Project Summary
Persistent pain secondary to tissue and nerve injury involves key roles for DRG neurons, macrophages, and
spinal microglia. Therapies targeting single components of this tripartite pain axis have not proven clinically
efficacious, suggesting a strategy is needed that addresses all three components in concert. We have exciting
new findings that suggest one such therapeutic strategy may be to target the subset of cellular membrane
cholesterol rich lipid rafts that contain Toll-like receptor 4 (TLR4-rafts). Membrane lipid rafts are a fundamental
organizational nidus in all cells for numerous membrane channels, receptors and enzymes that regulate reactivity
and excitability. The TLR4-raft subtype is special as these are specifically localized in the three key cellular
elements regulating the excitability of nociceptive signaling. Following tissue inflammation or nerve injury,
TLR4rafts transform from small diffuse labile membrane structures into markedly enlarged and persistent
complexes supporting the formation of homo and hetero dimers of several excitatory channels and receptors,
underpinning the transition from an acute to a persistent pain phenotype and to establishing a “primed state”
after an initial system activation. Specific disruption of these neuraxial TLR4-rafts has profound effects upon
injury induced pain behavior. While we have gained insight into mechanisms by which TLR4-rafts regulate
microglial function, we know little about the mechanisms by which TLR4-rafts regulate excitability of DRG
neurons and macrophages. To address this knowledge gap, we propose three specific aims: i) Define time
dependent changes in TLR4-rafts and pain behavior induced by trauma, inflammatory and neuropathic pain, and
the effects of targeted TLR4-raft disruption on pain behavior in these models; ii) Determine baseline molecular
architecture of DRG neuronal and macrophage TLR4-rafts and then how this architecture and function is altered
with tissue trauma, inflammation and nerve injury; and iii) Define role of DRG neuronal TLR4-raft localized
receptors in regulating excitatory phenotype in mouse, rat and human DRG neurons and effects of TLR4-raft
disruption in modifying this excitatory phenotype. This is a large multi-PI / multi-center study that defines
mechanisms by which neuraxial TLR4-rafts affect multiple pain-related signaling processes, integrated pain
behavior and the molecular anatomy underpinning this signaling and the degree to which these mechanisms are
conserved between rodents and humans, across differing pain phenotypes. This work will have a sustained
impact on understanding the neurobiology of a largely unexplored cellular element regulating post injury afferent
excitability. In sum, this proposal centers on the unifying thesis that neuraxial TLR4-rafts are a common
facilitatory element of the persistent pain facilitated states observed following tissue and nerve injury and that
this effect is mediated by TLR4 rafts not only on DRG macrophages, and spinal microglia but unexpectedly by
the TLR4-rafts expressed on the DRG nociceptor.