Lidia M. Yshii

Cocaine induces cell death and activates the transcription nuclear factor kappa-B in PC12 cells.

Cocaine is a worldwide used drug and its abuse is associated with physical, psychiatric and social problems. The mechanism by which cocaine causes neurological damage is very complex and involves several neurotransmitter systems. For example, cocaine increases extracellular levels of dopamine and free radicals, and modulates several transcription factors. NF-κB is a transcription factor that regulates gene expression involved in cellular death. Our aim was to investigate the toxicity and modulation of NF-κB activity by cocaine in PC 12 cells. Treatment with cocaine (1 mM) for 24 hours induced DNA fragmentation, cellular membrane rupture and reduction of mitochondrial activity. A decrease in Bcl-2 protein and mRNA levels, and an increase in caspase 3 activity and cleavage were also observed. In addition, cocaine (after 6 hours treatment) activated the p50/p65 subunit of NF-κB complex and the pretreatment of the cells with SCH 23390, a D1 receptor antagonist, attenuated the NF-κB activation. Inhibition of NF-κB activity by using PDTC and Sodium Salicilate increased cell death caused by cocaine. These results suggest that cocaine induces cell death (apoptosis and necrosis) and activates NF-κB in PC12 cells. This activation occurs, at least partially, due to activation of D1 receptors and seems to have an anti-apoptotic effect on these cells.

Time-dependent effects of training on cardiovascular control in spontaneously hypertensive rats: role for brain oxidative stress and inflammation and baroreflex sensitivity.

Baroreflex dysfunction, oxidative stress and inflammation, important hallmarks of hypertension, are attenuated by exercise training. In this study, we investigated the relationships and time-course changes of cardiovascular parameters, pro-inflammatory cytokines and pro-oxidant profiles within the hypothalamic paraventricular nucleus of the spontaneously hypertensive rats (SHR). Basal values and variability of arterial pressure and heart rate and baroreflex sensitivity were measured in trained (T, low-intensity treadmill training) and sedentary (S) SHR at weeks 0, 1, 2, 4 and 8. Paraventricular nucleus was used to determine reactive oxygen species (dihydroethidium oxidation products, HPLC), NADPH oxidase subunits and pro-inflammatory cytokines expression (Real time PCR), p38 MAPK and ERK1/2 expression (Western blotting), NF-κB content (electrophoretic mobility shift assay) and cytokines immunofluorescence. SHR-S vs. WKY-S (Wistar Kyoto rats as time control) showed increased mean arterial pressure (172±3 mmHg), pressure variability and heart rate (358±7 b/min), decreased baroreflex sensitivity and heart rate variability, increased p47phox and reactive oxygen species production, elevated NF-κB activity and increased TNF-α and IL-6 expression within the paraventricular nucleus of hypothalamus. Two weeks of training reversed all hypothalamic changes, reduced ERK1/2 phosphorylation and normalized baroreflex sensitivity (4.04±0.31 vs. 2.31±0.19 b/min/mmHg in SHR-S). These responses were followed by increased vagal component of heart rate variability (1.9-fold) and resting bradycardia (−13%) at the 4th week, and, by reduced vasomotor component of pressure variability (−28%) and decreased mean arterial pressure (−7%) only at the 8th week of training. Our findings indicate that independent of the high pressure levels in SHR, training promptly restores baroreflex function by disrupting the positive feedback between high oxidative stress and increased pro-inflammatory cytokines secretion within the hypothalamic paraventricular nucleus. These early adaptive responses precede the occurrence of training-induced resting bradycardia and blood pressure fall.

Amyloid β‐peptide activates nuclear factor‐κB through an N‐methyl‐D‐aspartate signaling pathway in cultured cerebellar cells

Amyloid β‐peptide (Aβ) likely causes functional alterations in neurons well prior to their death. Nuclear factor‐κB (NF‐κB), a transcription factor that is known to play important roles in cell survival and apoptosis, has been shown to be modulated by Aβ in neurons and glia, but the mechanism is unknown. Because Aβ has also been shown to enhance activation of N‐methyl‐D‐aspartate (NMDA) receptors, we investigated the role of NMDA receptor‐mediated intracellular signaling pathways in Aβ‐induced NF‐κB activation in primary cultured rat cerebellar cells. Cells were treated with different concentrations of Aβ1–40 (1 or 2 μM) for different periods (6, 12, or 24 hr). MK‐801 (NMDA antagonist), manumycin A and FTase inhibitor 1 (farnesyltransferase inhibitors), PP1 (Src‐family tyrosine kinase inhibitor), PD98059 [mitogen‐activated protein kinase (MAPK) inhibitor], and LY294002 [phosphatidylinositol 3‐kinase (PI3‐k) inhibitor] were added 20 min before Aβ treatment of the cells. Aβ induced a time‐ and concentration‐dependent activation of NF‐κB (1 μM, 12 hr); both p50/p65 and p50/p50 NF‐κB dimers were involved. This activation was abolished by MK‐801 and attenuated by manumycin A, FTase inhibitor 1, PP1, PD98059, and LY294002. Aβ at 1 μM increased the expression of inhibitory protein IκB, brain‐derived neurotrophic factor, inducible nitric oxide synthase, tumor necrosis factor‐α, and interleukin‐1β as shown by RT‐PCR assays. Collectively, these findings suggest that Aβ activates NF‐κB by an NMDA‐Src‐Ras‐like protein through MAPK and PI3‐k pathways in cultured cerebellar cells. This pathway may mediate an adaptive, neuroprotective response to Aβ.

Influence of N-methyl-D-aspartate receptors on ouabain activation of nuclear factor-κB in the rat hippocampus.

It has been shown that ouabain (OUA) can activate the Na,K‐ATPase complex and mediate intracellular signaling in the central nervous system (CNS). Inflammatory stimulus increases glutamatergic transmission, especially at N‐methyl‐D‐aspartate (NMDA) receptors, which are usually coupled to the activation of nitric oxide synthase (NOS). Nuclear factor‐κB (NF‐κB) activation modulates the expression of genes involved in development, plasticity, and inflammation. The present work investigated the effects of OUA on NF‐κB binding activity in rat hippocampus and the influence of this OUA‐Na,K‐ATPase signaling cascade in NMDA‐mediated NF‐κB activation. The findings presented here are the first report indicating that intrahippocampal administration of OUA, in a concentration that did not alter Na,K‐ATPase or NOS activity, induced an activation of NF‐κB, leading to increases in brain‐derived neurotrophic factor (Bdnf), inducible NOS (iNos), tumor necrosis factor‐α (Tnf‐α), and B‐cell leukemia/lymphoma 2 (Bcl2) mRNA levels. This response was not linked to any significant signs of neurodegeneration as showed via Fluoro‐Jade B and Nissl stain. Intrahippocampal administration of NMDA induced NF‐κB activation and increased NOS and α2/3‐Na,K‐ATPase activities. NMDA treatment further increased OUA‐induced NF‐κB activation, which was partially blocked by MK‐801, an antagonist of NMDA receptor. These results suggest that OUA‐induced NF‐κB activation is at least in part dependent on Na,K‐ATPase modulatory action of NMDA receptor in hippocampus. The interaction of these signaling pathways could be associated with biological mechanisms that may underlie the basal homeostatic state linked to the inflammatory signaling cascade in the brain.

CD8 T cell-mediated killing of orexinergic neurons induces a narcolepsy-like phenotype in mice.

Significance Narcolepsy with cataplexy is a sleep disorder characterized by excessive daytime sleepiness and sudden loss of muscle tone. These clinical manifestations are the result of selective loss of a neuronal population producing orexin. The etiology of the disease remains elusive, although converging evidence points to a key involvement of the immune system. We developed an animal model to study the autoimmune processes at play in narcolepsy. We demonstrate that cytotoxic CD8 T cells, but not Th1 CD4 cells, are able to target and destroy orexinergic neurons. This selective neuronal loss is responsible for clinical signs mimicking human narcolepsy. By identifying potential immune effectors of the immunopathological process in narcolepsy, these findings offer a rationale for the use of immunotherapies. Narcolepsy with cataplexy is a rare and severe sleep disorder caused by the destruction of orexinergic neurons in the lateral hypothalamus. The genetic and environmental factors associated with narcolepsy, together with serologic data, collectively point to an autoimmune origin. The current animal models of narcolepsy, based on either disruption of the orexinergic neurotransmission or neurons, do not allow study of the potential autoimmune etiology. Here, we sought to generate a mouse model that allows deciphering of the immune mechanisms leading to orexin+ neuron loss and narcolepsy development. We generated mice expressing the hemagglutinin (HA) as a “neo-self-antigen” specifically in hypothalamic orexin+ neurons (called Orex-HA), which were transferred with effector neo-self-antigen–specific T cells to assess whether an autoimmune process could be at play in narcolepsy. Given the tight association of narcolepsy with the human leukocyte antigen (HLA) HLA-DQB1*06:02 allele, we first tested the pathogenic contribution of CD4 Th1 cells. Although these T cells readily infiltrated the hypothalamus and triggered local inflammation, they did not elicit the loss of orexin+ neurons or clinical manifestations of narcolepsy. In contrast, the transfer of cytotoxic CD8 T cells (CTLs) led to both T-cell infiltration and specific destruction of orexin+ neurons. This phenotype was further aggravated upon repeated injections of CTLs. In situ, CTLs interacted directly with MHC class I-expressing orexin+ neurons, resulting in cytolytic granule polarization toward neurons. Finally, drastic neuronal loss caused manifestations mimicking human narcolepsy, such as cataplexy and sleep attacks. This work demonstrates the potential role of CTLs as final effectors of the immunopathological process in narcolepsy.

Pivotal role of endogenous tachykinins and the NK1 receptor in mediating leukocyte accumulation, in the absence of oedema formation, in response to TNFα in the cutaneous microvasculature

Tachykinins including substance P (SP) are well known to play a role in influencing oedema formation and leukocyte accumulation during tissue insult and inflammation. Cutaneous inflammatory models to characterize a TNFalpha-dependent mechanism where endogenous SP act via the NK1 receptor to promote leukocyte accumulation in the absence of oedema formation were used. We found that TNFalpha induced dose-dependent leukocyte accumulation at 4 h, which returned towards basal levels at 8 h in NK1+/+ mice. This response was absent in both the NK1+/+ mice treated with an NK1 receptor antagonist and NK1-/- mice. At the highest dose IL-6 induced a significant accumulation in NK1+/+ and NK1-/- mice but IL-12 was ineffective. SP induced skin oedema but none of the cytokines did. Either co-injection of SP with low dose of TNFalpha (0.3 pmol/site) or SP previously injected (30 min) to TNFalpha evoked a significant increase in MPO activity when compared with that induced by the cytokine alone. In contrast, SP injected i.d. 3.5 h after TNFalpha failed to produce additive response. Control, but not capsaicin-pretreated rats (to deplete sensory nerves), exhibited a marked increase in MPO activity in response to TNFalpha. Histological analysis showed that TNFalpha caused tissue infiltrate of leukocytes in NK1+/+ mice, whilst leukocytes accumulated at intravascular sites in NK1-/- mice, but did not appear to emigrate, suggesting a defect in trans-endothelial migration. Interestingly, monocytes in addition to neutrophils accumulated 4 h post TNFalpha injection. In conclusion, the NK1 receptor plays a functional role in mediating leukocyte accumulation independently of the historically important NK1 mediated oedema formation. It seems that TNFalpha directly activates sensory nerve in addition to its chemoattractant activity. The NK1 receptor agonist influences the accumulation of monocytes in addition to that of PMN by 4 h, thus revealing an important influence of the NK1 receptor on TNFalpha mediated events in mouse skin.

Characterization of the mechanisms underlying the inflammatory response to Polistes lanio lanio (paper wasp) venom in mouse dorsal skin

Stings by Polistes wasps can cause life-threatening allergic reactions, pain and inflammation. We examined the changes in microvascular permeability and neutrophil influx caused by the venom of Polistes lanio a paper wasp found in southeastern Brazil. The intradermal injection of wasp venom caused long-lasting paw oedema and dose-dependently increased microvascular permeability in mouse dorsal skin. SR140333, an NK(1) receptor antagonist, markedly inhibited the response, but the NK(2) receptor antagonist SR48968 was ineffective. The oedema was reduced in capsaicin-treated rats, indicating a direct activation of sensory fibres. Dialysis of the venom partially reduced the oedema and the remaining response was further inhibited by SR140333. Mass spectrometric analysis of the venom revealed two peptides (QPPTPPEHRFPGLM and ASEPTALGLPRIFPGLM) with sequence similarities to the C-terminal region of tachykinin-like peptides found in Phoneutria nigriventer spider venom and vertebrates. Wasp venom failed to release histamine from mast cells in vitro and spectrofluorometric assay of the venom revealed a negligible content of histamine in the usual dose of P. l. lanio venom (1nmol of histamine/7mug of venom) that was removed by dialysis. The histamine H(1) receptor antagonist pyrilamine, but not bradykinin B(1) or B(2) receptor antagonists, inhibited venom-induced oedema. In conclusion, P. l. lanio venom induces potent oedema and increases vascular permeability in mice, primarily through activation of tachykinin NK(1) receptors by substance P released from sensory C fibres, which in turn releases histamine from dermal mast cells. This is the first description of a neurovascular mechanism for P. l. lanio venom-mediated inflammation. The extent to which the two tachykinin-like peptides identified here contribute to this neurogenic inflammatory response remains to be elucidated.

Inflammatory CNS disease caused by immune checkpoint inhibitors: status and perspectives

Cancer treatment strategies based on immune stimulation have recently entered the clinical arena, with unprecedented success. Immune checkpoint inhibitors (ICIs) work by indiscriminately promoting immune responses, which target tumour-associated antigens or tumour-specific mutations. However, the augmented immune response, most notably the T cell response, can cause either direct neurotoxicity or, more commonly, indirect neurotoxic effects through systemic or local inflammatory mechanisms or autoimmune mechanisms. Consequently, patients treated with ICIs are susceptible to CNS disease, including paraneoplastic neurological syndromes, encephalitis, multiple sclerosis and hypophysitis. In this Opinion article, we introduce the mechanisms of action of ICIs and review their adverse effects on the CNS. We highlight the importance of early detection of these neurotoxic effects, which should be distinguished from brain metastasis, and the need for early detection of neurotoxicity. It is crucial that physicians are well informed of these neurological adverse effects, given the anticipated increase in the use of immunotherapies to treat cancer.

Migration of encephalitogenic CD8 T cells into the central nervous system is dependent on the α4β1-integrin.

Although CD8 T cells are key players in neuroinflammation, little is known about their trafficking cues into the central nervous system (CNS). We used a murine model of CNS autoimmunity to define the molecules involved in cytotoxic CD8 T‐cell migration into the CNS. Using a panel of mAbs, we here show that the α4β1‐integrin is essential for CD8 T‐cell interaction with CNS endothelium. We also investigated which α4β1‐integrin ligands expressed by endothelial cells are implicated. The blockade of VCAM‐1 did not protect against autoimmune encephalomyelitis, and only partly decreased the CD8+ T‐cell infiltration into the CNS. In addition, inhibition of junctional adhesion molecule‐B expressed by CNS endothelial cells also decreases CD8 T‐cell infiltration. CD8 T cells may use additional and possibly unidentified adhesion molecules to gain access to the CNS.

PAR2 and Temporomandibular Joint Inflammation in the Rat

The proteinase-activated receptor 2 (PAR2) is a putative therapeutic target for arthritis. We hypothesized that the early pro-inflammatory effects secondary to its activation in the temporomandibular joint (TMJ) are mediated by neurogenic mechanisms. Immunofluorescence analysis revealed a high degree of neurons expressing PAR2 in retrogradely labeled trigeminal ganglion neurons. Furthermore, PAR2 immunoreactivity was observed in the lining layer of the TMJ, co-localizing with the neuronal marker PGP9.5 and substance-P-containing peripheral sensory nerve fibers. The intra-articular injection of PAR2 agonists into the TMJ triggered a dose-dependent increase in plasma extravasation, neutrophil influx, and induction of mechanical allodynia. The pharmacological blockade of natural killer 1 (NK1) receptors abolished PAR2-induced plasma extravasation and inhibited neutrophil influx and mechanical allodynia. We conclude that PAR2 activation is pro-inflammatory in the TMJ, through a neurogenic mechanism involving NK1 receptors. This suggests that PAR2 is an important component of innate neuro-immune response in the rat TMJ.