A Narrative Review on Axonal Neuroprotection in Multiple Sclerosis.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) resulting in demyelination and neurodegeneration. The therapeutic strategy is now largely based on reducing inflammation with immunosuppressive drugs. Unfortunately, when disease progression is observed, no drug offers neuroprotection apart from its anti-inflammatory effect. In this review, we explore current knowledge on the assessment of neurodegeneration in MS and look at putative targets that might prove useful in protecting the axon from degeneration. Among them, Bruton's tyrosine kinase inhibitors, anti-apoptotic and antioxidant agents, sex hormones, statins, channel blockers, growth factors, and molecules preventing glutamate excitotoxicity have already been studied. Some of them have reached phase III clinical trials and carry a great message of hope for our patients with MS.

TGFß promotes low IL10-producing ILC2 with profibrotic ability involved in skin fibrosis in systemic sclerosis.

OBJECTIVE: Innate lymphoid cells-2 (ILC2) were shown to be involved in the development of lung or hepatic fibrosis. We sought to explore the functional and phenotypic heterogeneity of ILC2 in skin fibrosis within systemic sclerosis (SSc). METHODS: Blood samples and skin biopsies from healthy donor or patients with SSc were analysed by immunostaining techniques. The fibrotic role of sorted ILC2 was studied in vitro on dermal fibroblast and further explored by transcriptomic approach. Finally, the efficacy of a new treatment against fibrosis was assessed with a mouse model of SSc. RESULTS: We found that ILC2 numbers were increased in the skin of patients with SSc and correlated with the extent of skin fibrosis. In SSc skin, KLRG1- ILC2 (natural ILC2) were dominating over KLRG1+ ILC2 (inflammatory ILC2). The cytokine transforming growth factor-ß (TGFß), whose activity is increased in SSc, favoured the expansion of KLRG1- ILC2 simultaneously decreasing their production of interleukin 10 (IL10), which regulates negatively collagen production by dermal fibroblasts. TGFß-stimulated ILC2 also increased myofibroblast differentiation. Thus, human KLRG1- ILC2 had an enhanced profibrotic activity. In a mouse model of SSc, therapeutic intervention-combining pirfenidone with the administration of IL10 was required to reduce the numbers of skin infiltrating ILC2, enhancing their expression of KLRG1 and strongly alleviating skin fibrosis. CONCLUSION: Our results demonstrate a novel role for natural ILC2 and highlight their inter-relationships with TGFß and IL10 in the development of skin fibrosis, thereby opening up new therapeutic approaches in SSc.

Microglial Cell Morphology and Phagocytic Activity Are Critically Regulated by the Neurosteroid Allopregnanolone: A Possible Role in Neuroprotection.

Microglial cells are key players in neural pathogenesis and microglial function regulation appears to be pivotal in controlling neuroinflammatory/neurological diseases. Here, we investigated the effects and mechanism of action of neurosteroid allopregnanolone (ALLO) on murine microglial BV-2 cells and primary microglia in order to determine ALLO-induced immunomodulatory potential and to provide new insights for the development of both natural and safe neuroprotective strategies targeting microglia. Indeed, ALLO-treatment is increasingly suggested as beneficial in various models of neurological disorders but the underlying mechanisms have not been elucidated. Therefore, the microglial cells were cultured with various serum concentrations to mimic the blood-brain-barrier rupture and to induce their activation. Proliferation, viability, RT-qPCR, phagocytosis, and morphology analyzes, as well as migration with time-lapse imaging and quantitative morphodynamic methods, were combined to investigate ALLO actions on microglia. BV-2 cells express subunits of GABA-A receptor that mediates ALLO activity. ALLO (10µM) induced microglial cell process extension and decreased migratory capacity. Interestingly, ALLO modulated the phagocytic activity of BV-2 cells and primary microglia. Our results, which show a direct effect of ALLO on microglial morphology and phagocytic function, suggest that the natural neurosteroid-based approach may contribute to developing effective strategies against neurological disorders that are evoked by microglia-related abnormalities.

Laquinimod dampens IL-1ß signaling and Th17-polarizing capacity of monocytes in patients with MS.

OBJECTIVE: To assess the impact of laquinimod treatment on monocytes and to investigate the underlying immunomodulatory mechanisms in MS. METHODS: In this cross-sectional study, we performed in vivo and in vitro analyses of cluster of differentiation (CD14+) monocytes isolated from healthy donors (n = 15), untreated (n = 13), and laquinimod-treated patients with MS (n = 14). Their frequency and the expression of surface activation markers were assessed by flow cytometry and the viability by calcein staining. Cytokine concentrations in the supernatants of lipopolysaccharide (LPS)-stimulated monocytes were determined by flow cytometry. The messenger ribonucleic acid (mRNA) expression level of genes involved in cytokine expression was measured by quantitative PCR. The LPS-mediated nuclear factor kappa-light-chain-enhancer of activated B-cell (NF-κB) activation was determined by the quantification of the phosphorylation level of the p65 subunit. Laquinimod-treated monocytes were cocultured with CD4+ T cells, and the resulting cytokine production was analyzed by flow cytometry after intracellular cytokine staining. The interleukin (IL)-17A concentration of the supernatant was assessed by ELISA. RESULTS: Laquinimod did not alter the frequency or viability of circulating monocytes, but led to an upregulation of CD86 expression. LPS-stimulated monocytes of laquinimod-treated patients with MS secreted less IL-1ß following a downregulation of IL-1ß gene expression. Phosphorylation levels of the NF-κB p65 subunit were reduced after laquinimod treatment, indicating a laquinimod-associated inhibition of the NF-κB pathway. T cells primed with laquinimod-treated monocytes differentiated significantly less into IL-17A-producing T helper (Th)-17 cells. CONCLUSIONS: Our findings suggest that inhibited NF-κB signaling and downregulation of IL-1ß expression in monocytes contributes to the immunomodulatory effects of laquinimod and that the impairment of Th17 polarization might mediate its disease-modifying activity in MS.

Decreased CCN3 in Systemic Sclerosis Endothelial Cells Contributes to Impaired Angiogenesis.

Systemic sclerosis (SSc) is a rare and severe connective tissue disease combining autoimmune and vasculopathy features, ultimately leading to organ fibrosis. Impaired angiogenesis is an often silent and life-threatening complication of the disease. We hypothesize that CCN3, a member of the CCN family of extracellular matrix proteins, which is an antagonist of the profibrotic protein CCN2 as well as a proangiogenic factor, is implicated in SSc pathophysiology. We performed skin biopsies on 26 patients with SSc, both in fibrotic and nonfibrotic areas for 17 patients, and collected 18 healthy control skin specimens for immunohistochemistry and cell culture. Histological analysis of nonfibrotic and fibrotic SSc skin shows a systemic decrease of papillary dermis surface as well as disappearance of capillaries. CCN3 expression is systematically decreased in the dermis of patients with SSc compared with healthy controls, particularly in dermal blood vessels. Moreover, CCN3 is decreased in vitro in endothelial cells from patients with SSc. We show that CCN3 is essential for endothelial cell migration and angiogenesis in vitro. In conclusion, CCN3 may represent a promising therapeutic target for patients with SSc presenting with vascular involvement.

Disruption of Sema3A/Plexin-A1 inhibitory signalling in oligodendrocytes as a therapeutic strategy to promote remyelination.

Current treatments in multiple sclerosis (MS) are modulating the inflammatory component of the disease, but no drugs are currently available to repair lesions. Our study identifies in MS patients the overexpression of Plexin-A1, the signalling receptor of the oligodendrocyte inhibitor Semaphorin 3A. Using a novel type of peptidic antagonist, we showed the possibility to counteract the Sema3A inhibitory effect on oligodendrocyte migration and differentiation in vitro when antagonizing Plexin-A1. The use of this compound in vivo demonstrated a myelin protective effect as shown with DTI-MRI and confirmed at the histological level in the mouse cuprizone model of induced demyelination/remyelination. This effect correlated with locomotor performances fully preserved in chronically treated animals. The administration of the peptide also showed protective effects, leading to a reduced severity of demyelination in the context of experimental autoimmune encephalitis (EAE). Hence, the disruption of the inhibitory microenvironmental molecular barriers allows normal myelinating cells to exert their spontaneous remyelinating capacity. This opens unprecedented therapeutic opportunity for patients suffering a disease for which no curative options are yet available.

Immune-Mediated Repair: A Matter of Plasticity.

Though the immune system is generally defined as a system of defense, it is increasingly recognized that the immune system also plays a crucial role in tissue repair and its potential dysregulations. In this review, we explore how distinct immune cell types are involved in tissue repair and how they interact in a process that is tightly regulated both spatially and temporally. We insist on the concept of immune cell plasticity which, in recent years, has proved fundamental for the success/understanding of the repair process. Overall, the perspective presented here suggests that the immune system plays a central role in the physiological robustness of the organism, and that cell plasticity contributes to the realization of this robustness.

Platelets Induce Thymic Stromal Lymphopoietin Production by Endothelial Cells: Contribution to Fibrosis in Human Systemic Sclerosis.

OBJECTIVE: To investigate the relationship between vascular damage and fibrosis in systemic sclerosis (SSc) by testing the hypothesis that platelets contribute to skin fibrosis via the activation of human dermal microvascular endothelial cells (HDMECs) and subsequent production of profibrotic mediators. METHODS: A total of 203 SSc patients and 30 healthy donors were prospectively enrolled between 2012 and 2015 at the University Hospital of Bordeaux. Immunohistochemistry and immunofluorescence analyses were performed on skin biopsy sections from 18 SSc patients and 5 healthy donors. Serum thymic stromal lymphopoietin (TSLP) levels were measured by enzyme-linked immunosorbent assay in the entire cohort. HDMECs and fibroblasts were purified from biopsy sections. Extracellular matrix production by cultured fibroblasts was assessed by real-time quantitative polymerase chain reaction. RESULTS: Serum TSLP levels were significantly increased in SSc patients compared to healthy donors (P < 0.0001) and were associated with a higher frequency of vasculopathy (P = 0.02). The proportion of TSLP-positive dermal cells was increased in the skin of SSc patients compared with healthy donors (P < 0.0001) and was correlated with fibrosis (modified Rodnan skin thickness score) (r = 0.6146, P = 0.0001). In SSc dermis, TSLP was mainly expressed by CD31-positive endothelial cells. In vitro, activated platelets induced TSLP production by HDMECs in an interleukin-1ß-dependent manner. SSc fibroblasts responded differently according to their original TSLP environment. CONCLUSION: Taken together, these results identify HDMECs as contributors to TSLP production in SSc and suggest a potential mechanism by which platelets may profoundly affect the fibrotic process in SSc.

FTY720 (fingolimod) treatment tips the balance towards less immunogenic antigen-presenting cells in patients with multiple sclerosis.

OBJECTIVE: We aimed to clarify whether fingolimod has direct effects on antigen-presenting cells in multiple sclerosis patients. METHODS: Frequency and phenotype of directly ex vivo dendritic cells and monocytes were analyzed in 43 individuals, including fingolimod-treated and untreated multiple sclerosis patients as well as healthy subjects. These cells were further stimulated with lipopolysaccharide to determine functional effects of fingolimod treatment. RESULTS: Absolute numbers of CD1c+ dendritic cells and monocytes were not significantly reduced in fingolimod-treated patients indicating that fingolimod did not block the migration of antigen-presenting cells to peripheral blood. CD86 was upregulated on CD1c+ dendritic cells and thus their activation was not impaired under fingolimod treatment. Quantitative analyses of gene transcription in cells and protein content in supernatants from ex vivo CD1c+ dendritic cells and monocytes, however, showed lower secretion of TNFα, IL1-ß and IL-6 upon lipopolysaccharide-stimulation. These results could be matched with CD4+MOG-specific transgenic T cells exhibiting reduced levels of TNFα and IFN-γ but not IL-4 upon stimulation with murine dendritic cells loaded with MOG, when treated with fingolimod. CONCLUSIONS: Our data indicate that fingolimod - apart from trapping lymphocytes in lymph nodes - exerts its disease-modulating activity by rebalancing the immune tolerance networks by modulation of antigen-presenting cells.

Perivascular microglia promote blood vessel disintegration in the ischemic penumbra.

The contribution of microglia to ischemic cortical stroke is of particular therapeutic interest because of the impact on the survival of brain tissue in the ischemic penumbra, a region that is potentially salvable upon a brain infarct. Whether or not tissue in the penumbra survives critically depends on blood flow and vessel perfusion. To study the role of microglia in cortical stroke and blood vessel stability, CX3CR1(+/GFP) mice were subjected to transient middle cerebral artery occlusion and then microglia were investigated using time-lapse two-photon microscopy in vivo. Soon after reperfusion, microglia became activated in the stroke penumbra and started to expand cellular protrusions towards adjacent blood vessels. All microglia in the penumbra were found associated with blood vessels within 24 h post reperfusion and partially fully engulfed them. In the same time frame blood vessels became permissive for blood serum components. Migration assays in vitro showed that blood serum proteins leaking into the tissue provided molecular cues leading to the recruitment of microglia to blood vessels and to their activation. Subsequently, these perivascular microglia started to eat up endothelial cells by phagocytosis, which caused an activation of the local endothelium and contributed to the disintegration of blood vessels with an eventual break down of the blood brain barrier. Loss-of-microglia-function studies using CX3CR1(GFP/GFP) mice displayed a decrease in stroke size and a reduction in the extravasation of contrast agent into the brain penumbra as measured by MRI. Potentially, medication directed at inhibiting microglia activation within the first day after stroke could stabilize blood vessels in the penumbra, increase blood flow, and serve as a valuable treatment for patients suffering from ischemic stroke.

Biochemical characterization of a Caspase-3 far-red fluorescent probe for non-invasive optical imaging of neuronal apoptosis.

Apoptosis is a regulated process, leading to cell death, which is involved in several pathologies including neurodegenerative diseases and stroke. Caspase-3 is a key enzyme of the apoptotic pathway and is considered as a major target for the treatment of abnormal cell death. Sensitive and non-invasive methods to monitor caspase-3 activity in cells and in the brain of living animals are needed to test the efficiency of novel therapeutic strategies. In the present study, we have biochemically characterized a caspase-3 far-red fluorescent probe, QCASP3.2, that can be used to detect apoptosis in vivo. The specificity of cleavage of QCASP3.2 was demonstrated using recombinant caspases and protease inhibitors. The functionality of the probe was also established in cerebellar neurons cultured in apoptotic conditions. QCASP3.2 did not exhibit any toxicity and appeared to accurately reflect the induction and inhibition of caspase activity by H2O2 and PACAP, respectively, both in cell lysates and in cultured neurons. Finally, intravenous injection of the probe after cerebral ischemia revealed activation of caspase-3 in the infarcted hemisphere. Thus, the present study demonstrates that QCASP3.2 is a suitable probe to monitor apoptosis both in vitro and in vivo and illustrates some of the possible applications of this caspase-3 fluorescent probe.

Cladribine exerts an immunomodulatory effect on human and murine dendritic cells.

Cladribine is a purine nucleoside analog developed to treat lymphoid malignancies. Reported therapeutic benefits for the autoimmune disease multiple sclerosis indicate additional immunomodulatory effects beyond the well-characterized cytotoxic activity causing lymphopenia. Here, we demonstrate that cladribine reduces the secretion of inflammatory cytokines and chemokines by murine and human dendritic cells, the most potent antigen-presenting cells. This compound also modulates the expression of the activation markers CD86 and MHC II. Furthermore, cladribine affects the T cell priming capacity of dendritic cells, resulting in reduced induction of interferon-γ- and tumor necrosis factor-α-producing T cells and increased induction of interleukin-10-producing T cells. These effects, observed at cladribine concentrations in the therapeutically relevant range of serum steady-state concentrations for leukemia and multiple sclerosis, confirm the immunomodulatory activity of cladribine.

NG2 regulates directional migration of oligodendrocyte precursor cells via Rho GTPases and polarity complex proteins.

The transmembrane proteoglycan NG2 is expressed by oligodendrocyte precursor cells (OPC), which migrate to axons during developmental myelination and remyelinate in the adult after migration to injured sites. Highly invasive glial tumors also express NG2. Despite the fact that NG2 has been implicated in control of OPC migration, its mode of action remains unknown. Here, we show in vitro and in vivo that NG2 controls migration of OPC through the regulation of cell polarity. In stab wounds in adult mice we show that NG2 controls orientation of OPC toward the wound. NG2 stimulates RhoA activity at the cell periphery via the MUPP1/Syx1 signaling pathway, which favors the bipolar shape of migrating OPC and thus directional migration. Upon phosphorylation of Thr-2256, downstream signaling of NG2 switches from RhoA to Rac stimulation. This triggers process outgrowth through regulators of front-rear polarity and we show using a phospho-mimetic form of NG2 that indeed NG2 recruits proteins of the CRB and the PAR polarity complexes to stimulate Rac activity via the GEF Tiam1. Our findings demonstrate that NG2 is a core organizer of Rho GTPase activity and localization in the cell, which controls OPC polarity and directional migration. This work also reveals CRB and PAR polarity complexes as new effectors of NG2 signaling in the establishment of front-rear polarity.

Modulation of dendritic cell properties by laquinimod as a mechanism for modulating multiple sclerosis.

Laquinimod is an orally administered compound that is under investigation in relapsing-remitting multiple sclerosis. To understand the mechanism by which laquinimod exerts its clinical effects, we have performed human and murine studies assessing its immunomodulatory properties. In experimental autoimmune encephalomyelitis, the therapeutic administration of laquinimod beginning during the recovery of SJL mice, prevented further relapses as expected and strongly reduced infiltration of CD4+ and CD8+ T cells in the central nervous system. We hypothesized that this beneficial effect was mediated by dendritic cells, since we and others found a modulation of different dendritic cell subsets under treatment. According to the findings on antigen-presenting cells in the murine system, we found a reduced capacity of human monocyte-derived dendritic cells treated with therapeutic concentrations of laquinimod, upon maturation with lipopolysaccharide, to induce CD4+ T cell proliferation and secretion of pro-inflammatory cytokines. Furthermore, laquinimod treatment of mature dendritic cells resulted in a decreased chemokine production by both murine and human dendritic cells, associated with a decreased monocyte chemo-attraction. In laquinimod-treated patients with multiple sclerosis we consistently found reduced chemokine and cytokine secretion by conventional CD1c+ dendritic cells upon lipopolysaccharide stimulation. Similarly to the animal model of relapsing-remitting multiple sclerosis, dendritic cell subsets were altered in patients upon laquinimod treatment, as the number of conventional CD1c+ and plasmacytoid CD303+ dendritic cells were decreased within peripheral blood mononuclear cells. Moreover, laquinimod treatment in patients with multiple sclerosis and mice modified the maturation of dendritic cells demonstrated by an upregulation of CD86 expression in vivo. Our data suggest that inhibition of the NF-κB pathway is responsible for the changes observed in dendritic cell maturation and functions. These findings indicate that laquinimod exhibits its disease-modulating activity in multiple sclerosis by downregulating immunogenicity of dendritic cell responses. We suggest that monitoring dendritic cell properties in multiple sclerosis should be implemented in future therapeutic trials.

Neuroprotective effects of PACAP against ethanol-induced toxicity in the developing rat cerebellum.

The developing rat cerebellum is particularly sensitive to alcohol at the end of the first postnatal week, a period of intense neurogenesis. The neuropeptide Pituitary adenylate cyclase-activating polypeptide (PACAP) has previously been shown to prevent the death of cultured neurons in vitro. We have thus investigated the capacity of PACAP to counteract ethanol toxicity in 8-day-old rats. Behavioral studies revealed that PACAP reduces the deleterious action of alcohol in the negative geotaxis test. Administration of ethanol induced a transient increase of the expression of pro-apoptotic genes including c-jun or caspase-3 , which could be partially blocked by PACAP. Alcohol inhibited the expression of the α6 GABA ( A ) subunit while PACAP increased neuroD2 mRNA level, two markers of neuronal differentiation. Although gene regulations occurred rapidly, a third injection of ethanol was required to strongly reduce the number of granule cells in the internal granule cell layer, an effect which was totally blocked by PACAP. The action of PACAP was mimicked by D-JNKi1 and Z-VAD-FMK, indicating the involvement of the jun and caspase-3 pathways in alcohol toxicity. The present data demonstrate that PACAP can counteract in vivo the deleterious effect of ethanol. The beneficial action of PACAP on locomotor activity precedes its activity on cell survival, indicating that PACAP can block the detrimental action of ethanol on cell differentiation.

Latent fluorophores based on a self-immolative linker strategy and suitable for protease sensing.

The self-immolative spacer para-aminobenzyl alcohol (PABA) was used as a key component in the design of new protease-sensitive fluorogenic probes whose parent phenol-based fluorophore is released through an enzyme-initiated domino reaction. First, the conjugation of the phenylacetyl moiety to 7-hydroxycoumarin (umbelliferone) and 7-hydroxy-9 H-(9,9-dimethylacridin-2-one) (DAO) by means of the heterobifunctional PABA linker has led to pro-fluorophores 6a and 6d whose enzyme activation by penicillin amidase was demonstrated. The second part of this study was devoted to the extension of this latent fluorophore strategy to the caspase-3 protease, a key mediator of apoptosis in mammalian cells. Fluorogenic caspase-3 substrates 11 and 13 derived from umbelliferone and DAO, respectively, were prepared. It was demonstrated that pro-fluorophore 11 is a sensitive fluorimetric reagent for the detection of this cysteine protease. Furthermore, in vitro assays with fluorogenic probe 13 showed a deleterious effect of biological thiols on fluorescence of the released acridinone fluorophore DAO that, to our knowledge, had not been reported until now.

Inhibitory effect of PACAP on caspase activity in neuronal apoptosis: a better understanding towards therapeutic applications in neurodegenerative diseases.

Programmed cell death, which is part of the normal development of the central nervous system, is also implicated in various neurodegenerative disorders. Cysteine-dependent aspartate-specific proteases (caspases) play a pivotal role in the cascade of events leading to apoptosis. Many factors that inhibit cell death have now been identified, but the underlying mechanisms are not fully understood. Pituitary adenylate cyclase-activating polypeptide (PACAP) has been shown to exert neurotrophic activities during development and to prevent neuronal apoptosis induced by various insults such as ischemia. Most of the neuroprotective effects of PACAP are mediated through the PAC1 receptor. This receptor activates a transduction cascade of second messengers to stimulate Bcl-2 expression, which inhibits cytochrome c release and blocks the activation of caspases. The inhibitory effect of PACAP on the apoptotic cascade suggests that selective, stable, and potent PACAP derivatives could potentially be of therapeutic value for the treatment of post-traumatic and/or chronic neurodegenerative processes.

Molecular, cellular, and functional characterizations of pituitary adenylate cyclase-activating polypeptide and its receptors in the cerebellum of New and Old World monkeys.

The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) exerts trophic activities during cerebellar development, and a neuroprotective effect of PACAP has been demonstrated in pathological conditions such as stroke. However, all these data have been obtained in rodents, and neuroprotective effects of PACAP in primates remain unknown. Because of their evolutionary relationships with humans, monkeys represent powerful models for validating the therapeutic interest in PACAP. The objective of the present study was to characterize PACAP and its receptors in the cerebellum of two nonhuman primates. RT-PCR and in situ hybridization experiments revealed that PACAP is expressed in the cerebellum by Purkinje cells. Via immunohistochemistry, PACAP was detected in Purkinje cells and radial glial fibers. With regard to PACAP receptors, PAC1-R and VPAC1-R were detected by RT-PCR. In situ hybridization revealed a strong expression of PAC1-R and VPAC1-R in the granule cell layer (GCL), and VPAC1-R was also expressed in the Purkinje cell layer. A high density of PACAP binding sites was visualized in the GCL and the Purkinje cell layer. Competition studies indicated that, in the GCL, PACAP induced complete displacement of [(125)I]PACAP27 binding, whereas vasoactive intestinal polypeptide (VIP) was a weak competitor. In contrast, in the Purkinje cell layer, both PACAP and VIP displaced [(125)I]PACAP27 binding. Measurement of cAMP levels showed that PACAP is a powerful activator of adenylyl cyclase, whereas VIP is about 100-fold less potent. Altogether, these observations constitute the first demonstration of a functional PACAPergic system in monkey cerebellum. They strongly suggest that neuroprotective effects of PACAP can be transposed to primates, including human.

Neurotrophic effects of PACAP in the cerebellar cortex.

In the rodent cerebellum, PACAP is expressed by Purkinje neurons and PAC1 receptors are present on granule cells during both the development period and in adulthood. Treatment of granule neurons with PACAP inhibits proliferation, slows migration, promotes survival and induces differentiation. PACAP also protects cerebellar granule cells against the deleterious effects of neurotoxic agents. Most of the neurotrophic effects of PACAP are mediated through the cAMP/PKA signaling pathway and often involve the ERK MAPkinase. Caspase-3 is one of the key enzymes implicated in the neuroprotective action of PACAP but PACAP also inhibits caspase-9 activity and increases Bcl-2 expression. PACAP and functional PAC1 receptors are expressed in the monkey and human cerebellar cortex with a pattern of expression very similar to that described in rodents, suggesting that PACAP could also exert neurodevelopmental and neuroprotective functions in the cerebellum of primates including human.

Mechanisms underlying differential expression of interleukin-8 in breast cancer cells.

We have recently reported that interleukin-8 (IL-8) expression was inversely correlated to estrogen receptor (ER) status and was overexpressed in invasive breast cancer cells. In the present study, we show that IL-8 overexpression in breast cancer cells involves a higher transcriptional activity of IL-8 gene promoter. Cloning of IL-8 promoter from MDA-MB-231 and MCF-7 cells expressing high and low levels of IL-8, respectively, shows the integrity of the promoter in both cell lines. Deletion and site-directed mutagenesis of the promoter demonstrate that NF-kappaB and AP-1 and to a lesser extent C/EBP binding sites play a crucial role in the control of IL-8 promoter activity in MDA-MB-231 cells. Knockdown of NF-kappaB and AP-1 activities by adenovirus-mediated expression of an NF-kappaB super-repressor and RNA interference, respectively, decreased IL-8 expression in MDA-MB-231 cells. On the contrary, restoration of Fra-1, Fra-2, c-Jun, p50, p65, C/EBPalpha and C/EBPbeta expression levels in MCF-7 cells led to a promoter activity comparable to that observed in MDA-MB-231 cells. Our data constitute the first extensive study of IL-8 gene overexpression in breast cancer cells and suggest that the high expression of IL-8 in invasive cancer cells requires a complex cooperation between NF-kappaB, AP-1 and C/EBP transcription factors.