e A d d s t i c k y n o t e i c o n i n t h eA n n o t a t i o n s s e c t i o n .‚ C l i c k a t t h e p o i n t i n t h e p r o o f w h e r e t h e c o m m e n ts h o u l d b e i n s e r t e d .‚ T y p e t h e c o m m e n t i n t o t h e y e l l o w b o x t h a ta p p e a r s .

I n s e r t s a n i c o n l i n k i n g t o t h e a t t a c h e d f i l e i n t h ea p p r o p r i a t e p a c e i n t h e t e x t .‚ C l i c k o n t h e A t t a c h F i l e i c o n i n t h e A n n o t a t i o n ss e c t i o n .‚ C l i c k o n t h e p r o o f t o w h e r e y o u ’ d l i k e t h e a t t a c h e df i l e t o b e l i n k e d .‚ S e l e c t t h e f i l e t o b e a t t a c h e d f r o m y o u r c o m p u t e ro r n e t w o r k .‚ S e l e c t t h e c o l o u r a n d t y p e o f i c o n t h a t w i l l a p p e a ri n t h e p r o o f . C l i c k O K .

I n s e r t s a s e l e c t e d s t a m p o n t o a n a p p r o p r i a t ep l a c e i n t h e p r o o f .‚ C l i c k o n t h e A d d s t a m p i c o n i n t h e A n n o t a t i o n ss e c t i o n .‚ S e l e c t t h e s t a m p y o u w a n t t o u s e . ( T h e A p p r o v e ds t a m p i s u s u a l l y a v a i l a b l e d i r e c t l y i n t h e m e n u t h a ta p p e a r s ) .‚ C l i c k o n t h e p r o o f w h e r e y o u ’ d l i k e t h e s t a m p t oa p p e a r . ( W h e r e a p r o o f i s t o b e a p p r o v e d a s i t i s ,t h i s w o u l d n o r m a l l y b e o n t h e f i r s t p a g e ) .

A l l o w s s h a p e s , l i n e s a n d f r e e f o r m a n n o t a t i o n s t o b e d r a w n o n p r o o f s a n d f o rc o m m e n t t o b e m a d e o n t h e s e m a r k s . .‚ C l i c k o n o n e o f t h e s h a p e s i n t h e D r a w i n gM a r k u p s s e c t i o n .‚ C l i c k o n t h e p r o o f a t t h e r e l e v a n t p o i n t a n dd r a w t h e s e l e c t e d s h a p e w i t h t h e c u r s o r .‚

T o a d d a c o m m e n t t o t h e d r a w n s h a p e ,m o v e t h e c u r s o r o v e r t h e s h a p e u n t i l a na r r o w h e a d a p p e a r s .‚

D o u b l e c l i c k o n t h e s h a p e a n d t y p e a n yt e x t i n t h e r e d b o x t h a t a p p e a r s .

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REVIEW Matrix metalloproteinase-3 in the central nervous ... · addition, MMP-3 was also discovered intranuclearly, more speci cally in the nucleus of hepatocytes (Si-Tayeb et al