By crossing Huntington's disease (HD) R6/1 transgenic mice with ‘tissue’ transglutaminase (TG2) knock-out mice, we have demonstrated that this multifunctional enzyme plays an important role in the neuronal death characterising this disorder in vivo. In fact, a large reduction in cell death is observed in R6/1, TG2^−/− compared with R6/1 transgenic mice. In addition, we have shown that the formation of neuronal intranuclear inclusions (NII) is potentiated in absence of the ‘tissue’ transglutaminase. These phenomena are paralleled by a significant improvement both in motor performances and survival of R6/1, TG2^−/− versus R6/1 mice. Taken together these findings suggest an important role for tissue transglutaminase in the regulation of neuronal cell death occurring in Huntington's disease.

The vgf gene has been identified as an energy homeostasis regulator. Vgf encodes a 617-aa precursor protein that is processed to yield an incompletely characterized panel of neuropeptides. Until now, it was an unproved assumption that VGF-derived peptides could regulate metabolism. Here, a VGF peptide designated TLQP-21 was identified in rat brain extracts by means of immunoprecipitation, microcapillary liquid chromatography–tandem MS, and database searching algorithms. Chronic intracerebroventricular (i.c.v.) injection of TLQP-21 (15 μg/day for 14 days) increased resting energy expenditure (EE) and rectal temperature in mice. These effects were paralleled by increased epinephrine and up-regulation of brown adipose tissue β2-AR (β2 adrenergic receptor) and white adipose tissue (WAT) PPAR-δ (peroxisome proliferator-activated receptor δ), β3-AR, and UCP1 (uncoupling protein 1) mRNAs and were independent of locomotor activity and thyroid hormones. Hypothalamic gene expression of orexigenic and anorexigenic neuropeptides was unchanged. Furthermore, in mice that were fed a high-fat diet for 14 days, TLQP-21 prevented the increase in body and WAT weight as well as hormonal changes that are associated with a high-fat regimen. Biochemical and molecular analyses suggest that TLQP-21 exerts its effects by stimulating autonomic activation of adrenal medulla and adipose tissues. In conclusion, we present here the identification in the CNS of a previously uncharacterized VGF-derived peptide and prove that its chronic i.c.v. infusion effected an increase in EE and limited the early phase of diet-induced obesity.

In recent years a growing debate is about whether botulinum neurotoxins are retrogradely transported from the site of injection. Immunodetection of cleaved SNAP-25 (cl-SNAP-25), the protein of the SNARE complex targeted by botulinum neurotoxin serotype A (BoNT/A), could represent an excellent approach to investigate the mechanism of action on the nociceptive pathways at peripheral and/or central level. After peripheral administration of BoNT/A, we analyzed the expression of cl-SNAP-25, from the hindpaw’s nerve endings to the spinal cord, together with the behavioral effects on neuropathic pain. We used the chronic constriction injury of the sciatic nerve in CD1 mice as animal model of neuropathic pain. We evaluated immunostaining of cl-SNAP-25 in the peripheral nerve endings, along the sciatic nerve, in dorsal root ganglia and in spinal dorsal horns after intraplantar injection of saline or BoNT/A, alone or colocalized with either glial fibrillar acidic protein, GFAP, or complement receptor 3/cluster of differentiation 11b, CD11b, or neuronal nuclei, NeuN, depending on the area investigated. Immunofluorescence analysis shows the presence of the cl-SNAP-25 in all tissues examined, from the peripheral endings to the spinal cord, suggesting a retrograde transport of BoNT/A. Moreover, we performed in vitro experiments to ascertain if BoNT/A was able to interact with the proliferative state of Schwann cells (SC). We found that BoNT/A modulates the proliferation of SC and inhibits the acetylcholine release from SC, evidencing a new biological effect of the toxin and further supporting the retrograde transport of the toxin along the nerve and its ability to influence regenerative processes. The present results strongly sustain a combinatorial action at peripheral and central neural levels and encourage the use of BoNT/A for the pathological pain conditions difficult to treat in clinical practice and dramatically impairing patients’ quality of life.

In experimental and clinical pain studies, the sex of subjects was rarely taken into account, even if nociceptive inputs appear to be processed and modulated by partially distinct neural mechanisms in each sex. In this study we analysed, in male and female mice, behavioural and neuronal responses in developing, maintaining, and recovering from neuropathic pain. Experiments were carried out in adult CD1 mice by using Chronic Constriction Injury (CCI) as neuropathic pain model. We investigated the temporal trend of mechanical nociceptive threshold together with functional recovery of the injured paw, and the immunofluorescence staining of proteins associated with nerve injury and repair and with spinal gliosis, 7 and 121days after CCI. A proteomic analysis on proteins extracted from sciatic nerves was also performed. Male mice showed a gradual decrease of CCI-induced allodynia, the complete recovery occurring 81days after the sciatic nerve ligation. On the contrary, in female mice, allodynia was still present 121days after CCI. Sex-dependent differences also resulted from immunofluorescence experiments: in sciatic nerve, the expression of P0 and Neu200 is greater in neuropathic males than in neuropathic females, suggesting faster nerve regeneration. Proteomic analysis confirmed sex-related differences of proteins associated with nerve regenerative processes. In addition, the reactive gliosis induced by CCI at day 7, as revealed by colocalization of glial fibrillary acidic protein (astrocytes) and CD11b (microglia) with phosphorylated p38, disappeared 121 days after CCI in male but not in female mice. These results may have important therapeutic implications for the treatment of neuropathic pain.

Axonal degeneration in peripheral nerves after injury is accompanied by myelin degradation initiated by Schwann cells (SCs). These cells activate autophagy, a ubiquitous cytoprotective process essential for degradation and recycling of cellular constituents. Concomitantly to nerve insult and axonal degeneration, neuropathic pain (NeP) arises. The role of SC autophagy in the mechanisms underlying NeP is still unknown. In this study, we examined the role of the autophagy during the early phase of Wallerian degeneration in NeP induction and chronification by using a murine model of peripheral nerve lesion (chronic constriction injury). We demonstrate that the autophagy inducer rapamycin, administered in the first week after nerve damage, induces long-lasting analgesic and antiinflammatory effects, facilitates nerve regeneration, and prevents pain chronification. Conversely, when autophagy is altered, by means of autophagic inhibitor 3-methyladenine administration or as occurs in activating molecule in Beclin-1-regulated autophagy transgenic mice (Ambra1(+/gt)), NeP is dramatically enhanced and prolonged. Immunohistochemical and ultrastructural evaluations show that rapamycin is able to increase autophagic flux in SCs, to accelerate myelin compaction, and to reduce inflammatory and immune reaction. Proteomic analysis combined with bioinformatic analysis suggests that a redox-sensitive mechanism could be responsible for SC autophagy activation. These data suggest that a deficiency of autophagic activity in SCs can be an early event in the origin of NeP chronification and that autophagy modulation may represent a powerful pharmacological approach to prevent the onset and chronification of NeP in the clinical setting.

Nerve growth factor (NGF) is involved in pain transduction mechanisms and plays a key role in many persistent pain states, notably those associated with inflammation. On this basis, both the NGF ligand and its receptor TrkA (tyrosine kinase A) represent an eligible target for pain therapy. Although the direct involvement of NGF in pain modulation is well established, the effect of a direct functional block of the TrkA receptor is still unknown. In this study, we have demonstrated that MNAC13, the only anti-TrkA monoclonal antibody for which function neutralizing properties have been clearly shown both in vitro and in vivo , induces analgesia in both inflammatory and neuropathic pain models, with a surprisingly long-lasting effect in the latter. The formalin-evoked pain licking responses are significantly reduced by the MNAC13 antibody in CD1 mice. Remarkably, treatment with the anti-TrkA antibody also produces a significant antiallodynic effect on neuropathic pain: repeated i.p. injections of MNAC13 induce significant functional recovery in mice subjected to sciatic nerve ligation, with effects persisting after administration. Furthermore, a clear synergistic effect is observed when MNAC13 is administered in combination with opioids, at doses that are not efficacious per se . This study represents a direct demonstration that neutralizing antibodies directed against the TrkA receptor may display potent analgesic effects in inflammatory and chronic pain.