In response to peripheral nerve injury, Schwann cells adopt a migratory phenotype and modify the extracellular matrix to make it permissive for cell migration and axonal re-growth. Uridine 5'-triphosphate (UTP) and other nucleotides are released during nerve injury and activate purinergic receptors expressed on the Schwann cell surface, but little is known about the involvement of purine signalling in wound healing. We studied the effect of UTP on Schwannoma cell migration and wound closure and the intracellular signaling pathways involved. We found that UTP treatment induced Schwannoma cell migration through activation of P2Y2 receptors and through the increase of extracellular matrix metalloproteinase-2 (MMP-2) activation and expression. Knockdown P2Y2 receptor or MMP-2 expression greatly reduced wound closure and MMP-2 activation induced by UTP. MMP-2 activation evoked by injury or UTP was also mediated by phosphorylation of all 3 major mitogen-activated protein kinases (MAPKs): JNK, ERK1/2, and p38. Inhibition of these MAPK pathways decreased both MMP-2 activation and cell migration. Interestingly, MAPK phosphorylation evoked by UTP exhibited a biphasic pattern, with an early transient phosphorylation 5 min after treatment, and a late and sustained phosphorylation that appeared at 6 h and lasted up to 24 h. Inhibition of MMP-2 activity selectively blocked the late, but not the transient, phase of MAPK activation. These results suggest that MMP-2 activation and late MAPK phosphorylation are part of a positive feedback mechanism to maintain the migratory phenotype for wound healing. In conclusion, our findings show that treatment with UTP stimulates in vitro Schwannoma cell migration and wound repair through a MMP-2-dependent mechanism via P2Y2 receptors and MAPK pathway activation.
Amyloid beta (A?) aggregation and deposition is a key pathological hallmark of AD. Growing evidence suggests that neurotoxicity of this peptide is related to the formation of toxic oligomeric aggregates. Therefore, a deeply investigated therapeutic strategy comes at present from blocking the formation of these species to non-toxic aggregates. Among other considered strategies, the multi-target approach has been proposed as a more suitable potential therapy, precisely due to the multifactorial nature of AD. In this context, we recently identified ASS234, a novel compound that possesses a significant multipotent profile since it is able to inhibit cholinesterase and monoamine oxidase enzymes as well as to interfere in A? aggregation process. In this work, we investigated more in detail the effects of ASS234 on A? aggregation and toxicity in vitro as well as we explored its ability to penetrate to the CNS. We report that ASS234 inhibited A?1-42 self-aggregation more efficiently than that of A?1-40, limiting the formation of fibrillar and oligomeric species. Additionally, ASS234 completely blocked the aggregation mediated by AChE of both A?1-42 and A?1-40, showing a dual binding site to AChE. Interestingly, ASS234 significantly reduced A?1-42-mediated toxicity in SH-SY5Y human neuroblastoma cells through the prevention of the mitochondrial apoptosis pathway activation. Also importantly, we observed a significant ability of ASS234 to capture free-radical species in vitro as well as a potent effect in preventing the A?1-42-induced depletion of antioxidant enzymes (catalase and SOD-1). Finally, we report the capability of ASS234 to cross the bloodbrain barrier. Overall, our in vitro results show that ASS234 may have an impact on different processes involved in AD pathogenesis and provide evidences that it has encouraging attributes as a therapeutic lead compound.
Semicarbazide-sensitive amine oxidase (SSAO) metabolizes the oxidative deamination of primary aromatic and aliphatic amines. The final cytotoxic products of its catalysis contribute to diseases involving vascular degeneration. The increasing interest in measuring SSAO activity has led to the development of several different methods. Herein, we compare SSAO activity results obtained with radiometric and fluorimetric methods in 49 plasma samples. Although not interchangeable, a significant correlation was obtained between methods. Considering these limitations, the fluorimetric method might replace the radioisotopic one.
Dendrimers have been proved to interact with amyloids, although most of dendrimers assayed in amyloidogenic systems are toxic to cells. The development of glycodendrimers, poly(propyleneimine) (PPI) dendrimers decorated with maltose (Mal), represents the possibility of using dendrimers with a low intrinsic toxicity. In the present paper we show that fourth (PPI-G4-Mal) and fifth (PPI-G5-Mal) generation glycodendrimers have the capacity to interfere with Alzheimers amyloid peptide A?(1-40) fibrilization. The interaction is generation dependent: PPI-G5-Mal blocks amyloid fibril formation generating granular nonfibrillar amorphous aggregates, whereas PPI-G4-Mal generates clumped fibrils at low dendrimer-peptide ratios and amorphous aggregates at high ratios. Both PPI-G4-Mal and PPI-G5-Mal are nontoxic to PC12 and SH-SY5Y cells. PPI-G4-Mal reduces amyloid toxicity by clumping fibrils together, whereas amorphous aggregates are toxic to PC12 cells. The results show that glycodendrimers are promising nontoxic agents in the search for anti-amyloidogenic compounds. Fibril clumping may be an anti-amyloid toxicity strategy.
Genome wide association studies reported two single nucleotide polymorphisms in ANK3 (rs9804190 and rs10994336) as independent genetic risk factors for bipolar disorder. Another SNP in ANK3 (rs10761482) was associated with schizophrenia in a large European sample. Within the debate on common susceptibility genes for schizophrenia and bipolar disorder, we tried to investigate common findings by analyzing association of ANK3 with schizophrenia, bipolar disorder and unipolar depression.
Glial cells in the peripheral nervous system, such as Schwann cells, respond to nucleotides, which play an important role in axonal regeneration and myelination. Metabotropic P2Y receptor agonists are promising therapeutic molecules for peripheral neuropathies. Nevertheless, the proteomic mechanisms involved in nucleotide action on Schwann cells remain unknown. Here, we studied intracellular protein changes in RT4-D6P2T Schwann cells after treatment with nucleotides and Nucleo CMP Forte (CMPF), a nucleotide-based drug. After treatment with CMPF, 2-D DIGE revealed 11 differential gel spots, which were all upregulated. Among these, six different proteins were identified by MS. Some of these proteins are involved in actin remodelling (actin-related protein, Arp3), membrane vesicle transport (Rab GDP dissociation inhibitor ?, Rab GDI), and the endoplasmic reticulum stress response (protein disulfide isomerase A3, PDI), which are hallmarks of a possible P2Y receptor signalling pathway. Expression of P2Y receptors in RT4-D6P2T cells was demonstrated by RT-PCR and a transient elevation of intracellular calcium measured in response to UTP. Actin reorganisation was visualized after UTP treatment using phalloidin-FITC staining and was blocked by the P2Y antagonist suramin, which also inhibited Arp3, Rab GDI, and PDI protein upregulation. Our data indicate that extracellular UTP interacts with Schwann P2Y receptors and activates molecular machinery that induces changes in the glial cell cytoskeleton.
It has been extensively reported that diabetes mellitus (DM) patients have a higher risk of developing Alzheimers disease (AD), but a mechanistic connection between both pathologies has not been provided so far. Carbohydrate-derived advanced glycation endproducts (AGEs) have been implicated in the chronic complications of DM and have been reported to play an important role in the pathogenesis of AD. The earliest histopathological manifestation of AD is the apparition of extracellular aggregates of the amyloid beta peptide (Abeta). To investigate possible correlations between AGEs and Abeta aggregates with both pathologies, we have performed an immuhistochemical study in human post-mortem samples of AD, AD with diabetes (ADD), diabetic and nondemented controls. ADD brains showed increased number of Abeta dense plaques and receptor for AGEs (RAGE)-positive and Tau-positive cells, higher AGEs levels and major microglial activation, compared to AD brain. Our results indicate that ADD patients present a significant increase of cell damage through a RAGE-dependent mechanism, suggesting that AGEs may promote the generation of an oxidative stress vicious cycle, which can explain the severe progression of patients with both pathologies.
Alzheimer disease (AD) is a progressive dementia affecting a large proportion of the aging population. The histopathological changes in AD include neuronal cell death, formation of amyloid plaques and neurofibrillary tangles. There is also evidence that brain tissue in patients with AD is exposed to oxidative stress (e.g., protein oxidation, lipid oxidation, DNA oxidation and glycoxidation) during the course of the disease. Advanced glycation endproducts (AGEs) are present in amyloid plaques in AD, and its extracellular accumulation may be caused by an accelerated oxidation of glycated proteins. AGEs participate in neuronal death causing direct (chemical) and indirect (cellular) free radical production and consequently increase oxidative stress. The development of drugs for the treatment of AD that breaks the vicious cycles of oxidative stress and neurodegeneration offer new opportunities. These approaches include AGE-inhibitors, antioxidants and anti-inflammatory substances, which prevent free radical production.
Schwann cells (SCs) are peripheral myelinating glial cells that express the neuronal Ca(2+)-dependent cell adhesion molecule, neural cadherin (N-cadherin). N-cadherin is involved in glia-glia and axon-glia interactions and participates in many key events, which range from the control of axonal growth and guidance to synapse formation and plasticity. Extracellular UTP activates P2Y purinergic receptors and exerts short- and long-term effects on several tissues to promote wound healing. Nevertheless, the contribution of P2Y receptors in peripheral nervous system functions is not completely understood. The current study demonstrated that UTP induced a dose- and time-dependent increase in N-cadherin expression in SCs. Furthermore, N-cadherin expression was blocked by the P2 purinoceptor antagonist suramin. The increased N-cadherin expression induced by UTP was mediated by phosphorylation of mitogen-activated protein kinases (MAPKs), such as Jun N-terminal kinase, extracellular-regulated kinase and p38 kinase. Moreover, the Rho kinase inhibitor Y27632, the phospholipase C inhibitor U73122 and the protein kinase C inhibitor calphostin C attenuated the UTP-induced activation of MAPKs significantly. Extracellular UTP also modulated increased in the expression of the early transcription factors c-Fos and c-Jun. We also demonstrated that the region of the N-cadherin promoter between nucleotide positions -3698 and -2620, which contained one activator protein-1-binding site, was necessary for UTP-induced gene expression. These results suggest a novel role for P2Y purinergic receptors in the regulation of N-cadherin expression in SCs.
Alzheimers disease (AD) is a complex neurodegenerative disorder with a multifaceted pathogenesis. There are at present three Food and Drug Administration-approved drugs based on the "one drug, one target" paradigm (donepezil, galantamine and rivastigmine) that improve symptoms by inhibiting acetylcholinesterase. However, apart from the beneficial palliative properties, cholinergic drugs have shown little efficacy to prevent the progression of the disease evidencing the unsuitability of this strategy for the complex nature of AD. By contrast, the multifactorial nature of this neurodegenerative disorder supports the most current innovative therapeutic approach based on the "one drug, multiple targets" paradigm, which suggests the use of compounds with multiple activities at different target sites. Accordingly, the also called multitarget-directed ligand (MTDL) approach has been the subject of increasing attention by many research groups, which have developed a variety of hybrid compounds acting on very diverse targets. The therapeutic potential of monoamine oxidase inhibitors (MAOI) in AD has been suggested due to their demonstrated neuroprotective properties besides their enhancing effect on monoaminergic transmission. Especially, those containing a propargylamine moiety are of particular interest due to their reported beneficial actions. Therefore, targeting MAO enzymes should be considered in therapeutic interventions. This review makes a special emphasis on MTDLs that commonly target MAO enzymes. There is at present an urgent need for real disease-modifying therapies for AD and the MTDL approach makes a breakthrough for the development of new drugs capable of addressing the biological complexity of this disorder.
Semicarbazide-sensitive amine oxidase/vascular adhesion protein-1 (SSAO/VAP-1) is involved in vascular endothelial damage as well as in the vascular degeneration underlying diabetes mellitus and Alzheimers disease (AD). Recent evidence suggests that classic pathological features of AD are more pronounced in diabetic mellitus patients. To investigate the expression and distribution of SSAO/VAP-1 in the two pathologies, we have performed an immunohistochemical study in human hippocampal vessels of AD, AD with diabetic mellitus (ADD), diabetic mellitus (DM), and nondemented (ND) patients. The present results demonstrate major vessel accumulation of both SSAO/VAP-1 and amyloid-? immunolabeling intensity in ADD compared with AD patients. Interestingly, nearly damaged vessels with high levels of SSAO/VAP-1 also showed increased oxidative damage markers (AGE, RAGE, and SOD-1) and glial activation (GFAP and HLA). Overall, this work suggests that high vascular SSAO/VAP-1 levels in human hippocampus may contribute to vascular degeneration, which can explain the severe progression in patients with both pathologies.
Related JoVE Video
Journal of Visualized Experiments
What is Visualize?
JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.
How does it work?
We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.
Video X seems to be unrelated to Abstract Y...
In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.