The perineuronal net and the control of CNS plasticity

Perineuronal nets (PNNs) are reticular structures that surround the cell body of many neurones, and extend along their dendrites. They are considered to be a specialized extracellular matrix in the central nervous system (CNS). PNN formation is first detected relatively late in development, as the mature synaptic circuitry of the CNS is established and stabilized. Its unique distribution in different CNS regions, the timing of its establishment, and the changes it undergoes after injury all point toward diverse and important functions that it may be performing. The involvement of PNNs in neuronal plasticity has been extensively studied over recent years, with developmental, behavioural, and functional correlations. In this review, we will first briefly detail the structure and organization of PNNs, before focusing our discussion on their unique roles in neuronal development and plasticity. The PNN is an important regulator of CNS plasticity, both during development and into adulthood. Production of critical PNN components is often triggered by appropriate sensory experiences during early postnatal development. PNN deposition around neurones helps to stabilize the established neuronal connections, and to restrict the plastic changes due to future experiences within the CNS. Disruption of PNNs can reactivate plasticity in many CNSs, allowing activity-dependent changes to once again modify neuronal connections. The mechanisms through which PNNs restrict CNS plasticity remain unclear, although recent advances promise to shed additional light on this important subject.     

Extracellular matrix and perineuronal nets in CNS repair

A perineuronal net (PNN) is a layer of lattice-like matrix which enwraps the surface of the soma and dendrites, and in some cases the axon initial segments, in sub-populations of neurons in the central nervous system (CNS). First reported by Camillo Golgi more than a century ago, the molecular structure and the potential role of this matrix have only been unraveled in the last few decades. PNNs are mainly composed of hyaluronan, chondroitin sulfate proteoglycans, link proteins, and tenascin R. The interactions between these molecules allow the formation of a stable pericellular complex surrounding synapses on the neuronal surface. PNNs appear late in development co-incident with the closure of critical periods for plasticity. They play a direct role in the control of CNS plasticity, and their removal is one way in which plasticity can be re-activated in the adult CNS. In this review, we examine the molecular components and formation of PNNs, their role in maturation and synaptic plasticity after CNS injury, and the possible mechanisms of PNN action.     

Composition of perineuronal net extracellular matrix in rat brain: a different disaccharide composition for the net-associated proteoglycans

We developed a method to extract differentially chondroitin sulfate proteoglycans (CSPGs) that are diffusely present in the central nervous system (CNS) matrix and CSPGs that are present in the condensed matrix of perineuronal nets (PNNs). Adult rat brain was sequentially extracted with Tris-buffered saline (TBS), TBS-containing detergent, 1 m NaCl, and 6 m urea. Extracting tissue sections with these buffers showed that the diffuse and membrane-bound CSPGs were extracted in the first three buffers, but PNN-associated CSPGs remained and were only removed by 6 m urea. Most of the CSPGs were extracted to some degree with all the buffers, with neurocan, brevican, aggrecan, and versican particularly associated with the stable urea-extractable PNNs. The CSPGs in stable complexes only extractable in urea buffer are found from postnatal day 7-14 coinciding with PNN formation. Disaccharide composition analysis indicated a different glycosaminoglycan (GAG) composition for PGs strongly associated with extracellular matrix (ECM). For CS/dermatan sulfate (DS)-GAG the content of nonsulfated, 6-O-sulfated, 2,6-O-disulfated, and 4,6-O-disulfated disaccharides were higher and for heparan sulfate (HS)-GAG, the content of 6-O-sulfated, 2-N-, 6-O-disulfated, 2-O-, 2-N-disulfated, and 2-O-, 2-N-, 6-O-trisulfated disaccharides were higher in urea extract compared with other buffer extracts. Digestions with chondroitinase ABC and hyaluronidase indicated that aggrecan, versican, neurocan, brevican, and phosphacan are retained in PNNs through binding to hyaluronan (HA). A comparison of the brain and spinal cord ECM with respect to CSPGs indicated that the PNNs in both parts of the CNS have the same composition.     

Semaphorin 3A Binds to the Perineuronal Nets via Chondroitin Sulfate Type E Motifs in Rodent Brains

Chondroitin sulfate (CS) and the CS-rich extracellular matrix structures called perineuronal nets (PNNs) restrict plasticity and regeneration in the CNS. Plasticity is enhanced by chondroitinase ABC treatment that removes CS from its core protein in the chondroitin sulfate proteoglycans or by preventing the formation of PNNs, suggesting that chondroitin sulfate proteoglycans in the PNNs control plasticity. Recently, we have shown that semaphorin3A (Sema3A), a repulsive axon guidance molecule, localizes to the PNNs and is removed by chondroitinase ABC treatment (Vo, T., Carulli, D., Ehlert, E. M., Kwok, J. C., Dick, G., Mecollari, V., Moloney, E. B., Neufeld, G., de Winter, F., Fawcett, J. W., and Verhaagen, J. (2013) Mol. Cell. Neurosci. 56C, 186–200). Sema3A is therefore a candidate for a PNN effector in controlling plasticity. Here, we characterize the interaction of Sema3A with CS of the PNNs. Recombinant Sema3A interacts with CS type E (CS-E), and this interaction is involved in the binding of Sema3A to rat brain-derived PNN glycosaminoglycans, as demonstrated by the use of CS-E blocking antibody GD3G7. In addition, we investigate the release of endogenous Sema3A from rat brain by biochemical and enzymatic extractions. Our results confirm the interaction of Sema3A with CS-E containing glycosaminoglycans in the dense extracellular matrix of rat brain. We also demonstrate that the combination of Sema3A and PNN GAGs is a potent inhibitor of axon growth, and this inhibition is reduced by the CS-E blocking antibody. In conclusion, Sema3A binding to CS-E in the PNNs may be a mechanism whereby PNNs restrict growth and plasticity and may represent a possible point of intervention to facilitate neuronal plasticity.     

Extracellular Matrix in Neural Plasticity and Regeneration

The extracellular matrix (ECM) is a fundamental component of biological tissues. The ECM in the central nervous system (CNS) is unique in both composition and function. Functions such as learning, memory, synaptogenesis, and plasticity are regulated by numerous ECM molecules. The neural ECM acts as a non-specific physical barrier that modulates neuronal plasticity and axon regeneration. There are two specialized types of ECM in the CNS, diffuse perisynaptic ECM and condensed ECM, which selectively surround the perikaryon and initial part of dendritic trees in subtypes of neurons, forming perineuronal nets. This review presents the current knowledge about the role of important neuronal ECM molecules in maintaining the basic functions of a neuron, including electrogenesis and the ability to form neural circuits. The review mainly focuses on the role of ECM components that participate in the control of key events such as cell survival, axonal growth, and synaptic remodeling. Particular attention is drawn to the numerous molecular partners of the main ECM components. These regulatory molecules are integrated into the cell membrane or disposed into the matrix itself in solid or soluble form. The interaction of the main matrix components with molecular partners seems essential in molecular mechanisms controlling neuronal functions. Special attention is paid to the chondroitin sulfate proteoglycan 4, type 1 transmembrane protein, neural-glial antigen 2 (NG2/CSPG4), whose cleaved extracellular domain is such a molecular partner that it not only acts directly on neural and vascular cells, but also exerts its influence indirectly by binding to resident ECM molecules.

     

Matrix metalloproteinase‐3 in the central nervous system: a look on the bright side

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 extracellular 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 neurodegenerative 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 development 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.     

Differential expression of several molecules of the extracellular matrix in functionally and developmentally distinct regions of rat spinal cord

We have examined the regional distribution of several chondroitin sulfate proteoglycans (neurocan, brevican, versican, aggrecan, phosphacan), of their glycosaminoglycan moieties, and of tenascin-R in the spinal cord of adult rat. The relationships of these molecules with glial and neuronal populations, identified with appropriate markers, were investigated by using multiple fluorescence labeling combined with confocal microscopy. The results showed that the distribution of the examined molecules was similar at all spinal cord levels but displayed area-specific differences along the dorso-ventral axis, delimiting functionally and developmentally distinct areas. In the gray matter, laminae I and II lacked perineuronal nets (PNNs) of extracellular matrix and contained low levels of chondroitin sulfate glycosaminoglycans (CS-GAGs), brevican, and tenascin-R, possibly favoring the maintenance of local neuroplastic properties. Conversely, CS-GAGs, brevican, and phosphacan were abundant, with numerous thick PNNs, in laminae III-VIII and X. Motor neurons (lamina IX) were surrounded by PNNs that contained all molecules investigated but displayed various amounts of CS-GAGs. Double-labeling experiments showed that the presence of PNNs could not be unequivocally related to specific classes of neurons, such as motor neurons or interneurons identified by their expression of calcium-binding proteins (parvalbumin, calbindin, calretinin). However, a good correlation was found between PNNs rich in CS-GAGs and the neuronal expression of the Kv3.1b subunit of the potassium channel, a marker of fast-firing neurons. This observation confirms the correlation between the electrophysiological properties of these neurons and the specific composition of their microenvironment.     

Altered synaptic marker abundance in the hippocampal stratum oriens of Ts65Dn mice is associated with exuberant expression of versican

DS (Down syndrome), resulting from trisomy of chromosome 21, is the most common cause of genetic mental retardation; however, the molecular mechanisms underlying the cognitive deficits are poorly understood. Growing data indicate that changes in abundance or type of CSPGs (chondroitin sulfate proteoglycans) in the ECM (extracellular matrix) can influence synaptic structure and plasticity. The purpose of this study was to identify changes in synaptic structure in the hippocampus in a model of DS, the Ts65Dn mouse, and to determine the relationship to proteoglycan abundance and/or cleavage and cognitive disability. We measured synaptic proteins by ELISA and changes in lectican expression and processing in the hippocampus of young and old Ts65Dn mice and LMCs (littermate controls). In young (5 months old) Ts65Dn hippocampal extracts, we found a significant increase in the postsynaptic protein PSD-95 (postsynaptic density 95) compared with LMCs. In aged (20 months old) Ts65Dn hippocampus, this increase was localized to hippocampal stratum oriens extracts compared with LMCs. Aged Ts65Dn mice exhibited impaired hippocampal-dependent spatial learning and memory in the RAWM (radial-arm water maze) and a marked increase in levels of the lectican versican V2 in stratum oriens that correlated with the number of errors made in the final RAWM block. Ts65Dn stratum oriens PNNs (perineuronal nets), an extension of the ECM enveloping mostly inhibitory interneurons, were dispersed over a larger area compared with LMC mice. Taken together, these data suggest a possible association with alterations in the ECM and inhibitory neurotransmission in the Ts65Dn hippocampus which could contribute to cognitive deficits.     

The many faces of EMMPRIN—Roles in neuroinflammation

The central nervous system (CNS) is a relatively immune-privileged organ, wherein a well-instated barrier system (the blood-brain barrier) prevents the entry of blood cells into the brain with the exception of regular immune surveillance cells. Despite this tight security immune cells are successful in entering the CNS tissue where they result in states of neuroinflammation, tissue damage and cell death. Various components of the blood-brain barrier and infiltrating cells have been examined to better understand how blood cells are able to breach this secure barrier. Proteases, specifically matrix metalloproteinases (MMP), have been found to be the common culprits in most diseases involving neuroinflammation. MMPs secreted by infiltrating cells act specifically upon targets on various components of the blood-brain barrier, compromising this barrier and allowing cell infiltration into the CNS. Extracellular matrix metalloproteinase inducer (EMMPRIN) is an upstream inducer of several MMPs and is suggested to be the master regulator of MMP production in disease states such as cancer metastasis. EMMPRIN in the context of the CNS is still relatively understudied. In this review we will introduce EMMPRIN, discuss its ligands and roles in non-CNS conditions that can help implicate its involvement in CNS disorders, showcase its expression within the CNS in healthy and disease conditions, elucidate its ligands and receptors, and briefly discuss the emerging roles it plays in various diseases of the CNS involving inflammation.     

Perineuronal nets play a role in regulating striatal function in the mouse

The striatum is the primary input nucleus of the basal ganglia, a collection of nuclei that play important roles in motor control and associative learning. We have previously reported that perineuronal nets (PNNs), aggregations of chondroitin-sulfate proteoglycans (CSPGs), form in the matrix compartment of the mouse striatum during the second postnatal week. This period overlaps with important developmental changes, including the attainment of an adult-like gait. Here, we investigate the identity of the cells encapsulated by PNNs, characterize their topographical distribution and determine their function by assessing the impact of enzymatic digestion of PNNs on two striatum-dependent behaviors: ambulation and goal-directed spatial learning. We show PNNs are more numerous caudally, and that a substantial fraction (41%) of these structures surrounds parvalbumin positive (PV+) interneurons, while approximately 51% of PV+ cells are ensheathed by PNNs. The colocalization of these structures is greatest in dorsal, lateral and caudal regions of the striatum. Bilateral digestion of striatal PNNs led to an increase in both the width and variability of hind limb gait. Intriguingly, this also resulted in an improvement in the acquisition rate of the Morris water maze. Together, these data show that PNNs are associated with specific elements of striatal circuits and play a key role in regulating the function of this important structure in the mouse.     

Expression of matrix metalloproteinases and their tissue inhibitor during viral encephalitis

Matrix metalloproteinases (MMPs) participate in remodeling the extracellular matrix and facilitate entry of inflammatory cells into tissues. Infection of the murine central nervous system (CNS) with a neurotropic coronavirus induces encephalitis associated with increased levels of mRNA encoding MMP-3 and MMP-12. Whereas virus-induced MMP-3 expression was restricted to CNS resident astrocytes, MMP-12 mRNA was expressed by both inflammatory cells and CNS resident cells. Immunosuppression increased both MMP-3 and MMP-12 mRNA levels in CNS resident cells, suggesting that the presence of virus rather than inflammation induced protease up-regulation. MMP activity is partially regulated by a small family of genes encoding tissue inhibitors of matrix metalloproteinases (TIMPs); among the TIMPs, only TIMP-1 mRNA expression increased in the CNS following coronavirus infection. During inflammation TIMP-1 mRNA was most prominently expressed by infiltrating cells. By contrast, in the immunosuppressed host TIMP-1 mRNA was expressed by CNS resident cells. Analysis of cytokine and chemokine mRNA induction within the infected CNS of healthy and immunocompromised mice suggested a possible correlation between increased viral replication and increased levels of beta interferon, MMP-3, MMP-12, and TIMP-1 mRNA. CD4+ T cells which localize to the perivascular and subarachnoid spaces were identified as the primary source of TIMP-1 protein. By contrast, protein expression was undetectable in astrocytes or CD8+ T cells, the primary antiviral effectors that localize to the CNS parenchyma in response to infection. These data suggest that in contrast to the results seen with MMPs, inhibition of protease activity via TIMP-1 expression correlates with the differential tissue distribution of T-cell subsets during acute coronavirus-induced encephalitis.     

Thematic Minireview Series: Molecular Mechanisms of Synaptic Plasticity

The human brain contains ∼86 billion neurons, which are precisely organized in specific brain regions and nuclei. High fidelity synaptic communication between subsets of neurons in specific circuits is required for most human behaviors, and is often disrupted in neuropsychiatric disorders. The presynaptic axon terminals of one neuron release neurotransmitters that activate receptors on multiple postsynaptic neuron targets to induce electrical and chemical responses. Typically, postsynaptic neurons integrate signals from multiple presynaptic neurons at thousands of synaptic inputs to control downstream communication to the next neuron in the circuit. Importantly, the strength (or efficiency) of signal transmission at each synapse can be modulated on time scales ranging up to the lifetime of the organism. This “synaptic plasticity” leads to changes in overall neuronal circuit activity, resulting in behavioral modifications. This series of minireviews will focus on recent advances in our understanding of the molecular and cellular mechanisms that control synaptic plasticity.     

MMPs in the central nervous system: where the good guys go bad

Matrix metalloproteinases (MMPs) are expressed in the developing, healthy adult and diseased CNS. We emphasize the regulation of neurogenesis and oligodendrogenesis by MMPs during CNS development, and highlight physiological roles of MMPs in the healthy adult CNS, such as in synaptic plasticity, learning and memory. Nonetheless, MMPs as "the good guys" go bad in neurological conditions, likely aided by the sudden and massive upregulation of several MMP members. We stress the necessity of drawing a fine balance in the treatment of neurological diseases, and we suggest that MMP inhibitors do have therapeutic potential early after CNS injury.     

Activity dependent removal of agrin from synaptic basal lamina by matrix metalloproteinase 3

Agrin is a heparan sulfate proteoglycan, which plays an essential role in the development and maintenance of the neuromuscular junction. Agrin is a stable component of the synaptic basal lamina and strong evidence supports the hypothesis that agrin directs the formation of the postsynaptic apparatus, including aggregates of AChRs, and junctional folds. Changes in the distribution of agrin during synaptic remodeling, denervation and reinnervation reveal that agrin can be quickly and efficiently removed from the synaptic basal lamina in a regulated manner. In order to fully understand this mechanism we sought to identify those molecules that were responsible for the removal of agrin. Matrix Metalloproteinases (MMPs) were the most likely molecules since MMPs are involved in the regulation of the pericellular space, including the cleavage of matrix proteins. In particular, MMP3 has been shown to be effective in cleaving heparan sulfate proteoglycans. Antibodies to MMP3 recognize molecules concentrated in the extracellular matrix of perisynaptic Schwann cells. MMP3 specific phylogenic compounds reveal that active MMP3 is localized to the neuromuscular junction. Purified recombinant MMP3 can directly cleave agrin, and it can also remove agrin from synaptic basal lamina. MMP3 activity is itself regulated as activation of MMP3 is lost in denervated muscles. MMP3 null mutant mice have altered neuromuscular junction structure and function, with increased AChRs, junctional folds and agrin immunoreactivity. Altogether these results support the hypothesis that synaptic activity induces the activation of MMP3, and the activated MMP3 removes agrin from the synaptic basal lamina.     

Oxidative stress activates protein tyrosine kinase and matrix metalloproteinases leading to blood-brain barrier dysfunction

The blood-brain barrier (BBB) formed by brain microvascular endothelial cells (BMVEC) regulates the passage of molecules and leukocytes in and out of the brain. Oxidative stress is a major underlying cause of neurodegenerative and neuroinflammatory disorders and BBB injury associated with them. Using human BMVEC grown on porous membranes covered with basement membrane (BM) matrix (BBB models), we demonstrated that reactive oxygen species (ROS) augmented permeability and monocyte migration across BBB. ROS activated matrix metalloproteinases (MMP-1, -2, and -9) and decreased tissue inhibitors of MMPs (TIMP-1 and -2) in a protein tyrosine kinase (PTK)-dependent manner. Increase in MMPs and PTK activities paralleled degradation of BM protein and enhanced tyrosine phosphorylation of tight junction (TJ) protein. These effects and enhanced permeability/monocyte migration were prevented by inhibitors of MMPs, PTKs, or antioxidant suggesting that oxidative stress caused BBB injury via degradation of BM protein by activated MMPs and by PTK-mediated TJ protein phosphorylation. These findings point to new therapeutic interventions ameliorating BBB dysfunction in neurological disorders such as stroke or neuroinflammation.     

The Gut-Brain Axis,BDNF, NMDA and CNS Disorders

Gastro-intestinal (GI) microbiota and the ‘gut-brain axis’ are proving to be increasingly relevant to early brain development and the emergence of psychiatric disorders. This review focuses on the influence of the GI tract on Brain-Derived Neurotrophic Factor (BDNF) and its relationship with receptors for N-methyl-d-aspartate (NMDAR), as these are believed to be involved in synaptic plasticity and cognitive function. NMDAR may be associated with the development of schizophrenia and a range of other psychopathologies including neurodegenerative disorders, depression and dementias. An analysis of the routes and mechanisms by which the GI microbiota contribute to the pathophysiology of BDNF-induced NMDAR dysfunction could yield new insights relevant to developing novel therapeutics for schizophrenia and related disorders. In the absence of GI microbes, central BDNF levels are reduced and this inhibits the maintenance of NMDAR production. A reduction of NMDAR input onto GABA inhibitory interneurons causes disinhibition of glutamatergic output which disrupts the central signal-to-noise ratio and leads to aberrant synaptic behaviour and cognitive deficits. Gut microbiota can modulate BDNF function in the CNS, via changes in neurotransmitter function by affecting modulatory mechanisms such as the kynurenine pathway, or by changes in the availability and actions of short chain fatty acids (SCFAs) in the brain. Interrupting these cycles by inducing changes in the gut microbiota using probiotics, prebiotics or antimicrobial drugs has been found promising as a preventative or therapeutic measure to counteract behavioural deficits and these may be useful to supplement the actions of drugs in the treatment of CNS disorders.     

Matrix metalloproteinases and cellular motility in development and disease

The movement of cells and the accompanied remodeling of the extracellular matrix is a critical step in many developmental processes. The matrix metalloproteinases (MMPs) are well recognized as mediators of matrix degradation, and their activity as regulators of signaling pathways by virtue of the cleavage of nonmatrix substrates has been increasingly appreciated. In this review, we focus on the role of MMPs in altering processes that influence cellular motility. MMP involvement in cellular adhesion, lamellipodia-directed movement, invadopodial protrusion, axonal growth cone extension, and chemotaxis are discussed. Although not designed to be comprehensive, these examples clearly demonstrate that cellular regulation of the MMPs influences cell motility in a variety of ways, including regulating cell-cell interactions, cell-matrix interactions, matrix degradation, and the release of bioactive signaling molecules. Deregulation of these interactions can ultimately result in disorders including inflammatory diseases, vascular diseases, bone diseases, neurological disorders, and cancer.