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ABSTRACT: The retinal expression and distribution of pituitary adenylate cyclase-activating peptide (PACAP) and vasoactive intestinal peptide (VIP) and their receptors was investigated in early streptozotocin (STZ)-induced diabetic rats. Diabetes was induced in rats by STZ injection (60 mg/kg i.p.). PACAP, VIP and their receptors in nondiabetic control and diabetic retinas were assayed by quantitative real-time PCR and Western blot 1 and 3 weeks after STZ injection. Effects of intravitreal treatment with PACAP38 on the expression of the two apoptotic-related genes Bcl-2 and p53 were also evaluated. PACAP and VIP, as well as VPAC1 and VPAC2 receptors, but not PAC1 mRNA levels, were transiently induced in retinas 1 week following STZ. These findings were confirmed by immunoblot analyses. Three weeks after the induction of diabetes, significant decreases in the expression of peptides and their receptors were observed, Bcl-2 expression decreased and p53 expression increased. Intravitreal injection of PACAP38 restored STZ-induced changes in retinal Bcl-2 and p53 expression to nondiabetic levels. The initial upregulation of PACAP, VIP and related receptors and the subsequent downregulation in retina of diabetic rats along with the protective effects of PACAP38 treatment, suggest a role for both peptides in the pathogenesis of diabetic retinopathy.
Peptides 06/2012; 37(1):32-9. · 2.43 Impact Factor
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ABSTRACT: Recently, it has been proposed that neurofibromin (NF1) forms a binding complex with amyloid precursor protein (APP) that
interacts with the dopamine D3 receptor (D3R). In the present study we investigated whether the absence of the D3R is correlated to modifications in the expression of both NF1 and APP. Quantitative real-time PCR analyses of both transcripts
showed that NF1 mRNA levels were significantly reduced whereas APP levels were strikingly increased in D3R knock-out (D3R KO) as compared to wild type (WT) mice brains. Western blot analyses using mice whole brains produced comparable results
with those obtained by mRNA measurements. Moreover, immunohistochemical analyses revealed a similar brain regional distribution
of APP protein in the hippocampus, in the cerebral and cerebellar cortex of D3R KO mice. Conversely, hippocampal NF1 immunoreactivity did not seem to be affected by the absence of D3Rs. Further analyses confirmed that regional NF1 protein expression in the hippocampus was not affected by the absence of
the D3R, whereas APP levels were still increased in this specific brain region. In conclusion, these results show the existence
of a correlation among the D3R, NF1 and APP in mice brains and thus show the regional-specific regulation of NF1 in brains of D3R KO, which may contribute to gain insights into the comprehension of novel underlying mechanisms that regulate brain function.
KeywordsNeurofibromin–Amyloid precursor protein–Dopamine D3 receptor
Neurochemical Research 05/2012; 36(3):426-434. · 2.24 Impact Factor
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ABSTRACT: Amyloid ß peptide (Aß), generated by proteolytic cleavage of the amyloid precursor protein (APP), plays a pivotal role in
the pathogenesis of Alzheimer's disease (AD). The key step in the generation of Aß is cleavage of APP by ß-secretases (beta-site
APP-cleaving enzyme 1 (BACE1) and BACE2). There has been suggestion of interaction between aluminum and several AD-associated
pathways. However, the underlying mechanisms still remain unclear. Here, we report the effects of aluminum chloride (AlCl3) in Aß-induced toxicity using differentiated neuronal SH-SY5Y cells. The metal significantly enhances Aß-induced cell death
at concentrations ranging from 50 to 300µM after 24 and 48h. After 72 and 96 h treatment, cell death is increased already
at 10µM. Early coexposure of cells to 10µM AlCl3 and 2µM Aß differentially affected ß-secretase mRNA levels as compared to single Aß treatment after 1 and 3h. BACE1 levels
were slightly reduced after 1h and significantly increased after 3 h exposure, whereas BACE2 levels were increased at both
times considered. Both genes’ mRNA levels were downregulated at longer times (6, 12, and 24h). Although these results indicate
that aluminum toxicity is correlated to changes in both BACE1 and BACE2 expression levels, the subsequent common downregulation
observed suggests that aluminum involvement in the Aß cascade is subtle, and other underlying mechanisms might be involved.
KeywordsAluminum chloride-Alzheimer’s disease-BACE1-BACE2-Beta-amyloid
Cell Biology and Toxicology 04/2012; 26(4):367-377. · 2.51 Impact Factor
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ABSTRACT: Malignant peripheral nerve sheath tumors (MPNSTs) are sarcomas able to grow under conditions of metabolic stress caused by insufficient nutrients or oxygen. Both pituitary adenylate cyclase-activating polypeptide (PACAP) and activity-dependent neuroprotective protein (ADNP) have glioprotective potential. However, whether PACAP/ADNP signaling is involved in the resistance to cell death in MPNST cells remains to be clarified. Here, we investigated the involvement of this signaling system in the survival response of MPNST cells against hydrogen peroxide (H(2)O(2))-evoked death both in the presence of normal serum (NS) and in serum-starved (SS) cells. Results showed that ADNP levels increased time-dependently (6-48 h) in SS cells. Treatment with PACAP38 (10(-9) to 10(-5) M) dose-dependently increased ADNP levels in NS but not in SS cells. PAC(1)/VPAC receptor antagonists completely suppressed PACAP-stimulated ADNP increase and partially reduced ADNP expression in SS cells. NS-cultured cells exposed to H(2)O(2) showed significantly reduced cell viability (~50 %), increased p53 and caspase-3, and DNA fragmentation, without affecting ADNP expression. Serum starvation significantly reduced H(2)O(2)-induced detrimental effects in MPNST cells, which were not further ameliorated by PACAP38. Altogether, these finding provide evidence for the involvement of an endogenous PACAP-mediated ADNP signaling system that increases MPNST cell resistance to H(2)O(2)-induced death upon serum starvation.
Journal of Molecular Neuroscience 03/2012; 48(3):674-83. · 2.50 Impact Factor
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ABSTRACT: Hyperglycemia is implicated both in micro- and macro-vascular complications in diabetes mellitus. Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are two known nonclassic regulators of angiogenesis, although their biological role on endothelial cell proliferation remains poorly defined. In the present study we hypothesized that either peptides might play an inhibitory role on hyperglycemia-induced cell growth. To this end, we investigated the effect of both PACAP and VIP on cell proliferation in murine microvascular endothelial cells (H5V) cultured both under euglycemic and hyperglycemic conditions (5 and 25 mM glucose, respectively) for 24, 48 h, 7 and 15 days. Results demonstrated that high glucose treatment induced a time-dependent increase in cell viability after 48 h (p<0.05), which was much more evident after 7 and 15 days (p<0.001). Similar effects were observed in cell proliferation, although significant changes were obtained after prolonged exposures to high glucose (7 and 15 days; p<0.001). The proliferative response to the glucose-enriched environment was correlated to changes in the expression of PAC1 and, to a minor extent, to VPAC2, but not VPAC1 receptors, as measured by quantitative real-time PCR. These results were further confirmed by Western blot and immunofluorescence analyses. Interestingly, 10⁻⁷ M PACAP or VIP treatment significantly attenuated hyperglycemia-induced increase in cell viability and proliferation after 7 and 15 days. Taken together, our findings demonstrate that both PACAP and VIP peptides exert an inhibitory activity on hyperglycemia-induced endothelial cell proliferation, thus suggesting that the effect might be mediated by PAC1 and VPAC2 receptors.
Peptides 12/2010; 31(12):2276-83. · 2.43 Impact Factor
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ABSTRACT: Recently, it has been proposed that neurofi-bromin (NF1) forms a binding complex with amyloid precursor protein (APP) that interacts with the dopamine D 3 receptor (D 3 R). In the present study we investigated whether the absence of the D 3 R is correlated to modifica-tions in the expression of both NF1 and APP. Quantitative real-time PCR analyses of both transcripts showed that NF1 mRNA levels were significantly reduced whereas APP levels were strikingly increased in D 3 R knock-out (D 3 R KO) as compared to wild type (WT) mice brains. Western blot analyses using mice whole brains produced compara-ble results with those obtained by mRNA measurements. Moreover, immunohistochemical analyses revealed a sim-ilar brain regional distribution of APP protein in the hip-pocampus, in the cerebral and cerebellar cortex of D 3 R KO mice. Conversely, hippocampal NF1 immunoreactivity did not seem to be affected by the absence of D 3 Rs. Further analyses confirmed that regional NF1 protein expression in the hippocampus was not affected by the absence of the D 3 R, whereas APP levels were still increased in this spe-cific brain region. In conclusion, these results show the existence of a correlation among the D 3 R, NF1 and APP in mice brains and thus show the regional-specific regulation of NF1 in brains of D 3 R KO, which may contribute to gain insights into the comprehension of novel underlying mechanisms that regulate brain function.
Neurochemical Research 11/2010; · 2.24 Impact Factor
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ABSTRACT: Amyloid beta peptide (Abeta), generated by proteolytic cleavage of the amyloid precursor protein (APP), plays a pivotal role in the pathogenesis of Alzheimer's disease (AD). The key step in the generation of Abeta is cleavage of APP by beta-secretases (beta-site APP-cleaving enzyme 1 (BACE1) and BACE2). There has been suggestion of interaction between aluminum and several AD-associated pathways. However, the underlying mechanisms still remain unclear. Here, we report the effects of aluminum chloride (AlCl(3)) in Abeta-induced toxicity using differentiated neuronal SH-SY5Y cells. The metal significantly enhances Abeta-induced cell death at concentrations ranging from 50 to 300 microM after 24 and 48 h. After 72 and 96 h treatment, cell death is increased already at 10 microM. Early coexposure of cells to 10 microM AlCl(3) and 2 microM Abeta differentially affected beta-secretase mRNA levels as compared to single Abeta treatment after 1 and 3 h. BACE1 levels were slightly reduced after 1 h and significantly increased after 3 h exposure, whereas BACE2 levels were increased at both times considered. Both genes' mRNA levels were downregulated at longer times (6, 12, and 24 h). Although these results indicate that aluminum toxicity is correlated to changes in both BACE1 and BACE2 expression levels, the subsequent common downregulation observed suggests that aluminum involvement in the Abeta cascade is subtle, and other underlying mechanisms might be involved.
Cell Biology and Toxicology 08/2010; 26(4):367-77. · 2.51 Impact Factor
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ABSTRACT: In our previous study we have identified PACAP, VIP and their receptors in rat malignant peripheral nerve sheath tumor (MPNST) cells, thus showing anti-apoptotic roles. Recently it has been shown that the tumor suppressor neurofibromin, encoded by the Neurofibromatosis type I (NF1) gene, promotes MPNST cells sensitivity to apoptosis after serum withdrawal. In the present study we investigated whether PACAP or VIP negatively regulate NF1 expression under normal or serum-dependent pro-apoptotic culture conditions. Results indicated that serum itself significantly influenced gene and protein levels. In fact, the low NF1 levels of cells cultured in normal serum-containing medium were remarkably increased in cells switched to low- or no-serum after 24h and 48 h. Treatment with 100 nM PACAP or VIP did not affect NF1 expression when using normal amounts of serum, whereas it significantly inhibited transcript and protein levels both in low- or no-serum cultured cells. In particular, PACAP reduced NF1 levels already after 24h in low-serum cultured cells, while VIP showed a similar effect only after serum deprivation. However, both PACAP and VIP downregulated gene and protein levels within 48 h either in low-dose and serum-starved cells. Results were confirmed by fluorescence microscopy, showing that 100 nM PACAP or VIP attenuated neurofibromin cytoplasmic localization only in low- or no-serum cultured cells. The present study provides a comprehensive analysis of both neuropeptides effect on NF1 expression in normal, low- or serum-starved MPNST cells, ameliorating the hypothesis that resistance to apoptosis in serum-deprived cells might be correlated to PACAP-/VIP-induced NF1 inhibition.
Neuropeptides 11/2009; 44(1):45-51. · 1.55 Impact Factor
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ABSTRACT: Pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are structurally endogenous peptides showing rich profile of biological activities. These peptides bind specific membrane receptors belonging to the superfamily of G protein-coupled receptors, the PAC1 and VPAC type receptors. Although these receptors have been identified in oligodendrocytes progenitors cells, to date the effects of PACAP and VIP in Schwann cells are still unknown. In the present study we investigated the expression of these neuropeptides as well as their receptors in a schwannoma cell line. RT-PCR and western blot analysis demonstrated that both PAC1 and VPAC2 receptors, but also PACAP peptide were expressed. To study the physiological effects mediated by PAC1/VPAC receptors, we evaluated their role in preventing apoptotic cell death induced by serum deprivation. Treatment with 100 nM PACAP38 and 100 nM VIP increased survival of serum-deprived schwannoma cells. Anti-apoptotic effects of these peptides were correlated to changes in BCL2 and BAX gene expression. Our results suggested that both PACAP38 and VIP could act as trophic factors in Schwann cells.
Brain research 10/2008; 1241:29-35. · 2.46 Impact Factor
