Early Alterations of PACAP and VIP Expression in the Female Rat Brain Following Spinal Cord Injury.

Previous evidence shows that rapid changes occur in the brain following spinal cord injury (SCI). Here, we interrogated the expression of the neuropeptides pituitary adenylyl cyclase-activating peptide (PACAP), vasoactive intestinal peptides (VIP), and their binding receptors in the rat brain 24 h following SCI. Female Sprague-Dawley rats underwent thoracic laminectomy; half of the rats received a mild contusion injury at the level of the T10 vertebrate (SCI group); the other half underwent sham surgery (sham group). Twenty-four hours post-surgery, the hypothalamus, thalamus, amygdala, hippocampus (dorsal and ventral), prefrontal cortex, and periaqueductal gray were collected. PACAP, VIP, PAC1, VPAC1, and VPAC2 mRNA and protein levels were measured by real-time quantitative polymerase chain reaction and Western blot. In SCI rats, PACAP expression was increased in the hypothalamus (104-141% vs sham) and amygdala (138-350%), but downregulated in the thalamus (35-95%) and periaqueductal gray (58-68%). VIP expression was increased only in the thalamus (175-385%), with a reduction in the amygdala (51-68%), hippocampus (40-75%), and periaqueductal gray (74-76%). The expression of the PAC1 receptor was the least disturbed by SCI, with decrease expression in the ventral hippocampus (63-68%) only. The expression levels of VPAC1 and VPAC2 receptors were globally reduced, with more prominent reductions of VPAC1 vs VPAC2 in the amygdala (21-70%) and ventral hippocampus (72-75%). In addition, VPAC1 downregulation also extended to the dorsal hippocampus (69-70%). These findings demonstrate that as early as 24 h post-SCI, there are region-specific disruptions of PACAP, VIP, and related receptor transcript and protein levels in supraspinal regions controlling higher cognitive functions.

Altered Hippocampal and Striatal Expression of Endothelial Markers and VIP/PACAP Neuropeptides in a Mouse Model of Systemic Lupus Erythematosus.

Neuropsychiatric systemic lupus erythematosus (NPSLE) is one of the most common and severe manifestations of lupus; however, its pathogenesis is still poorly understood. While there is sparse evidence suggesting that the ongoing autoimmunity may trigger pathogenic changes to the central nervous system (CNS) microvasculature, culminating in inflammatory/ischemic damage, further evidence is still needed. In this study, we used the spontaneous mouse model of SLE (NZBWF1 mice) to investigate the expression of genes and proteins associated with endothelial (dys)function: tissue and urokinase plasminogen activators (tPA and uPA), intercellular and vascular adhesion molecules 1 (ICAM-1 and VCAM-1), brain derived neurotrophic factor (BDNF), endothelial nitric oxide synthase (eNOS) and Krüppel-like factor 4 (KLF4) and neuroprotection/immune modulation: pituitary adenylate cyclase-activating peptide (PACAP), vasoactive intestinal peptide (VIP), PACAP receptor (PAC1), VIP receptors 1 and 2 (VPAC1 and VPAC2). Analyses were carried out both in the hippocampus and striatum of SLE mice of two different age groups (2 and 7 months old), since age correlates with disease severity. In the hippocampus, we identified a gene/protein expression profile indicative of mild endothelial dysfunction, which increased in severity in aged SLE mice. These alterations were paralleled by moderate alterations in the expression of VIP, PACAP and related receptors. In contrast, we report a robust upregulation of endothelial activation markers in the striatum of both young and aged mice, concurrent with significant induction of the VIP/PACAP system. These data identify molecular signatures of endothelial alterations in the hippocampus and striatum of NZBWF1 mice, which are accompanied by a heightened expression of endogenous protective/immune-modulatory neuropeptides. Collectively, our results support the idea that NPSLE may cause alterations of the CNS micro-vascular compartment that cannot be effectively counteracted by the endogenous activity of the neuropeptides PACAP and VIP.

Identification of Dysregulated microRNA Networks in Schwann Cell-Like Cultures Exposed to Immune Challenge: Potential Crosstalk with the Protective VIP/PACAP Neuropeptide System.

Following peripheral nerve injury, dysregulations of certain non-coding microRNAs (miRNAs) occur in Schwann cells. Whether these alterations are the result of local inflammation and/or correlate with perturbations in the expression profile of the protective vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase-activating polypeptide (PACAP) system is currently unknown. To address these issues, we aimed at profiling the expression of selected miRNAs in the rat RT4 Schwann cell line. Cells exposed to lipopolysaccharide (LPS), to mimic the local inflammatory milieu, were appraised by real-time qPCR, Western blot and ELISAs. We found that upon LPS treatment, levels of pro-inflammatory cytokines (IL-1ß, -6, -18, -17A, MCP-1 and TNFα) increased in a time-dependent manner. Unexpectedly, the expression levels of VIP and PACAP were also increased. Conversely, levels of VPAC1 and VPAC2 receptors were reduced. Downregulated miRNAs included miR-181b, -145, -27a, -340 and -132 whereas upregulated ones were miR-21, -206, -146a, -34a, -155, -204 and -29a, respectively. Regression analyses revealed that a subset of the identified miRNAs inversely correlated with the expression of VPAC1 and VPAC2 receptors. In conclusion, these findings identified a novel subset of miRNAs that are dysregulated by immune challenge whose activities might elicit a regulatory function on the VIP/PACAP system.

Tackling dipeptidyl peptidase IV in neurological disorders.

Dipeptidyl peptidase IV (DPP-IV) is a serine protease best known for its role in inactivating glucagon-like peptide-1 (GLP-1), pituitary adenylate cyclase-activating polypeptide (PACAP) and glucose-dependent insulinotropic peptide (GIP), three stimulators of pancreatic insulin secretion with beneficial effects on glucose disposal. Owing to the relationship between DPP-IV and these peptides, inhibition of DPP-IV enzyme activity is considered as an attractive treatment option for diabetic patients. Nonetheless, increasing studies support the idea that DPP-IV might also be involved in the development of neurological disorders with a neuroinflammatory component, potentially through its non-incretin activities on immune cells. In this review article, we aim at highlighting recent literature describing the therapeutic value of DPP-IV inhibitors for the treatment of such neurological conditions. Finally, we will illustrate some of the promising results obtained using berberine, a plant extract with potent inhibitory activity on DPP-IV.

Mesenchymal stem cells-based therapy as a potential treatment in neurodegenerative disorders: is the escape from senescence an answer?

Aging is the most prominent risk factor contributing to the development of neurodegenerative disorders. In the United States, over 35 million of elderly people suffer from age-related diseases. Aging impairs the self-repair ability of neuronal cells, which undergo progressive deterioration. Once initiated, this process hampers the already limited regenerative power of the central nervous system, making the search for new therapeutic strategies particularly difficult in elderly affected patients. So far, mesenchymal stem cells have proven to be a viable option to ameliorate certain aspects of neurodegeneration, as they possess high proliferative rate and differentiate in vitro into multiple lineages. However, accumulating data have demonstrated that during long-term culture, mesenchymal stem cells undergo spontaneous transformation. Transformed mesenchymal stem cells show typical features of senescence, including the progressive shortening of telomers, which results in cell loss and, as a consequence, hampered regenerative potential. These evidences, in line with those observed in mesenchymal stem cells isolated from old donors, suggest that senescence may represent a limit to mesenchymal stem cells exploitation in therapy, prompting scholars to either find alternative sources of pluripotent cells or to arrest the age-related transformation. In the present review, we summarize findings from recent literature, and critically discuss some of the major hurdles encountered in the search of appropriate sources of mesenchymal stem cells, as well as benefits arising from their use in neurodegenerative diseases. Finally, we provide some insights that may aid in the development of strategies to arrest or, at least, delay the aging of mesenchymal stem cells to improve their therapeutic potential.

Emerging Role of PACAP as a New Potential Therapeutic Target in Major Diabetes Complications.

Enduring diabetes increases the probability of developing secondary damage to numerous systems, and these complications represent a cause of morbidity and mortality. Establishing the causes of diabetes remains the key step to eradicate the disease, but prevention as well as finding therapies to ameliorate some of the major diabetic complications is an equally important step to increase life expectancy and quality for the millions of individuals already affected by the disease or who are likely to develop it before cures become routinely available. In this review, we will firstly summarize some of the major complications of diabetes, including endothelial and pancreatic islets dysfunction, retinopathy, and nephropathy, and then discuss the emerging roles exerted by the neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) to counteract these ranges of pathologies that are precipitated by the prolonged hyperglycemic state. Finally, we will describe the main signalling routes activated by the peptide and propose possible future directions to focus on developing more effective peptide-based therapies to treat the major complications associated with longstanding diabetes.

Characterization of matrix metalloproteinase-2 and -9, ADAM-10 and N-cadherin expression in human glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most common and aggressive malignant primary brain tumor in humans, whose invasiveness and proliferation are associated with poor prognosis. Matrix metalloproteinases (MMPs) and the related family of "a disintegrin and metalloproteinase" (ADAM) both contribute to increase cell invasion, and its substrate N-cadherin is involved in proliferation and metastatic capacities of tumor cells. However, these molecular determinants of aggressiveness have not been adequately characterized in GBM. In an attempt to better define these pathogenetic signatures, in the present study we evaluated the comparative expression of two main MMPs (MMP-2 and -9), as well as of ADAM-10 and N-cadherin in surgical samples from patients diagnosed with WHO grade IV GBM (n = 25) and in cortical tissue specimens obtained from untreatable epileptic patients (controls, n = 8) through a series of histopathological, immunohistochemical and biochemical tests. Our studies revealed that both MMP-2 and -9 immunoreactivities (IRs) were upregulated in 13 of 25 (52 %) and 19 of 25 (76 %) GBMs, respectively, and the extent of the increase was highly significant with respect to controls (p < 0.001). ADAM-10 IR was also found to be increased (p < 0.001) in 16 of 25 GBM specimens (64 %). Conversely, N-cadherin IR was remarkably decreased (p < 0.001) in almost the totality of tumor samples (22 of 25, 88 %). A similar trend was also obtained at the mRNA and protein level by qPCR and western blot analyses, respectively. Collectively, the current study provides a comprehensive molecular portrayal of some of the major pathological hallmarks of GBM aggressiveness, which could be exploitable as potential targets for a new therapeutic approach.

Ameliorative effects of PACAP against cartilage degeneration. Morphological, immunohistochemical and biochemical evidence from in vivo and in vitro models of rat osteoarthritis.

Osteoarthritis (OA); the most common form of degenerative joint disease, is associated with variations in pro-inflammatory growth factor levels, inflammation and hypocellularity resulting from chondrocyte apoptosis. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide endowed with a range of trophic effects in several cell types; including chondrocytes. However; its role in OA has not been studied. To address this issue, we investigated whether PACAP expression is affected in OA cartilage obtained from experimentally-induced OA rat models, and then studied the effects of PACAP in isolated chondrocytes exposed to IL-1ß in vitro to mimic the inflammatory milieu of OA cartilage. OA induction was established by histomorphometric and histochemical analyses. Changes in PACAP distribution in cartilage, or its concentration in synovial fluid (SF), were assessed by immunohistochemistry and ELISA. Results showed that PACAP abundance in cartilage tissue and SF was high in healthy controls. OA induction decreased PACAP levels both in affected cartilage and SF. In vitro, PACAP prevented IL-1ß-induced chondrocyte apoptosis, as determined by MTT assay; Hoechst staining and western blots of apoptotic-related proteins. These changes were also accompanied by decreased i-NOS and COX-2 levels, suggesting an anti-inflammatory effect. Altogether, these findings support a potential role for PACAP as a chondroprotective agent for the treatment of OA.

PACAP interacts with PAC1 receptors to induce tissue plasminogen activator (tPA) expression and activity in schwann cell-like cultures.

Regeneration of peripheral nerves depends on the abilities of rejuvenating axons to migrate at the injury site through cellular debris and altered extracellular matrix, and then grow along the residual distal nerve sheath conduit and reinnervate synaptic targets. Considerable evidence suggest that glial cells participate in this process, although the mechanisms remain to be clarified. In cell culture, regenerating neurites secrete PACAP, a peptide shown to induce the expression of the protease tissue plasminogen activator (tPA) in neural cell types. In the present studies, we tested the hypothesis that PACAP can stimulate peripheral glial cells to produce tPA. More specifically, we addressed whether or not PACAP promoted the expression and activity of tPA in the Schwann cell line RT4-D6P2T, which shares biochemical and physical properties with Schwann cells. We found that PACAP dose- and time-dependently stimulated tPA expression both at the mRNA and protein level. Such effect was mimicked by maxadilan, a potent PAC1 receptor agonist, but not by the PACAP-related homolog VIP, suggesting a PAC1-mediated function. These actions appeared to be mediated at least in part by the Akt/CREB signaling cascade because wortmannin, a PI3K inhibitor, prevented peptide-driven CREB phosphorylation and tPA increase. Interestingly, treatment with BDNF mimicked PACAP actions on tPA, but acted through both the Akt and MAPK signaling pathways, while causing a robust increase in PACAP and PAC1 expression. PACAP6-38 totally blocked PACAP-driven tPA expression and in part hampered BDNF-mediated effects. We conclude that PACAP, acting through PAC1 receptors, stimulates tPA expression and activity in a Akt/CREB-dependent manner to promote proteolytic activity in Schwann-cell like cultures.

Distribution of p53, GST, and MTHFR polymorphisms and risk of cervical intraepithelial lesions in sicily.

OBJECTIVES: Host factors, including genetic polymorphisms, may explain some of the individual differences in cervical cancer occurrence, and susceptibility information may be useful to address effective and specific preventive strategies for different countries. The purpose of the present study was to investigate the role of p53 codon 72, glutathione S-transferase class mu (GSTM1), glutathione S-transferase class theta (GSTT1), and methylenetetrahydrofolate reductase (MTHFR) C677T polymorphisms on the risk for infection and/or of cervical intraepithelial lesions in women attending a colposcopy service in Catania, Sicily, with an already reported high prevalence of human papillomavirus. METHODS: To identify the association among individual genetic polymorphisms, human papillomavirus infection, and histological findings, a case-control study was designed. Furthermore, to assess the combined effects of these polymorphisms on cervical cancer risk, combined genotype frequencies were compared among case patients and controls. RESULTS: Women homozygous for the p53 codon 72 Arg genotype were at a 5.6-fold higher risk for developing cervical intraepithelial neoplasia (CIN) 2 or 3 compared with those showing homozygosity for the Pro genotype or heterozygosity for the Pro/Arg genotype. The GSTM1 and GSTT1 null genotypes were overrepresented in infected patients and in women with CIN 2 or 3, although without any significant associations. A decreased risk for CIN of individuals homozygous for the MTHFR T allele was shown. CONCLUSIONS: After multiple logistic analyses, the presence of the allele 677T of the MTHFR gene was the best explaining protective factor against cervical carcinogenesis, and the allelic distribution in the control group followed the Hardy-Weinberg equilibrium expectations. However, the findings of our study still remain to be confirmed by additional and larger population-based surveys.