The Adaptor Protein CD2AP Is a Coordinator of Neurotrophin Signaling-Mediated Axon Arbor Plasticity.

Growth of intact axons of noninjured neurons, often termed collateral sprouting, contributes to both adaptive and pathological plasticity in the adult nervous system, but the intracellular factors controlling this growth are largely unknown. An automated functional assay of genes regulated in sensory neurons from the rat in vivo spared dermatome model of collateral sprouting identified the adaptor protein CD2-associated protein (CD2AP; human CMS) as a positive regulator of axon growth. In non-neuronal cells, CD2AP, like other adaptor proteins, functions to selectively control the spatial/temporal assembly of multiprotein complexes that transmit intracellular signals. Although CD2AP polymorphisms are associated with increased risk of late-onset Alzheimer's disease, its role in axon growth is unknown. Assessments of neurite arbor structure in vitro revealed CD2AP overexpression, and siRNA-mediated knockdown, modulated (1) neurite length, (2) neurite complexity, and (3) growth cone filopodia number, in accordance with CD2AP expression levels. We show, for the first time, that CD2AP forms a novel multiprotein complex with the NGF receptor TrkA and the PI3K regulatory subunit p85, with the degree of TrkA:p85 association positively regulated by CD2AP levels. CD2AP also regulates NGF signaling through AKT, but not ERK, and regulates long-range signaling though TrkA(+)/RAB5(+) signaling endosomes. CD2AP mRNA and protein levels were increased in neurons during collateral sprouting but decreased following injury, suggesting that, although typically considered together, these two adult axonal growth processes are fundamentally different. These data position CD2AP as a major intracellular signaling molecule coordinating NGF signaling to regulate collateral sprouting and structural plasticity of intact adult axons. SIGNIFICANCE STATEMENT: Growth of noninjured axons in the adult nervous system contributes to adaptive and maladaptive plasticity, and dysfunction of this process may contribute to neurologic pathologies. Functional screening of genes regulated during growth of noninjured axons revealed CD2AP as a positive regulator of axon outgrowth. A novel association of CD2AP with TrkA and p85 suggests a distinct intracellular signaling pathway regulating growth of noninjured axons. This may also represent a novel mechanism of generating specificity in multifunctional NGF signaling. Divergent regulation of CD2AP in different axon growth conditions suggests that separate mechanisms exist for different modes of axon growth. CD2AP is the first signaling molecule associated with adult sensory axonal collateral sprouting, and this association may offer new insights for NGF/TrkA-related Alzheimer's disease mechanisms.

Delayed intramuscular human neurotrophin-3 improves recovery in adult and elderly rats after stroke.

There is an urgent need for a therapy that reverses disability after stroke when initiated in a time frame suitable for the majority of new victims. We show here that intramuscular delivery of neurotrophin-3 (NT3, encoded by NTF3) can induce sensorimotor recovery when treatment is initiated 24 h after stroke. Specifically, in two randomized, blinded preclinical trials, we show improved sensory and locomotor function in adult (6 months) and elderly (18 months) rats treated 24 h following cortical ischaemic stroke with human NT3 delivered using a clinically approved serotype of adeno-associated viral vector (AAV1). Importantly, AAV1-hNT3 was given in a clinically-feasible timeframe using a straightforward, targeted route (injections into disabled forelimb muscles). Magnetic resonance imaging and histology showed that recovery was not due to neuroprotection, as expected given the delayed treatment. Rather, treatment caused corticospinal axons from the less affected hemisphere to sprout in the spinal cord. This treatment is the first gene therapy that reverses disability after stroke when administered intramuscularly in an elderly body. Importantly, phase I and II clinical trials by others show that repeated, peripherally administered high doses of recombinant NT3 are safe and well tolerated in humans with other conditions. This paves the way for NT3 as a therapy for stroke.

Distinct subclassification of DRG neurons innervating the distal colon and glans penis/distal urethra based on the electrophysiological current signature.

Spinal sensory neurons innervating visceral and mucocutaneous tissues have unique microanatomic distribution, peripheral modality, and physiological, pharmacological, and biophysical characteristics compared with those neurons that innervate muscle and cutaneous tissues. In previous patch-clamp electrophysiological studies, we have demonstrated that small- and medium-diameter dorsal root ganglion (DRG) neurons can be subclassified on the basis of their patterns of voltage-activated currents (VAC). These VAC-based subclasses were highly consistent in their action potential characteristics, responses to algesic compounds, immunocytochemical expression patterns, and responses to thermal stimuli. For this study, we examined the VAC of neurons retrogradely traced from the distal colon and the glans penis/distal urethra in the adult male rat. The afferent population from the distal colon contained at least two previously characterized cell types observed in somatic tissues (types 5 and 8), as well as four novel cell types (types 15, 16, 17, and 18). In the glans penis/distal urethra, two previously described cell types (types 6 and 8) and three novel cell types (types 7, 14, and 15) were identified. Other characteristics, including action potential profiles, responses to algesic compounds (acetylcholine, capsaicin, ATP, and pH 5.0 solution), and neurochemistry (expression of substance P, CGRP, neurofilament, TRPV1, TRPV2, and isolectin B4 binding) were consistent for each VAC-defined subgroup. With identification of distinct DRG cell types that innervate the distal colon and glans penis/distal urethra, future in vitro studies related to the gastrointestinal and urogenital sensory function in normal as well as abnormal/pathological conditions may be benefitted.

Novel multi-system functional gains via task specific training in spinal cord injured male rats.

Locomotor training (LT) after spinal cord injury (SCI) is a rehabilitative therapy used to enhance locomotor recovery. There is evidence, primarily anecdotal, also associating LT with improvements in bladder function and reduction in some types of SCI-related pain. In the present study, we determined if a step training paradigm could improve outcome measures of locomotion, bladder function, and pain/allodynia. After a T10 contusive SCI trained animals (adult male Wistar rats), trained animals began quadrupedal step training beginning 2 weeks post-SCI for 1 h/day. End of study experiments (3 months of training) revealed significant changes in limb kinematics, gait, and hindlimb flexor-extensor bursting patterns relative to non-trained controls. Importantly, micturition function, evaluated with terminal transvesical cystometry, was significantly improved in the step trained group (increased voiding efficiency, intercontraction interval, and contraction amplitude). Because both SCI and LT affect neurotrophin signaling, and neurotrophins are involved with post-SCI plasticity in micturition pathways, we measured bladder neurotrophin mRNA. Training regulated the expression of nerve growth factor (NGF) but not BDNF or NT3. Bladder NGF mRNA levels were inversely related to bladder function in the trained group. Monitoring of overground locomotion and neuropathic pain throughout the study revealed significant improvements, beginning after 3 weeks of training, which in both cases remained consistent for the study duration. These novel findings, improving non-locomotor in addition to locomotor functions, demonstrate that step training post-SCI could contribute to multiple quality of life gains, targeting patient-centered high priority deficits.

Intramuscular AAV delivery of NT-3 alters synaptic transmission to motoneurons in adult rats.

We examined whether elevating levels of neurotrophin-3 (NT-3) in the spinal cord and dorsal root ganglion (DRG) would alter connections made by muscle spindle afferent fibers on motoneurons. Adeno-associated virus (AAV) serotypes AAV1, AAV2 and AAV5, selected for their tropism profile, were engineered with the NT-3 gene and administered to the medial gastrocnemius muscle in adult rats. ELISA studies in muscle, DRG and spinal cord revealed that NT-3 concentration in all tissues peaked about 3 months after a single viral injection; after 6 months NT-3 concentration returned to normal values. Intracellular recording in triceps surae motoneurons revealed complex electrophysiological changes. Moderate elevation in cord NT-3 resulted in diminished segmental excitatory postsynaptic potential (EPSP) amplitude, perhaps as a result of the observed decrease in motoneuron input resistance. With further elevation in NT-3 expression, the decline in EPSP amplitude was reversed, indicating that NT-3 at higher concentration could increase EPSP amplitude. No correlation was observed between EPSP amplitude and NT-3 concentration in the DRG. Treatment with control viruses could elevate NT-3 levels minimally resulting in measurable electrophysiological effects, perhaps as a result of inflammation associated with injection. EPSPs elicited by stimulation of the ventrolateral funiculus underwent a consistent decline in amplitude independent of NT-3 level. These novel correlations between modified NT-3 expression and single-cell electrophysiological parameters indicate that intramuscular administration of AAV(NT-3) can exert long-lasting effects on synaptic transmission to motoneurons. This approach to neurotrophin delivery could be useful in modifying spinal function after injury.

Early heart failure in the SMNDelta7 model of spinal muscular atrophy and correction by postnatal scAAV9-SMN delivery.

Proximal spinal muscular atrophy (SMA) is a debilitating neurological disease marked by isolated lower motor neuron death and subsequent atrophy of skeletal muscle. Historically, SMA pathology was thought to be limited to lower motor neurons and the skeletal muscles they control, yet there are several reports describing the coincidence of cardiovascular abnormalities in SMA patients. As new therapies for SMA emerge, it is necessary to determine whether these non-neuromuscular systems need to be targeted. Therefore, we have characterized left ventricular (LV) function of SMA mice (SMN2+/+; SMNΔ7+/+; Smn-/-) and compared it with that of their unaffected littermates at 7 and 14 days of age. Anatomical and physiological measurements made by electrocardiogram and echocardiography show that affected mouse pups have a dramatic decrease in cardiac function. At 14 days of age, SMA mice have bradycardia and develop a marked dilated cardiomyopathy with a concomitant decrease in contractility. Signs of decreased cardiac function are also apparent as early as 7 days of age in SMA animals. Delivery of a survival motor neuron-1 transgene using a self-complementary adeno-associated virus serotype 9 abolished the symptom of bradycardia and significantly decreased the severity of the heart defect. We conclude that severe SMA animals have compromised cardiac function resulting at least partially from early bradycardia, which is likely attributable to aberrant autonomic signaling. Further cardiographic studies of human SMA patients are needed to clarify the clinical relevance of these findings from this SMA mouse.

Skin incision induces expression of axonal regeneration-related genes in adult rat spinal sensory neurons.

UNLABELLED: Skin incision and nerve injury both induce painful conditions. Incisional and postsurgical pain is believed to arise primarily from inflammation of tissue and the subsequent sensitization of peripheral and central neurons. The role of axonal regeneration-related processes in development of pain has only been considered when there has been injury to the peripheral nerve itself, even though tissue damage likely induces injury of resident axons. We sought to determine if skin incision would affect expression of regeneration-related genes such as activating transcription factor 3 (ATF3) in dorsal root ganglion (DRG) neurons. ATF3 is absent from DRG neurons of the normal adult rodent, but is induced by injury of peripheral nerves and modulates the regenerative capacity of axons. Image analysis of immunolabeled DRG sections revealed that skin incision led to an increase in the number of DRG neurons expressing ATF3. RT-PCR indicated that other regeneration-associated genes (galanin, GAP-43, Gadd45a) were also increased, further suggesting an injury-like response in DRG neurons. Our finding that injury of skin can induce expression of neuronal injury/regeneration-associated genes may impact how clinical postsurgical pain is investigated and treated. PERSPECTIVE: Tissue injury, even without direct nerve injury, may induce a state of enhanced growth capacity in sensory neurons. Axonal regeneration-associated processes should be considered alongside nerve signal conduction and inflammatory/sensitization processes as possible mechanisms contributing to pain, particularly the transition from acute to chronic pain.

The many functions of nerve growth factor: multiple actions on nociceptors.

Nerve growth factor (NGF) initially interested neurobiologists because of its effects in the developing nervous system. It is now clear that NGF functions throughout the life of the animal with a wide repertoire of actions. In the sensory nervous system it primarily influences the structure and function of nociceptors. Here, we provide a brief review of these actions and raise the overriding biological questions of why these multiple actions occur and how they are carried out.

Mechanisms of sensitization of the response of single dorsal root ganglion cells from adult rat to noxious heat.

We investigated the regulation by nerve growth factor of the response of sensory neurons to noxious heat (>43 degrees C). In dissociated dorsal root ganglion neurons (<30 micro m) from adult rat we demonstrated, using perforated patch clamp recording, that the inward current elicited in response to noxious heating is enhanced by nerve growth factor and reduced by capsazepine. The tachyphylaxis observed in response to the second of two heat pulses was reversed in most cells when nerve growth factor was introduced into the medium during the 5 min between the two heat stimuli, similar to findings using capsaicin [X. Shu & L.M. Mendell (1999) Neurosci. Lett.274, 159-162]. The threshold temperature did not change systematically after nerve growth factor. Using antibodies to TRPV1 and trkA in a subset of cells from which we recorded, we found a virtually perfect correlation between expression of TRPV1 and sensitivity to noxious heat. In addition, trkA expression was perfectly correlated with the ability of nerve growth factor to reverse tachyphylaxis. Thus, this physiological test is a reliable measure of trkA expression in cells sensitive to noxious heat. In agreement with studies in heterologous cells expressing trkA and TRPV1, pharmacologically blocking phospholipase C abolished the effect of nerve growth factor on heat-evoked currents in cells verified to express trkA. We conclude that the response of dorsal root ganglion neurons to noxious heat is conditioned by nerve growth factor in the same way as their response to capsaicin and that these responses require the presence of trkA and TRPV1.