The role of endoproteolytic processing in neurodegeneration

Endoproteolysis is a normal post-translational process in the eukaryotic cell that had played a role early on in protein evolution allowing protein catabolism and the generation of amino acids. Endoproteolytic cleavage regulates many crucial cellular processes including the activity of many proteins, their protein-protein interactions and the amplification of cell signals. Not surprisingly, disruption or alternation of endoproteolytic cleavage maybe the root cause of many human diseases such as Alzheimer’s disease, Huntington’s disease and prion diseases. Most neurodegenerative diseases (ND) are caused by the build-up of misfolded proteins and the promotion of aggregation events. A common event that occurs in these ND is the alteration of endoproteolytic cleavage due to genetic mutations of the associated-proteases or in the target substrate. Endoproteolytic cleavage resulting in protein truncation has significant effects on the structure and function of a protein representing a common feature of ND. In this review, we will discuss the endoproteolytic cleavage events that lead to ND, namely Alzheimer’s disease, Huntington’s disease and prion diseases

Prevention of the β-amyloid peptide-induced inflammatory process by inhibition of double-stranded RNA-dependent protein kinase in primary murine mixed co-cultures

<p>Abstract</p> <p>Background</p> <p>Inflammation may be involved in the pathogenesis of Alzheimer's disease (AD). There has been little success with anti-inflammatory drugs in AD, while the promise of anti-inflammatory treatment is more evident in experimental models. A new anti-inflammatory strategy requires a better understanding of molecular mechanisms. Among the plethora of signaling pathways activated by β-amyloid (Aβ) peptides, the nuclear factor-kappa B (NF-κB) pathway could be an interesting target. In virus-infected cells, double-stranded RNA-dependent protein kinase (PKR) controls the NF-κB signaling pathway. It is well-known that PKR is activated in AD. This led us to study the effect of a specific inhibitor of PKR on the Aβ42-induced inflammatory response in primary mixed murine co-cultures, allowing interactions between neurons, astrocytes and microglia.</p> <p>Methods</p> <p>Primary mixed murine co-cultures were prepared in three steps: a primary culture of astrocytes and microglia for 14 days, then a primary culture of neurons and astrocytes which were cultured with microglia purified from the first culture. Before exposure to Aβ neurotoxicity (72 h), co-cultures were treated with compound C16, a specific inhibitor of PKR. Levels of tumor necrosis factor-α (TNFα), interleukin (IL)-1β, and IL-6 were assessed by ELISA. Levels of P_T451-PKR and activation of IκB, NF-κB and caspase-3 were assessed by western blotting. Apoptosis was also followed using annexin V-FITC immunostaining kit. Subcellular distribution of P_T451-PKR was assessed by confocal immunofluorescence and morphological structure of cells by scanning electron microscopy. Data were analysed using one-way ANOVA followed by a Newman-Keuls' post hoc test</p> <p>Results</p> <p>In these co-cultures, PKR inhibition prevented Aβ42-induced activation of IκB and NF-κB, strongly decreased production and release of tumor necrosis factor (TNFα) and interleukin (IL)-1β, and limited apoptosis.</p> <p>Conclusion</p> <p>In spite of the complexity of the innate immune response, PKR inhibition could be an interesting anti-inflammatory strategy in AD.</p

Intravitreal triamcinolone acetonide: Pattern of secondary intraocular pressure rise and possible risk factors

Ziad F Bashshur1, Abdallah M Terro1, Christelle P El Haibi1, Akaber M Halawi1, Alexandre Schakal2, Baha&amp;rsquo; N Noureddin11The Department of Ophthalmology, American University of Beirut, Lebanon; 2The Department of Ophthalmology, Hotel Dieu de France (St. Joseph University), LebanonPurpose: To determine the pattern of increase in intraocular pressure (IOP) following intravitreal triamcinolone acetonide (IVTA) and identify possible risk factors associated with this rise in IOP.Methods: We carried out a retrospective review of records for 185 patients (226 eyes) who received 4 mg of IVTA at the American University of Beirut Medical Center and Hotel Dieu de France eye clinics between 2003 and 2005.Results: Mean follow-up was 8.17 months (range 6 to 24 months). The mean number of IVTA injections per eye was 1.31 &amp;plusmn; 0.69. The mean IOP increased after the first IVTA injection from 15.04 &amp;plusmn; 3.18 mmHg at baseline to a mean maximum of 17.20 &amp;plusmn; 5.75 mmHg (p &amp;lt; 0.0001, paired t-test) at month 3 of follow-up with a return to mean baseline IOP (15.49 &amp;plusmn; 4.79 mmHg) at month 12. Fifty nine of 226 eyes showed IOP higher than 21 mmHg during follow-up. Nine eyes started to have IOP greater than 21 mmHg, 6 to 12 months after a single injection. Intraocular pressure lowering medications were started when IOP exceeded 25 mmHg in 15 of the 226 eyes studied. No risk factors have been found to predict this IOP rise.Conclusions: IOP elevation can occur in a significant number of eyes receiving 4 mg of IVTA. This phenomenon seems to be transient and a small number of eyes required treatment during this period. Eyes that received IVTA need to be monitored for IOP changes especially during the first 3 months, but the IOP may still rise 6 months and even 12 months after a single injection. This study did not show any risk factor that may predict this IOP rise.Keywords: intravitreal triamcinolone acetonide, intraocular pressure elevation, diabetic macular edema, choroidal neovascular membrane due to age-related macular degeneration, central retinal vein occlusion, Branch retinal vein occlusion, Uveiti

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Autophagy dysfunction and its link to Alzheimer's disease and type II diabetes mellitus.

Epidemiological data testifies the increasing incidence of Alzheimer's disease (AD) and type 2 diabetes mellitus (T2DM). Some associations were made between occidental lifestyle and development of these pathologies, moreover AD and T2DM are linked since each pathology is a causative risk factor for the other. Interestingly, autophagy, a catabolic pathway whose efficiency declines with age is importantly impaired in the affected tissues. Autophagy regulation is dependent of cell metabolic status and consequently on the 5'AMP-activated protein kinase (AMPK) and mammalian target of rapamycin signaling pathways. These pathways are altered with aging and molecular, pharmacological and physiological interventions increase lifespan in various organismal models and favours healthy aging diminishing the occurrence of age-related diseases such as diabetes, cancer, cardiovascular and neurodegenerative pathologies. Decreasing calorie intake has been known for a long time to have a beneficial effect on longevity and health. Some drug agonists of AMPK are known to mimic these effects such as metformin or resveratrol, a polyphenol extracted from plants and present in red wine, a component of the French paradox related diet. In this review, we present the epidemiological and pathogenesis links existing between AD and T2DM with an insight into the perturbations of the autophagic process highlighting the crucial role of the AMPK in development of age and metabolic related diseases. Hence, in a last part we will discuss the possible interventions susceptible to combat both T2DM and AD

In vivo activation and nuclear translocation of phosphorylated glycogen synthase kinase-3β in neuronal apoptosis: Links to tau phosphorylation

The roles of glycogen synthase kinase-3β (GSK-3β) and tau phosphorylation were examined in seven-day-old rats injected with the NMDA receptor antagonist (MK801) that is known to induce neuronal apoptosis. Immunoblot and immunohistochemical analysis of brain samples demonstrated a site-specific increase in tau phosphorylation associated with the relocalization of the protein to the nuclear/perinuclear region of apoptotic neurons. In addition, a tau 32-kDa fragment was detected, suggesting that tau was a target of intracellular proteolysis in MK801-treated brains. The proteolytically modified form of tau has reduced ability to bind to microtubules. GSK-3β kinase assay and immunoblottings of active (tyrosine-216) and inactive (serine-9) forms of GSK-3β revealed a rapid and transient increase in the kinase activity. Lithium chloride, a GSK-3β inhibitor, prevented tau phosphorylation suggesting that tau phosphorylation is mediated by the activation of GSK-3β. Confocal microscopy using double labelling of tau and GSK-3β revealed that the activation of GSK-3β in neurons was associated with early (2 h) nuclear translocation of tyrosine-216 GSK-3β. The execution phase of neuronal apoptosis was accompanied by a selective phosphorylation of serine-9 and dephosphorylation of tyrosine-216 GSK-3β. These findings demonstrate that in vivo, GSK-3β kinase activation and nuclear translocation are early stress signals of neuronal apoptosis.link_to_subscribed_fulltex

Aneuploidy studies in human cells exposed in vitro to GSM-900 MHz Radiofrequency Radiation using FISH

International audiencePURPOSE:Since previous research found an increase in the rate of aneuploidies in human lymphocytes exposed to radiofrequencies, it seems important to perform further studies. The objective of this study was then to investigate whether the exposure to RF (radiofrequency) radiation similar to that emitted by mobile phones of a second generation standard, i.e., Global System for Mobile communication (GSM) may induce aneuploidy in cultured human cells. MATERIALS AND METHODS:The potential induction of genomic instability by GSM-900 MHz radiofrequency (GSM-900) was investigated after in vitro exposure of human amniotic cells for 24 h to average-specific absorption rates (SAR) of 0.25, 1, 2 and 4 W/kg in the temperature range of 36.3-39.7°C. The exposures were carried out in a wire-patch cell (WPC). The rate of aneuploidy of chromosomes 11 and 17 was determined by interphase FISH (Fluorescence In Situ Hybridisation) immediately after independent exposure of three different donors for 24 h. At least 100 interphase cells were analysed per assay. RESULTS:No significant change in the rate of aneuploidy of chromosomes 11 and 17 was found following exposure to GSM-900 for 24 h at average SAR up to 4 W/kg. CONCLUSION:Our study did not show any in vitro aneuploidogenic effect of GSM using FISH and is not in agreement with the results of previous research

In vitro Cytogenetic effects of GSM-900 MHz on human cells

International audienc