Prevention of the foreign body response to implantable medical devices by inflammasome inhibition.

SignificanceImplantable electronic medical devices (IEMDs) are used for some clinical applications, representing an exciting prospect for the transformative treatment of intractable conditions such Parkinson's disease, deafness, and paralysis. The use of IEMDs is limited at the moment because, over time, a foreign body reaction (FBR) develops at the device-neural interface such that ultimately the IEMD fails and needs to be removed. Here, we show that macrophage nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activity drives the FBR in a nerve injury model yet integration of an NLRP3 inhibitor into the device prevents FBR while allowing full healing of damaged neural tissue to occur.

NCK1 Modulates Neuronal Actin Dynamics and Promotes Dendritic Spine, Synapse, and Memory Formation.

Memory formation and maintenance is a dynamic process involving the modulation of the actin cytoskeleton at synapses. Understanding the signaling pathways that contribute to actin modulation is important for our understanding of synapse formation and function, as well as learning and memory. Here, we focused on the importance of the actin regulator, noncatalytic region of tyrosine kinase adaptor protein 1 (NCK1), in hippocampal dependent behaviors and development. We report that male mice lacking NCK1 have impairments in both short-term and working memory, as well as spatial learning. Additionally, we report sex differences in memory impairment showing that female mice deficient in NCK1 fail at reversal learning in a spatial learning task. We find that NCK1 is expressed in postmitotic neurons but is dispensable for neuronal proliferation and migration in the developing hippocampus. Morphologically, NCK1 is not necessary for overall neuronal dendrite development. However, neurons lacking NCK1 have lower dendritic spine and synapse densities in vitro and in vivo EM analysis reveal increased postsynaptic density (PSD) thickness in the hippocampal CA1 region of NCK1-deficient mice. Mechanistically, we find the turnover of actin-filaments in dendritic spines is accelerated in neurons that lack NCK1. Together, these findings suggest that NCK1 contributes to hippocampal-dependent memory by stabilizing actin dynamics and dendritic spine formation.SIGNIFICANCE STATEMENT Understanding the molecular signaling pathways that contribute to memory formation, maintenance, and elimination will lead to a better understanding of the genetic influences on cognition and cognitive disorders and will direct future therapeutics. Here, we report that the noncatalytic region of tyrosine kinase adaptor protein 1 (NCK1) adaptor protein modulates actin-filament turnover in hippocampal dendritic spines. Mice lacking NCK1 show sex-dependent deficits in hippocampal memory formation tasks, have altered postsynaptic densities, and reduced synaptic density. Together, our work implicates NCK1 in the regulation of actin cytoskeleton dynamics and normal synapse development which is essential for memory formation.

Integrin-Driven Axon Regeneration in the Spinal Cord Activates a Distinctive CNS Regeneration Program.

The peripheral branch of sensory dorsal root ganglion (DRG) neurons regenerates readily after injury unlike their central branch in the spinal cord. However, extensive regeneration and reconnection of sensory axons in the spinal cord can be driven by the expression of α9 integrin and its activator kindlin-1 (α9k1), which enable axons to interact with tenascin-C. To elucidate the mechanisms and downstream pathways affected by activated integrin expression and central regeneration, we conducted transcriptomic analyses of adult male rat DRG sensory neurons transduced with α9k1, and controls, with and without axotomy of the central branch. Expression of α9k1 without the central axotomy led to upregulation of a known PNS regeneration program, including many genes associated with peripheral nerve regeneration. Coupling α9k1 treatment with dorsal root axotomy led to extensive central axonal regeneration. In addition to the program upregulated by α9k1 expression, regeneration in the spinal cord led to expression of a distinctive CNS regeneration program, including genes associated with ubiquitination, autophagy, endoplasmic reticulum (ER), trafficking, and signaling. Pharmacological inhibition of these processes blocked the regeneration of axons from DRGs and human iPSC-derived sensory neurons, validating their causal contributions to sensory regeneration. This CNS regeneration-associated program showed little correlation with either embryonic development or PNS regeneration programs. Potential transcriptional drivers of this CNS program coupled to regeneration include Mef2a, Runx3, E2f4, and Yy1. Signaling from integrins primes sensory neurons for regeneration, but their axon growth in the CNS is associated with an additional distinctive program that differs from that involved in PNS regeneration.SIGNIFICANCE STATEMENT Restoration of neurologic function after spinal cord injury has yet to be achieved in human patients. To accomplish this, severed nerve fibers must be made to regenerate. Reconstruction of nerve pathways has not been possible, but recently, a method for stimulating long-distance axon regeneration of sensory fibers in rodents has been developed. This research uses profiling of messenger RNAs in the regenerating sensory neurons to discover which mechanisms are activated. This study shows that the regenerating neurons initiate a novel CNS regeneration program which includes molecular transport, autophagy, ubiquitination, and modulation of the endoplasmic reticulum (ER). The study identifies mechanisms that neurons need to activate to regenerate their nerve fibers.

The roles of perineuronal nets and the perinodal extracellular matrix in neuronal function.

Perineuronal nets (PNNs) are extracellular matrix (ECM) chondroitin sulfate proteoglycan (CSPG)-containing structures that surround the soma and dendrites of various mammalian neuronal cell types. PNNs appear during development around the time that the critical periods for developmental plasticity end and are important for both their onset and closure. A similar structure - the perinodal ECM - surrounds the axonal nodes of Ranvier and appears as myelination is completed, acting as an ion-diffusion barrier that affects axonal conduction speed. Recent work has revealed the importance of PNNs in controlling plasticity in the CNS. Digestion, blocking or removal of PNNs influences functional recovery after a variety of CNS lesions. PNNs have further been shown to be involved in the regulation of memory and have been implicated in a number of psychiatric disorders.

Mllt11 Regulates Migration and Neurite Outgrowth of Cortical Projection Neurons during Development.

The formation of connections within the mammalian neocortex is highly regulated by both extracellular guidance mechanisms and intrinsic gene expression programs. There are two types of cortical projection neurons (CPNs): those that project locally and interhemispherically and those that project to subcerebral structures such as the thalamus, hindbrain, and spinal cord. The regulation of cortical projection morphologies is not yet fully understood at the molecular level. Here, we report a role for Mllt11 (Myeloid/lymphoid or mixed-lineage leukemia; translocated to chromosome 11/All1 Fused Gene From Chromosome 1q) in the migration and neurite outgrowth of callosal projection neurons during mouse brain formation. We show that Mllt11 expression is exclusive to developing neurons and is enriched in the developing cortical plate (CP) during the formation of the superficial cortical layers. In cultured primary cortical neurons, Mllt11 is detected in varicosities and growth cones as well as the soma. Using conditional loss-of-function and gain-of-function analysis we show that Mllt11 is required for neuritogenesis and proper migration of upper layer CPNs. Loss of Mllt11 in the superficial cortex of male and female neonates leads to a severe reduction in fibers crossing the corpus callosum (CC), a progressive loss in the maintenance of upper layer projection neuron gene expression, and reduced complexity of dendritic arborization. Proteomic analysis revealed that Mllt11 associates with stabilized microtubules, and Mllt11 loss affected microtubule staining in callosal axons. Taken together, our findings support a role for Mllt11 in promoting the formation of mature upper-layer neuron morphologies and connectivity in the cerebral cortex.SIGNIFICANCE STATEMENT The regulation of cortical projection neuron (CPN) morphologies is an area of active investigation since the time of Cajal. Yet the molecular mechanisms of how the complex dendritic and axonal morphologies of projection neurons are formed remains incompletely understood. Although conditional mutagenesis analysis in the mouse, coupled with overexpression assays in the developing fetal brain, we show that a novel protein called Mllt11 is sufficient and necessary to regulate the dendritic and axonal characteristics of callosal projection neurons in the developing mammalian neocortex. Furthermore, we show that Mllt11 interacts with microtubules, likely accounting for its role in neuritogenesis.

Improving adeno-associated viral (AAV) vector-mediated transgene expression in retinal ganglion cells: comparison of five promoters.

Recombinant adeno-associated viral vectors (AAVs) are an effective system for gene transfer. AAV serotype 2 (AAV2) is commonly used to deliver transgenes to retinal ganglion cells (RGCs) via intravitreal injection. The AAV serotype however is not the only factor contributing to the effectiveness of gene therapies. Promoters influence the strength and cell-selectivity of transgene expression. This study compares five promoters designed to maximise AAV2 cargo space for gene delivery: chicken ß-actin (CBA), cytomegalovirus (CMV), short CMV early enhancer/chicken ß-actin/short ß-globulin intron (sCAG), mouse phosphoglycerate kinase (PGK), and human synapsin (SYN). The promoters driving enhanced green fluorescent protein (eGFP) were examined in adult C57BL/6J mice eyes and tissues of the visual system. eGFP expression was strongest in the retina, optic nerves and brain when driven by the sCAG and SYN promoters. CBA, CMV, and PGK had moderate expression by comparison. The SYN promoter had almost exclusive transgene expression in RGCs. The PGK promoter had predominant expression in both RGCs and AII amacrine cells. The ubiquitous CBA, CMV, and sCAG promoters expressed eGFP in a variety of cell types across multiple retinal layers including Müller glia and astrocytes. We also found that these promoters could transduce human retina ex vivo, although expression was predominantly in glial cells due to low RGC viability. Taken together, this promoter comparison study contributes to optimising AAV-mediated transduction in the retina, and could be valuable for research in ocular disorders, particularly those with large or complex genetic cargos.

The extracellular matrix and perineuronal nets in memory.

All components of the CNS are surrounded by a diffuse extracellular matrix (ECM) containing chondroitin sulphate proteoglycans (CSPGs), heparan sulphate proteoglycans (HSPGs), hyaluronan, various glycoproteins including tenascins and thrombospondin, and many other molecules that are secreted into the ECM and bind to ECM components. In addition, some neurons, particularly inhibitory GABAergic parvalbumin-positive (PV) interneurons, are surrounded by a more condensed cartilage-like ECM called perineuronal nets (PNNs). PNNs surround the soma and proximal dendrites as net-like structures that surround the synapses. Attention has focused on the role of PNNs in the control of plasticity, but it is now clear that PNNs also play an important part in the modulation of memory. In this review we summarize the role of the ECM, particularly the PNNs, in the control of various types of memory and their participation in memory pathology. PNNs are now being considered as a target for the treatment of impaired memory. There are many potential treatment targets in PNNs, mainly through modulation of the sulphation, binding, and production of the various CSPGs that they contain or through digestion of their sulphated glycosaminoglycans.

Chondroitin 6-sulphate is required for neuroplasticity and memory in ageing.

Perineuronal nets (PNNs) are chondroitin sulphate proteoglycan-containing structures on the neuronal surface that have been implicated in the control of neuroplasticity and memory. Age-related reduction of chondroitin 6-sulphates (C6S) leads to PNNs becoming more inhibitory. Here, we investigated whether manipulation of the chondroitin sulphate (CS) composition of the PNNs could restore neuroplasticity and alleviate memory deficits in aged mice. We first confirmed that aged mice (20-months) showed memory and plasticity deficits. They were able to retain or regain their cognitive ability when CSs were digested or PNNs were attenuated. We then explored the role of C6S in memory and neuroplasticity. Transgenic deletion of chondroitin 6-sulfotransferase (chst3) led to a reduction of permissive C6S, simulating aged brains. These animals showed very early memory loss at 11 weeks old. Importantly, restoring C6S levels in aged animals rescued the memory deficits and restored cortical long-term potentiation, suggesting a strategy to improve age-related memory impairment.

Phosphite synthetic auxotrophy as an effective biocontainment strategy for the industrial chassis Pseudomonas putida.

The inclusion of biosafety strategies into strain engineering pipelines is crucial for safe-by-design biobased processes. This in turn might enable a more rapid regulatory acceptance of bioengineered organisms in both industrial and environmental applications. For this reason, we equipped the industrially relevant microbial chassis Pseudomonas putida KT2440 with an effective biocontainment strategy based on a synthetic dependency on phosphite, which is generally not readily available in the environment. The produced PSAG-9 strain was first engineered to assimilate phosphite through the genome-integration of a phosphite dehydrogenase and a phosphite-specific transport complex. Subsequently, to deter the strain from growing on naturally assimilated phosphate, all native genes related to its transport were identified and deleted generating a strain unable to grow on media containing any phosphorous source other than phosphite. PSAG-9 exhibited fitness levels with phosphite similar to those of the wild type with phosphate, and low levels of escape frequency. Beyond biosafety, this strategy endowed P. putida with the capacity to be cultured under non-sterile conditions using phosphite as the sole phosphorous source with a reduced risk of contamination by other microbes, while displaying enhanced NADH regenerative capacity. These industrially beneficial features complement the metabolic advantages for which this species is known for, thereby strengthening it as a synthetic biology chassis with potential uses in industry, with suitability towards environmental release.

Optimization of adeno-associated viral vector-mediated transduction of the corticospinal tract: comparison of four promoters.

Adeno-associated viral vectors are widely used as vehicles for gene transfer to the nervous system. The promoter and viral vector serotype are two key factors that determine the expression dynamics of the transgene. A previous comparative study has demonstrated that AAV1 displays efficient transduction of layer V corticospinal neurons, but the optimal promoter for transgene expression in corticospinal neurons has not been determined yet. In this paper, we report a side-by-side comparison between four commonly used promoters: the short CMV early enhancer/chicken ß actin (sCAG), human cytomegalovirus (hCMV), mouse phosphoglycerate kinase (mPGK) and human synapsin (hSYN) promoter. Reporter constructs with each of these promoters were packaged in AAV1, and were injected in the sensorimotor cortex of rats and mice in order to transduce the corticospinal tract. Transgene expression levels and the cellular transduction profile were examined after 6 weeks. The AAV1 vectors harbouring the hCMV and sCAG promoters resulted in transgene expression in neurons, astrocytes and oligodendrocytes. The mPGK and hSYN promoters directed the strongest transgene expression. The mPGK promoter did drive expression in cortical neurons and oligodendrocytes, while transduction with AAV harbouring the hSYN promoter resulted in neuron-specific expression, including perineuronal net expressing interneurons and layer V corticospinal neurons. This promoter comparison study contributes to improve transgene delivery into the brain and spinal cord. The optimized transduction of the corticospinal tract will be beneficial for spinal cord injury research.

Axonal Organelles as Molecular Platforms for Axon Growth and Regeneration after Injury.

Investigating the molecular mechanisms governing developmental axon growth has been a useful approach for identifying new strategies for boosting axon regeneration after injury, with the goal of treating debilitating conditions such as spinal cord injury and vision loss. The picture emerging is that various axonal organelles are important centers for organizing the molecular mechanisms and machinery required for growth cone development and axon extension, and these have recently been targeted to stimulate robust regeneration in the injured adult central nervous system (CNS). This review summarizes recent literature highlighting a central role for organelles such as recycling endosomes, the endoplasmic reticulum, mitochondria, lysosomes, autophagosomes and the proteasome in developmental axon growth, and describes how these organelles can be targeted to promote axon regeneration after injury to the adult CNS. This review also examines the connections between these organelles in developing and regenerating axons, and finally discusses the molecular mechanisms within the axon that are required for successful axon growth.

The Struggle to Make CNS Axons Regenerate: Why Has It Been so Difficult?

Axon regeneration in the CNS is inhibited by many extrinsic and intrinsic factors. Because these act in parallel, no single intervention has been sufficient to enable full regeneration of damaged axons in the adult mammalian CNS. In the external environment, NogoA and CSPGs are strongly inhibitory to the regeneration of adult axons. CNS neurons lose intrinsic regenerative ability as they mature: embryonic but not mature neurons can grow axons for long distances when transplanted into the adult CNS, and regeneration fails with maturity in in vitro axotomy models. The causes of this loss of regeneration include partitioning of neurons into axonal and dendritic fields with many growth-related molecules directed specifically to dendrites and excluded from axons, changes in axonal signalling due to changes in expression and localization of receptors and their ligands, changes in local translation of proteins in axons, and changes in cytoskeletal dynamics after injury. Also with neuronal maturation come epigenetic changes in neurons, with many of the transcription factor binding sites that drive axon growth-related genes becoming inaccessible. The overall aim for successful regeneration is to ensure that the right molecules are expressed after axotomy and to arrange for them to be transported to the right place in the neuron, including the damaged axon tip.

Transplantation of Neural Precursors Derived from Induced Pluripotent Cells Preserve Perineuronal Nets and Stimulate Neural Plasticity in ALS Rats.

A promising therapeutic strategy for amyotrophic lateral sclerosis (ALS) treatment is stem cell therapy. Neural progenitors derived from induced pluripotent cells (NP-iPS) might rescue or replace dying motoneurons (MNs). However, the mechanisms responsible for the beneficial effect are not fully understood. The aim here was to investigate the mechanism by studying the effect of intraspinally injected NP-iPS into asymptomatic and early symptomatic superoxide dismutase (SOD)1G93A transgenic rats. Prior to transplantation, NP-iPS were characterized in vitro for their ability to differentiate into a neuronal phenotype. Motor functions were tested in all animals, and the tissue was analyzed by immunohistochemistry, qPCR, and Western blot. NP-iPS transplantation significantly preserved MNs, slowed disease progression, and extended the survival of all treated animals. The dysregulation of spinal chondroitin sulfate proteoglycans was observed in SOD1G93A rats at the terminal stage. NP-iPS application led to normalized host genes expression (versican, has-1, tenascin-R, ngf, igf-1, bdnf, bax, bcl-2, and casp-3) and the protection of perineuronal nets around the preserved MNs. In the host spinal cord, transplanted cells remained as progenitors, many in contact with MNs, but they did not differentiate. The findings suggest that NP-iPS demonstrate neuroprotective properties by regulating local gene expression and regulate plasticity by modulating the central nervous system (CNS) extracellular matrix such as perineuronal nets (PNNs).

Aggrecan Directs Extracellular Matrix-Mediated Neuronal Plasticity.

In the adult brain, the extracellular matrix (ECM) influences recovery after injury, susceptibility to mental disorders, and is in general a strong regulator of neuronal plasticity. The proteoglycan aggrecan is a core component of the condensed ECM structures termed perineuronal nets (PNNs), and the specific role of PNNs on neural plasticity remains elusive. Here, we genetically targeted the Acan gene encoding for aggrecan using a novel animal model. This allowed for conditional and targeted loss of aggrecan in vivo, which ablated the PNN structure and caused a shift in the population of parvalbumin-expressing inhibitory interneurons toward a high plasticity state. Selective deletion of the Acan gene in the visual cortex of male adult mice reinstated juvenile ocular dominance plasticity, which was mechanistically identical to critical period plasticity. Brain-wide targeting improved object recognition memory.SIGNIFICANCE STATEMENT The study provides the first direct evidence of aggrecan as the main functional constituent and orchestrator of perineuronal nets (PNNs), and that loss of PNNs by aggrecan removal induces a permanent state of critical period-like plasticity. Loss of aggrecan ablates the PNN structure, resulting in invoked juvenile plasticity in the visual cortex and enhanced object recognition memory.

Targeting Chondroitin Sulfate Glycosaminoglycans to Treat Cardiac Fibrosis in Pathological Remodeling.

BACKGROUND: Heart failure is a leading cause of mortality and morbidity, and the search for novel therapeutic approaches continues. In the monogenic disease mucopolysaccharidosis VI, loss-of-function mutations in arylsulfatase B lead to myocardial accumulation of chondroitin sulfate (CS) glycosaminoglycans, manifesting as myriad cardiac symptoms. Here, we studied changes in myocardial CS in nonmucopolysaccharidosis failing hearts and assessed its generic role in pathological cardiac remodeling. METHODS: Healthy and diseased human and rat left ventricles were subjected to histological and immunostaining methods to analyze glycosaminoglycan distribution. Glycosaminoglycans were extracted and analyzed for quantitative and compositional changes with Alcian blue assay and liquid chromatography-mass spectrometry. Expression changes in 20 CS-related genes were studied in 3 primary human cardiac cell types and THP-1-derived macrophages under each of 9 in vitro stimulatory conditions. In 2 rat models of pathological remodeling induced by transverse aortic constriction or isoprenaline infusion, recombinant human arylsulfatase B (rhASB), clinically used as enzyme replacement therapy in mucopolysaccharidosis VI, was administered intravenously for 7 or 5 weeks, respectively. Cardiac function, myocardial fibrosis, and inflammation were assessed by echocardiography and histology. CS-interacting molecules were assessed with surface plasmon resonance, and a mechanism of action was verified in vitro. RESULTS: Failing human hearts displayed significant perivascular and interstitial CS accumulation, particularly in regions of intense fibrosis. Relative composition of CS disaccharides remained unchanged. Transforming growth factor-ß induced CS upregulation in cardiac fibroblasts. CS accumulation was also observed in both the pressure-overload and the isoprenaline models of pathological remodeling in rats. Early treatment with rhASB in the transverse aortic constriction model and delayed treatment in the isoprenaline model proved rhASB to be effective at preventing cardiac deterioration and augmenting functional recovery. Functional improvement was accompanied by reduced myocardial inflammation and overall fibrosis. Tumor necrosis factor-α was identified as a direct binding partner of CS glycosaminoglycan chains, and rhASB reduced tumor necrosis factor-α-induced inflammatory gene activation in vitro in endothelial cells and macrophages. CONCLUSIONS: CS glycosaminoglycans accumulate during cardiac pathological remodeling and mediate myocardial inflammation and fibrosis. rhASB targets CS effectively as a novel therapeutic approach for the treatment of heart failure.

EFA6 regulates selective polarised transport and axon regeneration from the axon initial segment.

Central nervous system (CNS) axons lose their intrinsic ability to regenerate upon maturity, whereas peripheral nervous system (PNS) axons do not. A key difference between these neuronal types is their ability to transport integrins into axons. Integrins can mediate PNS regeneration, but are excluded from adult CNS axons along with their Rab11 carriers. We reasoned that exclusion of the contents of Rab11 vesicles including integrins might contribute to the intrinsic inability of CNS neurons to regenerate, and investigated this by performing laser axotomy. We identify a novel regulator of selective axon transport and regeneration, the ARF6 guanine-nucleotide-exchange factor (GEF) EFA6 (also known as PSD). EFA6 exerts its effects from a location within the axon initial segment (AIS). EFA6 does not localise at the AIS in dorsal root ganglion (DRG) axons, and in these neurons, ARF6 activation is counteracted by an ARF GTPase-activating protein (GAP), which is absent from the CNS, ACAP1. Depleting EFA6 from cortical neurons permits endosomal integrin transport and enhances regeneration, whereas overexpressing EFA6 prevents DRG regeneration. Our results demonstrate that ARF6 is an intrinsic regulator of regenerative capacity, implicating EFA6 as a focal molecule linking the AIS, signalling and transport.This article has an associated First Person interview with the first author of the paper.

Neurochemical biomarkers in spinal cord injury.

STUDY DESIGN: This is a narrative review of the literature on neurochemical biomarkers in spinal cord injury (SCI). OBJECTIVES: The objective was to summarize the literature on neurochemical biomarkers in SCI and describe their use in facilitating clinical trials for SCI. Clinical trials in spinal cord injury (SCI) have been notoriously difficult to conduct, as exemplified by the paucity of definitive prospective randomized trials that have been completed, to date. This is related to the relatively low incidence and the complexity and heterogeneity of the human SCI condition. Given the increasing number of promising approaches that are emerging from the laboratory which are vying for clinical evaluation, novel strategies to help facilitate clinical trials are needed. METHODS: A literature review was conducted, with a focus on neurochemical biomarkers that have been described in human neurotrauma. RESULTS: We describe advances in our understanding of neurochemical biomarkers as they pertain to human SCI. The application of biomarkers from serum and cerebrospinal fluid (CSF) has been led by efforts in the human traumatic brain injury (TBI) literature. A number of promising biomarkers have been described in human SCI whereby they may assist in stratifying injury severity and predicting outcome. CONCLUSIONS: Several time-specific biomarkers have been described for acute SCI and for chronic SCI. These appear promising for stratifying injury severity and potentially predicting outcome. The subsequent application within a clinical trial will help to demonstrate their utility in facilitating the study of novel approaches for SCI.

The challenge of recruitment for neurotherapeutic clinical trials in spinal cord injury.

STUDY DESIGN: Narrative review by individuals experienced in the recruitment of participants to neurotherapeutic clinical trials in spinal cord injury (SCI). OBJECTIVES: To identify key problems of recruitment and explore potential approaches to overcoming them. METHODS: Published quantitative experience with recruitment of large-scale, experimental neurotherapeutic clinical studies targeting central nervous system and using primary outcome assessments validated for SCI over the last 3 decades was summarized. Based on this experience, potential approaches to improving recruitment were elicited from the authors. RESULTS: The rate of recruitment has varied between studies, depending on protocol design and other factors, but particularly inclusion/exclusion criteria. The recruitment rate also ranged over an order of magnitude between individual centers in a given study. In older multicenter studies, average recruitment rate was approximately one person per study center per month. More recent trials experienced lower rates of recruitment and potential reasons for this trend were examined. The current roles and potential of various stakeholder organizations in addressing problems of recruitment were explored. In addition, recent developments in methodology may help reduce the number of subjects required for well-powered studies. CONCLUSIONS: Several approaches are emerging to improve clinical trial design, efficacy outcome measures, and quantifiable surrogate markers, all of which should reduce the number of participants required for adequate statistical power. There is a growing sense of cooperation between various stakeholders but more should be done to bring together consumer and provider groups to improve recruitment and the effectiveness and relevance of neurotherapeutic clinical trials.

Neuronal Pentraxin 2 Binds PNNs and Enhances PNN Formation.

The perineuronal net (PNN) is a mesh-like proteoglycan structure on the neuronal surface which is involved in regulating plasticity. The PNN regulates plasticity via multiple pathways, one of which is direct regulation of synapses through the control of AMPA receptor mobility. Since neuronal pentraxin 2 (Nptx2) is a known regulator of AMPA receptor mobility and Nptx2 can be removed from the neuronal surface by PNN removal, we investigated whether Nptx2 has a function in the PNN. We found that Nptx2 binds to the glycosaminoglycans hyaluronan and chondroitin sulphate E in the PNN. Furthermore, in primary cortical neuron cultures, the addition of NPTX2 to the culture medium enhances PNN formation during PNN development. These findings suggest Nptx2 as a novel PNN binding protein with a role in the mechanism of PNN formation.

Expression of an Activated Integrin Promotes Long-Distance Sensory Axon Regeneration in the Spinal Cord.

UNLABELLED: After CNS injury, axon regeneration is blocked by an inhibitory environment consisting of the highly upregulated tenascin-C and chondroitin sulfate proteoglycans (CSPGs). Tenascin-C promotes growth of axons if they express a tenascin-binding integrin, particularly α9ß1. Additionally, integrins can be inactivated by CSPGs, and this inhibition can be overcome by the presence of a ß1-binding integrin activator, kindlin-1. We examined the synergistic effect of α9 integrin and kindlin-1 on sensory axon regeneration in adult rat spinal cord after dorsal root crush and adeno-associated virus transgene expression in dorsal root ganglia. After 12 weeks, axons from C6-C7 dorsal root ganglia regenerated through the tenascin-C-rich dorsal root entry zone into the dorsal column up to C1 level and above (>25 mm axon length) through a normal pathway. Animals also showed anatomical and electrophysiological evidence of reconnection to the dorsal horn and behavioral recovery in mechanical pressure, thermal pain, and ladder-walking tasks. Expression of α9 integrin or kindlin-1 alone promoted much less regeneration and recovery. SIGNIFICANCE STATEMENT: The study demonstrates that long-distance sensory axon regeneration over a normal pathway and with sensory and sensory-motor recovery can be achieved. This was achieved by expressing an integrin that recognizes tenascin-C, one of the components of glial scar tissue, and an integrin activator. This enabled extensive long-distance (>25 mm) regeneration of both myelinated and unmyelinated sensory axons with topographically correct connections in the spinal cord. The extent of growth and recovery we have seen would probably be clinically significant. Restoration of sensation to hands, perineum, and genitalia would be a significant improvement for a spinal cord-injured patient.