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

Abstract

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 ϩ /RAB5 ϩ 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.Key words: collateral sprouting; nerve growth factor; neural plasticity; signalosome; spared dermatome; transcriptomics Significance StatementGrowth 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.

Injury-free priming: induction of Primary Afferent Collateral Sprouting in uninjured sensory neurons in vivo primes them for enhanced axon outgrowth in vitro

“…In a previous study, to identify genes that were regulated in uninjured T11 DRG neurons during PACS, we performed microarray analysis upon RNA taken from T11 DRG from naïve rats (n=5) and from T11 DRG taken 7 days (n=7) or 14 days (n=7) after induction of PACS (Harrison et al, 2015;Harrison et al, 2016). Microarray analysis was also performed using RNA from those same rats from intact skin (n=6) or 7-day denervated skin (n=5) and 14-day denervated skin (n=5) (Flight et al, 2014).…”

“…We have previously performed microarray analysis using RNA from T11 DRG and from denervated skin taken from naïve rats or from rats 7 days or 14 days after induction of PACS in vivo (Flight et al, 2014;Harrison et al, 2015;Harrison et al, 2016). Tables 2 and 3 show the genes in DRG and skin (respectively) that were evaluated by real time qRTPCR; results obtained using dChip for microarray analysis are also presented for comparison.…”

“…We previously used microarray analysis to identify genes that are differentially regulated in uninjured T11 DRG housing neurons undergoing PACS (Harrison et al, 2015;Harrison et al, 2016) and in the corresponding denervated skin (Flight et al,. 2014).…”

“…For example, daily administration of antisera against beta NGF halts Primary Afferent Collateral Sprouting into denervated skin (Diamond et al 1987) whereas intradermal injections of NGF induce collateral sprouting de novo (Diamond et al 1992b). We previously showed that induction of PACS causes upregulation of CD2-associated protein (CD2AP) in DRG and that, in PC12 cells, overexpression of CD2AP enhances neurite outgrowth whilst siRNA-mediated knockdown of CD2AP reduces neurite outgrowth (Harrison et al 2016). In the future, conditional deletion of NGF or GDNF family ligands in skin (or their receptors in DRG) could be performed after induction of PACS.…”

Active mode of excretion across digestive tissues predates the origin of excretory organs

“…However, Cd2ap/Cin85, Zo1 and members of the Stomatin/Podocin are evolutionary ancient proteins present in all Metazoa (47,48,80) involved in a number of other functions apart of assembling filtration sites. For instance, stomatin membrane proteins have a role in ion channel regulation and membrane trafficking (81), the MAGUK proteins can be both structural components at the junction sites as well as functioning in signal transduction pathways related to gene expression and cell behavior (82,83) and the Cd2ap adaptor proteins are involved in cell signaling, cytoskeletal arrangement and receptor endocytic trafficking (84)(85)(86)(87).…”