Dynamic subtype- and context-specific subcellular RNA regulation in growth cones of developing neurons of the cerebral cortex.

Molecular mechanisms that cells employ to compartmentalize function via localization of function-specific RNA and translation are only partially elucidated. We investigate long-range projection neurons of the cerebral cortex as highly polarized exemplars to elucidate dynamic regulation of RNA localization, stability, and translation within growth cones (GCs), leading tips of growing axons. Comparison of GC-localized transcriptomes between two distinct subtypes of projection neurons- interhemispheric-callosal and corticothalamic- across developmental stages identifies both distinct and shared subcellular machinery, and intriguingly highlights enrichment of genes associated with neurodevelopmental and neuropsychiatric disorders. Developmental context-specific components of GC-localized transcriptomes identify known and novel potential regulators of distinct phases of circuit formation: long-distance growth, target area innervation, and synapse formation. Further, we investigate mechanisms by which transcripts are enriched and dynamically regulated in GCs, and identify GC-enriched motifs in 3' untranslated regions. As one example, we identify cytoplasmic adenylation element binding protein 4 (CPEB4), an RNA binding protein regulating localization and translation of mRNAs encoding molecular machinery important for axonal branching and complexity. We also identify RNA binding motif single stranded interacting protein 1 (RBMS1) as a dynamically expressed regulator of RNA stabilization that enables successful callosal circuit formation. Subtly aberrant associative and integrative cortical circuitry can profoundly affect cortical function, often causing neurodevelopmental and neuropsychiatric disorders. Elucidation of context-specific subcellular RNA regulation for GC- and soma-localized molecular controls over precise circuit development, maintenance, and function offers generalizable insights for other polarized cells, and might contribute substantially to understanding neurodevelopmental and behavioral-cognitive disorders and toward targeted therapeutics.

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.

Neuronal subtype-specific growth cone and soma purification from mammalian CNS via fractionation and fluorescent sorting for subcellular analyses and spatial mapping of local transcriptomes and proteomes.

During neuronal development, growth cones (GCs) of projection neurons navigate complex extracellular environments to reach distant targets, thereby generating extraordinarily complex circuitry. These dynamic structures located at the tips of axonal projections respond to substrate-bound as well as diffusible guidance cues in a neuronal subtype- and stage-specific manner to construct highly specific and functional circuitry. In vitro studies of the past decade indicate that subcellular localization of specific molecular machinery in GCs underlies the precise navigational control that occurs during circuit 'wiring'. Our laboratory has recently developed integrated experimental and analytical approaches enabling high-depth, quantitative proteomic and transcriptomic investigation of subtype- and stage-specific GC molecular machinery directly from the rodent central nervous system (CNS) in vivo. By using these approaches, a pure population of GCs and paired somata can be isolated from any neuronal subtype of the CNS that can be fluorescently labeled. GCs are dissociated from parent axons using fluid shear forces, and a bulk GC fraction is isolated by buoyancy ultracentrifugation. Subtype-specific GCs and somata are purified by recently developed fluorescent small particle sorting and established FACS of neurons and are suitable for downstream analyses of proteins and RNAs, including small RNAs. The isolation of subtype-specific GCs and parent somata takes ~3 h, plus sorting time, and ~1-2 h for subsequent extraction of molecular contents. RNA library preparation and sequencing can take several days to weeks, depending on the turnaround time of the core facility involved.

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.

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.

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.

A novel urodynamic model for lower urinary tract assessment in awake rats.

OBJECTIVES: To develop a urodynamic model incorporating external urethral sphincter (EUS) electromyography (EMG) in awake rats. MATERIALS AND METHODS: Bladder catheters and EUS EMG electrodes were implanted in female Sprague Dawley rats. Assessments were performed in awake, lightly restrained rats on postoperative day 12-14. Measurements were repeated in the same rat on day 16 under urethane anaesthesia. Urodynamics and EUS EMG were performed simultaneously. In addition, serum creatinine and bladder histology was assessed. RESULTS: No significant differences in urodynamic parameters were found between bladder catheter only vs bladder catheter and EUS EMG electrode groups. Urethane anaesthesia evoked prominent changes in both urodynamic parameters and EUS EMG. Serum creatinine was within the normal limits in all rats. Bladder weight and bladder wall thickness were significantly increased in both the bladder catheter only and the bladder catheter and EUS EMG group compared with controls. CONCLUSIONS: Our novel urodynamic model allows repetitive measurements of both bladder and EUS function at different time points in the same rat under fully awake conditions and opens promising avenues to investigate lower urinary tract dysfunction in a translational approach.