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Effects of familial Alzheimer's disease-linked presenilin 2 mutants on Ca2+ homeostasis of Golgi Apparatus sub-compartments
Full text linkAlzheimer's Disease (AD) is a progressive neurodegenerative disorder and the most common form of senile dementia. The characteristic histopathological hallmarks of AD are the intracellular neurofibrillary tangles and the amyloid plaques, made of aggregated amyloid peptides (Aß), that deposit in the extracellular matrix of the brain. Aß peptides are the result of two sequential cleavages of the amyloid precursor protein (APP); Aß is eventually released by the α-secretase enzyme. The most abundant Aß peptide species, both physiologically produced throughout life, are Aß40 and Aß42, which is more insoluble and aggregation-prone.
Although most AD cases are sporadic, a small percentage of patients is affected by the hereditary form of AD (Familial Alzheimer's Disease, FAD), caused by dominant mutations in one of three genes. These genes code for the APP, presenilin-1 (PS1) and presenilin-2 (PS2); PSs are the catalytic subunits of the α-secretase enzyme complex but they also function in a α-secretase indipendent manner.
FAD-linked mutations in PSs lead to an increased Aß42/Aß40 ratio, that promotes Aß plaques deposition. Beside this effect on Aß production, many mutations in PS1 and PS2 have been extensively demonstrated to cause alterations in the intracellular Ca2+ homeostasis, thus making neurons more sensitive to excitotoxic stimuli and apoptosis.
The Golgi apparatus (GA) represents, together with the endoplasmic reticulum (ER), the major IP3-sensitive, rapidly mobilizable, intracellular Ca2+ store and its functionality is thus important for shaping cytosolic Ca2+ responses. Increasing evidence suggests that the GA is an heterogeneous Ca2+ handling organelle, equipped with a diverse molecular Ca2+ toolkit compared to the one expressed in the ER. For example, as Ca2+ uptake mechanisms, the GA expresses the classical sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) but also an additional Ca2+ pump, the secretory pathway Ca2+ ATPase1, SPCA1.
The use of a specific Cameleon Ca2+ sensor targeted to the trans-Golgi, allowed us to directly demonstrate the functional GA heterogeneity by showing the distinct behavior of this sub-compartment: it takes up Ca2+ almost exclusively via SPCA1 (and not by SERCA); it does not release Ca2+ in response to IP3 generation, but rather accumulates the cation as a consequence of the cytoplasmic Ca2+ rise.
As regard to the other GA compartments, we generated a new FRET-based Ca2+ indicator fused to the cis/medial-Golgi targeting sequence of the enzyme 1,6 N-acetylglucosaminyltransferase (C2gnT). The new probe very nicely co-localizes with the cis/medial-Golgi marker Giantin and thus was used to study Ca2+ dynamics in this compartment at single cell level.
The data collected suggest that the GA is unique in terms of Ca2+ homeostasis, with compartments that are separated by a few microns, and in very rapid equilibrium with each other, that still maintain quite substantial differences in terms of ion concentration and response to external stimuli.
The differences between the two GA sub-compartments, the medial and the trans-one, are confirmed by the specific effect on Ca2+ homeostasis of the expression of the FAD-linked PS2 T122R mutation. Cells expressing the mutated form of the protein show a decreased Ca2+ content in the cis/medial-Golgi but no effects on trans-Golgi Ca2+ homeostasis. PS2-T122R seems to inhibiting Ca2+ uptake in the cis/medial-Golgi by inhibiting SERCA pump activity while does not affect Ca2+ uptake, mediated by SPCA1, in the trans-Golgi.
As a major Ca2+ store, the GA could play an important role in AD and understanding the contribution of GA Ca2+ dysfunction in AD will significantly impact our ability to develop more effective therapies for the disease.La malattia di Alzheimer's (AD) è un disordine neurodegenerativo e la forma più comune di demenza senile.
La caratteristica istopatologica di AD è la presenza di depositi neurofibrillari intracellulari e di placche amiloidi, costituite da aggregati di peptide amiloide (Aß), che si depositano nella matrice extracellulare del cervello. I peptidi Aß sono il risultato di due tagli sequenziali della Proteina Precursore dell'Amiloide (APP); Aß viene poi rilasciato dall'enzima α-secretasi. Le più abbondanti specie peptidiche di Aß, prodotte anche fisiologicamente per tutta la vita, sono Aß40 e Aß42, quest'ultimo più insolubile e più incline all'aggregazione.
Sebbene la maggior parte dei casi di AD siano sporadici, una piccola percentuale di pazienti è affetta dalla forma ereditaria di Alzheimer (malattia familiare di Alzheimer, FAD), causata da mutazioni dominanti in uno dei geni codificanti per APP, presenilina-1 (PS1) e presenilina-2 (PS2); le PSs sono le subunità catalitiche del complesso enzimatico della α-secretasi ma funzionano anche in maniera indipendente da tale attività enzimatica.
Le mutazioni in PSs legate a FAD portano ad un aumento nel rapporto Aß42/Aß40, che promuove la deposizione di placche amiloidi. Oltre a questo effetto, è stato ampiamente dimostrato che molte mutazioni in PS1 e PS2 provocano alterazioni della omeostasi del Ca2+ intracellulare, rendendo così i neuroni più sensibili agli stimoli eccitotossici e apoptotici.
L'apparato di Golgi (GA) rappresenta, insieme al reticolo endoplasmatico (ER), il principale deposito intracellulare di Ca2+, IP3 sensibile, e la sua funzionalità è fondamentale per il controllo delle risposte citosoliche di Ca2+.
Sempre maggiori evidenze suggeriscono che il GA sia un organello eterogeneo in termini di Ca2+ handling, essendo dotato di un diverso toolkit molecolare per il Ca2+ rispetto a quello espresso nell' ER. Ad esempio, come meccanismi di uptake per il Ca2+, il GA esprime la classica pompa SERCA (Sarco-Endoplasmic Reticulum Ca2+ ATPase) ma anche un ulteriore pompa, detta SPCA1 (Secretory Pathway Ca2+ ATPase1).
L'utilizzo di uno specifico sensore per il Ca2+ specificatamente indirizzato al trans-Golgi, ci ha precedentemente permesso di dimostrare direttamente la eterogeneità funzionale del GA, mostrando il comportamento distinto di questo sub-compartimento: i meccanismi di uptake di Ca2+ sono mediati esclusivamente dalla SPCA1 (e non dalla SERCA); non rilascia Ca2+ in risposta alla generazione IP3, ma piuttosto si accumula il catione come conseguenza dell'aumento di Ca2+ citoplasmatico.
Per quanto riguarda gli altri sub-compartimenti del GA, abbiamo generato un nuovo indicatore per il Ca2+ fuso alla sequenza di indirizzamento dell'enzima 1,6 N-acetylglucosaminyltransferasi (C2gnT) residente del cis/medial-Golgi. La nuova sonda co-localizza con il marcatore di cis/medial-Golgi Giantina e quindi è stata utilizzata per studiare le dinamiche di Ca2+ in questo sub-compartimento a livello di singola cellula.
Complessivamente i dati ottenuti suggeriscono che il GA sia unico in termini di omeostasi del Ca2+, con tali sub-compartimenti separati da pochi micron, e in equilibrio molto rapido tra loro, ma comunque in grado di mantenere differenze consistenti in termini di concentrazione dello ione e risposta a stimoli esterni .
Le differenze tra i due sub-compartimenti del GA sono confermate dall'effetto specifico sulla omeostasi del Ca2+ dell'espressione della forma mutata di PS2T122R legata alla malattia familiare di Alzheimer. Le cellule che esprimono tale proteina mostrano una diminuzione del contenuto di Ca2+ nel cis/medial-Golgi ma nessun effetto sull'omeostasi del Ca2+ nel trans-Golgi. PS2T122R sembra inibire l'assorbimento di Ca2+ nel cis/medial-Golgi, inibendo l'attività della pompa SERCA, mentre non influenza l'assorbimento di Ca2+, mediato dalla SPCA1, nel trans-Golgi.
Il GA sembra quindi giocare un ruolo importante nella patogenesi di AD e comprendere il contributo di tale organello nella patogenesi di AD e la sua base fisiopatologica potrà avere un forte impatto sulla possibilità di sviluppare terapie più efficaci per AD
Ca2+ signalling in the Golgi apparatus
No full textThe Golgi apparatus plays a central role in lipid and protein post-translational modification and sorting. Morphologically the organelle is heterogeneous and it is possible to distinguish stacks of flat cysternae (cis- and medial Golgi), tubular-reticular networks and vesicles (trans-Golgi). These morphological differences parallel a distinct functionality with a selective distribution and complementary roles of the enzymes found in the different compartments. The Golgi apparatus has been also shown to be involved in Ca(2+) signalling: it is indeed endowed with Ca(2+) pumps, Ca(2+) release channels and Ca(2+) binding proteins and is thought to participate in determining the spatio-temporal complexity of the Ca(2+) signal within the cell, though this role is still poorly understood. Recently, it has been demonstrated that the organelle is heterogeneous in terms of Ca(2+) handling and selective reduction of Ca(2+) concentration, both in vitro and in a genetic human disease, within one of its sub-compartment results in alterations of protein trafficking within the secretory pathway and of the entire Golgi morphology. In this paper we review the available information on the Ca(2+) toolkit within the Golgi, its heterogeneous distribution in the organelle sub-compartments and discuss the implications of these characteristics for the physiopathology of the Golgi apparatus
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Mitochondrial potassium channels in cell death
No full textMitochondria are intracellular organelles involved in several processes from bioenergetics to cell death. In the latest years, ion channels are arising as new possible targets in controlling several cellular functions. The discovery that several plasma membrane located ion channels have intracellular counterparts, has now implemented this consideration and the number of studies enforcing the understanding of their role in different metabolic pathways. In this review, we will discuss the recent updates in the field, focusing our attention on the involvement of potassium channels during mitochondrial mediated apoptotic cell death. Since mitochondria are one of the key organelles involved in this process, it is not surprising that potassium channels located in their inner membrane could be involved in modulating mitochondrial membrane potential, ROS production, and respiratory chain complexes functions. Eventually, these events lead to changes in the mitochondrial fitness that prelude to the cytochrome c release and apoptosis. In this scenario, both the inhibition and the activation of mitochondrial potassium channels could cause cell death, and their targeting could be a novel pharmacological way to treat different human diseases
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Heterogeneity of Ca2+ handling among and within Golgi compartments
No full textThe Golgi apparatus (GA) is a dynamic intracellular Ca2+ store endowed with complex Ca2+ homeostatic mechanisms in part distinct from those of the endoplasmic reticulum (ER). We describe the generation of a novel fluorescent Ca2+ probe selectively targeted to the medial GA. We demonstrate that in the medial-GA: i) Ca2+ accumulation takes advantage of two distinct pumps, the sarco-endoplasmic reticulum Ca2+ ATPase and the secretory pathway Ca2+ ATPase1; ii) activation of IP3 or ryanodine receptors causes Ca2+ release, while no functional two-pore channel was found; iii) luminal Ca2+ concentration appears higher than that of the trans-Golgi, but lower than that of the ER, suggesting the existence of a cis- to trans-Golgi Ca2+ concentration gradient. Thus, the GA represents a Ca2+ store of high complexity where, despite the continuous flow of membranes and luminal contents, each sub-compartment maintains its Ca2+ identity with specific Ca2+ homeostatic characteristics. The functional role of such micro-heterogeneity in GA Ca2+ handling is discussed
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Familial Alzheimer’s Disease-linked Presenilin mutants and intracellular Ca2+ handling: a single-organelle, FRET-based analysis
No full textAn imbalance in Ca 2+ homeostasis represents an early event in the pathogenesis of Alzheimer's disease (AD). Presenilin-1 and -2 (PS1 and PS2) mutations, the major cause of familial AD (FAD), have been extensively associated with alterations in different Ca 2+ signaling pathways, in particular those handled by storage compartments. However, FAD-PSs effect on organelles Ca 2+ content is still debated and the mechanism of action of mutant proteins is unclear. To fulfil the need of a direct investigation of intracellular stores Ca 2+ dynamics, we here present a detailed and quantitative single-cell analysis of FAD-PSs effects on organelle Ca 2+ handling using specifically targeted, FRET (Fluorescence/Förster Resonance Energy Transfer)-based Ca 2+ indicators. In SH-SY5Y human neuroblastoma cells and in patient-derived fibroblasts expressing different FAD-PSs mutations, we directly measured Ca 2+ concentration within the main intracellular Ca 2+ stores, e.g., Endoplasmic Reticulum (ER) and Golgi Apparatus (GA) medial- and trans-compartment. We unambiguously demonstrate that the expression of FAD-PS2 mutants, but not FAD-PS1, in either SH-SY5Y cells or FAD patient-derived fibroblasts, is able to alter Ca 2+ handling of ER and medial-GA, but not trans-GA, reducing, compared to control cells, the Ca 2+ content within these organelles by partially blocking SERCA (Sarco/Endoplasmic Reticulum Ca 2+ -ATPase) activity. Moreover, by using a cytosolic Ca 2+ probe, we show that the expression of both FAD-PS1 and -PS2 reduces the Ca 2+ influx activated by stores depletion (Store-Operated Ca 2+ Entry; SOCE), by decreasing the expression levels of one of the key molecules, STIM1 (STromal Interaction Molecule 1), controlling this pathway. Our data indicate that FAD-linked PSs mutants differentially modulate the Ca 2+ content of intracellular stores yet leading to a complex dysregulation of Ca 2+ homeostasis, which represents a common disease phenotype of AD
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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