Chondroitinase ABC-mediated Ocular Dominance Plasticity

Scuola Superiore di Studi Universitari e Perfezionamento Sant’Anna

Classe Accademica di Scienze Sperimental i

SETTORE DI SCIENZE MEDICHE

CHONDROITINASE ABC-MEDIATED

OCULAR DOMINANCE PLASTICITY

FROM MOLECULAR BASES TO THERAPEUTIC TARGETS

A LL IE VO : T U T OR :

F IL IPP O QU A T T RO N E ANT ON IO L’ABB A T E

A N NO A CCA D E MI CO 2009/2010

Chondroit inase ABC–mediated ocular dominance plastici ty 2

ABSTRACT

At birth, sensory cortical circuits are immature and shape their connections, ex-

perience-dependently, only during specific “critical periods” in early infancy.

Restoration of this neuronal plasticity in the adult central nervous system could

fix disorders due to a lack of environmental stimuli during critical periods. One

of these diseases is amblyopia, caused by a dominance of one eye stimulation in

primary visual cortex during its critical period. This situation leads to reduction

of visual acuity in the non-dominant eye and loss of stereopsis, the depth per-

ception given by binocular vision.

A molecular correlate of the closure of critical periods is the organization of the

extracellular matrix of the nervous tissue in condensed layers, known as peri-

neuronal nets, around cell bodies, dendrites and proximal segments of axons.

Chondroitinase-ABC is a bacterial enzyme that degrades chondroitin sulfate

glycosaminoglycans and hyaluronan, which are key constituents of the peri-

neuronal nets. This digestion induces a reopening of the critical period and thus

promotes recovery of ocular dominance and visual acuity in the animal model

of amblyopia.


CONTENTS

ABSTRACT 2

ABBREVIATIONS 5

INTRODUCTION 7

1 NEURONAL PLASTICITY IN VISUAL CORTEX 8

1.1 The concept of neuronal plasticity and its evolution 8

1.2 Critical periods: a limit to plasticity 9

1.3 Ocular dominance in visual cortex as model of neuronal plasticity 9

Figure 1. Experiments on ocular dominance plasticity 10

1.4 Molecular correlates of plasticity in visual cortex 11

2 EXTRACELLULAR ENVIRONMENT AND VISUAL CORTICAL PLASTICITY 12

2.1 The architecture of brain extracellular matrix 12

2.2 The role of extracellular matrix in ocular dominance plasticity 14

Figure 2. The perineuronal nets in adult and immature brain and after

chondroitinase ABC treatment 15


3 CHONDROITINASE ABC: BIOCHEMICAL ASPECTS 16

Figure 3: Molecular structure of chondroitinase-ABC and its active site 17

4 CHONDROITINASE ABC EFFECTS ON OCULAR DOMINANCE PLASTICITY 18

4.1 Experimental protocols to assess chondroitinase skills 18

Figure 4. Chondroitinase ABC reactivates ocular dominance plasticity 20

Figure 5. ChABC does not modify functional properties of cortical neurons 20

Figure 6. ChABC allows recovery of OD in adult RS rats 21

Figure 7. ChABC normalizes spine density in adult RS rats 21

4.2 Putative mechanisms of action of chondroitinase ABC 22

Figure 8. Anatomical plasticity and tPA: a mechanism model 23

5 CHONDROITINASE ABC-EFFECTS IN OTHER REGIONS: AN OVERVIEW 24

CONCLUSIONS 25

REFERENCES 28


ABBREVIATIONS

Arg Arginine

BDNF Brain derived neurotrophic factor

C-4-S Chondroitin-4-sulfate

C-6-S Chondroitin-6-sulfate

CaMKII Ca2+/calmodulin dependent protein kinases II

ChABC Chondroitinase ABC I

CNS Central nervous system

CREB cAMP response element-binding

Crtl1 Cartilage link protein 1

CS Chondroitin sulfate

DR Dark rearing

DS Dermatan sulfate

ECM Extracellular matrix

EGF Epidermal growth factor

ERK Extracellular-signal-regulated kinases

GABA γ-Aminobutyric acid

GAG Glycosaminoglycan

Glu Glutamic acid


HA Hyaluronan

His Histidine

HLT Hyaluronan–lectican-tenascin

MD Monocular deprivation

NCAM Neural Cell Adhesion Molecule

NMDA N-methyl-D-aspartic acid

OD Ocular dominance

P-ase Penicillinase

PG Proteoglycan

PKA Protein kinase A

PKC Protein kinase C

PNN Perineuronal nets

RPTP Receptor tyrosine phosphatase β

RS Reverse suture

tPA Tissue plasminogen activator

Tyr Tyrosine

V1 Primary visual cortex

VEP Visual evocated potential

INTRODUCTION

In occasion of my approach to chondroitinase-ABC (ChABC), I was impressed

by its property of freeing the brain from past experiences and restoring in

adulthood the malleability typical of the child. This skill could be useful not on-

ly to treat spinal lesions, traumas and amblyopia but also could help to cope

with aging, making for instance possible to learn easily lifelong.

This essay will show the results of the application of this tool in primary vis-

ual cortex (V1) where chABC treatment was able to induce in the animal model

a reorganization of ocular dominance digesting brain extracellular matrix

(ECM). This discovery could lead to a treatment for amblyopia, a visual disord-

er due to bad development of V1 during early infancy.

The choice of this topic was influenced by the courses on physiology of per-

ception and cellular biology held respectively by Nicoletta Berardi at Scuola

Normale, and by Gian Michele Ratto at Scuola Sant’Anna. My experience in his

laboratory at the NEST center in Pisa was an opportunity to better understand

the world of neurosciences and the experimental procedures used in this field.

After elucidating the concept of neuronal plasticity and critical period, the

peculiarities of ocular dominance plasticity and its possible molecular correlates

will be analyzed. A whole section will focus on the description of the ECM in

the brain and the role of some components, the chondroitin sulfate proteogly-

cans, in the closure of periods of plasticity. Afterwards chABC biochemical

properties will be outlined. Eventually attention will be conferred to results of

chABC application in V1 and, briefly, in other districts.


1 NEURONAL PLASTICITY I N VISUAL CORTEX

1.1 THE CONCEPT OF NEURONAL PLASTICITY AND ITS EVOLUTION

Neuronal plasticity is a term with a wide meaning referring to the “changes in

neural organization which may account for various forms of behavioral mod-

ifiability, either short-lasting or enduring, including maturation, adaptation to a

mutable environment, specific and unspecific kinds of learning, and compensa-

tory adjustments in response to functional losses from aging or brain damage”

(Berlucchi and Buchte, 2008). Neuronal plasticity was studied at the end of 19th

century by many scientists such as James, Tanzi, Lugaro and Cajal but its mod-

ern definition was given by Jerzy Konorski in 1948. A year after Donald Olding

Hebb in his The Organization of Behavior (1949) proposed that it is the synchronic

activity of pre and post synaptic element to give more stability to a synapse

(Berlucchi and Buchte, 2008). This concept is often summarized as “cells that

fire together wire together”.

Two are the forms of neuronal plasticity: synaptic plasticity, based on activi-

ty-dependent changing in synaptic strength of an existing synapse, and anatom-

ical plasticity (also known as structural plasticity or remodeling) based on for-

mation of new synapses (Galtrey and Fawcett, 2007). Recent studies (Chklovskii

et al., 2004) show that wiring changes and weight changes are not mutual exclu-

sive but that structural plasticity could cause a huge expansion of memory sto-

rage capacity, compared with weight plasticity alone. The anatomical correlates

of structural plasticity are not large-scale modifications of the dendritic arbor,

but modification of a small part of it: the dendritic spines, mobile tiny protru-

sions that emanate from dendritic shafts and contact axonal synaptic boutons.

According to this hypothesis many studies showed dendritic spines’ growth

and retraction are experience-dependent (Holtmaat and Svoboda, 2009).


1.2 CRITICAL PERIODS : A LIMIT TO PLASTICITY

Neuronal plasticity is not constant during life: not only decreases with aging

(Berardi et al., 2003) but is often confined in determinate time windows: the so

called “critical periods”. A critical period is a time in the early stages of an or-

ganism's life during which it displays a heightened sensitivity to certain envi-

ronmental stimuli, and its development is conditioned by experiences made at

this time. Critical periods were found in virtually every animal in the develop-

ment of visual, auditory and somatosensory systems and also in learning com-

plex skills such as language or song birds (Berardi et al., 2000). The role played

by experience in the development of neuronal circuits during critical periods is

probably based on a hebbian selection of appropriate synapses and elimination

of inappropriate ones (Berardi et al., 2000; Hensch and Stryker, 2004).

1.3 OCULAR DOMINANCE IN V ISUAL CORTEX

AS MODEL OF NEURONAL PLASTICITY

The first and most studied critical period is the development of ocular domin-

ance (OD) in primary visual cortex, called also striate cortex (because of the

presence of the stria of Gennari, a visible band of myelinated axon) or V1. It is

the first district where information from both eyes arrives, allowing binocular

vision. OD is the property of binocular neurons of V1 of being activated to dif-

ferent degrees by visual stimuli presented to one eye or the other. In human be-

ings the critical period lasts about two years (Banks et al., 1975) and its end

coincides with the gain of mature visual acuity. The concept itself of critical pe-

riod was first proposed by David Hunter Hubel and Torsten Wiesel studying

with electrophysiology V1 neurons shift of responsiveness when one eye was

deprived of vision during the critical period (Hubel and Wiesel, 1970). These

experiments based on monocular deprivation (MD) showed a reduction of neu-

rons excited by the sutured eye and an increase of neurons responding to the


non-deprived eye (Wiesel and Hubel, 1963; Hubel and Wiesel, 1970; Hubel et

al., 1977; Fagiolini et al., 1994). The consequence of this shift of OD, possible only

during the critical period, is reduction of visual acuity of the deprived eye and

loss of binocular vision similarly to human amblyopia (see figure 1). Another

experimental protocol to study V1 plasticity is dark rearing (DR) that delays on-

set of the critical period (Fagiolini et al., 1994) suggesting that the precritical pe-

riod contributes to the activation of the critical period (Hooks and Chen, 2007).

Recent studies, however, showed OD maturation, especially the formation of

OD columns, in contrast with Hubel and Wiesel view, is not a simple activity-

dependent event but it is also strongly gene-determined (Leamey, 2009; Katz

and Crowley, 2002; Hooks and Chen, 2007).

Figure 1. Experiments on ocular dominance plasticity

A) Ocular dominance distribution of single unit recordings from a large number of neurons in the prima-ry visual cortex of normal adult cats. Cells in group 1 were activated only by the contralateral eye, cells in group 7 by the ipsilateral eye. Diagrams below these graphs indicate procedure, and bars in-dicate duration of deprivation (purple). “Exp”= time when experimental observations were made.

B) Following closure of one eye from 1 week after birth until 2.5 months of age no cells could be acti-vated by the deprived eye. Some cells could not be activated by either eye (NR, no response).

C) A much longer period of monocular deprivation in an adult cat has little effect on ocular dominance (although overall cortical activity is diminished). In this case, the contralateral eye was closed from 12 to 38 months of age. (A after Hubel and Weisel, 1962; B after Wiesel and Hubel, 1963; C after Hubel and Wiesel, 1970.)

(from D. Purves et al., Neuroscience, Sinauer associates, 2001)


1.4 MOLECULAR CORRELATES OF PLASTICITY IN VISUAL CORTEX

Molecular mechanisms that regulate critical period in visual cortex are still un-

clear but many candidates have been proposed (Berardi et al., 2003).

Some studies show that NMDA (N-methyl-D-aspartic acid) receptors,

thought to be the neural implementation of hebbian hypothesis, switch their

subunits 2B to 2A in coincidence with the closure of the critical period (Berardi

et al., 2000) even if this changing is not essential for this process (Fagiolini et al.,

2003). Moreover block of NMDA receptors avoids the effects of monocular de-

privation (Bear et al., 1990).

NMDA receptors allow Ca 2+ ions to enter in the neuron activating a complex

network of kinases such as PKA, ERK, CaMKII and calcineurin phosphatase

(Beaver et al., 2001; Di Cristo et al., 2001; Taha et al., 2002; Yang et al., 2005) that

leads to activation of CREB transcription factor. Modification of this network

causes absence of OD shift after MD (Berardi et al, 2004).

Neurotrophins are secreted in response to electrical activity and enhance it.

They are, therefore, able to strengthen the most active synapses (Berardi et al.,

2003). In visual cortex BDNF (brain derived growth factor) overexpression acce-

lerates both the development of visual acuity and the time course of OD plastic-

ity. This is probably due to its action on intracortical inhibition.

The intracortical inhibition, actually, must surpass a threshold before the crit-

ical period can start (Berardi et al., 2004). If intracortical inhibition is increased,

either by early diazepam administration (Fagiolini and Hensch, 2000), that en-

hances the effect of the inhibitory neurotransmitter GABA, or by overexpres-

sion of BDNF (Hanover et al., 1999) the critical period starts and closes earlier.

Another key element that regulates neuronal plasticity, tightly bound to the

previous ones, is the extracellular matrix (ECM) and its principal components in

CNS, the chondroitin-sulfate proteoglycans (CSPGs) (Berardi et al., 2003).


2 EXTRACELLULAR ENVIRONMENT

AND VISUAL CORTICAL PLASTICITY

2.1 THE ARCHITECTURE OF BRAIN EXTRACELLULAR MATRIX

Histologically it is possible to identify in the brain three types of ECM: the first

is a diffuse matrix that exists throughout the CNS, the second is defined by

some cell surface-associated matrix molecules, the third is the dense organized

matrix of perineuronal nets (PNNs), the most involved in ocular dominance

plasticity (Sathyaseelan Deepa et al., 2006).

PNNs are lattice like structure of neuronal and glyal origin that ensheathe

neuronal bodies, proximal dendrites and axonal initial segments as far as the

beginning of the myelin sheath. They are fenestrated at sites of synaptic contact

(Fox and Caterson 2002, Karetko and Skangiel-Kramska, 2009). PNNs are

present around specific types of neurons in the cortex (above all in motor and

primary sensory areas), hippocampus, thalamus, brainstem and spinal cord. In

the cortex they are associated with GABAergic interneurons, especially parval-

bumin containing interneurons, and pyramidal cells (Murakami and Ohtsuka,

2003, Galtrey and Fawcett, 2007). PNNs were first described by Camillo Golgi

and object of debate by first neuroscientists. After a long period of stagnation

since 90s several studies have raised attention to PNNs (Celio et al., 1998).

PNNs structure is still mysterious and it is known that their composition va-

ries among different sets of neurons and between immature and mature brain.

The most credited hypothesis on their shape is the HLT (hyaluronan –lectican-

tenascin) matrix model (Yamaguchi, 2000) (see figure 2A). In this model PNNs

are constituted of a ternary complex of hyaluronan, lecticans and tenascin.

Hyaluronan (HA, also hyaluronic acid or hyaluronate) is a particular glyco-

saminoglycan (GAG). GAGs are usually large unbranched, negatively charged,

variably sulfated polymers composed of 20–200 repeating disaccharide units


usually bound to a proteic core to form proteoglycans (PGs). The principal

GAGs in PGs are chondroitin sulfate (CS), dermatan sulfate (DS), heparan sul-

fate, keratan sulfate. CSs show different patterns of sulfation: chondroitin-4-

sulfate (C-4-S) and chondroitin-6-sulfate (C-6-S) are the two most common va-

riants present on CSPG (Bandtlow and Zimmermann, 2000). HA, however, is

not sulfated and it is present, protein free, on cell surfaces and in the ECM.

Lecticans or hyalectans (hyaluronan plus lectin) are a group of CSPG charac-

terized by the presence of a hyaluronan-binding domain and a C-type lectin

domain in their core proteins (Yamaguchi, 2000). The interactions between hya-

luronan and CSPGs are stabilized via link proteins (Galtrey and Fawcett, 2007).

They are responsible, thanks to GAG chains, of the well-hydrated and strongly

anionic microenvironment surrounding the neurons (Karetko and Skangiel-

Kramska, 2009). Four lecticans have been identified in CNS, namely aggrecan,

versican, neurocan and brevican.

Tenascin, present in the two forms R and C, is a large molecular weight di-

meric or trimeric ECM glycoprotein expressed predominantly in CNS. It is the

physiological ligand for the C-terminal globular domain of lecticans: tenascin-R

shows higher affinity for brevican while tenascin-C binds strongly with neuro-

can. The presence of tenascin C in PNNs, however, is not essential for their sta-

bility (Yamaguchi, 2000; Galtrey and Fawcett, 2007).

Other CSPGs not included but compatible with HLT model were described

in CNS ECM: RPTPs and phosphacans, decorin and biglycan, NG2 and neu-

roglycan C (see figure 2B). RPTP (receptor-type protein-tyrosine phosphatase

) is a transmembrane protein that sometimes carries GAG chains (it is one of

the so called “part-time” PGs). Phosphacan is a RPTP secreted splice variant

that binds to tenascin and also to cell surface receptors such as neural cell adhe-

sion molecule (NCAM) through its CS GAG chains. Decorin and biglycan are

small leucine-rich proteoglycans. NG2 and neuroglycan C are other transmem-


brane CSPGs able to bind to tenascin (Bandtlow and Zimmermann, 2000; Fox

and Caterson, 2002; Galtrey and Fawcett, 2007).

To these molecules a heparan sulfate should be added: agrin. Essential for

the development and maintenance of the neuromuscular junction, agrin plays

an important role also in anatomical plasticity (Dityatev et al., 2010).

2.2 THE ROLE OF EXTRACELLULAR MATRIX

IN OCULAR DOMINANCE PLASTICITY

Nowadays it is common opinion that the ECM has not only a structural role but

participates in many cell-functions. In CNS, ECM contributes to cell migration,

axonal growth and neuronal plasticity both synaptic and anatomical (Bandtlow

and Zimmermann, 2000, Dityatev and Schachner, 2003). In OD plasticity, a case

of experience dependent structural plasticity, the role of CSPGs in the PNNs,

and of the Tissue plasminogen activator (tPA) was proved (Berardi et al., 2003),

but also other molecules of ECM, such as agrin, may play a role in this process

(Dityatev et al., 2010).

PNNs begin to condense during later development and are complete after

the end of the critical period. Moreover it was demonstrated that DR, treatment

that prevents the closure of the critical period, prevents also the formation of

PNNs. It was therefore proposed a role of PNNs in the end of ocular dominance

plasticity (Hockfield et al., 1990) (see figure 2C). The mechanism by which

CSPGs inhibits plasticity in the adult visual cortex is still unknown. The CSPGs

have an inhibitory action on axonal sprouting during development and in case

of spinal lesion, so they are probably also non-permissive substrates for rear-

rangement of synaptic connections (Berardi et al., 2004). It is important to say,

however, that the organization of CSPGs into perineuronal nets is the key event

in the control of CNS plasticity by ECM: knockout animals for Crtl1 (cartilage

link protein 1), a protein that binds lecticans to HA, despite having unchanged


levels of CSPGs and patterns of GAGs sulfation, retain juvenile levels of OD

plasticity and their visual acuity remains sensitive to MD (Carulli et al., 2010).

Moreover CS-chains are able to act as reservoir of many growth and inhibito-

ry factors. They control localization of these factors promoting or inhibiting sig-

naling and protect them from degradation (Crespo et al., 2007).

tPA activity in the visual cortex is increased during MD in response to the un-

balance between excitatory and inhibitory circuits. It was proposed that tPA

cleaves adhesion molecules of inactive synapses. This initially causes spine mo-

tility and eventually spine retraction. This hypothesis is confirmed by the resto-

ration of normal effects of MD in tPA-knockout mice by treatment with exogen-

ous enzyme (Berardi et al., 2004) (see figure 8).

Agrin is a heparan sulfate normally present in brain ECM. It is object of pro-

teolytic cleavage by neurotrypsin, a serine protease secreted in an inactive form

by the presynaptic terminal in case of action potential firing. The activation of

neurotrypsin is possible only in presence of a NMDA receptors-dependent

postsynaptic process. The cleavage of agrin unmasks a cryptic ECM-resident

signal, a carboxy-terminal 22-kDa fragment (agrin 22), essential for the sprout-

ing of filopodia, protrusions that can evolve in dendritic spines (Dityatev et al.,

2010).

One of the best ways of studying a biologic system is perturbing it. Therefore

the effects of extracellular environment on neuronal plasticity were studied

with an enzymatic tool able to deeply modify brain PNNs and, namely, GAG

chains: chondroitinase ABC (see figure 2D).


C.

Figure 2. The structure of perineuronal nets (PNNs) in adult and immature brain and after chondroitinase treatment

A) A schematic of the base pattern of mature PNNs based on a ternary complex of hyaluronan, tenascin-R and a member of the family of chondroitin sulfate proteoglycans (CSPGs) lecticans.

B) Several subclasses of CSPGs (CS glycosaminoglycans are depicted as red lines) and their relations with other elements of the mature PNNs are showed. Members of the lectican subfamily —neurocan (Nn) present with two cleavage isoforms neurocan-N, (Nn-N) and neurocan-C (Nn-C), versican (Vn), brevican (Bn), and aggrecan (An)— associates with matrix hyaluronan (HA, pink) through globular hya-luronan-binding domains at their N-termini (yellow circles), while their C-terminal globular domains (white circles) binds to the matrix glycoprotein tenascin (T, triangles). Phosphacan (Pn), a secreted CSPG that carries also heparan sulfate (black lines in the figure), as well as to cell surface CSPGs such as neurog-lycan C (NC) and NG2 takes part to PNNs binding to tenascin. Phosphacan can also bind to cell surface receptors such as neural cell adhesion molecule (NCAM).

C) A model of the immature PNNs: a favorable environment for cell migration, axon growth and synaptoge-nesis is given by a loose matrix with a high amount of hyaluronan.

D) Chondroitinase ABC is able to dramatically change extracellular environment degrading HA and CS gly-cosaminoglycans restoring the loose extracellular matrix present before the closure of the critical period.

(modified from Yamaguchi, 2000; Fox and Caterson, 2002)

D.

B. A.

.


3 CHONDROITINASE ABC: BIOCHEMICAL ASPECTS

Chondroitin Sulfate ABC lyase I, usually called Chondroitinase ABC (ChABC

sometimes also Chase), is a 997-amino-acid-residue endolytic enzyme from a

soil bacterium, Proteus vulgaris, that uses it to digest cartilages in animal car-

casses (Huang et al., 2003). ChABC shows chondroitin, C-4-S (in past called

chondroitin sulfate A), DS (chondroitin sulfate B), chondroitin-6-sulfate (chon-

droitin sulfate C), and HA as substrates. It cleaves GAG chains to tetrasaccha-

rides and disaccharides by -elimination of 1,4-hexosaminidic bond (Huang et

al., 2003).

Knowledge of its structure could be necessary to design modifications useful

to have a more effective enzyme in future therapeutic applications (Huang et al.,

2003). Nevertheless its mechanism of action is not still known also due to some

mistakes in its published sequence (Huang et al., 2003; Prabhakar and Capila et

al., 2005). Studies were not able to explain in a conclusive way, for example,

how chABC is able to degrade both epimers of the uronic acid: the glucoronic

acid of the CSs and the iduronic acid of DSs (Prabhakar and Capila et al., 2005;

Prabhakar et al., 2005). A persuasive hypothesis could be the presence of two

partially overlapping active sites catalyzing the respective reactions using the

same substrate-binding site (Shaya et al., 2008).

ChABC has three major domains. The N-terminal domain is the most mobile,

involved in the bond with the glycosaminoglycan chains and contains a NA+

ion. The central one has a horseshoe shape and has catalytic properties. The C-

terminal participates in giving to the molecule a C-shape and an extensive inte-

racting surface (Huang et al., 2003) (see figure 3A). Phylogenetic and site-

directed mutagenesis studies elucidated, in part, the structure of the active site,

showing the presence of a catalytic tetrad (see figure 3B and C) (Huang et al.,

2003; Prabhakar and Capila et al., 2005; Prabhakar et al., 2005).


Figure 3. Molecular structure of chondroitinase-ABC and its active site.

A) ChABC has a horseshoe structure and three domains: an N-terminal one (green), a central one (blue), a C-terminal one (yellow). The red sphere indicates a Na+ ion.

B) The active site consists of a tetrad of residues: Arg560 (stabilizes the charged intermediate); His 501 (necessary for the abstraction of the C-5 proton from the uronic acid moiety); Tyr 508 and Glu653 (both position the substrate via hydrogen bonding). Enzymes mutant for one of these resi-dues are unable to digest their substrates.

C) Schematic of the different amino acids in the active site and their proximity to a C-4-S molecule. The cleavable bond is between the −1 (non-reducing side) and +1 (reducing side) sites

The -elimination of 1,4-hexosaminidic bond needs (I) the abstraction of the C-5 proton from the uronic acid moiety (made by His 501), causing the formation of a double bond between C-4 and C-5, (II) the stabilization of the carbanion intermediate (mediated by Arg 560) (III) the protonation of the anomeric oxygen that breaks the glycosidic bond.

(modified from Huang et al., 2003; Prabhakar and Capila et al., 2005; Prabhakar et al., 2005)

A B

C


4 CHONDROITINASE ABC EFFECTS ON OCULAR

DOMINANCE PLASTICITY

Two studies of the same group (Pizzorusso et al., 2002 and Pizzorusso et al.,

2006) showed that treatment of the visual cortex of rats with ChABC was able to

reopen the OD critical period in adulthood and recover the effects of early MD.

Furthermore the procedure not only did not interfere with functional properties

of cortical neurons but also corrected them in case of early MD. There is no evi-

dence of inflammation of the treated part. It was showed a correlation between

these overall effects and increase of dendritic spine density.

4.1 EXPERIMENTAL PROTOCOLS TO ASSESS CHONDROITINASE SKI LLS

To demonstrate chondroitinase ABC is able to reopen the OD critical period,

three cohorts of adult rats (>100 postnatal days) were used: the first one had an

intracortical injection of chABC in binocular primary visual cortex every three

days and, at the moment of the first injection, was monocularly deprived sutur-

ing the eyelids of the eye contralateral to the treated cortex; the second one had

the same treatment but was given penicillinase, a control enzyme with no en-

dogenous substrate; the third had only MD. Shift in OD distribution was as-

sessed after 7 or 15 days of MD by extracellular recordings of single-unit activi-

ty in the treated cortex.

As expected, no shift was found in only monocularly deprived or penicilli-

nase treated rats, but, according to the hypothesis, a MD shift toward the ipsila-

teral, non-deprived, eye was found in chABC treated rats with no significant

differences between animals deprived for 7 or 15 days (see figure 4) .

Immunofluorescence studies using OX42, an antibody that binds microglia

and neutrophils, revealed an increase in the number of hypertrophic microglia

only in the injection site but not in the recording area (Pizzorusso et al., 2002).


Injection of chABC without MD did not induce a shift of OD toward the ipsi-

lateral eye. Functional properties (visual acuity, the measure of the spatial reso-

lution of sight, and receptive fields) of visual cortical neurons were studied with

visual evoked potentials (VEPs) protocol, based on recording of neuronal activi-

ty after presentation of a visual stimulus (a flashing light). The results were not

statically different from controls (Pizzorusso et al., 2002) (see figure 5).

Chondroitinase was also able to induce a recovery from effects of MD (Pizzo-

russo et al., 2006). The protocol used was based on reverse suturing (RS, depri-

vation of the previously open eye and opening of the previously deprived eye)

that during critical period is able to correct OD. In adult controls RS caused a

limited spontaneous recovery of OD but no improvement in visual acuity.

ChABC treatment allowed, on the contrary, complete recovery of ocular do-

minance and of visual acuity in adult RS rats (see figure 6).

Visual acuity was assessed both by recording VEPs (used also for receptive

field) and by a two-alternative forced-choice discrimination task (visual water

box). In this task a grating is displayed randomly on one of two monitors at the

end of a tank filled with water while in the other screen there is a gray-field.

Animals are trained to swim toward the screens and choose the screen display-

ing the grating. In this way they reach a submerged platform hidden below the

screen and escape from water (Pizzorusso et al., 2006). The spatial frequency of

the gratings is increased until the animals do not choose randomly between the

two monitors. This means that its maximal visual acuity was reached.

Dendritic spine density was assessed by immunolabelling of frozen brain

sections of rats treated with RS or RS and chABC. MD decreases spine density

of visual cortex, especially the contralateral due to the small decussation in the

visual system of the rodents. Treatment with chABC, not per se but after visual

experience and RS, made the dendritic spine density similar to normal animals

(Pizzorusso et al., 2006) (see figure 7).


Figure 4. Chondroitinase ABC reactivates ocular dominance plasticity

The first histogram shows the distribution of ocular dominance (OD) dominance classes in a non treated adult rat (NOR). Monocular deprivation (MD, black histogram) or MD with injection of penicillinase (P-ase, blue histogram) had no effects on OD in adult animals while chondroitinase ABC (chABC) treatment induced a shift toward the non deprived ipsilateral eye with no significant differences between the cohort treated for a week (red histogram) or for two weeks (green histogram).

(modified from Pizzorusso et al., 2002)

(modified from Pizzorusso et al. 2002)

Figure 5. ChABC does not modify functional properties of cortical neurons

A) Histogram distribution of the classes of ocular dominance in rat treated with chABC but without MD is the same of controls: chABC does not induce a shift in OD without MD.

B) Receptive fields (RF) were not altered by treatment with penicillinase or chABC in monocularly deprived or non deprived rats. (data presented by boxplots, square symbol denotes mean value).

C) Cell responsiveness was assessed as the ratio of peak response to baseline. There is no statistical dif-ference between the groups (data presented by boxplots, square symbol denotes mean value).

D) One week chABC treatment does not affect visual acuity. A representative example of visual acuity measurement by means of VEPs is shown. Estimated visual acuity is indicated on the abscissa by an arrowhead. Visual acuity of chABC-treated animals (0.95 ± 0.05 cycle/deg) is not different from normal values.



C

D

Figure 6. ChABC allows recovery of OD in adult RS rats

A) Ocular dominance distribution of RS chABC-treated rats (red histogram) is within the normal values (Nor, blue line). The photo on the right shows the abolishment of WFA staining (Wisteria Floribunda Agglutinin, a marker for perineuronal nets) after chABC treatment in visual cortex.

B) Treatment with penicillinase (P-ase) of RS rats does not induce any shift towards the first deprived eye while its distribution of OD class is similar to the one characteristic of monocularly deprived rats (MD, yellow line). On the right PNNs labeled with WFA appear intact after treatment with P-ase.

C) Visual acuity of the two eyes (in black the deprived one) in controls (nor and P-ase) and chABC treated rats was assessed with VEPs technique. Treatment with chABC promoted recovery of visual acuity of the formerly deprived eye. Asterisks indicate a statistically different group.

D) Also behavioral test (visual water box) gave similar results, showing a functional recovery of visual acuity of the amblyopic eye


Figure 7. ChABC normalizes spine density in adult RS rats

In control rats there is no difference in spine density between the two cortex (Nor, one blue column). RS causes a de-crease of spine density in the contralateral cortex to the formerly treated eye (RS, black columns). P-ase injection does not avoid this process (RS P-ase, green columns). ChABC-treated rats showed, however, a spine density not different from normal rats (RS chABC, red columns). The chABC treatment per se does not promote increase in spine density (chABC).


B

A


4.2 PUTATIVE MECHANISMS OF ACTION OF CHONDROITINASE ABC

There are different hypotheses on the way digestion of ECM with chABC in-

duces neuronal plasticity. It is likely that more factors play a role in such a

complex process (Pizzorusso et al., 2006; Berardi et al., 2004; Crespo et al., 2007).

Ocular dominance plasticity is thought be a case of anatomical plasticity

based on rearrangements of dendritic spines. This is confirmed by the stability

of spines underlined by the study of in vivo two-photon imaging in V1 (Grut-

zendler et al., 2002). It was shown that dynamic modifications of dendritic

spines require extracellular proteolysis (Mataga et al., 2004; Oray et al., 2004). So

ChABC, changing extracellular environment, could restore spine plasticity in

adult. This action is probably direct to either the two principal neuronal species

coated by PNNs: pyramidal neurons and GABAergic parvalbumin containing

inhibitory interneurons. ChABC treatment could restore in this way an intracor-

tical inhibition similar to the one present during critical period (Pizzorusso et al.

2006). In the loose matrix generated by chABC treatment also tPA is probably

able again to depress non-active synapses (Berardi et al., 2004) (see figure 8).

Other possible mechanisms that can take part in chABC effects are correlated

to the release of growth bound to CSPGs chains or effects of some of their diges-

tion products. Likely also the digestion of HA and its digestion products pro-

mote neuronal plasticity but not as much as CSPGs (Crespo et al., 2007).

It was also proposed that CSPGs inhibit directly neurons via a receptor and

this could be supported by the evidence of CSPG-mediated increases of intra-

cellular Ca2+ with inhibitory effects on growth cone. In vitro experiments

showed CSPGs activate protein kinase C (PKC) in cerebellar granule neurons in

vitro. Active PKC has an inhibitory effect on neurite growth mediated by the ac-

tivation of the small GTPase Rho. Recently, it has been discovered that suppres-

sion of the kinase activity of the epidermal growth factor (EGF) diminishes the


inhibitory effects of CSPGs that are able to phosphorylate the EGF-receptor in a

calcium-dependent manner (Crespo et al., 2007).

Figure 8. Anatomical plasticity and tPA: a mechanism model

A) In vivo studies show that in V1 during the critical period changes in spines (indicated by arrows) happen very often while in the adult they are very scarce. This changing is probably mediated by molecules of the ECM like tPA.

B) During the critical period, the imbalance in electrical activity caused by MD activates tPA release. Active syn-apses probably express adhesion molecules insensitive to tPA, while the ones present in inactive synapses are cleavable by it. tPA cleavage initially induces spine motility but eventually causes spine retraction. This ge-nerates a shift of OD in favor of the non-deprived eye (represented by the white eye on the schematic scale).

C) tPA knock-out mice have not a shift in OD even if monocularly deprived.

D) In the adult, the ECM is strongly non-permissive for morphological rearrangements, and tPA is no longer acti-vated following MD. As a result, no changes in spine dynamics or in OD are present.

E) ChABC changes the perineuronal environment promoting spine mobility and modifies the intracortical inhibito-ry circuits responsible of the detection of imbalance in electric activity. This could be a mechanism to explain how chABC induces shift in OD.

(modified from Berardi et al., 2004)


5 CHONDROITINASE ABC-EFFECTS IN OTHER REGIONS : AN OVERVIEW

PNNs, as previously stated, are widely diffuse in the CNS and in the last few

years several studies have explored the effects on neuronal plasticity of the di-

gestion of these structures in many different districts. The aim of these re-

searches is to study the role of ECM in plasticity in the hope to find a way of

promoting recovery from spinal injuries or traumas.

Following CNS injury (e.g. a spinal cord lesion) a glial scar develops, contain-

ing ECM molecules including CSPGs that act as an inhibitory substrate for

axonal growth. Treatment with chondroitinase ABC in rat spinal cord promotes

restoration of plasticity in both injured and intact axons of ascending sensory

projections and descending corticospinal tract (Bradbury et al., 2002; Barritt et

al., 2006; Karetko and Skangiel-Kramska, 2009). CSPGs digestion promoted the

regeneration of damaged dorsal root fibers and sprouting of intact dorsal ones

restoring sensory function (Cafferty et al., 2007, 2008; Karetko and Skangiel-

Kramska, 2009). These results make the use of chABC in the treatment of spinal

cord lesions the most promising application of this enzyme.

Also digestion of the PNNs present in the amygdale after fear conditioning

experiments gave interesting results. Fear conditioning in adult rats leads to the

formation of memories that cannot be erased: spontaneous recovery of condi-

tioned fear responses after extinction training shows that extinction involves

new learning and not an erasure of previous fear memories. In young rats,

however, extinction of conditioned fear induces memory erasure. This is proba-

bly due to the active protection against erasure given by PNNs that are present

in the adult amygdale. ChABC treatment digesting PNNS was able to make fear

memory erasable. These observations could lead to new strategies in treating

post-traumatic stress disorders (Gogolla et al., 2009; Pizzorusso, 2009).


CONCLUSIONS

Studies on ChABC effects on OD plasticity are a fascinating example on how

basic research could lead to findings of great clinical potential. They outline a

new paradigm in treating disorders of brain development based on the search

of factors promoting restoration of the plasticity typical of the immature brain.

They show, besides, a pivotal role of brain ECM in many essential neuronal

functions and so it is probable that alteration of PNNs could be involved in the

pathogenesis of some neurological and neuropsychiatric disorders.

Even if these results are very promising many questions have to be solved

before thinking of therapeutic application of ChABC.

Histological studies have to go on: the composition and the architecture of

brain ECM are still mysterious and our staining methods reveal probably only a

part of the PNNs present in the brain (Ajmo et al., 2008).

Besides, we know still too little about the role of ECM molecules in both syn-

aptic and anatomical plasticity and we do not understand the functional mean-

ing of the large variety of GAGs pattern of sulfation and of PGs splicing va-

riants. Use of other enzymes with different substrates and of knockout organ-

isms will help to comprehend such a complex issue.

An even more intriguing topic to solve is elucidating the role and possibili-

ties of the endogenous regulation of ECM represented by some enzymes as

neurotrypsin and metalloproteinases (Dityatev et al., 2010). Use of these en-

zymes could overwhelm immune response against chABC or its cleavage prod-

ucts and lead to more specific actions on ECM than the wide destruction oper-


ated by ChABC. Such a deep modification of neuronal environment could lead

to detrimental side effects such facilitation of tumor diffusion (Fox and Cater-

son, 2002).

Neuronal plasticity is a complex process that involves many different kinds

of molecules: it is likely that chABC treatment alone will not be able to give a

complete recovery from development disorders or CNS injuries, but should be

supported by supply of neurotrophins and other plasticity promoting factors.

Among these factors the effect on V1 of agrin 22 injection should be studied

with attention, due to its involvement in experience dependent plasticity.

Experiments combining different factors could assess the effectiveness of a

multiple approach to neuronal plasticity.

Studies should also be conducted about dosage, frequency, and timing of the

treatment to understand better its action and discover its best potentialities.

A caveat for the application of ChABC in the treatment of amblyopia is

represented by the deep differences between the almost monocular rodent vis-

ual system and the widely binocular human one. An important test for this the-

rapeutic target will be the use of primates as animal models in experiments on

OD plasticity.


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Chondroitinase ABC-mediated Ocular Dominance Plasticity

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