Daniela Carulli

Chondroitin sulfate proteoglycans in neural development and regeneration

Proteoglycans are of two main types, chondroitin sulfate (CSPGs) and heparin sulfate (HSPGs). The CSPGs act mainly as barrier-forming molecules, whereas the HSPGs stabilise the interactions of receptors and ligands. During development CSPGs pattern cell migration, axon growth pathways and axon terminations. Later in development and in adulthood CSPGs associate with some classes of neuron and control plasticity. After damage to the nervous system, CSPGs are the major axon growth inhibitory component of the glial scar tissue that blocks successful regeneration. CSPGs have a variety of roles in the nervous system, including binding to molecules and blocking their action, presenting molecules to cells and axons, localising active molecules to particular sites and presenting growth factors to their receptors.

Animals lacking link protein have attenuated perineuronal nets and persistent plasticity.

Chondroitin sulphate proteoglycans in the extracellular matrix restrict plasticity in the adult central nervous system and their digestion with chondroitinase reactivates plasticity. However the structures in the extracellular matrix that restrict plasticity are unknown. There are many changes in the extracellular matrix as critical periods for plasticity close, including changes in chondroitin sulphate proteoglycan core protein levels, changes in glycosaminoglycan sulphation and the appearance of dense chondroitin sulphate proteoglycan-containing perineuronal nets around many neurons. We show that formation of perineuronal nets is triggered by neuronal production of cartilage link protein Crtl1 (Hapln1), which is up-regulated in the visual cortex as perineuronal nets form during development and after dark rearing. Mice lacking Crtl1 have attenuated perineuronal nets, but the overall levels of chondroitin sulphate proteoglycans and their pattern of glycan sulphation are unchanged. Crtl1 knockout animals retain juvenile levels of ocular dominance plasticity and their visual acuity remains sensitive to visual deprivation. In the sensory pathway, axons in knockout animals but not controls sprout into the party denervated cuneate nucleus. The organization of chondroitin sulphate proteoglycan into perineuronal nets is therefore the key event in the control of central nervous system plasticity by the extracellular matrix.

Composition of perineuronal nets in the adult rat cerebellum and the cellular origin of their components

The decrease in plasticity that occurs in the central nervous system during postnatal development is accompanied by the appearance of perineuronal nets (PNNs) around the cell body and dendrites of many classes of neuron. These structures are composed of extracellular matrix molecules, such as chondroitin sulfate proteoglycans (CSPGs), hyaluronan (HA), tenascin‐R, and link proteins. To elucidate the role played by neurons and glial cells in constructing PNNs, we studied the expression of PNN components in the adult rat cerebellum by immunohistochemistry and in situ hybridization. In the deep cerebellar nuclei, only large excitatory neurons were surrounded by nets, which contained the CSPGs aggrecan, neurocan, brevican, versican, and phosphacan, along with tenascin‐R and HA. Whereas both net‐bearing neurons and glial cells were the sources of CSPGs and tenascin‐R, only the neurons expressed the mRNA for HA synthases (HASs), cartilage link protein, and link protein Bral2. In the cerebellar cortex, Golgi neurons possessed PNNs and also synthesized HASs, cartilage link protein, and Bral2 mRNAs. To see whether HA might link PNNs to the neuronal cell surface by binding to a receptor, we investigated the expression of the HA receptors CD44, RHAMM, and LYVE‐1. No immunolabelling for HA receptors on the membrane of net‐bearing neurons was found. We therefore propose that HASs, which can retain HA on the cell surface, may act as a link between PNNs and neurons. Thus, HAS and link proteins might be key molecules for PNN formation and stability. J. Comp. Neurol. 494:559–577, 2006.

Chondroitin 6-sulphate synthesis is up-regulated in injured CNS, induced by injury-related cytokines and enhanced in axon-growth inhibitory glia.

Chondroitin sulphate proteoglycans (CSPGs) are up‐regulated in the CNS after injury and inhibit axon regeneration mainly through their glycosaminoglycan (CS‐GAG) chains. We have analysed the mRNA levels of the CS‐GAG synthesizing enzymes and measured the CS‐GAG disaccharide composition by chromatography and immunocytochemistry. Chondroitin 6‐sulfotransferase 1 (C6ST1) is up‐regulated in most glial types around cortical injuries, and its sulphated product CS‐C is also selectively up‐regulated. Treatment with TGFα and TGFβ, which are released after brain injury, promotes the expression of C6ST1 and the synthesis of 6‐sulphated CS‐GAGs in primary astrocytes. Oligodendrocytes, oligodendrocyte precursors and meningeal cells are all inhibitory to axon regeneration, and all express high levels of CS‐GAG, including high levels of 6‐sulphated GAG. In axon growth‐inhibitory Neu7 astrocytes C6ST1 and 6‐sulphated GAGs are expressed at high levels, whereas in permissive A7 astrocytes they are not detectable. These results suggest that the up‐regulation of CSPG after CNS injury is associated with a specific sulphation pattern on CS‐GAGs, mediating the inhibitory properties of proteoglycans on axonal regeneration.

Distribution and synthesis of extracellular matrix proteoglycans, hyaluronan, link proteins and tenascin‐R in the rat spinal cord

Perineuronal nets (PNNs) are dense extracellular matrix (ECM) structures that form around many neuronal cell bodies and dendrites late in development. They contain several chondroitin sulphate proteoglycans (CSPGs), hyaluronan, link proteins and tenascin‐R. Their time of appearance correlates with the ending of the critical period for plasticity, and they have been implicated in this process. The distribution of PNNs in the spinal cord was examined using Wisteria floribunda agglutinin lectin and staining for chondroitin sulphate stubs after chondroitinase digestion. Double labelling with the neuronal marker, NeuN, showed that PNNs were present surrounding ∼ 30% of motoneurons in the ventral horn, 50% of large interneurons in the intermediate grey and 20% of neurons in the dorsal horn. These PNNs formed in the second week of postnatal development. Immunohistochemical staining demonstrated that the PNNs contain a mixture of CSPGs, hyaluronan, link proteins and tenascin‐R. Of the CSPGs, aggrecan was present in all PNNs while neurocan, versican and phosphacan/RPTPβ were present in some but not all PNNs. In situ hybridization showed that aggrecan and cartilage link protein (CRTL 1) and brain link protein‐2 (BRAL 2) are produced by neurons. PNN‐bearing neurons express hyaluronan synthase, and this enzyme and phosphacan/RPTPβ may attach PNNs to the cell surface. During postnatal development the expression of link protein and aggrecan mRNA is up‐regulated at the time of PNN formation, and these molecules may therefore trigger their formation.

Upregulation of aggrecan, link protein 1, and hyaluronan synthases during formation of perineuronal nets in the rat cerebellum

Extracellular matrix molecules accumulate around central nervous system neurons during postnatal development, forming so‐called perineuronal nets (PNNs). PNNs play a role in restricting plasticity at the end of critical periods. In the adult rat cerebellum, PNNs are found around large, deep cerebellar nuclei (DCN) neurons and Golgi neurons and are composed of chondroitin sulfate proteoglycans (CSPGs), tenascin‐R (TN‐R), hyaluronan (HA), and link proteins, such as cartilage link protein 1 (Crtl1). Granule cells and Purkinje cells are surrounded by a partially organized matrix. Both glial cells and neurons surrounded by PNNs are the site of synthesis of some CSPGs and of TN‐R, but only neurons produce HA synthetic enzymes (HASs), thus HA, and link proteins, which are scaffolding molecules for an organized matrix. To elucidate the mechanisms of formation of PNNs, we analyzed by immunohistochemistry and in situ hybridization which PNN components are upregulated during PNN formation in rat cerebellar postnatal development and what cell types express them. We observed that Wisteria floribunda agglutinin‐binding PNNs develop around DCN neurons from postnatal day (P)7 and around Golgi neurons from P14. At the same time as their PNNs start to form, these neurons upregulate aggrecan, Crtl1, and HASs mRNAs. However, Crtl1 is the only PNN component to be expressed exclusively in neurons surrounded by PNNs. The other link protein that shows a perineuronal net pattern in the DCN, Bral2, is upregulated later during development. These data suggest that aggrecan, HA, and, particularly, Crtl1 might be crucial elements for the initial assembly of PNNs. J. Comp. Neurol. 501:83–94, 2007.

Experience-Dependent Plasticity and Modulation of Growth Regulatory Molecules at Central Synapses

Structural remodeling or repair of neural circuits depends on the balance between intrinsic neuronal properties and regulatory cues present in the surrounding microenvironment. These processes are also influenced by experience, but it is still unclear how external stimuli modulate growth-regulatory mechanisms in the central nervous system. We asked whether environmental stimulation promotes neuronal plasticity by modifying the expression of growth-inhibitory molecules, specifically those of the extracellular matrix. We examined the effects of an enriched environment on neuritic remodeling and modulation of perineuronal nets in the deep cerebellar nuclei of adult mice. Perineuronal nets are meshworks of extracellular matrix that enwrap the neuronal perikaryon and restrict plasticity in the adult CNS. We found that exposure to an enriched environment induces significant morphological changes of Purkinje and precerebellar axon terminals in the cerebellar nuclei, accompanied by a conspicuous reduction of perineuronal nets. In the animals reared in an enriched environment, cerebellar nuclear neurons show decreased expression of mRNAs coding for key matrix components (as shown by real time PCR experiments), and enhanced activity of matrix degrading enzymes (matrix metalloproteinases 2 and 9), which was assessed by in situ zymography. Accordingly, we found that in mutant mice lacking a crucial perineuronal net component, cartilage link protein 1, perineuronal nets around cerebellar neurons are disrupted and plasticity of Purkinje cell terminal is enhanced. Moreover, all the effects of environmental stimulation are amplified if the afferent Purkinje axons are endowed with enhanced intrinsic growth capabilities, induced by overexpression of GAP-43. Our observations show that the maintenance and growth-inhibitory function of perineuronal nets are regulated by a dynamic interplay between pre- and postsynaptic neurons. External stimuli act on this interaction and shift the balance between synthesis and removal of matrix components in order to facilitate neuritic growth by locally dampening the activity of inhibitory cues.

In vitro modeling of perineuronal nets: hyaluronan synthase and link protein are necessary for their formation and integrity

J. Neurochem. (2010) 114, 1447–1459.

The chemorepulsive axon guidance protein semaphorin3A is a constituent of perineuronal nets in the adult rodent brain

In the adult rodent brain, subsets of neurons are surrounded by densely organised extracellular matrix called perineuronal nets (PNNs). PNNs consist of hyaluronan, tenascin-R, chondroitin sulphate proteoglycans (CSPGs), and the link proteins Crtl1 and Bral2. PNNs restrict plasticity at the end of critical periods and can be visualised with Wisteria floribunda agglutinin (WFA). Using a number of antibodies raised against the different regions of semaphorin3A (Sema3A) we demonstrate that this secreted chemorepulsive axon guidance protein is localised to WFA-positive PNNs around inhibitory interneurons in the cortex and several other PNN-bearing neurons throughout the brain and co-localises with aggrecan, versican, phosphacan and tenascin-R. Chondroitinase ABC (ChABC) was injected in the cortex to degrade glycosaminoglycans (GAGs) from the CSPGs, abolishing WFA staining of PNNs around the injection site. Sema3A-positive nets were no longer observed in the area devoid of WFA staining. In mice lacking the link protein Crtl1 in the CNS only vestigial PNNs are present, and in these mice there were no Sema3A-positive PNN structures. A biochemical analysis shows that Sema3A protein binds with high-affinity to CS-GAGs and aggrecan and versican extracted from PNNs in the adult rat brain, and a significant proportion of Sema3A is retrieved in brain extracts that are enriched in PNN-associated GAGs. The Sema3A receptor components PlexinA1 and A4 are selectively expressed by inhibitory interneurons in the cortex that are surrounded by Sema3A positive PNNs. We conclude that the chemorepulsive axon guidance molecule Sema3A is present in PNNs of the adult rodent brain, bound to the GAGs of the CSPGs. These observations suggest a novel concept namely that chemorepulsive axon guidance molecules like Sema3A may be important functional attributes of PNNs in the adult brain.

A change in the pattern of activity affects the developmental regression of the purkinje cell polyinnervation by climbing fibers in the rat cerebellum

Pattern of activity during development is important for the refinement of the final architecture of the brain. In the cerebellar cortex, the regression from multiple to single climbing fiber innervation of the Purkinje cell occurs during development between postnatal days (P) 5 and 15. However, the regression is hampered by altering in various ways the morpho-functional integrity of the parallel fiber input. In rats we disrupted the normal activity pattern of the climbing fiber, the terminal arbor of the inferior olive neurons, by administering harmaline for 4 days from P9 to P12. At all studied ages (P15-87) after harmaline treatment multiple (double only) climbing fiber EPSC-steps persist in 28% of cells as compared with none in the control. The ratio between the amplitudes of the larger and the smaller climbing fiber-evoked EPSC increases in parallel with the decline of the polyinnervation factor, indicating a gradual enlargement of the synaptic contribution of the winning climbing fiber synapse at the expense of the losing one. Harmaline treatment had no later effects on the climbing fiber EPSC kinetics and I/V relation in Purkinje cells (P15-36). However, there was a rise in the paired-pulse depression indicating a potentiation of the presynaptic mechanisms. In the same period, after harmaline treatment, parallel fiber-Purkinje cell electrophysiology was unaffected. The distribution of parallel fiber synaptic boutons was also not changed. Thus, a change in the pattern of activity during a narrow developmental period may affect climbing fiber-Purkinje cell synapse competition resulting in occurrence of multiple innervation at least up to 3 months of age. Our results extend the current view on the role of the pattern of activity in the refinement of neuronal connections during development. They suggest that many similar results obtained by different gene or receptor manipulations might be simply the consequence of disrupting the pattern of activity.