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ABSTRACT: Passive avoidance (PA) conditioning is a fear motivated task able to initiate a cascade of altered gene expression within the hippocampus, a structure critical to learning and memory. We have previously shown that neurofibromin (NF1) and amyloid precursor protein (APP), two genes implicated in cognitive function, are differentially expressed in brain of dopamine D3 receptor knock-out mice (D(3)R(-/-)), suggesting that the receptor might have a role in their trascriptional regulation. Here in this study, we hypothesized that during acquisition of PA conditioning the expression of NF1 and APP genes could be influenced by D(3)Rs. To address this issue, we analyzed the expression of NF1 and APP in the hippocampus of both wild-type (WT) and D(3)R(-/-) mice subjected to the single trial step-through PA paradigm. Our finding demonstrated that (1) D(3)R(-/-) mice exhibit increased cognitive performance as compared to WT mice in the step-through PA trial; (2) acquisition of PA increased D(3)R and NF1, but not APP expression in WT mice hippocampus; (3) PA-driven NF1 induction in WT was abrogated in D(3)R(-/-) mice and finally that (4) the heightened basal APP expression observed in naive D(3)R(-/-) mice was totally reversed by acquisition of PA. In conclusion, the present finding show for the first time that both D(3)R and NF1 genes are upregulated following PA conditioning and suggest that hippocampal D(3)Rs might be relevant to NF1 transcriptional regulation in the hippocampus.
Neurochemical Research 12/2012; · 2.24 Impact Factor
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ABSTRACT: Breakdown of outer blood retinal barrier (BRB) due to the disruption of tight junctions (TJs) is one of the main factors accounting for diabetic macular edema (DME), a major complication of diabetic retinopathy. Previously it has been shown that PACAP and VIP are protective against several types of retinal injuries. However, their involvement in the maintenance of outer BRB function during DME remains uncovered. Here, using an in vitro model of DME, we explored the effects of both PACAP and VIP. Human retinal pigment epithelial cells (ARPE19) were cultured for 26 days either in normal glucose (5.5mM, NG) or in high glucose (25mM, HG). In addition, to mimic the inflammatory aspect of the diabetic milieu, cells were also treated with IL-1β (NG+IL-1β and HG+IL-1β). Effects of PACAP or VIP on cells permeability were evaluated by measuring both apical-to-basolateral movements of fluorescein isothyocyanate (FITC) dextran and transepithelial electrical resistance (TEER). Expression of TJ-related proteins were evaluated by immunoblot. Results demonstrated that NG+IL-1β and, to a greater extent, HG+IL-1β significantly increased FITC-dextran diffusion, paralleled by decreased TEER. PACAP or VIP reversed both of these effects. Furthermore, HG+IL-1β-induced reduction of claudin-1 and ZO-1 expression was reversed by PACAP and VIP. Occludin expression was not affected in any of the conditions tested. Altogether, these finding show that both peptides counteract HG+IL-1β-induced damage in ARPE19 cells, suggesting that they might be relevant to the maintenance of outer BRB function in DME.
Peptides 12/2012; · 2.43 Impact Factor
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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: Patients affected by autosomic recessive juvenile parkinsonism (ARJP) exhibit parkin gene mutations with brain decrease in
dopamine D2/D3 binding sites. To date, there are no data indicating whether the reduction in dopamine D3 receptors (DRD3)
may be associated with the expression of specific parkin variants. In the present study we investigated parkin expression
profile in DRD3 knock-out mice brains. RT-PCR analysis was performed to assess qualitative changes in parkin isoforms’ distribution
pattern and in exons’ expression both in wild type controls and dopamine D3 receptor’s knock-out mice. Real-time PCR was performed
to quantify single exons mRNA. Results demonstrated that exons 1, 2, 4, 6, 7, 8, were more expressed in wild type compared
to dopamine D3 receptor KO mice brains while some other (3, 9, 10) were lower expressed. The expression levels of exons 5,
11 and 12 did not change in both animal groups. Our analysis was confirmed by western blot, which showed that parkin protein
levels were influenced by the absence of DRD3.
Neurochemical Research 04/2012; 34(2):327-332. · 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: The aim of the present study was to investigate the role of D₃ receptor on intraocular pressure regulation using WT and KO D₃R⁻/⁻ mice. Both mice were used with normal eye pressure or steroid-induced ocular hypertension. As measured by tonometry, the topical application of 7-OH-DPAT, a dopamine D₃-preferring receptor agonist, significantly decreased, in a dose-dependent manner, the intraocular pressure in WT mice both in an ocular normotensive group and an ocular hypertensive group. Pretreatment with U-99194A, a D₃ receptor antagonist, reverted 7-OH-DPAT induced ocular hypotension in WT mice. No change of intraocular pressure was observed after topical application of 7-OH-DPAT in KO D₃R⁻/⁻ mice. PCR analysis demonstrated the presence of all dopamine receptor genes in eye tissues obtained from WT mice, and the lack of D₃R mRNAs in KO mice. The present study identified the D₃R subtype as the most important receptor of the dopaminergic system to modulate intraocular pressure with relevant implications for glaucoma that represents one of the most crippling optic neuropathies.
Biochemical pharmacology 12/2011; · 4.25 Impact Factor
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ABSTRACT: We studied patients with degenerative disc disease (DDD) to demonstrate that i) remodeling of the extracellular matrix (ECM) in the intervertebral disc (IVD), particularly the elastic fiber system, of subjects with herniated discs is dysregulated and that ii) it is accompanied by accelerated elastin degradation due to increased expression of matrix metalloprotease-9 (MMP-9). Moreover we wanted to obtain a deeper insight into the pathogenesis of DDD through the study of ECM calcification, DNA fragmentation using TUNEL analysis, BAX, bcl-2 and vimentin immunopositive cells. We studied herniated discs from patients of three age groups (group 1=30-40 years; group 2=40-50 years; and group 3=50-65 years) to evaluate the oxytalan fiber systemMMP-9, apoptosis and vimentin immunopositive cells. The results demonstrated the presence of oxytalan fibers in the annulus fibrosus (AF) and the nucleus pulposus (NP) of herniated discs. In the AF oxytalan fibers replaced disrupted mature elastic fibers in calcified areas, while in the NP they were mostly found in nests at the periphery of chondrocytes. MMP-9 was prevalently observed in NP nests above all in group 1 and group 3 discs while group 2 exhibited a lower MMP-9 immunostaining. Activation of the apoptotic process was demonstrated by upregulated BAX expression in group 3. BAX immunopositivity was inversely mirrored by a significant decrease in bcl-2 expression. Intermediate filament protein vimentin was strongly expressed only in group 1 samples. A large number of apoptotic TUNEL+ cells was observed in group 3 specimens. The presence of oxytalan fibers may be the result of a process of incomplete elastogenesis, or a response to mechanical stress trying to functionally replace the lack of elastic fibers. MMP-9 expression seems to relate to disc damage, while chondrocyte BAX upregulation and TUNEL+ cell staining revealed apoptosis activation regardless of patient age. Vimentin immunopositivity was clearly detected in group 1 annulus fibrosus and nucleus pulposus cells. In conclusion, as demonstrated by the vimentin-positive cells, the injured IVD has endogenous resources that can stem the DDD damage, including substitution of damaged elastic fibers by oxytalan fibers. In addition, induction of apoptosis suggests an increased cell turnover in response to repair needs.
Annals of anatomy = Anatomischer Anzeiger: official organ of the Anatomische Gesellschaft 01/2011; 193(2):156-62. · 0.88 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
