Synaptic Autoregulation by Metalloproteases and -Secretase

The proteoloytic machinery comprising metalloproteases and γ-secretase, an intramembrane aspartyl protease involved in Alzheimer’s disease, cleaves several substrates besides the extensively studied amyloid precursor protein (APP). Some of these substrates, such as N-cadherin, are synaptic proteins involved in synapse remodeling and maintenance. Here we show, in rat and mice that metalloproteases and γ-secretase are physiologic regulators of synapses. Both proteases are synaptic, with γ-secretase tethered at the synapse by δ-catenin, a synaptic scafolding protein which also binds to N-cadherin and, through scaffolds, to α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor (AMPAR) and a metalloprotease. Activity-dependent proteolysis by metalloproteases and γ-secretase takes place at both sides of the synapse, with the metalloprotease cleavage being N-methyl-D-aspartic acid receptor (NMDAR)-dependent. This proteolysis decreases levels of synaptic proteins and diminishes synaptic transmission. Our results suggest that activity-dependent substrate cleavage by synaptic metalloproteases and γ-secretase modifies synaptic transmission, providing a novel form of synaptic autoregulation

Disruption of the IS6-AID Linker Affects Voltage-gated Calcium Channel Inactivation and Facilitation

Two processes dominate voltage-gated calcium channel (CaV) inactivation: voltage-dependent inactivation (VDI) and calcium-dependent inactivation (CDI). The CaVβ/CaVα1-I-II loop and Ca2+/calmodulin (CaM)/CaVα1–C-terminal tail complexes have been shown to modulate each, respectively. Nevertheless, how each complex couples to the pore and whether each affects inactivation independently have remained unresolved. Here, we demonstrate that the IS6–α-interaction domain (AID) linker provides a rigid connection between the pore and CaVβ/I-II loop complex by showing that IS6-AID linker polyglycine mutations accelerate CaV1.2 (L-type) and CaV2.1 (P/Q-type) VDI. Remarkably, mutations that either break the rigid IS6-AID linker connection or disrupt CaVβ/I-II association sharply decelerate CDI and reduce a second Ca2+/CaM/CaVα1–C-terminal–mediated process known as calcium-dependent facilitation. Collectively, the data strongly suggest that components traditionally associated solely with VDI, CaVβ and the IS6-AID linker, are essential for calcium-dependent modulation, and that both CaVβ-dependent and CaM-dependent components couple to the pore by a common mechanism requiring CaVβ and an intact IS6-AID linker

High-Resolution Mass Spectrometry for Human Exposomics: Expanding Chemical Space Coverage

In the modern "omics" era, measurement of the human exposome is a critical missing link between genetic drivers and disease outcomes. High-resolution mass spectrometry (HRMS), routinely used in proteomics and metabolomics, has emerged as a leading technology to broadly profile chemical exposure agents and related biomolecules for accurate mass measurement, high sensitivity, rapid data acquisition, and increased resolution of chemical space. Non-targeted approaches are increasingly accessible, supporting a shift from conventional hypothesis-driven, quantitation-centric targeted analyses toward data-driven, hypothesis-generating chemical exposome-wide profiling. However, HRMS-based exposomics encounters unique challenges. New analytical and computational infrastructures are needed to expand the analysis coverage through streamlined, scalable, and harmonized workflows and data pipelines that permit longitudinal chemical exposome tracking, retrospective validation, and multi-omics integration for meaningful health-oriented inferences. In this article, we survey the literature on state-of-the-art HRMS-based technologies, review current analytical workflows and informatic pipelines, and provide an up-to-date reference on exposomic approaches for chemists, toxicologists, epidemiologists, care providers, and stakeholders in health sciences and medicine. We propose efforts to benchmark fit-for-purpose platforms for expanding coverage of chemical space, including gas/liquid chromatography-HRMS (GC-HRMS and LC-HRMS), and discuss opportunities, challenges, and strategies to advance the burgeoning field of the exposome

Arachidonic acid inhibition of L-type calcium (CaV1.3b) channels varies with accessory CaVβ subunits

Arachidonic acid (AA) inhibits the activity of several different voltage-gated Ca2+ channels by an unknown mechanism at an unknown site. The Ca2+ channel pore-forming subunit (CaVα1) is a candidate for the site of AA inhibition because T-type Ca2+ channels, which do not require accessory subunits for expression, are inhibited by AA. Here, we report the unanticipated role of accessory CaVβ subunits on the inhibition of CaV1.3b L-type (L-) current by AA. Whole cell Ba2+ currents were measured from recombinant channels expressed in human embryonic kidney 293 cells at a test potential of −10 mV from a holding potential of −90 mV. A one-minute exposure to 10 µM AA inhibited currents with β1b, β3, or β4 58, 51, or 44%, respectively, but with β2a only 31%. At a more depolarized holding potential of −60 mV, currents were inhibited to a lesser degree. These data are best explained by a simple model where AA stabilizes CaV1.3b in a deep closed-channel conformation, resulting in current inhibition. Consistent with this hypothesis, inhibition by AA occurred in the absence of test pulses, indicating that channels do not need to open to become inhibited. AA had no effect on the voltage dependence of holding potential–dependent inactivation or on recovery from inactivation regardless of CaVβ subunit. Unexpectedly, kinetic analysis revealed evidence for two populations of L-channels that exhibit willing and reluctant gating previously described for CaV2 channels. AA preferentially inhibited reluctant gating channels, revealing the accelerated kinetics of willing channels. Additionally, we discovered that the palmitoyl groups of β2a interfere with inhibition by AA. Our novel findings that the CaVβ subunit alters kinetic changes and magnitude of inhibition by AA suggest that CaVβ expression may regulate how AA modulates Ca2+-dependent processes that rely on L-channels, such as gene expression, enzyme activation, secretion, and membrane excitability

Bovine proteins containing poly-glutamine repeats are often polymorphic and enriched for components of transcriptional regulatory complexes

peer-reviewedBackground: About forty human diseases are caused by repeat instability mutations. A distinct subset of these diseases is the result of extreme expansions of polymorphic trinucleotide repeats; typically CAG repeats encoding poly-glutamine (poly-Q) tracts in proteins. Polymorphic repeat length variation is also apparent in human poly-Q encoding genes from normal individuals. As these coding sequence repeats are subject to selection in mammals, it has been suggested that normal variations in some of these typically highly conserved genes are implicated in morphological differences between species and phenotypic variations within species. At present, poly-Q encoding genes in non-human mammalian species are poorly documented, as are their functions and propensities for polymorphic variation. Results: The current investigation identified 178 bovine poly-Q encoding genes (Q ≥ 5) and within this group, 26 genes with orthologs in both human and mouse that did not contain poly-Q repeats. The bovine poly-Q encoding genes typically had ubiquitous expression patterns although there was bias towards expression in epithelia, brain and testes. They were also characterised by unusually large sizes. Analysis of gene ontology terms revealed that the encoded proteins were strongly enriched for functions associated with transcriptional regulation and many contributed to physical interaction networks in the nucleus where they presumably act cooperatively in transcriptional regulatory complexes. In addition, the coding sequence CAG repeats in some bovine genes impacted mRNA splicing thereby generating unusual transcriptional diversity, which in at least one instance was tissue-specific. The poly-Q encoding genes were prioritised using multiple criteria for their likelihood of being polymorphic and then the highest ranking group was experimentally tested for polymorphic variation within a cattle diversity panel. Extensive and meiotically stable variation was identified. Conclusions: Transcriptional diversity can potentially be generated in poly-Q encoding genes by the impact of CAG repeat tracts on mRNA alternative splicing. This effect, combined with the physical interactions of the encoded proteins in large transcriptional regulatory complexes suggests that polymorphic variations of proteins in these complexes have strong potential to affect phenotype.Dairy Australia (through the Innovative Dairy Cooperative Research Center

The carboxy-terminal fragment of α1A calcium channel preferentially aggregates in the cytoplasm of human spinocerebellar ataxia type 6 Purkinje cells

Spinocerebellar ataxia type 6 (SCA6) is an autosomal dominant neurodegenerative disease caused by a small polyglutamine (polyQ) expansion (control: 4–20Q; SCA6: 20–33Q) in the carboxyl(C)-terminal cytoplasmic domain of the α1A voltage-dependent calcium channel (Cav2.1). Although a 75–85-kDa Cav2.1 C-terminal fragment (CTF) is toxic in cultured cells, its existence in human brains and its role in SCA6 pathogenesis remains unknown. Here, we investigated whether the small polyQ expansion alters the expression pattern and intracellular distribution of Cav2.1 in human SCA6 brains. New antibodies against the Cav2.1 C-terminus were used in immunoblotting and immunohistochemistry. In the cerebella of six control individuals, the CTF was detected in sucrose- and SDS-soluble cytosolic fractions; in the cerebella of two SCA6 patients, it was additionally detected in SDS-insoluble cytosolic and sucrose-soluble nuclear fractions. In contrast, however, the CTF was not detected either in the nuclear fraction or in the SDS-insoluble cytosolic fraction of SCA6 extracerebellar tissues, indicating that the CTF being insoluble in the cytoplasm or mislocalized to the nucleus only in the SCA6 cerebellum. Immunohistochemistry revealed abundant aggregates in cell bodies and dendrites of SCA6 Purkinje cells (seven patients) but not in controls (n = 6). Recombinant CTF with a small polyQ expansion (rCTF-Q28) aggregated in cultured PC12 cells, but neither rCTF-Q13 (normal-length polyQ) nor full-length Cav2.1 with Q28 did. We conclude that SCA6 pathogenesis may be associated with the CTF, normally found in the cytoplasm, being aggregated in the cytoplasm and additionally distributed in the nucleus

Les canaux calciques neuronaux (bases moléculaires de l'inactivation et implication dans l'ataxie spino-cérébelleuse)

MONTPELLIER-BU Médecine (341722104) / SudocMONTPELLIER-BU Médecine UPM (341722108) / SudocPARIS-BIUM (751062103) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Déterminisme de I'eutrophisation de la retenue de Grangent (Loire) : Etude des apports en nutriments, de la dynamique des populations phytoplanctoniques et des relations phyto-zooplancton en 1990-1991

Dans le cadre d'une approche pluridisciplinaire de l'eutrophisation de la retenue de Grangent (Loire), nous avons étudié, en 1990-91 : – les cycles biogéochimiques de divers éléments chimiques et notamment du phosphore et de l'azote; – la physico-chimie de la colonne d'eau; – la dynamique des populations phyto et zooplanctoniques; – le couplage développement de Microcystis aeruginosa (M.a.)/ rapport massique N/P. Les apports externes en azote ont trois sources diffuses : le lessivage des sols couverts de végétation, les épandages d'engrais et les rejets urbains. Les apports en orthophosphates ont eux, une origine ponctuelle et urbaine. L'évolution du rapport N/P confirme cette origine : il prend des valeurs très faibles (2,5-5) en fin de période d'étiage en été. Les flux d'entrée de phosphore ont été estimés à 159 t par an (99 t pour la Loire, 38 pour I'Ondaine et 22 pour le Lizeron et la Semène, affluents arrivant directement dans la retenue). Le flux de sortie est de 86 t par an. Le stockage interne est donc de 73 t pour 1990. S'agissant du sédiment, le rapport N/P mesuré sur le matériel particulaire oscille autour de 2,3 (faible). La matière organique (MO) produite dans la retenue ne constitue qu'une part minoritaire du flux de sédimentation, le phosphore est associé a des complexes minéraux. Le rôle du sédiment dans l'enrichissement de la colonne d'eau est faible lorsque I'hypolimnion est oxygéné. Dans les eaux surnageantes du sédiment, le phosphore est sous forme organique. Dans les eaux interstitielles, le P-PO4 est assez abondant. C'est une source potentielle de P biodisponible en cas d'anoxie. Mais de ce fait, cela ne constitue pas le facteur déterminant des blooms cyanobactériens, l'anoxie ne survenant qu'après leur développement. Toute tentative de restauration de la retenue par oxygénation de I'hypolimnion et/ou l'épandage de composés accepteurs d'électrons sur le sédiment, parait donc illusoire. A la confluence de I'Ondaine on note une forte teneur en métaux lourds dans les sédiments, directement apportés par la rivière. Dans la partie amont de la retenue, la colonne d'eau est très stratifiée en été. En juillet, la forte minéralisation des eaux en profondeur témoigne de la dégradation de la MO sédimentée. Le développement des fleurs d'eau phytoplanctoniques, constituant la manifestation la plus évidente de l'eutrophisation, dépend directement des apports externes en phosphore et en azote. Toutefois, l'apparition des blooms de M.a. résulte de la chute du rapport massique N/P en dessous du seuil exigé par les algues (soit 7 ). Lorsqu'il atteint des valeurs inférieures à 5, les algues périclitent et laissent la place aux cyanobactéries qui acceptent un N/P faible. Cependant dès que ce rapport réaugmente en automne, les algues réapparaissent et M.a. régresse sans doute à cause du brassage des eaux et de ses médiocres qualités de compétiteur. Lorsque M.a. se développe, le contenu énergétique de la biomasse zooplanctonique chute de façon importante. La plupart des animaux refuse cette cyanobactérie dans leur nourriture. Ils survivent alors en consommant les réserves accumulées tant que le phytoplancton était composé d'algues, pour compenser le jeûne partiel. Ceci conduit à un dysfontionnement de l'ensemble du réseau trophique et même à la disparition de certaines populations dont le maintien est lié à la constitution de réserves importantes, avant une époque précise (p. ex. pour se reproduire)