Apolipoprotein D synthesis progressively increases in frontal cortex during human lifespan

Apolipoprotein D (apo D) is a lipocalin present in the nervous system that may be related to processes of reinnervation, regeneration and neuronal cell protection. In the other way, apo D expression has been correlated, in some brain regions, with normal aging and neurodegenerative diseases. To elucidate the regional and cellular expression of apo D in normal human brain during aging, we performed a detailed and extensive study in samples of post-mortem human cerebral cortices. To achieve this study, slot blot techniques, for protein and mRNA, as well as immunohistochemistry and hybridohistochemistry methods were used. A positive correlation for apo D expression with aging was found; furthermore, mRNA levels, as well as the protein ones, were higher in the white than in the grey matter. Immunohistochemistry and non-isotopic HIS showed that apo D is synthesized in both neurons and glial cells. Apo D expression is notorious in oligodendrocytes but with aging the number of neurons that synthesize apo D is increased. Our results indicate that apo D could play a fundamental role in central nervous system aging and in the reduction of products derivated from lipid peroxidation. The increment in the expression of apo D with aging can be included in a global mechanism of cellular protection to prevent the deleterious effects caused by aging

NMDA receptors are selectively partitioned into complexes and supercomplexes during synapse maturation

How neuronal proteomes self-organize is poorly understood because of their inherent molecular and cellular complexity. Here, focusing on mammalian synapses we use blue-native PAGE and ‘gene-tagging’ of GluN1 to report the first biochemical purification of endogenous NMDA receptors (NMDARs) directly from adult mouse brain. We show that NMDARs partition between two discrete populations of receptor complexes and B1.5MDa supercomplexes. We tested the assembly mechanism with six mouse mutants, which indicates a tripartite requirement of GluN2B, PSD93 and PSD95 gate the incorporation of receptors into B1.5MDa supercomplexes, independent of either canonical PDZ-ligands or GluN2A. Supporting the essential role of GluN2B, quantitative gene-tagging revealed a fourfold molar excess of GluN2B over GluN2A in adult forebrain. NMDAR supercomplexes are assembled late in postnatal development and triggered by synapse maturation involving epigenetic and activity-dependent mechanisms. Finally, screening the quaternary organization of 60 native proteins identified numerous discrete supercomplexes that populate the mammalian synapse

Preserved dopaminergic homeostasis and dopamine-related behaviour in hemizygous TH-Cre mice

Cre-driver mouse lines have been extensively used as genetic tools to target and manipulate genetically defined neuronal populations by expression of Cre recombinase under selected gene promoters. This approach has greatly advanced neuroscience but interpretations are hampered by the fact that most Cre-driver lines have not been thoroughly characterized. Thus, a phenotypic characterization is of major importance to reveal potential aberrant phenotypes prior to implementation and usage to selectively inactivate or induce transgene expression. Here, we present a biochemical and behavioural assessment of the dopaminergic system in hemizygous tyrosine hydroxylase (TH)-Cre mice in comparison to wild-type (WT) controls. Our data show that TH-Cre mice display preserved dopaminergic homeostasis with unaltered levels of TH and dopamine as well as unaffected dopamine turnover in striatum. TH-Cre mice also show preserved dopamine transporter expression and function supporting sustained dopaminergic transmission. In addition, TH-Cre mice demonstrate normal responses in basic behavioural paradigms related to dopaminergic signaling including locomotor activity, reward preference and anxiolytic behaviour. Our results suggest that TH-Cre mice represent a valid tool to study the dopamine system, though careful characterization must always be performed to prevent false interpretations following Cre-dependent transgene expression and manipulation of selected neuronal pathways

Membrane-permeable C-terminal dopamine transporter peptides attenuate amphetamine-evoked dopamine release

The dopamine transporter (DAT) is responsible for sequestration of extracellular dopamine (DA). The psychostimulant amphetamine (AMPH) is a DAT substrate, which is actively transported into the nerve terminal, eliciting vesicular depletion and reversal of DA transport via DAT. Here, we investigate the role of the DAT C terminus in AMPH-evoked DA efflux using cell-permeant dominant-negative peptides. A peptide, which corresponded to the last 24 C-terminal residues of DAT (TAT-C24 DAT) and thereby contained the Ca2+-calmodulin-dependent protein kinase IIα (CaMKIIα) binding domain and the PSD-95/Discs-large/ZO-1 (PDZ)-binding sequence of DAT, was made membrane-permeable by fusing it to the cell membrane transduction domain of the HIV-1 Tat protein (TAT-C24WT). The ability of TAT-C24WT but not a scrambled peptide (TAT-C24Scr) to block the CaMKIIα-DAT interaction was supported by co-immunoprecipitation experiments in heterologous cells. In heterologous cells, we also found that TAT-C24WT, but not TAT-C24Scr, decreased AMPH-evoked 1-methyl-4-phenylpyridinium efflux. Moreover, chronoamperometric recordings in striatum revealed diminished AMPH-evoked DA efflux in mice preinjected with TAT-C24WT. Both in heterologous cells and in striatum, the peptide did not further inhibit efflux upon KN-93-mediated inhibition of CaMKIIα activity, consistent with a dominant-negative action preventing binding of CaMKIIα to the DAT C terminus. This was further supported by the ability of a peptide with perturbed PDZ-binding sequence, but preserved CaMKIIα binding (TAT-C24AAA), to diminish AMPH-evoked DA efflux in vivo to the same extent as TAT-C24WT. Finally, AMPH-induced locomotor hyperactivity was attenuated following systemic administration of TAT-C24WT but not TAT-C24Scr. Summarized, our findings substantiate that DAT C-terminal protein-protein interactions are critical for AMPH-evoked DA efflux and suggest that it may be possible to target protein-protein interactions to modulate transporter function and interfere with psychostimulant effects.Mattias Rickhag, William A. Owens, Marie-Therese Winkler, Kristine Nørgaard Strandfelt, Mette Rathje, Gunnar Sørensen, Bjørn Andresen, Kenneth L. Madsen, Trine Nygaard Jørgensen, Gitta Wörtwein, David P. D. Woldbye, Harald Sitte, Lynette C. Daws, and Ulrik Gethe

PICK1 Deficiency Impairs Secretory Vesicle Biogenesis and Leads to Growth Retardation and Decreased Glucose Tolerance

<div><p>Secretory vesicles in endocrine cells store hormones such as growth hormone (GH) and insulin before their release into the bloodstream. The molecular mechanisms governing budding of immature secretory vesicles from the trans-Golgi network (TGN) and their subsequent maturation remain unclear. Here, we identify the lipid binding BAR (Bin/amphiphysin/Rvs) domain protein PICK1 (protein interacting with C kinase 1) as a key component early in the biogenesis of secretory vesicles in GH-producing cells. Both PICK1-deficient <i>Drosophila</i> and mice displayed somatic growth retardation. Growth retardation was rescued in flies by reintroducing PICK1 in neurosecretory cells producing somatotropic peptides. PICK1-deficient mice were characterized by decreased body weight and length, increased fat accumulation, impaired GH secretion, and decreased storage of GH in the pituitary. Decreased GH storage was supported by electron microscopy showing prominent reduction in secretory vesicle number. Evidence was also obtained for impaired insulin secretion associated with decreased glucose tolerance. PICK1 localized in cells to immature secretory vesicles, and the PICK1 BAR domain was shown by live imaging to associate with vesicles budding from the TGN and to possess membrane-sculpting properties in vitro. In mouse pituitary, PICK1 co-localized with the BAR domain protein ICA69, and PICK1 deficiency abolished ICA69 protein expression. In the <i>Drosophila</i> brain, PICK1 and ICA69 co-immunoprecipitated and showed mutually dependent expression. Finally, both in a <i>Drosophila</i> model of type 2 diabetes and in high-fat-diet-induced obese mice, we observed up-regulation of PICK1 mRNA expression. Our findings suggest that PICK1, together with ICA69, is critical during budding of immature secretory vesicles from the TGN and thus for vesicular storage of GH and possibly other hormones. The data link two BAR domain proteins to membrane remodeling processes in the secretory pathway of peptidergic endocrine cells and support an important role of PICK1/ICA69 in maintenance of metabolic homeostasis.</p></div

PICK1 associates transiently with the Golgi compartment and is capable of tubular deformation of liposomes in vitro.

<p>(A) Brief (5 min) brefeldin A treatment traps PICK1 in the trans-Golgi. Confocal images of GH1 cells immunostained for PICK1, and the trans-Golgi marker TGN38, and the trans-Golgi/immature vesicle marker syntaxin 6 before (top) and after 5 min BFA treatment (bottom). Panels show from left signal from immunolabeled PICK1 (Alexa Fluor 568 signal), signal from TGN38 (Alexa Fluor 488 signal), signal from syntaxin 6 (Alexa Fluor 647 signal), and overlay of the three channels. Insets highlight an area with overlapping localization of PICK1 and syntaxin 6 but with PICK1 adjacent to TGN38 (top) and colocalization of PICK1 with both syntaxin and TGN38 (bottom). (B) Quantification of the PICK1 colocalization with TGN38 and syntaxin 6 after 5 min BFA treatment using Van Steensel's cross-correlation function, which reports the Pearson cross-correlation as a function of the relative movement of the two channels with respect to each other. Both syntaxin 6 and TGN38 show a sharp peak of similar height close to Δx = 0, indicating specific co-localization. Co-localization was quantified for 10–20 cells from three independent experiments, and data are means ± SE. Note that the drop in peak values compared to no BFA (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001542#pbio-1001542-g006" target="_blank">Figure 6F</a>) likely reflects the increased diffuse localization of both markers after BFA treatment. (C) Fast time-lapse dual color live confocal imaging showing transient association of YFP-PICK1 BAR with the Golgi marker GalT-Cerulean. Images are maximal intensity projections of representative time-lapse series for YFP-PICK1 BAR (left), YFP PICK1 BAR V121E-L125E (middle), and YFP-PICK1 BAR 3KE (right). The gray scale projections (left images) show the YFP channel alone, indicating highly dynamic behavior of YFP PICK1 BAR (left) and more static behaviour of YFP-PICK1 BAR V121E-L125E (middle) punctae, whereas no punctae are seen for YFP-PICK1 BAR 3KE (right). The dual color projections (right images, YFP channel in yellow, Cerulean channel in blue) show that most of the activity for YFP-PICK BAR (left) is lining the Golgi, whereas YFP-PICK1 BAR V121E-L125E (middle) shows activity throughout the cell. Circles indicate transiently appearing punctate structures (lasting for less than 10 frames, ∼30 s). Crosses indicate stable punctate structures lasting for more than 10 frames. (D) Time-lapse series of two transient punctate structures of YFP-PICK1 BAR, and (E) two stable punctate structures of YFP-PICK1 BAR V121E-L125E. Scale bar, large images 5 µm, time lapse 1 µm. (F) Tubulation of surface-immobilized artificial giant membrane vesicles by the PICK1 BAR domain. Purified and Alexa Fluor 488–labeled GST-PICK1 BAR or GST-PICK1 BAR V121E-L125E (control) with impaired membrane binding capacity. Protein was incubated at room temperature with surface immobilized and fluorescently labeled vesicles before confocal fluorescent imaging. No effect was seen for the mutant, whereas massive tubulation was seen with GST-PICK1 BAR. Arrow indicates a budding vesicle from a single GV.</p