Gunnar Dick

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 Golgimore 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 andsynaptic plasticity after CNS injury, and the possible mechanisms of PNN action.

Rab11 and Its Effector Rab Coupling Protein Contribute to the Trafficking of beta 1 Integrins during Axon Growth in Adult Dorsal Root Ganglion Neurons and PC12 Cells

Integrins play an important part in axon growth, but integrin traffic in neurons is poorly understood. Expression of the tenascin-C-binding integrin α9 promotes axon regeneration. We have therefore studied the mechanism by which α9 integrin and its partner β1 are trafficked along axons and at the growth cone using adult DRG neurons and PC12 cells. We have focused on the small GTPase Rab11 and its effector Rab coupling protein (RCP), as they are involved in the long-range trafficking of β1 integrins in other cells. Rab11 colocalizes with α9 and other α integrins and with β1 integrin in growth cones and axons, and immunopurified Rab11 vesicles contain α9 and β1. Endocytosed β1 integrins traffic via Rab11. However, Rab11 vesicles in axons are generally static, and α9 integrins undergo bouts of movement during which they leave the Rab11 compartment. In growth cones, α9 and β1 overlap with RCP, particularly at the growth cone periphery. We show that β1 integrin trafficking during neurite outgrowth involves Rab11 and RCP, and that manipulation of these molecules alters surface integrin levels and axon growth, and can be used to enhance α9 integrin-dependent neurite outgrowth. Our data suggest that manipulation of trafficking via Rab11 and RCP could be a useful strategy for promoting integrin-dependent axonal regeneration.

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

Background: Semaphorin3A (Sema3A) is an axon guidance molecule present in the CNS extracellular matrix and on the perineuronal nets (PNNs). Results: Sema3A interacts with chondroitin sulfate E (CS-E) for anchoring to the PNNs. Conclusion: The binding of Sema3A to CS in the PNNs presents a novel mechanism of PNNs in restricting plasticity. Significance: This finding suggests a novel candidate for intervention in promoting CNS recovery. 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.

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 proteoglycan undergoes different modifications en route to the apical and basolateral surfaces of Madin-Darby canine kidney cells

We have grown polarized epithelial Madin-Darby canine kidney II (MDCK II) cells on filters in the presence of [35S]sulfate, [3H]glucosamine, or [35S]cysteine/[35S]methionine to study proteoglycan (PG) synthesis, sorting, and secretion to the apical and basolateral media. Whereas most of the [35S]sulfate label was recovered in basolateral PGs, the [3H]glucosamine label was predominantly incorporated into the glycosaminoglycan chains of apical PGs, indicating that basolateral PGs are more intensely sulfated than their apical counterparts. Expression of the PG serglycin with a green fluorescent protein tag (SG-GFP) in MDCK II cells produced a protein core secreted 85% apically, which was largely modified by chondroitin sulfate chains. Surprisingly, the 15% of secreted SG-GFP molecules recovered basolaterally were more heavily sulfated and displayed a different sulfation pattern than the apical counterpart. More detailed studies of the differential modification of apically and basolaterally secreted SG-GFP indicate that the protein cores have been designated to apical and basolateral transport platforms before pathway-specific, post-translational modifications have been completed.

How many ways through the Golgi maze

The secretory route in eukaryotic cells has been regarded as one common pathway from the endoplasmic reticulum (ER) through the Golgi cisternae to the trans Golgi network where recognition, sorting and exit of cargo molecules are thought to occur. Morphologically, the ribosome‐coated ER is observed throughout the cytoplasm, while the Golgi apparatus usually is confined to a perinuclear position in mammalian cells. However, Golgi outposts have been observed in neuronal dendrites and dispersed Golgi elements in skeletal muscle myofibers. In insects, like in Drosophila melanogaster imaginal disc cells and epidermal cells of Tobacco and Arabidopsis leafs, individual Golgi stacks are distributed throughout the cytoplasm. Golgi stacks do not only differ in their intracellular localization but also in the number of stacks from one to several hundreds. Each stack consists of closely aligned, flattened, membrane‐limited cisternae. The number of cisternae in a Golgi stack is also variable, 2–3 in some ciliates, 10 in many plant cell types and up to 30 in certain euglenoids. The yeast Saccharomyces cerevisiae has a Golgi structure of minimal complexity with scattered solitary cisternae. It is assumed that the number of Golgi cisternae reflects the overall complexity of the enzymatic reactions that occur in their lumen, while the number of stacks reflects the load of macromolecules arriving at the cis side. In this review, we will focus on how the available morphological and biochemical data fit with the current view of protein sorting in the secretory pathway, particularly in polarized cells like neuronal and epithelial cells.

Removal of Perineuronal Nets Unlocks Juvenile Plasticity Through Network Mechanisms of Decreased Inhibition and Increased Gamma Activity

Perineuronal nets (PNNs) are extracellular matrix structures mainly enwrapping parvalbumin-expressing inhibitory neurons. The assembly of PNNs coincides with the end of the period of heightened visual cortex plasticity in juveniles, whereas removal of PNNs in adults reopens for plasticity. The mechanisms underlying this phenomenon remain elusive. We have used chronic electrophysiological recordings to investigate accompanying electrophysiological changes to activity-dependent plasticity and we report on novel mechanisms involved in both induced and critical period plasticity. By inducing activity-dependent plasticity in the visual cortex of adult rats while recording single unit and population activity, we demonstrate that PNN removal alters the balance between inhibitory and excitatory spiking activity directly. Without PNNs, inhibitory activity was reduced, whereas spiking variability was increased as predicted in a simulation with a Brunel neural network. Together with a shift in ocular dominance and large effects on unit activity during the first 48 h of monocular deprivation (MD), we show that PNN removal resets the neural network to an immature, juvenile state. Furthermore, in PNN-depleted adults as well as in juveniles, MD caused an immediate potentiation of gamma activity, suggesting a novel mechanism initiating activity-dependent plasticity and driving the rapid changes in unit activity. SIGNIFICANCE STATEMENT Emerging evidence suggests a role for perineuronal nets (PNNs) in learning and regulation of plasticity, but the underlying mechanisms remain unresolved. Here, we used chronic in vivo extracellular recordings to investigate how removal of PNNs opens for plasticity and how activity-dependent plasticity affects neural activity over time. PNN removal caused reduced inhibitory activity and reset the network to a juvenile state. Experimentally induced activity-dependent plasticity by monocular deprivation caused rapid changes in single unit activity and a remarkable potentiation of gamma oscillations. Our results demonstrate how PNNs may be involved directly in stabilizing the neural network. Moreover, the immediate potentiation of gamma activity after plasticity onset points to potential new mechanisms for the initiation of activity-dependent plasticity.

Proteoglycan Synthesis and Golgi Organization in Polarized Epithelial Cells

A large number of complex glycosylation mechanisms take place in the Golgi apparatus. In epithelial cells, glycosylated protein molecules are transported to both the apical and the basolateral surface domains. Although the prevailing view is that the Golgi apparatus provides the same lumenal environment for glycosylation of apical and basolateral cargo proteins, there are indications that proteoglycans destined for the two opposite epithelial surfaces are exposed to different conditions in transit through the Golgi apparatus. We will here review data relating proteoglycan and glycoprotein synthesis to characteristics of the apical and basolateral secretory pathways in epithelial cells.