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 (Crtll). 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, Crtll, and HASs mRNAs. However, Crtll 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, Crtll might be crucial elements for the initial assembly of PNNs.     

Bral2 is indispensable for the proper localization of brevican and the structural integrity of the perineuronal net in the brainstem and cerebellum

Perineuronal nets (PNNs) are pericellular coats of condensed matrix that enwrap the cell bodies and dendrites of many adult central nervous system (CNS) neurons. These extracellular matrices (ECMs) play a structural role as well as instructive roles in the control of CNS plasticity and the termination of critical periods. The cartilage link protein Crtl1/Hapln1 was reported to be a trigger for the formation of PNNs in the visual cortex. Bral2/Hapln4 is another link protein that is expressed in PNNs, mainly in the brainstem and cerebellum. To assess the role of Bral2 in PNN formation, we examined the expression of PNN components in targeted mouse mutants lacking Bral2. We show here that Bral2-deficient mice have attenuated PNNs, but the overall levels of chondroitin sulfate proteoglycans, lecticans, are unchanged with the exception of neurocan. Bral2 deficiency markedly affected the localization of brevican in all of the nuclei tested, and neurocan concomitant with Crtl1 in some of the nuclei, whereas no effect was seen on aggrecan even with the attenuation of Crtl1. Bral2 may have a role in the organization of the PNN, in association with brevican, that is independent of aggrecan binding. There was a heterogenous attenuation of PNN components, including glycosaminoglycans, indicating the elaborate molecular organization of the PNN components. Strikingly, a slight decrease in the number of synapses in deep cerebellar nuclei neurons was found. Taken together, these results imply that Bral2-brevican interaction may play a key role in synaptic stabilization and the structural integrity of the PNN.     

Postnatal development of GABAergic interneurons and perineuronal nets in mouse temporal cortex subregions

In human neuropsychiatric disorders, there are functional and anatomical abnormalities of GABAergic interneurons in each temporal cortex subregion. Furthermore, accumulation of amyloid-β is observed in the temporal cortex in the early stages of Alzheimer’s disease. Each subregion of the temporal cortex has an important role in coordinating the input and output of the hippocampus. When subregions of the temporal cortex are impaired, memory and learning ability decrease. GABAergic interneurons control excitatory neurons, forming the cortico-cortical and cortico-hippocampal networks. However, in temporal cortex subregions, details of the distribution and developmental processes of GABAergic interneurons and perineuronal nets (PNNs) have not been elucidated. Here we examined the development of GABAergic interneurons and PNNs in mouse temporal cortex subregions. Results indicate that temporal cortex GABAergic interneurons have developmental stages different to those of the primary sensory cortex. In addition, the density of PNNs in the temporal cortex is lower than that in the sensory cortex. Furthermore, we found that the Wisteria floribunda agglutinin-reactive extracellular matrix molecule is present in the upper level of layer 1 of the temporal cortex. These results support the idea that mouse temporal cortex subregions develop differently from other cortical regions and have region-specific characteristics after maturation. The present study results suggested that the structure of the temporal cortex is significantly different from the sensory cortex and that temporal cortex may be highly vulnerable to neuropsychiatric and neurodegenerative disorders.     

Modulation of semaphorin3A in perineuronal nets during structural plasticity in the adult cerebellum

In the adult central nervous system (CNS) subsets of neurons are enwrapped by densely organized extracellular matrix structures, called perineuronal nets (PNNs). PNNs are formed at the end of critical periods and contribute to synapse stabilization. Enzymatic degradation of PNNs or genetic deletion of specific PNN components leads to the prolongation of the plasticity period. PNNs consist of extracellular matrix molecules, including chondroitin sulfate proteoglycans, hyaluronan, tenascins and link proteins. It has been recently shown that the chemorepulsive axon guidance protein semaphorin3A (Sema3A) is also a constituent of PNNs, binding with high affinity to the sugar chains of chondroitin sulfate proteoglycans. To elucidate whether the expression of Sema3A is modified in parallel with structural plasticity in the adult CNS, we examined Sema3A expression in the deep cerebellar nuclei of the adult mouse in a number of conditions associated with structural reorganization of the local connectivity. We found that Sema3A in PNNs is reduced during enhanced neuritic remodeling, in both physiological and injury-induced conditions. Moreover, we provide evidence that Sema3A is tightly associated with Purkinje axons and their terminals and its amount in the PNNs is related to Purkinje cell innervation of DCN neurons, but not to glutamatergic inputs. On the whole these data suggest that Sema3A may contribute to the growth-inhibitory properties of PNNs and Purkinje neurons may directly control their specific connection pattern through the release and capture of this guidance cue in the specialized ECM that surrounds their terminals.     

Abnormal reorganization of preplate neurons and their associated extracellular matrix: An early manifestation of altered neocortical development in the reeler mutant mouse

The formation of the distinct layers of the cerebral cortex begins when cortical plate neurons take up positions within the extracellular matrix (ECM)-rich preplate, dividing it into the marginal zone above and the subplate below. We have analyzed this process in the reeler mutant mouse, in which cortical lamination is severely disrupted. The recent observation that the product of the reeler gene is an ECM-like protein that is expressed by cells of the marginal zone indicates a critical role for ECM in cortical lamination. We have found that preplate cells in normal cortex that are tagged during their terminal division with bromodeoxyuridine (BrdU) are closely associated with chondroitin sulfate proteoglycans (CSPGs), which were identified by immunolabeling; this association is maintained in the marginal zone and subplate after the preplate is divided by cortical plate formation. Cortical plate cells do not aggregate within the preplate in reeler; instead, preplate cells remain as an undivided superficial layer containing abundant CSPGs, and cortical plate neurons accumulate below them. These findings indicate that preplate cells are responsible for the formation of a localized ECM, because the association of CSPGs with preplate cells is maintained even when these cells are in abnormal positions. The failure of cortical plate neurons to aggregate within the framework of the preplate and its associated ECM and to divide it is one of the earliest structural abnormalities detectable in reeler cortex, suggesting that this step is important for the subsequent formation of cortical layers. J. Comp. Neurol. 378:173–179, 1997. © 1997 Wiley-Liss, Inc.     

Molecular heterogeneity of Vicia villosa-recognized perineuronal nets surrounding pyramidal and nonpyramidal neurons in the guinea pig cerebral cortex

Of three monoclonal antibodies (mAbs Cat 301, 1B5 and 473) which recognize epitopes on perineuronal nets (PnNs), only mAb 473 displayed considerably varied degrees of staining intensity on Vicia villosa-labeled pyramidal (P) neurons (5%, intensely; 54%, very weakly; and 41%, unstained). These results indicate the heterogeneity on molecular structure or composition of the terminal N-acetylgalactosamine-containing PnNs within P class as well as between P and nonpyramidal classes.     

Microglia/monocytes with NG2 expression have no phagocytic function in the cortex after LPS focal injection into the rat brain

While OX42(+) microglia/macrophages have been considered as a scavenger in the brain, NG2(+) cells are generally considered as oligodendrocyte progenitor cells or function-unknown glial cells. Recent evidence showed that under some pathological conditions, certain cells have become positive for both anti-NG2 and anti-OX42 antibodies. Our results suggested that some OX42(+) microglia or macrophages were induced to express NG2 proteins 3 and 5 days later after focal injection of lipopolysaccharide into the brain cortex of Sprague-Dawley rats. In consideration of the induction of NG2 expression may associate with gaining or losing functions of microglia/macrophages, we further showed that, while OX42(+) or ED1(+) microglia/macrophages presented active phagocytic function, NG2(+) /OX42(+) cells failed to engulf latex beads. The induced expression of NG2 protein may possibly indicate the functional diversity of activated microglia/macrophages in the brain.     

Saccadic omnipause and burst neurons in monkey and human are ensheathed by perineuronal nets but differ in their expression of calcium-binding proteins

The extracellular matrix of the brain contains large aggregates of chondroitin sulfate proteoglycans (CSPG), which form lattice-like cell coatings around distinct neuron populations and are termed perineuronal nets. The function of perineuronal nets is not fully understood, but they are often found around neurons containing the calcium-binding protein parvalbumin, suggesting a function in primarily highly active neurons. In the present paper the distribution of perineuronal nets was studied in two functional cell groups of the primate oculomotor system with well-known firing properties: 1) the saccadic omnipause neurons in the nucleus raphe interpositus (RIP) exhibit a high tonic firing rate, which is only interrupted during saccades; they are inhibitory and use glycine as a transmitter; and 2) premotor burst neurons for vertical saccades in the rostral interstitial nucleus of the medial longitudinal fascicle (RiMLF) fire with high-frequency bursts during saccades; they are excitatory and use glutamate and/or aspartate as a transmitter. In the macaque monkey, both cell populations were identified by their parvalbumin immunoreactivity and were studied for the presence of perineuronal nets using CSPG antibodies or lectin binding with Wisteria floribunda agglutinin. In addition, the expression of another calcium-binding protein, calretinin, was studied in both cell groups. Double- and triple-immunofluorescence methods revealed that both omnipause and burst neurons are selectively ensheathed with strongly labeled perineuronal nets. Calretinin was coexpressed in at least 70% of the saccadic burst neurons, but not in the omnipause neurons. Parallel staining of human tissue revealed strongly labeled perineuronal nets around the saccadic omnipause and burst neurons, in corresponding brainstem regions, which specifically highlighted these neurons within the poorly structured reticular formation. These findings support the hypothesis that perineuronal nets may provide a specialized microenvironment for highly active neurons to maintain their fast-spiking activity and are not related to the transmitter or the postsynaptic action of the ensheathed neurons.     

Chondroitin sulfate proteoglycan-immunoreactivity of lectin-labeled perineuronal nets around parvalbumin-containing neurons

Perineuronal nets represent highly specialized glial and glia-associated structures. In this study, a triple fluorescence labeling of chondroitin sulfate proteoglycan-immunoreactive (CSPG-ir) andN-acetylgalactosamine (GalNac)-specific plant lectinWisteria floribunda agglutinin (WFA) binding net components as well as parvalbumin-immunoreactivity (-ir) was performed. It was shown in the rat cortex, that the same nets frequently surrounding parvalbumin-ir neurons are stained by CSPG-ir as well as by the lectin binding method.     

tPA-mediated generation of plasmin is catalyzed by the proteoglycan NG2

Paralysis resulting from spinal cord injury is devastating and persistent. One major reason for the inability of the body to heal this type of injury ensues from the local increase of glial cells leading to the formation of a glial scar, and the upregulation of chondroitin sulfate proteoglycans (CSPGs) at the site of injury through which axons are unable to regenerate. Experimental approaches to overcome this problem have accordingly focused on reducing the inhibitory properties of CSPGs, for example by using chondroitinase to remove the sugar chains and reduce the CSPGs to their core protein constituents, although this step alone does not provide dramatic benefits as a monotherapy. Using in vitro and in vivo approaches, we describe here a potentially synergistic therapeutic opportunity based on tissue plasminogen activator (tPA), an extracellular protease that converts plasminogen (plg) into the active protease plasmin. We show that tPA and plg both bind to the CSPG protein NG2, which functions as a scaffold to accelerate the tPA-driven conversion of plg to plasmin. The binding occurs via the tPA and plg kringle domains to domain 2 of the NG2 CSPG core protein, and is enhanced in some settings after chondroitinase-mediated removal of the NG2 proteoglycan side chains. Once generated, plasmin then degrades NG2, both in an in vitro setting using recombinant protein, and in vivo models of spinal cord injury. Our finding that the tPA and plg binding is in some instances more efficient after exposure of the NG2 proteoglycan to chondroitinase treatment suggests that a combined therapeutic approach employing both chondroitinase and the tPA/plasmin proteolytic system could be of significant benefit in promoting axonal regeneration through glial scars after spinal cord injury.     

Brevican in the developing hippocampal fimbria: differential expression in myelinating oligodendrocytes and adult astrocytes suggests a dual role for brevican in central nervous system fiber tract development

Brevican is one of the most abundant extracellular matrix proteoglycans in the mammalian brain. We have previously shown that brevican produced by gray matter astrocytes constitutes a major component of perineuronal extracellular matrix in the adult brain. In this paper, we investigate the expression of brevican in the postnatal hippocampal fimbria to explore the role of the proteoglycan in central nervous system fiber tract development. We demonstrate that brevican is expressed by both oligodendrocytes and white matter astrocytes in the fimbria, but the expression of brevican in these two glial cell types is differently regulated during development. At P14, brevican immunoreactivity was observed throughout the fimbria, with particularly strong immunoreactivity in the developing interfascicular glial rows. In situ hybridization showed that oligodendrocytes in the glial rows strongly express brevican during the second and third postnatal weeks. Expression in oligodendrocytes was then down-regulated after P21. In the adult fimbria, no brevican expression was observed in oligodendrocytes. The time window of brevican expression coincides with the phase in which immature oligodendrocytes actively extend membrane processes and enwrap axon fibers. In contrast, the expression in astrocytes started around P21 as oligodendrocytes began to down-regulate the expression. In the adult fimbria, brevican expression was restricted to astrocytes. In situ hybridization with isoform-specific probes and RNase protection assays showed that the authentic, secreted form of brevican, not the glycosylphosphatidylinositol-anchored variant, is the predominant species expressed in the developing fimbria. Our results suggest that brevican plays a dual role in developing and adult fiber tracts.     

Morphology of perineuronal nets in tenascin-R and parvalbumin single and double knockout mice

Recently identified chondroitin sulphate proteoglycans in perineuronal nets include neurocan and phosphacan. However, the function and assembly of these components has yet to be resolved. In this study we show morphological alteration in Wisteria floribunda labelled nets around cortical interneurones both in tenascin-R knockout and tenascin-R/parvalbumin double knockout mice. This alteration reflects the loss of phosphacan and neurocan from cortical nets in mice deficient in tenascin-R. No effect on the membrane related cytoskeleton, as revealed by ankyrin(R), was observed in any of the mice. These results on mice lacking tenascin-R substantiate previously reported in vitro interactions between tenascin-R and phosphacan and neurocan.     

Transient compartmental expression of neurocan in the developing striatum of the rat

The expression of the chondroitin sulfate proteoglycan neurocan was examined in the developing striatum of the rat and compared with the distribution of dopaminergic terminals. Neurocan immunoreactivity shows a homogeneous pattern in the embryonic striatum. In the postnatal striatum, neurocan was first expressed within the matrix but not the patch compartments, and subsequently within both. These results suggest that chondroitin sulfate proteoglycans are involved in formation of connections between the substantia nigra and striatum. J. Neurosci. Res. 51:612–618, 1998. © 1998 Wiley-Liss, Inc.     

Schwann cell precursors transplanted into the injured spinal cord multiply, integrate and are permissive for axon growth

There is a strong current interest in the use of cell transplantation for the treatment of spinal cord injuries. We report here the novel and potentially useful properties of an early cell in the Schwann cell lineage, the Schwann cell precursor (SCP). The experiments reveal a striking difference between these cells and Schwann cells when transplanted into the CNS. Unlike Schwann cells, SCPs thrive in the CNS where they initially proliferate rapidly but then fall out of division, thus effectively filling up the large cystic cavities formed following crush injury, while avoiding tumor formation. By 8 weeks, SCPs had started to express S100beta protein, a marker that differentiates Schwann cells from SCPs and had formed an apparently stable, vascularized cell mass, which created a continuous cellular bridge across the cystic cavities. The formation of the surrounding glial scar was reduced by local spread of the transplanted cells into the surrounding CNS tissue, where the cells integrated intimately with astrocytes and attenuated the physical barrier they normally form. SCP transplantation also altered and reduced the expression of chondroitin sulfate proteoglycans around the injury site. Caudal to the SCP transplants there was a large increase in the number of axons, compared with that seen in nontransplanted control tissue, showing that the implants effectively support axonal growth or sprouting. SCPs have advantageous attributes for CNS repair, despite the fact that sticky tape removal and ladder crossing tests at 8 weeks did not reveal significant functional improvements when compared with control animals.     

NG2 upregulation in the denervated rat fascia dentata following unilateral entorhinal cortex lesion

The chondroitin sulfate proteoglycan NG2 is a component of the glial scar following brain injury. Because of its growth inhibiting properties, it has been suggested to impede axonal regeneration. To study whether NG2 could also regulate axonal growth in denervated brain areas, changes in NG2 were studied in the rat fascia dentata following entorhinal deafferentation and were correlated with the post-lesional sprouting response. Laser microdissection was employed to selectively harvest the denervated molecular layer and combined with quantitative RT-PCR to measure changes in NG2 mRNA (6 h, 12 h, 2 days, 4 days, 7 days post-lesion). This revealed increases of NG2 mRNA at day 2 (2.5-fold) and day 4 (2-fold) post-lesion. Immunocytochemistry was used to detect changes in NG2 protein (1 days, 4 days, 7 days, 10 days, 14 days, 30 days, 6 months post-lesion). NG2 staining was increased in the denervated outer molecular layer at day 1 post-lesion, reached a maximum 10 days post-lesion, and returned to control levels thereafter. Electron microscopy revealed NG2 immunoprecipitate on glial surfaces and in the extracellular matrix around neuronal profiles, indicating that NG2 is secreted following denervation. Double labeling of NG2-immunopositive cells with markers for astrocytes, microglia/macrophages, and mature oligodendrocytes suggested that NG2 cells are a distinct glial subpopulation before and after entorhinal deafferentation. BrdU labeling revealed that some of the NG2-positive cells are generated post-lesion. Taken together, our data revealed a layer-specific upregulation of NG2 in the denervated fascia dentata that coincides with the sprouting response. This suggests that NG2 could regulate lesion-induced axonal growth in denervated areas of the brain.     

In vivo and in vitro labelling of perineuronal nets in rat brain

Previous lectin-histochemical and immunocytochemical investigations using fixed tissue revealed perineuronal nets as lattice-like accumulations of extracellular matrix proteoglycans at the surface of several types of neurons. In the present study, perineuronal nets in the rat brain were labelled for the first time in vivo by stereotaxic injections of biotinylated Wisteria floribunda agglutinin (Bio-WFA), as well as in vitro, by incubation of unfixed brain slices with the same lectin. Six days after Bio-WFA injections into the parietal cortex, medial septum, reticular thalamic nucleus and red nucleus, the lectin remaining bound to perineuronal nets was detected by streptavidin/biotinylated peroxidase complexes or red fluorescent Cy3-streptavidin, respectively. Double-fluorescence labelling showed that Bio-WFA applied in vivo reacted with the chondroitin sulphate proteoglycan immunoreactive perineuronal nets in the injection zone. Labelling of perineuronal nets in unfixed slices was obtained with either Cy3-tagged WFA or Bio-WFA and subsequent visualization by Cy3-streptavidin which confirmed the region-dependent distribution patterns and the structural characteristics of perineuronal nets known from histochemical studies. These results provide support for the role of extracellular matrix proteoglycans to maintain a considerable chemical and, probably, spatial heterogeneity of the extracellular space in vivo. The ability of in vivo and in vitro labelling may promote the functional characterization of the extracellular matrix in various brain structures including its species-dependent neuronal association patterns.     

Distribution of parvalbumin-containing neurons and lectin-binding perineuronal nets in the rat basal forebrain

In sections of rat brain treated forWisteria floribunda agglutinin (WFA) labelling the occurrence of parvalbumin (PARV)-, calbindin (CALB)- or choline acetyltransferase (ChAT) immunoreactivity was analyzed in the basal forebrain using dual-peroxidase and double-fluorescence methods. Only PARV-immunoreactive (-ir) neurons were surrounded by WFA-labelled, i.e.N-acetylgalactosamine-containing, perineuronal lattice-like structures known as perineuronal nets. The distribution of these nets and PARV-ir cells in the rat basal forebrain was documented to obtain detailed data on their co-existence. A remarkable diversity distribution of both markers was observed, as PARV-ir neurons are only associated with nets in the medial septal nucleus, the nuclei of the diagonal band and the magnocellular preoptic nucleus, but not in the ventral pallidum or the substantia innominata/nucleus basalis complex. These differences in the neuronal microenvironment may reflect system-related specializations of neurons within the basal forebrain nuclei.