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.

Differential effects of brain-derived neurotrophic factor and neurotrophin-3 on hindlimb function in paraplegic rats.

We compared the effect of viral administration of brain-derived neurotrophic factor (BDNF) or neurotrophin 3 (NT-3) on locomotor recovery in adult rats with complete thoracic (T10) spinal cord transection injuries, in order to determine the effect of chronic neurotrophin expression on spinal plasticity. At the time of injury, BDNF, NT-3 or green fluorescent protein (GFP) (control) was delivered to the lesion via adeno-associated virus (AAV) constructs. AAV-BDNF was significantly more effective than AAV-NT-3 in eliciting locomotion. In fact, AAV-BDNF-treated rats displayed plantar, weight-supported hindlimb stepping on a stationary platform, that is, without the assistance of a moving treadmill and without step training. Rats receiving AAV-NT-3 or AAV-GFP were incapable of hindlimb stepping during this task, despite provision of balance support. AAV-NT-3 treatment did promote the recovery of treadmill-assisted stepping, but this required continuous perineal stimulation. In addition, AAV-BDNF-treated rats were sensitized to noxious heat, whereas AAV-NT-3-treated and AAV-GFP-treated rats were not. Notably, AAV-BDNF-treated rats also developed hindlimb spasticity, detracting from its potential clinical applicability via the current viral delivery method. Intracellular recording from triceps surae motoneurons revealed that AAV-BDNF significantly reduced motoneuron rheobase, suggesting that AAV-BDNF promoted the recovery of over-ground stepping by enhancing neuronal excitability. Elevated nuclear c-Fos expression in interneurons located in the L2 intermediate zone after AAV-BDNF treatment indicated increased activation of interneurons in the vicinity of the locomotor central pattern generator. AAV-NT-3 treatment reduced motoneuron excitability, with little change in c-Fos expression. These results support the potential for BDNF delivery at the lesion site to reorganize locomotor circuits.

Antagonism of nerve growth factor-TrkA signaling and the relief of pain.

Nerve growth factor (NGF) was originally discovered as a neurotrophic factor essential for the survival of sensory and sympathetic neurons during development. However, in the adult NGF has been found to play an important role in nociceptor sensitization after tissue injury. The authors outline mechanisms by which NGF activation of its cognate receptor, tropomyosin-related kinase A receptor, regulates a host of ion channels, receptors, and signaling molecules to enhance acute and chronic pain. The authors also document that peripherally restricted antagonism of NGF-tropomyosin-related kinase A receptor signaling is effective for controlling human pain while appearing to maintain normal nociceptor function. Understanding whether there are any unexpected adverse events and how humans may change their behavior and use of the injured/degenerating tissue after significant pain relief without sedation will be required to fully appreciate the patient populations that may benefit from these therapies targeting NGF.

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.

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.

Combined treatment with neurotrophin-3 and LSD facilitates behavioral recovery from double-hemisection spinal injury in neonatal rats.

We explored functional recovery in two spinal cord injury models following a novel combination treatment (NT-3 + LSD). One group of rats received a staggered double hemisection (DH) at postnatal day 2 (P2) of the left hemicord at T11 and the right hemicord at T12. Another group received complete transection (CT) at T11 on P2. A third group was sham operated. Each of these groups was also treated with the drug combination. Drugs were administered intrathecally above the lesion during surgery, and again s.c. at P4, P6, P8, and P10. Intracellular recording in an in vitro spinal cord preparation at P10-P12 in DH rats revealed weak polysynaptic connections to lumbar motoneurons through the injury region, but only in those receiving NT-3 + LSD; NT-3 or LSD alone had no effect. In behavioral experiments, the frequency of rearing in an open field and hindlimb kicks during swimming was assessed every 3-4 days from P9 to P58. Both CT and DH injury severely impaired rearing and hindlimb kicking during swimming. DH rats treated with NT-3 + LSD showed significantly more kicks during swimming than untreated DH or CT rats and treated CT rats beginning as early as P9 and lasting through the duration of testing. Rearing behavior was also improved by treatment but beginning only in the 3rd postnatal week, the time at which it normally develops. Rearing frequency reached sham control levels by P40. Our results suggest this combination treatment may be a promising new strategy for facilitating recovery from moderate spinal cord injury.

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.