Remodeling of extracellular matrix and ... - Wiley Online Library

Epilepsia, 51(Suppl. 3):61–65, 2010 doi: 10.1111/j.1528-1167.2010.02612.x

NOVEL THERAPEUTIC TARGETS AND APPROACHES

Remodeling of extracellular matrix and epileptogenesis Alexander Dityatev Department of Neuroscience and Brain Technologies, Italian Institute of Technology, Genova, Italy

supported by human genetic studies linking ECM molecules and epilepsy, by data showing altered epileptogenesis in mice deficient in ECM molecules, and by evidence that ECM may shape seizure-induced sprouting of mossy fibers, granule cell dispersion, and astrogliosis. Therefore, restraining seizure-induced remodeling of ECM or suppressing the signaling triggered by the remodeled ECM might provide effective therapeutic strategies to antagonize the progression of epileptogenesis. KEY WORDS: Epilepsy, Synapse, Glia, Homeostatic regulation, Hyaluronic acid, Tenascin.

SUMMARY Extracellular matrix (ECM) in the brain is composed of molecules synthesized and secreted by neurons and glial cells, which form stable aggregates of diverse composition in the extracellular space. In the mature brain, ECM undergoes a slow turnover and restrains structural plasticity while supporting multiple physiologic processes, including perisomatic c-aminobutyric acid (GABA)ergic inhibition, synaptic plasticity, and homeostatic regulations. Seizures lead to striking remodeling of ECM, which may be essentially engaged in different aspects of epileptogenesis. This view is

2007). In addition to the aforementioned core components, the ECM contains a wide variety of secreted growth factors and other matricellular proteins that regulate cell– cell and cell–matrix interactions. The ECM of perineuronal nets in the cerebral cortex and hippocampus is predominantly associated with c-aminobutyric acid (GABA)ergic interneurons expressing Ca2+-binding protein parvalbumin. Formation of perineuronal nets is an activity-dependent process that takes many days. In vitro experiments revealed that activities of Ca2+-permeable a-amino-3-hydroxyl-5-methyl4-isoxazole-propionate (AMPA) receptors and L-type voltage-dependent Ca2+ channels are necessary for perineuronal net formation (Dityatev et al., 2007). In vivo experiments with enzymatic digestion of perineuronal nets in adult rodents revealed that several months are needed for the complete recovery of these structures (Bruckner et al., 1998), thus demonstrating a slow turnover of ECM in mature brains. Another important property of ECM in mature brains is the provision of a nonpermissive environment that restrains structural remodeling of the neural network. For instance, the ECM contributes to the failure of axon regeneration in the CNS and to a lack of ocular dominance plasticity in the mature visual cortex. Strikingly, degradation of the chondroitin sulfate chains of CSPGs with chondroitinase ABC or remodeling of

There are multiple forms of extracellular matrix (ECM) in the central nervous system (CNS) (Dityatev & Fellin, 2008). These include: (1) perineuronal nets, which surround cell bodies, proximal dendrites, and axon initial segments of some neurons, in a mesh-like structure that interdigitates with synaptic contacts and astrocytic processes; (2) perisynaptic ECM in the neuropil; (3) the basal lamina, which are annexed to the capillary and postcapillary venules in the CNS, being an essential part of the blood–brain barrier in association with pericytes, perivascular microglia, and astrocytes; and (4) fractones in the lateral ventricle walls, which have been shown to promote growth factor activity in the neural stem cell niche. The structural and molecular organization of ECM in the CNS is heterogeneous and depends on the cell types and subcellular domains that associate with ECM. Commonly found components of neural ECM are chondroitin sulfate proteoglycans (CSPGs), heparan sulfate proteoglycans, glycosaminoglycan hyaluronan, glycoproteins of the tenascin family, and link proteins (Galtrey & Fawcett, Address correspondence to Alexander Dityatev, Department of Neuroscience and Brain Technologies, Italian Institute of Technology, via Morego 30, 16163 Genova, Italy. E-mail: alexander.dityatev@iit.it Wiley Periodicals, Inc. ª 2010 International League Against Epilepsy

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62 A. Dityatev ECM with extracellular proteinase tissue plasminogen activator (tPA) allows ocular dominance plasticity in the adult animals (Mataga et al., 2002; Pizzorusso et al., 2002). Although most studies point to nonpermissive roles of ECM in structural plasticity, many ECM components are beneficial for growth and regeneration. Therefore, a balance between repellent and growth-permissive cues may determine the net effect of ECM on structural plasticity. Although ECM inhibits structural plasticity, it promotes different forms of functional plasticity, including longterm potentiation (LTP), long-term depression (LTD), homeostatic plasticity, and metaplasticity. ECM regulates activity of N-methyl-D-aspartate (NMDA) receptors and L-type Ca2+ channels, perisomatic GABAergic inhibition, and number and properties of astrocytes (Dityatev et al., 2006; Dityatev & Fellin, 2008). The multiple roles played by ECM in neuroplasticity in healthy brains suggest that it could be an important factor for pathogenic plasticity associated with epileptogenesis; and evidence supporting this view is reviewed in subsequent text of this article.

The Role of ECM in Epileptogenesis Human genetic studies In humans, mutations in the leucine-rich, glioma-inactivated 1 gene (LGI1) cause autosomal-dominant lateral temporal lobe epilepsy with auditory features (Kalachikov et al., 2002) (Table 1). LGI1 is a secreted protein, which coimmunoprecipitates with the major component of the postsynaptic density, PSD-95, together with two transmembrane proteins, ADAM22 and stargazin (Fukata Table 1. ECM-related genes in epilepsy Gene (relevance to ECM) LGI1 LGI4

SRPX2 RELN SCN1B (binds to tenascin-R)

IDS (upregulates expression of chondroitin sulfate B and heparan sulfates) COL18A1 LAMA2

ECM1

Disease Autosomal-dominant lateral temporal lobe epilepsy with auditory features Childhood absence epilepsy, idiopathic generalized epilepsies, benign familial infantile convulsions Rolandic epilepsy Temporal lobe epilepsy Generalized epilepsy with febrile seizures plus type 1 and early onset absence seizures Hunter disease (in some cases seizures) Knobloch syndrome (in some cases generalized seizures) Merosin-deficient congenital muscular dystrophy type 1A (in some cases refractory epilepsy) Lipoid proteinosis (in some cases temporal lobe epilepsy)


et al., 2006). LGI1 belongs to a subfamily of leucine-rich repeat genes comprising four members (LGI1–LGI4) in mammals. LGI4, ADAM22, and stargazin have been implicated in epilepsy. The expression of mutated E383A LGI1 impairs its binding to ADAM22 and alters AMPAreceptor activity. Altogether these studies make a genetic link between ECM and epilepsy in humans and suggest that extracellular regulation of AMPA receptors by secreted proteins might be an important mechanism in epileptogenesis. Mutations in the SRPX2 (Sushi-Repeat Protein, Xlinked 2) gene cause rolandic epilepsy with speech impairment or with altered development of the speech cortex (bilateral perisylvian polymicrogyria) in humans. SRPX2 is a secreted protein that binds to urokinase-type plasminogen activator receptor (uPAR) (Royer-Zemmour et al., 2008). The p.Y72S mutation in SRPX2, which is associated with rolandic epilepsy and perisylvian polymicrogyria, leads to a six-fold gain-of-affinity of SRPX2 with uPAR. Knockout of uPAR results in the almost complete loss of parvalbumin-expressing GABAergic interneurons and in the epileptic phenotype, presumably due to a compromised development of inhibitory circuitry (Powell et al., 2003). Two more SRPX2 partners, the cysteine protease cathepsin B (CTSB, a well-known activator of uPA) and the metalloproteinase ADAMTS4, are also components of the extracellular proteolysis machinery. These findings suggest that a network of SRPX2-interacting proteins may be involved in the proteolytic remodeling of ECM during epileptogenesis. Mutations in the beta1 subunit of voltage-gated Na+ channels (SCN1B) are linked to generalized epilepsy with febrile seizures plus type 1 and early onset absence seizures. Na+ channel beta subunits modulate channel gating, regulate the level of channel expression at the plasma membrane, and function as cell adhesion molecules in terms of interaction with the ECM and regulation of cell migration. Particularly relevant is a beta subunit–mediated functional modulation of Na+ channels by the ECM glycoprotein tenascin-R (Isom, 2002). In several cases, mutations in genes encoding ECM molecules lead to complex disorders, with a minority of patients developing seizures. For instance, collagen type XVIII (COL18A1) gene mutations have been described in patients with Knobloch syndrome, which is characterized by high myopia, vitreoretinal degeneration, macular abnormalities, and occipital encephalocele. Some of these patients have epilepsy and develop myoclonic and other types of generalized seizures (Suzuki et al., 2002). Merosin (laminin-2, LAMA2)–deficient congenital muscular dystrophy type 1A is one of the most frequent forms of congenital muscular dystrophy in Western countries. In patients with cognitive deterioration, this disease is accompanied by epilepsy and focal cortical dysplasia, in addition to the classical diffuse white

63 Extracellular Matrix and Epileptogenesis matter abnormalities (Vigliano et al., 2009). Mutations in the extracellular matrix protein 1 (ECM1) gene are linked to lipoid proteinosis, with affected individuals displaying differing degrees of skin scarring and infiltration, variable signs of hoarseness and respiratory distress, and in some cases neurologic abnormalities such as temporal lobe epilepsy (Teive et al., 2004). Patients with Hunter disease, an X-linked lysosomal storage disease caused by deficiency of iduronate-2sulfatase (IDS), have accumulation of chondroitin sulfate B and heparan sulfates and develop seizures and mental retardation (Al Sawaf et al., 2008). Remodeling of ECM by seizures Epilepsies are characterized by recurrent seizures, which are the manifestation of an underlying transient abnormality of neuronal activity. Seizures have been shown to upregulate expression of multiple ECM molecules, including tenascin-C, tenascin-R, neuronal pentraxin NP2, hyaluronan, and hevin (perisynaptically), and downregulate expression of phosphacan, reelin, and hevin (somatically). Seizures also upregulate activity of extracellular proteinases, such as tissue plasminogen activator and metalloproteinase (MMP)-9. Importantly, extracellular proteinases may not only degrade the ECM but may also generate functionally active neoepitopes. For instance, cleavage of agrin by activity-dependent– released neurotrypsin generates 22-kDa fragments that stimulate filopodial motility. Therefore, the pattern of seizure-induced remodeling of ECM is rather complex and is brain subregion– and cellular subdomain–specific. The remodeled ECM may trigger numerous secondary long-term functional and structural changes in the CNS that could determine progression of the disease. Epileptogenesis in mice deficient in ECM molecules or extracellular proteinases Kindling progression is retarded in tenascin-R–deficient mice compared to wild-type littermate controls (Hoffmann et al., 2009). Specifically, tenascin-R–deficient mice remain significantly longer, compared to wildtypes, in stage 1 with mild focal clonus and stage 4 with bilateral forelimb clonus. Another important feature of tenascin-R–deficient mice is an increase in the number of S100B-expressing astrocytes (Hoffmann et al., 2009). This genotype-specific difference is not only detected under normal conditions but persists in the astrogliotic stage that follows the induction of generalized seizures in mice with implanted electrodes. Intriguingly, the kindling-induced increase in the number of S100B-expressing astrocytes in the dentate gyrus correlated negatively with the kindling rate on the individual animal basis. In line with this finding is a previous study reporting a more rapid onset and more severe seizures in kindled mice deficient in S100B as compared to wild-type mice (Dyck et al.,

2002). Taken together these data suggest that the complex interplay between ECM, astrocytes, and neurons may shape epileptogenesis. Spontaneous seizure-like behavior occurs in mice that are deficient in the major component of basement membrane, nidogen-1, which surrounds blood vessels in the CNS (Vasudevan et al., 2010). These episodes could develop into forelimb clonus, complex body distortions, and myoclonic jerks resembling full-blown seizures. Such severe, seizure-like events occurred in 30% of mutants. Seizures could be triggered audiogenically in 70% of mutants by exposing them to ultrasound. Seizures were accompanied by epileptiform spiking in hippocampal electroencephalograpic recordings. In vitro, a lack of nidogen-1 led to the appearance of spontaneous and evoked epileptiform activity and an increase of the basal synaptic transmission and paired-pulse facilitation. Although there were no changes in basement membrane and hippocampal morphology in nidogen-1 deficient mice, there was a reduction of laminin expression outside the basement membrane. It remains to be studied whether this reduction and changes in integrin signaling mediate higher excitability in the absence of nidogen-1. Seizure progression is significantly delayed in tPAdeficient mice. In addition, inhibition of tPA by the natural inhibitor of tPA, neuroserpin, within the hippocampus markedly delays the progression of seizure activity in both rats and wild-type mice (Yepes et al., 2002). Like tPA, MMPs are also implicated in epileptogenesis: Progression of pentylenetetrazole kindling–induced epileptogenesis is decreased in MMP-9 knockout mice, whereas it is increased in transgenic rats overexpressing MMP-9 (Wilczynski et al., 2008). Furthermore, MMP-9 deficiency diminishes seizure-evoked pruning of dendritic spines and decreases aberrant synaptogenesis after mossy fiber (MF) sprouting (Wilczynski et al., 2008). ECM and sprouting of mossy fibers and granule cell dispersion Seizure episodes induce the growth of axons and formation of new synapses. Particularly prominent is the seizure-induced sprouting of MFs, which culminates in the formation of aberrant structures in the CA3 infrapyramidal and the dentate supragranular layers. Experiments in vitro suggest that hyaluronan may promote MF sprouting. Application of kainic acid (KA) to organotypic hippocampal slice cultures induces robust MFs into the inner dentate molecular layer compared with vehicle-treated controls. Degradation of hyaluronan with hyaluronidase, however, significantly reduces KA-induced MF sprouting (Bausch, 2006). This finding is in agreement with the important permissive role that hyaluronan plays during development and with the increased hyaluronan expression associated with temporal lobe epilepsy. This increase in expression of permissive cue hyaluronan is accompanied by the proEpilepsia, 51(Suppl. 3):61–65, 2010 doi: 10.1111/j.1528-1167.2010.02612.x

64 A. Dityatev teolytic degradation of CSPG phosphacan (which is a repellent cue for MFs) in KA-injected mice. This degradation occurs via the tPA/plasmin system. In addition, MF sprouting is associated with an increase in the cleaved form of CSPG brevican and an increase in the deposition of neonatal full-length form of CSPG neurocan. This increase in permissive and decrease in repellent cues appear to trigger sprouting of MFs. Medial temporal lobe epilepsy is often accompanied by granule cell dispersion, i.e., widening of the granule cell layer and the appearance of ectopic dentate granule cells (DGCs), which integrate abnormally, are hyperexcitable, and thus may promote epileptogenesis. The ECM protein reelin is expressed in a subpopulation of interneurons, which are typically lost in human and experimental models of epilepsy, and this deficit underlies granule cell dispersion and appearance of hilar-ectopic DGCs in epileptic brains (for a review, see Dityatev & Fellin, 2008). Human mutations of the reelin gene (RELN) lead to cortical lissencephaly with cerebellar hypoplasia, severe epilepsy, and mental retardation.

ECM-Targeting Therapeutic Treatments The emerging links between the ECM and epilepsy are shown in the Fig. 1. I suggest that at least two types of treatments might be beneficial to prevent or slow down the progression of epilepsy. Because seizures elevate expression of some ECM molecules and extracellular proteinases, it is plausible to speculate that a combination of drugs counteracting these effects could be beneficial to prevent structural remodeling of ECM and thus epileptogenesis. Indeed, as discussed earlier, hyaluronidase treatment is known to inhibit MF sprouting. It would be of interest to test antiepileptic activities of hyaluronidase, inhibitors of hyaluronan synthases, the enzymes responsi-

Figure 1. The role of ECM in epileptogenesis and putative ECMtargeting antiepileptic treatments. Epilepsia ILAE Epilepsia, 51(Suppl. 3):61–65, 2010 doi: 10.1111/j.1528-1167.2010.02612.x

ble for production of hyaluronan, as well as inhibitors of tPA or MMPs. Another strategy would be to interfere with signaling triggered by the remodeled ECM. For instance, blocking of b3-integrin signaling might be beneficial to tune down erroneous upscaling of AMPA-receptor– mediated transmission during epileptogenesis. Most of the mentioned ECM-targeting compounds have already been developed as potential antitumor drugs and thus can be immediately tested in experimental models of epileptogenesis. To deal with abnormal ECM in patients carrying mutations in LGI1 and SRPX2, the peptides mimicking binding of LGI1 and ADAM22 or inhibiting interactions between SRPX2 with uPAR could be designed. Hopefully, further understanding of the functions of neural ECM molecules and development/testing of drugs targeting synthesis and degradation of ECM will lead to effective antiepileptic treatments.

Disclosure The author declares no conflict of interest. I confirm that I have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.

References Al Sawaf S, Mayatepek E, Hoffmann B. (2008) Neurological findings in Hunter disease: pathology and possible therapeutic effects reviewed. J Inherit Metab Dis 31:473–480. Bausch SB. (2006) Potential roles for hyaluronan and CD44 in kainic acid-induced mossy fiber sprouting in organotypic hippocampal slice cultures. Neuroscience 143:339–350. Bruckner G, Bringmann A, Hartig W, Koppe G, Delpech B, Brauer K. (1998) Acute and long-lasting changes in extracellular-matrix chondroitin-sulphate proteoglycans induced by injection of chondroitinase ABC in the adult rat brain. Exp Brain Res 121:300–310. Dityatev A, Frischknecht R, Seidenbecher CI. (2006) Extracellular matrix and synaptic functions. Results Probl Cell Differ 43:69–97. Dityatev A, Bruckner G, Dityateva G, Grosche J, Kleene R, Schachner M. (2007) Activity-dependent formation and functions of chondroitin sulfate-rich extracellular matrix of perineuronal nets. Dev Neurobiol 67:570–588. Dityatev A, Fellin T. (2008) Extracellular matrix in plasticity and epileptogenesis. Neuron Glia Biol 4:235–247. Dyck RH, Bogoch II, Marks A, Melvin NR, Teskey GC. (2002) Enhanced epileptogenesis in S100B knockout mice. Brain Res Mol Brain Res 106:22–29. Fukata Y, Adesnik H, Iwanaga T, Bredt DS, Nicoll RA, Fukata M. (2006) Epilepsy-related ligand/receptor complex LGI1 and ADAM22 regulate synaptic transmission. Science 313:1792–1795. Galtrey CM, Fawcett JW. (2007) The role of chondroitin sulfate proteoglycans in regeneration and plasticity in the central nervous system. Brain Res Rev 54:1–18. Hoffmann K, Sivukhina E, Potschka H, Schachner M, Lçscher W, Dityatev A. (2009) Retarded kindling progression in mice deficient in the extracellular matrix glycoprotein tenascin-R. Epilepsia 50:859– 869. Isom LL. (2002) Beta subunits: players in neuronal hyperexcitability? Novartis Found Symp 241:124–138. Kalachikov S, Evgrafov O, Ross B, Winawer M, Barker-Cummings C, Martinelli Boneschi F, Choi C, Morozov P, Das K, Teplitskaya E, Yu A, Cayanis E, Penchaszadeh G, Kottmann AH, Pedley TA, Hauser WA, Ottman R, Gilliam TC. (2002) Mutations in LGI1 cause autosomal-dominant partial epilepsy with auditory features. Nat Genet 30:335–341.

65 Extracellular Matrix and Epileptogenesis Mataga N, Nagai N, Hensch TK. (2002) Permissive proteolytic activity for visual cortical plasticity. Proc Natl Acad Sci U S A 99:7717–7721. Pizzorusso T, Medini P, Berardi N, Chierzi S, Fawcett JW, Maffei L. (2002) Reactivation of ocular dominance plasticity in the adult visual cortex. Science 298:1248–1251. Powell EM, Campbell DB, Stanwood GD, Davis C, Noebels JL, Levitt P. (2003) Genetic disruption of cortical interneuron development causes region- and GABA cell type-specific deficits, epilepsy, and behavioral dysfunction. J Neurosci 23:622–631. Royer-Zemmour B, Ponsole-Lenfant M, Gara H, Roll P, Leveque C, Massacrier A, Ferracci G, Cillario J, Robaglia-Schlupp A, Vincentelli R, Cau P, Szepetowski P. (2008) Epileptic and developmental disorders of the speech cortex: ligand/receptor interaction of wild-type and mutant SRPX2 with the plasminogen activator receptor uPAR. Hum Mol Genet 17:3617–3630. Suzuki OT, Serti AL, Der Kaloustian VM, Kok F, Carpenter M, Murray J, Czeizel AE, Kliemann SE, Rosemberg S, Monteiro M, Olsen BR, Passos-Bueno MR. (2002) Molecular analysis of collagen XVIII reveals novel mutations, presence of a third isoform, and possible genetic heterogeneity in Knobloch syndrome. Am J Hum Genet 71:1320–1329.

Teive HA, Pereira ER, Zavala JA, Lange MC, de Paola L, Raskin S, Werneck LC, Hamada T, McGrath JA. (2004) Generalized dystonia and striatal calcifications with lipoid proteinosis. Neurology 63:2168–2169. Vasudevan A, Ho MS, Weiergraber M, Nischt R, Schneider T, Lie A, Smyth N, Kohling R. (2010) Basement membrane protein nidogen-1 shapes hippocampal synaptic plasticity and excitability. Hippocampus 20:608–620. Vigliano P, Dassi P, Di Blasi C, Mora M, Jarre L. (2009) LAMA2 stopcodon mutation: merosin-deficient congenital muscular dystrophy with occipital polymicrogyria, epilepsy and psychomotor regression. Eur J Paediatr Neurol 13:72–76. Wilczynski GM, Konopacki FA, Wilczek E, Lasiecka Z, Gorlewicz A, Michaluk P, Wawrzyniak M, Malinowska M, Okulski P, Kolodziej LR, Konopka W, Duniec K, Mioduszewska B, Nikolaev E, Walczak A, Owczarek D, Gorecki DC, Zuschratter W, Ottersen OP, Kaczmarek L. (2008) Important role of matrix metalloproteinase 9 in epileptogenesis. J Cell Biol 180:1021–1035. Yepes M, Sandkvist M, Coleman TA, Moore E, Wu JY, Mitola D, Bugge TH, Lawrence DA. (2002) Regulation of seizure spreading by neuroserpin and tissue-type plasminogen activator is plasminogen-independent. J Clin Invest 109:1571–1578.
