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Background: Although the antidepressant potential of repetitive transcranial magnetic stimulation (rTMS), the pleiotropic effects in geriatric depression (GD) are poorly investigated. We tested rTMS on depression, cognitive performance, growth/neurotrophic factors, cerebral blood flow (CBF) to transcranial Doppler sonography (TCD), and motor-evoked potentials (MEPs) to TMS in GD., Methods: In this case series study, six drug-resistant subjects (median age 68.0 years) underwent MEPs at baseline and after 3 weeks of 10 Hz rTMS on the left dorsolateral prefrontal cortex. The percentage change of serum nerve growth factor, vascular endothelial growth factor, brain-derived growth factor, insulin-like growth factor-1, and angiogenin was obtained. Assessments were performed at baseline, and at the end of rTMS; psychocognitive tests were also repeated after 1, 3, and 6 months., Results: Chronic cerebrovascular disease was evident in five patients. No adverse/undesirable effect was reported. An improvement in mood was observed after rTMS but not at follow-up. Electrophysiological data to TMS remained unchanged, except for an increase in the right median MEP amplitude. TCD and neurotrophic/growth factors did not change., Conclusions: We were unable to detect a relevant impact of high-frequency rTMS on mood, cognition, cortical microcircuits, neurotrophic/growth factors, and CBF. Cerebrovascular disease and exposure to multiple pharmacological treatments might have contributed.

Chronic myeloid leukemia (CML), BCR-ABL1-positive, is classified as a myeloproliferative characterized by Philadelphia chromosome/translocation t(9;22) and proliferating granulocytes. Despite the clinical success of tyrosine kinase inhibitors (TKi) agents in the treatment of CML, most patients have minimal residual disease contained in the bone marrow microenvironment, within which stromal cells assume a pro-inflammatory phenotype that determines their transformation in cancer-associated fibroblasts (CAF) which, in turn can play a fundamental role in resistance to therapy. Insulin-like Growth Factor Binding Protein-6 (IGFBP-6) is expressed during tumor development, and is involved in immune-escape and inflammation as well, providing a potential additional target for CML therapy. Here, we aimed at investigating the role of IGFBP-6/SHH/TLR4 axis in TKi response. We used a CML cell line, LAMA84-s, and healthy bone marrow stromal cells, HS-5, in mono- or co-culture. The two cell lines were treated with Dasatinib and/or IGFBP-6, and the expression of inflammatory markers was tested by qRT-PCR; furthermore, expression of IGFBP-6, TLR4 and Gli1 were evaluated by Western blot analysis and immumocytochemistry. The results showed that both co-culture and Dasatinib exposure induce inflammation in stromal and cancer cells so that they modulate the expression of TLR4, and these effects were more marked following IGFBP-6 pre-treatment suggesting that this molecule may confer resistance through the inflammatory processes. This phenomenon was coupled with sonic hedgehog (SHH) signaling. Indeed, our data also demonstrate that HS-5 treatment with PMO (an inducer of SHH) induces significant modulation of TLR4 and overexpression of IGFPB-6 suggesting that the two pathways are interconnected with each other and with the TLR-4 pathway. Finally, we demonstrated that pretreatment with IGFBP-6 and/or PMO restored LAMA-84 cell viability after treatment with Dasatinib, suggesting that both IGFBP-6 and SHH are involved in the resistance mechanisms induced by the modulation of TLR-4, thus indicating that the two pathways may be considered as potential therapeutic targets.

Mature neurotrophic factors and their propeptides play key roles ranging from the regulation of neuronal growth and differentiation to prominent participation in neuronal survival and recovery after injury. Their signaling pathways sculpture neuronal circuits during brain development and regulate adaptive neuroplasticity. In addition, neurotrophic factors provide trophic support for damaged neurons, giving them a greater capacity to survive and maintain their potential to regenerate their axons. Therefore, the modulation of these factors can be a valuable target for treating or preventing neurologic disorders and age-dependent cognitive decline. Neuroregenerative medicine can take great advantage by the deepening of our knowledge on the molecular mechanisms underlying the properties of neurotrophic factors. It is indeed an intriguing topic that a significant interplay between neurotrophic factors and various metals can modulate the outcome of neuronal recovery. This review is particularly focused on the roles of GDNF, BDNF and NGF in motoneuron survival and recovery from injuries and evaluates the therapeutic potential of various neurotrophic factors in neuronal regeneration. The key role of metal homeostasis/dyshomeostasis and metal interaction with neurotrophic factors on neuronal pathophysiology is also highlighted as a novel mechanism and potential target for neuronal recovery. The progress in mechanistic studies in the field of neurotrophic factor-mediated neuroprotection and neural regeneration, aiming at a complete understanding of integrated pathways, offers possibilities for the development of novel neuroregenerative therapeutic approaches.

Proteasome malfunction parallels abnormal amyloid accumulation in Alzheimer's Disease (AD). Here we scrutinize a small library of pyrazolones by assaying their ability to enhance proteasome activity and protect neuronal cells from amyloid toxicity. Tube tests evidenced that aminopyrine and nifenazone behave as 20S proteasome activators. Enzyme assays carried out on an "open gate" mutant (α3ΔN) proteasome demonstrated that aminopyrine activates proteasome through binding the α-ring surfaces and influencing gating dynamics. Docking studies coupled with STD-NMR experiments showed that H-bonds and π-π stacking interactions between pyrazolones and the enzyme play a key role in bridging α1 to α2 and, alternatively, α5 to α6 subunits of the outer α-ring. Aminopyrine and nifenazone exhibit neurotrophic properties and protect differentiated human neuroblastoma SH-SY5Y cells from β-amyloid (Aβ) toxicity. ESI-MS studies confirmed that aminopyrine enhances Aβ degradation by proteasome in a dose-dependent manner. Our results suggest that some pyrazolones and, in particular, aminopyrine are promising compounds for the development of proteasome activators for AD treatment., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Nerve growth factor (NGF) is a protein necessary for development and maintenance of the sympathetic and sensory nervous systems. We have previously shown that the NGF N-terminus peptide NGF(1-14) is sufficient to activate TrkA signaling pathways essential for neuronal survival and to induce an increase in brain-derived neurotrophic factor (BDNF) expression. Cu 2+ ions played a critical role in the modulation of the biological activity of NGF(1-14). Using computational, spectroscopic, and biochemical techniques, here we report on the ability of a newly synthesized peptide named d-NGF(1-15), which is the dimeric form of NGF(1-14), to interact with TrkA. We found that d-NGF(1-15) interacts with the TrkA-D5 domain and induces the activation of its signaling pathways. Copper binding to d-NGF(1-15) stabilizes the secondary structure of the peptides, suggesting a strengthening of the noncovalent interactions that allow for the molecular recognition of D5 domain of TrkA and the activation of the signaling pathways. Intriguingly, the signaling cascade induced by the NGF peptides ultimately involves cAMP response element-binding protein (CREB) activation and an increase in BDNF protein level, in keeping with our previous result showing an increase of BDNF mRNA. All these promising connections can pave the way for developing interesting novel drugs for neurodegenerative diseases.

Several lines of evidence point to a compromised proteostasis associated with a reduction of the Ubiquitin Proteasome System (UPS) activity in patients affected by Alzheimer's Disease (AD) and suggest that the amyloid β peptide (Aβ) is an important player in the game. Inspired also by many reports, underlining the presence of ubiquitin (Ub) in the amyloid plaques of AD brains, here we set out to test whether Ub may bind the Aβ peptide and have any effect on its clearance pathways. By using an integrated array of MALDI-TOF/UPLC-HRMS, fluorescence, NMR, SPR, Microscale Thermophoresis (MST) and molecular dynamics studies, we consistently demonstrated that Aβ40 binds Ub with a 1 : 1 stoichiometry and K d in the high micromolar range. In particular, we show that the N-terminal domain of the Aβ peptide (through residues D1, E3 and R5) interacts with the C-terminal tail of Ub (involving residues K63 and E64), inducing the central region of Aβ ( 14 HQKLVFFAEDVGSNK 28 ) to adopt a mixed α-helix/β-turn structure. ELISA assays, carried out in neuroblastoma cell lysates, suggest that Aβ competitively binds Ub also in the presence of the entire pool of cytosolic Ub binding proteins. Ub-bound Aβ has a lower tendency to aggregate into amyloid-like fibrils and is more slowly degraded by the Insulin Degrading Enzyme (IDE). Finally, we observe that the water soluble fragment Aβ1-16 significantly inhibits Ub chain growth reactions. These results evidence how the non-covalent interaction between Aβ peptides and Ub may have relevant effects on the regulation of the upstream events of the UPS and pave the way to future in vivo studies addressing the role played by Aβ peptide in the malfunction of proteome maintenance occurring in AD.

Copper homeostasis is generally investigated focusing on a single component of the metallostasis network. Here we address several of the factors controlling the metallostasis for neuroblastoma cells (SH-SY5Y) upon treatment with 2,9-dimethyl-1,10-phenanthroline-5,6-dione (phendione) and 2,9-dimethyl-1,10-phenanthroline (cuproindione). These compounds bind and transport copper inside cells, exert their cytotoxic activity through the induction of oxidative stress, causing apoptosis and alteration of the cellular redox and copper homeostasis network. The intracellular pathway ensured by copper transporters (Ctr1, ATP7A), chaperones (CCS, ATOX, COX 17, Sco1, Sco2), small molecules (GSH) and transcription factors (p53) is scrutinised., Competing Interests: CONFLICTS OF INTEREST There are no conflicts to declare.

Human amylin (hA1-37) is a polypeptide hormone secreted in conjunction with insulin from the pancreatic β-cells involved in the pathogenesis of type 2 diabetes mellitus (T2DM). The shorter fragment hA17-29 than full-length peptide is capable to form amyloids "in vitro". Here, we monitored the time course of hA17-29 β-amyloid fibril and oligomer formation [without and with copper(II)], cellular toxicity of different amyloid aggregates, and involvement of specific receptors (receptor for advanced glycation end-products, RAGE; low-affinity nerve growth factor receptor, p75-NGFR) in aggregate toxicity. Fibril and oligomer formation of hA17-29 incubated at 37 °C for 0, 48, and 120 h, without or with copper(II), were measured by the thioflavin T fluorescence assay and ELISA, respectively. Toxicity of hA17-29 aggregates and effects of anti-RAGE and anti-p75-NGFR antibodies were evaluated on neuroblastoma SH-SY5Y viability. Fluorescence assay of hA17-29 indicates an initial slow rate of soluble fibril formation (48 h), followed by a slower rate of insoluble aggregate formation (120 h). The highest quantity of oligomers was recorded when hA17-29 was pre-aggregated for 48 h in the presence of copper(II) showing also the maximal cell toxicity (-44% of cell viability, p < 0.01 compared to controls). Anti-RAGE or anti-p75-NGFR antibodies almost abolished cell toxicity of hA17-29 aggregates. These results indicate that copper(II) influences the aggregation process and hA17-29 toxicities are especially attributable to oligomeric aggregates. hA17-29 aggregate toxicity seems to be mediated by RAGE and p75-NGFR receptors which might be potential targets for new drugs in T2DM treatment.

The nerve growth factor (NGF) N-terminus peptide, NGF(1-14), and its acetylated form, Ac-NGF(1-14), were investigated to scrutinize the ability of this neurotrophin domain to mimic the whole protein. Theoretical calculations demonstrated that non-covalent forces assist the molecular recognition of TrkA receptor by both peptides. Combined parallel tempering/docking simulations discriminated the effect of the N-terminal acetylation on the recognition of NGF(1-14) by the domain 5 of TrkA (TrkA-D5). Experimental findings demonstrated that both NGF(1-14) and Ac-NGF(1-14) activate TrkA signaling pathways essential for neuronal survival. The NGF-induced TrkA internalization was slightly inhibited in the presence of Cu 2+ and Zn 2+ ions, whereas the metal ions elicited the NGF(1-14)-induced internalization of TrkA and no significant differences were found in the weak Ac-NGF(1-14)-induced receptor internalization. The crucial role of the metals was confirmed by experiments with the metal-chelator bathocuproine disulfonic acid, which showed different inhibitory effects in the signaling cascade, due to different metal affinity of NGF, NGF(1-14) and Ac-NGF(1-14). The NGF signaling cascade, activated by the two peptides, induced CREB phosphorylation, but the copper addition further stimulated the Akt, ERK and CREB phosphorylation in the presence of NGF and NGF(1-14) only. A dynamic and quick influx of both peptides into PC12 cells was tracked by live cell imaging with confocal microscopy. A significant role of copper ions was found in the modulation of peptide sub-cellular localization, especially at the nuclear level. Furthermore, a strong copper ionophoric ability of NGF(1-14) was measured. The Ac-NGF(1-14) peptide, which binds copper ions with a lower stability constant than NGF(1-14), exhibited a lower nuclear localization with respect to the total cellular uptake. These findings were correlated to the metal-induced increase of CREB and BDNF expression caused by NGF(1-14) stimulation. In summary, we here validated NGF(1-14) and Ac-NGF(1-14) as first examples of monomer and linear peptides able to activate the NGF-TrkA signaling cascade. Metal ions modulated the activity of both NGF protein and the NGF-mimicking peptides. Such findings demonstrated that NGF(1-14) sequence can reproduce the signal transduction of whole protein, therefore representing a very promising drug candidate for further pre-clinical studies.

Semax is a heptapeptide (Met-Glu-His-Phe-Pro-Gly-Pro) that encompasses the sequence 4-7 of N-terminal domain of the adrenocorticotropic hormone and a C-terminal Pro-Gly-Pro tripeptide. N-terminal amino group acetylation (Ac-Semax) modulates the chemical and biological properties of parental peptide, modifying the ability of Semax to form complex species with Cu(II) ion. At physiological pH, the main complex species formed by Ac-Semax, [CuLH -2 ] 2- , consists in a distorted CuN 3 O chromophore with a weak apical interaction of the methionine sulphur. Such a complex differs from the Cu(II)-Semax complex system, which exhibits a CuN 4 chromophore. The reduced ligand field affects the [CuLH -2 ] 2- formal redox potential, which is more positive than that of Cu(II)-Semax corresponding species. In the amino-free form, the resulting complex species is redox-stable and unreactive against ascorbic acid, unlike the acetylated form. Semax acetylation did not protect from Cu(II) induced toxicity on a SH-SY5Y neuroblastoma cell line, thus demonstrating the crucial role played by the free NH 2 terminus in the cell protection. Since several brain diseases are associated either to Cu(II) or Zn(II) dyshomeostasis, here we characterized also the complex species formed by Zn(II) with Semax and Ac-Semax. Both peptides were able to form Zn(II) complex species with comparable strength. Confocal microscopy imaging confirmed that peptide group acetylation does not affect the Zn(II) influx in neuroblastoma cells. Moreover, a punctuate distribution of Zn(II) within the cells suggests a preferred subcellular localization that might explain the zinc toxic effect. A future perspective can be the use of Ac-Semax as ionophore in antibody drug conjugates to produce a dysmetallostasis in tumor cells., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Type-2 diabetes (T2D) is considered to be a potential threat on a global level. Recently, T2D has been listed as a misfolding disease, such as Alzheimer's and Parkinson's diseases. Human islet amyloid polypeptide (hIAPP) is a molecule cosecreted in pancreatic β cells and represents the main constituent of an aggregated amyloid found in individuals affected by T2D. The trace-element serum level is significantly influenced during the development of diabetes. In particular, the dys-homeostasis of Cu(2+) ions may adversely affect the course of the disease. Conflicting results have been reported on the protective role played by complex species formed by Cu(2+) ions with hIAPP or its peptide fragments in vitro. The histidine (His) residue at position 18 represents the main binding site for the metal ion, but contrasting results have been reported on other residues involved in metal-ion coordination, in particular those toward the N or C terminus. Sequences that encompass regions 17-29 and 14-22 were used to discriminate between the two models of the hIAPP coordination mode. Due to poor solubility in water, poly(ethylene glycol) (PEG) derivatives were synthesized. A peptide fragment that encompasses the 17-29 region of rat amylin (rIAPP) in which the arginine residue at position 18 was substituted by a histidine residue was also obtained to assess that the PEG moiety does not alter the peptide secondary structure. The complex species formed by Cu(2+) ions with Ac-PEG-hIAPP(17-29)-NH2 , Ac-rIAPP(17-29)R18H-NH2 , and Ac-PEG-hIAPP(14-22)-NH2 were studied by using potentiometric titrations coupled with spectroscopic methods (UV/Vis, circular dichroism, and EPR). The combined thermodynamic and spectroscopic approach allowed us to demonstrate that hIAPP is able to bind Cu(2+) ions starting from the His18 imidazole nitrogen atom toward the N-terminus domain. The stability constants of copper(II) complexes with Ac-PEG-hIAPP(14-22)-NH2 were used to simulate the different experimental conditions under which aggregate formation and oxidative stress of hIAPP has been reported. Speciation unveils: 1) the protective role played by increased amounts of Cu(2+) ions on the hIAPP fibrillary aggregation, 2) the effect of adventitious trace amounts of Cu(2+) ions present in phosphate-buffered saline (PBS), and 3) a reducing fluorogenic probe on H2 O2 production attributed to the polypeptide alone., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The sole role of bradykinin (BK) as an inflammatory mediator is controversial, as recent data also support an anti-inflammatory role for BK in Alzheimer's disease (AD). The involvement of two different receptors (B1R and B2R) could be a key to understand this issue. However, although copper and zinc dyshomeostasis has been demonstrated to be largely involved in the development of AD, a detailed study of the interaction of BK with these two metal ions has never been addressed. In this work, we have applied mass spectrometry, circular dichroism as well as computational methods in order to assess if copper and zinc have the ability to modulate the conformation and oligomerization of BK. In addition, we have correlated the chemical data with the effect of metals on the activity of BK analyzed in cell cultures by biochemical procedures. The biochemical analyses on monocyte/macrophage cell culture (THP-1 Cell Line human) in line with the effect of metals on the conformation of BK showed that the presence of copper can affect the signaling cascade mediated by the BK receptors. The results obtained show a further role of metal ions, particularly copper, in the development and outcome of neuroinflammatory diseases. The possible implications in AD are discussed.

Wound healing is a complex biological process that aims to repair damaged tissue. Even though many biological and biochemical mechanisms associated with the steps of physiological wound healing are known, there is still significant morbidity and mortality due to dysregulation of physiological mechanisms. It might be useful to revise the activity of old players and their links with new, often neglected, molecular entities. This review revises new findings supporting the hypothesis that copper ions regulate the activity and/or the expression of proteins crucially involved in the wound repair process. A better understanding of these interactions might suggest potential new targets for therapeutic intervention on scars or non-healing wounds., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Ever since the discovery of its neurite growth promoting activity in sympathetic and sensory ganglia, nerve growth factor (NGF) became the prototype of the large family of neurotrophins. The use of primary cultures and clonal cell lines has revealed several distinct actions of NGF and other neurotrophins. Among several models of NGF activity, the clonal cell line PC12 is the most widely employed. Thus, in the presence of NGF, through the activation of the transmembrane protein TrkA, these cells undergo a progressive mitotic arrest and start to grow electrically excitable neuritis. A vast number of studies opened intriguing aspects of NGF mechanisms of action, its biological properties, and potential use as therapeutic agents. In this context, identifying and utilizing small portions of NGF is of great interest and involves several human diseases including Alzheimer's disease. Here we report the specific action of the peptide encompassing the 1-14 sequence of the human NGF (NGF(1-14)), identified on the basis of scattered indications present in literature. The biological activity of NGF(1-14) was tested on PC12 cells, and its binding with TrkA was predicted by means of a computational approach. NGF(1-14) does not elicit the neurite outgrowth promoting activity, typical of the whole protein, and it only has a moderate action on PC12 proliferation. However, this peptide exerts, in a dose and time dependent fashion, an effective and specific NGF-like action on some highly conserved and biologically crucial intermediates of its intracellular targets such as Akt and CREB. These findings indicate that not all TrkA pathways must be at all times operative, and open the possibility of testing each of them in relation with specific NGF needs, biological actions, and potential therapeutic use.

Heptapeptide Semax, encompassing the sequence 4-7 of N-terminal domain of the adrenocorticotropic hormone (ACTH) and a C-terminal Pro-Gly-Pro tripeptide, belongs to a short regulatory peptides family. This compound has been found to affect learning processes and to exert marked neuroprotective activities on cognitive brain functions. Dys-homeostasis of metal ions is involved in several neurodegenerative disorders and growing evidences have showed that brain is a specialized organ able to concentrate metal ions. In this work, the metal binding ability and protective activity of Semax and its metal complexes were studied. The equilibrium study clearly demonstrated the presence of three complex species. Two minor species [CuL] and [CuLH-1]- co-exist together with the [CuLH-2]2- in the pH range from 3.6 to 5. From pH5 the [CuLH-2]2- species becomes predominant with the donor atoms around copper arranged in a 4N planar coordination mode. Noteworthy, a reduced copper induced cytotoxicity was observed in the presence of Semax by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay on a SHSY5Y neuroblastoma and RBE4 endothelial cell lines., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Amylin is a 37-residue peptide hormone produced by the islet β-cells of pancreas and the formation of amylin aggregates is strongly associated with β-cell degeneration in type 2 diabetes, as demonstrated by more than 95% of patients exhibiting amylin amyloid upon autopsy. It is widely recognized that metal ions such as copper(II) have been implicated in the aggregation process of amyloidogenic peptides such as Aβ and α-synuclein and there is evidence that amylin self-assembly is also largely affected by copper(II). For this reason, in this work, the role of copper(II) in the aggregation of amylin has been investigated by several different experimental approaches. Mass spectrometric investigations show that copper(II) induces significant changes in the amylin structure, which decrease the protein fibrillogenesis as observed by ThT measurements. Accordingly, solid-state NMR experiments together with computational analysis carried out on a model amylin fragment confirmed the non-fibrillogenic nature of the copper(II) induced aggregated structure. Finally, the presence of copper(II) is also shown to have a major influence on amylin proneness to be degraded by proteases and cytotoxicity studies on different cell cultures are reported.

Brain-derived neurotrophic factor (BDNF) is a neurotrophin essential for neuronal differentiation, growth, and survival; it is involved in memory formation and higher cognitive functions. The N-terminal domain of BDNF is crucial for the binding selectivity and activation of its specific TrkB receptor. Zn(2+) ion binding may influence BDNF activity. Zn(2+) complexes with the peptide fragment BDNF(1-12) encompassing the sequence 1-12 of the N-terminal domain of BDNF were studied by means of potentiometry, electrospray mass spectrometry, NMR, and density functional theory (DFT) approaches. The predominant Zn(2+) complex species, at physiological pH, is [ZnL] in which the metal ion is bound to an amino, an imidazole, and two water molecules (NH2, N(Im), and 2O(water)) in a tetrahedral environment. DFT-based geometry optimization of the zinc coordination environment showed a hydrogen bond between the carboxylate and a water molecule bound to zinc in [ZnL]. The coordination features of the acetylated form [AcBDNF(1-12)] and of a single mutated peptide [BDNF(1-12)D3N] were also characterized, highlighting the role of the imidazole side chain as the first anchoring site and ruling out the direct involvement of the aspartate residue in the metal binding. Zn(2+) addition to the cell culture medium induces an increase in the proliferative activity of the BDNF(1-12) peptide and of the whole protein on the SHSY5Y neuroblastoma cell line. The effect of Zn(2+) is opposite to that previously observed for Cu(2+) addition, which determines a decrease in the proliferative activity for both peptide and protein, suggesting that these metals might discriminate and modulate differently the activity of BDNF.

Antisense oligonucleotides are promising therapeutic agents against a variety of diseases. Effective delivery of these molecules is critical in view of their clinical application. Despite the richness of synthetic strategies addressed to the lipophilic modification of oligodeoxynucleotides (ODNs) for enhancing their pharmacokinetic behavior and trans-membrane delivery, the phosphatidyl group (1,2-di-O-acyl-sn-glycero-3-phosphoryl) has been never used as the lipophilic moiety of lipid-ODN conjugates. The present paper reports a general procedure for synthesizing 5'-phosphatidyl-ODNs. By this procedure, phosphatidyl conjugates of a VEGF antisense-ODN have been prepared, which differ in the fatty acid composition of their phosphatidyl moiety. These new lipid-ODN conjugates, which have been characterized on the basis of their physicochemical properties, showed an improved resistance to exonucleases and were able to lower the VEGF-mRNA expression in human SH-SY5Y neuroblastoma cells more effectively than the relevant free antisense-ODN did.

Brain-derived neurotrophic factor (BDNF) is a neurotrophin that influences development, maintenance, survival, and synaptic plasticity of central and peripheral nervous systems. Altered BDNF signaling is involved in several neurodegenerative disorders including Alzheimer's disease. Metal ions may influence the BDNF activity and it is well known that the alteration of Cu(2+) homeostasis is a prominent factor in the development of neurological pathologies. The N-terminal domain of BDNF represents the recognition site of its specific receptor TrkB, and metal ions interaction with this protein domain may influence the protein/receptor interaction. In spite of this, no data inherent the interaction of BDNF with Cu(2+) ions has been reported up to now. Cu(2+) complexes of the peptide fragment BDNF(1-12) encompassing the sequence 1-12 of N-terminal domain of human BDNF protein were characterized by means of potentiometry, spectroscopic methods (UV/Vis, CD, EPR), parallel tempering simulations and DFT-geometry optimizations. Coordination features of the acetylated form, Ac-BDNF(1-12), were also characterized to understand the involvement of the terminal amino group. Whereas, an analogous peptide, BDNF(1-12)D3N, in which the aspartate residue was substituted by an asparagine, was synthesized to provide evidence on the possible role of carboxylate group in Cu(2+) coordination. The results demonstrated that the amino group is involved in metal binding and the metal coordination environment of the predominant complex species at physiological pH consisted of one amino group, two amide nitrogen atoms, and one carboxylate group. Noteworthy, a strong decrease of the proliferative activity of both BDNF(1-12) and the whole protein on a SHSY5Y neuroblastoma cell line was found after treatment in the presence of Cu(2+). The effect of metal addition is opposite to that observed for the analogous fragment of nerve growth factor (NGF) protein, highlighting the role of specific domains, and suggesting that Cu(2+) may drive different pathways for the BDNF and NGF in physiological as well as pathological conditions., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The recent metal hypothesis represents an attempt of a new interpretation key of Alzheimer's disease (AD) to overcome the limits of amyloid cascade. Neurons need to maintain metal ions within a narrow range of concentrations to avoid a detrimental alteration of their homeostasis, guaranteed by a network of specific metal ion transporters and chaperones. Indeed, it is well known that transition metal ions take part in neuromodulation/neurotrasmission. In addition, they are prominent factors in the development and exacerbation of neurodegeneration. Neurotrophins are proteins involved in development, maintenance, survival and synaptic plasticity of central and peripheral nervous systems. A neurotrophin hypothesis of AD has been proposed, whereas the link between neurotrophic factor, the amyloid cascade and biometals has not been taken into account. As a matter of fact, there is a significant overlap between brain areas featured by metal ion dys-homeostasis, and those where the neurotrophins exert their biological activity. Metal ions can directly modulate their activities, through conformational changes, and/or indirectly by activating their downstream signaling in a neurotrophin-independent mode. The focus of this review is on the molecular aspects of Zn(2+) and Cu(2+) interactions with neurotrophins, with the aim to shed light on the intricate mechanisms involving metallostasis and proteostasis in AD., (Copyright © 2011 Elsevier Inc. All rights reserved.)

GHHPH is the peptide repeat present in histidine-proline rich glycoprotein (HPRG), a plasma glycoprotein involved in angiogenesis process. The copper(II) ions interaction with mono (Ac-GHHPHG-NH(2)) and its bis-repeat (Ac-GHHPHGHHPHG-NH(2)) was investigated by means of potentiometric and spectroscopic techniques. To single out the copper(II) coordination environments of different species formed with Ac-GHHPHG-NH(2), three single point mutated peptides were also synthesized and their ability to coordinate Cu(2+) investigated. Ac-GHHPHG-NH(2) binds Cu(2+) by the imidazole side chain and the amide nitrogen deprotonation that takes place towards the N-terminus. The bis-repeat is able to bind Cu(2+) more efficiently than Ac-GHHPHG-NH(2). This difference is not only due to the number of His residues in the sequence but also to the different binding sites. In fact, the comparison of the potentiometric and spectroscopic data of the copper(II) complexes with a bis-repeatPeg construct Ac-(GHHPHG)-Peg-(GHHPHG)-NH(2) and those of the metal complexes with Ac-HGHH-NH(2), indicates that the central HGHH amino acid sequence is the main copper(II) binding site., (Copyright © 2012 Elsevier Inc. All rights reserved.)

There is a significant overlap between brain areas with Zn(2+) and Cu(2+) pathological dys-homeostasis and those in which the nerve growth factor (NGF) performs its biological role. The protein NGF is necessary for the development and maintenance of the sympathetic and sensory nervous systems. Its flexible N-terminal region has been shown to be a critical domain for TrkA receptor binding and activation. Computational analyses show that Zn(2+) and Cu(2+) form pentacoordinate complexes involving both the His4 and His8 residues of the N-terminal domain of one monomeric unit and the His84 and Asp105 residues of the other monomeric unit of the NGF active dimer. To date, neither experimental data on the coordination features have been reported, nor has one of the hypotheses according to which Zn(2+) and Cu(2+) may have different binding environments or the Ser1 α-amino group could be involved in coordination been supported. The peptide fragment, encompassing the 1-14 sequence of the human NGF amino-terminal domain (NGF(1-14)), blocked at the C terminus, was synthesised and its Cu(2+) and Zn(2+) complexes characterized by means of potentiometric and spectroscopic (UV/Vis, CD, NMR, and EPR) techniques. The N-terminus-acetylated form of NGF(1-14) was also investigated to evaluate the involvement of the Ser1 α-amino group in metal-ion coordination. Our results demonstrate that the amino group is the first anchoring site for Cu(2+) and is involved in Zn(2+) coordination at physiological pH. Finally, a synergic proliferative activity of both NGF(1-14) and the whole protein on SHSY5Y neuroblastoma cell line was found after treatment in the presence of Cu(2+). This effect was not observed after treatment with the N-acetylated peptide fragment, demonstrating a functional involvement of the N-terminal amino group in metal binding and peptide activity., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Poly(ADP-ribose) polymerases (PARPs) play a crucial role in DNA damage surveillance through their nick sensor functions. Since PARPs' over activation leads to an excessive consumption of NAD(+) and ATP depletion, these enzymes also are involved in the early events of programmed cell death as well as in necrosis. In order to verify the protective action of L: -carnosine and trehalose against NO induced cell death, in the present study we examined their effects on the expression of PARP-1, PARP-2 and iNOS in primary rat astrocyte and oligodendrocyte cells, treated with lipopolysaccharide (LPS) and interferon gamma (INFγ), through semi-quantitative PCR and western analysis. To further characterize the molecular mechanisms underlying L-carnosine and trehalose action, we measured cell viability, nitrite production and LDH release. The data obtained clearly demonstrate that in the stress model employed L-carnosine and trehalose down regulate PARP-1 and PARP-2 expression in both cell phenotypes, thus suggesting their possible application in clinical trials.

Mildly denaturing conditions induce bovine alpha-crystallin, the major structural lens protein, to self-assemble into fibrillar structures in vitro. The natural dipeptide l-carnosine has been shown to have potential protective and therapeutic significance in many diseases. Carnosine derivatives have been proposed as potent agents for ophthalmic therapies of senile cataracts and diabetic ocular complications. Here we report the inhibitory effect induced by the peptide (l- and d-enantiomeric form) on alpha-crystallin fibrillation and the almost complete restoration of the chaperone activity lost after denaturant and/or heat stress. Scanning force microscopy (SFM), thioflavin T, and a turbidimetry assay have been used to determine the morphology of alpha-crystallin aggregates in the presence and absence of carnosine. DSC and a near-UV CD assay evidenced that the structural precursors of amyloid fibrils are polypeptide chain segments that lack stable structural elements. Moreover, we have found a disassembling effect of carnosine on alpha-crystallin amyloid fibrils. Finally, we show the ability of carnosine to restore most of the lens transparency in organ-cultured rat lenses exposed to similar denaturing conditions that were used for the in vitro experiments.

The enzyme poly(ADP-ribose)polymerase (PARP) has a leader role in the DNA damage survey mechanisms by its nick-sensor function, but it is also involved in the early events of the programmed cell death, particularly during inflammatory injury, as a coactivator of NF-kB. In the present study, we evaluated the PARP involvement in the mechanisms of protection and/or cell death in rat astroglial cell cultures during the early phase of proinflammatory commitment after lipopolysaccharide and interferon gamma treatment. According with the recent findings that PARP-1 phosphorylation by MAPK/ERK-2 pathway seems to modulate PARP activation, in time course experiments we demonstrated that a very early PARP activation and expression is able to trigger a cell death pathway, DNA damage independent, during strong proinflammatory insults, maintaining its role of guardian of the genome stability only during the normal cell cycling.

The neuropeptide carnosine (beta-amyloid peptide aggregation has been demonstrated. Carnosine protection against peroxynitrite damage is particularly relevant, but until now there has been no evidence of any direct interaction with nitric oxide. In this study we examined the protection that carnosine provides against nitric oxide (NO)-induced cell death in primary rat astroglial cell cultures treated with lipopolysaccharide (LPS) and interferon gamma (INFgamma), a well-known neurotoxic proinflammatory condition. A correlation was found between cell protection and NO free-radical scavenging activity of carnosine. Moreover, by competitive spectrophotometric measurement and electrospray mass spectrometry analysis in cell-free experiments, we demonstrated a direct interaction of the dipeptide with NO. A comparison of carnosine with its homologues or derivatives (homocarnosine and carcinine) as well as with its amino acid constituents (L-histidine and beta-alanine) highlighted that only histidine showed significant scavenging activity. Therefore, carnosine shows direct NO-trapping ability and may be a valuable multifunctional molecule in the treatment of neurodegenerative disorders., ((c) 2007 Wiley-Liss, Inc.)

alpha-Crystallin in its native state is a large, heterogeneous, low-molecular weight (LMW) aggregate that under certain conditions may progressively became part of insoluble high-molecular weight (HMW) systems. These systems are supposed to play a relevant role in eye lens opacification and vision impairment. In this paper, we report the effects of trehalose on alpha-crystallin aggregates. The role of trehalose in alpha-crystallin stress tolerance, chaperone activity and thermal stability is studied. The results show that trehalose stabilizes the alpha-crystallin native structure, inhibits alpha-crystallin aggregation, and disaggregates preformed LMW systems not affecting its chaperone activity.

Age-related increase of reactive oxygen species (ROS) is particularly detrimental in postmitotic tissues. Calorie restriction (CR) has been shown to exert beneficial effects, consistent with reduced ROS generation by mitochondria. Many antioxidant compounds also mimic such effects. N-acetyl cysteine (NAC) provides thiol groups to glutathione and to mitochondrial respiratory chain proteins; thus, it may counteract both ROS generation and effects. In the present study we investigated, in different rat brain areas during aging (6, 12, and 28 months), the effect of 1-year treatment with CR and dietary supplementation with NAC on the expression of subunit 39 kDa and ND-1 (mitochondrial respiratory complex I), subunit IV (complex IV), subunit alpha of F0F1-ATP synthase (complex V) and of adenine nucleotide translocator, isoform 1 (ANT-1). The observed age-related changes of expression were prevented by the dietary treatments. The present study provides further evidence for the critical role of mitochondria in the aging process.

Vascular endothelial growth factor (VEGF) has been shown to play a major role in intraocular neovascularisation in ischaemic retinal diseases. The aim of this study was to evaluate the concentration of VEGF in vitreous, aqueous and epiretinal membranes of diabetic and non-diabetic patients, with other pathological conditions requiring surgical intervention. Higher VEGF concentration were found in samples from the eyes of diabetic patients versus other pathologies as well as in epiretinal membranes versus the other eye compartments in diabetic patients. However, high VEGF levels were also found in retinal detachment and proliferative vitreoretinopathy of non-diabetic patients. We concluded that VEGF is produced locally and plays a fundamental, but not specific, role in diabetic retinal neovascularisation and proliferation.

A great deal of increasing evidence designs PARP as a multifunctional protein implicated in many cellular functions. Much interest is emerging to understand the precise mechanisms by which PARP mediates genome stabilization and protection against damage, as well as its involvement in cell death, either apoptotic or necrotic. Aside from the clearly established role of PARP hyperactivation in necrotic cell death, after excessive DNA damage and energy failure, it appears to be actively involved in the phenomenon of apoptosis. However, its exact role is still controversial. The identification of several enzymes sharing the poly(ADP-ribose) polymerase catalytic domain (PARPs), but with different features and subcellular localization, has opened a new perspective in the field of poly(ADP-ribosyl)ation. The picture of the role of PARP in the control of cell homeostasis became even more complex after demonstration of its implication in the regulation of gene transcription. The notion that energy failure is the sole mechanism by which PARP promotes cell death is therefore under reevaluation.

Over the last 20 years the JAK/STAT signal transduction pathway has been extensively studied. An enormous amount of data on different cell signal transduction pathways is now available. The JAK/STAT signal transduction pathway is one of the intracellular signaling pathways activated by cytokines and growth factors that was first studied in the hematopoietic system, but recent data demonstrate that this signal transduction is also greatly utilized by other systems. The JAK/STAT pathway is a signaling cascade that links the activation of specific cell membrane receptors to nuclear gene expression. This review is focused on the role of JAK/STAT signal transduction pathway activation in the central nervous system (CNS).

The production of nitric oxide by the calcium-independent inducible nitric oxide synthase (iNOS) in glial cells has been implicated in the neuropathogenesis of various diseases. It is well known that in response to lipopolysaccharide (LPS) and cytokines, such as IFN-gamma, glial cells are induced to synthesize large amount of nitric oxide (NO) (Bolaños et al., 1996; Nicoletti et al., 1998). The signaling transduction pathways for iNOS transcription in astroglial cells have however not yet been established. Because IFN-gamma receptor chains are associated with Janus tyrosine kinases (JAK1 and JAK2) (Darnell et al., 1994), we analyzed the involvement of the JAK/STAT signal transduction pathway in iNOS expression. Our study shows increased JAK2 and STAT1 alpha/beta tyrosine phosphorylation in primary astroglial cell culture after treatment with IFN-gamma and LPS. A temporal correlation was observed between JAK2 and STAT1 alpha/beta tyrosine phosphorylation, the appearance of interferon-regulatory factor-1 (IRF-1) mRNA and the iNOS expression. Inhibition experiments showed that JAK2 and STAT1 alpha/beta tyrosine phosphorylation were necessary for IFN gamma-mediated iNOS induction in astroglial cells. We conclude that JAK2 and STAT1 alpha/beta tyrosine phosphorylation is an early event involved in the expression of iNOS in astroglial cells., (Copyright 2001 Wiley-Liss, Inc.)

Glial cells in the nervous system can produce nitric oxide in response to cytokines. This production is mediated by the inducible isoform of nitric oxide synthase. Radical oxygen species (ROS) and nitric oxide (NO) derivatives have been claimed to play a crucial role in many different processes, both physiological such as neuromodulation, synaptic plasticity, response to glutamate, and pathological such as ischemia and various neurodegenerative disorders. In the present study we investigated the effects of NO synthase (iNOS) induction in astrocyte cultures on the synthesis of heat shock proteins, the activity of respiratory chain complexes and the oxidant/antioxidant balance. Treatment of astrocyte cultures for 18 hr with LPS and INFgamma produced a dose dependent increase of iNOS associated with an increased synthesis of hsp70 stress proteins. This effect was abolished by the NO synthase inhibitor L-NMMA and significantly decreased by addition of SOD/CAT in the medium. Time course experiments showed that iNOS induced protein expression increased significantly by 2 hr after treatment with LPS and INFgamma and reached a plateau at 18 hr; hsp70 protein synthesis peaked around 18 and 36 hr after the same treatment. Addition to astrocytes of the NO donor sodium nitroprusside resulted in a dose dependent increase in hsp70 protein that was comparable to that found after a mild heat shock. Additionally, a decrease in cytochrome oxidase activity, a marked decrease in ATP and protein sulfhydryl contents, an increase in the activity of the antioxidant enzymes mt-SOD and catalase were found which were abolished by L-NMMA. These findings suggest the importance of mitochondrial energy impairment as a critical determinant of the susceptibility of astrocytes to neurotoxic processes and point to a possible pivotal role of hsp70 in the signalling pathways of stress tolerance., (Copyright 2000 Wiley-Liss, Inc.)

GFAPbeta mRNA is an alternative transcript of the glial fibrillary acidic protein (GFAP) gene, whose transcriptional start site is located 169 nucleotides upstream to the classical GFAPalpha mRNA. By an RT-PCR method with primers on separate exons, we were able to confirm the presence of GFAP transcripts with a longer 5' untranslated region in all the examined areas of rat brain and in primary cultures of astroglial cells. Northern blot analysis, using an oligoprobe specific for the 5' region of GFAPbeta, revealed a single hybridization band of 2.9 kb in all the brain regions examined and in primary cultures of astroglial cells. The availability of the quantitative Northern blot assay allowed further studies on the regulation of GFAPbeta expression in vivo. Since it is well-known that neuronal brain injury is one of the most powerful inducers of GFAP, we examined the expression of GFAPalpha and beta after a neurotoxic lesion in the rat hippocampus. Results obtained show a parallel increase in both GFAP transcripts with an identical time-course, suggesting that regulatory regions of the gene influence in similar way the rate of transcription at the two different start sites (alpha and beta) or that a similar post-transcriptional mechanism is involved in regulating both mRNA isoforms.

The glial fibrillary acidic protein (GFAP) is expressed in a cell-specific manner and represents the major subunit of intermediate filaments of astroglial cells. The knowledge of the gene structure is an important step for further understanding the mechanisms of cell-specific expression. In the present study, we report the complete sequence of the rat GFAP gene and provide evidence for the existence, in the rat brain, of a novel alternative transcript. Since three different transcripts, indicated as GFAPalpha, beta, and gamma, have been previously reported (Feinstein et al. [1992] J. Neurosci. Res. 32:1-14; Zelenika et al. [1995] Mol. Brain Res. 30:251-258), we called this novel mRNA isoform GFAPdelta. It is generated by the alternative splicing of a novel exon located in the classic seventh intron. This alternative exon (called VII+) contains a 101-bp coding sequence in frame with exon VII and interrupted by a stop codon TAA at position +5451. Therefore, the novel GFAPdelta transcript encodes for an hypothetical GFAP where the forty-two carboxy-terminal amino acids encoded by exon VIII and IX are replaced by thirty-three amino acids encoded by exon VII+. Northern blot analysis with a specific probe for exon VII+ revealed a 4.2-kb mRNA, expressed in several brain areas, but absent in extracerebral tissues (lung, heart, kidney, liver, spleen). The previously discovered GFAP isoforms (alpha, beta, and gamma) produce hypothetical translation products differing in the amino-terminal Head domain. The present data suggest, for the first time, the possible existence of GFAP isoforms differing in the carboxy-terminal Tail domain.

In the present study we evaluated the effects of NO synthase (NOS) induction on the regulation of cytochrome c oxidase (CO) and F0F1-ATPase subunit expression in astroglial and mixed cortical cell cultures. In mixed cortical cell cultures, 18 h of treatment with lipopolysaccharide (LPS, 0.1 microgram/mL) plus interferon-gamma (INF-gamma, 10 U/mL) caused an increase of mRNAs for CO-I, F0F1-ATPase 6 and also for iNOS at 20 DIV. The induction of both CO-I and F0F1-ATPase 6 was abolished by the NOS inhibitor N-monomethyl-L-arginine (NMMA) or by the enzymatic scavenger superoxide dismutase/catalase (SOD/CAT). In primary astroglial cell cultures, treatment for 18 h with increasing concentrations of LPS and INF gamma, produced an increase in the amount of mitochondrial encoded CO-I and -II subunits, with no significant modifications of nuclear encoded subunit IV. An increase was also observed at level of transcription for CO-I and -II, and F0F1-ATPase 6 mRNAs. These effects were abolished by addition of NMMA or SOD/CAT. mRNA induction of CO-I was higher in mixed cortical than in astroglial cell cultures while that of F0F1-ATPase 6 was similar in both cell types. These results suggest that the expression of mitochondrial encoded subunits (CO-I, CO-II and F0F1-ATPase 6) is up-regulated in response to oxygen and NO reactive species. The activity of cytochrome c oxidase decreased after LPS/INF gamma treatment in both astroglial and mixed cortical cultures. The activity of ATP synthase was unmodified, while ATP content drastically decreased after LPS/INF gamma treatment, in both astroglial and mixed cortical cultures. The enzymatic activities of catalase and Mn-SOD (mitochondrial) showed a significant increase after LPS/INF gamma treatment, which was abolished by NMMA.

In the present study we analyzed the age-dependent changes of mRNA levels for cytochrome c oxidase and FoF1-ATP synthase subunits in rat cerebral cortex and cerebellum. To establish whether the regulation of expression is transcriptional or post-transcriptional, the results were compared to those related to protein subunits levels, of the same enzymatic complexes, previously observed. The different patterns of age-related changes of mRNA subunits, in particular the lower increments, compared with those related to protein subunits, indicate that post-transcriptional mechanisms of regulation might be involved in the coordinated expression of the various subunits of each complex. Northern blotting analyses of RNA from the cerebellum of rats at the various ages, showed also differences in age-dependent patterns of transcription between cerebral cortex and cerebellum. Moreover, the major age-dependent changes of mitochondrial-encoded subunits, compared with the nuclear-encoded ones, previously observed at proteins level, occur also during transcription.