Alternative splicing and gene polymorphism of the human TAP3/SEC14L4 gene

Three closely related human SEC14p-like proteins (hTAP1, hTAP2, hTAP3, or SEC14L2, SEC14L3, SEC14L4, respectively) have been described that are related to the Saccharomyces cerevisiae SEC14 protein. These proteins may participate in intracellular lipid transport and influence regulatory lipid-dependent events. Here we report the isolation of an alternatively spliced hTAP3 cDNA and a polymorphism within the coding region of the hTAP3/SEC14L4 gen

Selective inhibition of phosphodiesterase 4D increases tau phosphorylation at Ser214 residue

Tau is a protein that normally participates in the assembly and stability of microtubules. However, it can form intraneuronal hyperphosphorylated aggregates that are hallmarks of Alzheimer's disease and other neurodegenerative disorders known as tauopathies. Tau can be phosphorylated by multiple kinases at several sites. Among such kinases, the cAMP-dependent protein kinase A (PKA) phosphorylates tau at Ser214 (pTAU-S214), an event that was shown to reduce the pathological assembly of the protein. Given that the neuronal cAMP/PKA-activated cascade is involved in synaptic plasticity and memory, and that cAMP-enhancing strategies demonstrated promising therapeutic potential for the treatment of cognitive deficits, we investigated the impact of cAMP on pTAU-S214 in N2a cells and rat hippocampal slices. Our results confirm that the activation of adenylyl cyclase increases pTAU-S214 in both model systems and, more interestingly, this effect is mimicked by GEBR-7b, a phosphodiesterase 4D inhibitor with proven pro-cognitive efficacy in rodents

Alternative splicing and gene polymorphism of the human TAP3/SEC14L4 gene

Three closely related human SEC14p-like proteins (hTAP1, hTAP2, hTAP3, or SEC14L2, SEC14L3, SEC14L4, respectively) have been described that are related to the Saccharomyces cerevisiae SEC14 protein. These proteins may participate in intracellular lipid transport and influence regulatory lipid-dependent events. Here we report the isolation of an alternatively spliced hTAP3 cDNA and a polymorphism within the coding region of the hTAP3/SEC14L4 gene

Modulation of cAMP levels by highâ fat diet and curcumin and regulatory effects on CD36/FAT scavenger receptor/fatty acids transporter gene expression

Curcumin, a polyphenol from turmeric (Curcuma longa), reduces inflammation, atherosclerosis, and obesity in several animal studies. In Ldlrâ /â mice fed a highâ fat diet (HFD), curcumin reduces plasma lipid levels, therefore contributing to a lower accumulation of lipids and to reduced expression of fatty acid transport proteins (CD36/FAT, FABP4/aP2) in peritoneal macrophages. In this study, we analyzed the molecular mechanisms by which curcumin (500, 1000, 1500 mg/kg diet, for 4 months) may influence plasma and tissue lipid levels in Ldlrâ /â mice fed an HFD. In liver, HFD significantly suppressed cAMP levels, and curcumin restored almost normal levels. Similar trends were observed in adipose tissues, but not in brain, skeletal muscle, spleen, and kidney. Treatment with curcumin increased phosphorylation of CREB in liver, what may play a role in regulatory effects of curcumin in lipid homeostasis. In cell lines, curcumin increased the level of cAMP, activated the transcription factor CREB and the human CD36 promoter via a sequence containing a consensus CREB response element. Regulatory effects of HFD and Cur on gene expression were observed in liver, less in skeletal muscle and not in brain. Since the cAMP/protein kinase A (PKA)/CREB pathway plays an important role in lipid homeostasis, energy expenditure, and thermogenesis by increasing lipolysis and fatty acid βâ oxidation, an increase in cAMP levels induced by curcumin may contribute to its hypolipidemic and antiâ atherosclerotic effects. © 2016 BioFactors, 43(1):42â 53, 2017Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/136289/1/biof1307_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/136289/2/biof1307.pd

Glutathione-mediated antioxidant response and aerobic metabolism: two crucial factors involved in determining the multi-drug resistance of high-risk neuroblastoma

Neuroblastoma, a paediatric malignant tumor, is initially sensitive to etoposide, a drug to which many patients develop chemoresistance. In order to investigate the molecular mechanisms responsible for etoposide chemoresistance, HTLA-230, a human MYCN-amplified neuroblastoma cell line, was chronically treated with etoposide at a concentration that in vitro mimics the clinically-used dose. The selected cells (HTLA-Chr) acquire multi-drug resistance (MDR), becoming less sensitive than parental cells to high doses of etoposide or doxorubicin. MDR is due to several mechanisms that together contribute to maintaining non-toxic levels of H2O2. In fact, HTLA-Chr cells, while having an efficient aerobic metabolism, are also characterized by an up-regulation of catalase activity and higher levels of reduced glutathione (GSH), a thiol antioxidant compound. The combination of such mechanisms contributes to prevent membrane lipoperoxidation and cell death. Treatment of HTLA-Chr cells with L-Buthionine-sulfoximine, an inhibitor of GSH biosynthesis, markedly reduces their tumorigenic potential that is instead enhanced by the exposure to N-Acetylcysteine, able to promote GSH synthesis.Collectively, these results demonstrate that GSH and GSH-related responses play a crucial role in the acquisition of MDR and suggest that GSH level monitoring is an efficient strategy to early identify the onset of drug resistance and to control the patient's response to therapy

Memory-enhancing effects of GEBR-32a, a new PDE4D inhibitor holding promise for the treatment of Alzheimer\u2019s disease.

Memory loss characterizes several neurodegenerative disorders, including Alzheimer’s disease (AD). Inhibition of type 4 phosphodiesterase (PDE4) and elevation of cyclic adenosine monophosphate (cAMP) has emerged as a promising therapeutic approach to treat cognitive deficits. However, PDE4 exists in several isoforms and pan inhibitors cannot be used in humans due to severe emesis. Here, we present GEBR-32a, a new PDE4D full inhibitor that has been characterized both in vitro and in vivo using biochemical, electrophysiological and behavioural analyses. GEBR-32a efficiently enhances cAMP in neuronal cultures and hippocampal slices. In vivo pharmacokinetic analysis shows that GEBR-32a is rapidly distributed within the central nervous system with a very favourable brain/blood ratio. Specific behavioural tests (object location and Y-maze continuous alternation tasks) demonstrate that this PDE4D inhibitor is able to enhance memory in AD transgenic mice and concomitantly rescues their hippocampal long-term potentiation deficit. Of great relevance, our preliminary toxicological analysis indicates that GEBR-32a is not cytotoxic and genotoxic, and does not seem to possess emetic-like side effects. In conclusion, GEBR-32a could represent a very promising cognitive-enhancing drug with a great potential for the treatment of Alzheimer’s disease

Selective PDE4 subtype inhibition provides new opportunities to intervene in neuroinflammatory versus myelin damaging hallmarks of multiple sclerosis.

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) characterized by focal inflammatory lesions and prominent demyelination. Even though the currently available therapies are effective in treating the initial stages of disease, they are unable to halt or reverse disease progression into the chronic progressive stage. Thus far, no repair-inducing treatments are available for progressive MS patients. Hence, there is an urgent need for the development of new therapeutic strategies either targeting the destructive immunological demyelination or boosting endogenous repair mechanisms. Using in vitro, ex vivo, and in vivo models, we demonstrate that selective inhibition of phosphodiesterase 4 (PDE4), a family of enzymes that hydrolyzes and inactivates cyclic adenosine monophosphate (cAMP), reduces inflammation and promotes myelin repair. More specifically, we segregated the myelination-promoting and anti-inflammatory effects into a PDE4D- and PDE4B-dependent process respectively. We show that inhibition of PDE4D boosts oligodendrocyte progenitor cells (OPC) differentiation and enhances (re)myelination of both murine OPCs and human iPSC-derived OPCs. In addition, PDE4D inhibition promotes in vivo remyelination in the cuprizone model, which is accompanied by improved spatial memory and reduced visual evoked potential latency times. We further identified that PDE4B-specific inhibition exerts anti-inflammatory effects since it lowers in vitro monocytic nitric oxide (NO) production and improves in vivo neurological scores during the early phase of experimental autoimmune encephalomyelitis (EAE). In contrast to the pan PDE4 inhibitor roflumilast, the therapeutic dose of both the PDE4B-specific inhibitor A33 and the PDE4D-specific inhibitor Gebr32a did not trigger emesis-like side effects in rodents. Finally, we report distinct PDE4D isoform expression patterns in human area postrema neurons and human oligodendroglia lineage cells. Using the CRISPR-Cas9 system, we confirmed that pde4d1/2 and pde4d6 are the key targets to induce OPC differentiation. Collectively, these data demonstrate that gene specific PDE4 inhibitors have potential as novel therapeutic agents for targeting the distinct disease processes of MS

PKCδ Sensitizes Neuroblastoma Cells to L-Buthionine-Sulfoximine and Etoposide Inducing Reactive Oxygen Species Overproduction and DNA Damage

Neuroblastoma is a type of pediatric cancer. The sensitivity of neuroblastoma (NB) cancer cells to chemotherapy and radiation is inhibited by the presence of antioxidants, such as glutathione (GSH), which is crucial in counteracting the endogenous production of reactive oxygen species (ROS). We have previously demonstrated that cells depleted of GSH undergo apoptosis via oxidative stress and Protein kinase C (PKC) δ activation. In the present study, we transfected PKCδ in NB cells resistant to oxidative death induced by L-buthionine-S,R-sulfoximine (BSO), a GSH-depleting agent. Cell responses, in terms of ROS production, apoptosis and DNA damage were evaluated. Moreover, PKCδ activation was monitored by analyzing the phosphorylation status of threonine 505 residue, carrying out PKC activity assay and investigating the subcellular localization of the kinase. The cell responses obtained in BSO-resistant cells were also compared with those obtained in BSO-sensitive cells subjected to the same experimental protocol. Our results demonstrate, for the first time, that PKCδ induces DNA oxidation and ROS overproduction leading to apoptosis of BSO-resistant NB cells and potentiates the cytotoxic effects induced by BSO in sensitive cells. Moreover, PKCδ overexpression enhances the sensitivity of NB cells to etoposide, a well-characterised drug, commonly used in neuroblastoma therapy. Altogether our data provide evidence of a pro-oxidant role of PKCδ that might be exploited to design new therapeutic strategies aimed at selective killing of cancer cells and overcoming drug resistance. However, it becomes evident that a more detailed understanding of ROS-mediated signaling in cancer cells is necessary for the development of redox-modulated therapeutic approaches

The amyloid cascade hypothesis in Alzheimer’s disease: it’s time to change our mind

reserved2Since its discovery in 1984, the beta amyloid peptide has treaded the boards of neurosciences as the star molecule in Alzheimer’s disease pathogenesis. In the last decade, however, this vision has been challenged by evidence-based medicine showing the almost complete failure of clinical trials that experimented anti-amyloid therapies with great hopes. Moreover, data have accumulated which clearly indicate that this small peptide plays a key role in the physiological processes of memory formation. In the present review, we will discuss the different aspects of the amyloid cascade hypothesis, highlighting its pros and cons, and we will analyse the results of the therapeutic approaches attempted to date that should change the direction of Alzheimer’s disease research in the future.mixedRicciarelli, Roberta; Fedele, Ernesto.Ricciarelli, Roberta; Fedele, Ernest