CRMP-2 peptide mediated decrease of high and low voltage-activated calcium channels, attenuation of nociceptor excitability, and anti-nociception in a model of AIDS therapy-induced painful peripheral neuropathy

ABSTRACT: BACKGROUND: The ubiquity of protein-protein interactions in biological signaling offers ample opportunities for therapeutic intervention. We previously identified a peptide, designated CBD3, that suppressed inflammatory and neuropathic behavioral hypersensitivity in rodents by inhibiting the ability of collapsin response mediator protein 2 (CRMP-2) to bind to N-type voltage-activated calcium channels (CaV2.2) [Brittain et al..., Nature Medicine 17:822[EN DASH]829 (2011)]. RESULTS AND DISCUSSION: Here, we utilized SPOTScan analysis to identify an optimized variation of the CBD3 peptide (CBD3A6K) that bound with greater affinity to Ca2+ channels. Molecular dynamics simulations demonstrated that the CBD3A6K peptide was more stable and less prone to the unfolding observed with the parent CBD3 peptide. This mutant peptide, conjugated to the cell penetrating motif of the HIV transduction domain protein TAT, exhibited greater anti-nociception in a rodent model of AIDS therapy-induced peripheral neuropathy when compared to the parent TAT-CBD3 peptide. Remarkably, intraperitoneal administration of TAT-CBD3A6K produced none of the minor side effects (i.e. tail kinking, body contortion) observed with the parent peptide. Interestingly, excitability of dissociated small diameter sensory neurons isolated from rats was also reduced by TAT-CBD3A6K peptide suggesting that suppression of excitability may be due to inhibition of T- and R-type Ca2+ channels. TAT-CBD3A6K had no effect on depolarization-evoked calcitonin gene related peptide (CGRP) release by compared to vehicle control. CONCLUSIONS: Collectively, these results establish TAT-CBD3A6K as a peptide therapeutic with greater efficacy in an AIDS therapy-induced model of peripheral neuropathy than its parent peptide, TAT-CBD3. Structural modifications of the CBD3 scaffold peptide may result in peptides with selectivity against particular subset of voltage-gated calcium channels resulting in a multipharmacology of action on the target.     

3D Magnetic Holes in Collisionless Plasmas

Recent multispacecraft observations in the Earth’s magnetosphere have revealed an abundance of magnetic holes—localized magnetic field depressions. These magnetic holes are characterized by the plasma pressure enhancement and strongly localized currents flowing around the hole boundaries. There are several numerical and analytical models describing 2D configurations of magnetic holes, but the 3D distribution of magnetic fields and electric currents is studied poorly. Such a 3D magnetic field configuration is important for accurate investigation of charged particle dynamics within magnetic holes. Moreover, the 3D distribution of currents can be used for distant probing of magnetic holes in the magnetosphere. In this study, a 3D magnetic hole model using the single-fluid approximation and a spatial scale hierarchy with the distinct separation of gradients is developed. It is shown that such 3D holes can be obtained as a generalization of 1D models with the plasma pressure distribution adopted from the kinetic approach. The proposed model contains two magnetic field components and field-aligned currents. The magnetic field line configuration resembles the magnetic trap where hot charged particles bounce between mirror points. However, the approximation of isotropic pressure results in a constant plasma pressure along magnetic field lines, and the proposed magnetic hole model does not confine plasma along the field direction.     

Nerve Growth Factor Mediates a Switch in Intracellular Signaling for PGE2-Induced Sensitization of Sensory Neurons from Protein Kinase A to Epac

We examined whether nerve growth factor (NGF), an inflammatory mediator that contributes to chronic hypersensitivity, alters the intracellular signaling that mediates the sensitizing actions of PGE₂ from activation of protein kinase A (PKA) to exchange proteins directly activated by cAMP (Epacs). When isolated sensory neurons are grown in the absence of added NGF, but not in cultures grown with 30 ng/ml NGF, inhibiting protein kinase A (PKA) activity blocks the ability of PGE₂ to augment capsaicin-evoked release of the neuropeptide CGRP and to increase the number of action potentials (APs) evoked by a ramp of current. Growing sensory neurons in culture in the presence of increasing concentrations of NGF increases the expression of Epac2, but not Epac1. An intradermal injection of complete Freund''s adjuvant into the rat hindpaw also increases the expression of Epac2, but not Epac1 in the dorsal root ganglia and spinal cord: an effect blocked by intraplantar administration of NGF antibodies. Treating cultures grown in the presence of 30 ng/ml NGF with Epac1siRNA significantly reduced the expression of Epac1, but not Epac2, and did not block the ability of PGE₂ to augment capsaicin-evoked release of CGRP from sensory neurons. Exposing neuronal cultures grown in NGF to Epac2siRNAreduced the expression of Epac2, but not Epac1 and prevented the PGE₂-induced augmentation of capsaicin and potassium-evoked CGRP release in sensory neurons and the PGE₂-induced increase in the number of APs generated by a ramp of current. In neurons grown with no added NGF, Epac siRNAs did not attenuate PGE₂-induced sensitization. These results demonstrate that NGF, through increasing Epac2 expression, alters the signaling cascade that mediates PGE₂-induced sensitization of sensory neurons, thus providing a novel mechanism for maintaining PGE₂-induced hypersensitivity during inflammation.     

Role of the DNA Base Excision Repair Protein,APE1 in Cisplatin,Oxaliplatin, or Carboplatin Induced Sensory Neuropathy

Although chemotherapy-induced peripheral neuropathy (CIPN) is a dose-limiting side effect of platinum drugs, the mechanisms of this toxicity remain unknown. Previous work in our laboratory suggests that cisplatin-induced CIPN is secondary to DNA damage which is susceptible to base excision repair (BER). To further examine this hypothesis, we studied the effects of cisplatin, oxaliplatin, and carboplatin on cell survival, DNA damage, ROS production, and functional endpoints in rat sensory neurons in culture in the absence or presence of reduced expression of the BER protein AP endonuclease/redox factor-1 (APE1). Using an in situ model of peptidergic sensory neuron function, we examined the effects of the platinum drugs on hind limb capsaicin-evoked vasodilatation. Exposing sensory neurons in culture to the three platinum drugs caused a concentration-dependent increase in apoptosis and cell death, although the concentrations of carboplatin were 10 fold higher than cisplatin. As previously observed with cisplatin, oxaliplatin and carboplatin also increased DNA damage as indicated by an increase in phospho-H2AX and reduced the capsaicin-evoked release of CGRP from neuronal cultures. Both cisplatin and oxaliplatin increased the production of ROS as well as 8-oxoguanine DNA adduct levels, whereas carboplatin did not. Reducing levels of APE1 in neuronal cultures augmented the cisplatin and oxaliplatin induced toxicity, but did not alter the effects of carboplatin. Using an in vivo model, systemic injection of cisplatin (3 mg/kg), oxaliplatin (3 mg/kg), or carboplatin (30 mg/kg) once a week for three weeks caused a decrease in capsaicin-evoked vasodilatation, which was delayed in onset. The effects of cisplatin on capsaicin-evoked vasodilatation were attenuated by chronic administration of E3330, a redox inhibitor of APE1 that serendipitously enhances APE1 DNA repair activity in sensory neurons. These outcomes support the importance of the BER pathway, and particularly APE1, in sensory neuropathy caused by cisplatin and oxaliplatin, but not carboplatin and suggest that augmenting DNA repair could be a therapeutic target for CIPN.     

The Role of CHI3L1 (Chitinase-3-Like-1) in the Pathogenesis of Infections in Burns in a Mouse Model

In severe burn injury the unique setting of a depleted, dysfunctional immune system along with a loss of barrier function commonly results in opportunistic infections that eventually proof fatal. Unfortunately, the dynamic sequence of bacterial contamination, colonization and eventually septic invasion with bacteria such as Pseudomonas species is still poorly understood although a limiting factor in clinical decision making. Increasing evidence supports the notion that inhibition of bacterial translocation into the wound site may be an effective alternative to prevent infection. In this context we investigated the role of the mammalian Chitinase-3-Like-1 (CHI3L1) non-enyzmatic protein predominately expressed on epithelial as well as innate immune cells as a potential bacterial-translocation-mediating factor. We show a strong trend that a modulation of chitinase expression is likely to be effective in reducing mortality rates in a mouse model of burn injury with superinfection with the opportunistic PA14 Pseudomonas strain, thus demonstrating possible clinical leverage.