Silencing of Amyloid Precursor Protein Expression Using a New Engineered Delta Ribozyme

Alzheimer's disease (AD) etiological studies suggest that an elevation in amyloid-β peptides (Aβ) level contributes to aggregations of the peptide and subsequent development of the disease. The major constituent of these amyloid peptides is the 1 to 40–42 residue peptide (Aβ40−42) derived from amyloid protein precursor (APP). Most likely, reducing Aβ levels in the brain may block both its aggregation and neurotoxicity and would be beneficial for patients with AD. Among the several possible ways to lower Aβ accumulation in the cells, we have selectively chosen to target the primary step in the Aβ cascade, namely, to reduce APP gene expression. Toward this end, we engineered specific SOFA-HDV ribozymes, a new generation of catalytic RNA tools, to decrease APP mRNA levels. Additionally, we demonstrated that APP-ribozymes are effective at decreasing APP mRNA and protein levels as well as Aβ levels in neuronal cells. Our results could lay the groundwork for a new protective treatment for AD

Variability in Responses to Phoma medicaginis Infection in a Tunisian Collection of Three Annual Medicago Species

Spring black stem and leaf spot, caused by Phoma medicaginis, is an issue in annual Medicago species. Therefore, in this study, we analyzed the response to P. medicaginis infection in a collection of 46 lines of three annual Medicago species (M. truncatula, M. ciliaris, and M. polymorpha) showing different geographic distribution in Tunisia. The reaction in the host to the disease is explained by the effects based on plant species, lines nested within species, treatment, the interaction of species × treatment, and the interaction of lines nested within species × treatment. Medicago ciliaris was the least affected for aerial growth under infection. Furthermore, the largest variation within species was found for M. truncatula under both conditions. Principal component analysis and hierarchical classification showed that M. ciliaris lines formed a separate group under control treatment and P. medicaginis infection and they are the most vigorous in growth. These results indicate that M. ciliaris is the least susceptible in response to P. medicaginis infection among the three Medicago species investigated here, which can be used as a good candidate in crop rotation to reduce disease pressure in the field and as a source of P. medicaginis resistance for the improvement of forage legumes

Re-engineering a neuroprotective, clinical drug as a procognitive agent with high in vivo potency and with GABAA potentiating activity for use in dementia

Background Synaptic dysfunction is a key event in pathogenesis of neurodegenerative diseases such as Alzheimer’s disease (AD) where synapse loss pathologically correlates with cognitive decline and dementia. Although evidence suggests that aberrant protein production and aggregation are the causative factors in familial subsets of such diseases, drugs singularly targeting these hallmark proteins, such as amyloid-β, have failed in late stage clinical trials. Therefore, to provide a successful disease-modifying compound and address synaptic dysfunction and memory loss in AD and mixed pathology dementia, we repurposed a clinically proven drug, CMZ, with neuroprotective and anti-inflammatory properties via addition of nitric oxide (NO) and cGMP signaling property. Results The novel compound, NMZ, was shown to retain the GABAA potentiating actions of CMZ in vitro and sedative activity in vivo. Importantly, NMZ restored LTP in hippocampal slices from AD transgenic mice, whereas CMZ was without effect. NMZ reversed amnestic blockade of acetylcholine receptors by scopolamine as well as NMDA receptor blockade by a benzodiazepine and a NO synthase inhibitor in the step-through passive avoidance (STPA) test of learning and working memory. A PK/PD relationship was developed based on STPA analysis coupled with pharmacokinetic measures of drug levels in the brain: at 1 nM concentration in brain and plasma, NMZ was able to restore memory consolidation in mice. Conclusion Our findings show that NMZ embodies a promising pharmacological approach targeting synaptic dysfunction and opens new avenues for neuroprotective intervention strategies in mixed pathology AD, neurodegeneration, and dementia

Développement de nouvelles stratégies anti-amyloïdes ciblant la protéine précurseur amyloïde pour le traitement de la maladie d'alzheimer

La maladie d'Alzheimer (MA) est la forme la plus commune des maladies neurodegeneratives qui se caractérise par un déclin progressif et continu des fonctions cognitives et par des lésions histopathologiques caractéristiques : les plaques seniles et les dégénérescences neurofibrillaires. Avec une incidence qui augmente de façon vertigineuse, la MA représente un défi thérapeutique mais aussi un enjeu économique majeur. À ce jour, malgré les progrès réalisés dans la compréhension de la maladie au cours de 20 dernières années, aucun traitement efficace n'est disponible pour ralentir ou arrêter l'évolution de la maladie. Le peptide amyloïde (Aβ) principal constituant des plaques seniles, est issu du clivage amyloïdogénique de la protéine précurseur amyloïde (APP) suite à l'action des sécrétases bêta et gamma. L'accumulation extracellulaire de l'Aβ initie une cascade d'événements neuropathologiques, encore incomplètement comprise, connue sous le nom de la cascade amyloïde qui entraine un dysfonctionnent synaptique accompagné d'une mort neuronale intensive. Plusieurs évidences indiquent que l'APP joue un rôle déterminant dans l'étiologie de la MA. Les études génétiques menées à partir de formes familiales ont montré que toutes les mutations géniques pathogènes répertoriées à ce jour, avaient une incidence directe soit sur l'expression, soit sur le métabolisme de l'APP et, par conséquent, sur l'accumulation du peptide Aβ. La surexpression de l'ARNm cellulaire de l'APP étant étroitement et positivement corrélée à l'expression en aval de la protéine APP et le niveau de la production de l'Aβ. Ainsi, l'APP apparaît comme une cible pertinente pour le traitement de la MA. Son inhibition permettrait de bloquer l'accumulation et la toxicité liée à l'Aβ responsable de la pathogenèse observée dans la MA. Ce travail décrit le développement d'un outil d'inhibition moléculaire basé sur l'utilisation du ribozyme delta. Une nouvelle génération de ribozyme appelée SOFA-ð-Ribozyme a été spécialement construite pour cibler l'ARNm de l'APP. Nous avons démontré que ces APP-ð-Ribozymes assurent une correction efficace des niveaux de l'APP et permettent le rétablissement de la production aberrante d'Aβ dans les cellules neuronales. Cette intervention est donc considérée comme une étape importante et cruciale pour corriger les mécanismes qui animent l'augmentation des dépôts amyloïdes dans le but d'influencer les autres acteurs de cascade amyloïde. Nous nous sommes penchés ensuite sur la possibilité de trouver une approche alternative à l'inhibition des sécrétases impliquées dans la libération pleeà'A intervenant directement au niveau de leurs sites d'action sur l'APP. Pour cela un criblage d'une banque de peptides aléatoires à été réalisé grâce à l'utilisation du système double hybride chez la levure dans le but de sélectionner de nouvelles interactions in vivo peptides-APP capables interférer avec les activités sécrétases et d'empêcher la libération de l'Amyloïde. Cette approche est particulièrement pertinente, puisque ces enzymes possèdent différents substrats et leur inhibition directe peut avoir des effets secondaires indésirables voir fatals. Dans la mesure où la bêta-sécrétase (BACE) est l'enzyme limitant dans la génération de l'Aβ, la découverte d'une nouvelle génération d'inhibiteur agissant directement sur l'APP serait d'une importance majeure pour le développement d'une thérapie appropriée à la MA.

FANCC localizes with UNC5A at neurite outgrowth and promotes neuritogenesis

Abstract Objective The Uncoordinated 5A (UNC5A) protein is part of a family of receptors that play roles in axonal pathfinding and cell migration. We previously showed that the Fanconi anemia C protein (FANCC) interacts with UNC5A and delays UNC5A-mediated apoptosis. FANCC is a predominantly cytoplasmic protein that has multiple functions including DNA damage signaling, oxygen radical metabolism, signal transduction, transcriptional regulation and apoptosis. Given the direct interaction between FANCC and UNC5A and that FANCC interferes with UNC5A-mediated apoptosis, we explored the possibility that FANCC might play a role in axonal-like growth processes. Results Here we show that FANCC and UNC5A are localized to regions of neurite outgrowth during neuronal cell differentiation. We also show that absence of FANCC is required for neurite outgrowth. In addition, FANCC seems required for UNC5A expression. Results from this study combined with our previous report suggest that FANCC plays a role in tissue development through the regulation of UNC5A-mediated functions

The Fanconi anemia group C protein interacts with uncoordinated 5A and delays apoptosis.

The Fanconi anemia group C protein (FANCC) is one of the several proteins that comprise the Fanconi anemia (FA) network involved in genomic surveillance. FANCC is mainly cytoplasmic and has many functions, including apoptosis suppression through caspase-mediated proteolytic processing. Here, we examined the role of FANCC proteolytic fragments by identifying their binding partners. We performed a yeast two-hybrid screen with caspase-mediated FANCC cleavage products and identified the dependence receptor uncoordinated-5A (UNC5A) protein. Here, we show that FANCC physically interacts with UNC5A, a pro-apoptotic dependence receptor. FANCC interaction occurs through the UNC5A intracellular domain, specifically via its death domain. FANCC modulates cell sensitivity to UNC5A-mediated apoptosis; we observed reduced UNC5A-mediated apoptosis in the presence of FANCC and increased apoptosis in FANCC-depleted cells. Our results show that FANCC interferes with UNC5A's functions in apoptosis and suggest that FANCC may participate in developmental processes through association with the dependence receptor UNC5A

Overexpression of quality control proteins reduces prion conversion in prion-infected cells

Prion diseases are fatal infectious neurodegenerative disorders in humans and other animals and are caused by misfolding of the cellular prion protein (PrPC) into the pathological isoform PrPSc These diseases have the potential to transmit within or between species, including zoonotic transmission to humans. Elucidating the molecular and cellular mechanisms underlying prion propagation and transmission is therefore critical for developing molecular strategies for disease intervention. We have shown previously that impaired quality control mechanisms directly influence prion propagation. In this study, we manipulated cellular quality control pathways in vitro by stably and transiently overexpressing selected quality control folding (ERp57) and cargo (VIP36) proteins and investigated the effects of this overexpression on prion propagation. We found that ERp57 or VIP36 overexpression in persistently prion-infected neuroblastoma cells significantly reduces the amount of PrPSc in immunoblots and prion-seeding activity in the real-time quaking-induced conversion (RT-QuIC) assay. Using different cell lines infected with various prion strains confirmed that this effect is not cell type- or prion strain-specific. Moreover, de novo prion infection revealed that the overexpression significantly reduced newly formed PrPSc in acutely infected cells. ERp57-overexpressing cells significantly overcame endoplasmic reticulum stress, as revealed by expression of lower levels of the stress markers BiP and CHOP, accompanied by a decrease in PrP aggregates. Furthermore, application of ERp57-expressing lentiviruses prolonged the survival of prion-infected mice. Taken together, improved cellular quality control via ERp57 or VIP36 overexpression impairs prion propagation and could be utilized as a potential therapeutic strategy

A new type of scorpion Na+-channel-toxin-like polypeptide active on K+ channels.

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