Intranasal delivery of full-length anti-Nogo-A antibody: A potential alternative route for therapeutic antibodies to central nervous system targets.

Antibody delivery to the CNS remains a huge hurdle for the clinical application of antibodies targeting a CNS antigen. The blood-brain barrier and blood-CSF barrier restrict access of therapeutic antibodies to their CNS targets in a major way. The very high amounts of therapeutic antibodies that are administered systemically in recent clinical trials to reach CNS targets are barely viable cost-wise for broad, routine applications. Though global CNS delivery of antibodies can be achieved by intrathecal application, these procedures are invasive. A non-invasive method to bring antibodies into the CNS reliably and reproducibly remains an important unmet need in neurology. In the present study, we show that intranasal application of a mouse monoclonal antibody against the neurite growth-inhibiting and plasticity-restricting membrane protein Nogo-A leads to a rapid transfer of significant amounts of antibody to the brain and spinal cord in intact adult rats. Daily intranasal application for 2 wk of anti-Nogo-A antibody enhanced growth and compensatory sprouting of corticofugal projections and functional recovery in rats after large unilateral cortical strokes. These findings are a starting point for clinical translation for a less invasive route of application of therapeutic antibodies to CNS targets for many neurological indications.

Development of a Sensitive Enzyme-Linked Immunosorbent Assay with Three Amplification Antibodies to Detect Low-Abundant Mouse Antibodies in the Mouse Central Nervous System.

Recombinant Abs are gaining increasing importance for the treatment of certain cancers or immunological or neurologic disorders. The ELISA is one of the most used analytical tools for detecting and quantifying Abs of interest. However, the performance of ELISAs often varies because of nonstandard experimental procedures as well as inadequate data analysis. In our study, we standardized a procedure and statistical analysis for a highly sensitive ELISA of a mouse Ab in mouse (C57BL/6J) CNS tissue. The following steps are of crucial importance: 1) calculation of the limit of detection based on control tissue lysate samples in the same testing buffer as the testing samples; 2) calculation of the limit of quantification as measured with acceptable accuracy and precision; and 3) a five-parameter logistic regression model to interpolate the symmetric and asymmetric standard curves. We also show that three amplification Abs can significantly increase the sensitivity of the ELISA compared with a two amplification Ab setup. This standardized procedure may be a valuable tool to increase the sensitivity, reproducibility, and precision of ELISA studies in basic science and translational research.

Four-parameter analysis in modified Rotarod test for detecting minor motor deficits in mice.

BACKGROUND: The Rotarod test with commercial apparatus is widely used to assess locomotor performance, balance and motor learning as well as the deficits resulting from diverse neurological disorders in laboratory rodents due to its simplicity and objectivity. Traditionally, the test ends when rodents drop from the accelerating, turning rod, and the only parameter used commonly is "latency to fall". The values of individual animals can often vary greatly. RESULTS: In the present study, we established a procedure for mice with 4 consecutive days of training with 4 trials per day and modified the testing procedure by placing the mice back on the rod repeatedly after each fall until the trial ends (5 min). Data from the fourth training day as baseline results showed that the second, third and fourth trial were more consistent than the first, probably due to habituation or learning. There was no difference between the second, third and fourth trial, two trials may be sufficient in testing. We also introduced 3 additional read-outs: Longest duration on the rod (s), Maximal distance covered (cm), and Number of falls to better evaluate the motor capacity over the 5 min of testing. We then used this 4-parameter analysis to capture the motor deficits of mice with mild to moderate traumatic brain injuries (by a weight dropping on the skull (Marmarou model)). We found that normalization of data to individual baseline performance was needed to reduce individual differences, and 4 trials were more sensitive than two to show motor deficits. The parameter of Maximal distance was the best in detecting statistically significant long-term motor deficits. CONCLUSIONS: These results show that by making adjustments to the protocol and employing a more refined analysis, it is possible to expand a widely used routine behavioral test with additional accessible parameters that detect relevant deficits in a model of mild to moderate traumatic brain injury. The modified Rotarod test maybe a valuable tool for better preclinical evaluations of drugs and therapies.

The vascular gene Apold1 is dispensable for normal development but controls angiogenesis under pathological conditions.

The molecular mechanisms of angiogenesis have been intensely studied, but many genes that control endothelial behavior and fate still need to be described. Here, we characterize the role of Apold1 (Apolipoprotein L domain containing 1) in angiogenesis in vivo and in vitro. Single-cell analyses reveal that - across tissues - the expression of Apold1 is restricted to the vasculature and that Apold1 expression in endothelial cells (ECs) is highly sensitive to environmental factors. Using Apold1-/- mice, we find that Apold1 is dispensable for development and does not affect postnatal retinal angiogenesis nor alters the vascular network in adult brain and muscle. However, when exposed to ischemic conditions following photothrombotic stroke as well as femoral artery ligation, Apold1-/- mice display dramatic impairments in recovery and revascularization. We also find that human tumor endothelial cells express strikingly higher levels of Apold1 and that Apold1 deletion in mice stunts the growth of subcutaneous B16 melanoma tumors, which have smaller and poorly perfused vessels. Mechanistically, Apold1 is activated in ECs upon growth factor stimulation as well as in hypoxia, and Apold1 intrinsically controls EC proliferation but not migration. Our data demonstrate that Apold1 is a key regulator of angiogenesis in pathological settings, whereas it does not affect developmental angiogenesis, thus making it a promising candidate for clinical investigation.

Stimulation of the cuneiform nucleus enables training and boosts recovery after spinal cord injury.

Severe spinal cord injuries result in permanent paraparesis in spite of the frequent sparing of small portions of white matter. Spared fibre tracts are often incapable of maintaining and modulating the activity of lower spinal motor centres. Effects of rehabilitative training thus remain limited. Here, we activated spared descending brainstem fibres by electrical deep brain stimulation of the cuneiform nucleus of the mesencephalic locomotor region, the main control centre for locomotion in the brainstem, in adult female Lewis rats. We show that deep brain stimulation of the cuneiform nucleus enhances the weak remaining motor drive in highly paraparetic rats with severe, incomplete spinal cord injuries and enables high-intensity locomotor training. Stimulation of the cuneiform nucleus during rehabilitative aquatraining after subchronic (n = 8 stimulated versus n = 7 unstimulated versus n = 7 untrained rats) and chronic (n = 14 stimulated versus n = 9 unstimulated versus n = 9 untrained rats) spinal cord injury re-established substantial locomotion and improved long-term recovery of motor function. We additionally identified a safety window of stimulation parameters ensuring context-specific locomotor control in intact rats (n = 18) and illustrate the importance of timing of treatment initiation after spinal cord injury (n = 14). This study highlights stimulation of the cuneiform nucleus as a highly promising therapeutic strategy to enhance motor recovery after subchronic and chronic incomplete spinal cord injury with direct clinical applicability.

Nogo-A targeted therapy promotes vascular repair and functional recovery following stroke.

Stroke is a major cause of serious disability due to the brain's limited capacity to regenerate damaged tissue and neuronal circuits. After ischemic injury, a multiphasic degenerative and inflammatory response is coupled with severely restricted vascular and neuronal repair, resulting in permanent functional deficits. Although clinical evidence indicates that revascularization of the ischemic brain regions is crucial for functional recovery, no therapeutics that promote angiogenesis after cerebral stroke are currently available. Besides vascular growth factors, guidance molecules have been identified to regulate aspects of angiogenesis in the central nervous system (CNS) and may provide targets for therapeutic angiogenesis. In this study, we demonstrate that genetic deletion of the neurite outgrowth inhibitor Nogo-A or one of its corresponding receptors, S1PR2, improves vascular sprouting and repair and reduces neurological deficits after cerebral ischemia in mice. These findings were reproduced in a therapeutic approach using intrathecal anti-Nogo-A antibodies; such a therapy is currently in clinical testing for spinal cord injury. These results provide a basis for a therapeutic blockage of inhibitory guidance molecules to improve vascular and neural repair after ischemic CNS injuries.

Development of Neurogenic Detrusor Overactivity after Thoracic Spinal Cord Injury Is Accompanied by Time-Dependent Changes in Lumbosacral Expression of Axonal Growth Regulators.

Thoracic spinal cord injury (SCI) results in urinary dysfunction, which majorly affects the quality of life of SCI patients. Abnormal sprouting of lumbosacral bladder afferents plays a crucial role in this condition. Underlying mechanisms may include changes in expression of regulators of axonal growth, including chondroitin sulphate proteoglycans (CSPGs), myelin-associated inhibitors (MAIs) and repulsive guidance molecules, known to be upregulated at the injury site post SCI. Here, we confirmed lumbosacral upregulation of the growth-associated protein GAP43 in SCI animals with bladder dysfunction, indicating the occurrence of axonal sprouting. Neurocan and Phosphacan (CSPGs), as well as Nogo-A (MAI), at the same spinal segments were upregulated 7 days post injury (dpi) but returned to baseline values 28 dpi. In turn, qPCR analysis of the mRNA levels for receptors of those repulsive molecules in dorsal root ganglia (DRG) neurons showed a time-dependent decrease in receptor expression. In vitro assays with DRG neurons from SCI rats demonstrated that exposure to high levels of NGF downregulated the expression of some, but not all, receptors for those regulators of axonal growth. The present results, therefore, show significant molecular changes at the lumbosacral cord and DRGs after thoracic lesion, likely critically involved in neuroplastic events leading to urinary impairment.

The Gigantocellular Reticular Nucleus Plays a Significant Role in Locomotor Recovery after Incomplete Spinal Cord Injury.

Traditionally, the brainstem has been seen as hardwired and poorly capable of plastic adaptations following spinal cord injury (SCI). Data acquired over the past decades, however, suggest differently: following SCI in various animal models (lamprey, chick, rodents, nonhuman primates), different forms of spontaneous anatomic plasticity of reticulospinal projections, many of them originating from the gigantocellular reticular nucleus (NRG), have been observed. In line with these anatomic observations, animals and humans with incomplete SCI often show various degrees of spontaneous motor recovery of hindlimb/leg function. Here, we investigated the functional relevance of two different modes of reticulospinal fiber growth after cervical hemisection, local rewiring of axotomized projections at the lesion site versus compensatory outgrowth of spared axons, using projection-specific, adeno-associated virus-mediated chemogenetic neuronal silencing. Detailed assessment of joint movements and limb kinetics during overground locomotion in female adult rats showed that locally rewired as well as compensatory NRG fibers were responsible for different aspects of recovered forelimb and hindlimb functions (i.e., stability, strength, coordination, speed, or timing). During walking and swimming, both locally rewired as well as compensatory NRG plasticity were crucial for recovered function, while the contribution of locally rewired NRG plasticity to wading performance was limited. Our data demonstrate comprehensively that locally rewired as well as compensatory plasticity of reticulospinal axons functionally contribute to the observed spontaneous improvement of stepping performance after incomplete SCI and are at least partially causative to the observed recovery of function, which can also be observed in human patients with spinal hemisection lesions.SIGNIFICANCE STATEMENT Following unilateral hemisection of the spinal cord, reticulospinal projections are destroyed on the injured side, resulting in impaired locomotion. Over time, a high degree of recovery can be observed in lesioned animals, like in human hemicord patients. In the rat, recovery is accompanied by pronounced spontaneous plasticity of axotomized and spared reticulospinal axons. We demonstrate the causative relevance of locally rewired as well as compensatory reticulospinal plasticity for the recovery of locomotor functions following spinal hemisection, using chemogenetic tools to selectively silence newly formed connections in behaviorally recovered animals. Moving from a correlative to a causative understanding of the role of neuroanatomical plasticity for functional recovery is fundamental for successful translation of treatment approaches from experimental studies to the clinics.

The Spinal Transcriptome after Cortical Stroke: In Search of Molecular Factors Regulating Spontaneous Recovery in the Spinal Cord.

In response to cortical stroke and unilateral corticospinal tract degeneration, compensatory sprouting of spared corticospinal fibers is associated with recovery of skilled movement in rodents. To date, little is known about the molecular mechanisms orchestrating this spontaneous rewiring. In this study, we provide insights into the molecular changes in the spinal cord tissue after large ischemic cortical injury in adult female mice, with a focus on factors that might influence the reinnervation process by contralesional corticospinal neurons. We mapped the area of cervical gray matter reinnervation by sprouting contralesional corticospinal axons after unilateral photothrombotic stroke of the motor cortex in mice using anterograde tracing. The mRNA profile of this reinnervation area was analyzed using whole-genome sequencing to identify differentially expressed genes at selected time points during the recovery process. Bioinformatic analysis revealed two phases of processes: early after stroke (4-7 d post-injury), the spinal transcriptome is characterized by inflammatory processes, including phagocytic processes as well as complement cascade activation. Microglia are specifically activated in the denervated corticospinal projection fields in this early phase. In a later phase (28-42 d post-injury), biological processes include tissue repair pathways with upregulated genes related to neurite outgrowth. Thus, the stroke-denervated spinal gray matter, in particular its intermediate laminae, represents a growth-promoting environment for sprouting corticospinal fibers originating from the contralesional motor cortex. This dataset provides a solid starting point for future studies addressing key elements of the post-stroke recovery process, with the goal to improve neuroregenerative treatment options for stroke patients.SIGNIFICANCE STATEMENT We show that the molecular changes in the spinal cord target tissue of the stroke-affected corticospinal tract are mainly defined by two phases: an early inflammatory phase during which microglia are specifically activated in the target area of reinnervating corticospinal motor neurons; and a late phase during which growth-promoting factors are upregulated which can influence the sprouting response, arborization, and synapse formation. By defining for the first time the endogenous molecular machinery in the stroke-denervated cervical spinal gray matter with a focus on promotors of axon growth through the growth-inhibitory adult CNS, this study will serve as a basis to address novel neuroregenerative treatment options for chronic stroke patients.

Anti-Nogo-A Antibodies As a Potential Causal Therapy for Lower Urinary Tract Dysfunction after Spinal Cord Injury.

Loss of bladder control is common after spinal cord injury (SCI) and no causal therapies are available. Here we investigated whether function-blocking antibodies against the nerve-fiber growth inhibitory protein Nogo-A applied to rats with severe SCI could prevent development of neurogenic lower urinary tract dysfunction. Bladder function of rats with SCI was repeatedly assessed by urodynamic examination in fully awake animals. Four weeks after SCI, detrusor sphincter dyssynergia had developed in all untreated or control antibody-infused animals. In contrast, 2 weeks of intrathecal anti-Nogo-A antibody treatment led to significantly reduced aberrant maximum detrusor pressure during voiding and a reduction of the abnormal EMG high-frequency activity in the external urethral sphincter. Anatomically, we found higher densities of fibers originating from the pontine micturition center in the lumbosacral gray matter in the anti-Nogo-A antibody-treated animals, as well as a reduced number of inhibitory interneurons in lamina X. These results suggest that anti-Nogo-A therapy could also have positive effects on bladder function clinically.SIGNIFICANCE STATEMENT After spinal cord injury, loss of bladder control is common. Detrusor sphincter dyssynergia is a potentially life-threatening consequence. Currently, only symptomatic treatment options are available. First causal treatment options are urgently needed in humans. In this work, we show that function-blocking antibodies against the nerve-fiber growth inhibitory protein Nogo-A applied to rats with severe spinal cord injury could prevent development of neurogenic lower urinary tract dysfunction, in particular detrusor sphincter dyssynergia. Anti-Nogo-A therapy has entered phase II clinical trial in humans and might therefore soon be the first causal treatment option for neurogenic lower urinary tract dysfunction.

Pro- and antiangiogenic therapies: current status and clinical implications.

Blood vessels nurture every part of the human body. Consequently, abnormalities in the vasculature are closely associated with a variety of diseases, including cerebral stroke, heart disease, retinopathy, and cancer. Pro- or antiangiogenic therapies can influence these diseases by regulating the growth of new blood vessels from a pre-existing vascular network or dampening excessive blood growth. However, clinical translation of these approaches is slow and challenging. In this review, we discuss recent preclinical approaches to regulate angiogenesis and their potential and risks in a clinical setting.-Rust, R., Gantner, C., Schwab, M. E. Pro- and antiangiogenic therapies: current status and clinical implications.

Inactivation of sphingosine-1-phosphate receptor 2 (S1PR2) decreases demyelination and enhances remyelination in animal models of multiple sclerosis.

Multiple sclerosis is an inflammatory disease of the central nervous system (CNS) in which multiple sites of blood-brain barrier (BBB) disruption, focal inflammation, demyelination and tissue destruction are the hallmarks. Here we show that sphingosine-1-phosphate receptor 2 (S1PR2) has a negative role in myelin repair as well as an important role in demyelination by modulating BBB permeability. In lysolecithin-induced demyelination of adult mouse spinal cord, S1PR2 inactivation by either the pharmacological inhibitor JTE-013 or S1PR2 gene knockout led to enhanced myelin repair as determined by higher numbers of differentiated oligodendrocytes and increased numbers of remyelinated axons at the lesion sites. S1PR2 inactivation in lysolecithin-induced demyelination of the optic chiasm, enhanced oligodendrogenesis and improved the behavioral outcome in an optokinetic reflex test. In order to see the effect of S1PR2 inactivation on demyelination, experimental autoimmune encephalitis (EAE) was induced by MOG-peptide. S1PR2 inhibition or knockout decreased the extent of demyelinated areas as well as the clinical disability in this EAE model. Both toxin induced and EAE models showed decreased BBB leakage and reduced numbers of Iba1+ macrophages following S1PR2 inactivation. Our results suggest that S1PR2 activity impairs remyelination and also enhances BBB leakage and demyelination. The former effect could be mediated by Nogo-A, as antagonism of this factor enhances remyelination and S1PR2 can act as a Nogo-A receptor.

Enhancing rehabilitation and functional recovery after brain and spinal cord trauma with electrical neuromodulation.

PURPOSE OF REVIEW: This review discusses recent advances in the rehabilitation of motor deficits after traumatic brain injury (TBI) and spinal cord injury (SCI) using neuromodulatory techniques. RECENT FINDINGS: Neurorehabilitation is currently the only treatment option for long-term improvement of motor functions that can be offered to patients with TBI or SCI. Major advances have been made in recent years in both preclinical and clinical rehabilitation. Activity-dependent plasticity of neuronal connections and circuits is considered key for successful recovery of motor functions, and great therapeutic potential is attributed to the combination of high-intensity training with electrical neuromodulation. First clinical case reports have demonstrated that repetitive training enabled or enhanced by electrical spinal cord stimulation can yield substantial improvements in motor function. Described achievements include regaining of overground walking capacity, independent standing and stepping, and improved pinch strength that recovered even years after injury. SUMMARY: Promising treatment options have emerged from research in recent years using neurostimulation to enable or enhance intense training. However, characterizing long-term benefits and side-effects in clinical trials and identifying patient subsets who can benefit are crucial. Regaining lost motor function remains challenging.

RhoA regulates translation of the Nogo-A decoy SPARC in white matter-invading glioblastomas.

Glioblastomas strongly invade the brain by infiltrating into the white matter along myelinated nerve fiber tracts even though the myelin protein Nogo-A prevents cell migration by activating inhibitory RhoA signaling. The mechanisms behind this long-known phenomenon remained elusive so far, precluding a targeted therapeutic intervention. This study demonstrates that the prevalent activation of AKT in gliomas increases the ER protein-folding capacity and enables tumor cells to utilize a side effect of RhoA activation: the perturbation of the IRE1α-mediated decay of SPARC mRNA. Once translation is initiated, glioblastoma cells rapidly secrete SPARC to block Nogo-A from inhibiting migration via RhoA. By advanced ultramicroscopy for studying single-cell invasion in whole, undissected mouse brains, we show that gliomas require SPARC for invading into white matter structures. SPARC depletion reduces tumor dissemination that significantly prolongs survival and improves response to cytostatic therapy. Our finding of a novel RhoA-IRE1 axis provides a druggable target for interfering with SPARC production and underscores its therapeutic value.

Oligodendrocyte- and Neuron-Specific Nogo-A Restrict Dendritic Branching and Spine Density in the Adult Mouse Motor Cortex.

Nogo-A has been well described as a myelin-associated inhibitor of neurite outgrowth and functional neuroregeneration after central nervous system (CNS) injury. Recently, a new role of Nogo-A has been identified as a negative regulator of synaptic plasticity in the uninjured adult CNS. Nogo-A is present in neurons and oligodendrocytes. However, it is yet unclear which of these two pools regulate synaptic plasticity. To address this question we used newly generated mouse lines in which Nogo-A is specifically knocked out in (1) oligodendrocytes (oligoNogo-A KO) or (2) neurons (neuroNogo-A KO). We show that both oligodendrocyte- and neuron-specific Nogo-A KO mice have enhanced dendritic branching and spine densities in layer 2/3 cortical pyramidal neurons. These effects are compartmentalized: neuronal Nogo-A affects proximal dendrites whereas oligodendrocytic Nogo-A affects distal regions. Finally, we used two-photon laser scanning microscopy to measure the spine turnover rate of adult mouse motor cortex layer 5 cells and find that both Nogo-A KO mouse lines show enhanced spine remodeling after 4 days. Our results suggest relevant control functions of glial as well as neuronal Nogo-A for synaptic plasticity and open new possibilities for more selective and targeted plasticity enhancing strategies.

Axotomized Corticospinal Neurons Increase Supra-Lesional Innervation and Remain Crucial for Skilled Reaching after Bilateral Pyramidotomy.

Skilled upper limb function heavily depends on the corticospinal tract. After bilateral lesions to this tract, motor control is disrupted but can be partially substituted by other motor systems to allow functional recovery. However, the remaining roles of motor cortex and especially of axotomized corticospinal neurons (CSNs) are not well understood. Using the single pellet retrieval task in adult rats, we induced significant recovery of skilled reaching after bilateral pyramidotomy by rehabilitative reaching training, and show that reach-related motor cortex activity, recorded in layer V, topographically reappeared shortly after axotomy. Using a chemogenetic neuronal silencing technique, we found that axotomized CSNs retained a crucial role for the recovered pellet retrieval success. The axotomized CSNs sprouted extensively in the red nucleus supplying new innervation to its magnocellular and parvocellular parts. Specific silencing of the rubrospinal tract (RST) also strongly abolished the recovered pellet retrieval success, suggesting a role of this cervically projecting nucleus in relaying cortical motor control. In summary, our results show that after bilateral corticospinal axotomy, motor cortex still actively engages in forelimb motor control and axotomized CSNs are crucially involved in the recovered reaching movement, potentially by relaying motor control via the RST.

Changes of motor corticobulbar projections following different lesion types affecting the central nervous system in adult macaque monkeys.

Functional recovery from central nervous system injury is likely to be partly due to a rearrangement of neural circuits. In this context, the corticobulbar (corticoreticular) motor projections onto different nuclei of the ponto-medullary reticular formation (PMRF) were investigated in 13 adult macaque monkeys after either, primary motor cortex injury (MCI) in the hand area, or spinal cord injury (SCI) or Parkinson's disease-like lesions of the nigro-striatal dopaminergic system (PD). A subgroup of animals in both MCI and SCI groups was treated with neurite growth promoting anti-Nogo-A antibodies, whereas all PD animals were treated with autologous neural cell ecosystems (ANCE). The anterograde tracer BDA was injected either in the premotor cortex (PM) or in the primary motor cortex (M1) to label and quantify corticobulbar axonal boutons terminaux and en passant in PMRF. As compared to intact animals, after MCI the density of corticobulbar projections from PM was strongly reduced but maintained their laterality dominance (ipsilateral), both in the presence or absence of anti-Nogo-A antibody treatment. In contrast, the density of corticobulbar projections from M1 was increased following opposite hemi-section of the cervical cord (at C7 level) and anti-Nogo-A antibody treatment, with maintenance of contralateral laterality bias. In PD monkeys, the density of corticobulbar projections from PM was strongly reduced, as well as that from M1, but to a lesser extent. In conclusion, the densities of corticobulbar projections from PM or M1 were affected in a variable manner, depending on the type of lesion/pathology and the treatment aimed to enhance functional recovery.

Urodynamic measurements reflect physiological bladder function in rats.

AIMS: Our objective was to investigate and compare bladder function in rats assessed by metabolic cage and by urodynamic measurements in fully awake animals. METHODS: Bladder function of female Lewis rats was investigated in naïve animals by metabolic cage at baseline, 14-16 days after bladder catheter and external urethral sphincter electromyography electrode implantation in fully awake animals by urodynamics, and again by metabolic cage. RESULTS: Investigating the same animals (n = 8), voided volume, average flow, and duration of voiding were similar (P > 0.05) in naïve animals measured by metabolic cage and after catheter implantation by urodynamic measurements and by metabolic cage. In naïve animals measured by metabolic cage, voided volumes were significantly different in the light (resting phase) versus the dark (active phase) part of the 24 h cycle (mean difference 0.14 mL, 21%, P = 0.004, n = 27). CONCLUSIONS: Lower urinary tract function assessed by metabolic cage or by urodynamic meaurements in fully awake rats was indistinguishable. Thus, catheter implantation did not significantly change physiological bladder function. This shows that urodynamic measurements in awake animals are an appropriate approach to study lower urinary tract function in health and disease in animal models, directly paralleling the human diagnostic procedures.

Attractive and repulsive factors act through multi-subunit receptor complexes to regulate nerve fiber growth.

In the nervous system, attractive and repulsive factors guide neuronal growth, pathfinding and target innervation during development, learning and regeneration after injury. Repulsive and growth-inhibitory factors, such as some ephrins, semaphorins, netrins and myelin-associated growth inhibitors, restrict nerve fiber growth, whereas neurotrophins, and other ephrins, semaphorins and netrins attract fibers and promote neurite growth. Several of these guidance molecules also play crucial roles in vasculogenesis, and regulate cell migration and tissue formation in different organs. Precise and highly specific signal transduction in space and time is required in all these cases, which primarily depends on the presence and function of specific receptors. Interestingly, many of these ligands act through multi-subunit receptor complexes. In this Commentary, we review the current knowledge of how complexes of the receptors for attractive and repulsive neurite growth regulatory factors are reorganized in a spatial and temporal manner, and reveal the implications that such dynamics have on the signaling events that coordinate neurite fiber growth.

Tetraspanin-3 is an organizer of the multi-subunit Nogo-A signaling complex.

To ensure precision and specificity of ligand-receptor-induced signaling, co-receptors and modulatory factors play important roles. The membrane-bound ligand Nogo-A (an isoform encoded by RTN4) induces inhibition of neurite outgrowth, cell spreading, adhesion and migration through multi-subunit receptor complexes. Here, we identified the four-transmembrane-spanning protein tetraspanin-3 (TSPAN3) as a new modulatory co-receptor for the Nogo-A inhibitory domain Nogo-A-Δ20. Single-molecule tracking showed that TSPAN3 molecules in the cell membrane reacted to binding of Nogo-A with elevated mobility, which was followed by association with the signal-transducing Nogo-A receptor sphingosine-1-phosphate receptor 2 (S1PR2). Subsequently, TSPAN3 was co-internalized as part of the Nogo-A-ligand-receptor complex into early endosomes, where it subsequently separated from Nogo-A and S1PR2 to be recycled to the cell surface. The functional importance of the Nogo-A-TSPAN3 interaction is shown by the fact that knockdown of TSPAN3 strongly reduced the Nogo-A-induced S1PR2 clustering, RhoA activation, cell spreading and neurite outgrowth inhibition. In addition to the modulatory functions of TSPAN3 on Nogo-A-S1PR2 signaling, these results illustrate the very dynamic spatiotemporal reorganizations of membrane proteins during ligand-induced receptor complex organization.