Dysfunction of Mitochondria in Alzheimer's Disease: ANT and VDAC Interact with Toxic Proteins and Aid to Determine the Fate of Brain Cells.

Alzheimer's disease (AD), certainly the most widespread proteinopathy, has as classical neuropathological hallmarks, two groups of protein aggregates: senile plaques and neurofibrillary tangles. However, the research interest is rapidly gaining ground in a better understanding of other pathological features, first, of all the mitochondrial dysfunctions. Several pieces of evidence support the hypothesis that abnormal mitochondrial function may trigger aberrant processing of amyloid progenitor protein or tau and thus neurodegeneration. Here, our aim is to emphasize the role played by two 'bioenergetic' proteins inserted in the mitochondrial membranes, inner and outer, respectively, that is, the adenine nucleotide translocator (ANT) and the voltage-dependent anion channel (VDAC), in the progression of AD. To perform this, we will magnify the ANT and VDAC defects, which are measurable hallmarks of mitochondrial dysfunction, and collect all the existing information on their interaction with toxic Alzheimer's proteins. The pathological convergence of tau and amyloid ß-peptide (Aß) on mitochondria may finally explain why the therapeutic strategies used against the toxic forms of Aß or tau have not given promising results separately. Furthermore, the crucial role of ANT-1 and VDAC impairment in the onset/progression of AD opens a window for new therapeutic strategies aimed at preserving/improving mitochondrial function, which is suspected to be the driving force leading to plaque and tangle deposition in AD.

Dynamic structural determinants underlie the neurotoxicity of the N-terminal tau 26-44 peptide in Alzheimer's disease and other human tauopathies.

The intrinsically disordered tau protein plays a pivotal role in the pathogenesis of Alzheimer's disease (AD) and other human tauopathies. Abnormal post-translational modifications of tau, such as truncation, are causally involved in the onset/development of these neurodegenerative diseases. In this context, the AD-relevant N-terminal fragment mapping between 26 and 44 amino acids of protein (tau26-44) is interesting, being endowed with potent neurotoxic effects in vitro and in vivo. However, the understanding of the mechanism(s) of tau26-44 toxicity is a challenging task because, similarly to the full-length tau, it does not have a unique 3D structure but exists as dynamic ensemble of conformations. Here we use Atomic Force Spectroscopy, Small Angle X-ray Scattering and Molecular Dynamics simulation to gather structural and functional information on the tau26-44. We highlight the presence, the type and the location of its temporary secondary structures and we unveil the occurrence of relevant transient tertiary conformations that could contribute to tau26-44 toxicity. Data are compared with those obtained on the biologically-inactive, reverse-sequence (tau44-26 peptide) which has the same mass, charge, aminoacidic composition as well as the same overall unfolded character of tau26-44.

The Copper(II)-Assisted Connection between NGF and BDNF by Means of Nerve Growth Factor-Mimicking Short Peptides.

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. Cu2+ 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.

The Intersection of NGF/TrkA Signaling and Amyloid Precursor Protein Processing in Alzheimer's Disease Neuropathology.

Dysfunction of nerve growth factor (NGF) and its high-affinity Tropomyosin receptor kinase A (TrkA) receptor has been suggested to contribute to the selective degeneration of basal forebrain cholinergic neurons (BFCN) associated with the progressive cognitive decline in Alzheimer's disease (AD). The aim of this review is to describe our progress in elucidating the molecular mechanisms underlying the dynamic interplay between NGF/TrkA signaling and amyloid precursor protein (APP) metabolism within the context of AD neuropathology. This is mainly based on the finding that TrkA receptor binding to APP depends on a minimal stretch of ~20 amino acids located in the juxtamembrane/extracellular domain of APP that carries the α- and ß-secretase cleavage sites. Here, we provide evidence that: (i) NGF could be one of the "routing" proteins responsible for modulating the metabolism of APP from amyloidogenic towards non-amyloidogenic processing via binding to the TrkA receptor; (ii) the loss of NGF/TrkA signaling could be linked to sporadic AD contributing to the classical hallmarks of the neuropathology, such as synaptic loss, ß-amyloid peptide (Aß) deposition and tau abnormalities. These findings will hopefully help to design therapeutic strategies for AD treatment aimed at preserving cholinergic function and anti-amyloidogenic activity of the physiological NGF/TrkA pathway in the septo-hippocampal system.

4-Methylcoumarin derivatives with anti-inflammatory effects in activated microglial cells.

Inflammation contributes to the pathogenesis of neurodegenerative diseases and anti-inflammatory compounds may have a role in prevention or treatment of these pathologies. 4-Methylcoumarins are effective antioxidants with anti-inflammatory properties. In this study, the inhibitory effects of two 4-methylcoumarin derivatives, 7,8-dihydroxy-3-ethoxycarbonylmethyl-4-methylcoumarin (DHEMC) and 7,8-diacetoxy-3-ethoxycarbonylmethyl-4-methylcoumarin (DAEMC) were examined on the inflammatory processes induced by lipopolysaccharide (LPS) in activated primary rat microglial cultures. LPS-induced production of nitric oxide (NO, measured by Griess method) and other pro-inflammatory mediators, thromboxane (TX) B2 and prostaglandin (PG) E2 (both determined by radioimmunoassay (RIA)), as well as tumor necrosis factor (TNF)-α (determined by enzyme-linked immunosorbent assay (ELISA)) were inhibited in the presence of 100 µM DHEMC and DAEMC. DAEMC was able to significantly inhibit NO, TXB2 and TNF-α production also at 50 µM. Both compounds at 100 µM significantly lowered cyclooxygenase-2 (COX-2) protein expression in LPS-stimulated microglial cells measured by Western blot, but only DAEMC showed an inhibitory effect on inducible nitric oxide synthase (iNOS) protein expression at 100 µM. In conclusion, our findings show that 4-methylcoumarin derivatives can modulate inflammatory pathways in microglial cells, probably by acting at the protein expression level.

1-Phenil-6,7-dihydroxy-isochroman inhibits inflammatory activation of microglia.

Inflammation plays a central role in the pathogenesis of several brain disorders and neuronal injury, and it develops as a consequence of glial cell activation. Activated microglial cells generate potentially damaging nitric oxide, oxygen free radicals, prostanoids, and pro-inflammatory cytokines. Naturally occurring polyphenols have recently received attention for their potential protective effect on neurodegenerative disorders characterized by microglial activation, due to their anti-inflammatory and antioxidant properties. In the present study, we investigated, using an in vitro model of primary microglia, the ability of 1-phenyl-6,7-dihydroxy-isochroman (encoded L 137), a natural polyphenolic compound, to inhibit microglia activation induced by an inflammatory insult. So, L137 effects (1-100 µM) on production of pro-inflammatory mediators in lipopolysaccharide (LPS)-activated microglial cells were evaluated. The expression of inducible isoforms of nitric oxide synthase (iNOS) and cyclooxygenase (COX-2) as well as of the nuclear transcription Factor-kappa B (NF-κB) was also performed in cellular lysates by Immunoblot. L137 significantly reduced tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6 secretion, as well as nitric oxide (NO) and prostanoids [Thromboxane (TX)B2, prostaglandin (PG)E2] production in activated microglial cells. Western blot analyses showed an inhibitory effect of L137 on the iNOS and COX-2 expression, mediated by a modulation of redox-sensitive nuclear transcriptional factor (NF)-κB, known to control a wide array of genes involved in inflammation. In conclusion, this study demonstrate that L137 is able to inhibit the production of pro-inflammatory and neurotoxic mediators by LPS-activated microglial cells thus suggesting L137 as a potential lead compound for drug development for neurodegenerative disorders where microglia-mediated inflammatory responses play an important pathogenic role.

Beta-adrenoreceptor agonism influences retinal responses to hypoxia in a model of retinopathy of prematurity.

PURPOSE: In a mouse model of oxygen-induced retinopathy (OIR), a well-established model of retinopathy of prematurity (ROP), blocking beta-adrenoreceptors (ß-ARs), and, in particular, ß2-ARs, counteracts retinal responses to hypoxia. In the present work, we determined the effects of the ß-AR agonist isoproterenol on retinal angiogenesis and ß-AR signaling to better clarify the role of sympathetic transmission in ROP. METHODS: Isoproterenol was administered subcutaneously. Protein kinase A activity was determined by a colorimetric assay to assess drug effectiveness. Blood pressure and heart-to-body weight ratio were measured. Vascular endothelial growth factor (VEGF) and norepinephrine were measured with ELISA. Retinal neovascularization was assessed by CD31 immunohistochemistry. ß-AR-coupled adenylyl cyclase (AC) activity was measured with a competition assay. ß-ARs, G-protein-coupled receptor kinase (GRK)2, and ß-arrestins were determined by Western blot. Association of ß-arrestins with ß2-ARs was assessed by immunoprecipitation. RESULTS: Isoproterenol-induced modulation of protein kinase A activity suggests that the drug was effective at the receptor level. Isoproterenol did not affect cardiovascular parameters, but decreased retinal levels of VEGF and reduced pathogenic neovascularization, likely through an influence on sympathetic transmission. In fact, isoproterenol downregulated ß2-AR expression, recovered the hypoxia-induced increase in ß-AR-coupled AC activity, and increased GRK2 and ß-arrestins, which promote ß-AR desensitization through the uncoupling of G-protein-coupled receptors from G proteins. Immunoprecipitation studies demonstrated that ß-AR desensitization involved ß2-ARs. CONCLUSIONS: Our findings suggest that hypoxia-induced retinal neovascularization depends at least in part on increased sympathetic transmission, as reduction of sympathetic drive by agonist-induced ß2-AR desensitization inhibits some of the hallmarks of OIR.

Protective role of somatostatin receptor 2 against retinal degeneration in response to hypoxia.

In mouse retinal explants, octreotide, a somatostatin [somatotropin release-inhibiting factor (SRIF)] receptor 2 (sst(2)) agonist, prevents the hypoxia-induced vascular endothelial growth factor upregulation. In mice with oxygen-induced retinopathy (OIR), a model of retinopathy of prematurity, either sst(2) overexpression or octreotide have been found to limit hypoxia-induced angiogenic processes. Here, we investigated whether sst(2) influences retinal degeneration in response to hypoxia in wild-type (WT), sst(1)- and sst(2)-knockout (KO) mice. In retinal explants, we determined the role of sst(2) on apoptotic signals. In control condition, caspase-3 activity and the Bax/Bcl-2 ratio were lower in sst(1)-KO than in WT, but higher in sst(2)-KO than in WT retinas. In all strains, a comparable increase in caspase-3 activity and the Bax/Bcl-2 ratio was observed after hypoxia. The hypoxia-induced increase in apoptotic signals was recovered by octreotide in both WT and sst(1)-KO retinas. To investigate the role of sst(2) on retinal function, we recorded electroretinogram (ERG) in response to light flashes in OIR mice. ERG responses did not differ between WT and KO mice with the exception of oscillatory potentials (OPs) which, in sst(1)-KO mice, displayed much larger amplitude. In all strains, hypoxia drastically reduced a-, b-waves and OPs. In both WT and sst(1)-KO mice, octreotide recovered a- and b-waves, but did not recover OPs in sst(1)-KO mice. Neither apoptotic signals nor ERG was affected by octreotide in sst(2)-KO mice. These results show that sst(2) may protect retinal cells from hypoxia, thus implementing the background to establish potential pharmacological targets based on sst(2) pharmacology.

1-Phenyl-6,7-dihydroxy-isochroman suppresses lipopolysaccharide-induced pro-inflammatory mediator production in human monocytes.

Extra-virgin olive oil is an integral ingredient of the Mediterranean diet, and it has been suggested that its high consumption has beneficial effects on human health. Its protective effect, in particular against the development of CVD, has been related not only to the high content of oleic acid, but also to the antioxidant and anti-inflammatory properties of polyphenols. In order to verify the anti-inflammatory and anti-atherogenic properties of hydroxy-isochromans, a class of ortho-diphenols present in extra-virgin olive oil, we investigated the potential ability of 1-phenyl-6,7-dihydroxy-isochroman (L137) to modulate the production of key inflammatory mediators by human monocytes, by evaluating its in vitro effects on prostanoid (thromboxane A(2) and PGE(2)) and cytokine (TNF-α) production. Its effect on the protein expression of the inducible form of cyclo-oxygenase-2 (COX-2), a pro-inflammatory enzyme responsible for elevated prostanoid levels, was also explored. The results showed that L137 significantly inhibited both prostanoid and TNF-α production in lipopolysaccharide-primed human monocytes in a dose-dependent manner, by inhibiting the COX activity of COX-2. We also demonstrated that the effects of the isochroman are mediated, at least partly, through the suppression of NF-κB activation leading to the down-regulation of the synthesis of COX-2.

Cigarette smoke inhibits adenine nucleotide hydrolysis by human platelets.

Cigarette smoking is a recognized risk factor for cardiovascular diseases and has been implicated in the pathogenesis of atherosclerosis and thrombotic events. In athero-thrombotic diseases, the extracellular adenine nucleotides play an important role by triggering a range of effects such as the recruitment and activation of platelets, endothelial cell activation and vasoconstriction. NTPDase, a plasma membrane-bound enzyme, is the most relevant enzyme involved in the hydrolysis of extracellular tri- and di-phosphate nucleotides to adenosine monophosphate, which is further degraded by 5'ectonucleotidase to the anti-thrombotic and anti-inflammatory mediator adenosine. Thus, the preserved activity of these enzymes, regulating the extracellular concentrations of nucleotides, is critical in thromboregulatory functions. In the present in vitro study, performed on human platelets suspended in undiluted or diluted aqueous cigarette smoke extract (aCSE), we demonstrated that undiluted and 1 : 2 diluted aCSE is able to significantly reduce ADP hydrolysis (-24% and 12%, respectively) by intact human platelets. ATP degradation was also reduced (-31%) by undiluted aCSE. Conversely, aCSE did not alter platelet AMP hydrolysis. Results obtained by using N-acetylcysteine, a thiol-containing antioxidant, suggest that stable oxidants present in aCSE are responsible for the platelet NTPDase inhibition induced by aCSE. The decreased adenine nucleotide degradation could play a significant role in the extensive platelet activation and vascular inflammation observed in chronic smokers.