Substrate Specificity and Biochemical Characteristics of an Engineered Mammalian Chondroitinase ABC.

Chondroitin sulfate proteoglycans inhibit regeneration, neuroprotection, and plasticity following spinal cord injury. The development of a second-generation chondroitinase ABC enzyme, capable of being secreted from mammalian cells (mChABC), has facilitated the functional recovery of animals following severe spinal trauma. The genetically modified enzyme has been shown to efficiently break down the inhibitory extracellular matrix surrounding cells at the site of injury, while facilitating cellular integration and axonal growth. However, the activity profile of the enzyme in relation to the original bacterial chondroitinase (bChABC) has not been determined. Here, we characterize the activity profile of mChABC and compare it to bChABC, both enzymes having been maintained under physiologically relevant conditions for the duration of the experiment. We show that this genetically modified enzyme can be secreted reliably and robustly in high yields from a mammalian cell line. The modifications made to the cDNA of the enzyme have not altered the functional activity of mChABC compared to bChABC, ensuring that it has optimal activity on chondroitin sulfate-A, with an optimal pH at 8.0 and temperature at 37 °C. However, mChABC shows superior thermostability compared to bChABC, ensuring that the recombinant enzyme operates with enhanced activity over a variety of physiologically relevant substrates and temperatures compared to the widely used bacterial alternative without substantially altering its kinetic output. The determination that mChABC can function with greater robustness under physiological conditions than bChABC is an important step in the further development of this auspicious treatment strategy toward a clinical application

Brain ageing changes proteoglycan sulfation, rendering perineuronal nets more inhibitory.

Chondroitin sulfate (CS) proteoglycans in perineuronal nets (PNNs) from the central nervous system (CNS) are involved in the control of plasticity and memory. Removing PNNs reactivates plasticity and restores memory in models of Alzheimer's disease and ageing. Their actions depend on the glycosaminoglycan (GAG) chains of CS proteoglycans, which are mainly sulfated in the 4 (C4S) or 6 (C6S) positions. While C4S is inhibitory, C6S is more permissive to axon growth, regeneration and plasticity. C6S decreases during critical period closure. We asked whether there is a late change in CS-GAG sulfation associated with memory loss in aged rats. Immunohistochemistry revealed a progressive increase in C4S and decrease in C6S from 3 to 18 months. GAGs extracted from brain PNNs showed a large reduction in C6S at 12 and 18 months, increasing the C4S/C6S ratio. There was no significant change in mRNA levels of the chondroitin sulfotransferases. PNN GAGs were more inhibitory to axon growth than those from the diffuse extracellular matrix. The 18-month PNN GAGs were more inhibitory than 3-month PNN GAGs. We suggest that the change in PNN GAG sulfation in aged brains renders the PNNs more inhibitory, which lead to a decrease in plasticity and adversely affect memory

Full length talin stimulates integrin activation and axon regeneration.

Integrin function is regulated by activation involving conformational changes that modulate ligand-binding affinity and downstream signaling. Activation is regulated through inside-out signaling which is controlled by many signaling pathways via a final common pathway through kindlin and talin, which bind to the intracellular tail of beta integrins. Previous studies have shown that the axon growth inhibitory molecules NogoA and chondroitin sulfate proteoglycans (CSPGs) inactivate integrins. Overexpressing kindlin-1 in dorsal root ganglion (DRG) neurons activates integrins, enabling their axons to overcome inhibitory molecules in the environment, and promoting regeneration in vivo following dorsal root crush. Other studies have indicated that expression of the talin head alone or with kindlin can enhance integrin activation. Here, using adult rat DRG neurons, we investigate the effects of overexpressing various forms of talin on axon growth and integrin signaling. We found that overexpression of the talin head activated axonal integrins but inhibited downstream signaling via FAK, and did not promote axon growth. Similarly, co-expression of the talin head and kindlin-1 prevented the growth-promoting effect of kindlin-1, suggesting that the talin head acts as a form of dominant negative for integrin function. Using full-length talin constructs in PC12 cells we observed that neurite growth was enhanced by the expression of wild-type talin and more so by two 'activated' forms of talin produced by point mutation (on laminin and aggrecan-laminin substrates). Nevertheless, co-expression of full-length talin with kindlin did not promote neurite growth more than either molecule alone. In vivo, we find that talin is present in PNS axons (sciatic nerve), and also in CNS axons of the corticospinal tract.This work was funded by grants from the Medical Research Council (G1000864), the Henry Smith Charity, the Christopher and Dana Reeve Foundation, the John and Lucille van Geest Foundation, the European Union Framework 7 Programmes Spinal Cord Repair (201144) and Plasticise (223524), and the NIHR Cambridge Biomedical Research Centre. CLT was supported by the Merck, Sharpe and Dohme Fund. We thank Rienhardt Fassler for kindlin constructs and advice, David Critchley for talin antibodies and constructs and Mark Ginsberg for talin constructs.This is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.mcn.2015.03.01

New Model of Ventral Spinal Cord Lesion Induced by Balloon Compression in Rats.

Despite the variety of experimental models of spinal cord injury (SCI) currently used, the model of the ventral compression cord injury, which is commonly seen in humans, is very limited. Ventral balloon compression injury reflects the common anatomical mechanism of a human lesion and has the advantage of grading the injury severity by controlling the inflated volume of the balloon. In this study, ventral compression of the SCI was performed by the anterior epidural placement of the balloon of a 2F Fogarty's catheter, via laminectomy, at the level of T10. The balloon was rapidly inflated with 10 or 15 μL of saline and rested in situ for 5 min. The severity of the lesion was assessed by behavioral and immunohistochemical tests. Compression with the volume of 15 μL resulted in severe motor and sensory deficits represented by the complete inability to move across a horizontal ladder, a final Basso, Beattie and Bresnahan (BBB) score of 7.4 and a decreased withdrawal time in the plantar test (11.6 s). Histology and immunohistochemistry revealed a significant loss of white and gray matter with a loss of motoneuron, and an increased size of astrogliosis. An inflation volume of 10 μL resulted in a mild transient deficit. There are no other balloon compression models of ventral spinal cord injury. This study provided and validated a novel, easily replicable model of the ventral compression SCI, introduced by an inflated balloon of Fogarty´s catheter. For a severe incomplete deficit, an inflated volume should be maintained at 15 μL

Casting a Wide Net: Role of Perineuronal Nets in Neural Plasticity.

Perineuronal nets (PNNs) are unique extracellular matrix structures that wrap around certain neurons in the CNS during development and control plasticity in the adult CNS. They appear to contribute to a wide range of diseases/disorders of the brain, are involved in recovery from spinal cord injury, and are altered during aging, learning and memory, and after exposure to drugs of abuse. Here the focus is on how a major component of PNNs, chondroitin sulfate proteoglycans, control plasticity, and on the role of PNNs in memory in normal aging, in a tauopathy model of Alzheimer's disease, and in drug addiction. Also discussed is how altered extracellular matrix/PNN formation during development may produce synaptic pathology associated with schizophrenia, bipolar disorder, major depression, and autism spectrum disorders. Understanding the molecular underpinnings of how PNNs are altered in normal physiology and disease will offer insights into new treatment approaches for these diseases

Passive water control at the surface of a superhydrophobic lichen

Some lichens have a super-hydrophobic upper surface, which repels water drops, keeping the surface dry but probably preventing water uptake. Spore ejection requires water and is most efficient just after rainfall. This study was carried out to investigate how super-hydrophobic lichens manage water uptake and repellence at their fruiting bodies, or podetia. Drops of water were placed onto separate podetia of Cladonia chlorophaea and observed using optical microscopy and cryo-scanning-electron microscopy (cryo-SEM) techniques to determine the structure of podetia and to visualise their interaction with water droplets. SEM and optical microscopy studies revealed that the surface of the podetia was constructed in a three-level structural hierarchy. By cryo-SEM of water-glycerol droplets placed on the upper part of the podetium, pinning of the droplet to specific, hydrophilic spots (pycnidia/apothecia) was observed. The results suggest a mechanism for water uptake, which is highly sophisticated, using surface wettability to generate a passive response to different types of precipitation in a manner similar to the Namib Desert beetle. This mechanism is likely to be found in other organisms as it offers passive but selective water control

Chondroitin sulfates in the developing rat hindbrain confine commissural projections of vestibular nuclear neurons.

BACKGROUND: Establishing correct neuronal circuitry is crucial to proper function of the vertebrate nervous system. The abundance of chondroitin sulfate (CS) proteoglycans in embryonic neural environments suggests that matrix proteoglycans regulate axonal projections when fiber tracts have not yet formed. Among the early-born neurons, the vestibular nucleus (VN) neurons initiate commissural projections soon after generation at E12.5 and reach the contralateral target by E15.5 in the rat hindbrain. We therefore exploited 24-hour cultures (1 day in vitro (DIV)) of the rat embryos and chondroitinase ABC treatment of the hindbrain matrix to reveal the role of CS moieties in axonal initiation and projection in the early hindbrain. RESULTS: DiI tracing from the VN at E12.5(+1 DIV) showed contralaterally projecting fibers assuming fascicles that hardly reached the midline in the controls. In the enzyme-treated embryos, the majority of fibers were unfasciculated as they crossed the midline at 90°. At E13.5(+1 DIV), the commissural projections formed fascicles and crossed the midline in the controls. Enzyme treatment apparently did not affect the pioneer axons that had advanced as thick fascicles normal to the midline and beyond, towards the contralateral VN. Later projections, however, traversed the enzyme-treated matrix as unfasciculated fibers, deviated from the normal course crossing the midline at various angles and extending beyond the contralateral VN. This suggests that CSs also limit the course of the later projections, which otherwise would be attracted to alternative targets. CONCLUSIONS: CS moieties in the early hindbrain therefore control the course and fasciculation of axonal projections and the timing of axonal arrival at the target.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Long-Term Cultures of Spinal Cord Interneurons.

Spinal cord interneurons (SpINs) are highly diverse population of neurons that play a significant role in circuit reorganization and spontaneous recovery after spinal cord injury. Regeneration of SpIN axons across rodent spinal injuries has been demonstrated after modification of the environment and neurotrophin treatment, but development of methods to enhance the intrinsic regenerative ability of SpINs is needed. There is a lack of described in vitro models of spinal cord neurons in which to develop new regeneration treatments. For this reason, we developed a new model of mouse primary spinal cord neuronal culture in which to analyze maturation, morphology, physiology, connectivity and regeneration of identified interneurons. Isolated from E14 mice, the neurons mature over 15 days in vitro, demonstrated by expression of maturity markers, electrophysiological patch-clamp recordings, and formation of synapses. The neurons express markers of SpINs, including Tlx3, Lmx1b, Lbx1, Chx10, and Pax2. The neurons demonstrate distinct morphologies and some form perineuronal nets in long-term cultivation. Live neurons in various maturation stages were axotomized, using a 900 nm multiphoton laser and their fate was observed overnight. The percentage of axons that regenerated declined with neuronal maturity. This model of SpINs will be a valuable tool in future regenerative, developmental, and functional studies alongside existing models using cortical or hippocampal neurons

Antibody recognizing 4-sulfated chondroitin sulfate proteoglycans restores memory in tauopathy-induced neurodegeneration

Chondroitin sulfate proteoglycans (CSPGs) are the main active component of perineuronal nets (PNNs). Digestion of the glycosaminoglycan chains of CSPGs with chondroitinase ABC or transgenic attenuation of PNNs leads to prolongation of object recognition memory and activation of various forms of plasticity in the adult central nervous system. The inhibitory properties of the CSPGs depend on the pattern of sulfation of their glycosaminoglycans, with chondroitin 4-sulfate (C4S) being the most inhibitory form. In this study, we tested a number of candidates for functional blocking of C4S, leading to selection of an antibody, Cat316, which specifically recognizes C4S and blocks its inhibitory effects on axon growth. It also partly blocks binding of semaphorin 3A to PNNs and attenuates PNN formation. We asked whether injection of Cat316 into the perirhinal cortex would have the same effects on memory as chondroitinase ABC treatment. We found that masking C4S with the Cat316 antibody extended long-term object recognition memory in normal wild-type mice to 24 hours, similarly to chondroitinase or transgenic PNN attenuation. We then tested Cat316 for restoration of memory in a neurodegeneration model. Mice expressing tau with the P301S mutation showed profound loss of object recognition memory at 4 months of age. Injection of Cat316 into the perirhinal cortex normalized object recognition at 3 hours in P301S mice. These data indicate that Cat316 binding to C4S in the extracellular matrix can restore plasticity and memory in the same way as chondroitinase ABC digestion. Our results suggest that antibodies to C4S could be a useful therapeutic to restore memory function in neurodegenerative disorders.This work was supported by the ERC advanced grant ECM Neuro (294502), by a fellowship to SY from Alzheimer's Research UK (ARUK-RF2016A-1), and by the NIHR Cambridge Biomedical Research Centre

Identification of furfural resistant strains of Saccharomyces cerevisiae and Saccharomyces paradoxus from a collection of environmental and industrial isolates

Background Fermentation of bioethanol using lignocellulosic biomass as a raw material provides a sustainable alternative to current biofuel production methods by utilising waste food streams as raw material. Before lignocellulose can be fermented it requires physical, chemical and enzymatic treatment in order to release monosaccharides, a process that causes the chemical transformation of glucose and xylose into the cyclic aldehydes furfural and hydroxyfurfural. These furan compounds are potent inhibitors of Saccharomyces fermentation, and consequently furfural tolerant strains of Saccharomyces are required for lignocellulosic fermentation. Results This study investigated yeast tolerance to furfural and hydroxyfurfural using a collection of 71 environmental and industrial isolates of the baker’s yeast Saccharomyces cerevisiae and its closest relative Saccharomyces paradoxus. The Saccharomyces strains were initially screened for growth on media containing 100 mM glucose and 1.5 mg ml-1 furfural. Five strains were identified that showed a significant tolerance to growth in the presence of furfural and these were then screened for growth and ethanol production in the presence of increasing amounts (0.1-4 mg ml-1) of furfural. Conclusions Of the five furfural tolerant strains S. cerevisiae NCYC 3451 displayed the greatest furfural resistance, and was able to grow in the presence of up to 3.0 mg ml-1 furfural. Furthermore, ethanol production in this strain did not appear to be inhibited by furfural, with the highest ethanol yield observed at 3.0 mg ml-1 furfural. Although furfural resistance was not found to be a trait specific to any one particular lineage or population, three of the strains were isolated from environments where they might be continually exposed to low levels of furfural through the on-going natural degradation of lignocelluloses, and would therefore develop elevated levels of resistance to these furan compounds. Thus these strains represent good candidates for future studies of genetic variation relevant to understanding and manipulating furfural resistance and in the development of tolerant ethanologenic yeast strains for use in bioethanol production from lignocellulose processing